Metabolic Response in Patients With Post-treatment Lyme Disease Symptoms/Syndrome.

BACKGROUND: Post-treatment Lyme disease symptoms/syndrome (PTLDS) occurs in approximately 10% of patients with Lyme disease following antibiotic treatment. Biomarkers or specific clinical symptoms to identify patients with PTLDS do not currently exist and the PTLDS classification is based on the report of persistent, subjective symptoms for ≥6 months following antibiotic treatment for Lyme disease. METHODS: Untargeted liquid chromatography-mass spectrometry metabolomics was used to determine longitudinal metabolic responses and biosignatures in PTLDS and clinically cured non-PTLDS Lyme patients. Evaluation of biosignatures included (1) defining altered classes of metabolites, (2) elastic net regularization to define metabolites that most strongly defined PTLDS and non-PTLDS patients at different time points, (3) changes in the longitudinal abundance of metabolites, and (4) linear discriminant analysis to evaluate robustness in a second patient cohort. RESULTS: This study determined that observable metabolic differences exist between PTLDS and non-PTLDS patients at multiple time points. The metabolites with differential abundance included those from glycerophospholipid, bile acid, and acylcarnitine metabolism. Distinct longitudinal patterns of metabolite abundance indicated a greater metabolic variability in PTLDS versus non-PTLDS patients. Small numbers of metabolites (6 to 40) could be used to define PTLDS versus non-PTLDS patients at defined time points, and the findings were validated in a second cohort of PTLDS and non-PTLDS patients. CONCLUSIONS: These data provide evidence that an objective metabolite-based measurement can distinguish patients with PTLDS and help understand the underlying biochemistry of PTLDS.

Host Metabolic Response in Early Lyme Disease.

Lyme disease is a tick-borne bacterial illness that occurs in areas of North America, Europe, and Asia. Early infection typically presents as generalized symptoms with an erythema migrans (EM) skin lesion. Dissemination of the pathogen Borrelia burgdorferi can result in multiple EM skin lesions or in extracutaneous manifestations such as Lyme neuroborreliosis. Metabolic biosignatures of patients with early Lyme disease can potentially provide diagnostic targets as well as highlight metabolic pathways that contribute to pathogenesis. Sera from well-characterized patients diagnosed with either early localized Lyme disease (ELL) or early disseminated Lyme disease (EDL), plus healthy controls (HC), from the United States were analyzed by liquid chromatography-mass spectrometry (LC-MS). Comparative analyses were performed between ELL, or EDL, or ELL combined with EDL, and the HC to develop biosignatures present in early Lyme disease. A direct comparison between ELL and EDL was also performed to develop a biosignature for stages of early Lyme disease. Metabolic pathway analysis and chemical identification of metabolites with LC-tandem mass spectrometry (LC-MS/MS) demonstrated alterations of eicosanoid, bile acid, sphingolipid, glycerophospholipid, and acylcarnitine metabolic pathways during early Lyme disease. These metabolic alterations were confirmed using a separate set of serum samples for validation. The findings demonstrated that infection of humans with B. burgdorferi alters defined metabolic pathways that are associated with inflammatory responses, liver function, lipid metabolism, and mitochondrial function. Additionally, the data provide evidence that metabolic pathways can be used to mark the progression of early Lyme disease.

Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well-Characterized Serum Samples.

Standard two-tiered testing (STTT) is the recommended algorithm for laboratory diagnosis of Lyme disease (LD). Several limitations are associated with STTT that include low sensitivity in the early stages of disease, as well as technical complexity and subjectivity associated with second-tier immunoblotting; therefore, modified two-tiered testing (MTTT) algorithms that utilize two sequential first-tier tests and eliminate immunoblotting have been evaluated. Recently, a novel MTTT that uses a VlsE chemiluminescence immunoassay followed by a C6 enzyme immunoassay has been proposed. The purpose of this study was to evaluate the performance of the VlsE/C6 MTTT using well-characterized serum samples. Serum samples from the CDC Lyme Serum Repository were tested using three MTTTs, VlsE/C6, whole-cell sonicate (WCS)/C6, and WCS/VlsE, and three STTTs (immunoblotting preceded by three different first-tier assays: VlsE, C6, and WCS). Significant differences were not observed between the results of the MTTTs assessed; however, the VlsE/C6 MTTT resulted in the highest specificity (100%) when other diseases were tested and the lowest sensitivity (75%) for LD samples. Significant differences were present between the results for various MTTTs and STTTs evaluated. Specifically, all MTTTs resulted in higher sensitivities than the STTTs for all LD groups combined and were significantly more accurate (i.e., higher proportion of correct classifications) for this group, with the exception of the WCS/ViraStripe STTT. Additionally, when other diseases were tested, only the results of the VlsE/C6 MTTT differed significantly from those of the WCS/ViraStripe STTT, with the VlsE/C6 MTTT resulting in a 6.2% higher accuracy. Overall, the VlsE/C6 MTTT offers an additional laboratory testing algorithm for LD with equivalent or enhanced performance compared to that of the other MTTTs and STTTs evaluated in this study.

Evaluation of bioMérieux's Dissociated Vidas Lyme IgM II and IgG II as a First-Tier Diagnostic Assay for Lyme Disease.

The recommended laboratory diagnostic approach for Lyme disease is a standard two-tiered testing (STTT) algorithm where the first tier is typically an enzyme immunoassay (EIA) that if positive or equivocal is reflexed to Western immunoblotting as the second tier. bioMérieux manufactures one of the most commonly used first-tier EIAs in the United States, the combined IgM/IgG Vidas test (LYT). Recently, bioMérieux launched its dissociated first-tier tests, the Vidas Lyme IgM II (LYM) and IgG II (LYG) EIAs, which use purified recombinant test antigens and a different algorithm than STTT. The dissociated LYM/LYG EIAs were evaluated against the combined LYT EIA using samples from 471 well-characterized Lyme patients and controls. Statistical analyses were conducted to assess the performance of these EIAs as first-tier tests and when used in two-tiered algorithms, including a modified two-tiered testing (MTTT) approach where the second-tier test was a C6 EIA. Similar sensitivities and specificities were obtained for the two testing strategies (LYT versus LYM/LYG) when used as first-tier tests (sensitivity, 83 to 85%; specificity, 85 to 88%) with an observed agreement of 80%. Sensitivities of 68 to 69% and 76 to 77% and specificities of 97% and 98 to 99% resulted when the two EIA strategies were followed by Western immunoblotting and when used in an MTTT, respectively. The MTTT approach resulted in significantly higher sensitivities than did STTT. Overall, the LYM/LYG EIAs performed equivalently to the LYT EIA in test-to-test comparisons or as first-tier assays in STTT or MTTT with few exceptions.

Cardiac Tropism of Borrelia burgdorferi: An Autopsy Study of Sudden Cardiac Death Associated with Lyme Carditis.

Fatal Lyme carditis caused by the spirochete Borrelia burgdorferi rarely is identified. Here, we describe the pathologic, immunohistochemical, and molecular findings of five case patients. These sudden cardiac deaths associated with Lyme carditis occurred from late summer to fall, ages ranged from young adult to late 40s, and four patients were men. Autopsy tissue samples were evaluated by light microscopy, Warthin-Starry stain, immunohistochemistry, and PCR for B. burgdorferi, and immunohistochemistry for complement components C4d and C9, CD3, CD79a, and decorin. Post-mortem blood was tested by serology. Interstitial lymphocytic pancarditis in a relatively characteristic road map distribution was present in all cases. Cardiomyocyte necrosis was minimal, T cells outnumbered B cells, plasma cells were prominent, and mild fibrosis was present. Spirochetes in the cardiac interstitium associated with collagen fibers and co-localized with decorin. Rare spirochetes were seen in the leptomeninges of two cases by immunohistochemistry. Spirochetes were not seen in other organs examined, and joint tissue was not available for evaluation. Although rare, sudden cardiac death caused by Lyme disease might be an under-recognized entity and is characterized by pancarditis and marked tropism of spirochetes for cardiac tissues.

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.

Lyme Borreliosis Serology: Performance of Several Commonly Used Laboratory Diagnostic Tests and a Large Resource Panel of Well-Characterized Patient Samples.

The current recommendation for the laboratory confirmation of Lyme disease is serology-based diagnostics. Specifically, a standardized two-tiered testing (STTT) algorithm is applied that utilizes a first-tier immunofluorescence assay or enzyme immunoassay (EIA) that, if the result is positive or equivocal, is followed by second-tier immunoblotting. Despite the standardization and performance achievements, STTT is considered technically complex and subjective, as well as insensitive for early acute infection. These issues have prompted development of novel algorithms and testing platforms. In this study, we evaluated the performance of several commonly used assays for STTT. Several modified two-tiered testing (MTTT) algorithms, including a 2-EIA algorithm and modified criteria for second-tier IgG immunoblots, were also evaluated. All tests were performed on sera from a recently available, well-defined archive of positive- and negative-control patients. Our study demonstrates differences in the results between individual first- and second-tier tests, although the overall agreement of the different STTT algorithms used was strong. In addition, the MTTT algorithm utilizing 2-EIAs was found to be equivalent to all STTT algorithms tested, with agreement ranging from 94 to 97%. The 2-EIA MTTT algorithm slightly enhanced sensitivity in early disease compared to the STTT algorithms evaluated. Furthermore, these data add to the mounting evidence that a 2-EIA-based MTTT algorithm, where immunoblotting is replaced by the C6 EIA, performs as well or better than STTT.

Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia.

Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography-tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

Development of a metabolic biosignature for detection of early Lyme disease.

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 2-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. METHODS: Retrospective serum samples from patients with early Lyme disease, other diseases, and healthy controls were analyzed for small molecule metabolites by liquid chromatography-mass spectrometry (LC-MS). A metabolomics data workflow was applied to select a biosignature for classifying early Lyme disease and non-Lyme disease patients. A statistical model of the biosignature was trained using the patients' LC-MS data, and subsequently applied as an experimental diagnostic tool with LC-MS data from additional patient sera. The accuracy of this method was compared with standard 2-tier serology. 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. CONCLUSIONS: The data provide proof-of-concept that metabolic profiling for early Lyme disease can achieve significantly greater (P < .0001) diagnostic sensitivity than current 2-tier serology, while retaining high specificity.

Francisella tularensis subsp. tularensis group A.I, United States.

We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.

Collection and characterization of samples for establishment of a serum repository for lyme disease diagnostic test development and evaluation.

Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

Notes from the field: update on Lyme carditis, groups at high risk, and frequency of associated sudden cardiac death--United States.

On December 13, 2013, MMWR published a report describing three cases of sudden cardiac death associated with Lyme carditis. State public health departments and CDC conducted a follow-up investigation to determine 1) whether carditis was disproportionately common among certain demographic groups of patients diagnosed with Lyme disease, 2) the frequency of death among patients diagnosed with Lyme disease and Lyme carditis, and 3) whether any additional deaths potentially attributable to Lyme carditis could be identified. Lyme disease cases are reported to CDC through the Nationally Notifiable Disease Surveillance System; reporting of clinical features, including Lyme carditis, is optional. For surveillance purposes, Lyme carditis is defined as acute second-degree or third-degree atrioventricular conduction block accompanying a diagnosis of Lyme disease. During 2001-2010, a total of 256,373 Lyme disease case reports were submitted to CDC, of which 174,385 (68%) included clinical information. Among these, 1,876 (1.1%) were identified as cases of Lyme carditis. Median age of patients with Lyme carditis was 43 years (range = 1-99 years); 1,209 (65%) of the patients were male, which is disproportionately larger than the male proportion among patients with other clinical manifestations (p<0.001). Of cases with this information available, 69% were diagnosed during the months of June-August, and 42% patients had an accompanying erythema migrans, a characteristic rash. Relative to patients aged 55-59 years, carditis was more common among men aged 20-39 years, women aged 25-29 years, and persons aged ≥75 years.

Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection.

BACKGROUND: The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. METHODS: In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. RESULTS: Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. CONCLUSIONS: The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

Differential chitinase activity and production within Francisella species, subspecies, and subpopulations.

Genotyping of Francisella tularensis (A1a, A1b, A2, and type B) and Francisella novicida has identified multiple differences between species and among F. tularensis subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of F. tularensis and F. novicida to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among F. tularensis and F. novicida strains. The activity observed for F. tularensis strains was predominantly associated with whole-cell lysates, while the chitinase activity of F. novicida localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of F. tularensis and between the species. Bioinformatic analyses identified two new putative chitinase genes (chiC and chiD), as well as the previously described chiA and chiB. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between Francisella species and F. tularensis subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella.

Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay.

BACKGROUND: A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates. RESULTS: We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2). CONCLUSION: Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.

Metabolomic Insights into Human Arboviral Infections: Dengue, Chikungunya, and Zika Viruses.

The global burden of arboviral diseases and the limited success in controlling them calls for innovative methods to understand arbovirus infections. Metabolomics has been applied to detect alterations in host physiology during infection. This approach relies on mass spectrometry or nuclear magnetic resonance spectroscopy to evaluate how perturbations in biological systems alter metabolic pathways, allowing for differentiation of closely related conditions. Because viruses heavily depend on host resources and pathways, they present unique challenges for characterizing metabolic changes. Here, we review the literature on metabolomics of arboviruses and focus on the interpretation of identified molecular features. Metabolomics has revealed biomarkers that differentiate disease states and outcomes, and has shown similarities in metabolic alterations caused by different viruses (e.g., lipid metabolism). Researchers investigating such metabolomic alterations aim to better understand host⁻virus dynamics, identify diagnostically useful molecular features, discern perturbed pathways for therapeutics, and guide further biochemical research. This review focuses on lessons derived from metabolomics studies on samples from arbovirus-infected humans.

Evaluation of in vivo expressed Borrelia burgdorferi antigens for improved IgM serodiagnosis of early Lyme disease.

Improved serologic tests are needed for accurate diagnosis and proper treatment of early stage Lyme disease. We evaluated the 3 antigens currently used for 2-tiered IgM immunoblot testing (FlaB, OspC, and BmpA) in combination with 3 additional antigens (BBA65, BBA70, and BBA73) and measured the sensitivity and specificity against a serum repository of positive and negative controls. Using 3 statistical methods for positivity cutoff determinations and scoring criteria, we found increased sensitivities for early Lyme disease when 2 of 6 antigens were positive as compared with the 2 of 3 antigen IgM criteria currently used for second-tier immunoblot scoring. Specificities for negative controls were comparable or superior to using 2 of 3 antigens. These results indicate that IgM sensitivity and specificity of serological testing for Lyme disease in the early stages of illness can be improved by employing antigens that target the initial host antibody responses.

Immunoproteomic analysis of Borrelia miyamotoi for the identification of serodiagnostic antigens.

The tick-borne spirochete, Borrelia miyamotoi, is an emerging pathogen of public health significance. Current B. miyamotoi serodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B. miyamotoi antibodies can cross-react with C6 antigen testing for B. burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis of B. miyamotoi proteins to identify novel serodiagnostic antigens. Sera from mice infected with B. miyamotoi by subcutaneous inoculation or tick bite were collected for immunoblotting against B. miyamotoi membrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B. miyamotoi sera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B. burgdorferi serum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from a B. miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile of B. miyamotoi antigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen for B. miyamotoi serodetection.

Higher C6 enzyme immunoassay index values correlate with a diagnosis of noncutaneous Lyme disease.

The correlation between the Food and Drug Administration-cleared C6 enzyme immunoassay (EIA) C6 index values and a diagnosis of Lyme disease has not been examined. We used pooled patient-level data from 5 studies of adults and children with Lyme disease and control subjects who were tested with the C6 EIA. We constructed a receiver operating characteristic curve using regression clustered by study and measured the area under the curve (AUC) to examine the accuracy of the C6 index values in differentiating between patients with noncutaneous Lyme disease and control subjects. In the 4821 included patients, the C6 index value had excellent ability to distinguish between patients with noncutaneous Lyme disease and control subjects [AUC 0.99; 95% confidence interval (CI) 0.99-1.00]. An index value cut point of ≥3.0 had a sensitivity of 90.9% (95% CI, 87.8-93.3) and specificity of 99.0% (95% CI, 98.6-99.2%) for Lyme disease.

Evaluation of a sequential enzyme immunoassay testing algorithm for Lyme disease demonstrates lack of test independence but high diagnostic specificity.

To diagnose Lyme disease, a two-tier testing algorithm is used in which supplemental IgM and IgG immunoblots to detect antibody to Borrelia burgdorferi are reflexively performed if a first-tier assay, such as a whole-cell sonicate-based enzyme immunoassay (WCS EIA), is reactive. Recent data suggest that equal specificity is found by substituting the C6 peptide EIA for immunoblots. In this study using 3956 control sera, we demonstrated that although this two-tier testing algorithm does significantly improve diagnostic specificity compared with each of the EIAs individually, the WCS EIA and the C6 peptide EIA are not independent tests. Therefore, when the C6 peptide EIA is used as the second-tier test, it should be regarded as a supplemental rather than a confirmatory test.