Laboratory Diagnosis Of Lyme Borreliosis Research Articles

Evaluation of different standard and modified two-tier testing strategies for the laboratory diagnosis of lyme borreliosis in a European setting.

Diagnosis of Lyme borreliosis (LB) relies on clinical symptoms and detection of Borrelia-specific antibodies. Guidelines recommend a two-tier testing (TTT) strategy for disseminated LB: serological screening with a sensitive enzyme immunoassay (EIA) and confirmation with a specific immunoblot. Searching for the most sensitive and specific approach, this retrospective study evaluated standard (STTT) and modified (MTTT) strategies using a well-defined study population. Cases included patients with active Lyme neuroborreliosis (LNB; n = 29) or Lyme arthritis (LA; n = 17). Controls comprised patients treated for LNB (n = 36) or LA (n = 8), healthy individuals who were either untreated (n = 75) or treated for LB (n = 15) in the past, and patients with potentially cross-reactive diseases (n = 16). Sera were subjected to three EIAs and two immunoblots. Reactive screening results were confirmed by immunoblot (STTT) or EIA (MTTT). Solitary IgM results in the screening assay and effects of antibiotic treatment on isotype-specific seropositivity rates were also assessed. Sensitivities of STTT strategies ranged from 90%-97% for LNB and were 100% for LA. MTTT strategies were 100% sensitive. Specificities ranged from 89%-95% for STTT and from 88%-93% for MTTT strategies. Differences between STTT and MTTT strategies were not statistically significant. Solitary IgM reactivity was common among controls. Antibiotic treatment significantly reduced IgM/IgG positivity for LNB patients; for LA patients, a decline was only observed for IgM. In conclusion, MTTT strategies showed a slightly higher sensitivity and similar specificity compared to STTT strategies. Since EIAs are more time- and cost-efficient, MTTT strategies seem more favorable for clinical use. IgG testing enhances specificity with minimal sensitivity loss.

FEATURES OF THE ALGORITHM OF CLINICAL AND LABORATORY DIAGNOSIS OF LYME BORRELIOSIS IN CHILDREN

The aim of the study – to improve the algorithm of clinical and laboratory diagnosis of Lyme borreliosis in children. Materials and Methods. The search and analysis of available literature sources of the PubMed database is carried out, using a combination of keywords "Lyme disease in children", " Clinical and Laboratory diagnostic". A group of children (62) aged 1 to 17 years was observed to identify pathogens of haemotransmissible infections among children. C-reactive protein (CRP) was determined, which is a protein and reagent of the acute phase. To confirm the diagnosis of PM, determine the form of the lesion and identify the antigens of the pathogen, a routine two-step method of blood analysis was used. Results and Discussion. The algorithm for the diagnosis of Lyme borreliosis in children has been developed for use in making a diagnosis. Clinical: complaints – malaise, fatigue, headache, arthralgia, myalgia, subfebrile body temperature; anamnesis – tick bites, location of bites, time of appearance of symptoms after the bite; objective – lymphadenopathy, arthritis, pain syndrome, intoxication syndrome; laboratory and instrumental diagnostics: biochemical blood analysis (increased level of C-reactive protein, rheumatoid factor is rarely detected), ultrasound of joints, immunological examination (immunofluorescence analysis and blot test), detection of Borrelia by PCR method in synovial fluid. Conclusions. Proposed algorithm for diagnosing the Lyme disease in children.

Borrelia burgdorferi Is a Poor Inducer of Gamma Interferon: Amplification Induced by Interleukin-12.

Laboratory diagnosis of Lyme borreliosis (LB) is mainly based on serology, which has limitations, particularly in the early stages of the disease. In recent years there have been conflicting reports concerning a new diagnostic tool using the cytokine interferon-gamma (IFN-γ). Previous studies have generally found low concentrations of IFN-γ in early LB infection. The goal of this study is to investigate IFN-γ regulation during early LB and provide insights into the host response to B. burgdorferi. We performed in vitro experiments with whole blood assays and peripheral blood mononuclear cells (PBMCs) of LB patients and healthy volunteers exposed to B. burgdorferi and evaluated the IFN-γ response using ELISA and related interindividual variation in IFN-γ production to the presence of single nucleotide polymorphisms. IFN-γ production of B. burgdorferi-exposed PBMCs and whole blood was amplified by the addition of interleukin-12 (IL-12) to the stimulation system. This effect was observed after 24 h of B. burgdorferi stimulation in both healthy individuals and LB patients. The effect was highly variable between individuals, but was significantly higher in LB patients 6 weeks since the start of antibiotic treatment compared to healthy individuals. IL-12 p40 and IL-18 mRNA were upregulated upon exposure to B. burgdorferi, whereas IL-12 p35 and IFN-γ mRNA expression remained relatively unchanged. SNP Rs280520 in the downstream IL-12 pathway, Tyrosine Kinase 2, was associated with increased IFN-γ production. This study shows that IL-12 evokes an IFN-γ response in B. burgdorferi exposed cells, and that LB patients and healthy controls respond differently to this stimulation.

LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species.

Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system LoopTag for detection of Borrelia species. Advantages of the LoopTag system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. Results: Using the LoopTag probe system we were able to detect all nine tested European species belonging to the Borrelia burgdorferi (sensu lato) complex and differentiated them from relapsing fever Borrelia species. As few as 10 copies of Borrelia in one PCR reaction were detectable. Conclusion: We established a novel multiplex probe real-time PCR system, designated LoopTag, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the Borrelia burgdorferi s.l. complex.

Laboratory Diagnosis of Lyme Borreliosis.

Lyme borreliosis is caused by a growing list of related, yet distinct, spirochetes with complex biology and sophisticated immune evasion mechanisms. It may result in a range of clinical manifestations involving different organ systems, and can lead to persistent sequelae in a subset of cases. The pathogenesis of Lyme borreliosis is incompletely understood, and laboratory diagnosis, the focus of this review, requires considerable understanding to interpret the results correctly. Direct detection of the infectious agent is usually not possible or practical, necessitating a continued reliance on serologic testing. Still, some important advances have been made in the area of diagnostics, and there are many promising ideas for future assay development. This review summarizes the state of the art in laboratory diagnostics for Lyme borreliosis, provides guidance in test selection and interpretation, and highlights future directions.

Serological diagnostics of Lyme borreliosis: comparison of assays in twelve clinical laboratories in Northern Europe

Lyme borreliosis (LB), caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in Europe. Laboratory diagnosis of LB is mainly based on the patients’ medical history, clinical signs and symptoms in combination with detection of Borrelia-specific antibodies where indirect enzyme-linked-immunosorbent assay (ELISA) is the most widely used technique. The objective of the study was to evaluate and compare the diagnostic accuracy (sensitivities and specificities) of serological tests that are currently in use for diagnosis of LB in clinical laboratories in Northern Europe, by use of a large serum panel. The panel consisted of 195 serum samples from well-characterized and classified patients under investigation for clinically suspected LB (n = 59) including patients with Lyme neuroborreliosis, Lyme arthritis, acrodermatitis chronica atrophicans, erythema migrans or other diseases (n = 112). A total of 201 serum samples from healthy blood donors were also included. The panel (396 serum samples altogether) was sent to 12 clinical laboratories (using five different ELISA methods) as blinded for group affiliation and the laboratories were asked to perform serological analysis according to their routine procedure. The results from the study demonstrated high diagnostic concordance between the laboratories using the same diagnostic assay and lower diagnostic concordance between laboratories using different diagnostic assays. For IgG, the results were in general rather homogenous and showed an average sensitivity of 88% (range 85–91%) compared to IgM which showed lower average sensitivity of 59% (range 50–67%) and more heterogeneous results between assays and laboratories.

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.

Laboratory Diagnosis of Lyme Borreliosis in Scottish Patients: A Novel Approach

Traditional two-tier (enzyme immunoassay [EIA] screening and Western blot confirmation) testing for the laboratory diagnosis of Lyme borreliosis (LB) is expensive, lacks sensitivity in the diagnosis of early LB, cannot distinguish between current and past infection and cannot be used as a marker for treatment response. The aims of the present study is to investigate the role of the C6 EIA as a screening assay, as part of two-tier EIA test strategy, and its use as a marker of treatment response or resolving infection in a routine diagnostic laboratory. The C6 EIA was significantly less sensitive than the Enzygnost Lyme link VlsE/IgG EIA (169/249 vs. 190/249 reactive sera, respectively; P=0.0455, Fishers exact two-tailed test). The two-EIA strategy, utilising C6 EIA confirmation, was slightly more sensitive than traditional two-tier testing (82/151 vs. 67/151 positive sera). Twenty-seven patients were positive by the two-EIA strategy but negative by Western blot, raising questions of specificity, but 12 samples positive with the traditional two-tier testing were negative with the two-EIA strategy. There was no evidence to support the use of the C6 EIA for monitoring treatment response or resolving infection. The authors have devised a novel approach to detect LB in Scottish patients. For cases with a high clinical suspicion of disease, the C6 EIA could be incorporated into a two-EIA strategy, replacing the need for Western blot confirmation with a simpler, more cost-effective two-EIA strategy. Western blot confirmation would be reserved for those patients with discordant EIA results and whose clinical picture is more complex.

Multiplex Bead Based Immunoassays For The Serodiagnosis of Lyme Borreliosis

Laboratory diagnosis of Lyme borreliosis is based on the detection of specific IgG and IgM antibodies against relevant antigens of Borrelia burgdorferi. Serological diagnosis usually is performed by a two-step procedure. Where immunoassays for antibody screening exhibit high sensitivity, second-line tests, e.g. Western-blot or line-blot assays,show high specificity, and are used for confirmation of positive screening results. In addition to serology, in certain cases Borrelia spp. can be detected directly, for instance in synovial fluid from patients with Lyme arthritis by PCR or bacterial culture. With the development of multiplex technology, e.g. bead-based immunoassays, multiple antibodies to distinct bacterial antigens can be detected in a single run. Detection systems used in this context, e.g. analysers based on flow cytometry, can be highly standardised and automated. Furthermore, these analysers can be connected bidirectionally to an order-entry based laboratory information system, with random access for high throughput analysis. Thus, multiplex bead assays may have the power to replace the current two-step procedure.

Clinical and Serological Features of Patients with Suspected Lyme Borreliosis

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

Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of patients with clinical symptoms of Lyme borreliosis

Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies complex Borrelia burgdorferi sensu lato (s.l.) transmitted by Ixodes ticks. At present, serology remains the main diagnostic tool for laboratory diagnosis of Lyme borreliosis. Recently, the PCR technique has been applied for diagnosis of B. burgdorferi s.l., but, until now, a reliable, easy-to-perform and sensitive method has not been described. Here we present a new PCR-based method for the detection of both B. burgdorferi s.l. and Borrelia genospecies DNAs in serum samples collected from patients showing Lyme disease symptoms. Of 265 serum samples of patients included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were negative for antibodies against B. burgdorferi by enzyme-linked immunosorbent assay and Western blotting. The B. burgdorferi s.l. 16S rRNA gene was detected by PCR in all serum-positive and in two borderline samples. None of the serum-negative samples nor serum samples collected from healthy subjects gave positive PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for Borrelia afzelii, 18% for Borrelia garinii and 23% for two Borrelia species. Two samples (9%) were not identified to species level. The new protocol could be considered to be reliable as neither false-positive nor false-negative reactions were recorded, and to be sensitive as it detects DNA from one bacterial cell.

The Laboratory Diagnosis of Lyme Borreliosis: Guidelines from the Canadian Public Health Laboratory Network

Lyme borreliosis is uncommonly seen in Canada. Most cases have occurred in close proximity to small geographical areas where infected ticks have become established. Although few cases are seen, thousands of patients are tested yearly. Unless patients are carefully selected and an appropriately sensitive and specific testing algorithm is applied, large numbers of patients without Lyme borreliosis will be incorrectly diagnosed. The Canadian Public Health Laboratory Network has developed the present guidelines to assist physicians in assessing patients for Lyme borreliosis, and to help guide the choice and interpretation of laboratory testing.

Laboratory diagnosis of Lyme borreliosis at the Portuguese National Institute of Health (1990-2004)

Lyme borreliosis is considered to be an emerging infection in some regions of the world, including Portugal. The first Portuguese human case of Lyme borreliosis was identified in 1989. Since 1999, this disease is considered a notifiable disease (DDO) in Portugal, but only a few cases are reported each year, which does not allow consistent analysis of risk factors and the impact on public health. In this study the authors analyse the data available at the Centre for Vectors and Infectious Diseases Research (CEVDI) laboratory, at the Instituto Nacional de Saude Dr. Ricardo Jorge (National Institute of Health, INSA) during the past 15 years (1990-2004) and evaluate them against the registry of national reported cases (1999-2004). Serological tests were the basis for laboratory diagnosis. Data on year of diagnosis, sex, age, geographical origin and clinical signs are available for 628 well documented Portuguese positive cases. The number of cases per year varied between 2 and 78, with the highest number of cases reported in 1997. Of the positive cases, 53.5% were female and the age group most affected was 35-44 years old. Neuroborreliosis was the most common clinical manifestation (37.3%). Human cases were detected in 17 of the 20 regions of Portugal, and the highest number of laboratory confirmed cases were from the Lisbon district. The comparison of the number of notified cases and the number of positive cases confirmed by our laboratory show that Lyme borreliosis is clearly an underreported disease. Due to the scattered distribution of the positive cases and the low prevalence of the tick species Ixodes ricinus, the most effective prevention measure for Lyme borreliosis in Portugal is education of the risk groups on how to prevent tick bites.

Direct detection methods for Lyme Borrelia, including the use of quantitative assays.

Direct detection of Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, is the most reliable laboratory diagnostic tool. Several methods have been developed for direct detection of B. burgdorferi in infected vectors, host tissues, and clinical specimens from patients with Lyme borreliosis. These include microscope-based assays, antigen detection assays, in vitro cultivation, and nucleic acid-based detection of B. burgdorferi. The sensitivity and specificity of these methods depend on various factors and are also variable among laboratories. To date, only in vitro cultivation of B. burgdorferi has been widely accepted to confirm clinical diagnosis of Lyme borreliosis. Nevertheless, various polymerase chain reaction-based molecular assays have shown increasing significance in the laboratory diagnosis of Lyme borreliosis because of their high sensitivity, specificity, and capability for quantification and typing of spirochetes in clinical specimens. In this review, the currently available methods for direct detection of B. burgdorferi in clinical samples and quantitative analysis of spirochete load in different biological sources are discussed.

Lyme borreliosis

The spirochaete Borrelia burgdorferi (sensu lato) is the cause of Lyme borreliosis, a common tick-borne disease with a wide geographical distribution. B. burgdorferi s.l. was first isolated in 1982 after a clinical and epidemiological study of an unusual outbreak of arthritis in children in the town of Old Lyme, Connecticut, USA. Despite this recent discovery, there is evidence that Lyme borreliosis has existed for many years. Clinical manifestations are diverse, but antibiotic therapy is largely successful when initiated early in the disease. Molecular-based studies have improved the laboratory diagnosis of Lyme borreliosis and played a significant role in ecological and taxonomic investigations of the pathogen. B. burgdorferi s.l. is now divided into some nine genospecies and genomic groups, some of which have been associated with particular manifestations of disease, while the number of tick species found to be competent vectors has also increased. The emerging ecology of B. burgdorferi s.l. is more complex than originally described and, consequently, the assessment of risk of disease to an individual bitten by a tick should include establishing the identity of the tick, duration of attachment and degree of engorgement, as well as consideration of the individual's way of life and environment.

PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in the urine and breast milk of patients with Lyme borreliosis

Current laboratory diagnosis of Lyme borreliosis relies on tests for the detection of antibodies to Borrelia burgdorferi with known limitations. By using a simple extraction procedure for urine samples, B. burgdorferi DNA was amplified by a nested PCR with primers that target the specific part of the flagellin gene. To control possible inhibition of the enzyme (polymerase), a special assay using the same primers was developed. We examined 403 urine samples from 185 patients with skin manifestations of Lyme borreliosis. Before treatment, B. burgdorferi DNA was detected in 88 of 97 patients with Lyme borreliosis. After treatment, all but seven patients became nonreactive. Six of these seven persons suffered from intermittent migratory arthralgias or myalgias, and one from acrodermatitis chronica atrophicans. Two of 49 control patients with various dermatologic disorders and none out of 22 presumably healthy persons were reactive in the PCR. In addition to urine, breast milk from two lactating women with erythema migrans was tested and also found reactive. Borrelia burgdorferi DNA can be detected with high sensitivity (91%) by a nested PCR in urine of patients with Lyme borreliosis. In addition, this test can be a reliable marker for the efficacy of treatment.

The laboratory diagnosis of Lyme borreliosis

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Laboratory diagnosis and seroepidemiology of Lyme borreliosis

Laboratory diagnosis of Lyme borreliosis is performed by direct detection of Borrelia burgdorferi in body fluids and tissue samples. This can be achieved by cultivation of the organisms, staining techniques, or demonstration of parts of the genome. Although the best aetiologic proof in case of positivity, these methods cannot yet serve as routine techniques: they are too time consuming and expensive. Currently, the usual method for establishing the diagnosis of Lyme borreliosis is serologic testing (indirect detection). Immunofluorescence, hemagglutination, ELISA tests with whole cell antigen should be considered as screening methods. Assays with selected fractions of B. burgdorferi antigens or tests using selected recombinant antigens should be considered as more specific. Immunoblotting (Western blotting) may be considered as a confirmatory test. However, the interpretation of test results requires an experienced investigator. Laboratory diagnosis of B. burgdorferi infections of the central nervous systems (CNS) is the most highly developed method. Demonstration of intrathecally produced specific antibodies, and, moreover, demonstration of specific oligoclonal bands may very well prove the actual infection of the CNS and/or the nerve roots. Seroepidemiological investigations identify neurological manifestations as the most frequent ones among European cases of Lyme borreliosis. The true incidence and prevalence of Lyme borreliosis, however, cannot be determined with current diagnostic methods and must await the development of methods to identify actual infection.

The Laboratory Diagnosis of Lyme Borreliosis

The dependence on laboratory tests for the diagnosis of Lyme borreliosis has been increasing as the recognized clinical spectrum of disease manifestations has widened. Unfortunately, the state of standardization and proficiency testing for these tests is not well established and the quality of testing not optimum. It is extremely important in diagnosing Lyme borreliosis that clinical symptomatology and history be critically reviewed to make valid conclusions from the laboratory test results.