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.

The use of peptides for immunodiagnosis of human Chagas disease.

Chagas disease, caused by the protozoa Trypanosoma cruzi, continues to be a serious public health problem in Latin America, worsened by the limitations in its detection. Given the importance of developing new diagnostic methods for this disease, the present review aimed to verify the number of publications dedicated to research on peptides that demonstrate their usefulness in serodiagnosis. To this end, a bibliographic survey was conducted on the PubMed platform using the keyword "peptide" or "epitope" combined with "Chagas disease" or "Trypanosoma cruzi"; "diagno*" or "serodiagnosis" or "immunodiagnosis", without period restriction. An increasing number of publications on studies employing peptides in ELISA and rapid tests assays was verified, which confirms the expansion of research in this field. It is possible to observe that many of the peptides tested so far originate from proteins widely used in the diagnosis of Chagas, and many of them are part of commercial tests developed. In this sense, as expected, promising results were obtained for several peptides when tested in ELISA, as many of them exhibited sensitivity and specificity values above 90%. Furthermore, some peptides have been tested in several studies, confirming their diagnostic potential. Despite the promising results observed, it is possible to emphasize the need for extensive testing of peptides, using different serological panels, in order to confirm their potential. The importance of producing an effective assay capable of detecting the clinical stages of the disease, as well as new immunogenic antigens that enable new serological diagnostic tools for Chagas disease, is evident.

Evaluation of serological assays for the diagnosis of childhood tuberculosis disease: a study protocol.

BACKGROUND: Tuberculosis (TB) poses a major public health challenge, particularly in children. A substantial proportion of children with TB disease remain undetected and unconfirmed. Therefore, there is an urgent need for a highly sensitive point-of-care test. This study aims to assess the performance of serological assays based on various antigen targets and antibody properties in distinguishing children (0-18 years) with TB disease (1) from healthy TB-exposed children, (2) children with non-TB lower respiratory tract infections, and (3) from children with TB infection. METHODS: The study will use biobanked plasma samples collected from three prospective multicentric diagnostic observational studies: the Childhood TB in Switzerland (CITRUS) study, the Pediatric TB Research Network in Spain (pTBred), and the Procalcitonin guidance to reduce antibiotic treatment of lower respiratory tract infections in children and adolescents (ProPAED) study. Included are children diagnosed with TB disease or infection, healthy TB-exposed children, and sick children with non-TB lower respiratory tract infection. Serological multiplex assays will be performed to identify M. tuberculosis antigen-specific antibody features, including isotypes, subclasses, Fc receptor (FcR) binding, and IgG glycosylation. DISCUSSION: The findings from this study will help to design serological assays for diagnosing TB disease in children. Importantly, those assays could easily be developed as low-cost point-of-care tests, thereby offering a potential solution for resource-constrained settings. GOV IDENTIFIER: NCT03044509.

Deregulation of MicroRNA-146a and 155 expression levels might underlie complicated pregnancy in Toxoplasma Gondii seronegative women.

BACKGROUND: To evaluate the ability of the estimated plasma expression levels of genes of microRNA (MiR-) 146a and 155 to differentiate between samples of pregnant women suspected to be infected by T. gondii. 50 newly pregnant women who had at least one of the criteria of high risk for toxoplasma infection and 50 newly primigravida women free of these criteria gave blood samples for qualitative determination of serum toxoplasma antibodies and estimation of plasma expression levels of MiR-146a and 155 using the qRT-PCR. During the pregnancy course, the incidence of pregnancy complications was recorded. RESULTS: Twenty-six women were IgM-/IgG-, 17 women were IgM+/IgG- and 7 women were IgM+/IgG+. Thirty-two women had pregnancy complications with significantly lower incidence in IgM-/IgG- women. Plasma expression levels of MiR-146a and 155 were significantly higher in total patients compared to control levels and were significantly higher in samples of IgM+/IgG+ patients than in other samples. Statistical analyses defined a high plasma level of MiR-155 as the highly significant predictor for oncoming pregnancy complications and high levels of both microRNAs as predictors for the presence of toxoplasmosis despite seronegativity. Kaplan-Meier regression analysis defined increasing cumulative risk of having toxoplasmosis despite seronegativity with plasma levels of MiR-146a and MiR-155 of 1.2 and 3, respectively. CONCLUSION: The incidence of pregnancy complications is high, irrespective of the seronegativity of women at high risk of toxoplasmosis. Estimated plasma levels of MiR-155 might identify women liable to develop complications and differentiate seronegative women vulnerable to having T. gondii infection. TRIAL REGISTRATION: The study protocol was approved preliminarily by the Local Ethical Committee at Benha Faculty of Medicine. Before enrollment, the study protocol was discussed in detail with the study participants, and those accepted to participate in the study signed written fully informed consents. The final approval of the study protocol was obtained after the end of case collection and registered by RC: 5-11-2022.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

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.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

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.

Efficient generation and characterization of chimeric dengue viral-like particles.

Viral-like particles (VLPs) because of their non-infectious and high immunogenic properties have important applications in diagnostics, drug delivery, and vaccine production. They also serve as an attractive model system to study virus assembly and fusion processes. Unlike other flaviviruses, Dengue virus (DENV) is not very efficient in the production of VLPs on the expression of DENV structural proteins. On the other hand, the stem region and transmembrane region (TM) of G protein of Vesicular Stomatitis virus (VSV) alone are sufficient for budding. Here we generated chimeric VLPs replacing regions of stem and transmembrane domain (STEM) or only transmembrane domain (TM) of E protein of DENV-2 with corresponding regions of VSV G protein. Both chimeric proteins secreted VLPs at higher levels than the wild type (2-4 folds) without any significant change in the expression in the cell. Chimeric VLPs could be recognized by a conformational monoclonal antibody, 4G2. They were also found to interact with dengue-infected patient sera effectively thus implying that their antigenic determinants are preserved. In addition, they were able to bind to its putative receptor, heparin with similar affinity as the parent counterpart thus retaining its functional property. However, cell-cell fusion revealed that there is no significant increase in the fusion ability of chimeras as compared to the parent clone, whereas VSV G protein displayed high cell-cell fusion activity. Overall, this study suggests that chimeric dengue VLPs can be taken forward for their likely potential as vaccine production and serodiagnosis.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Dot-ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation.

Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter-rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.

High Incidence, Reinfections, and Active Syphilis in Populations Attending a Specialized HIV Clinic in Mexico, a Dynamic Cohort Study.

Syphilis, a sexually transmitted infection, has reemerged in many vulnerable groups around the world. The objective of the current study was to determine the prevalence and incidence of syphilis among people who attended a specialized HIV clinic in Mexico from 2011 to 2015. Databases from the laboratory were analyzed, and the following four groups were formed: people seeking HIV-1 voluntary counseling and testing (VCT), people in prison (PPr), people living with HIV (PLWH), and patients from primary care clinics (others). The diagnosis of syphilis was made using the reverse algorithm; antibody titers were examined to determine the stage of infection. Baseline data were analyzed and, with follow-up information, a retrospective dynamic cohort was formed. Factors associated with the seroprevalence of syphilis and active syphilis were evaluated by the chi-square test. Moreover, risk factors for the incidence of syphilis were described. A total of 81,863 baseline individuals were analyzed. The seroprevalence of syphilis was 9.9% in the VCT group, 8.2% in the PPr group, 37.0% in the PLWH group, and 8.7% in the others group; the prevalence of active syphilis was 1.7-13.1%. A total of 11,124 people were followed up. The incidence (cases per 100 person-years) was 3.5 among the VCT group, 16.0 among the PLWH group, and < 0.1 among both the PPr and others groups, respectively; moreover, the frequency of reinfections was 11.1-24.4%. The high prevalence and incidence of syphilis, active syphilis, and reinfections among men, transgender people, individuals aged 20-39 years, and people with a history of HIV or hepatitis B suggest that it is critical to improve prevention, diagnosis, and treatment measures to stop the reemergence of syphilis. There are also new factors such as methamphetamine use, group sex, or contacting partners over the internet that are associated with syphilis. In addition, HIV preexposure prophylaxis could contribute to the increased incidence of syphilis by providing false security in the prevention of STIs, thereby increasing risky sexual behaviors.

Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain.

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity.

When serology contradicts the experience of the disease: Ethics, research, and announcements about Ebola in Guinea / Quand la sérologie contredit le vécu de la maladie : Éthique, recherche et annonce à propos d'Ebola en Guinée

INTRODUCTION: In Guinea, serological tests have shown the absence of antibodies in people declared cured of Ebola Virus Disease (EVD), thus refuting their previous diagnosis; and the presence of antibodies in contact cases who had not been diagnosed. These findings have led to reflections on the implications of telling those affected. PURPOSE OF RESEARCH: The objectives of this study are to identify the stakes of announcing these results in the Guinean health context. 24 people, cured of Ebola or with expertise in ethics or health, were interviewed between November 2019 and February 2020 in Conakry. They presented their experiences in terms of medical announcements in Guinea, and their opinions on the relevance of announcing these discordant serological results. RESULTS: Although it is an important step in the care relationship, the medical announcement sometimes seems neglected in Guinea. In addition, the opinions of the interviewees are rather homogeneous and favorable to the announcement to people seropositive to the Ebola virus who had not been diagnosed. However, their views are varied regarding the announcement of a negative serology to people declared cured of the EVD. They follow two trends, between Ebola survivors who say the announcement is undesirable, and ethicists and healthcare professionals for whom it is preferable. CONCLUSIONS: This survey shows that certain biological results deserve critical reflection before being announced, especially when they indicate a new diagnosis. In order to decide on a course of action for the situations exposed, a second expertise would be useful, taking into account our results and new knowledge about the virus.

Introduction: En Guinée, des analyses sérologiques ont montré une absence d'anticorps chez des personnes déclarées guéries de la maladie à virus Ebola (MVE), réfutant ainsi leur diagnostic antérieur, ainsi que la présence d'anticorps chez des cas contacts qui n'avaient pas été diagnostiqués. Ces résultats ont entraîné des réflexions sur les implications que pourrait avoir leur annonce aux personnes concernées. But de l'étude: Les objectifs de cette étude sont d'identifier les enjeux de l'annonce de ces résultats dans le contexte sanitaire guinéen. Vingt-quatre personnes, guéries d'Ebola ou ayant une expertise en éthique ou en santé ont été sollicitées lors d'une série d'entretiens menés entre novembre 2019 et février 2020 à Conakry (Guinée). Elles ont présenté leur expérience en matière d'annonce médicale et leur avis quant à la pertinence de l'annonce de ces résultats sérologiques discordants. Résultats: Bien qu'elle soit une étape importante de la relation de soins, l'annonce médicale semble parfois négligée en Guinée. En outre, les opinions des interlocuteurs sont plutôt homogènes et favorables à l'annonce de leur maladie aux personnes séropositives au virus Ebola qui n'avaient pas été diagnostiquées. Mais leurs avis sont diversifiés quant à l'annonce d'une sérologie négative à des personnes déclarées guéries de la MVE. Ils suivent deux tendances, entre les survivants d'Ebola qui déclarent que l'annonce n'est pas souhaitable, et les spécialistes de l'éthique et les professionnels de santé, pour qui elle est préférable. Conclusions: Cette enquête montre que certains résultats biologiques méritent une réflexion critique avant d'être annoncés, notamment lorsqu'ils indiquent un nouveau diagnostic. Afin de décider d'une conduite à tenir face aux situations exposées, une deuxième expertise s'avérerait utile, en prenant en compte nos résultats et de nouvelles connaissances sur le virus.

Identification of Anti-Premembrane Antibody as a Serocomplex-Specific Marker To Discriminate Zika, Dengue, and West Nile Virus Infections.

Although transmission of Zika virus (ZIKV) in the Americas has greatly declined since late 2017, recent reports of reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection underscore a critical need for serological tests that can discriminate past ZIKV, DENV, and/or other flavivirus infections and improve our understanding of the immune interactions between these viruses and vaccine strategy in endemic regions. As serological tests for ZIKV primarily focus on envelope (E) and nonstructural protein 1 (NS1), antibodies to other ZIKV proteins have not been explored. Here, we employed Western blot analysis using antigens of 6 flaviviruses from 3 serocomplexes to investigate antibody responses following reverse transcription-PCR (RT-PCR)-confirmed ZIKV infection. Panels of 20 primary ZIKV and 20 ZIKV with previous DENV infection recognized E proteins of all 6 flaviviruses and the NS1 protein of ZIKV with some cross-reactivity to DENV. While the primary ZIKV panel recognized only the premembrane (prM) protein of ZIKV, the ZIKV with previous DENV panel recognized both ZIKV and DENV prM proteins. Analysis of antibody responses following 42 DENV and 18 West Nile virus infections revealed similar patterns of recognition by anti-E and anti-NS1 antibodies, whereas both panels recognized the prM protein of the homologous serocomplex but not others. The specificity was further supported by analysis of sequential samples. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be used to delineate current and past flavivirus infections in endemic areas. IMPORTANCE Despite a decline in Zika virus (ZIKV) transmission since late 2017, questions regarding its surveillance, potential reemergence, and interactions with other flaviviruses in regions where it is endemic remain unanswered. Recent studies have reported reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection, highlighting a need for better serological tests to discriminate past ZIKV, DENV, and/or other flavivirus infections and improved understanding of the immune interactions and vaccine strategy for these viruses. As most serological tests for ZIKV focused on envelope and nonstructural protein 1, antibodies to other ZIKV proteins, including potentially specific antibodies, remain understudied. We employed Western blot analysis using antigens of 6 flaviviruses to study antibody responses following well-documented ZIKV, DENV, and West Nile virus infections and identified anti-premembrane antibody as a flavivirus serocomplex-specific marker to delineate current and past flavivirus infections in areas where flaviviruses are endemic.

Genetic drift in the genome of SARS COV-2 and its global health concern.

The outbreak of the current coronavirus disease (COVID-19) occurred in late 2019 and quickly spread all over the world. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to a genetically diverse group that mutates continuously leading to the emergence of multiple variants. Although a few antiviral agents and anti-inflammatory medicines are available, thousands of individuals have passed away due to emergence of new viral variants. Thus, proper surveillance of the SARS-CoV-2 genome is needed for the rapid identification of developing mutations over time, which are of the major concern if they occur specifically in the surface spike proteins of the virus (neutralizing analyte). This article reviews the potential mutations acquired by the SARS-CoV2 since the pandemic began and their significant impact on the neutralizing efficiency of vaccines and validity of the diagnostic assays.

ELISA-based evaluation of antibody response to Leishmania in a region endemic for cutaneous leishmaniasis.

Leishmaniasis includes several clinical forms. While routine diagnosis of cutaneous leishmaniasis (CL) is by microscopy, an antibody response to CL has been reported in several recent studies. This study evaluated anti-leishmanial immunoglobulin G (IgG) antibody responses as a biomarker of active leishmaniasis and a measure of exposure to Leishmania. Sera from 50 untreated CL patients, 140 patients under treatment and 280 healthy individuals residing in endemic regions collected as part of an epidemiological survey, was analysed with an enzyme-linked immunosorbent assay established in-house using receiver operator characteristic curve at optimized cut-off value. The assay showed high performance as a diagnostic tool in identifying exposure in endemic individuals (sensitivity: 98%, specificity: 90.3%). All patients showed lower antibody levels over time since onset of lesion/s. Antibody levels were higher (p Ë‚ .01) and persisted for a longer period in untreated patients. In patients under treatment, the level of anti-IgG antibodies was negatively correlated with the total duration the patient had been on treatment. The anti-leishmanial IgG response in Leishmania donovani-induced CL is transient and is unlikely to confer protective immunity. Optimized serological assays may be useful in endemic settings for diagnosis and monitoring the treatment response in CL.

Development of a novel glycoprotein-based immunochromatographic test for the rapid serodiagnosis of bovine brucellosis.

AIMS: Bovine brucellosis is a worldwide zoonotic disease that causes important economic losses and public health concerns. Because control of the disease depends on vaccination, serodiagnosis and isolation of the infected animals, affordable, rapid and accurate point of care (POC) tests are needed. METHODS AND RESULTS: We developed and evaluated a novel glycoprotein-based immunochromatographic test for the detection of IgG antibodies against the O-polysaccharide of Brucella in bovine serum samples. Brucella GlycoStrip combines the power of immunochromatographic and bacterial glycoengineering technologies for the diagnosis of bovine brucellosis. The analysis of positive and negative reference samples indicated that the test has a diagnostic sensitivity and specificity of 96.9% (95% CI: 92.7%-100.0%) and 100%, respectively. CONCLUSIONS: Due to the recombinant glycoprotein-based antigen OAg-AcrA, which consists of the O-side chain of Brucella smooth lipopolysaccharide (sLPS) covalently linked to the carrier protein AcrA, the test is highly accurate, allows the differentiation of infected animals from those vaccinated with a rough strain or with a single dose of a smooth strain and fulfil the minimum diagnostic requirements established by the national and international regulations. SIGNIFICANCE AND IMPACT OF STUDY: This strip test could provide a rapid (10 min) and accurate diagnosis of bovine brucellosis in the field contributing to the control of the disease.

Assessment of recombinant antigens Tp0100 and Tp1016 of Treponema pallidum for serological diagnosis of syphilis.

OBJECTIVE: To discover novel serodiagnostic candidates for the serological diagnosis of syphilis. METHODS: Two recombinant Treponema pallidum proteins Tp0100 and Tp1016 were expressed, purified, and identified by Western Blotting. A total of 600 clinical serum samples were tested with the Tp0100-based ELISA, the Tp1016-based ELISA, and the commercial LICA Syphilis TP kit (ChIVD, Beijing, China). The sensitivities were determined by testing 340 samples from individuals with clinically diagnosed primary, secondary, latent, and tertiary syphilis. The specificities were determined by screening 260 samples from healthy controls and individuals with potentially cross-reactive infections, including leptospirosis, Lyme disease, hepatitis B, tuberculosis, rheumatoid arthritis, systemic lupus erythematosus. Kappa (κ) values were applied to compare the agreement between clinical syphilis diagnosis and the Tp0100-based ELISA, the Tp1016-based ELISA, or the LICA Syphilis TP test. RESULTS: Using clinical syphilis diagnosis as the gold standard, Tp0100 exhibited an overall sensitivity of 95.6% and specificity of 98.1% for testing IgG antibody while Tp1016 demonstrated only an overall sensitivity of 75.0% and specificity of 79.6%. In contrast, the LICA Syphilis TP test revealed an overall sensitivity of 97.6% and specificity of 96.2%. In addition, the overall percent agreement and corresponding κ values were 96.7% (95% CI 95.6%-97.8%) and 0.93 for the Tp0100-based ELISA, 77.0% (95% CI 74.3%-79.7%) and 0.54 for the Tp1016-based ELISA, and 97.0% (95% CI 96.0%-98.0%) and 0.94 for the LICA Syphilis TP test, respectively. CONCLUSION: The recombinant T. pallidum protein Tp0100 shows promise as a novel diagnostic antigen in the serological tests for syphilis.

Serological evaluation of the schistosome's secretory enzyme phytochelatin synthase and phosphoglycerate mutase for the detection of human Schistosoma japonicum infection.

Secretory enzymes from Schistosoma japonicum are promising candidate antigens in the diagnosis of schistosomiasis. Our previous studies have proven that thioredoxin peroxidase-1 (SjTPx-1) is useful for the detection of this parasitic disease in humans, water buffaloes, and dogs. In this study, we evaluated two more secretory enzymes namely phosphoglycerate mutase (SjPGM) and phytochelatin synthase (SjPCS) with SjTPx-1 as the reference antigen. SjPGM was shown to have good diagnostic potentials in animal samples in previous studies, whereas SjPCS was chosen because of its absence in the mammalian hosts. Serum samples including 96 endemic negative controls, 107 schistosomiasis japonica positive samples, and 31 samples positive for other parasitic trematode infections (Clonorchis sinensis, Opisthorchis viverrini, Paragonimus westermani) were tested with the antigens using enzyme-linked immunosorbent assay. Results showed that SjPCS detected more positive samples and had fewer cross-reactions than SjPGM. With 85.05% sensitivity and 93.55% specificity, SjPCS can therefore be used in the detection of human schistosomiasis.