BORRELIATESTIT

Asiantuntijana Soile Juvonen TTT

Valvojat: Bb, Sailairina, maranoma, Tiina

ViestiKirjoittaja soijuv » Ke Elo 29, 2012 11:45

Monilla borrelia-tartunnan saaneilla esiintyy ainoastaan IgM luokan vasta-aineita eikä myöhäis/kroonisessa vaiheessakaan IgG luokan vasta-aineita. Toisinaan heille sanotaan löydöksen olevan, erityisesti myöhäisvaiheen tapauksissa, merkityksetön.

Allaolevien näkemysten mukaan jatkuva IgM nousu merkitsee bakteerin selviytymistä elimistössä ja uuden immuunivasteen syntyä aika ajoin immuunijärjestelmän kohdatessa bakteerin. Toisen tutkimuksen mukaan IgM on negatiivinen varhaisessa ihomuutosvaiheessa mutta yleinen kroonisessa vaiheessa (ECM). Kohonneita IgM pitoisuuksia tavataan myös myöhäisvaiheessa ja se näyttäisi kertovan immuunipuolustuksen jatkuvasta stimulaatiosta bakteeria vastaan.


Joseph E. Craft, Duncan K. Fischer, Grant T. Shimamoto, and Allen C. Steere

J. Clin. Invest., Volume 78, October 1986, 934-939

?We report here the appearance of a new IgM response and the expansion of the IgG response late in the illness. These findings suggest that the Lyme spirochete persists for long periods in the host and triggers new immune responses during later attacks of arthritis.?
http://www.jci.org/articles/view/112683/pdf

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The Antibody Response in Lyme Disease
JOSEPH E. CRAFT, M.D., ROBERT L. GRODZICKI, M.S., MAHESH SHRESTHA, B.A., DUNCAN K. FISCHER, M.Phil., MARIANO GARCfA-BLANCO, M.Phil., AND ALLEN C. STEERE, M.D.

THE YALE JOURNAL OF BIOLOGY AND MEDICINE 57 (1984), 561-565
? We conclude from these data that the IgM response, although often absent in early localized Lyme disease, is generally present in patients with ECM who are more ill. The maximal response occurs after three to six weeks, and then declines. However, it may persist in later disease. Although the mechanism of the continued elevation of specific IgM is unclear, its persistence implies ongoing antigenic stimulation of the immune system, perhaps by an intact spirochete.? P.563   http://www.ncbi.nlm.nih.gov/pmc/article ... 0-0113.pdf   

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Useimmat aihetta käsittelevät tutkimukset ovat 1980 - luvulta jolloin asia vielä tunnustettiin.
Lisää tutkimuksia:

THE YALE JOURNAL OF BIOLOGY AND MEDICINE 57 (1984), 561-565
The Antibody Response in Lyme Disease
JOSEPH E. CRAFT, M.D., ROBERT L. GRODZICKI, M.S.,
MAHESH SHRESTHA, B.A., DUNCAN K. FISCHER, M.Phil.,
MARIANO GARCfA-BLANCO, M.Phil., AND ALLEN C. STEERE, M.D.
 
Antibody Response in Lyme Disease: Evaluation of Diagnostic Tests
Joseph E. Craft, Robert L. Grodzicki, Allen C. Steere
 The Journal of Infectious Diseases, Vol. 149, No. 5 (May, 1984), pp. 789-795
 
Antigens of Borrella burgdorferi Recognized during Lyme Disease
Appearance of a New Immunoglobulin M Response and Expansion of the
Immunoglobulin G Response Late in the Illness
Joseph E. Craft, Duncan K. Fischer, Grant T. Shimamoto, and Allen C. Steere
J. Clin. Invest.
Volume 78, October 1986, 934-939
 
Immunologic Aspects of Lyme Borreliosis
Raymond J. Dattwyler, David J. Volkman, Benjamin J. Luft:
Reviews of Infectious Diseases, Vol. 11, Supplement 6. Lyme Disease and Other Spirochetal Diseases (Sep. - Oct., 1989), pp. S1494-S1498
 
Treatment of Early Lyme Disease
MASSAROTTI, et.al.  April 1992 The American Journal of Medicine Volume 92 p. 396-403
 
LYME ARTHRITIS
Correlation of Serum and Cryoglobulin IgM with Activity, and Serum IgG with
Remission
ALLEN C. STEERE, JOHN A. HARDIN, SHAUN RUDDY, JON G. MUMMAW, and STEPHEN E. MALAWISTA
Arthritis and Rheumatism, Vol. 22, No. 5 (May 1979) p 471-483
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Syys 09, 2012 20:35

IgG luokan vasta-aineet vähentävät elimistön spirokeettakuormaa huomattavasti tehokkaammin kuin IgM luokan vasta-aineet. (2001)


Lyme borreliosis in rhesus macaques: effects of corticosteroids on
spirochetal load and isotype switching of anti-borrelia burgdorferi antibody.

Clin Diagn Lab Immunol 2001 Mar;8(2):225-32 (ISSN: 1071-412X)

Pachner AR; Amemiya K; Bartlett M; Schaefer H; Reddy K; Zhang WF

Department of Neurosciences, University of Medicine and Dentistry of New
Jersey-New Jersey Medical School, 185 S. Orange St., Newark, NJ 07103, USA.
pachner@umdnj.edu.

Lyme borreliosis in rhesus macaques: effects of corticosteroids on spirochetal load and isotype switching of anti-borrelia burgdorferi antibody.

Pachner AR, Amemiya K, Bartlett M, Schaefer H, Reddy K, Zhang WF

Experimental Borrelia burgdorferi infection of rhesus monkeys is an excellent model of Lyme
disease and closely parallels the infection in humans. Little is known about the interaction of host
immunity with the spirochete in patients with chronic infection.

We hypothesized that rapid development of anti-B. burgdorferi antibody in
immunocompetent nonhuman primates (NHPs) is the major determinant of the
reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by
measurement of the spirochetal load by PCR in association with characterization of the
anti-B. burgdorferi humoral immune response in immunocompetent NHPs versus that in
corticosteroid-treated NHPs.

Although anti-B. burgdorferi immunoglobulin G (IgG) antibody was effectively inhibited in
dexamethasone (Dex)-treated NHPs, anti-B. burgdorferi IgM antibody levels continued to rise
after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This
vigorous production of anti-B. burgdorferi IgM antibodies was also studied in vitro by
measurement of antibody produced by B. burgdorferi-stimulated peripheral blood mononuclear
cells.

Despite these high IgM antispirochetal antibodies in Dex-treated NHPs,
spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferi IgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » La Loka 06, 2012 20:01

Potentiaalinen uusi testi aktiivisen borrelioosin toteamiseksi? (2012)

http://about.gmu.edu/new-test-shows-pot ... e-disease/

New Test Shows Potential for Detecting Active Cases of Lyme Disease

By Michele McDonald

Alessandra Luchini

Worried that tick bite means Lyme disease? Mason researchers can find the answer well before the bite victim begins to show symptoms.

“If you are bit by a tick, you can’t be sure if you will get Lyme disease―that is the biggest problem right now,” says Mason researcher Alessandra Luchini of the Center for Applied Proteomics and Molecular Medicine.

Luchini and other Mason researchers are evaluating a new type of diagnostic test they developed for humans and their canine pals to pinpoint tiny signs of the bacteria that lead to Lyme disease. A study of the new type of test is underway. The test soon could be available commercially through privately held Ceres Nanosciences Inc., which partnered with Mason to develop the test and plans to market it to doctor’s offices and veterinary clinics.

The culprit is the blacklegged tick. It can carry the bacterium Borrelia burgdorferi, which leads to Lyme disease. To make matters worse, nymphs―about the size of the period at the end of this sentence―can bite unnoticed until the standard first sign of Lyme disease, a bull’s-eye rash, appears.

Joint and muscle aches, fatigue, fever, chills, headaches, and swollen lymph nodes typically come next, according to the Centers for Disease Control and Prevention.

Center for Proteomics and Molecular Medicine researchers Claudius Mueller and Lance Liotta in the lab. Photo by Creative Services.

A dose of antibiotics usually kills the bacteria, but sometimes symptoms persist. Patients return to their doctor months and even years later, convinced they still have Lyme disease, says Lance Liotta, the co-director of the center. Until now, there was no way of knowing definitively if the disease was still active or not.

Current blood tests only show if the body has created antibodies to fight the infection. Antibodies remain even after the infection is beaten.

But the active Lyme disease bacterium sheds a very small piece of itself called an antigen while it’s doing damage. In the past, these nanoparticles were too small to test. But thanks to technology developed at center, researchers can now use a “nanotrap” to capture the antigen in urine.

The patented nanotrap works much like a lobster trap, Liotta says. It’s an open meshwork with bait inside. The traps look like tiny white balls under the microscope. “The protein that we want goes in and gets stuck inside,” Liotta says. “It binds to that bait in the trap.”

The researcher plucks out the antigen, which is protected while in the trap. If the antigen shed by Borrelia burgdorferi is found, then the patient has an active case of Lyme disease, Luchini says.

“The antigen is a component of the toxic-causing agent itself,” Luchini says. “Instead of looking at the host response or whatever the human body does to fight the infection, we look at a piece of the infection-causing agent. Everyone measures the antibodies because it’s much easier.”

And it’s those antibodies that can cause problems, Liotta says. Antibodies fight infection and react to the proteins in the bacteria. But antibodies don’t stop with the infection—they move to attack proteins in the nerves, joints, and brain, Liotta says.

“The bacterium doesn’t directly cause the damage,” Liotta says. “It’s the immune response that’s doing the damage. The goal is to have a way to detect Lyme disease even before you make antibodies against it. Then you could treat the patient with antibiotics, and they wouldn’t get all those terrible symptoms. Or, if someone has joint problems and they’re convinced they have Lyme disease—and there are thousands of people who feel that way—it gives us a way to definitively say they do or don’t have Lyme disease.”

The inspiration for the test started about two and a half years ago when a high school student from Lucketts, Virginia, joined Mason’s Aspiring Scientists Summer Internship Program and worked with Luchini and Liotta. Temple Douglas, now a junior at Princeton University, had family members who suffered from Lyme disease.

She even collected the first round of ticks for the initial work on the test. “I lived in the countryside, so whenever people found ticks on their animals or crawling on their pants after they went hiking, I would take them with me to the lab,” Douglas says.

Ceres Nanosciences raised $1 million late last year, due in large part to the commercial potential of the Lyme disease diagnostic test, says Ross Dunlap, its chief executive officer. The results could do more than boost the company’s bottom line, he says.

“It would be good not to be flooding every tick bite with antibiotics,” Dunlap says.

This article originally appeared on the university’s News site.

To read more stories about Mason, check out the university’s News site.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Maalis 08, 2013 13:25

Laane 2013. "Olemme löytäneet yksinkertaisen menetelmän elävien borreliabakteerien havaitsemiseksi kroonista borrelioosia sairastavien verinäytteistä. Menetelmänä on perinteinen mikroskooppinen tutkimus jonka avulla havaitaan myös muita taudinaiheuttajia ja voidaan tarkastella esim. antibioottihoidon vaikutusta ."

A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques

http://www.biomedicalreports.org/index. ... %5B0%5D=98

From the page above, it is possible to download the pdf directly, from the link given at the bottom of the references.

Biological and Biomedical Reports
Vol 3, No 1 (2013)
A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques
Ivar Mysterud, Morten Motzfeldt Laane

Abstract

We have developed a simple method for the detection of live spirochaete stages in blood of patients where chronic borreliosis is suspected. Classic techniques involving phase-contrast and fluorescence microscopy are used. The method is also quite sensitive for detecting other bacteria, protists, fungi and other organisms present in blood samples. It is also useful for monitoring the effects of various antibiotics during treatment.

We also present a simple hypothesis for explaining the confusion generated through the interpretation of possible stages of Borrelia seen in human blood. We hypothesize that these various stages in the blood stream are derived from secondarily infected tissues and biofilms in the body with low oxygen concentrations.

Motile stages transform rapidly into cysts or sometimes penetrate other blood cells including red blood cells (RBCs). The latter are ideal hiding places for less motile stages that take advantage of the host’s RBCs blebbing-system. Less motile, morphologically different stages may be passively ejected in the blood plasma from the blebbing RBCs, more or less coated with the host’s membrane proteins which prevents detection by immunological methods.

References

References

Samuels, S. D.; Radolf, J. D. (eds), Borrelia. Molecular biology, host interaction and pathogenesis. Norfolk, Caister Academic Press. 547pp. (2010). ISBN 978-1-904455-58-5

Gray, J. S.; Kahl, O.; Lane, R. S.; Stanek, G. (eds), Lyme borreliosis. Biology,

epidemiology and control. New York, CABI Publishing, 347pp. (2002) ISBN 0851996329

Stricker, R. B.; Johnson, L., Lyme disease: the next decade. Infection and Drug Resistance 4 (2011), pp. 1-9. DOI: http:/dx.doi.org/10.2147/IDR.S15653

Burgdorfer, W., Lecture 12th Internat. Congr. On Lyme disease and other spirochetal and tick-borne disorders (1999). http://en.wikipedia.org/wiki/Willy_Burgdorfer

Hindle, E., On the life cycle of Spirochaeta gallinarum. Parasitology IV 1912, 463-477 (http://printfu.org/borrelia+burgdorferi+life+cycle).

Ferguson, J., Cure unwanted? Exploring the chronic Lyme disease controversy and why conflicts of interest in practice guidelines may be guidelines guiding us down the wrong path. American Journal of Law and Medicine 2012, 38, 196-224 .

Laane M. M., Ein Einfaches Mikroskopiesystem für Zeitrafferaufnahmen lebender Zellen. Mikrokosmos (Stuttgart) 2006, 95, 310-6. (A simple microscopical system for time-lapse records of living cells). In German

Laane, M. M.; Lie, T., Moderne mikroskopi med enkle metoder. Unipub forlag, Oslo. (2007), pp. 95-101. ISBN 978-82-7477-281-6. (Modern microscopy with simple methods). In Norwegian

Laane, M. M., The use of webcams in microscopy. “InFocus” Magazine, - The Proceedings of The Royal Microscopical Society, Oxford. Issue 2 (2007), pp. 42-54

http://www.rms.org.uk/OneStopCMS/Core/C ... 588aa7dd90

Laane, M. M.; Haugli, F. B., Illustrated guide to phase-contrast microscopy of nuclear events during mitosis and meiosis. In: H.C. Aldrich, J.W. Daniel (eds). Cell biology of Physarum and Didymium. New York, Academic Press Inc., Vol. 2 (1982), pp. 265-276.

ISBN 0-12-049602-X

Yu, L. W., Kjerneorganisering i Parabasalia. Et bidrag til forståelsen av mitosemekanismen som dobbeltsystem. (Nuclear organization in Parabasalids. A contribution to the understanding of the double nature of the mitotic mechanism). M.Sc. Thesis (supervisor M.M. Laane). University of Oslo, Norway. (2000)

Laane, M. M., YouTube video “SuperMikroskop” Borrelia live in human blood. (2011). http://www.youtube.com/watch?v=YxSHL9xG ... re=related

Laane, M. M.; Mysterud, I.; Longva, O.; Schumacher, T., Borrelia og Lyme-borreliose,- morfologiske studier av en farlig spirochet. Biolog 2009, 27, (2), 30-45. (Borrelia and Lyme borreliosis, - morphological studies of a dangerous spirochaete). In Norwegian. http://www.bio.ekanal.no/bio/vedlegg/borrelia.pdf

Laane, M. M.; Olsson, C. C.; Mysterud, I.; Longva, O.; Schumacher, T., Flått og hjortelusflue - viktige sykdomsvektorer under spredning i norsk natur. Biolog 2010, 28, (3-4), 70-85. (Tick and deerked – important disease vectors spreading in Norwegian ecosystems). In Norwegian.

http://www.borrelia-tbe.se/filer/2011/0 ... Biolog.pdf

Sens, P.; Gov, N., Force balance and membrane shedding at the red blood cell surface. Physical Review letters 2007, 98 018102, 1-4.

Margulis, L. J.; Ashen, B.; Sole, M.; Gurrero, R., Composite, large spirochetes from microbial mats: spirochetes structure review. Proc Natl Acad Sci USA 1993, 90, 6966-70.

Brorson, Ø.; Brorson, S.-H.; Schytes, J.; McAllister, J.; Wier, A.; Margulis, L., Destruction of the spirochaete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigeocycline. PNAS 2009, 106, (44), 18656-61.

Brorson, Ø.; Brorson, S.-H., A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes. APMIS 1998, 106, (12), 1131-41 .

The Lyme Info Net, see especially: Morphological transformation in Borrelia and other spirochetes: Observations of round forms and blebs 1905-2010, see also Survival in adverse conditions. http://www.lymeinfo.net/lymefiles.html

Brorson, Ø.; Brorson, S.-H., Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes. Infection 1997, 25, (4), 240-6.

Bozsik, B., YouTube video: Dark-field microscopy, DualDur (2009). http://www.youtube.com/watch?v=AiwRTu9zg5k

Moriarty, T. J.; Norman, M. U.; Colarusso, P.; Bankhead, T.; Kubes, P.; Chaconas, G., Real-time high-resolution 3D imaging of the Lyme disease spirochete adhering to and escaping from the vasculature of a living host. PLOS Pathogens 2008, 4, (6), e1000090. doi: 10.1371/journal.ppat.1000090.

Pohlod, D. J.; Mattman, L. H.; Tunsdall, L., Structures suggesting cell-wall deficient forms detected in circulating erythrocytes by fluorochrome staining. Applied Microbiology 1972, 23, 262-7.

Mattman, L., Lecture at Saginow, May 6th. 10 Ann. Int. Conf. NIH Bethesda. MD. (1997)

Badon, S. J.; Fister, R. D.; Cable, R. G., Survival of Borrelia burgdorferi in blood products. Transfusion 1989, 29, (7), 581-3.

Nadelmann, R. B.; Sherer, C.; Mack, L.; Pavia, C. S.; Wormser, G. P., Survival of Borrelia burgdorferi in human blood stored under blood banking conditions. Transfusion 1990, 30, (4), 298-301.

Margulis, L., (pers. comm.) letter to Dr. Todd LePine (2011).

Silvie, O.; Mota, M. M.; Matuschewski, K.; Prudencio, M., Interactions of the malaria parasite and its mammalian host. Current Opinion in Microbiology 2008, 11, 1-8.

MacDonald, A., A life-cycle for Borrelia spirochetes? Medical Hypotheses 2006, 67, (4), 810-8. .http://www.theoneclickgroup.co.uk/docum ... chetes.htm

MacDonald, A., Segmentation in the Borrelia genome. Genetic implications. (2012). http://lymeneteurope.org/forum/viewtopic.php?f=5&t=3686
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Maalis 08, 2013 14:27

Immunoblottauksen (western blot) tulkinta Kiinassa.

Biomed Environ Sci. 2010 Oct;23(5):341-9. doi: 10.1016/S0895-3988(10)60074-8.
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.

PMID:
21112481
[PubMed - indexed for MEDLINE]
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ke Maalis 13, 2013 18:55

Virheelliseen diagnoosiin voidaan päätyä esim virheellisen negatiivisen testituloksen johdosta. Virheellinen testitulos voi johtua mm siitä syystä että testissä käytetyt antigeenit eivät havaitse bakteerin alalajia esim. B.miyamotoita. Esim. USA:ssa löydetään yhä enenevässä määrin Euroopassa esiintyviä B.gariniita ja afzeliita mutta borreliatesteissä käytetään siellä antigeeninä ainoastaan B.burgdorferi B31. Se saattaa selittää osaltaan lukuisat virheelliset negatiiviset testitulokset.

Reconciliation of Published Facts with the Presence of Garinii type Borrelia on the North American Continent:

The presence of Garinii in the blood of USA patients ( N=5 patients) from the Advanced Labs paper by
Dr Eva Sapi, the additional patient with blood culture positive Afzelii, and two patients with
Kurtenbachii all reinforced the breakthrough idea that European Type Borrelia are now most likely
in the USA population - either by virtue of Travel to Europe on Vacation, or even because of exposure
to European ticks during military service [ the USA has a sizable ground force in Germany}

These Garinii " European strains" are also prominent in Mexican Lyme and are the exclusive agents
in Brazilian Lyme.
So we need to recalibrate our thinking about what strains might be in any individual's blood
from any continent.
Garinii will not be deteted in ELISA or WB manufactured from B31 USA borrelia burgdorferi.
So an an unknown % of "seronegative" patients may be " B31 diagnositic kit" negative but
seropositive with kits developed from "european" strains.

Further muddying the waters is the identification of Miyamotoi in Northeastern USA with a well
documented case of human disease now recorded. Miyamotoi test kits are not standardized
and are not commercially available. So here is another scenario for so called False negative but
in the body positive Borrelia infection of either the pure miyamotoi type or of the mixed
miyamotoi/burgdorferi type.

Further complicating the Serology unanswered questions :: the 92 genotypes {pyrG DNA sequencing} of
Burgdorferi type borrelia
now known to exist in the USA as subtypes of BB sensu stricto. The best genotyped among these are what
I refer to as the Schutzer "13 Strains" and among these is stran Bb ss Sz7.
One of the 92 isolates from Advanced labs was Sz7 Bb ss.
--------------------------
Whole-genome sequences of thirteen isolates of Borrelia burgdorferi.
http://www.ncbi.nlm.nih.gov/pubmed/20935092by SE Schutzer - 2011 - Cited by 20 - Related articles
Oct 8, 2010 – Schutzer SE, Fraser-Liggett CM, Casjens SR, Qiu WG, Dunn JJ, Mongodin EF, Luft BJ. ... The first complete genome sequence of B. burgdorferi strain 31, ... we determined the whole-genome sequences of 13 additional B.

Sapi,E., et al ,"Improved Culture conditions for the Growth and Detection of Borrelia from Human Serum",
International Journal of Medical Sciences, 2013, 10(4) 362-376, doi: 10.7150/ijms.5698
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ke Maalis 13, 2013 19:10

10 v vanha (2003) artikkeli Borrelioosin diagnostiikasta Euroopassa.

http://online.liebertpub.com/doi/pdfplu ... 3322662200

Diagnosis of Lyme Borreliosis in Europe

BETTINA WILSKE
ABSTRACT

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. Key Words: Lyme borreliosis—Borrelia burgdorferi—Diagnosis. Vector-Borne Zoonotic Dis.
3, 215–227.

INTRODUCTION

LYME BORRELIOSIS is a multisystem disease involving many organs such as the skin, the
nervous system, the joints, and the heart (Steere et al. 1989, Pfister et al. 1994). This condition is
the most frequent tick-borne disease in the northern hemisphere. Due to the diversity of
clinical symptoms, Lyme disease is often considered in a differential diagnosis. Examinations for antibodies against Borrelia burgdorferi are thus in high demand, and are among the most frequently requested serological tests
in microbiological laboratories. Microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae in Europe.

HETEROGENEITY OF LYME DISEASE BORRELIAE IN EUROPE AND ITS IMPACT FOR MICROBIOLOGICAL DIAGNOSIS

In Europe, Lyme borreliosis is caused by at
least three species: B. burgdorferi sensu stricto,
Max von Pettenkofer Institute, University of Munich, National Reference Center for Borreliae, Munich, Germany.
B. afzelii, and B. garinii. In contrast, B. burgdorferi
sensu stricto is the only human-pathogenic
species in the United States (Wang et al. al
1999b). The three human-pathogenic species
comprise at least seven OspA-serotypes in Europe
(Fig. 1) (Wilske et al. 1993c). Skin isolates
primarily belong to B. afzelii (OspA-type 2), especially
those from patients with acrodermatitis
chronica athrophicans, a chronic skin disease
not present in America (Canica et al. 1993,
Ohlenbusch et al. 1996, Wilske et al. 1993c) (see
also legend of Fig. 1). Isolates from CSF and
ticks are heterogeneous with a predominance
of B. garinii (Eiffert et al. 1995, van Dam et al.
1993, Wilske et al. 1996, Wilske et al. 1993c).
Sequence analysis of polymerase chain reaction
(PCR) ospA amplicons from synovial fluid
of Lyme arthritis patients revealed heterogeneity
(Eiffert et al. 1998, Vasiliu et al. 1998),
whereas other studies found mainly B.
burgdorferi s.s. using PCR based on the 5S/23S
rRNA intergenic spacer region (Lünemann et
al. 2001) or the flagellin gene (Jaulhac et al.
1996 and 2000). The most frequent genomic
groups in Europe B. afzelii and B. garinii occur
across the continent and the islands, whereas
the third frequent group B. burgdorferi s.s. has
only rarely been isolated in Eastern Europe (for
a survey, see Hubalek et al. 1997). Strains may
be very heterogeneous even within small areas
(Eiffert et al. 1995, Gern et al. 1999, Michel
et al. 2003, Rauter et al. 2002, Rijpkema et al.
1996). On the other side a focal prevalence of
certain species or subtypes was also observed
(Michel et al. 2003, Peter et al. 1995). Mixed infections
have been repeatedly observed in ixodid
ticks (for a survey, see Hubalek et al. 1997)
and sometimes also in specimens from patients
(Demaerschalck et al. 1995, Vasiliu et al. 1998,
Wilske et al. 1996). The heterogeneity of the
causative strains (Fig. 1) is a challenge for the
microbiological diagnosis of Lyme borreliosis
in Europe and must be kept in mind for development
of diagnostic tools such as PCR
primers and diagnostic antigens. For example,
ospA PCR has been widely used. Here, it is important
to be sure that not only representatives
of the three species are detected, but also the
different ospA-types of the heterogeneous B.
garinii group (Eiffert et al. 1995). In addition,
PCR should detect B. valaisiana and the recently
detected new genotype A14S since B.
valaisiana and genotype A14S might also be
pathogenic for humans, as suggested by positive
PCR results or cultures obtained from skin
biopsy specimens in a few studies (Rijpkema
et al. 1997, Wang et al. 1999a, Wilske et al.
2002). An ospA PCR for detection and differentiation
of the various European species and
OspA-types has been described by Michel
(2003).
Most of the proteins relevant for serodiagnosis
are heterogeneous. Interspecies amino
acid sequence identities are for example only
40–44% for DbpA (Osp17) and 54-68% for
OspC for representative strains of B. burgdorferi
sensu stricto, B. afzelii, and B. garinii
(strains B31, PKo, and PBi, respectively)
(Table 1). Especially DbpA has a much higher
amino acid sequence heterogeneity compared
to the DNA sequence heterogeneity indicating
immune selection. However, highly
heterogeneous proteins sometimes have conserved
immunogenic epitopes (e.g., the C6
peptide of VlsE) (Liang et al. 1999, Liang et
al. 2000).

GUIDELINES FOR THE
MICROBIOLOGICAL DIAGNOSIS
OF LYME BORRELIOSIS
The German Society of Hygiene and Microbiology
(DGHM) has recently published guidelines
for the microbiological diagnosis of Lyme
borreliosis written by an expert committee
(MiQ 12 Lyme-Borreliose) (Wilske et al. 2000).
The English version is accessible via internet
(www.dghm.org/red/index.html?cname5MI
Q). Except in cases with the pathognomic clinical
manifestation erythema migrans, the diagnosis
of Lyme borreliosis usually requires
confirmation by means of a microbiological diagnostic
assay. Antibody detection methods
mainly are used for this purpose, whereas detection
of the causative agent by culture isolation
and nucleic acid techniques is confined to
special situations, such as to clarify clinically
and serologically ambiguous findings. Application
of these methods should be reserved to
laboratories specialized in this type of examination.

DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 217
FIG. 1. Heterogeneity of Lyme disease Borrelia species and OspA-serotypes in Europe. Data for skin, CSF, and ticks are based on analysis of culture isolates; data
for synovial fluid are based on analysis of ospA PCR amplicons; source of skin specimens is known in 46 patients (30 cases with erythema migrans, thereof were 1,
26, 1, and 2 cases infected with OspA-types 1, 2, 4, and 6, respectively; 16 cases with ACA, thereof were 1 and 15 cases infected with OspA-types 1 and 2, respectively).
(Modified from Figures 5 and 6 in Wilske et al., 2002, with permission of the publisher.)
SPECIMENS FOR THE
MICROBIOLOGICAL DIAGNOSIS
For culture and PCR, skin biopsy samples are
the most promising specimens. In general poor
results are obtained from body fluids with the
exception of PCR of synovial fluid. Examination
of urine (PCR, antigen detection) is not recommended
nor the examination (PCR or IFA)
of ticks removed from patients in order to decide
antibiotic prophylaxis (Brettschneider et
al. 1998, Kaiser et al. 1998, Klempner et al. 2001,
Wilske et al. 2000). Examination of ticks should
be performed only for epidemiological or other
scientific studies. For antibody determination,
serum or CSF can be investigated. CSF examination
should always be done together with
serum antibody analysis (determination of the
CSF/serum antibody index).
DIRECT DETECTION METHODS
Culture
B. burgdorferi can be cultivated in modified
Kelly’s medium (Preac-Mursic et al. 1991,
Wilske and Schriefer 2002). This, however, is a
very time-consuming method (generation time
of B. burgdorferi is about 7–20 h) characterised by
low sensitivity, especially in body fluids (Arnez
et al. 2001, Åsbrink et al. 1985, Karlsson et al.
1990, Strle et al. 1999, Zore et al. 2002) (Table 2).
Culturing may be of help in individual cases if
the clinical picture suggests Lyme borreliosis despite
a negative antibody assay (seronegative
Lyme borreliosis), for example, in atypical erythema
migrans, suspected acute neuroborreliosis
without detection of intrathecal antibodies or
in the case of suspected Lyme borreliosis in patients
with immune deficiencies.
PCR
There is no standardized method for the
preparation of specimens nor for performing
the PCR itself. For DNA amplification under
experimental conditions various target sequences
have been used by specialised laboratories,
for example, from plasmid-borne genes
such as ospA and ospB, or chromosomal genes
such as the genes for the flagellar protein or
p66 (clone 2H1), or from gene segments of
the 16S rRNA or the 5S/23S rRNA intergenic
218 WILSKE
TABLE 1. SEQUENCE IDENTITIES AMONG MAJOR IMMUNODOMINANT PROTEINS OF THE THREE GENOSPECIES
OF B. BURGDORFERI SENSU LATO (COMPARISON OF STRAINS B31, PKO AND PBI)
DNA sequences, Amino acid sequences,
Protein range (in %) range (in %)
DbpAa 51–63 40–44
OspCa 61–77 54–68
OspAa 85–86 78–81
p35a 74–85 65–80
BmpA (p39)a 91–93 89–90
p58a 90–97 90–97
Flagellin 94–95 96–97
Flagellin fragment (aa 129–251) nd 93–96
p83/100a 87–89 81–87
aSequence identities were calculated without the leader sequence of the lipoproteins.
TABLE 2. SENSITIVITY OF DIRECT PATHOGEN DETECTION METHODS IN LYME BORRELIOSIS
Specimen Sensitivity
Skin (erythema migrans, acrodermatitis) 50–70% when using culture or PCR
CSF (neuroborreliosis, stage II) 10–30% when using culture or PCR
Synovial fluida (Lyme arthritis) 50–70% when using PCR (culture is only extremely
seldom positive)
aHigher sensitivity within synovial tissue compared to synovial fluid.
spacer region (for a survey, see Schmidt et al.
1997). Borrelia PCR should allow diagnosis of
the Borrelia species, that is, the medical report
should contain information as to which of the
three species pathogenic for humans has been
found. The diagnostic sensitivity of PCR is
about the same as the sensitivity of culture.
Borreliae are detected with much more difficulty
from body fluids than from tissue specimens
by either PCR or culture (Arnez et al.
2001, Jaulhac et al. 1996, Karlsson et al. 1990).
Solely PCR of synovial fluid seems to surpass
culture significantly in sensitivity (Nocton et
al. 1994).
Sensitivity of culture and PCR
Table 2 gives a survey about sensitivity of direct
detection methods in clinical specimens
from patients with Lyme borreliosis. Culture and
PCR have the highest detection rates (50–70%) in
skin biopsies from patients with erythema migrans
or acrodermatitis chronica atrophicans
(Åsbrink et al. 1985a, van Dam et al. 1993, von
Stedingk et al. 1995, Weber et al. 1990, Zore et al.
2002). In contrast borreliae are detected by PCR
or culture in the CSF of only 10–30% of patients
with neuroborreliosis (Eiffert et al. 1995, Karlsson
et al. 1990, Wilske and Preac-Mursic 1993b).
CSF isolates are more frequently obtained from
patients with short duration of disease than from
patients with disease of long duration (Karlsson
et al. 1990). It is surprising that borreliae are detected
by PCR in 50–70% in the synovial fluids
of Lyme arthritis patients, but culture is rarely
successful (Eiffert et al. 1998, Vassiliu et al. 1998).
The best PCR results are obtained from synovial
tissue, not fluid (Jaulhac et al. 1996).
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 219
FIG. 2. Two-step approach in serodiagnosis. For criteria for positive, borderline, and negative blot, see text. (Modified
from Figure 6 in Wilske et al., 2000.)
ANTIBODY DETECTION
It is generally accepted that serological examination
should follow the principles of a two
step approach (Centers for Disease Control and
Prevention 1995, Johnson et al. 1996, Wilske et
al. 2000, Wilske and Schriefer 2002): (1) A serological
screening assay and (2) in the event of
a positive or equivocal result a confirmatory
assay. A sensitive ELISA is recommended,
which—in case it is reactive—should be confirmed
by the immunoblot (Fig. 2).
ELISA
The ELISA tests used for screening should be
at least second generation tests (Wilske et al.
2000), which have been improved with respect
to cross reactivity with other bacteria (e.g., extract
antigen with previous Reiter treponeme
adsorption) (Wilske et al. 1993a) or purified intact
flagella as antigen (Hansen et al. 1988).
Strains used as antigen source should express
OspC the immunodominant antigen of the IgM
response and DbpA an immunodominant antigen
of the IgG response (Wilske et al. 2000). Recently
specific recombinant antigens (i.e., VlsE)
or synthetic peptides (i.e., the C6 peptide derived
from VlsE) have been successfully used
in the United States (Bacon et al. 2003, Lawrenz
et al. 1999, Liang et al. 1999) and in a study with
European sera from patients with erythema migrans,
acrodermatitis, and arthritis (C6 peptide)
(Liang et al. 2000). Very recently also patients
with neuroborreliosis stage II have been
investigated with the C6 ELISA (IgG test) and
compared to the recombinant immunoblot
(Fingerle et al. 2002). Of 36 sera 31 were positive
by immunoblot and 34 by the C6-ELISA.
Two of the 31 immunoblot positive sera were
only borderline in the C6-ELISA, these sera had
antibodies against recombinant DbpA and p58
and DbpA and VlsE respectively. The C6-
ELISA appears to be sufficiently sensitive as a
screening test for IgG antibodies in patients
with neuroborreliosis if also borderline results
are included. However, VlsE has other immunodominant
epitopes besides the C6 region
that could improve diagnostic sensitivity; heterogeneity
of those immunodominant epitopes
especially must be considered in Europe (Göttner
et al. 2002). The IgM and IgG immune responses
of Lyme borreliosis patients in recombinant
immunoblots should suggest the best
combination of antigens for the development
of recombinant ELISAs.
Immunoblot
As a confirmatory assay the immunoblot
should have high specificity (at least 95%). If a
whole cell lysate is used as antigen, diagnostic
bands must be defined by monoclonal antibodies
(Fig. 3). In case of recombinant antigens,
identification of diagnostic bands is much easier.
For the whole cell lysate blot, strains expressing
immunodominant variable antigens
(OspC, DbpA5Osp17) in culture should be
used (i.e., strain PKo) (Wilske et al. 2000).
The immunoblot criteria recommended by
the Centers of Disease Control (CDC) for use
in the United States can not be used for Europe
(Hauser et al. 1997, Hauser et al. 1998, Robertson
et al. 2000). Dressler et al. (1994) have
shown in an immunoblot study that the immune
response of European patients is re-
220 WILSKE
FIG. 3. Standardization of the whole cell lysate immunoblot
with monoclonal antibodies (antigen, B. afzelii
strain PKo; control sera, G5IgG, M5IgM; monoclonal antibodies
(1–11). Arrows indicate closely neighbored proteins
difficult to distinguish. (Modified from Figure 3 in
Wilske et al., 2000, with permission of the publisher.)
stricted to a narrower spectrum of Borrelia proteins,
compared with that shown by American
patients. Using different serum panels (first
serum panel from Germany, second serum
panel from various European countries),
Hauser et al. demonstrated in two studies that
strain-specific interpretation rules must be defined
(Hauser et al. 1997, Hauser et al. 1998).
Figure 4 shows that immunoblot antibody
binding patterns vary considerably by strain
used as antigen. Thus different interpretation
rules are required in order to achieve equal
sensitivity and specificity when different
genospecies of Borrelia are used in preparing
the blot antigen.
Interpretation criteria for the immunoblot
recommended by the DGHM are published
in the MiQ 12 Lyme-Borreliose (Wilske et al.
2000). These are if B. afzelii strain PKo is used
as antigen source the following: The IgG blot
is positive if $2 bands of the following are present:
p83/100, p58, p43, p39, p30, OspC, p21,
Osp17, p14; the IgM blot is positive if $1 band
of the following is present: p41 (strong), p39,
OspC, DbpA (Osp17). Further interpretation
criteria (other strains, recombinant blot) are
available via internet (www.dghm.org/red/index.
html?cname=MIQ). Borderline results are
reported if diagnostic bands are visible but the
criteria for a positive blot are not fulfilled. A
blot is negative if no diagnostic bands are visible.
Examples for IgM and IgG immunoblots are
shown in Figure 5. Patients with early manifestations
of acute neuroborreliosis have an
immune response restricted to only a few proteins.
Patients with late disease such as acrodermatitis
or arthritis have IgG antibodies to a
broad spectrum of antigens. Using recombinant
antigens for the immunoblot has several
advantages compared to the immunoblot using
whole cell lysate antigen: (a) specific antigens
can be selected (i.e., p83/100, BmpA), (b)
homologous antigens derived from different
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 221
FIG. 4. Heterogeneous IgG reactivity of sera from Lyme borreliosis patients in the immunoblot with different strains
of B. burgdorferi s.l as antigen. Strain PKa2 is B. burgdorferi s.s., strain PKo is B. afzelii, and strain PBi is B. garinii. (Modified
from Figure 2 in Hauser et al., 1997, with permission of the publisher.)
strains can be combined (i.e., DbpA (Osp17),
OspC, BmpA), (c) truncated antigens with
higher specificity can be designed (internal flagellin
fragments), and (d) antigens primarily
expressed in vivo can be used (i.e., DbpA, VlsE)
(Heikkilä et al. 2002, Schulte-Spechtel et al.
2002, Wilske et al. 1999). Commercial recombinant
antigen immunoblots are better standardised
than the conventional ones. If a broad
panel of recombinant antigens (including the
recently described VlsE) is used the recombinant
blot is at least as sensitive as the conventional
one. An in house recombinant IgG immunoblot
(Wilske et al. 1999) shown in Figure
6 could be significantly improved by addition
of recombinant VlsE and an additional DbpA
homologue (Schulte-Spechtel et al.2003). Purified
proteins and immunoblot reactivity with
sera from patients with acute neuroborreliosis
are shown in Figure 7. By addition of VlsE and
the DbpA homologue, sensitivity increased
from 52.7% to 86.1% in 36 cases of neuroborreliosis
stage II, while specificity remained unchanged.
Sensitivity was also increased compared
to the whole cell lysate immunoblot
(86.1% versus 63.8%). Thus the new recombinant
immunoblot is a considerable step towards
better standardisation and in addition is
more sensitive than the whole cell lysate blot
since homologous proteins from different
222 WILSKE
FIG. 5. Whole cell lysate immunoblot: IgM- and IgG immune response in patients with neuroborreliosis (lanes 1–7,
respectively), IgG immune response in patients with acrodermatitis (lanes 1-7). Lanes designated with 1 are IgG blots
to demonstrate a broad panel of diagnostic bands. (Modified from Figure 4 in Wilske et al., 2000, with permission of
the publisher.)
strains (especially those with low sequence
identities as DbpA, see Table 1) and in vivo expressed
proteins (as VlsE) are used as antigens.
Determination of the CSF/serum index
Methods taking into account potential dysfunction
of the blood-CSF barrier are suitable
for the detection of intrathecal antibody production
(Wilske et al. 1986, Hansen et al. 1990,
Hansen et al. 1991). Determination of the
CSF/serum index should be performed if neuroborreliosis
is considered, since a positive
CSF/serum index confirms involvement of the
CNS. It may be positive in some cases when
serum antibody tests are negative or equivocal,
especially if the patient’s illness has been of
short duration (Wilske et al. 2000). Depending
on the time elapsed since the first manifestation
of neurological symptoms, the IgG
CSF/serum index is positive for 80–90% of patients
(8–41 days after onset of the disease) up
to 100% of patients (.41 days after onset)
(Hansen et al. 1991). Detection of intrathecally
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 223
FIG. 6. Recombinant IgG immunoblot with sera from patients with neuroborreliosis stage II (old immunoblot); top
and bottoms panels are the same. (Modified from Figure 2 in Wilske et al., 1999, with permission of the publisher.)
FIG. 7. New recombinant antigens for an improved immunoblot:
VlsE from B. burgdorferi s.s. strain PKa2 and
DbpA from B. garinii strain PBr. (a) SDS- PAGE: A—recombinant
E. coli whole cell lysate; B—purified protein. (b)
Immunoblot with immune serum against E. coli, antigens as
in a. (c) Immunoblot with sera from three patients with acute
neuroborreliosis. (Modified from Figures 1 and 3 in Schulte-
Spechtel et al., 2003, with permission of the publisher.)
produced IgM antibodies shows a high degree
of sensitivity in neuroborreliosis with short duration
of symptoms, especially in children
(Christen et al. 1993, Hansen et al. 1991).
CSF/serum index determination is especially
important for diagnosis of chronic neuroborreliosis.
A positive IgG CSF/serum index
is essential for the diagnosis of chronic borreliosis
of the central nervous system (see
EUCALB case definitions, Stanek et al. 1996),
whereas chronic peripheral polyneuropathy is
usually negative for intrathecal antibody production
(Kristoferitsch et al. 1993).
Serological findings in various
stages of the disease
Interpretation of serological test results must
always be done in context with clinical data
(Table 3). Here case definitions are helpful
(Stanek et al. 1996, Wilske et al. 2000). In stage I
(erythema migrans), only 20–50% of the patients
are seropositive for IgM and/or IgG antibodies
(Åsbrink et al. 1985b, Hansen and Åsbrink 1989,
Weber et al. 1990). IgM antibodies usually prevail.
An exception might be the immune response
against the recently detected VlsE. In
American patients with erythema migrans IgG
responses against VlsE are observed earlier than
IgM responses (in acute erythema migrans, in
44% versus 19%, in convalescent erythema migrans
in 59% versus 43%) (Bacon et al. 2003). In
European patients with erythema migrans, an
early IgG response to VlsE was observed in
20 of 23 (87%) culture-confirmed EM cases, the
IgM response has not been investigated (Liang
et al. 2000). In stage II (acute neuroborreliosis)
seropositivity (IgM and/or IgG antibodies) increases
to 70–90% (Hansen et al. 1988, Wilske et
al. 1993a). In principle, patients with early manifestations
may be seronegative especially in case
of short duration of symptoms. Then serological
follow-up is recommended. Six weeks or more
after onset of symptoms, 100% of the patients
with stage II neuroborreliosis were seropositive
(Hansen et al.1988). In cases with late disease
(stage III, acrodermatis and arthritis), IgG antibodies
were detectable in all patients tested
(Hansen and Åsbrink 1989, Johnson et al. 1996,
Wilske et al. 1993a). A negative IgG test argues
against late Lyme borreliosis. Thus, a positive
IgM test without a positive IgG test is not diagnostic
for late disease manifestations (Wilske et
al. 2000). An exception could be the situation of
a patient who received inadequate antibiotic
therapy for early disease, but sufficient drug to
abrogate IgM to IgG class switch or very short
duration of clinical symptoms. Since serological
findings vary considerably and antibodies may
persist for long time in successfully treated individuals,
serological follow up is not suitable for
determining whether further antibiotic therapy
is warranted. The presence of specific antibodies
does not prove the presence of disease; a positive
antibody test may also be due to clinical or
subclinical infections in the past. The more nonspecific
the symptoms, the lower is the predictive
value of a positive serological test. Seropositivity
in the normal healthy population varies
with age and increased outdoor activities (e.g.,
in one study in Bavaria ,5% up to 20%) (Reimer
et al. 1999).
METHODS WHICH ARE NOT
RECOMMENDED FOR
MICROBIOLOGICAL DIAGNOSIS
Recently, various methods have been used in
commercially oriented laboratories that are not
224 WILSKE
TABLE 3. SENSITIVITY OF ANTIBODY DETECTION METHODS
IN THE DIAGNOSIS OF LYME DISEASE
Stage Sensitivity Remarks
I 20–50% Predominance of IgM
II 70–90% In cases of short disease duration
predominance of IgM, in cases of long
disease duration predominance of IgG
III Nearly 100% Usually solely IgGa
aThe presence of IgM antibodies without IgG antibodies is not diagnostic for
late disease; for possible exceptions, see text.
sufficiently evaluated for diagnostic purposes.
Among them are the antigen tests in body fluids,
PCR of urine, and lymphocyte transformation
tests. These tests are not recommended
for microbiological diagnosis. They are unreliable
and some of them are in addition very expensive,
especially if used for therapy control
(Brettschneider et al. 1998, Kalish et al. 2003,
Klempner et al. 2001).
REFERENCES
Arnez, M, Ruzic-Sabljic, E, Ahcan, J, et al. Isolation of Borrelia
burgdorferi sensu lato from blood of children with
solitary erythema migrans. Pediatr Infect Dis J 2001;
3:251–255.
Åsbrink, E, Hovmark, A. Successful cultivation of spirochetes
from skin lesions of patients with erythema
chronicum migrans Afzelius and acrodermatitis chronica
atrophicans. Acta Pathol Microbiol Immunol Scand
B 1985; 93:161–163.
Åsbrink, E, Hovmark, A, Hederstedt, B. Serologic studies
of erythema chronicum migrans Afzelius and acrodermatitis
chronica atrophicans with indirect immunofluorescence
and enzyme-linked immunosorbent assays.
Acta Derm Venereol 1985; 65:509–514.
Bacon, RM, Biggerstaff, BJ, Schriefer, M, et al. Improved
serodiagnosis of Lyme disease by kinetic ELISAs using
recombinant VlsE1 or peptide antigens of Borrelia
burgdorferi compared with two-tiered testing. J Infect
Dis 2003; 187:1187–1199.
Brettschneider, S, Bruckbauer, H, Klugbauer, N, et al. Diagnostic
value of PCR for detection of Borrelia burgdorferi
in skin biopsy and urine samples from patients with
skin borreliosis. J Clin Microbiol 1998; 36:2658–2665.
Canica, MM, Nato, F, Du Merle, L, et al. Monoclonal antibodies
for identification of Borrelia afzelii sp. nov. associated
with late cutaneous manifestations of Lyme
borreliosis. Scand J Infect Dis 1993; 25:441–448.
Centers for Disease Control and Prevention. Recommendations
for test performance and interpretation from the
second National Conference on Serologic Diagnosis of
Lyme Disease. Morbid Mortal Wkly Rprt 1995; 44:590.
Christen, H-J, Hanefeld, F, Eiffert, H, et al. Epidemiology
and clinical manifestations of Lyme borreliosis in childhood.
A prospective multicentre study with special regard
to neuroborreliosis. Acta Paediatr 1993; 386:1–76.
Demaerschalck, I, Messaoud, AB, De Kesel, M, et al. Simultaneous
presence of different Borrelia burgdorferi
genospecies in biological fluids of Lyme disease patients.
J Clin Microbiol 1995; 33:602–608.
Dressler, F, Ackermann, R, Steere, AC. Antibody responses
to the three genomic groups of Borrelia burgdorferi
in European Lyme borreliosis. J Infect Dis 1994;
169:313–318.
Eiffert, H, Karsten, A, Thomssen, R, et al. Characterization
of Borrelia burgdorferi strains in Lyme arthritis.
Scand J Infect Dis 1998; 30:265–268.
Eiffert, H, Ohlenbusch, A, Christen, H-J, et al. Nondifferentiation
between Lyme disease spirochetes from vector
ticks and human cerebrospinal fluids. J Infect Dis
1995; 171:476–479.
Gern, L, Hu, CM, Kocianova, E, et al. Genetic diversity of
Borrelia burgdorferi sensu lato isolates obtained from
Ixodes ricinus ticks collected in Slovakia. Eur J Epidemiol
1999; 15:665–669.
Göttner, G, Schulte-Spechtel, U, Wilske, B. Antigenic and
genetic heterogeneity of the immunodominant surface
protein VlsE among European B. burgdorferi s.l. strains.
Int J Med Microbiol 2002; 292:105.
Goossens, HAT, van den Bogaard, AE, Nohlmans, MKE.
Evaluation of fifteen commercially available serological
tests for diagnosis of Lyme borreliosis. Eur J Clin
Microbiol. Infect Dis 1999; 18:551–560
Fingerle, V, Schulte-Spechtel, U, Wilske, B. Evaluation of
an ELISA based on the C6-peptide of VlsE for diagnosis
of early neuroborreliosis. Int J Med Microbiol 2002;
292 (Suppl. 34): 217.
Hansen, K, Åsbrink, E. Serodiagnosis of erythema migrans
and acrodermatitis chronica atrophicans by the
Borrelia burgdorferi flagellum enzyme-linked immunosorbent
assay. J Clin Microbiol 1989; 27:545–551.
Hansen, K, Cruz, M, Link, H. Oligoclonal Borrelia burgdorferi-
specific IgG antibodies in cerebrospinal fluid in
Lyme neuroborreliosis. J Infect Dis 1990; 161:1194–1202.
Hansen, K, Hindersson, P, Pedersen, NS. Measurement of
antibodies to the Borrelia burgdorferi flagellum improves
serodiagnosis in Lyme disease. J Clin Microbiol 1988;
26:338–346.
Hansen, K, Lebech, A-M. Lyme neuroborreliosis: a new
sensitive diagnostic assay for intrathecal sythesis of Borrelia
burgdorferi–specific immunoglobulin G, A, and M.
Ann Neurol 1991; 30:197–205.
Hauser, U, Lehnert, G, Lobentanzer, R, et al. Interpretation
criteria for standardized western blots for three european
species of Borrelia burgdorferi sensu lato. J Clin
Microbiol 1997; 35:1433–1444.
Hauser, U, Lehnert, G, Wilske, B. Diagnostic value of proteins
of three Borrelia species (Borrelia burgdorferi sensu
lato) and implications for development and use of recombinant
antigens for serodiagnosis of Lyme borreliosis
in Europe. Clin Diagn Lab Immunol 1998; 5:456–462.
Heikkilä, T, Seppälä, I, Saxen, H, et al. Species-specific
serodiagnosis of Lyme arthritis and neuroborreliosis
due to Borrelia burgdorferi sensu stricto, B. afzelii, and B.
garinii by using decorin binding protein A. J Clin Microbiol
2002; 40:453–460.
Hubalek, Z, Halouzka, J. Distribution of Borrelia burgdorferi
sensu lato genomic groups in Europe, a review. Eur
J Epidemiol 1997; 13: 951–957.
Johnson, BJB, Robbins, KE, Balley, RE, et al. Serodiagnosis
of Lyme disease: accuracy of a two-step approach
using a flagella-based ELISA and immunoblotting. J Infect
Dis 1996; 174:346–353.
Jaulhac, B, Chary-Valckenaere, I, Sibilia, J, et al. Detection
of Borrelia burgdorferi by DNA amplification in synovial
tissue samples from patients with Lyme arthritis.
Arthritis Rheum 1996; 39:736–745.
Jaulhac, B, Heller, R, Limbach, FX, et al. Direct molecular
DIAGNOSIS OF LYME BORRELIOSIS IN EUROPE 225
typing of Borrelia burgdorferi sensu lato species in synovial
samples from patients with Lyme arthritis. J Clin
Microbiol 2000; 38:1895–1900.
Kaiser, R. Teilnehmer der Expertenkonferenz: Frühsommermeningoenzephalitis
und Lyme-Borreliose-Právention
vor und nach Zeckenstich. Dtsch med Wochenschr
1998; 123:847–853.
Karlsson, M, Hovind-Hougen, K, Svenungsson, B, et al.
Cultivation and characterization of spirochetes from
cerebrospinal fluid of patients with Lyme borreliosis J
Clin Microbiol 1990; 28:473–479.
Kalish, RS, Wood, J A, Golde, W, et al. Human T lymphocyte
response to Borrelia burgdorferi infection: no
correlation between human leukocyte function antigen
type 1 peptide response and clinical status. J Infect Dis
2003; 187:102–108.
Klempner, MS, Schmid, CH, Hu, L, et al. Intralaboratory
reliability of serologic and urine testing for Lyme disease.
Am J Med 2001; 110:217–219.
Kristoferitsch, W. Chronical peripheral neuropathy. In:
Weber, K, Burgdorfer, W, eds. Aspects of Lyme Borreliosis.
Berlin: Springer, 1993:219–227.
Lawrenz, MB, Hardham, JM, Owens, RT, et al. Human
antibody responses to VlsE antigenic variation protein
of Borrelia burgdorferi. J Clin Microbiol 1999; 37:3997–4004.
Liang, FT, Alvarez, AL, Gu, Y, et al. An immunodominant
conserved region within the variable domain of
VlsE, the variable surface antigen of Borrelia burgdorferi.
J Immunol 1999; 163:5566–5573.
Liang, FT, Aberer, E, Cinco, M, et al. Antigenic conservation
of an immunodominant invariable region of the
VlsE lipoprotein among european pathogenic genospecies
of Borrelia burgdorferi sl. J Infect Dis 2000;182:
1455–1462.
Lünemann, JD, Zarmas, S, Priem, S, et al. Rapid typing of
Borrelia burgdorferi sensu lato species in specimens from
patients with different manifestations of Lyme borreliosis.
J Clin Microbiol 2001; 39:1130–1133.
Michel, H, Wilske, B, Hettche, G, et al. An ospA-polymerase
chain reaction/restriction fragment length
polymorphism-based method for sensitive detection
and reliable differentiation of all European Borrelia
burgdorferi sensu lato species and OspA types. Med Microbiol
Immunol 2003 (in press).
Nocton, JJ, Dressler, F, Rutledge, BJ, et al. Detection of
Borrelia burgdorferi DNA by polymerase chain reaction
in synovial fluid from patients with Lyme arthritis.
N Engl J Med 1994; 330:229–234
Ohlenbusch, A, Matuschka, F-R, Richter, D, et al. Etiology
of the acrodermatitis chronica atrophicans lesion
in Lyme disease. J Infect Dis 1996; 174:421–423.
Péter, O, Bretz, AG, Bee, D. Occurence of different
genospecies of Borrelia burgdorferi sensu lato in ixodid
ticks of Valais, Switzerland. Eur J Epidemiol 1995;
11:463–467.
Pfister, H-W, Wilske, B, Weber, K. Lyme borreliosis: basic
science and clinical aspects. Lancet 1994; 343:1013–1016.
Preac-Mursic, V, Wilske, B, Reinhardt, S. Culture of Borrelia
burgdorferi on six solid media. Eur J Microbiol Infect
Dis 1991; 10:1076–1079.
Rauter, C, Oehme, R, Diterich, I, et al. Distribution of clinically
relevant Borrelia genospecies in ticks assessed by
a novel, single-run, real-time PCR. J Clin Microbiol
2002; 40:36–43.
Reimer, B, Marschang, A, Fingerle, V, et al. Epedimiology
of Lyme borreliosis in South-Eastern Bavaria (Germany).
Zent Bl Bakteriol 1999; 289:653–654.
Rijpkema SGT, Tazelaar, DJ, Molkenboer, MJCH, 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; 3:109–116.
Rijpkema, S, Golubic, D, Molkenboer, M, et al. Idenfication
of four genomic groups of Borrelia burgdorferi sensu
lato in Ixodes ricinus ticks collected in a Lyme borreliosis
endemic region of northern Croatia. Exp Appl Acarol
1996; 20:23–30.
Robertson, J, Guy, E, Andrews, N, et al. European multicenter
study of immunoblotting in serodiagnosis of
Lyme borreliosis. J Clin Microbiol 2000; 38:2097–
2102.
Schmidt, BL. PCR in laboratory diagnosis of human Borrelia
burgdorferi infections. Clin Microbiol Rev 1997;
10:185–201.
Schulte-Spechtel, U. Lehnert, G, Liegl, G, et al. Significant
improvement of the recombinant Borrelia IgG immunoblot
by addition of VlsE and a DbpA homologue
derived from B. garinii for the diagnosis of early neuroborreliosis.
J Clin Microbiol 2003; 41:1299–1303.
Stanek, G, O’Connell, S, Cimmino, M, et al. European
Union concerted action on risk assessment in Lyme borreliosis:
clinical case definitions for Lyme borreliosis.
Wien Klin Wochenschr 1996; 108/23:741–747.
Steere, AC. Medical progress—Lyme disease. N Engl J
Med 1989; 321:586–596.
Strle, F, Nadelman, RB, Cimperman, J, et al. Comparison
of culture-confirmed erythema migrans caused by Borrelia
burgdorferi sensu stricto in New York State and
by Borrelia afzelii in Slovenia. Ann Intern Med 1999;
130:32–36.
Van Dam, AP, Kuiper, H, Vos, K, et al. Different
genospecies of Borrelia burgdorferi are associated with
distinct clinical manifestations of Lyme borreliosis. Clin
Infect Dis 1993; 17:708–717.
Vasiliu, V, Herzer, P, Rössler, D, et al. Heterogeneity of
Borrelia burgdorferi sensu lato demonstrated by an ospAtype-
specific PCR in synovial fluid from patients with
Lyme arthritis. Med Microbiol Immunol 1998;
187:97–102.
Von Stedingk, LV, Olsson, I, Hanson, HS, et al. Polymerase
chain reaction for detection of Borrelia burgdorferi
DNA in skin lesions of early and late Lyme borreliosis.
Eur J Clin Microbiol Infect Dis 1995; 14:1–5.
Wang, G, van Dam, AP, Dankert, J. Phenotypic and genetic
characterization of a novel Borrelia burgdorferi
sensu lato isolate from a patient with Lyme borreliosis.
J Clin Microbiol 1999; 37:3025–3028.
Wang, G, van Dam, AP, Schwartz, I, et al. Molecular typing
of Borrelia burgdorferi sensu lato taxonomic, epi-
226 WILSKE
demiological, and clinical implications. Clin Microbiol
Rev 1999; 12:633–653.
Weber, K, Preac-Mursic, V, Wilske, B, et al. A randomized
trial of ceftriaxone versus oral penicillin for treatment
of early Lyme borreliosis. Infection 1990; 18:91–96
Wilske, B. Microbiological diagnosis in Lyme borreliosis.
Int J Med Microbiol 2002; 291:33:114–119.
Wilske, B, Busch, U, Eiffert, H, et al. Diversity of OspA
and OspC among cerebrospinal fluid isolates of Borrelia
burgdorferi sensu lato from patients with neuroborreliosis
in Germany. Med Microbiol Immunol 1996;
184:195–201.
Wilske, B, Fingerle, V, Herzer, P, et al. Recombinant immunoblot
in the serodiagnosis of Lyme borreliosis,
comparison with indirect immunofluoerescence and
enzyme-linked immnosorbent assay. Med Microbiol
Immunol 1993; 182:255–270.
Wilske, B, Habermann, C, Fingerle, V, et al. An improved
recombinant IgG immunoblot for serodiagnosis of
Lyme borreliosis. Med Microbiol Immunol 1999;
188:139–144.
Wilske, B, Preac-Mursic, V. Microbiological diagnosis of
Lyme borreliosis. In: Weber, K, Burgdorfer, W, eds. Aspects
of Lyme Borreliosis. Berlin: Springer; 1993:267–300.
Wilske, B, Preac-Mursic, V, Göbel, UB, et al. An OspA
serotyping system for Borrelia burgdorferi based on reactivity
with monoclonal antibodies and OspA sequence
analysis. J Clin Microbiol 1993; 31:340–350.
Wilske, B, Schierz, G, Preac-Mursic, V, et al. Intrathecal
production of specific antibodies against Borrelia
burgdorferi in patients with lymphocytic meningoradiculitis
(Bannwarth’s syndrome). J Infect Dis 1986;
153:304–314.
Wilske, B, Schriefer, M. Borrelia. Murray, PR, Baron, EJ,
Jorgensen, JH, eds. In: Manual of Clinical Microbiology,
8th ed. Washington, DC: ASM Press; 2003:937–954.
Wilske, B, Zöller, L, Brade, V, et al. MIQ 12 Lyme-borreliose.
In: Qualitätsstandards in der mikrobiologisch infektiologischen
Diagnostik. Mauch, H, Lütticken, R, eds. Munich:
Urban & Fischer Verlag, 2000.
Zore, A, Ruzic-Sabljic, E, Maraspin, V, et al. Sensitiviy of
culture and polymerase chain reaction for the etiologic
diagnosis of erythema migrans. Wien Klin Wochenschr
2002; 114:606–609.
Address reprint requests to:
Bettina Wilske, M.D., Ph.D.
Max von Pettenkofer Institute
University of Munich
National Reference Center for Borreliae
Pettenkofer-Strasse 9a
D 80336 Munich, Germany
E-mail: Bettina.Wilske@mvp-bak.med.unimuenchen.
de
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ke Maalis 13, 2013 20:03

Useita ihomuutoksia (2-3) siitä huolimatta 50% seronegatiivisia, ihosta otettu PCR kuitenkin positiivinen.

The Many Faces of Solitary and Multiple Erythema Migrans Acta Dermato-Venereologica

Accepted Nov 5, 2012; Epub ahead of print Feb 28, 2013

Acta Derm Venereol 2013; 93: XX–XX.

Pernilla Eriksson1, Marika T. Schröder1, Kirsi Niiranen1, Antti Nevanlinna2, Jaana Panelius1 and Annamari Ranki1

1Department of Dermatology and Allergology, Helsinki University Central Hospital, and 2Center for Information Technology, University of Helsinki, Helsinki, Finland


DOI: 10.2340/00015555-1549
Abstract:

Case definitions for European Lyme disease have been published. However, multiple erythema migrans may pose a diagnostic challenge. Therefore, we retrospectively reviewed the clinical and serological findings and response to therapy in a cohort of consecutive 54 patients with PCR-confirmed erythema migrans, referred to a university dermatology clinic. The proportion of patients with multiple erythema migrans lesions (usually 2 or 3) was almost equal (46%) to the proportion of patients with single erythema migrans lesions (54%). All patients, except for 2 multiple erythema migrans patients with a concomitant autoimmune disease, completely responded to treatment. In conclusion, multiple erythema migrans may be more common than anticipated, and since only 50% of the patients were seropositive when seeking medi-cal help, PCR testing of skin lesions is helpful to confirm the diagnosis in clinically atypical cases.

Full text and photos here:
http://www.medicaljournals.se/acta/cont ... &preview=1
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Maalis 24, 2013 09:43

Bb:n viljely kroonista borrelioosia sairastavilta. Myös aiemmin antibioottihoitoa saaneilta.

http://www.ncbi.nlm.nih.gov/pubmed/9861561

Infection. 1998 Nov-Dec;26(6):364-7.

A proposal for the reliable culture of Borrelia burgdorferi from patients with chronic Lyme disease, even from those previously aggressively treated.

Phillips SE, Mattman LH, Hulínská D, Moayad H.
Source

Greenwich Hospital, CT 06830, USA.
Abstract

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

Positive cultures were confirmed by fluorescent antibody immuno-electron microscopy using monoclonal antibody directed against Osp A, and Osp A PCR. 43/47 patients (91%) cultured positive. 23/23 controls (100%) cultured negative.


Although persistent infection has been, to date, strongly suggested in chronic Lyme disease by positive PCR and antigen capture, there are major problems with these tests. This new method for culturing B. burgdorferi from patients with chronic Lyme disease certainly defines the nature of the illness and establishes that it is of chronic infectious etiology. This discovery should help to reestablish the gold standard in laboratory diagnosis of Lyme disease.

PMID:
9861561
[PubMed - indexed for MEDLINE]
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Maalis 24, 2013 10:07

Borrelia miyamotoi ja valaisiana alalajeja löydettiin Ruotsissa jo v. 2002. Nykyisten borreliatestien kyky havaita kyseiset alalajit?

http://www.ncbi.nlm.nih.gov/pubmed/12202571

J Clin Microbiol. 2002 Sep;40(9):3308-12.
Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks.
Fraenkel CJ, Garpmo U, Berglund J.
Source
Department of Infectious Diseases, Blekinge Hospital, S-371 85 Karlskrona, Sweden. carl-johan.fraenkel@ltblekinge.se
Abstract
A total of 301 adult questing Ixodes ricinus ticks were collected at 15 different locations along the south and east coasts of Sweden to determine the Borrelia genospecies diversity. Thirty-two ticks (11%) were found to be positive by nested PCR with Borrelia burgdorferi sensu lato-specific primers. Species determination was based on partial sequencing of the 16S rRNA gene and the flagellin gene. Five different Borrelia species were found. The nucleotide sequence of the Borrelia DNA found in two ticks differed extensively from the nucleotide sequences of the Borrelia DNA found in the other ticks, and analysis revealed that they were closely related to the relapsing fever borrelia species Borrelia miyamotoi. This is the first report of a B. miyamotoi-like borrelia in I. ricinus and in Europe.

Moreover, the Borrelia DNA of two ticks (6%) clustered within the B. valaisiana complex. B. valaisiana has not previously been reported in Sweden.
B. afzelii DNA was found in 14 ticks (44%), and B. garinii DNA was found in 10 ticks (31%). B. burgdorferi sensu stricto DNA was found in four ticks (13%). We conclude that all of the known human-pathogenic species (B. garinii, B. afzelii, and B. burgdorferi sensu stricto) and B. valaisiana found elsewhere in Europe are also present in the Swedish host-seeking tick population and that a B. miyamotoi-like Borrelia species seems to be present in I. ricinus ticks in Europe.
PMID: 12202571 [PubMed - indexed for MEDLINE] PMCID: PMC130762 Free PMC Article
http://www.ncbi.nlm.nih.gov/pmc/article ... figure/F1/
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » To Huhti 11, 2013 22:58

http://suite101.com/article/vlse-and-ly ... WcSrzflTlY

VlsE and Lyme Disease
Posted by Albert Burchsted on Oct 20, 2008
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The VlsE Protein
VlsE Protein
How Cassettes are Interchanged

Bacteria and viruses are subject to intense attack by the immune systems of their hosts and often marshal elegant strategies to keep from being annihilated. The strategies used by Lyme Borrelia, B. burgdorferi and related spirochetes that cause Lyme disease (LD), are manifold and researchers are just beginning to understand the scope of this bacterium's arsenal. An important component of the Borrelia arsenal is a stealth protein: VlsE.

What is VlsE?

VlsE is a lipid-protein conjugate, found on the cell's outer surface during all Borrelia life stages. It is similar to a lipoprotein of the organism that causes African sleeping sickness.

Unlike most proteins, VlsE is produced in many forms. It is a complicated protein with several variable regions (VRs), and six invariable regions (IRs):

Five IRs serve as roots to lock the protein in the outer membrane of the bacteria.
One, C6, is exposed on the membrane surface and triggers antibody formation.

VlsE Evades the Immune System

Microbial surface proteins that are exposed to the host's immune system usually become targets by which the host eliminates the parasite. When synthesizing VlsE, Borrelia periodically replace the VRs with new sequences. This replacement presents fresh surface antigens, and helps Borrelia remain invisible to the immune system. Within four days of being transferred to a mammalian host, VlsE will be produced with more than one VR suite, reducing the strength of the immune response. In ticks, VlsE does not modify the VRs.

The VRs of VlsE form irregularly shaped loops that lie on the bacterial surface and cover both the invariable roots and surface portions of adjacent proteins, hiding them also from immune cells.

VlsE Antigenic Variation System

The loops containing the VRs are coded by pieces of DNA called antigenic cassettes. In a way similar to an audio or video tape cassette:

The loops can be put into and taken out of the protein.
One loop can be replaced by another loop.

The third image below shows this replacement in a schematic view.

Borrelia have fifteen different VlsE cassettes that can insert into any of the variable regions of VlsE. This interchanging of cassettes allows VlsE to appear as one thousand million billion trillion (that's 1 followed by 30 zeros) different antigens. Similar, but smaller, systems also operate for OSP-A, OSP-B, OSP-C, and other proteins. This heterogeneity allows Borrelia to confuse our immune systems by presenting an astronomical number of different antigens. Researchers are presently trying to determine control of cassettes activation.

The C6 Region

The sixth IR, IR6, of the VlsE protein constantly stimulates a strong immune response. It is possible this presentation is a red herring, or decoy, that misdirects the immune system from less protected sites. When antibodies bind to IR6, they do not damage the bacterium or reduce its effect on the host. It is also possible that VlsE is the protein that allows Borrelia to enter T-cells for the purpose of destroying them.

Since IR6 is invariable and found in all Lyme Borrelia, at all life stages, Dr. Phillipp, of Tulane Medical Center, synthesized this portion of VlsE and used it to devise the C6 ELISA one-step diagnostic test for LD. This test both separates Lyme disease from other conditions more accurately and provides a more consistent identification of the disease than the standard ELISA and Western Blot tests used alone, and about the same as the two used in combination.

How Often is the C6 ELISA Test Used?

The Infectious Diseases Society of America (IDSA) diagnostic criteria, which are used by the majority of doctors, reject use of the C6 test for LD. Rather, they use the two-step ELISA / WB sequence where the WB is prescribed only if the unreliable ELISA test first indicates the possible presence of Lyme disease.

Lyme literate doctors who recognize the limitations of the IDSA criteria usually know about and use the C6 test for diagnosis.

Disclaimer

The information in this article is believed to be accurate and is presented for the sole purpose of informing community members of information pertaining to Lyme Borreliosis. Any and all liability for the content or any omissions, inaccuracies, errors, or misstatements in such information is expressly disclaimed. Because the symptoms of Lyme disease may vary from person to person, if Lyme Disease is suspected, consult a qualified, Lyme disease literate doctor to discuss your symptoms and for medical advice. The author and Suite101.com are not liable for any direct or indirect damages or any damages whatsoever resulting from loss of function, use, data, or profits arising from or in connection with the application of information presented in this article.
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » To Huhti 11, 2013 23:09

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85865/

J Clin Microbiol. 1999 December; 37(12): 3997–4004.
PMCID: PMC85865
Human Antibody Responses to VlsE Antigenic Variation Protein of Borrelia burgdorferi
M. B. Lawrenz,1 J. M. Hardham,1,† R. T. Owens,2 J. Nowakowski,3 A. C. Steere,4 G. P. Wormser,3 and S. J. Norris1,*
Author information ► Article notes ► Copyright and License information ►
This article has been cited by other articles in PMC.
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Abstract

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the “whole-cell” ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

------------------------------------------------------
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183344/

Clin Immunol. Author manuscript; available in PMC 2012 October 1.
Published in final edited form as:
Clin Immunol. 2011 October; 141(1): 103–110.
Published online 2011 July 2. doi: 10.1016/j.clim.2011.06.005

PMCID: PMC3183344
NIHMSID: NIHMS310465
Epitope Mapping of Antibodies to VlsE Protein of Borrelia burgdorferi in Post-Lyme Disease Syndrome

Abhishek Chandra,a Norman Latov,a Gary P. Wormser,b Adriana R. Marques,c and Armin Alaedinia,*†

Abstract

The VlsE lipoprotein of Borrelia burgdorferi elicits a strong immune response during the course of Lyme disease. The present study was aimed at characterization of the epitopes of VlsE targeted by the antibody response in patients with post-Lyme disease syndrome, a condition characterized by persisting symptoms of pain, fatigue, and/or neurocognitive impairment despite antibiotic treatment of B. burgdorferi infection. Epitope mapping was carried out using microarrays that contained synthesized overlapping peptides covering the full sequence of VlsE from B. burgdorferi B31. In addition to the previously characterized IR6 region in the variable domain, specific sequences in the N- and C-terminal invariable domains of VlsE were found to be major B cell epitopes in affected patients. The crystal structure of VlsE indicated that the newly described epitopes form a contiguous region in the surface-exposed membrane-proximal part of the monomeric form of the protein.
Keywords: Lyme disease, post-Lyme disease syndrome, chronic Lyme disease, VlsE, epitope mapping, antibody



. RESULTS
3.1. Determination of seropositivity

All selected serum samples from PLDS patients and fully recovered post-Lyme healthy individuals were positive by IgG whole-cell ELISA, while none of the sera from the non-Lyme healthy control group was positive. 47 of 54 (87%) ELISA-positive PLDS subjects, 11 of 14 (79%) ELISA-positive post-Lyme healthy subjects, and none of the non-Lyme healthy subjects were found to be IgG seropositive for anti-borrelia antibodies by WB according to the CDC criteria.
3.2. Antibody response to VlsE protein of B. burgdorferi

3.2.1. Detection of antibodies to recombinant VlsE

45 of 54 (83%) whole-cell ELISA-positive PLDS subjects, 9 of 14 (64%) whole-cell ELISA-positive post-Lyme healthy subjects, and none of the non-Lyme healthy subjects were positive for IgG antibodies to recombinant VlsE.

3.2.2. C6 ELISA

Of the 54 whole-cell ELISA-seropositive PLDS serum specimens, 47 (87%) were positive (n=43) or equivocal positive (n=4) for antibody to the C6 peptide of the borrelial VlsE protein. In comparison, 9 of 14 (64%) whole-cell ELISA-seropositive post-Lyme healthy serum specimens were positive (n=9) or equivocal positive (n=0) for C6 antibody. None of the sera from the non-Lyme healthy control group was positive. The mean C6 antibody index value for the PLDS, post-Lyme healthy, and non-Lyme healthy groups were 3.91 ± 0.36 (SEM), 2.99 ± 0.71 (SEM), and 0.18 ± 0.01 (SEM), respectively.

3.2.3. Peptide microarray

Epitope mapping of the anti-VlsE antibody response in a randomly selected group of PLDS and post-Lyme healthy subjects (all were positive for anti-VlsE antibodies by immunoblotting) identified 14 individual VlsE peptides, comprising 6 unique contiguous amino acid sequences (Fig. 1B). Binding to these peptides (as quantified by the normalized fluorescence signal to noise ratio) for the PLDS and/or post-Lyme healthy serum antibodies was significantly higher than in the non-Lyme healthy control group (p<0.05) (Fig. 1C, Fig. 2). Figure 2 shows the level (Fig. 2A) and frequency of positivity (Fig. 2B) for antibodies to each of the 14 peptides in patient and control groups. Among these peptides were those that form the previously identified IR6 epitope of VlsE (VlsE271–291), which is utilized in the C6 assay. Among the 14 identified peptides, reactivity to 5 peptides covering 3 separate contiguous sequences of amino acids, including VlsE21 through VlsE31 (SQVADKDDPTNKFYQSVIQLGNGF), VlsE96 (SDISSTTGKPDSTG), and VlsE336 (LRKVGDSVKAASKE) was significantly higher in the PLDS group than in the post-Lyme healthy group.
Figure 1
Figure 1
Epitope mapping of VlsE
Figure 2
Figure 2
Level and frequency of antibody reactivity to differentially targeted peptides of VlsE, as determined by peptide microarray epitope mapping

3.2.4. ELISA for antibodies to differentially targeted VlsE epitopes

An ELISA protocol was developed to assess the reactivity of antibodies to the above three specific differentially targeted peptides of VlsE in all available specimens. By ELISA, the level of antibody reactivity to VlsE21–31 and VlsE336 (as measured by mean normalized absorbance) was significantly higher in the PLDS group than in the post-Lyme healthy and non-Lyme healthy groups (p<0.001) (Fig. 3). However, the difference in antibody reactivity towards VlsE96 did not reach statistical significance with the ELISA system. Similarly, the frequencies of antibody reactivity towards VlsE21–31 and VlsE336 were significantly greater in the PLDS group (30 of 54, 56%; 21 of 54, 39%, respectively) than the post-Lyme healthy (2 of 14, 14%; 1 of 14, 7%, respectively) (p<0.05 for VlsE21–31 and p<0.01 for VlsE336) and the non-Lyme healthy (0% for both peptides) (p<0.001) groups. When combining the samples that were positive for antibodies to either VlsE21–31 or VlsE336, the frequency of positivity was 65% (35 of 54) for the PLDS group versus 21% (3 of 14) for the post-Lyme healthy group (p<0.05) and 0% in the non-Lyme healthy group (p<0.0001).
Figure 3
Figure 3
Mean levels of antibodies to differentially targeted VlsE epitopes, as measured by ELISA

The amino acid sequence and 3-dimensional crystal structure of VlsE indicated that the newly identified epitopes are located in the two invariable domains of VlsE, appearing to form a single contiguous area in the surface-exposed membrane-proximal region of the monomeric form of the protein (Figs. 1B, ​,44).
Figure 4
Figure 4
Spatial position of epitopes of VlsE for which a differential antibody response is found in the PLDS patient group
Go to:
4. DISCUSSION

The VlsE protein of B. burgdorferi has emerged as a highly useful diagnostic entity in WB- and ELISA-format assays for active Lyme disease. It is used either as a whole recombinant protein or as a peptide representing its immunodominant epitope located in the IR6 region. While several studies have examined the immune response to VlsE in the course of acute B. burgdorferi infection, no systematic characterization of the targeted epitopes of the protein in the antibody response of PLDS patients has been attempted previously. In order to analyze the anti-VlsE immune response in PLDS, we carried out a detailed epitope mapping of the entire sequence of the VlsE protein of B. burgdorferi B31. Our data show that in addition to the IR6 epitope in the variable domain, PLDS patients have a strong antibody response to specific sequences in the N- and C-terminal invariable domains of VlsE.

We found good concordance between antibody reactivity to the recombinant VlsE molecule (WB) and the IR6 epitope (C6 ELISA). The epitope mapping data demonstrated that the IR6 region is the primary linear epitope in the anti-VlsE antibody response in PLDS patients, as well as in post-Lyme healthy individuals. Interestingly, earlier work had shown a lack of significant antibody reactivity in humans and non-human primates against constituent peptides of the IR6 epitope derived from the amino acid sequence of VlsE from the IP90 strain of B. garinii [25]. In contrast, our data indicate that patients with a history of Lyme disease express antibodies against the VlsE protein of the B. burgdorferi B31 strain that seem to recognize the IR6 region as multiple individual epitopes. The contradiction between our study and the earlier work may be attributed to differences in the amino acid sequences of the synthesized 14mer peptides.

In addition to the IR6 region, two additional sequences, covered by peptides VlsE21 through VlsE31 and by VlsE336 through VlsE343 were found to be major targets in the antibody response of PLDS patients. Specifically, antibodies to sequences covered by VlsE21 through VlsE31 and by VlsE336 were found at significantly lower level and frequency in the post-Lyme healthy group, which was also confirmed by ELISA. These two sequences are located at the N- and C-terminal ends in the invariable domains of VlsE. The 3-dimensional crystal structure of the protein indicates that the two sequences are spatially adjacent to one another, suggesting that they might form a single target region. In addition, they appear to be surface-exposed and located in the membrane-proximal part of the monomeric form of VlsE. Antibodies that bind to the membrane-proximal region of specific proteins in other organisms have been previously described, some of which exert neutralizing or lytic activity [26–28].

VlsE is a membrane protein with a high turnover rate and antigenic variation as a function of time [12]. The B cell memory immune response against specific epitopes in the invariable sections of the protein would be expected to become stronger the longer an infection is left untreated in an individual. Previous work from our group demonstrated increased antibody reactivity in PLDS patients towards borrelial proteins that are associated with later stages of Lyme disease [20]. The fact that PLDS patients in the current study exhibited significantly greater antibody response to VlsE21-31 and VlsE336 epitopes than the fully recovered individuals with a history of early localized or disseminated Lyme disease might indicate that antibodies to the membrane-proximal invariable domains of VlsE become more prominent in later phases of B. burgdorferi infection. Therefore, these antibodies may become useful in patient follow-up and for determination of the stage of active or antecedent infection in Lyme borreliosis and PLDS patients.

A limitation of this study is that it was focused on examining the antibody response to a single sequence variation of the VlsE molecule. It is therefore likely to have missed certain target epitopes in the protein's variable domain. However, in view of the rapid antigenic variation and sequence turnover in these regions, the associated antibody response is not expected to be significant. Nevertheless, follow-up work should consider such sequence variations of the protein, as well as sequence differences among the invariable regions of the various genospecies and strains of borrelia. Another issue to consider is that only seropositive PLDS patients were examined in this study. This was done in order to ensure that all samples had the minimal detectable anti-borrelia antibody response necessary for subsequent analyses. Therefore, our findings do not extend to the seronegative subset of individuals, which formed about 40% of affected PLDS patients in the original treatment study [9]. Future prospective analyses will help to determine whether the newly described antibodies could be useful in predicting the development of post-Lyme disease syndrome or in ascertaining if the treatment of early Lyme disease has been successful. Continuation of these studies, aimed at detailed examination of antigen and epitope specificity of the anti-borrelia immune response in PLDS, may lead to development of specific biomarkers for the condition and provide additional insights into its mode of pathogenesis and potential therapies.
soijuv
 
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Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Touko 05, 2013 18:13

Maailmalla esim. Augsburgin Borrelioosiklinikka, USA:n IGENEX laboratorio jne. käyttävät Borrelia Elispot LTT ( Lymfosyyttien transformaatio) - testiä. Testin sanotaan olevan vasta-ainetestejä luotettavampi ja monikäyttöisempi. Amerikan Lääkevirasto (FDA) ja Tartuntatautivirasto (CDC) ovat hyväksyneet testin esim. tuberkuloosin testaamiseen jo v.2011. Testiä käytetään sen lisäksi mm Borreliabakteerin, Klamydian, Anaplasman (Ehrlichian, Yersinian ja Epstein Barr-viruksen toteamiseen.
Testin käyttöalue:
- Kroonisen Borrelioosin diagnostiikka
- Akuutin Borrelioosin diagnostiikka
- Hoidon keston arviointiin
- Hoitotulosten arviointiin

Testi havaitsee jopa yhden Borrelialle-reaktiiivisen T-solun näytteestä. Testi tulee yleensä negatiiviseksi 6-8 viikon kuluttua onnistuneesta hoidosta.


Borrelia Elispot-LTT (LymphocyteTransformationsTest)

Actual news:

The Elispot-LTT method has been approved by the FDA in May 2011 for M. tuberculosis (Not ITT or MELISA) !!!

The FDA argues in this paper:

"... A positive result (in the Elispot-LTT) suggests that an infection is likely, a negative result that an infection is unlikely..."

"... Results (of the Elispot-LTT) can be available within 24 hours (ITT or MELISA not)..."

A Borrelia infection does not only activate the humoral immune response, but also activates T-lymphocytes at the same time. Once Borrelia bacteria are not active anymore, the T-cellular immune response is not present.

It is not possible to test the treatment success by Borrelia antibodies, because the 'titer" or antibodies can be measured in the blood over years. Furthermore, Lyme infections in Stage I (e.g. 'bulls-eye rash' or 'summer flu') only show antibodies in the blood after weeks and sometimes do not show them at all.

The Borrelia Elispot-LTT eliminates these problems. The test reflects the actual, current Borrelia burgdorferi activity of chronic and also acute Lyme infections. The Elispot-LTT is highly sensitive and can detect even one single Borrelia-reactive T-cell in the blood. The Elispot-LTT is very helpful when monitoring a chronic or acute Lyme therapy. The Elispot-LTT should usually become negative about 6 to 8 weeks after completion of an effective therapy.

Borrelia burgdorferi Elispot-LTT:

Material: 2 x 8.5 ml CPDA-Tubes (Do not centrifuge, keep at room temperature, do not cool or keep in a cool storage place)

Analytical test duration: 2 Days (Important remark: The laboratory report needs about 1 week because of additional pre- and postanalytical laboratory processes)

Indication:

to diagnose chronic Lyme disease
to diagnose acute Lyme disease
to determine the duration of therapy
to monitor treatment results after a Lyme therapy


Infectolab offers beneath the Borrelia burgdorferi-Elispot-LTT following bacterial Elispot-LTT:

- Chlamydia pneumoniae-Elispot-LTT

- Chlamydia trachomatis-Elispot-LTT

- Ehrlichia/Anaplasma-Elispot-LTT

- Yersinia-Elispot-LTT

- Epstein Barr-Virus (EBV)-Elispot-LTT


Material: 1 x 8.5 ml CPDA-Tubes for each bacterial Elispot-LTT (Do not centrifuge, keep at room temperature, do not cool or keep in a cool storage place)

Analytical test duration: 2 Days (Important remark: The laboratory report needs about 1 week because of additional pre- and postanalytical laboratory processes)

Indication:

to diagnose active Chlamydia pneumoniae, Chlamydia trachomatis, Ehrlichia/Anaplasma, Yersinia, EBV infections
to determine the duration of therapies
to monitor treatment results therapies


Sensitivity, Specificity and diagnostic Quality of the Lymphocyte-Transformations-Tests

Based on the displayed literature, the following tables show the diagnostic quality and effectiveness of the LTT as well as the sensitivity and specificity of the LTT. (compare Tabel 1 and 2 - click on the tables to enlarge them).

Publication about the Borrelia Lymphocyte-Transformations-Test (LTT) in chronological order, evaluated by the author.

+ positive

(+) positive with constraints (+/-) skeptical

(-) negative

* These tests are not done by Infectolab.
---------------------------------------------------------------------------------
http://benthamscience.com/open/toneuj/a ... TONEUJ.pdf


The Open Neurology Journal, 2012,6,(Suppl 1-M 5)
104-112

The Lymphocyte Transformation Test for Borrelia Detects Active Lyme Borreliosis and Verifies Effective Antibiotic Treatment
Volker von Baehr, Cornelia Doebis, Hans-Dieter Volk, Rüdiger von Baehr

Institute for Medical Diagnostics, Immunology Department, Nicolaistrasse 22, 12247 Berlin
Institute for Medical Immunology, Charité University Medicine Berlin, Campus Mitte, Charitéplatz 1, 10117 Berlin

Abstract:
Borrelia-specific antibodies are not detectable until several weeks after infection and even if they are present,
they are no proof of an active infection. Since the sensitivity
of culture and PCR for the diagnosis or exclusion of borrelio-
sis is too low, a method is required that
detects an active Borrelia infection as early as possible. For this purpose, a lym-
phocyte transformation test (LTT) using lysate antigens of Borrelia burgdorferi sensu
stricto, Borrelia afzelii and Borrelia garinii and recombinant OspC was developed and validated
through investigations of seronegative and seropositive
healthy individuals as well as of seropositive patients with clinically manifested
borreliosis. The sensitivity of the LTT in clinical borreliosis before antibiotic treatment was determined as 89,4% while the specificity was 98,7%. In 1480 patients
with clinically suspected borreliosis, results from serology
and LTT were comparable in 79.8% of cases. 18% were serologically positive and LTT-negative. These were mainly patients
with borreliosis after antibiotic therapy. 2.2% showed a
negative serology and a positive LTT result. Half of them had
an early erythema migrans. Following antibiotic treatment,
the LTT became negative or borderline in patients with early ma
nifestations of borreliosis, whereas in patients with late
symptoms, it showed a regression while still remaining positive. Therefore, we propose the follow-up monitoring of dis-
seminated Borrelia infections as the main indication for the Borrelia-LTT.
Keywords:
Borrelia serology, borreliosis, diagnostics, immune response, lymphocyte transformation test, T cells.

INTRODUCTION
Lyme borreliosis is the most common disease transmitted
by tick bite. Lyme borreliosis first manifests locally on the
skin at the site of the tick bite and then systemically, possibly
affecting one or more organs such as the skin, joints, mus-
cles, sense organs, nervous system and heart. In the latter
case, early (stage I and II) and late (stage III) manifestations
can be distinguished (1). Lyme borreliosis should be diag-
nosed by history and clinical
symptoms. If the clinical symp-
toms are clear, laboratory diag
nostics are of secondary im-
portance only. The difficulty is, however, that the tick bite
often goes unnoticed and the erythema migrans does not
necessarily occur or is not noticed. In these cases, the re-
quirement for early antibiotic treatment of borreliosis to pre-
vent the complications of systemic dissemination of the
pathogen, particularly of late borreliosis, cannot be met.
The symptoms associated w
ith the systemic phase of
Lyme borreliosis can be highly varied and ambiguous. In
these cases, the detection of Borrelia-specific antibodies (se-
rological laboratory diagnosis) becomes important for the
diagnosis and treatment decisi
on. The necessarily high qual-
ity demands cannot yet be completely fulfilled by Borrelia
serology due to the following reasons: 1) Borrelia-specific
*Address correspondence to this author
at the Institut für Medizinische
Diagnostik Berlin, Nicolaistraße 22, 12247 Berlin, Germany;
Tel: +49-30-77001220; Fax: +49-30-77001236;
E-mail: v.baehr@imd-berlin.de
IgM antibodies, and IgG antibodies in particular, cannot be
detected until several weeks after infection [1, 2].Seronega-
tive cases with late stage Lyme borreliosis have also been
recently described [3]. But these are becoming more rare
with the increasing quality of the assays following the intro-
duction of recombinant Borrelia antigens. 2) The heterogene-
ity of Borrelia species and strains within a species requires a
polymorphism of the Borrelia-s
pecific protein antigens [4,
5]. This is a difficult problem for the sensitivity of Borrelia
serology. 3) IgM antibodies against Borrelia OspC may be of
the nonspecific type [4, 5]. 4) A positive serological finding
alone is not proof of a current active Borrelia infection [1, 4,
5]. 5) Borrelia serology is not suitable for the monitoring of
therapy and evaluation of progress as IgG and IgM antibod-
ies may persist for years after borreliosis has been cured [6].
The direct detection of Borrelia by culture or PCR has a
high diagnostic value in the case of a positive result, but a
negative result does not rule out Lyme borreliosis [4, 5].
There is currently no method available which, in addition
to the serology, answers the question as to whether a specific
case is a status post Borrelia in
fection or active borreliosis.
Each humoral immune response to an infection requires a
specific cellular immune response with clonal proliferation
of various antigen-specific lymphocyte subpopulations. Of
central importance here are an
tigen-specific T helper lym-
phocytes (CD4
+
T
H
cells). In addition to effector T cells,
long-lived T and B memory lymphocytes are formed. In the
presence of antigen-presenting cells and protein antigens,

Lymphocyte Transformation Test for Borreliosis
The
Open Neurology Journal, 2012, Volume 6
105
specific CD4
+
T memory cells also proliferate
in vitro
. The
lymphocyte transformation test (LTT), also known as the
lymphocyte proliferation or lymphocyte activation test, is
based on this principle [7]. Shortly after the discovery of B.
burgdorferi, it was demonstrated that blood lymphocytes
from Lyme borreliosis patients proliferate in the presence of
Borrelia lysates [8, 9]. Published data on the diagnostic value
of the Borrelia-LTT, especially
in seronegative patients, can
be found beginning in 1988 [10-17]. However, false positive
LTT reactions have also been
described [18-20]. Important
for the motivation of our investigations presented here were
observations that positive LTT
reactions of blood lympho-
cytes to Borrelia antigens declined significantly or were neg-
ative after antibiotic treatment of Lyme borreliosis [12, 14,
16, 21]. This leads to the hypothesis that Borrelia-specific T
helper cells circulate in the blood in detectable numbers only
during an active immune response against Borrelia and per-
sist in a non-florid infection in lymphoid organs.
Using improved cell culture and measurement techniques
as well as our own experience in the development of antigen-
specific LTT applications, we will seek to answer using re-
evaluation of patient data the following questions:
1.
Is there a correlation between the results of Borrelia
serology and Borrelia-LTT?
2.
What are the Borrelia-LTT resu
lts in clinically healthy
seropositive subjects?
3.
Is it possible to obtain an indication of the respective
species involved from the L
TT reactions to antigens of
the three Borrelia species?
4.
Are Borrelia-LTT results influenced by antibiotic treat-
ment?
5.
How high are the sensitivity and specificity of the Bor-
relia-LTT?

In summary, the following conclusions can be made for
the use of the Borrelia-LTT:
1. Except for early manifestations of borreliosis (2 to 6
weeks after infection), the det
ection of Borrelia-specific an-
tibodies to confirm the clinical diagnosis of borreliosis is
Lymphocyte Transformation Test for Borreliosis
The
Open Neurology Journal, 2012, Volume 6
111
o
ften sufficient. Only in the cas
e of an unclear clinical pic-
ture of borreliosis coupled with negative or borderline serol-
ogy the Borrelia-LTT can be bene
ficial in order to make a
decision regarding the indication for antibiotic treatment
since it also exhibits a clear positive reaction in the case of
early manifestations.
2. In the case of infections in the more remote past with an
ambiguous clinical picture and positive Borrelia serology,
the Borrelia-LTT provides an important indication as wheth-
er an active Borrelia infection could exist. The Borrelia se-
rology is not suitable in this case, and the direct pathogen
detection methods (PCR or culture) are also not sufficient for
answering this question due to their low sensitivity. It must
be noted, however, that it is not only the results of the Borre-
lia-LTT that are important for
the indication for antibiotic
treatment, but also the medical
history and the current clini-
cal picture.
3. It has been shown that the Borrelia-LTT can be used to
evaluate the success of antibiotic treatment, although the
clinical course is also particularly important. However, the
symptoms of disseminated borreliosis may persist after suc-
cessful treatment for some time, however. An LTT follow-up
examination is reasonable, at
the earliest, 4 to 6 weeks after
the completion of therapy. This interval is necessary be-
cause, on the one hand, possibly surviving Borrelia can be-
come active during this time and, on the other hand, Borre-
lia-specific T cells persist in the blood for some time after
elimination of the antigens. The Borrelia-LTT should not be
performed during antibiotic ther
apy because, in our experi-
ence, the result will be negative, but soon after discontinua-
tion of treatment, it may again become positive.

Criticism is often based on
the frequency of false-
positive results of the Borrelia
-LTT (5). This problem con-
tinues to exist unless there is su
fficient testing of the antigen
specificity of the Borrelia-LTT.
In the case of frequently
false positive reactions, the dose
of the test antigens is too
high. Currently, since there is no available gold standard for
the laboratory diagnosis of borreliosis or its exclusion, the
Borrelia-LTT can only be validat
ed according to clinical and
serological findings. Nevertheless, there are still gaps in the
validation that we conducted. For instance, it was only pos-
sible in individual cases to examine patients with active
syphilis (n = 3) or leptospirosis infection (n = 2) for potential
cross reactivity. In these few cas
es, there was no evidence of
such cross-reactivity in the Bo
rrelia-LTT. Allergies, auto-
immune diseases and acute, persistent and latent viral infec-
tions (including HIV, EBV, CMV, VZV) have now been
excluded, by further investigations, as a possible cause of
false-positive reactions (unpublished data).
The Borrelia-LTT cannot pr
ovide any information on
whether a patient has ever had a Borrelia infection. This
question is largely answered only by serology. Our studies
presented here and the results of other authors [12, 14, 16,
21], particularly the work of Valentine-Thon
et al.
on LTT-
MELISA [31], provide good arguments that the positive Bor-
relia-LTT indicates an active
borreliosis which, however,
could only be definitely proven in conjunction with positive
detection of Borrelia. However, we succeeded in doing so in
only 3 seronegative patients with erythema migrans using
positive Borrelia-PCR in the blood (unpublished data).
The
in vitro
antigen-induced lymphocyte proliferation is
the approved cellular immunological method for detecting
antigen-specific memory T helper cells. In addition and prior
to clonal lymphocyte proliferation, however, a series of im-
munologically relevant genes
for both cell surface markers
and especially cytokines such as interleukin-2 and interferon-
γ
are activated, which in turn are used as the basis for newer
cellular immunological laboratory methods. An example is
the QuantiFERON test (interferon-
γ
stimulation) performed
to detect a Mycobacter tuberculosis infection [32]. However,
a distinction between a latent and florid infection is not pos-
sible with this test, however.
Currently, the ELISPOT test
(quantitative determination of
cytokine-producing lympho-
cytes) is used in vaccine research in particular, and is also
being tested in patients with a Borrelia infection. According
to the previously published results by Forsberg
et al.
and our
own experiences, even though seropositive individuals are
detected by the ELISPOT assay, a differentiation between
symptomatic and asymptomatic Borrelia infections was not
possible [33-35]. The advantag
e of antigen-specific cyto-
kine-stimulation assays would be that, while 6 days are re-
quired for an antigen-specific
LTT test, the incubation period
for the cytokine assay is only about 24 hours. It should be
not
ed, however, that the biology on which these cellular im-
munological test systems are based is very different. While
in the LTT setting T helper memo
ry cells are clearly identi-
fied as cytokine producers, also other lymphocyte subpopu-
lations like NK cells, and (depending on the cytokine) also
monocytes are being taken into consideration as cytokine
producers. However, cellular short-term stimulation methods
are rather sensitive to nonspecific activations, while, in the
LTT such influences dominate only during the first 48 to 72
hours, and thus have virtually disappeared after 6 days at the
time the cellular proliferation is
measured. For the particular
issue of florid or non-active Borrelia infection, the situation
is even more difficult since there is usually no gold standard
available (i.e., patients with positive detection of live Borre-
lia). In this difficult situation, a new method should always
be validated in comparison with an extensively proven
method. The aim of our investigations were therefore to
evaluate the results previously obtained with an optimized
Borrelia-LTT in order to prov
ide a basis for necessary com-
parative prospective studies with cellular immunological
methods based on antigen-specific gene activation or cyto-
kine stimulation. In our opinion, the Borrelia-LTT presented
here fulfills the associated requirements.
Finally, it should be emphasi
zed again that the primacy
for the diagnosis of borreliosis remains with the medical
history and clinical symptoms. In second place is the deter-
mination of Borrelia-specific antibodies in the blood or cere-
bral synovial fluid. With the Borrelia-LTT another diagnos-
tic tool is available that can help to clarify issues for the in-
dication of antibiotic treatment
. The Borrelia-LTT is particu-
larly important, however, for assessing the effects of therapy
if the initial findings are positive, as they should prove nega-
tive four weeks after the end of
therapy, or at least should
prove to have declined significantly
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Touko 05, 2013 19:36

Parannuksia Lymen taudin laboratoriodiagnostiikkaan

julkaistu 20.12.2002/Väitöskirjat

Lymen tauti (borrelioosi) on yleinen punkin levittämä infektio Euroopassa. Taudin määritys edellyttää yleensä myös laboratoriodiagnostisia menetelmiä. Kuitenkin rutiinikäytössä olevilla vasta-ainemäärityksilläkin on puutteita. Taudin alkuvaiheessa testit ovat usein negatiiviset ja vääriä positiivisia tuloksia voi liittyä mm. virusinfektioihin, reumaan ja kuppaan. Kohonneet vasta-ainetasot voivat säilyä veressä vuosia onnistuneenkin hoidon jälkeen, jolloin hoitotuloksen osoitus laboratoriomenetelmin on hankalaa. Lisäksi noin 5 prosentilla potilaista eivät vasta-ainetasot nouse lainkaan. Väitöskirjatyön tavoitteena oli parantaa Lymen taudin laboratoriodiagnostiikan herkkyyttä ja osuvuutta uusia rekombinantti- ja peptidiantigeenejä hyödyntämällä. Tämän lisäksi hoitotuloksen arvioimiseksi ja taudin aktiviteetin markkerin löytämiseksi verrattiin vasta-aineiden puoliintumisaikoja hoidon jälkeen tavanomaisia flagellaa ja kokosoluvalmistetta antigeeneinä käyttäen.

Tutkimuksessa käytettiin Borrelian viljelyä ja eristystä, polymeraasiketju-menetelmää, geenien kloonausta ja sekvensointia sekä rekombinanttiproteiineihin liittyviä puhdistus- ja biotinylaatiomenetelmiä. Tuotettuja rekombinantti- ja peptidiantigeenejä käytettiin enzyme-linked immunosorbent assay (ELISA)- ja Western blotting (WB)- menetelmissä vasta-aineiden osoittamiseksi.

Jokaisesta kolmesta Euroopassa esiintyvästä Borrelian alalajista (Borrelia burgdorferi sensu stricto, B. afzelii ja B. garinii) tuotettiin rekombinanttiproteiinit flagellin A (FlaA) ja ulkomembraaniproteiini C (OspC). FlaA:n aminohapposekvenssit olivat 95 prosenttisesti identtisiä, kun taas OspC oli heterogeenisempi (57-73% identtisyys). Neuroborrelioosin (71-74%) ja artriitin (86-79%) IgG serodiagnostiikassa (WB tai ELISA) FlaA osoittautui herkäksi antigeeniksi. IgM serologiassa FlaA:n käyttöä rajoittavat alhainen herkkyys Lymen taudin alkuvaiheessa ja risti-reagoivat vasta-aineet verrokkiaineistossa. Biotinyloitu rekombinantti OspC toimi IgM ELISA:lla paremmin taudin alkuvaiheessa, IgG ELISA taas paremmin taudin myöhäisvaiheessa. IgM serologiassa Epstein Barr –virusinfektioiden yhteydessä esiintyvää risti-reagointia kyettiin vähentämään käyttämällä tiosyanaattia seerumin laimennuspuskurissa. Sekä FlaA:n että OspC:n kohdalla B. afzelii- ja B. gariniiperäinen proteiini osoittautuivat immunoreaktiivisemmiksi kuin B. burgdorferi sensu strictosta saatu proteiini.

Kolmea variantti rekombinantti antigeeniä proteiineista DbpA (decorin-binding protein A), BBK32 ja OspC sekä IR6 peptidiä sovellettiin 89 neuroborrelioosia sairastavan potilaan selkäydinneste- ja seeruminäytteiden tutkimiseen. Näiden uusien antigeenien käyttäytymistä ELISA-metodissa verrattiin kaupalliseen flagella antigeeniin. Uudet antigeenit lisäsivät diagnostista herkkyyttä sekä selkäydinnesteen että seerumin osalta. Kuitenkin selkäydinnesteessä erottelukyky potilaiden ja kontrollinäytteiden välillä toimi paremmin. Tulokset viittaavat myös siihen, että vasta-aineet BBK32 proteiinia kohtaan voisivat toimia akuutin taudin markkerina.

Väitöskirjatyössä tutkittiin myös 68 myöhäisborrelioosia sairastavan potilaan seurantanäytteitä hoidon jälkeen ja todettiin, että flagella IgG vasta-ainetasojen (erityisesti IgG1) puoliintumisaika kliinisesti onnistuneen hoidon jälkeen oli tilastollisesti merkitsevästi lyhyempi kuin kokosoluvalmisteen puoliintumisaika.

Uusien rekombinantti- ja peptidiantigeenien käyttö yksin tai erityisesti rinnakkain lisää Lymen taudin laboratoriodiagnostiikan herkkyyttä ja osuvuutta. Euroopassa käytettäviin testeihin tarvitaan rekombinanttiproteiinit jokaisesta kolmesta Borrelian alalajista tai vähintään B. afzelii- ja B. garinii-peräiset proteiinit, jotta testin kattavuus olisi riittävä. Flagella IgG vasta-aineiden nopeaa laskua voidaan hyödyntää hoidon tehokkuuden mittarina Lymen taudissa.

Jaana Paneliuksen väitöskirja ”New recombinant and conventional antigens in the laboratory diagnosis of Lyme borreliosis” tarkastettiin Helsingin yliopistossa 20.12. 2002

http://www.terveyskirjasto.fi/terveyspo ... _hakusana=
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Touko 19, 2013 12:07

Tutkimuksen alla uusi testi - xenodiagnostiikka.

Verikokeet ovat aina enemmän tai vähemmän epäluotettavia menetelmiä. Borrelioosin diagnostiikassa tutkitaan parhaillaan (hiirikokeet) menetelmää jossa laboratorio-olosuhteissa kasvatettu "tautivapaa" punkki istutetaan hiireen joka on saanut antibioottihoitoa borrelioosiin. Punkki imee itseensä hiiressä mahdollisesti olevia borreliabakteereita ja siten on mahdollista osoittaa bakteerin olemassaolo edelleen hiiren elimistössä vaikka hiiri olisi hoitojen jälkeen oireetonkin.

Tri Fallonin mukaan testiä testataan parhaillaan ihmisiin ja tuloksia on odotettavissa kesän lopuun mennessä. Joidenkin mielestä borrelioosiin sairastuneiden kroonisten oireiden taustalla on jokin muu syy kuin borreliabakteeeri. Uusi testi saatta tuoda lisävaloa asiaan. Useissa eläinkokeissa on jo todettu borreliabakteerin väistävän usein eri tavoin antibiootteja.


http://www.thereddingpilot.com/10290/ly ... e-horizon/


So often public programs on Lyme disease focus on how devastating the disease can be, especially for those who suffer from a chronic form of the disease and those who cannot get a solid diagnosis as to whether they have the disease at all.

So it was to an appreciative audience at Wilton Library on Wednesday, May 1, that Dr. Brian Fallon said he would be delivering good news, speaking about new research in the field in the last five years and where it is going.
He was joined by Dr. Charles Alexander, who spoke about the benefits meditation can bring to those who suffer from the disease and a study the two doctors are hoping to conduct that would measure real benefits Lyme patients might derive from meditation.
The program was particularly timely given that Friday, May 10, is Worldwide Lyme Disease Awareness Day.
Perhaps it was not surprising that the audience for this event nearly filled the Brubeck Room. Over the past five years, there have been more cases of Lyme disease in Fairfield County than in any of the other counties in the state, except for New London County in the two most recent years.

Diagnostic test




Perhaps the biggest news Dr. Fallon related was a [b]new diagnostic test being conducted called the xenodiagnostic method.

“Blood tests are not all that sensitive,” he said, and for the 20% of Lyme patients who do not show the classic symptoms of a rash, arthritis-like pain and flu-like symptoms — and to a lesser extent meningitis or cranial nerve palsy — getting an accurate diagnosis can be frustrating.

The xenodiagnostic method has been tested with mice. When a “clean” tick — a non-Lyme carrier produced in a laboratory — is placed on a mouse that has been given antibiotics, it will suck up any Lyme spirochetes in that mouse. When the tick is tested, the spirochetes become apparent. It is then possible to say that mouse had been infected with Lyme even if it was presenting no symptoms.

Dr. Fallon said such testing is now being done with humans and results could be available by the end of the summer. If the results are conclusive, that would be front-page news, he said.
Although there are strong beliefs on both sides of the Lyme disease debate, some previously held views have changed.
For example, it was thought that any symptoms remaining after a course of antibiotics were from some other cause. From animal models it is known the Lyme organism can persist after antibiotics are given, Dr. Fallon said.
A positive Lyme test may or may not indicate an active infection. “It could just be an old disease,” Dr. Fallon said, and he would not recommend treating someone if they did not feel ill.


Psychiatric symptoms may respond to antibiotics, he said.

The infection may trigger changes in a person’s brain chemistry that might need to be addressed by drugs or psychotherapy.
Recent studies with animals have shown the Lyme organism can persist even with antibiotics, Dr. Fallon said. “We have more bacteria in our bodies than healthy cells,” he said, and trying to kill them all off would not only be foolhardy but would probably not be possible. “We just want our immune system to keep the spirochetes at bay.”
Scientists have also discovered in a study of Lyme and lupus patients that both had high levels of antibodies that attack the nervous system.
Lupus is a known autoimmune disease.
“There may be heightened immune responses contributing to Lyme,” he said, which has always been considered an infectious, not an autoimmune, disease. To that end, he said, people can take heart from the large amount of work being done in the field of multiple sclerosis that will help those with autoimmune diseases....
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Su Touko 19, 2013 12:25

http://newsdesk.gmu.edu/2013/05/local-legislators-meet-with-mason-lyme-disease-researchers/?fb_comment_id=fbc_506117209443666_5242624_506152012773519#f28e1a5cc

Kehitteillä on uusi testi varhaisvaiheen borrelioosiin, nimeltään NANOTRAP. Varhaisvaiheen diagnostiikka on vaikeaa koska vasta-aineita ei muodostu useisiin viikkoihin/kuukausiin. Mikäli laboratoriovastauksia jäädään odottelemaan, saattaa bakteeri ehtiä syvemmälle kudoksiin ja hoito vaikeutua entisestään. Nanotrap-testi on eräänlainen imuri joka imee infektiosta kertovia merkkiaineita 2000xherkemmin kuin tähänastiset testit. Testi vähentää vääriä negatiivisia tuloksia ja mahdollistaa varhaisemman diagnoosin. Tutkimustuloksia on odotettavissa kevään 2014 aikana.


By Sabena Moretz, associate director of State Government Relations
Lyme disease is at epidemic levels in Northern Virginia, so local elected officials gathered at George Mason University’s Prince William Campus last week to discuss the latest developments for detecting and treating the disease.
Legislators and staff from both the Virginia House of Delegates and the U.S. House of Representatives met with researchers from George Mason’s Center for Applied Proteomics and Molecular Medicine (CAPMM) and business partner Ceres Nanoscience to learn more about a new technology that will dramatically improve detection of the disease in order to facilitate earlier treatment and therefore much improved health outcomes. Mason has developed a breakthrough test for detection.
Mary Springer, chief of staff to Rep. Rob Wittman, Hannah Reynolds, legislative aide to Del. Tag Greason, Del. Tim Hugo, Del. Barbara Comstock, Dr. Alessandra Luchini, Dr. Lance Liotta, Del. David Ramadan, Dr. Chip Petricoin, Ross Dunlap, CEO of Ceres Nanoscience, Mary Ann Cannon, Director of Community Outreach for Rep. Frank Wolf
Meeting participants, from left: Mary Springer, chief of staff to Rep. Rob Wittman; Hannah Reynolds, legislative aide to Del. Tag Greason; Del. Tim Hugo; Del. Barbara Comstock; Alessandra Luchini, Mason researcher; Lance Liotta, Mason researcher; Del. David Ramadan; Chip Petricoin, Mason researcher; Ross Dunlap, CEO of Ceres Nanoscience; and Mary Ann Cannon, director of community outreach for Rep. Frank Wolf.
Lead researcher and Mason assistant professor Alessandra Luchini, recently named as one of the “Brilliant 10” scientists under the age of 40 by Popular Science magazine, explains, “Treatment of Lyme dsease is complicated by the fact that current tests are not effective at the first sign of symptoms of disease, and they have a high degree of false negatives. They measure the immune response to the disease, rather than the presence of the disease itself. But it can take months for those antibodies to show up in testing, all while the patient is experiencing symptoms and taking many different types of medical tests to rule out the possibilities of other diseases that might be causing the symptoms.”
Most physicians are hesitant to treat for Lyme disease without a definitive diagnosis, but by the time such a diagnosis is available, the disease is more entrenched and requires more aggressive treatment, Luchini says.
In the 2013 General Assembly session, a bill aimed to improve citizen understanding of the weaknesses of current testing was passed into law. Del. Barbara Comstock of Fairfax County sponsored the bill.
“The passage of House Bill 1933 is a great step in raising awareness about this terrible disease and the high incidence of patients receiving false negative tests for Lyme,” Comstock says. “I have heard from many of my constituents about how they or someone in their life suffers from Lyme disease. So often, they go untreated and undiagnosed for months and even years. I’m pleased that we were able to pass this bill, which focuses on getting information about testing problems directly to patients so they can seek additional testing, if necessary, as well as appropriate treatment.”
The bill was cosponsored by fellow Northern Virginia House of Delegates members Tim Hugo, Tag Greason and David Ramadan, as well as members from other regions in the state. Comstock, Hugo, Ramadan and an aide to Greason attended the meeting at Mason.
Congressmen Frank Wolf and Rob Wittman were also represented. Wolf, who chairs the House Lyme Disease Caucus, has hosted three Lyme disease forums in the 10th District to raise awareness about this debilitating disease. He has also recently cosponsored two bills, H.R. 610 and H.R. 611, to deal with this issue. The bills require the secretary of Health and Human Services to create a federal tick-borne diseases advisory committee charged with coordinating research and advising federal agencies on priorities related to Lyme and other tick-borne diseases, and authorize additional research.

Researchers at Mason and their partners at Ceres Nanoscience have been refining their work on a new technology called a Nanotrap that will dramatically improve detection of Lyme disease at much earlier stages.
Chip Petricoin, co-director of CAPMM and director of science at Ceres Nanoscience, explained the Nanotrap as a “vacuum cleaner for infectious disease markers,” able to “identify evidence of the disease when it is 2,000 times smaller” than what can be identified with current testing processes. Lance Liotta, co-director of CAPMM and a scientific advisor at Ceres Nanoscience, noted that use of the Nanotrap test will “dramatically reduce the false negatives of current testing processes and lead to earlier and greatly improved treatment outcomes for those suffering from Lyme disease.”


“Our singular focus is on getting this test out into commercial use,” Petricoin said. “This is not an academic exercise for us.”
Liotta emphasized that the Nanotrap technology originated at Mason with support from the Innovative Molecular Analysis Technologies grant funding program from the National Institutes of Health and is the subject of peer-reviewed publications and patents.
Legislators are interested in the relationship between Mason and Ceres Nanoscience as a model of how state investment in universities can facilitate and incubate businesses that have the potential to bring significant economic development to the region.
“Our collaboration with Mason on projects like the Nanotrap Lyme Test has been immensely successful,” said Ross Dunlap, CEO of Ceres. “It is a great example of how academic research and business development interests can align to provide economic growth, and most important, real patient benefit.”
The Nanotrap test is in the process of clinical trials and is expected to become routinely available by the spring of 2014.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Heinä 26, 2013 19:22

http://www.blackwellpublishing.com/eccm ... p?id=75892

Kahden borreliatestin vertailu; recomLine Borrelia (Mikrogen) and Borrelia Europe LINE (VIROTECH)


Evaluation of two line immunoassays for the detection of Borrelia antibodies

Abstract number: R2287

Verstreken I., Appeltans T., Verhaegen J., Lagrou K.

Objectives: Lyme borreliosis is a multisystemic disease with diverse clinical manifestations transmitted to humans by ticks. Clinical diagnosis is difficult. In routine practice a two step test strategy is recommended for the diagnosis of the disease. Several confirmation tests are available. We evaluated the performance characteristics of two different line immunoassays.

Methods: 62 sera submitted to the Laboratory Medicine unit of the University Hospitals Leuven were analyzed for the presence of Borrelia IgM and IgG antibodies with two line immunoassays: recomLine Borrelia (Mikrogen) and Borrelia Europe LINE (VIROTECH) and with an immunoblot assay recomBlot Borrelia (Mikrogen) as reference method. All tests were performed on the auto LIA II instrument (Innogenetics). Sensitivity was evaluated on 33 preselected sera from patients with Lyme borreliosis based on clinical symptoms and the presence of Borrelia IgM and/or IgG with recomBlot Borrelia assay (8 erythema migrans, 10 arthritis, 15 neuroborreliosis). Specificity was evaluated on 29 sera (10 Epstein Barr virus IgM positive sera, 9 rheumatoid factor positive sera and 10 sera positive for rapid plasma reagin antibodies).

Results: Specificity of both IgM line assays was 89.7%. 2/29 false positive IgM results were from patients with a primary EBV infection. Cross reaction due to EBV infection can be detected by the Europe LINE assay due to the presence of the EBV-VCA gp125 antigen on the strip. One IgG positive result was obtained with both line assays. This was probably due to a previous Borrelia infection in a rheumatoid factor positive patient. Sensitivity for both line assays was 97% if we combine IgM and IgG test results. IgG were present in 7/8 (87.5%) of erythema migrans sera with both line assay compared to 1/8 (12.5%) sera that was IgG positive with the recomBlot assay.

Conclusion: The performance characteristics of the two Borrelia antibody confirmation line immunoassays, recomLine Borrelia and Borrelia Europe LINE, were very good and similar in this evaluation. From early stage sera it seems that the sensitivity of both IgG line assays is higher compared to the Mikrogen IgG recomBlot assay.

---------------------------------------------------------------------------------------------------------------------------


http://www.hus.fi/ammattilaiselle/koulu ... kitaan.pdf

Milloin borreliavasta ainetutkimus –miten tulkitaan

Timo Walle
Kliinisen mikrobiologian erik.lääk.
HUSLAB, Kliininen mikrobiologia
Infektiosairauksien ja kliinisen
mikrobiologian alueellinen koulutuspäivä
25.4.2013

Borreliavasta
-
aineet
, seerumista
(
S
-
BorrAb
)
HUSLAB: 2
-
vaiheinen perustutkimus
S

BorrAb
Viitealue
Antigeeni
BorrAbG
< 9
-
11 VE/ml
(B. afzelii kokobakteeri + VlsE)
BorrAbM
< 9
-
11 VE/ml
(B. afzelii kokobakteeri)
Lisätestit, jos edellisistä saadaan positiivinen tulos (> 11 VE/ml):
VlsEAbG
< 10
-
15 AU/ml
(VlsE)
VlsEAbM
< 18
-
22 AU/ml
(VlsE + OspC)

Borreliavasta
-
aineet
, likvorista
(
Li
-
BorrAb
)
-
Tulisi ottaa samaan aikaan seeruminäyte (
S
-
BorrAb
)
-
Likvorin (ja
tarvittaessa
vertailuseerumin)
IgG
-
ja
IgM
-
vasta
-
aineet
tutkitaan
likvornäytteille
tarkoitelulla
(herkistetyllä)
ELISA
-
testillä
-
Mikäli
likvorin
borreliavasta
-
aineet
ovat koholla (>10
wME
), lasketaan
intratekaalisen
borreliavasta
-
ainetuotannon
indeksi
-
Kohonnut indeksi viittaa vasta
-
ainetuotantoon keskushermostossa
-
Lisäksi likvorin solut (ja/tai CXCL13
-
määritys)
-
Borreliavasta
-
aineiden
intratekaalinen
tuotanto jatkuu vuosia
keskushermostoinfektion päättymisen jälkeen

Milloin syytä pyytää
Varhaisvaihe (päiviä

viikkoja)
Erythema migrans (EM)
-
Kliininen diagnoosi ja hoito, ei laboratoriotutkimuksia
-
Vasta
-
aineet usein negatiiviset (ilmaantuvan EM:n aikana)
Lymfosytooma
-
Vasta
-
aineita ei aina alkuvaiheessa ole osoitettavissa
-
Hoito, tarvittaessa varmistus 3
-
4 viikon kuluttua (S
-
BorrAb)
Varhainen neuroborrelioosi
-
Lymfosytaarinen meningiitti, kasvohermohalvaus
-
Seerumin ja likvorin borreliavasta
-
aineet samaan aikaan otettuna
-
Likvorin solut (ja/tai CXCL13
-
sytokiini)
-
Alkuvaiheessa ei välttämättä vasta
-
aineita:
-
Lisäksi alkuvaiheessa BorrNhO likvorista (negatiivinen
tulos ei koskaan ole poissulkeva

Myöhäinvaiheen borrelioosi (kuukausia, joskus vuosia)
Acrodermatitis chronica atrophicans (ACA
)
-
Aina IgG
-
vasta
-
aineita seerumissa
Radikuloneuriitti, enkefalomyeliitti
-
Seerumin ja likvorin borreliavasta
-
aineet
-
Intratekaalinen vasta
-
ainetuotanto
Lymen artriitti
-
Tulisi löytyä aina IgG
-
vasta
-
aineita seerumista
-
NhO nivelnesteestä mahdollinen: positiivinen tulos varmistaa,
negatiivinen ei poissulje
Harvinaiset
silmä
-
, sydänmanifestaatiot
-
Tulisi löytyä IgG
-
vasta
-
aineita seerumista
-
Voi joskus olla alkuvaiheen manifestaatio (IgM)

Tulkinnassa otettava huomioon
-
Vähintään kohtalaisesti kohonnut IgG
-
vasta
-
ainetaso viittaa käynnissä
olevaan tai aiemmin sairastettuun borreliainfektioon:
kliininen arvio
-
Matalat tasot liittyvät usein vanhaan immuniteettiin tai alkuvaiheen
infektioon, näiden erottamiseksi
tarvittaessa
uusintatutkimus 3
-
4 viikon
kuluttua
-
IgM
-
vasta
-
aineiden merkitys toissijainen
-
Varhaisvaiheessa voi olla ilman IgG
-
vasta
-
aineita (esim.
varhainen neuroborrelioosi)
-
Ellei IgG
-
vasta
-
aineiden serokonversiota tule: epäspesifinen reaktio
tai vanha immuniteetti mahdolliset

Yksi tutkimus yleensä riittävä varsinkin myöhäisvaiheen borrelioosia
epäiltäessä, hoidonjälkeiset seurantatutkimukset eivät yleensä ole tarpeen
-
Perusteltu kliininen epäily disseminoituneesta infektiosta hoidon
jälkeen aihe uusintatutkimukselle
-
Vasta
-
aineet säilyvät selvästi kohonneella tasolla kuukausia

vuosia
onnistuneenkin hoidon jälkeen
-
Laboratorio ei useinkaan voi esittää selkeitä tulkintoja ilman mitään
kliinisiä tietoja: oireiden kesto (esim. artriitti 3 kk), mahdollisen hoidon
ajankohta varsinkin uusintatutkimuksessa

Borreliavasta
-
aineet
, jatkotutkimus
(
S
-
BorAbJt
)
-
Line blot ja ELISA (rekombinanttiantigeenit)
-
Pyydettävä erikseen joko IgG
-
tai IgM
-
vasta
-
aineet, ellei ole valittu
tehdään pääsääntöisesti IgG
-
vasta
-
aineet
-
Jos oireita yli 2
-
3 kk suositellaan IgG
-
vasta
-
aineiden jatkotutkimusta
-
Melko harvoin tarpeellinen perustutkimuksen ollessa 2
-
vaiheinen
-
Vasta
-
aineiden spesifisyyden varmistamiseksi esim. eri testien antaessa
ristiriitaisia tuloksia
-
Ei kerro immuniteetin tuoreutta
-
Infektion alkuvaiheessa perustutkimusta epäherkempi
-
Negatiivinen/raja
-
arvo/positiivinen + lausunto

BorrNhO (nukleiinihapon osoitus)
-
Herkkyys kohtuullinen ihobiopsiasta (ad 70 %),
-
Nivelnesteestä/
-
synoviaalibiopsiasta
-
Kudosbiopsioista mahdollinen
-
Verestä tehtynä epäherkkä vähäisen bakteerimäärän takia
-
Varhaisessa neuroborrelioosissa ennen vasta
-
aineiden ilmaantumista
-
Positiivinen tulos diagnostinen,
negatiivinen tulos ei ole koskaan
poissulkeva
-
Voi antaa positiivisessa tapauksessa tietoa borrelialajista

Muita tutkimuksia
-
Lymfoproliferaatio
tai ELISPOT
borreliantigeeneilla
: ei näyttöä hyödystä

Yhteenveto

Erythema
migrans
voidaan hoitaa ilman mikrobiologista
näyttöä

Alkuvaiheen infektiossa vasta
-
ainereaktiot voivat olla heikkoja
tai puuttua

Tarvittaessa uusintatutkimus 3
-
4 viikon kuluttua, hoito ei saa viivästyä
tarpeettomasti

Myöhäisvaiheen
borrelioosissa
tulisi löytyä vähintään
kohtuullisesti koholla olevat
IgG
-
vasta
-
aineet

II näyte ei yleensä tuo lisäinformaatiota
IgG
-
vasta-aineiden ollessa
jo
vahvasti koholla

Neuroborrelioosin
poissulku edellyttää myös likvorin
borreliavasta-aineiden tutkimista

Rutiininomainen hoidon seuranta vasta
-
aineilla ei ole tarpeen
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Heinä 26, 2013 21:10


Borreliatesteistä ja niiden luotettavuudesta esiintyy erilaisia näkemyksiä.
Artikkelin lopussa on esitetty Saksassa esim. BCA klinikalla käytössä olevia borreliatestejä. Monien kohdalla Suomessa suoritetut borreliatestit ovat olleet negatiiviset ja esim. Saksan testit positiiviset. Allalolevan artikkelin mukaan Suomen testit ovat hyvin kehittyneitä. http://www.mtv3.fi/uutiset/kotimaa.shtm ... linikoilla
".....Saksassa joillakin yksityisklinikoilla käytössä olevista testeistä. ...nimenomaan nämä testit ovat huonoja, koska ne antavat vääriä positiivisia tuloksia".


Useiden näkemysten mukaan kuitenkin nimenomaan perinteiset vasta-ainetestit ovat taudin kaikissa vaiheissa varsin epäluotettavia ja siksi tarvitaan uudenlaisia testejä. Vuonna 2011, FDA; Amerikan Elintarvike- ja Lääkevirasto ja CDC; Yhdysvaltojen tartuntatautien valvonta- ja ehkäisykeskus, hyväksyivät ELISPOT LTT tekniikan käytön. tuberkuloosiin. Nyt tätä tekniikkaa käytetään useiden eri taudinaiheuttajien tutkimiseen.
http://www.infectolab.com.au/Pages/LTT.aspx

Tekniikka ei ole käytössä Suomessa Borrelioosin diagnostiikassa ./size]

-----------------------------------------------------------------------------------------------

http://www.infectolab.de/index.php?id=51&L=1

Borrelia Elispot-LTT (LymphocyteTransformationsTest)

Actual news:

The Elispot-LTT method has been approved by the FDA in May 2011 for M. tuberculosis (Not ITT or MELISA)

The FDA argues in this paper:

"... A positive result (in the Elispot-LTT) suggests that an infection is likely, a negative result that an infection is unlikely..."

"... Results (of the Elispot-LTT) can be available within 24 hours (ITT or MELISA not)..."

A Borrelia infection does not only activate the humoral immune response, but also activates T-lymphocytes at the same time. Once Borrelia bacteria are not active anymore, the T-cellular immune response is not present.

It is not possible to test the treatment success by Borrelia antibodies, because the 'titer" or antibodies can be measured in the blood over years. Furthermore, Lyme infections in Stage I (e.g. 'bulls-eye rash' or 'summer flu') only show antibodies in the blood after weeks and sometimes do not show them at all.

The Borrelia Elispot-LTT eliminates these problems. The test reflects the actual, current Borrelia burgdorferi activity of chronic and also acute Lyme infections. The Elispot-LTT is highly sensitive and can detect even one single Borrelia-reactive T-cell in the blood. The Elispot-LTT is very helpful when monitoring a chronic or acute Lyme therapy. The Elispot-LTT should usually become negative about 6 to 8 weeks after completion of an effective therapy.

---------------------------------------

Artkkeli LTT-tekniikastta eri mikrobeja tutkittaessa:
Elispot®-LTT: FDA and CDC approved LTT technique in U.S.

Actual T-cellular activity in the blood against Borrelia burgdorferi, Chlamydia pneumoniae, Chlamydia trachomatis, Ehrlichia/Anaplasma, Epstein-Barr-Virus, 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 (ie: 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...", and "...results can be available within 24 hours..."


The Elispot®-LTT is available for the following infections:
- Borrelia burgdorferi
- Chlamydia pneumoniae
- Chlamydia trachomatis
- Ehrlichia/Anaplasma
- Epstein-Barr-Virus
- Yersinia species


LTT in Borrelia Testing

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 use Heparin for a stability of 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

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http://www.aacc.org/events/Annual_Meeti ... 9-B150.pdf

Chenggang Jin, MD, PhD/Bradley Bush, ND

[size=150]Development and Validation of a Novel Interferon-γ ELISPOT Assay for Sensitive and Specific Detection of Antigen-Specific T cell Response to Borrelia burgdorferi


The enzyme-linked immunospot (ELISPOT) technology has proven to be extremely sensitive in detecting antigen specific reactive T cells and has been applied in laboratory diagnostic for Tuberculosis approved by FDA. A novel T-cell based assay for diagnosis of Lyme disease -Lyme ELISPOT was successfully developed and validated.

ABSTRACT
Learning Objectives: After the presentation, an individual will be able to:
⦁ Describe ELISPOT assay technology
⦁ Contrast ELISPOT assays to conventional antibody detection methods utilized for the diagnosis of Lyme disease
⦁ Evaluate the potential application of ELISPOT as a diagnostic tool for Lyme disease
Presentation Outline:
Background: Lyme disease, caused by infection with the spirochete Borrelia burgdorferi, is an emerging infectious disease in the United States that has become an important public health problem. Both B-cell immunity and T-cell immunity develop in natural infection with Borrelia burgdorferi. Detection of specific antibody response mediated by B cells against Borrelia burgdorferi is utilized conventionally in aiding the clinical diagnosis of Lyme disease. However, the limitation of these antibody-based immunoassays is that they have low sensitivity and specificity, causing significant false negative and false positive results. Furthermore, Borrelia specific antibodies cannot be detected at the early stage of infection and in a fraction of seronegative Lyme patients who lack Borrelia specific antibody responses. In contrast, Borrelia specific T-cell based immune assays have not yet been well developed. Thus, highly sensitive and specific T-cell based clinical laboratory assays are needed to help in diagnosing Lyme disease in conjunction with antibody-based immunoassays. The enzyme-linked immunospot (ELISPOT) technology has proven to be extremely sensitive in detecting antigen specific reactive T cells and has been applied in laboratory diagnostic for Tuberculosis approved by FDA. We here explore the potential application of ELISPOT as diagnostic tool for Lyme disease.
Objective: The aim of this study is to develop and validate a novel T-cell based assay for diagnosis of Lyme disease using newly developed digitalized ELISPOT technology.
Methods: To develop the novel T-cell based diagnostic assay for Lyme disease, we detected the Borrelia antigen-specific memory T cells that were activated ex vivo by recombinant Borrelia specific antigens, using Th1 cytokine Interferon-γ ELISPOT at the single cell level. The human peripheral blood mononuclear cells (PBMC) were stimulated with single or a combination of recombinant Borrelia specific antigens, DbpA, OspC, p100 and VlsE. In addition, we added costimulatory cytokine IL-7 into the cell culture to increase the detection of T memory cells. The results of ELISPOT were analyzed using CTL S6 Ultimate-V Analyzer/BioSpot 5.0 Software and reported as IFN- γ Spot Forming Units (SFU). To validate the Lyme ELISPOT assay, a cohort of 21 clinically diagnosed Lyme patients and 45 healthy control subjects were further studied and compared with Western Blot test. The performance of the Lyme ELISPOT assay, including clinical sensitivity, clinical specificity, accuracy and precision, is also evaluated.
Results: The frequency of Borrelia specific T memory cells can be detected by Interferon- γ ELISPOT and therefore can be used as a biomarker for Borrelia infection. The detection of antigen specific T cells was significantly increased by a combination of recombinant Borrelia antigens and addition of constimulatory cytokine IL-7. The signal enhancing effect of IL-7 was observed even at saturating antigen concentration in terms of frequency, but IL-7 did not increase the amount of IFN- γ secreted by individual cells. A strong correlation was observed between ELISPOT and IFN- γ concentration measured by Bio-plex suspension system (R=0.8, P<0.0001). The Lyme ELISPOT assay cut-off value was determined using Receiver Operating Characteristic (ROC) curve analysis and it was found that a cut-off value of >25 PFU maximized assay sensitivity and specificity. It has a significantly higher specificity (96%) and sensitivity (76%) compared with Western blot (Sensitivity 24%). The results also demonstrated that there was dissociation between B cell response and T cell response during Borrelia infection, suggesting a comprehensive immunological diagnostic panel should include both B cell and T cell diagnostics. Further studies will include more Lyme patients, other related diseases and independent studies by other laboratories.
Conclusion: A novel T-cell based assay for diagnosis of Lyme disease -Lyme ELISPOT was successfully developed and validated. This newly developed Lyme ELISPOT assay may be a helpful laboratory diagnostic test for Lyme disease, especially for seronegative Lyme patients. A comprehensive evaluation of both antibody response and T cell response to Borrelia infection will provide new insights into the pathogenesis and diagnosis of Lyme disease.





Lymfosyyttitransformaatiotestistä/Borrelioosista lisää esim.

http://www.cdc.gov/lyme/stats/chartstab ... yyear.html
Yound, J.D. Underreporting of Lyme disease. N Engl J Med. 1998. 338(22):1629-1629.
http://www.cdc.gov/hiv/topics/surveillance/basic.htm
http://www.cdc.gov/ncidod/dvbid/westnil ... tailed.htm
http://www.who.int/influenza/human_anim ... 1cases.pdf
Maloney, E.L. The Need For Clinical Judgment in the Diagnosis and Treatment of Lyme Disease. Journal of American Physicians and Surgeons. 2009. 14(3):82-89.
Lehmann, P.V., Zhang, W. Unique Strengths of ELISPOT for T Cell Diagnostics. In: Alexander Kalyuzhny, E. Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 792. 2nd Ed. New York, NY: Spriner Science+Business Media, LLC; 2012: 3-23.
T-Spot.TB 96 [Package Insert]. Oxfordshire, UK: Oxford Immunotec Limited; 2009.
Tary-Lehmann M., Hamm, C.D., Lehmann, P.V. Validating reference samples for comparison in a regulated ELISPOT assay. In: Uma Orabhakar and Marian Kelley Eds. Validation of Cell-Based Assays in the GLP Setting: A Practical Guide. 1st Ed. West Sussex, England: John Wiley & Sons. Ltd; 2008: 127-146.
Forsberg, P., Ernerudh, J., Ekerfelt, C., et al. The outer surface of Lyme disease borrelia spirochetes stimulate T cells to secrete interferon-gamma (IFN-γ): diagnostic and pathogenic implications. Clin Exp Immunol. 1995; 101:453-460.
Ekerfelt C., Forsberg P., Svenvik, M., et al. Asymptomatic Borrelia-seropositive individuals display the same incidence of Borrelia-specific interferon-gamma (IFN-γ)-secreting cells in blood as patients with clinical Borrelia infection. Clin Exp Immunol. 1999. 115: 498-502.
Dattwyler, R.J., Volkman, D.J., Luft, B.J., et al. Seronegative Lyme Disease- Dissociation of Specific T- and B-Lymphocyte Responses to Borrelia burgdorferi. New England Journal of Med. 1988. 319(22): 1441-1446.
Dressler, F., Yoshinari, N.H., Steere, A.C. The T-Cell Proliferative Assay in the Diagnosis of Lyme Disease. Annals of Internal Medicine. 1991. 115:533-539.
Martinuzzi, E., Scotto, M., Enee, E., et al. Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISPOT detection. Journal of Immunol Meth. 2008. 333: 61-70.
Seriburi, V., Ndukwe, N., Chang, Z., et al. High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice.Clin Microbiol Infect. 2012 Dec;18(12):1236-40.
Kuerten, S., Batoulis, H., Recks, M.S., et al. Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays. Cells 2012, 1(3), 409-427


*Patent Pending (USPTO Application No: 61779064)

Conculsion

Lyme disease is an increasingly common condition that can have debilitating effects if not diagnosed and treated appropriately. A comprehensive approach to diagnosis will lead to the most positive outcomes and healthcare savings; however, testing options thus far have the potential for false negative results, making diagnosis difficult. NeuroScience’s testing options (iSpot Lyme™ including Western Blot analysis) can aid in the diagnosis of B. burgdorferi infection and allow for early detection leading to earlier treatment. iSpot Lyme™ is a highly sensitive T cell-based enzyme-linked immunospot (ELISPOT) method which enumerates the B. burgdorferi-specific activated effector/memory T cells. This test represents a breakthrough in diagnostic accuracy, and can increase the speed of diagnosis and treatment leading to improved clinical outcomes.
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http://www.mtv3.fi/uutiset/kotimaa.shtm ... linikoilla

Professori: Suomessa paremmat borrelioositestit kuin Saksan klinikoilla

Punkkitauti borrelioosista vuonna 1996 väitellyt professori kehuu Suomessa käytössä olevia borrelioositestejä.

Suomen testejä on kritisoitu julkisessa keskustelussa, ja ja ihmisten on jopa kerrottu matkustavan testeihin ulkomaille.

Turun yliopistollisen keskussairaalan infektiotautien ylilääkäri, infektiotautien professori Jarmo Oksi sanoo, että Suomessa käytössä olevat vasta-ainetestit borrelioosin diagnosoimiseksi ovat hyvin kehittyneitä.

– Väittäisin, että meillä käytetään todennäköisesti parempia testejä kuin suurimmassa osassa muuta Eurooppaa. Uskoisin, että juuri missään ei ole saatavilla parempia testejä, Oksi sanoo.


"Toinen testi antaa melkein kenelle tahansa positiivisen"

Oksi haluaa sanoa sanansa myös Saksassa joillakin yksityisklinikoilla käytössä olevista testeistä. Oksin mukaan nimenomaan nämä testit ovat huonoja, koska ne antavat vääriä positiivisia tuloksia.

– Joissakin Saksassa olevista yksityisklinikoista näytetään käytettävän niin sanottua lymfosyyttitransformaatiotestiä, joka on vähän eri asia kuin vasta-ainetesti ja selvästi ongelmallisempi. Tämä testi näyttää positiivista, jos on koskaan vähänkään törmännyt borreliabakteeriin, vaikkei bakteeria nykyään kehossa olisikaan, Oksi tyrmää.

Lymfosyytit kuuluvat ihmisen valkosoluihin, ja Oksin mukaan lymfosyytit voivat siis reagoida borreliabakteeriin, vaikka bakteeria ei ole elimistössä.

Oksi myöntää, että borrelioosin diagnosoiminen voi olla vaikeaa ja että Suomessa käytössä olevat vasta-ainetestitkään eivät ole täydellisiä.

– Ensimmäisen kahden kuukauden aikana punkin puremasta vasta-aineet eivät ole ehtineet vielä nousta. Toisaalta voi olla täysin terve, vaikka vasta-ainetesti olisi positiivinen. Vasta-aineet nousevat todennäköisesti kuitenkin hitaammin kuin lymfosyyttitunnistus, joka saattaa antaa melkein kenelle tahansa positiivisen tuloksen.

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Saksan Borrelioosiklinikan testit:

http://www.b-c-a.de/index.php?id=97\\\\\\\%27&L=1

http://www.b-c-a.de/fileadmin/img/bca/6 ... .07.11.pdf


Ihr kompetenter Laborpartner
http://www.infectolab.de
BCA-clinic
Betriebs
GmbH & Co. KG
Dr. med. Armin Schwarzbach
Facharzt für Labormedizin
Infecto
lab
, ein Geschäftsbereich der BCA-clinic Betriebs GmbH
& Co. KG
Geschäftsführer: Dr. med. Carsten Nicolaus, Dr. med
. Armin Schwarzbach
Amtsgericht Augsburg HRA 15697
·
Morellstraße 33
·
86159 Augsburg
·
Tel. +49/ 0821/ 455 074-0
·
Fax +49/ 0821/ 455 074-1
http://www.infectolab.de
·
e-mail: service@infectolab.de
·
Umsatzsteuer-Ident.-Nr. DE250941023
·
Bankverbindung:
Kreissparkasse Augsburg (BLZ 720 501 01)
·
Konto-Nr. 19885
·
IBAN: DE48 7205 0101 0000 0198 85
·
BIC: BYLANDEM1AUG
Patient information:
Laboratory services and diagnosis for Lyme disease
and possible
co-infections
- The detection of tick-borne infections makes high
demands on laboratory analysis in
combination with the diagnostic findings (anamnesis
) -
It is important to detect Borrelia infections at an
early stage. The earlier the detection the simpler
the treatment measures (usually antibiotics) and th
e shorter the ordeal of the infected patients.
This is how you can diagnose Lyme Disease

a Lyme disease infection progresses in 3 stages dep
ending on
symptoms and ailments
(time specification after the tick bite):
1.
Stage I
(after days up to weeks): “bull’s eye rash“ (“Eryth
ema chronicum
migrans“, only in 40-70% of all cases), Borrelia l
ymphocytoma,
headache, fever, sweating,
“Summer flue“ (app. 20% of all
cases), exhaustion and fatigue, facial palsy (espec
ially with
children/pupils).
2.
Stage II
(after weeks up to months): inflammation of the bra
in; meninges;
spinal marrow; any nerve in the human body, inflamm
ations of
the joints (“arthritis”), joint and muscle pain, in
flammation of the
eye, liver and kidneys, myocarditis, pericarditis,
Cardiac
arrhythmia
.
Borrelia burgdorferi – a
spirochete bacteria with a range
of over 300 proven species world
wide.
3.
Stage III
(after months up to years): Thinning of the skin at
the back of the hand, (“Acrodermatitis chronica
atrophicans“), Borrelia lymphocytoma (ear, nose, sc
rotum), lethargy, fatigue, paraesthesia, cognitive
dysfunction, muscle inflammations, joint inflammati
ons and swelling, tendon inflammations,
inflammation of the bursa, vasculitis, myocardinal
diseases, depression.
Symptoms of Lyme disease occur in thrusts with alte
rnating intensity and appearance in contrast
to a classic organic illness. Many patients also su
ffer from slightly higher temperature during those
thrusts. Co-infections with other bacteria and viru
ses have been increases over the past years and
often lead to a complicated course of the disease.
Often the tick bite is not detected early enough
or the acute treatment by the attending physician i
s not sufficient. The chronic Lyme disease
patients often go through a true “martyrdom” as the
y do not only suffer from actual physical and
mental ailments, but also from not receiving a reli
able diagnosis and the fact that their illness is n
ot
taken seriously.
There are three main reasons why a Lyme infection i
s not immediately detected in daily practice,
so that a treatment at an early stage is inhibited:
1. There is no
bull’s eye rash
(“Erythema chronicum migrans“)! Research has prov
en that
this classic symptom of Lyme disease only appears i
n 40% to a maximum of 70% of all
cases.
2.
No tick bite detected
! Such a bite can be induced even by very small tic
ks (larvae). Or it
was not detected because there was no specific skin
reaction. In addition, the scientific
community now assumes that Borrelia bacteria can al
so be transmitted by infected insects.
3.
Only conventional blood tests
have been performed. This was either too early
(antibodies can be tested positive only after a per
iod of up to six weeks after the tick bite)
or there have been absolutely no antibody productio
n in the body or the cellular stage was
not or not sufficiently tested (necessary tests are
: Elispot
®
-LTT and CD3-/CD57+ cells).

The BCA-clinic Augsburg (
BCA
) is specialised in the diagnosis and therapy of ti
ck-borne
diseases. In case of a suspected tick-borne disease
the diagnosis is performed by experienced
physicians of the “Medical Partnership” cooperating
with the BCA-clinic. The diagnosis is based on
an extensive anamnesis in which precise ailments an
d the antecedent are recorded and reviewed
along with a physical examination (=
clinical findings
). Additionally, the physician will initiate
specific laboratory tests of your blood, which will
be undertaken and analysed in the specialised
laboratory of the BCA-clinic.
First the
laboratory diagnosis
is crucial for the detection
of Borrelia. There is a difference between the test
s of the
humoral
(antibodies) and
cellular
level (lymphocytes,
NK-cells). Both levels have to be examined at the s
ame
time when a Lyme disease or an apparent illness is
suspected.
1. Laboratory testing of the humoral level (antibo-
dies):
Borrelia IgM- and IgG-EIA (Enzymimmuno-assay)
as well as Borrelia IgM- and IgG-Immunoblot
2. Laboratory-testing of the cellular level:
Elispot
®
-LTT (Borrelia Elispot Lymphocyte Transformation
Test), CD3-/CD57+ cells (NK-cells)
Explanations of these tests:
1. Antibodies – humoral level: In case of the Borre
lia antibodies
examinations up to 19% of the antibodies results in
the EIA are
falsely negative due to the minimal sensitivity (re
sponsiveness)
of the EIA in contrast to the Immunoblot. Therefore
it is
essential to check the Borrelia IgM- and IgG-Immuno
blot along
with the Borrelia IgM- and IgG-EIA (even with a neg
ative EIA)!
Important: The laboratory has to always tests for V
lsE (Variable
major protein-like sequence Expressed) in EIA and
Immunoblot. VlsE describes the characteristics of
the Borrelia
as a “chameleon“, which permanently changes the sur
face
structure VlsE in vivo to resist the detection via
the immune
system. VlsE has the highest sensibility for the an
tibody search!
Beware of positive antibodies constellations!
Armin Schwarzbach M.D.,
medical specialist for laboratory medicine,
head of the BCA laboratory and
specialist for Lyme and co-infections.
Borrelia IgM- as well as IgG-antibodies can remain
in the body for
months or years even without an active Lyme disease
infection.
Hence a positive Borrelia antibodies test does not
give any evidence
of the activity of a Lyme infection, but gives only
one conclusion: There must have been a tick bite o
r tick
bites in the past during which Borrelia bacteria ha
ve been transmitted – no more or less!
2. Cellular level:
a) The
Borrelia Elispot
®
-LTT
provides information about the current activity of
the Borrelia bacteria and is
20 to 200 fold more sensitive than a EIA-antibodies
test.
b) The
CD57+ cells
indicate the extent of immune suppression during a
chronic Lyme disease and are the
prognostic factor during and after the antibiotic t
reatment.
The complete laboratory diagnosis is very complex a
nd - considering the co-infections - has to be
put together like a mosaic. This demands experience
d Lyme disease analysts who can diagnose
and evaluate all other infections as well.
Armin Schwarzbach, M.D., PhD, is head of laboratory
medicine and diagnostics in the BCA.-clinic.
Dr. Schwarzbach is very experienced in laboratory m
edicine and has been a specialist in Lyme
disease and co-infections for years.
The medical laboratory diagnosis is primarily orien
tated on the guidelines of the German Society
for Hygiene and Medical Microbiology (MiQ12 Lyme-Bo
rreliose). However, the complexity of a
Lyme disease infection is not sufficiently covered
by this and needs additional laboratory testing.

herefore further internationally accepted methods
are applied which are of considerable
importance for the patient before and after the the
rapy (e.g. for the cellular level: Elispot
®
-LTT and
the CD3-/CD57+ cells).
The laboratory integrated in the BCA for the analys
is of vital parameters ensures, especially in the
case of the medical treatment (e.g. infusion therap
y), that the physicians of the medical
partnership make further therapy decision as soon
as possible according to the blood results.
In practice, the following laboratory constellation
s indicate a Borrelia infection:
(1) Positive antibody detection and positive cellul
ar results
(Elispot
®
-LTT and/or CD57+) – often active infection
(2) Positive antibody detection without positive ce
llular test findings – What kind of indications
are shown by the clinical findings (symptoms)?
(3) Negative antibodies detection, but positive cel
lular test results
- Indication for an active Lyme disease at an early
stage, but also chronic stage – what
kind of indications are shown by the clinical findi
ngs (symptoms)?
Attention: A negative antibody detection in the EI
A and/or Immunoblot does not give evidence of
a Lyme infection! Because: the antibodies productio
n of a Lyme disease in stage I needs several
weeks, 10 to 14 days at a minimum. Thus the immedia
te measurement of cellular activity in
Elispot
®
-LTT is a compulsory necessity, as normally the cel
lular activity precedes the humoral
activity in stage I of a Lyme disease infection.
In practice the following combinations of blood tes
ts have proven useful:
Laboratory diagnosis stage I
(Costs: 393,44 €
plus extra charges.
)
1. Borrelia IgG- and IgM-EIA incl. VlsE
2. Borrelia IgG- and IgM-Immunoblot
incl. VlsE
3. Borrelia Elispot
®
-LTT
Laboratory diagnosis stage II and
stage III
(Costs app.: 544 €
plus extra charges.
)
1. Borrelia IgG- and IgM-EIA incl. VlsE
2. Borrelia IgG- and IgM-Immunoblot
incl. VlsE
3. Borrelia Elispot
®
-LTT
4. CD3-/CD57+ cells
Necessary “
Staging
“ (evaluation of progression) of Lyme disease durin
g and after an antibiotic or
holistic approach to therapy: The above mentioned p
arameters also have to be checked during
Lyme therapy.
Recommended laboratory diagnosis during the therapy
:
Stage I
Performance of above mentioned 3 tests
4 weeks after beginning of therapy and 8
weeks after the end of therapy.
Stage II and III
Performance of above mentioned 4 tests after
beginning of therapy every 8 weeks as well as
8 weeks after end of therapy.
For explanation:
Borrelia antibodies or titer cannot be “eliminated”
, but they can exist in the
blood for months or even years. After a successful
antibiotic therapy Elispot
®
-LTT as well as
CD57+ cells should have come to a “normal” level 8
weeks after the end of therapy. But
: Inspite
of a symptom free status after treatment there migh
t be still positive findings of Elispot
®
-LTT
and/or CD57+ cells, so the patient should be consid
ered for a “monitoring” of the activity tests and
possible future symptoms. Otherwise there will be t
he risk of a relapse, a new infection or a co-
infection.
It should be noted that laboratory parameters alone
do not give a definite proof of Lyme disease,
but very important evidence and details about a pos
sible infection. The parameters are also very
important to make decisions about the duration and
success of therapy.

Adjoining is an overview
of relevant diagnostic
parameters of tick-borne
diseases.
These may be extended
or limited individually
during an extensive
consultation meeting with
your attending physician
(e.g. in case of pre-
findings).
Based on the
co-
infections questionnaire
indications are
established if such
additional laboratory tests
are necessary.
An overview of prices can
be found in the
attachment “Order for
laboratory tests with
declaration of consent“ for
patients.
Note: The laboratory analysis is only one part
of a comprehensive diagnosis. The
clinical
report
based on an extensive
anamnesis
and
a physical examination is the crucial part. The
experienced physicians of the cooperating
Medical Partnership know the broad range of
possible disease patterns, which can also
become noticeable as „
unspecific general
symptoms
“ (note adjoining chart).
Generally, the laboratory diagnosis causes the foll
owing costs:
Laboratory costs
1. for Borrelia IgM- and IgG- antibodies including
VLSE,
Borrelia Elispot
®
-LTT and CD3-/CD 57+ T-lymphocytes
with blood sampling and special tubes
app.
544 €
2. Basic laboratory (blood count, liver-kidney-clot
ting) app.
80 €
3.
Possible necessary additional examination when susp
icion of co-
infections (Each test app. 61 – 97 €):
up to app. 945 €
Note:

Elispot- and CD3-/CD57+ are principally not covered
by health insurances!

The unit prices for different laboratory tests resu
lt from the laboratory order form in the attachment
:
blood samplings and special tubes will be billed se
parately

Laboratory tests of possible co-infections
Laboratory tests of possible co-infections have an
increasing importance when adequate evidence
exists (BCA co-infections sheet). Especially Ehrlic
hia/Anaplasma, Babesia, Bartonella, Rickettsia,
Chlamydia and Mycoplasma are to be named.
Because: numerous symptoms of the co-pathogens over
lap the same symptoms of Lyme disease
patients. Without an exact knowledge of a patient’s
possible co-infections the therapist cannot
make a exact decision about the antibiotic therapy.
Not all co-infections can be treated by the
generally used antibiotics for Lyme disease. Howev
er, the testing for co-infections can induce
extensive additional laboratory costs (up to 945€).
But these are justified by the additional securit
y
in the diagnosis and especially by the correct anti
biotic decision, i.e. more success in antibiotic
therapies as well as generally less expensive costs
for medication during the antibiotic therapy
(vs. the “classical” antibiotic therapy of chronic
Lyme disease).
Apart from the LTT for Borrelia further cellular ac
tivity tests for Ehrlichia/Anaplasma, Chlamydia
pneunomiae and Chlamydia trachomatis have been deve
loped in the meantime (Babesia cellular
activity testing is coming soon) and can therefore
be determined with Elispot-LTT-technique. With
the help of this new Elispot
®
-LTT many activities of Chlamydia and Ehrlichia hav
e been detected
in the BCA! A serum examination concerning co-infec
tions is perfomed at the same time. There
are now well standardised antibody tests for Chlamy
dia, Mycoplasma, Ehrlichia, Bartonella,
Rickettsia, Babesia, Yersinia etc. The same applie
s here as well as with the Borrelia-LTT: the
antibodies alone do not have any significance towar
ds the activity of an infection – but the
Elispot
®
-LTT is significant as it attests the high-specific
interferon release against the respective
co-pathogen in the blood.
It should be noted that in some cases it is the co-
pathogen itself which is responsible for the
symptoms and not the Borrelia infection: e.g. Chla
mydia cause diseases patterns such as Morbus
Alzheimer, Multiple Sclerosis, fibromyalgia, Chroni
c Fatigue Syndrome (CFS), myocardinal
infarcts, strokes, vasculitides, visual disturbance
s.
The Lyme infection may well have been treated succe
ssfully with antibiotics, but the co-infection
might not have been destroyed by it. Therefore, an
exact anamnestic documentation of the
symptoms during the therapy is necessary before, du
ring and after a Borrelia infection.
Further information regarding the importance of co-
infections can be found in the separate
information for patients: „Increasing importance of
co-infections for Lyme disease patients” (article
by Armin Schwarzbach, M.D., PhD).
Laboratory transmittals
In case of a suspected tick-borne disease physician
s have the opportunity to execute specific
laboratory analyses in cooperation with the BCA. T
he blood kits contain special anamnestic
questionnaires by the BCA and a questionnaire to ch
eck for co-infections. The patient should fill
out these questionnaires and send them to the BCA t
ogether with the blood kits.
We are delighted to provide interested physicians w
ith further information about possibilities in
terms of collaboration and co-operation to provide
a successful treatment for patients suffering
from tick-borne diseases. Especially for the diagn
osis and therapy of chronically ill patients, who
are not living in the Augsburg area we can coordina
te the procedure of the treatment with the
attending physician at home if wanted. However, in
case of complicated symptoms and severe
illness patterns we recommend the physicians of the
Medical Partnership which cooperates with
the BCA for the first diagnosis and therapy plan pr
eparation. We also recommend the Lyme

Disease “Intensive Treatment and Rehabilitation” Pr
ogram of the BCA, which is a special Compact
Treatment clinic for chronically ill patients here
in Augsburg.
The BCA regularly offers seminars and workshops, as
well as exchange and sharing of
experiences to interested physicians and cooperatio
n partners.
Below you will find as
attachments
further information regarding the laboratory tests
on the
cellular level (Borrelia Elispot
®
-LTT, CD3-/CD57+ cells and the Elispots for Chlamyd
ia and
Ehrlichia) as well as the order form for laboratory
tests with declaration of consent for the BCA.
Attachments

Further information regarding the laboratory tests
on cellular levels: Borrelia Elispot
®
-LTT,
CD3-/CD57+ cells, Chlamydia Elispot
®
-LTT and Ehrlichia Elispot
®
-LTT

Order form for laboratory tests with declaration o
f consent
Borrelia Elispot
®
-LTT
-
Determination of actual activity in the blood regar
ding Borrelia burgdorferi
A Borrelia infection simultaneously leads to an act
ivation of T-lymphocytes lateral to the humoral
immune response. The T-cellular immune answer vanis
hes as soon as the Lyme disease infection
is not active anymore.
A control of success of a Lyme infection therapy is
not possible via Borrelia antibodies, as the
“titer” or the antibodies can still be found in the
blood for years after a cured infection. Addition
ally,
in the first stage of Lyme disease (e.g. “bull ́s ey
e rash” or “summer flu” after a tick bite) antibodi
es
can only be detected and measured after several wee
ks or not at all.
The diagnostic gaps are suggested to be Borrelia ac
cording to the Elispot, which measures the
actual activity regarding Borrelia burgdorferi with
chronic and also acute Lyme disease infections.
The Elispot is so sensitive, that it can even detec
t a single Borrelia reactive T-cell in the blood. T
he
Elispot is 20- to 200-fold more sensitive than an E
LISA-test on Borrelia and is able to find 1
reactive cell under 100.000 lymphocytes.
The Elispot of Borrelia is very important for contr
olling a therapy of a chronic or acute Lyme
infection. In general the Elispot is negative appr
oximately 6 to 8 weeks after the end of a
successful therapy.

Advantages of the Borrelia Elispot-LTT
(- as performed in the BCA -) in contrast to tradi
tional
lymphocyte transformation tests:

The result is obtainable within 2 days (LTT: 1 – 2
weeks) !

The use of cell stabilising CPDA tubes means a sta
bility of 3 days for the measured cells
(LTT: Heparin blood only 24 hours) !
This offers a significant improvement of the stabil
ity of the examined cells and a fast decision-
making possibility for Lyme disease therapists to e
xtend the duration of a therapy or start a new
treatment approach!
Borrelia Elispot:
Materials:
2 x 8.5 ml CPDA-tubes (kept at room temperature, d
o not
refrigerate)
Time required for analysis:
2 days
Billing according to GOÄ:
Number 3694 (3 different antigen approaches) factor
1.5 (3x
49.84 €) + number 4003 (Lymphocytes isolation) Fact
or 1.5 (34.97 €)
- total sum: 184.49 €
Indications:
- Diagnosis of a chronic Lyme disease
- Diagnosis of a acute Lyme disease
- Decision-making regarding the length of therapy
- Control of therapy after a Lyme disease therapy
Borrelia CD 57+ cells
-
Determination of chronic activity in the blood rega
rding Borrelia burgdorferi
A chronic progression (stage III) of a Lyme infecti
on leads to a weakening of the immune system.
This is reflected by the decrease of the CD3-/CD57+
NK-cells in case of chronic Lyme disease.
The CD3-/CD57+ cells are a subpopulation of the Nat
ural-Killer-Cells (NK-cells).
A decrease of the CD57+ cells indicates (an untreat
ed) chronic or not sufficiently treated chronic
Lyme disease and does not appear in cases of a acut
e Lyme infection (e.g. “bull’s eye rash“ or
“summer flu“ after a tick bite).
The CD57+ cells reflect the degree of activity of a
chronic Lyme disease and decrease to a normal
level after a successful Lyme disease therapy (afte
r the end of treatment).
In contrast there is no decrease of CD57+ cells wit
h clinical similar diseases, such as Multiple
Sclerosis (MS), Systemic Lupus Erythematosus (SLE)
or an Amyotrophic Lateral Sclerosis (ALS).
In addition there is no significant fluctuation of
CD57+ cells throughout the day.
CD57+ cells are appropriate laboratory parameters i
n cases where chronic Lyme disease is
suspected and for therapy monitoring. These should
be measured parallel to the Borrelia Elispot,
which reflects the actual T-cellular activity.
Advantages of the CD57+ cells determination of Borr
elia
:
1. The result is available within 2 days !
2. The use of cell-stabilising Heparin-tubes assure
s a stability of the measured NK-cells for 2
days!
In combination with the Borrelia Elispot this resul
ts in a significant improvement of the stability of
the examined cells and a fast decision-making possi
bility for Lyme disease therapists to extend
the duration of a therapy or start a new treatment
approach!
CD3-/CD57+ NK-cells:
Material:
1 x 10 ml Heparin-tube + 1 x EDTA-blood/blood coun
t-tube (kept
at room temperature, do not
refrigerate)
Time required for analysis:
2 days

Seite 9
Ihr kompetenter Laborpartner
Ehrlichia/Anaplasma Elispot
®
-LTT
-
Determination of actual activity in the blood regar
ding Ehrlichia/Anaplasma
Ehrlichia or Anaplasma are intracellular pathogens,
which can be found obligatory within the white
blood cells. Ehrlichia are transmitted to the human
by ticks contaminated with Ehrlichia
(approximately 6% of all ticks are contaminated wit
h the pathogen).
The symptoms of an Ehrlichia infection begin with f
lu like problems and express themselves with
strong headache, which are very often located “behi
nd the eyes”. Furthermore muscle aches and
numerous neurologic symptoms may be caused by Ehrli
chia. Rarely skin rashes on various parts
of the skin can be found, also on the palm of the h
and and soles of the feet.
An immune suppression is often the risk factor for
an Ehrlichia infection amongst others
depending on age, but also on chronic infections li
ke Lyme disease.
In the case that Ehrlichiosis (illness pattern of a
n infection with Ehrlichia) existing parallel to a
Lyme infection it is called co-infection. Accordin
g to the latest scientific literature additional co
-
infections with Ehrlichia should be taken into acco
unt in ca. 30% of all chronic Lyme disease
patients.
An infection with Ehrlichia leads to an activation
of the T-lymphocytes parallel to the humoral
immune answer (Ehrlichia-IgM- and Ehrlichia-IgG-ant
ibodies). The T-cellular immune answer
vanishes as soon as the Ehrlichia infection shows n
o activity.
The Elispot on Ehrlichia/Anaplasma measures the act
ual activity of this disease.
The Ehrlichia-Elispot helps with the diagnosis of t
his infection and also is a means of a successful
control during the course of the therapy.
Advantages of the Ehrlichia Elispot
:

The result is available within 2 days !

The use of cell stabilising CPDA tubes means a sta
bility of up to 3 days for the measured
T-cells !

The right choice of a specific antibiotic therapy
!
Ehrlichia Elispot:
Material:
2 x 8.5 ml CPDA-tubes (kept at room temperature, d
o not
refrigerate)
Time required for analysis:
2 days
Billing according to GOÄ:
Number 3694, factor 1.5 (49.84 €) + number 4003 (ly
mphocytes
isolation), factor 1.5 (34.97 €), total sum 84.81 €
Indications:
- Diagnosis of an infection with Ehrlichia
- Decision-making regarding the length of therapy
- Control of therapy success after an Ehrlichia-spe
cific therapy
Attachment: Order form for laboratory tests with de
claration of consent
-
This order is to be filled out by your physician a
nd has to be signed and dated by you in case of
an order to the infecto
lab
laboratory. The costs of the respective laboratory
order result from the
following individual prices (GOÄ), plus possible si
de costs for blood sampling, blood tubes,
shipment.

BCA klinikan testejä: http://www.b-c-a.de/index.php?id=97\\\\\\\%27&L=1
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » To Elo 15, 2013 10:22

Uusi testi neuroborrelioosin diagnostiikkaan 1.7.2013 alkaen.

Li-CXCL13 (13361)


Testi perustuu selkäydinnesteestä (likvorista) tehtyyn näytteeseen. CXCL13 on ns kemokiini jota tuottavat neuroborrelioosin yhteydessä keskushermoston immuunipuolustuksen solut. CXCL13 houkuttelee keskushermostoon B-lymfosyyttejä jotka tuottavat likvoritilaan borreliaspesifisiä vasta-aineita.

Testin luotettavuus?
Vasta-aineiden merkittävää nousua on tavattu esim. neurosyfiliksen, keskushermostolymfooman ja joidenkin aivokalvontuehduusten (Cryptococcus neoformans) yhteydessä. Virusinfektioissa ja MS-taudissa tapahtuu lievää nousua.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ke Syys 18, 2013 15:51

Tri A.McDonaldin mukaan (2013) Bb:n kystamuodot on mahdollista nähdä suoraan verinäytteistä. Piilossa (silmät, aivot jne) olevia Bb bakteereita ei löydetä ennenkuin näytteet otetaan kuoleman jälkeen kudoksista. Niitä ei löydetä millään nykyisellä testillä.

1. is there eve a circulating cystic borrelia form i human blood? answer: Yes - I can send to you a 200 slide lecture on Borrelia Cystic forms
if you wish to see it. It is also freely availabe on my website
www.alzheimerborreliosis.net
2. Can cysts of borrelia be demonstrated in direct analysis of human blood? answer --Yes --
see link to Round body (Cystic borrelia lecture-200 slides) - - DR Morten-Laane
3. Is the content of Cystic borrelia equal to the DNA content of spiral borrelia? answer --yes
4. Is a culture negative result ( of blood ) the end of the story as far as the possibility that cystic forms
might be present in tissue sites outside of blood? answer -- Negative in blood does not exclude the existence of cystic
forms elsewhere.
5. Does any lab testing method reflect the Status of :SANCTUARY SITES [ BRAIN,EYE,GONAD,FASCIA ETC] ?? Answer -- No Lab test other than the AUTOPSY
Is informative about the status of SANCTUARY SITES in the human body for the possible presence of any of the forms of borrelia
( Spiral, Straightened, Cystic,Granular, Cell Wall deficient, Biofilm communities,Liposomal forms of Borrelia)
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Syys 20, 2013 19:45

Neuroborrelioosi vaikka selkäydinnestenäyte negatiivinen. (2013)



Medicina (Kaunas). 2013;49(2):89-94.

Delayed diagnosis of lyme neuroborreliosis presenting with abducens neuropathy without intrathecal synthesis of borrelia antibodies.

Radzišauskienė D, Ambrozaitis A, Marciuškienė E.

Source

Department of Infectious, Chest Diseases, Dermatovenerology and Allergology, Vilnius University, Birutės 1, 08117 Vilnius, Lithuania. daiva730jvg@gmail.com.

Abstract

Lyme borreliosis is the most common tick-born infection in Europe. Global climate change expanding the range of tick vectors and an increase in the incidence suggest that this disease will remain an important health issue in the forthcoming decades. Lyme borreliosis is a multisystem organ disorder affecting the nervous system in 10% to 15% of cases. Lyme neuroborreliosis can present with any disorder of the central and peripheral nervous systems. The neuro-ophthalmological manifestations are a rare feature of the disease. The intrathecal synthesis of Borrelia burgdorferi antibodies is of diagnostic importance, but in rare cases, immunoglobulins against the Borrelia burgdorferi antigen may not be detected.
We report a case of possible Lyme neuroborreliosis presenting with sixth cranial nerve neuropathy at the onset of the disease further developing into typical meningoradiculitis and multiple mononeuropathy. Surprisingly, Borrelia burgdorferi antibodies were not detected in the cerebrospinal fluid.

PMID:
23888345
[PubMed - in process]

Free full text
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ti Marras 19, 2013 11:57

Immuunipuolustuksen kyky tunnistaa borreliabakteeri riippuu mm tiettyjen reseptoreiden, kuten TLR, tunnistamisesta.

Nykyään tunnetaan kymmenen erilaista ihmisen Tollin kaltaista reseptoria (TLR). Ne välittävät sytokiinien vapautumisen ja T-solujen aktivoitumisen. Jokainen TLR
tunnistaa erilaisen kirjon mikrobien pintarakenteita. Esimerkiksi TLR4 tunnistaa gramnegatiivisten bakteereiden lipopolysakkaridin (LPS),
TLR2 grampositiivisten bakteereiden lipoteikkohapon...

http://www.terveyskirjasto.fi/xmedia/duo/duo94134.pdf



PLoS One. 2011;6(10):e25998. doi: 10.1371/journal.pone.0025998. Epub 2011 Oct 5.

TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes


http://www.ncbi.nlm.nih.gov/pubmed/21998742

Oosting M, Ter Hofstede H, Sturm P, Adema GJ, Kullberg BJ, van der Meer JW, Netea MG, Joosten LA.

Source


Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Abstract

After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2.

Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production.

This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.


PMID:
21998742
[PubMed - indexed for MEDLINE]
PMCID:
PMC3187844

Free PMC Article
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » To Marras 21, 2013 13:54

KOTITESTI

Artikkelin mukaan testsi on tarkoitettu tuoreen tartunnan toteamiseen. Toisaalta nimenomaan myöhäis-/kroonistuneen borrelioosin diagostiikkaan kaivataan luotettavaa testiä.


Luotettavuus? Mielenkiintoinen lisä joka tapauksessa.


http://www.apteekkishop.fi/TikAlert-Bor ... eoen-1-kpl

Tik´Alert on uusi pikatesti tuoreen borrelioosin tunnistamiseen. Tuote on immunikromatografinen pikatesti, joka mittaa IgM vasta-aineita ihmisen sormenpäänäytteestä.

Testi tulee suorittaa 3 - 4 viikon kuluttua punkin puremasta. Tuote on CE hyväksytty.


http://www.diagnostica.fi/fileadmin/use ... 1_2011.pdf
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » To Marras 21, 2013 14:10


SPIROFIND - TESTI KROONISEEN BORRELIOOSIIN:


Boulder Diagnostics on kehittämässä uutta testiä krooniseen borrelioosiin nimeltään SpiroFind, joka tulee saataville Saksassa. Tämä uusi menetelmä tutkittiin Hollannissa viime vuonna ja on nyt kehittämisvaiheessa. Toivottavasti saatavilla pian.

Lisää tietoa: http://www.boulderdiagnostics.com/

Testi on nyt saatavilla. Saksan labra hoitaa koko EU-alueen. Testi sopii otettavaksi jo viikko punkin pureman jälkeen tai kroonisen borrelioosin toteamiseksi.

Hinta 217 Euroa plus toimituskulut.

http://boulderdiagnostics.com/eu/?page_id=32&lang=en

-----------------------------------------------------------------------------------
Ymmärtääkseni testi mittaa sytokiineja. Ilmeisesti monosyyttien (erilaistuu makrofageiksi eli syöjäsoluiksi) sytokiinivaste on kohonnut, kun aktiivinen Borre on paikalla. Testissä mitataan sytokiinia nimeltä interleukiini-1beeta eri borrekonsentraatioilla, jotta saadaan eroteltua pois muu immuunireaktio. (esim. Wikipediasta löytyy aika hyvin immuunivasteesta ja mitä noi kaikki siinä tekee :) )
ELISA ja Western blot käyttää vasta-aineita, PCR borren DNA:ta ja Saksan Elispot-LTT T-soluja.




[/size]

(Viestiketju on siirretty tänne yleistä foorumilta.)
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja thr0922 » Su Joulu 01, 2013 21:21

Understanding the EIA (ELISA)Test

http://www.cdc.gov/lyme/diagnosistestin ... oStep/EIA/

CDC:n sivuilta käy ilmi, että ELISA:n testin ykistyiskohdat ja sen mikä määrittelee positiivisen tuloksen päätettiin 1994! Eikä niitä ole muutettu sen jälkeen.

...For this reason, doctors want to verify any "positive" or “equivocal” (indeterminate) EIA results by performing an immunoblot test such as a Western blot. The Western blot or other FDA-approved type of immunoblot can help distinguish patients who have Lyme disease from those with other conditions.
...

New tests may be developed as alternatives to one or both steps of the two-step process. Before CDC will recommend new tests, their performance must be demonstrated to be equal to or better than the results of the two-test procedure. For more details, see Recommendations for Test Performance and Interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease.

Notice to Readers Recommendations for Test Performance and Interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease

The Association of State and Territorial Public Health Laboratory Directors, CDC, the Food and Drug Administration, the National Institutes of Health, the Council of State and Territorial Epidemiologists, and the National Committee for Clinical Laboratory Standards cosponsored the Second National Conference on Serologic Diagnosis of Lyme Disease held October 27-29, 1994. Conference recommendations were grouped into four categories: 1) serologic test performance and interpretation, 2) quality-assurance practices, 3) new test evaluation and clearance, and 4) communication of developments in Lyme disease (LD) testing. This report presents recommendations for serologic test performance and interpretation, which included substantial changes in the recommended tests and their interpretation for the serodiagnosis of LD.

A two-test approach for active disease and for previous infection using a sensitive enzyme immunoassay (EIA) or immunofluorescent assay (IFA) followed by a Western immunoblot was the algorithm of choice. All specimens positive or equivocal by a sensitive EIA or IFA should be tested by a standardized Western immunoblot. Specimens negative by a sensitive EIA or IFA need not be tested further. When Western immunoblot is used during the first 4 weeks of disease onset (early LD), both immuno- globulin M (IgM) and immunoglobulin G (IgG) procedures should be performed. A positive IgM test result alone is not recommended for use in determining active disease in persons with illness greater than 1 month's duration because the likelihood of a false-positive test result for a current infection is high for these persons. If a patient with suspected early LD has a negative serology, serologic evidence of infection is best obtained by testing of paired acute- and convalescent-phase serum samples. Serum samples from persons with disseminated or late-stage LD almost always have a strong IgG response to Borrelia burgdorferi antigens.

It was recommended that an IgM immunoblot be considered positive if two of the following three bands are present: 24 kDa (OspC) * , 39 kDa (BmpA), and 41 kDa (Fla) (1). It was further recommended that an that IgG immunoblot be considered positive if five of the following 10 bands are present: 18 kDa, 21 kDa (OspC) *, 28 kDa, 30 kDa, 39 kDa (BmpA), 41 kDa (Fla), 45 kDa, 58 kDa (not GroEL), 66 kDa, and 93 kDa (2).

The details of both plenary sessions and the work group deliberations are included in the publication of the proceedings, which is available from the Association of State and Territorial Public Health Laboratory Directors; telephone (202) 822-5227.

References

Engstrom SM, Shoop E, Johnson RC. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol 1995;33:419-22.

Dressler F, Whelan JA, Reinhart BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400.

The apparent molecular mass of OspC is dependent on the strain of B. burgdorferi being tested. The 24 kDa and 21 kDa proteins referred to are the same.
thr0922
 
Viestit: 65
Liittynyt: Ma Heinä 18, 2011 18:49
Paikkakunta: Helsinki

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Joulu 20, 2013 10:08

Przegl Epidemiol. 2009;63(4):539-43.
[Evaluation of the usefulness cerebrospinal fluid myelin basic protein (MBP)
concentration examination in patients with Lyme neuroborreliosis--preliminary
study]

[Article in Polish]

Kepa L.

Oddzial Chorob Zakaznych Slaskiego Uniwersytetu Medycznego w Bytomiu, przy
Klinice Chorob Pluc i Gruzlicy Slaskiego Uniwersytetu Medycznego w Zabrzu.

The aim of the study was evaluation of usefulness of cerebrospinal fluid (CSF)
myelin basic protein (MBP) level examination in diagnostics of Lyme
neuroborreliosis. The study was performed in 24 subjects. In all individuals CSF
MBP concentration was estimated on the 1st day of hospitalization. In patients
with depressive and cognitive impairments, proved in neuropsychological tests
(group I), mean CSF MBP concentration was 3.1 ng/mL, whereas in subjects without
abnormalities in tests (group II), respectively, 1.2 ng/mL.
The difference of mean CSF MBP levels was statistically significant (p<0.01).
The obtained results indicate usefulness of this CSF parameter, besides neuropsychological tests, in objective evaluation of clinical state in patients with chronic Lyme neuroborreliosis.

Publication Types:
English Abstract

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20120953 [PubMed - in process]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Joulu 20, 2013 10:28

Artikkelin mukaan uusi vasta-ainetesti on: Nopea, yksinkertainen ja erittäin herkkä.

Clin Vaccine Immunol. 2010 Apr 14; [Epub ahead of print]

Rapid, Simple, Quantitative, and Highly Sensitive Antibody Detection for Lyme
Disease.

Burbelo PD, Issa AT, Ching KH, Cohen JI, Iadarola MJ, Marques A.

Neurobiology and Pain Therapeutics Section, Laboratory of Sensory Biology,
National Institute of Dental and Craniofacial Research, Laboratory of Clinical
Infectious Diseases, National Institute of Allergy and Infectious Disease,
National Institutes of Health, Bethesda, MD 20892.

There is currently a need for improved serological tests for the diagnosis and
monitoring of Lyme disease, an infection caused by Borrelia burgdorferi (Bb).
Here, we evaluated Luciferase Immunoprecipitation Systems (LIPS) for profiling
antibody responses to a panel of Bb proteins for diagnosis of Lyme disease.
Initially, a training serum cohort of patients and controls (n=46) was profiled
using 15 different Bb antigen constructs. In the patient sera, antibody
responses to several Bb antigens including VlsE, Flagellin (FlaB), BmpA, DbpA,
and DbpB, showed high levels of immunoreactivity. However, the best diagnostic
performance was achieved with a synthetic protein, designated as VOVO,
consisting of a repeated antigenic VlsE-OspC-VlsE-OspC peptide sequence.
Analysis of an independent validation serum set (n=139) showed that the VOVO
LIPS test had 98% sensitivity (95% confidence interval [CI], 93%-100%; P<0.0001)
and 100% specificity (95% CI, 94%-100%; P<0.0001). Similarly,, the C6 peptide
ELISA also had 98% sensitivity (95% CI, 93%-100%; P<0.0001) and 98% specificity
(95% CI, 90%-100%; P<0.0001). Receiver operating characteristic analysis
revealed that the detection of patients by LIPS and the C6 ELISA were not
statistically different. However, the VOVO LIPS test displayed a wide dynamic
range of antibody detection spanning over 10,000-fold without the need of serum
dilution. These results suggest that LIPS screening using VOVO and other Bb
antigens offers an efficient quantitative approach for evaluation of antibody
responses in Lyme disease.

http://eutils.ncbi.nlm.nih.gov/entrez/e ... md=prlinks
PMID: 20392886 [PubMed - as supplied by publisher]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Joulu 20, 2013 10:57

USA: Uusi (2010) DNA testi


http://www.genomeweb.com/print/937302

Connecticut Pathologist Debuts Lyme Disease Test Based on Nested PCR, DNA Sequencing

March 30, 2010

By Kirell Lakhman

A scientist in Connecticut has launched a test that uses nested PCR and DNA sequencing to detect the presence of Borrelia burgdorferi, the bacterium that causes Lyme disease, according to a statement today.

"Most insurance companies have already agreed to cover the cost for their members," the statement said, without elaborating.

The test, called LoTemp, makes its debut ahead of the spring and summer tick season in the eastern US.

Other PCR-based tests for the indication exist, "[b]ut this is the first … one using nested PCR for detection and DNA sequencing to validate the molecular diagnosis in clinical laboratory medicine."

The assay, developed by Sin Hang Lee, a pathologist at Milford Hospital in Milford, Conn., it can be used before patients undergo traditional serology testing for the bug. More than 30,000 people in the US are suspected to be infected with the spirochete B. burgdorferi each year.

The test uses nested PCR to detect genomic DNA of B. burgdorferi in blood, and uses Sanger-based DNA sequencing and diagnostic signature sequences found in GenBank to validate the result, according to the statement.

**Up to 75 percent of patients with "acute-phase Lyme disease are negative for the characteristic antibodies, but in fact the percentage is higher," the statement said. However, a negative result does not rule out the presence of the bacteria because spirochetemia, or the presence of spirochetes in the blood, "is transient and its time points in Lyme [disease] vary from patient to patient."**

A paper co-authored about the test, which appears in the current of the American Journal of Clinical Pathology, said the test "may be a valuable supplement to the current serologic tests for Lyme disease."

"It is the marriage of [nested PCR and DNA sequencing] that minimizes false-negatives to the lowest possible and eliminates false-positives known to be associated with other Lyme disease DNA tests," it said.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Pe Joulu 20, 2013 11:16

Slovakia 2010.
32 potilasta. Kaikilla ELISA negatiiivinen mutta Western blot positiivinen. Kaikilta kroonisia oireita sairastavista tulee ottaa Wb vaikka ELISA on olut negatiivinen. Negatiivinen vasta-ainetulos on tutkijoiden mukaan todennäköisesti immuunipuutoksesta johtuvaa.

Suom. huom. Suomessa tehdään ensin vain ELISA. Mikäli se on negatiivinen jatkotutkimusta ei tehdä.
Useimmilla Suomessa negatiivisen testituloksen saaneista, Saksan testit ovat olleet positiiviset ja sen lisäksi immuunipuolustuksesta kertova CD 57 erittäin alhainen.


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

Abstract
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).
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ma Maalis 03, 2014 19:42

http://www.ebm-guidelines.com/xmedia/duo/duo70019.pdf


DIAGNOSTISIA ONGELMIA

Lymen borrelioosia on moninaisen oireistonsa
vuoksi nimitetty kupan jälkeen uudeksi »suu-
reksi matkijaksi». Tästä huolimatta Lymen
taudin diagnoosi on kliininen, ja anamneesi ja
laboratoriotestien tulokset useimmiten tukevat
sitä. Aiemmin tässä lehdessä on käsitelty paitsi
Suomen ensimmäisiä tapauksia (Kovanen ym.
1985, Marttila ja Panelius 1985), myös tautia
yleensä (Viljanen ja Lehtinen 1992, Wahlberg
1995) tai sen yksittäisiä ilmenemismuotoja (Kar-
ma ym. 1993, Peltomaa 1995). Infektion alku-
oire on usein erythema migrans (EM), päivien
tai viikkojen aikana punkinpuremakohdan ympä-
rille vähitellen leviävä rengasmainen tai homo-
geeninen punoitus (halkaisija usein yli 5 cm).
Veriteitse levitessään taudin korkkiruuvinmuotoi-
nen aiheuttajabakteeri Borrelia burgdorferi voi
myöhemmin aiheuttaa hermoston, nivelten, sy-
dämen, ihon tai silmän taholta tulevia oireita.
EM:n diagnoosi ja hoitopäätös tulee tehdä klii-
nisin perustein, koska tässä vaiheessa vasta-aine-
pitoisuus ei useinkaan vielä ole suurentunut mi-
tattavaksi.
Vaikka Lymen tauti on bakteeri-infektio, ovat
lasko ja C-reaktiivisen proteiinin pitoisuudet
useimmiten normaalit (Steere 1989). Poikkeuk-
sellisesti ne voivat kuitenkin kasvaa hyvinkin suu-
riksi. Kiertäviä immuunikomplekseja, lievästi
suurentuneita tumavasta-aine- ja aminotransfe-
raasiarvoja todetaan borrelioosipotilailla melko
usein. Tällaisista epäspesifisistä laboratoriotesteis-
tä ei kuitenkaan yleensä ole apua Lymen taudin
diagnostiikassa.
Vasta-ainemääritykset
Vasta-aineiden mittaus on ensisijainen labora-
toriokoe Lymen borrelioosia epäiltäessä. Vasta-
ainearvoja mitataan yleisimmin ELISA-menetel-
mällä. Tulosten ilmoitustavat ovat Suomessakin
eri laboratorioissa varsin erilaisia – osa ilmoittaa
tuloksen lukuna, osa puolikvantitatiivisesti mii-
nuksena tai plussina. Antigeenina ELISAssa voi-
daan käyttää joko koko borreliabakteeria, borre-
lian värekarvaa eli flagellaa, jotakin muuta bak-
teerin osaa, kuten ulkokalvoproteiineja, tai geeni-
Lymen taudin
laboratoriodiagnostiikka
Jarmo Oksi
Borrelioosi on kliininen diagnoosi, johon pääsemisessä anamneesi voi antaa huomattavaa
apua. Lukuun ottamatta erythema migransia, joka on Lymen taudille patognomoninen
varhaisvalhe, täytyy laboratoriokokeista saada kuitenkin vahvistus diagnoosille. Varmaan
diagnoosiin pääsy on usein vaikeaa ja edellyttää esimerkiksi neurologisen oireiston yhtey-
dessä useimmiten selkäydinnesteen tutkimista. Muiden syiden pois sulkeminen vaatii
kohtalaisen kattavia laboratorio- ja kuvantamistutkimuksia. Sen vuoksi Lymen tautia
epäiltäessä potilaat pitäisi varhaisvaihetta lukuun ottamatta kirjoittajan mielestä ohjata
sairaalan poliklinikan tutkimuksiin, jotta turhilta ja usein pitkiltä antibioottikuureilta
voitaisiin välttyä. Samasta syystä oireettomalta henkilöltä ei pitäisi tutkia borreliavasta-
aineita.
Duodecim
113:
69–73, 1997
70
J. Oksi
teknisesti tuotettuja antigeenejä. Borrelian flagel-
laa kohtaan alkaa infektion aikana muodostua
vasta-aineita ensimmäiseksi, minkä vuoksi väre-
karvaa antigeeninä käyttävät testit saattavat in-
fektion alussa olla herkempiä (Dressler ym.
1993). Infektion aikana vasta-aineita sitten muo-
dostuu myös monia muita bakteerin rakenteita
kohtaan ja esimerkiksi koko bakteeria antigeeni-
nä käyttävät testit ovat herkkiä. Toistaiseksi huo-
nosti tunnetun ongelman muodostavat Borrelia
burgdorferin eri alalajien (B. burgdorferi sensu
stricto, B. garinii, B. afzelii) antigeeniset eroavai-
suudet ja niiden vaikutukset vasta-ainetestin
herkkyyteen (Magnarelli ym. 1994, Dressler ym.
1994).
Vasta-aineiden esiintyminen, laatu ja määrä
riippuvat infektion kestosta. Tavallisimmin IgM-
luokan vasta-aineet lisääntyvät noin 3–4 viikon
kuluessa puremasta, jos borreliatartunta on ta-
pahtunut. Maksimissaan IgM-luokan vasta-aine-
pitoisuus on usein noin kahden kuukauden ku-
luttua, minkä jälkeen arvo alkaa yleensä pienetä.
IgG-luokan vasta-ainearvot alkavat kasvaa kuu-
kauden kuluttua, ja voivat pysyä suurina vuosia
riippumatta siitä, onko potilas saanut hoidon vai
ei (Dressler ym. 1993, Magnarelli 1995). Toisi-
naan kuitenkin IgG-luokan vasta-aineiden lisään-
tyminen saattaa jäädä kokonaan tapahtumatta.
Tällöin voidaan todeta taudin myöhäisvaiheessa-
kin vain IgM-luokan vasta-aineita. IgM-luokan
vasta-aineisiin on käytännön työssä kuitenkin
suhtauduttava varauksellisemmin kuin IgG-luo-
kan vasta-aineisiin, koska ensin mainitussa luo-
kassa väärän positiivisen tuloksen mahdollisuus
on suurempi. Infektion toisessa vaiheessa, borre-
lioiden levittyä elimistöön verenkierron välityk-
sellä, 70–90 % potilaista on seropositiivisia, ja kol-
mannessa vaiheessa seropositiivisten osuus on yli
90 % (Wilske ja Preac-Mursic 1993). Myöhäis-
vaiheenkin borrelioosi voi siis poikkeuksellisesti
olla täysin seronegatiivinen (Golightly 1993,
Liegner 1993, Oksi ym. 1995). On esitetty, että
varhaisvaiheessa annettu liian lyhyt antibiootti-
kuuri saattaisi toisinaan johtaa siihen, että vasta-
aineet eivät myöhemminkään lisäänny mitattavik-
si. Disseminoiduttuaan bakteerit voivat päästä
sellaisiin kudoksiin tai alueille, joissa ne ovat im-
muunipuolustukselta ja antibiooteilta kohtalaises-
sa suojassa. Mahdollista on sekin, että osa ihmi-
sistä ei jostain syystä kykene muodostamaan
vasta-aineita borreliaa kohtaan, vaikka bakteeri
olisi immuunipuolustukseen osallistuvien solujen
helposti tavoittamissa paikoissa.
Koska EM:n diagnoosi on kliininen, ei vasta-
ainemittausta tämän yhteydessä tai seurannassa
välttämättä tarvita. Vaikka varhaisvaiheen infek-
tioon annettu antibioottikuuri voikin vähentää
vasta-aineiden muodostumista, on hyödyllistä ot-
taa verinäyte vasta-ainetestiä varten joko ennen
tai jälkeen antibioottikuurin (noin yhden kuu-
kauden kuluttua puremasta), koska mahdollisten
myöhempien oireiden selvittelyssä on tärkeää
nähdä, onko vasta-aineita alkanut muodostua tai
onko niiden määrä muutoin kasvanut huomatta-
vasti seurannan aikana. Koska vasta-ainearvon
muutokset ovat hitaita, kannattaa testi uusia ai-
kaisintaan yhden ja mieluummin vasta kolmen
kuukauden kuluttua ensimmäisestä testistä. Myö-
häisvaiheen borrelioosiepäilyissä tai borrelioosin
hoidon jälkeen vasta-ainepitoisuuksien muutok-
sia seurataan vieläkin harvemmin.
Tavallisimmat syyt väärään positiiviseen tulok-
seen vasta-aineita mitattaessa ovat LED, reuma,
syfilis (aiheuttaja Treponema pallidum), suun spi-
rokeettojen (mm. Treponema denticola) aiheut-
tamat periodontaalitaudit ja Epstein–Barrin vi-
ruksen aiheuttama infektio (Steere 1989). Toi-
saalta on kuitenkin huomattava, että kuten muis-
sa kroonisluonteisissa infektioissa myös borreli-
oosipotilaille saattaa kehittyä tumavasta-aine- tai
reumatekijäpositiivisuus. Ristireaktiota epäiltäes-
sä saattaa olla hyödyllistä tutkia vasta-aineita
»Western blot» -menetelmällä, jolloin nähdään,
mitä antigeenisia rakenteita kohtaan vasta-aineita
on muodostunut. Parhaimmillaan menetelmä
paljastaa muulla menetelmällä – esimerkiksi risti-
reaktion pohjalta – saadun, väärän positiivisen
testituloksen. Yhdysvalloissa, jossa esiintyy vain
yhtä B. burgdorferin alalajia (B. b. sensu stricto),
on päädytty suosittamaan kaikkien borrelioosi-
epäilyjen seropositiivisuuden varmistamista »Wes-
tern blot» -menetelmällä (Aguero-Rosenfeld ym.
1996). Mainittu menetelmä on kuitenkin suh-
teellisen työläs rutiinikäyttöön otettavaksi. Siitä
huolimatta sen käyttömahdollisuuksia rutiinimai-
sessa borrelioosidiagnostiikassa on tulevaisuudes-
71
sa syytä harkita myös meillä. »Western blot» -me-
netelmän käyttökelpoisuutta Euroopassa rajoit-
taa lisäksi useamman alalajin esiintyminen, joten
täällä tarvitaan kattavia tutkimuksia serodiagnos-
tisten kriteerien luomiseksi.
Neuroborrelioosia epäiltäessä on aina syytä tut-
kia borreliavasta-aineiden intratekaalinen tuotto
selkäydinnesteestä. Käyttökelpoisin menetelmä
intratekaalisen vasta-ainetuotannon mittaamiseksi
on »antibody-capture»-ELISA (Hansen ja Lebech
1991). Mikäli intratekaalinen vasta-aineiden tuot-
to todetaan, neuroborrelioosin diagnoosi on var-
ma, mutta sen puuttuminen ei sulje pois neuro-
borrelioosin mahdollisuutta (Halperin ym. 1996).
Vaikka potilaalla todettu vasta-ainetestin posi-
tiivinen tulos olisi »todella positiivinen», on mel-
ko usein vaikea tietää varmasti potilaan oireiden
johtuvan käynnissä olevasta borreliainfektiosta,
sillä seropositiivisia henkilöitä on endeemisellä
alueella runsaasti. Koska diagnoosi usein on han-
kala, tulee mahdollisuuksien mukaan pyrkiä
osoittamaan borrelia epäsuorien menetelmien
(kuten vasta-ainepitoisuuden mittaus) lisäksi
myös suorilla laboratoriomenetelmillä (esim. po-
lymeraasiketjureaktio eli PCR ja viljely).
Yleensä vasta-aineet alkavat selvästi vähentyä
hoidon jälkeen (Wahlberg ym. 1994). Tällaisessa
tapauksessa voidaan muutos myös tulkita mer-
kiksi hoidon onnistumisesta. Erythema migran-
sin hoidon jälkeen vähenemä saatetaan todeta jo
parissa kuukaudessa, mutta myöhäisvaiheen in-
fektiota hoidettaessa muutos on yleensä havaitta-
vissa vasta 6–12 kuukauden kuluttua. Vasta-aine-
määrät voivat pysyä suurina tuloksellisesta hoi-
dosta huolimatta osalla potilaista jopa vuosien
ajan. Myös intratekaalinen vasta-ainetuotanto
saattaa jatkua pitkään riippumatta hoidon tulok-
sellisuudesta. Kaiken kaikkiaan vasta-ainearvojen
seuranta ei ainakaan kliinisesti tuloksellisen hoi-
don jälkeen ole tarpeellista.
Lymen taudin laboratoriodiagnostiikka
T a u l u k k o 1. Lymen taudin diagnostiikka.
Tutkimus Erythema migrans
1
Disseminoituneeseen borrelioosiin sopivat oireet
Testin Jatkotoimet Jatkotoimet
ottoindikaatio kun testitulos on kun testitulos on
positiivinen negatiivinen
Seerumin Voidaan ottaa ennen Aina Hoito, jos oireet klassiset Uusi näyte ja vasta-
vasta-aineet tai jälkeen hoidon tai voimakkaat. Seuranta aine-mittaus. PCR,
ja/tai jatkotutkimuksia, jos oireet klassiset
jos ei objektiivisia löydöksiä tai voimakkaat
Selkäydinnesteen Neurologisten Hoito, jos intratekaalinen Ei sulje pois neuro-
vasta-aineet oireiden yhteydessä vasta-ainetuotanto borrelioosia
PCR Tutkimuskäytössä Aina kun otetaan Hoito riippumatta vasta- Ei sulje pois Lymen
selkäydin- tai nivel- ainetasosta borrelioosia
nestenäyte. Selvien
yleisoireiden yhtey-
dessä myös plasmasta
tarvittaessa
Viljely Tutkimuskäytössä Tutkimuskäytössä Hoito Ei sulje pois Lymen
borrelioosia
Lymfosyytti- Seronegatiivisissa Hoito valikoiduissa Ei sulje pois Lymen
stimulaatio voimakasoireisissa tapauksissa borrelioosia
tapauksissa
1
Hoito kliinisen kuvan perusteella (vasta-ainearvoista riippumatta)
72
Viljely
Borreliaviljely onnistuu EM:stä kohtalaisen
usein mutta muista kliinisistä näytteistä vain hy-
vin harvoin. Viljely vaatii erikoiselatusaineen ja
pitkän (joskus yli kuukauden kestävän) inkubaa-
tion. Varsinkin selkäydinnestenäytteistä on hyvä
tehdä muiden borreliatutkimusten lisäksi myös
viljely, jos näyte saadaan kuljetetuksi tutkivaan
laboratorioon saman päivän aikana. Borrelian
osoitus viljelemällä onnistuu kuitenkin niin har-
voin, että viljelyä ei yleensä ole suositeltu muu-
hun kuin tutkimuskäyttöön.
Borrelian DNA:n osoitus PCR-
menetelmällä
Jos potilaalla epäillään vahvasti Lymen borre-
lioosia, vaikka vasta-ainetestien tulokset ovat tois-
tamiseen negatiiviset tai raja-arvoiset, PCR-me-
netelmällä voidaan etsiä elimistön nesteistä bor-
relian DNA:ta. Selkäydinneste- tai nivelneste-
näytteestä – jopa seerumista, EDTA-plasmasta tai
koepalasta – voidaan tehdä PCR-tutkimus tätä
ajatellen.
PCR-testi sopii myös seropositiivisen potilaan
käynnissä olevan infektion varmistukseen (Noc-
ton ym. 1994). On kuitenkin syytä muistaa, että
PCR-testin negatiivinen tulos ei milloinkaan sul-
je pois borreliainfektiota. Borrelioosissa aiheutta-
jamikrobia esiintyy elimistön nesteissä ja kudok-
sissa poikkeuksellisen pieniä määriä, joten PCR-
testikään ei herkkyydestään huolimatta riitä bak-
teerin DNA:n osoittamiseen silloin, kun vähäi-
seen näytteen osaan ei ole sattunut osumaan ai-
noatakaan bakteeria. Ihosta borrelia voi päästä
verenkiertoon aiheuttaen spiroketemian, jolloin
PCR-testillä saatetaan periaatteessa löytää plas-
masta borrelian DNA:ta. Erään tutkimuksen mu-
kaan plasmasta tehdyn PCR-testin tulos oli posi-
tiivinen 30 %:lla niistä EM-potilaista, joilla esiin-
tyi samanaikaisesti yleisoireita, ja 9 %:lla niistä,
joilla ei yleisoireita ollut (Goodman ym. 1995).
Käytännössä varsinaiseen EM:n diagnostiikkaan
ei kuitenkaan kuulu plasman tai edes ihopalan
borrelia-DNA:n osoitus. Tutkimustyössä nämä
kuitenkin ovat arvokkaita näytteitä.
PCR-testissä voidaan monistaa erilaisia etukä-
teen valittuja geenien DNA-alueita. B. burgdor-
ferin eri alalajit eroavat toisistaan luonnollisesti
myös geeniensä suhteen, joten laboratorion täy-
tyy määrittää mitä ja kuinka pitkää DNA-aluetta
testissä on järkevää käyttää. Yleensä on syytä vali-
ta sellainen DNA-alue, joka on sama kaikissa ky-
seisellä maantieteellisellä alueella esiintyvissä ala-
lajeissa (Pachner ja Delaney 1993). PCR:n käy-
tön etuna voidaan pitää sen suomaa mahdolli-
suutta todeta myös kuolleita organismeja. Eri
tutkimuksissa on saatu hyvin vaihtelevia tuloksia
PCR:n herkkyydestä kliinisten näytteiden tutki-
misessa. PCR:n käytön haitaksi borrelioosidiag-
nostiikassa on luettava se, että menetelmän herk-
kyyttä ei riittävästi tunneta. Toinen PCR:n käyt-
töä periaatteessa haittaava asia on äärimmäisen
tarkan laboratoriohygienian tarve. Mikäli tässä il-
menee puutteita, saadaan myös vääriä positiivisia
testituloksia. Tunnetuista borrelioosidiagnostii-
kan suorista menetelmistä PCR on lupaavin ja
vakiinnuttanee tulevaisuudessa asemansa. PCR-
testillä voidaan myös periaatteessa todeta ne ta-
paukset, joissa potilaalla on vielä hoidon jälkeen
bakteereita elimistössään ja joissa uusintahoidos-
ta on hyötyä.

Lymfosyyttien stimulaatiotesti
Jos on aihetta epäillä borrelioosia huolimatta
vasta-ainetestien negatiivisuudesta, on joskus
hyödyllistä tutkia perifeerisen veren lymfosyyttien
stimulaatiovaste borreliaan (Dressler ym. 1991).

Testin ongelmana on se, ettei vielä tiedetä riittä-
vän hyvin, kuinka yleistä normaaliväestössä on
vahva stimulaatiovaste borreliaa kohtaan.
Lymen tauti näyttää lisäävän Th1-solujen ja
vähentävän Th2-solujen aktiivisuutta (Oksi ym.
1996). Tämän seurauksena soluvälitteinen im-
muniteetti voimistuu ja vasta-ainetuotanto heik-
kenee. Tämä muutos on borrelian kannalta edul-
linen, koska elimistö hyökkää sitä vastaan pää-
asiassa vasta-aineita käyttäen. Periaatteessa lym-
fosyyttien selvä stimulaatiovaste borreliaan voisi
siis varmistaa erityisesti seronegatiiviseksi jääneen
tai sellaiseksi tulleen potilaan diagnoosin. Ai-
nakaan toistaiseksi tätä testiä ei kuitenkaan yleen-
sä ole mahdollista käyttää diagnoosin varmistuk-
sessa.
J. Oksi
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ma Maalis 03, 2014 20:10

http://personal.fimnet.fi/laakari/hannu ... us2014.htm


BORRELIA REVISITED

Jarmo Oksi Tyks:sta kertoi, että borreliainfektiot lisääntyvät jatkuvasti. Suomessa niitä todetaan laboratorioiden raporttien perusteella 1600 / vuosi. Oikeasti niitä esiintyy nelinkertainen määrä. Erythema migrans (EM) tulee hoidettua niin tehokkaasti, että laboratoriolöydökset vääristyvät.



Lymen Borrelioosin manifestaatiot

EM voi olla vaikea huomata. Se voi olla monimuotoinen. Aina se ei ole rengas. Joskus infektio voi olla jo EM:n esiintyessä levinnyt ja saattaa esiintyä meningiittikin. Jos potilaalla esiintyy useita EM yhtaikaa, meningiitti on 7,5 %:lla.

Borrelialymfosytooma esiintyy usein korvannipukassa:

Kuva

Acrodermatistis chronica atroficans



Disseminoitunut borrelioosi on hoitamattomana krooninen tauti ja sen kliininen kuva on vaikea tunnistaa. Selvittely vaatii mittavia tutkimuksia. Oireina voi olla meningiitti, radikuliitti, facialispareesi, muu keskushermosto-oire, artriitti, kardiitti jne.



Neuroborrelioosi

Akuutti neuroborrelioosi ilmaantuu 1-3 kuukautta infektion jälkeen. Meningiittiin liittyvä niskajäykkyys on usein vähäistä tai olematonta. Radikuliittioireen voi esiintyä raajaan tai vartaloon säteilevä toispuoleinen kipu. Kipu voi vaihtaa joskus puoltaan. Kasvohermon halvaus tai muun aivohermon pareesi voi olla toispuoleinen tai kummankin puolen halvaus. Siihen liittyy usein oireeton meningiitti.



Vuoden kuluessa voi ilmaantua vähittäisen neuroborrelioosin enkefalomyeliitti. Siihen liittyy löydöksenä MRI:ssa näkyvät valkean aineen pesäkkeet, myelopatia ja aivovaskuliitti. Joskus voi esiintyä Lymen enkefalopatiaa muistihäiriöineen ja persoonallisuusmuutoksineen. Ääreishermoston häiriöitä voi esiintyä.



Lymen neuroborrelioosin liquorlöydöksille on tyypillistä :

· Leuc 10 – 500

· Liquorin valkosolujen diffi: >90 % lymfosyytteja

· Proteiinit usein merkittävästi koholla vaikka leukosyyttien kohoaminen olisi vähäistä

· Usein oligoklonaalisia fraktioita

· Intratekaalinen borreliavasta-ainetuotanto riippuu taudin kestosta

· PCR positiivinen vain 10 %:lla

Neuroborrelioosissa CRP ja La voivat olla normaalit. Liquorlöydöksen normaalistuminen voi hoidon jälkeen kestää puolitoista vuotta.



Ahvenanmaalla 20-25 %:lla väestöstä löytyy verikokeissa borreliavasta-aineita. Osa ihmisistä on törmännyt elämässään borreliaan mutta parantunut. Syynä paranemiseen voi olla muuhun sairauteen annettu antibioottikuuri tai oman elimistön puolustusjärjestelmä. Oireettomia potilaita ei muutenkaan pitäisi tutkia. Osa veritesteistä antaa vastaukseksi väärän positiivisen.



Lymen borrelioosin diagnoosi on harvoin helppo tehdä. Diagnoosi ei edellytä tietoa punkinpuremasta tai erythema migransista. Diagnoosi perustuu kliiniseen taudinkuvaan jota tukee laboratorionäyttö. Negatiivinen PCR ei poissulje tautia koska tutkimus on epäsensitiivinen. Neuroborrelioosin diagnoosiin tarvitaan liquornäyte.

Antibioottihoito kannattaa kaikissa borrelioosin vaiheissa. Erythema migransin hoitona on kahden viikon hoito doksisykliinillä tai amoksisilliinilla. Disseminoituneessa taudissa hoidon tulos on sitä parempi mitä varhaisemmassa vaiheessa hoito aloitetaan. Suomessa perinteisesti disseminoituneen taudin hoito on ollut kolmen viikon mittainen intravenoosi keftriaksonihoito. Hoidon lopullisen onnistumisen arviointia voi tehdä vasta usean kuukauden, joskus jopa vuoden, kuluttua.



Mervi Kanerva Hyks:sta toi esitykseen KNK-lääkärin näkökulmaa. Hän kertoi, että borrelia on taustalla 30 %:ssa lasten kasvohermon halvauksista. Lasten kasvohermohalvauksen selvittelyyn kuuluu aina liquornäyte ja seeruminäyte. Aikuisten kasvohermon halvauksista vain 3 % liittyy borreliaan. Seeruminäyte aikuisilla riittää tavallisesti mutta epäilytilanteessa liquornäyte on tarpeen. Bellin pareesin hoidoksi aloitettu kortisonilääkitys ei ilmeisesti aiheuta ongelmia vaikka myöhemmin kasvohermon halvauksen syyksi osoittautuisikin borrelia.

Muut KNK-ilmentymät ovat harvinaisia. Periaatteessa äänihuulihalvaus ja sisäkorvaperäinen kuulonlasku voisivat tällaisia olla. Selvää vastausta siitä, pitäisikö serologiaa näissä selvittää, ei ole. Diagnostiikassa joskus ongelmaa voi aiheuttaa varicella zoster, joka ristireagoi borreliavasta-aineiden kanssa. Diagnostiikassa kannattaa ottaa kantaa jo ensimmäiseen vastaukseen vaikka laboratorio suositteleekin toista näytettä.
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ti Huhti 08, 2014 11:32

Gangliosidi vasta-ainetesti seerumista; 4116 S -GangAb

"..neuroborrelioosissa ja syfiliksessä tavataan IgM-luokan Gm1-vasta-aineita.."


GANGLIOSIDI, VASTA-AINEET

Gangliosidi Gm1, vasta-aineet

4116 S -GangAb
Tiedustelut

Asiakaspalvelu ja neuvonta, puh. 311 77800
Indikaatiot

Guillain-Barrén syndrooman ja demyelinoivien polyneuropatioiden diagnostiikka


Gangliosidi Gm1-vasta-aineita esiintyy varsinkin multifokaalisessa motorisessa neuropatiassa (MMN), Guillan-Barrén syndroomassa (GBS) ja proksimaalisen alemman motoneuronin taudeissa.

MMN:ssa yli 80 %:lla tavataan sekä IgG- että IgM-luokan vasta-aineita, kun taas erotusdiagnostisesti merkittävässä amyotrofisessa lateraaliskleroosissa vasta-aineita ei tavata tai titterit ovat matalia, IgM-luokan vasta-aineita tavataan myös muissa motorisissa ja sensorismotorisissa neuropatioissa.

IgG-luokan vasta-aineita löytyy n. 30 %:lla GBS-potilaita, varsinkin Campylobacter jejunii -infektioon liittyvässä akuutissa polyneuropatiassa. Vasta-ainetitterit korreloivat taudin aktiviteettiin.

Myös neuroborrelioosissa ja syfiliksessä tavataan IgM-luokan Gm1-vasta-aineita. Vasta-aineita esiintyy jonkin verran normaaliväestössäkin, varsinkin iäkkäämmillä henkilöillä, ilman selvää tautiassosiaatiota.
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ViestiKirjoittaja soijuv » Ti Touko 13, 2014 08:19

KOKO TUTKIMUS: https://dub124.mail.live.com/mail/ViewO ... n=72046399

Joulukuu 2013 | 40 sivua

Anna Karvonen ja Satu Leinonen

LIKVORIN CXCL13-MÄÄRITYKSEN VALIDOINTI – UUSI TYÖKALU NEUROBORRELIAINFEKTION DIAGNOSTIIKKAAN

Tämän opinnäytetyön tavoite on parantaa neuroborreliainfektion laboratoriodiagnostiikkaa. Tarkoituksena oli validoida likvorin CXCL13-määritys ja laatia validointiraportti Turun yliopiston lääketieteellisen mikrobiologian ja immunologian oppiaineen diagnostiselle palvelutoiminnalle, jotta CXCL13-määritystä voitaisiin käyttää osana neuroborreliainfektion diagnostiikkaa. Tutkimustehtävänä oli selvittää soveltuuko likvorin CXCL13-määritys neuroborreliainfektion diagnostiikkaan sekä antibioottihoidon vaikutus likvorin CXCL13-pitoisuuteen.

Neuroborreliainfektio aiheutuu Borrelia burgdorferi –bakteerista. Neuroborreliainfektiossa borreliabakteeri infektoi perifeeristä hermostoa tai keskushermostoa. Yleisimpiä neuroborreliainfektion oireita ovat aivokalvontulehdus, hermojuurentulehdus ja aivohermojen halvaus, erityisesti kasvohermohalvaus.

Tässä opinnäytetyössä käytettiin kaupallista (R&D Systems) ELISA-menetelmää. Kyseessä on immunologinen menetelmä, jossa hyödynnetään vasta-aineiden kykyä sitoa ja sitoutua spesifisti antigeeneihin. Opinnäytetyön tutkimusaineisto koostui likvornäytteistä. Näytteistä määritettiin CXCL13-pitoisuudet. Näytteiden avulla tehtiin CXCL13-määrityksen validointi, joka sisälsi spesifisyyden, toistettavuuden ja lineaarisuuden testaukset.

Tuoretta neuroborreliainfektiota sairastavien potilaiden likvoreiden CXCL13-pitoisuudet olivat 424 – 158030 pg/ml. Neurosyfilispotilaan likvorin CXCL13-pitoisuus oli 36998 pg/ml. MS-tautiin viittaavissa näytteissä ja keskushermoston virusinfektiota (VZV, HSV, HHV-6, entero ja TBE) sairastavien näytteissä CXCL13-pitoisuudet olivat välillä <7,8-406 pg/ml. Neuroborreliainfektioon viittaavana alarajana voidaan pitää likvorin CXCL13-pitoisuutta 500 pg/ml.

Tämän opinnäytetyön tulokset osoittavat, että likvorin CXCL13-määritys soveltuu neuroborreliainfektion diagnostiikkaan. CXCL13-pitoisuudet likvorissa laskevat antibioottihoidon myötä. Lisäksi voidaan todeta R&D Systemsin CXCL13-määrityksen olevan validi spesifisyyden, toistettavuuden ja lineaarisuuden suhteen.

ASIASANAT:

Neuroborreliainfektio, likvor, CXCL13 ja validointi
--------------------------

BACHELOR´S THESIS | ABSTRACT

TURKU UNIVERSITY OF APPLIED SCIENCES

Degree programme in nursing | Biomedical Laboratory Science

December 2013 | 40 pages

Anna Karvonen and Satu Leinonen

VALIDATION OF DETECTING CXCL13 FROM

CEREBROSPINAL FLUID – A NEW TOOL FOR DIAGNOSING NEUROBORRELIOSIS

The aim of this bachelor’s thesis is to improve laboratory diagnosis of neuroborreliosis. The purpose was to make a validation of detecting CXCL13 from cerebrospinal fluid and make a validation report to the department of medical microbiology and immunology of Turku University so that the detection of CXCL13 could become part of diagnosis of neuroborreliosis. The task was to find out if CXCL13 in cerebrospinal fluid is a diagnostic marker for neuroborreliosis and how CXCL13 levels in cerebrospinal fluid react to antibiotic treatment.

Neuroborreliosis is caused by Borrelia burgdorferi. In neuroborreliosis it infects peripheral or central nervous system. The most typical symptoms are meningitis, radiculoneuritis and cranial neuritis, particulary involving the facial nerve.

The assay used in this bachelor’s thesis was ELISA (enzyme-linked immunosorbent assay) by R&D System. ELISA assay is an immunological method in which an antibody’s ability to bind to specific antigen is exploited. The material of this bachelor’s thesis was cerebrospinal fluid samples. CXCL13 was detected from samples. Validation included testing of specificity, repeatability and linearity.

Concentrations of CXCL13 in acute neuroborreliosis were 424 – 158030 pg/ml. In neurosyfilis concentration of CXCL13 was 36998 pg/ml. In other inflammatory diseases of nervous system (MS-disease, VZV, HSV, HHV-6, entero and TBE) concentrations of CXCL13 were <7,8-406 pg/ml. A CXCL13 concentration of 500 pg/ml is a marker of neuroborreliosis.

The results of this bachelor’s thesis show that detection of CXCL13 can be part of diagnosing neuroborreliosis. Concentration of CXCL13 in cerebrospinal fluid decreases due to antibiotic treatment. It can also be shown that ELISA assay for detecting CXCL13 by R&D System is valid with regards to spesifility, repeatability and linearity.

KEYWORDS:

Neuroborreliosis, cerebrospinal fluid, CXCL13, validation
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ti Syys 02, 2014 14:11

Borrelioosin diagnostiikka ja hoito (Saksa):

http://www.borreliose-gesellschaft.de/T ... elines.pdf

Deutsche Borreliose-Gesellschaft e.V.
Diagnosis and Treatment of Lyme borreliosis (Lyme disease)
Guidelines of the German Borreliosis Society
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ma Syys 29, 2014 09:47

Jotkut laboratoriot, esim. artikkelin Labcorp, eivät tee tarkempaa immunoblottausta (western blot) mikäli edeltävä ELISA/EIA/IFA on ollut negatiivinen. Allaolevan, tri Cameronin, artikkelin mukaan käytäntö on väärä sillä immunoblottaus on tarkempi ja herkempi testi havaitsemaan borrelia-bakteerin. Myös Suomessa jätetään immunoblottaus usein tekemättä jos Elisa on ollut negatiivinen. Siksi monet teettävät testin yksityislaboratorioissa, esim. Mehiläisessä, tai esim. Saksan Infectolabissa.

http://danielcameronmd.com/labcorp-deny ... e-disease/

LabCorp to deny physicians access to western blot tests for Lyme disease

Labcorp will not offer a western blot test for individuals unless they are positive or equivocal for the Enzyme Immunoassay (EIA) or Immunoflorescense (IFA) screening tests for Lyme disease as of August 11, 2014.[1]

img-ldpr2Physicians have been disappointed by the poor sensitivity of the EIA or IFA screening tests for Lyme disease. The sensitivity of the whole-cell enzyme-linked immunosorbent assay (ELISA) to the B31 strain typically falls between 33-49% for patients presenting with an EM.[2-4] The sensitivity of the Food and Drug Administration (FDA) approved complement peptide C6 (C6-peptide) was 37% in 89 clinically well-defined individuals with LD [5] and 66.5% 403 sera from patients with an EM rash.[6]

Physicians have commonly ordered the western blot test for Lyme disease even in the absence of positive or equivocal testing. An IgM Western blot test can persist for at least 2 years in individuals with established Lyme disease infection. A IgM WB can persist for months to years in LD even if an individual is treated with antibiotics.[7-9] An IgG can be positive in individuals with a negative screening test.

LabCorp’s decision to deny physician assess to western blot test for Lyme disease only makes testing sensitive than it already is.

It important that LabCorp reverse their position and allow physicians to continue to order western blot tests for Lyme disease even if the EIA and/or IFA are negative. Until then, clinicians may have to direct their patients to other labs.

LabCorp newsletter for clients. Lyme disease testing now employs a two-tier antibody standard, Available from https://http://www.labcorp.com/wps/wcm/ ... 703d82366a Last accessed 8/16/14.
Aguero-Rosenfeld ME, Nowakowski J, Bittker S, Cooper D, Nadelman RB, Wormser GP. Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. J Clin Microbiol, 34(1), 1-9 (1996).
Trevejo RT, Krause PJ, Sikand VK et al. Evaluation of two-test serodiagnostic method for early Lyme disease in clinical practice. J Infect Dis, 179(4), 931-938 (1999).
Aguero-Rosenfeld ME, Nowakowski J, McKenna DF, Carbonaro CA, Wormser GP. Serodiagnosis in early Lyme disease. J Clin Microbiol, 31(12), 3090-3095 (1993).
Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T. Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots. Eur J Clin Microbiol Infect Dis, (2011).
Wormser GP, Schriefer M, Aguero-Rosenfeld ME et al. Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease. Diagn Microbiol Infect Dis, (2012).
Steere AC, Hardin JA, Ruddy S, Mummaw JG, Malawista SE. Lyme arthritis: correlation of serum and cryoglobulin IgM with activity, and serum IgG with remission. Arthritis Rheum, 22(5), 471-483 (1979).
Massarotti EM, Luger SW, Rahn DW et al. Treatment of early Lyme disease. Am J Med, 92(4), 396-403 (1992).
Craft JE, Grodzicki RL, Shrestha M, Fischer DK, Garcia-Blanco M, Steere AC. The antibody response in Lyme disease. Yale J Biol Med, 57(4), 561-565 (1984).
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » Ke Helmi 04, 2015 15:29

Kemokiinin CXCL13 merkityksestä lymen neuroborrelioosissa (LNB)

Daniel Bremellin väitöskirjan Lyme Neuroborreliosis , Diagnosis and Treatment yhtenä osatyönä oli seuraava :
Cerebrospinal fluid CXCL13 in Lyme neuroborreliosis and asymptomatic HIV infection

TAUSTA.
Lymen neuroborrelioosi (LNB) on tavallisin arthropoda-välitteinen keskushermostoinfektio Euroopassa ja USA.ssa. LNB- diagnooosi on kombinaatio anamnestisista tiedoista, kliinisistä löydöksistä ja laboratoriolöydöistä.
Eurooppalaisten ohjelinjojen mukaan vaaditaan täsmälliseen diagnoosiin LNB:lle johdonmukaiset kliiniset oireet ( kuten kivulias meningoradikuliitti), muiden diagnoosien poissulkeminen, aivoselkäydinnesteen pleosytoosi ja intratekaaliset Borrelia burgdorferi ( Bb) -vasta-aineitten muodostus.
Mutta aivoselkäydinnesteen solujen paljous ei ole spesifinen löytö Lymen neuroborrelioosissa , sillä sellaista tavataan muissakin infektioissa ja jopa sellaisissa keskushermostotaudeissa, joisa ei ole kyse infektiosta. Lisäksi jopa kuusi viikkoa oireitten alkamisesta voi intratekaaliset vasta-aineet viipyä ja toisaalta ne pysyvät positiivisena vuosia alkusairastumisen jälkeen.

Viime vuosina on alettu kiinnostua kemokiinista, joka voisi toimia mahdollisena Lymen neuroborrelioosin biomerkitsijänä. Tämä kemokiini on B-lymfosyyttien kemoatraktantti (BCL) eli CXCL13 . Tämä kemokiini saattaa vahvasti B-imusoluja liikkeelle tulehduskohtaan ja sen pitoisuus nousee varhain LNB taudinkulussa ja toisaalta sen pitoisuus vähenee, kun asianmukainen hoito on annettu. On havaittu lisäksi suurempia CXCL13-pitoisuuksia ´Lymen neuroborrelioosin (LNB) yhteydessä kuin muissa infektiivisissä ja tulehduksellisissa keskushermostotaudeissa. Niitä harvoja tauteja, joissa aivoselkäydinnesteen CXCL13 pitoisuudet ovat yhtä korkeita kuin Lymen neuroborrelioosissa (LNB) ovat kryptokokkoosi (cryptococcosis) ja neurosyfilis.

Jos on perifeerisen hermoston oireita Lymen neuroborrelioosissa (LNB), on yhtä tehokasta hoitoa siihen suun kautta otettu doksisykliini tai laskimoon annettu keftriaksoni. Mutta jos on kyse keskushermosto-oireisesta ( meningiitti, enkefaliitti) Lymen neuroborrelioosista (LNB) , on edelleen ensisijalla monissa hoitokeskuksissa laskimoon annettu kefttriaksoni.

HIV infektoi keskushermostoa jo primaari-infektiossa tai aivan pian infektion alkamisen jälkeen aiheuttaen kroonisen matala-asteisen tulehdusreaktion useimmilla kliinisesti oireettomilla potilailla.
Jos verrataan kontrollipotilaihin, niin HIV-potilailla seerumin kemokiinin CXCL13 pitoisuuksien on havaittu olevan merkitsevästi koholla .
Van Burgel tutkijaryhmänsä kanssa osoitti kuudella HIV- meningiittiä potevalla aivoselkäydinnesteen kohonneet CXCL13 pitoisuudet ja seitsemällä HIV-infektiota potevalla, joilla ei ollut intrathekaalista tulehdusta, vastaavat matalat CXCL13 pitoisuudet.
Mutta laajoista potilasryhmistä, jolla on oireeton HIV-infektio, ei ole aiemmin tutkittu aivoselkäydinnesteen CXCL13- pitoisuuksia.

Nyt tutkijat Daniel Bremell, Niklas Mattson, Mikael Edsbagge, Kaj Blennow , Ulf Andersson, Carsten Wikkelsö, Henrik Zetterberg ja Lars Hagberg ottivat analysoitavakseen potilasryhmiä, joilla oli LNB( Lymen neuroborrelioosi) tai HIV sekä terveitä kontrolleita. Näiltä katsottiin aivoselkäydinnesteen CXCL13 pitoisuudet , jotta voitaisiin määrittää, olisiko tämä kemokiini jotenkin LNB-spesifinen silloinkin, kun oli vertailtava HIV- infektioon.
Lisäksi tutkijaryhmä selvitti, miten doksisykliinihoito vaikuttaa likvorin CXCL13 kemokiinipitoisuuksiin ja likvorin solumääriin. Tämä määrittely tehtiin hyvin luonnehdituilta LNB-potilasryhmiltä ennen ja jälkeen lääkehoidon.

JOHTOPÄÄTÖS
Lymen neuroborrelioosipotilalla (LNB) on vahvasti kohonneet likvorin CXCL13-pitoisuudet.
Suun kautta annetun doxysykliinihoidon jälkeiset likvorin CXCL13-pitoisuudet erosivat satakertaisesti alkuarvoista Lymen borrelioosipotilailla, mikä tukee tämän hoitokaavan tehokkutta. Samansuuntainen vaikutus näkyi myös aivoselkäydinnesteen mononukleaaristen solujen laskussa . Tutkijat osoittivat myös, miten neurologisesti oireettomilla HIV- potilailla on myös likvorin kohonneita CXCL13-pitoisuuksia ja nämä pitoisuudet kattavat samoja alueita kuin LNB-potilaiden likvorin kemokiinipitoisuudet Eri tutkimuksissa saadut Lymen neuroborrelioosipotilaiden likvorin CXCL13 kemokiinipitoisuudet eroavat suuresti ( syitä on eritelty artikkelissa).

Edelleen on vielä tulevaisuudessa selvitettävä , mikä arvo CXCL13 kemokiinimäärityksillä on Lymen neuroborrelioosin diagnostiikassa
.
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Re: BORRELIATESTIT

ViestiKirjoittaja soijuv » La Kesä 04, 2016 19:09

Uusi nanoteknologiaan perustuva CERES LYME virtsatesti saamassa FDAn hyväksynnän (2016)

The new Ceres Lyme test, which is based on nanotechnology, is well on its way to getting FDA approval.The new urine test was developed by Temple Douglas at George Mason University three years ago, who pursued a test for Lyme disease because of personal interest.

Her idea for the test originated when Douglas was a high school intern at George Mason University in Virginia. She wondered whether a technique researchers were working on to test for microscopic cancer particles might be used to test for Lyme disease.

“I was in the right place at the right time,” she told CBS News. “I was aware of the issues with the Lyme disease test because of people in my family. Two people in my family at that time had Lyme disease.” Douglas is now a graduate student, and her brain child, “Nanotrap LA”, is the first commercial product for Ceres.

Lyme disease is difficult to diagnose not only because only a small percentage of people get the telltale rash after a tick bite, but also due to the lack of reliable testing. This is a compounding problem because the more people who fall through the cracks and fail to get sufficient treatment early on, the more people who develop persistent chronic Lyme which is far more difficult to treat.

So how does “Nanotrap LA” work?

According to Ceres (acommercial lab associated with George Mason University) the nano technology collects a part of the bacteria called Osp A (outer surface protein) which is shed after the bacteria is processed through the kidney, and evaluates those particles. The amount of bacteria in urine is very small, especially at the beginning of the infection, however, the beauty of this test is that the nano technology is able to find even the smallest amount of Osp A and process it. Even though this new test relies on the western blot assay to evaluate the findings, so far it does not seem to share the equivocal nature of the blood test, posting 100% accuracy in the published study.
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Re: BORRELIATESTIT

ViestiKirjoittaja Sailairina » Ti Loka 11, 2016 08:02

http://www.karjalainen.fi/uutiset/uutis ... a-tunnissa

KOTIMAA 10.10.2016 09:48
Uusi testi punkkitautien diagnosointiin - Uudella menetelmällä tulos neljässä tunnissa

Punkkitautien nykyistä parempaan tunnistamiseen on kehitetty uusi menetelmä. Menetelmän on kehittänyt Jyväskylän yliopiston tutkimusryhmä.

Tutkimushanketta johtavan bio- ja ympäristötieteiden laitoksen yliopistonlehtorin Leona Gilbertin mukaan uusi TICK-TAG testauspaketti tuodaan markkinoille ensi vuoden alussa.

– Tänä syksynä hankimme tuotteelle vielä CE ja ISO-hyväksynnät eli laatustandardit. Tavoitteena on saada tuote markkinoille tammikuun alussa, kertoo Gilbert.

Gilbertin mukaan punkkitautien, kuten borrelioosin, tunnistaminen tarkentuu uuden testin myötä. Gilbertin mukaan punkkien kautta leviää kymmeniä erilaisia mikrobeja, jotka aiheuttavat erilaisia infektioita.

– Tähän mennessä puutiaisten levittämistä taudeista on pystytty testaamaan vain yhtä taudinaiheuttajaa kerralla. Kehittämämme menetelmä mahdollistaa 20 eri mikrobin tunnistamisen yhdellä testauksella, kuvaa Gilbert.

– Testi kertoo, mikä infektio on kyseessä ja myös sen, onko sairaus vakava vai lievä. Kun tunnistamme, mikä mikrobi oireet on aiheuttanut, potilaita osataan hoitaa oikein, sanoo Gilbert.

Gilbertin mukaan ensi vaiheessa testiä tulevat hyödyntämään yksityiset laboratoriot Suomessa, Saksassa ja USA:ssa. Tavoitteena on hänen mukaansa saada testi Suomen julkisten sairaaloiden ja terveyskeskusten käyttöön vuoteen 2018 mennessä.

Uudella tavalla diagnoosi pystytään Gilbertin mukaan tekemään nopeasti, kun tähän saakka punkkitaudin diagnosointiin on usein tarvittu useita testejä.

– Nykyisin punkkien aiheuttamien tulehdusten diagnosointi voi kestää viikkoja. Uudella menetelmällä tulos saadaan neljässä tunnissa, kertoo Gilbert.

Uutta testiä on testattu tieteellisesti 1 500 potilaalla ympäri maailman. Heiltä on otettu 15 000 yksittäistä näytettä.

– Testeissämme kävi ilmi, että 74 prosentilta potilaista löytyi joku punkin välittämä infektio. Kun samoilta potilailta testattiin vain yhden mikrobin esiintymistä, sairaus löydettiin vain kahdeksalta prosentilta, vertaa Gilbert.

Gilbertin mukaan nykyisin paljon borrelioositapauksia jää piiloon puutteellisen diagnosoinnin takia.

Lue lisää maanantain Karjalaisesta.
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