Laboratory Diagnosis Of Lyme Disease Research Articles

Performance of two modified two-tier algorithms for the serologic diagnosis of Lyme disease.

The laboratory diagnosis of Lyme disease relies upon the detection of antibodies to Borrelia species. Standard two tier testing (STTT) methods rely upon immunoblots which have clinical and technical limitations. Modified two-tier testing (MTTT) methods have recently become available and are being widely adopted. There are limited independent data available assessing the performance of MTTT and STTT methods.

Linear Peptide Epitopes Derived from ErpP, p35, and FlaB in the Serodiagnosis of Lyme Disease.

Lyme disease is the most common vector-borne disease in the northern hemisphere. Current serodiagnostics are insensitive in early infection. Sensitivity in these seroassays is compromised by the necessity to preserve specificity in the presence of cross-reactive epitopes in Borrelia burgdorferi target antigens. We evaluated the efficacy of using synthetic peptides containing epitopes unique to B. burgdorferi as antigen targets in a Lyme disease seroassay. We performed linear B cell epitope mapping of the proteins p35 (BBH32) and ErpP to identify unique epitopes. We generated peptides containing these newly identified linear epitope sequences along with previously identified epitopes from the antigens FlaB and VlsE and evaluated their diagnostic capabilities via ELISA using large serum sets. Single-epitope peptides, while specific, demonstrated insufficient sensitivity. However, when epitopes from FlaB, ErpP, or p35 were combined in tandem with an epitope from VlsE, the sensitivity of the assay was significantly increased without compromising specificity. The identification of additional unique epitopes from other B. burgdorferi antigens and the further development of a combined multi-peptide-based assay for the laboratory diagnosis of Lyme disease offers a way to address the poor specificity associated with the use of whole protein antigen targets and thus significantly improve the laboratory diagnosis of Lyme disease.

Lyme disease: Modern approaches to prevention, diagnosis and treatment

Lyme disease (LD) or tick-borne borreliosis affects thousands of people every year in different regions of the world, primarily the United States and Europe. In endemic areas, early LD is a common disease that requires high medical vigilance. Considering the extreme relevance of this problem for public health, in November 2020, the committee of experts of three American scientific societies published an updated version of the clinical guidelines for the prevention, diagnosis and treatment of LD, the main provisions of which are presented in this article. It is emphasized that in the absence of vaccines, the risk of LD and other diseases transmitted by ticks can be reduced by using personal protective equipment and repellents. Antibiotic prophylaxis is carried out by a single oral administration of doxycycline. In the laboratory diagnosis of LD, the determination of antibodies to B. burgdorfery in the blood serum is a first-line study. At the second stage, serum samples are examined using an immunoblot for IgM and IgG. The basis of treatment of LD is rational antibiotic therapy. The choice of an antibiotic depends on a number of factors, including the presence of extracutaneous manifestations of LD (neuroborreliosis, carditis, arthritis). The most commonly used are doxycycline, amoxicillin, cefuroxime-axetil and ceftriaxone.

Expanding Access to Biospecimens for Lyme Disease Test Development.

The laboratory diagnosis of Lyme disease relies upon serologic testing. A standard or modified two-tiered testing algorithm is used to enhance the accuracy of antibody detection. However, this approach suffers from a lack of sensitivity in early Lyme disease. Ongoing efforts to develop more sensitive antibody detection technologies and other diagnostic approaches are dependent upon the availability of quality-assured biospecimens linked to reliable clinical data. In this issue of the Journal of Clinical Microbiology, Horn et al. (E. J. Horn, G. Dempsey, A. M. Schotthoefer, U. L. Prisco, et al., J Clin Microbiol 58:e00032-20, 2020, https://doi.org/10.1128/JCM.00032-20) described the development of the Lyme Disease Biobank. Clinically categorized case patients with early Lyme disease and healthy controls were identified (without laboratory diagnostic testing) from three sites where Lyme disease is endemic. Subjects provided whole blood and urine, which were processed and stored at a central biorepository. Whole blood, serum, and urine aliquots were prepared and are available to investigators developing laboratory diagnostics for Lyme disease. After obtaining samples, extensive laboratory testing was performed, including serologic and nucleic acid amplification testing for B. burgdorferi and other tick-borne pathogens. Direct detection methods yielded few positive results. Relative to the findings for another commonly used biorepository cohort, the results of this testing demonstrated a low seropositive rate, as determined by standard two-tiered testing. Additionally, relatively few subjects demonstrated seroconversion with testing of convalescent-phase samples. This clinical and serologically defined cohort of samples from Lyme disease and control cases from areas of Lyme disease endemicity offers an additional valuable resource for novel test development that includes alternate specimen types.

Multicenter Evaluation of the C6 Lyme ELISA Kit for the Diagnosis of Lyme Disease.

Lyme disease (LD), caused by infection with Borrelia burgdorferi, is the most common tick-borne infection in many regions of Eurasia. Antibody detection is the most frequently used laboratory test, favoring a two-step serodiagnostic algorithm; immunoenzymatic detection of antibodies to C6 has been shown to perform similarly to a standard two-step workflow. The aim of this study was the performance evaluation of the C6 Lyme ELISA kit compared to a standard two-step algorithm in three laboratories located in the northeastern region of Italy which cater to areas with different LD epidemiology. A total of 804 samples were tested, of which 695 gave concordant results between C6 testing and routine workflow (564 negative, 131 positive). Wherever available, clinical presentation and additional laboratory tests were analyzed to solve discrepancies. The C6 based method showed a good concordance with the standard two-step algorithm (Cohen’s κ = 0.619), however, the distribution of discrepancies seems to point towards a slightly lower specificity of C6 testing, which is supported by literature and could impact on patient management. The C6 ELISA, therefore, is not an ideal stand-alone test; however, if integrated into a two-step algorithm, it might play a part in achieving a sensitive, specific laboratory diagnosis of LD.

Measurement of Organ-Specific and Acute-Phase Blood Protein Levels in Early Lyme Disease.

Lyme disease results from infection of humans with the spirochete Borrelia burgdorferi. The first and most common clinical manifestation is the circular, inflamed skin lesion referred to as erythema migrans; later manifestations result from infections of other body sites. Laboratory diagnosis of Lyme disease can be challenging in patients with erythema migrans because of the time delay in the development of specific diagnostic antibodies against Borrelia. Reliable blood biomarkers for the early diagnosis of Lyme disease in patients with erythema migrans are needed. Here, we performed selected reaction monitoring, a targeted mass spectrometry-based approach, to measure selected proteins that (1) are known to be predominantly expressed in one organ (i.e., organ-specific blood proteins) and whose blood concentrations may change as a result of Lyme disease, or (2) are involved in acute immune responses. In a longitudinal cohort of 40 Lyme disease patients and 20 healthy controls, we identified 10 proteins with significantly altered serum levels in patients at the time of diagnosis, and we also developed a 10-protein panel identified through multivariate analysis. In an independent cohort of patients with erythema migrans, six of these proteins, APOA4, C9, CRP, CST6, PGLYRP2, and S100A9, were confirmed to show significantly altered serum levels in patients at time of presentation. Nine of the 10 proteins from the multivariate panel were also verified in the second cohort. These proteins, primarily innate immune response proteins or proteins specific to liver, skin, or white blood cells, may serve as candidate blood biomarkers requiring further validation to aid in the laboratory diagnosis of early Lyme disease.

Recombinant Antigens In Serological Diagnosis Of Lyme Borreliosis

Abstract Lyme borreliosis, an infectious disease caused by tick-borne spirochetes of the Borrelia burgdorferi sensu lato complex, is regarded as the most commonly reported vector-borne infection in the Northern Hemisphere. Currently, the basis for laboratory diagnosis of Lyme disease is a two-step serological examination. The first is an enzyme-linked immunosorbent assay (ELISA). If the test result is positive or questionable, a Western blot is used as the second phase test. In both methods, the total cell lysates of B. burgdorferi s.l. are used as the main source of antigens. However, the huge diversity of genospecies within B. burgdorferi s.l. and the low degree of preservation of the sequence of their proteins means that using the cell lysates of one of the species is not sufficient to correctly diagnose Lyme disease. Numerous literature reports show that the use of B. burgdorferi s.l. recombinant or chimeric antigens may be a potential solution to problems occurring in Lyme disease immunodiagnosis. However, for diagnostic tests based on recombinant proteins to be as effective as possible, carefully selected antigens or fragments should be used. With this approach, a test can be developed with a sensitivity that remains independent of the B. burgdorferi s.l. species which caused the disease. In addition, the exclusive use of protein fragments may definitely reduce the frequency of cross-reactions. 1. Introduction. 2. Characterization of selected B. burgdorferi s.l. antigens. 3. Diagnosis of Lyme disease. 4. Problems in Lyme disease serodiagnosis. 5. Use of recombinant antigens and synthetic peptides in the diagnosis of Lyme disease. 6. Summary

1760. Interferon Gamma Release Assay for Diagnosis of Lyme disease

BackgroundThe sensitivity of current antibody detection assays against Borrelia burgdorferi in the early stage of Lyme disease is very low. In children especially, who commonly have febrile viral illnesses, manifestations of early Lyme disease can be misdiagnosed. We previously demonstrated that IFNγ secretion could be detected in whole blood collected from Lyme disease patients at first clinical presentation following overnight incubation of the blood with peptides derived from B. burgdorferi. In the present study, we further evaluated the utility of IFNγ release for the laboratory diagnosis of Lyme disease in children with varying stages of the illness.MethodsChildren ages 2–18 years with no prior history of Lyme disease and with manifestations of Lyme disease at any stage were enrolled in the study. Sick and healthy controls were enrolled for comparison. We collected history and physical examination data and blood samples at the time of enrollment, at 1 month, and at 6 months. Standard 2-tier testing with ELISA (whole cell sonicate [WCS] and C6) and western blot were run in parallel to the IFNγ release assay for all blood samples. Sensitivity and specificity of the study assay were determined for presentation at all stages of Lyme disease. Clinical data were summarized.ResultsBlood samples from 22 patients with Lyme disease and 7 controls (4 sick, 3 healthy) were obtained at the first visit. The IFNγ release assay detected early and early disseminated Lyme disease with 78% sensitivity compared with 59% sensitivity of 2-tier testing in our study. For patients presenting with a single erythema migrans (EM) lesion, the IFNγ release assay detected Lyme disease with 63% sensitivity compared with 14% sensitivity with 2-tier testing. The IFNγ release assay had only 25% sensitivity for detecting late disease. A single control patient was positive for both the IFNγ release assay and 2-tier serology.ConclusionA novel IFNγ release assay demonstrated significantly increased sensitivity when compared with 2-tier testing in the laboratory diagnosis of Lyme disease in patients presenting with a single EM lesion. Future study is needed to determine its utility in detecting early Lyme disease in patients with nonspecific febrile illness in the absence of erythema migrans.Disclosures R. Dattwyler, Qiagen: Collaborator, Research support. P. Arnaboldi, Qiagen: Collaborator, research materials.

Pilot Study of Immunoblots with Recombinant Borrelia burgdorferi Antigens for Laboratory Diagnosis of Lyme Disease.

Accurate laboratory diagnosis of Lyme disease (Lyme borreliosis), caused by the spirochete Borrelia burgdorferi (BB), is difficult and yet important to prevent serious disease. The US Centers for Disease Control and Prevention (CDC) presently recommends a screening test for serum antibodies followed by confirmation with a more specific Western blot (WB) test to detect IgG and IgM antibodies against antigens in whole cell lysates of BB. Borrelia species related to BB cause tick-borne relapsing fever (TBRF). TBRF is increasingly recognized as a health problem in the US and occurs in areas where Lyme disease is prevalent. The two groups of Borrelia share related antigens. We have developed a modified WB procedure termed the Lyme immunoblots (IBs) using recombinant antigens from common strains and species of the BB sensu lato complex for serological diagnosis of Lyme disease. A reference collection of 178 sera from 26 patients with and 152 patients without Lyme disease were assessed by WB and IB in a blinded manner using either criteria for positive antibody reactions recommended by the CDC or criteria developed in-house. The sensitivity, specificity, positive and negative predictive values obtained with the reference sera suggest that the Lyme IB is superior to the Lyme WB for detection of specific antibodies in Lyme disease. The Lyme IB showed no significant reaction with rabbit antisera produced against two Borrelia species causing TBRF in the US, suggesting that the Lyme IB may be also useful for excluding TBRF.

Development of a sensitive PCR-dot blot assay to supplement serological tests for diagnosing Lyme disease

Laboratory diagnosis of Lyme disease is difficult and presently dependent on detecting Borrelia burgdorferi-specific antibodies in patient serum with the disadvantage that the immune response to B. burgdorferi can be weak or variable, or alternatively, the slow and inefficient culture confirmation of B. burgdorferi. PCR tests have previously shown poor sensitivity and are not routinely used for diagnosis. We developed a sensitive and specific Lyme Multiplex PCR-dot blot assay (LM-PCR assay) applicable to blood and urine samples to supplement western blot (WB) serological tests for detecting B. burgdorferi infection. The LM-PCR assay utilizes specific DNA hybridization to purify B. burgdorferi DNA followed by PCR amplification of flagellin and OspA gene fragments and their detection by southern dot blots. Results of the assay on 107 and 402 clinical samples from patients with suspected Lyme disease from Houston, Texas or received at the IGeneX laboratory in Palo Alto, California, respectively, were analyzed together with WB findings. The LM-PCR assay was highly specific for B. burgdorferi. In the Texas samples, 23 (21.5%) patients antibody-negative in WB assays by current US Centers for Disease Control (CDC) recommended criteria were positive by LM-PCR performed on urine, serum or whole blood samples. With IGeneX samples, of the 402 LM-PCR positive blood samples, only 70 met the CDC criteria for positive WBs, while 236 met IGeneX criteria for positive WB. Use of the LM-PCR assay and optimization of current CDC serological criteria can improve the diagnosis of Lyme disease.

Research progress in laboratory diagnosis of lyme disease

Research progress in laboratory diagnosis of lyme disease

Borrelia burgdorferi-specific IgA in Lyme Disease

The laboratory diagnosis of Lyme disease is currently dependent on the detection of IgM and IgG antibodies against Borrelia burgdorferi, the causative agent of the disease. The significance of serum IgA against B. burgdorferi remains unclear. The production of intrathecal IgA has been noted in patients with the late Lyme disease manifestation, neuroborreliosis, but production of antigen-specific IgA during early disease has not been evaluated. In the current study, we assessed serum IgA binding to the B. burgdorferi peptide antigens, C6, the target of the FDA-cleared C6 EIA, and FlaB(211-223)-modVlsE(275-291), a peptide containing a Borrelia flagellin epitope linked to a modified VlsE sequence, in patients with early and late Lyme disease. Specific IgA was detected in 59 of 152 serum samples (38.8%) from early Lyme disease patients. Approximately 50% of early Lyme disease patients who were seropositive for peptide-specific IgM and/or IgG were also seropositive for peptide-specific IgA. In a subpopulation of patients, high peptide-specific IgA could be correlated with disseminated disease, defined as multiple erythema migrans lesions, and neurological disease complications. These results suggest that there may be an association between elevated levels of antigen-specific IgA and particular disease manifestations in some patients with early Lyme disease.

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.

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

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

Evolutionary aspects of emerging Lyme disease in Canada.

In North America, Lyme disease (LD) is a tick-borne zoonosis caused by the spirochete bacterium Borrelia burgdorferi sensu stricto, which is maintained by wildlife. Tick vectors and bacteria are currently spreading into Canada and causing increasing numbers of cases of LD in humans and raising a pressing need for public health responses. There is no vaccine, and LD prevention depends on knowing who is at risk and informing them how to protect themselves from infection. Recently, it was found in the United States that some strains of B. burgdorferi sensu stricto cause severe disease, whereas others cause mild, self-limiting disease. While many strains occurring in the United States also occur in Canada, strains in some parts of Canada are different from those in the United States. We therefore recognize a need to identify which strains specific to Canada can cause severe disease and to characterize their geographic distribution to determine which Canadians are particularly at risk. In this review, we summarize the history of emergence of LD in North America, our current knowledge of B. burgdorferi sensu stricto diversity, its intriguing origins in the ecology and evolution of the bacterium, and its importance for the epidemiology and clinical and laboratory diagnosis of LD. We propose methods for investigating associations between B. burgdorferi sensu stricto diversity, ecology, and pathogenicity and for developing predictive tools to guide public health interventions. We also highlight the emergence of B. burgdorferi sensu stricto in Canada as a unique opportunity for exploring the evolutionary aspects of tick-borne pathogen emergence.

Decorin binding proteins A and B in the serodiagnosis of Lyme disease in North America.

The laboratory diagnosis of Lyme disease is based upon the detection of antibodies generated against Borrelia burgdorferi using a two-tier assay, typically consisting of an enzyme-linked immunosorbent assay (ELISA), followed by a Western blot. This system, put into place to address the nonspecificity associated with standalone first-tier assays, is insensitive for diagnosing early infection, when most people seek care. The use of bacterial lysates or whole-protein antigens as first-tier assay targets contributes to nonspecificity due, in part, to the presence of cross-reactive epitopes that are also found in other bacteria. This precludes their use as sensitive standalone assays. The use of peptides containing linear epitopes that are highly specific for B. burgdorferi offers a method for reducing this cross-reactivity. In the present study, we mapped the linear epitopes of the prominently expressed Borrelia adhesins decorin binding protein A (DbpA) and DbpB. We identified several epitopes in each protein that were highly conserved among North American strains of B. burgdorferi, and we screened peptides containing specific epitopes using serum panels from early and late Lyme disease patients. The individual peptides primarily detected IgM but not IgG, while the proteins efficiently detected both IgM and IgG. While no individual peptide demonstrated better utility for antibody detection than its respective whole protein, an assay containing a combination of a DbpA and a DbpB peptide adequately detected both IgM and IgG, accurately identifying 87.5% (84/96) of the early Lyme disease patients and 80.0% (16/20) of the late Lyme disease patients.

Editorial Commentary: Better Laboratory Testing for Lyme Disease: No More Western Blot

It seems unlikely, in routine clinical practice, that direct methods, including culture or polymerase chain reaction, will ever become the primary tests for the laboratory diagnosis of Lyme disease. Indirect, serologic tests will continue to be used to support this diagnosis [1]. Can serologic testing for Lyme disease be improved? The Centers for Disease Control and Prevention (CDC) currently recommends a 2-tiered algorithm for serodiag

P1499 Comparison between Enzygnost® Lyme link VlsE/IgG and LIAISON® Borrelia IgG for the laboratory diagnosis of Lyme disease

P1499 Comparison between Enzygnost® Lyme link VlsE/IgG and LIAISON® Borrelia IgG for the laboratory diagnosis of Lyme disease

Not So Good Old Summertime

Not So Good Old Summertime

Audit of the laboratory diagnosis of Lyme disease in Scotland

An audit was performed on the laboratory diagnosis of lyme disease in Scotland. The problem of a significant number of patients with clinical symptoms of lyme disease being reported as seronegative or equivocal by the confirmatory Western blot test was identified. Comparisons of current practice were made with American and European standards, and the Western blot scoring system revised. When applied retrospectively (April 2003 to March 2004), 39 (33 %) of 116 serum samples previously negative or equivocal became weak positive or stronger. Thirty-one (80 %) of these 39 samples were from patients with clinical details suggestive of early lyme disease. The changes were implemented and assessed prospectively for 6 months. There was a significant increase in the proportion of equivocal results, with fewer negatives compared to the same time period 1 year previously. This audit has helped clinicians in the diagnosis of lyme disease and the management of these patients in Scotland.