Serodiagnosis Of Disease Research Articles

A-299 Assessment of a new miniaturized line blot system for fully automated serodiagnosis of Borrelia infections

Abstract Background Serodiagnosis of Lyme disease caused by Borrelia burgdorferi commonly affords the combination of a sensitive antibody screening test followed by confirmatory immunoblot analysis. We developed and evaluated a new immunoblot system enabling the multiparametric analysis of anti-Borrelia antibodies by means of miniaturized line blots in a fully automated microplate format. Methods The EUROMicroblots Anti-Borrelia (IgG and IgM, EUROIMMUN) are based on diagnostically relevant Borrelia-specific antigens. Diagnostic sensitivity was determined by analyzing 203 pre-characterized samples from patients with Lyme disease stratified by clinical picture and antibiotic treatment. Specificity was assessed using 168 sera from patients clinically diagnosed with rheumatic diseases. The reference range was analyzed using a control panel of 211 (IgG) and 210 (IgM) samples from healthy individuals, comprising 151 (IgG) and 150 (IgM) blood donors as well as 60 pregnant women (IgG, IgM). The Microwell Imager and EUROLineScan software (EUROIMMUN) were used for image acquisition and evaluation. Results Depending on the clinical category, the diagnostic sensitivity of the EUROMicroblots ranged from 68.0% in the early phase of infection to 100% (IgG/IgM combined) at a specificity of 86.9% (IgG) and 94.0% (IgM). The analysis of the panel of healthy control subjects revealed a prevalence of 10.9% (IgG) and 2.8% (IgM). Conclusions The new EUROMicroblot system enables high-throughput serodiagnosis of Borrelia infections due to the microplate format and automated processing and evaluation. Depending on clinical symptoms and treatment, the assays provide excellent diagnostic sensitivity and specificity for the detection of anti-Borrelia IgG and IgM.

Rapid single-tier serodiagnosis of Lyme disease

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC’s LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.

Comparative Evaluation of Commercial Test Kits Cleared for Use in Modified Two-Tiered Testing Algorithms for Serodiagnosis of Lyme Disease.

Modified 2-tiered testing (MTTT) for Lyme disease utilizes automatable, high throughput immunoassays (AHTIs) in both tiers without involving western immunoblots, offering performance and practical advantages over standard 2-tiered testing (STTT; first-tier AHTI followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) western immunoblots). For MTTT, Centers for Disease Control and Prevention recommends using AHTI test kits that have been cleared by Food and Drug Administration (FDA) specifically for this intended use. We evaluated performance of FDA-cleared MTTT commercial test kits from 3 manufacturers by comparing with STTT results. We performed MTTT (total antibody AHTI with reflex to separate IgM and IgG AHTIs) using test kits from Diasorin, Gold Standard Diagnostics (GSD), and Zeus Scientific on 382 excess serum samples submitted to the clinical laboratory for routine Lyme disease serologic testing in July 2018, measuring agreement between MTTT and STTT using the κ statistic. Overall agreement with STTT was 0.87 (95% confidence interval [CI], .77-.97) using Diasorin assays (almost perfect agreement), 0.80 (95% CI, .68-.93) using GSD assays (substantial agreement) and 0.79 (95% CI, .68-.90) using Zeus assays (substantial agreement). For detection of IgM reactivity, agreement between MTTT and STTT was 0.70 (.51-.90; substantial), 0.63 (95% CI, .44-.82; substantial) and 0.56 (95% CI, .38-.73; moderate), respectively. For detection of IgG reactivity, MTTT/STTT agreement was 0.73 (95% CI,.58-.88), 0.78 (95% CI, .62-.94), and 0.75 (95% CI, .60-.90), respectively (substantial agreement in all cases). MTTT results obtained using commercial test kits from 3 different manufacturers had substantial to almost perfect agreement with STTT results overall and moderate to substantial agreement for IgM and IgG detection independently. Commercial MTTT tests can be used broadly for the diagnosis of Lyme disease.

In silico epitope prediction of Borrelia burgdorferi sensu lato antigens for the detection of specific antibodies

Despite many years of research, serodiagnosis of Lyme disease still faces many obstacles. Difficulties arise mainly due to the low degree of amino acid sequence conservation of the most immunogenic antigens among B. burgdorferi s.l. genospecies, as well as differences in protein production depending on the environment in which the spirochete is located. Mapping B-cell epitopes located on antigens allows for a better understanding of antibody-pathogen interactions which is essential for the development of new and more effective diagnostic tools. In this study, in silico B-cell epitope mapping was performed to determine the theoretical diagnostic potential of selected B. burgdorferi s.l. proteins (BB0108, BB0126, BB0298, BB0689, BB0323, FliL, PstS, SecD, EF-Tu). Bioinformatics software predicted 35 conserved linear and 31 conformational epitopes with the degree of identity among B. burgdorferi s.l. of at least 85%, which may prove to be useful in the development of a new tool for the diagnosis of Lyme disease.

861. Development and Clinical Validation of a Custom Modified Two-Tier Testing Algorithm for Serodiagnosis of Lyme Disease

Abstract Background Diagnosis of Lyme Disease has relied on measuring the serological response to Borrelia sp. Over the years the rigor of serological diagnosis has evolved to meet the needs of the clinic and vaccine manufacturers, where the latter require especially sensitive and specific tests to measure efficacy. Likewise, current US guidelines recommend a two-tier testing approach to diagnose Lyme Disease. Among a constellation of commercially available in vitro diagnostics, two highly sensitive immunoassays with Borrelia sp. antigenic diversity were characterized for utility as a custom Modified Two-Tier Testing (MTTT) algorithm. This custom algorithm consists of a first tier assay from Bio-Rad and a second tier assay from Zeus Scientific, and was developed to capture cases of Lyme Disease. Methods The BioPlex 2200 Lyme Total assay from BioRad and VlsE1/pepC10 IgG/IgM ELISA from Zeus Scientific were evaluated as the first and second tier assays in the custom MTTT, respectively. The MTTT was clinically assessed for diagnostic accuracy and compared to the highly characterized CDC Pre-Marketing Panel, which is composed of samples from US donors diagnosed with Lyme Disease, “look-alike” diseases or healthy individuals. Other clinically relevant sample populations were benchmarked off the predicate FDA-approved MTTT, also from Zeus Scientific. Results The custom MTTT, described here, was more clinically specific than either test alone and was more clinically sensitive than the only other FDA-approved MTTT. When matched against the FDA-approved predicate method, sensitivity and specificity was 100%. The custom MTTT also detected fewer “look-alike” disease samples than either test alone, or the FDA-approved MTTT. Furthermore, detection of early Lyme Disease in the CDC Pre-Marketing panel was 80% sensitive. In addition, the custom MTTT identified Lyme disease in 98.6% of Lyme Disease samples collected from samples that originated in Europe. Conclusion The clinically validated custom MTTT method, described here, has utility as a Lyme Disease diagnostic and is a highly specific tool for assessing efficacy of Lyme Disease vaccines currently in development. Disclosures Michelle A. Gaylord, PhD, Pfizer Inc.: Stocks/Bonds Danielle Baranova, PhD, Pfizer Inc: Stocks/Bonds Michael W. Pride, PhD, Pfizer: Stocks/Bonds Raphael Simon, PhD, Pfizer: Stocks/Bonds Annaliesa S. Anderson, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Warren Vincent. Kalina, PhD, Pfizer: Stocks/Bonds

Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches.

The current Centers for Disease Control and Prevention (CDC) interpretive criteria for serodiagnosis of Lyme disease (LD) involve a two-tiered approach, consisting of a first-tier EIA, IFA, or chemiluminescent assay, followed by confirmation of positive or equivocal results by either immunoblot or a second-tier EIA. To increase overall sensitivity, single-tier alternative immunoblot assays have been proposed, often utilizing antigens from multiple Borrelia burgdorferi strains or genospecies in a single immunoblot; including OspA and OspB in their antigen panel; requiring fewer positive bands than permitted by current CDC criteria; and reporting equivocal results. Published reports concerning alternative immunoblot assays have used relatively small numbers of LD patients and controls to evaluate novel multi-antigen assays and interpretive criteria. We compared the two most commonly used alternative immunoblot interpretive criteria (labeled A and B) to CDC criteria using data from multiple FDA-cleared IgG and IgM immunoblot test kits. These single-tier alternative interpretive criteria, applied to both IgG and IgM immunoblots, demonstrated significantly more false-positive or equivocal results in healthy controls than two-tiered CDC criteria (12.4% and 35.0% for Criteria A and B, respectively, versus 1.0% for CDC criteria). Due to limited standardization and high false-positive rates, the presently evaluated single-tier alternative immunoblot interpretive criteria appear inferior to CDC two-tiered criteria.

Antibody Cross-Reactivity in Serodiagnosis of Lyme Disease.

Lyme disease is a tick-borne disease caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex. The disease is characterized by a varied course; therefore, the basis for diagnosis is laboratory methods. Currently, a two-tiered serological test is recommended, using an ELISA as a screening test and a Western blot as a confirmatory test. This approach was introduced due to the relatively high number of false-positive results obtained when using an ELISA alone. However, even this approach has not entirely solved the problem of false-positive results caused by cross-reactive antibodies. Many highly immunogenic B. burgdorferi s.l. proteins are recognized nonspecifically by antibodies directed against other pathogens. This also applies to antigens, such as OspC, BmpA, VlsE, and FlaB, i.e., those commonly used in serodiagnostic assays. Cross-reactions can be caused by both bacterial (relapsing fever Borrelia, Treponema pallidum) and viral (Epstein-Baar virus, Cytomegalovirus) infections. Additionally, a rheumatoid factor has also been shown to nonspecifically recognize B. burgdorferi s.l. proteins, resulting in false-positive results. Therefore, it is necessary to carefully interpret the results of serodiagnostic tests so as to avoid overdiagnosis of Lyme disease, which causes unnecessary implementations of strong antibiotic therapies and delays in the correct diagnosis.

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.

Development and evaluation of NS1 protein based i-ELISA for serodiagnosis of Bluetongue disease

Bluetongue (BT) is an important viral disease of small ruminants and causes major economic losses in India. For serodiagnosis of BT, recombinant antigen-based assays are considered as alternatives to conventional approaches such as serum neutralization test. The present study aimed at the development of an indirect- enzyme-linked immunosorbent assay (i-ELISA) using bluetongue (BTV) recombinant NS1. NS1 gene of BTV encoding a mature NS1 protein was produced as a recombinant protein (rNS1) in Escherichia coli. The immunogenic potential of purified rNS1 was confirmed by detecting specific NS1 antibody responses in sera collected from immunized sheep using ELISA. An i-ELISA based on rNS1 was developed and optimized. In comparison to a commercially available c-ELISA kit, in the detection of BTV specific antibodies, this new assay exhibited a diagnostic sensitivity of 91.2%. This study highlighted the potential utility of rNS1 protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of serodiagnostic assay for surveillance of BTV infection in sheep and goats.

Optimizing use of multi-antibody assays for Lyme disease diagnosis: A bioinformatic approach.

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.

Evaluation of 2 Lateral Flow Rapid Tests in the Diagnosis of Chagas Disease in the Washington Metropolitan Area.

We compared the accuracy of the Stat-Pak and Chagas Detect Plus with a latent class analysis. Sensitivity values of 89.7% and 91.9% and specificities of 97.1% and 80.3%, respectively, were seen in the serodiagnosis of Chagas disease in Hispanic immigrants, revealing the limitations of these tests in diverse populations.

Linear B Cell Epitopes Derived from the Multifunctional Surface Lipoprotein BBK32 as Targets for the Serodiagnosis of Lyme Disease

BBK32 is a multifunctional surface lipoprotein expressed by Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. Previous studies suggested that BBK32 could be a sensitive antigen target of new, more effective, serodiagnostic assays for the laboratory diagnosis of Lyme disease. However, nonspecific antibody binding to full-length BBK32 has hampered its use as a target in clinical assays. Specificity can be improved by the use of peptides composed of linear B cell epitopes that are unique to B. burgdorferi, eliminating cross-reactive epitopes that bind to antibodies generated by non-B. burgdorferi antigens. In this study, we identified linear B cell epitopes in 2 regions, BBK32 amino acids 16 to 30 [BBK32(16-30)] and BBK32 amino acids 51 to 80 [BBK32(51-80)], by probing overlapping peptide libraries of BBK32 with serum from patients with early Lyme disease. We screened synthetic peptides containing these epitopes using a large panel of serum (n = 355) obtained from patients with erythema migrans lesions (early Lyme disease), Lyme arthritis, syphilis, rheumatoid arthritis, or healthy volunteers. BBK32(16-30) demonstrated a nearly universal antibody binding in serum from all patients, indicating that regions of BBK32 are highly cross-reactive. BBK32(51-80) was less cross-reactive, being able to distinguish serum from Lyme disease patients from control patient serum; however, an unacceptable level of antibody binding was still observed in control samples, resulting in a reduced specificity (94.7%). These results indicate that BBK32 contains cross-reactive epitopes that make it a poor antigen target for inclusion in a serodiagnostic assay for Lyme disease and highlight the difficulties in identifying highly sensitive and specific seroassay targets.IMPORTANCE Lyme disease is an infectious disease that has the potential to cause significant morbidity with damage to nervous and musculoskeletal systems if left untreated. Appropriate antibiotic treatment during early infection prevents disease progression. Unfortunately, currently available diagnostics are suboptimal in the detection of early disease. The inability to confirm Borrelia infection using laboratory methods during early disease is, in part, responsible for much of the controversy surrounding Lyme disease today. As a result, there has been significant investment in the identification of new antigen targets to generate diagnostic assays that are more sensitive for the detection of early infection. The importance of our research is that in our evaluation of BBK32, an antigen that was previously identified as a promising target for use in serodiagnostics, we found a high degree of cross-reactivity that could compromise the specificity of assays that utilize this antigen, leading to false-positive diagnoses.

Revisiting the Lyme Disease Serodiagnostic Algorithm: the Momentum Gathers.

Lyme disease is a tick-borne illness caused by Borreliella (Borrelia) burgdorferi, and it is the most common vector-borne disease in the United States, with an estimated incidence of 300,000 cases per year. The currently recommended approach for laboratory support of the diagnosis of Lyme disease is a standard two-tiered (STT) algorithm comprised of an enzyme-linked immunoassay (EIA) or immunofluorescence assay (IFA), followed by Western blotting (WB). The STT algorithm has low sensitivity in early infection, and there are drawbacks associated with the WB use in practice. Modified two-tiered (MTT) algorithms have been shown to improve the sensitivity of the testing in early disease while maintaining high specificity. In this issue of the Journal of Clinical Microbiology, A. Pegalajar-Jurado et al. (J Clin Microbiol 56:e01943-17, 2018, https://doi.org/10.1128/JCM.01943-17) report the results of their evaluation of the Liaison VlsE CLIA, the Captia B. burgdorferi IgG/IgM EIA, and the C6 B. burgdorferi (Lyme) EIA as MTT algorithms compared with results with the STT algorithm using the same tests as the first-tier test and the ViraStripe IgM and IgG WBs as the second-tier test. The results showed that all MTT algorithms had higher sensitivities than STT algorithms and were highly specific. These results showed that MTT approaches are a valid alternative to the currently recommended STT algorithm for serodiagnosis of Lyme disease, opening the door for the development of rapid diagnostics and point-of-care testing that can provide diagnostic information during the initial patient visit.

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Portable and power-free serodiagnosis of Chagas disease using magnetic levitating microbeads.

This work describes the detection of anti-T. cruzi antibodies in whole blood solutions using magnetic levitating microbeads (MLμBs). This simple diagnostic method can be easily performed by minimally trained personnel using an inexpensive and portable magnetic stage that requires no electricity. A multiphase test tube containing the MLμBs facilitates the sequential incubation, filtering, and reading of the immunoassays. The diagnostic method starts by adding a blood sample to the top phase of the test tube where the anti-T. cruzi antibodies present in the blood attach to the T. cruzi antigens on the surface of the MLμBs. Shaking the test tube after incubation mixes the top layer with a paramagnetic medium loaded with SiO2 microcrystals. The attachment of SiO2 microcrystals to those MLμBs bound to T. cruzi antibodies decreases their levitation height once the tube is placed between two antialigned permanent magnets. Measuring the levitation height of MLμBs enables the accurate detection and quantification of anti-T. cruzi antibodies in the blood across the clinically relevant range, with a detection limit of 5 μg mL-1. The small size of the test tubes facilitates the simultaneous analysis of over 50 different samples. MLμBs act as partial collimators for non-polarized light, facilitating their visual identification by the naked eye or by projecting incident light on a thin paper screen. A machine-vision algorithm was created to automatically interpret the results of the MLμB tests from a digital image, resulting in a rapid, accurate, and user-friendly assay for Chagas disease that can be used in resource-limited settings.

Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques.

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40-60%) followed by a gel filtration through Sephadex G-100 and DEAE- cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS-PAGE to be 22kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS-PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35-40°C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5mM) or iodoacetamide (10mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotype-specific serodiagnosis of Trypanosoma cruzi infection.

Distinct Trypanosoma cruzi genotypes have been considered relevant for patient management and therapeutic response of Chagas disease. However, typing strategies for genotype-specific serodiagnosis of Chagas disease are still unavailable and requires standardization for practical application. In this study, an innovative TcI/TcVI/TcII Chagas Flow ATE-IgG2a technique was developed with applicability for universal and genotype-specific diagnosis of T. cruzi infection. For this purpose, the reactivity of serum samples (percentage of positive fluorescent parasites-PPFP) obtained from mice chronically infected with TcI/Colombiana, TcVI/CL or TcII/Y strain as well as non-infected controls were determined using amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI in parallel batches of TcI, TcVI and TcII target antigens. Data demonstrated that “α-TcII-TRYPO/1:500, cut-off/PPFP = 20%” presented an excellent performance for universal diagnosis of T. cruzi infection (AUC = 1.0, Se and Sp = 100%). The combined set of attributes “α-TcI-TRYPO/1:4,000, cut-off/PPFP = 50%”, “α-TcII-AMA/1:1,000, cut-off/PPFP = 40%” and “α-TcVI-EPI/1:1,000, cut-off/PPFP = 45%” showed good performance to segregate infections with TcI/Colombiana, TcVI/CL or TcII/Y strain. Overall, hosts infected with TcI/Colombiana and TcII/Y strains displayed opposite patterns of reactivity with “α-TcI TRYPO” and “α-TcII AMA”. Hosts infected with TcVI/CL strain showed a typical interweaved distribution pattern. The method presented a good performance for genotype-specific diagnosis, with global accuracy of 69% when the population/prototype scenario include TcI, TcVI and TcII infections and 94% when comprise only TcI and TcII infections. This study also proposes a receiver operating reactivity panel, providing a feasible tool to classify serum samples from hosts infected with distinct T. cruzi genotypes, supporting the potential of this method for universal and genotype-specific diagnosis of T. cruzi infection.

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.

Polymorphism of 41 kD Flagellin Gene and Its Human B-Cell Epitope in Borrelia burgdorferi Strains of China.

The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi) is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease.

The Mycobacterium tuberculosis PPE protein Rv1168c induces stronger B cell response than Rv0256c in active TB patients

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a serious global health problem and is responsible for millions of deaths every year. For effective control of this dreadful disease, it is necessary to diagnose TB cases at the initial stages of infection. The serodiagnosis of disease represents simple, rapid and inexpensive method that can be used at the primary health care levels. In this study we have compared sensitivity of two PPE proteins of M. tuberculosis, i.e., Rv0256c and Rv1168c for their use as serodiagnostic markers in active tuberculosis patients. Employing a standardized enzyme immunoassay with these PPE proteins as candidate antigens we were able to successfully discriminate the TB patients' sera from the BCG-vaccinated healthy controls. Further, we observed that Rv1168c displayed higher sensitivity in detecting extrapulmonary and smear negative pulmonary TB cases which are difficult to diagnose by available diagnostic methods. Overall the study highlights that Rv1168c can be used as a potential serodiagnostic marker for the diagnosis of active tuberculosis disease.