Revisiting the antigen markers of vector-borne parasitic diseases identified by immunomics: identification and application to disease control.

INTRODUCTION: Protein microarray is a promising immunomic approach for identifying biomarkers. Based on our previous study that reviewed parasite antigens and recent parasitic omics research, this article expands to include information on vector-borne parasitic diseases (VBPDs), namely, malaria, schistosomiasis, leishmaniasis, babesiosis, trypanosomiasis, lymphatic filariasis, and onchocerciasis. AREAS COVERED: We revisit and systematically summarize antigen markers of vector-borne parasites identified by the immunomic approach and discuss the latest advances in identifying antigens for the rational development of diagnostics and vaccines. The applications and challenges of this approach for VBPD control are also discussed. EXPERT OPINION: The immunomic approach has enabled the identification and/or validation of antigen markers for vaccine development, diagnosis, disease surveillance, and treatment. However, this approach presents several challenges, including limited sample size, variability in antigen expression, false-positive results, complexity of omics data, validation and reproducibility, and heterogeneity of diseases. In addition, antigen involvement in host immune evasion and antigen sensitivity/specificity are major issues in its application. Despite these limitations, this approach remains promising for controlling VBPD. Advances in technology and data analysis methods should continue to improve candidate antigen identification, as well as the use of a multiantigen approach in diagnostic and vaccine development for VBPD control.

An ELISA Using Synthetic Mycolic Acid-Based Antigens with DIVA Potential for Diagnosing Johne's Disease in Cattle.

THE PROBLEM: Ante-mortem diagnosis of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne's disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne's disease in cattle. METHOD: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne's disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals. RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX. CONCLUSION: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.

A dot-blot ELISA preliminary evaluation using PvMSP1-42 recombinant protein as antigen for serological diagnosis of Plasmodium vivax infection in Thailand.

Plasmodium vivax is the most prevalent cause of malaria in Thailand and is predominant in malarial endemic areas worldwide. P. vivax infection is characterized by low parasitemia, latent liver-stage parasites, or asymptomatic infections leading to underreported P. vivax cases. These are significant challenges for controlling and eliminating P. vivax from endemic countries. This study developed and evaluated a dot-blot enzyme-linked immunosorbent assay (ELISA) using PvMSP1-42 recombinant antigen for serological diagnosis based on the detection of antibodies against P. vivax. The optimal PvMSP1-42 concentration and dilutions of anti-human IgG horseradish peroxidase (HRP)-conjugated antiserum were tested on 88 serum samples from P. vivax, Plasmodium falciparum and bacterial infection, including healthy individuals. A cut-off titer of 1:800 produced optimal values for sensitivity and specificity of 90.9 and 98.2%, respectively, with an accuracy of 95.5%. The positive and negative predictive values were 96.8 and 94.7% respectively. The results from microscopic examination and dot-blot ELISA showed strong agreement with the 0.902 kappa index. Thus, the dot-blot ELISA using PvMSP1-42 antigen provided high sensitivity and specificity suitable for serodiagnosis of P. vivax infection. The test is a simple and quick diagnostic assay suitable for field testing as it does not require specific equipment or particular skills.

An immunochromatographic test for serological diagnosis of scrub typhus.

A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O.tsutsugamushi were sprayed in another line across the membrane making this the "Control" line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the "Test" line if the sample contains antibodies to O.tsutsugamushi. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus.

Antibodies to Borrelia burgdorferi and Bartonella species in serum and synovial fluid from people with rheumatic diseases.

Vector-borne infections may underlie some rheumatic diseases, particularly in people with joint effusions. This study aimed to compare serum and synovial fluid antibodies to B. burgdorferi and Bartonella spp. in patients with rheumatic diseases. This observational, cross-sectional study examined paired synovial fluid and serum specimens collected from 110 patients with joint effusion between October 2017 and January 2022. Testing for antibodies to B. burgdorferi (using CDC criteria) and Bartonella spp. via two indirect fluorescent antibody (IFA) assays was performed as part of routine patient care at the Institute for Specialized Medicine (San Diego, CA, USA). There were 30 participants (27%) with positive two-tier B. burgdorferi serology and 26 participants (24%) with IFA seroreactivity (≥1:256) to B. henselae and/or B. quintana. Both B. burgdorferi IgM and IgG were detected more frequently in synovial fluid than serum: 27% of patients were either IgM or IgG positive in synovial fluid, compared to 15.5% in serum (P = 0.048). Conversely, B. henselae and B. quintana antibodies were detected more frequently in serum than synovial fluid; overall only 2% of patients had positive IFA titers in synovial fluid, compared to 24% who had positive IFA titers in serum (P < 0.001). There were no significant associations between B. burgdorferi or Bartonella spp. seroreactivity with any of the clinical rheumatological diagnoses. This study provides preliminary support for the importance of synovial fluid antibody testing for documenting exposure to B. burgdorferi but not for documenting exposure to Bartonella spp. IMPORTANCE: This study focuses on diagnostic testing for two common vector-borne diseases in an affected patient population. In it, we provide data showing that antibodies to B. burgdorferi, but not Bartonella spp., are more commonly found in synovial fluid than serum of patients with joint effusion. Since Lyme arthritis is a common-and sometimes difficult to diagnose-rheumatic disease, improving diagnostic capabilities is of utmost importance. While our findings are certainly not definitive for changes to practice, they do suggest that synovial fluid could be a useful sample for the clinical diagnosis of Lyme disease, and future prospective studies evaluating this claim are warranted.

Advances in diagnostic approaches to Fasciola infection in animals and humans: An overviews.

Fasciolosis, caused by Fasciola hepatica and F. gigantica, is an impediment to the livestock industry's expansion and has a massively negative socio-economic impact due to its widespread prevalence in livestock. It is a waterborne zoonosis affecting human populations in the countries where rural economies are associated with livestock rearing. Conventional diagnosis of Fasciola infection is done by detecting parasite eggs in the faeces of infected animals or by immunological methods. Accurate and quick immunodiagnosis of Fasciola infection in animals and humans is based on the detection of antibodies and specific antigens expressed in the prepatent stage of the parasite. Both molecular and serodiagnostic tests developed thus far have enhanced the reliability of Fasciola diagnosis in both man and animals but are not widely available in resource-poor nations. A pen-side diagnostic test based on a lateral flow assay or a DNA test like loop-mediated isothermal amplification (LAMP) would be simple, fast, and cost-effective, enabling clinicians to treat animals in a targeted manner and avoid the development of drug resistance to the limited flukicides. This review focuses on the recent advances made in the diagnosis of this parasite infection in animals and humans.

Comparative analysis of a novel N-butanol-prepared antigen vs thermo-resistant and sonicated antigens for human leptospirosis detection.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis.

Application of a recombinant novel trypsin from Trichinella spiralis for serodiagnosis of trichinellosis.

BACKGROUND: The excretory/secretory (ES) antigen of Trichinella spiralis muscle larvae (ML) is currently the most widely used diagnostic antigen to detect T. spiralis infection. However, this antigen has certain drawbacks, such as a complicated ES antigen preparation process and lower sensitivity during the early phase of infection. The aim of this study was to investigate the features of a novel T. spiralis trypsin (TsTryp) and evaluate its potential diagnostic value for trichinellosis. METHODS: The TsTryp gene was cloned and recombinant TsTryp (rTsTryp) expressed. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed to confirm the antigenicity of rTsTryp. The expression pattern and distribution signature of TsTryp at various life-cycle stages of T. spiralis were analyzed by quantitative PCR, western blotting and the immunofluorescence test. An ELISA with rTsTryp and ML ES antigens was used to detect immunoglobulins G and M (IgG, IgM) in serum samples of infected mice, swine and humans. The seropositive results were further confirmed by western blot with rTsTryp and ML ES antigens. RESULTS: TsTryp expression was observed in diverse T. spiralis life-cycle phases, with particularly high expression in the early developmental phase (intestinal infectious larvae and adults), with distribution observed mainly at the nematode outer cuticle and stichosome. rTsTryp was identified by T. spiralis-infected mouse sera and anti-rTsTryp sera. Natural TsTryp protease was detected in somatic soluble and ES antigens of the nematode. In mice infected with 200 T. spiralis ML, serum-specific IgG was first detected by rTsTryp-ELISA at 8 days post-infection (dpi), reaching 100% positivity at 12 dpi, and first detected by ES-ELISA at 10 dpi, reaching 100% positivity at 14 dpi. Specific IgG was detected by rTsTryp 2 days earlier than by ES antigens. When specific IgG was determined in serum samples from trichinellosis patients, the sensitivity of rTsTryp-ELISA and ES antigens-ELISA was 98.1% (51/52 samples) and 94.2% (49/52 samples), respectively (P = 0.308), but the specificity of rTsTryp was significantly higher than that of ES antigens (98.7% vs. 95.4%; P = 0.030). Additionally, rTsTryp conferred a lower cross-reaction, with only three serum samples in total testing positive from 11 clonorchiasis, 20 cysticercosis and 24 echinococcosis patients (1 sample from each patient group). CONCLUSIONS: TsTryp was shown to be an early and highly expressed antigen at intestinal T. spiralis stages, indicating that rTsTryp represents a valuable diagnostic antigen for the serodiagnosis of early Trichinella infection.

Evaluation of a minimal array of Treponema pallidum antigens as biomarkers for syphilis diagnosis, infection staging, and response to treatment.

IMPORTANCE: This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.

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.

Usefulness of receptor binding domain protein-based serodiagnosis of COVID-19.

Objectives: This study evaluated the performance of recombinant receptor binding domain (RBD) protein-based enzyme-linked immunosorbent assays (RBD-ELISAs) for detecting anti-SARS-CoV-2 immunoglobulin (Ig) G and IgM antibodies. Methods: In this study, 705 sera from SARS-CoV-2-infected individuals and 315 sera from healthy individuals were analyzed. Results: The RBD-ELISA IgG exhibited high specificity (99.1%) and moderate sensitivity (48.0%), with an overall diagnostic accuracy of 73.5%. RBD-ELISA IgM demonstrated specificity at 94.6% and sensitivity at 51.1%, with an accuracy of 72.8%. Both assays displayed improved performance when analyzing samples collected 15-21 days post-symptom onset, achieving sensitivity and accuracy exceeding 88% and 90%, respectively. Combining RBD-ELISA IgG and IgM in parallel analysis enhanced sensitivity to 98.6% and accuracy to 96.2%. Comparing these RBD-ELISAs with commercially available tests, the study found overlapping sensitivity and similar specificity values. Notably, the combined RBD-ELISA IgG and IgM showed superior performance. Cross-reactivity analysis revealed low false-positive rates (4.4% for IgG, 3.7% for IgM), primarily with viral infections. Conclusion: This research underscores the potential of RBD-based ELISAs for COVID-19 diagnosis, especially when assessing samples collected 15-21 days post-symptom onset and utilizing a parallel testing approach. The RBD protein's immunogenicity and specificity make it a valuable tool for serodiagnosis, offering an alternative to polymerase chain reaction-based methods, particularly in resource-limited settings.

IgM-specific linear epitopes on the E2 protein for serodiagnosis of Chikungunya.

Chikungunya virus (CHIKV) and Dengue virus (DENV) are vector-borne diseases transmitted by Aedes aegypti and Aedes albopictus that pose a significant threat to global public health. Cases of acute Chikungunya fever often present similar clinical symptoms to other vector-borne diseases, such as Dengue fever. In regions where multiple vector-borne diseases coexist, CHIKV is often overlooked or misdiagnosed as Dengue virus, West Nile virus, Zika virus or other viral infections, which delays its prevention and control. However, IgM antibodies directed against the E2 protein of CHIKV have not yet been generalized to clinical settings due to the low sensitivity and high cost in commercial kits. Indirect ELISA with peptides provides an effective supplementary tool for detecting CHIKV IgM antibodies. Our study aims at examining the potential of linear epitopes on the E2 glycoprotein that specifically bind to IgM antibodies as serodiagnostic tool for CHIKV. The sensitivity of the established peptide indirect ELISA method for detecting clinical samples is significantly better than that of commercial kits, realizing a beneficial supplement to the existing IgM antibody assay. It also established the groundwork for comprehending the biological mechanisms of the CHIKV E2 protein and the advancement of innovative epitope peptide vaccines.

Use of recombinant malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn] as antigens in indirect ELISA for diagnosis of bovine brucellosis.

The objective of this study was to validate an indirect enzyme-linked immunoassay (iELISA) using the recombinant proteins, malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn], as antigens and to evaluate its ability to discriminate antibodies produced by vaccination from those induced by infection in the diagnosis of bovine brucellosis. Sera from six groups were evaluated: G1 - culture-positive animals (52 serum samples) (naturally infected); G2 - non-vaccinated animals (28 serum samples) positive in RBT (Rose Bengal test) and 2ME (2-mercaptoethanol test) selected from brucellosis-positive herds; G3 - animals from a brucellosis-free area (32 serum samples); G4 - S19 vaccinated heifers (114 serum samples); G5 - RB51 vaccinated heifers (60 serum samples); G6 - animals inoculated with inactivated Yersinia enterocolitica O:9 (42 serum samples). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were estimated using the frequentist approach and the confidence interval (CI) (95%) calculated by the Clopper-Pearson (exact) method. The DSe for iELISA_MDH in the G1 group was 71.7% (CI 95%: 57.6-83.2%) and for the G2 100.0% (CI 95%: 87.7-100.0%), whereas the DSp was 84.4% in the G3 (CI 95%: 67.2-94.7%). For the iELISA_SOD the DSe was estimated 67.3% for the G1 (CI 95%: 52.9-79.7%) and 71.4% for G2 (CI 95%: 51.3-86.8%), while the DSp for G3 was 87.5% (CI 95%: 71.0-96.5%). iELISA_MDH and iELISA_SOD showed potential to be used in the diagnosis of infected animals, increasing the range of serological tests available for the diagnosis of bovine brucellosis, with the advantage of being S-LPS-free. However, none of the tests could differentiate between infection and vaccination.

Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

Strongyloidiasis is a neglected tropical disease that can cause fatal complications due to hyperinfection and disseminated strongyloidiasis in immunocompromised patients. We used two Strongyloides stercoralis recombinant antigenic proteins, L3NieAg.01 (NIE) and IgG-immunoreactive antigen (SsIR), to develop the recombinant antigen-based immunochromatography test (ICT) kit. We constructed and compared kits using either the NIE (NIE ICT kit) or the SsIR (SsIR ICT kit) antigens and a kit using a mixture of both (NIE-SsIR ICT kit) for detection of anti-Strongyloides IgG antibody in human serum samples. Serum samples from normal healthy individuals (Group I, n = 40), proven strongyloidiasis patients (Group II, n = 100), and those with other parasitic infections (Group III, n = 154) were evaluated. Sensitivity and specificity were 81.0% and 84.0% for the NIE ICT kit, 89.0% and 83.5% for the SsIR ICT kit, and 95.0% and 90.2% for the NIE-SsIR ICT kit, respectively. The NIE-SsIR ICT kit provided the best diagnostic results; it can supplement stool examination for clinical diagnosis and can be used to screen for asymptomatic S. stercoralis infection in people at risk in endemic areas. The NIE-SsIR ICT kit can also be used in large-scale sero-epidemiological investigations in endemic areas without the need for additional facilities or ancillary supplies.

Title: Amélioration de la sensibilité diagnostique de l'anguillulose humaine grâce à l'immunochromatographie à base d'antigènes recombinants mixtes sur le lieu d'intervention. Abstract: La strongyloïdose est une maladie tropicale négligée qui peut entraîner des complications mortelles dues à une hyperinfection et à une strongyloïdose disséminée chez les patients immunodéprimés. Nous avons utilisé deux protéines antigéniques recombinantes de Strongyloides stercoralis, L3NieAg.01 (NIE) et l'antigène immunoréactif IgG (SsIR), pour développer un kit de test d'immunochromatographie (TIC) basé sur les antigènes recombinants. Nous avons construit et comparé des kits utilisant les antigènes NIE (kit NIE ICT), SsIR (kit SsIR ICT) ou un mélange des deux (kit NIE-SsIR ICT) pour la détection des anticorps IgG anti-Strongyloides dans des échantillons de sérum humain. Des échantillons de sérum provenant d'individus normaux en bonne santé (groupe I, n = 40), de patients atteints d'anguillulose avérée (groupe II, n = 100) et de patients atteints d'autres infections parasitaires (groupe III, n = 154) ont été évalués. La sensibilité et la spécificité étaient respectivement de 81,0 % et 84,0 % pour le kit NIE ICT, 89,0 % et 83,5 % pour le kit SsIR ICT, et 95,0 % et 90,2 % pour le kit NIE-SsIR ICT. Le kit NIE-SsIR ICT a fourni les meilleurs résultats de diagnostic ; il peut compléter l'examen des selles pour le diagnostic clinique et peut être utilisé pour dépister une infection asymptomatique à S. stercoralis chez les personnes à risque dans les zones d'endémie. Le kit NIE-SsIR ICT peut également être utilisé dans des enquêtes séro-épidémiologiques à grande échelle dans les zones endémiques sans nécessiter d'installations supplémentaires ou de fournitures auxiliaires.

Liposomes: from August Wassermann to vaccines against COVID-19.

Background and Purpose: The development of vaccines against the SARS-CoV-2 virus has become a big challenge for many countries in 2020-2022. mRNA vaccines were shown to be effective and safe and have been widely used worldwide in the fight against the COVID-19 pandemic. The fundamental factor in creating mRNA vaccines, which ensures effective delivery of mRNA to the host cells, is the composition of lipid nanoparticles, namely the presence of ionized charged lipids, which ensures the binding of mRNA molecules. However, the significant role of liposomes in the development of liposomal vaccines and identification of immunochemical reactions involving lipids should be assessed in the context of the development of the pioneering idea of August Wassermann about the use of liposomal antigens in the diagnosis and immunoprophylaxis of serious human diseases. Experimental Approach: The review is devoted to the use of liposomal antigens as antigen-delivery systems for diagnosis and immunoprophylaxis. Key Results: Studies of cardiolipin antigen in serodiagnosis of syphilis became the foundation of antibodies in diagnosing various infectious diseases and pathological conditions, such as tuberculosis, lupus erythematosus, COVID-19, borreliosis, etc. Identification of antiphospholipid antibodies (mainly anticardiolipin) and today is the most important diagnostic tool for antiphospholipid syndrome. Conclusion: The liposomal system first proposed in 1906 for the diagnosis of syphilis evolved more than a century later into mRNA vaccines, which are used today in the fight against the COVID-19 pandemic.

Sensitivity and specificity of a syphilis rapid diagnostic test in blood donors' samples.

Rapid Diagnostic Tests (RDT) are useful to identify syphilis cases, particularly for hard-to-reach populations and if laboratory services are scarce. However, RDT performance may be suboptimal. We aimed to assess the sensitivity and specificity of a syphilis RDT using well-characterized blood donors' samples. We categorized samples from 811 blood donors into five groups: 1 - Samples with reactive Chemiluminescence (QML), FTA-Abs, and VDRL; 2 - Samples with reactive QML and FTA-Abs, and nonreactive VDRL; 3 - Samples with reactive QML, and nonreactive for other markers (false-positives); 4 - Controls with nonreactive QML; and 5 - Samples reactive for HIV, with nonreactive QML. Sensitivity was tested in groups 1 (overall and according to VDRL titers) and 2; specificity was tested in groups 3‒5. The RDT had high specificity, even in samples reactive for HIV. The sensitivity was high (91.9%) in samples with reactive VDRL but varied between 75.0%‒100% according to VDRL titers. The overall sensitivity was lower (81.3%) in samples with reactive FTA-Abs and nonreactive VDRL. The RDT is a useful tool to detect active syphilis but may be more limited for cases with very early or remote infection, or those with prior treatment. When higher sensitivity is needed, additional strategies including recurrent testing or laboratory-based tests may be required.

Recombinant TBEV Protein E of the Siberian Subtype Is a Candidate Antigen in the ELISA Test System for Differential Diagnosis.

The tick-borne encephalitis virus (TBEV) is one of the most common members of the Orthoflavivirus genus, which comprises the causative agents of severe diseases in humans and animals. Due to the expanding areas of orthoflavivirus infection, its differential diagnosis is highly demanded. Commercial test kits based on inactivated TBEV may not provide reliable differentiation between flaviviruses because of serological crossover in this genus. Application of recombinant domains (sE and dIII) of the TBEV Sukhar-strain protein E as antigens in an ELISA test system allowed us to identify a wide range of antibodies specific to different TBEV strains. We tested 53 sera from human patients with confirmed TBE diagnosis (the efficacy of our test system based on sE protein was 98%) and 56 sera from patients with other orthoflavivirus infections in which no positive ones were detected using our ELISA test system, thus being indicative of its 100% specificity. We also tested mouse and rabbit sera containing antibodies specific to 17 TBEV strains belonging to different subtypes; this assay exhibited high efficacy and differentiation ability in detecting antibodies against TBEV from other orthoflaviviruses such as Omsk hemorrhagic fever, Powassan, yellow fever, dengue, West Nile, Zika, and Japanese encephalitis viruses.

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.

Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value.

Lyme disease is a tick-borne zoonosis caused by Gram-negative bacteria belonging to the Borrelia burgdorferi sensu lato (s.l.) group. In this study, IgM- and IgG-specific linear epitopes of two B. burgdorferi sensu stricto (s.s.) antigens BmpA and BBK32 were mapped using a polypeptide array. Subsequently, two chimeric proteins BmpA-BBK32-M and BmpA-BBK32-G were designed to validate the construction of chimeras using the identified epitopes for the detection of IgM and IgG, respectively, by ELISA. IgG-ELISA based on the BmpA-BBK32-G antigen showed 71% sensitivity and 95% specificity, whereas a slightly lower diagnostic utility was obtained for IgM-ELISA based on BmpA-BBK32-M, where the sensitivity was also 71% but the specificity decreased to 89%. The reactivity of chimeric proteins with nondedicated antibodies was much lower. These results suggest that the identified epitopes may be useful in the design of new forms of antigens to increase the effectiveness of Lyme disease serodiagnosis. It has also been proven that appropriate selection of epitopes enables the construction of chimeric proteins exhibiting reactivity with a specific antibody isotype.