KROONINEN BORRELIOOSI

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Kroonisissa bakteeritaudeissa huomiota on alettu yhä enemmän kiinnittämään bakteerin soluseinää vailla olevaan muotoon (esim.kystamuoto) ja biofilmeihin. Tulevissa lääketutkimuksissa tulee kiinnittää huomiota nimenomaan näiden muotojen hoitoon. (2011)

Lyme disease: the next decade
Perspectives
(653) Article views
Authors: Raphael B Stricker, Lorraine Johnson
Published Date January 2011 , Volume 2011:4 Pages 1 - 9 DOI 10.2147/IDR.S15653

http://www.dovepress.com/articles.php?article_id=6013.

Raphael B Stricker, Lorraine Johnson
International Lyme and Associated Diseases Society, Bethesda, MD, USA

Abstract: Although Lyme disease remains a controversial illness, recent events have created an unprecedented opportunity to make progress against this serious tick-borne infection. Evidence presented during the legally mandated review of the restrictive Lyme guidelines of the Infectious Diseases Society of America (IDSA) has confirmed the potential for persistent infection with the Lyme spirochete, Borrelia burgdorferi, as well as the complicating role of tick-borne coinfections such as Babesia, Anaplasma, Ehrlichia, and Bartonella species associated with failure of short-course antibiotic therapy. Furthermore, renewed interest in the role of cell wall-deficient (CWD) forms in chronic bacterial infection and progress in understanding the molecular mechanisms of biofilms has focused attention on these processes in chronic Lyme disease. Recognition of the importance of CWD forms and biofilms in persistent B. burgdorferi infection should stimulate pharmaceutical research into new antimicrobial agents that target these mechanisms of chronic infection with the Lyme spirochete. Concurrent clinical implementation of proteomic screening offers a chance to correct significant deficiencies in Lyme testing. Advances in these areas have the potential to revolutionize the diagnosis and treatment of Lyme disease in the coming decade.

Keywords: Lyme disease, Borrelia burgdorferi, L-forms, cysts, biofilms, proteomics
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Solukalvoa vailla olevat

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Borrelia-bakeerit kykenevät vaikuttamaan taudin kulkuun muuttamalla antigeeniään ja pintaproteiinejaan. Proteiinien muuntelu vaikeuttaa immuunipuolustuksen yrityksiä tuhota bakteeri ja potilaalla voi esiintyä toistuvia kuumejaksoja.

Borrelia species are able to induce cycles of disease by varying antigen expression and by displaying new outer-surface proteins during the disease course. The proteins are named either variable small proteins or variable large proteins and are encoded within plasmid DNA. Alteration of these proteins prevents elimination of the spirochetes by the immune system, leading to recurrent febrile episodes.2 In 2008, Thein et al identified and described the first porin of relapsing fever, Oms38, which is present in the outer membranes of B hermsii, B turicatae, B duttonii, and B recurrentis.

http://emedicine.medscape.com/article/227272-overview

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Joukko vuosia aiemmin Borrelioosiin antibioottihoidon saaneita henkilöitä sai rytmihäiriöitä, fatiikkia ja kognitiivisia häiriöitä. Osalla esiintyi migreeniä, korkeaa verenpainetta, ahdistusta ja masennusta. Monet olivat oireiden vuoksi joutuneet jäämään pois työelämästä. (2011)


Cardiol J. 2011;18(1):63-6.

Postural orthostatic tachycardia syndrome following Lyme disease.

Kanjwal K, Karabin B, Kanjwal Y, Grubb BP.


Background: A subgroup of patients suffering from Lyme disease (LD) may
initially respond to antibiotics only to later develop a syndrome of fatigue,
joint pain and cognitive dysfunction referred to as 'post treatment LD
syndrome'. We report on a series of patients who developed autonomic dysfunction
in the form of postural orthostatic tachycardia syndrome (POTS).

Methods: All of
the patients in this report had suffered from LD in the past and were
successfully treated with antibiotics. All patients were apparently well, until
years later when they presented with fatigue, cognitive dysfunction and
orthostatic intolerance. These patients were diagnosed with POTS on the basis of
clinical features and results of the tilt table (HUTT) testing. Results: Five
patients (all women), aged 22-44 years, were identified for inclusion in this
study. These patients developed symptoms of fatigue, cognitive dysfunction,
orthostatic palpitations and either near syncope or frank syncope. The
debilitating nature of these symptoms had resulted in lost of the employment or
inability to attend school. Three patients were also suffering from migraine,
two from anxiety and depression and one from hypertension. All patients
demonstrated a good response to the employed treatment. Four of the five were
able to engage in their activities of daily living and either resumed employment
or returned to school.

Conclusions: In an appropriate clinical setting,
evaluation for POTS in patients suffering from post LD syndrome may lead to
early recognition and treatment, with subsequent improvement in symptoms of
orthostatic intolerance. (Cardiol J 2011; 18, 1: 63-66).

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 21305487 [PubMed - in process]

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Norja 2011: Noin puolella neuroborrelioosiin sairastuneista esiintyi huomattavia elämänlaatua heikentäviä tekijöitä esim. fatiikkia hoitojen jälkeenkin. Potilaita (50) seurattiin 30kk hoitojen päättymisestä.

"European persons treated for LNB have poorer health-related QoL and have more
fatigue than persons without LNB."



Acta Neurol Scand. 2011 Feb 9; [Epub ahead of print]

European neuroborreliosis: quality of life 30 months after treatment.

Eikeland R, Mygland A, Herlofson K, Ljostad U.

Department of Neurology, Sorlandet Hospital, Arendal, Norway Department of
Neurology, Sorlandet Hospital, Kristiansand, Norway Department of Clinical
Medicine, University of Bergen, Bergen, Norway Clinic of Rehabilitation,
Sorlandet Hospital, Kristiansand, Norway.

Eikeland R, Mygland A, Herlofson K, Ljostad U. European neuroborreliosis:
quality of life 30 months after treatment. Acta Neurol Scand: DOI:
10.1111/j.1600-0404.2010.01482.x. (c) 2011 John Wiley& Sons A/S.

Objectives -
The prognosis after Lyme neuroborreliosis (LNB) is debated. The aim of this
study was to assess health-related Quality of Life (QoL) and neurological
symptoms 30 months after treatment in European patients with LNB.

Materials and methods -
In a prospective case-control designed study, we investigated 50
well-characterized patients with LNB who had participated in a treatment trial
for LNB 30 months earlier and 50 matched control persons with the health QoL
questionnaire Short-Form 36 (SF-36), the Fatigue Severity Scale (FSS), the
Montgomery and Asberg Depression Rating Scale (MADRS), the Starkstein Apathy
Scale (SAS), and the Mini Mental State (MMS). Clinical and demographic data were
collected by semi-structured interviews and clinical neurological examination.

Results - Lyme neuroborreliosis-treated patients scored lower than control
persons in the SF-36 domains physical component summary (PCS) (44 vs 51 P<
0.001) and mental component summary (MCS) (49 vs 54 P = 0.010). They also scored
lower than control persons in all the SF-36 subscales, except for bodily pain,
and on FSS (3.5 vs 2.1 P< 0.001), but not on MMS (28 vs 29 P = 0.106). There
was a difference in MADRS (3.1 vs 0. 8 P = 0.003) and SAS (13 vs 11 P = 0.016),
but the scores were low in both groups. Fatigue was the most frequently reported
symptom among LNB-treated patients (50%). Patients who reported complete
recovery (56%) after LNB had similar QoL scores as the controls.

Conclusion -
European persons treated for LNB have poorer health-related QoL and have more
fatigue than persons without LNB. (c) 2011 John Wiley& Sons A/S.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 21303350 [PubMed - as supplied by publisher]

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http://www.duodecimlehti.fi/web/guest/u ... nnumero#s2

Duodecim
1999;115(4):413
Terho Heikkinen
Lehdistöreferaatit

Borrelia-artriitin pitkäaikaisennuste

Lymen taudin keksimisen jälkeen huomattava osa aiemmin reumana pidetyistä lasten niveltulehduksista onkin paljastunut borreliainfektion myöhäisiksi ilmentymiksi. Lymen artriitin pitkäaikaisennusteesta lapsilla on kuitenkin olemassa hyvin vähän tietoa. Yhdysvalloissa Connecticutin osavaltiossa tautia esiintyy runsaasti, ja siellä on nyt tehty selvitys vuosina 1982?91 Lymen artriittiin sairastuneiden 90 lapsen vaiheista 2?12 vuotta taudin toteamisen jälkeen (seuranta-ajan mediaani seitsemän vuotta). Vain 19:llä näistä lapsista oli todettu alkuvaiheen erythema migrans. Polvinivel oli ylivoimaisesti yleisimmin sairastunut nivel; 90 %:lla lapsista todettiin artriitti ainakin toisessa polvessa. Lapsista 85 oli hoidettu erilaisilla antibiooteilla, 17 suonensisäisillä. Viisi lasta ei ollut saanut lainkaan antibioottihoitoa. Tutkimuksessa todettiin, että noin puolella lapsista ensimmäinen artriittiepisodi jäi myös ainoaksi ja puolella esiintyi toistuvia artriitteja jopa useiden vuosien ajan. Kahdelle lapselle kehittyi krooninen niveltulehdus, ja heille tehtiin synovektomia. Tutkimusajankohtana yhdelläkään lapsella ei ollut enää todettavissa aktiivista niveltulehdusta. Neljällä lapsella esiintyi lieviä muskuloskeletaalisia vaivoja, mutta näiden yhteys sairastettuun Lymen artriittiin jäi avoimeksi. (Pediatrics 1998; 102: 905).

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Kroatia tapausselostus 2011: Kroonisen neuroborrelioosin oireena voi ilmetä vapinaa, kohtauksia ja psykoosi. Potilas sai iv + per os antibiootit ja hänen tilansa koheni huomattavasti, mutta hänelle jäi hoidon jälkeenkin useita systeemisiä ja neurologisia oireita jotka haittaavat arkielämää.

Coll Antropol. 2011 Jan;35 Suppl 1:313-8.

Tremor, seizures and psychosis as presenting symptoms in a patient with chronic lyme neuroborreliosis (LNB).

Markeljević J, Sarac H, Rados M.

University of Zagreb, School of Medicine, Zagreb University Hospital Centre, Department of Internal Medicine, Zagreb, Croatia. Jasenka-markeljevic@gmail.com

Abstract

Lyme borreliosis is a multisystem disorder caused by Borrelia burgdorferi (Bb). Neurological symptoms such as lymphocytic meningoradiculoneuritis (Bannwart's syndrome), cranial neuritis (II,III,IV,V,VI), encephalitis, transverse myelitis are found in about 10% of cases during the second phase of the disease.

In the chronic stage, many months or years after the initial infection, other neurologic complications may occur, such as encephalomyelitis, epileptic crises, cognitive impairment, peripheral neuropathy and psychiatric disturbances such as depression, anxiety, panicc attacks, catatonia, psychosis etc.
Some patients continue to experience symptoms of fatigue, insomnia or psychiatric disorder in the post borrelia syndrome.

We describe here a patient with a triad of unusual symptoms in chronic LNB including tremor, seizures and psychosis. Standardized medical interview, neurologic examination, neuroimaging, serum and CSF serology as well as EEG and EMNG evaluation were performed. The patient was treated with intravenous ceftriaxone and doxycycline and responded with rapid clinical and functional improvement. Nevertheless, he suffered from multiple systemic and neurologic sequelas that influenced his daily activities in post treatment period. Emphasis is placed on the atypical onset and evolution, the difficulties encountered in formulating diagnosis, early treatment and the uncertainties concerning the sequelae after treatment. In patients with non-specific long lasting symptoms in the absence of overt clinical signs suggesting CNS involvement, routine treatment with i.v. ceftriaxone is not to be encouraged.

PMID:
21648354
[PubMed - in process]

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Bb väistää immuunipuolustusta usein eri tavoin. Bakteeri menee imusolmukkeisiin ja huijaa immuunipuolustusta hyökkäämään infektiota vastaan voimakkaasti mutta riittämättömästi.

Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced Specific B Cell Activation

Acute Lyme Disease is one of the most important emerging diseases in the US. People with acute Lyme disease often develop swollen lymph nodes, or lymphadenopathy, but we do not know why this happens or what effect it has on the course of the disease. We show here that when mice are infected with live Borrelia burgdorferi spirochetes (the bacteria that cause Lyme disease), live spirochetes collect in the lymph nodes. These lymph nodes then swell up and start producing large numbers of antibody-producing cells.

Although many of these antibodies can recognize the bacteria, they apparently lack the quality to clear the infection. We hypothesize that by moving into the lymph node, usually a site in which strong immune responses are induced, Borrelia evades the immune response: it goes to the lymph nodes and tricks the immune system into making a very strong but inadequate response.


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Stefan S. Tunev1,2¤, Christine J. Hastey1,4, Emir Hodzic1, Sunlian Feng1, Stephen W. Barthold1,2,3,4, Nicole Baumgarth1,2,3,4*

1 Center for Comparative Medicine, University of California Davis, Davis, California, United States of America, 2 Graduate Group in Comparative Pathology, University of California Davis, Davis, California, United States of America, 3 Department of Pathology, Microbiology and Immunology, University of California Davis, Davis, California, United States of America, 4 Graduate Group in Microbiology, University of California Davis, Davis, California, United States of America
Abstract Top

Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.
Author Summary Top

Acute Lyme Disease is one of the most important emerging diseases in the US. People with acute Lyme disease often develop swollen lymph nodes, or lymphadenopathy, but we do not know why this happens or what effect it has on the course of the disease. We show here that when mice are infected with live Borrelia burgdorferi spirochetes (the bacteria that cause Lyme disease), live spirochetes collect in the lymph nodes. These lymph nodes then swell up and start producing large numbers of antibody-producing cells. Although many of these antibodies can recognize the bacteria, they apparently lack the quality to clear the infection. We hypothesize that by moving into the lymph node, usually a site in which strong immune responses are induced, Borrelia evades the immune response: it goes to the lymph nodes and tricks the immune system into making a very strong but inadequate response.

Citation: Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, et al. (2011) Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced Specific B Cell Activation. PLoS Pathog 7(5): e1002066. doi:10.1371/journal.ppat.1002066

Editor: Jenifer Coburn, Medical College of Wisconsin, United States of America

Received: October 29, 2010; Accepted: March 31, 2011; Published: May 26, 2011

Copyright: © 2011 Tunev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NIH grants T32 RR007038 (ST), T32-AI-0605 (CH), RO1 AI 26815 (SWB, EH, SF), and R01 AI073911 (NB, SWB, CH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: nbaumgarth@ucdavis.edu

¤ Current address: Medtronic Inc., Santa Rosa, California, United States of America
Introduction Top

Lyme borreliosis, caused by Borrelia burgdorferi transmitted by Ixodes spp. ticks, is the most common arthropod-borne illness in the US and Europe, and is increasing in prevalence and expanding in geographic distribution in the US [1], [2]. Clinical manifestations are highly varied, including involvement of the cutaneous, cardiovascular, musculoskeletal, and nervous systems [3]?[5]. A frequent, but largely under-studied manifestation is massive and systemic lymph node enlargement (lymphadenopathy), observed particularly in the regional lymph node near the site of infection in humans, and in experimentally-infected dogs [4], [6]. The lymph node enlargement that arises in both humans and dogs is characterized by increased cellularity and the accumulation of large pleomorphic IgM- and IgG-positive plasma cells [6]?[8]. Despite these unusual characteristics, the lymphadenopathy of Lyme borreliosis has not been well investigated.

Several in vitro studies have shown that culture-grown B. burgdorferi can act as mitogens when co-cultured with human or murine naive B cells [9]?[16]. Therefore, the unusual lymphadenopathy of Lyme borreliosis might be a manifestation of non-specific B cell activation. Massive lymph node enlargement has also been seen in wildtype but not TLR4 gene-targeted mice during infection with Salmonella typhimurium [17] and others have shown a role for TLR-independent, TNF-independent [18] or TNF-dependent [19] involvement of mast cells in non-specific induction of lymph node enlargement. Thus, innate immune activation might account for the lymphadenopathy observed during infection with B. burgdorferi.

On the other hand, there is ample evidence for the induction of specific immune responses following B. burgdorferi infection. Both following experimental and natural infections, B. burgdorferi-specific IgM and IgG antibodies are induced in the serum of infected humans [5], [20]?[24], dogs [25], and mice [26], among other host species. Importantly, passive transfer of immune-serum from chronically infected wildtype or T cell-deficient mice, from naturally infected dogs, and from human patients with chronic Lyme disease can protect mice from a challenge infection with B. burgdorferi [26]?[29], demonstrating that specific and protective antibodies are induced during the course of infection. However, once infection is established, the immune response is incapable of clearing infection [26], [30]. Thus, understanding the host immune response is critical to understanding and treating Lyme borreliosis.

The present study was undertaken to identify the mechanisms involved in the lymphadenopathy induced by infection with B. burgdorferi and to determine the nature and specificity of the reactive B cell response. Using a mouse model of infection with host-adapted spirochetes that faithfully recapitulates experimental and natural infections with ticks, we show that B. burgdorferi actively migrates into the lymph nodes, where it causes a largely specific, but unusual B cell response.
Materials and Methods Top
Mice and infections

Four to six week old female C3H/He, C57BL/6 and severe combined immunodeficient C57BL/B6.C-Prkdcscid (SCID) mice were obtained from The Jackson Laboratory, Bar Harbor, ME, and maintained at UC Davis in isolator cages under conventional housing conditions. Breeding pairs of C57BL/6.129P2/Ola-MyD88tm1Aki (MyD88 −/−) mice [31] were a generous gift of Richard Flavell (Yale University), given with kind permission from Shizuo Akira (Osaka University). The MyD88−/− mice were rederived and bred in the specific pathogen free barrier facility at UC Davis, and then transferred to conventional housing prior to experiment onset.

Mice were infected with B. burgdorferi in two ways: for tick-borne infections, five B. burgdorferi-infected nymphal ticks (or non-infected control ticks) were placed on the dorsal thoracic midline of mice and allowed to attach and feed to repletion. To generate host-adapted B. burgdorferi, SCID-mice were infected s.c. via syringe inoculation with 104 B. burgdorferi spirochetes grown to mid-log phase (day 5 of culture) in 0.1 ml of sterile medium. For infection with host-adapted spirochetes, 3 mm2 punch biopsies from infected SCID mice were obtained from the hairless, ethanol-cleaned ear pinnae. Biopsies were transplanted subcutaneously on the lateral side of the right tarsal joint of recipient naïve C57BL/6 mice. Ear transplants contained a mean of 1.8×104 spirochetes, based upon quantitative DNA analysis [32]. Control mice were transplanted at the same location with similar tissue from uninfected SCID mice (sham infection).
Ethics statement

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols involving animals were approved by the Animal Use and Care Committee at UC Davis (Permit Number: #15330).
Borrelia burgdorferi

A clonal strain of B. burgdorferi sensu stricto (cN40) was grown in modified Barbour-Stoenner-Kelly (BSK II) medium [33] at 33°C and enumerated with a Petroff-Hauser bacterial counting chamber (Baxter Scientific, McGaw Park, IL). Heat-inactivation of B. burgdorferi was done at 56°C for one hour followed by sonication. Aseptically collected samples of lymph nodes, spleen, inoculation site, and urinary bladder were taken at necropsy and cultured for 7 and 14 days in BSK II medium to assess the presence of spirochetes under dark field microscopy.
Ticks

Uninfected larval Ixodes scapularis ticks were obtained from field-collected adults in southern Connecticut (kindly provided by Durland Fish, Yale University). All larvae for the experiments described in this study were derived from a single cohort. A sample of the cohort was confirmed to be B. burgdorferi flaB negative by PCR. To generate infected nymphs, larvae were allowed to engorge on C3H mice that had been infected with B. burgdorferi for 2 weeks following syringe inoculation, as described previously [34]. Following feeding and molting, cohort analysis of the infected nymphal ticks revealed that 97% of the ticks were confirmed to be PCR positive for B. burgdorferi flaB as previously described [34].
Histology and immunohistochemistry

Lymph nodes were fixed in neutral buffered formalin, embedded in paraffin and sectioned at 4 mm and stained with hematoxylin and eosin or by immunohistochemistry. Sections for immunohistochemistry were processed at room temperature and placed on positively charged slides, air-dried, de-paraffinazed and re-hydrated. Endogenous peroxidase activity was eliminated by incubation in 3% H2O2 in methanol for 20 minutes. Non-specific binding was reduced with biotin blocking solution (Vector) for 15 minutes and Power Block (InnoGenex) for 15 minutes. Immunohistochemical labeling of B. burgdorferi was performed by treating sections with 0.5 mg/ml protease type VIII (Sigma Aldrich) for 10 minutes, followed by 30 minutes incubation with 1:1000 dilution of a polyclonal immune serum from B. burgdorferi-infected rabbits (infected for two months following inoculation with 104 spirochetes). Antigen detection utilized a three-step streptavidin-horseradish peroxidase technique with the substrate DAB (Vector). For other antigens, antigen retrieval was enhanced by microwaving tissue sections for 6 minutes in citrate buffer at pH 6.0. Sections were then incubated with antibodies to B220 (CD45R, RA3-6B2), CD138 (281-2, BD Biosciences), or Ki-67 (NeoMarkers), followed by incubation with biotinylated secondary antibodies (Vector), streptavidin conjugated Alexa 488 and Alexa 594 (Molecular Probes) or streptavidin-horseradish peroxidase followed by DAB (Vector), and mounting with Prolong Antifade (Molecular Probes).
Flow Cytometry

Live cell counts of single cell suspensions of lymph nodes were obtained using a hemocytometer and trypan blue exclusion of non-viable cells. Staining was performed using aliquots of 6.25×105 cells in ?staining medium? (buffered saline solution: 0.168 M NaCl, 0.168 M KCl, 0.112 M CaCl2, 0.168 M MsSO4, 0.168 M KH2PO4, 0.112 M K2HPO4, 0.336 M HEPES, 0.336 M NaOH, containing 3.5% heat-inactivated, filtered newborn calf serum and 1 mM EDTA) for 20 min on ice. The following antibody-conjugates were used at previously determined optimal concentrations: CD19-Cy5PE, CD3-APC Efluor780 (both e-biosciences), CD4-FITC, and CD8a-Cy5.5PE (both in-house generated) after blocking Fc receptor with anti-CD16/32 (2.4G2). Dead cells were discriminated with a live/dead violet staining kit (Invitrogen). Data acquisition was performed on a 13-color FACSAria instrument (BD Biosciences) [35]. Data were analyzed using FlowJo software (kind gift from Tree Star Inc.).
Elispot

To probe for B. burgdorferi-specific antibody-producing cells by ELISPOT, 96-well plates (#MAHAS4510, Mixed Cellulose Ester Membrane; Millipore) were coated with 2.5 µg/mL of four recombinant non-lipidated B. burgdorferi N40 proteins: decorin binding protein A (DbpA), outer surface protein C (OspC), arthritis-related protein (Arp), and borrelia membrane protein A (BmpA) in PBS overnight. After blocking with PBS/4% BSA, lymph node cell suspensions were 2-fold serially diluted in medium (RPMI 1640, 292 µg/mL L- glutamine, 100 µg/mL of penicillin and streptomycin, 10% heat inactivated FCS and 0.03 M 2-ME) and cultured overnight at 37°C with 5% CO2. Cells were lysed with water and binding was revealed by incubation with biotin conjugated anti-IgM (Southern Biotech) or anti-IgH+L (Southern Biotech) for 2 hours in 2% BSA in PBS. This was followed by SA-HRP incubation for 1 hour (Vector Laboratories) in PBS/2% BSA and by 3-amino-9-ethylcarbazole (Sigma-Aldrich). Plates were washed and dried and mean spots were counted in all wells with visible spots and calculated as mean spot numbers per input cell number.
Expression and purification of recombinant proteins

Genes encoding non-lipidated B. burgdorferi N40 proteins, previously identified by genomic expression library analysis to react with serum from B. burgdorferi-infected mice, as described [36], were amplified by PCR from B.burgdorferi N40 DNA using oligonucleotide primers based on their DNA sequences (Supplemental Table S1). Template DNA from the original reactive clone was denatured at 94°C for 1 min, annealed at 55°C for 1 min, and extended at 72°C for 1 min. This process was repeated for 30 cycles. The amplified genes were cloned in frame with the glutathione S-transferase (GT) gene into pMX, derived from a pGEX-2T vector (Pharmacia, Piscataway, N.J.) with a modified polylinker. The PCR-amplified DNA sequences were confirmed by sequence comparison with the original inserts.

E. coli DH5α cells transformed with the recombinant pMX vectors were grown to an optical density of 0.5 at 600 nm and the recombinant GT fusion proteins were induced with 1 mM IPTG for 2 h. Bacteria were centrifuged at 3,310 g for 20 min, pellets were washed with PBS and bacteria lysed with PBS/1% Triton X-100. The mixtures were sonicated and centrifuged at 35,000 g. Supernatants containing recombinant proteins were loaded onto glutathione-Sepharose 4B columns (Pharmacia), 25 U of thrombin was added to remove the GT partner, and purified proteins were eluted after 2 h.
B. burgdorferi lysate preparation

B. burgdorferi was grown to log-phase (8?10 days), pelletted by centrifugation, resuspended in cold PBS plus MgCl2 and centrifuged repeatedly for 5 min at 4°C 17,500 g. Samples were stored in aliquots at −20°C. Protein concentration was determined using Bradford assay (Bio-Rad).
Hybridoma generation

B cell hybridomas were created from enlarged lymph nodes collected at various times after infection with host-adapted B. burgdorferi. Three independent fusions were performed using standard protocols. Briefly, single cell suspensions from mechanically disrupted lymph nodes were fused with P3-X63Ag8.653 mouse myeloma cells (ATCC CRL-1580) using PEG 1450 (ATCC). Hybridomas were selected by incubating cells in HAT medium. Supernatants of all wells with visible cell growths were screened by ELISA for the presence of mouse Ig as previously described [37]. Hybridoma lines were established from all Ig-producers and tested further for reactivity against B. burgdorferi- specific recombinant antigens and whole B. burgdorferi lysate. Some hybridomas were then subcloned. The Ig heavy and light chain isotype profiles of the lines and clones were determined using the Mouse Immunoglobulin Cytometric Bead Array Kit, (BD Biosciences, Cat Number 550026).
Statistical analysis

Statistical analysis was performed using the two-way ANOVA or Student's t-test with help of Prism 5 software (GraphPad Software). A p-value of <0.05 was considered statistically significant.
Results Top
Mice infested with B. burgdorferi-infected ticks develop regional and distant lymphadenopathy

Since lymphadenopathy has not been documented in laboratory mice following B. burgdorferi infection, we first sought to determine if and when lymphadenopathy developed in laboratory mice infected experimentally with B. burgdorferi via the natural route, i.e. via tick-bite. For that, B. burgdorferi-genetically susceptible C3H/He mice [38] , were each infested with either 5 infected nymphal ticks or with 5 uninfected nymphal ticks (sham-infected). All ticks were placed on the dorsal cervico-thoracic midline. However, the ticks subsequently migrated and attached to different regions of the body, particularly the head and neck region. The most common tick attachment sites were the ear pinnae and face.

Axillary, brachial, lumbar and inguinal lymph nodes, among others, were collected at various times after infection and examined for visible signs of enlargement (not shown) and to determine cell number counts. Lymph node enlargement was noticed for all lymph nodes from mice exposed to B. burgdorferi infected ticks but not uninfected ticks (Figure 1A and data not shown). By day 14 following infestation with infected ticks, the lymph nodes closest to the tick-attachment site (axillary and brachial) were visibly enlarged and contained significantly increased numbers of cells in comparison to the same lymph nodes collected from the sham-exposed mice. Lymph nodes more distant from the attachment site (inguinal and lumbar) showed a slightly delayed increase in cellularity (Figure 1A). Thus, infection of laboratory mice with tick-borne B. burgdorferi faithfully recapitulates the lymphadenopathy observed in naturally infected humans and dogs, and suggests a relationship between time of lymph node enlargement and proximity to the site of infection.
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Figure 1. Tick-borne infection with B. burgdorferi causes systemic lymphadenopathy in mice.

C3H/HeN mice (n = 30) were each exposed to five B. burgdorferi-infected nymphal Ixodes scapularis ticks (or non-infected ticks). At indicated times after tick-attachment, groups of five mice were necropsied and (A) cellularity of four indicated lymph nodes and (B) total antibody-forming cells (AFC) of these lymph nodes were assessed by hemocytometer count and ELISPOT analysis, respectively. Shown are mean values ± SD per timepoint for each lymph node type. ELISPOT analysis of lymph nodes from sham-infected mice showed no significant induction of antibody production (data not shown).
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Rapid lymphadenopathy in regional lymph nodes of mice infected with host-adapted spirochetes

To directly assess the spatio-temporal relationship between the kinetics of the lymph node enlargement and the site of B. burgdorferi infection, a different infection modality was needed. Ticks change their attachment location in ways that varied significantly between mice, precluding targeted analysis of specific lymph nodes. Direct inoculation of mice with culture-grown B. burgdorferi, on the other hand, introduces untoward experimental variables due to the significant antigenic changes that B. burgdorferi undergoes as it adapts to the vertebrate host. One example is the antibody-response to the major outer surface protein A (OspA), which is strongly expressed in vitro and in ticks [39], but virtually absent in mice infected with B. burgdorferi via tick-infestation or following transplantation of tissue from infected mice containing host-adapted spirochetes [27]. Thus, infection via injection of culture-grown bacteria may favor distinct immune responses that differ from those seen after tick-infection. We therefore transplanted punch biopsies of ear pinnae from infected SCID mice, containing host-adapted spirochetes under the skin of the right tibiotarsus area of congenic, naïve C57BL/6 mice. The right inguinal lymph nodes were evaluated as the regional lymph nodes.

Infection of C57BL/6 mice with host-adapted B. burgdorferi resulted in a rapid enlargement of their regional inguinal lymph nodes (Figures 2A, 2B). These increases closely resembled the lymphadenopathy observed at the site of tick-attachment following tick-borne infection, albeit with somewhat faster kinetics (Figure 2C), possibly due to the increased time between tick-attachment and actual infection, and/or the time it takes for B. burgdorferi to adapt to the host-environment prior to dissemination [34]. Similar to tick-borne infection, infection with host-adapted spirochetes caused a generalized lymphadenopathy, with lymph nodes more distant from the infection-site increasing slower in cell numbers compared to those closest to the site of infection (Table 1). Thus, this infection model faithfully recapitulated tick-borne B. burgdorferi-induced lymphadenopathy with the advantage that we can consistently identify the lymph nodes draining the site of infection. The spleen was not increased in size or cellularity following either tick-borne infection (not shown) or following infection with host-adapted spirochetes (Table 1).
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Figure 2. Infection with host-adapted B. burgdorferi induces lymphadenopathy near the site of infection.

Lymphadenopathy of the right inguinal lymph node of C57BL/6 mice 10 days after infection via subcutaneous transplantation of small pieces of ear from B. burgdorferi-infected (A) or non-infected (B) congenic SCID mice into their right dorsal tarsal region. (C) Shown are mean cell numbers ± SD of right inguinal lymph nodes from groups of four mice per timepoint. (D) Comparison of lymph node celluarity (mean ± SD) obtained from draining lymph nodes in (C) and from the axillary lymph nodes of tick-infected mice as shown in Figure 1.
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Table 1. B. burgdorferi presence correlates with lymphadenopathy in mice infected with host-adapted spirochetes.
doi:10.1371/journal.ppat.1002066.t001
Lymphadenopathy is caused by the presence of extracellular B. burgdorferi in the lymph nodes

Since proximity to the infection site was correlated with an increase in lymph node cellularity, we investigated next if and when B. burgdorferi could be cultured from the lymph nodes. Within 24 h following infection with host-adapted spirochetes, B. burgdorferi was cultured from the closest draining right inguinal lymph nodes, but not any other lymph nodes (Table 1). By 48 h, the right lumbar lymph nodes became culture-positive. The right axillary lymph nodes yielded positive culture results two days later and before any of the contralateral lymph nodes on the left side of the mouse. A few days after the lymph nodes became culture-positive (about 4?6 days), a marked increase in cellularity of the lymph nodes was consistently observed for all lymph nodes, but not to the degree as the most proximal regional lymph nodes (Table 1). Once culture-positive, the lymph nodes remained so for the 90-day study period. Culture results from the spleen did not reveal B. burgdorferi until day 10 and then also only intermittently thereafter (Table 1).

These data suggested that the lymphadenopathy observed during Lyme borrreliosis is caused by a massive increase in lymph node cellularity triggered by the accumulation of live B. burdorferi spirochetes into the lymph nodes. Alternatively, it was possible that the culture results were a mere reflection of the presence of B. burgdorferi in the lymph node capsule, given that the spirochete travels along connective tissues. In that case, the increase in lymph node cellularity would be an indirect consequence of the infection-induced inflammation rather than the presence of the spirochetes in the lymph nodes. To distinguish between these possibilities, immunohistochemistry was utilized to determine the precise tissue-location of the spirochetes in lymph nodes of mice infected for 8 days with host-adapted B. burgdorferi. The results demonstrated the consistent presence of B. burgdorferi spirochetes in the sub-capsular sinus and superficial cortex of infected lymph nodes (Figures 3A, 3B). Interestingly, the spirochetes were found in the lymph nodes extracellularly appeared intact with characteristic spiral morphology. Together with the results from the culture experiments (Table 1), this suggests a degree of persistence of B. burgdorferi in lymph nodes.
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Figure 3. B. burgdorferi localizes to the subcapsular sinus of lymph nodes.

(A, B) Immunohistochemistry with polyclonal B. burgdorferi-specific rabbit serum, demonstrates the presence of extracellular B. burgdorferi spirochete (arrow) in the subcapsular sinus of the right inguinal lymph node of C57BL/6 mice infected by tissue-transplant eight days prior to analysis. (C) Shown are mean cell numbers of right inguinal lymph nodes of C57BL/6 mice (n = 2 per timepoint) either infected via injection of 104 culture-grown live (live) or heat-killed and sonicated B. burgdorferi in PBS. ?+? identifies positive B. burgdorferi cultures of lymph nodes (n = 2 per timepoint) from the same group of mice, ?−? shows lack of B. burgdorferi growth in culture. Results are from one of two independent experiments that gave similar results. (D?F) Comparison of mice infected as in (C) but injected with 106 inactivated B. burgdorferi for 8 days prior to analysis (D) Total numbers of cells recovered from lymph nodes. Each symbol represents results from one animal; horizontal bars indicate the mean for the group. (E) Data shown are total numbers CD4, CD8 T cells and CD19 B cells calculated from total cell counts and frequencies of cell subsets as determined by flow cytometry. (F) Shown are frequencies of IgG antibody-secreting cells in lymph nodes of mice given live or inactivated B. burgdorferi. Note data are normalized to B cell numbers. Each symbol represents the results from one mouse; the horizontal line indicates the mean for the group.
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Because lymphadenopathy was correlated with the presence of viable spirochetes, we determined next if lymphadenopathy could also be induced with inactivated spirochetes. For that, mice were either infected directly by subcutaneous inoculation in the right tarsal region with 104 viable cultured spirochetes or by the same number of spirochetes after inactivation by sonication. Inactivation was confirmed by culture of the sonicate. Right inguinal lymph nodes were collected at days 0, 10 and 21 days post infection and either cultured in BSK II medium or assessed for cellularity. At days 10 and 21 post infection, B. burgdorferi was cultured from the right inguinal lymph node of mice infected with live spirochetes but as expected, not from mice inoculated with inactivated spirochetes. Importantly, increases in lymph node cellularity were not observed in mice receiving inactivated B. burgdorferi, but were clearly induced in mice inoculated with viable B. burgdorferi (Figure 3C).

Since B. burgdorferi might replicate in vivo and thus the results might reflect application of differing amounts of bacteria or bacterial antigen between these two groups, the analysis was repeated by giving 100-fold higher amounts of inactivated bacteria (106 organisms). While the increased amount of inactivated bacteria resulted in lymph node enlargement compared to control mice (Figures 3C, 3D), the enlargement was significantly less (p = 0.001) than that seen with viable Borrelia (Figure 3D). Thus, we conclude that lymphadenopathy during B. burgdorferi infection is caused by the accumulation of viable spirochetes in lymph nodes.
Increase in lymph node cellularity is due to massive expansion of B cells

Next, the cause of the increase in cellularity of the lymph nodes was investigated. Immunohistochemistry on day 10 after infection demonstrated stark differences in lymph node organization compared to lymph nodes from uninfected mice (Figures 4A, 4B). Morphologically, the cortex of infected regional lymph nodes consisted of tightly packed extrafollicular lymphocytes and very few scattered, poorly demarcated germinal centers without a distinct mantel zone. Indeed follicular structures appear largely absent in these lymph nodes (Figures 4B, 4C). The majority of the lymphocytes in the cortex were positively labeled by B220 and thus identified as B cells (data not shown). The largest extrafollicular B cells were frequently arranged in distinct clusters interpreted as antibody forming foci. These large B cells were characterized by an open euchromatic nucleus with marginated chromatin and a large prominent nucleolus and moderate amounts of cytoplasm, characteristics of plasmablasts.
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Figure 4. B. burgdorferi-induced lymphadenopathy is caused by a massive proliferation and differentiation of lymph node B cells.

(A?C) H.E. staining of right inguinal lymph node from a C57BL/6 mouse (A) sham-infected or (B) infected by tissue-transplantation with host-adapted spirochetes 10 days prior. (C) Close-up image of (B). Note the complete lack of follicular and extrafollicular structure. (D) Immunohistochemistry on lymph nodes as in (B, C) reveals the presence of large numbers of Ki67+ proliferating cells in the lymph node cortex. (E) Three-color immunofluorescence identifies the Ki67+ (red) cells as B220+ (green) B cells. Blue color indicates nuclei as stained with DAPI. (F) Large numbers CD138+ plasma cells accumulate in the medullary cords of lymph nodes of infected mice as (in B/C) (G) Shown are mean cell numbers ± SD from 4 C57BL/6 mice per time point of CD4, CD8 T cells and CD19+ B cells in inguinal lymph nodes before (white) and at day 10 (black) of infection. Data were calculated from flow cytometric evaluation of live cell frequencies and total live cell counts by hemocytometer with trypan blue exclusion. Results are from one of >3 experiments performed that gave similar results. Statistical analysis was conducted by comparing results from each cell population using Student's t test; n.s. not significant. (H) Shown are mean numbers of antibody-secreting cells (AFC) ± SD for total (grey bars) and Borrelia-lysate (black bars)-specific antibodies in inguinal lymph nodes from 4 C57BL/6 mice per timepoint at the indicated times after infection with host-adapted B. burgdorferi. Horizontal bar indicates threshold levels of detection.
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To distinguish between increased trapping of migrating cells versus expansion of cells within the regional lymph nodes, cells were labeled for Ki-67 antigen to identify proliferating cells. The staining demonstrated large numbers of B cells in the cortex that were actively dividing (Figure 4D). Cells in the paracortex that were negative for B220 rarely expressed Ki-67, suggesting that cell division was restricted to the B cell population only (data not shown). Dual fluorescent labeling of Ki-67 and B220 identified the dividing cells as B cells (Figure 4E). The exclusive and extensive expansion of B cells was further confirmed by flow cytometry. Whereas the lymph nodes showed no significant increases in either CD4 or CD8 T cells compared to lymph nodes from non-infected mice, CD19+ B cells had expanded dramatically by day 10 of infection with live B. burgdorferi (Figure 4G). The massive increases in B cells were absent in mice injected with inactivated B. burgdorferi (Figure 3E).

In addition, the medullary cords of the lymph nodes from the infected mice showed the presence of large numbers of plasma cells, identified by staining with CD138 (Figure 4F), suggesting a strong induction of antibody secretion in the affected lymph nodes. Indeed, ELISPOT analysis on regional lymph nodes infected for up to 60 days with B. burgdorferi showed the presence of large numbers of antibody-forming cells (AFC), with peak responses noted around day 10 of infection (Figure 4H). Depending on the day of study between 1?3% of the AFC secreted antibodies were bound to the Borrelia lysate (Figure 4H). The strong antibody secretion within the lymph nodes following infection with host-adapted spirochetes was in magnitude and kinetics very similar to the induction seen following tick-borne infection with B. burgdorferi (Figure 1B), but was much larger than seen after injection of inactivated bacteria (Figure 3F). In summary, expansion of the lymph node cortex by reactive B cells and extrafollicular antibody forming foci constitutes the morphological basis of lymphadenopathy in Lyme borreliosis.
B cell expansion and differentiation following infection with B. burgdorferi is at least in part antigen-specific

The finding of active migration of B. burgdorferi into lymph nodes, i.e. an organ responsible for immune response induction, appeared counter-intuitive for an organism that aims to establish persistent infection. Therefore, we aimed to determine next whether B. burgdorferi might cause immune subversion in these lymph nodes. In particular we asked whether it was inducing massive non-specific B cell expansion and differentiation to antibody-secreting cells at the expense of an effective Borrelia-specific antibody response. Addressing this question is complicated by the fact that protein expression of culture-grown spirochetes does not fully resemble Borrelia in the host, i.e. the usefulness of protein lysates from culture-grown bacteria is limited as a source of antigen for ELISPOT analysis.

Initial studies were therefore conducted to identify a number of Borrelia antigens that are expressed in the host and induce robust antibody responses. A screen of available recombinant Borrelia-expressed antigens by ELISPOT analysis with lymph node cells from day 14 Borrelia-infected mice showed that lymph nodes had measurable reactivity against all of the recombinant antigens tested. Interestingly, DbpA had the highest level of reactivity, while the Borrelia lysate, included as a ?positive? control, identified a much smaller fraction of Borrelia-reactive AFC (Figure 5A). Further analysis showed that it was possible to pool various Borrelia antigens for ELISPOT analysis without losing sensitivity of reactivity against each antigen (data not shown). A pool of four recombinant antigens, consisting of DbpA, OspC, Arp, BmpA was used as a means of measuring the Borrelia-specific antibody response. While it clearly underestimates the number of total Borrelia-specific responses, testing with the pool of recombinant proteins that are expressed during infection was found to be more sensitive than testing with Borrelia lysate.
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Figure 5. Strong induction of B. burgdorferi-specific antibodies following infection.

(A) Indicated recombinant B. burgdorferi antigens and lysate were tested as coating reagents for ELISPOT to enumerate B. burgdoferi-specific antibody-secreting cells (AFC) in the inguinal lymph nodes of C57BL/6 mice infected for 14 days with host-adapted B. burgdorferi. Shown are mean numbers of antibody-secreting cells (AFC) ± SD from 5 mice. (B) A pool of recombinant proteins (DbpA, OspC, Arp and BmpA) was used to determine the frequencies of B. burgdorferi-specific antibody-secreting cells (black bars) by ELISPOT in C57BL/6 mice at indicated times after infection. Grey bars indicate the total number of IgG-AFC per lymph node. For each timepoint two infected mice and two sham-infected control mice were analyzed. Shown are the mean numbers B. burgdorferi-specific antibody-secreting cells ± SD assessed for the individual mice from which cell spots seen in sham-infected mice were subtracted. SD was calculated from titration curves of 3 replicates for each of the two mice analyzed. (C) Shown are the mean frequencies ± SD of B. burgdorferi-specific antibody-secreting cells expressing the indicated Ig-isoytypes or total Ig (black) on days 8 and 14 after infection. Data are from 4 mice per group and timepoint.
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A time course analysis of C57BL/6 mice infected for up to 90 days with host-adapted B. burgdorferi showed the robust induction of antibody-secretion within the regional lymph nodes (Figure 5B). The kinetics of the Borrelia-specific response was identical to that of the total antibody responses measured at the site (compare Figure 4H with Figure 5B). Depending on the day of analysis between 4?13% of AFC were shown to be specific for one of the four recombinant proteins included as antigens in the analysis. The isotype profile of the specific response showed a broad representation of all measured isotypes. More than half of the antibody-secreting cells appeared to generate IgM antibodies and IgG antibody isotypes classically associated with T-independent responses (IgG2b and IgG3, Figure 5C). Overall, the isotype profile of the Borrelia-specific response suggested that a considerable proportion of the B cell response might be T-independent, consistent with previous observations [29], [40].
Borrelia-specific B cell responses are strongly induced in regional lymph nodes following infection

The ELISPOT results suggested that a significant fraction of the induced B cell response was specific and directed against B. burgdorferi. However, given that we probed with only some of the many other Borrelia proteins that are potentially expressed selectively in vivo, assessment of the relative contribution of the specific over the non-specific response was difficult. Therefore, another series of experiments was conducted in which hybridomas were generated from the regional lymph nodes to assess the fraction of hybridomas directed against Borrelia-specific antigens with an expanded list of recombinant Borrelia proteins. Three successful fusions were conducted, including one on lymph nodes at day 8 of infection, and two on day 18. The overall results from these three fusions were similar (Figure 6A). Initial screening of roughly 1000 wells per fusion identified between 150?350 wells that showed antibody-secretion. Further screening of the antibody-secreting lines indicated that between 14?24% of the hybridoma lines generated antibodies that could be identified to react against an expanded list of recombinant Borrelia antigens (DbpA, OspC, Arp, BmpA, P23, P29, P32, P61 (defined in Supplemental Table S1) [41] and/or Borrelia lysate from cultured spirochetes.
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Figure 6. Lymph nodes of B. burgdorferi-infected mice contain large numbers borrelia-specific B cells.

Shown are the results of three independent hybridoma fusion experiments. (A) Shown are stacked bars with the total number of antibody-secreting cells (black) and the number of lines generating B. burgdorferi-specific antibody as assessed by ELISA with four recombinant B. Burgdorferi antigens. Cells for the fusion were from indicated lymph nodes and times after infection with host-adapted spirochetes. (B) Indicated are the Ig-isotype distribution among all B. burgdorferi-specific hybridoma lines generated.
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From these fusions, 132 hybridoma lines were cultivated. Of these, 45 were specific for B. burgdorferi antigens. The isotype profile of the 45 hybridoma lines matched very closely that observed by ELISPOT on Borrelia-infected lymph nodes, indicating that the hybridoma lines were recapitulating the responses in vivo (Figure 6B). Furthermore, the largest fraction of the 45 Borrelia-specific hybridomas (11/45) recognized DbpA (data not shown). This is consistent with the initial specificity screen by ELISPOT (Figure 5A). Given that the recombinant antigen pool was likely to underestimate the frequencies of antigen-specific B cells/hybridomas, we conclude that a sizable fraction of the massive B cell response induced during Lyme borreliosis is specific against B. burgdorferi.
Lymphadenopathy and B cell induction are independent of MyD88

Non-specific mitogenic stimulation of B cells with Borrelia lipoproteins in vitro has been reported previously [9]?[16]. OspA, a surface lipoprotein that is strongly expressed by Borrelia in culture, but down-regulated upon infection of a mammalian host, was shown to be responsible for at least some of the mitogenic activity [11], [14]. While host-adapted spirochetes are not expected to express significant amounts of OspA, other proteins or lipids may provide mitogenic signals to B cells in vivo. Therefore, we determined the role of the adaptor protein MyD88, important in TLR and IL-1-mediated innate signaling, in regulation of initial B cell activation and/or the lymph node enlargement. A previous study found impaired pathogen-clearance and alterations in the antibody-isotype profile of serum antibodies in mice lacking MyD88 [42]. MyD88−/− mice and congenic control mice were infected with host-adapted spirochetes for ten days. The analysis revealed no role for MyD88 in the quality or magnitude of the lymphadenopathy. Regional lymph nodes from MyD88−/− mice had similar cell numbers on day 10 of infection (Figure 7A), with similar predominance of CD19+ B cells compared to control mice (Figure 7B). Furthermore, there was no difference in the number of Borrelia-specific IgM or total Ig secreting cells in the lymph nodes (Figures 7C, 7D). Thus, MyD88-dependent innate signaling is not driving the induction of lymphadenopathy, nor the massive activation of B cell responses associated with Lyme borreliosis. Together with the strong antigen-specific B cell responses measured by hybridoma generation, the results suggest that Borrelia-infection induces a specific, albeit largely extrafollicular B cell response as a result of the accumulation of live B. burgdorferi in lymph nodes.
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Figure 7. Lymphadenopathy and lymph node B cell activation are independent of MyD88-signaling.

Control C57BL/6 (wildtype) and congenic MyD88−/− mice (n = 6 per group) were infected with host-adapted B. burgdorferi for 10 days. Lymph nodes were harvested and compared for (A) total cellularity and (B) numbers of CD4, CD8 and CD19 as assessed by flow cytometry. ELISPOT analysis was conducted to determine numbers of (C) borrelia-specific IgM or (D) all Ig-isotype secreting borrelia-specific cells. For scatter plots, each symbol represents the result from an individual animal. Lines indicate mean of the group. Bar chart shows the mean values ± SD. Results are pooled from two independent experiments. Statistical analysis for (A) (C) (D) was conducted by Student's t test and (B) by two-way ANOVA. None of the data showed significant differences between control and MyD88−/− mice.
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Discussion Top

This study provides new insights into the pathogenesis of lymphadenopathy during the early stages of human Lyme borreliosis. The results demonstrate for the first time the extracellular accumulation of B. burgdorferi in the cortical regions of lymph nodes and implicate the direct association of migrating B. burgdorferi spirochetes with a marked and specific but unusual B cell response in the lymph nodes, but not the spleens, of mice infected with tick-borne or host-adapted spirochetes. The strong accumulation of proliferating B cells in the cortical areas of the lymph nodes, in the absence of a simultaneous accumulation of CD4 T cells (Figures 3, 4), the lack of strongly demarcated lymph node follicles and germinal centers in the lymph nodes (Figures 4B, 4C), and the strongly IgM and IgG3/2b-driven specific antibody response (Figures 5, 6) indicate that this pathogen drives the borrelia-specific B cell response towards T cell-independence. From these results we hypothesize that these effects of B. burgdorferi on the Borrelia-specific B cell response constitute a novel immune-evasion strategy.

The active migration of B. burgdorferi into sites of immune induction appears counter-intuitive for an organism that aims to establish persistence. Their presence in the lymph nodes and the strong responses their presence evokes thus indicate the intricate balance this pathogen achieves between immune induction and immune evasion. The nature of the observed B cell response is clearly distinct from that observed following acute infections with other non-persistent pathogens, or following immunizations with various protein antigens that induce mainly T cell-dependent extrafollicular and germinal center responses [43], [44]. In particular we note a lack of clearly demarcated follicles, with it an apparent lack of germinal centers and the accumulation of proliferating B cells in the follicles. The presence of T-independent B cell responses to B. burgdorferi had previously been indicated by measurements of strong antibody-responses in the serum of T cell-deficient mice [29]. While we cannot fully exclude the possibility that it is the nature of the expressed Borrelia-antigens that drive the B cell response towards its extrafolicular nature (Figure 4C) and apparent T-independence, we believe that this cannot fully explain our observations. A major component of the B cell response induced to infection with host-adapted spirochetes was directed against decorin-binding protein (DbpA) (Figures 5, 6). When administered in adjuvant, a strong germinal center-response was observed in the draining lymph nodes and DbpA-specific antibody responses were strongly induced against this protein, suggesting that this major B. burgdorferi immunogen is capable of inducing T-dependent responses in the right context (unpubl. observations). Furthermore, immunization with DbpA does induce protective antibody responses [36].

Also, the strongly B cell-driven lymphadenopathy seen following B. burgdorferi infection was not observed following immunization of mice with culture-grown heat-killed and sonicated spirochetes (Figures 3C, 3E), although such immunization increased both lymph node size (Figure 3D) and induced moderate frequencies of B. burgdorferi-specific antibody-secreting cells (Figure 3F). Thus, either live infection and/or the presence of live extracellular bacteria and bacterial proteins in the cortex areas of the lymph nodes appear to trigger this unique B cell response to spirochetes, or alternatively, the response is triggered by an antigen(s) not present on the culture-grown bacteria used for immunization. Since neither the lymphadenopathy nor the B cell response were significantly different following infection of MyD88−/− mice compared to controls (Figure 7), TLR-mediated inflammatory responses can be excluded as potential triggers of this response, in contrast to apparently similar TLR-4-mediated alterations following S. typhimurium infection [17].

From this, it is tempting to speculate that it is the expression of specific immune-subversion antigens by B. burgdorferi in the mammalian host that induce overshooting and potentially aberrant T cell independent B cell responses that are neither of sufficient high-affinity nor induce memory responses able to combat primary and repeat infections. The analysis of candidate antigens must await the development of techniques that allow us to comprehensively compare protein expression by culture-grown and tissue-adapted spirochetes within the context of specific tissue sites, such as lymph nodes.

While the induced B cell response to B. burgdorferi is unable to clear the infection, it does provide immune protection from overt disease. This is indicated by studies in B cell- or CD40L-deficient mice that showed increased signs of tissue-inflammation and disease progression compared to controls [29], [40], [45]. Furthermore, passive transfer of immune serum from infected mice confers immune protection from infection when injected prior to pathogen challenge [26], [30]. Thus, understanding the mechanisms that induce and regulate the borrelia-specific B cell response is of importance. Assessing the specificity of the B cell response to B. burgdorferi is challenging, however, due to differences in the antigenic structure of B. burgdorferi cultured in artificial media versus those grown in the mammalian host [39], [46]?[50]. Thus, lysates or extracts from culture-grown spirochetes do not reflect antigens expressed in the mammalian host. Furthermore, B. burgdorferi differentially expresses antigens during the various stages of its life cycle in the flat tick, the feeding tick and the host [39] . We therefore utilized an infection protocol that mimics tick-borne infection and avoids induction of immune responses to Borrelia antigens not expressed in vivo, by infecting mice with mammalian host-adapted spirochetes via tissue transplant.

For detection of B. burgdorferi-specific antibodies by ELISA and ELISPOT, we used a cocktail of recombinant antigens, including OspC, DbpA, Arp and BmpA, each of which are expressed during infection of the mammalian host [36], [47], [49], [51]?[53]. Furthermore, each of these antigens have been shown to induce protective or disease-resolving immune responses in mice [41], [47], [54]?[57]. We did not include the VlsE protein in our studies, a surface-protein thought to subvert the immune response to B. burgdorferi through extensive genetic variation within the host. However, the N40 strain of B. burgdorferi, which we have used here, does not seem to express this protein, based on transcriptional analysis of the IR6 region of vlsE. Moreover, we found no evidence of seroconversion to the C6 antigen of vlsE from strain B31 (S. W. Barthold, unpublished). Recent sequence analysis of the N40 genome has confirmed that N40 vlsE and BBK01 are on different plasmids and that the vlsE locus is indeed significantly different compared to B31, the commonly used VlsE-expressing Borrelia-strain.

Using only a handful of such in vivo-expressed and immunodominant antigens, we demonstrated the induction of a strong B. burgdorferi-specific antibody response in the lymph nodes of infected mice (Figures 5, 6) in a manner that is independent of MyD88 (Figure 7). We furthermore showed that nearly a quarter of hybridomas generated from lymph nodes of acutely B. burgdorferi-infected mice are specific for this pathogen (Figure 6). Together with earlier studies that demonstrated the protective and disease-resolving capacity of immune sera from long-term infected mice [26], [30], [58], we can conclude that a strong and borrelia-specific B cell response is induced in these lymph nodes.

B. burgdorferi causes spirochetemia, but its primary means of dissemination is via migration through host connective tissues and extracellular matrix [59] . This is consistent with our finding of progressive involvement of the ipsilateral, but not the matching contralateral lymph nodes of the host (Table 1). Furthermore, the spleens of infected mice did not differ in size or in frequencies of B. burgdorferi-specific antibody-secreting cells compared to spleens from uninfected mice, and were only sporadically culture-positive for spirochetes (Table 1). In apparent contrast, a previous study in mice reported the involvement of marginal zone B cells in the response to B. burgdorferi infection [60]. This difference to our study might well be due to the difference in the route of infection, i.e. intra-cutaneously with culture-grown bacteria at the back of mouse versus infection with host-adapted spirochetes by tissue-transplantation in the tarsus region. It has been well documented that the course of infection and organ involvement varies with the site of inoculation in mice [61], [62]. Furthermore, it is notable that tick-borne infections also failed to induce a significant B cell response in the spleen (data not shown).

In conclusion, by accumulating in the extracellular cortical spaces of the lymph node, B. burgdorferi seems to both induce and subvert an important arm of the adaptive immune response. Rather than fully suppressing the activity of B cells, B. burgdorferi appears to shift the major B cell response towards the production of antibodies generated in extrafollicular foci. It thereby seems to support the production of antibodies that provide immune protection from disease, while subverting the induction of more strongly protective, possibly T-dependent B cell responses that could confer bacterial clearance.
Supporting Information Top

Table S1.

Primers for the generation of recombinant Borrelia burgdorferi N40 antigens used for detection of B cell responses.

(DOC)
Acknowledgments Top

The authors like to thank Kim Olsen and Jacqueline Dieter for expert technical help, Becky Elsner for comments on the manuscript, Adam Treistar (Treestar Inc) for Flow Jo software and Dr. Andy Fell (UC Davis News service) for help with writing the author summary.
Author Contributions Top

Conceived and designed the experiments: SST CJH SWB NB. Performed the experiments: SST CJH EH. Analyzed the data: SST CJH EH SWB NB. Contributed reagents/materials/analysis tools: SF SWB NB. Wrote the paper: SST CJH SWB NB.
References Top

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49. Schwan TG, Piesman J (2000) Temporal changes in outer s

[soijuv]

Osalla Borrelioosiin sairastuneista oireet jatkuvat hoidoista huolimatta.
Potilailla joiden oireita nimitetään "post-Lyme syndroomaksi" esiintyy poikkeavia vasta-aineita tiettyä proteiinia, VlsE, kohtaan. Proteiinia on borrelia-bakteerin pinnalla.
Mikäli akuuttia infektiota sairastava henkilö muodostaa tällaisia vasta-aineita, on mahdollista että hän tarvitsee normaalia pidemmän hoidon."



Published online 5 August 2011 | Nature | doi:10.1038/news.2011.463

News
Antibodies linked to long-term Lyme symptoms

Researchers find molecules that might mark elusive syndrome.

Amy Maxmen
Ticks spread the bacterium behind Lyme disease ? but symptoms can persist even when the microbe seems to have gone.Medical-on-Line/Alamy

Some patients with Lyme disease still show symptoms long after their treatment has finished. Now proteins have been discovered that set these people apart from those who are easily cured.


People who experience the symptoms of Lyme disease, which include fatigue, soreness and memory or concentration loss, after treatment for the disorder are sometimes diagnosed as having chronic Lyme disease or post-Lyme disease syndrome. But these diagnoses are difficult to make, because the individuals no longer seem to harbour the bacteria that cause Lyme disease. And the symptoms could instead be indicative of chronic fatigue syndrome or depression.

Now Armin Alaedini at Weill Cornell Medical College in New York and his colleagues have found that patients diagnosed with post-Lyme disease syndrome have antibodies that suggest they carried the infection for an unusually long time. The finding, published in Clinical Immunology1, might help the syndrome to be better understood, diagnosed and treated.

Alaedini's team looked at antibodies made in response to a protein called VlsE, which is found on the surface of Borrelia burgdorferi, the tick-borne bacterium that causes Lyme disease.


The antibodies recognize a snippet of the protein called an epitope, and recruit the immune system to attack the bacterium. The researchers found that post-Lyme sufferers have a greater variety of antibodies to this epitope than patients whose infection cleared up quickly.

This finding suggests that patients with chronic symptoms have experienced a prolonged infection, caused by microbes that have evaded the immune system by varying the epitopes they carry. As a result of these variations, the body makes new antibodies targeting the modified protein. The longer the microbe manages to keep changing, the more diverse its host's antibodies become.

Some post-Lyme sufferers had varied antibodies against VlsE epitopes despite being diagnosed and treated early, says Alaedini. "That could mean they naturally have a different antibody response to the infection than most people; it could mean they weren't treated properly; or it's possible they were reinfected and the second infection was never treated," he says.
Inflammatory role

"This is the first study I've seen that shows some immunologic difference between someone who resolves their Lyme and someone who develops post-Lyme disease syndrome," says Linda Bockenstedt, a rheumatologist and immunologist at Yale School of Medicine in New Haven, Connecticut.

The presence of varied antibodies hints that the chronic symptoms could be caused by an ongoing inflammatory response caused by antibodies mistakenly reacting to the body's own proteins, Bockenstedt suggests.

"The big question to me is whether this can lead to an autoimmune phenomenon," says Bockenstedt. "But if that were the case, I'd expect the disease to worsen without immune-modulating treatment, and it doesn't."

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Alaedini suggests that higher levels of antibodies could increase the body's levels of cytokines, immune-system proteins that can trigger the symptoms experienced by patients with post-Lyme disease syndrome. "Various cytokine profiles have been associated with fatigue, anxiety and depression," he explains.

If these antibodies are unique to people with chronic Lyme disease, it could lead to a test and treatments for the disorder, Alaedini says. It could also guide treatment of the disease itself. "If patients with an acute infection develop antibodies to these epitopes, perhaps they require a more aggressive course of therapy," he adds.

But a predictive marker won't be useful without new therapies for the persistent symptoms, says Henry Feder Jr, a physician specializing in infectious diseases at the University of Connecticut Health Center in Farmington. If an immune response problem leads to the syndrome, antibiotics won't help. "I guarantee you that if you tell a patient they won't feel better after antibiotics, they won't," Feder says. "We need to know what's going on.

[soijuv]

Pitkäaikainen altistuminen borrelia-bakteerille (pintaproteiini C) saattaa olla kroonisessa Borrelioosissa esiintyvien keskushermosto-oireiden taustalla. Bakteeri aiheuttaa mikroglia ja aksoni-vaurioita.(2011)

Long-Term Intrathecal Infusion of Outer Surface Protein C From Borrelia burgdorferi Causes Axonal Damage.

Tauber SC, Ribes S, Ebert S, Heinz T, Fingerle V, Bunkowski S, Kugelstadt D, Spreer A, Jahn O, Eiffert H, Nau R

J Neuropathol Exp Neurol 2011 09; 70 (9): 748-757

Lyme neuroborreliosis (LNB) is the most frequent tick-borne infectious disease of the central nervous system. In acute LNB and the rare chronic state of infection, patients can experience cognitive deficits such as attention and memory disturbances. During LNB, single compounds of Borrelia burgdorferi sensu lato are released into the subarachnoid space.To investigate the pathogenesis of neurologic dysfunction in LNB, we determined that the outer surface protein C (OspC), a major virulence factor of B. burgdorferi, stimulated mouse microglial cells in a dose-dependent manner to release nitric oxide (EC50 = 0.24 mg/L) in vitro. To mimic pathophysiologic conditions of long-term release of this bacterial component in vivo, we treated C57BL/6 mice with recombinant OspC from Borrelia garinii or buffer by intraventricular infusion and tested them for behavioral deficits. After 4weeks, brains were examined by routine histology and immunohistochemistry. Assessment of spatial learning and memory of treated mice during OspC exposure did not reveal significant differences from controls. Continuous exposure to intrathecal B. burgdorferi OspC led to activation of microglia and axonal damage without demonstrable cognitive impairment in experimental mice.

These results suggest that long-term intrathecal exposure to OspC resulted in axonal damage that may underlie the neurologic manifestations in chronic LNB.

[soijuv]

Lukuisia tutkimuksia joissa osoitetaan borrelia-bakteerin voivan aiheuttaa kroonisen infektion.

http://lymekick.com/chroniclyme.pdf

The Case For Chronic Infection: Evidential persistence of Borrelia
species post antibiotic exposure in vivo and in vitro.

Michael D. Parent & Erica Falkingham

Introduction Summary:
There is an abundance of evidence demonstrating that Borrelia Burgdorferi, the causative agent
of Lyme Disease, and related pathogenic species, can persist within specific body tissues and cells
of various mammals despite adequate antibiotic therapy: ponies [93.5, 111.5], non-human
primates [50, 86], dogs [65.5, 70, 80, 81, 82, 84], mice [44, 62, 88, 100, 107, 108, 110, 114], and
humans [all others]. There is also abundant evidence that Borrelia Burgdorferi has evolved in a
manner similar to other bacteria that evade the immune system via pleomorphic modification, in
other words, the bacteria can change its shape beyond the conventional spirochetal form [45, 55,
61, 64, 90, 105, 109, 113]. L-forms, and cystic Borrelia have been identified in a number of studies
[45, 68, 77, 87, 105, 109, 112, 113]. When these "forms" are exposed to the typical antibiotics,
such as Penicillin family antibiotics or Doxycycline, they are unaffected. When the antibiotic is
removed from the environment, the bacterium will alter its form once more, morphing back into
a spiral form, allowing ongoing mobility [45, 68, 87, 90, 105, 109].

I have taken the time to "bold" the conclusions and various other aspects that clearly indicate a
deviation from the point of view given by a number of physicians and researchers who deny the
possibility of ongoing chronic infection within the human host. The current guidelines issued by
the Infectious Disease Society Of America (IDSA) are consistently used to dismiss further
discussion regarding the subject of persistence. The guidelines are titled: ?The Clinical
Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis,
and Babesiosis? Clinical Infectious Diseases 2006; 43:1089?134.

Patients who receive a diagnosis of Lyme Disease, either based on clinical observation and/or
objective indicators often improve with antibiotic therapy [1, 4, 18, 19, 26, 33, 66].
However, if they have been undiagnosed and untreated for Lyme Disease for a long period of
time, it often takes longer to see progress in symptom reduction [15, 66, 73, 93, 105]. The U.S.
National Institute Of Health funded a number of randomized double-blind placebo-controlled
trials (RCT) regarding the long term treatment of Lyme Disease. However, these RCT's were 3
months in duration or less. Patients with documented medical records indicating Chronic Lyme
Disease or a Lyme-Like Illness who have been untreated often do not see improvement until after 4-6 months of treatment, and even still, the improvements are modest initially in many patients
and may require an ongoing open ended treatment regimen with antibiotics [66, 93].

It is well understood and agreed upon universally that the more time Borrelia Burdorferi has had
to disseminate into various ligaments, bones, collagen, muscles, and other tissues, then the higher
the probability of ongoing complications or symptoms post-antibiotic therapy. Presently, studies
indicate that antibiotics can not access many of the areas that Borrelia Burgdorferi disseminates
to unless the bacterium itself leaves the safe haven of a Fibroblast skin cell [11, 22, 23, 24, 25, 29,
35, 52, 64, 70, 72, 80, 81, 84, 94], or synovial tissue cells and fluid [1, 7, 9, 31, 34, 37, 42, 60, 61, 69,
70, 71, 102].

Introductory Conclusion:
Therefore, we have studies demonstrating abundant persistence. We have National Institute Of
Health funded studies that do not treat patients long enough to confirm whether the treatment
really is effective or not. The short term studies we do have contradict other studies as well as
those based on clinical reports from health care providers treating these patients with antibiotics
beyond the currently accepted time frame. It is unwise for the IDSA to claim that long-term
antibiotic therapy doesn't work when you've only performed a study for 3 months, when the vast
majority of the patients in the study have had the infection for many years and require at least 3-6
months of oral antibiotic before clinical improvements are seen. IV antibiotics may demonstrate
minor to moderate symptomatic improvement after 1- 3 months, but if that treatment is only
given for 3 months and then discontinued, then it will be equally ineffective and the symptoms
will return to pre-treatment levels. Coincidentally, that's exactly what happened in Dr. Brian
Fallon's study. Some symptoms improved, but then returned upon discontinuing therapy.

I have discussed merely one specific possibility for the failure of patients to thrive and improve
during the currently available randomized double-blind placebo-controlled clinical trials (RCT).
Dr. Daniel J. Cameron writes in the Journal Of Medical Hypothesis that a number of limitations
exist within the currently structured (RCTs), that strongly support the position I've laid forth.
Med Hypotheses. 2009 Jun;72(6):688-91. Epub 2009 Mar 5. Insufficient evidence to deny
antibiotic treatment to chronic Lyme disease patients. First Medical Associates, Medicine, 175
Main Street, Mount Kisco, NY 10549, USA. Cameron@LymeProject.com
"Evidence for the hypothesis: There are eight limitations that support the hypothesis: (1) the
power of the evidence is inadequate to draw definite conclusions, (2) the evidence is too
heterogeneous to make strong recommendations, (3) the risk to an individual of facing a
long-term debilitating illness has not been considered, (4) the risk to society of a growing
chronically ill population has not been considered, (5) treatment delay has not been considered as
a confounder, (6) co-infections have not been considered as a confounder, (7) the design of RCTs did not address the range of treatment options in an actual practice, and (8) the findings cannot
be generalized to actual practice. Implications of the hypothesis: This hypothesis suggests that
physicians should consider the limitations of the evidence before denying antibiotic treatment for
Chronic Lyme Disease (CLD). Physicians who deny antibiotic treatment to CLD patients might
inform their patients that there are some clinicians who disagree with that position, and then
offer to refer them for a second opinion to a doctor who could potentially present a different
point of view. The hypothesis also suggests that health care insurers should consider the
limitations of the evidence before adopting policies that routinely deny antibiotic treatment for
CLD patients and should expand coverage of CLD to include clinical discretion for specific
clinical situations."

There is more than enough information to justify at least a neutral position in respect to whether
Borrelia Burgdorferi and related infectious species persist in human beings despite the Infectious
Disease Society Of America's recommendations. Due to this uncertainty, treating physicians can
not conclusively deny that persistence in human beings may be more problematic than assumed.
The scientific studies available on Lyme Disease contradict each other to a significant degree.
Many study authors state in no uncertain terms that the discussion of Lyme Disease is a closed
case. I disagree. The evidence disagrees. The Chief Medical Officer in the United Kingdom
echoed the sentiments of the IDSA in 2009 stating: "There is no biological evidence of
symptomatic chronic Lyme disease amongst those who have received the recommended
treatment regimen." - CMO, Autum 2009, Issue 49, pg. 4. The IDSA states: "To date, there is no
convincing biologic evidence for the existence of symptomatic chronic B. burgdorferi infection
among patients after receipt of recommended treatment regimens for Lyme disease." - Clin Infect
Dis 2006 Nov 1;43(9):1089-134

Skepticism is the heart of science. Cynicism is the death of reason.
The following studies are organized by year, page, and study title within the Study table index.

3

Study Table Index:
Year Page Study Title
1986 18 Ann Intern Med. 1986 Jun;104,6:798-800. Borrelia burgdorferi in
joint fluid in chronic Lyme arthritis. Snydman DR, Schenkein DP,
Berardi VP, Lastavica CC, Pariser KM.
1986 18 J Am Acad Dermatol. 1986 Sep;15,3:459-63.Treating erythema
chronicum migrans of Lyme disease. Berger BW.
1987 18 Arthritis Rheum. 1987 Apr;30,4:448-50.Failure of tetracycline
therapy in early Lyme disease. Dattwyler RJ, Halperin JJ.
1987 19 Arthritis Rheum. 1987 Jun;30,6:705-8. Lyme meningoencephalitis:
report of a severe, penicillin-re sistant case. Diringer MN, Halperin
JJ, Dattwyler RJ.
1988 19 Pediatr Infect Dis J. 1988 Apr;7,4:286-9. Borrelia burgdorferi in a
newborn despite oral penicillin for Lyme borreliosis during
pregnancy. Weber K, Bratzke HJ, Neubert U, Wilske B, Duray PH.
1988 19 Ann N Y Acad Sci. 1988;539:346-51. Treatment of erythema
chronicum migrans of Lyme disease. Berger BW. Department of
Dermatology, New York University School of Medicine, New York
10016.
1988 20 Arthritis Rheum. 1988 Apr;31,4:487-95. Spirochetal antigens and
lymphoid cell surface markers in Lyme synovitis. Comparison with
rheumatoid synovium and tonsillar lymphoid tissue. Steere AC,
Duray PH, Butcher EC.
1988 20 AMA. 1988 May 13;259,18:2737-9 Fatal adult respiratory distress
syndrome in a patient with Lyme disease. Kirsch M, Ruben FL,
Steere AC, Duray PH, Norden CW, Winkelstein A.
1988 20 J Infect Dis. 1988 Oct;158,4:905-6. Cultivation of Borrelia
burgdorferi from joint fluid three months after treatment of facial
palsy due to Lyme borreliosis. Schmidli J, Hunziker T, Moesli P,
Schaad UB.
Year Page Study Title
4
1988 21 N Engl J Med. 1988 Dec 1;319,22:1441-6. Comment in: N Engl J Med.
1989 May 11;320,19:1279-80.Seronegative Lyme disease.
Dissociation of specific T- and B-lymphocyte responses to Borrelia
burgdorferi. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ,
Thomas J, Golightly MG.
1989 21 Am J Clin Pathol. 1989 Jan;91,1:95 7. Spirochetes in the spleen of a
patient with chronic Lyme disease. Cimmino MA, Azzolini A, Tobia
F, Pesce CM Istituto Scientifico di Medicina Interna, Università di
Genova, Italy.
1989 22 Conn Med. 1989 Jun;53,6:335-7. Treatment of Lyme disease. Schoen
RT.
1989 22 Infection. 1989 Jul-Aug;17,4:216-7. High-dose intravenous
penicillin G does not prevent further progression in early
neurological manifestation of Lyme borreliosis. Kohler J, Schneider
H, Vogt A.
1989 22 Dtsch Med Wochenschr. 1989 Oct 20;114,42:1602-6.
Neuro-borreliosis or intervertebral disk prolapse? [Article in
German] Dieterle L, Kubina FG, Staudacher T, Büdingen HJ.
1989 23 Infection. 1989 Nov-Dec;17,6:355-9.Survival of Borrelia
burgdorferi in antibiotically treated patients with Lyme
borreliosis. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross
B, Baumann A, Prokop J. Neurologische Klinik Grosshadern,
München, FR Germany.
1990 23 Acta Trop. 1990 Dec;48, 2:89-94.Clinical implications of delayed
growth of the Lyme borreliosis spirochete, Borrelia burgdorferi.
MacDonald AB, Berger BW, Schwan TG.
Department of Pathology, Southampton Hospital, New York 11968.
1991 24 Infect Immun. 1991 Feb;59,2:671-8. Intracellular localization of
Borrelia burgdorferi within human endothelial cells. Ma Y,
Sturrock A, Weis JJ.
1991 24 1991: Journal of Infectious Diseases, Feb;163,2:311-8 Randomized
comparison of ceftriaxone and cefotaxime in Lyme
neuroborreliosis. Pfister HW, Preac-Mursic V, Wilske B, Schielke E,
SÃrgel F, Einhäupl KM.
Year Page Study Title
5
1991 25 Medicine, Baltimore. 1991 Mar;70,2:83-90. Lyme disease: clinical
features, classification, and epidemiology in the upper midwest.
Agger W, Case KL, Bryant GL, Callister SM.
1991 25 N Engl J Med. 1991 Apr 18;324(16):1137. Chronic neurologic
manifestations of Lyme disease. Logigian EL, Kaplan RF, Steere AC.
Department of Neurology, Tufts University School of Medicine,
Boston, MA 02111.
1991 26 Arthritis Rheum. 1991 Aug;34,8:1056-60. Treatment of refractory
chronic Lyme arthritis with arthroscopic synovectomy. Schoen RT,
Aversa JM, Rahn DW, Steere AC.
1992 26 Clin Exp Rheumatol. 1992 Jul-Aug;10,4:387-90. Molecular detection
of persistent Borrelia burgdorferi in a man with dermatomyositis.
Fraser DD, Kong LI, Miller FW.
1992 27 J Infect Dis. 1992 Aug;166,2:440-4.Fibroblasts protect the Lyme
disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.
Georgilis K, Peacocke M, Klempner MS. Department of Medicine,
New England Medical Center, Boston, Massachusetts.
1993 27 J Am Acad Dermatol. 1993 Feb;28,2 Pt 2:312-4. Recurrent erythema
migrans despite extended antibiotic treatment with minocycline in
a patient with persisting Borrelia burgdorferi infection. Liegner
KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L.
1993 27 J Infect Dis. 1993 May;167,5:1074-81.Invasion of human skin
fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.
Klempner MS, Noring R, Rogers RA.
1993 28 Infection. 1993 Mar-Apr;21,2:83-8. Azithromycin versus doxycycli
ne for treatment of erythema migrans: clinical and microbiological
findings. Strle F, Preac-Mursic V, Cimperman J, Ruzic E, Maraspin
V, Jereb M.
1993 29 J Neurol. 1993 May;240,5:278-83. Borrelia burgdorferi myositis:
report of eight patients. Reimers CD, de Koning J, Neubert U,
Preac-Mursic V, Koster JG, Müller-Felber W, Pongratz DE, Duray
PH.
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1993 30 Arthritis Rheum. 1993 Nov;36,11:1621 6. Persistence of Borrelia
burgdorferi in ligamentous tissue from a patient with chronic
Lyme borreliosis. Häupl T, Hahn G, Rittig M, Krause A, Schoerner
C, Schönherr U, Kalden JR, Burmester GR.
1993 30 J Clin Neuroophthalmol. 1993 Sep;13,3:155-61; discussion 162. 59:
First isolation of Borrelia burgdorferi from an iris biopsy.
Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B, Reinhardt
S, Böhmer R.
1993 31 Cent Eur J Public Health. 1993 Dec;1,2:81-5. Electron microscopy
and the polymerase chain reaction of spirochetes from the blood of
patients with Lyme disease. Hulínská D, Krausová M, Janovská D,
Rohácová H, Hancil J, Mailer H.
1993 32 Neurology. 1993 Dec;43,12:2705-7. Stroke due to Lyme disease. Reik
L Jr. Department of Neurology, University of Connecticut Health
Center, Farmington 06030-1845.
1994 32 N Engl J Med. 1994 Jan 27; 330,4:282-3.Detection of Borrelia
burgdorferi DNA by polymerase chain reaction in synovial fluid
from patients with Lyme arthritis. Nocton JJ, Dressler F, Rutledge
BJ, Rys PN, Persing DH, Steere AC.
1994 33 J Clin Microbiol. 1994 Mar;32,3:715-20.Isolation of Borrelia
burgdorferi from biopsy specimens taken from healthy-looking
skin of patients with Lyme borreliosis. Kuiper H, van Dam AP,
Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo
I, Vos K, Dankert J. Department of Medical Microbiology, Academic
Medical Centre, University Hospital, University of Amsterdam, The
Netherlands.
1994 33 J Rheumatol. 1994 Mar;21,3:454-61. Lyme disease: an infectious and
postinfectious syndrome.Asch ES, Bujak DI, Weiss M, Peterson MG,
Weinstein A.
1994 34 Ann Intern Med. 1994 Mar 15;120,6:487-9. The persistence of
spirochetal nucleic acids in active Lyme arthritis. Bradley JF,
Johnson RC, Goodman JL.
1994 34 Ann Intern Med. 1994 Oct 15;121,8:560-7.The long-term clinical
outcomes of Lyme disease. A population-based retrospective
cohort study. Shadick NA, Phillips CB, Logigian EL, Steere AC,
Kaplan RF, Berardi VP, Duray PH, Larson MG, Wright EA, Ginsburg
KS, Katz JN, Liang MH.
7
1994 35 Infect. 1994 Nov;29,3:255-61.Treatment of late Lyme borreliosis.
Wahlberg P, Granlund H, Nyman D, Panelius J, Seppälä I.
1994 36 Late complaints after erythema migrans Herta Klade, MD and
Elizabeth Aberer, MD. JSTD 1994; 1:52-56.
1994 36 Borrelia burgdorferi - Seek and ye shall find. Expanding the
envelope Kenneth Liegner, MD. JSTD 1994; 1:79-81.
1994 37 Psychiatric aspects of Lyme disease in children and adolescents: A
community epidemiologic study in Westchester, New York Brian
A. Fallon, MD, MPH; Hector Bird, MD; Christina Hoven, DrPH;
Daniel Cameron, MD, MPH; Michael R. Liebowitz, MD; and David
Shaffer, MD. JSTD 1994; 1:98-100.
1994 37 Persistence of Borrelia burgdorferi despite antibiotic treatment
Michael A. Patmas, MD. JSTD 1994; 1:101.
1994 38 J Infect Dis. 1994 Nov;170,5:1312-6 Comment in: J Infect Dis. 1995
May;171,5:1379-80. Fate of Borrelia burgdorferi DNA in tissues of
infected mice after antibiotic treatment. Malawista SE, Barthold
SW, Persing DH. Department of Internal Medicine, Yale University
School of Medicine, New Haven, Connecticut.
1995 39 Antimicrob Agents Chemother. 1995 May;39,5:1127-33. Effects of
penicillin, ceftriaxone, and doxycycline on morphology of Borrelia
burgdorferi. Kersten A, Poitschek C, Rauch S, Aberer E.
1995 40 Persistent PCR positivity in a patient being treated for Lyme
disease. Kornelia Keszler, MD and Richard C. Tilton, PhD. JSTD
1995; 2:57-58.
1995 40 Neuroborreliosis in Texas Audrey Stein Goldings, MD. JSTD 1995;
2:59-61.
1995 40 Vartiovaara I. 1995 Living with Lyme. Lancet, 345:842-4 A Finnish
physician ?s account of his experiences that beginning with a tick
bite in Vancouver in 1987.
Year Page Study Title
1995 40 J Neuropsychiatry Clin Neurosci. 1995 Summer;7,3:345-7. Rapidly
progressive frontal-type dementia associated with Lyme disease.
Waniek C, Prohovnik I, Kaufman MA, Dwork AJ.
8
1995 41 Ann Neurol. 1995 Oct;38,4:667-9. Comment in: Ann Neurol. 1995
Oct;38,4:560-2.Neuroborreliosis in the nonhuman primate:
Borrelia burgdorferi persists in the central nervous system.
Pachner AR, Delaney E, O'Neill T.
1995 41 Eur Neurol. 1995;35,2:113-7. Comment in: Eur Neurol.
1996;36,6:394-5. Seronegative chronic relapsing
neuroborreliosis.Lawrence C, Lipton RB, Lowy FD, Coyle PK.
1996 42 Infection. 1996 Jan-Feb;24,1:64-8. Azithromycin and doxycycline
for treatment of Borrelia culture-positive erythema migrans. Strle
F, Maraspin V, Lotric-Furlan S, Ruzi·-Sablji· E, Cimperman J.
1996 42 Infection. 1996 Jan-Feb;24,1:9-16. Erratum in: Infection 1996
Mar-Apr;24,2:169.Kill kinetics of Borrelia burgdorferi and
bacterial findings in relation to the treatment of Lyme borreliosis.
Preac Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Max
v. Pettenkofer Institut, Ludwig-Maximilians-Universität München,
Germany.
1996 43 Infection. 1996 Jan-Feb;24,1:73-5. Treatment failure in erythema
migrans--a review. Weber K. Dermatologische Privatpraxis,
München, Germany.
1996 43 Infection. 1996 May-Jun;24,3:218-26. Erratum in: Infection 1996
Jul-Aug;24,4:335. Formation and cultivation of Borrelia
burgdorferi spheroplast-L-form variants. Mursic VP, Wanner G,
Reinhardt S, Wilske B, Busch U, Marget W.
1996 44 JAMA. 1996 Jun 5; 275,21, :1657-60. Concurrent Lyme disease and
babesiosis. Evidence for increased severity and duration of illness.
K rause PJ, Telford SR 3rd, Spielman A, Sikand V, Ryan R,
Christianson D, Burke G, Brassard P, Pollack R, Peck J, Persing DH.
1996 44 Antimicrob Agents Chemother. 1996 Jun;40,6:1552-4. Eucaryotic
cells protect Borrelia burgdorferi from the action of penicillin and
ceftriaxone but not from the action of doxycycline and
erythromycin. Brouqui P, Badiaga S, Raoult D. Unité des Rickettsies,
Faculté de Médecine, Centre National de la Recherche Scientifique,
Marseille, France.
Year Page Study Title
9
1996 45 Infection. 1996 Sep-Oct;24,5:347-53.Borrelia burgdorferi DNA in
the urine of treated patients with chronic Lyme disease symptoms.
A PCR study of 97 cases. Bayer ME, Zhang L, Bayer MH. Fox Chase
Cancer Center, Philadelphia, PA 19111, USA.
1996 45 Hum Pathol. 1996 Oct;27,10:1025-34.Ultrastructural demonstration
of spirochetal antigens in synovial fluid and synovial membrane in
chronic Lyme disease: possible factors contributing to persistence
of organisms. Nanagara R, Duray PH, Schumacher HR Jr.
Allergy-Immunology-Rheumatology Division, Department of
Medicine, Faculty of Medicine, KhonKaen University, Thailand.
1996 46 Rheumatol Int. 1996;16,3:125-32.Intracellular persistence of
Borrelia burgdorferi in human synovial cells. Girschick HJ,
Huppertz HI, Rüssmann H, Krenn V, Karch H.
1996 47 Antimicrob Agents Chemother. 1996 Nov;40 11 :2632-6.In vivo
activities of ceftriaxone and vancomycin against Borrelia spp in the
mouse brain and other sites. Kazragis RJ, Dever LL, Jorgensen JH,
Barbour AG.
1996 47 Brain. 1996 Dec;119, Pt 6:2143-54. Inflammatory brain changes in
Lyme borreliosis. A report on three patients and review of
literature. Oksi J, Kalimo H, Marttila RJ, Marjamäki M, Sonninen P,
Nikoskelainen J, Viljanen MK.
1996 48 Am J Dermatopathol. 1996 Dec;18,6:571-9. Heterogeneity of
Borrelia burgdorferi in the skin. Aberer E, Kersten A, Klade H,
Poitschek C, Jurecka W.
1997 48 328: Semin Neurol. 1997 Mar;17,1:25-30.Peripheral nervous system
Lyme borreliosis. Logigian EL.
1997 49 J Clin Microbiol. 1997 January; 35(1): 111?116. Persistence of
Borrelia burgdorferi in experimentally infected dogs after
antibiotic treatment. R K Straubinger, B A Summers, Y F Chang,
and M J Appel Institute for Animal Health, College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853, USA.
rks4@cornell.edu
1997 49 Clin Infect Dis. 1997 Jul;25 Suppl 1:S52-6. Tetracycline therapy for
chronic Lyme disease. Donta ST.
Year Page Study Title
10
1997 50 Clin Infect Dis. 1997 Jul;25 Suppl 1:S64-70.Why is chronic Lyme
borreliosis chronic? Aberer E, Koszik F, Silberer M.
1997 51 American College of Rheumatology, Vol 40,9, Branigan P; Rao J;
1997 PCR evidence for Borrelia burgdorferi DNA in synovium in
absence of positive serology. Suppl, Rao J; Gerard H; Sept, p.S270
Hudson A; Williams W; Arayssi T; Pando J; Bayer M; Rothfuss S;
.PCR evidence for Borrelia has been identified in synovial biopsies of
patients with clinical pictures that had not initially suggested Lyme
disease. Clayburne G; Sieck M; Schumacher HR.
1997 51 Journal of Spirochetal & Tick-borne Diseases, Vol. 4, No. 1/2 Two
lessons from the canine model of Lyme Disease: migration of
Borrelia burgdorferi in tissues and persistence after antibiotic
treatment. Straubinger RK; 1997 Straubinger AF; Jacobson RH;
Chang Y; Summer BA;
1998 51 Ann Rheum Dis. 1998 Feb;57,2:118-21.Detection of Borrelia
burgdorferi by polymerase chain reaction in synovial membrane,
but not in synovial fluid from patients with persisting Lyme
arthritis after antibiotic therapy. Priem S, Burmester GR, Kamradt
T, Wolbart K, Rittig MG, Krause A.
1998 52 Med J Aust. 1998 May 18;168,10:500-2. Comment in: Med J Aust.
1998 May 18;168,10:479-80. Culture-positive Lyme borreliosis.
Hudson BJ, Stewart M, Lennox VA, Fukunaga M, Yabuki M,
Macorison H, Kitchener-Smith J.
1998 52 Acta Clin Belg. 1998 Jun;53,3:178-83.Lyme borreliosis--a review of
the late stages and treatment of four cases. Petrovic M, Vogelaers D,
Van Renterghem L, Carton D, De Reuck J, Afs chrift M. Department
of Internal Medicine, University Hospital Ghent, Belgium.
1998 53 Eur J Clin Microbiol Infect Dis. 1998 Oct;17,10:715-9.Comparison of
oral cefixime and intravenous ceftriaxone followed by oral
amoxicillin in disseminated Lyme borreliosis. Oksi J, Nikoskelainen
J, Viljanen MK. Department of Medicine, Turku University Central
Hospital, Finland.
Year Page Study Title
11
1998 53 Neurology. 1998 Nov;51,5:1489-91. Comment in: Neurology. 1999
Sep 11;53,4:895-6. Clinical and serologic follow-up in patients with
neuroborreliosis. Treib J, Fernandez A, Haass A, Grauer MT, Holzer
G, Woessner R.
1998 54 Infection. 1998 Nov-Dec; 26,6:364-7.A proposal for the reliable
culture of Borrelia burgdorferi from patients with chronic Lyme
disease, even from those previously aggressively treated. Phillips
SE, Mattman LH, Hulínská D, Moayad H. Greenwich Hospital, CT
06830, USA.
1998 54 Klin Monatsbl Augenheilkd. 1998 Dec;213,6:351-4. Pars plana
vitrectomy in Borrelia burgdorferi endophthalmitis [Article in
German] Meier P, Blatz R, Gau M, Spencker FB, Wiedemann P.
1999 55 Ann Med. 1999 Jun; 3,3:225-32. Borrelia burgdorferi detected by
culture and PCR in clinical relapse of disseminated Lyme
borreliosis. Oksi J, Marjamäki M, Nikoskelainen J, Viljanen MK.
1999 55 Zentralbl Bakteriol. 1999 Jul;289,3:301-18. Persistence of Borrelia
garinii and Borrelia afzelii in patients with Lyme arthritis.
Hulínská D, Votýpka J, Valeso vá M.
2000 56 J Infect Dis. 2000 Mar;181,3:1069-81. Status of Borrelia burgdorferi
infection after antibiotic treatment and the effects of
corticosteroids: An experimental study. Straubinger RK,
Straubinger AF, Summers BA, Jacobson RH.
2000 57 J Clin Microbiol. 2000 Jun; 38,6, :2191-9. PCR-Based quantification
of Borrelia burgdorferi organisms in canine tissues over a 500-Day
postinfection period. Straubinger RK. James A. Baker Institute for
Animal Health, College of Veterinary Medicine, Cornell University,
Ithaca, New York 14853, USA. rks4@cornell.edu
2001 59 Br J Dermatol. 2001 Feb;144,2:387-92. Is olation and polymerase
chain reaction typing of Borrelia afzelii from a skin lesion in a
seronegative patient with generalized ulcerating bullous lichen
sclerosus et atrophicus. Breier F, Khanakah G, Stanek G, Kunz G,
Aberer E, Schmidt B, Tappeiner G.
Year Page Study Title
12
2001 59 Epidemiol Mikrobiol Imunol. 2001 Feb;50,1:10-6.Persistence of
Borrelia burgdorferi sensu lato in patients with Lyme borreliosis
[Article in Czech] Honegr K, Hulínská D, Dostál V, Gebouský P,
Hanková E, Horácek J, Vyslouzil L, Havlasová J. Infekcní klinika,
Fakultní nemocnice, Hradec Králové.
2001 60 Ann Neurol. 2001 Sep;50,3, :330-8.Central and peripheral nervous
system infection, immunity, and inflammation in the NHP model
of Lyme borreliosis. Pachner AR, Cadavid D, Shu G, Dail D, Pachner
S, Hodzic E, Barthold SW. Department of Neurosciences,
UMDNJ-New Jersey Medical School, Newark 07103, USA.
pachner@umdnj.edu
2002 60 Wien Klin Wochenschr. 2002 Jul 31;114,13-14:574-9. Cystic forms of
Borrelia burgdorferi sensu lato: induction, development, and the
role of RpoS. Murgia R, Piazzetta C, Cinco M.
2002 61 Acta Neurol Scand. 2002 Oct;106(4):205-8. Chronic symptoms are
common in patients with neuroborreliosis -- a questionnaire
follow-up study. Vrethem M, Hellblom L, Widlund M, Ahl M,
Danielsson O, Ernerudh J, Forsberg P.
2002 62 J Infect Dis. 2002 Nov 15;186,10:1430-7. Epub 2002 Oct 23.
Detection of attenuated, noninfectious spirochetes in Borrelia
burgdorferi-infected mice after antibiotic treatment. Bockenstedt
LK, Mao J, Hodzic E, Barthold SW, Fish D.
2002 62 Antimicrob Agents Chemother. 2002 Nov;46,11:3637-40.
Erythromycin resistance in Borrelia burgdorferi. Terekhova D,
Sartakova ML, Wormser GP, Schwartz I, Cabello FC.
2002 63 Przegl Epidemiol. 2002;56 Suppl 1:57-67.New aspects of the
pathogenesis of lyme disease [Article in Polish] Zajkowska JM,
Hermanowska-Szpakowicz T. Klinika Chorób Zaka·nych i
Neuroinfekcji AM w Bia ymstoku.
2003 63 Neurology. 2003 Jun 24;60,12:1923-30. Comment in: Neurology.
2003 Jun 24;60,12:1888-9.Study and treatment of post Lyme di
sease, STOP-LD: a randomized double masked clinical trial. Krupp
LB, Hyman LG, Grimson R, Coyle PK, Melville P, Ahnn S, Dattwyler
R, Chandler B.
Year Page Study Title
13
2003 64 Med Sci Monit. 2003 Nov;9,11:PI136-42. Macrolide therapy of
chronic Lyme Disease. Donta ST.
2005 65 Vet Microbiol. 2005 May 20;107(3-4):285-94 Antibiotic treatment of
experimentally Borrelia burgdorferi-infected ponies. Chang YF,
Ku YW, Chang CF, Chang CD, McDonough SP, Divers T, Pough M,
Torres A. College of Veterinary Medicine, Cornell University, Ithaca,
NY 14853, USA. yc42@cornell.edu
2005 65 Int J Antimicrob Agents. 2005 Jun;25,6:474-8. Susceptibility of
Borrelia afzelii strains to antimicrobial agents. Ruzi·-Sablji· E,
Podreka T, Maraspin V, Strle F.
2006 66 Int J Med Microbiol. 2006 May;296 Suppl 40:233-41. Epub 2006 Mar
10.Risk of culture-confirmed borrelial persistence in patients
treated for erythema migrans and possible mechanisms of
resistance. Hunfeld KP, Ruzi·-Sablji· E, Norris DE, Kraiczy P, Strle F.
Institute of Medical Microbiology, University Hospital of Frankfurt,
Paul-Ehrlich Str. 40, D-60596 Frankfurt/Main, Germany.
K.Hunfeld@em.uni-frankfurt.de
2006 67 Eur J Pediatr. 2006 Jun;165,6:420-1. Epub 2006 Mar 4. Persistent
synovitis in two children with Lyme arthritis linked with
HLA-DRB1*1104. Hendrickx G, Demanet C, Vandenplas Y.
Department of Paediatrics, Paediatric Orthopaedic and
Rheumatology Unit, Academisch Ziekenhuis -Vrije Universiteit
Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium.
g.hendrickx@st-anna.nl
2006 67 Int J Immunopathol Pharmacol. 2006 Jul-Sep;19,3:545-9. In vitro
susceptibility of isolates of Borrelia burgdorferi s.l. to
antimicrobial agents. Santino I, Scazzocchio F, Ciceroni L,
Ciarrocchi S, Sessa R, Del Piano M. Department of Public Health
Sciences, La Sapienza University, Rome, Italy.
iolanda.santino@uniroma1.it
2006 68 Microbes Infect. 2006 Nov-Dec; 8,14-15:2832-40. Epub 2006 Sep
22.Invasion of human neuronal and glial cells by an infectious
strain of Borrelia burgdorferi. Livengood JA, Gilmore RD Jr.
Year Page Study Title
14
2007 68 Acta Radiologica, Volume 48, Issue 7 2007 , pages 755 - 762 Brain
Magnetic Resonance Imaging Does Not Contribute to the Diagnosis
of Chronic Neuroborreliosis. Aalto A, Sjöwall J, Davidsson L,
Forsberg P, Smedby O. Division of Radiology, Department of
Medicine and Care, Faculty of Health Sciences, Linköping University,
Linköping, Sweden. anne.aalto@imv.liu.se
2007 69 Pol Merkur Lekarski. 2007 Apr;22,130:275-9. Related Articles,
Concentrations of pro-inflammatory cytokines IFN-gamma, IL-6,
IL-12 and IL-15 in serum and cerebrospinal fluid in patients with
neuroborreliosis undergoing antibiotic treatment. Article in Polish.
Pancewicz SA, Kondrusik M, Zajkowska J, Grygorczuk S. Akademia
Medyczna w Bialymstoku, Klinika ChorÃ3b Zakaznych i
Neuroinfekcji.20spancewicz@interia.pl
2007 69 J Infect Dis. 2007 May 15;195,10:1489-96. Epub 2007 Apr
6.Anti-tumor necrosis factor-alpha treatment activates Borrelia
burgdorferi spirochetes 4 weeks after ceftriaxone treatment in
C3H/He mice. Yrjänäinen H, Hytönen J, Song XY, Oksi J, Hartiala K,
Viljanen MK. Department of Medical Microbiology, University of
Turku, Turku, 20520, Finland. heta.yrjanainen@utu.fi
2007 70 Adv Med Sci. 2007;52:174-8. Concentration of TGF-beta1 in the
supernatant of peripheral blood mononuclear cells cultures from
patients with early disseminated and chronic lyme borreliosis.
Grygorczuk S, Chmielewski T, Zajkowska J, Swierzbi·ska R,
Pancewicz S, Kondrusik M, Tylewska-Wierzbanowska S,
Hermanowska-Szpakowicz T. Department of Infectious Diseases and
Neuroinfections, Medical University of Bia·ystok, ul. Zurawia 14,
15-540 Bia·ystok, Poland. neuroin@amb.edu.pl
2007 71 Rheumatol Int. 2007 Sep;27,11:1091-3. Epub 2007 Apr 4.
Seronegative Lyme arthritis. Holl-Wieden A, Suerbaum S, Girschick
HJ. Children's hospital, Section of Pediatric Rheumatology,
Immunology and Infectious diseases, University of Wuerzburg,
Josef-Schneider-Str. 2, 97090 Wuerzburg, Germany.
Year Page Study Title
15
2007 71 Pol Merkur Lekarski. 2007 Sep;23,135:174-8. Concentration of
soluble forms of selectins in serum and in cerebrospinal fluid in
group of patients with neuroborreliosis--a preliminary study
Moniuszko AM, Pancewicz SA, Ko ndrusik M, Zajkowska J,
Grygorczuk S, Swierzbi·ska R. Akademia Medyczna w Bia·ymstoku,
Klinika Chorób Zaka·nych i Neuroinfekcji.
2008 72 Volume 358:428-431 January 24, 2008 Number An Appraisal of
"Chronic Lyme Disease" To the Editor: Feder et al., Oct. 4 issue,
2008 75 Persistence of Borrelia burgdorferi Following Antibiotic
Treatment in Mice Antimicrobial Agents and Chemotherapy,
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Sunlian Feng, Kevin Holden, Kimberly J. Freet, and Stephen W.
Barthold
2008 75 Antimicrobial Agents and Chemotherapy, May 2008, p. 1728-1736,
Vol. 52, No. 50066-4804 Persistence of Borrelia burgdorferi
following Antibiotic Treatment in Mice Emir Hodzic, Sunlian Feng,
Kevin Holden, Kimberly J. Freet, and Stephen W. Barthold*
2008 76 Pol Arch Med Wewn. 2008 May;118 5:314-7. : Neuroborreliosis with
extrapyramidal symptoms: a case report. Biesiada G, Czapiel J,
Sobczyk-Krupiarz I, Garlicki A, Mach T. Department of Infectious
Diseases, Division of Gastroenterology, Hepatology, and Infectious
Diseases, Jagiellonian University School of Medicine, Kraków,
Poland. gbiesiada@op.pl
2008 76 J Neuroinflammation. 2008 Sep 25;5:40. Persisting atypical and
cystic forms of Borrelia burgdorferi and local inflammation in
Lyme neuroborreliosis. Miklossy J, Kasas S, Zurn AD, McCall S, Yu
S, McGeer PL.
2008 77 Microb Pathog. 2008 Sep 20. Borrelia burgdorferi expression of the
bba64, bba65, bba66, and bba73 genes in tissues during persistent
infection in mice. Gilmore RD Jr, Howison RR, Schmit VL, Carroll
JA.
2008 78 Med Hypotheses. 2008;70,5:967-74. Epub 2007 Nov 5. The
association between tick-borne infections, Lyme borreliosis and
autism spectrum disorders. Bransfield RC, Wulfman JS, Harvey
WT, Usman AI.
Year Page Study Title
16
2008 79 Journal of Veterinary Diagnostic Investigation Vol. 20 Issue 3,
321-324 Copyright © 2008 by the American Association of
Veterinary Laboratory Diagnosticians: Validation of an in-clinic
enzyme-linked immunosorbent assay kit for diagnosis of Borrelia
burgdorferi infection in horses. Amy L. Johnson1, Thomas J. Divers
and Yung-Fu Chang
2009 80 J Antimicrob Chemother. 2009 Jun;63 6:1163-72. Epub 2009 Apr 17.
Assessment of methylthioadenosine/S-adenosylhomocysteine
nucleosidases of Borrelia burgdorferi as targets for novel
antimicrobials using a novel high-throughput method. Cornell KA,
Primus S, Martinez JA, Parveen N.
2009 81 Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18656-61. Epub 2009
Oct 20. Destruction of spirochete Borrelia burgdorferi round-body
propagules (RBs) by the antibiotic tigecycline. Brorson Ø, Brorson
SH, Scythes J, MacAllister J, Wier A, Margulis L.
2010 81 Persistence of borrelial DNA in the joints of Borrelia
burgdorferi-infected mice after ceftriaxone treatment HETA
YRJÄNÄInen 1 , JUKKA HYTÖNen 1 , PAULIINA HARTIALA 1 ,
JARMO OKSI 2 and MATTI K. VILJANEN Departments of
1Medical Microbiology and Immunology and 2 Medicine, University
of Turku, Turku, Finland
Evidential support for the case of Chronic Infection:
17
1: Ann Intern Med. 1986 Jun;104,6:798-800. Borrelia burgdorferi in joint fluid in chronic
Lyme arthritis. Snydman DR, Schenkein DP, Berardi VP, Lastavica CC, Pariser KM.
Although indirect evidence suggests that chronic Lyme arthritis is caused by persistent
infection with Borrelia burgdorferi, direct visualization has been lacking. We report the
demonstration of B. burgdorferi from synovial fluid aspirated from the right knee of a
31-year-old man with Lyme arthritis for more than 1 year. After 6 days, culture medium
inoculated with synovial fluid showed one motile and several nonmotile spirochetes. Direct
immunofluorescence staining showed reactivity with anti-B. burgdorferi serum. Spirochetes were
not seen in subcultured material. The patient's arthritis improved with high-dose intravenous
penicillin. Identification of B. burgdorferi from the joint fluid of a patient with long-standing
arthritis supports the concept that the arthritis is due to persistent infection.
2: J Am Acad Dermatol. 1986 Sep;15,3:459-63.Treating erythema chronicum migrans of Lyme
disease. Berger BW.
The efficacy of antibiotic treatment of 117 patients with erythema chronicum migrans of Lyme
disease was evaluated in terms of the necessity for retreatment and the prevention of the late
manifestations of Lyme disease.Fifty-six patients with a minor form of the illness did not
require retreatment and did not develop late manifestations following antibiotic treatment.
Three pregnant patients were included in this group.Fourteen of sixty-one patients with a major
form of the illness required retreatment, and five developed posttreatment late
manifestations of Lyme disease consisting of Bell's palsy and persistent joint pain. Although
the preferred antibiotic for treating erythema chronicum migrans of Lyme disease has not been
conclusively established, tetracycline and penicillin proved effective. The use of probenecid plus
penicillin may be of benefit to patients with the major form of the illness.
3: 1: Arthritis Rheum. 1987 Apr;30,4:448-50.Failure of tetracycline therapy in early Lyme
disease. Dattwyler RJ, Halperin JJ.
We describe the clinical courses of 5 patients with Lyme disease who developed significant late
complications, despite receiving tetracycline early in the course of their illness. All 5 patients
had been treated for erythema chronicum migra ns with a course of tetracycline that met or
exceeded current recommendations. The late manifestations of Lyme disease included arthritis,
cranial nerve palsy, peripheral neuropathy, chronic fatigue, and changes in mental function. Our
findings suggest that the use of tetracycline at a dosage of 250 mg, 4 times a day for 10 days, as a
treatment for early Lyme disease should be reconsidered. To determine optimal therapy for early
Lyme disease, a study that compares an increased dosage of tetracycline with alternative
18
treatments is indicated.
4: Arthritis Rheum. 1987 Jun;30,6:705-8. Lyme meningoencephalitis: report of a severe,
penicillin-re sistant case. Diringer MN, Halperin JJ, Dattwyler RJ.
Although Lyme disease frequently attacks the central nervous system, this involvement is rarely
severe, and high-dose intravenous penicillin usually is adequate treatment. The patient we
describe developed severe Lyme meningoencephalitis despite receiving a full course of
penicillin, and his condition continued to deteriorate after reinstitution of this treatment.
Intravenous chloramphenicol was used successfully and resulted in a substantial improvement.
5: Pediatr Infect Dis J. 1988 Apr;7,4:286-9. Borrelia burgdorferi in a newborn despite oral
penicillin for Lyme borreliosis during pregnancy. Weber K, Bratzke HJ, Neubert U, Wilske B,
Duray PH.
Department of Medicolegal Medicine, Dermatology and Microbiology, University of Munich,
Federal Republic of Germany. "We now demonstrate B. burgdorferi in the brain and liver of a
newborn whose mother had been treated with oral penicillin for LB [Lyme borreliosis] during
the first trimester of pregnancy. ..The death of the newborn was probably due to a respiratory
failure as a consequence of perinatal brain damage.?
6: Ann N Y Acad Sci. 1988;539:346-51. Treatment of erythema chronicum migrans of Lyme
disease. Berger BW. Department of Dermatology, New York University School of Medicine, New
York 10016.
Between June 1981 and July 1987 the efficacy of antibiotic treatment of 215 patients with
erythema chronicum migrans of Lyme disease was evaluated in terms of the necessity for
retreatment and the prevention of the late manifestations of Lyme disease. The principal
antibiotics utilized to treat 161 patients through 1986 were varying doses of tetracycline, or
penicillin alone or in combination with probenecid. Two of 8 0 patients with a minor form of the
illness and 17 of 81 patients with a major form of the illness required retreatment. There were
four patients who did not respond to retreatment with their original medication. A 15- to 30-day
course of amoxicillin, 500 mg q.i.d., and probenecid, 500 mg q.i.d., or doxycycline, 100 mg t.i.d.,
and on three occasions ceftriaxone, 2-4 g/day i.v., were used to treat 54 patients in 1987.
Although it is too early to judge the efficacy of treatment in these patients, increases in the
incidence of Herxheimer reactions and drug eruptions were observed. Strict compliance with
treatment protocols and the possibility of reactions to medications should be thoroughly
discussed with patients.
19
7: 1: Arthritis Rheum. 1988 Apr;31,4:487-95. Spirochetal antigens and lymphoid cell surface
markers in Lyme synovitis. Comparison with rheumatoid synovium and tonsillar lymphoid
tissue. Steere AC, Duray PH, Butcher EC.
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.
Using monoclonal antibodies to spirochetal antigenes and lymphoid cell surface markers, we
examined the synovial lesions of 12 patients with Lyme disease, and compared them with
rheumatoid synovium and tonsillar lymphoid tissue. The synovial lesions of Lyme disease
patients and rheumatoid arthritis patients were similar and often consisted of the elements found
in normal organized lymphoid tissue. In both diseases, T cells, predominantly of the
helper/inducer s ubset, were distributed diffusely in subsynovial lining areas, often with nodular
aggregates of tightly intermixed T and B cells. IgD-bearing B cells were scattered within the
aggregates, and a few follicular dendritic cells and activated germinal center B cells were
sometimes present. Outside the aggregates, many plasma cells, high endothelial venules, scattered
macrophages, and a few dendritic macrophages were found. HLA-DR and DQ expression was
intense throughout the lesions. In 6 of the 12 patients with Lyme arthritis, but in none of those
with rheumatoid arthritis, a few spirochetes and globular antigen deposits were seen in and
around blood vessels in areas of lymphocytic infiltration. Thus, in Lyme arthritis, a small
number of spirochetes are probably the antigenic stimulus for chronic synovial inflammation.
8: AMA. 1988 May 13;259,18:2737-9 Fatal adult respiratory distress syndrome in a patient with
Lyme disease. Kirsch M, Ruben FL, Steere AC, Duray PH, Norden CW, Winkelstein A.
Department of Medicine, Montefiore Hospital, University of Pittsburgh School of Medicine, PA
15213.
A dry cough, fever, generalized maculopapular rash, and myositis developed in a 67-year-old
woman; she also had markedly abnormal liver function test results. Serologic tests proved that
she had an infection of recent onset with Borrelia burgdorferi, the agent that causes Lyme
disease. During a two-month course of illness, her condition remained refractory to
treatment with antibiotics, salicylates, and steroids. Ultimately, fatal adult respiratory distress
syndrome developed; this was believed to be secondary to Lyme disease.
9: J Infect Dis. 1988 Oct;158,4:905-6. Cultivation of Borrelia burgdorferi from joint fluid three
months after treatment of facial palsy due to Lyme borreliosis. Schmidli J, Hunziker T, Moesli
P, Schaad UB.
Attacks typically are intermittent and last from 3 days to 12 months. The knees are affected most
20
often, but migratory arthritis is common and other large and small joints may be involved. Only
very few Borrelia strains have been cultured from joint specimens worldwide However, a high
percentage of patients with Lyme arthritis, 85%, have evidence of B burgdorferi DNA,
detected by PCR, in the synovial fluid The local persistence of B burgdorferi in the joint over a
long period of time might be related to the exacerbations of symptoms after chondrocyte cell
transplantation. B burgdorferi is difficult to detect in synovial fluid, and cultures are positive only
rarely
10: 1: N Engl J Med. 1988 Dec 1;319,22:1441-6. Comment in: N Engl J Med. 1989 May
11;320,19:1279-80.Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte
responses to Borrelia burgdorferi. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J,
Golightly MG.
Department of Medicine, State University of New York, School of Medicine, Stony Brook
11794-8161.
The diagnosis of Lyme disease often depends on the measurement of serum antibodies to Borrelia
burgdorferi, the spirochete that causes this disorder.Although prompt treatment with
antibiotics may abrogate the antibody response to the infection, symptoms persist in some
patients. We studied 17 patients who had presented with acute Lyme disease and received
prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently
developed. Although these patients had clinically active disease, none had diagnostic levels of
antibodies to B. burgdorferi on either a standard enzyme-linked immunosorbent assay or
immunofluorescence assay. On Western blot analysis, the level of immunoglobulin reactivity
against B. burgdorferi in serum from these patients was no greater than that in serum from
normal controls. The patients had a vigorous T-cell proliferative response to whole B.
burgdorferi, with a mean, +/- SEM, stimulation index of 17.8 +/- 3.3, similar to that, 15.8 +/- 3.2,
in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of
both groups was greater than that of a control group of healthy subjects, 3.1 +/- 0.5; P less than
0.001.We conclude that the presence of chronic Lyme disease cannot be excluded by the
absence of antibodies against B. burgdorferi and that a specific T-cell blastogenic response to
B. burgdorferi is evidence of infection in seronegative patients with clinical indications of
chronic Lyme disease.
11: 1: Am J Clin Pathol. 1989 Jan;91,1:95 7. Spirochetes in the spleen of a patient with chronic
Lyme disease. Cimmino MA, Azzolini A, Tobia F, Pesce CM Istituto Scientifico di Medicina
Interna, Università di Genova, Italy.
A 54-year-old man had intermittent evening fever, arthralgia, transient erythematous macular
21
eruption on the skin, and splenomegaly of two year's duration. Immunofluorescence tests for
Borrelia burgdorferi serum antibodies had positive results, but G-penicillin treatment was
ineffective. Splenectomy with lymph node biopsy was performed to rule out lymphoproliferative
disorders. Borrelia-like spirochetes were identified histologically in the spleen; this finding
was consistent with persistence of B. burgdorferi organisms in inner organs in chronic Lyme
disease.
12: 1: Conn Med. 1989 Jun;53,6:335-7. Treatment of Lyme disease. Schoen RT.
Lyme disease, a tick-transmitted spirochetal infection, can be divided into three stages that can
overlap or occur alone. The goals of antibiotic therapy in stage one are to shorten the duration of
early disease and to prevent the development of later stages20of the illness. This can usually be
accomplished with oral antibiotic therapy. Later stages of the illness are frequently more
difficult to treat, requiring prolonged oral or intravenous antibiotic therapy.
13: Infection. 1989 Jul-Aug;17,4:216-7. High-dose intravenous penicillin G does not prevent
further progression in early neurological manifestation of Lyme borreliosis. Kohler J,
Schneider H, Vogt A.
Neurologische Universitätsklinik und Poliklinik, Freiburg.
We report two cases of Lyme borreliosis, LB, with erythema migrans, EM, and simultaneous
meningopolyneuritis with radicular pain and lymphocytic pleocytosis in the cerebrospinal fluid,
CSF. EM and pain disappeared completely under high-dose penicillin G therapy within few a
days. Pathological findings in CSF improved. Nevertheless, during and after therapy,
neurological signs of LB developed: cranial nerve palsies as well as paresis of extremity
muscles with radicular distribution.
14: 1: Dtsch Med Wochenschr. 1989 Oct 20;114,42:1602-6. Neuro-borreliosis or intervertebral
disk prolapse? [Article in German] Dieterle L, Kubina FG, Staudacher T, Büdingen HJ.
Abteilung für Neurologie und klinische Neurophysiologie, St.-Elisabethen-Krankenhaus
Ravensburg.
Between September 1986 and November 1988, 17 patients were hospitalized and treated for
neuro-borreliosis. Ten of them had been admitted with suspected lumbar or cervical root or
compression syndrome. Only four patients recalled a tick bite, only three an erythema migrans.
Uni- or bilateral facial paresis was a prominent feature in six patients. Three of 14 patients had no
IgG antibodies against Borrelia, either in serum or cerebrospinal fluid at the initial examination,
22
two had positive titres in serum only. Despite antibiotic treatment, usually 10 mega U
penicillin three times daily, six patients had a recurrence by April, 1989, treated with
penicillin again or with twice daily 100 mg doxycycline or 2 g ceftriaxon. In four of them a
residual painful polyneuropathy remains.
15: 1: Infection. 1989 Nov-Dec;17,6:355-9.Survival of Borrelia burgdorferi in antibiotically
treated patients with Lyme borreliosis. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross
B, Baumann A, Prokop J. Neurologische Klinik Grosshadern, München, FR Germany.
The persistence of Borrelia burgdorferi in patients treated with antibiotics is described. The
diagnosis of Lyme disease is based on clinical symptoms, epidemiology and specific IgG and IgM
antibody titers to B. burgdorferi in serum. Antibiotic therapy may abrogate the antibody response
to the infection as shown in our patients. B. burgdorferi may persist as shown by positive culture
in MKP-medium; patients may have subclinical or clinical disease without diagnostic antibody
titers to B. burgdorferi.We conclude that early stage of the disease as well as chronic Lyme
disease with persistence of B. burgdorferi after antibiotic therapy cannot be excluded when
the serum is negative for antibodies against B. burgdorferi.
[Persistence:] However, some patients later developed symptoms of the disease despite
antibiotic treatment, 9-11. Because of these observations it has become questionable if a
definite eradication of B. burgdorferi with antibiotics is possible, p.357. ..The central nervous
system invasion by spirochetes and a persistence of Treponema pallidum after penicillin G
therapy is common in neurosyphilis, 22,23, p.358.[Treatment:] In view of the hitherto failure of
treatment, low CSF concentration of penicillin G, survival of B. burgdorferi in patients treated
with antibiotics, the moderate penicillin G susceptibility o f the organism and unpredictable
progression of the disease, it seems appropriate to treat patients with substantially larger
doses of antibiotics and/or longer than is provided in present treatment regimens.
p.358.[Seronegativity:] As shown, negative antibody-titers do not provide evidence for successful
therapy; antibody-titers may become negative despite persistence.
16: Acta Trop. 1990 Dec;48, 2:89-94.Clinical implications of delayed growth of the Lyme
borreliosis spirochete, Borrelia burgdorferi. MacDonald AB, Berger BW, Schwan TG.
Department of Pathology, Southampton Hospital, New York 11968.
Lyme borreliosis, a spirochetal infection caused by Borrelia burgdorferi, may become clinically
active after a period of latency in the host.Active cases of Lyme disease may show clinical relapse
following antibiotic therapy. The latency and relapse phenomena suggest that the Lyme disease
spirochete is capable of survival in the host for prolonged periods of time. We studied 63 patients
with erythema migrans, the pathognomonic cutaneous lesion of Lyme borreliosis, and examined
23
in vitro cultures of biopsies from the active edge of the erythematous patch. Sixteen biopsies
yielded spirochetes after prolonged incubations of up to 10.5 months, suggesting that Borrelia
burgdorferi may be very slow to divide in certain situations. Some patients with Lyme
borreliosis may require more than the currently recommended two to three week course of
antibiotic therapy to eradicate strains of the spirochete which grow slowly.
17: Infect Immun. 1991 Feb;59,2:671-8. Intracellular localization of Borrelia burgdorferi within
human endothelial cells. Ma Y, Sturrock A, Weis JJ.
Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.
The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are
characterized by the persistence of the organism in individuals possessing a strong
anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue
protected from the immune system of the host or there is a reservoir of the organism residing
within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into
human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B.
burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1
resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular
location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi
into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining
which differentiated intracellular from extracellular organisms. Actin-containing microfilaments
were required for the intracellular localization, indica ting that the host cell participates in the
internalization process. Activation of endothelial cells by agents known to increase the expression
of several adhesion molecules had no effect on the interaction of B. burgdorferi with the
endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is
constitutively expressed and that internalization is not dependent upon adhesion molecules
whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi
within endothelial cells suggest that intracellular localization may be a potential mechanism by
which the organism escapes from the immune response of the host and may contribute to
persistence of the organism during the later stages of Lyme disease.
18: 1991: Journal of Infectious Diseases, Feb;163,2:311-8 Randomized comparison of
ceftriaxone and cefotaxime in Lyme neuroborreliosis. Pfister HW, Preac-Mursic V, Wilske B,
Schielke E, SÃrgel F, Einhäupl KM.
Neurological Department, Klinikum Grosshadern, University of Munich, Federal Republic of
Germany.
24
In this prospective, randomized, open trial, 33 patients with Lyme neuroborreliosis were
assigned to a 10-day treatment with either ceftriaxone, 2 g intravenously, iv, every 24 h, n = 17,
or cefotaxime, 2 g iv every 8 h, n = 16. Of the 33 patients, 30 were eligible for analysis of
therapeutic efficacy. Neurologic symptoms improved or even subsided in 14 patients of the
cefotaxime group and in 12 patients of the ceftriaxone group during the treatment period. At
follow-up examinations after a mean of 8.1 months, 17 of 2 7 patients examined were clinically
asymptomatic. In one patient Borrelia burgdorferi was isolated from the cerebrospinal fluid, CSF,
7.5 months after ceftriaxone therapy. CSF antibiotic concentrations were above the MIC 90 level
for B. burgdorferi in nearly all patients examined. Patients with Lyme neuroborreliosis may
benefit from a 10-day treatment with ceftriaxone or cefotaxime.However, as 10 patients were
symptomatic at follow-up and borreliae persisted in the CSF of one patient, a prolongation of
therapy may be necessary.
19: Medicine, Baltimore. 1991 Mar;70,2:83-90. Lyme disease: clinical features, classification,
and epidemiology in the upper midwest. Agger W, Case KL, Bryant GL, Callister SM.
Section of Infectious Disease, La Crosse Lutheran Hospital, Wisconsin.
Lyme disease can be classified using the terminology of syphilis. In this series of 95 cases from
the upper midwest, early cases, defined as an illness of less than 2 months, were more likely to
have lived in or recently visited a highly endemic area. Unlike late cases, early cases presented
entirely in the nonwinter months, p less than .001. Early disease was further subdivided into
primary and secondary disease. Ninety percent of primary and 43% of secondary cases had
erythema migrans, while no late cases had active erythema migrans, p less than .001. Clinical
manifestations of nonspecific inflammation, except for arthralgia, were more common in early
than late disease, p less than .01. In secondary cases, monoarticular arthritis was slightly more
common than polyarticular arthritis, with the reverse occurring in late disease, p less than .05.
Indirect fluorescent antibody testing revealed a ratio of IgM to IgG antibodies to be helpful in
distinguishing early from late disease. Antibacterial therapy in early, primary cases caused
Jarisch-Herxheimer reaction 7% of the time. Despite longer and more frequent parenteral
therapy, late Lyme disease frequently required retreatment, owing to poor clinical response, p
less than .05.
19.5: N Engl J Med. 1991 Apr 18;324(16):1137. Chronic neurologic manifestations of Lyme
disease. Logigian EL, Kaplan RF, Steere AC. Department of Neurology, Tufts University School
of Medicine, Boston, MA 02111.
BACKGROUND AND METHODS. Lyme disease, caused by the tick-borne spirochete Borrelia
burgdorferi, is associated with a wide variety of neurologic manifestations. To define further the
25
chronic neurologic abnormalities of Lyme disease, we studied 27 patients, age range, 25 to 72
years, with previous signs of Lyme disease, current evidence of immunity to B. burgdorferi, and
chronic neurologic symptoms with no other identifiable cause. Eight of the patients had been
followed prospectively for 8 to 12 years after the onset of infection. RESULTS. Of the 27 patients,
24, 89 percent, had a mild encephalopathy that began 1 month to 14 years after the onset of the
disease and was characterized by memory loss, mood changes, or sleep disturbance. Of the 24
patients, 14 had memory impairment on neuropsychological tests, and 18 had increased
cerebrospinal fluid protein levels, evidence of intrathecal production of antibody to B.
burgdorferi, or both. Nineteen of the 27 patients,70 percent, had polyneuropathy with radicular
pain or distal paresthesias; all but two of these patients also had encephalopathy. In 16 patients
electrophysiologic testing showed an axonal polyneuropathy. One patient had leukoencephalitis
with asymmetric spastic diplegia, periventricular white-matter lesions, and intrathecal production
of antibody to B. burgdorferi. Among the 27 patients, associated symptoms included fatigue, 74
percent, headache, 48 percent, arthritis, 37 percent, and hearing loss, 15 percent. At the time of
examination, chronic neurologic abnormalities had been present from 3 months to 14 years,
usually with little progression. Six months after a two-week course of intravenous ceftriaxone, 2 g
daily, 17 patients, 63 percent, had improvement; 6, 22 percent, had improvement but then
relapsed; and 4,15 percent, had no change in their condition. CONCLUSIONS. Months to years
after the initial infection with B. burgdorferi, patients with Lyme disease may have chronic
encephalopathy, polyneuropathy, or less commonly, leukoencephalitis. These chronic
neurologic abnormalities usually improve with antibiotic therapy.
20: Arthritis Rheum. 1991 Aug;34,8:1056-60. Treatment of refractory chronic Lyme arthritis
with arthroscopic synovectomy. Schoen RT, Aversa JM, Rahn DW, Steere AC.
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Of 20 patients who underwent arthroscopic synovectomy for refractory chronic Lyme
arthritis of the knee, 16, 80%, had resolution of joint inflammation during the first month
after surgery or soon thereafter, and they have remained well during the 3-8-year followup
period. Three of these 16 patients who were more disabled preoperatively, still had mild
functional limitation at long-term followup. The remaining 4 patients, 20%, had persistent or
recurrent synovitis. We conclude that arthroscopic synovectomy is effective in treating chronic
Lyme arthritis in patients in whom the disease does not respond to antibiotic therapy.
21: 1: Clin Exp Rheumatol. 1992 Jul-Aug;10,4:387-90. Molecular detection of persistent Borrelia
burgdorferi in a man with dermatomyositis. Fraser DD, Kong LI, Miller FW.
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
26
Health, Bethesda, Maryland.
A 40-year-old white man with a several year history of various immunologic disorders, including
anti-Jo-1 autoantibody positive dermatomyositis, developed clinical Lyme disease after being
biten by a tick. The patient was treated with oral tetracycline and his initial symptoms
resolved; however, he suffered an exacerbation of his muscle disease which was difficult to
control despite cytotoxic therapy. Antibiotic therapy was reinstituted after Borrelia
burgdorferi was detected in the patient's peripheral blood leukocytes by the polymerase chain
reaction, PCR. All serologic, T-cell stimulation, and western blot analyses, however, were
negative. The patient's disease responded to oral ampicillin, p robenecid therapy and concurrent
cytotoxic therapy. Subsequent leukocyte PCR testing has been negative for the causative agent of
Lyme disease. This case may provide an example of the in vivo immuno-modulatory effects of
spirochetes in human autoimmune disease. In addition, this case emphasizes the potential
clinical utility of PCR technology in evaluating the persistent sero-negative Lyme disease
which may occur in immunocompromised individuals.
22: 1:20 J Infect Dis. 1992 Aug;166,2:440-4.Fibroblasts protect the Lyme disease spirochete,
Borrelia burgdorferi, from ceftriaxone in vitro. Georgilis K, Peacocke M, Klempner MS.
Department of Medicine, New England Medical Center, Boston, Massachusetts.
The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial
infection, even from antibiotic-treated patients, indicating that it resists eradication by host
defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible
protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease,
ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal
action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of
fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by
glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The
ability of the organism to survive in the presence of fibroblasts was not related to its
infectivity.Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone.
Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective
effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective
environment contributing to its long-term survival.
23: J Am Acad Dermatol. 1993 Feb;28,2 Pt 2:312-4. Recurrent erythema migrans despite
extended antibiotic treatment with minocycline in a patient with persisting Borrelia
burgdorferi infection. Liegner KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L.
Department of Medicine, Northern Westchester Hospital Center, Mount Kisco, NY.
27
Erythema migrans recurred in a patient 6 months after a course of treatment with
minocycline for Lyme disease. Polymerase chain reaction on heparinized peripheral blood at
that time demonstrated the presence of Borrelia burgdorferi-specific DNA. The patient was
seronegative by Lyme enzyme-linked immunosorbent assay but showed suspicious bands on
Western blot. Findings of a Warthin-Starry stain of a skin biopsy specimen of the eruption
revealed a Borrelia-compatible structure. Reinfection was not believed to have occurred.
Further treatment with minocycline led to resolution of the erythema migrans.
24: 1: J Infect Dis. 1993 May;167,5:1074-81.Invasion of human skin fibroblasts by the Lyme
disease spirochete, Borrelia burgdorferi. Klempner MS, Noring R, Rogers RA.
Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts
University School of Medicine, Boston, Massachusetts 02111.
The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by
scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi
were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell
surface by treatment with ceftriaxone, 1 microgram/mL, for 5 days. Despite the absence of
visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were
isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear
region within human fibroblasts by laser scanning confocal microscopy.Intracellular spirochetes
specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent
laser scanning confocal microscopy. These observations suggest that B. bu

[soijuv]

Vastaus kollegoille Saxén ym.


"Olen eri mieltä kirjoittajien kanssa ehdotuksesta aloittaa lyhyellä hoidolla ja pidentää hoitoa vain in casu. Objektiivisia keinoja hoitovasteen ennakoimiseksi ei ole, ja oireiden perusteellakaan ei voida arvioida etukäteen, ketkä potilaat olisivat näitä in casu -tapauksia. Epäonnistuminen voi ilmetä vasta viikkojen, ehkä kuukausien, kuluttua ensimmäisen hoidon lopettamisesta.2


Objektiivisia merkkejä reaktiivisesta immunologisesta häiriöstä ei meidän myöhäisborrelioosipotilaillamme ole todettu.

Olen täysin samaa mieltä pitkän seurannan välttämättömyydestä. Muun muassa 14 vuorokauden keftriaksonihoitojemme epäonnistuminen todettiin usein vasta pitkän ajan kuluttua hoidon jälkeen, vaikka alkutulos olisikin näyttänyt lupaavalta.


Lääketieteellinen Aikakauskirja Duodecim
1995;111(22):2184
Peter Wahlberg
Kirjeet

Kiitän asiantuntevista ja maltillisista kommenteista; olen pitkälti samaa mieltä kanssanne. "Yksioikoista" kantaa myöhäisborrelioosin hoitokaavaan ei kukaan vielä voi ottaa, siksi käytin sanamuotoa "mitkään kokeilemamme lyhyet hoitojaksot eivät meillä ole olleet kyllin tehokkaita?-". Tämä on kokemani tosiseikka, ei muuta.

Pitkän hoidon tarpeellisuutta ei ole yleisesti vahvistettu, kuten katsauksessani toteankin, mutta lyhyitäkin hoitoja koskee sama asia. Sigalin katsaus käsittelee Yhdysvaltain oloja, ja tiedämme, että niitä ei voida rinnastaa suoraan Euroopan oloihin mm. siksi, että tautien alaryhmä- ja oireistokirjot ovat erilaisia. Taudin pitkittymisen tai kroonistumisen selitys on ajankohtainen keskustelun asia; joko reaktiivinen immunologinen häiriö tai persistoiva infektio voi olla kyseessä eri potilailla. Objektiivisia merkkejä reaktiivisesta immunologisesta häiriöstä ei meidän myöhäisborrelioosipotilaillamme ole todettu.

Olen täysin samaa mieltä pitkän seurannan välttämättömyydestä. Muun muassa 14 vuorokauden keftriaksonihoitojemme epäonnistuminen todettiin usein vasta pitkän ajan kuluttua hoidon jälkeen, vaikka alkutulos olisikin näyttänyt lupaavalta.

Satunnaistetut tutkimukset sisältävät huomattavia eettisiä ongelmia, kun käytettävissä on hoitomenetelmä, joka näyttää tehoavan. Tutkimme paraikaa seuraavaa noin 100 potilaan ryhmää. Hoito-ongelmien laatu ilmenee mm. siteeraamastani Steeren ym. kirjoituksesta (1994), jossa Lymen artriitin näennäisesti onnistuneen hoidon jälkeen todetaan: "patients may still develop neuroborreliosis".

Tietysti olisi pyrittävä mahdollisimman yksinkertaiseen ja lyhyeen hoitoon. Todella satunnaistettuja hoitokokeiluja varten tarvitsisimme enemmän taustatietoa taudista. Olisi eriteltävä eri borrelia-alalajien aiheuttamien infektioiden kliiniset piirteet, ja nämä tutkimukset ovatkin käynnissä. Tarvitsemme myös nopeita laboratoriomenetelmiä eri alalajien toteamiseksi potilailla.

Olen eri mieltä kirjoittajien kanssa ehdotuksesta aloittaa lyhyellä hoidolla ja pidentää hoitoa vain in casu. Objektiivisia keinoja hoitovasteen ennakoimiseksi ei ole, ja oireiden perusteellakaan ei voida arvioida etukäteen, ketkä potilaat olisivat näitä in casu -tapauksia. Epäonnistuminen voi ilmetä vasta viikkojen, ehkä kuukausien, kuluttua ensimmäisen hoidon lopettamisesta. Näin ollen meidän on ollut pakko toistaiseksi antaa kaikille potilaille tehokkainta hoitoa, vaikka joillekuille heistä ehkä riittäisikin lyhyempi hoito, jos vain tietäisimme ennakolta, keitä tämä koskee.

Kommentoivista kollegoista neljä on lastenlääkäreitä. Omat kokemukseni lasten borrelioosin hoidosta ovat vaatimattomat. Olen ollut huomaavinani, että lapsilla?varsinkin neuroborrelioosin ollessa kyseessä?hoito onnistuu helpommin kuin aikuisilla.

Katsaukseni kappaleessa "Haasteita ja odotuksia" mainitsin joitakin tutkimuskohteita. Nämä sopivat mielestäni hyvin yhteen kirjoittajien ehdotusten kanssa. Seuraajani johtavat täällä näitä jatkotutkimuksia.

Kirjoittaja: PETER WAHLBERG, professori Åsvägen 14-B, 22100 Mariehamn
Artikkelin tunnus: duo50502 (95222184)
© 2011 Suomalainen Lääkäriseura Duodecim

http://www.duodecimlehti.fi/web/guest/e ... usinnumero

[soijuv]

2011. Terveeseen verrokkiryhmään verrattuna kroonista Borrelioosia sairastavilla esiintyi korkeampia vasta-ainepitoisuuksia esim. p28, p30, p31 ja p34 proteiinien kohdalla.

suom.huom. Koska yksi tutkimuksen tekijöistä on IDSA:n ajttelumallia edustava Gary Wormser, käyttää hän potilaiden kroonisista oireista nimitystä Post Lyme Syndrooma (PLDS) eli Borrelioosin jälkeiset oireet.

Anti-Borrelia burgdorferi antibody profile in post-Lyme disease syndrome.

Chandra A, Wormser GP, Marques AR, Latov N, Alaedini A

Clin. Vaccine Immunol. 2011(May); 18(5): 767-71.


Patients with post-Lyme disease syndrome (PLDS) report persistent symptoms of pain, fatigue, and/or concentration and memory disturbances despite antibiotic treatment for Lyme borreliosis. The etiopathogenesis of these symptoms remains unknown and no effective therapies have been identified. We sought to examine the antiborrelia antibody profile in affected patients with the aim of finding clues to the mechanism of the syndrome and its relationship to the original spirochetal infection. Serum specimens from 54 borrelia-seropositive PLDS patients were examined for antibodies to Borrelia burgdorferi proteins p18, p25, p28, p30, p31, p34, p39, p41, p45, p58, p66, p93, and VlsE by automated immunoblotting and software-assisted band analysis. The presence of serum antibodies to the 31-kDa band was further investigated by examination of reactivity against purified recombinant OspA protein. Control specimens included sera from 14 borrelia-seroposit ive individuals with a history of early localized or disseminated Lyme disease who were symptom free (post-Lyme healthy group), as well as 20 healthy individuals without serologic evidence or history of Lyme disease.

In comparison to the post-Lyme healthy group, higher frequencies of antibodies to p28 (P < 0.05), p30 (P < 0.05), p31 (P < 0.0001), and p34 (P < 0.05) proteins were found in the PLDS group. Assessment of antibody reactivity to recombinant OspA confirmed the presence of elevated levels in PLDS patients (P < 0.005).


The described antiborrelia antibody profile in PLDS offers clues about the course of the antecedent infection in affected patients, which may be useful for understanding the pathogenic mechanism of the disease.

[soijuv]

"Antibioottihoidon jälkeiset krooniset oireet ovat jatkuvan kiistelyn kohteena. Tutkijat selvittivät antibioottihoidon tehokkuutta Borrelioosia sairastavilla makaki apinoilla. Osalle apinoista annettiin 4-6kk infektiosta voimakas antibioottihoito. He saivat kuukauden suonensisäistä antibioottia (keftriaksoni) ja kaksi kuukautta doksisykliiniä suun kautta.

Antibioottihoidosta huolimatta apinoilta löydettiin pieniä määriä eläviä borrelia-bakteereita hoidon jälkeen. Tulokset osoittavat borrelia-bakteerin selviävän antibioottihoidoista. Vaikka borrelia-bakteerin on osoitettu tuhoutuvan antibioottihoidoilla koeputkiolosuhteissa, se näyttää omaavan ominaisuuksia jotka mahdollistavat sen selviämisen elimistössä antibiooteista huolimatta."
http://www.plosone.org/article/fetchArt ... ne.0029914


Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection

Monica E. Embers1*, Stephen W. Barthold4, Juan T. Borda2, Lisa Bowers1, Lara Doyle3, Emir Hodzic4, Mary B. Jacobs1, Nicole R. Hasenkampf1, Dale S. Martin1, Sukanya Narasimhan5, Kathrine M. Phillippi-Falkenstein3, Jeanette E. Purcell3¤, Marion S. Ratterree3, Mario T. Philipp1*

1 Divisions of Bacteriology & Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America, 2 Comparative Pathology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America, 3 Veterinary Medicine, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America, 4 Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California Davis, Davis, California, United States of America, 5 Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America



The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4?6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals.

Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.


Citation: Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, et al. (2012) Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection. PLoS ONE 7(1): e29914. doi:10.1371/journal.pone.0029914

Editor: Jean Louis Herrmann, Hopital Raymond Poincare - Universite Versailles St. Quentin, France

Received: July 22, 2011; Accepted: December 6, 2011; Published: January 11, 2012

Copyright: © 2012 Embers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NIAID grant R01-AI042352 (MTP), R01-AI26815 (SWB and EH), a TNPRC Pilot Study Grant (MEE), and NCRR grant RR00164. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: members@tulane.edu (MEE); Philipp@tulane.edu (MTP)

¤ Current address: Biologic Resources Laboratory, University of Illinois, Chicago, Illinois, United States of America
Introduction Top

Lyme borreliosis is caused by the spirochetes of the Borrelia burgdorferi sensu lato species complex. The clinical progression of Lyme borreliosis may be divided into early-localized, early-disseminated, and late stages. During the early-localized phase, the disease's most prevalent sign is an erythematous skin rash known as erythema migrans. Subsequently, patients may develop early-disseminated disease with dermatologic, rheumatologic, cardiac, and neurologic involvement. Patients with late disease present chiefly with arthritis or with neurologic manifestations [1]. The Infectious Diseases Society of America (IDSA) has issued guidelines for Lyme borreliosis therapy [2]. Signs and symptoms are usually successfully ameliorated with antimicrobial therapy. However, some patients continue to have persistent subjective complaints [3], [4] while a few patients fail to respond to antibiotic therapy, as made evident by signs of persistent infection [2], [5]. The response to treatment in patients with late manifestations is typically slower [2] and sometimes remains incomplete.

Post-treatment Lyme disease syndrome (PTLDS) is a condition that occurs in some patients after treatment for Lyme borreliosis. The cause of PTLDS is currently unknown but prolonged antibiotic therapy does not seem to be helpful [6], [7]. Objective evaluation of this phenomenon in humans is complicated by the difficulty in obtaining a patient population with confirmed Lyme borreliosis treated post-dissemination, and the vague, non-specific symptoms (fatigue, headache, memory and concentration difficulties, myalgias and arthralgias) with which PTLDS patients present. In addition, reliable procedures to determine that infection has been cleared from Lyme disease patients have not been established.

The C6 ELISA detects antibodies to a region of the B. burgdorferi VlsE lipoprotein that is immunogenic in infected individuals and common to all infectious variants tested thus far. Not only is the C6 test among the most reliable in terms of accuracy, but it is also a serologic test for Lyme disease that has been used experimentally as a predictor of treatment outcome [8]. In patients with PTLDS, anti-C6 titers were found to generally persist at a low level compared to acute patient titers [9]. To date, the experimental assessment, in animals, of antibiotic treatment effectiveness, measured by the presence or absence of spirochetes, correlated with C6 serologic test reactivity has not been reported.

Signs and symptoms of putative failure of antibiotic treatment in late disease or ineffectiveness of repeated treatment in patients with PTLDS may be formally attributed to several causes, including: 1) spirochetes that persist in the tissues, likely in small numbers, inaccessible or impervious to antibiotic; 2) inflammatory responses to residual antigens from dead organisms; or 3) autoimmune responses, possibly elicited by antigenic mimicry [10].

In an effort to gain insight into the viability of these hypotheses, we designed two experiments in which we respectively assessed the efficacy of two regimens of ceftriaxone and/or doxycycline treatment in rhesus macaques commencing at 4?7 months of infection with B. burgdorferi. Rhesus macaques were chosen because of the ability of this animal model to reproduce many of the key signs of human Lyme disease, including neuroborreliosis [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] and because of the similarity between the available pharmacokinetics data for ceftriaxone and doxycycline in rhesus macaques and in humans [23], [24], [25], [26]. Our results confirm that spirochetes are capable of persisting in treated nonhuman primate hosts. We discuss the possible mechanisms and need for further inquiry into this phenomenon.
Results Top

We performed two separate experiments to assess post-treatment persistence by B. burgdorferi in nonhuman primates with treatment administered at different phases of disseminated infection. Both experiments involved infection, treatment post-dissemination, serology and detection of spirochetes in tissues. The two varied in the number of animals, B. burgdorferi strain used, time interval prior to treatment, antibiotic treatment regimen, and detection methods. The first (Experiment 1) was aimed at evaluating animals treated at the late disseminated phase of infection and the treatment regimen was chosen to correspond to the regimen used to treat human PTLDS patients in a clinical evaluation of treatment for this population [6]. The outline for Experiment 1 is depicted in Figure 1.
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Figure 1. Experimental design for assessment of treatment efficacy in the late, disseminated phase of infection (Experiment 1).

A) diagram of animal groups, showing inoculation (B. burgdorferi or sham) and treatment groups (treated with antibiotics or untreated); and B) the time line of the study.
doi:10.1371/journal.pone.0029914.g001
Experiment 1: Antibiotic treatment in the late, disseminated phase of infection
Evidence of spirochetal infection.

During the first 4 weeks post-inoculation (PI), skin biopsies were taken weekly to assess infection by culture. All of the B. burgdorferi-inoculated animals yielded culture-positive skin biopsies. In addition, antibody in serum samples that were obtained just prior to the initiation of antibiotic treatment yielded both a positive B. burgdorferi whole-cell antigen ELISA, and a positive C6 ELISA in all of the inoculated animals (not shown).
Evidence that doxycycline had reached the MIC in serum of treated animals.

Serum specimens were obtained from 7 rhesus macaques that were treated with doxycycline as per the regimen of Experiment 1, and 50 µL of each specimen was tested, as described in the Materials and Methods section, for relative concentration. The mean doxycycline concentration at the peak time (2 h) was 2.071±0.431 mg/L or 6.7-fold the MIC of 0.31 mg/L. The median concentration was 2.2 mg/L (range 1.5?2.7). In another experiment, B. subtilis was used as the indicator strain. Serum specimens were obtained from 5 additional doxycycline-treated animals both at peak and trough times. The mean doxycycline concentration at the peak was 0.630±0.125 mg/L (median 0.60 mg/L, range 0.45?0.75) and at the trough (12 h) it was 0.160±0.082 mg/L (median 0.15, range 0.10?0.30). The overall median value of the peak doxycycline concentration was 1.35 mg/L and ranged from 0.63?2.07 mg/L). Thus, at the peak, the mean concentration of antibiotic exceeded the MIC in these animals by a factor of 2, but not at the trough, when it was 0.52-fold the MIC. The serum concentration of ceftriaxone was not determined. However, the published concentration of ceftriaxone in rhesus plasma after a single intravenous dose of 20 mg/Kg varies from over 200 mg/L at time zero, to about 50 mg/L at 6 h [25]. The published MIC and MBC for ceftriaxone with B. burgdorferi sensu stricto (B31) are 0.013 mg/L and 0.050 mg/L, respectively [27]. Therefore, it is likely that the concentration of ceftriaxone in the rhesus circulation remained above the MIC and the MBC for a significant portion, if not all of the 24-h interval between consecutive doses of this antibiotic.
Distinct patterns in the antibody response to C6.

The antibody response to C6 was measured in serially collected serum specimens. In all of the infected animals, the C6 antibody index rose steeply within the first 5?8 weeks post-inoculation (PI) (Figure 2). Thereafter, the responses fit into three patterns, depending on whether the animals were or were not treated with antibiotics. In the treated group, the response declined steadily during the treatment period and reached background levels at the endpoint in all animals (Figure 2A, Table 1). In contrast, the responses of the untreated group remained either largely unchanged (5 out of 12 animals, Figure 2C, scored positive (+) in Table 1), or returned to background levels (7 out of 12 animals, scored negative (−) in Table 1) but not in parallel with the kinetics of the treated group's decline in specific antibody (Figure 2B). The vertical lines in the figures denote the treatment intervals with ceftriaxone and doxycycline (Fig. 2A), or sham (Fig. 2B, C). A comparison of the number of animals with a negative C6 response postmortem in the treated group (11/11) with those in the untreated group (7/12) yielded a statistically significant difference (p = 0.0373).
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Figure 2. Patterns of antibody response to the C6 peptide in infected animals.

The C6 index measured as a function of time PI with B. burgdorferi followed three distinct patterns in Experiment 1. Each graph is the longitudinal antibody response to C6 from one representative animal for each pattern. In treated (n = 12) animals (A) the response declined sharply, almost to background levels, within the antibiotic administration periods (solid vertical lines, weeks 27?31 for ceftriaxone, weeks 31?39 for doxycycline). In sham-treated animals (B, C) the response either declined, but independently from the sham treatment intervals, to eventually reach background level (B, n = 7), or oscillated throughout the study period without ever declining steadily (C, n = 5). The pattern in (C) corresponds to the C6-positive animals, indicated in Table 1. The dotted lines in B and C indicate the sham-treatment intervals. The index cut-off value was calculated as described in Material and Methods.
doi:10.1371/journal.pone.0029914.g002
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Table 1. Detection of B. burgdorferi DNA (PCR), antigen by immunofluorescence assay (IFA), anti-C6 serum antibody (C6), B. burgdorferi RNA (RT-PCR), and inflammatory lesions (by Hematoxylin and Eosin stain (H&E)) in all animals at postmortem, 6 to 12 months after the end of the antibiotic treatment (Experiment 1).
doi:10.1371/journal.pone.0029914.t001
Monitoring of infection status postmortem.

Infection status postmortem was evaluated by culture, PCR, RT-PCR, gross pathology, histology, and immunofluorescence. All of the animals were assessed, except one of the animals in the treated group (AK21), which died due to anesthetic shock 3 months PI. With regard to culture of spirochetes, one of the treated animals yielded a positive culture and only one of the untreated animals was culture-positive (lung tissue).

With regard to PCR, tissues were assessed for spirochetal DNA by PCR that amplified both the flaB and ospA genes of B. burgdorferi. Among the untreated group, 2 animals were positive (Table 1). Here, spirochetal DNA was found in the dorsal root ganglia for one animal, and in the heart for the other. One of the animals of the treated group was PCR positive (Table 1), and in several organs, including the meninges, bladder, spleen, and lungs. The difference between treated and untreated groups was not statistically significant (p = 1.000).

For detection of B. burgdorferi transcript, RNA was extracted from heart and brain specimens. Two of the animals that were not treated with antibiotics were positive, one in heart and the other in brain. Two of the treated animals had detectable spirochetal RNA in the heart, and one additional treated animal was positive for B. burgdorferi RNA in both heart and brain (Table 1). The difference was not statistically significant (p = 0.6464).

In the assessment of gross pathology, histology, and immunofluorescence, no gross lesions were observed in any of the animals. Fragments of heart and meninges were collected postmortem, and fixed, sectioned and stained for histology and immunofluorescence. Three animals, all of them treated, had moderate to severe inflammatory lesions in the heart (Table 1, Figure 3A). Positive immunofluorescent staining for B. burgdorferi appeared as fragments. Serial sections of tissue were cut at 6 µm width, so only bacteria lying perfectly parallel to the section would appear as 12?17 µm in length (the reported length of B31 spirochetes [28] (Figure 3B). We did not find samples of this type. However, there were numerous immunofluorescence-positive specimens both in the untreated and the treated groups (Table 1). The difference was not statistically significant (p = 0.6668). The four animals that were not inoculated with B. burgdorferi were negative by this test (Table 1).
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Figure 3. Images of tissue inflammation and B. burgdorferi antigen in tissues from animals treated in the late, disseminated phase (Experiment 1).

For antigen detection, samples of tissue were stained for fluorescent detection (IFA) with anti-B. burgdorferi monoclonal (see Materials and Methods) antibody. (A) Hematoxylin &Eosin stain showing monocytic and lymphocytic infiltrate in a heart section (20×) of a treated animal (AM38). (B) Image of positive IFA staining from the heart tissue of the same animal.
doi:10.1371/journal.pone.0029914.g003
Experiment 2: Antibiotic treatment in the early, disseminated phase of infection

The second experiment (Experiment 2) focused on animals treated at the early disseminated phase and the duration of the treatment regimen was chosen by the IDSA guidelines [29]. The dose of doxycycline given was markedly higher than in Experiment 1. In addition to molecular methods, xenodiagnosis was used for detection of infection. The experimental outline is depicted in Figure 4.
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Figure 4. Experimental outline and sample collection for assessment of treatment efficacy in the disseminated phase of infection (Experiment 2).
doi:10.1371/journal.pone.0029914.g004
Confirmation of B. burgdorferi infection.

In Experiment 2, skin biopsies and blood were taken at 1?4 weeks PI. Skin samples were cultured in BSK-H medium and subjected to DNA extraction for Borrelia-specific PCR. Seroconversion was tested by ELISA for antibody reactivity to the C6 peptide [30], the Ct peptide (a 51-mer synthetic peptide that reproduces the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31) [31] and outer surface protein C (OspC) [32]. Infection was indicated by Borrelia DNA detection in skin biopsies and by serology (Table S1). Cultures were kept 12?15 weeks, but none were positive for spirochete growth.
Decrease in anti-C6 antibody titers with treatment.

We measured the responses to C6 longitudinally to include before, during, and after treatment. The endpoint dilution titers were also determined for the final time point (week 47 for Experiment 2). The C6-specific antibody levels declined in all animals treated with antibiotics (Figure S1). In untreated animals, the anti-C6 responses either leveled off or slowly declined. To determine if antibody titers declined to baseline, the endpoint dilution titers for each of the five animals in Experiment 2 were determined for sera collected before, during, and after treatment. For all of the untreated animals, the titers remained high and the titers from the treated animals declined markedly, and remained at low level even at 27 weeks post-treatment (Table 2).
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Table 2. Reciprocal end-point dilution titers of anti-C6 antibodies in antibiotic-treated* (bold) and untreated macaques infected with B. burgdorferi (Experiment 2).
doi:10.1371/journal.pone.0029914.t002
Detection of spirochetes in treated animals by xenodiagnosis.

At 7 and 11 months PI, all monkeys were fed upon by Ixodes scapularis nymphs for the uptake of persisting spirochetes by xenodiagnosis. The number of nymphs that fed to repletion varied considerably between animals. For the first round, a total of 7, 8, and 11 ticks, respectively, fed on the treated animals, whereas only 5 ticks fed on each of the untreated animals (Table S2). Tick midguts were split into 3 parts for culture, direct fluorescence and for DNA extraction. The probability of recovering spirochetes would be higher from animals upon which more ticks feed. As such, intact spirochetes were detected from the cultured midgut contents (Figure 5A) or directly from tick midgut smears (Figure 5B) of two animals at 7 months PI, both of which had been treated. These animals (GB56 and GA59) also had the most xenodiagnostic ticks feed (Table S2).
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Figure 5. Spirochetes recovered by xenodiagnosis from animals treated in the disseminated phase of infection.

Images from direct fluorescent staining of B. burgdorferi spirochetes found in xenodiagnostic tick midgut culture (A) or tick midgut preparation (B) from treated animals GA59 and GB56, respectively.
doi:10.1371/journal.pone.0029914.g005
Detection of spirochetal nucleic acids in tissues.

For Experiment 2, several B. burgdorferi genes were used both for detection of spirochetes (by standard PCR) and for detection of metabolically active spirochetes (by RT-PCR). The detection of genes throughout the course of infection, in tissues, by standard PCR/RT-PCR is shown in Figure 6.
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Figure 6. Nucleic acid detection of B. burgdorferi (Experiment 2).

Detection by PCR or RT-PCR using primers for the indicated genes from: A) skin biopsy samples; B) xenodiagnostic ticks; C) organ tissue culture pellets; and D) directly from tissues known to harbor the spirochetes. Animal numbers in bold are of those that were treated. Asterisks indicate clear positive amplimers.sk = skin; h t = heart; bl = bladder; spl = pleen. Labels include: M = marker (100 bp ladder); (+) = B.burgdorferi, strain B31 DNA/RNA; ssc = spirochete strain negative control (Bb strain JD1 DNA); ntc = no template control; tnc = tick negative control; uim = uninfected monkey DNA/RNA.
doi:10.1371/journal.pone.0029914.g006

The ospC gene was amplified from skin biopsy tissue from each animal in at least one of the four samplings (Figure 6A) shortly after inoculation. These results confirmed infection and specific detection by the ospC PCR method. The detection of B. burgdorferi nucleic acids by PCR was also performed on xenodiagnostic tick midgut samples (Figure 6B). For treated animal FK38, spirochetal DNA was detected by amplification of ospC from midgut contents when organisms were not detected by culture or DFA (Figure 6B).

A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after >9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals. We also hypothesized that persistent spirochetes may lose linear plasmid 28-1 (lp28-1), which encodes the VlsE antigen bound by the anti-C6 antibody. Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D).
Discussion Top

In some cases, patients who have been treated for Lyme disease experience persistent symptoms. The assertion that further antibiotic treatment is warranted in these cases is a matter of contention and considerable debate [33], [34], [35], [36]. Our results indicate that disseminated spirochetes of two different B. burgdorferi strains can persist in the primate host following high dose, or long-lasting antibiotic therapy. In terms of disease, only objective signs of disease post-therapy may be measurable in an animal model. While we did not find gross signs of disease postmortem, in Experiment 1 we did identify heart sections with inflammatory infiltrates in three of the treated animals. In addition, several animals, both treated and untreated showed sections of heart and meninges that were positive by immunofluorescence for B. burgdorferi. At the molecular level, B. burgdorferi DNA would indicate the presence of organisms, live or dead. The detection of RNA, however, should indicate that those present are metabolically active and thus alive. In Experiment 1, spirochetal DNA and RNA were detected in the tissues of a few animals, independent of treatment. This may reflect a low spirochetal burden, lack of flaB transcription [37], and/or seclusion in untested tissues.

In Experiment 2, we were able to recover spirochetes by xenodiagnosis in two of the three treated animals. The inability to recover spirochetes from all five macaques could be in part attributed to the number of ticks that fed, which was markedly smaller for untreated animals (Supplementary Table S2). However, a few slow-growing organisms were recovered by culture from each animal. We detected the ospA transcript in culture pellets from tissues of four animals and directly from at least one tissue from each animal. We chose ospA because this gene has been shown to be transcribed by host-adapted B. burgdorferi [38], in disseminated infection [39], and because of the induction of an anti-OspA response in patients [40] post-dissemination.

The nature of the persistent organisms and the acquisition of tolerance to antibiotics are questions that need to be addressed. The B. burgdorferi spirochete is known to invade collagenous tissue as a possible mechanism of immune evasion. It has been postulated that the joint tissues provide a protective niche during antibiotic treatment [41]. Our studies and others [37], [42], however, have not demonstrated a specific predilection for spirochete presence in joints of treated animals. Antibiotic tolerance has been demonstrated in vitro with several bacterial species, both gram negative (E. coli) and gram positive (Staphylococcus spp.). The fact that organisms can persist in the presence of antibiotics such as penicillin and cephalosporins (ceftriaxone) that interfere with cell wall synthesis appears to stem from their ability to enter a dormant, non-dividing state [43], [44], thus avoiding the need for cell wall synthesis to continue growth. The other antibiotic that was used in these experiments is doxycycline. The tetracycline class of antibiotics corrupts translation at the ribosome; therefore, minimal gene expression from dormant organisms may be unaffected. A ?persister? phenotype may possibly be responsible for the recalcitrance of persisting spirochetes made evident by previous studies in mice and dogs [37], [42], [45], and by those presented in this report. Perhaps incomplete clearance of bacteria following antibiotic treatment is not a phenomenon unique to B. burgdorferi, but one that occurs with other bacterial infections as well. In this case, xenodiagnosis enables detection of otherwise inconspicuous live organisms through acquisition by the natural vector.

To date, the C6 ELISA is the only test in which a decline in antibody titer is statistically associated with outcome of antibiotic treatment [8], [46], [47]. In accord with this finding, we observed a distinct and rapid decline in C6 titer in all antibiotic-treated animals. Not all animals, however, were spirochete-free, so the question of what facet of infection is indicated by anti-C6 antibody titers was brought to the fore. Also, the C6 titers declined in some untreated animals over a long period of time, but not in others, though presence of spirochetes was indicated in C6-negative untreated animals by IFA or PCR/RT-PCR. This is likely due in part to genetic differences in outbred animals. Possible explanations for the lack of correlation between C6 response and presence of spirochetes include: (1) the anti-C6 titer is an indicator of treatment efficacy and the infection is cleared despite the presence of spirochetal genetic material/antigen; (2) organisms persist and the anti-C6 titer does not reflect their presence, perhaps due to loss of plasmid lp28-1 (which encodes VlsE, the parent molecule of C6); or (3) anti-C6 titer declines with a significant reduction in spirochetal burden, but a low number of organisms reside in the host; if these organisms are dormant, then transcription of vlsE also may be negligible, minimizing re-stimulation with antigen. The detection of intact organisms ruled out the first explanation and detection of transcript (bbf26) from lp28-1 disproves that explanation #2 may be operating exclusively. We therefore favor explanation #3 and seek to determine the level of transcriptional/metabolic activity, and pathogenicity of persistent organisms. If, for example, spirochetes that are recovered by xenodiagnosis from treated animals turned out to be non-pathogenic, this would validate the decline in C6 titer as a measure of successful treatment outcome.

In infected mice treated with tigecycline, transcription of the chromosomally-encoded B. burgdorferi oppA2 gene was detected in 8 of 9 treated mice, indicating spirochetal viability [37]. Importantly, these studies also indicated that DNA copies of another gene, ospA, were present in treated mice, but were significantly fewer than in untreated mice. This result likely reflects a much lower spirochetal burden with treatment, supporting the notion that anti-C6 titer may decline as a function of reduced numbers. The C6 titers in these mice were not determined. Due to the relatively small quantities of bacteria over a large amount of tissue, we were unable to reliably quantify DNA or transcript levels in nonhuman primates. Similarly, the recovery of few spirochetes by tissue culture is aptly a reflection of the rhesus model and not necessarily the treatment [48].

The most pressing question in terms of human disease is whether or not spirochetes remain pathogenic after antimicrobial therapy. Similarly, do spirochetes persist long-term, or are they eventually cleared by the host? Clearly, the phenotype of persistent organisms needs to be elucidated. These studies support the use of the C6 test for diagnosis and measurement post-treatment; however, the absolute quantification of antibody levels may be essential in determining treatment efficacy for PTLDS patients, as low levels (yet above baseline) may indicate presence of residual spirochetes or antigen. Finally, the use of variable and pulse-dosing regimens of antibiotics may improve efficacy [43] and this warrants testing in an appropriate model.

Finally, in these studies we used an artificial mode of inoculation and spirochetal dose. The experimental results must be confirmed with tick-mediated infection, which is our intent. Our studies do however offer proof of the principle that intact spirochetes can persist in an incidental host comparable to humans, following antibiotic therapy. Additionally, our experiments uncover residual antigen associated with inflammatory foci. Whether persistent spirochetes or spirochetal antigen can cause PTLDS remains unanswered.
Materials and Methods Top
Ethics statement

Practices in the housing and care of animals conformed to the regulations and standards of the PHS Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. The Tulane National Primate Research Center (Animal Welfare Assurance # A4499-01) is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care-International. The Institutional Animal Care and Use Committee (IACUC) of the Tulane National Primate Research Center approved all animal-related protocols, including the infection, treatment, and tick-feeding procedures used with nonhuman primates. All animal procedures were overseen by veterinarians and their staff. For blood collection, skin biopsy collection and the tick capsule procedures, monkeys were anesthetized with ketamine (10 mg/kg) by intramuscular injection. Animals were humanely euthanized by the veterinary staff at the TNPRC in accordance with endpoint policies. Euthanasia was conducted by anesthesia with ketamine hydrochloride (10 mg/kg) followed by an overdose with sodium pentobarbital. This method is consistent with the recommendation of the American Veterinary Medical Association guidelines.
Experimental design

In the first experiment (Experiment 1), the treatment regimen was initiated at 27 weeks PI. It included the administration of ceftriaxone for 30 days, followed by 60 days of doxycycline. The latter antibiotic is used primarily to treat early localized and disseminated disease, whereas ceftriaxone is used to treat late disease [2]. Animals were kept for a minimum of 6 months after treatment termination. Tissue samples were collected for 1) in vitro culture of B. burgdorferi; 2) quantitative real-time PCR and reverse transcriptase (RT)-PCR, for detection of B. burgdorferi nucleic acids; and 3) histopathology/immunofluorescence to localize inflammatory lesions and spirochetes in tissues, respectively. The C6 test was also performed on serum specimens collected serially from all of the animals.

In the second experiment (Experiment 2), the treatment regimen began 4 months after inoculation with B. burgdorferi and consisted of a 28-day regimen of oral doxycycline, as per the IDSA guidelines for disseminated infection [2]. At 7 and 11 months PI, xenodiagnosis with I. scapularis ticks was performed. Serum was collected throughout the study period and tested for reactivity against the C6 peptide. Following euthanasia, tissues were cultured and tested by PCR and RT-PCR for detection of spirochetes and spirochetal nucleic acids.
Animals, spirochetal inoculation, animal groups, and antibiotic treatment
Experiment 1.

Twenty-eight rhesus macaques (Macaca mulatta) of Chinese origin were used in this study. Their median age when assigned to the study was 2.22 years (range 1.25?4.32). Three of the animals were females and the rest males. All were singly caged, fed commercial monkey chow (Purina Mills 5037 R), and had water available ad libitum.

For inoculation, B. burgdorferi spirochetes of the JD1 strain [49] were grown in BSK-H medium (Sigma-Aldrich, St. Louis, MO) up to stationary phase (~108 cells per mL). Twenty-four animals were given an inoculation of 3.2×108 spirochetes each via needle and syringe, with 108 organisms given intraperitoneally in the lower right quadrant, 108 subcutaneously in the same injection site, and 2×107 intradermally, also in the same site. An additional 108 spirochetes were given subcutaneously in the dorsal cervical midline. Four animals were sham-inoculated as uninfected controls. The inoculation and treatment design is summarized in Figure 1A.

At 27 weeks PI, 12 of the infected animals and two of the controls were treated with ceftriaxone, followed by doxycycline. Animals received intravenous ceftriaxone, 25 mg/kg, once per day for 30 days (Ceftiofur sodium, Pfizer Animal Health, Kalamazoo, MI) followed by 60 days of oral doxycycline, 2 mg/kg, twice per day (Bio-Serv, Frenchtown, NJ). Twelve infected and 2 control animals were sham-treated. Animal euthanasia began approximately six months post-termination of treatment. Within an additional six-month period all of the animals had been euthanized. The complete time line of the study is depicted in Figure 1B.
Experiment 2.

Five male rhesus macaques (Chinese origin), 3?4 years of age, were used for the second experiment (outlined in Figure 4C). Four animals were pair-housed after observation under protected contact, and one (GB56) was housed alone. Each animal was given 3 inoculations in the ventral midline of in vitro-cultured late log-phase B. burgdorferi strain B31, isolate 5A19 [50]: two subcutaneous 1.0 mL injections and one intradermal 0.1 mL injection, each containing 1×108 organisms diluted in sterile Hanks Balanced Salt Solution (HBSS), for a total inoculum of 3×108.

At four months PI, three of the five animals received antibiotic treatment. Animals were trained to accept treats in which the tablets were concealed. Each of the three treated macaques was given one 50 mg tablet of doxycycline (Bio-Serv) twice/day for 28 consecutive days. This dose corresponded to >12 mg/kg/day to ensure that an effective blood level was achieved.
Determination of the serum concentration of doxycyline in vivo

In vitro-cultured B. burgdorferi do not grow well in the presence of monkey serum, so antibiotic concentrations in serum had to be tested with alternative reporter strains of bacteria. A 0.5 McFarland suspension of bacteria (Staphylococcus aureus, ATCC #29213 or Bacillus subtilis, ATCC #6633), made in Mueller-Hinton broth (BD, Franklin Lakes, NJ), was streaked onto Mueller-Hinton agar plates (BD). The streaking was done so that a confluent ?lawn? of bacteria would grow after 18?24 hours of incubation at 37°C. To determine antibiotic concentration in serum, 50 µL of serum from treated animals was applied to 6-mm diameter plain paper discs (BD) and allowed to dry for 30 minutes. Standards were made by dissolving doxycycline hyclate (Sigma-Aldrich) into normal monkey serum; these were applied to the discs in the same manner. Standard doxycycline concentrations were 2, 5 and 10-fold the B. burgdorferi minimum inhibitory concentration (MIC) when S. aureus was used, and 0.5, 1, and 2-fold the MIC when B. subtilis was used. Both indicator strains were used in order to expand the range of detection. The doxycycline MIC for the JD1 strain of B. burgdorferi was determined to be 0.310 mg/L, which was approximately the same as the mean value of 0.327 mg/L that had previously been obtained for several strains and species of B. burgdorferi sensu lato [27].

The test discs were placed onto the prepared agar plates and incubated overnight at 37°C. The zones of inhibition were measured in radial mm. A standard curve was generated using the measurements of the control/standards. The test measurements were compared to the standard curve giving an approximate doxycycline concentration from the test serum. Serum from treated animals was obtained both at the times of peak and trough concentrations for doxycycline, namely, at 2 h and 12 h post-dose administration, during the 8th week of doxycycline treatment. Peak and trough times for rhesus macaques were as per Kelly et al. [51], who in turn based their estimations on pharmacokinetic data from humans.
Culture of skin biopsy and organ specimens

In both experiments, 4 mm skin biopsy specimens were collected from all animals, under anesthesia, weekly during the first four weeks PI and were cultured in BSK-H medium as described [11]. Skin biopsy samples were thus collected from live animals before antibiotic treatment so as to confirm infection. Organ specimens were also collected at necropsy. In Experiment 1, the following organ specimens, obtained postmortem, were cultured: brain (20 samples per monkey), meninges (10), dorsal root ganglia (5), sciatic nerve (10), skin (10), heart (10), synovial membrane (2), peritoneal membrane (10), lung (5), bladder (5), and spleen (5). In Experiment 2, specimens of skin, heart, bladder, spleen, knee joint synovial membrane, and shoulder tendon were cultured. Two ~2 mm3 tissue sections from each organ were cultured.
Detection of spirochetal DNA

In Experiment 1, real time PCR targeted at the ospA and flaB genes was performed following a protocol described previously [52]. DNA extracted from up to ten specimens each of brain, meninges, dorsal root ganglia, sciatic nerve, skin, heart, synovial membrane, peritoneal membrane, lung, bladder, and spleen was amplified for all animals postmortem. Only those specimens that were positive for both the ospA and flaB genes were considered positive.

In Experiment 2, standard PCR was used for detection of B. burgdorferi from tick midguts, culture pellets and animal tissues. DNA was extracted from ~60 mg (30 mg×2) of each tissue (heart, bladder, spleen, knee joint synovium, shoulder tendon and skin) with the DNeasy® Blood and Tissue kit (Qiagen, Valencia, CA). Primer sets that target the flaB and ospC genes were used and are published [53]. The Taq DNA polymerase Core Kit (Qiagen) was utilized according to the manufacturer's protocol. For each primer, 0.4 µM concentration was used. PCR was performed with 35 cycles of denaturation (94°, 30 sec.), annealing (43?53°, 45 sec.) and extension (72°, 1 min.). Negative controls included DNA extracted from uninfected monkey tissue (from Experiment 1) and DNA extracted from a pool of 5 ticks derived from the same egg mass as those used for xenodiagnosis.
Detection of spirochetal transcripts by reverse transcriptase PCR

For Experiment 1, total RNA from heart and brain specimens from all of the animals at postmortem was isolated using the Trizol method (Invitrogen, Carlsbad, CA) and contaminating DNA was removed from 2?5 µg of total RNA specimens using the DNA-free? kit, according to the manufacturer's protocol (Applied Biosystems/Ambion, Austin, TX). cDNA was subsequently synthesized from 1 µg of DNA-free RNA using the iScript cdNA synthesis kit (Bio-Rad, Hercules, CA). No amplicon was obtained by the procedure described below when the cDNA synthesis step was omitted. One µl of cDNA from each of the monkey tissue samples served as template to PCR amplify a 275 bp fragment of the B. burgdorferi flagellin (flaB) transcript. The forward primer was 5′-TTGCTGATCAAGCTCAATATAACCA-3′ and the reverse primer was 5′-CACCGGTTCAAGAGGGTGTT-3′. PCR was performed using Taq polymerase (Roche, CA). The cycling conditions were as follows: 1 cycle of denaturation at 94°C for 3 min followed by 35 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 60°C and 30 sec extension at 72°C and one cycle of 5 min extension at 72°C. PCR products were electrophoresed on a 1.5% agarose gel and the gel processed for Southern blotting as described [54]. Separated amplicons were capillary transferred to Hybond N+ nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK) as described [54]. A 22-mer oligonucleotide corresponding to a sequence within this amplicon (5′-CTGCTTCTCAAAATGTAAGAACAGCTGAAGAGCTTG-​3′) was synthesized at the W. M. Keck Facility at Yale University and non-radioactively labeled using the Gene Images AlkPhos Direct Labeling and Detection system (Amersham Biosciences, UK). The labeled probe was hybridized to the nitrocellulose membrane at 55°C overnight and washed as described in the manual (Amersham Biosciences, UK). Hybridization and washings were performed in hybridization bottles in a hybridization oven (Labnet International, Woodbridge, NJ). Bound probe was detected using the CDP-Star detection reagent for chemiluminescent detection of alkaline phosphatase (Amersham Biosciences, UK). Two independent RNA preparations were obtained from heart and brain tissues. Tissue specimens were considered positive only when they were positive in both preparations.

In Experiment 2, standard RT-PCR detection was used. RNA was extracted from the tissues with the RNeasy® Fibrous Tissue Mini Kit and treated with the RNase-free DNase set (Qiagen) to remove any residual DNA. The ospA and bbf26 genes were amplified, using the following primers: (1) ospA forward primer 5′-AATGTTAGCAGCCTTGACGA-3′; reverse primer 5′-TCGTACTTGCCGTCTTTGTT -3′; (2) bbf26 forward primer 5′-TGCCTCTAATTGTGAACACC-3′; reverse primer 5′-TCAAATCTTGAACAATACACTCA-3′. RT-PCR was performed with the Qiagen® OneStep RT-PCR kit. A quantity of 100?250 ng total RNA was used as template, with primer concentrations of 0.8 µM (forward) and 1.2 µM (reverse). The cycling was carried out per manufacturer's suggestion with the GeneAmp PCRSystem 9700 (Applied Biosystems, Carlsbad, CA). Each set produces a ~100 bp amplicon. Samples of RNA from monkey tissue and B. burgdorferi cultures were tested for DNA contamination by PCR amplification using Taq polymerase and none was detected (Figure S2).
Detection of anti-C6 antibody

Anti-C6 serum antibody levels were quantified in all animals starting prior to inoculation and then serially thereafter until post-euthanasia. The ELISA procedure used has been described [55]. In Experiment 1, monkey serum was used at a dilution of 1:800 for all time points. The cut-off ELISA value for each plate was set as the mean of the optical density at 450 nm (OD450) of duplicate determinations of pre-immune serum specimens from 7 animals plus 3× the standard deviation (SD). To correct for plate-to plate variations, a reference positive control serum pool was also included in each plate, in triplicate. This pool was made by combining equal volumes of serum that was obtained from 24 animals inoculated with B. burgdorferi. The aliquots for the pool were from serum specimens that were obtained from each animal on two consecutive collection dates just prior to the initiation of antibiotic treatment. The mean OD450 of the triplicate determination for each plate was used as a divisor for all of the OD450 values obtained with that plate, including the cut-off value, and values were reported as a quotient (index).

In Experiment 2, the same peptide antigen (C6), here derived from the strain B31 sequence, and ELISA procedure as in Experiment 1 was used. However, all responses could be compared on one plate, so the conversion to an index value was not required. Blood was collected at the following time points: 0, 2, 6, 10, 14, 16, 18, 22, 26, 28, 34, 40 and 47 weeks PI. Monkey serum was used at a 1:400 dilution for standard ELISA and at 2-fold dilution (in 5% pre-immune serum) from 1:40 through 1:2560 for endpoint dilution titers. The mean OD450 value of triplicate pre-immune serum samples from each individual animal was subtracted from the value at each time point or dilution for that animal. The reported end-point dilution titer is the dilution at which the sample reached the highest OD450 value for pre-immune serum+2× the SD obtained from that animal. In addition, serum from 2 weeks PI was tested for antibody to the Ct peptide [56] and OspC by ELISA, as described [56], [57]. Recombinant OspC was kindly provided by Robert Gilmore (CDC, Fort Collins, CO).
Detection of antibody to B. burgdorferi whole-cell antigen extract (Experiment 1)

B. burgdorferi antigen was extracted from spirochete cells by sonication in PBS, and dispensed onto 96-well ELISA plates (Corning® Costar®, Lowell, MA) at 100 µL/well (0.1 µg protein) in 50 mM carbonate buffer pH 9.6 overnight at 4°C. Plates were washed with 0.1% Tween 20 in PBS (PBS/T) and blocked for 1 h with 200 µL of 5% nonfat dried milk in PBS/T in a rotating plate set at 100?150 rpm. Serum specimens from bleeds obtained prior to inoculation, immediately prior to antibiotic treatment initiation, and at necropsy were diluted in PBS/T and tested at dilutions of 1:800 and 1:3200. Antibody bound to the plate was reacted with horseradish peroxidase-labeled goat anti-monkey IgG (gamma chain-specific, Kirkegaard and Perry Labs, (KPL), Gaithersburg, MD) at a dilution of 1:5000, for 1 h, followed by TMB Microwell Peroxidase Substrate System (KPL). The color reaction was allowed to proceed for 8?9 min, and stopped by adding 100 µL per well of 1.0 M phosphoric acid. OD450 was measured on a plate reader (Biotek Instruments, Winooski, VT).
Gross pathology assessment (Experiment 1)

Several organs were assessed for gross pathology by a veterinary pathologist, including heart, lungs, spleen, liver, musculoskeletal system, bladder, kidneys, peripheral lymph nodes, brain and meninges.
Detection of inflammatory lesions and spirochetal antigen in tissues (Experiment 1)

To detect inflammatory lesions in heart and meninges that were collected postmortem, tissue fragments were fixed in Z-fix (Anatech Ltd., Battle Creek, MI), embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin. Stained sections were evaluated under a light microscope using 4?40× magnification and a Leica microscope with a SPOT Insight digital camera (Digital Instruments Inc., Michigan, USA). To detect spirochetal antigen a monoclonal antibody (MAb 240.1) was used. This IgG1 MAb reacts with a 7.5 kDa B. burgdorferi lipoprotein [58] and had been employed before by us to detect B. burgdorferi antigen by immunohistochemistry [13]. Deparaffinized sections were washed with PBS containing 0.2% fish-skin gelatin and 0.1% Triton X-100 (PBS-FSG-Tx100) at room temperature for 10 minutes. Sections were then blocked with 10% normal goat serum (NGS) diluted in phosphate buffered saline containing 0.2% fish skin gelatin (PBS-FSG) in a humidified chamber, also at room temperature, for 40 minutes. The primary antibody was diluted in NGS and incubated for 1 hour at room temperature in a humidified chamber protected from light. Following incubation the slides were washed twice with PBS-FSG-Tx100 for ten minutes each. B. burgdorferi immunofluorescence was revealed using a species-specific goat anti-mouse secondary antibody coupled with Alexa 488 (Molecular Probes, Eugene, OR). The secondary antibody was also diluted in 10% NGS and incubated in a humidified chamber for 40 minutes at room temperature protected from light. Following incubation, the slides were washed twice with PBS-FSG-Tx100 for ten minutes each and followed with PBS-FSG. Upon completion of immunofluorescence staining, the sections were rinsed in doubly distilled water and mounted in an anti-quenching solution composed of 0.33 g/mL glycerol, analar grade, 0.133 g/mL MOWIOL 4?88 (Calbiochem), 33.33% double distilled water, 66.66% 0.2 M Tris Buffer, and 25 mg/mL DABCO (1.4-diazobicyclo-[2.2.2.]-octane) (Sigma-Aldrich) and coverslipped.
Xenodiagnosis

In Experiment 2, xenodiagnosis was performed at 7 and 11 months PI, which translated to 2 and 6 months after cessation of treatment. At 27 weeks PI, nylon-mesh jackets that zip in the back (Lomir Biomedical, NY) were placed on each of the five animals for acclimatization. At 28 weeks PI, 10 laboratory-reared nymphal Ixodes scapularis ticks were placed on the back of each animal through a containment capsule by a procedure that has been described [11]. Briefly, the area of the back to be used for tick placement was shaved and wiped with SkinPrep (Smith & Nephew Medical, UK). The tick containment capsule was fixed to the skin at its base with Super Glue and at the edges with SkinBond contact adhesive (Smith & Nephew Medical). The capsule was further secured into place with Hypafix tape (Smith & Nephew Medical). The ticks were placed inside the capsule on the skin and sealed over with a piece of nylon mesh and screw cap lid, so that ticks could not escape from the capsule. The average feeding time for ticks is 72 hours, so to allow time for them to feed to repletion, they were removed 4 days later. For tick removal, the capsules were opened and ticks were collected with a paintbrush. Ticks that had not detached from the skin were removed carefully with forceps. The capsules were gently removed by dissolving the adhesive with UniSolve (Smith & Nephew Medical) so as not to irritate the skin.

Ticks were washed with 1% sodium hypochlorite, 0.5% benzalkonium chloride and 70% ethanol for 1 min each and crushed in 45 µL PBS with a microfuge pestle. The contents were split into three 15-µL portions for: (1) direct fluorescence assay (DFA); (2) culture; and (3) PCR. For DFA, the samples were smeared on microscope slides, dried and fixed with acetone. The samples were stained with an anti-Borrelia-FITC antibody (KPL) and examined by fluorescence microscopy to detect the presence of spirochetes [59]. For culture, samples were added to ~4 mL BSK-H medium and incubated for 8 weeks at 34°C, in the presence of 5% CO2 and influx of N2 to produce a microaerophilic environment.
Statistical analysis (Experiment 1)

Differences between treated and untreated animals in detection results of spirochetal DNA, RNA, antigen, and in the C6 antibody response at postmortem were evaluated with Fisher's exact test (two-tailed). Differences were considered significant with p≤0.5.
Supporting Information Top

Table S1.

Evidence of Productive Infection in Experimental Animals (Experiment 2).

(DOC)

Table S2.

Xenodiagnostic Ticks Recovered (Experiment 2).

(DOC)

Figure S1.

The decline in C6 antibodies that accompanied antibiotic treatment in Experiment 2. Animal designations in bold (black lines) indicate those that were treated. Antibody levels indicated by gray lines are from the untreated animals.

(TIF)

Figure S2.

RNA samples tested for DNA contamination with Taq polymerase PCR. Samples included the 100 bp ladder (M), no template control (ntc), B. burgdorferi DNA (Bb DNA), B. burgdorferi RNA (Bb RNA), monkey DNA (Mk DNA) and monkey RNA (Mk RNA).

(TIF)
Acknowledgments Top

We thank Dr. Xavier Alvarez and Cecily Midkiff for help with immunofluorescence.
Author Contributions Top

Conceived and designed the experiments: MEE MTP SWB. Performed the experiments: MEE EH LCB MBJ NRH DSM SN JEP MSR LAD. Analyzed the data: MEE SWB JTB KMP-F MTP. Contributed reagents/materials/analysis tools: SWB EH MBJ. Wrote the paper: MEE MTP.
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Matsui H, Komiya M, Ikeda C, Tachibana A (1984) Comparative pharmacokinetics of YM-13115, ceftriaxone, and ceftazidime in rats, dogs, and rhesus monkeys. Antimicrobial Agents & Chemotherapy 26: 204?207. Find this article online
Maesen FP, Davies BI, van den Bergh JJ (1989) Doxycycline and minocycline in the treatment of respiratory infections: a double-blind comparative clinical, microbiological and pharmacokinetic study. Journal of Antimicrobial Chemotherapy 23: 123?129. Find this article online
Baradaran-Dilmaghani R, Stanek G (1996) In vitro susceptibility of thirty Borrelia strains from various sources against eight antimicrobial chemotherapeutics. Infection 24: 60?63. Find this article online
Hovind-Hougen K (1984) Ultrastructure of spirochetes isolated from Ixodes ricinus and Ixodes dammini. Yale Journal of Biology & Medicine 57: 543?548. Find this article online
Wormser G, Nadelman R, Dattwyler R, Dennis D, Shapiro E, et al. (2000) Practice Guidelines for the Treatment of Lyme Disease. Clinical Infectious Diseases 31: 1?14. Find this article online
Liang FT, Steere AC, Marques AR, Johnson BJ, Miller JN, et al. (1999) Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE. Journal of Clinical Microbiology 37: 3990?3996. Find this article online
Liang FT, Jacobs MB, Philipp MT (2001) C-terminal invariable domain of VlsE may not serve as target for protective immune response against Borrelia burgdorferi.[erratum appears in Infect Immun 2001 Aug;69(8):5216]. Infection & Immunity 69: 1337?1343. Find this article online
Gilmore RD Jr, Kappel KJ, Dolan MC, Burkot TR, Johnson BJ (1996) Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC. Infection & Immunity 64: 2234?2239. Find this article online
Auwaerter P (2007) Point: Antibiotic Therapy Is Not the Answer for Patients with Persisting Symptoms Attributable to Lyme Disease. Clinical Infectious Diseases 45: 143?148. Find this article online
Stricker R (2007) Counterpoint: Long-Term Antibiotic Therapy Improves Persistent Symptoms Associated with Lyme Disease. Clinical Infectious Diseases 45: 149?157. Find this article online
Feder HM Jr, Johnson BJ, O'Connell S, Shapiro ED, Steere AC, et al. (2007) A critical appraisal of ?chronic Lyme disease?. New England Journal of Medicine 357: 1422?1430. Find this article online
Mayer L, Merz S (2008) An appraisal of ?chronic Lyme disease?.[comment]. New England Journal of Medicine 358: 428; author reply 430?421. Find this article online
Barthold SW, Hodzic E, Imai DM, Feng S, Yang X, et al. (2010) Ineffectiveness of tigecycline against persistent Borrelia burgdorferi. Antimicrobial Agents & Chemotherapy 54: 643?651. Find this article online
Crowley H, Huber BT (2003) Host-Adapted Borrelia burgdorferi in Mice Expresses OspA during Inflammation. Infect Immun 71: 4003?4010. Find this article online
Narasimhan S, Caimano MJ, Liang FT, Santiago F, Laskowski M, et al. (2003) Borrelia burgdorferi transcriptome in the central nervous system of non-human primates.[erratum appears in Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1426 Note: Camaino MJ [corrected to Caimano MJ]]. Proceedings of the National Academy of Sciences of the United States of America 100: 15953?15958. Find this article online
Kalish RA, Leong JM, Steere AC (1993) Association of treatment-resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi. Infect Immun 61: 2774?2779. Find this article online
YrjÄNÄInen H, HytÖNen J, Hartiala P, Oksi J, Viljanen MKPersistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment. APMIS 118: 665?673. Find this article online
Hodzic E, Feng S, Holden K, Freet KJ, Barthold SW (2008) Persistence of Borrelia burgdorferi following Antibiotic Treatment in Mice. Antimicrob Agents Chemother 52: 1728?1736. Find this article online
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Straubinger RK, Summers BA, Chang YF, Appel MJ (1997) Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. Journal of Clinical Microbiology 35: 111?116. Find this article online
Philipp MT, Bowers LC, Fawcett PT, Jacobs MB, Liang FT, et al. (2001) Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans. Journal of Infectious Diseases 184: 870?878. Find this article online
Philipp MT, Marques AR, Fawcett PT, Dally LG, Martin DS (2003) C6 test as an indicator of therapy outcome for patients with localized or disseminated lyme borreliosis. Journal of Clinical Microbiology 41: 4955?4960. Find this article online
Pachner AR, Zhang WF, Schaefer H, Schaefer S, O'Neill T (1998) Detection of active infection in nonhuman primates with Lyme neuroborreliosis: comparison of PCR, culture, and a bioassay. Journal of Clinical Microbiology 36: 3243?3247. Find this article online
Piesman J, Hicks TC, Sinsky RJ, Obiri G (1987) Simultaneous transmission of Borrelia burgdorferi and Babesia microti by individual nymphal Ixodes dammini ticks. Journal of Clinical Microbiology 25: 2012?2013. Find this article online
Purser JE, Norris SJ (2000) Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proceedings of the National Academy of Sciences of the United States of America 97: 13865?13870. Find this article online
Kelly D, Chulay JD, Mikesell P, F

[soijuv]

Neuroborrelioosi on yksi Borrelioosin kroonisista ilmenemismuodoista. Oireen aiheuttaa borrelia burgdorferi spirokeetta. Keskushermosto-ongelma voi esiintyä Borrelioosin toisessa ja kolmannessa vaiheessa. Toisessa vaiheessa voi esiintyä aivokalvontulehduksia ja esim radikuliitteja l. hermojuuritulehduksia. Kolmannessa eli kroonisessa vaiheessa voi esiintyä useiden eri sairauksien kaltaisia oireita kuten MS-oireita, aivokasvaimia, aivotulehduksia, psykiatrisia oireita, myelopatiaa l. luuydin-, selkäydinsairauden, aivojen verisuonitulehduksia jne.
10-vuotiaalla lapsella havaittiin borrelia-bakteerin aiheuttama neuroborrelioosi. Sen seurauksena lapsella ilmeni kallonsisäistä kudosmassaa.

Lyme and tumors
Neurosurgery. 1992 May;30(5):769-73.
Lyme neuroborreliosis manifesting as an intracranial mass lesion.
Murray R, Morawetz R, Kepes J, el Gammal T, LeDoux M.
Source

Department of Surgery, University of Alabama, School of Medicine, Birmingham.
Abstract
Lyme neuroborreliosis is one of the chronic manifestations of Lyme disease and is caused by the neurotropic spirochete, Borrelia burgdorferi. Two of the three stages of Lyme disease potentially involve the central nervous system: a second stage that may manifest as meningitis, cranial neuritis, or radiculoneuritis; and a third stage, or chronic neuroborreliosis, with parenchymal involvement. The tertiary stage may mimic many conditions, including multiple sclerosis, polyneuropathy, viral encephalitis, brain tumor, vasculitis, encephalopathy, psychiatric illness, and myelopathy.

We report a 10-year-old child with signs, symptoms, and radiological manifestations of intracranial mass lesions, without previously recognized manifestations of Lyme disease. This proved to be Lyme neuroborreliosis, documented by histological and serological examination, which responded well to antibiotic therapy. The need to establish a tissue diagnosis of intracranial mass lesions is emphasized, and the utility of a computed tomographic-guided stereotactic system for this purpose is discussed.
PMID:
1584393
[PubMed - indexed for MEDLINE]
Pseudotumor cerebri
http://www.pseudotumorcerebri.net/

[soijuv]

Nykyisillä testeillä borreliabakteereita löytyy vain harvoin kroonista borrelioosia sairastavilta. Siksi potilaiden jatkuvien oireiden syyn selvittäminen on ollut vaikeata.

Seuraavassa tutkimuksessa esitetään normaaleista vasta-aine- ja PCR-tutkimuksista poikkeava menetelmä infektion toteamiseksi luotettavammin. Vasta-aine- ja PCR-tutkimukset ovat osoittautuneet useissa tutkimuksissa epäluotettaviksi borrelioosin diagnosoinnissa. Vain muutamalla tutkimukseen osallistuneella oli positiivinen ELISA-testitulos.

Tutkimuksessa näytteet tutkittiin elektronimikroskoopilla fluoresoivaa vasta-ainevärjäystä (monoklooninen vasta-aine AspA ja Asp A Pcr ) käyttäen. 43/47 kroonista borrelioosia sairastavista oireiden aiheuttajiksi osoittautui borreliabakteeri. (Suom. huom. Elektronimikroskooppi on mikroskooppi, jossa käytetään näkyvän valon sijasta elektronisuihkua. Tämä mahdollistaa tavallista valomikroskooppia huomattavasti pienempien yksityiskohtien havaitsemisen.)

Kaikkien tutkimukseen osallistuneiden oireet olivat palanneet vaikka he olivat saaneet pitkäaikaisen antibioottihoidon suunkautta ja suonensisäisillä antibiooteilla. Potilaiden jatkuvien oireiden väitetään usein johtuvan ns. Post-Lyme syndroomasta. Väite on kuitenkin perusteeton sillä potilaiden tila paranee useimmiten antibiooteilla ja pahenee uudelleen, kun antibiootit lopetettiin. Tämän uuden menetelmän perusteella voitiin todeta, että potilaiden oireet johtuivat jatkuvasta infektiosta.

Menetelmä tulisi ottaa käyttöön borrelioosin laboratoriodiagnostiikkaan. ?Tällä menetelmällä borreliabakteereita kyettiiin viljelemään luotettavasti ja toistuvasti kroonista borrelioosia sairastavien verestä. Löysimme borreliabakteereita useimmiten niiden L-muodossa (ilman soluseinämää)."

Tutkimus todistaa kroonisen borrelioosin oireiden johtuvan jatkuvasta infektiosta, ja että suositushoitoja pidemmät antibioottihoidot eivät välttämättä paranna infektiota.

http://www.ncbi.nlm.nih.gov/entrez/quer ... t=Abstract

http://www.cbc.ca/ideas/features/Aids/phillips.html

A Proposal for the Reliable Culture of Borrelia burgdorferi from Patients with Chronic Lyme Disease, Even from Those Previously Aggressively Treated

S. E. Phillips, L. H. Mattman, D. Hulinska, H. Moayad

Infection 26 (1998) 364-367

Summary: Since culture of Borrelia burgdorferi from patients with chronic Lyme disease has been an extraordinarily rare event, clarification of the nature of the illness and proving its etiology as infectious have been difficult. A method for reliably and reproducibly culturing B. burgdorferi from the blood of patients with chronic Lyme disease was therefore sought by making a controlled blood culture trial studying 47 patients with chronic Lyme disease. All had relapsed after long-term oral and intravenous antibiotics. 23 patients with other chronic illness formed the control group.

Positive cultures were confirmed by fluorescent antibody immuno-electron microscopy using monoclonal antibody directed against Asp A, and Asp A PCR.

43/47 patients (91%) cultured positive. 23/23 controls (100%) cultured negative. Although persistent infection has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen capture, there are major problems with these tests.

This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness and establishes that it is of chronic infectious etiology. This discovery should help to reestablish the gold standard in laboratory diagnosis of Lyme disease.

INTRODUCTION
Lyme disease is a multi-system illness caused by infection with Borrelia burgdorferi. Its manifestations can be myriad. This. coupled with problems in current serologic assays, leads to frequent misdiagnosis at all stages of the illness. Some investigators believe that Lyme borreliosis is overdiagnosed, while others maintain that it is underdiagnosed. To further confuse matters, a significant percentage of patients with Lyme disease relapse despite antibiotic therapy [1, 2].

Chronic Lyme disease is a controversial topic. Even after extended antibiotic treatment, persistent infection in chronic Lyme disease has been strongly suggested by the persistence of borrelial antigen, as demonstrated by polymerase chain reaction [3, 4]. However, these diagnostic tests are plagued by the absence of a gold standard. The gold standard for laboratory diagnosis in the field of infectious diseases has usually involved culturing the causative organism from the infected host. In the case of Lyme disease, attempts to do so have been disheartening.

The organism has seldom been cultured from cases of treated. Late-stage disease, and if so, primarily from cerebrospinal or synovial fluid [5-8]. Culture of the organism from blood has been a rarity, with successful cultures primarily from cases of untreated, early disease [9, 10].

We set out to demonstrate a methodology by which we could reliably and reproducibly culture B. burgdorferi from the blood of patients with chronic Lyme disease even though they had had extended antibiotic therapy. If this were successful, it would also provide a unique opportunity to compare the serologic diagnostic criteria set forth by The Centers for Disease Control (CDC) in conjunction with The Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) to what is potentially a gold standard diagnostic test.

PATIENTS AND METHODS
The study was a multi-center, controlled blood culture trial with an approximately 2:1 ratio of cases to controls. Patients were selected from private practices in areas both hyper-endemic and non-endemic for Lyme disease. All cases had a diagnosis of Lyme disease and had failed or relapsed after extended oral and intravenous antibiotic therapy. The diagnosis of Lyme disease was made primarily on clinical grounds. Although almost all cases had serologic evidence suggestive of infection with B. burgdorferi, few had positive ELISAs and only a little over half met CDC serologic criteria for Western blot positivity. 4/47 (9%) were positive by Lyme ELISA. 3/47 (6%) were equivocal by ELISA. 26/47 (55%) were positive by CDC criteria for Lyme Western blot. of these, 20/26 (77% ) were IgM positive, 10/26 (38% ) were IgG positive, and 4/26 (15%) were positive for both IgM and IgG.

To participate in the study, all patients had to have had at least 6 consecutive weeks of therapy with an intravenous third-generation cephalosporin and a subsequent relapse. Some patients had had as long as 6 months of intravenous therapy, with the mean being approximately 3 months. Controls resided in non-endemic areas and consisted of patients with chronic illnesses other than Lyme disease.

The following MPM medium was used for this study: To 1 l of Detroit tap water was added: proteose peptone 20g, beef infusion from 1,000g, dextrose 10g, sodium chloride 10g, dipotassium phosphate 4g, sodium thioglycollate 1g, purified agar 1g, bacto methylene blue .004g, sucrose 100g, soluble starch 5g. This was autoclaved for 15 min at 120 degrees C. For the medium to be used in tube or slide culture, it had to be refrigerated for 24 h before final preparation.

For the medium to be used in tubes, 10ml of medium were boiled to dissolve the agar just before use and the following was added to each tube: 1 ml separately autoclaved yeast extract from a 10% solution to give a final concentration of 1%, and 1 ml of sterile 10% NaHC03. Since yeast extract may contain heat-resistant bacilli, it was separately autoclaved for 30 min at 124 degrees C and batch-tested for sterility. The inoculum was 0.1 ml of blood in EDTA to 4 ml of medium in a slender screw-top tube. Incubation was at 30 degrees C under normal atmospheric conditions for a period of 1-3 weeks.

For the medium to be used in slide culture, it was sterilized in 30ml amounts in screw-top tubes. Just before use, the medium was boiled to melt the agar and. when cool but not solidified, the following was added: 3ml of separately autoclaved 10% yeast extract and 10ml of sterile 10% NaHC03. The broth was then poured aseptically into a sterile plastic Coplin jar. Slides were smeared with the patient's chosen body fluid. The slides had to be specialized so as not to require fixative. The smears were dried in an aseptic environment before being placed in the Coplin jar. Once they were inside, the lid was tightly closed and incubation was at 30 degrees C under normal atmospheric conditions for a period of 1-3 weeks.

For the medium to be used for blood agar plates, the broth medium was modified by adding a total of 16 g of agar. Sixty ml of sheep's blood was added as soon as the medium was removed from the autoclave, resulting in ."chocolate agar." At this point, separately autoclaved 10% yeast extract was added to give a final concentration of 1%. The medium was then poured into sterile plastic Petri dishes and stored under refrigeration for 24 h once solidified. The inoculum was 0.5ml of blood in EDTA with incubation at 30 degrees C under normal atmospheric conditions for a period of 1-3 weeks.

Two blood samples of 5 ml each were collected in EDTA lavender-top test tubes from each patient and control. From these, seven cultures were processed from each participant. All positive cultures were stained with acridine orange at pH 3.5 4.0 and then confirmed by our laboratory with affinity-adsorbed polyclonal fluorescent antibody to B. burgdorferi (02-97-91, Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA).

Further confirmation of positive culture results was accomplished by electron microscopy. immuno-electron microscopy utilizing monoclonal antibody directed against Asp A (monoclonal antibody no. 181, courtesy of Prof. B. Left. Stony Brook, NY, USA), and plasmid PCR with Asp A primer. The methods employed in these processes have been previously reported [11, 12].

RESULTS
Of the 47 patients with chronic Lyme disease, 43 (91%) cultured positive for B. burgdorferi. while 23/23 (100%) of
the controls cultured negative. Many of the cultures were clearly spirochetes when examined under light microscopy (Figures 1-3). Immuno-electron microscopy and Osp A PCR confirmation provided additional confirmatory evidence as to the identity of the spirochetes (Figures 4-7). The slide cultures consistently demonstrated the fastest and most abundant yields. With this technique, placement in the Coplin jar allows for varying gradations of oxygen tension. Sometimes spirochetal growth can be seen after as little as 20 h. appearing as a band near the upper end of the smear.

DISCUSSION
An attempt to culture B. burgdorferi from the blood of previously aggressively treated chronic Lyme disease patients seemed at first a monumental task. Before undertaking this effort, we therefore had to be as sure as possible that the organisms were indeed present in the blood of these patients. As a first step, we scrutinized a report where B. burgdorferi had been cultured from the blood of patients with early untreated disease. From this group of patients it had been noted in follow-up that subsequent blood cultures became routinely negative after antibiotic therapy, despite 71% of the patients remaining symptomatic [9]. Three possibilities readily come to mind for the explanation of this paradox: either 1) the infection is cleared, but a post-infectious process continues, or 2) the organism is cleared from the blood rapidly. but finds a pathogenic harbor elsewhere, or 3) the organism is maintained in the blood in an altered state which cannot be cultured on routine media.

In response to the first possibility, the notion of a post-Lvme syndrome has countless flaws. A post-infectious syndrome could not explain the observation that patients with "post-Lyme" or "post-Lyme fibromyalgia" responded to re-treatment with antibiotics, only to relapse with its discontinuation [13-15]. With the advent of PCR, antigen capture, and the benefit of those rare successful culture experiments even in the face of prior "curative treatment" [3-8]. the notion of "post-Lyme" should have been dismissed long ago. In response to the second possibility, given the common finding of circulating immune complexes with Lyme disease, we thought this unlikely [16]. Thus we were left with the third and most logical possibility. Specifically, we chose to pursue the organism in its cell wall-deficient state, i.e. L-forms, as previously reported [17].

Although L-forms will complex with fluorescent antibody to B. burgdorferi, only as they revert to classic parent forms can the typical spirochetal morphology be seen. There has been a considerable spectrum of cell wall deficiency demonstrated in our laboratory. B. burgdorferi may exist in various forms depending on its environment. In addition to the spirochetal form, we have demonstrated its growth both as amorphous L-forms and rounded giant L-bodies which have been previously described as cystic forms [11, 18]. As B. burgdorferi reverts from cell wall deficiency with the rebuilding of its cell wall, classic spirochetal forms can be seen. Most often, in our cultures, B. burgdorferi can be seen in varying stages of reversion, i.e. some L-dependent spirochetal forms within an L-form colony.

The L-form variants, osmotically fragile by nature, require precise conditions to grow in culture. our medium and methodology are specifically designed for the fostering of cell wall-deficient organisms and their reversion to classic parent forms. In most instances, the methods must be followed precisely. Even small variations produce no growth. For example, 2% yeast extract instead of 1% is inhibitory. or if the yeast extract is autoclaved with the rest of the medium instead of separately. that too will be inhibitory. However, there is one aspect of B. burgdorferi's growth characteristics which we found to be remarkably non-fastidious. The organism can be easily grown throughout a wide range of pH, from 6.8-7.8. This explains the different ratios of NaHC03 used in the various types of culture mediums. We are still not sure about the optimal pH for culture. Future research will address that question more specifically.

It should be noted from this study that currently accepted standards for serologic diagnosis seem to be inadequate. Only a small minority of participants in the study had positive Lyme ELISAs. Under the current recommendations for two-tier testing by the CDC/ASTPHLD, 91% of the patients in the study would have been misdiagnosed as not having Lyme borreliosis.

It is hoped that our work will help to end a medical controversy which has been going on for far too long. This study proves that chronic Lyme disease is of chronic infectious etiology, and that even antibiotic treatment well in excess of current recommendations is not necessarily curative. Given the flaws in currently accepted serologic diagnostic criteria, it is likely that Lyme borreliosis is vastly underdiagnosed. May this research help to focus the scientific community on effective curative therapies for patients with chronic Lyme disease.

It should also be noted that, in addition to its utility in growing B. burgdorferi, the MPM medium may be useful for culturing a variety of other spirochetes from patients.

REFERENCES

1 Krupp. L. B../ Maser, V../ Schwartz, J., Doyle, P. K., Langenback, L. J., Fernquist, S. R.: Cognitive functioning in late Lyme borreliosis Arch. Neurol. 48 (1991) 1125-1129
2. Logigian. E. L../ Kaplan. R. F., Steere, A. C.: Chronic necrologic manifestations of Lyme disease N. Engl J. Med. 323 (1990) 143~1444. 3 Bayer. M. E../ Zhang, L../ Bayer, M. H.: Borrelia burgdorferi DNA in the urine of treated patients with chronic Lyme disease symptoms A PCR study of 97 cases Infection 24 (1996) 347-353
4 Nocton, J. J../ Dressier. F../ Rutledge, B. J., Rys, P. N., Persing, D. H., Steere, A. C.: Detection of Borrelia burgdorferi DNA by polymerase chain reaction in svnovial fluid from patients with Lyme arthritis N. Engl. J. Med. 330 (1994) 229-234
5 Preac Mursic, V., Weber, K., Pfister, H. W., Wilske, B../ Gross, B., Baumann, A., Prokop, J.: Survival of Borrelia burgdorferi in anti-biotically treated patients with Lyme borreliosis Infection 17 (1989) 355-369 6 Schmidli, J., Hunzicker, T., Moesli, P., Schasd, U. B.: Cultivation of Borrelia burgdorferi from joint fluid three months after treatment of facial palsy due to Lyme borreliosis J. Infect Dis 158 (1988) 905-906
7 Pfister, H. W../ Preac Mursic. V../ Wilske, B../ Schielke, E../ Sorgel, F., Einhaupl, K. M.: Randomized comparison of ceftriaxone and cefotaxime in Lyme neuroborreliosis. J. Infect. Dis 163 (1991) 311-318.
8 Hassler, D../ Riedel. K., Zorn, J., Preac Mursic, V.: Pulsed high-dose cefotaxime therapy in refractory Lyme borreliosis (letter). Lancet 338(1991) 193
9 Nadelman, R. B../ Pavia, C. S., Magnarelle, L. A., Wormser, G. P.: Isolation of Borreha burgdorferi from the blood of seven patients with Lyme disease. Am. J. Med. 88 (1990) 21-26.
10 Berger, B. W., Johnson. R. C., Kodner, C., Coleman, L.: Cultivation of Borrelia burgdorferi from the blood of two patients with erythema migrans lesions lacking extracutaneous signs and symptoms of Lyme disease. J. Am. Acad Dermatol. 30 (1994) 48-51
11. Hulinska, D., Bartak, P., Hercogova, J., Hancil, J., Basta, l., Schramlova, J.: Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients. Zbl. Bakt 280 (1994) 348-359 12 Hulinska, D., Krausova, M., Janovska, D., Rohacova, H../ Hancil, J., Mailer, H.: Electron microscopy and the polymerase chain reaction of spirochetes from the blood of patients with Lyme disease. Cent Eur. J. Public Health 1 (1993) 81-85
13 Sigal, L. H., Patella. S. J.: Lyme arthritis as the incorrect diagnosis in pediatric and adolescent fibromyalgia Pediatrics 90 (1992) 523-528 14 Dinerman, H., Steere, A. C.: Lyme disease associated with fibromyalgia. Ann Intern Med. 117 (1992) 281-285
15 Steere, A. C., Taylor, E., McHugh, G. L., Logigian, E. L.: The over-diagnosis of Lyme disease JAMA 269 (1993) 1812-1816
16. Schutzer, S. E., Coyle, P. K., Belman, A. L../ Golightly, M. G., Drulle, J: Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease Lancet 33S (1990) 312-315
17 Preac Mursis V../ Wanner, G., Reinhardt, S., Wilske. B../ Busch, U., Marget, W.: Formation and cultivation of Borrelia burgdorferi spheroplast L-form variants Infection 24 (1996) 218-226
18 Brorson, 0, Brorson, S. H.: Transformation of cystic forms of Borrelia burgdorferi to normal mobile spirochetes Infection 2S (1997) 240-246

Received: 27 September 1997/Revision accepted: 3 September 1998

S. E Phillips. M. D../ Greenwich Hospital, S Perry Ridge Rd., Greenwich, CT 06830: L. H. Mattman, Ph. D., Spirotech Institute, Empire State Bldg., 350 Fifth Ave../ Suite 6101, New York, NY 10118: H. Monyad. D. O., Columbia North Hills Medical Center, 4401 Booth Calloway Rd., North Richland Hills, TX 76180, USA: Dagmar Hulinska. Ph. D., National Institute of Public Health. GEM-ELM. Srobarova 48. 10042 Praha 10. Czech Republic.

Correspondence to: Dr. S. E. Phillips. 10 Roberts Lane. Suite 2. Ridgefield. CT 06877. USA.

[soijuv]

Monien kroonisten tulehdustautien syynä on bakteeri tai virus. Yksi tällainen on borrelia-bakteeri joka voi aiheuttaa ihmisille ja eläimille kroonisia tulehduksia ja kudosvaurioita esim. niveliin, keskushermostoon, sydämeen ja muualle elimistöön.
Borrelia-bakteeri selviää immuunipuolustuksesta. Bakteeri kykenee kasvattamaan immuunipuolustukselle resistenttejä kantoja ja siten kroonistumaan. (2012)

http://www.ncbi.nlm.nih.gov/pubmed/22470370

Front Microbiol. 2012;3:104. Epub 2012 Mar 22.

Population Dynamics of Borrelia burgdorferi in Lyme Disease.

Binder SC, Telschow A, Meyer-Hermann M.

Department of Systems Immunology, Helmholtz Centre for Infection Research Braunschweig, Germany.

Abstract
Many chronic inflammatory diseases are known to be caused by persistent bacterial or viral infections. A well-studied example is the tick-borne infection by the gram-negative spirochaetes of the genus Borrelia in humans and other mammals, causing severe symptoms of chronic inflammation and subsequent tissue damage (Lyme Disease), particularly in large joints and the central nervous system, but also in the heart and other tissues of untreated patients.

Although killed efficiently by human phagocytic cells in vitro, Borrelia exhibits a remarkably high infectivity in mice and men. In experimentally infected mice, the first immune response almost clears the infection. However, approximately 1 week post infection, the bacterial population recovers and reaches an even larger size before entering the chronic phase. We developed a mathematical model describing the bacterial growth and the immune response against Borrelia burgdorferi in the C3H mouse strain that has been established as an experimental model for Lyme disease.

The peculiar dynamics of the infection exclude two possible mechanistic explanations for the regrowth of the almost cleared bacteria. Neither the hypothesis of bacterial dissemination to different tissues nor a limitation of phagocytic capacity were compatible with experiment.

The mathematical model predicts that Borrelia recovers from the strong initial immune response by the regrowth of an immune-resistant sub-population of the bacteria. The chronic phase appears as an equilibration of bacterial growth and adaptive immunity. This result has major implications for the development of the chronic phase of Borrelia infections as well as on potential protective clinical interventions.

[soijuv]

Tutkimuksen mukaan borrelia-bakteeri selviytyy antibioottihoidoista. Borrelioosin hoidossa käytetyt antibiootit kuten doksisykliini ja keftriaksoni tuhoavat bakteerin koeputkiolosuhteissa (in vitro), mutta se on huomattavasti selviytymiskykyisempi ihmisen tai eläimen elimistössä. Tutkimus suoritettiin makaki apinoilla. (2012)

Persistence of Borrelia burgdorferi in Rhesus Macaques
following Antibiotic Treatment of Disseminated Infection

Monica E. Embers1*, Stephen W. Barthold4, Juan T. Borda2, Lisa Bowers1, Lara Doyle3, Emir Hodzic4,
Mary B. Jacobs1, Nicole R. Hasenkampf1, Dale S. Martin1, Sukanya Narasimhan5, Kathrine M. Phillippi-
Falkenstein3, Jeanette E. Purcell3¤, Marion S. Ratterree3, Mario T. Philipp1*

1 Divisions of Bacteriology & Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of
America, 2 Comparative Pathology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America,
3 Veterinary Medicine, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, Louisiana, United States of America, 4 Center for
Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California Davis, Davis, California, United States of America, 5 Section of Rheumatology,
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America

Abstract

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a
matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus
macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4?6 months later. Multiple
methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys
(xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic
peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were
detected in the tissues of treated animals.
Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys.

These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered postdissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate
host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not
they can contribute to symptoms post-treatment.




Citation: Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, et al. (2012) Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment
of Disseminated Infection. PLoS ONE 7(1): e29914. doi:10.1371/journal.pone.0029914
Editor: Jean Louis Herrmann, Hopital Raymond Poincare - Universite Versailles St. Quentin, France
Received July 22, 2011; Accepted December 6, 2011; Published January 11, 2012
Copyright: 2012 Embers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIAID grant R01-AI042352 (MTP), R01-AI26815 (SWB and EH), a TNPRC Pilot Study Grant (MEE), and NCRR grant RR00164.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: members@tulane.edu (MEE); Philipp@tulane.edu (MTP)
¤ Current address: Biologic Resources Laboratory, University of Illinois, Chicago, Illinois, United States of America

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Tutkimuksen mukaan henkilöillä joiden oireet jatkuvat antibioottihoitojen jälkeen esiintyy vasta-aineita jotka viittaavat pitkään jatkuneeseen infektioon. Vasta-aineita esiintyi borrelia-bakteerin pinnalla esiintyvää VlsE proteiinia kohtaan. Kroonisista oireista kärsivillä esiintyi erilaisia vasta-aineita proteiinia kohtaan. Sen seurauksena immuunipuolustus aktivoitui hyökkäämään bakteeria vastaan.
Näyttää siis siltä että potilaat kärsivät pitkittyneestä mikrobin aiheuttamasta infektiosta. Bakteeri on paennut immuunipuolustustusta muuntamalla pintaproteiinejaan.

Erilaisten vasta-aineiden olemassaolo voi viitata siihen että krooniset oireet johtuvat jatkuvasta vasta-aineiden aiheuttamasta tulehduksellisesta reaktiosta eloimistön omia proteiineja kohtaan.

http://www.nature.com/news/2011/110805/ ... rticles%29

Published online 5 August 2011 | Nature | doi:10.1038/news.2011.463

News
Antibodies linked to long-term Lyme symptoms

Researchers find molecules that might mark elusive syndrome.

Amy Maxmen
Ticks spread the bacterium behind Lyme disease ? but symptoms can persist even when the microbe seems to have gone. Medical-on-Line/Alamy

Some patients with Lyme disease still show symptoms long after their treatment has finished. Now proteins have been discovered that set these people apart from those who are easily cured.

People who experience the symptoms of Lyme disease, which include fatigue, soreness and memory or concentration loss, after treatment for the disorder are sometimes diagnosed as having chronic Lyme disease or post-Lyme disease syndrome. But these diagnoses are difficult to make, because the individuals no longer seem to harbour the bacteria that cause Lyme disease. And the symptoms could instead be indicative of chronic fatigue syndrome or depression.

Now Armin Alaedini at Weill Cornell Medical College in New York and his colleagues have found that patients diagnosed with post-Lyme disease syndrome have antibodies that suggest they carried the infection for an unusually long time. The finding, published in Clinical Immunology1, might help the syndrome to be better understood, diagnosed and treated.


Alaedini's team looked at antibodies made in response to a protein called VlsE, which is found on the surface of Borrelia burgdorferi, the tick-borne bacterium that causes Lyme disease.

The antibodies recognize a snippet of the protein called an epitope, and recruit the immune system to attack the bacterium. The researchers found that post-Lyme sufferers have a greater variety of antibodies to this epitope than patients whose infection cleared up quickly.

This finding suggests that patients with chronic symptoms have experienced a prolonged infection, caused by microbes that have evaded the immune system by varying the epitopes they carry. As a result of these variations, the body makes new antibodies targeting the modified protein. The longer the microbe manages to keep changing, the more diverse its host's antibodies become.

Some post-Lyme sufferers had varied antibodies against VlsE epitopes despite being diagnosed and treated early, says Alaedini. "That could mean they naturally have a different antibody response to the infection than most people; it could mean they weren't treated properly; or it's possible they were reinfected and the second infection was never treated," he says.
Inflammatory role

"This is the first study I've seen that shows some immunologic difference between someone who resolves their Lyme and someone who develops post-Lyme disease syndrome," says Linda Bockenstedt, a rheumatologist and immunologist at Yale School of Medicine in New Haven, Connecticut.

The presence of varied antibodies hints that the chronic symptoms could be caused by an ongoing inflammatory response caused by antibodies mistakenly reacting to the body's own proteins, Bockenstedt suggests.

"The big question to me is whether this can lead to an autoimmune phenomenon," says Bockenstedt. "But if that were the case, I'd expect the disease to worsen without immune-modulating treatment, and it doesn't."

ADVERTISEMENT

Alaedini suggests that higher levels of antibodies could increase the body's levels of cytokines, immune-system proteins that can trigger the symptoms experienced by patients with post-Lyme disease syndrome. "Various cytokine profiles have been associated with fatigue, anxiety and depression," he explains.

If these antibodies are unique to people with chronic Lyme disease, it could lead to a test and treatments for the disorder, Alaedini says. It could also guide treatment of the disease itself. "If patients with an acute infection develop antibodies to these epitopes, perhaps they require a more aggressive course of therapy," he adds.

But a predictive marker won't be useful without new therapies for the persistent symptoms, says Henry Feder Jr, a physician specializing in infectious diseases at the University of Connecticut Health Center in Farmington. If an immune response problem leads to the syndrome, antibiotics won't help. "I guarantee you that if you tell a patient they won't feel better after antibiotics, they won't," Feder says. "We need to know what's going on."

References
Chandra A. et al. Clin. Immunol. http://dx.doi.org/10.1016/j.clim.2011.06.005 (2011).

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http://www.ncbi.nlm.nih.gov/pubmed/22470370

Saksa 2012: Bakteerit ja virukset aiheutavat useita kroonisia tulehdustauteja, esimerkiksi Borrelioosin. Borrelioosin aiheuttaa gram-negatiivinen bakteeri joka voi hoitamattomana aiheuttaa ihmisille ja eläimille kroonisen tulehduksen ja kudosvaurioita esim. nivelissä ja keskushermostossa, sydämessä jne.

Immuunipuolustuksesta huolehtivat fagosyytit tuhoavat Bb:n tehokkaasti koeputkiolosuhteissa (in vitro) mutta ihmisen tai eläimen elimistössä bakteeri toipuu nopeasti. Jo viikon kuluttua infektion alkamisesta bakteerien määrä on korkeampi kuin tartunnan alussa.

Tutkijat kehittivät aiheesta matemaattisen mallin ja sen perusteella he tulivat siihen tulokseen, että Bb selviää elimistön immuunivasteesta kasvattamalla immuunipuolustukselle vastustuskykyisiä bakteereja. Taudin kroonistuminen on näin ollen seurausta bakteerien kasvusta ja vastustuskyvyn kehittymisestä.

Front Microbiol. 2012;3:104. Epub 2012 Mar 22.

Population Dynamics of Borrelia burgdorferi in Lyme Disease.

Binder SC, Telschow A, Meyer-Hermann M.

Department of Systems Immunology, Helmholtz Centre for Infection Research
Braunschweig, Germany.


Abstract
Many chronic inflammatory diseases are known to be caused by persistent bacterial or viral infections. A well-studied example is the tick-borne infection by the gram-negative spirochaetes of the genus Borrelia in humans and other mammals, causing severe symptoms of chronic inflammation and subsequent tissue damage (Lyme Disease), particularly in large joints and the central nervous system, but also in the heart and other tissues of untreated patients.

Although killed efficiently by human phagocytic cells in vitro, Borrelia
exhibits a remarkably high infectivity in mice and men. In experimentally infected mice, the first immune response almost clears the infection.
However, approximately 1 week post infection, the bacterial population recovers and reaches an even larger size before entering the chronic phase.


We developed a mathematical model describing the bacterial growth and the immune response against Borrelia burgdorferi in the C3H mouse strain that has been established as an experimental model for Lyme disease.

The peculiar dynamics of the infection exclude two possible mechanistic explanations for the regrowth of the almost cleared bacteria. Neither the hypothesis of bacterial dissemination to different tissues nor a limitation of phagocytic capacity were compatible with experiment.

The mathematical model predicts that Borrelia recovers from the strong initial immune response by the regrowth of an immune-resistant sub-population of the bacteria. The chronic phase appears as an equilibration of bacterial growth and adaptive immunity. This result has major implications for the development of the chronic phase of Borrelia infections as well as on potential protective clinical interventions.

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2012. Krooninen Borrelioosi yhteydessä ADHD oireisiin.

Medscape Medical News from: The American Psychiatric Association's 2012 Annual Meeting

Chronic Lyme Disease Linked to ADHD in Adults


May 8, 2012 (Philadelphia, Pennsylvania) ? Chronic lyme disease (CLD) has been linked to attention-deficit hyperactivity disorder (ADHD) in adults, new research shows.

"The association between ADHD and CLD has not been identified previously," principal investigator Joel L. Young, MD, medical director, Rochester Center for Behavioral Medicine, Rochester, Minnesota, told Medscape Medical News. The survey results also corroborate earlier findings of a relationship between CLD and anxiety and depression, he said.

Dr. Young presented his research here at the American Psychiatric Association's 2012 Annual Meeting.

Participants for the survey were drawn from the 2009 Michigan Lyme Disease Association Conference. A total of 58 adults with CLD and a control group of 26 adults without CLD participated. The mean age was 48 to 49 years in both groups.

On the AD/HD Self-Report Scale (ASRS), adults with CLD endorsed more ADHD symptoms than control participants. Results were significant for both inattentive and hyperactive subunits and the combined type, Dr. Young reported.


Novel Finding

"Cognitive deficits associated with CLD have been demonstrated before," Dr. Young said, "although this is the first survey to identify a linkage between these two conditions."

As expected, the CLD group had statistically significantly higher scores on the Fatigue Severity Scale (FSS) than the control group. The CLD group also had "dramatically higher" rates of dysthymia, generalized anxiety, major depression, and somatization.

Dr. Young said the correlation between ADHD and CLD "is novel in the research literature. Symptoms of CLD include persistent fatigue and unexplainable generalized pain. We conclude that many individuals who are diagnosed with CLD might have ADHD (inattentive type). We believe that many are diagnosed with CLD inaccurately and that ADHD symptoms might better explain their persistent pain and fatigue," he added.

Dr. Young emphasized that there is currently "little consensus about the validity of CLD. Most clinicians agree that there is a phenomenon of acute Lyme disease, but there is no consensus about whether it is a chronic condition. I believe that patients who have these symptoms often get the diagnosis of CLD because there is no other explanation for their chronic fatigue and pain."

"Many times," Dr. Young added, "the neuropsychiatric complications associated with CLD are the most problematic for individuals. My research indicates that individuals with CLD should be evaluated for ADHD. It is unclear if treating ADHD will help these individuals' symptoms of pain and fatigue."

Interpret With Caution

Commenting on the study for Medscape Medical News, Brian Fallon, MD, MPH, director of the Lyme and Tick-Borne Diseases Research Center, Columbia University, New York City, cautioned against drawing any firm conclusions from this survey. Surveys are "notorious for elevating psychiatric complaints," he said.

"Most carefully conducted neurocognitive studies have identified problems with memory, processing speed, and verbal fluency in Lyme patients ? and not attention problems. Attention problems are primarily seen in depression," added Dr. Fallon, who was not involved in the study.

Dr. Young is the author of the book ADHD Grown Up: A Guide to Adolescent and Adult ADHD (W.W. Norton, 2009). He has disclosed relationships with Cyberonics Inc, Eli Lily, Novartis, Otsuka, Pfizer, Shire, Forest, Merck, Bristol Myers Squibb Co, and Shionogi Inc. Dr. Fallon has disclosed no relevant financial relationships.

The American Psychiatric Association's 2012 Annual Meeting. Abstract NR8-30. Presented May 8, 2012.

http://www.medscape.com/viewarticle/763458?src=nl_topic


article/763458?src=nl_topic

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Krooninen Borrelioosi/neuropsykiatrinen Borrelioosi

http://www.mentalhealthandillness.com/tnaold.html

The Neuropsychiatric Assessment of Lyme Disease
Robert Bransfield, M.D.

Objective: A structured clinical interview is proposed to assist in the overall clinical assessment when late state Lyme disease is suspected.

Method: From a combination of clinical experience, journal review, and discussion with colleagues, a structured interview was developed. Information from patients with late stage neuropsychiatric Lyme disease (NPLD) was entered into a database to serve as a reference point for diagnosis and tracking the patient?s status after diagnosis.

Results: An analysis of symptoms acquired from a thorough history and mental status exam can be quite helpful towards the total clinical assessment when suspecting late stage Lyme disease. Details are provided in the text of this article.

Conclusion: When NPLD is a diagnostic possibility, a detailed, well-focused interview and mental status exam is proposed, and a database of symptoms seen in NPLD is established. It is recommended to continue perfecting the assessment as well as expanding the database. If diagnostic accuracy is improved, there would be better consensus regarding treatment strategies.

Objective

There are many unanswered questions regarding chronic Lyme disease. They remain unanswered as a result of our inability to accurately diagnose the presence or absence of the causative agent B. burgdorferi. The usual laboratory tests alone are not totally reliable to confirm or refute the diagnosis of Lyme disease (1). When we combine the current laboratory tests with a very thorough history, physical, and mental status exam, the accuracy of diagnosis is greatly increased. If we can improve the accuracy of diagnosing the presence of Lyme disease, there would then be more agreement regarding treatment guidelines. In an effort to improve diagnostic accuracy, I have developed a psychiatric diagnostic and tracking system.

Background

There are an increasing number of patients with chronic Lyme disease (neuroboreliosis) presenting in psychiatric offices. Lyme disease does not begin as a psychiatric illness. Other symptoms occur in early stage disease. Late in the progression of this disease neurological, cognitive, and psychiatric symptoms predominate. If not well understood, these symptoms are sometimes viewed as non-specific and bizarre. Actually the symptoms can be quite specific with a clear physiological basis, but far too often a routine evaluation is insufficient to adequately evaluate these patients. When the evaluation is not property targeted, key symptoms can be overlooked and these patients may be mistakenly diagnosed with chronic fatigue syndrome, fibromyalgia, M.S., lupus, Epstein barr, as well as many other medical and psychiatric symptoms. (2) They are considered by some to be "hypochondriacal" or "crazy." As a result, many of these patients feel alienated from the mainstream of the health care system. (3,4,5). The recent work of Drs. Fallon and Nields drew attention to the significance of the psychiatric component of chronic Lyme disease. (2,6,7,8,9,10).

As a psychiatrist practicing in a Lyme endemic area, I have evaluated and treated many of these patients from a psychiatric perspective over a period of years. Most of these patients were previously diagnosed with Lyme disease and many were considered to have been cured by prior adequate antibiotic treatment. I would like to share some of my observations, experience, and impressions from working with this population. Clinical experience is critical to add towards our total understanding of chronic Lyme disease (11).

We can view Lyme disease as a stealth Phoenix - it is difficult to find, and even more difficult to eradicate after it has penetrated deep into body tissue. (1,11) Once late stage disease exists, it is impossible, with current technology, to prove that B. burgdorferi has been eradicated. Constant vigilance is, therefore, required. Years of failure to recognize, diagnose, and adequately treat these patients has led to an ever expanding epidemic of chronic Lyme disease. (12,13,14,15,16).

All involved with late state Lyme disease agree there is a large amount of inaccurate information on this subject. This disagreement exists at every level - journals, scientific meetings, clinical practice, media outlets, etc. (17,18,19) Some of this disagreement can best be viewed as the normal difference of opinion seen when scientists approach a very complex problem from a very different perspective. To fuel the intensity of these disputes, some approach these issues with a significant bias. The full recognition of this illness has implications, which could effect tourism, real estate values, disability, insurance company/managed care liability, workman?s compensation cases, motor vehicle issues, some criminal cases, and political issues. Bias issues can adversely effect patient care, research funding, and medical regulatory issues. Some of those previously impacted by bias now have difficulty approaching this disease with full-unhampered objectivity.

As physicians, it is our responsibility to protect life and quality of life. A tenacious pro-active stand prevents bias from obstructing access and quality of care.

Lyme disease is clearly a very complex disease. When considering a similar spirochete disease, syphilis, it has been said, "To know syphilis is to know medicine." However, to know Lyme disease is not only to know medicine but also neurology, psychiatry, politics, economics, and law. The complexity of this disease and all that surrounds it challenges our scientific as well as our ethical capabilities. I shall not address every aspect of this disease but I shall focus on diagnosis, in particular from a psychiatric perspective.

The diagnosis of Lyme disease and in particular late stage Lyme disease is a total clinical diagnosis (20). We need to return to basics. In my opinion, a very thorough history, mental status exam, neurological, and physical are the most significant components of the examination. We, in psychiatry, are uniquely trained to perform the type of exam that is needed for these patients. Laboratory tests, which are highly controversial, are also helpful, but the interpretation of the findings is complex and requires clinical correlation.

The commonly used blood, urine, and spinal fluid tests have a significant rate of false negatives in the chronic neuropsychiatric Lyme disease population. (See addendum - Seronegative Lyme)

Other tests that may be useful include P.E.T. and SPECT. Brain SPECT scans in Lyme patients sometimes show a swiss cheese pattern of hypoperfusion. (10)

In my experience, the location of the hypoperfusion may correlate with brain areas noted to be dysfunctional on the clinical exam. Although often reliable, not every neuropsychiatric Lyme disease patient demonstrated SPECT findings.

Some patients show findings on MRI?s, but more commonly after illness has persisted for a few years. HTL tissue typing may be useful in showing who is more vulnerable to a more severe form of the illness. (21,22,23) Spinal taps (24) and nerve conduction studies can provide additional information. Psychological testing by an examiner who is experienced with evaluating Lyme disease may also be helpful.(2) Lyme urine antigen tests and PCR are being used with more frequency in the assessment of chronic Lyme disease. In addition to B. burgdorferi infections, coinfections with other agents leading to interactive infections are a significant issue. Some of these coinfections are other tick borne diseases, such as babesiosis, human granulocytic ehrlichiosis, human monocytic ehrlichiosis, etc. Other non-tick borne coinfections may also be significant, such as CMV, strep, etc. None of these tests can, however, exclude the diagnosis of Lyme disease. Many of our colleagues in internal medicine find the evaluation of this disease to be highly frustrating since compared to other illnesses there are less so called "objective" findings which are present. In both psychiatry and primary care, we are trained to contend with this gray zone of diagnostic criteria in which a skillful interviewer can recognize that subjective symptoms are valid. We are more accepting of Sir William Osler?s quote - "If you listen long enough, the patient will give you the answer." Effective communication with the patient is critical in considering the diagnosis. For us, Lyme disease presents with more objective symptoms than we commonly see with other mental disorders. There has been a recent trend to incorrectly view so called "objective" signs and symptoms as more valid than those which are "subjective." Often a machine or lab test is perceived as giving validity to these "objective" signs. Many of these "objective" tests are far less valid and are based on questionable techniques, faulty assumptions, and flawed logic. On the other hand, "subjective" complains are sometimes viewed with excessive suspicion. When a credible patient describes a symptom that challenges our medical capability, it is an error to assume without the proper supporting evidence that they are lying, delusional, or hypochondriacal. In an effort to create predictability, reliance upon cookbook medicine has given us a recipe for disaster. Algorithms should be viewed as teaching tools and very rough guidelines, but should never be given more significance than a detailed thorough exam and sound clinical judgment. We, as physicians, owe it to our patients to always retain our courage and never defer our sound clinical judgment to dictatorial guidelines. When considering a diagnosis of Lyme disease, a distinction is made between the following groups:

1. Never infected by B. burgdorferi.

2. Infected by a Lyme like bacteria. (i.e., some other spirochete)

3. Infected by a B. burgdorferi and manifesting.

Subclinical illness
Minimal self-limiting illness
Early stage disease
Disseminated disease

4. Infected, never manifesting late stage disease, and currently either cured or in remission.

5. Infected, having manifested late stage disease and:

Not previously diagnosed with Lyme disease.
Previously diagnosed and treated with:

a. Possible cure

b. Remission

c. Subsequent progression of Lyme disease symptoms.

d. Current illness unrelated to Lyme disease.

e.Progression of Lyme disease symptoms and comorbid illness (es)

Late stage neuropsychiatric Lyme disease can best be conceptualized as a disseminated and progressive, (predominately sub-cortical), encephalopathy. Animal studies and autopsies have contributed to our understanding of the disease process. (25,26,27,28). As symptoms progress, additional symptoms occur and increase in severity.

These symptoms may be categorized in the following manner:

1. Brain Stem

Cranial Nerve symptoms
Autonomic symptoms. Dysautonomia may be the result of involvement of brain stem, involvement of other parts of the autonomic nervous system, or end organ pathology - i.e.: migraine, temperature dysregulation, sexual dysfunction, bright light sensitivity, mitral valve prolapse, irregular pulse, neutrally mediated hypotension, asthma, non-ulcerative dyspepsia, irritable bowel, and irritable bladder
Hormonal symptoms: From the result of either hypothalamus or end organ involvement, i.e., thyroid disease, HPA axis dysregulation, decline of sex hormone functioning, hypoglycemia
Long track disconnection syndromes (very late in the progression of the disease)
Cerebellar symptoms

2. Limbic system, greater limbic system, extended amygdala

Altered attention, emotional, and behavioral modulation
Pathological psychiatric syndromes

3. Cortical (May be from either cortical or sub-cortical nuclei involvement)

Signature syndromes
Processing difficulties

4. Peripheral Neuropathy

The Structured Interview

When evaluating a patient, I recommend completing the following structured interview. I have found it to be quite helpful and cost effective in evaluating the possibility of neuropsychiatric Lyme disease. I have gradually developed this examination after interviewing many patients with neuropsychiatric Lyme disease. Since these patients have multiple deficits, which impair effective communication, we cannot expect the patient to volunteer the relevant information. Therefore, we must methodically ask the appropriate questions in order to acquire an accurate history. The thoroughness of this exam combined with sound clinical judgment helps us to differentiate between who may have had Lyme disease, who may be in remission, who is showing active disease, and who may have some closely related condition. When the diagnosis is unclear, I suggest repeating the exam at a later date to consider whether the symptoms are increasing, stable or improving. The exam should also be repeated during and after treatment to further track the patient?s status.

On the structured interview, at the left is a database for each item. The first data base column shows the incidence of a given symptom based on history prior to infection to serve as a control. The second column shows the incidence of these symptoms in the same population after infection in cases with significant clinical and laboratory findings. I am still in the process of completing this database. Many of the patients I have seen with LD are children, teenagers, and young adults. As a group, it has been my experience to find their pre-morbid status to be healthier than the average, both mentally and physically. This is a disease that more commonly attacks "lovers of life." (29) - young, healthy active individuals who engage in more outdoor activities, particularly those living in suburban and rural areas. Many report they have never had any significant illness prior to the onset of Lyme disease. After becoming ill, most of the patients report they had previously been diagnosed with Lyme disease and were given, what was considered by some standards, to be an adequate treatment. Subsequently, their symptoms progressed with increasing cognitive, psychiatric, and neurological components. Most of the patients tested positive at some point in the course of their illness. Some were not seropositive until after treatment had progressed. Some were seronegative which resulted in a significant delay in treatment and a progression to a greater number and more severe symptoms. When comparing the symptoms, there appeared to be no significant difference between the strongly seropositive (some of whom sell their blood to be used as a reference for labs), and the patients with more moderate laboratory findings and the seronegative group. The longer the interval between initial infection and effective treatment, the worse the prognosis.

Some symptoms are more specific than others. The control database is useful as a reference point. By looking at the patient?s profile and comparing it to the database, we can see that some symptoms are more prevalent than others. Symptoms with an asterisk correlate with high diagnostic significance. Any single symptom may be seen in other illnesses, but the cluster of symptoms combined with our total knowledge of psychiatry, neurology, and medicine helps us to make the appropriate diagnosis. Patients with NPLD show significant number of positive responses. Patients with a different diagnosis do not demonstrate a large number of positive responses.

(Neuropsychiatric Assessment of Lyme Disease - Assessment Form)

Assessment

A Review of Items in the Intervie

To review each item in sequence, refer to the flow sheet. Some of these times are self-explanatory whereas other require clarification. Recurrent erythema migrans rash is sometimes seen in the chronic Lyme population and has diagnostic significance. On rare occasions, chicken pox and other conditions can bring out the bull?s eye rash. For example, patients with a chronic Lyme infection who become infected with chickenpox sometimes show the bull?s eye rash around the chickenpox lesions.

The cognitive symptoms are particularly noteworthy when NPLD is suspected. When we attempt to correlate SPECT or PET findings with areas of deficit which are demonstrated in a clinical exam, cognitive deficits are easier to localize than emotional one. Many of these patients give a history of an acquired attention deficit disorder. Auditory hyperacusis is particularly noteworthy.

Memory deficits are more selective for working memory, short-term memory, slowness of retrieval, and sequential memory (2). Long-term memory for information prior to the onset of the encelopathy is usually relatively preserved until very last in the course of the illness. Memory encoding errors sometimes exist with the creating of some false memories, some of which occur during dissociative episodes. Working spatial memory can be impaired, i.e.: patients may have difficulty with the spatial awareness of where their front and back doors are in their house. One patient had a panic attack when they were lost in their garage and had lost the spatial awareness of the location of the door. The slowness of retrieval is evidenced as these patients grope to retrieve words and names. In the later stage of this disease they also have difficulty recalling motor sequences, (2,30) i.e., a recall of the sequential steps needed to tie their shoelaces. They are able to tie their shoelaces but must think out each sequential step. Many Lyme patients state "I feel like I have become dyslexic." Impairment of reading comprehension is an earlier sign with the later addition of auditory comprehension difficulties. Acquired left/right confusion is seen with some of these patients displaying what appears to be an acquired Gerstmann?s Syndrome or some variant of this syndrome. They have problems with calculations and often complain of errors when trying to calculate their checkbooks. Fluency of speech is a very significant problem. When interviewing these patients, this is a clearly evident symptom. Stuttering is seen in many of these patients, a finding which may correlate with left caudate/striatum involvement. Slurred speech is significant and can lead to the false impression that these patients are intoxicated which has, on occasion, led to motor vehicle charges. Handwriting declines and a comparison of writing samples can be useful to confirm this finding. Optic ataxia (41) is another important finding. Sometimes it can be bilateral or unilateral. This is an upper parietal function involving the contra-lateral side. When this symptom is present, patients have trouble targeting, and they may bump into doorways, place objects incorrectly, and have problems driving in congested traffic.

Agnosia is a late appearing symptom. One patient was unable to recognize her own car in a parking lot. After receiving IV antibiotic treatment, she could recognize her car but was not able to recognize which key unlocked the car and which one started her car. Some of these patients display intrusive images which are more commonly of an aggressive nature but sometimes can be of a sexual or other nature. Occasionally these images are of a homicidal nature. The hallucinations are quite different from those commonly seen in schizophrenia. NPLD hallucinations are correlated with better reality testing.

Executive function and thought process are severely impaired with Lyme patients. This is very significant in contributing to the disability, which is seen in these patients, especially those accustomed to doing five things at once in their usual professional capacities. A small impairment in a patient who assumes a high level of responsibility can result in severe consequences. Cognitive tracking deficits, commonly called brain fog is a symptom that most of these patients describe. It is very different from the concentration impairment seen in clinical depression. Brain fog is a slowness and sluggishness of internal thought. Patients describe this as though their thought processes are shrouded in a fog.

The emotional and behavioral symptoms caused by Lyme disease are more complex to understand than the cognitive impairments. Let?s first review the physiology of emotion. The different emotional functions have a hierarchy of circuitry, which includes stimulatory pathways, opposing inhibitory pathways, and a hierarchy of modulation centers. The basic hierarchy is pre-frontal cortex, para limbic association areas, limbic structures, and brain stems - hypothalamus. Lyme encephalopathy can result in dysfunction of the modulation centers, inhibitory pathways, and stimulatory pathways. Autopsies, animal studies, and brain imaging tests have contributed to this understanding. The presenting symptoms of NPLD are sometimes emotional in nature, and include obsessive-compulsive disorder, depression, and aggression, panic disorder, and other phobic disorders.

In considering the behavioral symptoms, these patients can become suddenly suicidal and there have been completed suicides attributed to Lyme disease. Homicidal ideation, urges, and behavior occur in some of these patients. Some adult patients describe struggling to not act on these urges. When these patients act on a homicidal urge, more commonly it is a child becoming assaultive to a sibling. Dissociative episodes sometimes occur with these patients occasionally accompanied with aggressive behavior and loss of memory.
Compensatory compulsions are common in an effort to compensate for the memory deficits. NPLD can imitate a number of common psychiatric syndromes. It can be difficult to differentiate Lyme disease from rapid cycling Bipolar illness or Posttraumatic Stress Disorder. Eating disorders are common. Invariably these patients either gain or lose weight. Sometimes massive weight gain is also seen.

Neurological symptoms have been previously recognized as a component of Lyme disease (31-46). Cranial nerve findings begin before the cognitive changes are seen and can intensify again late in the course of the illness. There are times when the cranial nerve findings are more evident late in the day when the patient becomes tired and they acquire double vision, choke on food, or lose their ability to talk. Grand mal seizures are more significant with congenital Lyme cases, while complex partial seizures are seen in a notable percent of other NPLD patients. These seizures are effectively controlled with both anticonvulsants and antibiotics. Some neurological findings are common such as numbness, tingling, sensory loss, burning, weakness, tremors, myoclonic jerks. torticollis, and fainting. Ataxia is common in these patients who are often clumsy, which leads to frequent accidents. Myotonia is uncommon but I have been this in a few patients, and Parkinson?s syndrome caused by Lyme disease can also seen, although it is uncommon. A number of these patients have herniated discs after having Lyme disease for several years. I suspect, but cannot prove, there is a causal relationship between Lyme disease and herniated discs. Burning is quite specific to NPLD, but is also seen in herpes infections. The patient describes a sensation that a blowtorch is burning the skin. It can affect any part of the body. In some patients the burning migrates, while in others it remains in a given area. Both antibiotics and anticonvulsants relieve this symptom.

Joint pain, swelling, and tightness is an earlier manifestation of Lyme disease and is often more effectively treated than the central nervous system symptoms. Knees are the joints most commonly involved (47). Bone pain as a result of periostitis affects certain bones, such as the tibias. The periostium is spongy on examination. Chronic fatigue and fibromyalgia may be seen as part of Lyme disease (48). Of course, these two syndromes can be caused by other conditions as well. Chondritis of the ear and nose and costochondritis are sometimes seen with these patients.

The cardiac symptoms (49-57) are quite prominent early on in the disease although more commonly with alternating episodes of racing pulse and bradycardia. Conduction defects can be fatal in some cases. Other cardiac complications including cardiomyopathy can be a later manifestation of this disease. Irritable bladder is common. Chronic Lyme patients frequently acquire a number of autonomic nervous system problems which were not present prior to the onset of the disease. Alcohol intolerance is common and most patients state "I don?t drink any more." Some other physical manifestations are quite uncommon.

The Jarish Herxheimer reaction is seen when antibiotics are having a therapeutic effect. There can be a worsening in the symptoms, which may include the periostitis, and the psychiatric and cognitive symptoms. Some patients become very impulsive, aggressive, depressed, and suicidal during a Herxheimer reaction and may require close monitoring during these times.

Progression of symptoms is a significant item. After working with these patients, it is clear there are common patterns in which different symptoms appear in a different sequence. This item is checked when the symptoms are appearing in a sequence that is seen in the progression of Lyme disease, i.e.: it begins with a tick bite, then a bull?s eye rash associated with a flu like illness, then there may be some of the disseminated symptoms such as the joint pain. The cranial nerve symptoms may be seen. Later there is the development of the cognitive symptoms that gradually increase over time. Then the psychiatric symptoms develop later in the course of the illness with an intensification of the cognitive and neurological symptoms. Not every stage is seen in all patients. Although many similarities exist between patients, no two patients display the exact same symptoms; and there are many variants in the manner in which the disease presents. There is some evidence that different clusters of symptoms are associated with different strains of the bacteria, and there are many variants in the manner in which this disease presents.

After completing the interview and relevant mental status, neurological, and physical exams, we now review the pattern of symptoms that exist. Some of the symptoms may be difficult to localize, i.e., in a given patient is light sensitivity a result of meningeal irritation, cornea involvement, iritis, cranial nerve involvement, brain stem involvement, dysautonmia, or some other pathological process? Similar questions could be raised regarding other symptoms as well. The broad spectrum of symptoms helps to make the diagnosis. Can the symptoms be explained on some other basis? Is there some comorbid illness present?



The Role of the Psychiatrist in Treatment

Although diagnosis is the major focus of this article, I would like to say a few words about the role of psychiatry in the treatment of Neuropsychiatric Lyme Disease. The effective treatment of psychiatric symptoms in these patients helps improve their overall prognosis and reduce their need for antibiotic treatment. Let?s review the physiology.

In a state of stress, the body?s resources are allocated away from immune and regenerative functions towards stress related functions (58). If we reduce the symptoms related to the state of stress, the body will then allocate more resource towards immune and regenerative functions. Therefore, the treatment of sleep disorder, depression, anxiety, ADD, etc. associated with neuropsychiatric Lyme disease helps the patient recover. The treatment of sleep disorder is particularly significant. Chronic fatigue and fibromyalgia whether or not caused by Lyme disease are associated with a deficiency of slow wave sleep (62). Improvement of sleep quality, particularly slow wave sleep is strongly correlated with improvement of the chronic fatigue and fibromyalgia components of Lyme disease, which in turn benefits overall prognosis. In a milder case, this psychiatric treatment may lead to a total remission. In more severe cases this treatment merely buys time and gradually becomes less effective as the infection progresses, and the psychiatric symptoms become increasingly difficult to treat. When this trend exists, antibiotics and other treatments need to be combined with the psychiatric treatment. Some patients clearly need extended and repeated courses of antibiotic treatment (l, 59, 60). Although it is sometimes stated that most patients respond to conservative courses of antibiotic treatment, many of the patients I see have shown inadequate responses to such approaches and some have responded better to more aggressive, well-monitored antibiotic treatments. As with any treatment, the administration of antibiotics is an individualized risk verses benefit clinical decision. For effective treatment often intramuscular and intravenous antibiotics are needed, sometimes for extended period of time. It is well recognized such treatments have potential risks and a methodical risk vs. benefit assessment is needed. This decision should only be made by physicians who have assumed clinical responsibility, have personally examined the patient, and who have adequate knowledge of the illness and the therapeutic agents. Other treatments include nutritional approaches and physical therapy. Hyperbaric oxygen is a treatment that is showing increasing potential in the treatment of Lyme disease. It is currently speculated that many of the symptoms seen in chronic disease are attributable to an inflammatory process rather than active infection. Although it is clear some inflammatory symptoms exist (61), it is difficult to accept this belief to explain the majority of the progressive symptoms seen in these patients. The Jarish Herxheimer reaction appears to be inflammatory in nature and is of fairly brief duration after antibiotic treatment is discontinued. Many of the symptoms perceived as "inflammatory" improve in response to antibiotic treatment. The inflammatory view is not without risk when steroids are administered which can increase the progression of active infection.

As with any other invisible disability, many of the chronic patients feel demoralized after being exposed to stigma and abuse from those who cannot understand or those who are biased for a variety of reasons. Neuropsychiatric Lyme disease patients are the recipients of double stigmas - that of Lyme disease as well as mental disorder. Some patients are contending with illness while also stating there is a lack of support from family, health care providers, employers, and/or insurance companies. When such conditions exist, the stress level is magnified and the progression of the illness tends to increase.

We can also assist our patients by helping to understand the nature of this condition, resolving conflict, and offering input towards advocacy efforts. Lyme disease is an illness that has proven managed care to be a catastrophic failure. Early, effective treatment is critical. Managed care short-term cost containment techniques have resulted in very expensive long-term direct and indirect costs. Most of the indirect costs have been shifted to the general public.

To emphasize this point, I have seen too many examples of young people who have been denied needed treatment. Their condition deteriorates, they develop more symptoms, and health care expenditures increase, which in some cases leads to lifetime disability. In addition to the human cost, the extra financial burden for health care and disability expenses are shifted to the general public through various public tax funded programs. A significant amount of advocacy is needed to correct the many system failures seen with this disease.



Future Research Issues

In summary, my experience with Neuropsychiatric Lyme Disease has led me to ask the question - How much of mental disorders are caused by a chronic infectious process? Nervous system tissue is of ectodermal embryological origin and has similarities to skin that are also of ectodermal origin. Herpes simplex lies dormant in skin for extended periods of time and becomes symptomatic during times of stress causing fever blisters.

Might a similar process occur in the central nervous system with this or other infectious agents? Does an expending bull?s eye, erythema migrans process occur in the central nervous system as it does in skin? Why is this a benign disease in some people, while malignant in others?

Microbes evolve faster than people. For this reason, infectious disease will always exist. Many poorly understood diseases were later found to have an infectious disease basis. Infectious agents are continually evolving. New organisms are being recognized, and old ones develop new capabilities. As we develop new therapeutic agents, microbes evolve defenses against this technology. We are seeing increasing problems with infectious disease in humans and animals. Why? Are we losing ground in the "arms war"? Is this due to increased exposure to otherwise remote part of the globe? Is it a natural cycle of infectious disease? Is it a result of a declining global environment? Has the irresponsible use of technology contributed to this problem? Why is Lyme disease more prevalent now? How much of what is called "Lyme disease" is some other infectious disease? Could some of these patients be infected with seronegative syphilis? What can we do to reduce the number of infected ticks in our environment?

The study of this illness yields more questions than answers. It blurs the boundaries between psychiatry and other medical disciplines. It challenges our ethical capability. We need to continue approaching this with an open mind while listening very carefully to our patients.



Conclusion

When neuropsychiatric Lyme disease is a diagnostic possibility, a well-focused interview can assist toward the diagnosis and the evaluation of any change in status over time. Such a tracking system can assist toward clinical decision making and research. Once the reference point database is established, it shall constantly be updated.

References:

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Tri Fallonin kommentti tutkimuksista joissa Bb:n todettiin selviävän antibiooteista. Yksi tutkimuksista on Heta Yrjänäisen.

Persistence of Borrelia burgdorferi in mice after antibiotic therapy.

Two studies have recently been published which reveal that Bb may persist in the mouse despite antibiotic therapy. These studies support much earlier work by Straubinger et al in the dog model (1997) and Bockenstedt et al (2002) in the mouse model. Bockenstedt et al (2002) showed that Bb persistence can occur after antibiotic treatment and that these spirochetes could be acquired by ticks (xenodiagnosis) but that the infected ticks could not transmit infection to naïve hosts ? suggesting that the spirochetes were attenuated in that they had become non-infectious. Straubinger et al (1997) had shown that even after 30 days of antibiotic treatment, Bb spirochetes could be demonstrated in 3/12 dogs by culture, and DNA could be demonstrated by PCR in 9/12 dogs long after treatment.
More recently, Hodzic et al from UC Davis in California reported in Antimicrob Agents Chemother. (2008;52(5):1728-36) a study which examined the effectiveness of antibiotic treatment using ceftriaxone or saline for 1 month. Mice were treated either early in the infection (3 weeks) or later (4 months). Tissues were tested by immunohistochemistry, PCR, culture, transplantation of allografts, and xenodiagnosis at 1 and 3 months after treatment. Tissues from the mice treated with antibiotics were culture negative, but tissues from some of the mice remained PCR positive and intact antigen-positive organisms with spirochetal morphology were visualized in collagen-rich tissues. Xenodiagnosis demonstrated that uninfected larval ticks after feeding on the antibiotic-treated mice were able to acquire spirochetes (confirmed by PCR) and then transmit these spirochetes to naïve SCID mice which became PCR positive but culture negative. This study therefore demonstrated that antibiotic treatment in the mouse model does not result in eradication of the Bb spirochetes and that some of these spirochetes were infectious, although attenuated in activity.
Yrjanainen et al from Univ of Turku in Finland reported in J Infectious Disease (2007; 195(10):1489-96) a study which examined whether anti-tumor necrosis factor-alpha would have a beneficial effect on Bb-infected mice. C3H/He mice were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. In study 1 (with B. garinii) and in study 2 (with Bb SSN40), 2 weeks after infection, 10 mice were treated with ceftriaxone only for 5 days and 10 mice were treated with anti-TNF-alpha only. In another group of 10 mice, anti-TNF was added simultaneous to the ceftriaxone at 2 weeks after infection while in another group of 10 mice anti-TNF was added at 6 weeks after infection (ie, 4 weeks after ceftriaxone). Finally, a fifth group of mice was treated with saline as a sham treatment. For the group that received ceftriaxone only, no samples were positive by culture or by PCR at 2 weeks after infection. However, among those mice treated with anti-TNF-alpha either at 2 weeks or 6 weeks after infection, spirochetes grew from one-third of the mice. Contrary to earlier findings by Bockenstedt et al (2002) in which the spirochetes detected after antibiotic treatment were attenuated in activity, the recovered spirochetes in this study did not appear to be attenuated, as ceftriaxone sensitivity rates, plasmid profiles, and virulence rates were similar to those of bacteria used to infect the mice. This study demonstrated that a portion of B. burgdorferi-infected mice still have live spirochetes in their body, which are activated by anti-TNF-alpha treatment.

Commentary. These two studies demonstrate that Bb spirochetes can persist in the mouse after ceftriaxone therapy. The Finish study was remarkable in that culture and PCR were negative after ceftriaxone but, after additional treatment with anti-TNF-alpha, viable spirochetes were recovered. TNF is a pro-inflammatory cytokine which, when blocked, typically results in a reduction in clinical inflammation; for this reason, such treatment is used for patients with rheumatoid arthritis. To the surprise of the authors, viable spirochetes were recovered in these PCR- and culture-negative mice after TNF blocking treatment was given. Also interesting is that anti-TNF treatment did not result in the expected finding of a reduction of joint swelling.
The Finnish study was the first study to demonstrate that immunomodulatory treatment of animals infected with Bb could convert them from culture negative to culture positive. The California study was remarkable in that only tick-feeding was capable of extracting infectious but non-replicating attenuated spirochetes; without having done that step of xenodiagnosis and then transferring the tick to feed on naïve SCID mice, the authors? conclusion would have been that infectious spirochetes do not persist in the mouse model as culture was negative. The authors further concluded that negative culture and PCR can not be relied upon as markers of treatment success.
We do not know the extent to which these findings can be translated to the human situation. Nevertheless, the activation of infectious spirochetes after anti-TNF therapy in mice should alert clinicians to the possibility that anti-cytokine therapy may result in a similarly increased risk of activating latent infection among patients with a history of treated Lyme disease. At this point, we do not know whether attenuated spirochetes are capable of inducing illness-symptoms in mice or humans; while it is possible that spirochetal mRNA may be producing surface lipoproteins that stimulate systemic symptoms, this hypothesis needs to be tested in the next phase of this important research.

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Riskitekijöitä jotka johtavat epätoivottuun hoitotulokseen eurooppalaisilla neuroborrelioosiin sairastuneilla. (Norja 2012)
Borreliahoitojen viivästynyt aloitus aiheutti potilaille enemmän oireita ennen hoitojen aloitusta. Oireet eivät myöskään parantuneet kunnolla pitkälläkään aikavälillä. Esim. fatiikkia esiintyi vielä n. 3 vuoden kuluttua hoitojen aloituksesta.

Risk factors for a non-favorable outcome after treated European neuroborreliosis.

Authors: Eikeland R, Mygland A, Herlofson K, Ljøstad U

Citation: Acta Neurol Scand 2012(Jun)

Location: Department of Neurology, Sørlandet Hospital, Arendal, Norway.

DOI: 10.1111/j.1600-0404.2012.01690.x


AIM: To identify possible risk factors for reduced health-related quality of life (HRQoL) and fatigue after treated Lyme neuroborreliosis (LNB).

METHODS: We included 50 patients with LNB and analyzed associations between their demographic, clinical, and laboratory characteristics at baseline and outcome at 30 months assessed by the self-report questionnaires Short Form-36 (SF-36) and Fatigue Severity Scale (FSS).

RESULTS: Lower scores in the SF-36 domain Physical Component Summary were associated with pretreatment symptom duration >6 weeks (B = -11.0, P = 0.001) and non-complete recovery at 4 months (B = -5.5, P = 0.037) (R(2) = 0.35). Lower scores in the SF-36 domain Mental Component Summary were associated with non-complete recovery at 4 months (B = -8.9, P = 0.01 (R(2) = 0.14). Higher FSS scores were associated with pretreatment symptom duration >6 weeks (B = 1.4, P = 0.006), high scores on the composite clinical score pretreatment (B = 0.1, P = 0.003), and non-complete recovery at 4 months (B = 1.6, P = 0.005) (R(2) = 0.46). No laboratory test results were associated with these predefined outcomes.

CONCLUSIONS: Delayed treatment start, more symptoms and findings before treatment, and non-complete recovery at 4 months after treatment are possible predictors of a poorer HRQoL and more fatigue 30 months after treated LNB.
We did not find age, gender, educational level, involvement of the central nervous system, coexisting diseases, or cerebrospinal fluid findings to be associated with reduced HRQoL or fatigue. Our findings should be replicated in future studies before any conclusions can be drawn.
© 2012 John Wiley & Sons A/S.

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Spirokeettojen "osaset" saattavat olla syynä siihen miksi borrelia-bakteerin aiheuttamat niveltulehdukset eivät häviä antibioottihoidoista huolimatta.

Nat Rev Rheumatol. 2012 Jul 17;8(8):440. doi: 10.1038/nrrheum.2012.115. Epub 2012 Jul 17.

Lyme arthritis: Spirochaete remnants could explain antibiotic-refractory Lyme arthritis.

Onuora S.

PMID:
22801980
[PubMed - in process]

NO ABSTRACT AVAILABLE

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Tri Eva Sapi sairastui Borrelioosiin. Hän on nykyisin arvostettu borrelioositutkija ja tekee tutkimusta esim. bakteerien biofilmien merkityksestä Borrelioosissa. Hän kertoo aiheesta allaolevalla videolla. Biofilmit ovat todennäköisesti yksi merkittävä syy infektiotautien kroonistumiseen.

Dr. Eva Sapi - Bacterial Biofilms and Lyme Disease

This video is a 10 minute clip, part of a 70 minute interview with Dr. Sapi from the University of New Haven. She is credited with being the first researcher to demonstrate that Lyme spirochetes can actually create their own complex biofilm community to survive indefinitely within their hosts; both human and animal.

Interview excerpts & videos with Bacterial Biofilm Experts (doctors & researchers):
http://www.biofilmcommunity.org/expert-interviews/

The documentary web site for the film:
http://www.whyamistillsick.com/

The DVD is now available and it includes TWO films. See: http://www.whyamistillsick.com/buy/

http://www.youtube.com/watch?feature=en ... IN_8c&NR=1

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Tri Mirkin; Miksi Borrelioosi kroonistuu?
Antibioottihoidolla saadaan tuhottua ainoastaan solujen ulkopuolella olevia bakteereita - ei solujen sisällä olevia.

http://www.drmirkin.com/archive/6937.html

Why Lyme Disease Persists
Report #6937

Doctors usually treat arthritis caused by Lyme disease with massive intravenous doses of the antibiotic, ceftriaxone, for one month. This cures less than 75 percent of those treated. A report in the medical journal, Rheumatology International, tells us why (1).

The bacteria that causes Lyme disease can live both inside and outside human cells. Massive doses of antibiotics can kill the Lyme spirochetes outside cells, but not those inside cells (1). A study in Infection, showed that people unsuccessfully treated for Lyme arthritis continue to excrete the Lyme spirochete in their urines (2). These experiments help to explain why short term-treatment with antibiotics fails to cure Lyme and other infectious arthritides. It takes long-term treatment with antibiotics to cure a sexually transmitted arthritis caused by mycoplasma. The vast majority of doctors feel that rheumatoid arthritis cannot be treated with long-term antibiotics. However, several recent papers show that antibiotics can help to lessen the pain of rheumatoid arthritis.

Lyme disease is caused by a tick bite. Several days later, the person may feel sick and a bull's eye: a red dot surrounded by a red circle, forms at the site of the bite. If a person is treated with the antibiotic, doxycycline, at this point, he has a 99% chance of being cured. However, if he is not treated, the bull's eye disappears and the patient gets better. However, many months later, the person can develop joint pains or nerve damage. At this point, he may not be able to be cured. All tick bites followed by illness should be treated.

More on Lyme disease

By Gabe Mirkin, M.D., for CBS Radio News
Checked 10/20/12

1) HJ Girschick, HI Huppertz, H Russmann, V Krenn, H Karch. Intracellular persistence of Borrelia burgdorferi in human synovial cells. Rheumatology International 16: 3 (SEP 1996): 125-132.

2) ME Bayer, L Zhang, MH Bayer. Borrelia burgdorferi DNA in the urine of treated patients with chronic lyme disease symptoms. A PCR study of 97 cases. Infection 24: 5 (SEP-OCT 1996): 347-353.

3) R Gasser, E Reisinger, B Sedaj, R Horvarth, G Seinost, A Keplinger, I Wendelin, W Klein. Oral treatment of late lyme borreliosis with a combination of roxithromycin and co-trimoxazole - A pilot study on 18 patients. Acta Medica Austriaca 23: 3 (1996): 99-101.

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Yksi Borrelioosihoitosuositusten laatijoista oli tri Linda Bockenstedt. Hän teki jokin aika sitten tutkimuksen jossa totesi antibioottihoidon jälkeen löydettyjen borrelia-bakteerien olleen kuolleita (hiirikoe) eikä siten enää kykeneviä aiheuttamaan oireita - eikä siis kroonisia oireita. Tri MacDonald pyysi Bockenstedtiä lähettämään bakteerinäytteet hänelle jotta hän voisi tutkijaryhmineen tutkia kyseisiä "kuolleita bakteereita". Bockectedt kieltäytyi lähettämästä näytteitä tutkijoille rikkoen näin vakavasti normaaleja tutkijakäytänteitä. Tri MacDonald epäilee Bockenstedtin näytteissä olevien bakteerien olevan osin elossa ja/tai osa borrelia-bakteerien muodostamasta biofilmiyhdyskunnasta. MacDonald kirjoitti Bockenstedtille henkilökohtaisesti ja pyysi tätä tekemään näytteille tietyn yksinkertaisen, halvan värjäystestin jolla pystytään erottelemaan elävät ja kuolleet bakteerit. Bockenstedt kieltäytyi tekemästä testiä eikä antanut MacDonaldinkaan tehdä testiä näytteistä. Hän ei halua muiden tutkijoiden varmentavan ovatko bakteerit elossa vai kuolleita.

Tri MacDonald kirjoitti asiasta artikkelin kliinisiä tutkimuksia käsittelevään julkaisuun (Journal of Clinical Investigation). Artikkeli löytyy sivun loppupuolelta.

http://flash.lymenet.org/scripts/ultima ... 121833;p=0

Lyme borrelia biofilms expert Dr. Alan Macdonald writes to the Journal of Clinical Investigation to protest as...

IDSA Guidelines Author Bockenstedt Contravenes Regulations to Evade Verification of her Findings


by Elena Cook
Background

Dr Linda Bockenstedt is a Yale-based author of the deeply-flawed Lyme Disease guidelines issued by IDSA in 2006. Recently, she published a study purporting to show that signs of persisting Borrelia burgdorferi in antibiotic-treated mice (and, by implication, in humans with chronic Lyme Disease), were nothing more than the remnants of dead bacteria (1). She did not explain why this "debris" should, exceptionally, resist all normal immune mechanisms for clearing dead microbe remnants in a mammalian host.

Images accompanying her publication attracted the attention of Dr Alan B. Macdonald, who is currently researching Biofilms of Borrelia along with Dr Eva Sapi and her team at University of New Haven, Connecticut. This collaboration recently resulted in the first-ever study demonstrating Biofilm formation by Borrelia burgdorferi in vitro. (2).

Biofilms are highly organised structures produced by microbes on both living and non-living surfaces. One of their main functions is protective; they are notorious for shielding bacteria from harmful environmental influences, including the presence of antibiotics.

Dr. Macdonald believes that the bacteria, clearly seen glowing green in Dr. Bockenstedt's images (due to Green Fluorescent Protein DNA recombinantly inserted into the Lyme bacteria's infectivity plasmid Cp26) represents a mixture of live and possibly dead Borrelia biofilm community members in the mouse tissue, as opposed to all completely dead, as she alleges.The term "amorphous globs" used by Dr. Bockenstedt to describe the fluorescent material is unscientific. It has never appeared in any published article in any language describing Borreliosis in any living host.

In her article, Bockenstedt uses arrows to indicate her "amorphous globs" as constituents of "fields or carpets" of Borrelia in the tissues of the mouse, whilst ignoring the fact that spherical (atypical) forms of Borrelia can clearly be seen within!

It is well known that the matrix (non-living structural material of the living biofilm community) is derived from microbes - once living but now dead - which contribute their outer surface glycoprotein "slime layer" and their DNA, (extracellular DNA), possibly also incorporating other microbes within the extracellular matrix. (See image gallery in paper by Sapi et al, referenced below.)

The Borrelia in a biofilm consist of organisms which vary in shape and structure, reflecting the specialisation that takes place within the structure. Spherical forms, such as those visible in the photo published by Bockenstedt, have been shown to be an integral part of a Borrelia biofilm community. Yet Bockenstedt labels this material "amorphous" (ie without shape). That is not what we see when we look carefully at the image material she herself has published (see image included with Dr. Macdonald's letter below).

Because her images so strikingly reproduce the known features of a Borrelia biofilm, Dr. Macdonald wrote to her privately, requesting that she carry out a very simple, dye-based test on the material. The test is inexpensive and easily distinguishes between dead and live micro-organisms. She refused.

Dr. Macdonald then asked her to share some of the mouse tissue from her experiment so that he could perform this test in his own lab.
Once again, Dr. Bockenstedt refused - in flagrant denial of US public health agency regulations, which require taxpayer-funded scientists to make their material available to other researchers for independent verification.

Dr. Bockenstedt's study was funded by the National Institutes for Health (NIH) and the Centre for Disease Control (CDC), but also by the non-profit National Research Fund for Tick-Borne Diseases. Her refusal to share tissue or to perform the simple dye test calls into question her integrity as a scientist.

If Dr. Bockenstedt has nothing to hide,why will she not allow anyone to determine if her "dead" persisting Borrelia are truly "dead"?

Is she afraid that her study will negate the validity of her own recommendations, made in conjunction with the other IDSA Lyme committee members, denying the reality of chronic Lyme Disease? Is she worried that what her own published material shows is actually a biofilm of Borrelia (which now has a precedent in the peer- reviewed literature) and that she may now be obliged to issue a retraction of her manuscript, with its accompanying press releases releases to the national and international media?

Below is the e-letter submitted to the Journal of Clinical Investigation by Dr. Alan Macdonald.

(1)Bockenstedt L. et al Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy DOI 10.1172/JCI 58813
(2)Sapi E. et al. (2012) Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro. PLoS ONE 7(10): e48277. doi:10.1371/journal.pone.0048277
http://www.plosone.org/article/info%3Ad ... ne.0048277

Letter Submitted to Journal of Clinical Invesigations by Dr. Alan B. Macdonald
Oct 2012


Title: Biofilms as a differential diagnosis for "Amorphous Globs of Dr Linda Bockenstedt"
Letter:


Dear Dr Bockenstedt,

I remark with great interest your inclusion of the brief discussion of Biofilms of Borrelia burgdorferi in your Discussion section, in connection with possible etiologies for the GFP-emitting "amorphous blobs" of Borrelia burgdorferi in deep dermal sites closely apposed to the articular cartilage of the murine arthritic Lyme arthritis mice.

Our paper [6] describing for the very first time the entity of in vitro Biofilms of Borrelia burgdorferi, and accepted for publication release on the PLOS ONE website during the week Of October 24, 2012, is the first peer reviewed manuscript on Biofilm formation by borrelia of any species. It is indeed fortuitous that we, and you were working with borrelia burgdorferi.

We and you were curious to understand the implications of possible in vivo Borrelia biofilm formation. At the level of verbal definition to fulfill the requisite (Costerton)[Ref 5] definitions for a biofilm community are the following:

1. Specialization of member microbes within the community which set these specialized forms apart from Planktonic forms of the establishing microbe.

2. Investment of the microbial members of the biofilm community by a self generated Extracellular matrix, usually including structural constituents from once living but now dead members of the biofilm community. The presence of free Extracellular DNA derived from once living now dead members of the biofilm community.

3. The existence of microbe density which exceeds the density of Planktonic microbes on an ordinary bacteriologic solid phase medium.

4. The existence of "plumbing networks" (i.e. water channels and channels for waste product elimination) in the biofilm community. (Ref 4)

5. The Resistance to antibiotic killing among specialized members of the biofilm community. (Ref4)

Now which of items 1-5 above are or might be "in play" in your so called "Amorphous Blobs" of GFP-labeled Borrelia burgdorferi in the Lyme arthritic mice?

1. Specialization:

Morphology of the microbes within the so-called "amorphous globs" differs significantly from the corkscrew-shaped forms (Planktonic forms-motile forms) (Ref 4) of Borrelia burgdorferi, which are nicely demonstrated in your supplementary Videos and in Figures 1-5 (Especially in your figures 5A and 5B). You clearly demonstrate "Round bodies" among the members of the Amorphous blobs.

Round form metamorphosis from pre-existing spiral forms of Borrelia burgdorferi has been elegantly demonstrated in the long list of peer reviewed publications by Drs Oystein Brorson and his cousin Dr. Sverre Henning Brorson (Ref 7) using electron Microscopy and Phase contrast microscopy. Round bodies were embraced by the late Dr. Lynn Margulis.(Ref 8)

You specifically took the time and effort to demonstrate the formation of borrelia Round bodies in your attached supplementary videos in still photographs in Figure 5 (5A and 5B) in your manuscript.Non-spiral borrelia - i.e.round body borrelia are indeed legitimate shape shifted forms of the Borrelia genus, and represent a specialization by the well known spiral MOTILE form of Borreliae (Planktonic from of Borrelia).

Round bodies, granular forms, Cell wall deficient forms, and finally spiral or straightened forms of Borrelia are specialized forms of the Planktonic (spiral form). Thanks to the works of Dr Alban and colleagues (Ref 1) at the University of Rhode Island, these vital round body borreliae demonstrate a diverse protein repertoire which differ from spiral forms in their protein "fingerprint" in two-dimensional SDS PAGE gel electrophoresis.(Ref 1)

2. The overall density of borrelia microbes in your "Amorphous blobs" in the murine deep dermis,exceeds by several orders of magnitude, the density of Planktonic (spiral/motile) forms of Borrelia burgdorferi when these are grown on solid media in the Microbiology laboratory ( Preac-Mursic, V. et al)(Ref 9)

3. The assertion that All of the borrelia microbes in the "Amorphous Blobs in murine deep dermis in laboratory induced Chronic Lyme Arthritis" are ALL DEAD and merely represent "debris"....is just that: an assertion, buttressed by what you say are corroborative mRNA supportive data...( not presented in your paper).

I have suggested to you in a personal private communication that the way to establish Live versus Dead borrelia in your "Amorphous Blobs" is to pour some Dye ( Invitrogen ::Live Dead Assay) (Ref 10)(Red=dead, and Green = alive).
You have steadfastly refused to accommodate this quality control procedure, which is fast, cheap, reliable and easily accomplished.

You have refused me tissue from your "amorphous Globs" dermal tissue from murine subjects, so that I might perform this simple quality control step in my own laboratory. Refusal to provide tissue to an outside scientist, is in violation of the guidelines of the NIH and other Federal funding agencies.

Your Research activities were accomplished with the use of public funds. You have an incumbent obligation to provide tissue to outside scientists upon request,to verify your experimental findings.


4. A review of all previously published manuscripts since the 1982 NEJM articles announcing the Spirochetal (Borrelial) etiology of Lyme Disease, performed by me and by pathology colleagues (Ref3) interested in the published peer-reviewed Borrelia pathology manuscripts , which specifically demonstrate with figure illustrations Borrelia burgdorferi in diseased tissues-- Such a comprehensive 30-year literature review by Board Certified Anatomic and Clinical Pathologists--reveals that there has NEVER been published a manuscript which contains images of Borrelia burgdorferi - either Living Borrelia or Dead Borrelia - which are present in mammalian tissue in the densities which were illustrated in your figures 5A and 5B. (Ref 3)

By Density criteria ALONE, these "Amorphous globs" are Biofilm communities in murine tissue, UNTIL PROVEN OTHERWISE.(Ref 4)

I therefore challenge you to voluntarily participate in Quality Control microscopic exercise -- to establish with the LIVE DEAD Invitrogen Kit that ALL of the Borrelia inside of your "Amorphous Blobs" stain Red(=Dead) and that none of the borrelia within your "Amorphous Globs" stains Green in the Invitrogen Live Dead assay.(Ref 10) If you are correct, there is nothing to lose in this quality control exercise.

Respectfully,
Alan B.MacDonald MD, FCAP, FASCP

References:

1. Microbiology. 2000 Jan: 146 ( Pt1):119-27. Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Alban PS, Johnson PW, Nelson DR. Source Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston R.I. 02881, USA
2.Burgdorfer, W,., Barbour, A.G., Hayes,S.F., Benach, J.L. et al, "Lyme Disease - A Tick borne
Spirochetosis" , Science, 1982:216: 1317-9
3. Duray, Paul Harrison, MD, Complete Bibliography, Pub Med.
4. Montana State University, Center for Biofilm Studies: http://www.biofilm.montana.edu
5. Costerton, William , J. , Complete bibliography ( 600 peer reviewed Biofilm manuscripts), Pub
Med.
6. Sapi, Eva, J, et al, PONE-D-12-11352R2 Characterization of biofilm formation by Borrelia
burgdorferi in vitro :PLOS ONE , 2012, In Press, To be released on the Internet PLOS ONE website in the week of October 24, 2012.
7. Brorson, Oystein and Brorson, Sverre Henning, Complete bibliography, PubMED
8. Brorson, O,... Margulis, Lynn, et al, "Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline ", Oystein Brorson, Sverre-Henning Brorson, John Scythes, James MacAllister, Andrew Wiere, and Lynn Margulis
http://www.pnas.org/cgi/content/full/0908236106
9. Preac-Mursic V, Wilske B, Reinhardt S. Eur J Clin Microbiol. Infect Dis. 1991 Dec;10 (12):1076-9.
“Culture of Borrelia burgdorferi on six solid media.-, Source Max von Pettenkofer Institut, Ludwig-Maximillian-Universitat, Munich, Germany.
10. Invitrogen Inc-
http://www.invitrogen.com/site/us/en/ho ... wcytometry‐ protocol/cell‐viability/live‐dead‐fixable‐dead‐cell‐stain‐kits.html
11. Eisendle K, Grabner T, Zelger B.Focus floating microscopy: "Gold standard" for cutaneous borreliosis?, Am J Clin Pathol. 2007 Feb;127(2):213-22.

(Note: Use of boldface, extra spacing and other minor formatting changes mine -EC)

Above: Set of images included by Dr. Macdonald in his letter to Dr. Bockenstedt, sent to JCI, showing the striking resemblance between what she dismissed as shapeless globs and a known Borrelia biofilm. As some resolution may have been lost in transferring this image, interested readers can view the original published by Bockenstedt at http://www.jci.org/articles/view/58813/figure/5. There many round forms can clearly be distinguished in the so-called "glob" labelled "A". (All the green in her image is Borrelia, genetically altered to fluoresce for easy identification.)
Below: The same known Borrelia biofilm reproduced below showing more detail. The use of spectral imaging has clearly brought out the shapes of the Borrelia organisms in photo "A", which appear in red. A few spiral, very many round forms are distinguished from the extracellular matrix (here coloured green) which envelopes them.


IMAGES

A supplementary atlas of Biofilms of Borrelia and related information are available on Dr. Macdonald's two websites at the following URL's:

http://www.alzheimerborreliosis.net


http://www.molecularalzheimer.org

The articles by Elena Cook on this website may be distributed as long as they are reproduced without changes, attributing the author, and the link to the original URL is included.

Disclaimer: Material on this website is intended for informational purposes only. It is not intended as medical advice. For all questions relating to your own health, please consult a qualified medical professional.The site owner is not responsible for the content of external sites.

An attempt has been made to render this website accessible to people with a variety of disabilities. If you are having difficulty using this site, or have suggestions for improving the site's accessibility, please contact me.
Copyright © 2012-2013Elena Cook
http://www.elenacook.org/macdonaldjci2012.html

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http://www.columbia-lyme.org/research/scientific.html

2011. Tri Costerton ja Sapi: Biofilmi saattaa osaltaan selittää Borrelioosia sairastavien oireiden jatkumisen ja antibioottihoitojen epäonnistumisen.

2011 Lyme and Tick Borne-Diseases National Conference

Scientific Chairs: Brian Fallon, MD, Columbia University

Richard Marconi, PhD, Virginia Commonwealth University

Co-sponsored by Columbia University & The Lyme Disease Association

Oct 1 & 2, 2011, Philadelphia

This year’s conference was once again an exciting and stimulating meeting bringing together researchers, clinicians, community leaders, patients, and public health officials. Below we summarize the talks for the benefit of those who were not able to attend.

Dr. J. William Costerton’s riveting talk on “The Role of Biofilms in Chronic Bacterial Infections” reviewed the history of the discovery of biofilms, demonstrating that these biofilms enable micro-organisms to resist host defenses and antibiotics, enabling infections to become chronic. Biofilm forms when bacteria adhere to surfaces in moist environments by excreting a slimy, glue-like substance. Sites for biofilm formation include natural materials, metals, plastics, medical implant materials—even plant and body tissue. Biofilms are held together by sugary molecular strands, collectively termed "extracellular polymeric substances" or "EPS." The cells produce EPS and are held together by these strands, allowing them to develop complex three-dimensional, resilient, attached communities. Biofilms can be as thin as a few cell layers or many inches thick, depending on environmental conditions. Over 500 bacterial species have been identified in typical dental plaque biofilms. Dr. Costerton described how the capillary bed in the knee is a trap for bacteria, pointing out that septic arthritis in children settles in the knee (not the hip) and Treponema denticola (from periodontitis) also settles in the osteoarthritic knee (not the hip); this raises questions about the potential role of biofilms in chronic Lyme arthritis. Finally, emerging knowledge on biofilm dispersants was reviewed. For more information about biofilms, check out www.erc.montana.edu.

Dr. Eva Sapi’s talk on “Killing Borrelia – an impossible job?” addressed various mechanisms associated with Borrelia burgdorferi that may help it to survive despite antibiotic treatment. B. burgdorferi is a known pleomorphic species, able to adopt alternative, defensive morphologies to evade the immune response and perhaps to increase antibiotic resistance. One of these morphologies is a cyst form, which Dr. Sapi’s research suggests is resistant to the front line antibiotic treatment; alternative antibiotics were suggested. Another possible explanation for persistent symptoms might be the formation of a biofilm. Her group has employed several modes of microscopy to characterize biofilm morphology. Among optical microscopy techniques, dark field microscopy was used to observe the interaction of peripheral spirochetes with the biofilm, DIC microscopy revealed the heterogeneity of the biofilm matrix, and fluorescence microscopy enabled observation of the sessile internal biofilm population in a GFP-expressing population. A relatively new technique, atomic force microscopy, was used to directly scan the topography of the biofilm. The ability of B. burgdorferi to assume a biofilmic morphology may partly explain the continuing presence of symptoms in chronic Lyme sufferers. Dr. Sapi’s group is examining different agents that may help to reduce biofilms, such as the antibiotics doxycycline and tinidazole as well as the herb Banderol. Dr. Sapi concluded with the hypothesis that the B. burgdorferi biofilm likely provides a refuge for chronic Lyme infection, and offers an additional avenue of attack for potential treatments for Lyme disease.

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Oksi ym 1999. 165 edennyttä borrelioositapausta. 32:lla oireet jatkuivat antibioottihoidon jälkeen. ".. yli 3kk:n pituinenkaan antibioottihoito ei välttämättä pysty hävittämään kaikkia borreliabakteereita."

Ann Med. 1999 Jun;31(3):225-32.
Borrelia burgdorferi detected by culture and PCR in clinical relapse of disseminated Lyme borreliosis.
Oksi J, Marjamäki M, Nikoskelainen J, Viljanen MK.
Source

Department of Medicine, Turku University Central Hospital, Finland. jarmo.oksi@utu.fi
Abstract

A total of 165 patients with disseminated Lyme borreliosis (diagnosed in 1990-94, all seropositive except one culture-positive patient) were followed after antibiotic treatment, and 32 of them were regarded as having a clinically defined treatment failure. Of the 165 patients, 136 were tested by polymerase chain reaction (PCR) during the follow-up. PCR was positive from the plasma of 14 patients 0-30 months after discontinuation of the treatment, and 12 of these patients had a clinical relapse. In addition, Borrelia burgdorferi was cultured from the blood of three patients during the follow-up. All three patients belonged to the group with relapse, and two of them were also PCR positive. This report focuses on the 13 patients with clinical relapse and culture or PCR positivity. Eight of the patients had culture or PCR-proven initial diagnosis, the diagnosis of the remaining five patients was based on positive serology only. All 13 patients were primarily treated for more than 3 months with intravenous and/or oral antibiotics (11 of them received intravenous ceftriaxone, nine for 2 weeks, one for 3 weeks and one for 7 weeks, followed by oral antibiotics). The treatment caused only temporary relief in the symptoms of the patients. All but one of them had negative PCR results immediately after the first treatment. The patients were retreated usually with intravenous ceftriaxone for 4-6 weeks. None of them was PCR positive after the retreatment. The response to retreatment was considered good in nine patients.
We conclude that the treatment of Lyme borreliosis with appropriate antibiotics for even more than 3 months may not always eradicate the spirochete. By using PCR, it is possible to avoid unnecessary retreatment of patients with 'post-Lyme syndrome' and those with 'serological scars' remaining detectable for months or years after infection.

PMID:
10442678
[PubMed - indexed for MEDLINE]

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TUTKIMUKSIA http://www.lymeinfo.net/lymefiles.html

LYME DISEASE MEDICAL LITERATURE SUMMARIES

We are very grateful to the contributer of these files for organizing these excellent summaries and providing them for this website. The files must be viewed using Adobe Acrobat Reader. If this program is not already on your computer, it can be downloaded for free at: www.adobe.com.

Persistence File:
77 peer-reviewed studies showing that Lyme disease can persist or relapse despite antibiotic therapy. 18 pages. Last modified: May 2012

Symptoms:
Lyme disease is a multi-systemic infection, infecting multiple parts of the body and causing extensive symptoms, as demonstrated by these abstracts. This file now includes a table of contents. 51 pages. Last modified: May 2012

Symptoms Supplement:
Abstracts on additional topics related to Lyme disease, such as tick bites, co-infections, and the immune response to Lyme disease. 8 pages. December 2003

Seronegativity:
These abstracts demonstrate that the tests for Lyme disease and other spirochetal infections can be falsely negative. 17 pages. Last modified: September 2003

Cystic Form of Bb: An Introduction:
These abstracts shed light on how Borrelia burgdorferi (Bb) is able to survive antibiotic therapy, and on a primary mechanism underlying post-treatment clinical relapses. They document the ability of Bb to change from a spirochete-form to a "cystic" or coccoid form in response to adverse environmental conditions. The cystic forms can later regenerate spirochetes. 17 pages. Last modified: September 2003

Cystic Form of Bb & Other Spirochetes: Advanced:
*Highly Recommended*. This file juxtaposes photographs and quotations from studies dating as far back as the early 1900's to show how spirochetes can transform to and from cystic or coccoid forms. The ability of Bb and other spirochetes to exist in coccoid forms provides a cogent explanation for phenomenon such as latency, persistent infection, and seronegativity. A picture is worth a thousand words! 30 pages. Last modified: September 2003

Cystic Forms of Spirochetes: A Complete Bibliography, 1905-Present:
Especially helpful for researchers. This file contains a complete bibliography of over 260 studies with information/observations on round forms of spirochetes (including 63 studies pertaining to Lyme disease), dating from the early 1900's to the present. Relevant quotations from the studies are included.
53 pages. Last modified: November 2010

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http://wwwnc.cdc.gov/eid/article/10/9/0 ... rticle.htm

Neuroborrelioosi on yleisin nivejalkaisten (esim. puutiaiset) aiheuttama keskushermostoinfektio Euroopassa ja USA:ssa. Borrelioosia on vaikea diagnosoida vasta-ainetesteillä/ immunoblottauksella. Borrelia valaisiana - bakteeria löydettiin 10 v kävelyvaikeuksista jne kärsineen miehen selkäydinnesteestä. Potilaan vasta-aineet olivat alhaiset. Syynä on todennäköisesti vasta-ainetestissä käyetty antigeeni, B.burgdorferi ss, joka ei kyennyt tunnistamaan B.valaisianaa.

CDC; Emerging infectious diseases

Volume 10, Number 9—September 2004

Borrelia valaisiana in Cerebrospinal Fluid

To the Editor: Lyme borreliosis is the most common tickborne human disease in the Northern Hemisphere. The incidence of the disease in not the same throughout Europe; in southern Europe, the incidence ranges from 43% in Croatia to 1.1% in Greece. Suspected borreliosis cases have been reported in Greece, none were confirmed. Ixodes ricinus, the principal tick vector of Borrelia burgdorferi in Europe, is found in northern Greece. A low prevalence of B. burgdorferi antibodies was found in healthy persons in Greece (1,2); a frequency of 7.3% was found in arthritis patients (1), while a frequency of 16.9% was found in patients with neurologic disorders (A. Papa, unpub. data).

Polymerase chain reaction (PCR) has been used to detect B. burgdorferi DNA in humans and to determine genospecies (3). Isolates found in the United States have constituted a homogeneous group. In Europe, five different genospecies from the original B. burgdorferi, now called burgdorferi sensu lato complex, have been described: B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae. Pathogenicity for humans remains uncertain for B. valaisiana and B. lusitaniae (4).

Neuroborreliosis, the most serious manifestation of disseminated Lyme disease, has become the most frequently recognized arthropodborne infection of the nervous system in the United States and Europe. B. garinii, B. afzelii, and B. burgdorferi sensu stricto are confirmed causes of neuroborreliosis (5); however, B. valaisiana has not been isolated from cerebrspinal fluid (CSF) until this report.


We report the genetic detection of B. valaisiana in the CSF of a 61-year-old man with a history of spastic paraparesis, which is strong clinical evidence of advanced neuroborreliosis. Symptoms, mainly difficulty in walking, began approximately 10 years earlier, with a slow progressive course of neuroborreliosis. His medical history showed an unidentified sexually transmitted disease in 1982, an undefined episode of arthritis in the lower limbs in 1990, and a nonspecific rash in the genitals in 1995. The patient lived in South Africa from 1961 to 1997 and visited Thassos Island in northern Greece every year. The neurologic examination demonstrated an intense pyramidal spasticity in the lower limbs and moderate weakness (Medical Research Council grade 3) of the proximal muscles. Serial magnetic resonance imaging (MRI) of the brain showed small hyperintensities in the periventricular area on T2-weighted images; MRI of the spinal cord showed no abnormalities. Multiple sclerosis, B12 deficiency, human T-cell lymphotrophic virus-1 infection, structural inflammatory lesions of the spinal cord, motor neuron disease, and hereditary spastic paraplegia have been excluded. The patient was treated occasionally with intravenous penicillin G, as well as with corticosteroids, but no clinical improvement was achieved. Venereal disease reaction level was negative and all tests for syphilis in CSF were negative.

DNA was extracted from CSF, and a region of the chromosomal flagellin gene of B. burgdorferi was amplified by nested PCR (3). B. afzelii (VS461) DNA was used as a positive control. All precautions were taken to avoid contamination. The amplified PCR product was sequenced, and the sequence (Th1) was deposited in GenBank with the accession no. AY270021. Phylogenetic analysis showed that strain Th1 was clustering with strains belonging to B. valaisiana genomic group. Specifically, a nucleotide difference of 0.38% was observed among Th1 and isolates Ku10 and To76 (accession no. AYO83505 and AYO83504, respectively), which belong to B. valaisiana genomic group and were isolated from ricinus in Sweden (6). A genetic difference of 0.77% was observed between Th1 and B. valaisiana strain Tr29 (accession no. ABO91805) isolated from I. ricinus in Turkey (7), while the genetic difference between Th1 and B. burgdorferi (X15661) was much greater, 6.83%.

This report is the first of genetic detection of B. valaisiana in CSF, which indicates a probable association of this genospecies with disease in humans. B. valaisiana has been isolated from I. ricinus ticks collected from vegetation and from ticks engorged on birds, in several European countries, including Turkey (7). The pathogenic capabilities of B. valasiana are still uncertain; it has been detected by PCR and restriction fragment length polymorphism analysis in skin biopsy specimens from two erythema migrans patients and from patients with mixed infection (erythema migrans and acrodermatitis chronica atrophicans) (4). Indirect evidence suggests that B. valaisiana is involved in some chronic clinical manifestations (8).

Borreliosis is difficult to diagnose by serologic evaluation and Western blot interpretation. In our patient, no intrathecal antibodies were produced to support clinical suspicion of disease. The low antibody titers could be attributed to antigenic variation between B. valaisiana and B. burgdorferi sensu stricto, which was used as antigen because no commercial kit is specific for B. valaisiana. Differences between the strain causing infection and the antigen may play a role in the false-negative results (9). The low antibody response in our patient could be caused by antimicrobial drugs and corticosteroid medication.


The high homology of the nucleotide sequence from our patient and respective B.valaisiana sequences from other European countries suggests that he likely was infected in Greece. The status of Lyme disease in southern Africa is unknown, but Ixodes spp. ticks have been found there, and preliminary evidence indicates that the disease may occur in humans in South Africa (10).

We detected B. valaisiana DNA in CSF of a patient with slow progressive spastic paraparesis, which suggests that this microorganism might be the causative agent of the disease. Nucleotide sequence information of Borrelia strains from clinical cases and ticks from different countries will elucidate the molecular epidemiology of the disease.
Eudoxia Diza*, Anna Papa*Comments to Author , Eleni Vezyri*, Stefanos Tsounis*, Ioannis Milonas*, and Antonis Antoniadis*
Author affiliations: *Aristotle University of Thessaloniki, Thessaloniki, Greece
Acknowledgment

We thank O. Peter in Switzerland for providing DNA samples.
References

Settas L, Diza E, Kyriazopoulou V, Dimitriadis G, Souliou E, Sfetsios T. Detection of anti-Borrelia burgdorferi antibodies in patients with arthritis from Northern Greece (Macedonia and Thrace). Helliniki Rheumatologia. 1996;7:11–20.
Stamouli M, Totos G, Braun HB, Michel G, Gizaris V. Very low seroprevalence of Lyme borreliosis in young Greek males. Eur J Epidemiol. 2000;16:495–6. DOIExternal Web Site IconPubMedExternal Web Site Icon
Schmidt B, Muelleger RR, Stockenhuber C, Soyer PH, Hoedl S, Luger A, Detection of Borrelia burgdorferi-specific DNA in urine specimens from patients with erythema migrans before and after antibiotic therapy. J Clin Microbiol. 1996;34:1359–63.PubMedExternal Web Site Icon
Rijpkema SG, Tazelear DJ, Molkeboer HJ, Noordhoek GT, Plantinga G, Schouls LM, Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin Microbiol Infect. 1997;3:109–16. DOIExternal Web Site IconPubMedExternal Web Site Icon
Ornstein K, Berglund J, Bergstrom S, Norrby R, Barbour AG. Three major Lyme Borrelia genospecies (Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii) indentified by PCR in cerebrospinal fluid from patients with neuroborreliosis in Sweden. Scand J Infect Dis. 2002;34:341–6. DOIExternal Web Site IconPubMedExternal Web Site Icon
Fraenkel CJ, Garpmo U, Berglund J. Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. J Clin Microbiol. 2002;40:3308–12. DOIExternal Web Site IconPubMedExternal Web Site Icon
Guner ES, Hashimoto N, Takada N, Kaneda K, Imai Y, Masuzawa T. First isolation and characterization of Borrelia burgdorferi sensu lato strains from Ixodes ricinus ticks in Turkey. J Med Microbiol. 2003;52:807–13. DOIExternal Web Site IconPubMedExternal Web Site Icon
Ryffel K, Peter O, Rutti B, Suard A, Dayer E. Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B.garinii, B. afzelii and B. valaisiana in humans. J Clin Microbiol. 1999;37:4086–92.PubMedExternal Web Site Icon
Kaiser R. False negative serology in patients with neuroborreliosis and the value of employing of different borrelial strains in serological assays. J Med Microbiol. 2000;49:911–5.PubMedExternal Web Site Icon
Fivaz BH, Petney TN. Lyme disease—a new disease in southern Africa? J S Afr Vet Assoc. 1989;60:155–8.PubMedExternal Web Site Icon

Suggested citation for this article: Diza E, Papa A, Vezyri E, Tsounis S, Milonas I, Antioniadis A. Borrelia valaisiana in cerebrospinal fluid [letter]. Emerg Infect Dis [serial on the Internet]. 2004 Sep [date cited]. Available from: http://wwwnc.cdc.gov/eid/article/10/9/03-0439.htm

DOI: 10.3201/eid1009.030439

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Artikkeli + mikroskooppikuvia borreliabakteerin selviytymiskeinoista:

Evidence-Based Lyme Disease Practice

Keith Berndtson, MD

parkridgemultimed.com/site/wp-content/uploads/2013/02/Evidence-Based-Lyme-Disease-Practice.pdf

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(2013) Onko krooninen borrelioosi kroonisen infektion aiheuttama? Kolmenvuosikymmenen tutkimustyön perusteella lopullista vastausta haetaan edelleen. Yhä enenevässä määrin tutkimuksissa on kuitenkin havaittu borreliabakteerin selviävän sekä antibiootihoidoista että immuunipuolustuksesta.

Review of evidence for immune evasion and persistent infection in Lyme disease

Review
(6568) Total Article Views

Authors: Berndtson K
Published Date April 2013 Volume 2013:6 Pages 291 - 306
DOI: http://dx.doi.org/10.2147/IJGM.S44114
Received: 17 February 2013 [These are actual dates the paper was submitted to, accepted for, and published in the journal.<br />These dates are only available for papers published since January 1, 2012]
Accepted: 18 March 2013
Published: 23 April 2013
Keith Berndtson

Park Ridge MultiMed, Park Ridge, IL, USA

Abstract: Is chronic illness in patients with Lyme disease caused by persistent infection? Three decades of basic and clinical research have yet to produce a definitive answer to this question. This review describes known and suspected mechanisms by which spirochetes of the Borrelia genus evade host immune defenses and survive antibiotic challenge. Accumulating evidence indicates that Lyme disease spirochetes are adapted to persist in immune competent hosts, and that they are able to remain infective despite aggressive antibiotic challenge. Advancing understanding of the survival mechanisms of the Lyme disease spirochete carry noteworthy implications for ongoing research and clinical practice.

Keywords: Lyme disease, Borrelia, biofilm, bacterial persistence, antibiotic tolerance

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"...Borrelia use host proteases to spread from the infection focus
to blood, and to invade distant organs.
... RF Borrelia species tested in this project has a great ability
to pass the blood-placenta barrier. As many as 70% of the foetus are
infection positive d18 after plug formation"

Reactivation and immune evasion of Borrelia infection

April 2005

Consortium leader: Professor SVEN BERGSTRÖM,
Department of Molecular Biology, Umeå University, SE-901 87 Umeå,
Sweden, Tel.: +46 (0)90 7856726, sven.bergstrom@...

Other project leader of the consortium:
Professor Matti Viljanen,
Department of Medical Microbiology, University of Turku,
Kiinamyllynkatu 13, FIN-20520 Turku, Finland, Tel.: +358-2-3337330,
email: matti.viljanen@... , homepage:
http://www.utu.fi/research/tic/viljanen/
Key words: Borreliosis, immune defense, immune evasion, and
reactivation

Abstract
The aim of this project is to gain knowledge of the
interactions between Borrelia spirochetes and the host during
infection. We are using both the Lyme borreliosis and the relapsing
fever borreliosis spirochetes as model organisms. We are
investigating how the Borrelia spirochetes can circumvent the
immunological defence, how they spread form the infectious focus,
reach various sites in the mammalian body, and how the spirochetes
can live in these tissues at a dormant state, reactivate, return to
the circulatory system and cause acute disease again. We also aim to
characterize and define the components involved in the interactions
between Borrelia and human cells, including the cells of the innate
and adaptive immunity.

The current status of the project is presented below according to
the questions and aims of the original research program

Adhesion of Borrelia to endothelium is mediated by specific integrin
binding.

This project will further extended to identify outer membrane
proteins, i.e. P66, P13, OspA-C etc in pore formation, adhesion to
host cells, and tissue tropism. Structural characteristics of some
of these proteins are known and will be used to design inhibitory
compounds for the binding and interaction process. Additionally, the
Borrelia species that causes relapsing fever binds and aggregates
erythrocytes as a possible additional mechanism for evasion of the
immune system. A potential adhesin involved in this aggregation has
been identified. Pore forming assays and aggregation assays will be
used to measure the inhibitory effects of test compounds chemical
inhibitors that block the function of Borrelia species. Thus, we
have demonstrated in earlier studies that some species of relapsing
fever Borrelia adhere to erythrocytes, causing the formation of
erythrocyte rosettes. This aggregate of Borrelia and red blood cells
may protect the spirochetes and contribute to the delayed immune
response seen in rosette-forming strains compared non-rosette-
forming strains. Mice infected with a rosette-forming strain
exhibited more severe pathology and reduced blood flow compared to
mice infected with a non-rosette-forming strain. Therefore, adhesins
and receptors involved in this interaction would lead to possible
therapeutic tools against relapsing fever. We have now identified a
27 kDa Borrelia protein that binds to a component of human
erythrocyte membranes. We are in the process of purifying this
protein in order to capture the erythrocyte receptor by affinity
chromatography and subsequently identify it by mass spectrometry. A
Borrelia library will be constructed and screened using the purified
receptor.

Borrelia use host proteases to spread from the infection focus
to blood, and to invade distant organs.

The initial studies concerning this part were published in 2001
(Nordstrand A., et al. "Delayed kidney and brain invasion by
Borrelia crocidurae in plasminogen knock-out mice". Infect Immun
2001, 69: 5832-5839). Further protease evaluation is described
below, where B. crocidurae, B. hermsii and B. duttoni are
investigated in relation to invasion capability and characteristics.
A murine model has been established to test Borrelia-mediated
activation of host proteases and modulation of the immune responses
in the induction of abortion associated with relapsing fever
borrelisosis. Immune cell subsets and the role of proteases are
being investigated (Andersson M, Larsson C, Bergström S and
Nordstrand A. Invasion, inflammation and host proteases in a murine
model of Borrelia-induced complications during pregnancy. 2005
Manuscript in preparation). So far matrix metalloproteases and
plasmin have been tested. However, no upregulation have been
documented so far, although other proteases will be tested.

Borrelia can evade the first line defense and direct the later
specific immune response by interfering with the function of
neutrophils and dendritic cells.

We have previously shown that contact with B. burgdorferi
induces maturation and IL-8 production of immature dendritic cells
(DCs) in a similar manner as contact with LPS, a known maturation
inducer. These observations suggest that the interplay between
borreliae and DCs is similar to the interplay of DCs with other
microbes. However, gene expression studies are essential to
investigate in more detail the possibility that borreliae are
somehow manipulate DCs to their benefit. We have now completed the
microarray experiments where we determined the gene expression
profiles of borrelia-stimulated and -unstimulated DCs, and compared
the borrelia-induced changes in DC gene expression to the effects of
LPS. Changes in gene expression were analysed in four time points,
and each time point was done in triplicate resulting in 36
microarray hybridizations with two technical repeats. A computer
algorithm has been set up to finalise the array results. As a
result, we have identified the differential expression of up to
several hundred genes, many of which are important regulators of
immune response. Confirmatory experiments with quantitative PCR and
protein arrays are underway.

We have also studied the role of borrelial outer surface
proteins (Osps) in the phagocytosis of borrelia by neutrophils. In
theses studies, we have used B. burgdorferi strains B31 and B313,
which is a mutant of B31 lacking OspA and OspB surface proteins,
among others. Flow cytometry and Baclight bacterial viability assays
have been used to assess the amount and viability of bacteria
ingested by neutrophils. We have found that B31 is phagocytized more
efficiently than B313. Thus, OspA and/or OspB seem to play a role in
the phagocytosis of Borrelia by neutrophils, but this has to be
confirmed by using an OspAB complemented B313 strain, which we have
just cloned. In addition, we have expanded our experiments
concerning Borrelia-neutrophil interaction into a new direction by
setting up assays where neutrophil phagocytosis of Borrelia is
manipulated with antibodies and other reagents interfering with
neutrophil receptors and signal transduction.

Toll-like receptor expression is modulated in immune cells as
an effect of spirochete invasion. The Borrelia-TLR interaction is
central for clinical outcome.

Organ tissues available have been evaluated by
immunohistochemistry (included in PhD project, M Andersson). B.
crocidurae, B. hermsii and B. duttoni are evaluated regarding
invasion characteristics. Preliminary results indicate differential
ability of the species to invade as well as by the invaded
spirochetes to trigger an inflammatory response in situ. This will
be further investigated by Real-Time PCR methodology.

Severe symptoms are associated with a certain type of T-helper
response. Manipulation of the Th1/Th2 ratio and attenuation of
inflammatory responses may be used to prevent severe manifestations
of borreliosis.

We are currently preparing a manuscript on one part of this
project, where we describe the immune response in brain during the
early process leading to neuroborreliosis. We report herein on a
macrophage-dominated response, with IL-10, IL-15 and IFNg as the
most important cytokines. Interestingly following IL-10 increase,
the inflammation subsides to a low level, but does not result in
eradication of the bacterium from the tissue. These results indicate
that during infection IL-10 down regulates the intense macrophage
dominated response, but this may occur at the expense of complete
eradication of the bacteria (Nordstrand A, Anderssojn M, Shamaei-
Tousi A, Jansson A and Bergström S. In situ immune response in brain
and other organs at the early stage of murine neuroborreliosis. 2005
Manuscript in preparation).

Whether this mechanism reflects the initial explanation to
bacterial persistence or not remains to be addressed, as does the
role of IL-15, a cytokine that so far has not been investigated
during borreliosis. This will be further investigated by Real-Time
PCR methodology.

As a new approach, we have investigated the effects of
treatment with anti-TNF-a antibody and/or antibiotics on the
development and persistence of borrelia arthritis in mice. The
results suggest that the administration of anti- TNF-a with or
without antibiotics does not lead to amelioration of arthritis.
However, during these experiments we have observed that anti- TNF-a
given after ceftriaxone treatment leads to activation of a latent
form of borrelial infection. Characterization of the cellular and
molecular biology of this latent form of borrelial infection is
currently underway.

We have also analysed the effect of immunomodulator Linomide on
borrelia arthritis in mice. Linomide caused a mild reduction in
joint swelling of borrelia infected mice, but there was no decrease
in lymphocyte infiltration in Linomide-treated animals.

Borrelia may, by crossing very tight barriers, such as the
blood-testis and blood-brain barriers, invade organs and use these
as reservoirs for an extensive length of time. Reactivation of
infection can occur from these sites.

Results indicate that B. duttoni are able to reside in the
immune privileged organ brain an extensive time after their
disappearance from blood, and can be reactivated to appear in blood
by immunosuppresing conditions. This appears to be a unique feature
of this species. The result from this work is expected to result in
submission of a manuscript during 2005. Interestingly, we have found
that the RF Borrelia species tested in this project has a great
ability to pass the blood-placenta barrier. As many as 70% of the
foetus are infection positive d18 after plug formation if infected
at day 9 indicating a effective mechanism to also pass this
important physical barrier to an immune privileged site.

Publications

a.. Suhonen J, Komi J, Soukka J, Lassila O, Viljanen MK.
Interaction between Borrelia burgdorferi and immature human
dendritic cells. Scand J Immunol 2003;58:67-75.
b.. Mäkinen J, Vuorinen I, He Q, Oksi J, Peltomaa M,
Marjamäki M, Viljanen MK. Prevalence of Granulocytic Ehrlichia and
Borrelia burgdorferisensu lato in Ixodes ricinus ticks collected
from Southwestern Finland and from Island. APMIS 2003;111:355-362.
c.. Ekerfelt E, Jarefors S, Tynngård N, Hedlund M, Sander B,
Bergström S, Forsberg P, and Enerudh J. Phenotypes indicating
cytolytic properties of Borrelia-specific interferon-g secreting
cells in chronic Lyme neuroborreliosis. J. Neuroimmunol 2003.
145:115-126.
d.. Widhe M, Jarefors S, Ekerfelt C, Vrethem M, Bergström S,
Forsberg P and Ernerudh J. Borrelia specific IFN-g and IL-4
secretion in CSF and blood during the course of Human Lyme
borreliosis: relation to clinical outcome. J Inf Dis 2004, 189: 1881-
1891.
e.. Östberg Y., Carrol JM, Pinne M, Rosa P and Bergström
S.Pleiotropic effects of inactivating a carboxyl-terminal protease,
CtpA, in Borrelia burgdorferi.J. Bacteriol. 2004 186: 2074-2084
f.. Pinne M, Östberg Y, Comstedt P, and Bergström S.
Molecular analysis of the channel-forming protein P13 and its
paralog family 48 from different Lyme disease Borrelia species.
Microbiology. 2004, 150: 549-559
g.. Östberg Y, Bunikis I, Bergström S and Johansson J The
etiological agent of Lyme disease, Borrelia burgdorferi, appears to
contain only a few small RNA molecules. J Bacteriol. 2004 186:8472-
8477.
Degrees

a.. Suhonen Juha. "The role of neutrophils and dendritic
cells in Lyme borreliosis". Thesis, University of Turku, 2003.
An abstract of the research plan (January 2003)
http://es.groups.yahoo.com/group/lyme_y ... ssage/1828

[soijuv]

The Case For Chronic Infection: Evidential persistence of Borrelia species post antibiotic exposure in vivo and in vitro

Michael D.Parent & Erica Falkingham

83 sivua tutkimuksia joissa osoitetaan borreliabakteerin selviävän kudoksissa ja soluissa ns.riittävien antibioottihoitojen jälkeen:

http://www.lymekick.com/chroniclyme.pdf

Introduction Summary:

There is an abundance of evidence demonstrating that Borrelia Burgdorferi , the causative agent of Lyme Disease, and related pathogenic species, can persist within specific body tissues and cells of various mammals despite adequate antibiotic therapy: ponies [93.5, 111.5], non-human primates [50, 86], dogs [65.5, 70, 80, 81, 82, 84], mice [44, 62, 88, 100, 107, 108, 110, 114], and
humans [all others]. There is also abundant evidence that Borrelia Burgdorferi has evolved in a manner similar to other bacteria that evade the immune system via pleomorphic modification, in other words, the bacteria can change its shape beyond the conventional spirochetal form [45, 55,61, 64, 90, 105, 109, 113]. L-forms, and cystic Borrelia have been identified in a number of studies
[45, 68, 77, 87, 105, 109, 112, 113]. When these "forms" are exposed to the typical antibiotics, such as Penicillin family antibiotics or
Doxycycline , they are unaffected. When the antibiotic is removed from the environment, the bacterium will alter its form once more, morphing back into a spiral form, allowing ongoing mobility [45, 68, 87, 90, 105, 109].

The current guidelines issued by the Infectious Disease Society Of America (IDSA) are consistently used to dismiss further
discussion regarding the subject of persistence. The guidelines are titled: “The Clinical
Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic
Anaplasmosis and Babesiosis ” Clinical Infectious Diseases 2006; 43:1089–134.

[soijuv]

Borreliabakteeri selviää antibiooteista. Aihetta käsitteleviä tutkimuksia on runsaasti.

Vast.ott.: Soile Juvonen
Yhteyshenkilön Juvonen Jukka kuva

The Case For Chronic Infection: Evidential persistence of Borrelia

species post antibiotic exposure in vivo and in vitro.

Michael D. Parent & Erica Falkingham

Introduction Summary:

There is an abundance of evidence demonstrating that Borrelia Burgdorferi, the causative agent

of Lyme Disease, and related pathogenic species, can persist within specific body tissues and cells

of various mammals despite adequate antibiotic therapy: ponies [93.5, 111.5], non-human

primates [50, 86], dogs [65.5, 70, 80, 81, 82, 84], mice [44, 62, 88, 100, 107, 108, 110, 114], and

humans [all others]. There is also abundant evidence that Borrelia Burgdorferi has evolved in a

manner similar to other bacteria that evade the immune system via pleomorphic modification, in

other words, the bacteria can change its shape beyond the conventional spirochetal form [45, 55,

61, 64, 90, 105, 109, 113]. L-forms, and cystic Borrelia have been identified in a number of studies

[45, 68, 77, 87, 105, 109, 112, 113]. When these "forms" are exposed to the typical antibiotics,

such as Penicillin family antibiotics or Doxycycline, they are unaffected. When the antibiotic is

removed from the environment, the bacterium will alter its form once more, morphing back into

a spiral form, allowing ongoing mobility [45, 68, 87, 90, 105, 109].

I have taken the time to "bold" the conclusions and various other aspects that clearly indicate a

deviation from the point of view given by a number of physicians and researchers who deny the

possibility of ongoing chronic infection within the human host. The current guidelines issued by

the Infectious Disease Society Of America (IDSA) are consistently used to dismiss further

discussion regarding the subject of persistence. The guidelines are titled: “The Clinical

Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis,

and Babesiosis” Clinical Infectious Diseases 2006; 43:1089–134.

Patients who receive a diagnosis of Lyme Disease, either based on clinical observation and/or

objective indicators often improve with antibiotic therapy [1, 4, 18, 19, 26, 33, 66].

However, if they have been undiagnosed and untreated for Lyme Disease for a long period of

time, it often takes longer to see progress in symptom reduction [15, 66, 73, 93, 105]. The U.S.

National Institute Of Health funded a number of randomized double-blind placebo-controlled

trials (RCT) regarding the long term treatment of Lyme Disease. However, these RCT's were 3

months in duration or less. Patients with documented medical records indicating Chronic Lyme

Disease or a Lyme-Like Illness who have been untreated often do not see improvement until after

1

4-6 months of treatment, and even still, the improvements are modest initially in many patients

and may require an ongoing open ended treatment regimen with antibiotics [66, 93].

It is well understood and agreed upon universally that the more time Borrelia Burdorferi has had

to disseminate into various ligaments, bones, collagen, muscles, and other tissues, then the higher

the probability of ongoing complications or symptoms post-antibiotic therapy. Presently, studies

indicate that antibiotics can not access many of the areas that Borrelia Burgdorferi disseminates

to unless the bacterium itself leaves the safe haven of a Fibroblast skin cell [11, 22, 23, 24, 25, 29,

35, 52, 64, 70, 72, 80, 81, 84, 94], or synovial tissue cells and fluid [1, 7, 9, 31, 34, 37, 42, 60, 61, 69,

70, 71, 102].

Introductory Conclusion:

Therefore, we have studies demonstrating abundant persistence. We have National Institute Of

Health funded studies that do not treat patients long enough to confirm whether the treatment

really is effective or not. The short term studies we do have contradict other studies as well as

those based on clinical reports from health care providers treating these patients with antibiotics

beyond the currently accepted time frame. It is unwise for the IDSA to claim that long-term

antibiotic therapy doesn't work when you've only performed a study for 3 months, when the vast

majority of the patients in the study have had the infection for many years and require at least 3-6

months of oral antibiotic before clinical improvements are seen. IV antibiotics may demonstrate

minor to moderate symptomatic improvement after 1- 3 months, but if that treatment is only

given for 3 months and then discontinued, then it will be equally ineffective and the symptoms

will return to pre-treatment levels. Coincidentally, that's exactly what happened in Dr. Brian

Fallon's study. Some symptoms improved, but then returned upon discontinuing therapy.

I have discussed merely one specific possibility for the failure of patients to thrive and improve

during the currently available randomized double-blind placebo-controlled clinical trials (RCT).

Dr. Daniel J. Cameron writes in the Journal Of Medical Hypothesis that a number of limitations

exist within the currently structured (RCTs), that strongly support the position I've laid forth.

Med Hypotheses. 2009 Jun;72(6):688-91. Epub 2009 Mar 5. Insufficient evidence to deny

antibiotic treatment to chronic Lyme disease patients. First Medical Associates, Medicine, 175

Main Street, Mount Kisco, NY 10549, USA. Cameron@LymeProject.com

"Evidence for the hypothesis: There are eight limitations that support the hypothesis: (1) the

power of the evidence is inadequate to draw definite conclusions, (2) the evidence is too

heterogeneous to make strong recommendations, (3) the risk to an individual of facing a

long-term debilitating illness has not been considered, (4) the risk to society of a growing

chronically ill population has not been considered, (5) treatment delay has not been considered as

a confounder, (6) co-infections have not been considered as a confounder, (7) the design of RCTs

2

did not address the range of treatment options in an actual practice, and (8) the findings cannot

be generalized to actual practice. Implications of the hypothesis: This hypothesis suggests that

physicians should consider the limitations of the evidence before denying antibiotic treatment for

Chronic Lyme Disease (CLD). Physicians who deny antibiotic treatment to CLD patients might

inform their patients that there are some clinicians who disagree with that position, and then

offer to refer them for a second opinion to a doctor who could potentially present a different

point of view. The hypothesis also suggests that health care insurers should consider the

limitations of the evidence before adopting policies that routinely deny antibiotic treatment for

CLD patients and should expand coverage of CLD to include clinical discretion for specific

clinical situations."

There is more than enough information to justify at least a neutral position in respect to whether

Borrelia Burgdorferi and related infectious species persist in human beings despite the Infectious

Disease Society Of America's recommendations. Due to this uncertainty, treating physicians can

not conclusively deny that persistence in human beings may be more problematic than assumed.

The scientific studies available on Lyme Disease contradict each other to a significant degree.

Many study authors state in no uncertain terms that the discussion of Lyme Disease is a closed

case. I disagree. The evidence disagrees. The Chief Medical Officer in the United Kingdom

echoed the sentiments of the IDSA in 2009 stating: "There is no biological evidence of

symptomatic chronic Lyme disease amongst those who have received the recommended

treatment regimen." - CMO, Autum 2009, Issue 49, pg. 4. The IDSA states: "To date, there is no

convincing biologic evidence for the existence of symptomatic chronic B. burgdorferi infection

among patients after receipt of recommended treatment regimens for Lyme disease." - Clin Infect

Dis 2006 Nov 1;43(9):1089-134

Skepticism is the heart of science. Cynicism is the death of reason.

The following studies are organized by year, page, and study title within the Study table index.

3

Study Table Index:

Year Page Study Title

1986 18 Ann Intern Med. 1986 Jun;104,6:798-800. Borrelia burgdorferi in

joint fluid in chronic Lyme arthritis. Snydman DR, Schenkein DP,

Berardi VP, Lastavica CC, Pariser KM.

1986 18 J Am Acad Dermatol. 1986 Sep;15,3:459-63.Treating erythema

chronicum migrans of Lyme disease. Berger BW.

1987 18 Arthritis Rheum. 1987 Apr;30,4:448-50.Failure of tetracycline

therapy in early Lyme disease. Dattwyler RJ, Halperin JJ.

1987 19 Arthritis Rheum. 1987 Jun;30,6:705-8. Lyme meningoencephalitis:

report of a severe, penicillin-re sistant case. Diringer MN, Halperin

JJ, Dattwyler RJ.

1988 19 Pediatr Infect Dis J. 1988 Apr;7,4:286-9. Borrelia burgdorferi in a

newborn despite oral penicillin for Lyme borreliosis during

pregnancy. Weber K, Bratzke HJ, Neubert U, Wilske B, Duray PH.

1988 19 Ann N Y Acad Sci. 1988;539:346-51. Treatment of erythema

chronicum migrans of Lyme disease. Berger BW. Department of

Dermatology, New York University School of Medicine, New York

10016.

1988 20 Arthritis Rheum. 1988 Apr;31,4:487-95. Spirochetal antigens and

lymphoid cell surface markers in Lyme synovitis. Comparison with

rheumatoid synovium and tonsillar lymphoid tissue. Steere AC,

Duray PH, Butcher EC.

1988 20 AMA. 1988 May 13;259,18:2737-9 Fatal adult respiratory distress

syndrome in a patient with Lyme disease. Kirsch M, Ruben FL,

Steere AC, Duray PH, Norden CW, Winkelstein A.

1988 20 J Infect Dis. 1988 Oct;158,4:905-6. Cultivation of Borrelia

burgdorferi from joint fluid three months after treatment of facial

palsy due to Lyme borreliosis. Schmidli J, Hunziker T, Moesli P,

Schaad UB.

Year Page Study Title

4

1988 21 N Engl J Med. 1988 Dec 1;319,22:1441-6. Comment in: N Engl J Med.

1989 May 11;320,19:1279-80.Seronegative Lyme disease.

Dissociation of specific T- and B-lymphocyte responses to Borrelia

burgdorferi. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ,

Thomas J, Golightly MG.

1989 21 Am J Clin Pathol. 1989 Jan;91,1:95 7. Spirochetes in the spleen of a

patient with chronic Lyme disease. Cimmino MA, Azzolini A, Tobia

F, Pesce CM Istituto Scientifico di Medicina Interna, Universita di

Genova, Italy.

1989 22 Conn Med. 1989 Jun;53,6:335-7. Treatment of Lyme disease. Schoen

RT.

1989 22 Infection. 1989 Jul-Aug;17,4:216-7. High-dose intravenous

penicillin G does not prevent further progression in early

neurological manifestation of Lyme borreliosis. Kohler J, Schneider

H, Vogt A.

1989 22 Dtsch Med Wochenschr. 1989 Oct 20;114,42:1602-6.

Neuro-borreliosis or intervertebral disk prolapse? [Article in

German] Dieterle L, Kubina FG, Staudacher T, Budingen HJ.

1989 23 Infection. 1989 Nov-Dec;17,6:355-9.Survival of Borrelia

burgdorferi in antibiotically treated patients with Lyme

borreliosis. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross

B, Baumann A, Prokop J. Neurologische Klinik Grosshadern,

Munchen, FR Germany.

1990 23 Acta Trop. 1990 Dec;48, 2:89-94.Clinical implications of delayed

growth of the Lyme borreliosis spirochete, Borrelia burgdorferi.

MacDonald AB, Berger BW, Schwan TG.

Department of Pathology, Southampton Hospital, New York 11968.

1991 24 Infect Immun. 1991 Feb;59,2:671-8. Intracellular localization of

Borrelia burgdorferi within human endothelial cells. Ma Y,

Sturrock A, Weis JJ.

1991 24 1991: Journal of Infectious Diseases, Feb;163,2:311-8 Randomized

comparison of ceftriaxone and cefotaxime in Lyme

neuroborreliosis. Pfister HW, Preac-Mursic V, Wilske B, Schielke E,

SArgel F, EinhA.upl KM.

Year Page Study Title

5

1991 25 Medicine, Baltimore. 1991 Mar;70,2:83-90. Lyme disease: clinical

features, classification, and epidemiology in the upper midwest.

Agger W, Case KL, Bryant GL, Callister SM.

1991 25 N Engl J Med. 1991 Apr 18;324(16):1137. Chronic neurologic

manifestations of Lyme disease. Logigian EL, Kaplan RF, Steere AC.

Department of Neurology, Tufts University School of Medicine,

Boston, MA 02111.

1991 26 Arthritis Rheum. 1991 Aug;34,8:1056-60. Treatment of refractory

chronic Lyme arthritis with arthroscopic synovectomy. Schoen RT,

Aversa JM, Rahn DW, Steere AC.

1992 26 Clin Exp Rheumatol. 1992 Jul-Aug;10,4:387-90. Molecular detection

of persistent Borrelia burgdorferi in a man with dermatomyositis.

Fraser DD, Kong LI, Miller FW.

1992 27 J Infect Dis. 1992 Aug;166,2:440-4.Fibroblasts protect the Lyme

disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

Georgilis K, Peacocke M, Klempner MS. Department of Medicine,

New England Medical Center, Boston, Massachusetts.

1993 27 J Am Acad Dermatol. 1993 Feb;28,2 Pt 2:312-4. Recurrent erythema

migrans despite extended antibiotic treatment with minocycline in

a patient with persisting Borrelia burgdorferi infection. Liegner

KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L.

1993 27 J Infect Dis. 1993 May;167,5:1074-81.Invasion of human skin

fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.

Klempner MS, Noring R, Rogers RA.

1993 28 Infection. 1993 Mar-Apr;21,2:83-8. Azithromycin versus doxycycli

ne for treatment of erythema migrans: clinical and microbiological

findings. Strle F, Preac-Mursic V, Cimperman J, Ruzic E, Maraspin

V, Jereb M.

1993 29 J Neurol. 1993 May;240,5:278-83. Borrelia burgdorferi myositis:

report of eight patients. Reimers CD, de Koning J, Neubert U,

Preac-Mursic V, Koster JG, Muller-Felber W, Pongratz DE, Duray

PH.

Year Page Study Title

6

1993 30 Arthritis Rheum. 1993 Nov;36,11:1621 6. Persistence of Borrelia

burgdorferi in ligamentous tissue from a patient with chronic

Lyme borreliosis. Haupl T, Hahn G, Rittig M, Krause A, Schoerner

C, Schonherr U, Kalden JR, Burmester GR.

1993 30 J Clin Neuroophthalmol. 1993 Sep;13,3:155-61; discussion 162. 59:

First isolation of Borrelia burgdorferi from an iris biopsy.

Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B, Reinhardt

S, Bohmer R.

1993 31 Cent Eur J Public Health. 1993 Dec;1,2:81-5. Electron microscopy

and the polymerase chain reaction of spirochetes from the blood of

patients with Lyme disease. Hulinska D, Krausova M, Janovska D,

Rohacova H, Hancil J, Mailer H.

1993 32 Neurology. 1993 Dec;43,12:2705-7. Stroke due to Lyme disease. Reik

L Jr. Department of Neurology, University of Connecticut Health

Center, Farmington 06030-1845.

1994 32 N Engl J Med. 1994 Jan 27; 330,4:282-3.Detection of Borrelia

burgdorferi DNA by polymerase chain reaction in synovial fluid

from patients with Lyme arthritis. Nocton JJ, Dressler F, Rutledge

BJ, Rys PN, Persing DH, Steere AC.

1994 33 J Clin Microbiol. 1994 Mar;32,3:715-20.Isolation of Borrelia

burgdorferi from biopsy specimens taken from healthy-looking

skin of patients with Lyme borreliosis. Kuiper H, van Dam AP,

Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo

I, Vos K, Dankert J. Department of Medical Microbiology, Academic

Medical Centre, University Hospital, University of Amsterdam, The

Netherlands.

1994 33 J Rheumatol. 1994 Mar;21,3:454-61. Lyme disease: an infectious and

postinfectious syndrome.Asch ES, Bujak DI, Weiss M, Peterson MG,

Weinstein A.

1994 34 Ann Intern Med. 1994 Mar 15;120,6:487-9. The persistence of

spirochetal nucleic acids in active Lyme arthritis. Bradley JF,

Johnson RC, Goodman JL.

1994 34 Ann Intern Med. 1994 Oct 15;121,8:560-7.The long-term clinical

outcomes of Lyme disease. A population-based retrospective

cohort study. Shadick NA, Phillips CB, Logigian EL, Steere AC,

Kaplan RF, Berardi VP, Duray PH, Larson MG, Wright EA, Ginsburg

KS, Katz JN, Liang MH.

7

1994 35 Infect. 1994 Nov;29,3:255-61.Treatment of late Lyme borreliosis.

Wahlberg P, Granlund H, Nyman D, Panelius J, Seppala I.

1994 36 Late complaints after erythema migrans Herta Klade, MD and

Elizabeth Aberer, MD. JSTD 1994; 1:52-56.

1994 36 Borrelia burgdorferi - Seek and ye shall find. Expanding the

envelope Kenneth Liegner, MD. JSTD 1994; 1:79-81.

1994 37 Psychiatric aspects of Lyme disease in children and adolescents: A

community epidemiologic study in Westchester, New York Brian

A. Fallon, MD, MPH; Hector Bird, MD; Christina Hoven, DrPH;

Daniel Cameron, MD, MPH; Michael R. Liebowitz, MD; and David

Shaffer, MD. JSTD 1994; 1:98-100.

1994 37 Persistence of Borrelia burgdorferi despite antibiotic treatment

Michael A. Patmas, MD. JSTD 1994; 1:101.

1994 38 J Infect Dis. 1994 Nov;170,5:1312-6 Comment in: J Infect Dis. 1995

May;171,5:1379-80. Fate of Borrelia burgdorferi DNA in tissues of

infected mice after antibiotic treatment. Malawista SE, Barthold

SW, Persing DH. Department of Internal Medicine, Yale University

School of Medicine, New Haven, Connecticut.

1995 39 Antimicrob Agents Chemother. 1995 May;39,5:1127-33. Effects of

penicillin, ceftriaxone, and doxycycline on morphology of Borrelia

burgdorferi. Kersten A, Poitschek C, Rauch S, Aberer E.

1995 40 Persistent PCR positivity in a patient being treated for Lyme

disease. Kornelia Keszler, MD and Richard C. Tilton, PhD. JSTD

1995; 2:57-58.

1995 40 Neuroborreliosis in Texas Audrey Stein Goldings, MD. JSTD 1995;

2:59-61.

1995 40 Vartiovaara I. 1995 Living with Lyme. Lancet, 345:842-4 A Finnish

physician ’s account of his experiences that beginning with a tick

bite in Vancouver in 1987.

Year Page Study Title

1995 40 J Neuropsychiatry Clin Neurosci. 1995 Summer;7,3:345-7. Rapidly

progressive frontal-type dementia associated with Lyme disease.

Waniek C, Prohovnik I, Kaufman MA, Dwork AJ.

8

1995 41 Ann Neurol. 1995 Oct;38,4:667-9. Comment in: Ann Neurol. 1995

Oct;38,4:560-2.Neuroborreliosis in the nonhuman primate:

Borrelia burgdorferi persists in the central nervous system.

Pachner AR, Delaney E, O'Neill T.

1995 41 Eur Neurol. 1995;35,2:113-7. Comment in: Eur Neurol.

1996;36,6:394-5. Seronegative chronic relapsing

neuroborreliosis.Lawrence C, Lipton RB, Lowy FD, Coyle PK.

1996 42 Infection. 1996 Jan-Feb;24,1:64-8. Azithromycin and doxycycline

for treatment of Borrelia culture-positive erythema migrans. Strle

F, Maraspin V, Lotric-Furlan S, Ruzi・-Sablji・ E, Cimperman J.

1996 42 Infection. 1996 Jan-Feb;24,1:9-16. Erratum in: Infection 1996

Mar-Apr;24,2:169.Kill kinetics of Borrelia burgdorferi and

bacterial findings in relation to the treatment of Lyme borreliosis.

Preac Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Max

v. Pettenkofer Institut, Ludwig-Maximilians-Universitat Munchen,

Germany.

1996 43 Infection. 1996 Jan-Feb;24,1:73-5. Treatment failure in erythema

migrans--a review. Weber K. Dermatologische Privatpraxis,

Munchen, Germany.

1996 43 Infection. 1996 May-Jun;24,3:218-26. Erratum in: Infection 1996

Jul-Aug;24,4:335. Formation and cultivation of Borrelia

burgdorferi spheroplast-L-form variants. Mursic VP, Wanner G,

Reinhardt S, Wilske B, Busch U, Marget W.

1996 44 JAMA. 1996 Jun 5; 275,21, :1657-60. Concurrent Lyme disease and

babesiosis. Evidence for increased severity and duration of illness.

K rause PJ, Telford SR 3rd, Spielman A, Sikand V, Ryan R,

Christianson D, Burke G, Brassard P, Pollack R, Peck J, Persing DH.

1996 44 Antimicrob Agents Chemother. 1996 Jun;40,6:1552-4. Eucaryotic

cells protect Borrelia burgdorferi from the action of penicillin and

ceftriaxone but not from the action of doxycycline and

erythromycin. Brouqui P, Badiaga S, Raoult D. Unite des Rickettsies,

Faculte de Medecine, Centre National de la Recherche Scientifique,

Marseille, France.

Year Page Study Title

9

1996 45 Infection. 1996 Sep-Oct;24,5:347-53.Borrelia burgdorferi DNA in

the urine of treated patients with chronic Lyme disease symptoms.

A PCR study of 97 cases. Bayer ME, Zhang L, Bayer MH. Fox Chase

Cancer Center, Philadelphia, PA 19111, USA.

1996 45 Hum Pathol. 1996 Oct;27,10:1025-34.Ultrastructural demonstration

of spirochetal antigens in synovial fluid and synovial membrane in

chronic Lyme disease: possible factors contributing to persistence

of organisms. Nanagara R, Duray PH, Schumacher HR Jr.

Allergy-Immunology-Rheumatology Division, Department of

Medicine, Faculty of Medicine, KhonKaen University, Thailand.

1996 46 Rheumatol Int. 1996;16,3:125-32.Intracellular persistence of

Borrelia burgdorferi in human synovial cells. Girschick HJ,

Huppertz HI, Russmann H, Krenn V, Karch H.

1996 47 Antimicrob Agents Chemother. 1996 Nov;40 11 :2632-6.In vivo

activities of ceftriaxone and vancomycin against Borrelia spp in the

mouse brain and other sites. Kazragis RJ, Dever LL, Jorgensen JH,

Barbour AG.

1996 47 Brain. 1996 Dec;119, Pt 6:2143-54. Inflammatory brain changes in

Lyme borreliosis. A report on three patients and review of

literature. Oksi J, Kalimo H, Marttila RJ, Marjamaki M, Sonninen P,

Nikoskelainen J, Viljanen MK.

1996 48 Am J Dermatopathol. 1996 Dec;18,6:571-9. Heterogeneity of

Borrelia burgdorferi in the skin. Aberer E, Kersten A, Klade H,

Poitschek C, Jurecka W.

1997 48 328: Semin Neurol. 1997 Mar;17,1:25-30.Peripheral nervous system

Lyme borreliosis. Logigian EL.

1997 49 J Clin Microbiol. 1997 January; 35(1): 111–116. Persistence of

Borrelia burgdorferi in experimentally infected dogs after

antibiotic treatment. R K Straubinger, B A Summers, Y F Chang,

and M J Appel Institute for Animal Health, College of Veterinary

Medicine, Cornell University, Ithaca, New York 14853, USA.

rks4@cornell.edu

1997 49 Clin Infect Dis. 1997 Jul;25 Suppl 1:S52-6. Tetracycline therapy for

chronic Lyme disease. Donta ST.

Year Page Study Title

10

1997 50 Clin Infect Dis. 1997 Jul;25 Suppl 1:S64-70.Why is chronic Lyme

borreliosis chronic? Aberer E, Koszik F, Silberer M.

1997 51 American College of Rheumatology, Vol 40,9, Branigan P; Rao J;

1997 PCR evidence for Borrelia burgdorferi DNA in synovium in

absence of positive serology. Suppl, Rao J; Gerard H; Sept, p.S270

Hudson A; Williams W; Arayssi T; Pando J; Bayer M; Rothfuss S;

.PCR evidence for Borrelia has been identified in synovial biopsies of

patients with clinical pictures that had not initially suggested Lyme

disease. Clayburne G; Sieck M; Schumacher HR.

1997 51 Journal of Spirochetal & Tick-borne Diseases, Vol. 4, No. 1/2 Two

lessons from the canine model of Lyme Disease: migration of

Borrelia burgdorferi in tissues and persistence after antibiotic

treatment. Straubinger RK; 1997 Straubinger AF; Jacobson RH;

Chang Y; Summer BA;

1998 51 Ann Rheum Dis. 1998 Feb;57,2:118-21.Detection of Borrelia

burgdorferi by polymerase chain reaction in synovial membrane,

but not in synovial fluid from patients with persisting Lyme

arthritis after antibiotic therapy. Priem S, Burmester GR, Kamradt

T, Wolbart K, Rittig MG, Krause A.

1998 52 Med J Aust. 1998 May 18;168,10:500-2. Comment in: Med J Aust.

1998 May 18;168,10:479-80. Culture-positive Lyme borreliosis.

Hudson BJ, Stewart M, Lennox VA, Fukunaga M, Yabuki M,

Macorison H, Kitchener-Smith J.

1998 52 Acta Clin Belg. 1998 Jun;53,3:178-83.Lyme borreliosis--a review of

the late stages and treatment of four cases. Petrovic M, Vogelaers D,

Van Renterghem L, Carton D, De Reuck J, Afs chrift M. Department

of Internal Medicine, University Hospital Ghent, Belgium.

1998 53 Eur J Clin Microbiol Infect Dis. 1998 Oct;17,10:715-9.Comparison of

oral cefixime and intravenous ceftriaxone followed by oral

amoxicillin in disseminated Lyme borreliosis. Oksi J, Nikoskelainen

J, Viljanen MK. Department of Medicine, Turku University Central

Hospital, Finland.

Year Page Study Title

11

1998 53 Neurology. 1998 Nov;51,5:1489-91. Comment in: Neurology. 1999

Sep 11;53,4:895-6. Clinical and serologic follow-up in patients with

neuroborreliosis. Treib J, Fernandez A, Haass A, Grauer MT, Holzer

G, Woessner R.

1998 54 Infection. 1998 Nov-Dec; 26,6:364-7.A proposal for the reliable

culture of Borrelia burgdorferi from patients with chronic Lyme

disease, even from those previously aggressively treated. Phillips

SE, Mattman LH, Hulinska D, Moayad H. Greenwich Hospital, CT

06830, USA.

1998 54 Klin Monatsbl Augenheilkd. 1998 Dec;213,6:351-4. Pars plana

vitrectomy in Borrelia burgdorferi endophthalmitis [Article in

German] Meier P, Blatz R, Gau M, Spencker FB, Wiedemann P.

1999 55 Ann Med. 1999 Jun; 3,3:225-32. Borrelia burgdorferi detected by

culture and PCR in clinical relapse of disseminated Lyme

borreliosis. Oksi J, Marjamaki M, Nikoskelainen J, Viljanen MK.

1999 55 Zentralbl Bakteriol. 1999 Jul;289,3:301-18. Persistence of Borrelia

garinii and Borrelia afzelii in patients with Lyme arthritis.

Hulinska D, Votypka J, Valeso va M.

2000 56 J Infect Dis. 2000 Mar;181,3:1069-81. Status of Borrelia burgdorferi

infection after antibiotic treatment and the effects of

corticosteroids: An experimental study. Straubinger RK,

Straubinger AF, Summers BA, Jacobson RH.

2000 57 J Clin Microbiol. 2000 Jun; 38,6, :2191-9. PCR-Based quantification

of Borrelia burgdorferi organisms in canine tissues over a 500-Day

postinfection period. Straubinger RK. James A. Baker Institute for

Animal Health, College of Veterinary Medicine, Cornell University,

Ithaca, New York 14853, USA. rks4@cornell.edu

2001 59 Br J Dermatol. 2001 Feb;144,2:387-92. Is olation and polymerase

chain reaction typing of Borrelia afzelii from a skin lesion in a

seronegative patient with generalized ulcerating bullous lichen

sclerosus et atrophicus. Breier F, Khanakah G, Stanek G, Kunz G,

Aberer E, Schmidt B, Tappeiner G.

Year Page Study Title

12

2001 59 Epidemiol Mikrobiol Imunol. 2001 Feb;50,1:10-6.Persistence of

Borrelia burgdorferi sensu lato in patients with Lyme borreliosis

[Article in Czech] Honegr K, Hulinska D, Dostal V, Gebousky P,

Hankova E, Horacek J, Vyslouzil L, Havlasova J. Infekcni klinika,

Fakultni nemocnice, Hradec Kralove.

2001 60 Ann Neurol. 2001 Sep;50,3, :330-8.Central and peripheral nervous

system infection, immunity, and inflammation in the NHP model

of Lyme borreliosis. Pachner AR, Cadavid D, Shu G, Dail D, Pachner

S, Hodzic E, Barthold SW. Department of Neurosciences,

UMDNJ-New Jersey Medical School, Newark 07103, USA.

pachner@umdnj.edu

2002 60 Wien Klin Wochenschr. 2002 Jul 31;114,13-14:574-9. Cystic forms of

Borrelia burgdorferi sensu lato: induction, development, and the

role of RpoS. Murgia R, Piazzetta C, Cinco M.

2002 61 Acta Neurol Scand. 2002 Oct;106(4):205-8. Chronic symptoms are

common in patients with neuroborreliosis -- a questionnaire

follow-up study. Vrethem M, Hellblom L, Widlund M, Ahl M,

Danielsson O, Ernerudh J, Forsberg P.

2002 62 J Infect Dis. 2002 Nov 15;186,10:1430-7. Epub 2002 Oct 23.

Detection of attenuated, noninfectious spirochetes in Borrelia

burgdorferi-infected mice after antibiotic treatment. Bockenstedt

LK, Mao J, Hodzic E, Barthold SW, Fish D.

2002 62 Antimicrob Agents Chemother. 2002 Nov;46,11:3637-40.

Erythromycin resistance in Borrelia burgdorferi. Terekhova D,

Sartakova ML, Wormser GP, Schwartz I, Cabello FC.

2002 63 Przegl Epidemiol. 2002;56 Suppl 1:57-67.New aspects of the

pathogenesis of lyme disease [Article in Polish] Zajkowska JM,

Hermanowska-Szpakowicz T. Klinika Chorob Zaka・nych i

Neuroinfekcji AM w Bia ymstoku.

2003 63 Neurology. 2003 Jun 24;60,12:1923-30. Comment in: Neurology.

2003 Jun 24;60,12:1888-9.Study and treatment of post Lyme di

sease, STOP-LD: a randomized double masked clinical trial. Krupp

LB, Hyman LG, Grimson R, Coyle PK, Melville P, Ahnn S, Dattwyler

R, Chandler B.

Year Page Study Title

13

2003 64 Med Sci Monit. 2003 Nov;9,11:PI136-42. Macrolide therapy of

chronic Lyme Disease. Donta ST.

2005 65 Vet Microbiol. 2005 May 20;107(3-4):285-94 Antibiotic treatment of

experimentally Borrelia burgdorferi-infected ponies. Chang YF,

Ku YW, Chang CF, Chang CD, McDonough SP, Divers T, Pough M,

Torres A. College of Veterinary Medicine, Cornell University, Ithaca,

NY 14853, USA. yc42@cornell.edu

2005 65 Int J Antimicrob Agents. 2005 Jun;25,6:474-8. Susceptibility of

Borrelia afzelii strains to antimicrobial agents. Ruzi・-Sablji・ E,

Podreka T, Maraspin V, Strle F.

2006 66 Int J Med Microbiol. 2006 May;296 Suppl 40:233-41. Epub 2006 Mar

10.Risk of culture-confirmed borrelial persistence in patients

treated for erythema migrans and possible mechanisms of

resistance. Hunfeld KP, Ruzi・-Sablji・ E, Norris DE, Kraiczy P, Strle F.

Institute of Medical Microbiology, University Hospital of Frankfurt,

Paul-Ehrlich Str. 40, D-60596 Frankfurt/Main, Germany.

K.Hunfeld@em.uni-frankfurt.de

2006 67 Eur J Pediatr. 2006 Jun;165,6:420-1. Epub 2006 Mar 4. Persistent

synovitis in two children with Lyme arthritis linked with

HLA-DRB1*1104. Hendrickx G, Demanet C, Vandenplas Y.

Department of Paediatrics, Paediatric Orthopaedic and

Rheumatology Unit, Academisch Ziekenhuis -Vrije Universiteit

Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium.

g.hendrickx@st-anna.nl

2006 67 Int J Immunopathol Pharmacol. 2006 Jul-Sep;19,3:545-9. In vitro

susceptibility of isolates of Borrelia burgdorferi s.l. to

antimicrobial agents. Santino I, Scazzocchio F, Ciceroni L,

Ciarrocchi S, Sessa R, Del Piano M. Department of Public Health

Sciences, La Sapienza University, Rome, Italy.

iolanda.santino@uniroma1.it

2006 68 Microbes Infect. 2006 Nov-Dec; 8,14-15:2832-40. Epub 2006 Sep

22.Invasion of human neuronal and glial cells by an infectious

strain of Borrelia burgdorferi. Livengood JA, Gilmore RD Jr.

Year Page Study Title

14

2007 68 Acta Radiologica, Volume 48, Issue 7 2007 , pages 755 - 762 Brain

Magnetic Resonance Imaging Does Not Contribute to the Diagnosis

of Chronic Neuroborreliosis. Aalto A, Sjowall J, Davidsson L,

Forsberg P, Smedby O. Division of Radiology, Department of

Medicine and Care, Faculty of Health Sciences, Linkoping University,

Linkoping, Sweden. anne.aalto@imv.liu.se

2007 69 Pol Merkur Lekarski. 2007 Apr;22,130:275-9. Related Articles,

Concentrations of pro-inflammatory cytokines IFN-gamma, IL-6,

IL-12 and IL-15 in serum and cerebrospinal fluid in patients with

neuroborreliosis undergoing antibiotic treatment. Article in Polish.

Pancewicz SA, Kondrusik M, Zajkowska J, Grygorczuk S. Akademia

Medyczna w Bialymstoku, Klinika ChorA3b Zakaznych i

Neuroinfekcji.20spancewicz@interia.pl

2007 69 J Infect Dis. 2007 May 15;195,10:1489-96. Epub 2007 Apr

6.Anti-tumor necrosis factor-alpha treatment activates Borrelia

burgdorferi spirochetes 4 weeks after ceftriaxone treatment in

C3H/He mice. Yrjanainen H, Hytonen J, Song XY, Oksi J, Hartiala K,

Viljanen MK. Department of Medical Microbiology, University of

Turku, Turku, 20520, Finland. heta.yrjanainen@utu.fi

2007 70 Adv Med Sci. 2007;52:174-8. Concentration of TGF-beta1 in the

supernatant of peripheral blood mononuclear cells cultures from

patients with early disseminated and chronic lyme borreliosis.

Grygorczuk S, Chmielewski T, Zajkowska J, Swierzbi・ska R,

Pancewicz S, Kondrusik M, Tylewska-Wierzbanowska S,

Hermanowska-Szpakowicz T. Department of Infectious Diseases and

Neuroinfections, Medical University of Bia・ystok, ul. Zurawia 14,

15-540 Bia・ystok, Poland. neuroin@amb.edu.pl

2007 71 Rheumatol Int. 2007 Sep;27,11:1091-3. Epub 2007 Apr 4.

Seronegative Lyme arthritis. Holl-Wieden A, Suerbaum S, Girschick

HJ. Children's hospital, Section of Pediatric Rheumatology,

Immunology and Infectious diseases, University of Wuerzburg,

Josef-Schneider-Str. 2, 97090 Wuerzburg, Germany.

Year Page Study Title

15

2007 71 Pol Merkur Lekarski. 2007 Sep;23,135:174-8. Concentration of

soluble forms of selectins in serum and in cerebrospinal fluid in

group of patients with neuroborreliosis--a preliminary study

Moniuszko AM, Pancewicz SA, Ko ndrusik M, Zajkowska J,

Grygorczuk S, Swierzbi・ska R. Akademia Medyczna w Bia・ymstoku,

Klinika Chorob Zaka・nych i Neuroinfekcji.

2008 72 Volume 358:428-431 January 24, 2008 Number An Appraisal of

"Chronic Lyme Disease" To the Editor: Feder et al., Oct. 4 issue,

2008 75 Persistence of Borrelia burgdorferi Following Antibiotic

Treatment in Mice Antimicrobial Agents and Chemotherapy,

published online ahead of print on 3 March 2008 Emir Hodzic,

Sunlian Feng, Kevin Holden, Kimberly J. Freet, and Stephen W.

Barthold

2008 75 Antimicrobial Agents and Chemotherapy, May 2008, p. 1728-1736,

Vol. 52, No. 50066-4804 Persistence of Borrelia burgdorferi

following Antibiotic Treatment in Mice Emir Hodzic, Sunlian Feng,

Kevin Holden, Kimberly J. Freet, and Stephen W. Barthold*

2008 76 Pol Arch Med Wewn. 2008 May;118 5:314-7. : Neuroborreliosis with

extrapyramidal symptoms: a case report. Biesiada G, Czapiel J,

Sobczyk-Krupiarz I, Garlicki A, Mach T. Department of Infectious

Diseases, Division of Gastroenterology, Hepatology, and Infectious

Diseases, Jagiellonian University School of Medicine, Krakow,

Poland. gbiesiada@op.pl

2008 76 J Neuroinflammation. 2008 Sep 25;5:40. Persisting atypical and

cystic forms of Borrelia burgdorferi and local inflammation in

Lyme neuroborreliosis. Miklossy J, Kasas S, Zurn AD, McCall S, Yu

S, McGeer PL.

2008 77 Microb Pathog. 2008 Sep 20. Borrelia burgdorferi expression of the

bba64, bba65, bba66, and bba73 genes in tissues during persistent

infection in mice. Gilmore RD Jr, Howison RR, Schmit VL, Carroll

JA.

2008 78 Med Hypotheses. 2008;70,5:967-74. Epub 2007 Nov 5. The

association between tick-borne infections, Lyme borreliosis and

autism spectrum disorders. Bransfield RC, Wulfman JS, Harvey

WT, Usman AI.

Year Page Study Title

16

2008 79 Journal of Veterinary Diagnostic Investigation Vol. 20 Issue 3,

321-324 Copyright c 2008 by the American Association of

Veterinary Laboratory Diagnosticians: Validation of an in-clinic

enzyme-linked immunosorbent assay kit for diagnosis of Borrelia

burgdorferi infection in horses. Amy L. Johnson1, Thomas J. Divers

and Yung-Fu Chang

2009 80 J Antimicrob Chemother. 2009 Jun;63 6:1163-72. Epub 2009 Apr 17.

Assessment of methylthioadenosine/S-adenosylhomocysteine

nucleosidases of Borrelia burgdorferi as targets for novel

antimicrobials using a novel high-throughput method. Cornell KA,

Primus S, Martinez JA, Parveen N.

2009 81 Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18656-61. Epub 2009

Oct 20. Destruction of spirochete Borrelia burgdorferi round-body

propagules (RBs) by the antibiotic tigecycline. Brorson O, Brorson

SH, Scythes J, MacAllister J, Wier A, Margulis L.

2010 81 Persistence of borrelial DNA in the joints of Borrelia

burgdorferi-infected mice after ceftriaxone treatment HETA

YRJANAInen 1 , JUKKA HYTONen 1 , PAULIINA HARTIALA 1 ,

JARMO OKSI 2 and MATTI K. VILJANEN Departments of

1Medical Microbiology and Immunology and 2 Medicine, University

of Turku, Turku, Finland

Evidential support for the case of Chronic Infection:

17

1: Ann Intern Med. 1986 Jun;104,6:798-800. Borrelia burgdorferi in joint fluid in chronic

Lyme arthritis. Snydman DR, Schenkein DP, Berardi VP, Lastavica CC, Pariser KM.

Although indirect evidence suggests that chronic Lyme arthritis is caused by persistent

infection with Borrelia burgdorferi, direct visualization has been lacking. We report the

demonstration of B. burgdorferi from synovial fluid aspirated from the right knee of a

31-year-old man with Lyme arthritis for more than 1 year. After 6 days, culture medium

inoculated with synovial fluid showed one motile and several nonmotile spirochetes. Direct

immunofluorescence staining showed reactivity with anti-B. burgdorferi serum. Spirochetes were

not seen in subcultured material. The patient's arthritis improved with high-dose intravenous

penicillin. Identification of B. burgdorferi from the joint fluid of a patient with long-standing

arthritis supports the concept that the arthritis is due to persistent infection.

2: J Am Acad Dermatol. 1986 Sep;15,3:459-63.Treating erythema chronicum migrans of Lyme

disease. Berger BW.

The efficacy of antibiotic treatment of 117 patients with erythema chronicum migrans of Lyme

disease was evaluated in terms of the necessity for retreatment and the prevention of the late

manifestations of Lyme disease.Fifty-six patients with a minor form of the illness did not

require retreatment and did not develop late manifestations following antibiotic treatment.

Three pregnant patients were included in this group.Fourteen of sixty-one patients with a major

form of the illness required retreatment, and five developed posttreatment late

manifestations of Lyme disease consisting of Bell's palsy and persistent joint pain. Although

the preferred antibiotic for treating erythema chronicum migrans of Lyme disease has not been

conclusively established, tetracycline and penicillin proved effective. The use of probenecid plus

penicillin may be of benefit to patients with the major form of the illness.

3: 1: Arthritis Rheum. 1987 Apr;30,4:448-50.Failure of tetracycline therapy in early Lyme

disease. Dattwyler RJ, Halperin JJ.

We describe the clinical courses of 5 patients with Lyme disease who developed significant late

complications, despite receiving tetracycline early in the course of their illness. All 5 patients

had been treated for erythema chronicum migra ns with a course of tetracycline that met or

exceeded current recommendations. The late manifestations of Lyme disease included arthritis,

cranial nerve palsy, peripheral neuropathy, chronic fatigue, and changes in mental function. Our

findings suggest that the use of tetracycline at a dosage of 250 mg, 4 times a day for 10 days, as a

treatment for early Lyme disease should be reconsidered. To determine optimal therapy for early

Lyme disease, a study that compares an increased dosage of tetracycline with alternative

18

treatments is indicated.

4: Arthritis Rheum. 1987 Jun;30,6:705-8. Lyme meningoencephalitis: report of a severe,

penicillin-re sistant case. Diringer MN, Halperin JJ, Dattwyler RJ.

Although Lyme disease frequently attacks the central nervous system, this involvement is rarely

severe, and high-dose intravenous penicillin usually is adequate treatment. The patient we

describe developed severe Lyme meningoencephalitis despite receiving a full course of

penicillin, and his condition continued to deteriorate after reinstitution of this treatment.

Intravenous chloramphenicol was used successfully and resulted in a substantial improvement.

5: Pediatr Infect Dis J. 1988 Apr;7,4:286-9. Borrelia burgdorferi in a newborn despite oral

penicillin for Lyme borreliosis during pregnancy. Weber K, Bratzke HJ, Neubert U, Wilske B,

Duray PH.

Department of Medicolegal Medicine, Dermatology and Microbiology, University of Munich,

Federal Republic of Germany. "We now demonstrate B. burgdorferi in the brain and liver of a

newborn whose mother had been treated with oral penicillin for LB [Lyme borreliosis] during

the first trimester of pregnancy. ..The death of the newborn was probably due to a respiratory

failure as a consequence of perinatal brain damage.”

6: Ann N Y Acad Sci. 1988;539:346-51. Treatment of erythema chronicum migrans of Lyme

disease. Berger BW. Department of Dermatology, New York University School of Medicine, New

York 10016.

Between June 1981 and July 1987 the efficacy of antibiotic treatment of 215 patients with

erythema chronicum migrans of Lyme disease was evaluated in terms of the necessity for

retreatment and the prevention of the late manifestations of Lyme disease. The principal

antibiotics utilized to treat 161 patients through 1986 were varying doses of tetracycline, or

penicillin alone or in combination with probenecid. Two of 8 0 patients with a minor form of the

illness and 17 of 81 patients with a major form of the illness required retreatment. There were

four patients who did not respond to retreatment with their original medication. A 15- to 30-day

course of amoxicillin, 500 mg q.i.d., and probenecid, 500 mg q.i.d., or doxycycline, 100 mg t.i.d.,

and on three occasions ceftriaxone, 2-4 g/day i.v., were used to treat 54 patients in 1987.

Although it is too early to judge the efficacy of treatment in these patients, increases in the

incidence of Herxheimer reactions and drug eruptions were observed. Strict compliance with

treatment protocols and the possibility of reactions to medications should be thoroughly

discussed with patients.

19

7: 1: Arthritis Rheum. 1988 Apr;31,4:487-95. Spirochetal antigens and lymphoid cell surface

markers in Lyme synovitis. Comparison with rheumatoid synovium and tonsillar lymphoid

tissue. Steere AC, Duray PH, Butcher EC.

Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.

Using monoclonal antibodies to spirochetal antigenes and lymphoid cell surface markers, we

examined the synovial lesions of 12 patients with Lyme disease, and compared them with

rheumatoid synovium and tonsillar lymphoid tissue. The synovial lesions of Lyme disease

patients and rheumatoid arthritis patients were similar and often consisted of the elements found

in normal organized lymphoid tissue. In both diseases, T cells, predominantly of the

helper/inducer s ubset, were distributed diffusely in subsynovial lining areas, often with nodular

aggregates of tightly intermixed T and B cells. IgD-bearing B cells were scattered within the

aggregates, and a few follicular dendritic cells and activated germinal center B cells were

sometimes present. Outside the aggregates, many plasma cells, high endothelial venules, scattered

macrophages, and a few dendritic macrophages were found. HLA-DR and DQ expression was

intense throughout the lesions. In 6 of the 12 patients with Lyme arthritis, but in none of those

with rheumatoid arthritis, a few spirochetes and globular antigen deposits were seen in and

around blood vessels in areas of lymphocytic infiltration. Thus, in Lyme arthritis, a small

number of spirochetes are probably the antigenic stimulus for chronic synovial inflammation.

8: AMA. 1988 May 13;259,18:2737-9 Fatal adult respiratory distress syndrome in a patient with

Lyme disease. Kirsch M, Ruben FL, Steere AC, Duray PH, Norden CW, Winkelstein A.

Department of Medicine, Montefiore Hospital, University of Pittsburgh School of Medicine, PA

15213.

A dry cough, fever, generalized maculopapular rash, and myositis developed in a 67-year-old

woman; she also had markedly abnormal liver function test results. Serologic tests proved that

she had an infection of recent onset with Borrelia burgdorferi, the agent that causes Lyme

disease. During a two-month course of illness, her condition remained refractory to

treatment with antibiotics, salicylates, and steroids. Ultimately, fatal adult respiratory distress

syndrome developed; this was believed to be secondary to Lyme disease.

9: J Infect Dis. 1988 Oct;158,4:905-6. Cultivation of Borrelia burgdorferi from joint fluid three

months after treatment of facial palsy due to Lyme borreliosis. Schmidli J, Hunziker T, Moesli

P, Schaad UB.

Attacks typically are intermittent and last from 3 days to 12 months. The knees are affected most

20

often, but migratory arthritis is common and other large and small joints may be involved. Only

very few Borrelia strains have been cultured from joint specimens worldwide However, a high

percentage of patients with Lyme arthritis, 85%, have evidence of B burgdorferi DNA,

detected by PCR, in the synovial fluid The local persistence of B burgdorferi in the joint over a

long period of time might be related to the exacerbations of symptoms after chondrocyte cell

transplantation. B burgdorferi is difficult to detect in synovial fluid, and cultures are positive only

rarely

10: 1: N Engl J Med. 1988 Dec 1;319,22:1441-6. Comment in: N Engl J Med. 1989 May

11;320,19:1279-80.Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte

responses to Borrelia burgdorferi. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J,

Golightly MG.

Department of Medicine, State University of New York, School of Medicine, Stony Brook

11794-8161.

The diagnosis of Lyme disease often depends on the measurement of serum antibodies to Borrelia

burgdorferi, the spirochete that causes this disorder.Although prompt treatment with

antibiotics may abrogate the antibody response to the infection, symptoms persist in some

patients. We studied 17 patients who had presented with acute Lyme disease and received

prompt treatment with oral antibiotics, but in whom chronic Lyme disease subsequently

developed. Although these patients had clinically active disease, none had diagnostic levels of

antibodies to B. burgdorferi on either a standard enzyme-linked immunosorbent assay or

immunofluorescence assay. On Western blot analysis, the level of immunoglobulin reactivity

against B. burgdorferi in serum from these patients was no greater than that in serum from

normal controls. The patients had a vigorous T-cell proliferative response to whole B.

burgdorferi, with a mean, +/- SEM, stimulation index of 17.8 +/- 3.3, similar to that, 15.8 +/- 3.2,

in 18 patients with chronic Lyme disease who had detectable antibodies. The T-cell response of

both groups was greater than that of a control group of healthy subjects, 3.1 +/- 0.5; P less than

0.001.We conclude that the presence of chronic Lyme disease cannot be excluded by the

absence of antibodies against B. burgdorferi and that a specific T-cell blastogenic response to

B. burgdorferi is evidence of infection in seronegative patients with clinical indications of

chronic Lyme disease.

11: 1: Am J Clin Pathol. 1989 Jan;91,1:95 7. Spirochetes in the spleen of a patient with chronic

Lyme disease. Cimmino MA, Azzolini A, Tobia F, Pesce CM Istituto Scientifico di Medicina

Interna, Universita di Genova, Italy.

A 54-year-old man had intermittent evening fever, arthralgia, transient erythematous macular

21

eruption on the skin, and splenomegaly of two year's duration. Immunofluorescence tests for

Borrelia burgdorferi serum antibodies had positive results, but G-penicillin treatment was

ineffective. Splenectomy with lymph node biopsy was performed to rule out lymphoproliferative

disorders. Borrelia-like spirochetes were identified histologically in the spleen; this finding

was consistent with persistence of B. burgdorferi organisms in inner organs in chronic Lyme

disease.

12: 1: Conn Med. 1989 Jun;53,6:335-7. Treatment of Lyme disease. Schoen RT.

Lyme disease, a tick-transmitted spirochetal infection, can be divided into three stages that can

overlap or occur alone. The goals of antibiotic therapy in stage one are to shorten the duration of

early disease and to prevent the development of later stages20of the illness. This can usually be

accomplished with oral antibiotic therapy. Later stages of the illness are frequently more

difficult to treat, requiring prolonged oral or intravenous antibiotic therapy.

13: Infection. 1989 Jul-Aug;17,4:216-7. High-dose intravenous penicillin G does not prevent

further progression in early neurological manifestation of Lyme borreliosis. Kohler J,

Schneider H, Vogt A.

Neurologische Universitatsklinik und Poliklinik, Freiburg.

We report two cases of Lyme borreliosis, LB, with erythema migrans, EM, and simultaneous

meningopolyneuritis with radicular pain and lymphocytic pleocytosis in the cerebrospinal fluid,

CSF. EM and pain disappeared completely under high-dose penicillin G therapy within few a

days. Pathological findings in CSF improved. Nevertheless, during and after therapy,

neurological signs of LB developed: cranial nerve palsies as well as paresis of extremity

muscles with radicular distribution.

14: 1: Dtsch Med Wochenschr. 1989 Oct 20;114,42:1602-6. Neuro-borreliosis or intervertebral

disk prolapse? [Article in German] Dieterle L, Kubina FG, Staudacher T, Budingen HJ.

Abteilung fur Neurologie und klinische Neurophysiologie, St.-Elisabethen-Krankenhaus

Ravensburg.

Between September 1986 and November 1988, 17 patients were hospitalized and treated for

neuro-borreliosis. Ten of them had been admitted with suspected lumbar or cervical root or

compression syndrome. Only four patients recalled a tick bite, only three an erythema migrans.

Uni- or bilateral facial paresis was a prominent feature in six patients. Three of 14 patients had no

IgG antibodies against Borrelia, either in serum or cerebrospinal fluid at the initial examination,

22

two had positive titres in serum only. Despite antibiotic treatment, usually 10 mega U

penicillin three times daily, six patients had a recurrence by April, 1989, treated with

penicillin again or with twice daily 100 mg doxycycline or 2 g ceftriaxon. In four of them a

residual painful polyneuropathy remains.

15: 1: Infection. 1989 Nov-Dec;17,6:355-9.Survival of Borrelia burgdorferi in antibiotically

treated patients with Lyme borreliosis. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross

B, Baumann A, Prokop J. Neurologische Klinik Grosshadern, Munchen, FR Germany.

The persistence of Borrelia burgdorferi in patients treated with antibiotics is described. The

diagnosis of Lyme disease is based on clinical symptoms, epidemiology and specific IgG and IgM

antibody titers to B. burgdorferi in serum. Antibiotic therapy may abrogate the antibody response

to the infection as shown in our patients. B. burgdorferi may persist as shown by positive culture

in MKP-medium; patients may have subclinical or clinical disease without diagnostic antibody

titers to B. burgdorferi.We conclude that early stage of the disease as well as chronic Lyme

disease with persistence of B. burgdorferi after antibiotic therapy cannot be excluded when

the serum is negative for antibodies against B. burgdorferi.

[Persistence:] However, some patients later developed symptoms of the disease despite

antibiotic treatment, 9-11. Because of these observations it has become questionable if a

definite eradication of B. burgdorferi with antibiotics is possible, p.357. ..The central nervous

system invasion by spirochetes and a persistence of Treponema pallidum after penicillin G

therapy is common in neurosyphilis, 22,23, p.358.[Treatment:] In view of the hitherto failure of

treatment, low CSF concentration of penicillin G, survival of B. burgdorferi in patients treated

with antibiotics, the moderate penicillin G susceptibility o f the organism and unpredictable

progression of the disease, it seems appropriate to treat patients with substantially larger

doses of antibiotics and/or longer than is provided in present treatment regimens.

p.358.[Seronegativity:] As shown, negative antibody-titers do not provide evidence for successful

therapy; antibody-titers may become negative despite persistence.

16: Acta Trop. 1990 Dec;48, 2:89-94.Clinical implications of delayed growth of the Lyme

borreliosis spirochete, Borrelia burgdorferi. MacDonald AB, Berger BW, Schwan TG.

Department of Pathology, Southampton Hospital, New York 11968.

Lyme borreliosis, a spirochetal infection caused by Borrelia burgdorferi, may become clinically

active after a period of latency in the host.Active cases of Lyme disease may show clinical relapse

following antibiotic therapy. The latency and relapse phenomena suggest that the Lyme disease

spirochete is capable of survival in the host for prolonged periods of time. We studied 63 patients

with erythema migrans, the pathognomonic cutaneous lesion of Lyme borreliosis, and examined

23

in vitro cultures of biopsies from the active edge of the erythematous patch. Sixteen biopsies

yielded spirochetes after prolonged incubations of up to 10.5 months, suggesting that Borrelia

burgdorferi may be very slow to divide in certain situations. Some patients with Lyme

borreliosis may require more than the currently recommended two to three week course of

antibiotic therapy to eradicate strains of the spirochete which grow slowly.

17: Infect Immun. 1991 Feb;59,2:671-8. Intracellular localization of Borrelia burgdorferi within

human endothelial cells. Ma Y, Sturrock A, Weis JJ.

Department of Pathology, University of Utah School of Medicine, Salt Lake City 84132.

The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are

characterized by the persistence of the organism in individuals possessing a strong

anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue

protected from the immune system of the host or there is a reservoir of the organism residing

within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into

human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B.

burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1

resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular

location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi

into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining

which differentiated intracellular from extracellular organisms. Actin-containing microfilaments

were required for the intracellular localization, indica ting that the host cell participates in the

internalization process. Activation of endothelial cells by agents known to increase the expression

of several adhesion molecules had no effect on the interaction of B. burgdorferi with the

endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is

constitutively expressed and that internalization is not dependent upon adhesion molecules

whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi

within endothelial cells suggest that intracellular localization may be a potential mechanism by

which the organism escapes from the immune response of the host and may contribute to

persistence of the organism during the later stages of Lyme disease.

[soijuv]

18: 1991: Journal of Infectious Diseases, Feb;163,2:311-8 Randomized comparison of

ceftriaxone and cefotaxime in Lyme neuroborreliosis. Pfister HW, Preac-Mursic V, Wilske B,

Schielke E, SArgel F, EinhA.upl KM.

Neurological Department, Klinikum Grosshadern, University of Munich, Federal Republic of

Germany.

24

In this prospective, randomized, open trial, 33 patients with Lyme neuroborreliosis were

assigned to a 10-day treatment with either ceftriaxone, 2 g intravenously, iv, every 24 h, n = 17,

or cefotaxime, 2 g iv every 8 h, n = 16. Of the 33 patients, 30 were eligible for analysis of

therapeutic efficacy. Neurologic symptoms improved or even subsided in 14 patients of the

cefotaxime group and in 12 patients of the ceftriaxone group during the treatment period. At

follow-up examinations after a mean of 8.1 months, 17 of 2 7 patients examined were clinically

asymptomatic. In one patient Borrelia burgdorferi was isolated from the cerebrospinal fluid, CSF,

7.5 months after ceftriaxone therapy. CSF antibiotic concentrations were above the MIC 90 level

for B. burgdorferi in nearly all patients examined. Patients with Lyme neuroborreliosis may

benefit from a 10-day treatment with ceftriaxone or cefotaxime.However, as 10 patients were

symptomatic at follow-up and borreliae persisted in the CSF of one patient, a prolongation of

therapy may be necessary.

19: Medicine, Baltimore. 1991 Mar;70,2:83-90. Lyme disease: clinical features, classification,

and epidemiology in the upper midwest. Agger W, Case KL, Bryant GL, Callister SM.

Section of Infectious Disease, La Crosse Lutheran Hospital, Wisconsin.

Lyme disease can be classified using the terminology of syphilis. In this series of 95 cases from

the upper midwest, early cases, defined as an illness of less than 2 months, were more likely to

have lived in or recently visited a highly endemic area. Unlike late cases, early cases presented

entirely in the nonwinter months, p less than .001. Early disease was further subdivided into

primary and secondary disease. Ninety percent of primary and 43% of secondary cases had

erythema migrans, while no late cases had active erythema migrans, p less than .001. Clinical

manifestations of nonspecific inflammation, except for arthralgia, were more common in early

than late disease, p less than .01. In secondary cases, monoarticular arthritis was slightly more

common than polyarticular arthritis, with the reverse occurring in late disease, p less than .05.

Indirect fluorescent antibody testing revealed a ratio of IgM to IgG antibodies to be helpful in

distinguishing early from late disease. Antibacterial therapy in early, primary cases caused

Jarisch-Herxheimer reaction 7% of the time. Despite longer and more frequent parenteral

therapy, late Lyme disease frequently required retreatment, owing to poor clinical response, p

less than .05.

19.5: N Engl J Med. 1991 Apr 18;324(16):1137. Chronic neurologic manifestations of Lyme

disease. Logigian EL, Kaplan RF, Steere AC. Department of Neurology, Tufts University School

of Medicine, Boston, MA 02111.

BACKGROUND AND METHODS. Lyme disease, caused by the tick-borne spirochete Borrelia

burgdorferi, is associated with a wide variety of neurologic manifestations. To define further the

25

chronic neurologic abnormalities of Lyme disease, we studied 27 patients, age range, 25 to 72

years, with previous signs of Lyme disease, current evidence of immunity to B. burgdorferi, and

chronic neurologic symptoms with no other identifiable cause. Eight of the patients had been

followed prospectively for 8 to 12 years after the onset of infection. RESULTS. Of the 27 patients,

24, 89 percent, had a mild encephalopathy that began 1 month to 14 years after the onset of the

disease and was characterized by memory loss, mood changes, or sleep disturbance. Of the 24

patients, 14 had memory impairment on neuropsychological tests, and 18 had increased

cerebrospinal fluid protein levels, evidence of intrathecal production of antibody to B.

burgdorferi, or both. Nineteen of the 27 patients,70 percent, had polyneuropathy with radicular

pain or distal paresthesias; all but two of these patients also had encephalopathy. In 16 patients

electrophysiologic testing showed an axonal polyneuropathy. One patient had leukoencephalitis

with asymmetric spastic diplegia, periventricular white-matter lesions, and intrathecal production

of antibody to B. burgdorferi. Among the 27 patients, associated symptoms included fatigue, 74

percent, headache, 48 percent, arthritis, 37 percent, and hearing loss, 15 percent. At the time of

examination, chronic neurologic abnormalities had been present from 3 months to 14 years,

usually with little progression. Six months after a two-week course of intravenous ceftriaxone, 2 g

daily, 17 patients, 63 percent, had improvement; 6, 22 percent, had improvement but then

relapsed; and 4,15 percent, had no change in their condition. CONCLUSIONS. Months to years

after the initial infection with B. burgdorferi, patients with Lyme disease may have chronic

encephalopathy, polyneuropathy, or less commonly, leukoencephalitis. These chronic

neurologic abnormalities usually improve with antibiotic therapy.

20: Arthritis Rheum. 1991 Aug;34,8:1056-60. Treatment of refractory chronic Lyme arthritis

with arthroscopic synovectomy. Schoen RT, Aversa JM, Rahn DW, Steere AC.

Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.

Of 20 patients who underwent arthroscopic synovectomy for refractory chronic Lyme

arthritis of the knee, 16, 80%, had resolution of joint inflammation during the first month

after surgery or soon thereafter, and they have remained well during the 3-8-year followup

period. Three of these 16 patients who were more disabled preoperatively, still had mild

functional limitation at long-term followup. The remaining 4 patients, 20%, had persistent or

recurrent synovitis. We conclude that arthroscopic synovectomy is effective in treating chronic

Lyme arthritis in patients in whom the disease does not respond to antibiotic therapy.

21: 1: Clin Exp Rheumatol. 1992 Jul-Aug;10,4:387-90. Molecular detection of persistent Borrelia

burgdorferi in a man with dermatomyositis. Fraser DD, Kong LI, Miller FW.

National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of

26

Health, Bethesda, Maryland.

A 40-year-old white man with a several year history of various immunologic disorders, including

anti-Jo-1 autoantibody positive dermatomyositis, developed clinical Lyme disease after being

biten by a tick. The patient was treated with oral tetracycline and his initial symptoms

resolved; however, he suffered an exacerbation of his muscle disease which was difficult to

control despite cytotoxic therapy. Antibiotic therapy was reinstituted after Borrelia

burgdorferi was detected in the patient's peripheral blood leukocytes by the polymerase chain

reaction, PCR. All serologic, T-cell stimulation, and western blot analyses, however, were

negative. The patient's disease responded to oral ampicillin, p robenecid therapy and concurrent

cytotoxic therapy. Subsequent leukocyte PCR testing has been negative for the causative agent of

Lyme disease. This case may provide an example of the in vivo immuno-modulatory effects of

spirochetes in human autoimmune disease. In addition, this case emphasizes the potential

clinical utility of PCR technology in evaluating the persistent sero-negative Lyme disease

which may occur in immunocompromised individuals.

22: 1:20 J Infect Dis. 1992 Aug;166,2:440-4.Fibroblasts protect the Lyme disease spirochete,

Borrelia burgdorferi, from ceftriaxone in vitro. Georgilis K, Peacocke M, Klempner MS.

Department of Medicine, New England Medical Center, Boston, Massachusetts.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial

infection, even from antibiotic-treated patients, indicating that it resists eradication by host

defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible

protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease,

ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal

action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of

fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by

glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The

ability of the organism to survive in the presence of fibroblasts was not related to its

infectivity.Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone.

Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective

effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective

environment contributing to its long-term survival.

23: J Am Acad Dermatol. 1993 Feb;28,2 Pt 2:312-4. Recurrent erythema migrans despite

extended antibiotic treatment with minocycline in a patient with persisting Borrelia

burgdorferi infection. Liegner KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L.

Department of Medicine, Northern Westchester Hospital Center, Mount Kisco, NY.

[soijuv]

27

Erythema migrans recurred in a patient 6 months after a course of treatment with

minocycline for Lyme disease. Polymerase chain reaction on heparinized peripheral blood at

that time demonstrated the presence of Borrelia burgdorferi-specific DNA. The patient was

seronegative by Lyme enzyme-linked immunosorbent assay but showed suspicious bands on

Western blot. Findings of a Warthin-Starry stain of a skin biopsy specimen of the eruption

revealed a Borrelia-compatible structure. Reinfection was not believed to have occurred.

Further treatment with minocycline led to resolution of the erythema migrans.

24: 1: J Infect Dis. 1993 May;167,5:1074-81.Invasion of human skin fibroblasts by the Lyme

disease spirochete, Borrelia burgdorferi. Klempner MS, Noring R, Rogers RA.

Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts

University School of Medicine, Boston, Massachusetts 02111.

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by

scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi

were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell

surface by treatment with ceftriaxone, 1 microgram/mL, for 5 days. Despite the absence of

visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were

isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear

region within human fibroblasts by laser scanning confocal microscopy.Intracellular spirochetes

specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent

laser scanning confocal microscopy. These observations suggest that B. burgdorferi can

adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

25: Infection. 1993 Mar-Apr;21,2:83-8. Azithromycin versus doxycycli ne for treatment of

erythema migrans: clinical and microbiological findings. Strle F, Preac-Mursic V, Cimperman

J, Ruzic E, Maraspin V, Jereb M.

Department of Infectious Diseases, University Medical Center, Ljubljana, Slovenia.

The effectiveness of azithromycin and doxycycline in the treatment of erythema migrans was

compared in a prospective randomized trial. One hundred seven adult patients with typical

erythema migrans, examined in the Lyme Borreliosis Outpatients' Clinic, University

Department of Infectious Diseases in Ljubljana, were included in the study. Fifty-five patients

received azithromycin, 500 mg twice daily for the first day, followed by 500 mg once daily for

four days, and 52 patients received doxycycline, 100 mg twice daily for 14 days. The mean

duration of skin lesions after the beginning of treatment was 7.5 +/- 5.9 days, median value 5,

28

range 2-28 days, in the azithromycin group and 11.4 +/- 7.8 days, median value 9, range 2 days--8

weeks, in the doxycycline group, p < 0.05. Borrelia burgdorferi was isolated from erythema

migrans in 28 patients before therapy: in 13 out of 52 in the doxycycline group and in 15 out of 55

in the azithromycin group. Three months after therapy, the culture was positive in four out of

13 patients treated with doxycycline and in one of the 15 patients who received azithromycin.

A biopsy was repeated in all the patients with a positive isolation from the first skin specimen.

During the first 12 months' follow-up, three patients treated with doxycycline but none in the

azithromycin group developed major manifestations of Lyme borreliosis, while 15

doxycycline recipients and 10 azithromycin recipients developed minor consecutive

manifestations.


26: 1: J Neurol. 1993 May;240,5:278-83. Borrelia burgdorferi myositis: report of eight patients.

Reimers CD, de Koning J, Neubert U, Preac-Mursic V, Koster JG, Muller-Felber W, Pongratz DE,

Duray PH.

Friedrich-Baur-Institute, Clinic for Internal Medicine Innenstadt, Munich, Germany.

Myositis is a rare manifestation of Lyme disease of unknown pathogenesis. This study describes

the course of disease in eight patients with Lyme disease, aged 37-70 years, all of whom were

suffering from histologically proven myositis. The clinical, electrophysiological, and

myopathological findings are reported. One patient showed signs and symptoms of myositis of all

limbs. In six patients myositis was localized in the vicinity of skin lesions, arthritis or

neuropathy caused by Borrelia burgdorferi. In another patient suffering from pronounced

muscle weakness of the legs and cardiac arrest, inflammation of the myocardium, the conducting

system and skeletal muscles was revealed at autopsy. Muscle biopsy revealed

lymphoplasmocellular infiltrates combined with few fibre degenerations in three patients. The

lymphoplasmocellular infiltrates were found predominantly in the vicinity of small vessels.

Several spirochetes were stained in six of seven muscle biopsy samples by means of the

immunogold-silver technique. Culturing of20B. Burgdorferi from the muscle biopsy samples

was, however, unsuccessful. Antibiotic treatment succeeded in curing the myositis in four of

six patients. In one patients signs and symptoms improved. One patient died from cardiac arrest

caused by myocarditis and Guillain-Barre syndrome. The outcome is unknown in one patient.

Clinical and myopathological findings indicate that Lyme myositis can be caused either by local

spreading of B. burgdorferi or an unknown antigen or toxin from adjacent tissues or

haematogenously.

27: Zhonghua Yan Ke Za Zhi. Sep;29,5:271-3. Lyme disease in China and its ocular

manifestations [Article in Chinese] Liu AN.

29

Department of Ophthalmology, Chinese Navy General Hospital, Beijing.

The authors report 30 chinese patients of ocular Lyme borreliosis, which is a tick-borne

spirochaetal disease involving multiple organ systems. The ocular manifestations begin as

conjunctivitis, and then as uveitis, choroidoretinitis, keratitis and vitritis. Diagnosis is based on

case history and clinical and laboratory findings. Early cases may be cured by oral antibiotics

while intravenous drip of large dosage is needed for advanced cases, with a relapsing rate of

16%. Prolonged systemic corticosteroids may predispose the patient to antibiotic failure;

however, topical corticosteroids in combination with antibiotics may minimize ocular

inflammation and complications.

28: Arthritis Rheum. 1993 Nov;36,11:1621 6. Persistence of Borrelia burgdorferi in

ligamentous tissue from a patient with chronic Lyme borreliosis. Haupl T, Hahn G, Rittig M,

Krause A, Schoerner C, Schonherr U, Kalden JR, Burmester GR.

Department of Medicine III, University of Erlangen-Nuremberg, Germany.

OBJECTIVE. To document the persistence of Borrelia burgdorferi in ligamentous tissue

samples obtained from a woman with chronic Lyme borreliosis. METHODS. Spirochetes were

isolated from samples of ligamentous tissue, and the spirochetes were characterized

antigenetically and by molecular biology techniques. The ligamentous tissue was examined by

electron microscopy. Humoral and cellular immune responses were analyzed. RESULTS.

Choroiditis was the first recognized manifestation of Lyme disease in this patient. Despite

antibiotic therapy, there was progression to a chronic stage, with multisystem manifestations.

The initially significant immune system activation was followed by a loss of the specific humoral

immune response and a decrease in the cellular immune response to B burgdorferi over the

course of the disease. "Trigger finger" developed, and a portion of the flexor retinaculum obtained

at surgery was cultured. Viable spirochetes were identified. Ultramorphologically, the spirochetes

were situated between collagen fibers and along fibroblasts, some of which were deeply

invaginated by these organisms. The cultured bacteria were identified as B burgdorferi by

reactions with specific immune sera and monoclonal antibodies, and by polymerase chain

reaction amplification and Southern blot hybridization techniques. CONCLUSION. To our

knowledge, this is the first report of the isolation of B burgdorferi from ligamentous tissue. This

suggests that tendon tissues serve as a specific site of spirochete residence in human hosts.

29: J Clin Neuroophthalmol. 1993 Sep;13,3:155-61; discussion 162. 59: First isolation of Borrelia

burgdorferi from an iris biopsy. Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B,

Reinhardt S, Bohmer R.

30

Max v. Pettenkofer Institut fur Hygiene u. Medizinische Mikrobiologie, LM-Universitat

Munchen, Germany.

The persistence of Borrelia burgdorferi in six patients is described. Borrelia burgdorferi has

been cultivated from iris biopsy, skin biopsy, and cerebrospinal fluid also after antibiotic therapy

for Lyme borreliosis. Lyme Serology: IgG antibodies to B. burgdorferi were positive, IgM

negative in four patients; in two patients both IgM and IgG were negative. Antibiotic therapy may

abrogate the antibody response to the infection as shown by our results. Patients may have

subclinical or clinical disease without diagnostic antibody titers. Persistence of B. burgdorferi

cannot be excluded when the serum is negative for antibodies against it.

30: Repeat

31: Cent Eur J Public Health. 1993 Dec;1,2:81-5. Electron microscopy and the polymerase chain

reaction of spirochetes from the blood of patients with Lyme disease. Hulinska D, Krausova M,

Janovska D, Rohacova H, Hancil J, Mailer H.

Department of Electron Microscopy, National Institute of Public Health, Prague, Czech Republic.

Results of studies using direct antigen detection suggest that seronegative Lyme borreliosis is

not rare and support the hypothesis that Borrelia antigens can persist in humans. We report

three successful cultures from blood out of 30 attempts from 96 Lyme disease patients. The

proof of borreliaemia in early or late phases of Lyme disease by immuno-capture electron

microscopy has practical importance for subsequent cultivation. The polymerase chain reaction

with oligonucleotide sequences directed against 16S rRNA identified two of our blood isolates as

Borrelia burgdorferi genospecies III., VS 461 group, and one as Borrelia garinii sp. nov. All of the

three isolates were reactive with monoclonal antibody H9724 against flagellin and with antibody

against main extracellular protein at 83 kDa. Borrelia garinii had a single predominant protein

OspA at 33.5 kDa and reacted with monoclonal antibody H5332 in contrast to two isolates of the

VS 461 group with two major proteins OspA and OspB at 32.5 and 35 kDa. We conclude that

isolation of spirochetes from the blood might prove successful in clinically selected cases of

Lyme borreliosis. Immuno-capture electron microscopy has proved to be a sensitive assay for

monitoring and studying Lyme borreliosis.Clin Orthop Relat Res. 1993 Dec;,297:238-41. Chronic

septic arthritis caused by Borrelia burgdorferi. Battafarano DF, Combs JA, Enzenauer RJ,

Fitzpatrick JE.

Department of Medicine, Fitzsimons Army Medical Center, Aurora, Colorado 80045-5001.

Chronic arthritis occurs in 10% of Lyme disease patients. A patient had chronic septic Lyme

31

arthritis of the knee for seven years despite multiple antibiotic trials and multiple

arthroscopic and open synovectomies. Spirochetes were documented in synovium and

synovial fluid, SF. Polymerase chain reaction, PCR, analysis of the SF was consistent with

Borrelia infection. Persistent infection should be excluded with silver stains and cultures in any

patient with chronic monoarticular arthritis and a history of Lyme disease.

32: 1: Neurology. 1993 Dec;43,12:2705-7. Stroke due to Lyme disease. Reik L Jr. Department of

Neurology, University of Connecticut Health Center, Farmington 06030-1845.

A 56-year-old Connecticut woman suffered multiple strokes 18 months after antibiotic

treatment for early Lyme disease with facial palsy. Pleocytosis, intrathecal synthesis of

anti-Borrelia burgdorferi antibody, and the response to antibiotic treatment substantiated the

diagnosis of neuroborreliosis. This is the first report of stroke caused by Lyme disease

acquired in North America.

33: Lancet, Vol 345: 1436-37 Lopez-Andreu JA; Salcede-Vivo J; . Our patient received during 2

years seven short-term antibiotic treatments, achieving transitory improvements. Nonetheless,

his condition greatly deteriorated. In October, 1993, he started a different antibiotic regimen,

ceftriaxone, 2 g per day intravenously for 12 months, oral roxithromycin 150 mg per day for 2

months, and oral ciprofloxacin, 500 mg per 12 hours for 2 months. After ceftriaxone he has

continued with oral minocycline, 100 mg per 12 hours for 7 months. His quality of life has

greatly improved and the treatment is more tolerable than the borreliosis. We add, however,

in accord with the advice of others that antibiotics should be continued in the long term, until

we achieve cure or delay the progression of the disease.

34: N Engl J Med. Jan 27; 330,4:282-3.Detection of Borrelia burgdorferi DNA by polymerase

chain reaction in synovial fluid from patients with Lyme arthritis. Nocton JJ, Dressler F,

Rutledge BJ, Rys PN, Persing DH, Steere AC.

Division of Rheumatology/Immunology, New England Medical Center, Boston, MA 02111.

BACKGROUND. Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our

understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists

despite antibiotic therapy. METHODS. Using the polymerase chain reaction, PCR, we

attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period.

The samples were tested in two separate laboratories with four sets of primers and probes, three

of which target plasmid DNA that encodes outer-surface protein A, OspA. RESULTS. B.

burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in

none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the

32

results were moderately concordant in the two laboratories, kappa = 0.54 to 0.73. Of 73 patients

with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics,

70, 96 percent, had positive PCR results. In contrast, of 19 patients who received either

parenteral antibiotics or long courses of oral antibiotics, > or = 1 month, only 7, 37 percent,

had positive tests, P < 0.001. None of these seven patients had received more than two months of

oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10

patients with chronic arthritis, continuous joint inflammation for one year or more, despite

multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months

to two years after treatment. CONCLUSIONS. PCR testing can detect B. burgdorferi DNA in

synovial fluid. This test may be able to show whether Lyme arthritis that persists after

antibiotic treatment is due to persistence of the spirochete.

35: 1: J Clin Microbiol. 1994 Mar;32,3:715-20.Isolation of Borrelia burgdorferi from biopsy

specimens taken from healthy-looking skin of patients with Lyme borreliosis. Kuiper H, van

Dam AP, Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo I, Vos K, Dankert

J. Department of Medical Microbiology, Academic Medical Centre, University Hospital,

University of Amsterdam, The Netherlands.

Erythematous skin lesions due to infection with Borrelia burgdorferi will often disappear without

antibiotic treatment. The aim of the study was to assess whether after disappearance of the

erythematous skin lesion B. burgdorferi is still present in the healthy-looking skin of untreated

patients. In six patients, a skin biopsy specimen was taken at the site of a previous erythematous

skin lesion 1 to 6 months after disappearance of the lesion. Four of them presented with early

disseminated Lyme borreliosis. In one additional patient with early disseminated Lyme

borreliosis, the site of a previous tick bite was biopsied. None of these patient s had been treated

with antibiotics before presentation.The cultures of the skin biopsy specimens of the seven

patients showed growth of Borrelia species. By rRNA gene restriction analysis and

genospecies-specific PCR, six isolates were classified as Borrelia garinii and one as Borrelia group

VS461.These results show that B. burgdorferi can still be cultured from the skin after

disappearance of the erythematous skin lesion or at the site of a previous tick bite.

36: J Rheumatol. 1994 Mar;21,3:454-61. Lyme disease: an infectious and postinfectious

syndrome.Asch ES, Bujak DI, Weiss M, Peterson MG, Weinstein A.

Department of Medicine, New York Medical College, Valhalla 10595.

OBJECTIVE. To determine chronic morbidity and the variables that influence recovery in

patients who had been treated for Lyme disease. METHODS. Retrospective evaluation of 215

patients from Westchester County, NY, who fulfilled Centers for Disease Control case definition

33

for Lyme disease, were anti-Borrelia antibody positive and were diagnosed and treated at least

one year before our examination. RESULTS. Erythema migrans had occurred in 70% of patients,

neurological involvement in 29%, objective cardiac problems in 6%, arthralgia in 78% and

arthritis in 41%. Patients were seen at a mean of 3.2 years after initial treatment. A history of

relapse with major organ involvement had occurred in 28% and a history of reinfection in 18%.

Anti-Borrelia antibodies, initially present in all patients, were still positive in 32%. At followup,

82, 38%, patients were asymptomatic and clinically active Lyme disease was found in 19, 9%.

Persistent symptoms of arthralgia, arthritis, cardiac or neurologic involvement with or

without fatigue were present in 114, 53%, patients. Persistent symptoms correlated with a

history of major organ involvement or relapse but not the continued presence of

anti-Borrelial antibodies. Thirty-five of the 114, 31%, patients with persistent symptoms had

predomina ntly arthralgia and fatigue. Antibiotic treatment within 4 weeks of disease onset was

more likely to result in complete recovery. Children did not significantly differ from adults in

disease manifestations or in the frequency of relapse, reinfection or complete recovery.

CONCLUSION. Despite recognition and treatment, Lyme disease is associated with significant

infectious and postinfectious sequelae.

[soijuv]

27

Erythema migrans recurred in a patient 6 months after a course of treatment with

minocycline for Lyme disease. Polymerase chain reaction on heparinized peripheral blood at

that time demonstrated the presence of Borrelia burgdorferi-specific DNA. The patient was

seronegative by Lyme enzyme-linked immunosorbent assay but showed suspicious bands on

Western blot. Findings of a Warthin-Starry stain of a skin biopsy specimen of the eruption

revealed a Borrelia-compatible structure. Reinfection was not believed to have occurred.

Further treatment with minocycline led to resolution of the erythema migrans.

24: 1: J Infect Dis. 1993 May;167,5:1074-81.Invasion of human skin fibroblasts by the Lyme

disease spirochete, Borrelia burgdorferi. Klempner MS, Noring R, Rogers RA.

Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts

University School of Medicine, Boston, Massachusetts 02111.

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by

scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi

were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell

surface by treatment with ceftriaxone, 1 microgram/mL, for 5 days. Despite the absence of

visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were

isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear

region within human fibroblasts by laser scanning confocal microscopy.Intracellular spirochetes

specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent

laser scanning confocal microscopy. These observations suggest that B. burgdorferi can

adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

25: Infection. 1993 Mar-Apr;21,2:83-8. Azithromycin versus doxycycli ne for treatment of

erythema migrans: clinical and microbiological findings. Strle F, Preac-Mursic V, Cimperman

J, Ruzic E, Maraspin V, Jereb M.

Department of Infectious Diseases, University Medical Center, Ljubljana, Slovenia.

The effectiveness of azithromycin and doxycycline in the treatment of erythema migrans was

compared in a prospective randomized trial. One hundred seven adult patients with typical

erythema migrans, examined in the Lyme Borreliosis Outpatients' Clinic, University

Department of Infectious Diseases in Ljubljana, were included in the study. Fifty-five patients

received azithromycin, 500 mg twice daily for the first day, followed by 500 mg once daily for

four days, and 52 patients received doxycycline, 100 mg twice daily for 14 days. The mean

duration of skin lesions after the beginning of treatment was 7.5 +/- 5.9 days, median value 5,

28

range 2-28 days, in the azithromycin group and 11.4 +/- 7.8 days, median value 9, range 2 days--8

weeks, in the doxycycline group, p < 0.05. Borrelia burgdorferi was isolated from erythema

migrans in 28 patients before therapy: in 13 out of 52 in the doxycycline group and in 15 out of 55

in the azithromycin group. Three months after therapy, the culture was positive in four out of

13 patients treated with doxycycline and in one of the 15 patients who received azithromycin.

A biopsy was repeated in all the patients with a positive isolation from the first skin specimen.

During the first 12 months' follow-up, three patients treated with doxycycline but none in the

azithromycin group developed major manifestations of Lyme borreliosis, while 15

doxycycline recipients and 10 azithromycin recipients developed minor consecutive

manifestations.


26: 1: J Neurol. 1993 May;240,5:278-83. Borrelia burgdorferi myositis: report of eight patients.

Reimers CD, de Koning J, Neubert U, Preac-Mursic V, Koster JG, Muller-Felber W, Pongratz DE,

Duray PH.

Friedrich-Baur-Institute, Clinic for Internal Medicine Innenstadt, Munich, Germany.

Myositis is a rare manifestation of Lyme disease of unknown pathogenesis. This study describes

the course of disease in eight patients with Lyme disease, aged 37-70 years, all of whom were

suffering from histologically proven myositis. The clinical, electrophysiological, and

myopathological findings are reported. One patient showed signs and symptoms of myositis of all

limbs. In six patients myositis was localized in the vicinity of skin lesions, arthritis or

neuropathy caused by Borrelia burgdorferi. In another patient suffering from pronounced

muscle weakness of the legs and cardiac arrest, inflammation of the myocardium, the conducting

system and skeletal muscles was revealed at autopsy. Muscle biopsy revealed

lymphoplasmocellular infiltrates combined with few fibre degenerations in three patients. The

lymphoplasmocellular infiltrates were found predominantly in the vicinity of small vessels.

Several spirochetes were stained in six of seven muscle biopsy samples by means of the

immunogold-silver technique. Culturing of20B. Burgdorferi from the muscle biopsy samples

was, however, unsuccessful. Antibiotic treatment succeeded in curing the myositis in four of

six patients. In one patients signs and symptoms improved. One patient died from cardiac arrest

caused by myocarditis and Guillain-Barre syndrome. The outcome is unknown in one patient.

Clinical and myopathological findings indicate that Lyme myositis can be caused either by local

spreading of B. burgdorferi or an unknown antigen or toxin from adjacent tissues or

haematogenously.

27: Zhonghua Yan Ke Za Zhi. Sep;29,5:271-3. Lyme disease in China and its ocular

manifestations [Article in Chinese] Liu AN.

29

Department of Ophthalmology, Chinese Navy General Hospital, Beijing.

The authors report 30 chinese patients of ocular Lyme borreliosis, which is a tick-borne

spirochaetal disease involving multiple organ systems. The ocular manifestations begin as

conjunctivitis, and then as uveitis, choroidoretinitis, keratitis and vitritis. Diagnosis is based on

case history and clinical and laboratory findings. Early cases may be cured by oral antibiotics

while intravenous drip of large dosage is needed for advanced cases, with a relapsing rate of

16%. Prolonged systemic corticosteroids may predispose the patient to antibiotic failure;

however, topical corticosteroids in combination with antibiotics may minimize ocular

inflammation and complications.

28: Arthritis Rheum. 1993 Nov;36,11:1621 6. Persistence of Borrelia burgdorferi in

ligamentous tissue from a patient with chronic Lyme borreliosis. Haupl T, Hahn G, Rittig M,

Krause A, Schoerner C, Schonherr U, Kalden JR, Burmester GR.

Department of Medicine III, University of Erlangen-Nuremberg, Germany.

OBJECTIVE. To document the persistence of Borrelia burgdorferi in ligamentous tissue

samples obtained from a woman with chronic Lyme borreliosis. METHODS. Spirochetes were

isolated from samples of ligamentous tissue, and the spirochetes were characterized

antigenetically and by molecular biology techniques. The ligamentous tissue was examined by

electron microscopy. Humoral and cellular immune responses were analyzed. RESULTS.

Choroiditis was the first recognized manifestation of Lyme disease in this patient. Despite

antibiotic therapy, there was progression to a chronic stage, with multisystem manifestations.

The initially significant immune system activation was followed by a loss of the specific humoral

immune response and a decrease in the cellular immune response to B burgdorferi over the

course of the disease. "Trigger finger" developed, and a portion of the flexor retinaculum obtained

at surgery was cultured. Viable spirochetes were identified. Ultramorphologically, the spirochetes

were situated between collagen fibers and along fibroblasts, some of which were deeply

invaginated by these organisms. The cultured bacteria were identified as B burgdorferi by

reactions with specific immune sera and monoclonal antibodies, and by polymerase chain

reaction amplification and Southern blot hybridization techniques. CONCLUSION. To our

knowledge, this is the first report of the isolation of B burgdorferi from ligamentous tissue. This

suggests that tendon tissues serve as a specific site of spirochete residence in human hosts.

29: J Clin Neuroophthalmol. 1993 Sep;13,3:155-61; discussion 162. 59: First isolation of Borrelia

burgdorferi from an iris biopsy. Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B,

Reinhardt S, Bohmer R.

30

Max v. Pettenkofer Institut fur Hygiene u. Medizinische Mikrobiologie, LM-Universitat

Munchen, Germany.

The persistence of Borrelia burgdorferi in six patients is described. Borrelia burgdorferi has

been cultivated from iris biopsy, skin biopsy, and cerebrospinal fluid also after antibiotic therapy

for Lyme borreliosis. Lyme Serology: IgG antibodies to B. burgdorferi were positive, IgM

negative in four patients; in two patients both IgM and IgG were negative. Antibiotic therapy may

abrogate the antibody response to the infection as shown by our results. Patients may have

subclinical or clinical disease without diagnostic antibody titers. Persistence of B. burgdorferi

cannot be excluded when the serum is negative for antibodies against it.

30: Repeat

31: Cent Eur J Public Health. 1993 Dec;1,2:81-5. Electron microscopy and the polymerase chain

reaction of spirochetes from the blood of patients with Lyme disease. Hulinska D, Krausova M,

Janovska D, Rohacova H, Hancil J, Mailer H.

Department of Electron Microscopy, National Institute of Public Health, Prague, Czech Republic.

Results of studies using direct antigen detection suggest that seronegative Lyme borreliosis is

not rare and support the hypothesis that Borrelia antigens can persist in humans. We report

three successful cultures from blood out of 30 attempts from 96 Lyme disease patients. The

proof of borreliaemia in early or late phases of Lyme disease by immuno-capture electron

microscopy has practical importance for subsequent cultivation. The polymerase chain reaction

with oligonucleotide sequences directed against 16S rRNA identified two of our blood isolates as

Borrelia burgdorferi genospecies III., VS 461 group, and one as Borrelia garinii sp. nov. All of the

three isolates were reactive with monoclonal antibody H9724 against flagellin and with antibody

against main extracellular protein at 83 kDa. Borrelia garinii had a single predominant protein

OspA at 33.5 kDa and reacted with monoclonal antibody H5332 in contrast to two isolates of the

VS 461 group with two major proteins OspA and OspB at 32.5 and 35 kDa. We conclude that

isolation of spirochetes from the blood might prove successful in clinically selected cases of

Lyme borreliosis. Immuno-capture electron microscopy has proved to be a sensitive assay for

monitoring and studying Lyme borreliosis.Clin Orthop Relat Res. 1993 Dec;,297:238-41. Chronic

septic arthritis caused by Borrelia burgdorferi. Battafarano DF, Combs JA, Enzenauer RJ,

Fitzpatrick JE.

Department of Medicine, Fitzsimons Army Medical Center, Aurora, Colorado 80045-5001.

Chronic arthritis occurs in 10% of Lyme disease patients. A patient had chronic septic Lyme

31

arthritis of the knee for seven years despite multiple antibiotic trials and multiple

arthroscopic and open synovectomies. Spirochetes were documented in synovium and

synovial fluid, SF. Polymerase chain reaction, PCR, analysis of the SF was consistent with

Borrelia infection. Persistent infection should be excluded with silver stains and cultures in any

patient with chronic monoarticular arthritis and a history of Lyme disease.

32: 1: Neurology. 1993 Dec;43,12:2705-7. Stroke due to Lyme disease. Reik L Jr. Department of

Neurology, University of Connecticut Health Center, Farmington 06030-1845.

A 56-year-old Connecticut woman suffered multiple strokes 18 months after antibiotic

treatment for early Lyme disease with facial palsy. Pleocytosis, intrathecal synthesis of

anti-Borrelia burgdorferi antibody, and the response to antibiotic treatment substantiated the

diagnosis of neuroborreliosis. This is the first report of stroke caused by Lyme disease

acquired in North America.

33: Lancet, Vol 345: 1436-37 Lopez-Andreu JA; Salcede-Vivo J; . Our patient received during 2

years seven short-term antibiotic treatments, achieving transitory improvements. Nonetheless,

his condition greatly deteriorated. In October, 1993, he started a different antibiotic regimen,

ceftriaxone, 2 g per day intravenously for 12 months, oral roxithromycin 150 mg per day for 2

months, and oral ciprofloxacin, 500 mg per 12 hours for 2 months. After ceftriaxone he has

continued with oral minocycline, 100 mg per 12 hours for 7 months. His quality of life has

greatly improved and the treatment is more tolerable than the borreliosis. We add, however,

in accord with the advice of others that antibiotics should be continued in the long term, until

we achieve cure or delay the progression of the disease.

34: N Engl J Med. Jan 27; 330,4:282-3.Detection of Borrelia burgdorferi DNA by polymerase

chain reaction in synovial fluid from patients with Lyme arthritis. Nocton JJ, Dressler F,

Rutledge BJ, Rys PN, Persing DH, Steere AC.

Division of Rheumatology/Immunology, New England Medical Center, Boston, MA 02111.

BACKGROUND. Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our

understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists

despite antibiotic therapy. METHODS. Using the polymerase chain reaction, PCR, we

attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period.

The samples were tested in two separate laboratories with four sets of primers and probes, three

of which target plasmid DNA that encodes outer-surface protein A, OspA. RESULTS. B.

burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in

none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the

32

results were moderately concordant in the two laboratories, kappa = 0.54 to 0.73. Of 73 patients

with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics,

70, 96 percent, had positive PCR results. In contrast, of 19 patients who received either

parenteral antibiotics or long courses of oral antibiotics, > or = 1 month, only 7, 37 percent,

had positive tests, P < 0.001. None of these seven patients had received more than two months of

oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10

patients with chronic arthritis, continuous joint inflammation for one year or more, despite

multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months

to two years after treatment. CONCLUSIONS. PCR testing can detect B. burgdorferi DNA in

synovial fluid. This test may be able to show whether Lyme arthritis that persists after

antibiotic treatment is due to persistence of the spirochete.

35: 1: J Clin Microbiol. 1994 Mar;32,3:715-20.Isolation of Borrelia burgdorferi from biopsy

specimens taken from healthy-looking skin of patients with Lyme borreliosis. Kuiper H, van

Dam AP, Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo I, Vos K, Dankert

J. Department of Medical Microbiology, Academic Medical Centre, University Hospital,

University of Amsterdam, The Netherlands.

Erythematous skin lesions due to infection with Borrelia burgdorferi will often disappear without

antibiotic treatment. The aim of the study was to assess whether after disappearance of the

erythematous skin lesion B. burgdorferi is still present in the healthy-looking skin of untreated

patients. In six patients, a skin biopsy specimen was taken at the site of a previous erythematous

skin lesion 1 to 6 months after disappearance of the lesion. Four of them presented with early

disseminated Lyme borreliosis. In one additional patient with early disseminated Lyme

borreliosis, the site of a previous tick bite was biopsied. None of these patient s had been treated

with antibiotics before presentation.The cultures of the skin biopsy specimens of the seven

patients showed growth of Borrelia species. By rRNA gene restriction analysis and

genospecies-specific PCR, six isolates were classified as Borrelia garinii and one as Borrelia group

VS461.These results show that B. burgdorferi can still be cultured from the skin after

disappearance of the erythematous skin lesion or at the site of a previous tick bite.

36: J Rheumatol. 1994 Mar;21,3:454-61. Lyme disease: an infectious and postinfectious

syndrome.Asch ES, Bujak DI, Weiss M, Peterson MG, Weinstein A.

Department of Medicine, New York Medical College, Valhalla 10595.

OBJECTIVE. To determine chronic morbidity and the variables that influence recovery in

patients who had been treated for Lyme disease. METHODS. Retrospective evaluation of 215

patients from Westchester County, NY, who fulfilled Centers for Disease Control case definition

33

for Lyme disease, were anti-Borrelia antibody positive and were diagnosed and treated at least

one year before our examination. RESULTS. Erythema migrans had occurred in 70% of patients,

neurological involvement in 29%, objective cardiac problems in 6%, arthralgia in 78% and

arthritis in 41%. Patients were seen at a mean of 3.2 years after initial treatment. A history of

relapse with major organ involvement had occurred in 28% and a history of reinfection in 18%.

Anti-Borrelia antibodies, initially present in all patients, were still positive in 32%. At followup,

82, 38%, patients were asymptomatic and clinically active Lyme disease was found in 19, 9%.

Persistent symptoms of arthralgia, arthritis, cardiac or neurologic involvement with or

without fatigue were present in 114, 53%, patients. Persistent symptoms correlated with a

history of major organ involvement or relapse but not the continued presence of

anti-Borrelial antibodies. Thirty-five of the 114, 31%, patients with persistent symptoms had

predomina ntly arthralgia and fatigue. Antibiotic treatment within 4 weeks of disease onset was

more likely to result in complete recovery. Children did not significantly differ from adults in

disease manifestations or in the frequency of relapse, reinfection or complete recovery.

CONCLUSION. Despite recognition and treatment, Lyme disease is associated with significant

infectious and postinfectious sequelae.

borrelia burgdorferi despite lengthy antibiotic treatment were noted.

Case number: In October 1991, a 35 year old Caucasian female, registered nurse, was referred for

evaluation. She had reported a lesion compatible with Erythema Chronicum Migrans about one

year earlier. After a short course of oral antibiotics, she noted fatigue, myalgia, and arthralgias

and was given 2 weeks of intravenous ceftriaxone 1 g daily with resolution of her symptoms.

Over the next several months, however her symptoms gradually returned. An ELISA titer was

elevated, and she was started on ceftriaxone 2 g intravenously daily. After 10 days, the patient

developed a vigorous Jarisch-Herxheimer reaction and was referred to the author. The patient

was switched to cefotaxime 3 g intravenously every 12 hours with improvement in symptoms.

After 6 weeks, the intravenous cefotaxime was changed to oral Clarithromycin 500mg daily for 6

37

more weeks, with complete resolution of all signs and symptoms. One week later the patient

discovered that she was 1 month pregnant, and after normal gestation, delivered a health male

infant. The placenta was examined at Brigham and Women's Hospital in Boston

Massachusetts, where several spirochetes were noted in perivascular and intervillous spaces

on modified dieterle silver stain.

Case Number 2: A 47 year-old caucasian female was well until an untreated tick bite in 1985. She

subsequently developed a progressive arthritis diagnosed as Rheumatoid. After failed treatment

with nonsterodal anti-inflammatories and remittive agents, the author saw the patient for the first

time in 1990. Aspiration of fluid from the right knee was positive by specific antibody ratio for

Lyme Disease as the University of Medicine and Dentistry of New Jersey -- Robert Wood

Johnson University Hospital Lyme Disease Research Center. The patient was started on

Ceftriaxone 2 g i ntravenously daily for 4 weeks. She had a significant objective response to

treatment, but quickly relapsed after it was discontinued. A second 4 week course of

ceftriaxone was given with only moderate improvement. The patient then sought treatment at

several university center where she received experimental treatment for rheumatoid arthritis

including monoclonal antibody therapy. There was no improvement in her condition. By July

1992, the patient developed bilateral aseptic necrosis of her hips. A right total hip

replacement was performed and a histopathologic examination revealed several spirochetes

on modified dieterle silver stain of synovial tissue performed at the Brigham and Women's

Hospital. The patient was then started on continous oral antibiotic treatment with

Azithromycin 250mg daily. Approximately 6 months later, the patient underwent left total

knee replacement and once again spirochete-like structures were observed in synovial tissue

on modified dieterle silver stain.

These two cases suggest that despite lengthy courses of aboth intravenous and oral antibiotics,

Borrelia burgdorferi may persist. The presumption that residual symptoms are due to

Fibromyalgia may not always be true and is not assured simply because a patient has received 30

days of treatment. Careful histopathologic examination by modified dieterle silver stain my

suggest otherwise.

44: J Infect Dis. 1994 Nov;170,5:1312-6 Comment in: J Infect Dis. 1995 May;171,5:1379-80. Fate

of Borrelia burgdorferi DNA in tissues of infected mice after antibiotic treatment. Malawista

SE, Barthold SW, Persing DH. Department of Internal Medicine, Yale University School of

Medicine, New Haven, Connecticut.

Persistence of Borrelia burgdorferi DNA in tissues following antibiotic treatment was

evaluated in C3H mice inoculated intradermally with 10,3, B. burgdorferi N40 or sterile medium.

Half of the infected mice and all of the uninfected mice were treated with ceftriaxone 15 days after

38

inoculation for 5 days. Ear and urinary bladder samples were collected on days 20, 30, and 60

after inoculation for culture and for extraction of DNA and amplification of specific spirochetal

DNA by polymerase chain reaction, PCR. PCR primers were specific for a 280-bp portion of a

highly conserved region of the gene encoding outer surface protein, Osp, A of B. burgdorferi and

for a 328-bp part of the OspB gene. There was excellent concordance between culture and PCR

for ears, 35/36 mice, and bladders, 33/36. Both tissues became uniformly negative at the earliest

interval tested after antibiotic treatment. Thus, the ability to amplify B. burgdorferi DNA quickly

disappeared from tissues that had become culture-negative after antibiotic treatment, suggesting

that serial study of PCR-positive tissues and fluids may be useful for evaluating the efficacy of

antibiotic therapy in human Lyme disease. 2 out of 5 mice tested 60 days after treatment were

found to be positive on culture; 1 of these mice was also positive by PCR. The authors speculate

that this could be due to:, a, reinfection, which they consider .highly unlikely.,, b,

contamination, or, c, the .resurgence of spirochetes in animals not completely sterilized by

antibiotics. This last possibility will bear further scrutiny because late recurrences of Lyme

disease without obvious reinfection may occur in humans.[Diagnosis:] Positive PCR results

were found to suggest active infection. .Unless some patients with Lyme disease have a defect

in their ability to degrade spirochetal DNA, these results suggest that persisting PCR

positivity indicates persisting infection.

45: Antimicrob Agents Chemother. 1995 May;39,5:1127-33. Effects of penicillin, ceftriaxone,

and doxycycline on morphology of Borrelia burgdorferi. Kersten A, Poitschek C, Rauch S,

Aberer E.

Department of Dermatology, University of Vienna, Austria.

Antibiotic therapy with penicillin, doxycycline, and ceftriaxone has proven to be effective for the

treatment of Lyme borreliosis. In some patients, however, it was noticed that borreliae can

survive in the tissues in spite of seemingly adequate therapy. For a better understanding of this

phenomenon, we investigated the different modes of degeneration of Borrelia burgdorferi

suspensions during a 96-h exposure to various antibiotics. By dark-field microscopy and

ultrastructural investigations, increasing blebbing and the gradual formation of granular and

cystic structures could be followed during the exposure time. Although antibiotic concentrations

at the MIC at which 90% of organisms are inhibited after 72 h were 80% or even greater, motile

organisms were still present after incubation with penicillin and doxycycline but not after

incubation with ceftriaxone. By transmission electron microscopy, intact spirochetal parts,

mostly situated in cysts, were seen up to 96 h after exposure with all three antibiotics tested.

According to experiences from studies with other spirochetes it is suggested that encysted

borreliae, granules, and the remaining blebs might be responsible for the ongoing antigenic

stimulus leading to complaints of chronic Lyme borreliosis.

39

46: Persistent PCR positivity in a patient being treated for Lyme disease. Kornelia Keszler, MD

and Richard C. Tilton, PhD. JSTD 1995; 2:57-58.

A 30-year-old white female presented with worsening clinical symptoms suggestive of Lyme

disease while on antibiotic therapy. Results of enzyme-linked immunosorbent assay, ELISA, and

of western blot tests for IgG and IgM antibody were equivocal. However, Borrelia burgdorferi

DNA detected by the polymerase chain reaction, PCR, was detected in whole blood on two

separate occasions, 1 month apart, while the patient was on oral doxycycline, 100 mg b.i.d.

This report questions the significance of persistent Borrelia burgdorferi DNA in a patient who is

not responding to antibiotic therapy.

47: Neuroborreliosis in Texas Audrey Stein Goldings, MD. JSTD 1995; 2:59-61.

Chronic persistent symptoms after treatment for Lyme disease, LD, are common. Early effective

treatment is the only known way to avoid this possibility. despite early recognition of the

infection, patients still may not do well due to failure to eradicate the spirochete. This is a case

study of one such patient.

48: Vartiovaara I. 1995 Living with Lyme. Lancet, 345:842-4 A Finnish physician ’s account of

his experiences that beginning with a tick bite in Vancouver in 1987.

Dr. Vartiovaara resigned from his position with the Finnish Medical Journal in 1992, due to

disabilities caused by Lyme disease. [Persistence:] After that [a positive result on a T-cell

proliferation test at Stony Brook Hospital] I had two months’ heavy treatment with oral

doxycycline 300mg a day. I was a little better after it, but only for about two months. Then it

started all over again, and got worse. ..We sent blood and spinal fluid to Dr. Oksi and they

turned out to be positive [by PCR]--in other words, the spirochaete was still alive in my body

after six years, despite the antibiotics. Dr. Vartiovaara was then treated aggressively with a

combination of antibiotics, including four weeks of ceftriaxone, for six months. Some time after

the cessation of treatment however, he found that .My symptoms are on the move again.

[Diagnosis:] What should be done when a patient has the typical Lyme disease history but

negative serology? This is still a hot question especially in the USA. My strong opinion is that

oral antibiotics should be given in such cases. Ordinary laboratory tests cannot be relied upon

and the PCR is too expensive for routine use. When the whole picture leans towards Lyme

borreliosis it is both ethically and medically right to treat.

49: J Neuropsychiatry Clin Neurosci. 1995 Summer;7,3:345-7. Rapidly progressive frontal-type

dementia associated with Lyme disease. Waniek C, Prohovnik I, Kaufman MA, Dwork AJ.

40

New York State Psychiatric Institute, NY 10032, USA.

The authors report a case of fatal neuropsychiatric Lyme disease, LD, that was expressed

clinically by progressive frontal lobe dementia and pathologically by severe subcortical

degeneration. Antibiotic treatment resulted in transient improvement, but the patient

relapsed after the antibiotics were discontinued. LD must be considered even in cases with

purely psychiatric presentation, and prolonged antibiotic therapy may be necessary.

50: Ann Neurol. 1995 Oct;38,4:667-9. Comment in: Ann Neurol. 1995

Oct;38,4:560-2.Neuroborreliosis in the nonhuman primate: Borrelia burgdorferi persists in

the central nervous system. Pachner AR, Delaney E, O'Neill T.

Department of Neurology, Georgetown University School of Medicine, Washington, DC 20007,

USA.

Neurological involvement in Lyme disease is common, and is frequently difficult to diagnose and

treat. Little is known about the fate of the causative spirochete Borrelia burgdorferi in the ce ntral

nervous system, CNS. To determine the frequency of parenchymal infection and to determine

localization of the organism, polymerase chain reaction/hybridization assays were performed in a

newly described model of Lyme neuroborreliosis in nonhuman primates infected with B.

burgdorferi. Polymerase chain reaction/hybridization of CNS tissues from 5 infected nonhuman

primates was performed.Substantial amounts of B. burgdorferi DNA were detected in the CNS

in all infected animals, with a predilection toward subtentorial structures. These data suggest

that Lyme neuroborreliosis represents persistent infection with B. burgdorferi.

51: 1: Eur Neurol. 1995;35,2:113-7. Comment in: Eur Neurol. 1996;36,6:394-5. Seronegative

chronic relapsing neuroborreliosis.Lawrence C, Lipton RB, Lowy FD, Coyle PK.

Department of Medicine, Albert Einstein College of Medicine, New York, N.Y., USA.

We report an unusual patient with evidence of Borrelia burgdorferi infection who

experienced repeated neurologic relapses despite aggressive antibiotic therapy. Each course of

therapy was associated with a Jarisch-Herxheimer-like reaction. Although the patient never had

detectable free antibodies to B. burgdorferi in serum or spinal fluid, the CSF was positive on

multiple occasions for complexed anti-B. burgdorferi antibodies, B. burgdorferi nucleic acids

and free antigen.

52: Infection. 1996 Jan-Feb;24,1:64-8. Azithromycin and doxycycline for treatment of Borrelia

41

culture-positive erythema migrans. Strle F, Maraspin V, Lotric-Furlan S, Ruzi・-Sablji・ E,

Cimperman J.

Dept. of Infectious Diseases, University Medical Centre Ljubljana, Japlijeva, Slovenia.

Adult patients with typical solitary erythema migrans, participating in prospective therapeutic

studies on early Lyme borreliosis at the Lyme borreliosis Outpatient's Clinic, University

Department of Infectious Diseases in Ljubljana, in 1991 to 1993, and followed up for 1 year,

were included in the study. Only patients who were treated with azithromycin or doxycycline

and in whom Borrelia burgdorferi was isolated from the border of the skin lesion prior to

institution of antibiotic treatment were selected for presentation in this report. Fifty-eight

patients received azithromycin, 500 mg twice daily for the first day, followed by 500 mg once

daily for 4 days, and 42 patients received doxycycline, 100 mg twice daily for 14 days. The median

duration of skin lesions after the beginning of treatment was 6.5, 2-30, days in the azithromycin

group and 8, 2-35, days in the doxycycline group, non-significant difference. During the

follow-up of 12 months one patient in each group developed major later manifestations of

Lyme borreliosis and in 19 patients minor manifestations appeared: in nine, 20, 15.5%,

treated with azithromycin and in ten, 23.8%, receiving doxycycline. In one patient in the

azithromycin group and in one patient in the doxycycline group B. burgdorferi was isolated

from normal appearing skin at the site of previous erythema migrans 2 months after the

institution of antibiotic therapy. Five, 8.6%, patients receiving azithromycin and nine, 21.4%,

patients receiving doxycycline reported mild to moderate gastrointestinal discomfort. In

addition, five patients treated with doxycycline developed photosensitivity.

53: 1: Infection. 1996 Jan-Feb;24,1:9-16. Erratum in: Infection 1996 Mar-Apr;24,2:169.Kill

kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme

borreliosis. Preac Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Max v. Pettenkofer

Institut, Ludwig-Maximilians-Universitat Munchen, Germany.

For a better understanding of the persistence of Borrelia burgdorferi sensu lato, s.l., after

antibiotic therapy the kinetics of killing B. burgdorferi s.l. under amoxicillin, doxycycline,

cefotaxime, ceftriaxone, azithromycin and penicillin G were determined.The killing effect was

investigated in MKP medium and human serum during a 72 h exposure to antibiotics. Twenty

clinical isolates were used, including ten strains of Borrelia afzelii and ten strains of Borrelia

garinii. The results show that the kinetics of killing borreliae differ from antibiotic to antibiotic.

The killing rate of a given antibiotic is less dependent on the concentration of the antibiotic than

on the reaction time. Furthermore , the data show that the strains of B. afzelii and B. garinii have

a different reaction to antibiotics used in the treatment of Lyme borreliosis and that different

reactions to given antibiotics also exist within one species.The B. garinii strains appear to be

42

more sensitive to antibiotics used in therapy. Furthermore, the persistence of B. burgdorferi

s.l. and clinical recurrences in patients despite seemingly adequate antibiotic treatment is

described. The patients had clinical disease with or without diagnostic antibody titers to B.

burgdorferi.

54: Infection. 1996 Jan-Feb;24,1:73-5. Treatment failure in erythema migrans--a review. Weber

K. Dermatologische Privatpraxis, Munchen, Germany.

Patients with erythema migrans can fail to respond to antibiotic therapy. Persistent or

recurrent erythema migrans, major sequelae such as meningitis and arthritis, survival of Borrelia

burgdorferi and significant and persistent increase of antibody titres against B. burgdorferi

after antibiotic therapy are strong indications of a treatment failure. Most, if not all, antibiotics

used so far have been associated with a treatment failure in patients with erythema migrans.

Roxithromycin and erythromycin are definitely or probab ly ineffective. However, doxycycline,

amoxicillin, cefuroxime, ceftriaxone, azithromycin and high-dose penicillin V perform

comparably well.

55: Infection. 1996 May-Jun;24,3:218-26. Erratum in: Infection 1996 Jul-Aug;24,4:335.

Formation and cultivation of Borrelia burgdorferi spheroplast-L-form variants. Mursic VP,

Wanner G, Reinhardt S, Wilske B, Busch U, Marget W.

Max von Pettenkofer-Institut, Ludwig-Maximilians-Universitat Munchen, Germany.

As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs

despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants

and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the

variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated

from antibiotically treated and untreated patients with the clinical diagnosis of definite and

probable Lyme borreliosis and from patient specimens contaminated with bacteria were

investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in

vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron

microscopy revealed diverse alterations. Persisters isolated from a great number of patients,

60-80%, after treatment with antibiotics had an atypical form. The morphological alterations in

culture with penicillin G developed gradually and increased with duration of incubation.

Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells

to replicate, the long period of adaptation to growth in MKP-medium and the

mycoplasma-like colonies after growth in solid medium, PMR agar, suggest that B.

burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of

Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in

43

the organism for a long time, probably with all beta-lactam antibiotics, [corrected] and the

cell-wall-dependent antibody titers disappear and emerge after reversion.

56: JAMA. 1996 Jun 5; 275,21, :1657-60. Concurrent Lyme disease and babesiosis. Evidence for

increased severity and duration of illness. K rause PJ, Telford SR 3rd, Spielman A, Sikand V,

Ryan R, Christianson D, Burke G, Brassard P, Pollack R, Peck J, Persing DH.

Department of Pediatrics, University of Connecticut School of Medicine, Farmington 06030,

USA.

OBJECTIVE--To determine whether patients coinfected with Lyme disease and babesiosis in sites

where both diseases are zoonotic experience a greater number of symptoms for a longer period of

time than those with either infection alone. DESIGN--Community-based, yearly serosurvey and

clinic-based cohort study. SETTING--Island community in Rhode Island and 2 Connecticut

medical clinics from 1990 to 1994. STUDY PARTICIPANTS--Long-term residents of the island

community and patients seeking treatment at the clinics. MAIN OUTCOME

MEASURES--Seroreactivity to the agents of Lyme disease and babesiosis and number and

duration of symptoms. RESULTS--Of 1156 serosurvey subjects, 97, 8.4%, were seroreactive

against Lyme disease spirochete antigen, of whom 14, 14%, also were seroreactive against babesial

antigen. Of 240 patients diagnosed with Lyme disease, 26, 11%, were coinfected with babesiosis.

Coinfected patients experienced fatigue, P = .002, headache, P < .001, sweats, P < .001, chills, P =

.03, anorexia, P = .04, emotional lability, P = .02, nausea, P = .004, conjunctivitis, P = .04, and

splenomegaly, P = .01, more frequently than those with Lyme disease alone. Thirteen, 50%, of 26

coinfected patients were symptomatic for 3 months or longer compared with 7,4%, of the 184

patients with Lyme disease alon e from whom follow-up data were available, P < .001.Patients

coinfected with Lyme disease experienced more symptoms and a more persistent episode of

illness than did those, n = 10, experiencing babesial infection alone. Circulating spirochetal

DNA was detected more than 3 times as often in coinfected patients as in those with Lyme

disease alone, P = .06.

CONCLUSIONS-- Approximately 10% of patients with Lyme disease in southern New

England are coinfected with babesiosis in sites where both diseases are zoonotic. The number of

symptoms and duration of illness in patients with concurrent Lyme disease and babesiosis are

greater than in patients with either infection alone. In areas where both Lyme disease and

babesiosis have been reported, the possibility of concomitant babesial infection should be

considered when moderate to severe Lyme disease has been diagnosed.

57: 1: Antimicrob Agents Chemother. 1996 Jun;40,6:1552-4. Eucaryotic cells protect Borrelia

burgdorferi from the action of penicillin and ceftriaxone but not from the action of

44

doxycycline and erythromycin. Brouqui P, Badiaga S, Raoult D. Unite des Rickettsies, Faculte de

Medecine, Centre National de la Recherche Scientifique, Marseille, France.

Despite appropriate antibiotic treatment, Lyme disease patients may have relapses or may

develop chronic manifestations. The intracellular location of Borrelia burgdorferi suggests that

antibiotics that penetrate cells will have greater efficiency. Doxycycline or erythromycin was more

effective than penicillin or ceftriaxone in killing B. burgdorferi when the organism was grown in

the presence of eucaryotic cells

58: Repeat

59: 1: Infection. 1996 Sep-Oct;24,5:347-53.Borrelia burgdorferi DNA in the urine of treated

patients with chronic Lyme disease symptoms. A PCR study of 97 cases.

Bayer ME, Zhang L, Bayer MH. Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

The presence of Borrelia burgdorferi DNA was established by PCR from urine samples of 97

patients clinically diagnosed as presenting with symptoms of chronic Lyme disease. All patients

had shown erythema chronica migrans following a deer tick bite. Most of the patients had been

antibiotic-treated for extended periods of time. We used three sets of primer pairs with DNA

sequences for the gene coding of outer surface protein A, OspA, and of a genomic sequence of B.

burgdorferi to study samples of physician-referred patients from the mideastern USA. Controls

from 62 healthy volunteers of the same geographic areas were routinely carried through the

procedures in parallel with patients' samples.Of th e 97 patients, 72, 74.2%, were found with

positive PCR and the rest with negative PCR. The 62 healthy volunteers were PCR negative. It

is proposed that a sizeable group of patients diagnosed on clinical grounds as having chronic

Lyme disease may still excrete Borrelia DNA, and may do so in spite of intensive antibiotic

treatment.

The urine of 74.2% of patients previously treated with antibiotics for Lyme disease was found

to be positive for B. burgdorferi DNA using PCR testing. All patients, n=97, had prior

documented EM rash and had received a minimum of 3 weeks to 2 months oral or intravenous

antibiotics. In 4 patients, PCR results were temporarily negative after treatment, but became

positive again 4-6 weeks later. All patients suffered continuing, often gradually worsening

Lyme disease-like symptoms. ..it seems to be characteristic for most of the patients in our

study that, after antibiotic-free periods of a few months, they had again become increasingly

ill with neurological and arthritic symptoms, so that treatment had been resumed.

60: Hum Pathol. 1996 Oct;27,10:1025-34.Ultrastructural demonstration of spirochetal antigens

in synovial fluid and synovial membrane in chronic Lyme disease: possible factors

45

contributing to persistence of organisms. Nanagara R, Duray PH, Schumacher HR Jr.

Allergy-Immunology-Rheumatology Division, Department of Medicine, Faculty of Medicine,

KhonKaen University, Thailand.

To perform the first systematic electronmicroscopic, EM, and immunoelectron microscopy, IEM,

study of the pathological changes and the evidence of spirochete presence in synovial membranes

and synovial fluid, SF, cells of patients with chronic Lyme arthritis. EM examination was

performed on four synovial membrane and eight SF cell samples from eight patients with chronic

Lyme disease. Spirochetal antigens in the samples were sought by IEM using monoclonal

antibody to Borrelia burgdorferi outer surface protein A, OspA, as the immunoprobe. Prominent

ultrastructural findings were surface fibrin-like material, thickened synovial lining cell layer and

signs of vascular injury. Borrelia-like stru ctures were identified in all four synovial membranes

and in two of eight SF cell samples. The presence of spirochetal antigens was confirmed by IEM

in all four samples studied, one synovial membrane and three SF cell samples. OspA labelling was

in perivascular areas, deep synovial stroma among collagen bundles, and in vacuoles of fibroblasts

in synovial membranes; and in cytophagosomes of mononuclear cells in SF cell samples.Electron

microscopy adds further evidence for persistence of spirochetal antigens in the joint in

chronic Lyme disease. Locations of spirochetes or spirochetal antigens both intracellulary and

extracellulary in deep synovial connective tissue as reported here suggest sites at which

spirochaetes may elude host immune response and antibiotic treatment.

61: Rheumatol Int. 1996;16,3:125-32.Intracellular persistence of Borrelia burgdorferi in

human synovial cells. Girschick HJ, Huppertz HI, Russmann H, Krenn V, Karch H.

Children's Hospital, University of Wurzburg, Germany.

To investigate if Borrelia burgdorferi can persist in resident joint cells, an infection model using

cell cultures of human synovial cells was established and compared to the interaction of Borrelia

burgdorferi and human macrophages. Borrelia burgdorferi were found attached to the cell

surface or folded into the cell membrane of synovial cells analysed by transmission electron and

confocal laser scanning microscopy. In contrast to macrophages, morphologically intact Borrelia

burgdorferi were found in the cytosol of synovial cells without engulfment by cell membrane

folds or phagosomes. Borrelia burgdorferi were isolated from parallel cultures.Treatment with

ceftriaxone eradicated extracellular Borrelia burgdorferi, but spirochetes were reisolated after

lysis of the synovial cells. Borrelia burgdorferi persisted inside synovial cells for at least 8

weeks. These data suggested that Borrelia burgdorferi might be able to persist within resident

joint cells in vivo.

62: Antimicrob Agents Chemother. 1996 Nov;40 11 :2632-6.In vivo activities of ceftriaxone and

46

vancomycin against Borrelia spp in the mouse brain and other sites. Kazragis RJ, Dever LL,

Jorgensen JH, Barbour AG.

Department of Medicine Infectious Diseases, University of Texas Health Science Center at San

Antonio 78284, USA.

Borrelia burgdorferi, the agent of Lyme disease, and B. turicatae, a neurotropic agent of

relapsing fever, are susceptible to vancomycin in vitro, with an MIC of 0.5 microgram/ml. To

determine the activity of vancomycin in vivo, particularly in the brain, we infected adult

immunocompetent BALB/c and immunodeficient CB-17 scid mice with B. burgdorferi or B.

turicatae. The mice were then treated with vancomycin, ceftriaxone as a positive control, or

normal saline as a negative control. The effectiveness of treatment was assessed by cultures of

blood and brain and other tissues. Ceftriaxone at a dose of 25 mg/kg of body weight administered

every 12 h for 7 to 10 days eliminated cultivable B. burgdorferi or B. turicatae from all BALB/c or

scid mice in the study.Vancomycin at 30 mg/kg administered every 12 h was effective in

eliminating infection from immunodeficient mice if treatment was started within 3 days of

the onset of infection. If treatment with vancomycin was delayed for 7 days or more,

vancomycin failed to eradicate infection with B. burgdorferi or B. turicatae from

immunodeficient mice. The failure of vancomycin in eradicating established infections in

immunodeficient mice was associated with the persistence of viable spirochetes in the brain

during antibiotic treatment.

63: Brain. 1996 Dec;119, Pt 6:2143-54. Inflammatory brain changes in Lyme borreliosis. A

report on three patients and review of literature. Oksi J, Kalimo H, Marttila RJ, Marjamaki M,

Sonninen P, Nikoskelainen J, Viljanen MK.

Department of Internal Medicine, Turku University Central Hospital, Finland.

Despite a rapid increase in the number of patients with Lyme neuroborreliosis, LNB, its

neuropathological aspects are poorly understood. The objective of this study was evaluation of

neuropathological, microbiological, and magnetic resonance imaging, MRI, findings in three

patients with the Borrelia burgdorferi infection and neurological disease from whom brain

tissue specimens were available. Perivascular or vasculitic lymphocytic inflammation was

detected20in all specimens. Large areas of demyelination in periventricular white matter were

detected histologically and by MRI in one patient. The disease had a fatal outcome in this

patient. Brain MRI suggested malignancies in two patients before histopathological studies

were carried out. One of these two patients was a child with sudden hemiparesis. Another was

a 40-year-old man presenting with epileptic seizures and MRI-detected multifocal lesions,

which disappeared after repeated courses of antibiotics. We conclude that cerebral lymphocytic

[soijuv]

47

vasculitis and multifocal encephalitis may be associated with B. burgdorferi infection. The

presence of B. burgdorferi DNA in tissue samples from areas with inflammatory changes

indicates that direct invasion of B. burgdorferi may be the pathogenetic mechanism for focal

encephalitis in LNB.

"In one of the six analysed brain tissue specimens [from a patient who had received more than six

months of antibiotic treatment prior to death, including two 3-week courses of IV ceftriaxone], B.

burgdorferi DNA was detected by PCR."

64: Am J Dermatopathol. 1996 Dec;18,6:571-9. Heterogeneity of Borrelia burgdorferi in the

skin. Aberer E, Kersten A, Klade H, Poitschek C, Jurecka W.

Department of Dermatology, University of Vienna, Austria.

The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still

unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia

genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin

biopsies of erythema chronicum migrans, from which borrelia later was cultured, of

acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted

stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin"

model; identical to agar-embedded borreliae; and identical to cultured borreliae following

exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive

structures represent various forms of B. burgdorferi rather than staining artifacts. These

"atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this

bacterium.

Neuralgias arising 6 months after ECM in spite of antibiotic therapy were evident in a

seronegative patient who showed perineural rod-like borrelia structures.

65: 328: Semin Neurol. 1997 Mar;17,1:25-30.Peripheral nervous system Lyme borreliosis.

Logigian EL.

Harvard Medical School, Clinical Neurophysiology Laboratory, Brigham and Women's Hospital,

Boston, Massachusetts 02115, USA.

There are acute and chronic Lyme neuropathies. The seasonal acute syndromes of cranial neuritis

or radiculoneuritis are generally quite distinctive, but may cause diagnostic difficulty when one

syndrome occurs without the other, when erythema migrans is absent or missed, and when

meningeal signs are minimal or absent. The chronic Lyme radiculoneuropathies are less severe,

48

and less distinctive. Their recognition depends on eliciting a history of earlier classical

manifestations of Lyme disease and by labor atory testing. In both acute and chronic Lyme

radiculoneuropathy, electrophysiologic testing often proves the presence of a sensorimotor, axon

loss polyradiculoneuropathy. Both acute and chronic Lyme radiculoneuropathy have similar

pathologic features and can be classified as a nonvasculitic mononeuritis multiplex. The

pathogenesis is uncertain; both direct infection as well as parainfectious mechanisms may play a

role.The treatment with which we have the most experience is intravenous ceftriaxone 2 g/day

for 2 to 4 weeks. Improvement occurs rapidly over days to weeks in early Lyme

neuroborreliosis, but slowly over many months in chronic neuroborreliosis.

65.5: J Clin Microbiol. 1997 January; 35(1): 111–116. Persistence of Borrelia burgdorferi in

experimentally infected dogs after antibiotic treatment. R K Straubinger, B A Summers, Y F

Chang, and M J Appel Institute for Animal Health, College of Veterinary Medicine, Cornell

University, Ithaca, New York 14853, USA. rks4@cornell.edu

In specific-pathogen-free dogs experimentally infected with Borrelia burgdorferi by tick exposure,

treatment with high doses of amoxicillin or doxycycline for 30 days diminished but failed to

eliminate persistent infection. Although joint disease was prevented or cured in five of five

amoxicillin- and five of six doxycycline-treated dogs, skin punch biopsies and multiple tissues

from necropsy samples remained PCR positive and B. burgdorferi was isolated from one

amoxicillin- and two doxycycline-treated dogs following antibiotic treatment. In contrast, B.

burgdorferi was isolated from six of six untreated infected control dogs and joint lesions were

found in four of these six dogs.Serum antibody levels to B. burgdorferi in all dogs declined

after antibiotic treatment. Negative antibody levels were reached in four of six doxycyclineand

four of six amoxicillin-treated dogs. However, in dogs that were kept in isolation for 6

months after antibiotic treatment was discontinued, antibody levels began to rise again,

presumably in response to proliferation of the surviving pool of spirochetes. Antibody levels

in untreated infected control dogs remained high.

66: Clin Infect Dis. 1997 Jul;25 Suppl 1:S52-6. Tetracycline therapy for chronic Lyme disease.

Donta ST.

Boston University Medical Center and Boston Veterans Affairs Medical Center, Massachusetts

02118, USA.

Two hundred seventy-seven patients with chronic Lyme disease were treated with tetracycline

for 1 to 11 months, mean, 4 months; the outcomes for these patients were generally good.

Overall, 20% of the patients were cured; 70% of the patients' conditions improved, and treatment

failed for 10% of the patients. Improvement frequently did not take place for several weeks; after 2

49

months of treatment, 33% of the patients' conditions were significantly improved, degree of

improvement, 75%-100%, and after 3 months of treatment, 61% of the patients' conditions were

significantly improved. Treatment outcomes for seronegative patients, 20% of all patients, were

similar to those for seropositive patients. Western immunoblotting showed reactions to one or

more Borrelia burgdorferi-specific proteins for 65% of the patients for whom enzyme-linked

immunosorbent assays were negative. Whereas age, sex, and prior erythema migrans were not

correlated with better or worse treatment outcomes, a history of longer duration of symptoms

or antibiotic treatment was associated with longer treatment times to achieve improvement

and cure. These results support the use of longer courses of treatment in the management of

patients with chronic Lyme disease. Controlled trials need to be conducted to validate these

observations.

67: 1: Clin Infect Dis. 1997 Jul;25 Suppl 1:S64-70.Why is chronic Lyme borreliosis chronic?

Aberer E, Koszik F, Silberer M.

Department of Dermatology, University of Graz Medical School, Austria.

Chronic Lyme borreliosis, CLB, can present not only in different organs but also in different

patterns. Although many theories exist about the mechanisms leading to CLB, it is known

that viable Borrelia burgdorferi can persist for decades and cause late skin manifestations of

acrodermatitis chronica atrophicans, ACA. Thus, the immunopathogenetic findings in ACA

can serve as a model for studying the chronic course of Lyme borreliosis. Recent findings indicate

that the most important cell for antigen presentation, the epidermal Langerhans cell, LC, is

invaded by B. burgdorferi in early Lyme borreliosis. Therefore, LCs were stained

immunohistochemically with different markers to investigate their functional activity. Numbers

of CD1a+ LCs were reduced in erythema migrans but normal or slightly elevated in ACA. In both

diseases there was also a marked downregulation of major histocompatibility complex class II

molecules on LCs, as measured by staining of human leukocyte antigen DR.This phenomenon

might be a mechanism that protects against the presentation of autoantigens and may be the

cause of the impaired capacity of LCs to eliminate B. burgdorferi antigens, thus explaining

why CLB is chronic.

68: 1: Infection. 1997 Jul-Aug;25,4:240-6.Transformation of cystic forms of Borrelia

burgdorferi to normal, mobile spirochetes. Brorson O, Brorson SH.

Dept. of Microbiology, Ulleval University Hospital, Oslo, Norway.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under

controlled conditions. The occurrence o f cystic forms of Borrelia burgdorferi in vitro was

50

noted, and these cysts were able to be transformed to normal, mobile spirochetes. B.

burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes

multiplied only slowly in this medium, and transformation to encysted forms was observed after

1 week. When these cysts were transferred to the same culture medium with rabbit serum, the

encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration

time was normal. Examination of these cysts in the transmission electron microscope revealed

transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under

conditions unfavourable for spirochetes. These observations may help to explain why diagnosis

and treatment of B. burgdorferi infections in humans can be difficult.

69: American College of Rheumatology, Vol 40,9, Branigan P; Rao J; 1997 PCR evidence for

Borrelia burgdorferi DNA in synovium in absence of positive serology. Suppl, Rao J; Gerard H;

Sept, p.S270 Hudson A; Williams W; Arayssi T; Pando J; Bayer M; Rothfuss S; .PCR evidence for

Borrelia has been identified in synovial biopsies of patients with clinical pictures that had not

initially suggested Lyme disease. Clayburne G; Sieck M; Schumacher HR.

All [6 PCR-positive] patients were negative for antibodies to Borrelia and some were PCR

positive in synovium despite previous treatment with antibiotics.

70: Journal of Spirochetal & Tick-borne Diseases, Vol. 4, No. 1/2 Two lessons from the canine

model of Lyme Disease: migration of Borrelia burgdorferi in tissues and persistence after

antibiotic treatment. Straubinger RK; 1997 Straubinger AF; Jacobson RH; Chang Y; Summer

BA;

[Persistence:] In two studies, antibiotic treatment with amoxicillin or doxycycline for 30 days

failed to eliminate persistent infection in 11 dogs. Immediately after treatment, borreliae could

not be demonstrated, antibody levels declined, and joint lesions were prevented or cured. Live

spirochetes, however, persisted in the tissue of at least three dogs as B. burgdorferi DNA was

detected in all 11 treated dogs for up to 6 months after treatment, at which time antibody levels

again began to rise.

[Diagnostic issues:] In the dog model, we detected B. burgdorferi reliably in skin but infrequently

in blood by culture and polymerase chain reaction, PCR. We found the organism in the

synovium of joints but not in synovial fluids, and in meninges but not in cerebrospinal fluid.

71: 1: Ann Rheum Dis. 1998 Feb;57,2:118-21.Detection of Borrelia burgdorferi by polymerase

chain reaction in synovial membrane, but not in synovial fluid from patients with persisting

Lyme arthritis after antibiotic therapy. Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig

MG, Krause A.

51

Charite University Hospital, Department of Medicine III, Rheumatology and Clinical

Immunology, Berlin, Germany.

OBJECTIVES: To identify possible sites of bacterial persistence in patients with treatment

resistant Lyme arthritis. It was determined whether Borrelia burgdorferi DNA may be

detectable by polymerase chain reaction, PCR, in synovial membrane, SM, when PCR results

from synovial fluid, SF, had become negative after antibiotic therapy. METHODS: Paired SF

and SM specimens and urine samples from four patients with ongoing or recurring Lyme

arthritis despite previous antibiotic therapy were investigated. A PCR for the detection of B

burgdorferi DNA was carried out using pri mer sets specific for the ospA gene and a p66 gene of

B burgdorferi. RESULTS: In all four cases, PCR with either primer set was negative in SF and

urine, but was positive with at least one primer pair in the SM specimens. In all patients arthritis

completely resolved after additional antibiotic treatment. CONCLUSIONS: These data suggest

that in patients with treatment resistant Lyme arthritis negative PCR results in SF after

antibiotic therapy do not rule out the intraarticular persistence of B burgdorferi DNA.

Therefore, in these patients both SF and SM should be analysed for borrelial DNA by PCR as

positive results in SM are strongly suggestive of ongoing infection.

72: Med J Aust. 1998 May 18;168,10:500-2. Comment in: Med J Aust. 1998 May 18;168,10:479-80.

Culture-positive Lyme borreliosis. Hudson BJ, Stewart M, Lennox VA, Fukunaga M, Yabuki M,

Macorison H, Kitchener-Smith J.

Microbiology Department, Royal North Shore Hospital, Sydney, NSW.

bhudson@med.usyd.edu.au

We report a case of Lyme borreliosis. Culture of skin biopsy was positive for Borrelia garinii,

despite repeated prior treatment with antibiotics. The patient had travelled in Europe 17

months before the onset of symptoms, but the clinical details indicate that the organism could

have been acquired in Australia. The results of conventional serological and histopathological

tests were negative, despite an illness duration of at least two years.

73: Acta Clin Belg. 1998 Jun;53,3:178-83.Lyme borreliosis--a review of the late stages and

treatment of four cases. Petrovic M, Vogelaers D, Van Renterghem L, Carton D, De Reuck J, Afs

chrift M. Department of Internal Medicine, University Hospital Ghent, Belgium.

Difficulties in diagnosis of late stages of Lyme disease include low sensitivity of serological testing

and late inclusion of Lyme disease in the differential diagnosis.Longer treatment modalities may

have to be considered in order to improve clinical outcome of late disease stages. These

52

difficulties clinical cases of Lyme borreliosis. The different clinical cases illustrate several aspects

of late borreliosis: false negative serology due to narrow antigen composition of the used ELISA

format, the need for prolonged antibiotic treatment in chronic or recurrent forms and typical

presentations of late Lyme disease, such as lymphocytic meningo-encephalitis and

polyradiculoneuritis.

A five-week treatment with doxycycline at a dose of 200 mg daily was prescribed. Fatigue,

arthralgia en myalgia seemed to respond positively to Carton D; et al. the initiated

therapy.However, they reappeared two weeks after cessation of doxycycline. ..it was decided to

treat with ceftriaxone IM 2 g daily for three weeks. This resulted in a complete resolution of

the general symptoms. However, three weeks later arthralgia of the knees and myalgia in both

legs recurred. Symptoms and signs may improve only temporarily shortly after treatment, but

re-emerge within weeks or months.

74: 1: Eur J Clin Microbiol Infect Dis. 1998 Oct;17,10:715-9.Comparison of oral cefixime and

intravenous ceftriaxone followed by oral amoxicillin in disseminated Lyme borreliosis. Oksi J,

Nikoskelainen J, Viljanen MK. Department of Medicine, Turku University Central Hospital,

Finland.

Two treatment regimens for disseminated Lyme borreliosis, mainly neurologic and

musculoskeletal manifestations, were compared in a randomized trial. A group of 30 patients

received oral cefixime 200 mg combined with probenecid 500 mg three times daily for 100 days.

Another group of 30 patients received intravenous ceftriaxone 2 g daily for 14 days followed by

oral amoxicillin 500 mg combined with probenecid 500 mg three times daily for 100 days. There

was no statistically significant difference in the outcome of infection between the two groups.

However, the total number of patients with relapses or no response at all and the number of

positive polymerase chain reaction findings after therapy were greater in the cefixime group. The

general outcomes of infection in patients with disseminated Lyme borreliosis after 3-4 months of

therapy indicate that prolonged courses of antibiotics may be beneficial in this setting, since

90% of the patients showed excellent or good treatment responses.

75: Neurology. 1998 Nov;51,5:1489-91. Comment in: Neurology. 1999 Sep 11;53,4:895-6.

Clinical and serologic follow-up in patients with neuroborreliosis. Treib J, Fernandez A, Haass

A, Grauer MT, Holzer G, Woessner R.

Department of Neurology, University of the Saarland, Homburg, Germany.

The authors performed a clinical and serologic follow-up study after 4.2 +/- 1.2 years in 44

patients with clinical signs of neuroborreliosis and specific intrathecal antibody production. All

53

patients had been treated with ceftriaxone 2 g/day for 10 days. Although neurologic deficits

decreased significantly, more than half the patients had unspecific complaints resembling a

chronic fatigue syndrome and showed persisting positive immunoglobulin M serum titers for

Borrelia in the Western blot analysis.

76: 1: Infection. 1998 Nov-Dec; 26,6:364-7.A proposal for the reliable culture of Borrelia

burgdorferi from patients with chronic Lyme disease, even from those previously aggressively

treated. Phillips SE, Mattman LH, Hulinska D, Moayad H. Greenwich Hospital, CT 06830, USA.

Since culture of Borrelia burgdorferi from patients with chronic Lyme disease has been an

extraordinarily rare event, clarification of the nature of the illness and proving its etiology as

infectious have been difficult. A method for reliably and reproducibly culturing B. burgdorferi

from the blood of patients with chronic Lyme disease was therefore sought by making a

controlled blood culture trial studying 47 patients with chronic Lyme disease. All had relapsed

after long-term oral and intravenous antibiotics. 23 patients with other chronic illness formed the

control group. Positive cultures were confirmed by fluorescent antibody immuno-electron

microscopy using monoclonal antibody directed against Osp A, and Osp A PCR. 43/47 patients,

91%, cultured positive. 23/23 controls, 100%, cultured negative. Although persist ent infection

has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen

capture, there are major problems with these tests.This new method for culturing B.

burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness

and establishes that it is of chronic infectious etiology. This discovery should help to reestablish

the gold standard in laboratory diagnosis of Lyme disease.

77: Klin Monatsbl Augenheilkd. 1998 Dec;213,6:351-4. Pars plana vitrectomy in Borrelia

burgdorferi endophthalmitis [Article in German] Meier P, Blatz R, Gau M, Spencker FB,

Wiedemann P.

Klinik und Poliklinik fur Augenheilkunde der Universitat Leipzig.

BACKGROUND: Ocular manifestations of Lyme borreliose present with unusual forms of

conjunctivitis, keratitis, optic nerve disease, uveitis, vitritis and rarely endophthalmitis. CASE

REPORT: A 57-year-old man working as logger in Sax-ony-Anhalt suffering from an

endophthalmitis on his left eye was referred to us. The vision of his left eye was intact light

perception and hand motions. The slit-lamp examination revealed severe inflammation of the

anterior chamber with hypopyon, posterior synechiae, and opacity of the posterior lens capsule.

Funduscopy showed no red reflex, no retinal details. In the local hospital serum analysis was

performed and showed in Western-Blot IgM- and IgG-antibodies against Borrelia burgdorferi.

Despite of intravenous application of ceftriaxon for 14 days panuveitis persisted, and

54

endophthalmitis developed when antibiotic therapy was finished. RESULTS: During pars plana

vitrectomy a sharply delineated cystic lesion containing yellowish fluid was revealed, and creamy

yellow fluid was aspirated. Microscopically in hematoxylineosin stained slides of the aspirate

structures consistent with Borrelia burgdorferi were found. Postoperatively vision increased to

1/15. Despite of a second intravenous ceftriaxon treatment for 14 days we observed a retinal

vasculitis in the follow up of 6 months. CONCLUSIONS: Despite intravenous

ceftriaxon-therapy borrelia burgdorferi must have survived in the vitreous body. Further

investigations are required with respect to the use of other antibiotics or immunosuppressives.

78: Ann Med. 1999 Jun; 3,3:225-32. Borrelia burgdorferi detected by culture and PCR in

clinical relapse of disseminated Lyme borreliosis. Oksi J, Marjamaki M, Nikoskelainen J,

Viljanen MK.

Department of Medicine, Turku University Central Hospital, Finland. jarmo.oksi@utu.fi

A total of 165 patients with disseminated Lyme borreliosis, diagnosed in 1990-94, all

seropositive except one culture-positive patient, were followed after antibiotic treatment, and 32

of them were regarded as having a clinically defined treatment failure. Of the 165 patients,

136 were tested by polymerase chain reaction, PCR, during the follow-up. PCR was positive

from the plasma of 14 patients 0-30 months after discontinuation of the treatment, and 12 of

these patients had a clinical relapse. In addition, Borrelia burgdorferi was cultured from the

blood of three patients during the follow-up. All three patients belonged to the group with

relapse, and two of them were also PCR positive. This report focuses on the 13 patients with

clinical relapse and culture or PCR positivity. Eight of the patients had culture or PCR-proven

initial diagnosis, the diagnosis of the remaining five patients was based on positive serology

only.All 13 patients were primarily treated for more than 3 months with intravenous and/or

oral antibiotics,11 of them received intravenous ceftriaxone, nine for 2 weeks, one for 3 weeks

and one for 7 weeks, followed by oral antibiotics. The treatment caused only temporary relief

in the symptoms of the patients.All but one of them had negative PCR results immediately after

the first treatment. The patients were retreated usually with intravenous ceftriaxone for 4-6

weeks. None of them was PCR positive after the retreatment.The response to retreatment was

considered good in nine patients. We conclude that the treatment of Lyme borreliosis with

appropriate antibiotics for even more than 3 months may not always eradicate the

spirochete.By using PCR, it is possible to avoid unnecessary retreatment of patients with

'post-Lyme syndrome' and those with 'serological scars' remaining detectable for months or years

after infection.

79: Zentralbl Bakteriol. 1999 Jul;289,3:301-18. Persistence of Borrelia garinii and Borrelia

afzelii in patients with Lyme arthritis. Hulinska D, Votypka J, Valeso va M.

55

National Institute of Public Health, Prague, Czech Republic.

We repeatedly detected DNA of Borrelia garinii or B. afzelii and Borrelia-like structures in

the blood, joint fluid or in the synovium of 10 patients with Lyme arthritis by means of the

polymerase chain reaction and immunoelectron microscopy at 2-4-month intervals in the

course of two years. All samples were analyzed using primers which amplified the 16S rRNA

gene sequence of Borrelia burgdorferi sensu lato and nucleotide sequences for the OspA gene. No

cross hybridization occurred with DNA from human cells and with DNA from other bacteria.

Capture and labelling with monoclonal antibodies of aggregated antigens, membranes and

flagellae were evident in the blood of 7 patients, in 4 synovial membranes and 2 synovial

fluids. Borreliae were found in blood capillaries, in collagen and in clusters surrounding

inflammatory cells in the synovium of patients with recurrent infections who carried IgM and

IgG antibodies to OspA and to 83 kDa core protein. After significant improvement for several

weeks after treatment, arthritis recurred in six patients. Synoviocyte hyperplasia, inflammatory

infiltration and concentric adventitial fibroplasia were seen in the synovium of the patients with

persisting borreliae. Only two patients were infected with B. afzelii, the others with B. garinii.

80: J Infect Dis. 2000 Mar;181,3:1069-81. Status of Borrelia burgdorferi infection after

antibiotic treatment and the effects of corticosteroids: An experimental study. Straubinger RK,

Straubinger AF, Summers BA, Jacobson RH.

James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University,

Ithaca NY, 14853, USA. rks4@cornell.edu

Sixteen specific-pathogen-free beagles were infected with Borrelia burgdorferi. Three groups of 4

dogs were treated with antibiotics for 30 consecutive days starting 120 days after tick exposure; 4

dogs were untreated controls. At day 420 after tick exposure and again before euthanasia, 2 dogs

of each group were treated with prednisone for 14 days. All dogs contracted infection and 11

developed acute arthritis 50-120 days after exposure. After day 120, one of 12 antibiotic-treated

dogs and 2 of 4 untreated dogs became lame. Antibiotic therapy reduced the freq uency of

Borrelia-positivity in subsequent skin biopsy samples. After prednisone treatment, both control

dogs developed severe polyarthritis. At euthanasia, single tissues of the antibiotic-treated dogs

and multiple tissues of all control dogs were Borrelia-positive by polymerase chain reaction.

Viable spirochetes were not recovered from antibiotic-treated dogs. Two antibiotic-treated dogs

showed histologic evidence of minimal lesions, whereas all control dogs had mild polyarthritis

with periarteritis.

16 dogs were infected with Borrelia burgdorferi. 120 days after tick exposure, 12 dogs were

56

treated with antibiotics for 30 days; 4 control dogs were not treated. .At euthanasia, single tissues

of the antibiotic-treated dogs and multiple tissues of all control dogs were Borrelia-positive by

polymerase chain reaction.[Persistence:] .Do the data indicate an ongoing persistent infection in

these animals or only the presence of DNA remnants of dead Borrelia..? From this study and our

previous investigations, 20, it appears likely that B. burgdorferi maintains a persistent infection

with live organisms albeit at a very low level, p.1079, [Diagnosis:] As demonstrated by the

injection of heat-killed B. burgdorferi organisms into the skin of an uninfected animal, DNA of

dead organisms was detectable in our hands only for 3 weeks. These results are in concordance

with a study in which persistent experimental infection with Treponema pallidum, the spirochetal

agent of syphilis, was identified by PCR, 21. Wicher et al.[1998] discovered that DNA of dead

Treponema organisms was removed from or degraded within rabbit tissue within 15-30 days

after syringe inoculation, p.1079, Our studies show that at least in the dog, blood is an unreliable

tissue to demonstrate B. burgdorferi infection., p.1080

81: J Clin Microbiol. 2000 Jun; 38,6, :2191-9. PCR-Based quantification of Borrelia burgdorferi

organisms in canine tissues over a 500-Day postinfection period. Straubinger RK. James A.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca,

New York 14853, USA. rks4@cornell.edu

Borrelia burgdorferi infection in beagle dogs was studied quantitatively with skin punch biopsy

samples and blood samples collected at 4- and 2-week intervals, respectively, over a 500-day

period. Thereafter, 25 tissue samples of each dog were collected for further analysis. Starting at

day 120 after tick challenge, 12 dogs were treated with antibiotics, azithromycin, ceftriaxone,

or doxycycline, for 30 consecutive days. Four dogs received no antibiotic therapy.

Quantification of B. burgdorferi DNA was done with an ABI Prism 7700 Sequence Detection

System with oligonucleotide primers and a fluorescence-labeled probe designed to specifically

amplify a fragment of the ospA gene of B. burgdorferi strain N40. All 16 dogs became infected

with B. burgdorferi after tick challenge. In skin biopsy samples, spirochete numbers peaked at

day 60 postinfection, <1.5 x 10, 6, organisms per 100 microgram of extracted DNA, at the same

time when clinical signs of arthritis developed in 11 of 16 dogs, and decreased to almost

undetectable levels during the following 6 months. The number of B. burgdorferi organisms

detected in skin biopsy samples was inversely correlated with the antibody levels measured by

enzyme-linked immunosorbent assay. Antibiotic treatment reduced the amount of detectable

spirochete DNA in skin tissue by a factor of 1,000 or more. At the end of the experiment, B.

burgdorferi DNA was detectable at low levels, 10, 2, to 10, 4, organisms per 100 microgram of

extracted DNA, in multiple tissue samples regardless of treatment. However, more tissue

samples of untreated dogs than of antibiotic-treated dogs were positive, and tissue samples of

untreated dogs also were positive by culture. Only 1.6% of 576 blood samples of all dogs were

positive for B. burgdorferi by PCR.

[soijuv]

57

82: Straubinger RK, Straubinger AF, Summers BA, Jacobson RH, Erb HN. James A. Baker

Institute for Animal Health, Ithaca, New York, USA. rks4@cornell.edu

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, infects humans and

animals. In humans, the disease primarily affects the skin, large joints, and the nervous system

days to months after infection. Data generated with approp riate animal model help to

understand the fundamental mechanisms of the disease. OBJECTIVE: 1, More clearly define the

clinical manifestation and pathogenetic mechanisms of Lyme disease in dogs; 2, evaluate the

effect of antibiotics in dogs infected with B. burgdorferi; 3, describe the effects of corticosteroids

on dogs persistently infected with B. burgdorferi. DESIGN: Specific-pathogen-free beagles were

infected with B. burgdorferi using ticks collected in an endemic Lyme disease area. Clinical signs

were recorded daily. Antibody titers were measured by ELISA at two-week intervals. B.

burgdorferi organisms were detected in tissues by culture and PCR. Synovial fluids were

evaluated microscopically and with a chemotaxis cell migration assay. Histological sections were

examined for pathological lesions. Specific cytokine up-regulation in tissues was detected by

RT-PCR. INTERVENTIONS: In three separate experiments, B. burgdorferi-infected dogs

received antibiotic treatment, amoxicillin; azithromycin; ceftriaxone; doxycycline, for 30

consecutive days. Two subclinical persistently infected dogs received oral prednisone for 14

consecutive days starting at day 420 post-infection. RESULTS: Dogs developed acute arthritis in

the joints closest to the tick bites after a median incubation period of 68 days. Synovial

membranes of lame and non-lame dogs produced the chemokine IL-8 in response to B.

burgdorferi.Antibiotic treatment prevented or resolved episodes of acute arthritis, but failed

to eliminate the bacterium from infected dogs. Corticosteroid treatment reactivated Lyme

disease in pe rsistently infected dogs, which had not received antibiotics previously.

CONCLUSIONS: B. burgdorferi disseminates through tissue by migration following tick

inoculation, produces episodes of acute arthritis, and establishes persistent infection. The

spirochete survives antibiotic treatment and disease can be reactivated in immunosuppressed

animals.

83: Kaiser R. Neurologische Klinik, Stadtisches Klinikum Pforzheim. K

aiser.Neurologische_Klinik@Stadt-Pforzheim.de

Between 1990 and 2000, a total of 101 patients with acute, n=86, or chronic, n=15,

neuroborreliosis, proven by clinical data, pleocytosis in the CSF, and elevated Borrelia

burgdorferi-specific antibody indices, were treated with 2 g of ceftriaxone per day for either 2

or 3 weeks. The patients were reexamined clinically and serologically after 3, 6, and 12 months.

Six, 12, months after the antibiotic treatment, about 93%, 95%, of the patients with acute

neuroborreliosis and 20%, 66%, of the patients with chronic neuroborreliosis were cured. One

58

year after treatment, four patients with acute neuroborreliosis still suffered from facial palsy and

five with chronic neuroborreliosis still had moderate spastic ataxic gait disturbance.The

prognosis of facial palsy in neuroborreliosis is quite similar to that in idiopathic facial palsy,

while that in chronic neuroborreliosis largely depends on the time elapsed before diagnosis.

84: 1: Br J Dermatol. 2001 Feb;144,2:387-92. Isolation and polymerase chain reaction typing of

Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating

bullous lichen sclerosus et atrophicus. Breier F, Khanakah G, Stanek G, Kunz G, Aberer E,

Schmidt B, Tappeiner G.

Department of Dermatology, Lainz Municipal Hospital, Wolkersbergenstrasse 1, A-1130 Vienna,

Austria. brf@der.khl.magwien.gv.at

A 64-year-old woman presented with bullous and ulcerating lichen sclerosus et atrophicus,

LSA, on the neck, trunk, genital and perigenital area and the extremities. Histology of lesional

skin showed the typical manifestations of LSA; in one of the biopsies spirochaetes were

detected by silver staining. Despite treatment with four courses of ceftriaxone with or without

methylprednisone for up to 20 days, progression of LSA was only stopped for a maximum of 1

year. Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions.

These spirochaetes were identified as Borrelia afzelii by sodium dodecyl

sulphate--polyacrylamide gel electrophoresis and polymerase chain reaction, PCR, analyses.

However, serology for B. burgdorferi sensu lato was repeatedly negative. After one further 28-day

course of ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished. At

this time cultures for spirochaetes and PCR of lesional skin for B. afzelii DNA remained negative.

These findings suggest a pathogenetic role for B. afzelii in the development of LSA and a

beneficial effect of appropriate antibiotic treatment.

[From the article:] The relapses she repeatedly suffered despite initially successful antibiotic

treatment could be related to the observation that Borrelia may possibly be able to remain

dormant in certain tissue compartments, thus escaping bactericidal antibiotic activity. This

would be consistent with the fact that these relapses were always able to be treated successfully

with a course of the same antibiotics as before; this is corroborated by a recent report that Bb

may persist in experimentally infected dogs despite antibiotic treatment with doxycycline or

amoxycillin.

85: Epidemiol Mikrobiol Imunol. 2001 Feb;50,1:10-6.Persistence of Borrelia burgdorferi sensu

lato in patients with Lyme borreliosis [Article in Czech] Honegr K, Hulinska D, Dostal V,

Gebousky P, Hankova E, Horacek J, Vyslouzil L, Havlasova J. Infekcni klinika, Fakultni

nemocnice, Hradec Kralove.

59

In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia

burgdorferi sensu lato by detection of the causal agent by immune electron microscopy or of

its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months. Clinical

manifestations common in Lyme borreliosis were present in only half the patients, in the

remainder non-specific symptoms were found. In nine subjects with confirmed Borrelia

burgdorferi sensu lato in the cerebrospinal fluid the cytological and biochemical finding was

normal. Examination of antibodies by the ELISA method was negative in 7 of 18 patients during

the first examination and in 12 of 18 during the second examination. In all negative examinations

the specific antibodies were assessed by the Western blot or ELISA method after liberation from

the immunocomplexes. In the authors' opinion it is advisable to examine repeatedly plasma and

other biological material from potentially affected organs by PCR and subjects with persisting or

relapsing complaints after the acute form of Lyme borreliosis as well as to examine cerebrospinal

fluid in case on non-specific symptoms and concurrent pathic EEG or MR findings.

86: 1: Ann Neurol. 2001 Sep;50,3, :330-8.Central and peripheral nervous system infection,

immunity, and inflammation in the NHP model of Lyme borreliosis. Pachner AR, Cadavid D,

Shu G, Dail D, Pachner S, Hodzic E, Barthold SW. Department of Neurosciences, UMDNJ-New

Jersey Medical School, Newark 07103, USA. pachner@umdnj.edu

The relationship between chronic infection, antispirochetal immunity, and inflammation is

unknown in Lyme neuroborreliosis. In the nonhuman primate model of Lyme neuroborreliosis,

we measured spirochetal density in the nervous system and othe r tissues by polymerase chain

reaction and correlated these values to anti-Borrelia burgdorferi antibody in the serum and

cerebrospinal fluid, and to inflammation in tissues. Despite substantial presence of Borrelia

burgdorferi, the causative agent of Lyme borreliosis, in the central nervous system, only minor

inflammation was present there, though skeletal and cardiac muscle, which contained similar

levels of spirochete, were highly inflamed. Anti-Borrelia burgdoferi antibody was present in the

cerebrospinal fluid but was not selectively concentrated. All infected animals developed

anti-Borrelia burgdorferi antibody in the serum, but increased amplitude of antibody was not

predictive of higher levels of infection. These data demonstrate that Lyme neuroborreliosis is a

persistent infection, that spirochetal presence is a necessary but not sufficient condition for

inflammation, and that antibody measured in serum may not predict the severity of infection.

87: Wien Klin Wochenschr. 2002 Jul 31;114,13-14:574-9. Cystic forms of Borrelia burgdorferi

sensu lato: induction, development, and the role of RpoS. Murgia R, Piazzetta C, Cinco M.

Dipartimento di Scienze Biomediche, sez. Microbiologia, Universita degli Studi di Trieste,

Trieste, Italy. rmurgia@dsbmail.units.it

60

It has been demonstrated recently that cells of Borrelia burgdorferi sensu lato, the etiological

agent of Lyme disease, transform from mobile spirochetes into nonmotile cystic forms in the

presence of certain unfavourable conditions, and that cystic forms are able to reconvert to

vegetative spirochetes in vitro and in vivo. T he purpose of this study was to investigate the

kinetics of conversion of borreliae to cysts in different stress conditions such as starvation

media or the presence of different antibiotics. Using the same experimental conditions we also

investigated the possible role in cyst formation of RpoS, an alternative sigma factor that controls a

regulon in response to starvation and transition to stationary phase. We observed that

beta-lactams penicillin G and ceftriaxone, the antibiotics of choice in Lyme borreliosis

treatment, favoured the production of cysts when used with serum-depleted BSK medium. In

contrast, we observed a low level of cyst formation in the presence of macrolides and

tetracyclines. In order to elucidate the role of the rpoS gene in cyst formation we analyzed the

reaction of the rpoS mutant strain in comparison with its wild-type in different conditions.

Under the same stimuli, both the wild-type borrelia and the rpoS knock-out isogenic strain

produced cystic forms with similar kinetics, thus excluding the participation of the gene in this

phenomenon. Our findings suggest that cyst formation is mainly due to a physical-chemical

rearrangement of the outer membrane of Borrelia burgdorferi sensu lato leading to

membrane fusion and controlled by different regulation mechanisms.

87.5: Acta Neurol Scand. 2002 Oct;106(4):205-8. Chronic symptoms are common in patients

with neuroborreliosis -- a questionnaire follow-up study. Vrethem M, Hellblom L, Widlund M,

Ahl M, Danielsson O, Ernerudh J, Forsberg P.

Division of Neurology, University Hospital, Linkoping, Sweden, Division of Neurophysiology,

University Hospital, Linkoping, Sweden. magnus.vrethem@lio.se

OBJECTIVES: The existence of chronic neuroborreliosis is controversial. The aim of our study

was to investigate the existence and kind of persistent symptoms in patients previously

treated because of neurological symptoms as a result of neuroborreliosis. MATERIALS AND

METHODS: A total of 106 patients with neuroborreliosis, according to established criteria, and a

control group of 123 patients with Borrelia induced erythema migrans diagnosed in a general

practitioner office were studied. A questionnaire was sent to patients and controls concerning

their health situation. Time from onset of neurological symptoms to the questionnaire send out

was 32 months, mean, for the patients with neuroborreliosis and 33 months , mean, for the

controls. RESULTS: Fifty per cent of the individuals in the patient group compared with 16%

of the individuals in the control group showed persistent complaints after their Borrelia

infection, P < 0.0001. The most significant differences between the groups were the presence

of neuropsychiatric symptoms such as headache, attention problems, memory difficulties and

61

depression. Paresthesia, pain and persistent facial palsy was also significantly more common

in patients treated because of neuroborreliosis. CONCLUSION: Our study shows that

persisting neurological symptoms are common after a neuroborreliosis infection. The

pathological mechanisms that lay behind the development of chronic symptoms, however, are

still uncertain.

88: J Infect Dis. 2002 Nov 15;186,10:1430-7. Epub 2002 Oct 23. Detection of attenuated,

noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment.

Bockenstedt LK, Mao J, Hodzic E, Barthold SW, Fish D.

Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

06520-8031, USA. linda.bockenstedt@yale.edu

Xenodiagnosis by ticks was used to determine whether spirochetes persist in mice after 1 month

of antibiotic therapy, Doxycyline and Ceftriaxone, for vectorborne Borrelia burgdorferi

infection. Immunofluorescence and polymerase chain reaction, PCR, were used to show that

spirochetes could be found in Ixodes scapularis ticks feeding on 4 of 10 antibiotic-treated mice up

to 3 months after therapy. These spirochetes could not be transmitted to naive mice, and some

lacked genes on plasmids correlating with infectivity. By 6 months, antibiotic-treated mice no

longer tested positive by xenodiagnosis, and cortisone immunosuppression did not alter this

result. Nine months after treatment, low levels of spirochete DNA could be detected by

real-time PCR in a subset of antibiotic-treated mice. In contrast to sham-treated mice,

antibiotic-treated mice did not have culture or histopathologic evidence of persistent infection.

These results provide evidence that noninfectious spirochetes can persist for a limited

duration after antibiotics but are not associated with disease in mice.

89: Antimicrob Agents Chemother. 2002 Nov;46,11:3637-40. Erythromycin resistance in

Borrelia burgdorferi. Terekhova D, Sartakova ML, Wormser GP, Schwartz I, Cabello FC.

Departments of Microbiology and Immunology, New York Medical College, Valhalla, New York

10595, USA.

Susceptibility testing of laboratory strains and clinical isolates of Borrelia burgdorferi

indicates that resistance to erythromycin is present in them. Evaluation of the MICs, minimal

bactericidal concentrations, and kinetics of bacterial killing of erythromycin suggests that this

resistance is increased by preexposure to the antibiotic, is dependent on inoculum size, and may

be the result of selection of subpopulations of bacterial cells with increased resistance.

62

90: 1: Przegl Epidemiol. 2002;56 Suppl 1:57-67.New aspects of the pathogenesis of lyme disease

[Article in Polish] Zajkowska JM, Hermanowska-Szpakowicz T. Klinika Chorob Zaka・nych i

Neuroinfekcji AM w Bia ymstoku.

Morphological changes of B. burgdorferi as well as changes in expression of surface proteins

caused by environmental determinants are essential in pathogenesis of Lyme disease.Cysts,

spherical form, spheroplasts, L-form, and "blebs", gemmae, can be responsible for long lasting

antigenic stimulation, signs of chronic borreliosis, and even probably connected with MS and

Alzheimer disease. Mechanisms to avoid elimination and persistence in the host include:

expression of low heterogenic Osp A, B replaced by polymorphic in sequence and antigenic

reactivity OspC, the hindrance of access to some membrane proteins by other proteins on the

spirochete's surface, effects of tick saliva proteins action.Hiding of spirochetes is possible by

invagination into fibrocytes membrane as well as, coating by antigens derived from

lymphocytes B. Distribution of spirochetes is facilitated by binding to platelets through integrin

aIIb b3, and to the endothelial cells through integrins av b3 i a5b1, recognition of decorin by

lipoproteins DbpA i DbpB, receptor for NAG, N-acetyl glucosamina. Endothelial cells, toxic

products of granulocytes, monocytes, macrophages as well as phagocytosis counterpart in

pathogenesis. Induced cytokines are connected with activation subsets of T lymphocytes involved

in inflammatory response. Cytokines produced by Th1 as cytotoxic CD8 accompany the disease.

Important are also dendritic cells regarded as initiators of Th1 response with participation of

IL-12. In pathogenesis of Lyme disease participation of autoimmunity is notified, e specially

molecular similarities between OspA and human lymphocytic antigen, hLFA-1. Neurotoxin,

produced by B. burgdorferi Bbtox1 was identified. Encephalopathy signs in Lyme borreliosis

could be result of releasing toxico-metabolic products, ability of spirochetes to pass the

blood-brain barrier as well as, effect of lymphocytes migration. Active invasion of brain

endothelium as ability to adherence to endothelial wall could be the source of focused or

disseminated inflammation of brain vessels. Antiaxonal antibodies could disturb axon

conduction without damaging. But damage of white matter could be connected with damage of

mielin production cells, probably by antibodies, induced in cross reaction.

91: Repeat

92: Neurology. 2003 Jun 24;60,12:1923-30. Comment in: Neurology. 2003 Jun

24;60,12:1888-9.Study and treatment of post Lyme di sease, STOP-LD: a randomized double

masked clinical trial. Krupp LB, Hyman LG, Grimson R, Coyle PK, Melville P, Ahnn S,

Dattwyler R, Chandler B.

Department of Neurology, Stony Brook University Medical Center, Stony Brook, NY 11794-8121,

USA. lkrupp@notes.cc.sunysb.edu

63

OBJECTIVE: To determine whether post Lyme syndrome, PLS, is antibiotic responsive.

METHODS: The authors conducted a single-center randomized double-masked

placebo-controlled trial on 55 patients with Lyme disease with persistent severe fatigue at least 6

or more months after antibiotic therapy. Patients were randomly assigned to receive 28 days of IV

ceftriaxone or placebo. The primary clinical outcomes were improvement in fatigue, defined by a

change of 0.7 points or more on an 11-item fatigue questionnaire, and improvement in cognitive

function, mental speed, defined by a change of 25% or more on a test of reaction time. The

primary laboratory outcome was an experimental measure of CSF infection, outer surface protein

A, OspA. Outcome data were collected at the 6-month visit. RESULTS: Patients assigned to

ceftriaxone showed improvement in disabling fatigue compared to the placebo group, rate

ratio, 3.5; 95% CI, 1.50 to 8.03; p = 0.001. No beneficial treatment effect was observed for

cognitive function or the laboratory measure of persistent infection. Four patients, three of whom

were on placebo, had adverse events associated with treatment, which required hospitalization.

CONCLUSIONS: Ceftriaxone therapy in patients with PLS with severe fat igue was associated

with an improvement in fatigue but not with cognitive function or an experimental

laboratory measure of infection in this study. Because fatigue, a nonspecific symptom, was the

only outcome that improved and because treatment was associated with adverse events, this

study does not support the use of additional antibiotic therapy with parenteral ceftriaxone in

post-treatment, persistently fatigued patients with PLS.

93: Med Sci Monit. 2003 Nov;9,11:PI136-42. Macrolide therapy of chronic Lyme Disease.

Donta ST.

Boston University Medical Center, 650 Albany Street-8th Floor, Boston, MA 02118, U.S.A.

sam.donta@bmc.org

BACKGROUND: Macrolide antibiotics are highly active in vitro against B.burgdorferi, but have

limited efficacy in the treatment of patients with Lyme Disease. As macrolides are less active at a

low pH, their poor clinical activity might be due to localization of borrelia to an acidic endosome,

and their activity improved by alkalinization of that compartment with hydroxychloroquine.

MATERIAL/METHODS: 235 patients with a multi-symptom complex typical of chronic Lyme

disease, ie fatigue, musculoskeletal pain, and neurocognitive dysfunction and with serologic

reactivity against B.burgdorferi were treated with a macrolide antibiotic, eg clarithromycin, and

hydroxychloroquine. RESULTS: Eighty% of patients had self-reported improvement of 50% or

more at the end of 3 months. After 2 months of treatment, 20% of patients felt markedly

improved, 75-100% of normal; after 3 months of treatment, 45% were markedly improved.

Improvement frequently did not begin until after several weeks of therapy. There were no

differences among the three macrolide antibiotics used. Patients who had been on

64

hydroxychloroquine or macrolide antibiotic alone had experienced little or no improvement.

Compared to patients ill for less than 3 years, the onset of improvement was slower, and the

failure rate higher in patients who were ill for longer time periods. CONCLUSIONS: These

results support the hypothesis that the Lyme borrelia reside in an acidic endosome and that the

use of a lysosomotropic agent augments the clinical activity of macrolide antibiotics in the

treatment of patients with chronic Lyme Disease. In contrast, the efficacy of tetracycline in such

patients is not affected by hydroxychloroquine.

93.5: 1: Vet Microbiol. 2005 May 20;107(3-4):285-94 Antibiotic treatment of experimentally

Borrelia burgdorferi-infected ponies. Chang YF, Ku YW, Chang CF, Chang CD, McDonough

SP, Divers T, Pough M, Torres A. College of Veterinary Medicine, Cornell University, Ithaca, NY

14853, USA. yc42@cornell.edu

The objective of this study is to determine whether doxycycline, ceftiofur or tetracycline could be

effectively used to treat equine Lyme disease. Ponies experimentally infected with Borrelia

burgdorferi by tick exposure were treated with doxycycline, ceftiofur or tetracycline for 4

weeks, 28 days. Doxycyline and ceftiofur treatment were inconsistent in eliminating

persistent infection in this experimental model. However, tetracycline treatment seems to

eliminate persistent infection. Although serum antibody levels to B. burgdorferi in all ponies

declined gradually after antibiotic treatment, three out of four ponies treated with doxycline and

two out of four ponies treated with ceftiofur, serum KELA titers were raised again 3 month after

treatment was discontinued.Five months after antibiotic treatment, tissues aseptically collected

at necropsy from ponies with increased antibody levels after antibiotic treatment also showed

culture positive to B. burgdorferi in various post-mortem tissues.However, all

four-tetracycline treatment ponies showed a negative antibody level and culture negative from

post-mortem tissues. Untreated infected ponies maintained high KELA titers throughout the

study and were tissue culture positive.

94: Int J Antimicrob Agents. 2005 Jun;25,6:474-8. Susceptibility of Borrelia afzelii strains to

antimicrobial agents. Ruzi・-Sablji・ E, Podreka T, Maraspin V, Strle F.

Institute of Microbiology and Immunology, Medical Faculty Ljubljana, Slovenia.

eva.ruzic-sabljic@mf.uni-lj.si

The aim of the present study was to determine the susceptibility of Borrelia afzelii strains to

antibiotics, and to test the hypothesis that persistence of borrelia in skin, after therapy, is a

consequence of resistance to the antibiotic used for treatment. Ten B. afzelii strains isolated

from skin of seven adult patients, two with acrodermatitis chronica atrophicans, five with

erythema migrans, were studied. In three patients B. afzelii was isolated from erythema

65

migrans lesion before antibiotic therapy and 2-3 months after treatment with cefuroxime

axetil, two patients, or with ceftriaxone, one patient. MICs and MBCs for amoxicillin,

azithromycin, ceftriaxone, cefuroxime, doxycycline and amikacin were measured. There was total

resistance to amikacin but isolates were susceptible to all other antibiotics except one isolate that

was resistant to cefuroxime, MIC > 4 mg/L. Comparison of MBC values after 3 and 6 weeks'

incubation revealed comparable results for azithromycin and ceftriaxone while for amoxicillin,

cefuroxime and doxycycline, some differences were found. In one of the patients from whom

there were borrelia isolated before and after treatment with cefuroxime axetil, both isolates

were resistant to cefuroxime. In the other two patients, the paired isolates were susceptible to the

antibiotic used for therapy.

95: Int J Med Microbiol. 2006 May;296 Suppl 40:233-41. Epub 2006 Mar 10.Risk of

culture-confirmed borrelial persistence in patients treated for erythema migrans and possible

mechanisms of resistance. Hunfeld KP, Ruzi・-Sablji・ E, Norris DE, Kraiczy P, Strle F. Institute of

Medical Microbiology, University Hospital of Frankfurt, Paul-Ehrlich Str. 40, D-60596

Frankfurt/Main, Germany. K.Hunfeld@em.uni-frankfurt.de

Erythema migrans, EM, develops at the site of the tick bite in 77-90% of Lyme borreliosis, LB,

patients and is therefore a common manifestation of early disease.Clinical treatment failures

have been reported in early LB cases for almost every suitable antimicrobial agent. The exact

risk of resistance to antibiotic treatment in patients with EM, however, is not known and

there are few published cases of culture-proven treatment failure. Moreover, currently

available diagnostic techniques cannot reliably discriminate between possible reinfection, true

endogenous relapse and co-infection with other tick-borne pathogens. These drawbacks together

with the phenomenon of r esistance to therapy in individual patients undoubtedly contribute to

the inconsistencies surrounding the optimal treatment regimens for LB and are often

misinterpreted and misused to support prolonged antibiotic treatment regimens. The question

for the underlying mechanisms of possible antimicrobial resistance in Borrelia burgdorferi sensu

lato remains unresolved but a better understanding of such genetic or phenotypic mechanisms

would be helpful for the treatment of LB and other spirochetal diseases. Investigations on this

issue, at best, should start with borrelial isolates cultured from patients before the start of

antibiotic therapy and again after the conclusion of treatment. This task, however, remains

challenging insofar, as culture is rarely successful under routine laboratory conditions after

antimicrobial therapy.Here, we review recent clinical and experimental data on treatment

resistance in EM patients suggesting that, although rare, borrelial persistence does occur at

the site of the infectious lesion after antibiotic treatment. Borrelial persistence, however, is

unlikely to result from acquired resistance against antimicrobial agents that were used for initial

specific chemotherapy.

66

96: Eur J Pediatr. 2006 Jun;165,6:420-1. Epub 2006 Mar 4. Persistent synovitis in two children

with Lyme arthritis linked with HLA-DRB1*1104. Hendrickx G, Demanet C, Vandenplas Y.

Department of Paediatrics, Paediatric Orthopaedic and Rheumatology Unit, Academisch

Ziekenhuis -Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium.

g.hendrickx@st-anna.nl

We report on two patients with a persistent Lyme arthritis. In addition both had a peculiar

disease history. The first patient had oligoarticular juvenile idiopathic arthritis in remission. Five

months after an infected tick bite, she developed a relapse of arthritis in the same knee. We

considered Lyme borreliosis as the possible trigger for this reactivation. The disease history of

the second patient was that of a classical non-responder. After extensive antibiotic treatment

osteolytic lesions became visible. MRI images suggested an erosive arthropathy and arthroscopy

was used to investigate possible erosive arthritis. Studies on collected material made us consider

the following hypothesis. Despite demonstration of a spirochete fragment in a synovial

biopsy, the patient recovered without additional antibiotic treatment. Conclusion: delay of

antibiotic treatment after appearance of erythema migrans may cause systemic spread of the

antigen and predispose to Lyme arthritis. If intra-articular steroids are considered when

spontaneous resolution of Lyme arthritis does not occur, magnetic resonance imaging of the

affected joint, prior to administration, may provide additional information. The success of

synovectomy may be related to removal of undegraded antigenic material which may prolong the

inflammation.

97: Int J Immunopathol Pharmacol. 2006 Jul-Sep;19,3:545-9. In vitro susceptibility of isolates of

Borrelia burgdorferi s.l. to antimicrobial agents. Santino I, Scazzocchio F, Ciceroni L,

Ciarrocchi S, Sessa R, Del Piano M. Department of Public Health Sciences, La Sapienza

University, Rome, Italy. iolanda.santino@uniroma1.it

In the present study, we investigate the in vitro antimicrobial activity of macrolides,

beta-lactams and tetracycline against Borrelia burgdorferi s.l. clinical and tick isolates.

Minimal inhibitory concentrations, MICs, were determined in normal growth condition and after

pre-exposure of the strains to sub-MIC of the founder of each drug family. All the classes of

tested antibiotics showed good antibacterial activity against all the borreliae isolates and there

were no significant susceptibility differences among clinical and tick isolates. After pre-exposure

of the strains to sub-MIC of erythromycin, cefoxitin and tetracycline, we observed that some

strains of B. burgdorferi s.l. showed higher MIC values to both the pre-exposed drug and

drugs of the same family. The less susceptibility of borreliae, in the last growth condition in

vitro, could be one of the justifications of clinical results indicating the limited efficacy of

these antibiotics in treatment of B. burgdoferi infections.

[soijuv]

67

98: 1: Microbes Infect. 2006 Nov-Dec; 8,14-15:2832-40. Epub 2006 Sep 22.Invasion of human

neuronal and glial cells by an infectious strain of Borrelia burgdorferi. Livengood JA, Gilmore

RD Jr.

Centers for Disease Control and Prevention, Divi sion of Vector-borne Infectious Diseases,

3150 Rampart Road, CSU Foothills Campus, Fort Collins, CO 80522, USA.

Human infection by Borrelia burgdorferi, the etiological agent for Lyme disease, can result in

serious acute and late-term disorders including neuroborreliosis, a degenerative condition of the

peripheral and central nervous systems.To examine the mechanisms involved in the cellular

pathogenesis of neuroborreliosis, we investigated the ability of B. burgdorferi to attach to

and/or invade a panel of human neuroglial and cortical neuronal cells. In all neural cells tested,

we observed B. burgdorferi in association with the cell by confocal microscopy. Further analysis

by differential immunofluorescent staining of external and internal organisms, and a gentamicin

protection assay demonstrated an intracellular localization of B. burgdorferi. A

non-infectious strain of B. burgdorferi was attenuated in its ability to associate with these

neural cells, suggesting that a specific borrelial factor related to cellular infectivity was responsible

for the association. Cytopathic effects were not observed following infection of these cell lines

with B. burgdorferi, and internalized spirochetes were found to be viable. Invasion of neural

cells by B. burgdorferi provides a putative mechanism for the organism to avoid the host's

immune response while potentially causing functional damage to neural cells during infection of

the CNS.

98.5: Acta Radiologica, Volume 48, Issue 7 2007 , pages 755 - 762 Brain Magnetic Resonance

Imaging Does Not Contribute to the Diagnosis of Chronic Neuroborreliosis. Aalto A, Sjowall J,

Davidsson L, Forsberg P, Smedby O. Division of Radiology, Department of Medicine and Care,

Faculty of Health Sciences, Linkoping University, Linkoping, Sweden. anne.aalto@imv.liu.se

BACKGROUND: Borrelia infections, especially chronic neuroborreliosis, NB, may cause

considerable diagnostic problems. This diagnosis is based on symptoms and findings in the

cerebrospinal fluid but is not always conclusive. PURPOSE: To evaluate brain magnetic

resonance imaging, MRI, in chronic NB, to compare the findings with healthy controls, and to

correlate MRI findings with disease duration. MATERIAL AND METHODS: Sixteen

well-characterized patients with chronic NB and 16 matched controls were examined in a 1.5T

scanner with a standard head coil. T1-, with and without gadolinium, T2-, and

diffusion-weighted imaging plus fluid-attenuated inversion recovery, FLAIR, imaging were used.

RESULTS: White matter lesions and lesions in the basal ganglia were seen in 12 patients and 10

controls, no significant difference. Subependymal lesions were detected in patients down to the

68

age of 25 and in the controls down to the age of 43. The number of lesions was correlated to age

both in patients, rho = 0.83, P<0.01, and in controls, rho = 0.61, P<0.05, but not to the duration of

disease. Most lesions were detected with FLAIR, but many also with T2-weighted imaging.

CONCLUSION: A number of MRI findings were detected in patients with chronic NB,

although the findings were unspecific when compared with matched controls and did not

correlate with disease duration.

99: Pol Merkur Lekarski. 2007 Apr;22,130:275-9. Related Articles, Concentrations of

pro-inflammatory cytokines IFN-gamma, IL-6, IL-12 and IL-15 in serum and cerebrospinal

fluid in patients with neuroborreliosis undergoing antibiotic treatment. Article in Polish.

Pancewicz SA, Kondrusik M, Zajkowska J, Grygorczuk S. Akademia Medyczna w Bialymstoku,

Klinika ChorA3b Zakaznych i Neuroinfekcji.20spancewicz@interia.pl

Pathogenesis of Lyme disease, including neuroborreliosis, remains unclear. However,

pro-inflammatory cytokines seem to be involved and might be used to monitor the course of the

disease. It has been also shown that B. burgdorferi protects itself from elimination by modulating

function of the host's immune system. THE AIM OF THIS STUDY: The purpose of this study

was to evaluate the serum and cerebrospinal fluid, CSF, concentrations of selected cytokines in

patients with neuroborreliosis and their change during antibiotic treatment. MATERIAL AND

METHODS: The group of 25 patients was examined, all undergoing antibiotic therapy due to

meningitis caused by Borrelia burgdorferi infection. The group included 10, 40%, females and 15,

60%, males in the mean age x = 42,3 years. The control group for serum measurements consisted

of 25 healthy individuals, mean age x =43, 1, while control group for CSF study included 10

patients, aged x = 53,5 years, from whom CSF with normal parameters was taken during

diagnostic procedures neurosurgical. We examined serum and CSF before and after antibiotics

for concentrations of interferon-gamma, INF-gamma, interleukin-6, IL-6, interleukin-12, IL-12,

and interleukin-15, IL-15. RESULTS: In the first examination the significant increase of

IFN-gamma, IL-6, IL-2, IL-15 serum and CSF concentration was detected in comparison to

control group. After 4-weeks antibiotic treatment the concentrations of studied cytokines

decreased significantly in serum as well as in CSF but remained increased in compa rison with

controls. CONCLUSIONS: Although antibiotic treatment leads to withdrawal of clinical

symptoms of neuroborreliosis and normalization of CSF general parameters, pro-inflammatory

cytokines' concentrations in serum and CSF remain elevated.It may be explained by the

persistence of inflammatory conditions, perhaps related to surviving of a fraction of Borrelia

burgdorferi spirochetes within CNS tissue. This phenomenon might lead to development of

chronic CNS lesions.

100: 1: J Infect Dis. 2007 May 15;195,10:1489-96. Epub 2007 Apr 6.Anti-tumor necrosis

factor-alpha treatment activates Borrelia burgdorferi spirochetes 4 weeks after ceftriaxone

69

treatment in C3H/He mice. Yrjanainen H, Hytonen J, Song XY, Oksi J, Hartiala K, Viljanen MK.

Department of Medical Microbiology, University of Turku, Turku, 20520, Finland.

heta.yrjanainen@utu.fi

BACKGROUND: The effect of anti-tumor necrosis factor, TNF,-alpha treatment in Borrelia

burgdorferi-infected and ceftriaxone-treated C3H/He mice was evaluated. METHODS: Mice

were infected with B. garinii A218 or B. burgdorferi sensu stricto N40. At 2 weeks of infection,

one group was treated simultaneously with ceftriaxone and anti-TNF-alpha, whereas another

received ceftriaxone at 2 weeks and anti-TNF-alpha 4 weeks later. One group received ceftri

axone treatment only. Infected and noninfected control groups were sham treated. RESULTS: At

14 weeks of infection, B. burgdorferi could not be detected by cultivation or by polymerase chain

reaction in tissue samples of any mouse treated with ceftriaxone only. However, spirochetes grew

from the tissue samples of one-third of the mice treated with anti-TNF-alpha simultaneously

or 4 weeks after ceftriaxone. These activated spirochetes showed ceftriaxone sensitivity rates,

plasmid profiles, and virulence rates similar to those of bacteria used to infect the mice. All

infected control mice and mice given anti-TNF-alpha only were culture positive.

CONCLUSIONS: This report shows that, after ceftriaxone treatment for 5 days, a portion of B.

burgdorferi-infected mice still have live spirochetes in their body, which are activated by

anti-TNF-alpha treatment.

101: Adv Med Sci. 2007;52:174-8. Concentration of TGF-beta1 in the supernatant of

peripheral blood mononuclear cells cultures from patients with early disseminated and

chronic lyme borreliosis. Grygorczuk S, Chmielewski T, Zajkowska J, Swierzbi・ska R, Pancewicz

S, Kondrusik M, Tylewska-Wierzbanowska S, Hermanowska-Szpakowicz T. Department of

Infectious Diseases and Neuroinfections, Medical University of Bia・ystok, ul. Zurawia 14, 15-540

Bia・ystok, Poland. neuroin@amb.edu.pl

PURPOSE: The aberrant inflammatory response is probably involved in the pathogenesis of

chronic Lyme borreliosis, including chronic Lyme arthritis and neuroborreliosis. Transforming

growth factor-beta 1, TGF-beta1, is an important anti-inflammatory and immunomodulatory

cytokine and its deficient synthesis is linked to exaggerated inflammation and immune response.

MATERIAL AND METHODS: Peripheral blood mononuclear cells, PBMC, from 25 patients

with Lyme borreliosis and 6 controls were incubated for 7 days with suspension of Borrelia afzeli,

B. garinii and B. burgdorferi sensu stricto spirochetes. TGF-beta1 concentration in culture

supernatants was measured with ELISA. Results were analyzed according to disease duration,

group I--chronic borreliosis, n=20; group II--early borreliosis, n=5, and clinical form,

LA--arthritis, NB--neuroborreliosis. RESULTS: TGF-beta1 concentration was increased in

supernatants of PBMC cultures of patients with early neuroborreliosis, in comparison with

chronic borreliosis and controls. In chronic, but not in early borreliosis, there was a tendency

70

for decrease of TGF-beta1 synthesis under stimulation with B. burgdorferi spirochetes.

CONCLUSIONS: Impaired synthesis of TGF-beta1 by mononuclear cells seems to be present

in patients with chronic forms of Lyme borreliosis when compared to those with early stage of

the disease. It may be a factor contributing to the persistence of inadequate inflammatory

response in patients in whom chronic form of the disease develops.

102: 1: Rheumatol Int. 2007 Sep;27,11:1091-3. Epub 2007 Apr 4. Seronegative Lyme arthritis.

Holl-Wieden A, Suerbaum S, Girschick HJ. Children's hospital, Section of Pediatric

Rheumatology, Immunology and Infectious diseases, University of Wuerzburg,

Josef-Schneider-Str. 2, 97090 Wuerzburg, Germany.

We present a 10-year-old girl who had been diagnosed with juvenile idiopathic arthritis 5 years

before and who experienced a flare of arthritis affecting one knee while she was off medication

for almost 3 years. Seronegative Lyme arthritis had to be diagnosed based on the detection of

Borrelia burgdorferi DNA in synovial fluid. No humoral immune response to Borrelia

burgdorferi was detectable before, at the time of diagnosis and up to 3 years later.

103: Pol Merkur Lekarski. 2007 Sep;23,135:174-8. Concentration of soluble forms of selectins

in serum and in cerebrospinal fluid in group of patients with neuroborreliosis--a preliminary

study Moniuszko AM, Pancewicz SA, Ko ndrusik M, Zajkowska J, Grygorczuk S, Swierzbi・ska R.

Akademia Medyczna w Bia・ymstoku, Klinika Chorob Zaka・nych i Neuroinfekcji.

The results of the research already done, suggest an important role of selectins in inflammatory

process of various etiology. Lack of selectins or their ligands causes severe complications, such as

chronic inflammatory processes. The aim of this study was to analyze the role of selectins sL, sE

and sP in the development and course of neuroborreliosis in the form of meningitis. We have also

analyzed the influence of treatment on changes of selectins' concentration in serum and

cerebrospinal fluid. MATERIAL AND METHODS: We have analyzed 17 patients with

neuroborreliosis presenting as meningitis, in whom we measured by immunoenzymatic method

concentration of selectins sL, sP and sE in blood and cerebrospinal fluid before and after 4-week

therapy with cefotaxim. We used Human sL-selectin, Human sE-selectin and Human sP-selectin

kits produced by Bender Med. Systems, Austria. Control group for measurement of

concentration of selectins in serum consisted of 8 healthy patients. Control group for

measurement of concentration of selectins in cerebrospinal fluid consisted of 8 patients, in whom

lumbar puncture excluded inflammatory disease of the central nervous system. RESULTS: In

serum concentration of selectins sL and sP was significantly higher comparing to control group.

After treatment concentration of these selectins decreased, but still was significantly higher than

in control group. Only con centration of selectin sE was significantly lower than in control group

and after treatment decreased further remaining lower comparing to control group. In

71

cerebrospinal fluid concentration of selectin sL was significantly higher comparing to control

group and increased after treatment. Concentration of selectins sE and sP increased before

treatment and decreased after treatment, but still remained elevated comparing to control group.

CONCLUSIONS: Persistence of increased concentration of selectins sP and sL in serum and

also of selectin sE in cerebrospinal fluid in patients with neuroborreliosis after completed

antibiotic therapy and regression of clinical symptoms can suggest permanence of chronic

inflammatory state in consequence of survival of B. burgdorferi spirochetes in affected

tissues.

104: Volume 358:428-431 January 24, 2008 Number An Appraisal of "Chronic Lyme Disease" To

the Editor: Feder et al., Oct. 4 issue,1 review the great controversy surrounding "chronic Lyme

disease."v For most patients with this diagnosis, the authors advocate against the use of

antibiotics. But before the decision is made not to use antibiotics for patients with

post–tick-bite symptoms, anaplasma, babesia, bartonella, 2 and ehrlichia must be ruled out.

These tick-borne 2 intracellular pathogens are difficult to diagnose and can establish

long-term, persistent infection. 3,4,5 Anaplasma, babesia, and bartonella are underdiagnosed:

the nonspecific symptoms of infections with these organisms tend to be ascribed to the more

easily identifiable Lyme disease, which often accompanies them.2,3,4,5,6 Indeed, when studied

prospectively, 65 of 161 patients with Lyme disease, 40%, were coinfected with babesia, and 11 of

161, 7%, with anaplasma.6 Accurate diagnosis of these infections helps steer successful

treatment: babesia 3 and bartonella 5 are especially difficult to eradicate. Accurate diagnosis is

also important, since babesia 3 and anaplasma 4 can spread through blood transfusion. As Feder

et al. note, "chronic Lyme disease" is often unrelated to borrelia. If symptoms occur after a tick

bite in the absence of evidence of active borrelia infection or if they persist despite

anti-borrelia treatment, another tick-borne infection should be suspected. If such an infection

is found, the patient may indeed benefit from appropriate antibiotics.

1 Feder HM Jr, Johnson BJB, O'Connell S, et al. A critical appraisal of "chronic Lyme disease." N

Engl J Med 2007;357:1422-1431.[Free Full Text]

2 Adelson ME, Rao RV, Tilton RC, et al. Prevalence of Borrelia burgdorferi, Bartonella spp.,

Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis ticks collected in northern

New Jersey. J Clin Microbiol 2004;42:2799-2801.[Free Full Text]

3 Krause PJ, Spielman A, Telford SR III, et al. Persistent parasitemia after acute babesiosis. N Engl

J Med 1998;339:160-165.[Free Full Text]

4 Dumler JS. Is human granulocytic ehrlichiosis a new Lyme disease? Review and comparison of

clinical, laboratory, epidemiological, and some biological features. Clin Infect Dis 1997;25:Suppl

1:S43-S47.[CrossRef][ISI][Medline]

5 Rolain JM, Brouqui P, Koehler JE, Maguina C, Dolan MJ, Raoult D. Recommendations for

treatment of human infections caused by Bartonella species. Antimicrob Agents Chemother

72

2004;48:1921-1933.[Free Full Text]

6 Krause PJ, McKay K, Thompson CA, et al. Disease-specific diagnosis of coinfecting tickborne

zoonoses: babesiosis, human granulocytic ehrlichiosis, and Lyme disease. Clin Infect Dis

2002;34:1184-1191.[CrossRef][ISI][Medline]

105: To the Editor: Feder et al. fail to adequately inform readers about the science underlying the

"chronicity" debate. Multiple researchers have documented Borrelia burgdorferi's ability to

penetrate human cells. In demonstrating the presence of the organism inside neurons and glial

cells, Livengood and Gilmore established that it can exist in an intracellular state within a

protected site, 1 characteristics favoring persistence and necessitating longer courses of

antibiotics. B. burgdorferi's pleomorphic abilities also favor persistence. One study suggested

that penicillin, ceftriaxone, and doxycycline are ineffective against the bacteria in its cystic form.2

The study by Yrjanainen et al. revealed that B. burgdorferi can survive standard therapy,

lending further credence to the theory of bacterial persistence.3 Krupp et al. found that, as

compared with 23% of the placebo group, had significant improvement in fatigue.4 "Clinical

assessment remains the most important method for determining the efficacy of treatment."5

Persistent symptoms in patients with late Lyme disease suggest treatment failure and the need

for a new approach.

Elizabeth L. Maloney, M.D.

25611 West Comfort Dr.

Wyoming, MN 55092

References

1 Livengood JA, Gilmore RD Jr. Invasion of human neuronal and glial cells by an infectious strain

of Borrelia burgdorferi. Microbes Infect 2006;8:2832-2840.[CrossRef][ISI][Medline]

2 Kersten A, Poitschek C, Rauch S, Aberer E. Effects of penicillin, ceftriaxone, and doxycycline on

morphology of Borrelia burgdorferi. Antimicrob Agents Chemother

1995;39:1127-1133.[Abstract]

3 Yrjanainen H, Hytonen J, Song XY, Oski J, Hartiala K, Viljanen MK. Anti-tumor necrosis

factor-alpha treatment activates Borrelia burgdorferi spirochetes 4 weeks after ceftriaxone

treatment in C3H/HE mice. J Infect Dis 2007;195:1489-1496.[CrossRef][ISI][Medline]

4 Krupp LB, Hyman LG, Grimson R, et al. Study and treatment of post Lyme disease, STOP-LD:

a randomized double masked clinical trial. Neurology 2003;60:1923-1930.[Free Full Text]

5 Moellering R Jr, Eliopoulos G. Monitoring the response of the patient to antimicrobial therapy.

In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett's principles and practice

of infectious diseases. 6th ed. Vol. 1. Philadelphia: Elsevier, 2005.

73

106: To the Editor: The article by Feder et al. on the proper therapy of chronic Lyme disease

addresses a very timely concern. Unfortunately, the authors' statement that there are no

"scientific data" that support persistent B. burgdorferi infection in the face of negative

serologic test results is erroneous. In 1988, we reported on 17 patients who had all had

erythema migrans, received inadequate antibiotic therapy, had vigorous T-cell blastogenesis

to borrelia antigens, and were seronegative on the basis of enzyme-linked immunoassay. 1,2

The majority of these patients had improvement after definitive antibiotic therapy.

Seronegative infection was confirmed by other laboratories using polymerase-chain-reaction,

PCR, assays to document the presence of microbes in seronegative patients. 3,4 Abrogation of

a humoral response by removal of the bulk of microbial antigens has been seen in other settings,

including infection with Treponema pallidum. Although the use of repeated courses of antibiotics

for a putative borrelia infection is unsupported and may cause serious morbidity,5 persons with

evidence of previously inadequately treated Lyme disease may be seronegative and may benefit

from adequate antibiotic therapy. Fortunately, erythema migrans is now more readily recognized,

and occult Lyme disease is rarer. In the absence of antibiotic treatment, most persons become

seropositive.

David J. Volkman, M.D., Ph.D.

State University of New York at Stony Brook

Stony Brook, NY 11794

volkmans@optonline.net

References

1. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative late

Lyme borreliosis: dissociation of specific T- and B-lymphocyte responses to Borrelia burgdorferi.

N Engl J Med 1988;319:1441-1446.[Abstract]

&nbs p; 2. Volkman D. Prophylaxis after tick bites. Lancet Infect Dis

2007;7:370-371.[CrossRef][ISI][Medline]

3. Keller TL, Halperin JJ, Whitman M. PCR detection of Borrelia burgdorferi DNA in

cerebrospinal fluid of Lyme neuroborreliosis patients. Neurology 1992;42:32-42.[Free Full Text]

4. Oksi J, Uksila J, Marjamaki M, Nikoskelainen J, Viljanen MK. Antibodies against whole

sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients

with PCR- or culture-proven late Lyme borreliosis. J Clin Microbiol

1995;33:2260-2264.[Abstract]

5. Klempner MS, Hu LT, Evans J, et al. Two controlled trials of antibiotic treatment in patients

with persistent symptoms and a history of Lyme disease. N Engl J Med 2001;345:85-92.[Free Full

Text]

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107: Persistence of Borrelia burgdorferi Following Antibiotic Treatment in Mice

Antimicrobial Agents and Chemotherapy, published online ahead of print on 3 March 2008

Emir Hodzic, Sunlian Feng, Kevin Holden, Kimberly J. Freet, and Stephen W. Barthold

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis.

Mice were treated with ceftriaxone or saline for one month, commencing during the early, 3

weeks, or chronic, 4 months, stages of infectionwith Borrelia burgdorferi. Tissues from mice

were tested for infection by culture, polymerase chain reaction, PCR, xenodiagnosis, and

transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues

were examined for spirochetes by immunohistochemistry.

In contrast to saline-treated mice, mice treated with antibiotic were consistently

culture-negative, but tissues from some of the mice remained PCR-positive, and spirochetes

could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic treated

mice were fed upon by Ixodes scapularis ticks, xenodiagnosis, spirochetes were acquired by the

ticks, based upon PCR, and ticks from those cohorts transmitted spirochetes to naive SCID mice,

which became PCR-positive, but culture-negative.

Results indicated that following antibiotic treatment, mice remained infected with

non-dividing but infectious spirochetes, particularly when antibiotic treatment was commenced

during the chronic stage of infection.

108: Antimicrobial Agents and Chemotherapy, May 2008, p. 1728-1736, Vol. 52, No. 50066-4804

Persistence of Borrelia burgdorferi following Antibiotic Treatment in Mice Emir Hodzic,

Sunlian Feng, Kevin Holden, Kimberly J. Freet, and Stephen W. Barthold*

Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of

California at Davis, One Shields Avenue, Davis, California 95616 Received 9 August 2007/

Returned for modification 1 November 2007/ Accepted 26 December 2007

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis.

Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early,

3 weeks, or chronic, 4 months, stages of infection with Borrelia burgdorferi. Tissues from mice

were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1

and 3 months after completion of treatment. In addition, tissues were examined for the presence

of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice

treated with antibiotic were consistently culture negative, but tissues from some of the mice

remained PCR positive, and spirochetes could be visualized in collagen-rich tissues.

75

Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks,

xenodiagnosis, spirochetes were acquired by the ticks, as determined based upon PCR results, and

ticks from those cohorts transmitted spirochetes to naive SCID mice, which became PCR positive

but cult ure negative. Results indicated that following antibiotic treatment, mice remained

infected with nondividing but infectious spirochetes, particularly when antibiotic treatment

was commenced during the chronic stage of infection.

109: Pol Arch Med Wewn. 2008 May;118 5:314-7. : Neuroborreliosis with extrapyramidal

symptoms: a case report. Biesiada G, Czapiel J, Sobczyk-Krupiarz I, Garlicki A, Mach T.

Department of Infectious Diseases, Division of Gastroenterology, Hepatology, and Infectious

Diseases, Jagiellonian University School of Medicine, Krakow, Poland. gbiesiada@op.pl

The disease of Lyme is a tick-borne infection. It involves skin, the nervous system, joints and the

heart. Spirochaeta Borrelia burgdorferi is the etiologic agent of the disease. In the majority of

cases, clinical symptoms, like migrating erythema, occur from 3 to 30 days, sometimes to 3

months after a bite from a tick. The early disseminated infection involves multiple migrating

erythema, neuroborreliosis, arthritis, myocarditis and other organ-related symptoms. The late

stage of chronic infection involves chronic atrophic leg dermatitis, neurological and

rheumatological symptoms, and other organ-related symptoms which persist for above 12

months. The diagnosis of the disease of Lyme is based upon specific clinical symptoms confirmed

by serologic tests. The two-step diagnostic protocol including the ELISA method, confirmed by

the Western-blot test, is optimal. The present article describes a case of a 59-year-old man, a

computer specialist, who often spends his free time walking in woods for recreation, and who

was bitten by a tick 3 years before hospitalization. The bite resulted in migrating erythema that

subsided without antimicrobial treatment. In spite of this, the man had not changed his hobby

exposing himself to bites from ticks. One year later, multiple migrating erythema and

extrapyramidalis symptoms appeared without any other organ malfunctions. In the current

year, the patient was admitted to the Infectious Diseases Hospital, and received antibiotics

ceftriaxon with following neurological improvement. Several months later, extrapyramidal

symptoms increased. On the day of admission to the hospital, the neurologic examination

showed abnormalities of upper and lower limbs movements propulsive walking and the right

lower leg traction), the right hand tremor, pouts of the face, and sleepiness.

109: 1: J Neuroinflammation. Sep 25;5:40. Persisting atypical and cystic forms of Borrelia

burgdorferi and local inflammation in Lyme neuroborreliosis. Miklossy J, Kasas S, Zurn AD,

McCall S, Yu S, McGeer PL.

Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, BC,

76

Canada. judithmiklossy@bluewin.ch

BACKGROUND: The long latent stage seen in syphilis, followed by chronic central nervous

system infection and inflammation, can be explained by the persistence of atypical cystic and

granular forms of Treponema pallidum. We investigated whether a similar situation may occur in

Lyme neuroborreliosis. METHOD: Atypical forms of Borrelia burgdorferi spirochetes were

induced exposing cultures of Borrelia burgdorferi, strains B31 and ADB1, to such unfavorable

conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and

Congo red. We also analyzed whether these forms may be induced in vitro, following infection of

primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed

whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three

patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence

of neuroinflammation in the form of reactive microglia and astrocytes. RESULTS: Under these

conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We

characterized these abnormal forms by histochemical, immunohistochemical, dark field and

atomic force microscopy, AFM, methods. The atypical and cystic forms found in the brains of

three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those

induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the

TUNEL metho d. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes

were present in the cerebral cortex. CONCLUSION: The results indicate that atypical extraand

intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local

neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of

these more resistant spirochete forms, and their intracellular location in neurons and glial

cells, may explain the long latent stage and persistence of Borrelia infection. The results also

suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection

and recognition of atypical, cystic and granular forms in infected tissues is essential for the

diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

110: Microb Pathog. 2008 Sep 20. Borrelia burgdorferi expression of the bba64, bba65, bba66,

and bba73 genes in tissues during persistent infection in mice. Gilmore RD Jr, Howison RR,

Schmit VL, Carroll JA.

Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, 3150

Rampart Rd, Fort Collins, CO 80521, USA.

Borrelia burgdorferi, the etiological agent of Lyme disease in humans, is vectored between

mammalian hosts in nature by Ixodes ticks. The organism adapts to diverse environments

encountered throughout the enzootic cycle by differentially expressing essential gene

products to survive the specialized conditions, whether in ticks or warm-blooded hosts. Ho

[soijuv]

77

wever, little is known regarding the identity and/or function of B. burgdorferi genes expressed

during colonization of tissues during mammalian infection. Experimental evidence has shown

that a group of genes, formerly classified as paralogous gene family 54, contiguously localized on

the 54-kilobase linear plasmid of B. burgdorferi, are among the most highly regulated by in vitro

conditions resembling mammalian infection. In this study, we employed quantitative reverse

transcription-PCR to measure temporal gene expression of a subset of this B. burgdorferi gene

family, bba64, bba65, bba66, and bba73, in tissues during chronic murine infection. The goal was

to gain insight into the role of these genes in infectivity and pathogenesis by identifying when the

genes are induced and whether they are expressed in specific target tissues. B. burgdorferi bba64,

bba65, bba66, and bba73 expression was measured from infected mouse tissues relative to

expression in in vitro culture conditions at specific times post-infection. bba64 expression was

highly upregulated in bladder, heart, and spleen tissues throughout the infection period,

contrasting with the sharp downregulation previously observed in ear tissues. bba65, bba66, and

bba73 demonstrated upregulated differential expression in various tissues over 1year

post-infection. These results suggest an essential role for these genes in borrelial survival,

persistence, and/or pathogenesis.

111: Med Hypotheses. 2008;70,5:967-74. Epub 2007 Nov 5. The association between tick-borne

infections, Lyme borreliosis and autism spectrum disorders. Bransfield RC, Wulfman JS,

Harvey WT, Usman AI.

Department of Psychiatry, Riverview Medical Center, 225 State Route 35, Red Bank, NJ, United

States. bransfield@comcast.net

Chronic infectious diseases, including tick-borne infections such as Borrelia burgdorferi may

have direct effects, promote other infections and create a weakened, sensitized and

immunologically vulnerable state during fetal development and infancy leading to increased

vulnerability for developing autism spectrum disorders. A dysfunctional synergism with other

predisposing and contributing factors may contribute to autism spectrum disorders by provoking

innate and adaptive immune reactions to cause and pe rpetuate effects in susceptible individuals

that result in inflammation, molecular mimicry, kynurenine pathway changes, increased

quinolinic acid and decreased serotonin, oxidative stress, mitochondrial dysfunction and

excitotoxicity that impair the development of the amygdala and other neural structures and

neural networks resulting in a partial Kluver-Bucy Syndrome and other deficits resulting in

autism spectrum disorders and/or exacerbating autism spectrum disorders from other causes

throughout life. Support for this hypothesis includes multiple cases of mothers with Lyme

disease and children with autism spectrum disorders; fetal neurological abnormalities

associated with tick-borne diseases; similarities between tick-borne diseases and autism spectrum

disorder regarding symptoms, pathophysiology, immune reactivity, temporal lobe pathology, and

78

brain imaging data; positive reactivity in several studies with autistic spectrum disorder

patients for Borrelia burgdorferi, 22%, 26% and 20-30%, and 58% for mycoplasma; similar

geographic distribution and improvement in autistic symptoms from antibiotic treatment. It

is imperative to research these and all possible causes of autism spectrum disorders in order to

prevent every preventable case and treat every treatable case until this disease has been eliminated

from humanity.

111.5: Journal of Veterinary Diagnostic Investigation Vol. 20 Issue 3, 321-324

Copyright c 2008 by the American Association of Veterinary Laboratory Diagnosticians:

Validation of an in-clinic enzyme-linked immunosorbent assay kit for diagnosis of Borrelia

burgdorferi infection in horses. Amy L. Johnson1, Thomas J. Divers and Yung-Fu Chang

Correspondence: 1Corresponding Author: Amy L. Johnson, Department of Clinical Studies,

University of Pennsylvania, New Bolton Center, 382 West Street Road, Kennett Square, PA

19348, e-mail: aljdvm03@gmail.com

Confirmation of Borrelia burgdorferi infection in horses has required enzyme-linked

immunosorbent assay (ELISA) or Western blot tests performed by reference laboratories. An

in-clinic C6 ELISA SNAP kit has been marketed for dogs. This canine kit was evaluated for horses

using serum from experimentally infected ponies. Serum samples originated from 2 previous

studies. In the first study, 7 ponies were exposed to B. burgdorferi–infected ticks; 4 ponies served

as uninfected controls. Serum samples were obtained bimonthly for 9 months. In the second

study, 16 ponies were exposed to B. burgdorferi–infected ticks. After confirmation of infection by

skin culture, polymerase chain reaction (PCR), and serology, the ponies were allocated to 4

groups that received tetracycline, doxycycline, ceftiofur, or no treatment. Serum samples were

obtained monthly, both before and after antibiotic treatments, for 11 months. For the current

study, selected samples (n = 220) from both studies were tested with IDEXX SNAP Heartworm

Ab/Borrelia burgdorferi Ab/Ehrlichia canis Ab Test Kits. Tested samples included samples taken

before infection, from various times postinfection, and after antibiotic treatments. Results from

confirmed positive or negative samples were used to determine sensitivity and specificity of the

assay. Results indicate that the test kits have fair sensitivity (63%) and very high specificity (100%)

for horses recently infected with B. burgdorferi. Validation of this test provides equine

practitioners with an inexpensive, in-clinic method to confirm infection, although its moderate

sensitivity may result in a moderate chance of a false negative test.

Finally, recent reports 14,15 indicate that C6 technology may allow evaluation of successful

antibiotic treatment of Lyme disease based on a decreasing titer. Human studies indicate that a

decreasing titer is usually seen with successful treatment of early infection but not in cases of

chronic infection despite extensive antibiotic treatment.7 SNAP testing of experimentally

79

infected ponies successfully treated with antibiotics (based on negative culture and PCR results by

the end of the study) revealed that all ponies became negative on SNAP test. Practitioners in

Lyme-endemic areas often see horses with persistently positive ELISA results despite

long-term antibiotic treatment. It is not known whether those cases represent failure of

antibiotic therapy to eliminate the organism, reinfection with B. burgdorferi, or a persistent

immune response despite successful treatment. Further research is needed to determine

whether C6 technology will better define the infection status of these horses

112: J Antimicrob Chemother. 2009 Jun;63 6:1163-72. Epub 2009 Apr 17. Assessment of

methylthioadenosine/S-adenosylhomocysteine nucleosidases of Borrelia burgdorferi as targets for

novel antimicrobials using a novel high-throughput method. Cornell KA, Primus S, Martinez JA,

Parveen N.

Department of Chemistry and Biochemistry, Boise State University, IA 83725-1520, USA.

Abstract

BACKGROUND: Lyme disease is the most prevalent tick-borne disease in the USA with the

highest number of cases (27 444 patients) reported by CDC in the year 2007, representing an

unprecedented 37% increase from the previous year. The haematogenous spread of Borrelia

burgdorferi to various tissues results in multisystemic disease affecting the heart, joints, skin,

musculoskeletal and nervous system of the patients. OBJECTIVES: Although Lyme disease can

be effectively treated with doxycycline, amoxicillin and cefuroxime axetil, discovery of novel

drugs will benefit the patients intolerant to these drugs and potentially those suffering from

chronic Lyme disease that is refractory to these agents and to macrolides. In this study, we

have explored 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase as a drug target for

B. burgdorferi, which uniquely possesses three genes expressing homologous enzymes with two

of these proteins apparently exported. METHODS: The recombinant B. burgdorferi Bgp and Pfs

proteins were first used for the kinetic analysis of enzymatic activity with both substrates and

with four inhibitors. We then determined the antispirochaetal activity of these compounds using

a novel technique. The method involved detection of the live-dead B. burgdorferi by fluorometric

analysis after staining with a fluorescent nucleic acids stain mixture containing Hoechst 33342

and Sytox Green. RESULTS: Our results indicate that this method can be used for

high-throughput screening of novel antimicrobials against bacteria. The inhibitors formycin A

and 5'-p-nitrophenythioadenosine particularly affected B. burgdorferi adversely on prolonged

treatment. CONCLUSIONS: On the basis of our analysis, we expect that structure-based

modification of the inhibitors can be employed to develop highly effective novel antibiotics

against Lyme spirochaetes.

113: Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18656-61. Epub 2009 Oct 20. Destruction of

80

spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic tigecycline.

Brorson O, Brorson SH, Scythes J, MacAllister J, Wier A, Margulis L.

Department of Microbiology, Sentralsykehuset i Vestfold HF, N-3116 Tonsberg, Norway.

Abstract

Persistence of tissue spirochetes of Borrelia burgdorferi as helices and round bodies (RBs)

explains many erythema-Lyme disease symptoms. Spirochete RBs (reproductive propagules also

called coccoid bodies, globular bodies, spherical bodies, granules, cysts, L-forms, sphaeroplasts, or

vesicles) are induced by environmental conditions unfavorable for growth. Viable, they grow,

move and reversibly convert into motile helices. Reversible pleiomorphy was recorded in at least

six spirochete genera (>12 species). Penicillin solution is one unfavorable condition that induces

RBs. This antibiotic that inhibits bacterial cell wall synthesis cures neither the second "Great

Imitator" (Lyme borreliosis) nor the first: syphilis. Molecular-microscopic techniques, in

principle, can detect in animals (insects, ticks, and mammals, including patients) helices and RBs

of live spirochetes. Genome sequences of B. burgdorferi and Treponema pallidum spirochetes

show absence of >75% of genes in comparison with their free-living relatives. Irreversible

integration of spirochetes at behavioral, metabolic, gene product and genetic levels into animal

tissue has been documented. Irreversible integration of spirochetes may severely impair

immunological response such that they persist undetected in tissue. We report in vitro inhibition

and destruction of B. burgdorferi (helices, RBs = "cysts") by the antibiotic Tigecycline (TG;

Wyeth), a glycylcycline protein-synthesis inhibitor (of both 30S and 70S ribosome subunits).

Studies of the pleiomorphic life history stages in response to TG of both B. burgdorferi and

Treponema pallidum in vivo and in vitro are strongly encouraged.

114: Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after

ceftriaxone treatment HETA YRJANAInen 1 , JUKKA HYTONen 1 , PAULIINA HARTIALA 1

, JARMO OKSI 2 and MATTI K. VILJANEN Departments of 1Medical Microbiology and

Immunology and 2 Medicine, University of Turku, Turku, Finland

Correspondence to Heta Yrjanainen, Department of Medical Microbiology and Immunology,

University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland. e-mail:

heta.yrjanainen@utu.fi Copyright Journal compilation c 2010 APMIS

We have earlier shown that Borrelia burgdorferi-infected and ceftriaxone-treated mice have

viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be

detected by culture. However, the niche of the persisting spirochetes remained unknown. In the

present study, we analyzed the tissues of B. burgdorferi-infected and ceftriaxone-treated mice by

culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via

81

intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i)

short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after

either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and

were killed 21 weeks after the treatment. All samples of ceftriaxone-treated mice were culture

negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA

was detected in the joints of 30–100% of the treated mice. In conclusion, these results combined

with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of

persisting B. burgdorferi in ceftriaxone-treated mice.

82

1

2

3

4

5

6

"We conclude that the treatment of Borrelia burgdorferi (Lyme Disease) with appropriate antibiotics for even more than 3

months may not always eradicate the spirochete." - Ann Med. 1999 Jun; 3,3:225-32.

Michael Parent Sept 1, 2010 12:10 PM

"The Lyme disease spirochete, can be recovered long after initial infection, even from antibiotic-treated patients,

indicating that it resists eradication by host defense mechanisms and antibiotics".- 1:20 J Infect Dis. 1992 Aug;166,2:440-4

Michael Parent Sept 1, 2010 12:15 PM

This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the

illness and establishes that it is of chronic infectious etiology." - Infection. 1998 Nov-Dec; 26,6:364-7

Michael Parent Sept 1, 2010 12:18 PM

"These data demonstrate that Lyme neuroborreliosis is a persistent infection."- Ann Neurol. 2001 Sep;50,3, :330-8

Michael Parent Sept 1, 2010 12:21 PM

"In one of the six analysed brain tissue specimens [from a patient that received more than six months of antibiotic

treatment prior to death, including two 3-week courses of IV ceftriaxone], B. burgdorferi DNA was detected by PCR." - Brain.

1996 Dec;119, Pt 6:2143-54

Michael Parent Sept 1, 2010 12:43 PM

"Two hundred seventy-seven patients with chronic Lyme disease were treated with tetracycline for 1 to 11 months,

These results support the use of longer courses of treatment in the management of patients with chronic Lyme

disease." - Clin Infect Dis. 1997 Jul;25 Suppl 1:S52-6.

Michael Parent Sept 1, 2010 12:12 PM

[soijuv]

Osalle borrelioosiin sairastuneista, erityisesti geneettisesti alttiille (HLA-DR4) henkilöille kehittyy krooninen niveltulehdus joka saattaa aiheuttaa esim. rustojen ja luiden eroosiota.


Live Borrelia burgdorferi Preferentially Activate Interleukin-1f. Gene Expression and Protein Synthesis over the lnterleukin-1 Receptor Antagonist


Laurie C. Miller, Sana Isa, Edouard Vannier, Kostis Georgilis, Allen C. Steere, and Charles A. Dinarello

Departments ofPediatrics and Medicine, New England Medical Center and

Tufts University School ofMedicine, Boston, Massachusetts 02111

Abstract

Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1,8 and the IL-1 receptor antagonist (IL-lra) in human pe-ripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1l@ than IL-la and sevenfold more IL-1l8 than IL-lra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1,B was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-lra was seen between these strains. The marked induction of IL-1iB was partially di-minished by heat-treatment and abrogated by sonication; IL-lra was not affected. This suggested that a membrane compo-nent(s) accounted for the preferential induction ofIL-1i6. How-ever, recombinant outer surface protein ,B induced little IL-1i6.

By 4 h after stimulation, B. burgdorferi induced sixfold more

IL-1,8 protein than LPS. In contrast to LPS-induced IL-l/i mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1A6 mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-lra mRNA peaked at 12 h, whereas LPS-induced IL-lra mRNA peaked at 9 h. IL-1,8 synthesis increased in response to increasing numbers ofspiro-chetes, whereas IL-lra synthesis did not. The preferential in-duction by B. burgdorferi ofIL-1i8 over IL-lra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion ofLyme arthritis. (J. Clin. Invest. 1992. 90:906-912.) Key words: an-tagonist * cytokine * lipopolysaccharide * Lyme arthritis

Introduction

Lyme disease is a multisystem disease caused by infection with the tick-borne spirochete Borrelia burgdorferi ( 1). Arthritis is a prominent feature ofthe disorder. Early in the illness, the spir-ochete probably spreads hematogenously to joints and may cause vague, migratory joint pain. After several months, many

Address reprint requests to Dr. Miller, Box 67, New England Medical

Center, 750 Washington Street, Boston, MA 021 1 1.

Receivedfor publication 3 October 1991 and in revisedform 21 February 1992.

patients begin to have brief attacks of arthritis in large joints (2). During the second and third years ofillness, a small subset ofpatients, primarily those with HLA-DR4 or HLA-DR2 (3, 4), develop chronic arthritis which may lead to erosion ofcarti-lage and bone (5). The synovial lesion in these patients is simi-lar to that in rheumatoid arthritis. The development ofchronic arthritis coincides with the appearance of a humoral immune response to two prominent outer surface proteins (Osp)' ofB. burgdorferi, Osp A and B, (6).

Cytokines, in particular interleukin-1 (IL-1) and tumor ne-crosis factor-a (TNF), are thought to contribute to the patho-genesis of the synovial lesion in rheumatoid arthritis (7-9). The potent biological effects ofthese cytokines are tightly regu-lated at many levels, including gene transcription, translation, protein processing and secretion, as well as naturally occurring inhibitors such as the IL-1 receptor antagonist (IL-1 ra) (10, 11) which occupies IL-1 receptors without inducing signal transduction.

Because Lyme arthritis is one of the few forms of chronic inflammatory arthritis in which the cause is known with cer-tainty (1), this illness presents a unique opportunity to study the direct effects ofthe pathogen on cytokine gene expression. In contrast to IgG, granulocyte/macrophage colony-stimulat-ing factor, and experimental endotoxemia which preferentially induce IL-1ra ( 12-15), we now demonstrate that live B. burg-dorferi preferentially induce synthesis of the agonist IL-1,8 compared to the antagonist IL-lra. This preferential induction was most marked in response to a low-passage, virulent prepa-ration of B. burgdorferi. Furthermore, the selective induction of IL- 1: appears to reside in a membrane component of B. burgdorferi whose activity is partially diminished by heat treat-ment but abrogated by sonication.

Methods

Sample collection. Whole heparinized blood was obtained from 23 healthy volunteers, age 22-40 yr, who had not ingested nonsteroidal anti-inflammatory drugs ( 16) within the previous 2 wk, and had no history or clinical symptoms suggestive ofLyme disease. The investiga-tion was approved by the Human Investigation Review Board ofNew England Medical Center. Informed consent was obtained from each donor.

All reagents and glassware were sterile and endotoxin free. Culture media and sterile water used to prepare Ficoll-Hypaque were passed through a hollow-fiber polysulfone capillary ultrafilter to remove mi-crobial products and endotoxin (17). RPMI 1640 was supplemented only with L-glutamine (10 mM) (Gibco Laboratories, Grand Island, NY) and 10 mM Hepes (M.A. Biologicals, Walkersville, MD). No antibiotics were included in any media used. In preliminary experi-

1. Abbreviations used in this paper: HP and LP, high and low passage (strains); Osp, outer surface protein; ra, receptor antagonist; TNF, tu-mor necrosis factor-a.

906 Miller et al.

J. Clin. Invest.

© The American Society for Clinical Investigation, Inc.

0021-9738/92/09/0906/07 $2.00

Volume 90, September 1992, 906-912

ments, the effects on cytokine induction ofheat-inactivated (56°C for 1 h) 10% FCS (Hyclone Laboratories, Inc., Logan, UT) and 1% human

AB serum were compared. Human serum alone induced IL-Ira, whereas no induction ofeither IL-I or IL-Ira was detected in the pres-ence of 10% FCS (data not shown). Therefore, for these experiments,

FCS was used as a culture supplement.

B. burgdorferi. B. burgdorferi were grown in Barbour-Stoenner-

Kelly medium at 32°C in a humidified atmosphere containing 5%

CO2. Three different strains were used: 297, isolated from the cerebro-spinal fluid of a patient with Lyme disease; N40, an Ixodes dammini tick midgut isolate; and G39/40, initially isolated from an L dammini tick, but passaged for many years in the laboratory. Both high-passage (HP) and low-passage (LP) preparations of strain 297 were used; only the LP 297 strain retained infectivity in mouse inoculation studies as previously reported (18). Organisms were used at log phase growth. The spirochetes were pelleted by centrifugation at 10,000 g for 20 min at room temperature, then washed three times in RPMI and resus-pended at the desired concentration. In some experiments, spirochetes killed by sonication or heat (57°C for 2 h) were used. After pelleting by centrifugation at 10,000 g for 20 min at 4°C and four washes in cold 0.01 M PBS/5 mM magnesium chloride (pH 7.3), organisms were sonicated on ice by eight 15-s pulses, setting 6, ofa cell sonicator (Bran-son Sonic Power Co., Danbury, CT). The supernatant was clarified by

centrifugation at 10,000 gfor 30 min at4°C; protein content was deter-mined by optical density at 280 nm in a spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, OH). Aliquots were stored at

-70°C. In other experiments, recombinant Osp B protein (a gift of

Drs. John Leong and Robert Kalish, New England Medical Center, Boston, MA) was used to stimulate cytokine production from PBMC. To investigate whether a soluble product of B. burgdorferi could con-tribute to the marked induction of IL-1I, the spirochetes were exten-sively washed, then cultured (25 x 106) in antibiotic-free RPMI for 24 h at 37°C or 32°C. After ultracentrifugation, the spirochete-free super-natant was filtered twice through 0.22-jim filters. The absence ofspiro-chetes or spirochetal fragments was verified by darkfield microscopy. The spirochete-conditioned media was then incubated with PBMC for 24 h and resulting cytokine production measured by radioimmunoas-says (RIAs).

PBMC cultures. Heparinized whole blood was fractionated by den-sity gradient centrifugation using Ficoll (Sigma Chemical Co., St. Louis, MO)-Hypaque (Winthrop Laboratories, New York, NY). Mononuclear cells were cultured (2.5 x 106 cells/ml) in 1.0 ml of complete RPMI with 10% heat-inactivated endotoxin-free FCS in 2.5-ml polypropylene tubes (Falcon Plastics, Oxnard, CA) with B. burg-dorferi or LPS (Escherichia coli 055:B5, Sigma Chemical Co.), or me-dium alone. Cultures were incubated for 24 h in a humidified, 5% CO2 atmosphere at 37°C. In experiments measuring total cytokine produc-

tion, cells were lysed by three freeze/thaw cycles (19). After centrifuga-tion at 400 gfor 15 min, the supernatants were removed, and the pellets containing cellular and spirochetal debris were discarded. In experi-ments measuring secreted vs. cell-associated cytokines, cell-free culture supernatant (containing secreted cytokines) was removed and replaced by an equal volume offresh media. The cell-containing tubes were then subjected to three freeze/thaw cycles to release cell-associated cyto-kines, centrifuged at 400 g for 15 min, and harvested as above.

Cvtokine RIAs. Specific RIAs for IL-1IB, TNF, IL-la, IL-6, and

IL-Ira were used (14, 19-22). lodination of IL-1,B and IL-Ira used the

Bolton-Hunter method; all other cytokines were iodinated by the chloramine-T method. Sensitivities for each of the RIAs was< 80 pg/ ml except for the IL-lra RIA which had a sensitivity of 300 pg/ml.

RNA isolation and Northern analysis. After incubation with B. buirgdorferi (five spirochetes per PBMC), LPS (10 ng/ml), or control media, total cellular RNA was extracted by lysis with 4 M guanidine isothiocyanate, followed by ultracentrifugation on a 5.7 M cesium chlo-ride cushion. Total RNA (20,g) was subjected to electrophoresis in

6.6% formaldehyde (Sigma Chemical Co.) and 1.2% agarose (Interna-tional Biotechnologies Inc., New Haven, CT), and then transferred to nylon membranes (Hybond-N, Amersham Corp., Arlington Heights,

IL) by capillary blotting. For quantitation ofmRNA levels, serial dilu-tions of RNA (2.50, 1.25, and 0.62 jug) were directly applied to nylon membranes using a filtration manifold apparatus (Schleicher & Schuell, Inc., Keene, NH). The membranes were exposed to short-wave UV light for 5 min to fix the RNA to the nylon matrix, and treated at 42°C for 2 h with prehybridization solution containing 10 mg/ml salmon sperm DNA. Membranes were then treated overnight with prehybridization solution containing 10 mg/ml ofsalmon sperm DNA and 32P-labeled nucleic acid probe. The probes used were a

1,075-bp fragment of human IL-I/3 precursor cDNA subcloned into pGEM2, 800-bp fragment of human IL-Ira precursor cDNA sub-cloned into pUC8, and the full length (2,000-bp) chicken ,B-actin cDNA subcloned in pGEM3. The DNA was labeled using [32P]dCTP (3,000 Ci/mmol, New England Nuclear, Boston, MA) and a random primed DNA labeling kit (Boehringer Mannheim, Mannheim, FRG).

After incubation, membranes were washed in 0.1% SDS, 1 X SSC at

42°C. Washed membranes were exposed overnight to Kodak KAR5

X-ray film (Eastman Kodak Co., Rochester, NY) at -70°C with an intensifying screen.

Statistical analysis. Total cytokine levels were expressed as mean±SEM of the indicated number ofdonors. Differences were ana-lyzed for significance using Student's t test for paired samples or analy-sis of variance using the computer program StatView (BrainPower, Inc., Calabasas, CA) on a Macintosh SE computer.

Results

Cytokine synthesis induced by B. burgdorferi. Live B. burgdor-feri (297 LP), sonicated B. burgdorferi, and LPS were com-pared for their ability to induce cytokine synthesis from PBMC (Fig. 1). Live B. burgdorferi induced 65±11 ng/ml of IL-1I. Large amounts of TNF were also induced (82±18 ng/ml), whereas production of IL-ia and IL-6 were stimulated to a lesser extent (12±2 and 2±1 ng/ml, respectively). Live B. burgdorferi induced significantly more IL-113 (P < 0.01) and TNF (P <0.001 ) than sonicated B. burgdorferi or LPS. Synthe-sis of IL-1 a, IL-6, and IL-1 ra induced by all three stimuli was similar. Unexpectedly, live B. burgdorferi stimulated fivefold more IL-113 than IL-1 a (P < 0.0001 ) and sevenfold more IL-1I3

10

60

44

2(

_ 0I**L-1

* *~~~~~~0IL-1cx

3- M TNF

'r t L

0 IL-6

0- E3 ~~~~~IL-1ira

10

n-

A. .

live sonicated LPS

B. burgdorferi

Figure 1. Induction of IL-1I3, IL-la, TNF, IL-6, and IL-lra by live B. burgdorferi (LP 297), sonicated B. burgdorferi, or LPS. PBMC from 23 donors were cultured for 24 h, and the resulting supernatants tested in cytokine-specific RIAs. Live B. burgdorferi induced signifi-cantly more IL-1IB (*P < 0.01) and TNF (**P < 0.001) than the amounts induced by sonicated B. burgdorferi or LPS.

Borrelia burgdorferi Preferentially Induce IL-1j3 over IL-I Receptor Antagonist 907

8(

**

U IL-1 p

150- [l IL-lra

U 100-

50

297 LP 297 HP N40 G39/40

Figure 2. Induction of IL-1I13 and IL-Ira by four preparations of B. burgdorferi, strains 297 (LP and HP), N40, and G39/40. Data are mean±SEM of six donors. Significantly more IL-1I3 was induced by the virulent strain 297 LP compared to the other three strains (** vs. *, P < 0.001I); no strain differences in IL-Ira induction were seen.

than IL-Ira (P < 0.001I). In contrast, the ratios of IL- 113 to IL- 1 a and IL-113l to IL-lIra induced by sonicated B. burgdorferi or LPS were close to 1.

Strain differences and Borrelial factors. Because of the markedly high levels of IL- 113 induced by LP 297 live B. burg-dorfieri, the cytokine-inducing capacity ofother strains was also tested. LP 297 live B. burgdorferi induced 148±16 ng/ml IL-I11, significantly more than the avirulent strains HP 297,

N40, or G39/40 (P < 0.001I) (Fig. 2). However, no difference was observed in the amount ofIL- 1ra induced by these strains.

To elucidate further the microbial factor(s) responsible for the preferential induction ofIL- 113l, four preparations oflive vs. heat-treated B. burgdorferi were compared. Heating signifi-cantly decreased induction of IL-1I3 by all four B. burgdorferi preparations (P < 0.05) (Fig. 3, left), whereas no differences were seen after heating in the induction of IL- Ira (Fig. 3, right).

The possibility that live B. burgdorferi, particularly LP 297, produced a soluble factor which could stimulate IL-113l produc-tion was investigated. Because BSK medium (which contains neopeptone, yeastolate, tryptone, gelatin, bovine serum albu-min, and rabbit serum) is itself a potent inducer of cytokines (20-30 ng/ml ofIL-1I3 or TNF), we prepared spirochete-con-ditioned RPMI to test as a stimulant for cytokine induction.

svv * ~~~~IL-1, O v

80 - O heated

W-J

- 20

n

w _

.0 40* ; i

0

20 -V-

F.4

V-J

Spirochete-conditioned RPMI was incubated with PBMC from four donors. Levels of IL-113, IL-Ira, and TNF by this conditioned media were below limits of detection by RIAs (data not shown).

Because immunoaffinity purified B. burgdorferi lipopro-teins (including a mixture ofOsp A and B) induce TNF (23), the contribution ofOsp B, a major outer surface protein ofB. burgdorferi, to the induction ofIL-I,B and IL-Ira was then ex-amined (Fig. 4). At the highest concentrations tested, Osp B induced 1.0±0.2 ng/ml of IL-1,B, whereas at lower concentra-tions, Osp B induced <0.10 ng/ml IL-1,B. In contrast, the highest concentration ofOsp B tested induced 4.1± 1.1 ng/ml IL-Ira, whereaslowerconcentrationsinduced 1-2 ng/ml. Poly-myxin B had no effect on the response to this stimulus. Thus, Osp B made little contribution to the marked induction of

IL-11, but could contribute to some extent to induction of

IL-Ira.

Secretion ofIL-1a, IL-1I1, and IL-i ra. The proportions of cell-associated vs. secreted IL-I a, IL-113, and IL-Ira induced by five spirochetes per PBMC (live LP 297) were compared to those induced by LPS (10 ng/ml) in PBMC of 18 donors. After stimulation with B. burgdorferi, 34±2% of total IL-la and

78±1% of total IL-11 were secreted. After LPS stimulation,

21±4% (P = 0.01) of IL-la and 63±3% of total IL-1I8 (P

< 0.001) were secreted. No differences were seen in the amounts or proportions ofsecreted IL-Ira induced by B. burg-dorferi or LPS (72±3% and 76±5%).

Dose-dependent effect ofB. burgdorferi on cytokineproduc-tion. Increasing the number of spirochetes (live LP 297) per PBMC (from 0.08 to 60 organisms per PBMC) resulted in a dose response for IL- 11 synthesis (Fig. 5, top). In contrast, increasing the number of spirochetes from 0.08 to 0.74 per PBMC resulted in a shallow dose-response curve for IL-Ira, which reached a plateau thereafter despite an increase to 60 spirochetes per PBMC. No dose response was seen for IL-Ia. The ratio of IL-13 to IL-Ira increased to 14:1 (inset). When LPS (0.001-1000 ng/ml) was used as a stimulus, production ofIL-1I3 and IL-Ira increased to maximal amounts in response to 10 ng/ml LPS, then decreased at higherconcentrations (Fig. 5, bottom). The ratio of IL-1,8 to IL-Ira increased to a maxi-mum at a concentration of 1 ng/ml LPS, then remained at a plateau (inset). IL-I a production increased linearly in re-sponse to LPS concentrations from 0.001 to 0.10 ng/ml, then

297LP 297 HP N40 G39/40 297 LP 297 HP N40 G39/40

Figure 3. Induction ofIL-1I3 (left) and IL-Ira (right) by paired samples offour preparations oflive or heated B. burgdorferi in PBMC from five donors. For all preparations of B. burgdorferi, heating significantly decreased the synthesis of IL-1B (all pairs, P < 0.05), but had no effect on induction ofIL-Ira.

908 Miller et al.

600

W-

5

4001 200

500 ng/ml 50 ng/ml 5 ng/ml 500 pg/ml

[Osp BI

Figure 4. Induction of IL-1:3 and IL-lra by recombinant Osp B pro-tein. Log dilutions ofrecombinant Osp B were incubated with PBMC of four donors (mean±SEM).

remained unchanged despite increasing the concentration of

LPS to 1,000 ng/ml.

Kinetics ofIL-IA, IL-la, and IL-Ira synthesis. No differ-ences were seen in the time of production of IL-la or IL-Ira induced by LP 297 B. burgdorferi or LPS (Fig. 6). In contrast, by 4 h, B. burgdorferi induced 24±5 ng/ml of IL- fl, whereas

40

0-1 P-

*^ 'o

30

20

10

LPS induced 2±3 ng/ml (P < 0.03). Compared to LPS, more striking differences in B. burgdorferi-induced IL-l1B protein synthesis were seen at 12 h (95±18 vs. 23±13 ng/ml, P < 0.01) and 24 h (109±6 vs. 19±8 ng/ml, P < 0.006). By 4 h, B. burgdorferi induced the synthesis of more than fivefold more

IL-iI3 than IL-Ira, whereas no difference was seen in LPS-in-duced synthesis of IL- 3l and IL-Ira. For both stimulants, ra-tios of IL- to IL- ra remained unchanged at 12 and 24 h.

Accumulation ofmRNAfor IL-JI andIL-Ira. The kinetics ofsteady-state IL- 1l3 and IL- ra mRNA induced by LP 297 B. burgdorferi or LPS were compared by Northern and dot blot analysis. Levels ofmRNA for IL- (l: and IL- ra were nondetec-table in unstimulated cells. The time course for IL- 13 mRNA levels were similar during the first 4 h after B. burgdorferi or

LPS stimulation; IL- 1:3 mRNA accumulation reached a peak at 4 h (Fig. 7). Protein production showed eightfold more IL- 13 stimulated by B. burgdorferi compared to LPS at 4 h ( 17 vs. 2.1 ng/ml). In a longer time course study, IL-1(3 mRNA reached a peak at 3 h and declined until 12 h after stimulation by B. burgdorferi or LPS (Fig. 8 A). However, IL-1B mRNA levels again increased 18 h after exposure to B. burgdorferi, and remained elevated at 24 h. In contrast, LPS-induced IL-1,B mRNA continued to decline until 24 h. B. burgdorferi-in-duced IL- 1: protein continued to rise at 24 h; LPS-induced IL-1O protein reached near-peak levels by 6 h.

.01 .1 1 10 100 spirochetes/PBMC

15

10.-N

P-

C1)

.001 .01 .1 1

LPS (ng/ml)

Figure 5. Dose-dependent effect of B. burgdor-feri on the synthesis ofIL-lIB. (Top) Synthesis

of IL- I,, IL- a, and IL- I ra in response to in-

IL-la creasing numbers of live spirochetes per

PBMC. (Bottom) Synthesis of IL- Id, IL- a, and IL- I ra in response to increasing concen-trations of LPS. Data are mean±SEM of six

10 100 1000 donors. Insets show the ratios of IL-1,B to IL-

Ira over these different concentrations of stimuli.

Borrelia burgdorferi Preferentially Induce IL-1f3 over IL-I Receptor Antagonist 909

M IL-1i

_ IL-lra

[L-N

A

I BILI3I I I I

0 4 8 12 16 20 24 C

Time (h)

120. 100.

80 .

60.

40.

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cn_

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w *

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Figure 7. Early kinetics of IL- 1,B protein and mRNA induced by LPS and B. burgdorferi. (A) The synthesis ofIL-1O protein at 30 min and

1, 2, and 4 h. (B) The corresponding IL-lI3 mRNA. (C) The same blot probed for (-actin.

Time (h)

Cd

"4

Time (h)

Figure 6. Kinetics of production of IL-la, IL-113, and IL-Ira in re-sponse to live B. burgdorferi or LPS. Data (mean±SEM) for three donors are shown. No differences were seen in the kinetics of pro-duction of IL-lca or IL-Ira, whereas significantly more IL-I was in-duced by live B. burgdorferi at 4, 12, and 24 h than LPS (*P < 0.03, **P < 0.01, and ***P < 0.006).

Similar to IL-1( mRNA, the accumulation of IL-1ra mRNA induced by B. burgdorferi or LPS were comparable until 4 h (data not shown). Thereafter, LPS-induced IL-1ra

mRNA continued to increase until reaching peak levels at 9 h, then decreasing gradually until 24 h (Fig. 8 B). In contrast, B. burgdorferi-induced IL- 1ra mRNA peaked at 12 h, and then gradually declined. No difference in IL- 1 ra protein induced by the two stimuli was seen over 24 h.

Discussion

By using specific RIAs, we have shown a remarkable preferen-tial induction ofIL- 1l: and TNF by B. burgdorferi. Unlike LPS, live B. burgdorferi preferentially induced IL- over the IL-I ra, as well as IL-Ia. This selectivity was most dramatic when the virulent strain LP297 was used. Habicht et al. (24) reported that B. burgdorferi stimulate marked IL-1 biologic activity from murine macrophages, P388Dl cells, and human PBMC. However, they used nonspecific fibroblast and T cell prolifera-tion assays which do not distinguish between IL- 1(3 and IL-I a, and may respond to other cytokines such as IL-2, IL-4, IL-6, or TNF, alone or in combination (25-27). Furthermore, it is known that the bioassays for IL- 1 are affected by cytokine an-tagonists such as IL- 1 ra and soluble cytokine receptors (28-30).

The present studies show that the component(s) ofB. burg-dorferi associated with the preferential induction of IL- 1l: is heat resistant but eliminated during the sonication procedure. We were unable to demonstrate that spirochete-conditioned

910 Miller et al.

A

B

B. burgdorfersf

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*/ ~~~~LPS

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LPS B. burgdorferi

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* ., 1.25

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X * -S@- - *------2.50

Figure 8. Late kinetics of IL-1O protein and mRNA induced by LPS and B. burgdorferi. (A) The synthesis ofIL-1O protein at 3, 6, 9, 12, 18, and 24 h, with the corresponding levels of IL- 1O mRNA shown by dilutional analysis below. (B) IL-Ira protein and mRNA induced by LPS and B. burgdorferi. (C) The same blot probed for /-actin.

medium contained soluble borrelial product(s) which induced IL-1:. Therefore, we conclude that the component(s) of B. burgdorferi responsible for IL-1/3 induction resides in the cell wall, and is not readily secreted, but rather is associated with the structural integrity ofthe wall. This suggests a requirement for intact cells. Although one could speculate that such a puta-tive spirochetal wall structure satisfies a simple "particulate size" requirement for monocyte stimulation, the data on strain and passage differences suggest another mechanism. The com-ponent(s) which preferentially trigger IL-1/3 gene expression and protein synthesis may be a complex ofcomponents which varies among strains and/or is lost with repeated in vitro pas-sage ofB. burgdorferi. Extensive passage of strain 297 renders the spirochete noninfectious ( 18) and as we show, its ability to induce high levels of IL-1/3. Preferential induction of IL-i over IL- Ira may relate to infectivity and production ofdisease in vivo.

The onset of chronic arthritis in Lyme disease coincides with the appearance of a humoral immune response to Osp A and Osp B (6). Furthermore, Osp B may be lost during serial laboratory passage ofB. burgdorferi; in some strains, this corre-

sponds to loss of infectivity in mice (31, 32). We found that recombinant Osp B induced little IL-1o.

The relative synthesis of the related but distinct genes IL-

13, IL-1 a, and IL-1 ra is stimulus dependent ( 12, 33, 34). Solu-ble stimuli such as LPS, phytohemagglutinin, and toxic shock syndrome toxin-1 induce nearly equal amounts of IL-l1a, IL-1/, and TNF (5-15 ng/ml) with somewhat more IL-la pro-duced by most donors (35, 36). Particulate stimuli such as Staphylococcus epidermidis induce threefold more IL-1O than

IL-1a. However, there appears to be more IL-1ra than IL-1: synthesized by PBMC using various stimuli (37). Soluble IgG or granulocyte/macrophage colony-stimulating factor induce IL-lra but not IL-la or IL-l1/ ( 12-14). More IL-lra than IL-I is found in children with systemicjuvenile rheumatoid arthritis (38). In experimental human endotoxemia, at least 100-fold more IL-Ira is found in the circulation than IL-1/3( 15). Thus, live B. burgdorferi, ofany stimuli studied to date, appears to be unique in its marked preferential activation of IL-1/. In our dose-response experiments, increasing amounts of LPS in-duced parallel increases in IL-1/ and IL-1 ra. In contrast, B. burgdorferi induced larger amounts of the agonist IL- I than the antagonist IL-1 ra. By 4 h after stimulation with B. burgdor-feri, IL-1/ protein exceeded IL-1 ra protein by greater than five-fold. Some reports suggest that a 10-50 molar excess ofIL-Ira is needed to inhibit 50% of IL-I binding to T cells (39). Changes in the relative proportions of cytokines and their an-tagonists in vivo at different sites in the body or different times after infection clearly has important biological implications.

The kinetics of IL-1/ mRNA production induced by B. burgdorferi resemble the pattern observed with LPS (40) within the first 12 h after stimulation. Nonetheless, increased protein production is seen as early as 4 h after stimulation, suggesting that B. burgdorferi enhances translational efficiency ofIL- /3 rather than affecting transcription. The second peak of IL-1/ mRNA induced by B. burgdorferi may reflect failure of B. burgdorferi to induce a repressor which down-regulates or destabilizes this mRNA, as described after LPS or PMA stimu-lation (41 ).

We believe our findings are relevant to the pathogenesis of Lyme disease. Early in the illness, low-grade fever, malaise, and marked fatigue are common symptoms, which may be me-diated by IL-I and other cytokines (42). However, the specific immune response to the organism seems to be suppressed early in the illness, and patients commonly experience only vague joint pain despite the presumed early spread of B. burgdorferi to synovial tissue. After several months, as the specific cellular and humoral immune responses expand to multiple spiroche-tal polypeptides, patients have briefattacks ofarthritis in large

joints (43).

Alterations in the balance of cytokines and their antagonists are likely involved in the sudden turning on and offofthe inflammatory response in these patients, but the mechanisms controlling such alterations are not yet known. At the time of maximal expansion of the immune response, which usually occurs during the second or third years ofillness, a genetically susceptible subset of patients, particularly those with HLA-DR4, may develop chronic arthritis (4). As in rheumatoid arthritis, erosion of cartilage and bone with elevated levels of collagenase and PGE2 may occur (5). The preferential induc-tion by B. blurgdorferi ofthe agonist IL-1/ , over the antagonist IL-1 ra, may contribute to the destructive lesion of Lyme ar-thritis.


Borrelia burgdorferi Preferentially Induce IL-ifi over IL-1 Receptor Antagonist 911

..LL

The contributions ofB. Reinhardt, J. Mitchell, Dr. J. G. Schaller, and

Dr. M. S. Klempner are gratefully acknowledged. This work was sup-ported in part by the Charles A. Hood Foundation and Massachusetts Arthritis Foundation (Dr. Miller), and National Institutes of Health grants AR-20358 andAR-40576 (Dr. Steere) and AI-15614 (Dr. Dina-rello).

References

1. Steere, A. C., R. L. Grodzicki, A. N. Komblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista. 1983. The spiro-chetal etiology ofLyme disease. N. Engl. J. Med. 308:733-40.

2. Steere, A. C., R. T. Schoen, and E. Taylor. 1987. The clinical evolution of Lyme arthritis. Ann. Intern. Med. 107:725-3 1.

3. Steere, A., A. Gibosky, M. Patarroyo, R. Winchester, J. Hardin, and S. Malawista. 1979. Chronic Lyme arthritis: clinical and immunogenetic differen-tiation from rheumatoid arthritis. Ann. Intern. Med. 90:896-901.

4. Steere, A., E. Dwyer, and R. Winchester. 1990. Association of chronic Lyme arthritis with HLA-DR4 and HLA-DR2 alleles. N. Engi. J. Med. 323:219-223.

5. Steere, A., C. Brinckerhoff, D. Miller, H. Drinker, E. Harris, and S. Mala-wista. 1980. Elevated levels ofcollagenase and prostaglandin E2 from synovium associated with erosion of cartilage and bone in a patient with chronic Lyme arthritis. Arthritis Rheum. 23:591-9.

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43. Steere, A. 1989. Lyme disease. N.

[soijuv]

Toistuva kurkunpäähermon halvaus

Rev Med Interne. 2010 Jan 13; [Epub ahead of print]
[Recurrent nerve palsy due to Lyme disease: Report of two cases.]

[Article in French]

Martzolff L, Bouhala M, Dukic R, Saraceni O, Wilhelm JM, Bombaron P, Kieffer P.

Service de medecine interne et endocrinologie, hopital Emile-Muller, 87, avenue
d'Altkirch, BP 1070, Mulhouse cedex, France.

INTRODUCTION: Neuroborreliosis can be a difficult diagnosis which requires
epidemiologic, clinical and biologic arguments. CASE REPORTS: We report two
patients who presented with a recurrent laryngeal nerve palsy with positive Lyme
serology and favorable outcome after antibiotic therapy. In one case, a
lymphocytic meningitis with intrathecal production of specific antibodies was
evidenced. CONCLUSION: Recurrent laryngeal nerve palsy is an uncommon
manifestation of neuroborreliosis. Lyme serology is an important tool when
neurologic disorder occurs because of an atypical course of Lyme disease.
Copyright (c) 2009 Societe nationale francaise de medecine interne (SNFMI).
Published by Elsevier SAS. All rights reserved.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20079561 [PubMed - as supplied by publisher]

[soijuv]

Osa borreliabakteereista muuntuu antibiooteille vastustuskykyisiksi bakteereiksi. Se saattaa selittää hoitojen epäonnistumisen ja taudin kroonistumisen.


Abstract Title: Persister Formation in Borrelia burgdorferi

Author Block: B. Sharma, A. Brown, K. Lewis;
Northeastern Univ., Boston, MA

Presentation Number: 1047

Poster Board Number: 1047

Keywords: Borrelia burgdorferi,persisters,Lyme disease

Abstract: Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne infection in North America and Europe. In 10-20% of cases, patients develop chronic Lyme disease after completing antibiotic treatment. The cause of these chronic symptoms is, however, poorly understood. We have previously shown that high-persister mutants are selected for over the course of relapsing chronic infections of Pseudomonas aeruginosa in cystic fibrosis patients and Candida albicans in oral thrush patients. It seems likely that these high persister mutants may contribute to the recalcitrance of the infection. Persister cells are drug-tolerant phenotypic variants of normal cells and may cause recurrent bacterial infections by resuming growth once antibiotic treatment has ceased. We hypothesize that persister cells play a role in the treatment failure that leads to chronic Lyme disease.

Here, using time-dependent and dose dependent survival assays, we show that B. burgdorferi forms persister cells to the antibiotics commonly used for treatment of Lyme disease. Our results indicate that in a B.burgdorferi population, 0.001% to 1% of the cells can survive lethal doses of various antibiotics in vitro.

These persister cells may contribute to treatment failure in chronic Lyme patients. Future experiments are aimed at screening for a better antimicrobial therapy to eradicate persisters in B. burgdorferi.

[soijuv]

Tri Liegnerin lausunto kroonisesta/kuolemaan johtavasta Borrelioosista.

http://www.aapsonline.org/testimony/liegtest.htm

Kenneth B. Liegner, M.D., P.C.
Internal & Critical Care Medicine
Lyme Borreliosis & Related Disorders
8 Barnard Road
Armonk, New York 10504
273-2121

REMARKS BEFORE THE NYS ASSEMBLY COMMITTEE ON HEALTH, CHAIRED BY RICHARD GOTTFRIED, ALBANY, NOVEMBER 27, 2001.
Chronic Lyme Disease

I wish to take the opportunity to thank Richard Gottfried and the Assembly Committee on Health for convening this hearing.

I'd like to briefly tell you of my background. I am a born and bred New Yorker and I love the beautiful state of New York. I graduated from Columbia College in New York City and from New York Medical College in Valhalla, New York where I was elected to Alpha Omega Alpha Honor Medical Society in my Junior year. I graduated second in my class and was awarded the Conrad Engerud Thuraldsen Prize in Anatomy. I am board certified in Internal Medicine and was trained and certified in Critical Care Medicine. During the course of my training I did a full year of Anatomic Pathology and performed 20-30 complete autopsies.

I went into private practice in Armonk in 1985, not realizing at the time that I had plunked myself down in the midst of what was to be a burgeoning epidemic of Lyme disease. Like many other internists, I began seeing patients with Lyme disease.

Lyme disease is a tick-transmissible infectious disease caused by the spirochete, Borrelia burgdorferi. Best known for the "bull's eye" rash, swollen knee, and Bell's palsy, it is turning out to be a very complex infectious disease with systemic effects as well as varied manifestations affecting individual organ systems such as the skin, visual and auditory systems, joints, nervous system and heart. To give you a sense of the wide scope of manifestations associated with the illness, please refer to Document 1, an outline of a talk I gave in Chicago in 1998.

The range of manifestations associated with Lyme disease has been continually expanding. It can be difficult to distinguish Lyme disease from a number of other disorders which it can greatly resemble including rheumatoid arthritis, multiple sclerosis, lupus, the chronic fatigue syndrome, fibromyalgia, Lou Gehrig's disease, Alzheimer's disease and others.

Many persons who prove to have Lyme disease have no recollection of a tick attachment and estimates varying between 20 and 80% of those with the disease having recollection of the rash associated with the illness. Testing for Lyme disease can sometimes be quite clear cut and conclusive but false positive as well as false negative test results may occur. Thus the treating physician's judgment in making a clinical diagnosis is crucial. Over-reliance on imperfect tests can result in failure to treat true Lyme disease when it is present, which can have disastrous consequences for patients.

Patients who test negative on standard tests but who really have the disease (the seronegative subset) may be most ill. There is ample precedent for variable expression of severity in infectious diseases depending on a patient's immune response. In leprosy, for example, patients having a vigorous immune response are able to contain the illness with resultant mild disease. Those with an ineffective immune response have more devastating leprosy, with deformity and loss of appendages.

For the most part, academic medical centers have restricted their studies to the seropositive subset only. At the same time assays available in certain research centers have been able to demonstrate active infection in patients who test negative on standard tests; such testing is not generally available to practicing physicians.

When I first started seeing patients with Lyme disease in the mid-1980s I found they didn't always behave the way the books said they should. Some would respond favorably to antibiotic treatment but when treatment was stopped they relapsed only to respond again to reinstitution of antibiotic therapy and relapse once again when treatment was stopped. It began to dawn on me that this might be a chronic infection that could be treated but perhaps not cured with antibiotics. Because of this I gradually lengthened the duration of treatment I offered patients.

Many reputable workers from around the world have independently proven that the Lyme disease organism is capable of surviving in both animal and human hosts despite application of antibiotic therapy, including intensive intravenous antibiotics (Documents 2,3,4,5,6,7). It seems in some cases the infection can endure for months or years and even the natural life of the host despite application of antimicrobial treatment.

Such chronic infection can induce a wide variety of pathologic manifestations both directly and mediated through the immune response. Antibiotic treatment may be viewed, then, as suppressive rather than truly curative in such cases. The outcome of the infection depends on the patients immunogenetics, the virulence of the strain of borrelia, and effects of other tick-borne co-infections. Individual response is very variable and blanket statements about what constitutes sufficient treatment can not be made. Some individuals may be cured with treatment. As in other spirochetal diseases, some may enter a latent phase of the infection which does not necessarily require continuation of treatment. However, relapse and need for re-treatment needs is a possibility which must be borne in mind and persons who have had Lyme disease need to be followed carefully over long periods of time.

Due to my background and training I began to see the more complex and more seriously ill persons with chronic and neurologic illness. I would like to share with you the case of one of the most seriously ill patients I have cared for. The case exemplifies some of the problems associated with diagnosis and treatment of Lyme disease. Also I am presenting the case succinctly in a few minutes. To have a more in depth understanding of the case please refer to my article on chronic meningoencephalomyelitis which presents the case in detail (Document 7, Case 2). The patient's wife, who will be a speaker later today, has given me consent to disclose his identity.

Case of Martin Eisenhardt:

A 61 year old outdoorsman, a resident of Cairo, NY, developed a grapefruit-sized red rash on one thigh Fall of 1985. Its significance was not appreciated and no treatment was given. The following Winter and Spring he developed a serious neurologic symptoms and spinal fluid examination showed lymphocytic meningitis. A Lyme ELISA was negative for which reason the possibility of Lyme disease was discounted. He was diagnosed with "vasculitis" (an inflammation of blood vessels) and treated with prednisone and immunosuppressive (cancer) chemotherapy agents with progressive deterioration to just above a vegetative state. In 1988 he had a positive Lyme ELISA at SUNY Stony Brook and was treated with two weeks of intramuscular Rocephin (ceftriaxone) with slight benefit. In 1992 he was transferred to Northern Westchester Hospital Center in Mount Kisco, New York in order to be re-evaluated by me. He was found to be in status epilepticus (continuous seizures). (Slide 1: CT scan of brain). Based on his history I felt it likely that he had chronic neurologic Lyme disease. I had to defend my decision to admit him to my hospital and treat him (Document 8). Initial laboratory tests were inconclusive. One of my colleagues expressed to me his opinion that the best thing that could happen to the patient was to die and have an autopsy. His wife certainly didn't feel that way. She felt he deserved a chance to be treated, and I agreed. He was treated for one month with daily intravenous Rocephin and for about one year with once weekly "pulse" Claforan (cefotaxime).

Although he was already very severely brain damaged when he was placed under my care he still improved modestly as vouched for by Greene County Public Health Nursing Service which had cared for him for years (Document 9). Treatment with intravenous antibiotics was discontinued in the late Spring of 1993 after which he was treated with oral antibiotics. He succumbed to his illness July 1993. A complete autopsy was performed by Jeff Hubbard, M.D. of Bender Laboratory, right here in Albany. Here are photographs from the autopsy showing a picture of the cut brain showing hydrocephalus (loss of brain substance)(Slide 2) and a microscopic view of brain tissue with florid chronic meningoencephalomyelitis (Slide 3). Electron microscopy of brain tissue showed structures compatible with borreliae (Slide 4); PCR of brain was positive for detection of the DNA of the Lyme organism (Slide 5) [both by Dagmar Hulinska, Ph.D. of the Borrelia Reference Laboratory of the Czech Republic], and his spinal fluid was very strongly positive for detection of OspA antigen and Lyme-specific immune complexes at neurologist Patricia Coyle's research lab at the State University of New York at Stony Brook, indicating the presence of active infection despite the treatment he had received.

This patient's illness was extremely severe, however it is not an isolated case. I have dealt over the past 15 years with many other very seriously ill patients whose nervous systems are being damaged or destroyed by the infection.

I have had a number of other fatalities due directly to Lyme disease in my practice, including in a 7 year-old child (see Document 10). This child was improving on intravenous antibiotic treatment. A successful appeal to Cigna's IntraCorp review physician resulted in extension of treatment from 3 to 6 months (Document 11). When the physician reviewer (following CIGNA's corporate policies) denied further intravenous treatment, uncontrollable status epilepticus recurred despite maximal anti-convulsant therapy. She died within one month of cessation of intravenous antibiotic treatment.

The world literature also contains numerous reports of fatal outcomes in Lyme disease (see References), but this information has not received emphasis and public health authorities (Document 12), insurance companies and their paid physician consultants insist that Lyme disease is not a fatal illness.

I should hasten to add that in contrast to these devastating cases, when Lyme disease is correctly diagnosed and treated appropriately intensively before irreversible neurologic injury has occurred, recovery to a greater or less extent is the rule. I have had innumerable cases in my practice where intensive treatment has restored very compromised individuals to normal or near normal status where they can enjoy a satisfactory and satisfying quality of life as opposed to one of utter misery and suffering.

It is vital that treating physicians be enabled to exercise their individual clinical judgment as to choice and method of administration of antimicrobial agent (e.g. oral, intramuscular or intravenous) and duration of treatment unencumbered by third party interference.

The controversies about the nature of Lyme disease and what constitutes appropriate treatment for it need to be put into perspective. At the turn of the 20th century, bitter debate raged within the medical profession about another spirochetal disease, syphilis. There was bitter controversy over what constituted appropriate treatment, duration of treatment, and criteria for cure. There was a chaos of treatment regimens and private preferences (Document 13). It has taken medical science more than 400 years to understand syphilis as a chronic multi-system infectious disease which evolves over time. We are but 25 years into understanding the spirochetal infection known as Lyme disease.

In syphilis it is a well-accepted dictum that progressing neurologic symptoms in patients with a prior diagnosis of syphilis and in the absence of a clear alternate cause for symptoms should be treated further with antibiotics, regardless of test results. There is ample precedence in the syphilis literature for seronegativity and chronic persistent infection despite prior application of antibiotic therapy (see Documents 14,15).

A major difference between the syphilis controversy of the turn of the 20th Century and the Lyme controversy at the turn of the 21th Century is the existence and influence of the insurance industry. Metropolitan Life Insurance Company had an important formative role in the creation of the National Institutes of Health (Document 16). This raises the issue of possible ongoing undue influence of the insurance industry in setting national public health priorities.

Spring of 2000, the Infectious Diseases Society of America (IDSA) published Practice Guidelines for the Treatment of Lyme Disease. It asserted that there was no significant evidence that 'chronic Lyme disease' exists as a separate diagnostic entity and that there is no role for treatment with antibiotics beyond one or at most two months for any case of Lyme disease (Document 17). Insurance companies have "glommed on" to these guidelines and routinely deny reimbursement to insureds for oral and intravenous antibiotic treatment extending beyond 60 days. Insurance company pharmacy benefit managers keep track of physician prescribing patterns. Physicians whose prescribing patterns do not conform to IDSA guidelines have been targeted and reported to State Departments of Health for investigations for medical misconduct. It is a "no lose" proposition for the insurance industry. This enmeshes such physicians in a costly, stressful and time consuming administrative process that pits them individually against the vast power and resources of the State and jeopardizes their professional reputations, practices and financial solvency. Even if they win, they lose. It sends a chilling message to rank and file physicians and undermines physicians' professional autonomy. Not surprisingly, persons with chronic Lyme disease are having increasing difficulty finding any physician anywhere willing to see them.

At the very least the IDSA guidelines are highly scientifically biased; at worst they may be frankly fraudulent. The document omits scores of articles from the worldwide peer-reviewed literature demonstrating the reality of chronic persistent infection despite prior antibiotic treatment. No clinicians who actually care for the majority of patients having chronic Lyme disease were invited to participate in drafting the guidelines. The sole academician in the IDSA known to advocate the existence of chronic Lyme disease was purged from the committee drafting the document. That some committee members involved in the creation of the guidelines had long-standing financial relationships with the insurance industry was not disclosed in the publication. Lack of disclosure of potential conflicts of interest in peer-reviewed research and publications has been the subject of a recent editorial in the Journal of the American Medical Association, decrying such practices on grounds of medical ethics (Document 18).

The single greatest obstacle to badly needed progress in development of improved methods of diagnosis and treatment for Lyme disease is the chronic persistent denial of chronic persistent infection in the illness. Such denial, in the face of so much objective evidence to the contrary, must be viewed as a type of social pathology.

The question has been raised, what is tertiary syphilis without the spirochetal bacterium Treponema pallidum? Similarly one ought to ask, what is chronic Lyme disease without the spirochete Borrelia burgdorferi?

What is being promoted as the "standard of care" for chronic Lyme disease is medical neglect, a vast de facto and unintended Tuskegee experiment, whose hapless subjects are your constituents.

Ladies and gentlemen of the committee, you can do something about it(Document 19).

Thank you for your attention.

Kenneth B. Liegner, M.D.



Summary of Documents:

Document 1

Liegner KB. Disseminated Lyme Disease: Diagnosis & Treatment(Talk Outline). Lyme Disease Foundation, Illinois Department of Health, Illinois Academy of Family Practice & Cook County Department of Health. Chicago March 23, 1998.

Document 2

Liegner KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L. Recurrent Erythema Migrans Despite Extended Antibiotic Treatment with Minocycline in a Patient with Persisting B. burgdorferi Infection. J Amer Acad Derm 1993;28:312-4.

Document 3

Straubinger RK. PCR-Based Quantification of Borrelia burgdorferi Organisms in Canine Tissues over a 500-Day Postinfection Period. J Clin Microbiol 2000;38:2191-2199.

Document 4

Liegner KB, Rosenkilde CE, Campbell GL, Quan TJ, Dennis DT. Culture-confirmed Treatment Failure of Cefotaxime and Minocycline in a Case of Lyme Meningoencephalomyelitis in the United States. Program and Abstracts. V International Conference on Lyme Borreliosis. Abstr. 63 P. A11, Arlington, VA. May/June 1992.

Document 5

Liegner KB, Agricola MD, Bayer ME, Duray PH. Chronic Lyme Disease (CLD): A Costly Dilemma. Program and Abstracts, VI International Conference on Lyme Borreliosis. Abstr. P012M. Bologna, Italy, June 19-22, 1994.

Document 6

Liegner KB. Lyme Disease: The Sensible Pursuit of Answers. (Guest Commentary). J Clin Microbiol 1993;31:1961-1963.

Document 7

Liegner KB, Duray P, Agricola M, Rosenkilde C, Yannuzzi L, Ziska M, Tilton R, Hulinska D, Hubbard J, Fallon B. Lyme Disease and the Clinical Spectrum of Antibiotic-Responsive Chronic Meningoencephalomyelitides. J Spirochetal and Tick-borne Dis 1997;4:61-73.

Document 8

Liegner KB. Letter to Vice President for Medical Affairs, Northern Westchester Hospital Center defending admission and treatment of Mr. Eisenhardt, August 4, 1992.

Document 9

Letter from Greene County Public Health Nursing Service regarding Martin Eisenhardt, status in response to treatment.

Document 10

Liegner KB & Jones CR. Fatal progressive encephalitis following an untreated deer tick attachment in a 7 year-old Fairfield County, Connecticut child. [Abstract] VIII International Conference on Lyme Disease and other Emerging Tick-borne Diseases, Munich, Germany, June 1999.

Document 11

Liegner KB. Letter to CIGNA IntraCorp review physician.

Document 12

CDC, Division of Vector-borne Infectious Diseases. Lyme Disease Home Page: "Lyme disease is rarely, if ever, fatal."

Document 13

Brandt AM. No Magic Bullet. A Social History of Venereal Disease in the United States Since 1880. Oxford University Press, 1985.

Document 14

Hotson JR. Modern Neurosyphilis: A Partially Treated Chronic Meningitis(Medical Progress). West J Med 1981; 135:191-200.

Document 15

Dibbern DA & Ray SC. Recrudescence of Treated Neurosyphilis in a Patient With Human Immunodeficiency Virus. Mayo Clin Proc 1999;74:53-56.

Document 16

Harden VA. Inventing the NIH. Federal Biomedical Research Policy, 1887-1937. The Johns Hopkins University Press.Baltimore.1986.

Document 17

Wormser GP, et. al. Practice Guidelines for the Treatment of Lyme Disease. Guidelines from the Infectious Diseases Society of America. Clin Infect Dis 2000;31:1-14.

Document 18

Davidoff et. al. Sponsorship, Authorship, and Accountability.[Editorial].JAMA 2001;286:1232-1234.

Document 19

Liegner KB.(Preface) in Murray P. The Widening Circle. A Lyme Pioneer Tells Her Story. St. Martin's Press, New York 1996.



REFERENCES: CHRONIC AND NEUROLOGIC LYME DISEASE

(INCLUDING FATAL CASES)

Straubinger RK. PCR-Based Quantification of Borrelia burgdorferi Organisms in Canine Tissues over a 500-Day Postinfection Period. J Clin Microbiol 2000;38:2191-2199.

Liegner KB. Lyme Disease: The Sensible Pursuit of Answers (Guest Commentary). J Clin Microbiol 31:1961-1963, 1993.

Weder B, Wiedersheim P, Matter L, Steck A, Otto F. Chronic progressive neurological involvement in Borrelia burgdorferi infection. J Neurology 1987;234:40-43.

Ackermann R, Gollmer E, Rehse-Kupper B. Progressive Borrelien-Enzephalomyelitis. Chronische Manifestation der Erythema-migrans Krankheit am Nervensystem. Dtsh. Med. Wochenschr.110(26)(1985)1039-1042.

Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross B, Baumann A, Prokop J. Survival of Borrelia burgdorferi in Antibiotically Treated Patients with Lyme borreliosis. Infection 1989;17:355-359.

Liegner KB, Shapiro JR, Ramsay D, Halperin AJ, Hogrefe W, Kong L. Recurrent erythema migrans despite extended antibiotic treatment with minocycline in a patient with persisting Borrelia burgdorferi infection. J Amer Acad Derm 1993;28:312-4.

Lawrence C, Lipton RB, Lowy FD, Coyle PK. Seronegative Chronic Relapsing Neuroborreliosis. Eur Neurol 1995;35:113-117.

Liegner KB, Duray P, Agricola M, Rosenkilde C, Yannuzzi L, Ziska M, Tilton R, Hulinska D, Hubbard J, Fallon B. Lyme Disease and the Clinical Spectrum of Antibiotic-Responsive Chronic Meningoencephalomyelitides. J Spirochetal and Tick-borne Dis 1997;4:61-73.

Liegner KB & Jones CR. Fatal progressive encephalitis following an untreated deer tick attachment in a 7 year-old Fairfield County, Connecticut child. [Abstract] VIII International Conference on Lyme Disease and other Emerging Tick-borne Diseases, Munich, Germany, June 1999.

Fallon BA, Tager F, Fein L, Liegner K, Keilp J, Weiss N, Liebowitz MR. Repeated Antibiotic Treatment in Chronic Lyme Disease. J Spirochetal and Tick-borne Dis 1999;6:94-102.

Miklossy J, Kuntzer T, Bogousslavsky J, Regli F, Janzer RC. Meningovascular form of neuroborreliosis: Similarities between neuropathological findings in a case of Lyme disease and those occurring in tertiary Neurosyphilis. Acta Neuro Pathol 1990;80:568-572.

Oksi J, Uksila J, Marjamaki M, Nikoskelainen J, Viljanen MK. Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis. J Clin Microbiol 1995;33:2304-15.

Bertrand E, Szpak GM, Pilkowski E, Habib N, Lipczynska-Lojkowska W, Rudnicka A, Tylewska-Wierzbanowska S, Kulczycki J. Central Nervous System Infection Caused by Borrelia burgdorferi. Clinico-Pathological Correlation of Three Post-Mortem Cases. Folia Neuropathol 1999;37:43-51.

Haass Anton. Lyme Neuroborreliosis. Current Opinion in Neurology. 1998;11:253-258.

Kohler J, Kern U, Kasper J, Rhese-Kupper B, Thoden U. Chronic central nervous system involvement in Lyme borreliosis. Neurology 1988;38:863-867.

-Kollikowski HH, Schwendemann G, Schulz M, Wilhelm H, Lehmann HJ. Chronic borrelia encephalomyeloradiculitis with severe mental disturbance: immunosuppressive versus antibiotic therapy. J Neurol 1988;235:140-142.

Petrovic M, Vogelaers D, Van Renterghem L, De Reuck J, Afschrift M. Lyme Borreliosis - A Review of the Late Stages and Treatment of Four Cases. Acta Clinica Belgica 1998;53-3:178-1+83.

Straubinger RK, Summers BA, Chang Y-F, Appel MJG. Persistence of Borrelia burdgorferi in Experimentally Infected Dogs after Antibiotic Treatment. J Clin Microbiol 1997;35:111-116.

Liegner KB, Rosenkilde CE, Campbell GL, Quan TJ, Dennis DT. Culture-confirmed Treatment Failure of Cefotaxime and Minocycline in a Case of Lyme Meningoencephalomyelitis in the United States. Program and Abstracts. V International Conference on Lyme Borreliosis. Abstr. 63 P. A11, Arlington, VA. May/June 1992.

Omasits M, Seiser A, Brainin M. Zur rezidivierenden und schubhaft verlaufenden Borreliose des Nervensystems. Wiener clinische Wochenschrift1990;102:4-12.

Liegner KB, Ziska M, Agricola MD, Hubbard JD, Klempner MS, Coyle PK, Bayer ME, Duray PH. Fatal Chronic Meningoencephalomyelitis (CMEM) With Massive Hydrocephalus, In A New York State Patient With Evidence of Borrelia Burgdorferi Exposure. Program and Abstracts, VI International Conference on Lyme Borreliosis. Abstr. P041T. Bologna, Italy, June 19-22, 1994.

Merlo A, Weder B, Ketz E, Matter L. Locked-in state in Borrelia burgdorferi meningitis. J Neurol 1989;236:305-306.

Oksi J, Kalimo H, Marttila RJ, Marjamaki M, Sonninen P, Nikoskelainen J, Viljanen MK. Inflammatory brain changes in Lyme borreliosis. A report on three patients and review of literature. Brain 1996;119:2143-2154.

Wokke JHJ, van Gijn J, Elderson A, Stanek G. Chronic forms of Borrelia burgdorferi infection of the nervous system. Neurology 1987;37:1031-1034.

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[Sailairina]

http://www.northeastern.edu/cos/2015/06 ... e-disease/

"by Greg St. Martin

North­eastern Uni­ver­sity researchers have found that the bac­terium that causes Lyme dis­ease forms dor­mant per­sister cells, which are known to evade antibi­otics. This sig­nif­i­cant finding, they said, could help explain why it’s so dif­fi­cult to treat the infec­tion in some patients.

“It hasn’t been entirely clear why it’s dif­fi­cult to treat the pathogen with antibi­otics since there has been no resis­tance reported for the causative agent of the dis­ease,” explained Uni­ver­sity Dis­tin­guished Pro­fessor Kim Lewis, who led the North­eastern research team.

In other chronic infec­tions, Lewis’ lab has tracked the resis­tance to antibi­otic therapy to the pres­ence of per­sister cells—which are drug-​​tolerant, dor­mant vari­ants of reg­ular cells. These per­sister cells are exactly what they’ve iden­ti­fied here in Bor­relia burgdor­feri, the bac­terium that causes Lyme disease.

The researchers have also reported two approaches—one of them quite promising—to erad­i­cate Lyme dis­ease, as well as poten­tially other nasty infections.

Lewis and his col­leagues pre­sented their find­ings in a paper pub­lished online last week in the journal Antimi­cro­bial Agents and Chemotherapy. He co-​​authored the paper with North­eastern doc­toral stu­dents Bijaya Sharma and Autumn Brown, both PhD’16; recent grad­uate Nicole Matluck, S’15, who received her Bach­elor of Sci­ence in Behav­ioral Neu­ro­science; and Linden T. Hu, a pro­fessor of mol­e­c­ular biology and micro­bi­ology at Tufts University.

The research was sup­ported by grants from the Lyme Research Alliance and the National Insti­tutes of Health.

Lyme dis­ease affects 300,000 people annu­ally in the U.S., according to the Cen­ters for Dis­ease Con­trol and Pre­ven­tion, and is trans­mitted to people via bites from infected black­legged ticks. If caught early, patients treated with antibi­otics usu­ally recover quickly. How­ever, about 10 to 20 per­cent of patients, par­tic­u­larly those diag­nosed later, who have received antibi­otic treat­ment may have per­sis­tent and recur­ring symp­toms including arthritis, muscle pain, fatigue, and neu­ro­log­ical prob­lems. These patients are diag­nosed with Post-​​treatment Lyme Dis­ease Syndrome.

In addi­tion to iden­ti­fying the pres­ence of these per­sister cells, Lewis’ team also pre­sented two methods for wiping out the infection—both of which were suc­cessful in lab tests. One involved an anti-​​cancer agent called Mit­o­mycin C, which com­pletely erad­i­cated all cul­tures of the bac­terium in one fell swoop. How­ever, Lewis stressed that, given Mit­o­mycin C’s tox­i­city, it isn’t a rec­om­mended option for treating Lyme dis­ease, though his team’s find­ings are useful to helping to better under­stand the disease.

The second approach, which Lewis noted is much more prac­tical, involved pulse-​​dosing an antibi­otic to elim­i­nate per­sis­ters. The researchers intro­duced the antibi­otic a first time, which killed the growing cells but not the dor­mant per­sis­ters. But once the antibi­otic washed away, the per­sis­ters woke up, and before they had time to restore their pop­u­la­tion the researchers hit them with the antibi­otic again. Four rounds of antibi­otic treat­ments com­pletely erad­i­cated the per­sis­ters in a test tube.

“This is the first time, we think, that pulse-​​dosing has been pub­lished as a method for erad­i­cating the pop­u­la­tion of a pathogen with antibi­otics that don’t kill dor­mant cells,” Lewis said. “The trick to doing this is to allow the dor­mant cells to wake up.”

He added: “This gives you an idea that you could, in prin­ciple, estab­lish a sim­ilar reg­i­ment for treating patients for this and other chronic diseases.”

Lewis is a fac­ulty member in the biology depart­ment and directs Northeastern’s Antimi­cro­bial Dis­covery Center. Over the past decade he has led pio­neering work on this spe­cial­ized class of cells pro­duced by all pathogens known as per­sis­ters. Ear­lier this year, Lewis, biology pro­fessor Slava Epstein, and other col­leagues pub­lished ground­breaking research in Nature pre­senting a new antibi­otic that kills pathogens without encoun­tering any detectable resistance.

In pre­vious work, Lewis’ lab iden­ti­fied a com­pound called ADEP that causes dor­mant per­sister cells in MRSA to self-​​destruct. This com­pound was among the first options the researchers tried out to combat Lyme dis­ease. But it didn’t work, and nei­ther did com­bi­na­tions of stan­dard antibi­otics used to treat the dis­ease. The team thought it had hit a dead end yet remained vig­i­lant in its quest to iden­tify promising alter­na­tive options.

“What we came up with was the pulse-​​dosing reg­imen, which worked beau­ti­fully,” Lewis explained. “I think this could be very useful, espe­cially for antibi­otics for which resis­tance doesn’t rapidly develop.”

Though the researchers iden­ti­fied the pres­ence of these per­sister cells, they also note in their paper that the mech­a­nisms by which the per­sis­ters are able to sur­vive remain unknown. More work in this area will be required, they wrote.

Originally published in news@Northeastern on June 1, 2015"




http://www.northeastern.edu/cos/2016/03 ... e-disease/
“I find it amazing that when you show up at the doctor’s office you are not told that there is a 10 to 20 per­cent chance that your life as you know it has ended,” says Lewis. “Nobody seems to be focusing on the next step: How to prevent the subsequent rise of the chronic condition.”

"by Thea Singer


The ticks that transmit Lyme dis­ease have mul­ti­plied aggres­sively over the past 20 years, and now thrive in half of all coun­ties in the U.S., according to a recent study in the Journal of Med­ical Entomology.

So it’s no sur­prise that when North­eastern researchers reported last May how the bac­terium that causes the dis­ease evades antibi­otics, sug­gesting new treat­ments, the media and the gen­eral public took notice.

Uni­ver­sity Dis­tin­guished Pro­fessor Kim Lewis, who leads the Lyme dis­ease research team, is now expanding that ther­a­peutic reach with the help of a $1.5 mil­lion grant from the Steven and Alexandra Cohen Foundation.

The team is pur­suing four arms of treatment-​​related research at Northeastern’s Antimi­cro­bial Dis­covery Center, which Lewis directs.

All four show exciting promise. The grant, Lewis says, “will give us the flex­i­bility to test our approaches in par­allel, which will save us an enor­mous amount of time.”

Pre­venting chronic disease

Time is of the essence. According to the Cen­ters for Dis­ease Con­trol and Pre­ven­tion, about 300,000 people are diag­nosed with Lyme dis­ease in the U.S. each year. The dis­ease is trans­mitted by ticks pri­marily car­rying the bac­terium Bor­relia burgdor­feri, though a new less preva­lent bac­te­rial species, Bor­relia may­onii, was iden­ti­fied by Mayo Clinic researchers in February.

If Lyme is caught early, patients gen­er­ally recover quickly when treated with antibi­otics, pri­marily doxy­cy­line. How­ever, 10 to 20 per­cent of patients go on to develop a debil­i­tating chronic con­di­tion called Post-​​Treatment Lyme Dis­ease Syn­drome, or PTLDS, with symp­toms that include extreme fatigue, arthritis, muscle pain, and cog­ni­tive difficulties.

“I find it amazing that when you show up at the doctor’s office you are not told that there is a 10 to 20 per­cent chance that your life as you know it has ended,” says Lewis. “Nobody seems to be focusing on the next step: How to pre­vent the sub­se­quent rise of the chronic condition.”

A new regimen

Lewis and his col­leagues are pro­viding that focus. A sub­pop­u­la­tion of B. burgdor­feri cells, they dis­cov­ered ear­lier, are “per­sister” cells—they are alive but lie dor­mant, in a spore­like state. Because antibi­otics attack only actively func­tioning bac­te­rial cells, per­sis­ters escape the onslaught. How­ever, once the antibi­otic has been flushed from the system, the per­sis­ters “wake up,” says Lewis, dividing and mul­ti­plying until an army of progeny infect the host.

That’s where “pulse dosing” comes in. Lewis’ team, in col­lab­o­ra­tion with researchers studying B. burgdor­feri in mice at Tufts University’s Sackler School of Bio­med­ical Sci­ences, has been ana­lyzing the effect of giving the mice an antibi­otic that kills all the actively func­tioning bac­te­rial cells and then—using the timing that erad­i­cated the pathogen in the test tube—giving addi­tional doses to quash the per­sister cells as they begin to wake up but before they reproduce.

Plans are in the works for the first pulse-​​dosing human trials with med­ical schools.

Drugs com­bined and discovered

Doxy­cy­cline may be stan­dard first-​​line treat­ment for Lyme, but, says Lewis, it doesn’t even kill B. burgdor­feri, it just sup­presses its growth, leaving the rest of the work to the immune system. “We simply asked the ques­tion: ‘Is it pos­sible to com­bine existing antibi­otics to treat not only chronic Lyme but any stage of Lyme if the diag­nosis is unambiguous?”

The researchers have already found com­bi­na­tions that are effec­tive against the B. burgdor­feri in the test tube and will move on to animal studies next.

They are tack­ling new-​​drug dis­covery on two fronts: Plumbing the 200,000-plus com­pounds in their col­lec­tion at North­eastern to find the ones that act solely against B. burgdor­feri to avoid unwanted side effects and, in col­lab­o­ra­tion with Novo­bi­otic Phar­ma­ceu­ti­cals, extracting drugs from bac­teria that live in soil using the iChip, a device devel­oped by Slava Epstein, Dis­tin­guished Pro­fessor at North­eastern, in col­lab­o­ra­tion with Lewis. The iChip pro­vides access to the 99 per­cent of microbes in the envi­ron­ment that hereto­fore could not be grown in the lab.

“So far we have iden­ti­fied two lead com­pounds that kill B. burgdor­feri and have no activity against other bac­teria,” says Lewis.

The researchers are also exploring whether the micro­biome has “shifted” in those with PTLDS, to see whether intro­ducing cer­tain microor­gan­isms might shift it back. Animal studies have shown that manip­u­lating the micro­biome com­po­si­tion alle­vi­ates symp­toms of autoim­mune dis­eases such as rheuma­toid arthritis, which share many char­ac­ter­is­tics with PTLDS.

“We are going at Lyme dis­ease with every­thing we have,” says Lewis.

Originally published in news@Northeastern on March 29, 2016."

[soijuv]

Tri Phillipsin 81 sivuinen artikkeli kroonisesta borrelioosista:

https://drive.google.com/file/d/0B-c32z ... ef=2&pli=1

[soijuv]

Borrelia - bakteeri selviää elimistössä monin eri tavoin. Allaolevan tutkimuksen mukaan Bb piiloutuu mm parasiittien sisään ja aiheuttaa esim. vakavia neurologisia sairauksia kuten MS taudin

https://www.morgellonssurvey.org/news/l ... -diseases/