BORRELIATESTIT

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Int J Immunopathol Pharmacol. 2009 Apr-Jun;22(2):537-41.

Detection of different Borrelia burgdorferi genospecies in serum of people with
different occupational risks: short report.

Santino I, Sessa R, Pantanella F, Tomao P, Di Renzi S, Martini A, Nicoletti M,
Del Piano M.

Department of Public Health Sciences, Sapienza University of Rome, Rome, Italy.

This study is aimed at applying a previously described PCR-based method to
detect B. burgdorferi sensu lato and different Borrelia genospecies in total DNA
preparations of serum samples collected from people with different occupational
risks for tick bite and with serological evidence of borreliosis. Among the
seropositive samples, the PCR for B. burgdorferi confirmed the positivity in 65
percent of the forestry workers and in 60 percent of the subjects living in the
same area. None of the seronegative subjects belonging to the control group
showed the presence of B. burgdorferi sensu lato DNA. Results on genospecies
distribution show that B. afzelii was the predominant species, followed by B.
garinii and finally by B. valaisiana.

PMID: 19505407 [PubMed - in process]

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Suurella osalla borrelioosipotilaista joilla esiintyi niveltulehdusta esiintyi voimakas IgG-vaste borreliabakteerin glykolipideille.


Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patients with Lyme arthritis, a late manifestation of the infection

Clinical Immunology
Volume 132, Issue 1, July 2009, Pages 93-102

Kathryn L. Jones, Robert J. Seward, Gil Ben-Menachem, Lisa J. Glickstein, Catherine E. Costello and Allen C. Steere


Abstract
In this study, the membrane lipids of B. burgdorferi were separated into 16 fractions; the components in each fraction were identified, and the immunogenicity of each fraction was determined by ELISA using sera from Lyme disease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG, BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic. Early in the infection, 24 of 84 patients (29%) who were convalescent from erythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weak IgG responses to purified MgalD, and a smaller percentage of patients had early responses to synthetic ACG. However, almost all of 75 patients with Lyme arthritis, a late disease manifestation, had strong IgG reactivity with both glycolipids. Thus, almost all patients with Lyme arthritis have strong IgG antibody responses to B. burgdorferi glycolipid antigens.

Keywords: Lyme disease;; Bacterial infection;; Borrelia burgdorferi;; Antibodies;; Glycolipid antigens

[soijuv]

Onko mahdollista kehittää luotettava borrelioositesti?


Will There Ever Be An Accurate Test for Lyme Disease?

http://www.canlyme.com/flawedtest.html

By Tom Grier MS

Lyme Disease is a complex systemic disease that is caused by highly motile bacterium in the spirochete family. The bacteria Borrelia burgdorferi was first isolated from the skin of a Lyme patient with the distinctive bull?s-eye rash in the early 1980s. Since that time, culturing the elusive bacterium has been difficult, frustratingly unpredictable, and miserably inconsistent.

Borrelia burgdorferi can very quickly move from the site of a tick bite into the circulatory system, where it can circulate throughout the body. This unique bacterium has a distinct and insidious method for passing through the capillary walls of blood vessels and nestling its way deep inside many organs and tissues of the human body. In animal models, the Lyme spirochete within mere hours of a tick bite, can cause a breakdown of the blood-brain-barrier.

It is the bacteria?s ability to exit the blood stream, hide, and survive that makes Lyme disease so difficult to detect with any one test. It is the unique microbiology of the bacteria that gives it the ability to hide and survive undetected within the human body. That is why using other bacterial diseases as a model for Lyme disease is difficult and leads to misunderstandings in the medical community on how to diagnose and treat Lyme disease. Infections causd by the family of bacteria known as Borrelia are unique in their microbiology and cannot be dismissed with a rubber stamped one-treatment-fits-all approach.

There are five main methods of testing for Lyme disease:

1. Antibody tests (either the Elisa or Western Blot serology tests)

2. Bacterial DNA detection by polymerase-chain-reaction test (PCR)

3. Live culture

4. Antigen detection: Finding particles and proteins of the bacteria in blood, CSF, urine, or

tissue samples.

5. Direct observation by microscope: This can employ the use of biopsy and stain, or

centrifuged blood, spinal fluid, and urine.

By far, the most commonly used tests for Lyme disease are antibody serologies. These tests are indirect measurements of your body?s response to the bacterial pathogen. Antibody serology tests are most often employed not for their accuracy, but more for their convenience, low risks, and low costs. Essentially, these tests look for your antibody response to an infection in the blood stream.

The most common of the antibody tests is the Enzyme Linked Immune Sera Assay test (ELISA). This test is often the first test given to Lyme patients and can only give you a very general idea of the presence or absence of anti-Lyme antibodies.

The Main Drawbacks of Using Elisa Tests for Lyme Disease:

First, each lab can have their own version of an ELISA test, using different enzyme-linked antigens that are used to trap specific anti-Lyme antibodies in the patient?s blood. When an enzyme is in the presence of the correct anti-Lyme antibody, there is an enzymatic color change that occurs which is read by a machine. If the lab chooses antigens that do not reflect the same set of antibodies that the patient is producing then the test will fail to detect Lyme disease. The individual that tests negative with one lab?s test may have a have detectable Lyme antibodies if another lab?s ELISA test is given employing a slightly different set of antigens.

Think of it like this: If you take a gold fish bowl and drop a paperclip and a penny into the bowl and then use a magnet to find the paper clip, you cannot conclude that there is no other metal in the bowl. The magnet is simply incapable of detecting the penny. The same is true if you look for the wrong antibodies in a Lyme patient.

Another problem is consistent accuracy within labs in interpreting results. his lack of consistency in laboratory accuracy was borne out in Lori Bakken?s ELISA test study. This study showed that more than 50% of the time, labs could not correctly or consistently replicate identical results in identical triple-paired serum samples highly positive for Lyme antibodies. This is despite claims by labs of being 99% specific and accurate. However, what is accurate by laboratory definition has nothing to do with accuracy as a diagnostic test to determine the presence or absence of active infection in a patient.

· · Bakken LL, Callister SM, Wand PJ, Schell RF. Interlaboratory Comparison of Test Results for the Detection of Lyme Disease by 516 Participants in the Wisconsin State Lab of Hygiene/College of American Pathologists Proficiency Testing Program. J Clin Microbiol 1997; Vol 35, No. 3:537-543

· · Bakken LL, Case KL, Callister SM et al. Performance of 45 Laboratories Participating in a Proficiency Testing Program for Lyme Disease Serology. JAMA 1992;268:891-895

You see, when companies marketing their products use buzzword terms like specific, sensitive, or accurate, these marketing terms do not mean diagnostically accurate!

The next problem for both the Western Blot and the ELISA tests is timing. In Lyme disease, timing is everything! Timing is everything for both successful diagnosis and successful treatment.

In most infections of the human body, the body?s immune system releases early and late antibody responses. However, there are some caveats to this general assumption. First, the immune system is most effective when infection is in the blood stream, or places where blood can travel to easily. This is because several white blood cells in the blood stream must interact with the pathogen and then with each other to create the proper immune response.

About four to six weeks after first detecting a harmful pathogen within the blood-stream, a white blood cell called the B-cell lymphocyte expands and becomes a plasma cell and produces immunoglobulin type M (IgM) antibody. Then, in another four weeks, if the infection is still in the blood stream, a new set of plasma cells creates IgG antibodies. Therefore, in general, the presence of IgM antibody and the absence of IgG antibody mean an early infection. (Early means the initial exposure to the bacteria was probably within eight to twelve weeks.) Unfortunately, in Lyme disease this general rule of the lone presence IgM antibody as an indicator of early infection does not apply: IgM antibody can and will appear throughout the infection.

Within mere days after an infected tick bite, the Lyme bacteria can already find its way into the brain and joints of patients. Since Borrelia burgdorferi is prefers other locations within the human body other than the blood, it is equipped to seek out those environs more suitable to long-term survival of the bacteria

The migration of the Lyme spirochete from the human blood stream into the tissues has the same effect as muting the patient?s antibody response. This happens because the fewer bacteria that remain in the bloodstream, the less stimulation there is to evoke and provoke a B-Cell to become a plasma cell. If the initial infection never makes a strong presence in the blood stream, the host?s antibody response may be muted or never initiated. In primate studies a strong antibody response was dependent on the infection-load that the animal was given. The more bacteria injected the greater the antibody titer.

The next problem with timing is this: If you give an antibody serology test too early, it will always be negative. This is simply by virtue of not allowing the immune system enough time to clone enough of the correct plasma cells to create a measurable antibody response. The immune system must manufacture exact copies of the cells that can fight the disease. In some patients it is thought that they may not have the correct B-cells to even make an effective assault against this highly variable pathogen. It is akin to trying to fix a broken motor with the wrong tools. You do the best you can but will it be good enough?

Finally, since the infection can already be beyond the reach of the blood stream, the antibody serology tests are worthless for early-sequestered infections of the brain, joints, bladder, skin, and tendons. These are sites where the immune system has little effect. The assumption that the absence of antibodies means the absence of infection could prove disastrous if the infection has already established itself in the brain. There is evidence from looking at spinal taps on dozens of ?Early? Lyme patients with a bull?s-eye rash that the infection can penetrate into the CNS early and without symptoms! If not treated effectively a trapped infection within the CNS is hard to detect and very hard to treat. Unlike other infections of the brain, Lyme does not routinely produce immune cells and antibodies in the spinal fluid.

Further Considerations:

IgM antibodies in Lyme disease can occur at any point in the infection, early or late. This is most probably because of the infection seeding from one site to another via the breach of capillaries in that area. In other words, if the Lyme spirochete can escape out of the capillaries during early infection by stimulating the blood vessel cells to produce digestive enzymes, then it also reasons that the sequestered spirochetes can occasionally seed back into the circulatory system by a similar mechanism.

The ELISA test is sometimes used to test cerebrospinal fluid (CSF) in order to detect antibodies in the central nervous system (CNS). While the presence of Lyme antibodies in the CNS is highly significant, no accurate conclusion whatsoever can be made by the absence of Lyme antibodies in CSF. Short of an autopsy, an infection of the brain with Borrelia burgdorferi is almost impossible to rule out. The brain is a very poor producer of immune responses. This is why Lyme infections of the brain appear to be aseptic (the absence of white blood cells in the spinal fluid or brain.).

ELISA Recap:

The ELISA test is useless within the first four weeks of a tick bite.

The ELISA may not detect late infection because the bacteria can find immune privileged sites in which to hide.

The ELISA test is not a standardized test. The design of the test can vary greatly from lab to lab.

The choice of antigens used in the test is derived from a laboratory strain B-31 instead of the naturally occurring wild strains. The B-31 strain is proving to be highly variable and changing. Using a high passage lab strain may be cheap and convenient, but not an accurate representation of the various strains of Borrelia found in nature.

The accuracy of the test varies even on identical samples, meaning that even the labs themselves introduce a variable of inaccuracy by poor procedure, interpretation, or quality control.

Western Blot:

The Western Blot antibody test has only two slight advantages over the ELISA test. First, it is slightly more sensitive, probably due to the inclusion of more bacterial antigens. Second, it tells us which bacterial proteins are eliciting an antibody response in that patient. However, in the end, the Western Blot still suffers from all the same downfalls as the ELISA, with one additional disadvantage. In Dearborn, Michigan, a group of state epidemiologists decided to standardize the interpretation of the Western Blot test: The IgM Western Blot must have two significant bands to be positive and the IgG Western Blot must have 5 out of 10 possible bands present. These numbers are somewhat arbitrary, because the presence of the single band 39 kda is specific for only Borrelia burgdorferi, and no other bacteria yet discovered.

In essence, the conference in Dearborn, Michigan, chose to eliminate the reporting of a half dozen significant bacterial proteins from the test. Even if antibodies are found present against these significant excluded bacterial proteins the Western Blot using these new reporting criteria cannot be interpreted as a positive test.

The IgG Western Blot must show the strong presence of five out of ten pre-selected bacterial proteins, and the presence of any other highly significant bands is to be ignored. In fact, most state labs are prohibited from even reporting these other significant bands. They are allowed only to report the test as positive or negative, thus taking the ability to interpret the test away from the physicians.

How badly did the new reporting criteria bootstrap the Western Blot accuracy? Compare these results in a 66-patient study on children with physician-diagnosed bull?s-eye Lyme skin rashes:

h · 19 Rheumatology Symposia Abstract # 1254 Dr. Paul Fawcett et al. This abstract shows that, under the old criteria, all 66 pediatric patients with a history of a tick bite and bull?s-eye rash who were symptomatic were accepted as positive under the old Western Blot interpretation. Under the newly proposed criteria, only 20 were now considered positive. This means 46 children, all symptomatic, would probably be denied treatment. That?s a success rate of only 31%!

Note: A misconception about Western Blots and ELISA tests is that they have as many false positives as false negatives. This is not true. False positives are rare. Negative serologies despite a rash or a positive culture is routine. Remember words like sensitivity, specificity, and accuracy DO NOT MEAN DIAGNOSTICALLY ACCURATE to determine if a patient has an infection. A NEGATIVE TEST CANNOT RULE OUT AN INFECTION THAT HAS ESCAPED THE BLOODSTREAM.

The conclusion of the researchers in this pediatric study was: ?The proposed Western Blot Reporting Criteria are inadequate, because it excluded 69% of the infected children?

Finally, the failings of all antibody tests are that they can only capture uncomplexed antibody. Once an antibody finds something to latch on to in the bloodstream, it is no longer available to be detected with our current tests.

Free antibodies are detectable with the current Lyme tests, but antigen/antibody complexes are undetectable using either the ELISA or Western Blot tests. It has been suggested that complexes are the biggest weakness of both the ELISA and Western Blot tests and yet they are completely ignored when Lyme tests are given.

ScShutzer SE, Coyle PK, Belman AL, Golightly MG, Drulle J. Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Lancet 1990;335:312-315

When the ELISA or Western Blot tests are positive, they are significant. However, when they are negative, they are incapable of determining the complete absence of active infection somewhere in the body. All it takes to validate this is a single case history of a patient who is sero-negative for Lyme disease (the absence of Lyme antibodies), but is culture positive for the bacteria. Here are a few such examples of patients found to be actively infected despite having no measurable antibodies.

· · Masters EJ, Lynxwiler P, Rawlings J. Spirochetemia after Continuous High Dose Oral Amoxicillin Therapy. Infect Dis Clin Practice 1994;3:207-208

· · Schmidli J, Hunzicker T, Moesli P, et al. Cultivation of Bb from Joint Fluid Three Months After Treatment of Facial Palsy Due to Lyme Borreliosis. J Infect Dis 1988;158:905-906

· · Liegner KB, Shapiro JR, Ramsey D, Halperin AJ, Hogrefe W, and Kong L. Recurrent erythema migrans despite extended antibiotic treatment with minocycline in a patient with persisting Borrelia burgdorferi infection. J. American Acad Dermatol. 1993;28:312-314

The biggest misunderstanding physicians have about Lyme antibody tests is thinking that a high titer of antibody in a patient means that the patient is more ill than a patient with a low titer of antibodies. What these tests are really measuring is the body?s natural immunity against the bacteria. A patient who is able to mount a strong antibody defense against this pathogen is far better off than a patient who makes little or no antibody. It only stands to reason that a patient with a high infection load and no antibodies is going to be more symptomatic than a patient with a high natural immunity. Unfortunately, most physicians look at higher titers and assume those patients are the most ill, but their reasoning is backwards. Low titers in a highly symptomatic Lyme patient is a very bad thing.

DNA Amplification or PCR (Polymerase Chain Reaction) Tests:

Every living cell has DNA that is unique to that species of organism. This is true from the simplest bacteria to the most complex mammal. The polymerase chain reaction test is a test that can search for a specific sequence of DNA and multiply that sequence a billion times in less than a day. It is often said that the PCR test is the most sensitive and specific diagnostic test available. However, PCR tests are not without their drawbacks.

Specificity is a big problem with today?s Lyme tests. It sounds good to hear things like, ?This is the most specific test on the market!? Those buzz words meant to promote and sell a test, however, should throw up a red flag to anyone who understands this family of bacteria. The family of bacteria that Lyme disease spirochetes belong to is the borrelia family. Borrelia is the same group of bacteria that cause tick-borne-relapsing-fever. There are more than 40 different disease-causing subspecies of borrelia that are closely related to the Lyme spirochete.

Variability in the borrelia family of bacteria is not only routine; it is actually built into the genetics of the bacteria to add variations its proteins when stressed during division. This is what causes relapses of symptoms in relapsing fever. The bacteria presents one set of proteins to your immune system and then six weeks later, the bacteria adapt and present new variations of surface proteins to your immune system. In turn, your own immune system is what makes you feel sick, sweaty, feverish, and ill with each new set of antigens presented.* The Lyme bacteria uses a slightly more subtle form of this morphogenic antigen variability, but it, too, like its cousins, is capable of changing its outward appearance like a criminal changing his disguise.

Escaping the immune system is very important if you want to survive as a bacterium. Borrelia has several mechanisms to do this, but this mechanism of gene variability and variable proteins make both serology antibody tests and PCR tests less effective.

One could easily make a case that Lyme disease and all of its Lyme-like cousins are nothing more than variations of Relapsing-Fever. In America, we now have at least two geno-species that cause Lyme disease, which are not Borrelia burgdorferi. The truth is, we may have a situation with Lyme disease that is similar to relapsing fever, in that there are dozens of yet to be discovered species of borrelia that cause disease but are not Borrelia burgdorferi.

Would you want to be denied medical treatment, medical coverage, and your good health, all because the test you are given is so specific that it excludes all other borrelia? What we need in America and other Lyme-endemic countries is not a hundred different species-specific tests, but rather one general screening test for Borrelia. This is true for the antibody tests as well as the PCR DNA tests. Suddenly the buzz-word SPECIFICITY has become a dirty word. Specific also can mean exclusionary!

*NOTE: There is no substantial evidence that a Herxheimer reaction during antibiotic treatment is due to the release of bacterial toxins. There is good evidence that a true Herxheimer reaction is the rapid synthesis of cytokines from your immune cells. This increases when internal bacterial antigens are exposed when the bacteria ruptures with treatment. This was established in Relapsing Fever patients in the late 1960s. To date the evidence of Borrelia burgdorferi producing significant toxins in symptomatic patients is virtually non-existent.

How a PCR test works:

The first step is to create a template or primer for the PCR test that is specific to the organism you are looking for. Second, you have to be sure that the test sample has a high probability of containing the DNA sequence for which you are searching. Another concern is inhibitory factors in the samples. Finally, you have to be sure there are no false positives from lab contamination due to DNA floating around like dust in the air.

In Lyme disease, the PCR test really has limited value. The main problem is that doctors and labs like to use blood, urine, and spinal fluid to test simply because of the ease of collection. In the case of Lyme disease, however, the borrelia spirochete does not like the blood stream, and PCR tests of fluids are frequently negative while the same PCR tests of skin biopsies in the same patient are positive. This indicates that finding a sample with the bacterium?s DNA is a bit like finding a needle in a haystack.

The example I like to use to demonstrate this is the parable about the bucket and the hailstorm. If it is hailing outside, you could run around all day with a teacup, trying to catch just one hailstone. Just one hailstone in the teacup would be proof that it is hailing outside: But your chance of success is low. If, however, you used a bucket to catch the hailstones, you would have a slightly better chance to make your case. If you had an entire parking lot to catch hail you would have even a better chance!

However, remember, just because it is hailing in New Jersey doesn?t mean it?s hailing in New York. If the hail represents borrelia DNA, it is clear the bigger and better the sample you collect the better the result. However, if New Jersey represents your blood and New York represents your brain, you cannot use a hailstone from New Jersey to prove whether it?s hailing in New York. To make matters worse, the tests we have now are not as good as they could be, so it compares to using teacups with holes in the bottom to collect hailstones! Absence of proof is not proof of absence of infection.

It has been reported that there are inhibitory enzymes in the urine that can affect the PCR tests. Most labs using PCR tests don?t address this but the truth is the whole organism rarely shows up in the urine so a PCR test like serology tests can be inconclusive if negative.

Cerebrospinal fluid has been reported PCR positive in only 7% of the cases of documented neurological Lyme disease. This indicates that the organism is highly tissue bound in the CNS and that bacterial DNA in the spinal fluid is the exception, not the rule, during infection.

The biggest problem with the PCR test in Lyme disease isn?t its accuracy or sensitivity, but the squabbling over patents. Every lab wants its own patent on its own test and will often present data on their tests in a favorable way to sell the test, but is misrepresentative of the test?s abilities to diagnose Lyme disease at any stage.

Most labs use the OSP-A gene as a primer for its PCR test, but, in truth, a PCR test that can use multiple primers is probably better, there are some species of Borrelia burgdorferi don?t even have a gene for OSP-A. However, using multiple primers can mean paying royalties to many different labs for the use of proprietary patents. This raises costs and means cooperation between competitors.

PCR Recap:

No single sample from a patient can be diagnostic for Lyme disease using PCR as the test. Even the University of Minnesota reported a mere 18% success rate in early Lyme when skin biopsies were obtained from culture positive bull?s-eye rashes.

Patent disputes often result in poor PCR tests being marketed with little or no sharing of technology between competitors. This competition often leads to over-hyped performance and over hyped accuracy claims by manufacturers.

Often the best tissues for PCR tests are tissue biopsies, which are rarely done because of costs, risk, and inconvenience. In truth, PCR tests are best used in the post-mortem exam on tissues such as the brain, bladder, and heart. As a diagnostic tool, it can be useful when positive, but tells us nothing when negative. I see few patients lining up for a brain biopsy.

· · Appel MJ, Allan S, Jacobson RH, Lauderdale TL, Chang YF et al. Experimental Lyme disease in dogs produces arthritis and persistent infection. J Infect Dis, March 1993;167(3):651-4

Culturing Borrelia burgdorferi:

Culturing spirochetes can be difficult (see my article on the Lyme Alliance web site). Some spirochetes, such as T. pallidum, which causes syphilis, have never been successfully cultured in culture media, and are maintained only in live animal models. Borrelia burgdorferi is difficult to culture. The University of Minnesota, when comparing culturing of erythema migrans rashes to their early PCR test, reported culture success rates as low as 4%.

As discussed earlier, culturing blood is frustrating because the bacteria do not like the blood stream. The same is true for the spinal fluid and urine. Therefore, once again we are left with the option to biopsy at great expense and risk, or to use other, less costly, tests. Culturing is the gold standard for confirmation of an infection, and a positive culture is highly important. However, as a diagnostic tool in Lyme diagnosis, culturing has a poor success rate, and the cost and time often make it prohibitive except as a research tool.

The technique of culturing the non-classical form of the spirochete known as spheroid-forms or L-forms is controversial and has yet to be corroborated and commercially reproducible. L-forms seem to be shed by the spirochetes when they are stressed and moribund. They appear to be able to transform back into viable spirochete indicating that the vesicles retain a full compliment of bacterial genetic material. Until the link can be established between these L-forms and disease, and the cultures can be corroborated by PCR and consistently reproducible by other labs the presence of L-forms will be debated by opponents as artifacts and aberrations.

Just as in the case of PCR and antibody tests, culturing can also be affected by the species of bacteria that is present. Some laboratory strains of Lyme disease have been known to take months to culture. Most hospital labs don?t even stock culture media that is suitable for culturing Borrelia, and the choices for culture media are extremely limited. Until better techniques and culture medias are made available, culturing spirochetes is a techniques best used as a research tool. But the inexperience of hospital labs with spirochete culturing techniques is by far the biggest hurdle in seeing this test flourish.

Microscopic observation of the organism:

Using a microscope to find the Lyme bacteria is quite literally like looking for a needle in a haystack. The Borrelia burgdorferi organism is found in such low numbers in fluids and tissue that it requires hours and hours of lab time to do an adequate search of just a few millimeters of tissue. As a research tool biopsy and stain is useful, but to use microscopy to diagnose Lyme is too slow, too costly, and quite unreliable.

The modern variation of spirochete microscopy is to use a fluorescent stain attached to an antibody that will latch on to the bacteria. Under a fluorescent microscope, the bacteria stand out as brightly colored spirals.

The final method is simply to take a bodily fluid, such as CSF or urine, and ultra centrifuge it, then look for spirochetes under a light microscope with a drop of acrodine orange dye.

All of these techniques are direct observation of the organism, but it is impossible to determine the species just by looking at the bacterium?s gross morphology. The most useful place for this tool is in the post-mortem exam, where deep tissues can be collected, preserved in paraffin, stained, and studied months later. Any autopsy performed on a Lyme patient should include silver stain sections of paraffin- fixed brain biopsies. However, using microscopic exam to diagnose Lyme would be expensive and time consuming, and would yield poor results. (In a post-mortem exam the presence of spirochetes is significant. Spirochetes should never be found in the brain. One wonders what percentage of dementia patients would have spirochetes in the brain if we looked for them?

Conclusion:

One of the biggest misconceptions about Lyme disease antibody tests, which has led to years of unnecessary morbidity and mortality for Lyme disease patients, is the insistence that the absence of Lyme antibodies means the absence of active infection. You cannot equate the absence of antibodies with the absence of infection, nor can you use antibody serologies as an endpoint in treatment studies to determine the effectiveness of any treatment regimen. It isn?t just a bad idea - it is just plain bad science.

Will there ever be a Lyme test that can be used dependably to diagnose Lyme disease? I don?t believe such a test is within our current technology, and until such a test exists denying patients treatment using our current tests is bad medicine.

Key words and concepts used in this article:

Systemic: Borrelia burgdorferi and other spirochetes in the tick-borne relapsing fever family of bacteria circulate through the blood stream to the entire body. They can find passage through blood vessel walls and invade other tissues and organs, including known target tissues such as skin, tendons, joints, heart, nerves, and brain.

Sequestered: The Lyme spirochete can find haven in areas of the body that are poorly protected by the immune system (the brain), have poor areas of blood circulation (tendons), or where the bacteria can lay metabolically inactive for lengths of time and resist antibiotic penetration (the skin and the brain).

Multi-systemic: The bacteria has affected more than one system of the body, such as: Peripheral nervous system, skin, joints and connective tissues, cardiovascular system and circulatory system, eyes, muscles, liver and spleen, and central nervous system.

Blood Brain Barrier (BBB): The network of capillaries surrounding the brain that selectively let things in and out of the brain. A healthy BBB prevents infections, white blood cells, and many medicines from entering the brain. A breakdown of the BBB is not a good thing, and occurs very early in most animal models of Lyme disease.

Antigen: A protein usually on the surface of the bacteria that stimulates the immune system to make antibodies against that protein. Antigens can also stimulate other immune responses, such as T-cell and macrophage responses.

Serologies: Using the centrifuged serum extracted from blood to look for antibodies against the Lyme bacterium.

Titer: A measurement of antibody content in the serum. Titers are usually reported as dilution series. The higher the dilution of serum where antibodies are still detectable means there are more antibodies present in that patient?s serum. The cut off for reporting a positive is a bit arbitrary and varies from lab to lab. Institutions conservative on the diagnosis of Lyme have raised their cutoffs from 1:256 to 1:1024 Which is rare to see in most Lyme patients.

Infection Load: The actual number of bacteria in a host. If the bacteria remain in the bloodstream, the number of bacteria per unit of blood can be calculated, but this number is meaningless if the infection has moved beyond the bloodstream. In Lyme disease, there is no accurate way of determining true infection load. If the bacteria out pace antibody production then there is no FREE ANTIBODY only ANTIBODY COMPLEXES.

References:

· · Cimmino MA, Azzolini A, Tobia F, Pesce CM. Spirochetes in the Spleen of a Patient with Chronic Lyme Disease. American J Clin Pathol 1989;91(1):95-97

· · DeKoning J, Hoogkamp-Korstanje JAA, van der linde MR, Crjins HJGM. Demonstration of Spirochetes in Cardiac Biopsies of Patients with Lyme Disease. J Infect Dis 1989;160:150-153

· · Waniek C, Prohovnik I, Kaufman MA. Rapid Progressive Frontal-Type Dementia and Death with Subcortical Degeneration Associated with Lyme Disease. A case report/abstract/poster presentation. The Lyme bacterium is isolated from the brain of a deceased Lyme patient treated with antibiotics. LDF State of the Art Conference, with emphasis on neurological Lyme. April 1994, Stanford, CT*

· · Lawrence C, Lipton RB, Lowy FD, and Coyle PK. Seronegative Chronic Relapsing Neuroborreliosis. European Neurology. 1995;35(2):113-117

· · Cleveland CP, Dennler PS, Durray PH. Recurrence of Lyme Disease Presenting as a Chest Wall Mass: Borrelia burgdorferi was present despite five months of IV ceftriaxone 2g, and three months of oral Cefixime 400 mg BID. The husband was seronegative for Lyme and highly symptomatic. The wife had a high tier of antibodies, but had mild symptoms. The excised fibrous mass was culture positive for Borrelia burgdorferi despite eight months of continuous high dose antibiotics. Poster presentation, LDF International Conference on Lyme Disease Research, Stamford, CT, April 1992 *

· · · · Preac-Mursic V, Wilske B, Schierz G, et al. Repeated Isolation of Spirochetes From the Cerebrospinal Fluid of an Antibiotic-treated Patient with Meningoradiculitis Bannwarth? Syndrome. Eur J Clin Microbiol 1984;3:564-565

· · Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross B, Baumann A, and Prokop J. Survival of Borrelia Burgdorferi in Antibiotically-treated Patients with Lyme Borreliosis. Infection 1989;17:335-339

· · Schmidli J, Hunzicker T, Moesli P, et al. Cultivation of Bb from Joint Fluid Three Months After Treatment of Facial Palsy Due to Lyme Borreliosis. J Infect Dis 1988;158:905-906

· · Stanek G, Klein J, Bittner R, Glogar D. Isolation of Borrelia Burgdorferi from the Myocardium of a Patient with Long-Standing Cardiomyopathy.

· · Abstract # D654: J. Nowakowski, et al. Culture-Confirmed Treatment Failures of Cephalexin Therapy for Erythema Migrans. Two of six patients biopsied had culture-confirmed Borrelia burgdorferi infections despite up to 21 days of Cephalexin (500 mg TID) antibiotic treatment.

· · Abstract # D655: Nowakowski, et al. Culture-confirmed Infection and Reinfection with Borrelia Burgdorferi. A patient, despite antibiotic therapy, had a recurring Erythema Migrans rash on three separate occasions. On each occasion, it was biopsied and revealed the active presence of Borrelia burgdorferi on two separate occasions, indicating reinfection had occurred.

· · Abstract # D657: J. Cimperman, F. Strle, et al. Repeated Isolation of Borrelia Burgdorferi from the CSF of Two Patients Treated for Lyme Neuroborreliosis. Patient 1 was a twenty-year-old woman who presented with meningitis, but was seronegative for Borrelia burgdorferi. Subsequently, six weeks later, Bb was cultured from her CSF and she was treated with IV Rocephin, 2 grams a day for 14 days. Three months later, the symptoms returned, and Bb was once again isolated from the CSF. Patient 2 was a 51-year-old female who developed an EM rash after tick bite. Within two months, she had severe neurological symptoms, but her serology was negative. She was denied treatment until her CSF was culture positive, nine months post-tick bite. She was treated with 2 grams of Rocephin for 14 days. Two months post-antibiotic treatment, Bb was once again cultured from her CSF. In both of these cases, the patients had negative antibodies but were culture positive, suggesting that the antibody tests are not reliable predictors of neurological Lyme disease. In addition, standard treatment regimens are insufficient when infection of the CNS is established, and Bb can survive in the brain despite intravenous antibiotic treatment.

· · Abstract # D658: F. Strle et al. Reinfection with Borrelia Burgdorferi in Endemic Areas. Conclusion: Even despite high antibody titers, as seen in ACA patients, 7% of 2273 patients with previous Lyme disease became reinfected and presented with an EM rash and late symptoms after a recent tick bite.

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Magneettikuvauksessa (MRI) 392:lla henkilöllä epäiltiin oireiden perusteella neuroborrelioosia. Kliinisten, serologisten ja hoitovasteen perusteella pääteltiin että 66 heistä sairasti tautia varmuudella. Seitsemällä heistä esiintyi aivojen valkean aineen muutoksia ym. keskushermostomuutoksia esim. hermojuurissa.

Radiology. 2009 Jul 8; [Epub ahead of print] Neuro-Lyme Disease: MR Imaging Findings. Agarwal R, Sze G. Department of Diagnostic Radiology, Yale University School of Medicine, 333Cedar St, PO Box 208042, New Haven, CT 06520. Purpose: To describe the neuroimaging manifestations of Lyme disease at magneticresonance (MR) imaging of the brain. Materials and Methods: Institutional reviewboard approval was obtained and HIPAA compliance was followed. This studyretrospectively reviewed the MR imaging findings of all patients seen from 1993to 2007 in whom neuro-Lyme disease was suspected and who were referred for MRimaging of the brain for the evaluation of neurologic symptoms.

Results: Of 392patients suspected of having neuro-Lyme disease, 66 patients proved to have thedisease on the basis of clinical criteria, serologic results, and response totreatment. Seven of these 66 patients showed foci of T2 prolongation in thecerebral white matter, one had an enhancing lesion with edema, and threedemonstrated nerve-root or meningeal enhancement. Of the seven patients withfoci of T2 prolongation in the white matter, three were an age at which whitematter findings due to small-vessel disease are common. Conclusion: In cases ofnerve-root or meningeal enhancement, Lyme disease should be considered in thedifferential diagnosis in the proper clinical setting. (c) RSNA, 2009. PMID: 19587309 [PubMed - as supplied by publisher]

[soijuv]

Uusi PCR testi, nested PCR, osoittautui melko spesifiseksi testiksi (2009).


Pak J Biol Sci. 2009 Mar 1;12(5):463-6. Rapid detection of Borrelia burgdorferi strains by nested polymerase chainreaction. Sorouri R, Ranjbar R, Jafari NJ, Karami A. Faculty of Medicine, Zanjan Medical Sciences University, Zanjan, Iran. This study was carried out to evaluate the nested PCR for specific detection ofdifferent strains of B. burgdorferi. Five strains of B. burgdorferi includingACA-1, B-31, 2B45, 3B45, 7B49 obtained from different countries were used inthis study. The strains of B. hermsii, Escherichia. coli and T. pallidum werealso included as control strains. Two pairs of nested PCR primers were used toamplify the gene encoding the Osp-A protein of B. burgdorfer under standard PCRcondition. In a two stage procedure, nested PCR yielded a positive reaction forfive tested strains of B. burgdorferi. None the strains including B. hermsii, E.coli and T. pallidum showed positive reaction when used as control strains inPCR.
In conclusion, nested PCR showed acceptable specificity for rapid detectionofB. burgdorferi. PMID: 19579990 [PubMed - in process]

[soijuv]

Ruotsalaiset tutkivat 150 neuroborrelioositapausta jälkikäteen. Tapausten määrä on ollut nousussa vuosina 2000 - 2005. Potilaista 17 %:lla borreliavasta-aineet olivat negatiiviset veressä MUTTA positiiviset selkäydinnesteessä.

Clin Microbiol Infect. 2009 Sep 29; [Epub ahead of print]

Neuroborreliosis - an epidemiological, clinical and health economical study from an endemic area in the south-east of Sweden.

Henningsson AJ, Malmvall BE, Ernerudh J, Matussek A, Forsberg P. Department of Infectious Diseases, Ryhov County Hospital, Jonkoping, Sweden.


We studied retrospectively the medical records of all patients diagnosed with neuroborreliosis (NB) by cerebrospinal fluid (CSF) analysis in Jonkoping County,Sweden, during 2000-2005 (n=150). The number of NB cases increased from 5 to10/100,000 inhabitants/year. In 17% of the patients, Borrelia-antibodies werefound in CSF but not in serum at the time of diagnosis. Facial palsy, headacheand fever were frequent manifestations in children, whereas unspecific muscleand joint pain were the most commonly reported symptoms in older patients.Symptoms persisting more than six months post-treatment occurred in 13%, andthese patients were significantly older, had longer duration of symptoms priorto treatment, higher levels of Borrelia-specific IgG in CSF and more oftenradiculitis.

The total cost for NB-related health care was estimated to 500,000EUR for the entire study group (3,300 EUR/patient), and the cost for socialbenefits to 134,000 EUR (2,000 EUR/patient). CSF analysis is necessary for NBdiagnosis since some patients develop antibodies in serum later than in CSF. Early diagnosis of borreliosis would result in reduced human suffering andeconomic gain.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... rlinksPMID: 19793326 [PubMed - as supplied by publisher]

[soijuv]

Borreliabakteereita nähdään veressä pimeäkenttämikroskooppisessa tutkimuksessa. Allaolevilla videoilla esitetään tutkimuksen periaatteita ja videokuvia bakteerista ja sen kystamuotoon muuntumisesta.

http://www.youtube.com/watch?v=Mg21897IW_4&feature=fvw¨

live blood analysis dark field examination video clip
http://www.youtube.com/watch?v=E8R16Sq1AtM&feature=fvw

Borreliabakteereita
http://www.youtube.com/watch?v=rFxiV_C8 ... re=related
http://www.youtube.com/watch?v=O0y7X5ac ... re=related

Borreliabakteerin muuntuminen kystamuotoon yksityiskohtaisesti kuvattuna

http://www.youtube.com/watch?v=lVmCa70b ... re=related

[soijuv]

Borreliabakteerin aiheuttaman niveltulehduksen erottaa septisestä niveltulehduksesta esim. valkosoluarvojen perusteella. Borrelioosissa esim. jalan päälle pystyy osittain varaamaan, kuumetta ei välttämättä esiinny ja valkosoluarvot ovat alhaiset.
http://www.medscape.com/viewarticle/712343

Low White Blood Cell Count Distinguishes Lyme Arthritis From Septic Arthritis
Crina Frincu-Mallos, PhD

Authors and Disclosures

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Review data November 13, 2009 (Washington, DC) ? The odds that a child living in a Lyme-endemic area of the United States who presents with a joint effusion will be diagnosed as having Lyme arthritis is 29%. The odds are even higher (44%) if the affected joint is the knee. The leukocyte count is useful in distinguishing between septic and Lyme arthritis, researchers announced here.

?There was an increase in the number of cases in the United States by 101% over the past 15 years, possibly due to increased recognition of Lyme disease,? said Aristides I. Cruz Jr., MD, resident in the Department of Orthopedics and Rehabilitation at Yale University in New Haven, Connecticut. During his presentation, he noted that 93% of all Lyme disease cases arise from 10 states, most in the Northeast United States.

?Children are more likely to present with arthritis as initial manifestation of Lyme disease,? Dr. Cruz told the audience.

?Children with Lyme arthritis presenting with a limp and a swollen joint will typically have a lower peripheral white blood cell count,? Dr. Cruz added. "They are less likely to have complete non?weight-bearing on the affected limb, less likely to have a fever, and [arthritis symptoms are] more likely to involve the knee joint compared with children with septic arthritis.?

The findings were reported here at the American Academy of Pediatrics 2009 National Conference & Exhibition.

Basic Diagnostic Tools Help Distinguish Lyme From Septic Arthritis

?In the Northeast, we see a lot of Lyme disease,? said Yi-Meng Yen, MD, PhD, instructor in orthopaedic surgery, Harvard Medical School, Children?s Hospital Boston in Massachusetts. ?It is hard to distinguish whether [it] is Lyme disease or whether [it] is septic arthritis,? Dr. Yen agreed.

?Septic arthritis mandates that we take the patient to the operating room and do a surgery, whereas Lyme arthritis theoretically can be treated with antibiotics,? he told Medscape Pediatrics in an interview. For instance, he said, ?Our institution has been looking at MRIs [magnetic resonance images] as a way to reliably distinguish between the two, because it takes several days sometimes for the lab tests to come back to definitely tell you whether you have Lyme disease or not. So, in those few days, if you have septic arthritis, that?s a bad thing.?

?To reliably, quickly diagnose what the patient has can help us determine the treatment quickly,? added Dr. Yen, who was not involved in this study.

?If you are clinically susceptible for septic arthritis, it pays to go to the operating room,? Dr. Cruz said in answer to a question from the audience. ?In the past, almost all these patients automatically went to the operating room.?

However, if the clinical presentation is consistent with Lyme arthritis, treatment with antibiotics should suffice, he added. ?The point of this study was to come up with some clinically useful criteria to arm ourselves with more tools to diagnose the disease.?

Dr. Cruz and his team sought to evaluate clinical parameters that could eventually be used to differentiate Lyme arthritis from septic arthritis in children and help with diagnosis and subsequent treatment.

In this retrospective analysis, the investigators reviewed data from children who underwent lower-extremity joint aspiration at Yale University Medical Center, a tertiary care children?s hospital in a Lyme disease endemic area.

Between August 2002 and August 2008, more than 200 children underwent a total of 212 aspirations for a joint effusion. Cell count, culture, hematologic inflammatory markers, and subsequent surgical intervention were available for 170 of the 212 aspirates.

Dr. Cruz's team compared findings from 50 children with serologically confirmed Lyme disease with data from 21 patients with culture-positive septic arthritis.

They found statistically significant differences between the 2 cohorts. For instance, the peripheral white blood cell count was 9.5 x 1000/μL (range, 3.0 ? 14.9 x 1000/μL) in the aspirates from children with Lyme disease vs 12.5 (range, 5.5 ? 30.1) in children with septic arthritis (P = .002).

Other parameters, such as joint fluid cell count, erythrocyte sedimentation rate, and C-reactive protein levels, were not significantly different between the 2 groups and could not be used to differentiate between septic and Lyme arthritis.

Interestingly, said Dr. Cruz, of all the children presenting with a joint effusion at their hospital, 29% were likely to be diagnosed as having Lyme arthritis overall compared with 44% if the aspirate was a knee aspirate.

?Is it worthwhile to develop something that?s very reliable? Absolutely!? said Dr. Yen. ?Especially in the Northeast centers. It is a growing healthcare problem and a lot more study should be put into it.?

Dr. Cruz and Dr. Yen have disclosed no relevant financial relationships.

American Academy of Pediatrics (AAP) 2009 National Conference & Exhibition (NCE): Abstract 5806. Presented October 17, 2009.

[CLOSE WINDOW]
Authors and Disclosures
Journalist
Crina Frincu-Mallos, PhD
Crina Frincu-Mallos is a freelance writer for Medscape Medical News.

[soijuv]

Euroopassa useat eri borreliabakteerin alalajit voivat aiheuttaa borrelioosin. Mahdollisia testeja ovat koepalat, nesteista otetut PCR -testit esim. virtsasta, lymfosyyttien transformaatiotesti (LTT). Selkaydinnesteesta tehdyt testit ovat vain harvoin positiivisia (10 - 30 %).


Vector Borne Zoonotic Dis. 2003 Winter;3(4):215-27. Diagnosis of Lyme borreliosis in Europe. Wilske B. Max von Pettenkofer Institute, University of Munich, National Reference Center for Borreliae, Pettenkofer-Stresse 9a, D 80336 Munich, Germany. Bettina.Wilske@mvp-bak.med.uni-muenchen.de In Europe, Lyme borreliosis is caused by at least three species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. Thus microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae for development of diagnostic tools such as PCR primers and diagnostic antigens. According to guidelines of the German Society of Hygiene and Microbiology, the serological diagnosis should follow the principle of a two-step procedure. A sensitive ELISA differentiating IgM and IgG is recommended as the first step. In case the ELISA is reactive, it is followed by immunoblots (IgM and IgG) as the second step. The reactive diagnostic bands should be clearly identified, which is easy if recombinant antigens are used. The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens instead of whole cell lysates. Improved sensitivity resulted from use of recombinant proteins that are expressed primarily in vivo (e.g., VlsE) and combination of homologous proteins from different strains of borrelia (e.g., DbpA). It also appears promising to use recombinant proteins (DbpA, VlsE, others) or synthetic peptides (the conserved C6 peptide derived from VlsE) as ELISA antigens. At present, detection rates for serum antibodies are 20-50% in stage I, 70-90% in stage II, and nearly 100% in stage III Lyme disease. The main goals for the future are to improve specificity in general and sensitivity for diagnosis of early manifestations (stage I and II). Detection of the etiological agent by culture or PCR should be confined to specific indications and specialised laboratories. Recommended specimens are skin biopsy specimens, CSF and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50-70%) and synovial tissue or fluid (50-70% with PCR). CSF yields positive results in only 10-30% of patients. Methods that are not recommended for diagnostic purposes are antigen tests in body fluids, PCR of urine, and lymphocyte transformation tests. PMID: 14733674 [PubMed - indexed for MEDLINE]

[soijuv]

Perinteiseen kaksivaiheiseen vasta-ainetestiin (IgM ja IgG) verrattuna
onuusi IgG -testi jossa on huomioitu VlsE,testimenetelmää kehittävien
tutkijoiden mukaan, aiempaa herkempi.

Suom.huom. Tutkimuksen luotettavuutta tarkasteltaessa on
huomioitava esim. tutkimuksen suorittajat ja heidän mahdolliset
taloudelliset kytköksensä. Tämän tutkimuksen suorittajista ainakin
kaksi, Steere ja Wormser, ovat ajaneet Amerikassa voimakkaasti
kantaa jonka mukaan kroonista borrelioosia ei ole olemassakaan
ja lyhyt antibioottihoito riittää hoidoksi. He olivat mm. laatimassa
parhaillaan tutkinnan alla olevia IDSA:n hoitosuosituksia.


Clin Infect Dis. 2009 Nov 30; [Epub ahead of print]

2-Tiered Antibody Testing for Early and Late Lyme Disease Using Only an
Immunoglobulin G Blot with the Addition of a VlsE Band as the Second-Tier Test.

Branda JA, Aguero-Rosenfeld ME, Ferraro MJ, Johnson BJ, Wormser GP, Steere AC.

Departments of 1Pathology and 2Medicine, Massachusetts General Hospital and
Harvard Medical School, Boston, Massachusetts; Departments of 3Pathology and
4Medicine, Division of Infectious Diseases, New York Medical College, Valhalla,
New York; 5Division of Vector-Borne Infectious Diseases, Centers for Disease
Control and Prevention, Fort Collins, Colorado.

Background. Standard 2-tiered immunoglobulin G (IgG) testing has performed well
in late Lyme disease (LD), but IgM testing early in the illness has been
problematic. IgG VlsE antibody testing, by itself, improves early sensitivity,
but may lower specificity. We studied whether elements of the 2 approaches could
be combined to produce a second-tier IgG blot that performs well throughout the
infection.

Methods. Separate serum sets from LD patients and control subjects
were tested independently at 2 medical centers using whole-cell enzyme
immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG
blots. The results from both centers were combined, and a new second-tier IgG
algorithm was developed.

Results. With standard 2-tiered IgM and IgG testing,
31% of patients with active erythema migrans (stage 1), 63% of those with acute
neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late
neurologic involvement (stage 3) had positive results. Using new IgG criteria,
in which only the VlsE band was scored as a second-tier test among patients with
early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage
3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those
with stage 3 infection had positive responses. Both new and standard testing
achieved 100% specificity.

Conclusions. Compared with standard IgM and IgG
testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM
testing; it provides comparable or better sensitivity, and it maintains high
specificity.


http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 19947857 [PubMed - as supplied by publisher]

[soijuv]

Uudet, erittäin herkät, diagnostiset testausmenetelmät ovat osoittaneet, että punkkien välittämät taudit kuten anaplasmoosi ja babesioosi aiheuttavat useimmiten pitkäkestoisen infektiotaudin. Taudinaiheuttajat muuntavat jatkuvasti pintarakenteitaan selviytyäkseen.

Onderstepoort J Vet Res. 2009 Mar;76(1):53-8.

Persistence mechanisms in tick-borne diseases.

Barbet AF.

Department of Infectious Diseases & Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

The use of new, highly sensitive diagnostic methods has revealed persistent infections to be a common feature of different tick-borne diseases, such as babesiosis, anaplasmosis and heartwater. Antigenic variation can contribute to disease persistence through the continual elaboration of new surface structures, and we know in several instances how this is achieved. Known or suspected mechanisms of persistence in babesial parasites include cytoadhesion and rapid variation of the adhesive ligand in Babesia bovis and genetic diversity in several merozoite stage proteins of different Babesia spp. In Anaplasma, extensive variation in the pfam01617 gene family accompanies cycling of organism levels in chronic infection. One result from the pioneering research at Onderstepoort is the definition of a related polymorphic gene family that is likely involved in immunity against heartwater disease. We are beginning to understand the sizes of the antigenic repertoires and full definition is close, with the possibility of applying simultaneous high-throughput sequencing to the order of 1000 small genomes. We also, for the first time, can consider modifying these genomes and looking at effects on persistence and virulence. However, important biological questions remain unanswered; for example, why we are seeing a new emerging Anaplasma infection of humans and is infection of endothelial cells by Anaplasma significant to persistence in vivo.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 19967928 [PubMed - in process]

[soijuv]

Borreliabakteeria löydettiin PCR-testillä muutamista morphea, lichen sclerosus ja granuloma annulare -ihonäyttteistä. Saksa 2009.

Borrelia in granuloma annulare, morphea and lichen sclerosus: a PCR-based study and review of the literature

Journal of Cutaneous Pathology, 12/21/09
10.1111/j.1600-0560.2009.01493.x

Therese Zollinger 1 , Kirsten D. Mertz 1 , Mirka Schmid 1 , Anja Schmitt 1,2 , Madeleine Pfaltz 1 and Werner Kempf 1,3
1 Kempf und Pfaltz Histologische Diagnostik, Zürich, Switzerland , 2 Department of Pathology, Institute for Surgical Pathology, University Hospital Zürich, Zürich, Switzerland , and 3 Department of Dermatology, University Hospital Zürich, Zürich, Switzerland

Correspondence to Werner Kempf, MD, Kempf and Pfaltz Histologic Diagnostics, Schaffhauserplatz 3, CH-8006, Zürich, Switzerland
Tel: +41 44 233 3377
Fax: +41 44 233 3378
e-mail: kempf@kempf-pfaltz.ch

ABSTRACT

Background: Morphea, granuloma annulare (GA) and lichen sclerosus et atrophicans (LSA) have also been suggested to be linked to Borrelia infection. Previous studies based on serologic data or detection of Borrelia by immunohistochemistry and polymerase chain reaction (PCR) reported contradictory results. Thus, we examined skin biopsies of morphea, GA and LSA by PCR to assess the prevalence of Borrelia DNA in an endemic area and to compare our results with data in the literature.

Methods: Amplification of DNA sequences of Borrelia burgdorferi sensu lato by nested PCR from formalin-fixed and paraffin-embedded skin biopsies of morphea, GA and LSA, followed by automated sequencing of amplification products. PCR-based studies on Borrelia species in these disorders published until July 2009 were retrieved by a literature search.

Results: Borrelia DNA was detected in 3 of 112 skin biopsies (2.7%) including one of 49 morphea biopsies (2.0%), one of 48 GA biopsies (2.1%) and one of 15 LSA biopsies (6.6%). Amplification products belonged to B. burgdorferi sensu stricto in two cases available for sequence analysis.

Conclusions: The results of our and most of other PCR-based studies do not argue for a significant association of B. burgdorferi sensu lato with morphea, GA, LSA.

[soijuv]

"Dilatoiva kardiomyopatia on kolmanneksi yleisin sydänkuolleisuuden ja sydänsiirtojen syy. Sen syynä saattaa olla esim. borreliabakteeri. Serologisten testien perusteella borrelioosidiagnoosia ei voida tehdä. Ne kykenivät osoittamaan IgG-luokan vasta-aineita ainoastaan kahdessa tapauksessa eivätkä IgM luokan vasta-aineita yhdessäkään tapauksessa. 21 %:lla dilatoivaa kardiomyopatiaa sairastavista potilaista löydettiin koepalatutkimuksessa borreliabakteereita sydänlihaksesta.(Tsekki 2010)."


"Serological examination does not provide a tool for diagnosing the disease [Lyme]"

: Med Microbiol Immunol. 2010 Jan 6; [Epub ahead of print]
Presence of Borrelia burgdorferi in endomyocardial biopsies in patients with new-onset
unexplained dilated cardiomyopathy.

Palecek T, Kuchynka P, Hulinska D, Schramlova J, Hrbackova H, Vitkova I, Simek
S, Horak J, Louch WE, Linhart A.

1st Medical Faculty, 2nd Medical Department - Clinical Department of Cardiology
and Angiology, Charles University of Prague, Prague, Czech Republic,
tpalec@lf1.cuni.cz.

Dilated cardiomyopathy (DCM) represents the third most common cause of heart
failure and the most frequent cause of heart transplantation. Infectious, mostly
viral, and autoimmune mechanisms, together with genetic abnormalities, have been
reported as three major causes of DCM. We hypothesized that Lyme disease (LD),
caused by spirochete Borrelia burgdorferi (Bb), might be an important cause of
new-onset unexplained DCM in patients living in a highly endemic area for LD
such as the Czech Republic.

We performed endomyocardial biopsy (EMB) in 39
consecutive patients presenting with symptomatic unexplained left ventricular
(LV) systolic dysfunction lasting no more than 12 months. In eight subjects
(21%), Bb was detected in the EMB sample by polymerase chain reaction or by
electron microscopy. None of these patients exhibited any form of
atrioventricular block or other extracardiac manifestation of Bb infection.

Serological testing identified IgG antibodies against Bb in only two cases and
IgM antibodies in none. All affected patients were treated with intravenous
ceftriaxone for 3 weeks. At 6 months follow-up, LV morphology and function as
well as functional status of these patients significantly improved.
In conclusion, Bb infection may represent an important cause of new-onset
unexplained DCM in patients living in endemic regions such as the Czech
Republic. Because the antibiotic treatment appears to be markedly effective and
serological examination does not provide a tool for diagnosing the disease, EMB
focused on the detection of Bb should be performed in all patients from endemic
areas with new-onset unexplained DCM not responding to conventional therapy.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20052487 [PubMed - as supplied by publisher]

[soijuv]

Uusi (2010) testi useiden eri taudinaiheuttajien testaamiseen (DNA) esim. borreliabakteerin.
Suom. huom. Testi ei todennäköisesti ole käytössä Suomessa.

(WO/2008/147879) AUTOMATED METHOD AND DEVICE FOR DNA ISOLATION, SEQUENCE DETERMINATION, AND IDENTIFICATION

WO 2008147879 20081204

AUTOMATED METHOD AND DEVICE FOR DNA ISOLATION, SEQUENCE DETERMINATION, AND IDENTIFICATION

Related Information This application claims the benefit of U.S. Provisional Patent Application Serial

No. 60/931,285, filed May 22, 2007, the contents of this and any patents, patent applications, references, cited throughout this specification are hereby incorporated by reference in their entireties.

Background of the Invention

The rapid identification of the nucleic acid sequences present in a complex biological sample has many practical applications. For example, the ability to rapidly identify the presence of pathogens in a biological sample, via their DNA or RNA signature, would be of enormous importance for the identification of hazardous bioagents or the diagnosis of disease in human patients.

The majority of current methods for pathogen identification require specimen culturing or detection with pathogen-specific antibodies, both of which are not possible for all types of infections. Molecular diagnostic methods involve detecting the hybridization of pathogen DNA or RNA present in the sample to known probes using DNA chips. Such methods are limited to the detection of known pathogens thus, as pathogens mutate, the pathogenic DNA may no longer hybridize to existing probes and new probes must be developed. Alternative methods of pathogen identification include nucleic acid sequencing of DNA or RNA present in the sample. However, current sequencing methodologies for pathogen identification are based on Sanger DNA sequencing which requires both amplification of the target nucleic acid and allows only a single nucleotide sequence to be identified from each sequencing reaction. Sanger sequencing is performed on a single known DNA fragment of interest. Thus, amplification and sequencing of the target nucleic acid implies a priori knowledge of the pathogen contained within the sample. Moreover, none of these current detection methods are capable of seamless, integrated operation.

There is therefore a need in the art for alternative methods and devices for the rapid identification of nucleic acid sequences present in biological samples.

Summary of the Invention The present invention provides novel methods, software and devices for the rapid identification of any nucleic acid sequence or nucleic acid-containing bioagent present in a biological sample. The present invention involves: a) isolating nucleic acid from a biological sample; b) sequencing the nucleic acid within the sample using single-

molecule sequencing technology; and c) analyzing the derived nucleic acid sequences by comparison to reference sequence(s), for example, in a database.

The present invention has many uses in areas that would require a rapid and integrated molecular diagnostic identification system. The present invention allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. Thus, the invention is useful in a wide variety of fields, including, but not limited to, medical diagnosis and pharmacogenetic analysis (including: diagnosis of infectious diseases and conditions; cancer diagnosis based on mutations and polymorphisms; drug resistance and susceptibility testing; screening for and/or diagnosis of genetic diseases and conditions), germ warfare (allowing immediate identification of the bioagent and appropriate treatment), environmental testing (e.g., detection and discrimination of pathogenic vs. non-pathogenic bacteria in soil, water or other samples), agricultural testing (e.g., detection of livestock infection, produce contamination), veterinary testing, and forensics (e.g., rapid detection of bioagents for molecular fingerprinting).

The present invention can be used to detect and classify any bioagent containing nucleic acid (e.g., DNA), including bacteria, viruses, fungi and toxins. As one example, where the bioagent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling), DNA mutations (preventing DNA chip or primer hybridization) and emerging infectious diseases. Accordingly, the invention has several advantages that include, but are not limited to, the following, providing integrated methods for the rapid identification of any nucleic acid sequence or nucleic acid-containing biological organisms present in a complex biological sample directly from the sample without the need for amplification of the nucleic acid; providing software for the identifying the source organism of any deduced nucleic acid sequence; and providing devices capable of performing the integrated processing of complex biological samples to determine the identity and predicted source of any nucleic acid present.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Figures

Figure 1. Depicts an environment suitable for practicing an embodiment of the present invention;

Figure 2. Depicts an alternative distributed environment suitable for practicing an embodiment of the present invention;

Figure 3. Depicts a flowchart of a sequence of steps that may be followed by an embodiment of the present invention to predict bioagents present in a nucleic acid sequence isolated from a biological sample and subjected to a single molecule sequencing operation.

Detailed Description of the Invention

In order to provide a clear understanding of the specification and claims, the following definitions are provided below.

Definitions So that the invention may be more readily understood, certain terms are first defined.

The term "bioagent" refers to any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.

The term "sample" refers to any form of matter capable of containing a bioagent. Examples of samples include, but are not limited to, blood, animal tissue, sputum, urine, cell culture medium, water, leaf spot, soil, plant tissue, paleontology samples, forensic samples, water, food, and powders.

The term "nucleic acid" and "single-stranded nucleic acid" refers to RNA or RNA containing molecules as well as DNA or DNA containing molecules. The term RNA refers to a polymer of ribonucleotides. The term "DNA" or "DNA molecule" or deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single- stranded (i.e., ssRNA and

ssDNA, respectively), or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively), i.e., duplexed or annealed.

The term "nucleic acid sequence" refers to the ordering of the individual nucleotides in a DNA or RNA polymer. The term "single-molecule sequencing" refers to any method of determining the sequence of an individual nucleic acid molecule without the need for prior amplification. The term "compare", when used with respect to nucleic acid sequences, refers to the alignment of one or molecule nucleic acid sequences to establish a percentage identity or similarity (identity and similarity will be used interchangeably) using, for example, a mathematical algorithm. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions (+ # of substitutions for bases or amino acids)/total # of positions x 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced. The alignment can be generated over a certain portion of the sequence (i.e., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. ScL USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. ScL USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) /. MoI. Biol. 215:403-10. The alignment can be optimized by introducing appropriate gaps and percentage identity determined over the length of the aligned sequence (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. Another global alignment algorithm is that of Needleman-Wunsch, (1970) /. MoI. Biol. 48:443-453.

Various aspects of the invention are described in further detail in the following subsections.

/. Overview The present invention provides novel methods, software algorithms and devices for the rapid identification of any nucleic acid sequence or nucleic acid-containing bioagent present in a biological sample. The present invention involves: a) isolating nucleic acid from a biological sample; b) sequencing the totality of nucleic acid within the sample using single-molecule sequencing technology; and c) analyzing the derived nucleic acid sequences by comparison to a database.

In one aspect, the invention provides methods for the identification of any nucleic acid sequence or nucleic acid-containing bioagent present in a biological sample. In one embodiment, a sample suspected of containing a bioagent capable of causing a disease or disorder is obtained. In a related embodiment, a blood sample is obtained from a human patient suspected of having contracted an infectious, bioagent- induced disease. The total nucleic acid content of the either sample is extracted from the sample by art-recognized means and subject to a single-molecule sequencing reaction. The resultant nucleic acid sequence data is then searched against reference sequences in databases using a software algorithm and the predicted source of the nucleic acid reported.

In another aspect, the invention provides a physical medium that holds computer- executable instructions for identifying bioagents present in a biological sample. The medium holds instructions for receiving at least one result of a single molecule sequencing reaction conducted on nucleic acid in a biological sample. The medium further holds computer-executable instructions for comparing the received nucleic acid sequence obtained from the single molecule sequencing reaction to one or more reference sequences contained in a database in order to predict at least one bioagent present in the biological sample.

In another aspect, the invention provides devices for the identification of any nucleic acid sequence or nucleic acid-containing bioagent present in a biological sample. In one embodiment a device is contacted with a sample and said device performs all the combined functions of the invention in an integrated manner i.e., nucleic acid extraction, single-molecule sequencing, database searching and source organism reporting.

In another aspect, the invention provides a means to acquire patient- specific, as well as general, population-based data concerning the genetic basis of diseases and disorders.

In another aspect, the invention provides a means to acquire gene expression analysis data indicative of a change in physiological status of an organism.

In another aspect, the invention provides a means to acquire epidemiological data.

In another aspect, the invention provides methods performing pharmacogenomics. In another aspect, the invention provides a means for testing livestock animals for diseases such as foot and mouth, and mad cow disease.

2. Selecting a Biological Sample

The present invention provides methods and devices for the identification of nucleic acid molecules contained within a biological sample. Exemplary samples include, but are not limited to, blood, animal tissue, sputum, urine, cell culture medium, water, leaf spot, soil, plant tissue, paleontology samples, forensic samples, water, food or any form of matter capable of containing bioagents or nucleic acid. Several independent sources of nucleic acid may exist in the sample. In the case of human blood, human DNA and RNA will be present in white blood cells, in addition to the nucleic acid present in any infectious bioagents that may be present.

3. Bioagents

The present invention provides methods and devices for the identification of bioagents via the presence of their nucleic acids. In the context of the present invention, a "bioagent" is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered.

Bacterial biological warfare bioagents capable of being detected by the present methods include, but are not limited to, Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders),

Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, Borrelia burgdorferi (Lyme disease), and Vibrio cholerae.

Biological warfare fungus bioagents include, but are not limited to, coccidioides immitis (Coccidioidomycosis).

Biological warfare toxin genes capable of being detected by the methods of the present invention include but not limited to botulism, T- 2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin, and saxitoxin. Biological warfare viral bioagents are mostly RNA viruses (positive-strand and negative- strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases.

Examples of (-)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbilli virus).

Examples of (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue viruses, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus).

4. Nucleic Acid Extraction

The present invention can employ at least partial purification of target nucleic acid molecules. All methods of art recognized nucleic acid extraction and purification are contemplated. Exemplary methods include those commercialized by QIAGEN or PROMEGA. Nucleic acid purification on nanoengineered surfaces, as exemplified in U.S. patent application US20060166223), is also contemplated. In cases where biological samples are desiccated, where necessary, the sample with be solublized using appropriate art recognized solvents to facilitate nucleic acid extraction.

5. Single Molecule Sequencing

The present invention involves nucleic sequencing at the single molecule level. Several art-recognized methods of single-molecule sequencing have been developed (see U.S. patent application US2006000400730 and U.S. patents 7,169,560; 6,221,592; 6,905,586; 6,524,829; 6,242,193; 6,221,592; and 6,136,543). Single molecule sequencing is a powerful tool capable of elucidating sequence-specific information on a single nucleic acid template. The ability to conduct single template sequencing allows the identification of subtle, often rare event, changes in nucleic acids that are important as the underlying basis for diseases such as cancer and others.

Single molecule sequencing also provides the ability to rapidly analyze a multitude of single nucleic acid templates, from a single sample, in parallel and with a high degree of precision. Using an isolated nucleic acid sequence as the substrate, individual labeled nucleotides are added sequentially by a polymerase to a growing complement strand. A label is detected as each nucleotide is added to the strand and the template sequence is determined.

In one embodiment, the invention comprises exposing a nucleic acid primer to a template sequence in the presence of a polymerase and at least one labeled nucleotide base that is capable of hybridizing with a template nucleic acid downstream of the hybridized primer. Nucleotide bases may be selected from the common Watson-Crick bases, adenine, thymine, cytosine, guanine, and uracil, or may be modifications of those bases, such as peptide nucleic acids, ribonucleotides, or nucleotides modified to incorporate a detectable label (e.g., with linkers or adapters). As each nucleotide is added to the growing complement strand, its label is detected and its position on the template is noted. Once a sufficient number of nucleotides have been incorporated, a sequence is determined. Methods of the invention facilitate rapid whole genome sequencing. Methods of the invention, however, also contemplate partial genome sequencing to obtain template or fingerprint sequences, thereby facilitating even more rapid sequence comparisons. Suitable nucleic templates include DNA, RNA and RNA/DNA hybrids.

In another embodiment, the invention comprises passing a single-stranded nucleic acid through a nano-pore. As the ssDNA travels through the nano-pore, the ssDNA passes over 4 nano-probes each containing one of the four nucleotide bases. Each time a probe hybridizes with the ssDNA, the signal is detected and the template sequence is determined.

6. Devices

In another aspect, the present invention provides devices for the identification of nucleic acid molecules and nucleic acid-containing bioagents contained within a biological sample. In one embodiment, the device contains an integrated means of nucleic acid purification, single molecule sequencing, and sequence analysis.

Embodiments where any one or more of the aforementioned functions are performed outside or remote from the device are also contemplated. In another embodiment the device is portable, preferably handheld. In another embodiment, the device may also include a microfabricated biopsy instrument as exemplified in U.S. patent application 2003/0119176A1. In another embodiment, the device connects wirelessly to a computer. In another embodiment, the device is part of a remotely controlled vehicle. In another embodiment, the device is capable of being operated by remote control. In another embodiment, the device is disposable. In another embodiment the device is biodegradable. In another embodiment, the device is designed and/or packaged for home use, hospital use, or police/military use.

7. Genomic DNA Analysis

In another aspect, the present invention allows for the acquisition of patient- specific, as well as general, population-based data concerning the genetic basis of diseases and disorders. Cancer is an example of a disease or disorder that has a strong genetic basis. Complete sequencing of large numbers of tumors using single molecule sequencing provides a catalog of somatic cell mutations (including, without limitation, deletions, additions, amplifications, rearrangements, substitutions, losses, translocations, methylation, and other alterations of genomic DNA) that are useful to diagnose, evaluate, prognose, and treat patients. A catalog of disease-related mutations and other alterations is a powerful diagnostic tool useful to rapidly categorize samples sequenced from future patients. Moreover, single molecule sequencing allows one to identify previously-unknown mutations that may be associated with cancer. Finally, single molecule sequencing on pooled samples allows rapid identification of deletions, amplifications, and other changes that are indicative of cancer, even if the specific mutational change is not known.

Analysis of genomic DNA using single molecule sequencing provides an approach that allows rapid identification of a genomic change present in a sample in low amounts. The ability to quickly and accurately perform rare-event detection is of great significance for the early diagnosis of cancer. Many cancers, if detected early, are treatable, and if detected too late may not be treatable. Cancer begins as somatic cell mutations accumulate in a very small initial population of cells. In samples typically obtained for genomic analysis, cancer or precancer cells are in very low abundance

compared to healthy somatic cells. Bulk mutation detection mechanisms typically fail to detect these rare event changes. A digital technique, such as single molecule sequencing, allows the sequencing through mutations in multiple single templates rapidly. This, in turn, allows the detection of the rare-event mutations underlying cancer or precancer. In one embodiment of the invention, tumor DNA is obtained and prepared using standard methods. Approximately 10 times coverage of each genomic region is sequenced. Using single molecule sequencing, the genome of the cancer tissue is rapidly sequenced. Mutations, insertions, deletions, rearrangements, and other alterations present in the tumor DNA are detected. Sequence assembly is accomplished using standard alignment techniques, such as BLAST (www.ncibi.nlm.nih.gov), incorporated by reference herein. Tumor sequences are compared to known sequences for either normal or cancer tissue or to consensus sequences in order to identify changes associated with cancer. Newly discovered genomic changes (i.e., those not previously associated with cancer) are cataloged and become known to be associated with a particular disease over time. Thus, patients are rapidly and accurately diagnosed based upon their individual genomic complement, either before or at the time of symptomatic- presentation of a disease.

In another embodiment of the invention, DNA is isolated from a patient's tumor or other diseased sample and is compared to normal DNA from the same patient. Whole genome sequencing of both the tumor and normal DNA may be done rapidly on a parallel basis using single molecule sequencing as described above. Alternatively, only portions of the genome are sequenced and compared. Genome portions of interest include, for example, sequences associated with a known or candidate tumor suppressor gene or oncogene, or intronic sequences containing repeats that are susceptible to amplification by defective cellular machinery. Following sequence determination, a comparison is made between tumor and normal sequence. Differences between the tumor and normal sequences are identified as tumor-related mutations. In effect, any difference between the two likely is indicative of disease because all somatic cells should have the same sequence. Detection of a variation from the normal somatic cell sequence, indicating that a population of cells containing abnormal sequences is present, results in a positive diagnosis. Alternatively, patient tumor sequence may be compared to a normal banked or consensus sequence instead of the patient's own normal DNA.

In another embodiment broad-based disease susceptibility testing is performed using single molecule sequencing on pooled genomic samples. For example, in a large population, the number of positive samples (i.e., those with a mutation present) is relatively small. Bulk sequencing likely would not detect mutations in pooled samples. Using high-resolution single molecule sequencing, however, any positive sample is detected with digital precision. Thus, according to the invention, genomic samples from

a predetermined number of patients (the number of patients does not matter for purposes of the invention) are collected, pooled and sequenced using single molecule sequencing techniques as described above. Single molecule sequencing is done through large tracts of the genome, and mutations derived from any source are detected in the pooled sample. To determine the source of a mutation or mutations, the original collection of individual patient samples is divided in half, re-pooled, and resequenced. This process continues until a unique identification of the affected patient or patients is possible. Due to the rapidity of single molecule sequencing, it is possible to perform multiple sequencing steps in a matter of minutes, hours or days. Using single molecule sequencing, pooled sequences, when compared to a consensus sequence, readily identify losses or amplifications in genomic DNA. All somatic cells will have not only the same sequence but will also be present in the same amounts. Deviations are detected using single molecule sequencing with fewer cells than in bulk sequencing because individual DNA molecules are sequenced instead of an amalgam of cells that typically provide the basis for bulk sequencing assays as, for example, in assays for loss of heterozygosity. In a related embodiment, data from a pooled experiment is useful for determining the frequency and distribution of mutations in a given population, without identifying the owners of specific mutations.

The rapid results provided by the invention also allow sequencing to detect familial mutations. For example, if it is determined that a patient has a mutation indicative of a cancer, certain forms of which have a strong familial link (e.g., breast cancer, colon cancer), primary siblings typically are not tested unless specified criteria are met. Single molecule sequencing not only identifies the underlying mutation in the primary patient, but allows rapid, cost-effective sequencing of relatives who also might carry the mutation.

The invention is also useful to perform tumor typing. Tumor typing may involve determining a genetic profile for a particular patient's tumor in order to guide treatment or other decisions. For example, the standard treatment for patients with colon cancer is the drug 5-Fluorouracil (5FU). Although 5FU works to reduce tumors in many colon cancer patients, it actually accelerates tumor growth in a class of patients who have Hereditary Non-Polyposis Colorectal Cancer (HNPCC). HNPCC is a familial form of colon cancer with a distinct genetic profile that is ascertainable by sequencing cellular DNA. Thus, to avoid tumor acceleration in potential HNPCC patients, it is particularly important to know a colon cancer patient's genetic profile in order to determine the most effective treatment for that patient. Single molecule sequencing is useful to make that determination because it is rapid, reliable, and effectively digital, therefore promptly indicates the presence or absence of the relevant genetic event(s). Methods of the

invention make possible the rapid and accurate identification of tumor-related mutations, thus an appropriate treatment may be selected or an inappropriate treatment avoided.

8. Expression Analysis In another aspect, the invention provides gene expression analysis data.

Alteration in expression constructs is often indicative of a change in physiological status. Changes in expression patterns reflect cellular activities as well as disease state. Expression sequence analysis provides insight into the specialized activities of cells from different organs or of different types. Thus, expression analysis reveals aspects of the immune repertoire that are not apparent on a gross level. In one embodiment of the invention, a sequence determination is made with respect to the total antibody repertoire expressed by B-cells. In another embodiment of the invention, a sequence determination is made with respect to the T-cell receptor repertoire expressed by T-cells. Single molecule sequencing offers rapid, high-throughput sequencing that reveals specific detail as to which immune cells are active, and the likely epitopes against which they function. Single molecule sequencing also provides an immune fingerprint that is used to identify an infection based upon the specifics of a patient's immune response. The immune fingerprint generated using single molecule sequencing is compared to a database of collected immune sequence data in order to identify an infection. New infections are tracked through the appearance of new sequence specificities either alone or in combination with other diagnostic techniques. Isolation of immune cells is well- known in the art, and application of the present invention to sequencing a patient's immune cell complement is contemplated by the present invention.

9. Epidemiology

In another aspect the invention also provides epidemiological data. In a preferred embodiment, an appropriate patient sample is obtained and DNA in the sample is sequenced. Optionally, the patient's genomic DNA is excluded. A catalog is compiled comprising a fingerprint of the DNA (or RNA in other preferred embodiments) present in samples obtained from a multiplicity of patients. Each patient's disease status then is correlated with specific sequence information obtained from the patient's sample. In this way, diagnostic accuracy and verifiability is improved, as a patient's disease status is confirmed by comparing the patient's DNA to sequences in the database. As mentioned above, whole genome sequencing is optional. In some circumstances, it is necessary only to sequence sufficient nucleic acid to establish a fingerprint for comparison with future samples. In one embodiment, ubiquitous epidemiology is performed. In this case patient DNA is routinely sequenced and stored for disease identification and comparison with future samples to identify and track new disease outbreaks. For example, a patient

who presents with a new DNA profile (i.e., containing a sequence that is not in the database) may be diagnosed with a new condition. Future patients presenting with the same nucleic acid profile are tracked. In this way, potential epidemic outbreaks are controlled. With respect to new diseases, no a priori assumptions are necessary. A novel sequence will immediately be identified as such, and appropriate monitoring can be put in place.

10. Pharmacogenomics

In another aspect, the invention provides methods and devices for performing pharmacogenomics. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a therapeutic agent as well as tailoring the dosage and/or therapeutic regimen of treatment with a therapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266 In one embodiment, the methods of the invention provide information regarding patient genome sequence which is used to select patients or patient subpopulations for treatment with FDA-approved therapies e.g., antibody, small molecule or peptide therapies.

//. Hardware and Software Environment

As noted above, the embodiments of the present invention programmatically analyze the results of a single molecule sequencing reaction in order to predict bioagents present in a biological sample. Figures 1-3 discuss aspects of the hardware and software environment utilized by the present invention to perform the bioagent prediction.

Figure 1 depicts an environment suitable for practicing an embodiment of the present invention. A computing device 102 holds a database 104 or other storage structure containing reference sequences 105 and an analysis facility 106. The computing device 102 may be a server, workstation, laptop, personal computer, PDA or other computing device equipped with one or more processors and able to execute the analysis facility 106 discussed herein. The analysis facility 106 is preferably implemented in software although in an alternate implementation, the logic may be also be implemented in hardware. The analysis facility 106 operates on and analyzes results

of single molecule sequencing reactions 122 that are received from a biological sample acquisition apparatus 120. The biological sample acquisition apparatus conducts single molecule sequencing operations on nucleic acid isolated from a biological sample. In one embodiment, the biological sample acquisition apparatus 120 is a handheld device in wireless communication with the computing device 102. The analysis facility 106 programmatically compares the results of the single molecule sequencing reaction 122 to the reference sequences 105 contained in the database 104 in order to generate a listing of predicted bioagents 144 that are present in the biological sample under consideration. In one implementation, the comparison of the results of the single molecule sequencing operation 122 to the reference sequences 105 in order to predict bioagents present in a biological sample is performed programmatically without any user input. In alternate implementations, the analysis facility 106 prompts a user for parameters controlling the comparison via the user interface 142.

The listing of the predicted bioagents 144 may be displayed to a user via a user interface 142 displayed on a display device 140 that is in communication with the computing device 102. It will be appreciated that the listing of predicted bioagents 144 may also be stored for later use and/or display to a user. The user interface 142 may also be utilized to enable a user to configure the parameters of the comparison operation performed by the analysis facility 106. Those skilled in the art will recognize that many other configurations are also possible within the scope of the present invention. Figure 2 depicts an alternative distributed environment 200 suitable for practicing an embodiment of the present invention. A first computing device 202 may be used to execute an analysis facility 204. The first computing device 202 may communicate over a network 250 with a second computing device 210 holding reference sequences 212. The network 250 may be the Internet, a local area network (LAN), a wide area network (WAN), an intranet, an internet, a wireless network or some other type of network over which the first computing device 202 and the second computing device 210 can communicate. The analysis facility 204 on the first computing device 202 may communicate over the network 250 with a biological sample acquisition apparatus 230 that generates results data 232 from a single molecule sequencing reaction performed on nucleic acid isolated from a biological sample. The analysis facility 204 may store a listing of predicted bioagents that is generated by a comparison of the results of the single molecule sequencing reaction and the reference sequences 212. The storage may occur on the first computing device 202 or at a location remote from the first computing device that is accessible over the network 250. Alternatively, the listing of predicted bioagents may be displayed to a user. It should be recognized that Figure 2 depicts only a single distributed configuration and many other distributed configurations are possible within the scope of the present invention.

Figure 3 is a flowchart of a sequence of steps that may be followed by an embodiment of the present invention to predict bioagents present in a biological sample. The sequence begins by providing a biological sample (step 302). The sample may be a previously acquired sample or may be a sample that is obtained immediately in advance of the bioagent prediction process that is discussed herein being performed. Nucleic acid is then isolated from the biological sample (step 304) and a single molecule sequencing reaction is conducted on the isolated nucleic acid (step 306) as discussed above. The results of the single molecule sequencing reaction are compared to reference sequences (step 308) and a listing of predicted bioagents that are present in the biological sample is generated. The listing of predicted bioagents may then be displayed to a user or stored for later retrieval (step 310).

Embodiments of the present invention may be provided as one or more computer-readable programs embodied on or in one or more mediums. The mediums may be a floppy disk, a hard disk, a compact disc, a digital versatile disc, a flash memory card, a PROM, an MRAM, a RAM, a ROM, or a magnetic tape. In general, the computer-readable programs may be implemented in any programming language. Some examples of languages that can be used include FORTRAN, C, C++, C#, Python, Perl or Java. The software programs may be stored on or in one or more mediums as object code. Hardware acceleration may be used and all or a portion of the code may run on a FPGA, an Application Specific Integrated Processor (ASIP), or an Application Specific Integrated Circuit (ASIC). The code may run in a virtualized environment such as in a virtual machine. Multiple virtual machines running the code may be resident on a single processor.

Since certain changes may be made without departing from the scope of the present invention, it is intended that all matter contained in the above description or shown in the accompanying drawings be interpreted as illustrative and not in an exclusive sense. Practitioners of the art will realize that the sequence of steps and architectures depicted in the figures may be altered without departing from the scope of the present invention and that the illustrations contained herein are singular examples of a multitude of possible depictions of the present invention.

12. Other Applications of the Technology of the Invention

In another aspect, the methods and devices disclosed herein can be used to screen fetal mRNA or DNA, present in maternal blood, for disease-associated mutations as an alternative to amniocentesis.

In another aspect, the invention provides nucleic acid sequence information for making diagnostic kits, or chips.

In another aspect, the nucleic acid sequence information or methodology disclosed herein can be used for forensic applications.

In another aspect, the methods and devices disclosed herein can be used for research purposes, for example genetic research on the distribution or migration of human populations.

In another aspect, the methods and devices disclosed herein can be used in paleontology, for example to identify and catalogue nucleic acid sequences contained in ancient biological samples.

In another aspect, the methods and devices disclosed herein can be used for environmental analysis to determine the bioagent profile of a particular of ecosystem.

In another aspect, the methods and devices disclosed herein can be used in agriculture. In one embodiment the methods and devices of the invention are used to determine the bioagent profile of soil. In another embodiment the methods and devices of the invention are used to determine the nucleic acid sequences present in plant samples and thereby assess whether they have been infected with disease-causing bioagents or have been modified by genetic engineering.

In another aspect, the methods and devices disclosed herein are used to determine genetic fingerprinting information about a subject and thereby uniquely identify them. In another aspect, the invention provides business methods for commercializing nucleic acid sequences suitable for use in, for example, the making of devices, diagnostic chips, kits, networks, and pharmaceuticals for diagnosing and treating disease.

Exemplification

Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods

In general, the practice of the present invention can employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, PCR technology, immunology, cell culture, and any necessary computer or electronic related technology that are within the skill of the art and are explained in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning, VoIs. 1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (MJ. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al, Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., John Wiley & Son Ltd (1996); The PCR Technique: RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998).

EXAMPLE 1

A METHOD FOR DETECTING A BIOAGENT IN A PATIENT SUSPECTED OF HAVING CONTRACTED AN INFECTIOUS BIOAGENT-INDUCED DISEASE

The following example describes a novel method for determining the presence of an unknown bioagent in a patient sample. A patient sample is obtained. If necessary several types of sample encompassing all potential areas of infection may be obtained and processed together.

Nucleic acid is extracted from the sample(s) using art recognized means. A single- nucleotide sequencing of the nucleic acid is then performed to determine the sequences of nucleic acids present in the sample. The deduced nucleic acid sequence data is compared against databases of known nucleic acid sequences, for example, using a mathematical algorithm and the percentage identity with known sequences is reported.

From these sequence identities all bioagents present in the sample are deduced and known infectious bioagents identified.

EXAMPLE 2.

A DEVICE FOR DETECTING A BIOAGENT IN A PATIENT SUSPECTED OF HAVING CONTRACTED AN INFECTIOUS BIOAGENT-INDUCED DISEASE The following example describes a novel device for determining the presence of an unknown bioagent in a patient sample.

A patient sample is obtained. If necessary several types of sample encompassing all potential areas of infection may be obtained and processed together. The sample is contacted with a device which proceeds to perform the following steps in an integrated operation: a) isolate nucleic acid from the sample in sufficient purity to perform single - nucleotide sequencing; b) perform single-nucleotide sequencing to determine the sequences of all nucleic acids isolated from the sample; c) compare the deduced nucleic acid sequences against databases of known nucleic acid sequences using, for example, a mathematical algorithm to determine the percentage identity of the deduced nucleic acid with known sequences; d) report the best sequence matches for all known (infectious) bioagents.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[soijuv]

Tällä hetkellä käytössä olevat DNA-testit (PCR) eivät ole standardoituja. Uuden tutkimuksen mukaan bakteerin flagella-geenin löytyminen elimistöstä vahvistaa borreliabakteeritartunnan. (2010)

Suom.huom. Suomessa on vuosien ajan käytetty jonkunlaista PCR-testiä. Testitulosten luotettavuus?

J Med Microbiol 59 (2010), 309-314; DOI: 10.1099/jmm.0.013508-0
A comparative analysis of molecular markers for the detection and identification of Borrelia spirochaetes in Ixodes ricinus

Beata Wodecka, Agata Leonska and Bogumila Skotarczak

Department of Genetics, University of Szczecin, 71-065 Szczecin, Poland

Correspondence
Agata Leonska
agataleonska@op.pl

Received June 10, 2009
Accepted December 9, 2009

Borrelia burgdorferi sensu lato, carried by Ixodes ticks, is one of the most significant human pathogens, causing Lyme disease. As there is no standardized PCR method for detection and identification of spirochaete DNA, we carried out a comparative analysis using a set of complementary primers for three regions in the genomic DNA of these bacteria (genes fla and rrs and the non-coding rrs?rrlA region). DNA extracted from 579 Ixodes ricinus ticks was subjected to nested PCR. DNA of the examined spirochaetes was detected in 43 (7.4 %) lysates when the fla gene was used as a molecular marker, in 7 (1.2 %) lysates when using primers complementary to the rrs gene, and in 12 (2.1 %) lysates using primers complementary to the non-coding rrs?rrlA sequence. RFLP analysis based on the fla gene helped identify species from the B. burgdorferi sensu lato complex (B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana), detect co-infections, and also identify Borrelia miyamotoi. Therefore, the fla gene is the most sensitive and specific molecular marker for the detection and identification of Borrelia spirochaetes in I. ricinus.

The GenBank/EMBL/DDBJ accession numbers for the fla gene sequence of B. burgdorferi sensu stricto G112-04, B. afzelii D131-07, B. garinii G23-07, B. valaisiana G43-0507 and B. miyamotoi D110-07 are FJ518808, FJ518805, FJ518806, FJ518807 and FJ518804, respectively.

[Sailairina]

HUS luopui 1.2.2010 alkaen eräästä PCR-testistä sen epäluotettavuuden vuoksi...


http://huslab.fi/ohjekirjan_liitteet/tu ... alkaen.pdf

Uusi tutkimus 20697
B BorrNhO B Borrelia burgdorferi, nukleiinihapon osoitus

Lopetettava tutkimus
9399 S BorrNhO S Borrelia burgdorferi,osoitus PCRreaktiolla



"Tutkimuksen BorrNhO herkkyys on seeruminäytteestä huono. Seerumissa esiintyy vain harvoin vapaata mikrobin DNA:ta. Tutkimus S BorrNhO (9399) korvataan kokoveren tutkimuksella, B ?BorrNhO (20697)".

"Borrelia burgdorferi on kudoshakuinen ja esiintyy veressä vain harvoin runsaana."

[soijuv]

Patentin mukaan kyseessä on Flagella proteiini 41kDa fragmentteihin perustuva tutkimus joka soveltuu borrelioosin diagnostiikkaan ja hoitoon. Havaitsee bakteerin ihmisissä ja eläimissä. Mikäli testi on positiivinen henkilö sairastaa borrelioosia mikäli suun sairaudet ja kuppa on poissuljettu.


Patent on antibodies against 41 kDa Flaggellar protein fragments

TECHNICAL FIELD OF THE INVENTION

This invention relates to flagellin-based compositions and methods useful for the diagnosis and treatment of Lyme disease. More particularly, this invention relates to flagellin polypeptides which are useful to detect the presence of B. burgdorferi in humans and other animals, and which are useful to diagnose and treat neuroborreliosis, arthritis, carditis and other manifestations of Lyme disease. Also within the scope of this invention are antibodies directed against the flagellin polypeptides and diagnostic kits comprising the antibodies or polypeptides.

BACKGROUND OF THE INVENTION

Lyme borreliosis is the most common vector-borne infection in the United States [S. W. Barthold, et al., "An Animal Model For Lyme Arthritis", Ann. N.Y. Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans (ECM), which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.

The causative agent of Lyme disease is a spirochete known as Borrelia burgdorferi, transmitted primarily by ixodes ticks that are part of the Ixodes ricinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.

Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (ECM) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al., "Isolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migrans", J. Am. Acad. Dermatol., 3, pp. 444-49 (1985)]. Flu-like or meningitis-like symptoms are common at this time. Stage two occurs within days or weeks, and involves spread of the spirochete through the patient's blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., "Borrelia burgdorferi--Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitis", Ann Neurol., 24, pp 509-16 (1988)].

The neurologic manifestations of Lyme disease are protean and include weakness, peripheral nerve palsy, radiculitis, meningitis and encephalitis. The pathogenesis of neuroborreliosis, however, is unclear. Examination of the cerebrospinal fluid (CSF) of patients with neuroborreliosis shows a mononuclear pleocytosis and production of B. burgdorferi specific antibody, suggesting that inflammation is involved in disease. In some cases, spirochetes have been cultured from the CSF of patients with neurologic symptoms, implicating the organism directly with disease. Experiments using mice suggest that spirochete virulence may play a role in the development of neurologic infection.

Infection with B. burgdorferi induces a strong humoral immune response. Early in human infection, antibodies are generated primarily against the 41-kDa flagellar protein. In later stages, antibodies to the outer surface proteins OspA and OspB, among others, appear [J. E. Craft et al., "Antigens Of Borrelia burgdorferi Recognized During Lyme Disease", J. Clin. Invest., 78, pp. 934-39 (1986)].

Antibodies to the B. burgdorferi flagellin antigen remain prominent in patient serum during infection, and local CSF antibody production to spirochetal antigens, including flagellin, occurs. Further, the sera from patients with neurologic manifestations of Lyme disease have IgM antibodies that bind human axons; binding is weak or absent in patients without neurologic disease. The presence of high antibody titers in the CSF correlates with clinical signs of neuroborreliosis.

At present, all stages of Lyme disease are treated with antibiotics. Treatment of early disease is usually effective, however the cardiac, arthritic and nervous system disorders associated with the later stages often do not respond to therapy [A. C. Steere, "Lyme Disease", N. Engl. J. Med., 321, pp. 586-96 (1989)].

At present, Lyme disease is diagnosed primarily by serology. The enzyme-linked immunosorbent assay (ELISA) is one method of detection, using sonicated whole spirochetes as the antigen [J. E. Craft et al., "The Antibody Response In Lyme Disease: Evaluation Of Diagnostic Tests", J. Infect. Dis., 149, pp. 789-95 (1984)]. However, serologic testing is not yet standardized, and results may vary between laboratories and commercial kits, causing false negative and, more commonly, false positive results [S. Luger and E. Kraus, "Serologic Tests For Lyme Disease: Interlaboratory Variability," Arch. Int. Med., 150, pp. 761-63 (1990)]. In addition, available serologic tests have limited usefulness early in infection prior to the development of a measurable antibody response [R. Grodzickiano and A. C. Steere, "Comparison Of Immunoblotting And Enzyme Linked Immunosorbent Assay Using Different Antigen Preparations For Diagnosing Early Lyme Disease," J. Inf. Dis., 157, pp. 790-97 (1988)].

Because the B. burgdorferi flagellar antigen is detectable early in infection, it could potentially prove useful in a diagnostic test. However, the flagellar protein is known to contain epitopes which are conserved among other spirochetes [J. L. Coleman and J. L. Benach, "Identification And Characterization Of An Endoflagellar Antigen Of Borrelia burgdorferi", J. Clin. Invest., 84, pp. 322-330 (1989)], and the flagellin cross-reacts with antibodies directed against other bacterial flagellins [L. A. Magnarelli et al., "Cross-Reactivity Of Nonspecific Treponemal Antibody In Serologic Tests For Lyme Disease", J. Clin. Microbiol., 28, pp. 1276-1279 (1990)]. As a result, antibodies to this protein do not provide a specific marker for Lyme disease.

In view of the above, there exists an urgent need for a highly specific and sensitive laboratory test for detection of B. burgdorferi infection and diagnosis of Lyme disease. There also exists a need for therapeutic agents and methods of diagnosis and treatment that are useful in later stages of Lyme disease.

DISCLOSURE OF THE INVENTION

The present invention solves the problems referred to above by providing in one preferred embodiment, flagellin polypeptides that comprise an immunodominant region of the B. burgdorferi flagellin antigen. The flagellin polypeptides of this invention are recognized by sera from Lyme disease patients but have substantially no homology to other bacterial flagellins and thus do not result in false-positive diagnostic test results.

In another embodiment, this invention provides diagnostic means and methods characterized by flagellin polypeptides and antibodies directed against those polypeptides. These means and methods are useful for the diagnosis of Lyme disease and the detection of B. burgdorferi infection. They are also useful in following the course of treatment against such infection.

In another embodiment, this invention provides neuroborreliosis-associated antigens and pharmaceutically effective compositions and methods comprising those antigens, which are useful for the treatment of prevention of the later stages of Lyme disease. The preferred compositions and methods of this embodiment are characterized by antigens that are recognized by antibodies in the serum of patents with neuroborreliosis, which antibodies also bind to antigens associated with neuronal tissue.

BRIEF DESCRIPTION OF THE FIGURES

Op zaterdag 10-04-2010 om 00:38 uur [tijdzone -0700], schreef Dottie: Dr. Piccirillo,

If the patient does not have periodontal disease or syphilis then they have a borrelia infection.

IgG Band 41 ++ IgM Band 41++ <pos
Yale Uni owns a patent on that and won't let anyone use it.

This is the only scientifically valid test for Lyme disease per the
FDA rules for validating a disease.
US PATENT #5,618,533
Flagellin-based polypeptides for the diagnosis of lyme disease
US PATENT OFFICE -

To View Complete Abstract

http://patft1.uspto.gov/netacgi/nph-Par ... PN/5618533

[soijuv]

APMIS. 2010 Apr;118(4):313-23.
Simultaneous use of serum IgG and IgM for risk scoring of suspected early Lyme
borreliosis: graphical and bivariate analyses.

Dessau RB, Ejlertsen T, Hilden J.

Department of Clinical Microbiology, Naestved Hospital, Region Zealand,
Naestved, Denmark. ramd@regionsjaelland.dk

The laboratory diagnosis of early disseminated Lyme borreliosis (LB) rests on
IgM and IgG antibodies in serum. The purpose of this study was to refine the
statistical interpretation of IgM and IgG by combining the diagnostic evidence
provided by the two immunoglobulins and exploiting the whole range of the
quantitative variation in test values. ELISA assays based on purified flagella
antigen were performed on sera from 815 healthy Danish blood donors as negative
controls and 117 consecutive patients with confirmed neuroborreliosis (NB). A
logistic regression model combining the standardized units of the IgM and IgG
ELISA assays was constructed and the resulting disease risks graphically
evaluated by receiver operating characteristic and 'predictiveness' curves. The
combined model improves the discrimination between NB patients and blood donors.
Hence, it is possible to report a predicted risk of disease graded for each
individual patient, as is theoretically preferable. The predictiveness curve,
when adapted to the local pretest probability of LB, allows high-risk and
low-risk thresholds to be defined instead of cut-offs based on the laboratory
characteristics only, and it allows the extent of under- and over-treatment to
be assessed. It is shown that an example patient with low ELISA results in IgM
and IgG, considered negative by the conventional cut-off, has a relatively high
risk of belonging to the truly diseased population and a low risk of being false
positive. Using a 20% high-risk threshold for advising the clinician to consider
treatment, the sensitivity of the assay is increased from 76% to 85%, while the
specificity is maintained at around 95%.

[Sailairina]

Tämäkin tutkimus kertoo borrelioositestien epäluotettavuudesta.

http://www.cdc.gov/ncidod/eid/vol10no9/03-0439.htm

Borrelia valaisiana in Cerebrospinal Fluid

Eudoxia Diza,* Anna Papa,* Eleni Vezyri,* Stefanos Tsounis,* Ioannis Milonas,* and Antonis Antoniadis*
*Aristotle University of Thessaloniki, Thessaloniki, Greece

To the Editor: Lyme borreliosis is the most common tickborne human disease in the Northern Hemisphere. The incidence of the disease is 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 (E. Diza, 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. valaisiana 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.


Acknowledgment
We thank O. Peter for providing DNA control 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.
Schmidt B, Muelleger RR, Stockenhuber C, Soyer PH, Hoedl S, Luger A, et al. 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.
Rijpkema SG, Tazelear DJ, Molkeboer HJ, Noordhoek GT, Plantinga G, Schouls LM, et al. 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.
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.
Fraenkel CJ, Garpmo U, Berglund J. Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. J Clin Microbiol. 2002;40:3308?12.
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.
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.
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.
Fivaz BH, Petney TN. Lyme disease?a new disease in southern Africa? J S Afr Vet Assoc. 1989;60:155?8.

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Uusi testi, Lawrence Livermore Microbial Detection Array (LLMDA), havaitsee minkä tahansa bakteerin, viruksen, sienen tms. vuorokaudessa. Testiä on kehitetty vuodesta 2003 lähtien. Sitä kehitellään edelleen:

New LLNL detection technology identifies bacteria, viruses, other organisms within 24 hours
array

https://publicaffairs.llnl.gov/news/new ... 05-02.html


LIVERMORE, Calif. ? Law enforcement authorities seeking to detect bioterrorism attacks, doctors diagnosing diseases and regulatory agencies checking product safety may find a new ally in a Lawrence Livermore National Laboratory (LLNL) detection technology.

The advance, known as the Lawrence Livermore Microbial Detection Array (LLMDA), could enable law enforcement, medical professionals and others to detect within 24 hours any virus or bacteria that has been sequenced and included among the array?s probes.

Developed between October 2007 and February 2008, the LLMDA detects viruses and bacteria with the use of 388,000 probes that fit in a checkerboard pattern in the middle of a one-inch wide, three-inch long glass slide.

The current operational version of the LLMDA contains probes that can detect more than 2,000 viruses and about 900 bacteria.

?The ability to detect the major bacterial and viral components of any sample can be used in countless different ways,? said Tom Slezak, LLNL?s associate program leader for Informatics. ?This is important because it fills a cost-performance gap that is relevant to many missions: biodefense, public health and product safety.?

In the area of biodefense, current systems are centered upon the detection of smaller prioritized sets of high-risk pathogens, rather than testing for a much broader spectrum of organisms.

?The LLMDA allows us to not only identify the biological pathogens on a priority screening list, but also any other already-sequenced bacteria or virus in a sample that you might not have been expecting to find, including possible novel or emerging pathogens,? Slezak said.

Current plans call for the detection array to be evaluated for operational bioforensic use at the Frederick, Md.-based National Biodefense Analysis and Countermeasures Center of the U.S. Department of Homeland Security.
Array

Shown is the one-inch wide by three-inch long Lawrence Livermore Microbial Detection Array that contains 388,000 probes that are used to detect viruses and bacteria.

Photos by Jacqueline McBride/LLNL
Click for high resolution image

As the cost of the array is reduced, the LLMDA technology could be used to improve public health diagnostics, Slezak said, adding that dozens of bacteria and viruses can be detected in a single test from the entire spectrum of sequenced organisms.

One advantage of the Livermore array is that it provides researchers with the capability of detecting pathogens over the entire range of known viruses and bacteria. Current multiplex polymerase chain reaction (PCR) techniques can at most offer detection from among 50 organisms in one test.

In April, in a Journal of Virology article, Livermore researchers working with a scientist from the San Francisco-based Blood Systems Research Institute said they used the LLMDA technology to confirm the presence of an apparently benign pig virus in a vaccine.

The pig virus, porcine circovirus-1 (PCV-1), was unexpectedly found in GlaxoSmithKline?s Rotarix vaccine, which is used to prevent diarrhea in babies.

?One result of this research is that it demonstrates how modern technologies could change and drastically improve product safety,? Slezak said.

While product safety rules require demonstrating that a list of known contaminants is not present, Slezak said the use of modern advances in DNA sequencing and arrays would allow manufacturers to identify the potential presence of contaminating biological material present in quantities large enough to be of potential concern.

?For each bacteria or virus that has been sequenced anywhere in the world, we have several dozen squares on the checkerboard that will identify sequences from that organism,? Slezak explained.

Currently, Slezak?s team is testing a next-generation LLMDA that boasts 2.1 million probes. This version contains probes representing about 178,000 viral sequences from some 5,700 viruses, and about 785,000 bacterial sequences from thousands of bacteria.

The latest LLMDA version also encompasses fungi and protozoa ? with probes representing about 237,000 fungal sequences from thousands of fungi and about 202,000 protozoa sequences from 75 protozoa.

As a screening tool, Slezak sees the LLMDA as occupying niche roles between PCR machines and sequencing.

The LLMDA process starts with the purification of DNA or RNA from a sample, such as sputum or blood. The sample is next labeled with a fluorescent dye and hybridized on the microarray at 42 degrees C or about 107.6 degrees Fahrenheit. In turn, a fluorescent scanner and analysis software are used to detect the probes that have lit up, identifying the presence of viral or bacterial sequences.

The Livermore team plans to update probes on the array with new sequences of bacteria, viruses and other microorganisms from GenBank and other public databases about once per year, in addition to using sequences obtained from collaborators for their probes.

LLNL?s current collaborators include the University of California, San Francisco; the Blood Systems Research Institute; the University of Texas Medical Branch (Galveston); the National Institute for Public Health and the Environment of Bilthoven, the Netherlands; the Statens Serum Institut of Copenhagen, Denmark; the University of California, Davis; Imigene; the U.S. Food & Drug Administration; and the Marine Mammal Center of Sausalito, Calif.

A computer scientist and the team?s leader, Slezak came up with the idea for the LLMDA in 2003. His team includes biologist Crystal Jaing, who leads the microarray lab work and manages the collaborations; bioinformaticist Shea Gardner, who designed the array; biostatistician Kevin McLoughlin, who designed the analysis software; and James B. Thissen, who performs the microarray experiments.

Founded in 1952, Lawrence Livermore National Laboratory is a national security laboratory that develops science and engineering technology and provides innovative solutions to our nation's most important challenges. Lawrence Livermore National Laboratory is managed by Lawrence Livermore National Security, LLC for the U.S. Department of Energy's National Nuclear Security Administration.

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2010: Bakteerin DNA:n löytämiseen perustuva 16S rDNA - testi.

Increased Sensitivity and Specificity of Borrelia burgdorferi 16S Ribosomal DNA Detection

1. Sin Hang Lee, MD,
2. Veronica S. Vigliotti, CMIAC,
3. Jessica S. Vigliotti,
4. William Jones and
5. Suri Pappu, MD

+ Author Affiliations

1.
From the Department of Pathology, Milford Hospital, Milford, CT

1. Address reprint request to Dr Lee: 300 Seaside Ave, Milford, CT 06460.

Abstract

The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

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Slovakia 2010: Potilaiden ELISA testi oli negatiivinen, mutta immunoblottaus positiivinen. Syynä siihen on sairastuneiden elimistön immuunipuolustuksen heikentynyt tila. Siksi erityisesti kroonisista oireista kärsivien kohdalla tulee tehdä nimenomaan Western blot -tutkimus.

Bratisl Lek Listy. 2010;111(3):153-5.
Our experience with examination of antibodies against antigens of Borrelia burgdorferi in patients with suspected lyme disease.

Durovska J, Bazovska S, Ondrisova M, Pancak J.

1st Department of Neurology, Faculty of Medicine, Comenius University, Bratislava, Slovakia. durovska@borelioza.sk

BACKGROUND: Lyme borreliosis is a multisystemic disease which affects several organs such as skin, nervous system, joints and the heart. The presented study focused on patients with persisting symptoms of the disease, which could be in correlation with Lyme disease but antiborrelial antibodies were not confirmed by screening tests.

MATERIAL AND METHODS: 32 patients with anamnestic data and suspected clinical signs of lyme borreliosis were tested for the presence of antiborrelia antibodies by using ELISA and westernblot analysis and the state of cellular and humoral immunity.

RESULTS: All patients had specific antiborrelial antibodies confirmed by using the westernblot in spite of negative ELISA.

Immunological investigations revealed a deficiency of cellular immunity in all patients and in a part of them (15.6%) a deficiency of humoral immunity was also found. The presence of different types of autoantibodies was detected in 17 (53.1%) patients.

CONCLUSION: In patients with persisting difficulties that could be associated with Lyme disease, it is necessary to use the westernblot test which could prove the presence of specific antibodies. It is probably due to the very low production of specific antibodies caused also by the status of immune deficiency detected in all our patients (Tab. 1, Ref. 11).

Publication Types:
Research Support, Non-U.S. Gov't

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20437826 [PubMed - in process]

[soijuv]

Borrelioosi vaikuttaa eri puolille elimistöä esim. niveliin. Ultraäänitutkimus saattaa olla käyttökelpoinen tutkimusmenetelmä esim. polvinivelen tulehdustapauksia tutkittaessa.

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Przegl Epidemiol. 2006;60 Suppl 1:34-8.

[Ultrasonography in diagnosing Lyme arthritis of the knee joints in correlation with anti-CCP antibodies]
[Article in Polish]

* Czeczuga A,
* Targonski A,
* Zajkowska J,
* Hermanowska-Szpakowicz T,
* Swierzbinska R.

Zaklad Diagnostyki Obrazowej Wojewodzkiego Szpitala Specjalistycznego im. K. Dluskiego w Bialymstoku.

Lyme disease, a multi-system disorder may be associated with arthritis. Lyme arthritis most commonly affects the knee joints. Ultrasonography can show the inflammation changes of the knee joint and can be a usefull method in diagnosis of Lyme arthritis. The most freguent ultrasonographic finding was knee joint effusion. Because of Lyme arthritis similarities to rheumatoid arthritis, a serologic test antibodies against cyclic cytrulinated peptid (anty CCP) can be helpfull in distinguishing of these two diseases.

PMID: 16909773 [PubMed - in process]

[soijuv]

"Borrelioosin diagnostiikka on lääkäreille haaste. Varhaisoireiden jättäminen huomioimatta johtaa viivästyneeseen diagnoosiin, tehottomiin antibioottihoitoihin ja taudin myöhäisoireisiin."

http://www.biomedcentral.com/content/pd ... 4-9-79.pdf

Diagnostic challenges of early Lyme disease: Lessons from a community case series

BMC Infectious Diseases 2009, 9:79 doi:10.1186/1471-2334-9-79
John Aucott (jaucott2@jhmi.edu)
Candis Morrison (candismorrison@comcast.net)
Beatriz Munoz (bmunoz@jhmi.edu)
Peter C Rowe (prowe@jhmi.edu)
Alison Schwarzwalder (alison.schwarzwalder@gmail.com)
Sheila K West (shwest@jhmi.edu)

Conclusions
For community physicians practicing in high-risk geographic areas, the diagnosis of Lyme disease remains a challenge. Failure to recognize erythema migrans or alternatively, viral-like presentations without a rash, can lead to missed or delayed diagnosis of Lyme disease, ineffective antibiotic treatment, and the potential for late manifestations.

[soijuv]

Borrelia-bakteereita tutkittaessa viljelyalustaan laitettiin vähäinen määrä sinkkiä ja kuparia jotta bakteeri nähtiin liikkuvana. Peter Kemp lisäsi aineet norjalaisen tri Brorsonin ohjeiden mukaan.

http://www.counsellingme.com/microscopy ... ture2.html

Peter Kemp added traces of zinc and copper to his cultures, on the advice of Dr Brorson.
For the first time he was able to see a spirochaete moving.

[soijuv]

Uusi bakteerin DNA testi: Igenex Lyme Multiplex PCR-testi (2010)


IGeneX?s Lyme Multiplex PCR Test

The Lyme Multiplex PCR is more sensitive than nested PCR and has the same specificity as DNA sequencing of the nested PCR product, because of the following reasons.

· The Lyme multiplex PCR assay detects both genomic and plasmid sequences of all B. burgdorferi strains, B. afzelii, B. andersonii and B. garinii.



· Borrelia DNA targets are hybrid-selected from clinical sample with Borrelia specific probes.

This step allows the use of a large volume of sample (100 fold more than standard techniques) of clinical sample for PCR.

· The selected Borrelia genomic and plasmid targets are amplified by PCR using Borrelia burgdorferi specific primers.

· The PCR products are confirmed by Southern Dot-blot using B. burgdorferi specific probes.

· The assay works well in all types of clinical samples including serum, blood, urine and tissues. Unlike most PCR protocols, the urine and whole blood PCR works very well as demonstrated by the following study that was presented at 104th General ASM Meeting, New Orleans, LA.

[soijuv]

Saxenin luento lääkäripäivillä.
http://www.infektiofoorumi.fi/tapahtuma ... paivat.pdf

Borreliosin diagnostiikka ja pari potilastapausta. Artikkelissa mainitaan mm.
1.Mikään yksittäinen metodi tai antigeeni ei ole osoittautunut ylivertaiseksi.
2.Testin tai testikombinaation tulee "perustua paikallisiin kokemuksiin". Artikkelista huomaa että Suomessakin käytetään erilaisia testejä. Luotettavuuskysymykset?
3.75%:ssa tapauksista ihomuutos oli tasainen, vain kolmasosassa tapauksista ihomuutos oli rengasmainen.

http://www.ecdc.europa.eu/en/healthtopi ... urope.aspx

[soijuv]

Tulehdusvälittäjäaineiden, sytokiinien, seuraaminen saattaa antaa diagnostista ja prognostista tietoa Borrelioosia sairastavien kohdalla.

Zh Mikrobiol Epidemiol Immunobiol. 2010 May-Jun;(3):67-71.
[Features of cytokine levels in serum of patients with tick-borne borreliosis
with different clinical signs]

[Article in Russian]

[No authors listed]

AIM: To assess levels of several cytokines in blood of patients with tick-borne
borreliosis (Lyme disease) with different clinical variants of the disease.
MATERIALS AND METHODS: Study of complex of proinflammatory cytokines (IFNalpha,
IL-1beta, IFNgamma, IL-2, IL-4, IL-8) during course of the disease was performed
by solid-phase ELISA using domestic diagnostic kits (Scientific Manufacturing
Organization "Proteinovyi Contur", "Cytokine" Ltd., Saint Petersburg). Levels of
TNFalpha was determined by ELISA using commercial kits "Boehringer Manheim"
(Austria). RESULTS: Performed comparative clinico-laboratory analysis
demonstrated increased levels of LL-2, IL-4, and IL-8 in patients during acute
phase of tick-borne borreliosis that could point to host's response on bacterial
infection. It should be noted that in patients with arthritis levels of LL-4 and
IL-2 remained high during recovery phase that probably determined by possible
persistence of Borrelia burgdorferi. CONCLUSION: Further research of cytokines
during Lyme borreliosis could have important diagnostic and prognostic value.
Publication Types:
English Abstract

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20734722 [PubMed - in process]

[soijuv]

Tutkimuksessa selvitettiin Borrelioosia sairastavien/ sairastaneiden naisten ja miesten välisiä diagnostisia eroja. Tapaukset olivat jo aiemmin varmennettuja borrelioositapauksia.
Eroja löytyi vasta-ainetesti ELISAN:n, IgG:n kohdalla. Tutkimuksen mukaan 50%:lla miehistä ja n.70%:lla naisista borrelioositesti oli virheellisesti negatiivinen.

Merkittävää oli että testi oli virheellisesti negatiivinen suuressa osassa tapauksia.


http://www.prohealth.com/library/showar ... ibid=15636

Sex differences in the clinical and serologic presentation of early Lyme disease: Results from a retrospective review ? Source: Gender Medicine, Aug 2010
by Alison Schwrzwalder, et al.
September 29, 2010

[Note: To read the full text PDF of this article click HERE ]

Background: Lyme disease is the most common vector-borne disease in the United States, and the number of reported cases has more than doubled between 1992 and 2008.

Few studies have explicitly examined sex-based differences in the clinical presentation of or serologic response to early Lyme disease. It is unknown whether the sex-based variability observed in other infectious diseases is relevant to this clinical setting.

Objective: This study retrospectively examined clinical and serologic differences by sex among a community case series of patients with a current or past episode of confirmed early Lyme disease.

Methods: This was a retrospective, consecutive case series of adult patients in Maryland enrolled from August 2002 to August 2007 meeting criteria for a current or past episode of confirmed early Lyme disease.

Clinical variables and patients' self-report surrounding illness onset were abstracted through chart review. All serologic tests drawn within 3 months of illness onset were interpreted using Centers for Disease Control and Prevention criteria.

Results: In a total of 125 patients, there were no significant differences in clinical presentation by sex.

The initial self-misdiagnosis rates for men and women were 10% and 18%, respectively (P = NS).

Among the 62 patients with a serologic test as part of their clinical evaluation:

? 50% of men had a positive, 2-tier result

? Compared with 32% of women (P = NS).

Among the 41 patients with a positive ELISA, median ELISA values (3.4 vs 2.0; P = 0.03) and median number of immunoglobulin G (IgG) bands (4 vs 2; P = 0.03) were significantly higher among men.

Conclusions: In this small, retrospective sample, we found evidence for sex-based differences in the magnitude of ELISA and IgG serologic response to early Lyme disease.

Such differences could have implications for appropriate diagnosis, treatment, and disease classification.

Larger, prospective studies are needed to replicate the results found in this study and to examine their relationship to sex-based immunologic variability.

Source: Gender Medicine, Aug 2010;7(4):320-9. PMID: 20869632, by Schwarzwalder A, Schneider MF, Lydecker A, Aucott JN.The Lyme Disease Research Foundation of Maryland, Lutherville, Maryland, USA.

[soijuv]

C6-testin spesifisyys oli allaolevan tutkimuksen mukaan 83% neuroborrelioosin diagnostiikassa (selkäydinneste).

http://onlinelibrary.wiley.com/doi/10.1 ... 8F6.d03t01>

High sensitivity and specificity of C6-peptide ELISA on CSF in Lyme neuroborreliosis patients

1. Nathalie D. van Burgel1,*,
2. Afke Brandenburg2,
3. Hendrik-Jan Gerritsen1,
4. Aloys C.M. Kroes1,
5. Alje P. van Dam3

DOI: 10.1111/j.1469-0691.2011.03459.x

Copyright © 2011 European Society of Clinical Microbiology and Infectious Diseases

Abstract

Lyme neuroborreliosis (LNB) is a serious but treatable disease. Diagnosing LNB poses a challenge to clinicians and improved tests are needed. C6-peptide ELISA is frequently used on serum but not on cerebrospinal fluid (CSF). Data about sensitivity of C6-peptide ELISA in CSF in patients suffering from LNB has been conflicting.

Serum-CSF pairs from 59 LNB patients, 36 Lyme non-neuroborreliosis cases, 69 infectious meningitis/encephalitis controls and 74 neurological controls were tested in a C6-peptide ELISA.

Using the optimal cut-off of 1.1, sensitivity of the C6-peptide ELISA for LNB patients in CSF was 95% and specificity was 83% in the Lyme non-neuroborreliosis patients, 96% in the infectious controls and 97% in the neurologic controls

These results suggest that C6-peptide ELISA has a high sensitivity and good specificity for diagnosing Lyme neuroborreliosis patients in CSF. The C6-peptide ELISA can be used on CSF in a clinical setting to screen for Lyme neuroborreliosis.

[soijuv]

Selkäydinnesteen CXCL13-testiä voidaan käyttää neuroborrelioosin diagnostiikassa.


http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 21325326 [PubMed - as supplied by publisher]

2: Acta Neurol Scand. 2011 Feb 15; [Epub ahead of print]

CXCL13 chemokine in pediatric and adult neuroborreliosis.

Wutte N, Berghold A, Loffler S, Zenz W, Daghofer E, Krainberger I, Kleinert G,
Aberer E.

Department of Dermatology Institute for Medical Informatics, Statistics and
Documentation Department of General Pediatrics Institute for Hygiene and
Microbiology Department of Neurology, Medical University of Graz, Graz, Austria.

Acta Neurol Scand: DOI: 10.1111/j.1600-0404.2010.01477.x. (c) 2011 John Wiley& Sons A/S.

Objectives - Diagnosis of Lyme neuroborreliosis (NB) depends on the proof of
intrathecal antibody production against Borrelia burgdorferi. CXCL13 has been
seen to be elevated early in NB, before antibody production has started. In this
study, we determined the diagnostic role of the CXCL13 chemokine in
cerebrospinal fluid (CSF) and serum for the first time in pediatric NB patients
as well as in adults, compared to controls and blood donors (BD).

Material and methods - CXCL13 levels were measured in CSF and serum of 33 children and 42
adult patients. Serum CXCL13 was measured in 300 BD.

Results - CSF CXCL13 levels were significantly elevated in definite and probable
acute NB in children and adults compared to seropositive and seronegative neurological
controls (P< 0.001). Serum CXCL13 levels showed great fluctuations and were not
significantly elevated in NB patients.
Conclusions - Our study suggests that CSF CXCL13 can
be used as a diagnostic marker for NB in children as well. In contrast, CXCL13
serum levels show great variance even in the healthy population and are not
indicative of active NB. (c) 2011 John Wiley& Sons A/S.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 21320077 [PubMed - as supplied by publisher]

[soijuv]

2011. Kroonista Borrelioosia ja kroonista väsymysoireyhtymää sairastavien selkäydinesteen valkuaisaineet olivat kohonnneet mutta poikkesivat keskenään toisistaan.

February 23, 2011
Breaking News
CFS will be featured tonight on "The CBS Evening News with Katie Couric." The report focuses on a new research study on biological markers found in the cerebrospinal fluid of CFS patients described below. The University of Medicine and Dentistry of New Jersey circulated an embargoed press release http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... ZQTbuh-4M= and the CFIDS Association helped media outlets, including CBS News http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... qaloDOK8M= , to interview patients and experts about CFS and the study. The embargo lifts at 5:00 p.m. (EST) today and we are pleased to bring you immediate analysis of this study http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... e14USS1b3u and its results. We'll recap media coverage in the next issue of CFIDSLink.

The Promise of the Proteome
Analysis of the PLoS ONE study
(abridged; click here http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... e14USS1b3u to read the detailed version with links to resources and definitions of terms)

In an article titled, "Distinct Cerebrospinal Fluid Proteomes Differentiate Post-Treatment Lyme Disease and CFS," Steven Schutzer and colleagues from six institutions tested samples collected by lumbar puncture from 43 CFS subjects, 25 subjects with documented Lyme disease who did not recover after standard antibiotic treatment (referred to in this paper as "nPTLS") and 11 healthy controls.

Figure 1: Venn diagram illustrating distribution of proteins identified in the three groups

Using mass spectroscopy and liquid chromatography, the team was able to generate a comprehensive list of 30,000 peptides in the cerebrospinal fluid (CSF) samples pooled from subjects in each disease group. Of these 30,000 peptides, 738 proteins were found only in CFS subjects. The nPTLS samples had 692 unique proteins and the normal controls had 724 unique proteins. Differences in the amounts of various proteins were detected between groups and CFS and nPTLS had more proteins in common than with the healthy controls.

Looking at the biological pathways implicated by the different proteins identified, the team found that proteins in the complement cascade were elevated in abundance in the pooled nPTLS and CFS samples compared to controls, but at different levels for the two disease groups. The complement system is a part of the immune system that helps to clear infectious pathogens. Proteins involved in the CDK5 signaling pathway were significantly enriched in the CFS samples. Alterations in the CDK5 signaling pathway have been linked to Parkinson's disease and Alzheimer's disease.

By analyzing individual CSF samples, they determined that nPTLS patients are distinct from CFS patients. The ability to distinguish CFS and nPTLS on the basis of these proteins has important diagnostic implications. It also suggests different treatment approaches may be warranted. Some have proposed that nPTLS represents a subset of CFS; however, the authors of this paper conclude that their data does not support that concept.

In the final discussion section, the authors state, "CSF proteome analysis may provide important and meaningful insights into the biological processes modulated as a function of disease and facilitate the identification of protein candidates for further investigation." They suggest that by uncovering these candidates in cerebrospinal fluid, a targeted search in blood for these proteins is now possible.

This team of researchers provided the comprehensive list of proteins they identified as part of the open access paper http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... Q1tv6Yow== . This will enable these and other investigators to explore and interrogate the data further. The collaborative effort across six institutions and multiple funding partners, use of new technologies for discovery and willingness to openly share data to advance the field represent an inspiring 21st century research initiative worthy of high hopes.

Schutzer SE, Angel TE, Liu T, Schepmoes AA, Clauss TR, Adkins NJ, Camp DG, Holland BK, Bergquist J, Coyle PK, Smith RD, Fallon BA, Natelson BH. (2011) Distinct cerebrospinal fluid proteomes differentiate post-treatment Lyme disease from chronic fatigue syndrome. PLoS ONE 6(2): e17287. doi:10.1371/journal.pone.0017287

Click here http://r20.rs6.net/tn.jsp?llr=eqsp8ocab ... e14USS1b3u to read the unabridged version of "The Promise of the Proteome"

[soijuv]

Tutkimuksessa (Hollanti 2011) selvitettiin kahdeksan käytössä olevan ELISA vasta-ainetestin luotettavuutta ja viittä immunoblottausta. Osa immunobloteista antoi positiivisen tuloksen vaikka 8 ELISAA oli aiemmin antanut negatiivisen tuloksen.

(suom.huom. Suomessa immunoblottausta ei yleensä tehdä jos ELISA on ollut negatiivinen. Joidenkin asiantuntijoiden mukaan immunoblottaus on ELISAA luotettavampi. On kuitenkin muistettava että kummassakin tutkimuksessa kyseessä on vasta-aineiden muodostumista kuvaava testi, ei bakteerin olemassaoloa).

ELISA-testien positiiviset tulokset vaihtelivat 34 - 59%. Immunoblottauksissa esiintyi suurta hajontaa ja testituloksien luotettavuus keskenään oli korkeintaan kohtalainen. Positiivinen immunoblottaustulos riippui suuresti siitä mitä ELISA/immunoblottaus-kombinaatiotestiä käytettiin. Siksi laboratorioiden välisten testitulosten vertaileminen on erittäin sattumanvaraista.


Eur J Clin Microbiol Infect Dis. 2011 Jan 27; [Epub ahead of print]

Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.

Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T.

VUMC, Amsterdam, The Netherlands,w.ang@vumc.nl.

We investigated the influence of assay choice on the results in a two-tier
testing algorithm for the detection of anti-Borrelia antibodies.

Eighty-nine serum samples from clinically well-defined patients were tested in eight
different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell
antigens, whole-cell antigens supplemented with VlsE and assays using
exclusively recombinant proteins.

A subset of samples was tested in five immunoblots: one whole-cell blot, one
whole-cell blot supplemented with VlsE and three recombinant blots.

The number of IgM- and/or IgG-positive ELISA results in
the group of patients suspected of Borrelia infection ranged from 34 to 59%.

The percentage of positives in cross-reactivity controls ranged from 0 to 38%.

Comparison of immunoblots yielded large differences in inter-test agreement and
showed, at best, a moderate agreement between tests.

***Remarkably, some immunoblots gave positive results in samples that had been
tested negative by all eight ELISAs.***

The percentage of positive blots following a positive ELISA
result depended heavily on the choice of ELISA-immunoblot combination.

We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity.

The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 21271270 [PubMed - as supplied by publisher]

[soijuv]

EU:ssa kehitteillä uusi borreliatesti, HILYSENS lab-on-a-chip testi. Tiedotteen mukaan kaikki nykyiset testit jättävät virheellisesti huomioimatta lukuisia tapauksia. Uuden testin mainostetaan olevan herkempi ja tarkempi sekä akuutin, kroonisen että autoimmuunitilojen diagnostiikassa.

One tick bite can cause many diseases. Lyme Disease is the most prevalent tick-borne infection in Europe and North America. Patients with acute Lyme disease need prompt diagnosis to prevent the development of chronic infections. Current commercial laboratory methods fail to detect a huge amount of cases. As consequence, patients receive delayed or inadequate treatment, leading to more serious symptoms which can be disabling.

HILYSENS will be the first lab-on-a-chip tool allowing specific and sensitive detection of acute, chronic and autoimmunity associated Lyme Disease and ensuring non-invasive, fast, specific and easy diagnosing, prognosis and monitoring. Optimisation of resources, reduction of the associated costs and an increase in the quality of care are some of the potential impacts of HILYSENS .

HILYSENS is a 2 year R+D project funded by the European Commission's Seventh Framework Programme (FP7) under Research for the Benefit of the SMEs. The project officially started on November 1st 2010. The Kick-off Meeting was held in Barcelona on November 8th and was hosted by AROMICS at the PCB.
http://www.hilysens.eu/

[soijuv]

"Serologisten testien luotettavuus on alhainen mikäli testi suoritetaan ilman kliinistä kuvaa. ELISA tulee aina varmistaa immunoblottauksella (western blot)."

Postgrad Med J 1999;75:650-656 doi:10.1136/pgmj.75.889.650

Overdiagnosis and overtreatment of Lyme neuroborreliosis are preventable

1. Avinash Prasad,
2. Douglas Sankar

Department of Neurology, New York University Medical Center, New York, NY 10016, USA

1. Avinash Prasad MD, 212 Copeley Road #3, Charlottesville, VA 22903, USA

* Accepted 7 April 1999

Abstract

The problems of diagnosis and treatment of Lyme neuroborreliosis can be minimised by strictly following the clinical diagnostic criteria, and understanding the pitfalls of laboratory tests. The diagnosis is based solely on objective clinical findings, with serologic test results used only to confirm the diagnosis. It must be underscored that serologic testing, when ordered without regard for clinical presentation (ie, used as a screen), may be misleading due to its extremely low positive predictive value. Enzyme-linked immunosorbent assay should always be confirmed by Western blot.

The cerebrospinal fluid Borrelia burgdorferiantibody index is more meaningful than simple titres of specific antibody. Polymerase chain reaction is still a research tool and should not be utilised without clinical correlation. All serologic tests and polymerase chain reaction may remain positive long after successful treatment. Overdiagnosis and overtreatment can be minimised by following these guidelines.

[soijuv]

Kuudella kortisonia saaneella elinsiirtopotilaalla borreliatestit (ELISA) olivat negatiiviset toistuvasti. Siitä huolimatta bakteeria löydettiin ihosta (PCR).

CID 2006:42 (15 June) ? 1751

Erythema Migrans in Solid-Organ
Transplant Recipients
Vera Maraspin,1 Jozˇe Cimperman,1 Stanka Lotricˇ -Furlan,1 Mateja Logar,1
Eva Ruzˇic´ -Sabljic´ ,2 and Franc Strle1
1Department of Infectious Diseases, University Medical Centre Ljubljana,
and 2Institute for Microbiology and Immunology, University of Ljubljana,
Ljubljana, Slovenia

Six adult solid-organ transplant recipients who had chronic
drug-induced immunosuppression and who presented with
solitary erythema migrans were treated with antibiotics administered
at the same dosage and for the same duration
used for the treatment of early, localized Lyme borreliosis in
immunocompetent patients. The patients had a smooth
course of illness and a favorable outcome but did not develop
a measurable borrelial serum antibody response.

[soijuv]

Selkäydinnestetutkimus:
Neuroborrelioosia sairastavien likvorissa kohonneet leukosyytit. Osalla korkea laktaattipitoisuus.Tällaisilla potilailla esiintyi kuumetta ja päänsärkyä. Virusmeningiitteihin verrattuna borrelioosiin sairastuneilla esiintyi poikkeavuutta likvorin proteiineissa ja likvor/seerumi suhteessa.
Suurimmalla osalla esiintyi IgM synteesiä (70,2%) ja/tai IgG synteesiä (70,3%).


Cerebrospinal fluid findings in adults with acute Lyme neuroborreliosis.

Djukic M, Schmidt-Samoa C, Lange P, Spreer A, Neubieser K, Eiffert H, Nau R, Schmidt H

J Neurol 2011 09 6

Presence of BB-specific antibodies in the cerebrospinal fluid (CSF) with evidence of their intrathecal production in conjunction with the white cell count in the CSF and typical clinical symptoms is the traditional diagnostic gold standard of Lyme neuroborreliosis (LNB). Few data are available on the CSF lactate concentration in European adults with the diagnosis of acute LNB. The objective of the study was to investigate the CSF changes during acute LNB. Routine CSF parameters [leukocyte count, protein, lactate and albumin concentrations, CSF/serum quotients of albumin (Q(Alb)), IgG, IgA and IgM, and oligoclonal IgG bands] and the Borrelia burgdorferi (BB)-specific antibody index were retrospectively studied in relation to the clinical presentation in patients diagnosed with acute LNB.

A total of 118 patients with LNB were categorized into the following groups according to their symptoms at presentation;
group 1: polyradiculoneuritis (Bannwarth's syndrome),
group 2: isolat ed facial palsy and
group 3: predominantly meningitic course of the disease.

In addition to the CSF of patients with acute LNB, CSF of 19 patients with viral meningitis (VM) and 3 with neurolues (NL) were analyzed. There were 97 patients classified with definite LNB, and 21 as probable LNB. Neck stiffness and fever were reported by 15.3% of patients. Most of these patients were younger than 50 years. Polyradiculoneuritis was frequently found in patients older than 50 years. Lymphopleocytosis was found in all patients. Only 5 patients had a CSF lactate ≥3.5 mmol/l, and the mean CSF lactate level was not elevated (2.1 ± 0.6 mmol/l). The patients with definite LNB had significantly higher lactate levels than patients with probable LNB. Elevated lactate levels were accompanied by fever and headache.

In the Reiber nomograms, intrathecal immunoglobulin synthesis was found for IgM in 70.2% followed by IgG in 19.5%. Isoelectric focussing detected an intrathecal IgG synthesi s in 83 patients (70.3%). Elevated BB AIs in the CSF were found in 97 patients (82.2%). Patients with VM showed lower CSF protein concentration and CSF/serum quotients of albumin than LNB patients.


In acute LNB, all patients had elevated cerebrospinal fluid (CSF) leukocyte counts. In contrast to infections by other bacteria, CSF lactate was lower than 3.5 mmol/l in all but 5 patients. The CSF findings did not differ between polyradiculoneuritis, facial palsy, and meningitis. The CSF in LNB patients strongly differed from CSF in VM patients with respect to protein concentration and the CSF/serum albumin quotient.

[soijuv]

2011 tutkimuksen mukaan borrelioosin diagnostiikassa yleisimmin käytettyihin serologisiin testeihin tulee suhtautua varauksella. Testitulosten lisäksi tulee ottaa huomioon kliininen kuva ja oireet.



Clinical and serological features of patients with suspected Lyme borreliosis.

Santino I, Longobardi V

Int J Immunopathol Pharmacol 2011 10; 24 (3): 797-801

Serology is currently the method of choice for the laboratory diagnosis of Lyme Borreliosis, but it must be interpreted with caution. A total of 954 patients with suspected Lyme borreliosis were evaluated on the basis of clinical and serological data.
**The seroprevalence of Borrelia burgdorferi antibodies was 4.4 percent (42 of the 954 serum samples).**
The most frequent clinical manifestation was erythema migrans which occurred in 50 percent of the seropositive patients, followed by neuroborreliosis (16.6 percent) and arthritis (11.9 percent). Carditis was rare.
**Our findings suggest that for the diagnosis of Lyme borreliosis, serologic tests need to be combined with clinical signs and symptoms.**

[soijuv]

Uusi borreliatesti jossa bakteeri viljellään verestä. Nykyisin käytetään epäluotettavaa vasta-aineiden muodostumiseen perustuvaa testiä joka antaa usein virheellisiä negatiivisia tai raja-arvoisia testituloksia.

Uuden testin kehittäneen laboratorion mukaan: "Borreliatestit ovat tunnettuja epäluotettavuudestaan. Sen seurauksena on esiintynyt runsaasti virheellisiä diagnooseja ja hoitoja. Nyt laboratorio, "Advanced Laboratory Services Inc. ", on kehittänyt borrelia-testin bakteerin viljelemiseksi verestä. Nykyiset seulonnoissa käytettävät edulliset vastainetatestit kertovat ainoastaan mahdollisesta tartunnasta jossakin vaiheessa elämää ja elimistön kyvystä tunnistaa ja muodostaa vasta-aineita. Tutkimusten mukaan nämä testit jättävät havaitsematta 30 - 70% borreliatapauksista.
Uusi testi kertoo näytteenottohetken tilanteen. Testi voi olla positiivinen myös sellaisissa tapauksissa joissa vasta-ainetesti on negatiivinen."

Alla myös erään amerikkalaisen Borrelioosiin erikoistuneen lääkärin näkemyksiä uudesta testistä. "Borrelia-bakteeri hakeutuu kudoksiin mutta ilmestyy aika ajoin verenkiertoon. Mahdollisimman luotettavan tuloksen saamiseksi verinäytteen tulle olla laboratoriossa 24 tunnin sisällä. Kuljetuksen aikana lämpötila ei saa vaihdella suuresti. Näistä syistä testi on tällä hetkellä käytössä vain USA:ssa. Näyte on parasta ottaa varhain iltapäivällä sillä suurin osa sairastuneista saa silloin oireita esim. kasvojen punoitus, lämmön lievä nousu, uupumus jne. Antibiootteja ei tule käyttää 4 viikkoon ennen näytteenottoa. Koska testitulos kertoo todellisen tilanteen juuri sillä hetkellä, hoitava lääkäri ei voi kieltää tilannetta. Borrelia-tartunta on tosiasia."


PRESS RELEASE
Sharon Hill, PA, September 5, 2011
BORRELIA CULTURE NOW AVAILABLE TO EVALUATE
LYME DISEASE PATIENTS
Research breakthrough promises a new Gold Standard in Lyme Disease testing

Lyme Disease blood testing has been notorious for its unreliability This has been responsible for misdiagnoses and inappropriate patient care, as well as confusion on the part of both patient and physician alike. Now, as a result of intensive research, Advanced Laboratory Services Inc. is able to offer what will rapidly become the new gold standard of Lyme tests, the Borrelia Culture.


Until now, the most widely used confirmatory tests for Lyme disease have been serologies. Being indirect tests, at best they can only indicate possible exposure to this organism at some previous point in time. It has been variously reported that the sensitivity of these assays is low and may miss anywhere from 30% to as many as 70% of cases of Lyme. Once positive, these serologic tests tend to remain positive for variable periods, even years, even after treatment. Therefore they do not and cannot be used as a marker for progress during treatment or for success of treatment. In addition, some acute viral infections may potentially give a false positive result.
How are most other infectious diseases diagnosed? Traditionally, a culture is taken to see if an infection is present, and if so, what specific bacterium is causing it. Unfortunately, because Lyme Borrelia are symbionts, meaning that they need a living host to survive, trying to get them to thrive in vitro has been a nearly impossible task. In addition, they are noted for their very slow growth. Because of these difficulties, Borrelia culture until now has not been available to clinicians.
Advanced Laboratory Services Inc. is proud to announce that they have overcome many of these technical difficulties and is able to offer Borrelia cultures. While still considered investigational, the new methods employed allow these advantages:

By definition, culture is a direct test and if positive, indicates that an infection was present at the time the specimen was taken
Cultures may be positive even in a patient who is seronegative
In theory, any fluid or tissue that is infected can be cultured
All known strains of Borrelia burgdorferi sensu lato can be detected
When combined with PCR and DNA sequencing, the exact identity of the Borrelia can be ascertained
Advanced methods have the promise to increase yield and decrease turn-around time
Culture positivity fulfills even the strict CDC surveillance case definition
Will become the new Gold Standard for laboratory testing
\
Currently, Advanced Labs is offering two Borrelia blood culture panels. The Basic Panel consists of culturing, with confirmation of identity by histology and by specific immunostaining. The report, if positive, will include a picture of the actual immunostained culture result. The expected turnaround time for this is approximately ten to fourteen days. However, some do grow more slowly, so all cultures will be held in the lab for a minimum of six weeks.
We also offer the Extended Panel. In addition to histology and immunostaining, the identity of all positive cultures will be further studied by polymerase chain reaction (PCR) and by nucleic acid sequencing. These extra steps will generally require an additional ten to fourteen days. However, if the initial histology with immunostaining is positive, a preliminary report will be issued while the nucleic acid studies are pending.
Bb culture nuts and bolts
Sensitivity exceeds 80% if the patient is symptomatic at the time of blood draw and not on antibiotics for at least four weeks
All negative controls have remained negative so far
As with all blood cultures, may need several sets to be sure
Any positive test is significant, even if it is the only positive out of a set of three

We have found that the success of culturing Borrelia can be increased by following these simple recommendations:
The patient should not have been exposed to any antibiotics, even those not known to affect this organism, for a minimum of four weeks prior to the blood sample being drawn.
Borrelia are more likely to be recovered from patients who are symptomatic at the time of blood sampling.
A higher yield may be seen if the blood is drawn in the early afternoon, when most infected patients feel especially ill.

Advanced Laboratory Services is continually refining its processes, so we welcome your feedback! Please feel free to share your ideas with us.
Advanced Laboratory Services, Inc. 501 Elmwood Avenue - Sharon Hill, PA 19079
Web: www.advanced-lab.com email: info@advanced-lab.com
Toll Free- 855-238-4949 Fax- 855-238-4946
---------------------------------------------------------------------------------------------------------

.....the biology of Bb in people is quite complex and still not fully understood.
Bb do seek out and persist in deep tissues, but they regularly transit through the circulation throughout the infection. I have seen some very impressive vital videos of this, and my extensive experience with MacDonald's blood Bb cultures 25 years ago confirmed this.
To assure maximal test sensitivity, the patient must have been off antibiotics for four weeks, be symptomatic from Lyme, and if at all possible, have the blood drawn in the early afternoon. I have observed that in active Lyme, many of not most patients get a wave of fatigue, increased symptoms, flushed face, and slightly elevated temperature at that time of day. If blood is drawn then, MacDonald was able to get a positive culture in nearly every one of my patients (probably about 100 cultures were done then). So I have to assume that that is when the spirochetemia seems most likely to be present.
Current lab experiments have revealed growth of Borrelia from many different patients from all across the USA. Because the ability to culture Borrelia is broad, the test is not reported as positive for Bb unless it is identified by growth characteristics, histology and at a minimum direct immunostaining by monoclonal and polyclonal antibodies.
Further testing is optional, and consists of PCR testing by a variety of primers and then by nucleic acid sequencing.
If a non-Borrelia spirochete is seen, it will be reoorted as such.
---What I specifically mean is that the lab has tested people who do not have Lyme and they never got a positive culture from any of them. In other words, if a patient has a positive culture, it is real, and not a contaminant etc. Therefore a positive culture cannot be dismissed, just as, for example, one would not ignore a positive culture of meningococcus on a spinal tap.
In other words, this data indicates that a doc should not dismiss a positive culture. It is real.

[salamanteri]

Tietääkö kukaan, ketkä lääkärit pk seudulla ovat perehtyneet borrelioosiin?

Tytärtäni puri punkki, ja hänen igm -arvonsa oli ensin kuukauden kuluttua puremasta 11 ja kahden kuukauden kuluttua 12, mikä on raja-arvo. Sormi kipeä ja pureman ympärille tuli pieni, borrelioosin kannalta epätyypillinen ihottuma, joka hävisi muutamassa viikossa. Terveyskeskuslääkärin kanssa pitäisi jäädä seuraamaan tilannetta ja odotella, paraneeko sormi itsestään. Yritin kysellä, eikö voisi tehdä toisenlaisia laboratoriotutkimuksia, mutta hänen mielestään se ei ole tarpeen. Olisiko mahdollista, että tyttären hengitettävä kortisonivalmiste, jota hän käyttää lievään astmaan, sotkisi hänen tutkimustuloksiaan?

Kun kerran tytöllä on särkyjä, pitäisi asia mielestäni tutkia tarkemmin, vaikkeivat raja-arvot ylitykään. Mistähän tuollainen raja-arvotulos voisi johtua? Myös reumavasta-aineet tutkittiin, mutta niissä ei näkynyt mitään poikkeavaa.

Eli nyt täytyisi löytää borrelioosiin perehtynyt lääkäri mieluiten pääkaupunkiseudulta. Ainoa, jonka itse tiedän, on Ville Valtonen, jonka seuraava vapaa aika on 12.1., eli liian myöhään. Umpimähkään en haluaisi mennä lääkäriin, olen kuullut paljon kertomuksia siitä, kuinka huonosti sisätautien erikoislääkäritkin tuntevat tautia. Toivoisin, että ei tarvitsi käydä kovin monilla lääkäreillä ja että saisimme pian oikean diagnoosin.

Kiitos etukäteen!

[soijuv]

(2010) Kiinalaiset tutkivat 13 borrelia-bakteerin proteiinia; 14 - 100kD väliltä. Tutkijat päätyivät siihen tulokseen että positiivisen borrelianäytteen tulee sisältää vähintään yksi seuraavista proteiineista:
IgG; P83/100, P58, P39, P30, OspC, P17, P66, ja OspA.
IgM; P83/100, P58, OspA, P30, OspC, P17 tai P41.
Sen lisäksi tulee poissulkea esim. syfiliksen, leptospiroosin jne mahdollisuus.



Biomed Environ Sci. 2010 Oct;23(5):341-9.

Interpretation criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China.
Jiang Y, Hou XX, Geng Z, Hao Q, Wan KL.
Source
State Key Laboratory for Infectious Diseases Prevention and Control, National Institute of Communicable Disease Control & Prevention, Chinese Center of Disease Control & Prevention, Beijing 102206, China.
Abstract
OBJECTIVE:
Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for its performance and interpretation have been established in China. The present study was designed to determine the criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China, in which WB was produced with strain PD₉₁ as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato.
METHODS:
Approximately 13 bands between 14 and 100 kD were differentiated for strain PD₉₁ by using Gel-Pro analysis software. In a study with 631 serum samples (taken from 127 patients with Lyme borreliosis and 504 controls), all observed bands were documented. To establish criteria for a positive WB result for strain PD₉₁, receiver operating characteristic (ROC) curves were used.

RESULTS:
The following interpretation criteria were recommended: for IgG, at least one band of P83/100, P58, P39, P30, OspC, P17, P66, and OspA; for IgM, at least one band of P83/100, P58, OspA, P30, OspC, P17 or P41. In addition, syphilis, leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM. For IgG criteria, the sensitivity is 73.2%, the specificity is 99.4% and Youden index is 0.726; for IgM criteria, the sensitivity is 50.6%, the specificity is 93.1% and Youden index is 0.437.

CONCLUSION:
Standardization of WB assays is necessary for comparison of results from different laboratories. Moreover, the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.
Copyright © 2010 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

[soijuv]

Turun seudulla Borrelia vasta-ainetestin (verestä tai selkäydinnesteestä) tulos merkitään +/- :lla. Vasta-ainetestien luotettavuudesta esiintyy maailmalla runsaasti allaolevasta näkemyksestä poikkeavia näkemyksiä.
Monien sairastuneiden kohdalla Suomessa tehty testi on ollut positiivinen vasta useiden mittausten jälkeen
tai vasta tarkemmassa immunoblottauksessa (Western blot)
tai esim. ulkomailla tehdyissä testeissä (esim. Saksan Melisa testi tai USA:n Igenexin testit).

http://www.med.utu.fi/tiedostot/ylab/pd ... AINEET.pdf

UTULab info@utulab.fi www.utulab.fi

BORRELIA BURGDORFERI, VASTA-AINEET
Lyhenne: S-BorrAb (3552), Li-BorrAb (3975)

Tiedustelut: Laboratorio: (02) 333 7401, lääkäri: (02) 333 7428

Yleistä: Borrelia burgdorferi -spirokeetta aiheuttaa Lymen borrelioosia. Tartunta saadaan yleensä puutiaisen (punkin) pureman kautta. Ensioireet kehittyvät viikosta kahteen kuukauteen. Tavallisimmat varhaisvaiheen oireet ovat erythema migrans (EM; n. puolella potilaista) ja joskus flunssan kaltainen oireisto. Yleensä ihomuutos häviää muutaman viikon kuluessa. Tauti voi edetä myöhäisvaiheen borrelioosiksi, johon kuuluu iho-oireiden lisäksi nivel- ja lihasoireita (20?50 %), keskushermoston ja perifeerisen hermoston oireita (30?50 %; meningiitti, meningoradikuliitti, enkefalomyeliitti, aivohermopareesit kuten fasiaalispareesi). Kardiittia ja AV-johtumishäiriötä esiintyy alle 10 %:lla potilaista. Myös erilaisia silmäoireita on kuvattu myöhäisborrelioosin yhteydessä.

IgG luokan vasta-aineet nousevat hitaasti, IgM-vaste on no-peampi, mutta epäspesifimpi. Osalla potilaista nähdään pitkittynyt IgM-vaste ilman IgG-vasta-aineiden nousua. Neurologisten oireiden yhteydessä on aina tutkittava myös likvorin vasta-aineet. Serologian sensitiivisyys on 30-70 % EM-tapauksissa ja yli 90 % disseminoituneessa borrelioosissa.

Lähete: Immunologisten tutkimusten lähete tai asiakkaan oma lähete.
Näyte: 2 ml verta tai 1 ml seerumia
Neurologisten oireiden yhteydessä 1 ml likvoria ja samanaikaisesti (48 tunnin sisällä) otettettu seeruminäyte.

Menetelmä: Kaksi entsyymi-immunologiseen menetelmään perustuvaa testiä. Seulontatestissä antigeenina käytetään B. burgdorferi -tyyppikannasta B31 sonikoimalla irroitettua antigeenia ja toisen vaiheen testissä, joka tehdään
UTULab info@utulab.fi www.utulab.fi
seulonnan positiivisista näytteistä, B. burgdorferin flagella-antigeenia (IDEIATM Borrelia burgdorferi IgM/IgG, Oxoid).
Tekotiheys: Kaksi kertaa viikossa.

Tulkinta: Positiivista löydöstä seuraa aina lausunto löydöksen merkityksestä ja suositelluista jatkotutkimuksista.

Yleistä: Matalaa IgM-luokan vasta-ainepositiivisuutta todetaan seulontatestissä kohtalaisen usein. Näiden vasta-aineiden spesifisyys tulee varmentaa S-BorAbJt ?jatkotutkimuksella tai toisena vaihtoehtona seurata mahdollista vasta-ainetasojen nousua pariseerumi-näytteellä.

Positiivinen IgG-vasta-ainelöydös (> +) on useimmiten spesifinen ja tukee siten borrelioosidiagnoosia.

negatiivinen: ei viitettä borrelioosista. Tulee kuitenkin muistaa, että vasta-aineet ovat usein negatiiviset EM-vaiheessa, joten negatiivinen löydös tässä tilanteessa ei sulje pois borreliainfektiota. Jos EM-diagnoosi pyritään varmentamaan serologisesti, voidaan tutkia uusi näyte n. kuukauden kuluttua akuuttivaiheesta.

IgM-vasta-aineet:

+/- = rajatapaus:
1) näyte suositellaan tutkitavaksi S-BorAbJt ?jatkotutkimuksella (ks. Borrelia burgdorferi, vasta-aineet, jatkotutkimus (S-BorAbJt), tai
2) suositellaan pariseerumin tutkimista n. kuukauden kuluttua

+ = todennäköisesti patologinen löydös
vasta-aineiden spesifisyys suositellaan varmistettavaksi S-BorAbJt ?jatkotutkimuksella (ks. Borrelia burgdorferi, vasta-aineet, jatkotutkimus (S-BorAbJt)

++ ja +++ = sopii borrelioosiin

[soijuv]

Oulun Yliopistollinen sairaala

http://oyslab.fi/ohjekirja/3975.html


Borrelia, vasta-aineet, aivo-selkäydinnesteestä
3975 Li-BorrAb

Osatutkimukset

Li-BorGInd, Li-BorrAbG, Li-BorrAbM, S -BorrAbG ja S -BorrAbM
Tiedustelut

Mikrobiologia/ Serologian laboratorio (OML), puh. 08-3156328.
Asiantuntijat

lääkäri Jaana Kauppila: jaana.kauppilaatppshp.fi / 08-3154001 / 040-5127203,
sairaalamikrobiologi Irma Ikäheimo: irma.ikaheimoatppshp.fi / 08-315 4005 ja
sairaalamikrobiologi Hanna Tuokko: hanna.tuokkoatppshp.fi / 08-3154086
Indikaatiot

Epäily Borrelia-spirokeetan aiheuttamasta infektiosta (Lymen borrelioosi). Borreliat tarttuvat punkin välityksellä.

Esivalmistelu

Mikrobiologian lähete, OML http://oyslab.fi/cgi-bin/lahete/oml_mlah.doc Läheteessä tulee mainita: kliininen diagnoosi, kysymyksenasettelu. Matkustusanamneesi.
Näyteastia

Steriili kierrek. muoviputki 7 ml
Näyte

1 ml likvoria
Menetelmä

Entsyymi-immunologinen vasta-ainemääritys ( EIA ).

Tekotiheys

Kerran viikossa
Tulkinta

Seulontavaiheen testit Li -BorrAbG ja Li -BorrAbM ovat herkkiä, mutta ei täysin spesifisiä osoittamaan Borrelia burgdorferi-infektiota. Seulonnassa positiiviseksi tullut näyte tutkitaan intratekaalisen vasta-ainesynteesin osoittamiseksi, edellyttäen että samanaikainen seeruminäyte on käytettävissä. OYS:n WbLabin kautta tilattuna Li-BorrAb-tutkimus sisältää automaattisesti myös S-BorrAb pyynnön.

Positiivisen tuloksen tulkinnassa tulee ottaa huomioon ristireaktiivisen immuniteetin mahdollisuus muun immuunireaktion aiheuttamana. Tyypillisiä ristireaktion aiheuttajia IgG-vasta-ainemäärityksessä ovat syfilis ja lievemmin tuberkuloosi, IgM-vasta-ainemäärityksessä erityisesti mykoplasma tai herpes simplex-virusinfektio. Näyte lähetetään tarvittaessa referenssilaboratorioon vasta-aineen spesifiteetin selvittämistä varten. Jatkotutkimus veloitetaan erikseen.

IgM-luokan vasta-aineet häviävät hoidon jälkeen aivo-selkäydinnesteestä selvästi nopeammin kuin IgG-vasta-aineet, jotka häviävät vasta kuukausien tai vuosien kuluessa. Siten aivo-selkäydinnesteiden kohdalla vasta-aineiden väheneminen hoidon jälkeen on säännönmukaisempaa kuin seerumin kohdalla.

Negatiivinen vasta-ainetulos ei poissulje tartuntamahdollisuutta. Osassa tuoreista neuroborrelioositapauksista voi bakteerigenomin osoitus geeninmonistuksella tulla positiiviseksi (Li-BorrNhO), kun vasta-aineita ei vielä ole osoitettavissa. Vastaukseen liitetään lausunto.

TULOKSET VALMIINA: Viikon kuluessa. Jatkotutkimukset yleensä n.2vk sisällä näytteen lähettämisestä referenssilaboratorioon.

Päivitetty 07.04.2011 / RK

[soijuv]

Ahvenanmaalla tehdään likvorista immunoblottaus neuroborrelioosia epäiltäessä seerumista ja likvorista + C6 seerumista

http://www.bimelix.ax/provtagningsanvis ... orr_fi.pdf

Borrelia burgdorferi, vasta-aineet selkäydinnesteestä

(Intratekaalisen vasta-ainetuotannon osoittaminen)
Li-Borrelia Ab

Indikaatio: Keskushermoston neuroborrelioosin tunnistus

Analyysipakettiin sisältyvät tutkimukset:

recomBead IgG Immunoblot selkäydinnesteestä
recomBead IgG Immunoblot seerumista
C6 seerumista

Lisäksi tarvitaan tiedot arvoista S-Alb, Li-Alb, S-IgG ja Li-IgG

Menetelmä: Analyyysissa tehdään recomBead IgG seerumista ja selkäydinnesteestä, joista
lasketaan intratekaalinen vasta-ainemuodostus jokaisesta syntyneestä vastaaineesta
erikseen.

Päivystys: Ei

Aikataulu: Analysoidaan n. 3-4 kertaa viikossa.
Näyte: 1 ml selkäydinnestettä sterilissä putkessa ilman lisäaineita.
1 ml seerumia, putki ilman geeliä, punainen tulppa.
Tiedot arvoista S-Alb, Li-Alb, S-IgG ja Li-IgG. (kts. alla)
Säilyvyys jääkaapissa korkeintaa 3 päivää.
Kuljetus huoneenlämmössä.

Lähettäjä vastaa pakkauksesta, näytteen merkinnöistä ja kuljetuksesta.
Huom.: Laskutoimitusten vuoksi tarvitaan lähetteeseen tiedot arvoista S-Alb,
Li-Alb, S-IgG ja Li-IgG. Jos arvot puuttuvat lähetteestä, Bimelix huolehtii
analyyseista tilaajan laskuun, jolloin vastaus voi myöhästyä.

Viitearvot: Li-IgG: Positiivinen tai negatiivinen.
Mikäli vastaus on positiivinen, ilmoitetaan intratekaalisesti syntyneet vastaaineet
jokainen erikseen.

S-Borr IgG Immunoblot ja S-Borr C6: Kts. asianomaista analyysiohjetta.

Tulkinta: Positiivinen Li-recomBead likvorista ja samalla likvorin mononukleaarinen
pleosytoosi puhuu vahvasti Lymen neuroborrelioosin puolesta.
Negatiivinen serologinen tulos vaikka mononukleaarine pleositoosi havaittaisiin
ei sulje pois neuroborrelioosia, varsinkaan jos oireiden alkamisesta kulunut aika
on lyhyt. Tällöin voi uusintatutkimus olla aiheellinen.
Spesifiset vasta-aineet voivat joskus olla osoitettavissa vasta 2 viikkoa
neurologisten oireiden alkamisesta.

[soijuv]

Melisa testin voi teettää nykyään myös Australiassa.

www.melisa.org

One of our labs Australian Biologics is now able to offer this testing for Borrelia and heavy metals

We are pleased to inform you that the MELISA test (Memory Lymphocyte Immunostimulation Assay) is now available through our laboratory.

The MELISA test was developed by Professor Vera Stejskal during her time as Head of Toxicology at AstraZeneca in Stockholm. The test is a cell culture test utilizing memory Lymphocytes that detects Type IV allergy to metals and chemicals and is also being used to detect immune responses to infections such as Borrelia.

See www.melisa.org

[soijuv]

Borrelia-bakteeri voi olla dilatoivan kardiopatian aiheuttaja. Bakteeria löydettiin potilaiden sydänlihaksesta otetuista koepaloista. (2012)

Eur J Heart Fail 2012(Feb)

Detection of Borrelia burgdorferi sensu lato in endomyocardial biopsy specimens in individuals with recent-onset dilated cardiomyopathy.

Kubánek M, Sramko M, Berenová D, Hulínská D, Hrbácková H, Malusková J, Lodererová A, Málek I, Kautzner J

Department of Cardiology, Institute for Clinical and Experimental Medicine, Videňská 1958/9, Prague, Czech Republic.

# DOI: 10.1093/eurjhf/hfs027

AIMS: Recent studies in patients with dilated cardiomyopathy (DCM) have detected the genome of Borrelia burgdorferi sensu lato (BBSL) in endomyocardial biopsy (EMB) specimens using a qualitative polymerase chain reaction (PCR), suggesting a causal link between Lyme disease and DCM in areas in which Lyme disease is endemic. We aimed to study this relationship using a comprehensive molecular analysis detecting BBSL in EMB samples.

METHODS AND RESULTS: We performed a comprehensive histopathological, immunohistochemical, ultrastructural, and molecular analysis targeting cardiotropic viruses and BBSL in EMB specimens of 41 individuals with recent-onset DCM and 15 controls with end-stage coronary artery disease. Specifically, quantitative PCR and electron microscopy of EMB specimens were employed. In addition, autoantibodies and manifestation of autoimmune diseases were evaluated in both groups. Individuals with recent-onset DCM presented more frequently with myocar dial BBSL persistence as compared with the control group (24% vs. 0%, P = 0.035). In contrast, the prevalence of parvovirus B19 and cytomegalovirus was similar in both groups. Sequence analysis of borrelial DNA revealed the following genospecies: Borrelia burgdorferi sensu stricto in three patients (30%), Borrelia afzelii in two patients (20%), and Borrelia garinii in four patients (40%), the results being inconclusive in one case. BBSL-positive DCM patients had a higher prevalence of organ-specific autoimmune diseases in comparison with the remaining DCM patients (50% vs. 16%, P = 0.030).

CONCLUSION: Myocardial persistence of BBSL may be involved in the pathophysiology of DCM in individuals living in areas in which Lyme disease is endemic.

[soijuv]

Tutkijat selvittivät Belgiassa käytettyjä borreliatestejä. Tutkimuksen mukaan borrelioosin diagnostiikka serologisten testien perusteella on edelleen haasteellista. Esim. malariaa sairastavilla esiintyi positiivisia testituloksia.

Evaluation of commercial screening tests and blot assays for the diagnosis of Lyme borreliosis.

Authors: Busson L, Reynders M, Van den Wijngaert S, Dahma H, Decolvenaer M, Vasseur L, Vandenberg O

Citation: Diagn Microbiol Infect Dis 2012(May)

Location: Department of Microbiology, Porte de Hal Laboratory, Saint-Pierre University Hospital & Jules Bordet Institute, 1000 Brussels, Belgium.

DOI: 10.1016/j.diagmicrobio.2012.04.001

The performance of 4 screening tests and 10 blot assays for the serologic diagnosis of Lyme borreliosis in a Belgian population was evaluated.

A total of 196 sera were tested: 36 Lyme borreliosis at different stages of the disease, 50 healthy blood donors, and 110 representing various clinical circumstances. The DiaSorin Liaison and Euroimmun Anti-Borrelia screening tests were evaluated. The tested blot assays were Virotech Borrelia LINE tests WE222, WE225, and WE224, as well as Mikrogen recomLine Borrelia and Viramed ViraStripe. The specificity of IgG was acceptable for the different assays. For IgM, DiaSorin Liaison Borrelia IgM Quant, Mikrogen recomLine, and Viramed ViraStripe lacked specificity.

Interestingly, a higher rate of falsely reactive samples was observed in the group of patients suffering from malaria. Serological diagnosis of Lyme borreliosis remains challenging; assays should be evaluated in the population where they are intended to be used.
Copyright © 2012 Elsevier Inc. All rights reserved.

[soijuv]

http://www.sciencedirect.com/science/ar ... 9312001186


Dolores Claesson, FL

Comparison of five diagnostic modalities for direct detection of Borrelia burgdorferi in patients with early Lyme disease ☆

Dionysios Liverisa,
Ira Schwartza, b,
Donna McKennab,
John Nowakowskib,
Robert Nadelmanb,
Joseph DeMarcoa,
Radha Iyera,
Susan Bittkerb,
Denise Cooperb,
Diane Holmgrenb,
Gary P. Wormserb, Corresponding author contact information, E-mail the corresponding author

a Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA
b Department of Medicine, Division of Infectious Diseases, New York Medical College, Valhalla, NY 10595, USA

Received 6 January 2012. Revised 21 March 2012. Accepted 28 March 2012. Available online 7 May 2012.

http://dx.doi.org/10.1016/j.diagmicrobio.2012.03.026, How to Cite or Link Using DOI

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1. Methods
2. Results
3. Discussion
References
Abstract
Lyme disease, the most commonly reported tick-borne infection in North America, is caused by infection with the spirochete Borrelia burgdorferi. Although an accurate clinical diagnosis can often be made based on the presence of erythema migrans, in research studies microbiologic or molecular microbiologic confirmation of the diagnosis may be required. In this study, we evaluated the sensitivity of 5 direct diagnostic methods (culture and nested polymerase chain reaction [PCR] of a 2-mm skin biopsy specimen, nested PCR and quantitative PCR (qPCR) performed on the same 1-mL aliquot of plasma and a novel qPCR?blood culture method) in 66 untreated adult patients with erythema migrans. Results of one or more of these tests were positive in 93.9% of the patients. Culture was more sensitive than PCR for both skin and blood, but the difference was only statistically significant for blood samples (P < 0.005). Blood culture was significantly more likely to be positive in patients with multiple erythema migrans skin lesions compared to those with a single lesion (P = 0.001). Positive test results among the 48 patients for whom all 5 assays were performed invariably included either a positive blood or a skin culture. The results of this study demonstrate that direct detection methods such as PCR and culture are highly sensitive in untreated adult patients with erythema migrans. This enabled microbiologic or molecular microbiologic confirmation of the diagnosis of B. burgdorferi infection in all but 4 (6.1%) of the 66 patients evaluated.
Keywords

Lyme disease;
Borrelia burgdorferi;
qPCR;
Molecular diagnostics

Figures and tables from this article:

Table 1. Comparison of PCR and culture tests in adult patients with EM.
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Table 2. Frequency of positive test results for the 52 patients with EM for whom all 5 assays were performed
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☆
This work was supported by NIH grant AR41511 to IS.

Corresponding author contact information
Corresponding author. Tel:+1-914-493-8865; fax:+1-914-493-7289.

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[soijuv]

Virtsatesti:

Easier way to detect Lyme disease may be on horizon
http://www.wtop.com/?nid=41&sid=2842963
...........
"I believe that many people are over-treated for Lyme disease, even though they have not been adequately diagnosed," says Dr. Donald Poretz, a clinical professor of medicine at Georgetown University and past president of the Infectious Disease Society of America.
"That can lead to using antibiotics unnecessarily, which is expensive. People can have bad reactions and the bacteria are becoming resistant to drugs."
The new test, being developed by researchers at George Mason University and Ceres Nanosciences, a diagnostic product development company, would detect the disease in urine samples - sometimes before symptoms appear.

"Our test will say, 'Yes, you still have the infection,' or, 'No, you don't,'" says Ross Dunlap, chief executive officer of Ceres. "With this, you're able to detect things that you were never able to detect before."
........... Currently, the only approved method of diagnosing the disease is by conducting an ELISA test on a blood sample. If that test is positive, then a Western blot test is done, Poretz says. If that is also positive, doctors can diagnose Lyme disease.

[soijuv]

Elispot-LTT testi (FDA:N ja CDC:n hyväksymä LTT tekniikka myös USA:ssa). Tutkimuksen mukaan 100% spesifisyys esim. borrelia-bakteerin aiheuttamissa infektioissa. Testiä suoritetaan Euroopassa mm. Saksan Borrelioosiklinikoilla.


Elispot®-LTT: FDA and CDC approved LTT technique in U.S.
Actual T-cellular activity in the blood against Borrelia burgdorferi, Chlamydia
pneumoniae/trachomatis, Ehrlichia/Anaplasma, EBV, Yersinia
In May 2011 the U.S. Food and Drug Administration (FDA) and the Centers for Disease Control
and Prevention (CDC) have approved the Elispot®-LTT (T-Spot) technique beneath the
QuantiFERON® TB Gold In-Tube test.
Both tests represent Interferon-Gamma Release Assays (IGRAs) in form of Lymphocyte
Transformation Tests (LTT).
No other laboratory T-cell tests have been approved (f.e. MELISA® or ITT® techniques) in the
field of all Lymphocyte Transformation Tests (LTT) by the FDA/CDC yet.
In the paper of the CDC regarding Interferon-Gamma Release Assays (IGRAs) from May
2011 the CDC says:
?... A positive result suggests that an infection is likely, a negative result that an infection is unlikely...?
?...Results can be available within 24 hours...?
Brandnew study for the specificity of Borrelia-Elispot-LTT:
? Borrelia antibody positive asymptomatic children (n=20), children with previous clinical

LB (n=24), and controls (n=20). Blood samples were analyzed for Borrelia-specific interferon-
gamma?by ELISPOT?.
?We found no significant differences in cytokine secretion between groups?
Skogman et al.: ?Adaptive and Innate Immune Responsiveness to Borrelia burgdorferi sensu lato?
in Exposed Asymptomatic Children and Children with Previous Clinical Lyme Borreliosis, Clincal and Development Immunology,
Vol. 2012, Article ID 294587, 10 pages

According this study:
100 % Specificity of Borrelia-Elispot-LTT

Actually the Elispot®-LTT is available for following tick-borne or other infections:

Borrelia burgdorferi
Chlamydia pneumoniae
Chlamydia trachomatis
Ehrlichia/Anaplasma
Epstein-Barr-Virus
Yersinia species

More informations:
Center for Disease Control and Prevention. Updated Guidelines for Using Interferon Gamma
Release Assays to Detect Mycobacterium tuberculosis Infection, United States. MMWR
2010; 59 (No.RR-5)
http://www.cdc.gov/mmwr/pdf/rr/rr5905.pdf

A Borrelia infection leads to a vitalisation of T-lymphocytes apart from the humoral immune
answer. The T-cellular immune response vanishes as soon as the Lyme disease is not active
anymore.
A therapy success control of a Lyme infection is not possible by the Borrelia antibodies, as
the ?titer? of antibodies in the blood can be found for years after an infection. Additionally in
stage I (e.g. ?bull´s eye rash? or ?summer flue? after a tick bite) antibodies can be found after
several weeks or in stage III they cannot be found in every case (weak immune system).
These diagnostic gaps are closed by the Elispot-LTT for Borrelia, which detects the actual
cellular activity against Borrelia burgdorferi in chronic and also acute Lyme disease. The
Elispot is so sensitive, that even a single Borrelia can reactivate T-cells in the blood. The
Elispot is 20- to 200-fold more sensitive than an ELISA-test on Borrelia and will already find 1
reactive T-cell in 100.000 lymphocytes.
The Elispot for Borrelia is very important for controlling a therapy of a chronic or acute Lyme
infection. In general the Elispot-LTT is going to be negative app. 6 to 8 weeks after the end of
a successful therapy.
Advantages of the Elispot-LTT for Borrelia (- as performed by infectolab -) in contrast to
other lymphocyte transformation or proliferation tests from other laboratories are:
· The result is available within 5 days (Other similar tests: 2 ? 3 weeks)
· The use of cell stabilising CPDA-tubes means a stability of 3 days for the measured
cells after taking the blood (Others: Heparin blood only 24 hours)!
· Better reliability than the different "unspecific" tests using the proliferation of T-cells
(e.g. ITT®)
This offers a significant improvement of the stability of the T-cells and a very quick decision
possibility for Lyme treating physicians to extend a therapy or to switch to a new treatment
option for Lyme disease!
Elispot-LTT for Borrelia:
Material: 3 x 9 ml CPDA-tubes (yellow cap, kept at room temperature, do not cool or
centrifuge)
Time required for analysis: 2 days (Results in 5 days)
Indications: - Diagnosis of chronic Lyme disease
- Diagnosis of acute Lyme disease
- Decision for the length of Lyme disease therapies
- Success control of therapies after Lyme treatment