Diagnostic Tests For Lyme Disease Research Articles

Challenges of laboratory diagnosis of Lyme disease ‒ Questionnaire survey results.

The accuracy in diagnostic testing for Lyme disease in the early stages of infection is an important factor necessary for delivering proper treatment to patients. Atotal of 173 individuals with confirmed Lyme disease or with laboratory testing underway participated in the quantitative survey. ELISA was the first test conducted in 51% of the respondents, 28% of whom yielded positive findings of both IgM and IgG antibody classes. The positivity of ELISA test findings was confirmed by Western blot in 100% of results. Negative results of ELISA were consistent with Western blot only in less than half of the patients. More than half of the respondents had not been tested for any bacterial or viral co-infections. The results of serological testing were not consistent with clinical findings in all cases, including those with clinically discernible skin manifestation of erythema migrans. The comparison of results obtained by ELISA and Western blot revealed significant discrepancies. Simultaneous infections by vectors with several pathogens were detected (Tab. 3, Fig. 2, Ref.15).

Adaptive Immune Response Investigation in Lyme Borreliosis.

To diagnose Lyme Borreliosis, it is advised to use an enzyme-linked immunosorbent test to check for serum antibodies specific for Lyme and all tests with positive or ambiguous enzyme-linked immunosorbent assay (ELISA) results being confirmed by immunoblot. This method of measuring the humoral immunity in human fluids (e.g., by ELISA) has provided robust and reproducible results for decades and similar assays have been validated for monitoring of B cell immunity. These immunological tests that detect antibodies to Borrelia burgdorferi are useful in the diagnosis of Borreliosis on a routine basis. The variety of different Borrelia species and their different geographic distributions are the main reasons why standards and recommendations are not identical across all geographic regions of the world. In contrast to humoral immunity, the T cell reaction or cellular immunity to the Borrelia infection has not been well elucidated, but over time with more studies a novel T cell-based assay (EliSpot) has been developed and validated for the sensitive detection of antigen-specific T cell responses to B. burgdorferi. The EliSpot Lyme assay can be used to study the T cell response elicited by Borrelia infections, which bridges the gap between the ability to detect humoral immunity and cellular immunity in Lyme disease. In addition, detecting cellular immunity may be a helpful laboratory diagnostic test for Lyme disease, especially for seronegative Lyme patients. Since serodiagnostic methods of the Borrelia infection frequently provide false positive and negativeresults, this T cell-based diagnostic test (cellular assay) may help in confirming a Lyme diagnosis. Many clinical laboratories are convinced that the cellular assay is superior to the Western Blot assay in terms of sensitivity for detecting the underlying Borrelia infection. Research also suggests that there is a dissociation between the magnitude of the humoral and the T cell-mediated cellular immune responses in the Borrelia infection. Lastly, the data implies that the EliSpot Lyme assay may be helpful to identify Borrelia infected individuals when the serology-based diagnostic fails to do so. Here in this chapter the pairing of humoral and cellular immunity is employed to evaluate the adaptive response in patients.

Biomarker selection and a prospective metabolite-based machine learning diagnostic for lyme disease

We provide a pipeline for data preprocessing, biomarker selection, and classification of liquid chromatography–mass spectrometry (LCMS) serum samples to generate a prospective diagnostic test for Lyme disease. We utilize tools of machine learning (ML), e.g., sparse support vector machines (SSVM), iterative feature removal (IFR), and k-fold feature ranking to select several biomarkers and build a discriminant model for Lyme disease. We report a 98.13% test balanced success rate (BSR) of our model based on a sequestered test set of LCMS serum samples. The methodology employed is general and can be readily adapted to other LCMS, or metabolomics, data sets.

A Review of Post-treatment Lyme Disease Syndrome and Chronic Lyme Disease for the Practicing Immunologist.

Lyme disease is an infection caused by Borrelia burgdorferi sensu lato, which is transmitted to humans through the bite of an infected Ixodes tick. The majority of patients recover without complications with antibiotic therapy. However, for a minority of patients, accompanying non-specific symptoms can persist for months following completion of therapy. The constellation of symptoms such as fatigue, cognitive dysfunction, and musculoskeletal pain that persist beyond 6months and are associated with disability have been termed post-treatment Lyme disease syndrome (PTLDS), a subset of a broader term "chronic Lyme disease." Chronic Lyme disease is a broad, vaguely defined term that is used to describe patients with non-specific symptoms that are attributed to a presumed persistent Borrelia burgdorferi infection in patients who may or may not have evidence of either previous or current Lyme disease. The diagnoses of chronic Lyme disease and of PTLDS have become increasingly relevant to the practice of immunologists due to referrals for consultation or for intravenous immunoglobulin (IVIG) treatment. This review aims to explore the relationship between chronic Lyme disease, post-treatment Lyme disease syndrome, and the immune system. Here, we review the current literature on (1) issues in conventional and alternative diagnostic testing for Lyme disease, (2) the hypothesis that B. burgdorferi infection can persist despite appropriate use of recommended antibiotics, (3) current theories regarding B. burgdorferi's role in causing both immune dysregulation and protracted symptoms, and (4) the use of IVIG for the treatment of Lyme disease.

Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species.

Lyme disease, initially described as Lyme arthritis, was reported before nucleic-acid based detection technologies were available. The most widely used diagnostic tests for Lyme disease are based on the serologic detection of antibodies produced against antigens derived from a single strain of Borrelia burgdorferi. The poor diagnostic accuracy of serological tests early in the infection process has been noted most recently in the 2018 Report to Congress issued by the U.S. Department of Health and Human Services Tick-Borne Disease Working Group. Clinical Lyme disease may be caused by a diversity of borreliae, including those classified as relapsing fever species, in the United States and in Europe. It is widely accepted that antibiotic treatment of Lyme disease is most successful during this critical early stage of infection. While genomic sequencing is recognized as an irrefutable direct detection method for laboratory diagnosis of Lyme borreliosis, development of a molecular diagnostic tool for all clinical forms of borreliosis is challenging because a “core genome” shared by all pathogenic borreliae has not yet been identified. After a diligent search of the GenBank database, we identified two highly conserved segments of DNA sequence among the borrelial 16S rRNA genes. We further developed a pair of Borrelia genus-specific PCR primers for amplification of a segment of borrelial 16S rRNA gene as a “core genome” to be used as the template for routine Sanger sequencing-based metagenomic direct detection test. This study presented examples of base-calling DNA sequencing electropherograms routinely generated in a clinical diagnostic laboratory on DNA extracts of human blood specimens and ticks collected from human skin bites and from the environment. Since some of the tick samples tested were collected in Ireland, borrelial species or strains not known to exist in the United States were also detected by analysis of this 16S rRNA “core genome”. We recommend that hospital laboratories located in Lyme disease endemic areas begin to use a “core genome” sequencing test to routinely diagnose spirochetemia caused by various species of borreliae for timely management of patients at the early stage of infection.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease.

Next-generation sequencing approaches have revolutionized genomic medicine and enabled rapid diagnosis for several diseases. These approaches are widely used for pathogen detection in several infectious diseases. Lyme disease is a tick-borne infectious disease, which affects multiple organs. The causative organism is a spirochete, Borrelia burgdorferi, which is transmitted by ticks. Lyme disease can be treated easily if detected early, but its diagnosis is often delayed or is incorrect leading to a chronic debilitating condition. Current confirmatory diagnostic tests for Lyme disease rely on detection of antigens derived from B. burgdorferi, which are prone to both false positives and false negatives. Instead of focusing only on the human host for the diagnosis of Lyme disease, one could also attempt to identify the vector (tick) and the causative organism carried by the tick. Since all ticks do not transmit Lyme disease, it can be informative to accurately identify the tick from the site of bite, which is often observed by the patient and discarded. However, identifying ticks based on morphology alone requires a trained operator and can still be incorrect. Thus, we decided to take a molecular approach by sequencing DNA and RNA from a tick collected from an individual bitten by the tick. Using next-generation sequencing, we confirmed the identity of the tick as a dog tick, Dermacentor variabilis, and did not identify any pathogenic bacterial sequences, including Borrelia species. Despite the limited availability of nucleotide sequences for many types of ticks, our approach correctly identified the tick species. This proof-of-principle study demonstrates the potential of next-generation sequencing in the diagnosis of tick-borne infections, which can also be extended to other zoonotic diseases.

The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research.

There has been an increasing incidence of Lyme disease (LD) in Canada and the United States corresponding to the expanding range of the Ixodes tick vector and Lyme disease agent (Borrelia burgdorferi sensu stricto). There are many diagnostic tests for LD available in North America, all of which have some performance issues, and physicians are concerned about the appropriate use and interpretation of these tests. The objective of this systematic review is to summarize the North American evidence on the accuracy of diagnostic tests and test regimes at various stages of LD. Included in the review are 48 studies on diagnostic tests used in North America published since 1995. Thirteen studies examined a two-tier serological test protocol vs. clinical diagnosis, 24 studies examined single assays vs. clinical diagnosis, 9 studies examined single immunoblot vs. clinical diagnosis, 7 studies compared culture or PCR direct detection methods vs. clinical diagnosis, 22 studies compared two or more tests with each other and 8 studies compared a two-tiered serological test protocol to another test. Recent studies examining the sensitivity and specificity of various test protocols noted that the Immunetics® C6 B. burgdorferi ELISA™ and the two tier approach have superior specificity compared to proposed replacements, and the CDC recommended western blot algorithm has equivalent or superior specificity over other proposed test algorithms. There is a dramatic increase in test sensitivity with progression of B. burgdorferi infection from early to late LD. Direct detection methods, culture and PCR of tissue or blood samples were not as sensitive or timely compared to serological testing. It was also noted that there are a large number of both commercial (n = 42) and in-house developed tests used by private laboratories which have not been evaluated in the primary literature.

Microfluidics-based point-of-care test for serodiagnosis of Lyme Disease.

Currently, diagnostic testing for Lyme disease is done by determination of the serologic responses to Borrelia burgdorferi antigens, with the exception of the early localized phase of disease where diagnosis must be done clinically. Here, we describe the use of microfluidics technology to develop a multiplexed rapid lab-on-a-chip point of care (POC) assay for the serologic diagnosis of human Lyme disease. Following ELISA screening of 12 candidate antigens, we tested 8 on a microfluidic diagnostic system, called mChip-Ld, using a set of 60 serological samples. The mChip-Ld test, which can be performed in 15 minutes at the point of care, showed promising performance for detection of antibodies to B. burgdorferi using the PPO triplex test (rP100 + PepVF + rOspC-K, AUC of 0.844) compared to a gold-standard reference of culture confirmed clinical samples. The performance is comparable to the commonly used C6 peptide by lab-based ELISA. In addition, the mChip-Ld test showed promising performance for early-stage diagnosis of the disease using the antigen OspC-K (sensitivity and specificity of 84% and 92%, respectively; AUC of 0.877). Overall, this study underscores the potential of using microfluidics to aid the diagnosis of Lyme disease at the point of care.

Current Guidelines, Common Clinical Pitfalls, and Future Directions for Laboratory Diagnosis of Lyme Disease, United States.

In the United States, Lyme disease is caused by Borrelia burgdorferi and transmitted to humans by blacklegged ticks. Patients with an erythema migrans lesion and epidemiologic risk can receive a diagnosis without laboratory testing. For all other patients, laboratory testing is necessary to confirm the diagnosis, but proper interpretation depends on symptoms and timing of illness. The recommended laboratory test in the United States is 2-tiered serologic analysis consisting of an enzyme-linked immunoassay or immunofluorescence assay, followed by reflexive immunoblotting. Sensitivity of 2-tiered testing is low (30%-40%) during early infection while the antibody response is developing (window period). For disseminated Lyme disease, sensitivity is 70%-100%. Specificity is high (>95%) during all stages of disease. Use of other diagnostic tests for Lyme disease is limited. We review the rationale behind current US testing guidelines, appropriate use and interpretation of tests, and recent developments in Lyme disease diagnostics.

A concise critical analysis of serologic testing for the diagnosis of lyme disease.

Diagnostic testing for Lyme disease in the clinical setting primarily relies on assessment of serologic responses to infection, with the exception of the early localized phase of disease, in which the diagnosis must be made clinically, due to the recognized insensitivity of serologic testing at this phase of disease. For the diagnosis of early disseminated and late disease, the Centers for Disease Control and Prevention (CDC) recommends a two-tiered approach to testing consisting of initial IgM and IgG quantitative enzyme-linked immunosorbent assay (ELISA), followed by confirmation of all indeterminate or positive ELISA tests with separate IgG and IgM Western blots. This critical analysis addresses the sensitivity, specificity, and predictive value of serologic testing for Lyme disease in early localized, early disseminated, and late disease. Other testing modalities currently under evaluation are also discussed, including IgG vlsE C6 peptide ELISA, other two-tiered testing strategies, rapid diagnostics, and PCR. An understanding of the strengths and limitations of currently available testing for Lyme disease is critical for appropriate diagnosis.

Laboratory diagnostics for Lyme disease.

Lyme disease is on the rise in Canada. It is a notifiable disease, and when infection is disseminated, serological testing provides supplemental evidence to confirm a case. To describe the current diagnostic tests for Lyme disease, review the recommended approach to laboratory testing for Lyme disease and identify future research priorities for Lyme disease laboratory diagnostics in Canada. A review of the literature was carried out. We then summarized parameters to consider before Lyme disease testing is conducted, described the current best practice to use a two-tiered diagnostic algorithm for the laboratory confirmation of disseminated Lyme disease, and analyzed the advantages and disadvantages of the supplemental tests for Lyme disease. Diagnostic testing is indicated in people who have symptoms of disseminated disease and a history of exposure to vector ticks. To maximize sensitivity and specificity, a two-tiered serological approach is recommended, consisting of an enzyme immunoassay (EIA) screening test followed by confirmation with Western blot (WB) testing. A number of other diagnostic tests are available; however, these are largely for research purposes. Two-tiered serology is currently the best approach available to assist doctors when they are making a diagnosis of disseminated Lyme disease. The Public Health Agency of Canada (the Agency) will seek to improve on this approach through standardization of the Lyme disease diagnostics used across laboratories in Canada, evaluation of test performance characteristics of current and new diagnostic platforms and development of a process to secure robust serum panels to assist in the development and evaluation of new diagnostic tests for Lyme disease.

The Pain of Chronic Lyme Disease: Moving the Discourse Backward?

Phillip Baker has taken “Lyme denialism” to new depths (1). According to the Institute of Medicine, there are at least 116 million people living with acute and chronic pain in the United States. According to Baker, not one of those individuals suffers from chronic Lyme disease as a result of persistent infection with the Lyme spirochete, Borrelia burgdorferi. What is the basis for his opinion? Baker claims that “there is no evidence to indicate that chronic Lyme disease is due to a persistent infection” or that “extended antibiotic therapy is beneficial and safe”. This denialist statement about a disease that causes pain and suffering equivalent to that of diabetes or congestive heart failure ignores a growing body of evidence from the peer-reviewed medical literature that contradicts his opinion (2–5). Baker starts by attacking “Lyme-literate” physicians who use a “Lyme disease specialty laboratory” to diagnose tick-borne disease in a manner that is inconsistent with the surveillance case definition established by the Centers for Disease Control and Prevention (CDC). What Baker fails to tell us is that the CDC admits that its surveillance case definition “was developed for national reporting of Lyme disease” and was “not intended to be used in clinical diagnosis” (6). Thus, the diagnostic approach that Baker endorses is inappropriate for diagnosis of Lyme disease. Furthermore, the 46 patented commercial laboratory tests that Baker recommends for Lyme disease diagnosis have a sensitivity of only 46% and appear to yield results that are biased against women (7–9). Consequently, these commercial tests miss more than one-half of the patients with chronic Lyme disease in the United States. In contrast, the maligned “Lyme disease specialty laboratory” uses diagnostic criteria based on evidence from the peerreviewed medical literature (10, 11), and its “genderneutral” testing has a sensitivity and specificity of 90% (12). Clearly, this testing is preferable for the diagnosis of Lyme disease. As for Baker’s pat statement that “there is no evidence to indicate that chronic Lyme disease is due to a persistent infection,” numerous reports of veterinary cases and animal models confirm persistent infection with the Lyme spirochete in gerbils, hamsters, mice, dogs, monkeys, birds, and horses (13– 15). Among these cases, persistent pathology was seen in mice, dogs, and horses after the animals failed short-course treatment for their infection. Furthermore, there are at least 27 reports of persistent symptoms and failure to eradicate B. burgdorferi infection in humans treated with short-course antibiotic therapy for their tick-borne disease (5). Why does Baker deny these reports? He does so because they do not fit his limited view of Lyme disease. Baker also attacks the safety and efficacy of prolonged antibiotic therapy for chronic Lyme disease, stating that “all of the evidence obtained thus far...indicates no significant benefit as well as serious safety problems” with extended antibiotic therapy for these patients. What Baker fails to tell us is that the number of patients in controlled trials of Lyme disease treatment totals a mere 221 highly selected subjects who do not represent the vast majority of patients with chronic Lyme disease (3–5). Further analysis of these studies reveals that they were “of questionable quality”, lacked the power to detect potentially positive treatment effects, and failed to report predefined endpoints (16). Even so, in two of the four controlled trials, a significant benefit was seen in fatigue and cognition with the limited antibiotic regimen that was used, and safety problems were minimal (17, 18). In larger studies of extended antibiotic therapy, the safety of this treatment was shown to be acceptable, and the benefit of extended therapy was significant in terms of cognition, fatigue, and myalgic pain, although it took 6 –12 months of i.v. therapy to achieve this benefit (19, 20). In summary, Baker’s denialist view of the Lyme disease epidemic ignores significant evidence from the peer-reviewed medical literature that contradicts his opinion. It follows that practitioners who subscribe to his narrow view are abandoning the multitude of patients with acute and chronic pain who would benefit from treatment for their persistent spirochetal infection.

Isolation of OspC Recombinant Proteins and FlaB (f-FlaB) Fragments from Western-Siberian Borrelia garinii NT29 Isolates and Investigation of Their Immunochemical Properties

The structural OspC and FlaB proteins of the Lyme disease (LD) agent are known to be the main antigens that induce a humoral immune response at the initial stage of the disease. It is the purpose of this paper to obtain the recombinant OspC protein and a fragment of FlaB protein (f-FlaB) from the western Siberian Bor- relia Garinii NT29 isolates and to evaluate the possibility of their use for LD diagnosis. Encoding regions of the ospC and f-FlaB genes were amplified using PCR, inserted into pREB expressive vectors and cloned in the BL21 and C-600 E. coli strains, respectively. The recombinant OspC and f-FlaB proteins were purified by affin- ity chromatography on Ni-NTA sepharose 6B. Their ability to bind serum antibodies from patients with LD was then tested using western-blot and ELISA methods. The results of analyses allow one to consider these proteins to be promising components for the elaboration of diagnostic tests for LD. A prototype of the ELISA diagnostic test was designed on the basis of OspC and FlaB recombinant antigens. This test system was shown to provide satisfactory parameters of diagnostic specificity (70%) and sensitivity (85%).

Culture-confirmed reinfection of a person with different strains of Borrelia burgdorferi sensu stricto.

In recent years, the utility of serum-based diagnostic testing for Lyme disease has improved substantially; however, recovery by culture of the bacterium from skin biopsies of suspected patients is still the only definitive laboratory test. Reinfection of patients has been assumed to occur but as yet has not been documented by serial isolates from the same person. We present a case of culture-confirmed reinfection of a patient in Menominee County, Michigan. Borrelia burgdorferi was isolated from the skin punch biopsy specimens during each episode of erythema migrans (EM) and was subjected to molecular strain typing, genetic analysis of two outer surface protein genes, protein profile analysis, and serum antibody response testing. Results show that these isolates are distinct strains of the bacterium and that the two episodes of EM were caused by independent infections. This report describes the documented, culture-confirmed reinfection of a human by two different strains of B. burgdorferi.

False positive seroreactivity to Borrelia burgdorferi in systemic lupus erythematosus: the value of immunoblot analysis

The object of this study was to determine the incidence of seropositivity to B. burgdorferi by the commonly available enzyme-linked immunosorbent assay (ELISA) in patients with SLE and other rheumatic diseases and to evaluate immunoblot analysis as a tool to differentiate true from false positive ELISA. Sera were obtained from patients with SLE (n = 35), rheumatoid arthritis (n = 26), seronegative arthritis (n = 28) and Lyme disease (n = 18). Reactivity to B. burgdorferi antigens was analysed by two available diagnostic techniques: ELISA and immunoblot. Correlations were made between seroreactivity to B. burgdorferi and standard serological tests of autoimmunity: antibodies to nuclear antigens, dsDNA, cardiolipin, SSA and SSB. Seroreactivity to B. burgdorferi antigens by the ELISA system was detected in 40% of patients with SLE, 8% of patients with rheumatoid arthritis and 4% with seronegative arthritis. Among patients seropositive by ELISA, immunoblots were negative in all cases. However, eight of 14 patients with rheumatoid arthritis (57%) showed cross-reactivity to multiple borreli antigens. No significant correlations were found between Lyme seropositivity by ELISA and other autoantibodies except IgM rheumatoid factor (r = 0.61, P < 0.01) in patients with rheumatoid arthritis. a positive ELISA for Lyme disease was found in up to 40% of patients with established SLE and also in other rheumatic diseases. However, specific serum antibodies to Borrelia were not confirmed by the more specific immunoblot technique.(ABSTRACT TRUNCATED AT 250 WORDS)

5279938 Sensitive diagnostic test for Lyme disease

5279938 Sensitive diagnostic test for Lyme disease

5279938 Sensitive diagnostic test for Lyme disease

5279938 Sensitive diagnostic test for Lyme disease

5279938 Sensitive diagnostic test for Lyme disease

5279938 Sensitive diagnostic test for Lyme disease

Identification of species-specific, non-cross-reactive proteins of Borrelia burgdorferi

The low specificity of diagnostic tests for Lyme disease is due to the fact that Borrelia burgdorferi possesses many antigenic proteins that are cross-reactive with other spirochetes and bacteria. The low sensitivity is a result of high (⩾1:100) dilutions used for patient sera during testing to eliminate non-specific cross-reactivity. The present study was conducted to identify species-specific non-cross-reactive protein(s) of B. burgdorferi that might be used as antigen(s) in serologic tests. Whole-cell sonicates of B. burgdorferi were tested against pooled sera from patients with symptoms, signs, and serologic features diagnostic of Lyme disease (LD), rheumatoid arthritis, infectious mononucleosis, systemic lupus erythematosus, Rocky Mountain spotted fever, secondary syphilis, and from healthy individuals. Different LD pools were also tested against whole-cell sonicates of Treponema pallidum, Treponema phagedenis, Leptospira interrogans, and Escherichia coli. Comparison among patterns obtained by each serum pool revealed that IgM antibodies to species-specific 39-, 23-, and 22-kD proteins and IgG antibodies to 34- and 31-kD proteins were present only in the patients with LD and absent from patients with rheumatoid arthritis, infectious mononucleosis, systemic lupus erythematosus, Rocky Mountain spotted fever, secondary syphilis, and healthy individuals pools. These results suggest that 39-, 23-, and 22-kD proteins may be used in an IgM immunoassay for diagnosis of LD.