If you are new to Lyme & looking for testing options check out our main site at: http://www.ticktalkireland.org/suspectlyme.html
Also check out our good news section for updates on testing & related issues..
Cautions on Immuno Blots by Mayo Clinic
The immunoblot should not be used as a screening assay. In addition, immunoblot may be negative in specimens that are weakly-positive by EIA, or in patients with early Lyme disease.
Test results should be used in conjunction with clinical evaluation and information related to tick exposure.
A negative test result does not necessarily rule out current or recent infection. The specimen may have been drawn before demonstrable antibody developed. Patients with early disease often have serum antibody titers below the diagnostic threshold for several weeks following disease onset.
Test results from immunosuppressed patients and pregnant women may be difficult to interpret.
Positive test results may not be valid in persons who have received blood or blood product transfusions within the past several months.
Antibiotic therapy administered early in the first-stage of disease may suppress the antibody response to the point that diagnostic threshold levels are never attained.
False-positive reactions may occur with patients with other spirochetal diseases (syphilis, yaws, pinta, relapsing fever, or leptospirosis), influenza, autoimmune disorders, multiple sclerosis, or amyotrophic lateral sclerosis.
*Updated 26 Sep 2016
Please note that while it is reassurring to have a greater choice of diagnostics out there (some of which are listed below), the American CDC, IDSA et al still insist on 2 tier testing as standard which continues to miss a subset of patients – I feel it’s high time we moved on from this outdated form of testing..
Development of a multi-antigen panel for improved detection of Borrelia burgdorferi infection in early Lyme disease
Lauren J. Laheya,b, Michael W. Panasc, Rong Maoa,b, Michelle Delanoyd, John Flanagand, Steven R. Binderd, Alison W. Rebmane, Jose G. Montoyaf, Mark J. Soloskie, Allen C. Steereg, Raymond J. Dattwylerh,i, Paul M. Arnaboldih,i, John N. Aucotte and William H. Robinsona
The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi (Bb). The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm, however this scheme has limited sensitivity in detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage when antibiotic treatment is highly efficacious.
We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers, while simultaneously maintaining high specificity by requiring a positive test at two markers.
Ten markers were selected from our initial analysis of 62 Bb surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls.
In a validation set, this 10-antigen panel identified a higher proportion of early Lyme disease patients as positive at the baseline or post-treatment visit compared to two-tiered testing (87.5% and 67.5% respectively, P<0.05).
Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans.
The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early Bb infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.
Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis
Ruben Magni, Benjamin H. Espina, Ketul Shah, Benjamin Lepene, Christine Mayuga, Temple A. Douglas, Virginia Espina, Sally Rucker, Ross Dunlap, Emanuel F. III Petricoin, Mary Frekko Kilavos, Donald M. Poretz, Gilbert R. Irwin, Samuel M. Shor, Lance A. Liotta and Alessandra Luchini
Journal of Translational Medicine201513:346
DOI: 10.1186/s12967-015-0701-z© Magni et al. 2015
Published: 4 November 2015
Urinary OspA in patients suspected of having early stage LB
In this study, 100 % of pre and post treatment samples that had active symptoms were found to be positive for detectable urinary OspA (40/40, Table 2). Urinary OspA outcome (positive) was significantly associated with presence of clinical symptoms (EM rash, Chi squared p value <2.2e−16; significance level = 5 %, power = 100 %).
Of these 40 patients, 22 were serology positive by CDC standards for early stage disease . This is in keeping with prior studies showing positive serology in approximately 50 % of early stage cutaneous LB patients . Early prompt treatment of LB is known to blunt the serology response because the infection and immune response is being interrupted at an early stage [101, 102].
Urinary OspA in patients suspected of having “chronic” LB
In this study, we evaluated the level of urinary OspA protein in 100 previously or currently treated patients with joint, neurologic, and other objective symptoms (Additional file 1: Table S6). This group of patients were being evaluated for the potential of recurrent or persistent infection with Borrelia. Our analysis was blinded to outcome. 41 of 100 (41 %) patients were positive for urinary OspA protein. This percentage of positive urinary OspA, assuming that it reflects a specific infection by Bb that is shedding OspA C-terminal fragments, is in keeping with prior studies.
Biomedical engineering student developed new Lyme disease test, now working in cancer detection
BLACKSBURG, Va – Temple Douglas of Lucketts, Virginia, is one of several doctoral students working in a Virginia Tech microfluidics lab helping discover new cancer detection methods. The task is well suited for her. While in high school, Douglas developed a new test for Lyme disease that uses urine.
“Antibodies form weeks after the tick bite, after which you have lost a window of time when antibiotics could have been started,” said Douglas.
“Antibody reactions can be incomplete, often leading to false positives or false negatives. By detecting bacterial protein rather than the patient’s antibody response, it is possible to know earlier and more accurately whether someone has contracted Lyme disease.”
Douglas’ mentor at George Mason, Alessandra Luchini, had developed nanoparticles to use for the concentration and detection of low molecular weight proteins and biomarkers. Temple thought the same nanoparticles could detect Borrelia burgdorferi antigen fragments – that’s the bacteria that forms Lyme disease — in urine, allowing for a more sensitive diagnostic test. George Mason allowed Temple to work on the method for her high school senior research project.
After graduation, before Princeton, Douglas continued working on the Lyme disease test in the summer and it went into clinical testing soon afterward.
George Mason researchers carried the tests onward, and announced this past spring tests in early-stage Lyme disease patients had a near perfect detection rate.
The test is now commercially available and being used in some hospitals in Northern Virginia.
The timing is fortunate. Virginia and several other Southern states have reported increases in Lyme disease cases. The disease can be brutal, moving from early stage headaches and fatigue to achy joints, heart and central nervous system issues, and stroke, according to the Centers for Disease Control.
Researchers honored for Lyme disease biomarker breakthrough
June 14, 2015
The Bay Area Lyme Foundation has recognized Nira Pollock, MD, PhD, and John Branda, MD, for their research on a potential biomarker for Lyme disease with a grant to support future development of a novel diagnostic urine test, according to a press release.
Pollock and Branda, both of Harvard Medical School, recently identified a Borrelia burgdorferi biomarker in the urine of Lyme patients, according to the release. Their method used techniques previously developed to identify biomarkers for tuberculosis and leishmaniasis, and may provide an advantage over currently used Lyme diagnostics.
“Without an accurate and reliable diagnostic, patients suffer with unexplained symptoms and miss the opportunity for early treatment,” Bonnie Crater, co-founder and vice president of the Bay Area Lyme Foundation, said in the release. “We are hopeful that the approach of Drs. Pollock and Branda, which taps learning from other diseases, will lead to a more direct way to detect the bacteria that causes Lyme disease than the current methods.”
The nonprofit organization’s 2015 Emerging Leader Award comes with a $100,000 grant, which will fund further research adapting this finding into a novel urine test to detect the infection.
“Our hope is that findings from this round of research will bring us closer to developing an accurate diagnostic test,” Branda said in the release.
TGen developing quick, affordable and accurate test to diagnose debilitating Lyme disease
Focus On Lyme funds TGen research; plans scientific conference promoting better diagnostics and treatments for infected patients
GILBERT, Ariz. – July 20, 2015 – Focus On Lyme, an initiative sponsored by the Leadership Children’s Foundation of Gilbert, Ariz., has donated $75,000 to the Translational Genomics Research Institute (TGen) to support research into the development of a quick, affordable and accurate method of diagnosing Lyme disease.
..Today, no perfect test for Lyme disease exists due to three main barriers: multiple strains of Lyme bacteria often confound test results, the existence of related bacteria may cause false positive test results and most Lyme infections typically present at a level not detectable by current testing methods.
Scientists at TGen’s Pathogen Genomics Division in Flagstaff, Ariz. – TGen North – will use the power of targeted DNA sequencing to develop and validate a test to measure the presence and severity of tick-borne Lyme disease at the genomic level.
By analyzing a sample’s DNA, the new test should be able to pinpoint Lyme disease, identify multiple Lyme strains, detect other tick-related infections, and show non-Lyme causes of disease.
“With recent advances at TGen and genomics overall, we can finally develop a diagnostic test that will put more actionable information into the hands of the physician than previously possible. We are thrilled to be working with Focus On Lyme on this project,” said Dr. Paul Keim, Professor and Director of TGen North and Director of the Center for Microbial Genetics & Genomics at Northern Arizona University (NAU), which will help develop the test.
Lyme Disease DNA Test
From the site: The Molecular Diagnostics Department of Milford Medical Laboratory is the first, and at present, the only laboratory in the United States to offer a combined DNA Sequencing-based diagnostic test for Borrelia burgdorferi, the infectious agent for Lyme disease and Borrelia miyamotoi, the infectious agent of a similar Lyme disease-like illness found recently in the United States.
Milford Medical Laboratory can produce a diagnosis within five days of a sample’s receipt, ensuring the fastest, most conclusive test for Lyme disease available anywhere. The diagnosis fee is $150.00 per sample.
Clinical “Lyme disease” is in fact a group of infectious diseases caused by many species of spirochetes, the corkscrew-shaped parasitic bacteria of the genus Borrelia. As for all infectious diseases, the laboratories can test for the antibodies generated by the patient in response to the infectious agent by ELISA or Western blots, or perform a molecular test for the DNA or RNA of the casual agent. The difference is that a positive molecular test result indicates a current, active infection, and a positive antibody test indicates an exposure to the infective agent in the past.
Hilysens reaches stage II of project
Further down the page we covered a new test being developed with EU funding called Lab on a Chip – the chip card has been sent to some centres for vailidation checking – more info available at: http://hilysensproject.eu/pdfs_others/PRESS-RELEASE_Lyme_Card_Validation_start.pdf
For a video of the process see the link below:
T2 Lyme Panel
Approximately 3.4 million tests 1,2 are run for Lyme disease each year including serology tests, polymerase chain reaction (PCR) techniques and blood culture. These tests are labor-intensive, can take weeks to process and are subject to high false negative rates due to their inability to detect the disease. Because of these limitations, patients are frequently misdiagnosed or face delayed diagnosis. Up to 90% 1,2 of patients with Lyme disease go undetected altogether.
Because of the poor diagnostic testing currently available for Lyme, the CDC recommends that the diagnosis remain a clinical one, based on a patient’s symptoms and history, and that blood tests should only be used to provide supporting evidence for the diagnosis.
The T2Lyme Panel, which is being developed through a partnership between T2 Biosystems and Canon US Life Sciences is being designed to identify the bacteria that cause Lyme disease directly from a patient’s blood, without the need for blood culture. The test panel is expected to be run on the FDA-cleared T2Dx Instrument, the same instrument currently used to run our FDA-cleared T2Candida Panel and our future T2Bacteria Panel.
We anticipate the T2Lyme Panel will benefit from similar advantages provided by our T2MR technology as the T2Candida Panel, including high sensitivity, high specificity, ease of use and rapid time to result.
We are developing the T2Lyme Panel to provide accurate and timely diagnosis of Lyme disease, which may help prevent the evolution of the disease to its later stages to avoid the associated neurological and musculoskeletal diseases and reduce or eliminate the significant costs associated with these effects.
Development of a metabolic biosignature for detection of early Lyme disease
Clin Infect Dis. (2015)
First published online: March 11, 2015
Claudia R. Molins1, Laura V. Ashton2, Gary P. Wormser3, Ann M. Hess4, Mark J. Delorey1, Sebabrata Mahapatra2, Martin E. Schriefer1, and John T. Belisle2,*
Background. Early Lyme disease patients often present to the clinic prior to developing a detectable antibody response to Borrelia burgdorferi, the etiologic agent. Thus, existing two-tier serology-based assays yield low sensitivities (29-40%) for early infection. The lack of an accurate laboratory test for early Lyme disease contributes to misconceptions about diagnosis and treatment, and underscores the need for new diagnostic approaches.
Results. Metabolic biosignature development selected 95 molecular features that distinguished early Lyme disease patients from healthy controls. Statistical modeling reduced the biosignature to 44 molecular features, and correctly classified early Lyme disease patients and healthy controls with a sensitivity of 88% (84-95%), and a specificity of 95% (90-100%). Importantly, the metabolic biosignature correctly classified 77-95% of the of serology negative Lyme disease patients.
Diagnostic use of the lymphocyte transformation test-memory lymphocyte immunostimulation assay in confirming active Lyme borreliosis in clinically and serologically ambiguous cases
Basant K Puri,1 Daniel RM Segal,2 and Jean A Monro2
Int J Clin Exp Med. 2014; 7(12): 5890–5892.
Published online 2014 Dec 15.
The aim of this study was to carry out an independent evaluation of the proposition that the lymphocyte transformation test-memory lymphocyte immunostimulation assay (LTT-MELISA) may be diagnostically useful in the confirmation of active Lyme borreliosis in clinically and serologically ambiguous cases.
Blood samples from 54 patients consecutively presenting to a British center with clinical suspicion of Lyme borreliosis were tested for this disease by immunoglobulin M (IgM) and immunoglobulin G (IgG) Western blots and by LTT-MELISA. Forty-five of these patients had Western blot results which were negative for both IgM and IgG by the criteria of the Centers for Disease Control and Prevention (CDC); of these patients, 19 (42%) were LTT-MELISA-positive. Two of the patients who had IgM positive results by the CDC criteria were LTT-MELISA-negative.
It is concluded that, for putative European-acquired Lyme borreliosis infections, it would be sensible to carry out both the LTT-MELISA and Western blot assay.
Testing T-Cell Response for the Improved Clinical Diagnosis and Treatment of Lyme Disease
by Mark Menolascino, MD, MS, ABIHM, ABAARM
Townsend Letter July 2014
Evaluating Lyme Disease
The current CDC-recommended evaluation for diagnosis of Lyme is a two-tier testing protocol, including both ELISA and western blot analyses. These tests are serological assays that detect antibodies to B. burgdorferi. Such tests (known as B cell-based or humoral tests) are often not sensitive enough to detect the antibody response (as in the brothers’ cases), or do not detect it quickly enough to prevent chronic infection.
This two-tier testing method has very low sensitivity and specificity, with a significant number of false positive and negative tests. The high false-negative rates are of particular concern, as many Lyme patients are left undiagnosed and untreated. Due to these factors, many Lyme specialists believe that more sensitive T cell-based laboratory tests should be developed.
The ELISPOT Assay
One such T cell-based test is the enzyme-linked immunosorbent spot (ELISPOT) assay. Newly available in a test called iSpot Lyme, this test is showing promise as a new, innovative method for assessment of the magnitude and quality of T cell immunity through the measurement of stimulated antigen-specific T cells. Requiring a blood sample, iSpot Lyme was developed by Pharmasan Labs, a CLIA-approved lab with expertise in testing for tick-borne diseases.
The test uses a method already approved and sanctioned by the FDA and CDC for assessing a tuberculosis infection and simply applies it to the Lyme bacteria B. burgdorferi. While other tests are predicated on a B cell antibody response, the iSpot Lyme test is unique in that it examines the primary mediator of the cell-mediated branch of the immune system: the T cells.
As such, it is a highly sensitive technique for detecting immune cells that secrete signature proteins (such as a given cytokine). With a sensitivity of 84% and a specificity of 94% for the detection of B. burgdorferi, it is the most sensitive technology currently available for detection, measurement, and functional analysis of these immune cells. Thus, it is an excellent complement to the current two-tiered antibody methods.
Prolonged antibiotic therapy in PCR confirmed persistent Lyme disease
Scientific Study, 2011, 19 Pages
Dr. med. Bernt-Dieter Huismans
Surprisingly, only 57% of patients had a positive Borrelia serology, even though all had PCR confirmed disease. The serologic findings of our sample are shown in figure 3. The IgG western blot exhibited the highest degree of sensitivity (58%).
Only 43,52% (37/85) of patients had both a positive ELISA test and a positive Western blot, while in 10,85% (9/85) the positive ELISA result was not confirmed by the Western blot.
In 24,70% (21/85) of cases the ELISA test remained negative despite a positive Western blot, even though, to be useful as a screening tool, the ELISA test should theoretically exhibit higher sensitivity.
Effects of Borrelia on host immune system: Possible consequences for diagnostics
Mualla McManus, Ann Cincotta
Tick Borne Diseases Unit, School of Medical Sciences (Pharmacology), University of Sydney, 2006 NSW, Australia
..Indirect diagnostics are the most common method of testing for Borreliosis as they are cheap and convenient. However due to wide variation in antigenicity of genospecies, the sensitivity and specificity of diagnostics can be questioned. Evidence is accumulating which suggests that immune dysregulation induced by Borrelia (and other tick borne infections) can impact the indirect diagnostics, especially in Stage 3.
The direct detection of Borrelia using nucleotide amplification method is possible but wider usage of this method is difficult as it has high specificity and narrow sensitivity. In vitro culturing is ideal but difficult as Borrelia has fastidious growth requirements.
The immune status of the borreliosis patient needs to be considered, especially in Stage 3 in conjunction with clinical symptoms in the diagnosis.
Borrelia has the ability to manipulate both the innate and active immunity and alter the cytokines secreted hence alter the path of the immune response. Immune parameters such as IFN-gamma/IL-10, lymphocyte markers, complement C3a, C4a, and total immunoglobulin levels may help to discriminate between stages and monitor treatment outcomes.
The level of immune dysfunction in Stage 3 may depend on the number of co-infections delivered by a tick bite, such as Babesia, and Rickettsia, the genospecies of Borrelia, other pathogens, the patients’ biome and immunogenetics.
4 Reasons A Lyme Test Will Come Back Negative Even If A Person Truly Has Lyme Disease
Reason 1: You Have A Different Strain Of Borrelia Than The Strain Of Borrelia The Test Looks For
Reason 2: The Lab Performing Your Lyme Disease Test
Reason 3: The Type Of Test Used
Reason 4: You Don’t Have Lyme Disease, But A Co-Infection
Check out the following web pages for more details:
This site (which we list further down the page) is another useful resource on serongegative Lyme: http://www.mentalhealthandillness.com/seronegativelymedisease.html
*Updated 28 Apr 2015
Check out our new article examining issues surrounding antibody testing:
*Updated 2 Feb 2015
DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infections
Int J Mol Sci. 2014 Jul; 15(7): 11364–11386.
Published online 2014 Jun 25. doi: 10.3390/ijms150711364
Sin Hang Lee,1,* Jessica S. Vigliotti,1 Veronica S. Vigliotti,1 William Jones,1 Thomas A. Moorcroft,2 and Katherine Lantsman3
From the Abstract: This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.
From the Article: Full-blown spirochetemia in Lyme borreliosis is a transient phenomenon and occurs within the first 30 days of the disease . Any free bacterial DNA in the circulating blood left over from an early full-blown spirochetemia would be degraded or excreted within 42 h  after the spirochetes vanished from the circulating blood, and certainly would have been excluded when the bacteria were pelleted by differential centrifugation to be tested according to our laboratory procedures.
Since the spirochetemia detected in these 14 patients was unlikely to be the result of a recent infection and some of the patients had received multiple courses of antibiotics for the treatment of the disease up to the date of blood testing, we interpret these 14 patients as cases of undiagnosed Lyme or related borrelioses, or as cases of “Lyme disease” not completely cured by the standard courses of antibiotic treatment.
*Updated 3 Dec 2014
Contribution of HLA alleles in the regulation of antibody production in Lyme disease.
Front Biosci. 2001 Sep 1;6:B10-6.
Wang P1, Hilton E.
A small subset of patients infected with Borrelia burgdorferi (Bb) does not produce Bb specific antibody. Our research provides additional evidence of a genetic predisposition for seronegativity in some individuals with Lyme disease.
Because human leukocyte antigen (HLA) class II, a heterodimeric glycoprotein, plays an essential role in the regulation of antibody production, we investigated the difference in HLA genes between seropositive and seronegative patients with Lyme disease (LD). Our results show that HLA-DR7 was associated with anti-Bb antibody production. Nine out of the 22 seropositive LD patients (40.9%) had HLA-DRB1*0701, *0703, *0704 (HLA-DR7); only 1 out of the 18 seronegative LD patients (5.6%) had HLA-DR7 (odds ratio (OR)=11.8, P=0.0126). HLA-DRB1*01021 and HLA-DRB1*0101, *0104, *0105 (HLA-DR1) contributed negatively to anti-Bb antibody production. Seven of 18 seronegative LD patients had HLA-DR1, only 1 of 22 seropositive LD patients had HLA-DR1 (38.9% vs. 4.5%, OR=13.4, P=0.0138).
These results suggest that the presence and or lack of production of specific antibody to Bb infection may be associated with particular HLA specificities of the Class II.
A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques
Biological and Biomedical Reports, 201
Morten M. Laane 1, Ivar Mysterud 2,*
1 Department of Molecular Biosciences, University of Oslo, P.O. Box 1041 Blindern, 0316 Oslo, Norway
2 Department of Biology, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway
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 prevent detection by immunological methods.
Full paper available in PDF: http://www.worldwide-lyme-protest.org.uk/downloads/98-446-2-PB.pdf
A Patent looking at culture – Culture media for growing spirochetes
Application number: 20010036658
Filed: Dec 01, 2000
Issued: Nov 01, 2001
Inventors: Steven E. Phillips (Ridgefield, CT), Hamid Moayad (Bedford, TX), Lida Mattman (Grosse Pointe, MI)
Mentions culture techniques, persistence of Lyme & links to MS
Divergent opinions of proper Lyme disease diagnosis and implications for children co-morbid with autism spectrum disorder
Received: February 1, 2014; Accepted: June 6, 2014; Published Online: June 16, 2014
DOI: http://dx.doi.org/10.1016/j.mehy.2014.06.005 Mason Kuhnemail, Robert Bransfield
This paper proposes that some children with an autism spectrum disorder (ASD) in the United States have undiagnosed Lyme disease and different testing criteria used by commercial laboratories may be producing false negative results.
Two testing protocols will be evaluated; first, the Centers for Disease Control (CDC) and Infectious Disease Society of America (IDSA) approved two-tiered Enzyme Immunoassay (EIA) or Immunofluorescence Assay (IFA) followed by an IgM and/or IgG Western Blot test. Second, a clinical diagnosis (flu like symptoms, joint pain, fatigue, neurological symptoms, etc.) possibly followed by a Western Blot with a broader criteria for positive bands  . The hypothesis proposes that the former criteria may be producing false negative results for some individuals diagnosed with an ASD. Through an online survey parents of 48 children who have a diagnosis of an ASD and have been diagnosed with Lyme disease were asked to fill out the Autism Treatment Evaluation Checklist (ATEC) before they started antibiotic therapy and after treatment.
Of the 48 parents surveyed 45 of them (94%) indicated their child initially tested negative using the two-tiered CDC/IDSA approved test. The parents sought a second physician who diagnosed their child with Lyme disease using the wider range of Western Blot bands. The children were treated with antibiotics and their scores on the ATEC improved. Anecdotal data indicated that some of the children achieved previously unattained developmental milestones after antibiotic therapy began. Protein bands OSP-A and/or OSP-B (Western Blot band 31) and (Western Blot band 34) were found in 44 of 48 patients. These two bands are so specific to Borrelia burgdorferi that they were targeted for use in vaccine trials, yet are not included in the IDSA interpretation of the Western Blot.
Tick-specific borrelial antigens appear to be upregulated in American but not European patients with Lyme arthritis, a late manifestation of Lyme borreliosis.
J Infect Dis. 2013 Sep;208(6):934-41. doi: 10.1093/infdis/jit269. Epub 2013 Jun 12.
Li X1, Strle K, Wang P, Acosta DI, McHugh GA, Sikand N, Strle F, Steere AC.
Borrelia burgdorferi (Bb) sensu lato, the etiologic agent of Lyme borreliosis, adapts to distinct environments in the mammalian host and the tick vector by differential gene expression. As a result, infected mice are not exposed to and rarely make antibodies to the set of antigens that are preferentially expressed in the tick, including outer surface protein A (OspA), Borrelia iron and copper-binding protein A (BicA), and OspD. Surprisingly, however, antibodies to OspA and BicA have been noted in American patients with Lyme arthritis.
Here, we examined serum samples from 210 American patients and 66 European patients with a range of early or late manifestations of Lyme borreliosis and found that only American patients with Lyme arthritis commonly had antibody responses to OspA, BicA, and OspD.
This suggests that infection with American but not European Borrelia strains often leads to concerted upregulation or derepression of tick-specific spirochetal antigens in these patients.
Characteristics of seroconversion and implications for diagnosis of post-treatment Lyme disease syndrome: acute and convalescent serology among a prospective cohort of early Lyme disease patients
Clinical Rheumatology Date: 13 Jun 2014
Alison W. Rebman, Lauren A. Crowder, Allison Kirkpatrick, John N. Aucott
Two-tier serology is often used to confirm a diagnosis of Lyme disease. One hundred and four patients with physician diagnosed erythema migrans rashes had blood samples taken before and after 3 weeks of doxycycline treatment for early Lyme disease. Acute and convalescent serologies for Borrelia burgdorferi were interpreted according to the 2-tier antibody testing criteria proposed by the Centers for Disease Control and Prevention. Serostatus was compared across several clinical and demographic variables both pre- and post-treatment. Forty-one patients (39.4 %) were seronegative both before and after treatment. The majority of seropositive individuals on both acute and convalescent serology had a positive IgM western blot and a negative IgG western blot. IgG seroconversion on western blot was infrequent. Among the baseline variables included in the analysis, disseminated lesions (p < 0.0001), a longer duration of illness (p < 0.0001), and a higher number of reported symptoms (p = 0.004) were highly significantly associated with positive final serostatus, while male sex (p = 0.05) was borderline significant.
This variability, and the lack of seroconversion in a subset of patients, highlights the limitations of using serology alone in identifying early Lyme disease. Furthermore, these findings underline the difficulty for rheumatologists in identifying a prior exposure to Lyme disease in caring for patients with medically unexplained symptoms or fibromyalgia-like syndromes.
Is there a place for xenodiagnosis in the clinic?
Informa Healthcare: November 2014, Vol. 12, No. 11, Pages 1307-1310 (doi:10.1586/14787210.2014.966084)
Sam R Telford, Linden T Hu, and Adriana Marques
1 Tufts University Cummings School of Veterinary Medicine, North Grafton, MA, USA
2 Tufts Medical Center, Boston, MA 02115, USA
3 National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA
Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist after antibiotic therapy is an area of ongoing controversy. In animal models, B. burgdorferi DNA can be detected in tissues after antibiotic therapy as well as by using the natural tick vector to acquire the organism through feeding (xenodiagnosis). Vector arthropods have been successfully used in xenodiagnosis to describe the etiology of infections such as malaria, typhus and Chagas disease. Our recent safety trial of xenodiagnosis demonstrates that ticks may be successfully fed on patients and may help determine the biological basis for post-treatment Lyme disease syndrome.
An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi
Published: November 03, 2014 / DOI: 10.1371/journal.pone.0111809
Jie Feng, Ting Wang, Shuo Zhang, Wanliang Shi, Ying Zhang
..Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader.
The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.
Breakthrough test evaluates drugs for Lyme treatment
Supported in part by a research grant from LymeDisease.org, scientists at Johns Hopkins have developed a test to help determine which drugs are most effective against Lyme disease. – See more at: http://lymedisease.org/news/lyme_disease_views/news-breakthrough-test-evaluates-drugs-for-lyme-treatment.html#sthash.1eKu0zIZ.dpuf
*Updated 18 Jun 2014
Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells.
Tanguay S1, Killion JJ. – Lymphokine Cytokine Res. 1994 Aug;13(4):259-63.
A direct comparison was made between the insoluble ELISPOT, solubilized ELISPOT, and ELISA assays, to detect cytokine secretion by cells, using sterile ELISA plates and commercially available monoclonal antibodies. We evaluated the IL-6 secretion by resident peritoneal macrophages of BALB/c mice and the secretion of IL-2, IL-4, IL-5, and IL-6 by the murine T helper clone, D10.G4.1 cells. Our results demonstrated that ELISPOT can detect cytokine secretion at the single cell level in either adherent or nonadherent cells.
The level of detection by ELISPOT was 10 to 200 times more sensitive than ELISA performed on culture supernatants. We also demonstrated that the solubilized ELISPOT can detect cytokine secretion by cells with greater sensitivity than conventional ELISA. These ELISPOT assays can be used to characterize the cytokine secretion pattern of different cell populations in a simple, reproducible, and reliable manner.
PMID: 7999925 [PubMed – indexed for MEDLINE]
Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity
Mark J. Soloski, Lauren A. Crowder, Lauren J. Lahey, Catriona A. Wagner, William H. Robinson equal contributor,
John N. Aucott equal contributor – Published: April 16, 2014DOI: 10.1371/journal.pone.0093243
To study the immunological processes that are initiated following B. burgdorferi infection, we have utilized samples generated from a large cohort of Lyme disease patients that have been followed longitudinally for two years from the time of diagnosis and treatment. In this report, we measured the levels of a comprehensive panel of cytokines and chemokines to identify inflammatory mediators associated with acute Lyme disease as well as long-term outcomes of B. burgdorferi infection. Interestingly, mediator levels allow us to distinguish two populations of Lyme disease patients that display significant differences in the number of disease symptoms, seroconversion rates, lymphopenia and serum liver enzyme levels. Several T cell chemokines were coordinately upregulated while chemokines that drive other immune cell types were not. This work suggests that the complexity and levels of mediators present in the serum may be informative in understanding the various pathophysiological outcomes that occur in acute B. burgdorferi infection and that are associated with subsequent development of PTLDS.
In this patient cohort, 35.7% of Lyme patients fail to test positive either at diagnosis or through seroconversion following antibiotic treatment (Table 1). This is consistent with previous studies that demonstrated that a significant fraction of Lyme patients that exhibit EM along with other symptoms of infection do not seroconvert when assayed by the current two-tiered testing.
Evidence from mouse and primate models supports either the persistence of live bacteria or antigens after antibiotic treatment following Lyme borreliosis –. Convincing validation of these studies in the human setting is not available. Nevertheless, our observations suggest that there is an ongoing process that is selectively driving IL-6 production, the mechanistic basis of this awaits further study. Future studies need to examine if there is any relationship of persistently elevated IL-6 levels or other inflammatory markers with long-term outcomes such as PTLDS.
Simple objective detection of human Lyme disease infection using immuno-PCR and a single recombinant hybrid antigen.
Micah D. Halpern1, Claudia R. Molins2, Martin Schriefer2 and Mollie W. Jewett1#
+ Author Affiliations
1Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, 6900 Lake Nona Blvd Orlando, FL 32827, USA
2Diagnostic and Reference Laboratory, Bacterial Diseases Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, 3150 Rampart Road, Fort Collins, CO 80521.
A serology-based, tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease but lacks sensitivity in early disease and is often dependent on subjectively scored immunoblots. We recently demonstrated use of immuno-PCR (iPCR) for detection of B. burgdorferi antibodies in Lyme disease patient serum. To better understand the performance of the Lyme disease iPCR assay, the repeatability and the variability of the background of the assay across a healthy population (n=36) was analyzed. Both of these parameters were found to have coefficients of variation of less than 3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis of Lyme disease patient sera (n=12) demonstrated strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach, using a single hybrid antigen and detection of IgG antibodies only, confirmed the 2-tier analysis diagnosis of Lyme disease patient sera (n=12).
Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient sera (n=92) resulted in a sensitivity of 69% (95% CI: 50%-84%) compared to 2-tier analysis at 59% (95% CI: 41%-76%) and a specificity of 98% (95% CI: 91%-100%) as compared to 2-tier analysis at 97% (95% CI: 88%-100%).
A single tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host generated antibodies against B. burgdorferi.
Immune responses to borrelial VlsE IR6 peptide variants.
Sillanpää H1, Lahdenne P, Sarvas H, Arnez M, Steere A, Peltomaa M, Seppälä I.
Int J Med Microbiol. 2007 Feb;297(1):45-52. Epub 2007 Jan 17.
Laboratory confirmation of Lyme borreliosis (LB) relies mainly on the demonstration of anti-borrelial antibodies. In recent studies, a novel VlsE protein IR(6) peptide-based assay has been introduced. Our aim was to evaluate the IR(6) peptides from three Borrelia burgdorferi sensu lato genospecies in the serodiagnosis of European and North American patients. Five VlsE protein IR(6) peptide variants representing sequences from B. burgdorferi sensu stricto, B. garinii, and B. afzelii were used as antigens in both IgG and IgM enzyme-linked immunosorbent assays (ELISA). Serum antibodies of 187 patients at different stages of LB from Europe and the United States were evaluated for serodiagnosis. For comparison samples were tested with one of the commercial IR(6) ELISAs. Three B. afzelii IR(6) variant peptides revealed antibodies that were concordant with each other. B. burgdorferi sensu stricto peptide antibodies mostly paralleled B. afzelii peptide antibodies, and positive values were also obtained in the majority of European sera. For several sera, B. garinii IR(6) peptide antibodies were discordant to B. afzelii peptide antibodies.
The commercial IR(6) peptide antibody assay (C6 ELISA) results correlated better with B. burgdorferi sensu stricto IR(6) than with B. garinii IR(6) peptide IgG results, especially in sera from patients with facial palsy. Thus, antibody specificity to IR(6) peptides may vary according to the infecting Borrelia species. In some manifestations of the disease, C6 ELISA may not cover all LB cases. Evidently, the methodological aspects in ELISA design for peptide antibody measurements are important as well as the amino acids sequence of the antigen.
PMID: 17234451 [PubMed – indexed for MEDLINE]
Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.
Wormser GP1, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I. – Clin Infect Dis. 2008 Oct 1;47(7):910-4. doi: 10.1086/591529.
A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide.
C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.
The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes.
The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P=.07); this relationship was not observed with C6 testing.
Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.
PMID: 18724824 [PubMed – indexed for MEDLINE] PMCID: PMC2773679
*Updated 10 Jun 2014
Damage of Collagen and Elastic Fibres by Borrelia Burgdorferi – Known and New Clinical and Histopathological Aspects (study used LTT for diagnosis)
Kurt E Müller – Open Neurology Jnl 2012
The sensitivity of LTT was superior to serological investigation of antibodies in the ELISA or immunoblot tests and correlated well with clinical symptoms. FFM was able to detect the pathogen directly in numerous skin conditions associated with LB, regardless of the results of serology or of PCR. LTT is a useful addition to the haematological diagnostic spectrum, especially for the diagnostic of LB of tendons and ligaments, as histopathology in those cases rarely can be done.
An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi
Chenggang Jin, Diana R. Roen, […], and Gottfried H. Kellermann
The standard two-tier tests used to detect specific antibodies to B. burgdorferi include an enzyme-linked immunosorbent assay (ELISA) and a Western Blot assay (WB) . However, the limitation of these assays is that they have low sensitivity and specificity, frequently producing false negative and false positive results. For example, nearly 30% of results from a Western Blot IgM test are false positive . Furthermore, Borrelia-specific antibodies cannot be detected at the early stage of the infection, and a subgroup of Lyme patients lack detectable Borrelia-specific antibodies [15,16,17], in both cases providing a false negative result. Borrelia-specific T cell immunity has not yet been studied sufficiently due to the lack of highly sensitive and specific T cell-based assays that would be suited for the clinical laboratory. Several attempts have been made to study T cell reactivity against Borrelia, but the results were not consistent from different studies [18,19,20].
There is increasing evidence, however, that T cell assays have potential advantages over antibody-based assays in the detection of Borrelia infections.
Firstly, patients with erythema chronicum migrans (ECM), a clinical manifestation of B. burgdorferi infection, displayed specific T cell responses before antibodies to this organism become detectable by ELISA [21,22] and Lastavica et al. reported a case in which seroconversion did not occur until 18 months after the onset of the illness .
Secondly, a number of patients who received antibiotics for ECM had low or undetectable levels of anti-Borrelia antibodies suggesting that the antibody response can be decreased or aborted by early antibiotic intervention .
Thirdly, antibody titers often drop to levels below the cutoff value for positivity by ELISA, in particular for untreated subjects or patients with chronic Borrelia infection.
Fourth, changes in IgM/IgG titers and ratios cannot be used to monitor progress and treatment of Borrelia infection since they may stay constant for as long as 20 years [25,26]. Thus, there is a definite need for complementary T cell assays that may help overcome the aforementioned shortcomings of serological assays for diagnosing and monitoring the progress and treatment of Borrelia infection.
The enzyme-linked immunospot assay (ELISPOT) has emerged as a superior method for assessment of the magnitude and the quality of T cell immunity. It enumerates at the single cell level the frequency and cytokine signature of activated antigen-specific T cells [27,28]. The sensitivity of ELISPOT for detecting cytokine producing T cells is 20 to 200 fold higher than that of ELISA or flow cytometry-based intracellular staining . The ELISPOT technology has proven to be extremely sensitive in detecting even low frequencies of antigen reactive T cells and has been approved by the FDA for use in the diagnosis of tuberculosis [30,31]. Here, we explore the potential application of our newly developed Lyme ELISPOT assay, iSpot Lyme, as a diagnostic tool for the detection of Lyme Borreliosis.
*Updated 03 Jun 2014
Latent, dormant, subclinical, or asymptomatic Lyme disease
An Annotated Bibliography
This page contains citations and highlighted extracts for medical and scientific articles from the National Institutes of Health (NIH), National Library of Medicine (NLM) MEDLINE database about latent, dormant, subclinical, or asymptomatic Lyme disease. Citations are sorted by date within categories.
Detection of Borreliae in Archived Sera from Patients with Clinically Suspect Lyme Disease
Int. J. Mol. Sci. 2014, 15(3), 4284-4298; doi:10.3390/ijms15034284
Sin Hang Lee,* Jessica S. Vigliotti, Veronica S. Vigliotti, William Jones and David M. Shearer
From the PDF full article…
Since the rate of borrelial clearance from the blood and the immunological responses to the highly complex antigenic compositions of the borrelia species in terms of antibody production may vary from patient to patient, it is reasonable to expect that the blood samples drawn at any one time from the Lyme disease patients may show different antibody characteristics and harbor a variable density of the borrelial bacteria. It is also
reasonable to expect that the blood of a Lyme disease patient may be positive for the diagnostic antibody bands, but negative for the causative agent, or vice versa, at any time point..
Testing 52 blind-coded serum samples, including 12 post-treatment and 20 pre-treatment sera from patients with clinically suspect Lyme disease, showed that 1 of 12 post-treatment sera contained residual bacteria of a novel borrelia and 3 of 20 pre-treatment sera were positive for known borrelial bacteria, 1 identified as B. miyamotoi and 2 as B. burgdorferi based on 16S rDNA sequencing. Only in 1 of the 4 split samples which were PCR-positive, the pattern of the antibody bands of the 2-tier serology test matched the infectious agent detected in the same serum and was confirmed by DNA sequencing. Based on these findings, we conclude that a sensitive and reliable DNA-based test is needed to complement the 2-tier serology assay to support the clinical diagnosis of Lyme disease and Lyme disease-like borreliosis.
A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis
J. Clin. Microbiol. June 2000 vol. 38 no. 6 2097-2102
J. Robertson1, E. Guy2, N. Andrews3, B. Wilske4, P. Anda5, M. Granström6, U. Hauser4, Y. Moosmann7, V. Sambri8, J. Schellekens9, G. Stanek10, and J. Gray11,*
A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays…
The use of EIA and immunoblotting in a two-step testing strategy has gained wide acceptance, and the greater specificity of immunoblotting, also shown by the present study, has led to the view that it may be used as a confirmatory test. However, immunoblotting still has many problems, including the rationale and predictive value of tests, which have prompted recent reevaluations of their use (1, 3, 7, 26, 30). Several studies have reported that the use of different species and strains of B. burgdorferi sensu lato as antigen leads to inconsistency in blotting results because of variations in the expression of immunogenic proteins (4, 6, 15,22, 23, 27, 33). This aspect of serodiagnosis of LB in Europe has led to the publication of several different recommendations for blot interpretation. Further difficulties result from the subjectivity of interpreting band strength, from problems with band resolution and identification, and from differences in the immune response to the various clinical presentations of LB (2, 9, 10, 13, 20, 25,32).
*Updated 14 Feb 2014
Course of Antibody Response in Lyme Borreliosis Patients before and after Therapy
Elisabeth Aberer1 and Gerold Schwantzer2
1Department of Dermatology and Venereology, Medical University of Graz, Auenbruggerplatz 8, 8036 Graz, Austria
2Institute for Medical Informatics, Statistics and Documentation, Graz, Medical University of Graz, 8036 Graz, Austria
Received 27 September 2011; Accepted 27 October 2011
The early immune response (IR) in European Lyme borreliosis patients has not yet been studied in detail. The aim of the study was to analyse retrospectively the antibody development in 61 erythema migrans (EMs) patients depending on the duration of infection from tick bite by using a whole-cell lysate B. garinii immunoblot. The evolution of antibodies proved to be undulatory in untreated patients with two peaks for IgM at weeks 5 and 9 and for IgG at weeks 4 and 8. The analysis of IR courses after therapy identified patients constantly seropositive or seronegative and patients with repeated seroconversions with a switch, disappearance, or reappearance of anti-23 kD or anti-39 kD antibodies during the one-year period. We suggest that the antibody production in EM patients may be missed due to an undulatory IR. This phenomenon might be an as yet insufficiently researched aspect in Lyme borreliosis.
(more from article…)
The development of the IR to an infection with B. burgdorferi (Bb) s.s. in EM patients from the United States was studied by Aguero-Rosenfeld and showed that 43% of the patients had positive IgM antibodies before therapy . Due to the heterogeneity of borrelia strains in Europe, differences between IR on the two continents are expected since the Bb s.s. species provokes a stronger immune reaction than the dominant European species B. afzelii. Patients with culture-confirmed B. burgdorferi s.s. erythemas from the USA were more often seropositive (35.3%) at presentation compared with 22.4% culture-confirmed B. afzelii erythemas . In a another European study with recombinant ELISA, German Lyme borreliosis patients yielded positive IgG antibodies in 22% and IgM antibodies in 61.5% for phase I of LB .
Both the IgM and IgG IR showed an undulatory distribution with antibodies coming and going in untreated patients over weeks. A similar periodicity is known in other bacterial infections, like relapsing fever with recurrent bacteraemias . Syphilis can be reactivated by different conditions like HIV infection, showing seroconversion of the unspecific VDRL .
With respect to the course of IR after therapy, 21 of 61 (34%) patients did not show IgM seroconversion (constantly negative), whereas 12 (20%) were constantly positive. In the remaining 28 patients, different kinds of IgM seroconversion occurred. Nine patients (15%) (Figure 2, patients 1–9) seroconverted from positive to negative and 6 (11%) (Figure 2, patients 10–15) from negative to positive during the observation period. Seven seronegative patients (11%) (Figure 2, patients 16–22) seroconverted to positive and than back to seronegative during the 12 months. Six patients (10%) (Figure 2, patients 23–28) showed repeated seroconversions of IgM antibodies that presented as a switch of anti-23 kD to anti-39 kD antibodies and vice versa or the dis- or reappearance of either anti-23 kD or anti-39 kD antibodies.
*Updated 12 Nov 2013
Audio interview with Robert Giguere from IgeneX:
It’s an hour & a half long but VERY interesting, he talks candidly about the Dearborn convention, their quality controls, strains, immune systems, WB bands, co-infections, treatment, cysts, lyme vaccine & so on. Well worth a listen! http://www.lymeresearchalliance.org/Video/Bob%20Guigere-Edit.mp3
Testing for borrelia / Research by Lyme Research UK & Ireland..
These are summaries based on an extensive review of published research. However, since this is a fast moving area of medicine, use of this information should be supplemented with a full review of the literature where required.
The effect of use of antibiotics on test results
Persistence of Borrelia and sero-negativity
The two tier test for Lyme antibodies (accuracy)
Factors affecting test results (eg immune, genetic etc)
*Updated 16 Aug 2013
New I Spot test on market using same T cell technology as FDA approved test for TB. (LTT/elispot tests use similar technique)
LTT study: The Lymphocyte Transformation Test for Borrelia Detects Active Lyme Borreliosis and Verifies Effective Antibiotic Treatment
LTT study: A novel lymphocyte transformation test (LTT-MELISA) for Lyme borreliosis.
This study uses Elispot technique:
The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT.
*Updated 8 Mar 2013
Improved Culture Conditions for the Growth and Detection of Borrelia from Human Serum
Eva Sapi1,2 Corresponding address, Namrata Pabbati1, Akshita Datar1, Ellen M Davies1, Amy Rattelle1, Bruce A Kuo1
1. Research Division of Advanced Laboratory Services Philadelphia PA, USA;
2. Department of Biology and Environmental Science, University of New Haven, West Haven CT, USA.
Source: Int J Med Sci 2013; 10(4):362-376. doi:10.7150/ijms.5698.
In this report we present a method to cultivate Borrelia spirochetes from human serum samples with high efficiency. This method incorporates improved sample collection, optimization of culture media and use of matrix protein. The method was first optimized utilizing Borrelia laboratory strains, and later by demonstrating growth of Borrelia from sera from fifty seropositive Lyme disease patients followed by another cohort of 72 Lyme disease patients, all of whom satisfied the strict CDC surveillance case definition for Lyme disease. The procedure resulted in positive cultures in 47% at 6 days and 94% at week 16. Negative controls included 48 cases. The positive identification of Borrelia was performed by immunostaining, PCR, and direct DNA sequencing.
Full report available from: http://www.medsci.org/v10p0362.htm
For more on the new Advanced Labs culture test go to: http://lymedisease.org/news/lyme_disease_views/culture-test.html
*Updated 12 Sep 2012
Check out Tick Talk Ireland’s Western Blot for IgG Comparison Chart looking at CDC (US), IgeneX, MiQ 12 2000 for Germany, MiQ 12 2000 for rest of Europe plus Scotland:-
An interesting article about the different types of blood tests and the difference between IgM and IgG – good stuff!!
Dark field microscopy can expose spirochetes:
Samples used are from 4 people, all with chronic illness longer than 15 years and with wide-ranging symptoms and severe disability. All have been diagnosed at some time with M.E. 3 have had negative ELISA/WB tests for Lyme disease (1 not tested). All were very high risk for tick bite before becoming ill due to occupation and/or other activities. 3 have had more than 3 years antibiotic treatment; 2 of these include treatment specifically for borrelia.
Reasons for seronegative Lyme Disease:
SERONEGATIVE LYME DISEASE
1. Recent infection before immune response
2. Antibodies are in immune complexes
3. Spirochete encapsulated by host tissue (i.e.: lymphocytic cell walls)
4. Spirochete is deep in host tissue (i.e.: fibroblasts, neurons, etc.)
5. Blebs in body fluid, no whole organisms needed for PCR
6.No spirochetes in body fluid on day of test
7.Genetic heterogeneity (300 strains, 100 in U.S.)
9.Surface antigens change with temperature
10.Utilization of host protease instead of microbial protease
11.Spirochete in dormancy phase (L-form) with no cell walls
12.Recent antibiotic treatment
13.Recent anti-inflammatory treatment
14.Concomitant infection with babesia may cause immunosuppression
15.Other causes of immunosuppression
16.Lab with poor technical capability for Lyme disease
17.Lab tests not standardized for late stage disease
18.Lab tests labeled “for investigational use only”
19.CDC criteria is epidemiological not a diagnostic criteria
20.Lack of standardized control
21.Most controls use only a few strains as reference point
22.Few organisms are sometimes present
23.Encapsulated by glycoprotein “S-layer” which impairs immune recognition
24. “S”- layer binds to IgM
26.Possible down regulation of immune system by cytokines
27.Revised W.B. criteria fails to include most significant antigens
Grant available for Improved Diagnostics for Lyme Borreliosis (U01)
Center for Disease Contrcol (CDC)
Deadline(s): May 14, 2010
Applications are due April 14, 2010. NOTE: On-time submission requires that applications be successfully submitted to Grants.gov and validated no later than 11:59 PM ET. Refer to this program’s guidelines for detailed submission information.
The purpose of the program is to bring into clinical practice laboratory tests that are superior to two-tiered serologic testing in their utility for diagnosing Lyme borreliosis and are capable of distinguishing active from prior infection. Examples of the types of proposals that might be considered favorably include, but are not limited to, work aimed at developing and evaluating with clinical samples (1) serologic tests that out-perform two-tiered testing, (2) tests capable of detecting evidence of B. burgdorferi infection during early and later stages of Lyme disease, and (3) tests capable of detecting markers of active infection. The purpose and objectives of this program are very specific in nature. The distinct subject matter of this funding opportunity necessitates a thorough review of the official program guidance referenced at the URL provided in the contact section of this summary.
History of Funds
None is available.
Name: Valerie McCloud
Address: U.S. Department of Health and Human Services
Address 2: 2920 Brandywine Road, Mailstop K14
Fax: (770) 488-2044
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. email@example.com
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).
PMID: 20437826 [PubMed – in process]
Interpreting Test Results by Dr Tedde Rinker
First of all, the diagnosis of Lyme disease is a clinical diagnosis. That means that there is no one certain diagnostic test that will give us a “yes/no” answer in every case whether an individual has the disease or not, and that the diagnosis depends not only on the lab, but more importantly, on the clinical history, the environmental likelihood of exposure and the signs and symptoms of disease.
Lyme disease, or borreliosis, is caused by a spiral shaped bacteria (spirochete) called Borrelia Burgdorferi. There are several other species of borrelia (some say over 100 strains in the USA alone!) that also cause the same or similar symptoms throughout the world, but in the U.S., this is the most common but by no means the exclusive pathogen causing what is known as “Lyme disease”. This may be why some people have all the symptoms and have “negative” test results. Another reason is because the bacteria can compromise the immune system over time, and effectively hide from the immune system. The longer one has the infection, the more compromised the immune system, and the more likely it is that antibodies will not be strongly reactive to the bacteria.
For more information go to PDF article at:
Will there ever be an accurate test for Lyme?
Tom Grier examines all the problems with testing including Elisa, WB, DNA/PCR & microscopic examination. At the bottom he has a summary on key words. It is lengthy but I added a shortened summary below:
* The ELISA test is useless within the first four weeks of a tick bite.
* The ELISA may not detect late infection because the bacteria can find immune privileged sites in which to hide.
* The ELISA test is not a standardized test. The design of the test can vary greatly from lab to lab.
* The choice of antigens used in the test is derived from a laboratory strain B-31 instead of the naturally occurring wild strains. The B-31 strain is proving to be highly variable and changing. Using a high passage lab strain may be cheap and convenient, but not an accurate representation of the various strains of Borrelia found in nature.
* The accuracy of the test varies even on identical samples, meaning that even the labs themselves introduce a variable of inaccuracy by poor procedure, interpretation, or quality control.
The Western Blot antibody test has only two slight advantages over the ELISA test. First, it is slightly more sensitive, probably due to the inclusion of more bacterial antigens. Second, it tells us which bacterial proteins are eliciting an antibody response in that patient. However, in the end, the Western Blot still suffers from all the same downfalls as the ELISA.
* Note: A misconception about Western Blots and ELISA tests is that they have as many false positives as false negatives. This is not true. False positives are rare. Negative serologies despite a rash or a positive culture is routine. Remember words like sensitivity, specificity, and accuracy DO NOT MEAN DIAGNOSTICALLY ACCURATE to determine if a patient has an infection. A NEGATIVE TEST CANNOT RULE OUT AN INFECTION THAT HAS ESCAPED THE BLOODSTREAM.
The biggest misunderstanding physicians have about Lyme antibody tests is thinking that a high titer of antibody in a patient means that the patient is more ill than a patient with a low titer of antibodies. What these tests are really measuring is the body’s natural immunity against the bacteria. A patient who is able to mount a strong antibody defense against this pathogen is far better off than a patient who makes little or no antibody. It only stands to reason that a patient with a high infection load and no antibodies is going to be more symptomatic than a patient with a high natural immunity. Unfortunately, most physicians look at higher titers and assume those patients are the most ill, but their reasoning is backwards. Low titers in a highly symptomatic Lyme patient is a very bad thing.
DNA Amplification or PCR (Polymerase Chain Reaction) Tests:
In Lyme disease, the PCR test really has limited value. The main problem is that doctors and labs like to use blood, urine, and spinal fluid to test simply because of the ease of collection. In the case of Lyme disease, however, the borrelia spirochete does not like the blood stream, and PCR tests of fluids are frequently negative while the same PCR tests of skin biopsies in the same patient are positive. This indicates that finding a sample with the bacterium’s DNA is a bit like finding a needle in a haystack.
No single sample from a patient can be diagnostic for Lyme disease using PCR as the test. Even the University of Minnesota reported a mere 18% success rate in early Lyme when skin biopsies were obtained from culture positive bull’s-eye rashes.
Patent disputes often result in poor PCR tests being marketed with little or no sharing of technology between competitors. This competition often leads to over-hyped performance and over hyped accuracy claims by manufacturers.
Often the best tissues for PCR tests are tissue biopsies, which are rarely done because of costs, risk, and inconvenience. In truth, PCR tests are best used in the post-mortem exam on tissues such as the brain, bladder, and heart. As a diagnostic tool, it can be useful when positive, but tells us nothing when negative. I see few patients lining up for a brain biopsy.
Microscopic observation of the organism:
Using a microscope to find the Lyme bacteria is quite literally like looking for a needle in a haystack. The Borrelia burgdorferi organism is found in such low numbers in fluids and tissue that it requires hours and hours of lab time to do an adequate search of just a few millimeters of tissue. As a research tool biopsy and stain is useful, but to use microscopy to diagnose Lyme is too slow, too costly, and quite unreliable.
One of the biggest misconceptions about Lyme disease antibody tests, which has led to years of unnecessary morbidity and mortality for Lyme disease patients, is the insistence that the absence of Lyme antibodies means the absence of active infection. You cannot equate the absence of antibodies with the absence of infection, nor can you use antibody serologies as an endpoint in treatment studies to determine the effectiveness of any treatment regimen. It isn’t just a bad idea – it is just plain bad science.
Will there ever be a Lyme test that can be used dependably to diagnose Lyme disease? I don’t believe such a test is within our current technology, and until such a test exists denying patients treatment using our current tests is bad medicine.
Key words and concepts used in this article:
Systemic: Borrelia burgdorferi and other spirochetes in the tick-borne relapsing fever family of bacteria circulate through the blood stream to the entire body. They can find passage through blood vessel walls and invade other tissues and organs, including known target tissues such as skin, tendons, joints, heart, nerves, and brain.
Sequestered: The Lyme spirochete can find haven in areas of the body that are poorly protected by the immune system (the brain), have poor areas of blood circulation (tendons), or where the bacteria can lay metabolically inactive for lengths of time and resist antibiotic penetration (the skin and the brain).
Multi-systemic: The bacteria has affected more than one system of the body, such as: Peripheral nervous system, skin, joints and connective tissues, cardiovascular system and circulatory system, eyes, muscles, liver and spleen, and central nervous system.
Blood Brain Barrier (BBB): The network of capillaries surrounding the brain that selectively let things in and out of the brain. A healthy BBB prevents infections, white blood cells, and many medicines from entering the brain. A breakdown of the BBB is not a good thing, and occurs very early in most animal models of Lyme disease.
Antigen: A protein usually on the surface of the bacteria that stimulates the immune system to make antibodies against that protein. Antigens can also stimulate other immune responses, such as T-cell and macrophage responses.
Serologies: Using the centrifuged serum extracted from blood to look for antibodies against the Lyme bacterium.
Titer: A measurement of antibody content in the serum. Titers are usually reported as dilution series. The higher the dilution of serum where antibodies are still detectable means there are more antibodies present in that patient’s serum. The cut off for reporting a positive is a bit arbitrary and varies from lab to lab. Institutions conservative on the diagnosis of Lyme have raised their cutoffs from 1:256 to 1:1024 Which is rare to see in most Lyme patients.
Infection Load: The actual number of bacteria in a host. If the bacteria remain in the bloodstream, the number of bacteria per unit of blood can be calculated, but this number is meaningless if the infection has moved beyond the bloodstream. In Lyme disease, there is no accurate way of determining true infection load. If the bacteria out pace antibody production then there is no FREE ANTIBODY only ANTIBODY COMPLEXES.
For a more detailed view hop along to:
Lyme disease isn’t always diagnosed by a test
says Linda Olley – registered nurse and Lyme sufferer for 26 years..
Early Lyme disease with spirochetemia – diagnosed by DNA sequencing
Sin Hang Lee email, Veronica S Vigliotti email, Jessica S Vigliotti email, William Jones email, Jessie Williams email and Jay Walshon email
BMC Research Notes 2010, 3:273
Published: 1 November 2010
A sensitive and analytically specific nucleic acid amplification test (NAAT) is valuable in confirming the diagnosis of early Lyme disease at the stage of spirochetemia.
Venous blood drawn from patients with clinical presentations of Lyme disease was tested for the standard 2-tier screen and Western Blot serology assay for Lyme disease, and also by a nested polymerase chain reaction (PCR) for B. burgdorferi sensu lato 16S ribosomal DNA. The PCR amplicon was sequenced for B. burgdorferi genomic DNA validation. A total of 130 patients visiting emergency room (ER) or Walk-in clinic (WALKIN), and 333 patients referred through the private physicians’ offices were studied. While 5.4% of the ER/WALKIN patients showed DNA evidence of spirochetemia, none (0%) of the patients referred from private physicians’ offices were DNA-positive. In contrast, while 8.4% of the patients referred from private physicians’ offices were positive for the 2-tier Lyme serology assay, only 1.5% of the ER/ WALKIN patients were positive for this antibody test. The 2-tier serology assay missed 85.7% of the cases of early Lyme disease with spirochetemia. The latter diagnosis was confirmed by DNA sequencing.
Nested PCR followed by automated DNA sequencing is a valuable supplement to the standard 2-tier antibody assay in the diagnosis of early Lyme disease with spirochetemia. The best time to test for Lyme spirochetemia is when the patients living in the Lyme disease endemic areas develop unexplained symptoms or clinical manifestations that are consistent with Lyme disease early in the course of their illness.
Trinity Biotech states that a negative result should NOT be used to exclude diagnosis:
‘The diagnosis of Lyme disease should be made based on history and symptoms (such as erythema migrans), and other laboratory data, in addition to the presence of antibodies to Borrelia afzelii, Borrelia garinii and B.burgdorferi(B-31) including the VlsE protein. Negative results (either first or second-tier) should not be used to exclude Lyme disease.
Patients in the early stage of disease and a portion of patients with late manifestations may not have detectable antibodies. Early antimicrobial treatment, after appearance of EM may lead to diminished antibody concentrations. Serologic tests have been shown to have low sensitivity and specificity and, therefore, cannot be relied upon for establishing a diagnosis of Lyme disease.’
Links on seronegative Lyme can be found at:
Blood tests may not pick up all strains & variations of Lyme:
For instance borrelia lonetstari (STARI):
Master’s Disease (aka STARI) was described in the early 1990’s by Dr. Ed Masters of Cape Girardeau, MO, when a number of people were bitten by lone star ticks and developed Lyme disease symptoms. Many patients had a classic rash, nearly identical to the one described in people infected with the more well known strain of Lyme, however, the standard Lyme disease tests were not able to detect evidence of the infection in humans.
In addition Lyme has many strains involved which may not be picked up in current testing:
Benjamin Luft, M.D., Professor of Medicine, Stony Brook University Medical Center, and a team of medical researchers have determined the genetic blueprint of 13 strains of the bacteria that cause Lyme disease.
“By characterizing every gene in the Lyme disease agents family, we have a blueprint of every possible characteristic of the organism,” says Dr. Luft, senior author on the study. “This is the building block to developing more accurate and effective diagnostic tests, therapeutic agents and vaccines.”
Dr. Luft and colleagues point out in the study that improved diagnostics are needed because the best clinical sign of Lyme disease, the erythema migrans skin rash, does not always occur in patients. In addition, diagnostic assays and vaccines developed before their blueprint of the entire genome of B. burdorferi have had less than satisfactory results.
Scottish lab identifies that Western Blot bands 20, 28 and 48 kDa bands should be regarded as Lyme specific.
Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity
J Clin Pathol 2009;62:552-554 doi:10.1136/jcp.2008.063461
1.National Lyme Borreliosis Testing Laboratory, Microbiology Department, Raigmore Hospital, Inverness, UK
1. S Mavin, National Lyme Borreliosis Testing Laboratory, Microbiology Department, Raigmore Hospital, Old Perth Road, Inverness IV2 3UJ, UK; firstname.lastname@example.org
* Accepted 11 February 2009
* Published Online First 23 February 2009
Aims: This study evaluates the use of local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen for in-house IgG western blots in the routine diagnostic setting by comparing it with the current in-house protocol.
Methods: Sera from 233 patients from areas of Scotland with low and high prevalence for Lyme borreliosis were tested by western blots prepared from reference strain antigen (B burgdorferi sensu stricto) and mixed antigen (B burgdorferi sensu stricto and B afzelii). Results were scored using original and revised criteria, and results were compared.
Results: The mixed antigen produced significantly more bands than the reference antigen. Using the original interpretation criteria the mixed antigen produced more positive results than the reference antigen (90 versus 85). When the revised criteria were applied to the mixed antigen there were 14 more patients with positive results than with the reference antigen (99 versus 85); this difference was significant.
Conclusions: The mixed antigen and revised interpretation criteria have been successfully incorporated into the routine diagnostic testing service, increasing the sensitivity of the in-house IgG western blot test for Scottish patients.
An extremely good site comparing different labs for Lyme & co-infection testing. PS one lab not mentioned is BCA labs in Germany who do Lyme, co-infections & CD57..
Serodiagnosis of Borreliosis: Indirect Immunofluorescence Assay, Enzyme-Linked Immunosorbent Assay and Immunoblotting.
Arch Immunol Ther Exp (Warsz). 2011 Jan 22. [Epub ahead of print]
Wojciechowska-Koszko I, Mączyńska I, Szych Z, Giedrys-Kalemba S.
Department of Microbiology and Immunology, Pomeranian Medical University, Powstańców Wielkopolskich 72, 70-111, Szczecin, Poland, IwonaKoszko@interia.pl.
Lyme disease is an infectious, multi-system, tick-borne disease caused by genospecies of Borrelia burgdorferi bacteria sensu lato, characterized by remarkable heterogeneity. In this situation choosing an optimal antigen array for diagnostic tests seems problematic. The serological tests for borrelia routinely done in laboratories often produce ambiguous results, which makes a proper diagnosis rather complicated and thus delays the implementation of an appropriate treatment regimen. Thirty-seven outpatients and eight inpatients with suspected borreliosis diagnosis hospitalized at the Clinics of the Pomeranian Medical University (Szczecin, Poland), participated in the study. In order to detect the antibodies against Borrelia sensu lato three kinds of serological tests were used: indirect immunofluorescence assay (IIFA), enzyme-linked immunosorbent assay (ELISA), and immunoblot. The IIFA and immunoblot tests conducted on 45 patients (100%) produced positive results for both the IgM and IgG antibody types. In the case of ELISA, positive or borderline results were observed in only 24 patients (53.3%). The immunoblot test for IgM most frequently detected antibodies against the outer surface protein C (OspC) antigen (p25), and, in the case of IgG, against the recombinant variable surface antigen (VlsE). The IIFA screening test used for diagnosing Lyme borreliosis produced the highest percentage of positive results, which were then confirmed by immunoblot, but not by ELISA. Therefore using only ELISA as a screening test or for diagnosing Lyme borreliosis seems debatable.
PMID: 21258869 [PubMed – as supplied by publisher]
CD57: Critical update 2nd December 2010
We now know we are specifically interested in
CD3- CD57+ “NK” cells
Please note there are also CD57+ T cells but these are helper cells
Normal CD57 NK cell count is >180
>120 as treatment ends has a good prognosis
20-60 chronic Lyme disease
<20 severe illness
This data is quoted form the following presentation:-
LYME DISEASE THE NUTS & BOLTS
BY JOSEPH T. BURRASCANO, JR, MD
presented July 2010 in DePere, WISCONSIN
CD-57 COUNT (Natural Killer Cells) • Low counts seen in Chronic Lyme when the infection has been active > 1 year
• Reflects degree of infection
• CAN BE A SCREENING TEST!
• Predicts a relapse if low when antibiotics end
• Must use method of LabCorp (normal is >180)
– 60- Lyme activity minimal
– >120- Relapse NOT likely after treatment ends
Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.
Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T.
Eur J Clin Microbiol Infect Dis. 2011 Jan 27. [Epub ahead of print]
VUMC, Amsterdam, The Netherlands, email@example.com.
We investigated the influence of assay choice on the results in a two-tier testing algorithm for the detection of anti-Borrelia antibodies. Eighty-nine serum samples from clinically well-defined patients were tested in eight different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell antigens, whole-cell antigens supplemented with VlsE and assays using exclusively recombinant proteins. A subset of samples was tested in five immunoblots: one whole-cell blot, one whole-cell blot supplemented with VlsE and three recombinant blots. The number of IgM- and/or IgG-positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34 to 59%. The percentage of positives in cross-reactivity controls ranged from 0 to 38%. Comparison of immunoblots yielded large differences in inter-test agreement and showed, at best, a moderate agreement between tests. Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISAs. The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISA-immunoblot combination. We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity. The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.
PMID: 21271270 [PubMed – as supplied by publisher]
Doubts shared on Western Blot Lyme test:
HIGHLY SENSITIVE AND SPECIFIC LOW-COST LAB-ON-A-CHIP SYSTEM FOR LYME DISEASE DIAGNOSIS
The aim of HILYSENS is to develop a novel lab-on-chip diagnostic tool to improve clinical diagnostic, disease monitoring and treatment of Lyme Disease by enabling specific and sensitive detection of the human serological response against its causative agent: Borrelia burgdoferi. Lyme Disease is the most common tick-borne infection in Europe with around 85,000 new cases per year and its incidence is increasing due to climate change. Current laboratory diagnostic methods lack sensitivity and specificity to detect early cases as well as late manifestations of the disease such as chronic or autoimmune-related infections. For this reasons, disease incidence is underestimated as many cases go mis- or undiagnosed. Late, delayed, or inadequate treatment can lead to serious symptoms such as neuroborreliosis or arthritis, which can be disabling and difficult to treat.
HILYSENS will develop a specific multi-antigen assay in a lab-on-a-chip device to detect Borrelia infections.
HILYSENS will provide a compact and robust lab-on-chip system, designed to work with small volumes and without the need of expert operators. This tool together with a portable reader and user-friendly software will enable more precise, accurate and reproducible testing making it possible to become the standard tool for the disease diagnosis. A novel production approach for the lab-on-a-chip will be developed which will enable to target really low mass-production costs, overcoming one of the main issues of the lab-on-a-chip technology and opening great market opportunities for the participant SMEs. Sensitive and reliable patient diagnosis provided by HILYSENS device will optimise resources available to medical practitioners, heavily reducing the current costs of the disease, increasing profitability and most importantly, patients’ quality of life.
NB: A new site Hilysens II is available inc a video of the new technique at: http://hilysensproject.eu/
The Complexities of Lyme Disease
A Microbiology Tutorial: Part 1
By Thomas M. Grier, MS
There’s a Lyme test, so what’s the problem? There are several Lyme tests, but most of them are dependent on the body’s ability to make antibody against this bacteria. As we have seen, this may be a problem. There is the S-layer protecting the bacteria; the surface antigens are not readily exposed; there may be a blocking antibody; the bacteria might be inside a human cell; the bacteria might be down regulating the immune system through cytokines; the bacteria might have altered its antigenic appearance to fool the immune system; the bacteria might be cloaked in B-cell membrane; the bacteria might be hiding in joints, tendons, white blood cells, skin cells or the brain. Remember, if even just one spirochete survives, it could cause a relapse. Then there is another problem – the tests that detect antibody can only detect free uncomplexed antibody. (23,25,55,70)
When an antibody is formed, it is meant to latch on to something and never let go until it is destroyed. Like a lock and key, antibodies fit their associated antigens. Once the antibody attaches to the antigen it is no longer is a detectable antibody, because it has now become an antibody-antigen complex. This complex is not measurable using today’s commercially available tests. Also, as the amount of antigen increases, the amount of antibody can decrease, because the antigen will trap out the available antibody and sequester it. So, a person who has a bad infection but is making a limited amount of antibody can be overwhelmed by antigen, thus making antibody detectable only if you can detect the complex.
This means: People who have the worst infections may have the lowest antibody titers, and test negative. Note: It takes four weeks from the tick bite to test positive.
There are two main categories of Lyme tests. The most common and least specific is the Enzyme Linked Immune Sera Assay or ELISA, the other is an Immuno Blot or Western Blot. The Western Blot essentially makes a map of the different antibodies we make to the bacteria. The map separates the antibodies by size and weight, and is reported in units called kilo daltons or kDa. For example, a Western Blot may report bands at 22, 25, 31, 34, 39, and 41 kDa. Each of these bands represents an antibody response to a specific protein found on the spirochete. The 41 band indicates an antibody to the flagella protein, and is non-specific. The 31-kDa band represents the OSP-A protein and is specific for Borrelia, as is the 34 band OSP-B and 25 kDa OSP-C.
In 1994, the NIH decided that there should be consistency between labs reporting Lyme Disease Western Blots, and that a specific reporting criteria should be established. This sounds good, but one could argue they made a bad situation worse. The consensus committee decided to set the standards for a positive test based on the number of bands that appear. Whereas every lab prior to the hearing had accepted bands 25, 31, and 34 as specific and significant, the NIH, without any clear reasoning, disqualified those bands from being reportable. The result was that what had been a fair good test had now become poor or even useless. (90)
How badly did the NIH bootstrap this test? The following is an analysis of the new guidelines presented as an abstract and lecture at the 1995 Rheumatology Conference in Texas. (1995 Rheumatology Symposia Abstract # 1254 Dr. Paul Fawcett et al.)
This was a study designed to test the recently proposed changes to Western Blot Interpretation. At the Second National Conference on Serological Testing for Lyme Disease, sponsored by the NIH, the committee proposed limiting the bands that could be reported in a Western Blot for diagnosis of Lyme Disease. An IgG Western Blot must have five or more of these bands: 18, 23,28, 30, 39, 41, 45, 58, 66, and 93 kDa. An IgM Western Blot must have two or more bands of the following three bands: 23, 39, 41. Conspicuously absent are the most important bands, 22, 25, 31 and 34, which include OSP-A, OSP-B and OSP-C antigens – the three most widely accepted and recognized antigens. These antigens are so immuno-reactive that they were the antigens chosen for human vaccine trials. Yet they are not considered important enough to include in the diagnostic criteria. Why?
This abstract showed that, under the old criteria, all of 66 pediatric patients with a history of a tick bite and Bull’s Eye rash who were symptomatic were accepted as positive under the old Western Blot interpretation. Under the newly proposed criteria, only 20 were now considered positive. That means 46 children who were all symptomatic would probably be denied treatment! That’s a success rate of only 31 %. The number of false positives under both criteria was ZERO %. * Note: A misconception about Western Blots is that they have as many false positives as false negatives. This is not true. False positives are rare. The conclusion of the researchers was: “the proposed Western Blot Reporting Criteria are grossly inadequate, because it excluded 69% of the infected children.”
We are told by manufacturers, health departments, and clinics that the Lyme ELISA tests are good and that they are useful, but in two blinded studies that tested laboratories accuracy, they failed miserably. In the latest study, 516 labs were tested. The overall result: 55% inaccurate! You are actually better off to flip a coin! (98, 99)
Repeatedly, there have been patients who are seronegative for antibodies, yet culture positive. Despite this, our medical community is dependent on these tests and relies upon them as though they were 100 % accurate. No matter how bad the tests are, as long as we have them doctors will use them. This is why doctor Samual Donta, M.D., called for a complete ban of the Lyme ELISA test at the 1996 LDF Lyme Conference. He found that, in some cases, Lyme ELISAs were more than 75 % inaccurate, yet it was relied upon as though it were the last word – and all too often it is.
The worst problem for chronic Lyme patients is that, after they are treated with antibiotics, they are told they are cured even if they have a recurrence of symptoms. There is a persistent dogma in medicine that 28 days of IV antibiotics cures all Lyme Disease. In fact, the ongoing six-year-old Nantucket Island Lyme Treatment Study showed IV antibiotics to have the highest relapse rate in late Lyme disease! This was because doctors put too much faith in IV antibiotics as being so powerful, that they did not follow up IV’s with oral antibiotics. The key to treating late Lyme appears to be the length of antibiotic treatment, not the method. If IV’s are followed up by six months or more of oral antibiotics, the relapse rate dropped to 13%. (Dr. Leslie Fein MD, MPH, Magnarelli MD, MPH 96 LDF Conference)
I have included in the references several published studies, case histories and abstracts that deal with culture positive patients who had been previously treated aggressively with antibiotics, often including intravenous antibiotics. Most of these cases are patients who are seronegative for any Lyme antibodies, yet are culture positive. If we are repeatedly culturing this bacteria out of patients who have been treated and who are negative by all other tests, we need to rethink our understanding of this disease! We need to treat symptoms, not tests; we need to recognize that, while Lyme is a treatable disease, in some cases it appears to be incurable. I would not like to be the doctor who under treats this disease, now knowing that relapses are potentially more dangerous than treating until the symptoms are gone. (4,6,42,49,67,68,70-96) (Lawrence C, Lipton RB, Lowy FD, and Coyle PK. Seronegative Chronic Relapsing Neuroborreliosis. European Neurology. 1995; 35(2): 113-117)
Too often, I have seen the word cured used in Lyme Disease Studies, only to find that the researchers have redefined the word cure to mean seronegative. Seronegativity is not synonymous with cure. The numerous culture positive cases in recent years should have negated that kind of logic years ago, and yet, in 1997, researchers are still publishing studies that use antibodies and PCR as the end point for cure. It’s time to ask the patients one simple question: How are you feeling?
So, let’s say hypothetically you are bitten by an infected tick, you get a rash, you get sick, and you have a positive test. So you get 2-4 weeks of antibiotics and you get better, but then you get sick again. No problem! You go back to your doctor, and he says, “Well, we’d better give you another Lyme test” – and its negative. Why? Even though you have an active infection, the antibiotics cleared that infection from your blood stream. That is where your immune system is. The rest of the pathogens are hiding from the immune system inside your joints, your tendons, and your brain. Only now you don’t have antibiotics to fight the infection, or any antibodies!
In a study by Dr. Musher, M.D., he looked at incompletely treated Tertiary Syphilis patients, and compared them to those Tertiary Syphilis patients who never got antibiotics. He found that the incompletely treated group went into dementia faster.
Why? Because they had no natural immunity left. Their ability to make sufficient antibody was diminished, because the antibiotics eliminated the stimulus from the blood stream, but the infection was still hidden in the brain! (35,61,62,65,74,83)
Conclusion: Lyme is an extremely complex disease that can cause long term chronic infections. Patients can be seronegative, yet culture positive (even after aggressive antibiotic therapy). The infection enters the brain early in the infection (within days). The sequestered bacteria within the CNS can be so different from the initial infection that serum antibodies are ineffective. Incomplete antibiotic treatment of Lyme Encephalitis can harm the patient.
Seronegative chronic relapsing neuroborreliosis.
Eur Neurol. 1995;35(2):113-7.
Lawrence C, Lipton RB, Lowy FD, Coyle PK.
Department of Medicine, Albert Einstein College of Medicine, New York, N.Y., USA.
* Eur Neurol. 1996;36(6):394-5.
We report an unusual patient with evidence of Borrelia burgdorferi infection who experienced repeated neurologic relapses despite aggressive antibiotic therapy. Each course of therapy was associated with a Jarisch-Herxheimer-like reaction. Although the patient never had detectable free antibodies to B. burgdorferi in serum or spinal fluid, the CSF was positive on multiple occasions for complexed anti-B. burgdorferi antibodies, B. burgdorferi nucleic acids and free antigen.
PMID: 7796837 [PubMed – indexed for MEDLINE]
Interpretation criteria in Western blot diagnosis of Lyme borreliosis
ORIGINAL ARTICLES: Br J Biomed Sci 2011; 68(1); 5-10
S. Mavin, S. McDonagh, R. Evans, R. M. Milner, J. M. W. Chatterton, D. O. Ho-Yen
National Lyme Borreliosis Testing Laboratory, Microbiology Department, Raigmore Hospital, Old Perth Road, Inverness IV2 3UJ, UK
This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P<0.0001). In total, 76 patients who were negative/equivocal became positive, which may have led to a change in their management. Conversely, 33 patients who were weak-positive became equivocal but their management may not have been affected. The authors believe that the revised criteria have simplified blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients.
Letter to the Editor
American College of Physicians
Independence Mall West
6th St. At Race
To Whom It May Concern,
We have several concerns with the report entitled Guidelines for Laboratory Evaluation in the Diagnosis of Lyme Disease (Annals Int Med 127, 1106-1123. 1997)
* The Centers for Disease Control have developed a set of clinical and diagnostic criteria for surveillance purposes. The authors of these “Guidelines” state, with no substitution, that this criteria is also applicable to the clinical diagnosis of Lyme disease. To our knowledge, no such evidence exists. It would appear that the published “Guidelines” have as a basis, a clinical criteria for Lyme disease diagnosis which has never been tested except for clinical studies published by the authors themselves. There is no external validation to support the claim of equivalence between clinical diagnostic criteria and the CDC surveillance definition.
* The authors state that “testing with ELISA is the cornerstone of laboratory diagnosis for Lyme disease”. In fact, it is not. The commercially available ELISA assays for Lyme Disease do not meet acceptable criteria according to the group that is responsible for much of the United States Laboratory proficiency testing program in Lyme Disease. The data, from a recent study by Bakken et.al. (J Clin Microbiol 35:537-543,1997), indicated “that” the sensitivity and specificity of the currently used tests for Lyme Disease are not adequate to meet the two-tier test approach being recommended by the CDC/ASTPHLD group”. Bakken et. al. (1997) also stated that a screening test must have >95% sensitivity to adequately screen for Lyme disease and that the currently available ELISA tests do not perform at that level.
* The authors have not followed the CDC/ASTPHLD recommendation for 2-tiered testing. That is, all indeterminate and reactive ELISAs should be reflexed to Western blot (WB), not just indeterminate ELISAs as the authors of the “Guidelines” suggest. Certainly the authors realize that reactive Lyme ELISA results may be non-specific because of a number of cross reacting antibodies (e.g. antibody to the 41 Kda flagellin protein).
The authors have missed some important studies of Western blotting, especially those that may be critical of the recommendations of Dressler et. al. (J. Infect. Dis. 167, 392-400,1993) and the CDC/ASTPHLD. The report by Engstrom et al (J Clin Microbiol 33:419-427, 1995) found, for example, that 20% of their Lyme patients remained seronegative throughout the study and that fewer bands on the IgG WB could be appropriately used for interpretation. They noted that the WB was more specific and more sensitive than the ELISA. Their study also showed that only 19% of patients treated with antibiotics for 20 days still had a positive ELISA antibody response after one year, yet almost 60% of their patients continued to be WB positive at the end of the first year. The study by Aguero- Rosenfeld et al (J Clin Microbiol 34:1-9, 1996) reported that 89% of patients, with culture-confirmed erythema migrans (EM), developed specific IgG antibodies by WB, but only 22% of these patients were positive by the interpretive criteria proposed by the CDC/ASTPHLD. They further reported that the duration of the antibody response was related to the duration of the EM. Tilton et. al. (1997) stated that a highly sensitive and specific Western blot is desirable for a 2 tiered test approach or as a primary test. Despite the CDC/ASTPHLD recommendations, many physicians who treat patients for L.D. do not believe that an ELISA is an appropriate screening test and consequently use the Western blot as a primary test.
The authors state that patients not be tested for Lyme Disease unless the pretest probability of disease is between 0.20 and 0.80. These recommendations will:
a) rule out any laboratory detection of B. burgdorferi antibodies, antigens, and/or DNA in non-endemic areas
b) require physicians to screen patients based on epidemiological data which may not be available to them outside their own local area require physicians to know the performance characteristics of a wide variety of Lyme disease tests.
The authors in their comment on non-antibody based tests have chosen to overlook a number of publications on the utility of direct detection tests for Lyme disease. A comprehensive review of molecular techniques for diagnosis of Lyme Disease has recently been published by Schmidt (Clin Micro Rev 10, 185-201, 1997). They state “evidence is growing that a positive PCR test can be associated with active disease after adequate therapy, PCR results are usually negative” Manak et. al. (J Spirochet Tick Borne Dis 4., 11-20. 1997) in a well controlled study using the CDC criteria for selection of patients indicated that PCR on serum, plasma, or buffy coat could be effectively used to monitor the efficacy of therapy. Similarly, Harris et. al. (J Spirochet Tick Borne Dis 237, 1995) have validated the Lyme urinary antigen test (LUAT) in more than 700 LD patients and controls. The LUAT has a specificity of >95%. These “Guidelines” only complicate an already complex disease diagnostic process.
Richard C. Tilton, Ph.D.
Diplomate American Board Medical Microbiology
BBI Clinical Laboratories, Inc.
75 North Mountain Rd.
New Britain, CT 06053
Mary N. Sand, Ph.D.
BBI Clinical Laboratories, Inc.
75 North Mountain Rd.
New Britain, CT 06053
Nick S Harris, Ph.D.,
Diplomate American Board Medical Laboratory Immunology
IGeneX Inc. Reference Laboratory
797 San Antonio Road
Palo Alto, CA
The difference between Elisa & Western Blot illustrated:
Lyme treating doctor explains more on blood testing including the Elisa & WB:
Great list of seronegativity in Lyme Disease
ELISA and Western Blot: Lies that can kill you? Avon, Ohio
By Tom Grier
Undertstanding Lyme Tests (Elisa & WB explained)
Seronegative or False Negative Lyme Disease – An Annotated Bibliography
Tick talk’s file on seronegativity & persistence
Check out our blood testing information at: