Antigen ELISA Research Articles

Random X-Chromosome Inactivation in Adult Hemophilia B Carriers Does Not Attenuate Bleeding Risk

Introduction: The X-linked bleeding disorder Hemophilia B (HB) results from deficiency in coagulation Factor IX (FIX). In contrast to Hemophilia A, genotype-phenotype correlations in HB males are complex, as some F9 variants in males are associated with low plasma FIX activity (FIX:C) but milder than expected bleeding phenotypes. At least 75% of FIX is extravascular and not adequately measured from plasma. Hemostasis is likely influenced by this extravascular FIX which is bound reversibly to type IV collagen in the subendothelial basement membrane. We predict that genotype-phenotype correlations in female carriers of F9 mutations may have additional complexity as the mutant FIX can competitively interfere with wild type (WT) FIX for extravascular binding sites. F9 expression in female HB carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that randomly inactivate either the X carrying the mutant or wild type F9, although skewed XCI (>80:20) is found in ~25% of the general population. XCI skewing in HB carriers can lead to preferential expression of the X carrying the F9 mutation, which in turn influences plasma and extravascular levels of FIX and bleeding tendency. How frequently XCI is skewed in HB carriers, and the role of XCI in modulating FIX levels and bleeding phenotypes, remains poorly understood. We sought to determine whether XCI skewing may further differentiate bleeding risk in HB carriers. Methods: Subjects were enrolled in a prospective, observational cross-sectional study with informed consent. Both the Condensed MCMDM-1 bleeding assessment tool (BAT) and Pictorial Blood-loss Assessment Chart (PBAC) were completed at the time of the visit. F9 mutation analysis confirmed carrier status. FIX:C and FIX antigen ELISA assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci. Results: A total of 15 subjects (11 adults) were confirmed F9 mutation carriers; six missense mutations and one frameshift mutation were identified. Abnormal bleeding is frequent in adult HB carriers, with 5/11 (45%) having BAT scores >4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC > 100, and 8/11 with BAT HMB subcategory score >1. FIX assessment by activity (mean 56% ± 28) and antigen (mean 0.55 IU/mL ± 0.2) assays are highly correlated (r 2= 0.83, p < 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels < 50%, while two HB carriers with FIX levels < 50% have BAT scores < 4, indicating that neither FIX:C nor FIX antigen levels adequately predict bleeding risk in all HB carriers. Indeed, for all 11 adult HB carriers, no significant differences in either FIX:C or FIX antigen were observed between those with BAT scores ≥ 4 and < 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (> 70:30); only one is extremely XCI skewed (> 80:20), and of the individuals with skewed XCI, 3/5 have BAT scores ≥ 4. Although random XCI is proposed to attenuate HB carrier bleeding phenotype, 2/6 HB carriers with random XCI also have BAT scores ≥ 4. XCI weakly correlated with FIX antigen (n = 15; r 2= 0.27, p < 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C>T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p < 0.05) but not with FIX antigen or bleeding phenotype. The lack of correlation between XCI and FIX antigen in individuals with an identical F9 mutation argues for additional influences on intravascular, and likely extravascular, FIX levels and will require further investigation. Conclusions: Bleeding tendency in HB carriers is not rare and does not correlate with plasma FIX:C or FIX antigen. Random XCI is not fully protective against abnormal bleeding. Future studies are needed to better understand bleeding phenotypes in HB carriers that consider XCI, F9 mutation type and influences on circulating levels of WT and mutant FIX.

Effects of royal jelly on the antisenescence, mitochondrial viability and osteogenic differentiation capacity of umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells that have the ability to self-renew and regulate paracrine signalling and immune system processes. MSCs have extensive clinical applications in regeneration, functional reconstruction and cellular therapies. However, studies are needed to discover ways to improve the properties of MSCs, such as differentiation, and prevent senescence in culture, which are both very important for cell therapies. Royal jelly (RJ) is a nutritional substance produced by worker bees that contains a substantial amounts of proteins that are beneficial for cell growth and proliferation. RJ is widely used in traditional medicine today, and due to the specific components in its content, it has been reported to have antioxidant, antiproliferative, antimicrobial, neuroprotective, anti-inflammatory, immunomodulatory and anti-ageing properties. In our study, human Wharton's jelly mesenchymal stem cells (WJ-MSCs) derived from umbilical cord matrix were grown in culture medium supplemented with RJ. The control group comprised minimum essential medium (MEM) and 10% foetal bovine serum (FBS); RJ groups were formed using MEM, 10% FBS and 0.075mg/ml or 0.150mg/ml RJ. In our study, we evaluated the effect of RJ on WJ-MSC growth by MTT assay, proliferating cell nuclear antigen ELISA, β-galactosidase activity assay, MitoTracker Green staining and differentiation tests in adipogenic, osteogenic and chondrogenic cell lines. It was observed that the number of mitochondria increased, senescence decreased and osteogenic differentiation increased after differentiation induction after the addition of RJ to MSC culture. In general, the results of this study indicate that WJ-MSCs enhance mitochondrial numbers and important cellular activities, such as antisenescence and osteogenic differentiation, and with increasing evidence from further studies, RJ supplementation may be found beneficial for the use of MSCs in bone engineering regenerative medicine or cell therapy.

Epizootiology and seroprevalence of Crimean-Congo hemorrhagic fever virus in ruminant population of East Afghanistan

Crimean Congo hemorrhagic fever (CCHF) is a tick-borne, zoonotic disease which is characterized by fever and hemorrhages in humans but is asymptomatic in livestock. In the present investigation, research was conducted to determine the seroprevalence of CCHF virus IgG antibodies, intrinsic and extrinsic factors associated with the distribution of the disease in cattle, goats, and sheep in two provinces (Nangarhar and Kunar) of Afghanistan. The blood samples (n ​= ​768), and data related to intrinsic and extrinsic risk factors were collected, and the seroprevalence was determined using double antigen (DA) ELISA. Based on the ELISA S/P% value (sample to positive control ratio percent), the overall seroprevalence of CCHF virus IgG antibodies among the animal species screened in the two provinces was 19% (146 out of 768). A non-significant association was observed in the seropositivity of CCHF in the study regions (p ​> ​0.05). The intrinsic (animal species, sex, age, and breed) and extrinsic (husbandry practices, animal body condition score, and tick infestation) potential risk factors were found to be significantly (p ​< ​0.05) correlated with the frequency distribution of CCHF virus IgG antibodies. The early detection of this lethal virus in the animal host will aid in marking the areas where the CCHF virus infection is endemic and in developing control strategies for public safety.

Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen.

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.

Seroprevalence of bovine viral diarrhoea in organized herds in India

Bovine viral diarrhoea (BVD) is an important infectious viral disease affecting cattle populations all over the world. In addition to direct loss caused by the disease, the virus causes immunosuppression thereby predisposing the host to other diseases. A cross-sectional study was undertaken to detect the prevalence of BVD in 14 well-organized herds located in different parts of India. A total of 880 serum samples (646 cattle and 234 buffaloes) were screened by a commercial ELISA kit, detecting antibodies towards the p80 (NS3) region of BVDV. The overall true prevalence was 56.67% (95% CI: 53.26-60.02%) and within herds, it ranged from 0-99.99%. The prevalence rate was higher in cattle (65.42%) than in buffaloes (32.49%) and the difference was statistically significant. Further, a significant difference in prevalence among cattle breed types was recorded, with the lowest in indigenous cattle (16.49%) followed by crossbreeds (16,97% and exotic breeds (87.80%). Higher positivity was detected among females (68.87%) than males (48.83%) but this difference was not significant, as revealed by multivariate regression analysis. Of the 10 semen stations studied, the prevalence varied from 9.72% to 72.68%. However, none of the animals from these semen stations turned positive in the antigen ELISA test, suggesting the antibodies detected in this study were from past infections. On the two dairy farms/bull mother farms showing very high positivity, two (one each) persistently infected cows were detected during whole herd screening by antigen ELISA test. One bull mother farm was free of BVD antibodies suggesting it is possible to maintain BVDV-free herds. The present study indicates the endemicity of BVDV in Indian organized herds, and therefore a suitable testing strategy and management should be adopted in response to testing to control the introduction and further transmission of the disease on farms.

Diagnostic Overview of Foot-And-Mouth Disease Viruse (FMDV)

Foot-and-mouth disease (FMD) is one of the highly contagious diseases of domestic animals. Sensitive, specific, and quick detection of FMD virus antigens and antibodies is an essential corner stone diagnostic tool at each tier of control strategy. Continued development of molecular and genetic technologies combined with novel analytical techniques will benefit many areas of FMD research and understanding. Genome sequencing provides excellent insight into the evolutionary and epidemiological dynamics of such viruses. The advent of next generation sequencing (NGS) technologies enables evolutionary studies of the entire viral swarm. Sequencing technologies have advanced uses of viral genomic data; which helps to understand the global distribution and Trans-boundary movements of Foot-and-mouth virus disease. Also sequencing information can be used to understand antigenic change within virus strains; therefore, the aim of this review is to assess various diagnostic techniques including conventional and advanced techniques both from agent and host response sides. Among the agent side virus isolation, RT-PCR, antigen ELISA and immune-chromatography strips are commonly used diagnostic tests. Whereas from the host side: infrared thermo gram, antibody ELISA and Virus neutralization are some mentions. Owing to current technological advancement: DNA sequencing and microarray, expressing a luciferase reporter and quantitative proteomics were reviewed. Therefore, potential confirmatory diagnostic test should be in combination with each other.

Immunodiagnostic Detection of Angiostrongylus cantonensis Exposure on Hawaii Island Using Isogeographic 31-kDa Antigen.

Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis worldwide, and east Hawaii Island is a hotspot for the disease in the United States. A combination of glycoproteins with molecular weight of 31 kDa has been used as antigen to evaluate antibody response in human serum samples in Thailand with high specificity and sensitivity. In a previous pilot study, the Thailand-isolated 31-kDa proteins showed efficacy in dot-blot tests using serum samples from 435 human volunteers on Hawaii Island. However, we hypothesized that native antigen isolated from Hawaii A. cantonensis may exhibit higher specificity than the Thailand-isolated 31-kDa antigen due to potential minor variation in epitopes between isolates. In this study, 31-kDa glycoproteins were isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis from adult A. cantonensis nematodes collected from rats captured on east Hawaii Island. The resultant proteins were purified by electroelution, pooled, bioanalyzed, and quantified. A subset of 148 samples from human participants of the original cohort of 435 was consented for this study, including 12 of the original 15 clinically diagnosed participants. Results of ELISA using the Hawaii-isolated 31-kDa antigen were compared with results of the same serum samples previously tested with both crude Hawaii antigen ELISA and Thailand 31-kDa antigen dot blot. This study shows a seroprevalence in the general population of East Hawaii Island of 25.0%, similar to previous findings of 23.8% seroprevalence in this cohort using crude antigen from Hawaii A. cantonensis and 26.5% using Thailand 31-kDa antigen.

Evaluation of a point-of-care test for the diagnosis of Taenia solium neurocysticercosis in rural southern Tanzania: a diagnostic accuracy study

Neurocysticercosis is a common cause of epilepsy in Taenia solium-endemic areas in sub-Saharan Africa but is often undiagnosed because of an absence of affordable diagnostic tools. This study evaluated the diagnostic accuracy of a T solium cysticercosis antibody-detecting lateral-flow point-of-care assay (TS POC test) for the neuroimaging-based diagnosis of neurocysticercosis. Patients with epileptic seizures or severe progressive headache were recruited consecutively from three hospitals in southern Tanzania. All patients were tested with the TS POC test. All patients positive for cysticercosis on the TS POC test and every tenth patient who was negative for cysticercosis received a brain CT examination and underwent reference testing for T solium cysticercosis (ie, rT24H-EITB, LLGP-EITB, and antigen ELISA). The primary outcome of the study was the sensitivity of the TS POC test for the diagnosis of neurocysticercosis. Of the 601 recruited participants, 102 (17%) tested positive for cysticercosis with the TS POC test. Overall, 48 (62%) of the 77 patients positive for cysticercosis and five (17%) of the 29 patients negative for cysticercosis on the TS POC test had CT-confirmed neurocysticercosis. The TS POC test yielded a sensitivity of 49% (uncertainty interval [UI] 41-58) for neurocysticercosis. Sensitivity was similar to that of the rT24H-EITB (44%, UI 37-51) and the antigen ELISA (50%, 43-56). For the subset of neurocysticercosis cases with at least one active (ie, vesicular) lesion, sensitivity was above 98% for the TS POC test, the rT24H-ETIB, and the antigen ELISA. The TS POC test showed promising results for the diagnosis of neurocysticercosis in patients with vesicular lesions, which need to be confirmed in a larger study. This test could be considered to support policies on screening patients with suspected neurocysticercosis in clinical settings, which would allow appropriate referral for neuroimaging and early treatment. German Federal Ministry of Education and Research and the European & Developing Countries Clinical Trials Partnership. For the Swahili translation of the abstract see Supplementary Materials section.

Long-term acute infections during a bovine viral diarrhea virus (BVDV) outbreak in dairy farm from Galicia (NW Spain)

An observational study describes an outbreak of bovine viral diarrhea virus (BVDV) in a dairy herd in Spain. The herd was subjected to a voluntary control program. In a sampling carried out in June 2020, bulk tank milk antibody levels increased compared to the previous sampling. Additionally, serum samples from 4 young heifers also tested positive for antibodies. Since the results were consistent with a recent infection, we proceeded to detect possible persistently infected (PI) animals using antigen ELISA (on serum/ear-notch samples), following the program guidelines. From this moment on, 42 animals tested positive for BVDV antigen, of which 17 were under typical acute infection (AI), 13 were deemed as PI, and eight died early on the farm before having information to determine their status. The remaining 4 showed intriguing test results consistent with a long-term AI since they tested BVDV positive in at least two antigen tests more than 3 weeks apart. Thus, one animal was positive until 80 days of age in serum, and others even for longer periods in ear-notch samples, until they finally tested negative for BVDV. Based on these results, longer follow-up may be necessary in BVDV positive animals to accurately confirm persistent infection.

Mild-to-moderate COVID-19 does not predispose to the development of autoimmune rheumatic diseases or autoimmune-based thrombosis.

An infection with severe acute respiratory syndrome coronavirus (SARS-CoV-2) may have a significant impact on the human immune system. Interactions between the virus and defence mechanisms may promote the development of autoimmune processes which manifest as antinuclear antibody (ANA) synthesis. Since many different viruses are suspected to take part in the pathogenesis of different systemic autoimmune rheumatic diseases (SARDs), we examined whether coronavirus disease 2019 convalescents who suffer from mild to moderate disease have a higher risk of developing ANA and anti-β2-glicoprotein I IgG antibodies (β2 GPI). In a retrospective study, we examined 294 adults among volunteer blood donors divided into convalescents (N = 215) and healthy controls (N = 79). For ANA detection, we used line-blotting, a type of indirect immunofluorescence assay (IF), to determine antigenic specificity and ELISA for β2 GPI. We found a lower incidence of ANA in convalescents than in healthy controls, with the majority of these antibodies directed to antigens with no known clinical significance. Additionally, no participants were positive for β2 GPI in either group. Our results show that COVID-19 with mild to moderate symptoms in the generally healthy population does not induce the development of ANA or anti-β2 GPI antibodies for at least 6 months following the disease.

Utility of CDC DENV1-4 real time PCR assay and trioplex assay for the diagnosis of dengue in patients with acute febrile illness.

Nucleic acid amplification tests (NAATs) have revolutionized reliable detection of dengue virus (DENV) during acute phase of infection. The study evaluated performance of CDC DENV-1-4 real-time assay, trioplex RT-PCR and heminested conventional RT-PCR assay in the diagnosis of DENV. The three NAATs were performed on 107 consecutive samples collected from patients suspected of DENV infection during acute phase of illness. Their performance was compared against composite reference standard, consisting of DENV NS1 antigen ELISA and DENV IgM ELISA. 88/107 study samples were positive by DENV ELISA, either NS1Ag (80), IgM (3) or both (5). The overall sensitivity of CDC DENV-1-4 RT-PCR assay, trioplex RT-PCR assay and conventional multiplex RT-PCR was 68.18%, 54.55% and 38.64%, respectively in diagnosing dengue during acute phase, with an area under the curve of 0.841, 0.773 and 0.693 respectively when compared against composite reference standard. The sensitivity was 82.93%, 73.17% and 51.22%, respectively within three days of illness and 60%, 42.86% and 28.57%, respectively between 4 and 5th day of illness. All the three molecular assays had 100% specificity. Maximum concordance values of 86.9% were recorded among CDC DENV-1-4 rRT-PCR assay and trioplex assay with kappa value of 0.74, suggestive of substantial agreement. CDC DENV-1-4 rRT-PCR assay can be used as a reliable and accurate test for diagnosis of DENV during acute phase of illness.

Evaluation of immunoserological detection of anti-liver kidney microsomal, anti-soluble liver antigen and anti-mitochondrial antibodies

Autoantibodies are the diagnostic hallmark of autoimmune liver diseases. Indirect immunofluorescence (IFT) is the reference method for the detection of anti-mitochondrial antibodies (AMA) and anti-liver kidney microsomal type-1 (anti-LKM1) antibodies, and inhibition ELISA (iELISA) for anti-soluble liver antigen (anti-SLA) antibodies. Given the complexity of these techniques, commercial ELISAs have emerged as a practical alternative, but without head-to-head validations. This study evaluated the agreement between three commercial ELISAs and the reference techniques and the impact of polyreactive immunoglobulin G (pIgG), a recently described phenomenon in autoimmune hepatitis, on commercial ELISAs. Inter-rater reliability was assessed using Cohen-Kappa coefficient (κ). Forty-eight, 46, and 66 samples were analyzed for AMA, anti-LKM1, and anti-SLA, respectively. For AMA, one commercial assay showed high agreement (κ = 0.91 (0.78–1.00)) with the reference method, while the other two showed weak or moderate agreement. For anti-LKM1, only one commercial assay showed high agreement (κ = 0.86 (0.71–1.0)). For anti-SLA antibodies only moderate agreement was achieved (κ up to 0.71 (0.52–0.89)). There was a trend towards higher pIgG levels in false-positives in the commercial ELISAs. Patients with high suspicion of autoimmune liver diseases should be referred to reference laboratories with the capacity of performing gold standard methods if the initial ELISA-based screening was performed.

Prokaryotic expression of the V protein of the peste des petits ruminants virus and development of an indirect ELISA

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

Production and characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and development of a sandwich ELISA for virus antigen detection.

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O.

Serological Status of Viral Hepatitis B and Associated Factors Among Sex Workers in Douala (Littoral-Cameroon)

<i>Introduction</i>: Viral Hepatitis B (VHB) is a real concern for international organisations in general and the Cameroonian authorities in particular; our work aimed to determine the serological status of VHB as well as the associated factors among female sex workers (FSWs) in the city of Douala. <i>Method</i>: We conducted a descriptive cross-sectional study over a period of two months from 01 August to 02 October 2022. For the detection of serological markers, we used immuno-chromatographic tests [HBV 5-in-1 Hepatitis B Virus markers Rapid Test Panel (batch number; China, 2022)] and enzyme-linked immunosorbent assays [Hepatitis B Virus surface Antigen (HBsAg) ELISA Test Kit, batch number: HBSG37310B, China, 2022], both of HIGHTOP brand. The markers investigated by the immuno-chromatographic tests were HBsAg, HBsAb, HBeAg, HBeAb, and cHbAb. Logistic regression analysis was performed to determine the degree of association with HBsAg carriage at the 5% significance level. Results: A total of 87 participants were included in the present work, with a mean age of 29.66 ± 6.24 years, ranging from 18 to 43 years; furthermore, the serological profiles obtained in these TS were distributed as follows: 1% (1/87) of the participants were infected with the presence of viral activity; 7% (6/87) were infected with the absence of viral activity; 19% (17/87) had received a vaccine; 13% (11/87) had been in contact with the virus; 7% (6/87) had been in contact with the virus in the past and were cured; and 53% (46/87) were not infected; However, univariate regression analyses showed that primary education (OR= 0.042; 95% CI = 0.002-0.973; P=0.048); secondary education (OR= 0.034; 95% CI = 0.003-0.459; P=0.011); barmaid activity (OR= 0.030; 95% CI = 0.002-0.495; P=0.014) and shopkeeper activity (OR= 0.056; 95% CI = 0.003-0.923; P=0.044) were significantly associated with HBsAg carriage <i>Conclusion</i>: The present study shows that a variety of serological profiles of HBV were found among SWs in the city of Douala; however, the implementation of large-scale sensitization campaigns (via the media, social networks, and within schools) and expanded vaccination programmes against HBV throughout the country would help reduce its prevalence.

Unmasking Arterial-resident Autoreactive B cells involved in Atherosclerosis and Peripheral Artery Disease Progression

Abstract Atherosclerosis activates the immune system, including B cells which secrete antibodies that bind antigens released by necrotic cells within plaques and may promote inflammation. To identify these antibodies, we isolated activated B cells from atherosclerotic ApoE-deficient mice aortas and human tibial arteries with peripheral artery disease post-amputation. Single-cell sequencing of the B cell receptors identified 35 mouse and 48 human overrepresented disease-relevant heavy and light chain pairings, which were sequentially expressed as antibody. Epitope determination was performed by human proteome array, and 14 mouse and 21 human protein targets were found with significant affinity and specificity. Mouse antigens were scrutinized by immunohistochemistry to confirm presence in inflamed plaques, and expression of these proteins were used to interrogate autoreactive antibody titers in diseased sera by ELISA. ApoE-deficient mice were immunized with the 6 most promising antigens. Serum antibody measurement showed strong, rapid, and early onset of antigen-specific antibody responses in the immunized mice compared to an adjuvant only control group. Strikingly, we saw decreased plaque accumulation in the immunized animals, suggesting antigen responses may clear pathogenic antigens before disease develops. Likewise, human antigen ELISAs comparing antigen-specific serum antibody titers to patient Framingham score for cardiovascular risk supported a role for two antigens in disease. To confirm these antigens as potential biomarkers, we will characterize the emergence of their antibody counterparts using a historical library of serum samples from individuals enrolled in the Baltimore Longitudinal Study on Aging. This work is directly funded by the National Institutes of Health – National Institute on Aging’s Intramural Research Program in the Lab of Molecular Biology and Immunology

Heartworm (Dirofilaria immitis) in carnivores kept in zoos in Texas, USA: risk perception, practices, and antigen detection

BackgroundDirofilaria immitis is the causative agent of heartworm disease in wild and domestic canids, felids, and mustelids. Recent studies demonstrate that additional families in the order Carnivora are also susceptible to infection. Therefore, the objectives of this study were to (1) better understand current practices surrounding heartworm prevention and diagnostics in zoological facilities located in the state of Texas, USA, and (2) assess archival serum samples of carnivores kept in these facilities for the presence D. immitis antigen and/or antibody.MethodsA questionnaire was completed by veterinarians or veterinary technicians representing 10 zoological facilities across Texas. This questionnaire was designed at the taxonomic family level, encompassing the 12 terrestrial carnivore families Ailuridae, Canidae, Eupleridae, Felidae, Herpestidae, Hyaenidae, Mephitidae, Mustelidae, Prionodontidae, Procyonidae, Ursidae, and Viverridae. The second objective was achieved with the use of archival serum samples made available by six zoo facilities.ResultsRisk perception varied across facilities for every family, including among species belonging to Canidae. All facilities used monthly heartworm prevention in canids and felids, with more variation existing in the other families. The use of diagnostic testing and type and route of administration of preventive varied by facility, with oral ivermectin the most commonly used preventive. A total of 217 archival serum samples, belonging to 211 individual animals encompassing 11 families and 39 species, were tested with a commercial heartworm antigen ELISA test, pre- and post-immune-complex dissociation. A subset of samples was also assessed for the presence of feline anti-heartworm antibodies using a commercial ELISA test. Two animals, both of which were Asian small-clawed otters from the same facility, had antigen detected (0.95%).ConclusionsThis study demonstrates that while the zoo veterinary community is aware of the risk and health impact of heartworm disease in canids and felids, there is still a great deal of uncertainty regarding the risks and ideal strategies for prevention in other carnivore families. The low proportion of antigen detection may serve as a baseline for future prevalence studies across the southern United States, where there is an emerging concern of macrocyclic lactone resistance in heartworm.Graphical

Epidemiological and demographic characteristics of dengue at a tertiary care center in Kutch region during the year 2019

Dengue is the most rapidly spreading mosquito borne viral disease of mankind, with a 30 fold increase in global incidence over the last five decades. It is a major public health concern throughout the tropical and subtropical regions of the world. The present study was conducted with objective to study epidemiological and demographic characteristics of dengue infection during the year 2019 in Kutch region, Gujarat, India.The study was carried out from January 2019 to December 2019 at Department of Microbiology, Gujarat Adani Institute of Medical Sciences, G.K. General Hospital, Bhuj. The patients having suspected dengue fever was based on standard criteria like presentation of febrile illness of 2-7 days duration were included in this study.A total of 1509 blood samples were collected during study period and serologically tested for dengue NS1 antigen and IgM antibody by capture ELISA testing method. Total 1509 blood samples were tested by ELISA for NS1 antigen and/or IgM antibody as per the protocols and personal, demographic and clinical details of each patient was recorded. Out of 1509 cases 761 tested positive for dengue. Amongst all positive cases 533(47%) were tested positive for NS1 antigen which helped in early diagnosis of dengue. Rise of dengue cases started after the month of October and falls down by the end of December. Analysis of this data revealed that age group 00-10 years had maximum dengue cases. Male cases 418(55%) were more than females 343(45%). Dengue cases were high in urban area.The present study reported that dengue mainly affected children, males and urban population. Perennial occurrence with seasonal increase during monsoon and post monsoon moths was reported. Effective implementation of vector control measures through efforts toward vector breeding source reduction and with the use of personal prophylactic measures against mosquito bites will help in reducing the dengue prevalence in the community.

Serological Status of Hepatitis B Virus Among Pregnant Women in the Mifi District (West Cameroon)

<i>Introduction</i>: Mother-to-child transmission is a major route of hepatitis B virus (HBV) transmission, particularly in highly endemic areas. The aim of our work was to determine the extent of HBV serological markers in pregnant women in the Mifi district (West Cameroon). <i>Method</i>: We conducted a descriptive cross-sectional study over a period of two months from 01 August to 02 October 2022. For the search for serological markers, we used immuno-chromatographic tests [HBV 5-in-1 Hepatitis B Virus markers Rapid Test Panel (serum/plasma), China, 2022] and enzyme-linked immunosorbent assays [Hepatitis B Virus surface Antigen (HBsAg) ELISA Test Kit, lot: HBSG37310B, China, 2022], both of which are from HIGHTOP. The markers investigated by the immuno-chromatographic tests were HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb. Logistic regression analysis was performed to determine the degree of association with HBsAg carriage at the 5% significance level. <i>Results</i>: A total of 109 pregnant women were enrolled in the present study, the mean age was 26.89 ± 6 years with a range of 16 to 42 years. The following serological profiles were obtained in 27 (24.77%) participants and distributed as follows 19% (5/27) of the pregnant women were surface antigen positive (AgHbs +; AcHbc +); 37% (10/27) had been in contact with the virus in the past and were cured (AcHbs +; AcHbc +), 7% (2/27) had been vaccinated against hepatitis B virus (AgHBs -; Ac Hbs +; Ac anti Hbc -) and 37% (10/27) were in contact with the virus (AcHbc +). Logistic regression analyses revealed no association between socio-demographic variables and HBsAg carriage. <i>Conclusion</i>: In sum, the present work resulted in a prevalence of HBsAg of 4.59% (5/50); with an estimated marker positivity of 11% (12/50); 22.94% (25/50); 7.34% (8/50) and 0% for HBsAg, cAbAg, HBeAg and HBeAg respectively. Furthermore, no socio-demographic variables showed an association with HBsAg carriage.

Role of recombinant Histoplasma capsulatum 100-kilodalton antigen in the diagnosis of histoplasmosis among HIV/AIDS patients: Antigenuria and antibodies detection.

Diagnosing progressive disseminated histoplasmosis (PDH) is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or by cytology/histopathology but both procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has a low sensitivity in immunocompromised individuals. Commercially available antigen detection assays have high sensitivity in PDH cases; however, they are expensive and only performed in few laboratories. To describe the potential use of a novel ELISA for antibodies testing and a dot blot assay for antigen testing for diagnosing PDH using the recombinant 100 kDa protein of Histoplasma capsulatum (Hcp100) and their polyclonal antibodies as novel reagents, respectively. Serum and urine samples from a cohort of patients with HIV/AIDS and proven PDH were studied for the detection of anti-Hcp100 antibodies by ELISA and Hcp100 antigen by dot blot, respectively. Sensitivity, specificity and cross-reactions with other diseases were estimated for each assay and compared with those obtained using histoplasmin (HMN) as a reagent for antibodies detection by ELISA and immunodiffusion, and using a commercial antigenuria test. Antibodies detection by the Hcp100 ELISA demonstrated 78.6% sensitivity and 88.4% specificity, versus 85.7% sensitivity and 81.0% specificity for the HMN ELISA and 26.1% sensitivity and 100% specificity for the immunodiffusion assay. Antigen detection by the Hcp100 dot blot demonstrated 89.3% sensitivity and 97.0% specificity versus 82.1% sensitivity and 90.9% specificity for the commercial test. The immunoassays described herein based on Hcp100 would be a valuable screening tool for diagnosing PDH.