Journal of Clinical Virology Plus

Introduction: Hantavirus disease is an emerging and potentially severe zoonosis of global distribution. In Uruguay, it is transmitted by rodents inhabiting peridomestic, suburban, and rural areas. Global incidence is estimated at 150,000 to 200,000 cases per year, with up to 300 annual cases in the Americas. Since 1997, Uruguay's Ministry of Public Health (MPH) has monitored Hantavirus cardiopulmonary syndrome (HCPS), the most common clinical presentation in the region. By 2019, a total of 271 cases had been identified in the country, with an estimated mortality rate of nearly 50%. Objectives: To describe the clinical, epidemiological, and occupational characteristics of patients with Hantavirus disease in Uruguay during the pre-pandemic (2018-2019) and pandemic (2020-2021) periods. Methods: A descriptive, cross-sectional, observational study was conducted, including all serologically confirmed cases of Hantavirus infection reported to the MPH between 2018 and 2021. Clinical and demographic data were extracted from the mandatory reporting form for zoonotic diseases. Incidence and case fatality rates were calculated, and factors associated with fatal outcomes were analyzed. Results: A total of 58 confirmed cases were identified between 2018 and 2021. Most patients were male (62%), with a mean age of 36.5 years (SD 16). A decline in incidence was observed during 2020-2021, with no significant change in case fatality. Direct rodent exposure was the most frequently associated risk factor. Montevideo and Canelones were the most affected departments. Renal and pulmonary involvement were significantly associated with mortality. Conclusion: Hantavirus remains a relevant public health concern in Uruguay. Although a decrease in incidence was observed during the COVID-19 pandemic years, case fatality rates remained high. The findings underscore the need for sustained surveillance and early recognition, particularly in urbanizing regions.

Rabies remains a significantly underreported fatal zoonosis in India, where the Arctic-like 1a (AL1a) lineage predominates in dog populations. While atypical clinical presentations in dogs can delay diagnosis and increase human exposure risk, genomic and clinical data on neonatal canine rabies remain limited. This study reports an exceptional case of rabies in a 54-day old unvaccinated German shepherd puppy which presented with severe pruritus and self-biting behaviour. The puppy was euthanized due to poor clinical response. Post-mortem testing revealed viral antigen (by Direct Fluorescent Antibody Test) and viral RNA (by real-time RTPCR) in the brain tissue. Whole-genome sequencing recovered a near-complete rabies virus genome (11,947 nucleotides; 99.5% genome coverage), classified within the AL1a_A1.1 sublineage. Phylogenetic analysis revealed close genetic relatedness to contemporary Indian rabies virus strains. Comparative genomic analysis identified 4, 3, 6, and 8 non-synonymous substitutions in the phosphoprotein, matrix, glycoprotein, and polymerase genes, respectively. This case is one of the youngest documented cases of canine rabies with atypical manifestations, caused by the AL1a viral clade. Our findings highlight the risks associated with neonatal canine rabies, the need for heightened clinical suspicion in atypical cases, and the importance of genomic surveillance to monitor evolving rabies virus lineages in endemic regions.

Background: ChAdOx1 nCoV-19 remains a cornerstone COVID-19 vaccine in sub-Saharan Africa, yet population-specific molecular responses are understudied. We examined peripheral blood ACE2 and TMPRSS2 expression, total RNA concentration, and coagulation indices in Nigerians >=6 months post-vaccination. Methods: In a case-control study in Port Harcourt, Nigeria, 51 ChAdOx1-vaccinated adults and 51 age/sex-matched unvaccinated controls provided venous blood for RNA extraction, qRT-PCR, and coagulation assays. Multivariable linear models assessed effects of vaccination, sex, and age on molecular parameters. Results: Vaccinated participants had 37% lower total RNA concentration than controls (4.02 +/- 0.09 vs 6.38 +/- 0.14 ng/uL, p<0.0001). ACE2 and TMPRSS2 expression did not differ by vaccination status overall. However, TMPRSS2 showed a significant sex-by-treatment interaction (p=0.011): vaccinated females had higher expression than vaccinated males. GAPDH expression varied by vaccination status and showed a three-way interaction with sex and age (p=0.027). Coagulation indices were unchanged. Conclusions: At >=6 months post-ChAdOx1, Nigerians show reduced peripheral blood RNA without sustained ACE2/TMPRSS2 upregulation. The sex-specific TMPRSS2 pattern suggests hormone and vaccine interactions previously unreported in African cohorts and highlights the need for sex-disaggregated molecular surveillance. Region-specific reference gene validation is recommended for Nigerian transcriptomic studies.

The recent shift in Bangladesh from tradition backyard rearing system to modern commercial layer farming has made the birds immune to infectious diseases. Pediculosis, however, continues to pose a challenge in modern production system due to its invasive nature, often going unnoticed and neglected as it is typically non-lethal, yet capable of causing significant production losses. Lice infestation is a persistent threat in poultry production; however, its implications in battery-caged commercial layer hens in Bangladesh remain insufficiently characterized. The study aimed to identify the causative louse species and evaluate its associations with clinical pathology, hematological alteration and productive performance in 30 white-feathered (15 infested + 15 non-infested) and 30 brown-feathered (15 infested + 15 non-infested) laying birds from two commercial farms in Tangail. Morphological characterization confirmed the parasite as Menacanthus stamineus, distinguished by a dorsoventrally flattened body, parabolicallly rounded head wider than long, concealed club-shaped antennae, an oblong-oval abdomen with fine setae and three pairs of short legs each bearing paired claws. Infested birds exhibited consistent clinical pathology, including pale combs, petechial hemorrhages around the vent, severe feather damage with alopecic and exudative areas and incidence of irregular and broken-shelled eggs. Production performance analysis revealed significant reduction in hen-day egg production, egg weight, and feed intake, accompanied by significantly increased feed conversion ratios. Hematological evaluation demonstrated significantly reduced hemoglobin concentration, hematocrit and erythrocyte counts in infested hens, indicating mild anemia and compromised oxygen-carrying capacity. Collectively, pediculosis was strongly associated with lice-induced self-inflicted injury and cannibalism, systemic physiological stress, impaired erythropoiesis, reduced production efficiency and compromised welfare in caged laying hens. To best of our knowledge, it was the first integrative reports from Bangladesh documenting M. stramineus infestation in battery-caged commercial layer system with concurrent evidence of hematological disruption and measurable productivity losses, underscoring its epidemiological and economic significance and urgent need for targeted, evidence-based ectoparasite control strategies.

Background: Lung cancer is leading cause of cancer related death globally. It is second most prevalent cancer among women worldwide and ranks third among females in Nepal. Contributing factors are smoking, tobacco use, air pollution, and delayed diagnosis. Image-guided fine needle aspiration cytology (FNAC) is rapid diagnostic technique for evaluating lung lesions. It is minimally invasive procedure with less complications. This study examine histocytologic makeup of lung lesions and link the results. Materials and Methods: This cross-sectional observational study included 65 patients irrespective of age and sex presenting with lung masses at Chitwan Medical College and Teaching Hospital from April 2023 to September 2024. After clinical and radiologic evaluation, all cases underwent image-guided FNAC and biopsy. Only specimens with unequivocal malignant features were classified positive. Histopathology served as diagnostic reference standard. Results: FNAC diagnosed 90.8% as malignant and 9.2% as benign. Biopsy confirmed malignancy in 92.3% of cases. FNAC demonstrated a sensitivity of 98.33%, specificity of 100%, positive predictive value(PPV) of 100%, and negative predictive value (NPV) of 83.33%. Concordance between FNAC and histopathological subtyping was 98.46%. Adenocarcinoma was most common subtype, followed by Squamous cell carcinoma(SCC) and small cell carcinoma. Smoking was most common contributing factor associated with malignancy. Conclusion and implications: Image-guided FNAC is an excellent diagnostic accuracy tool which possess higher level of concordance with biopsy in evaluating lung masses. It should be considered as frontline diagnostic tool, especially in resource limited settings. Keywords: FNAC, Lung cancer, Biopsy, SCC, Adenocarcinoma, Small cell carcinoma, Nepal

Background Otitis media is a leading cause of childhood morbidity and presents a significant healthcare burden worldwide. Streptococcus pneumoniae is a major aetiological agent of OM, and the introduction of pneumococcal conjugate vaccines into the Malaysian National Immunisation Programme in 2020 would have altered pneumococcal carriage and serotype distribution. This study aimed to determine pneumococcal carriage, serotype distribution, and associated risk factors among children with OM in the early post-PCV era in Peninsular Malaysia. Methods and Findings A total of 360 children with OM were recruited from hospitals on the east and west coasts of Peninsular Malaysia between 2023 and 2025. Nasopharyngeal and middle ear fluid samples were collected for Spn isolation by culture, followed by serotyping using multiplex PCR. Sociodemographic, environmental, and medical history data were analysed for associations with pneumococcal carriage using chi-square, Fishers exact tests, and logistic regression. Pneumococcal carriage was detected in 26.7% of children in either NP or MEF samples, with carriage rates of 25.6% and 1.9% in NP and MEF samples, respectively. The most prevalent serotypes were 23A, 15B/15C, non-typable strains, 19F, and 11A/11D. Daycare attendance (p = 0.012, aOR [95% CI]: 2.177 [1.186 - 3.995] and residence in rural areas (p = 0.019, aOR [95% CI]: 2.476 [1.159 - 5.292] were significantly associated with pneumococcal carriage. The main limitation of the study was the reliance on self-reported questionnaire data, which may have introduced recall bias and reporting errors. Conclusions The predominance of non-vaccine serotypes and non-typable Spn indicates ongoing serotype replacement and the emergence of phase-variant strains in the early post-PCV era. Continued surveillance is essential to monitor these changes and inform the development of next-generation pneumococcal vaccines. This study was registered under clinical trial registration number NCT05429541.

Background: The N-terminal ectodomain (NTE) of the SARS-CoV-2 membrane (M) glycoprotein is a short, flexible region that remains exposed on the virion surface and exhibits immunogenic potential across multiple coronaviruses. Despite its small size and conformational plasticity, this region contains conserved linear epitopes that may serve as practical surrogates for full-length proteins in serological diagnostics. Objective: To develop and evaluate a synthetic peptide-based diagnostic assay targeting the NTE of the SARS-CoV-2 M protein. Methods: Epitope prediction, peptide synthesis, and antibody affinity assays were performed to design homomultivalent peptide analogs that exploit avidity effects through disulfide polymerization. The resulting peptide antigens were tested in an enzyme-linked immunosorbent assay (ELISA) using clinical samples from RT-PCR-confirmed COVID-19 patients and biobanked controls. Results: The selected peptide analogs (M1, M1i, M1s) corresponded to a conserved surface-exposed motif of the SARS-CoV-2 M protein. Polymeric M1 exhibited a twofold gain in apparent affinity (Kdapp = 4.33 nM) compared with the monomeric form (Kdapp = 8.00 nM). Clinical validation using 1,222 patient samples yielded a sensitivity of 95.26% and specificity of 52.27%, with an overall diagnostic accuracy of 88.70%. Conclusion: The M peptide analogs demonstrate that synthetic peptide antigens can serve as stable, high-sensitivity surrogates for whole-protein assays. This design principle may be applied to other emerging pathogens where rapid assay development and scalability are critical. Keywords: Peptides, Antibodies, COVID-19, Enzyme-Linked Immunosorbent Assay, Protein Binding

Background SARS-CoV-2 genomic surveillance is vital for public health, but whole-genome sequencing (WGS) remains costly and inaccessible in many resource-limited settings. We developed and validated a multiplex real-time RT-PCR assay for rapid, economical detection of key mutations associated with variants of interest (VOI) and concern (VOC). Methodology Two multiplex mixes (M1, M2) targeting eight mutations in the ORF1a and Spike genes were designed. Analytical validation included sensitivity, specificity, reproducibility, and limit of detection (LoD) using WHO international standards and a respiratory pathogen panel. In parallel, an in silico analysis evaluated oligonucleotide efficacy against 10.4 million SARS-CoV-2 genomes from GISAID/NCBI, assessing inclusivity, target-site secondary structure (RNAalifold), and hybridization energy (Primer3Plus). Results The assay demonstrated 100% clinical sensitivity among samples with valid RT-PCR results (41/42 samples yielded interpretable results, with one inhibited sample excluded from sensitivity calculation), a LoD of 5.7 log10 IU/mL, and 100% analytical specificity against 32 non-SARS-CoV-2 respiratory pathogens. Six out of eight oligonucleotide sets showed >96% inclusivity; two sets exhibited reduced inclusivity (94.03%, 90.14%) and structural features potentially affecting binding against emerging variants. The assay enables direct identification of major VOCs (Alpha, Beta, Gamma, Delta, Omicron) and indirect detection of multiple VOIs (P.2, Epsilon, Kappa, Eta, Iota, Lambda). Conclusion This standardized multiplex assay provides a rapid, sensitive, and low-cost alternative for SARS-CoV-2 variant surveillance in Cuba and similar settings. The integration of experimental and in silico validation offers a robust, adaptable framework to sustain diagnostic accuracy amid viral evolution, optimizing the allocation of scarce sequencing resources.

The increasing incidence of H5N1 influenza virus transmission from animal species to humans has heightened concerns about an imminent H5N1 pandemic. Prior studies using recombinant hemagglutinin and neuraminidase proteins have reported age-dependent cross-reactivity to H5N1, attributed to immune imprinting from an individual's first influenza virus exposure. However, whether this pattern holds when using whole inactivated virus (WIV), capturing antibodies against diverse viral proteins, and is stable over time remains unknown. We therefore aimed to determine whether H5N1 cross-reactivity of pre-existing antibodies to whole virus follows an age-dependent or imprinting-specific pattern, and whether this pattern is stable over a five-year period. To this end, we measured serum antibody levels in adolescents, adults and seniors by ELISA using whole inactivated H5N1 virus as antigen rather than purified proteins. Detectable, albeit generally low, levels of H5N1-reactive antibodies were present in most individuals, irrespective of age. Comparison of antibody levels against H5N1 with those to five historical influenza virus strains revealed a consistent positive correlation between H5N1-reactive antibodies and responses to the H1N1pdm09 strain A/California/7/2009 (CA), across all age groups. Using unbiased clustering of antibody titers against H5N1, CA, and the H3N2 strain A/Perth/16/2009 (PE), we identified seven distinct age-transcending antibody profiles. These profiles covered individuals with varying titers to all three included viruses but also identified individuals with high anti-CA levels, yet low anti-H5N1 levels and vice versa. Moreover, despite stable antibody levels over a five-year interval in the study population, individual antibody levels and profiles fluctuated considerably over this period. Taken together, our results confirm the presence of H5N1-reactive antibodies in human sera and their association with previously circulating strains. However, they also caution against inferring antibody levels against a new strain based solely on responses to antigenically related strains and highlight the limitations of extrapolating immune status from single timepoint measurements.

Background. Direct livestock exposure is a risk factor for zoonotic influenza, including H5N1 highly pathogenic avian influenza (HPAI) A virus. But whether living in regions of high poultry and swine production intensity (PPI, SPI) increases risk of exposure to zoonotic influenza viruses independent of occupational livestock contact remains unclear. Objectives. To determine whether livestock workers and community members with no occupational livestock exposure in North Carolina, where poultry and swine production are increasingly co-located, are at higher risk of exposure to zoonotic influenza. Methods. Saliva samples from industrial livestock operation worker (ILO-W), ILO neighbor (ILO-N) and metropolitan area (Metro) households were analyzed for mucosal influenza A (H5N1, H1N1, and H3N2) hemagglutinin (HA) IgA and IgG antibodies to determine associations of PPI, SPI, exposure group, and detection of a swine-specific fecal contamination marker (Pig-2-Bac DNA) with influenza A antibody levels. Results. Residing in the highest PPI and SPI tertile was associated with significantly higher mucosal H5 and H1 HA IgA levels, including among residents without occupational livestock exposure. Households with occupational poultry or swine contact had significantly higher H5 IgA and IgG and H1 IgA levels compared to Metro households. In regression models accounting for clustering at the participant level, log10 anti-H5 HA mucosal IgA increased 0.16 (95% CI: 0.06, 0.27, p<0.005) and 0.10 (95% CI: 0.03, 0.17, p<0.005), per log10 increase in PPI and SPI, respectively, and 0.16 (95% CI: 0.03, 0.19, p<0.02) when Pig-2-Bac DNA was detected on household surfaces. Conclusions. Mucosal H5 HA IgA and IgG and H1 HA IgA were consistently elevated across different metrics of livestock exposure intensity, including residential exposure, occupational contact within a household, and a molecular marker of household swine fecal contamination in a state with intensive poultry and swine production.

Objectives: In some resource-limited settings the case-fatality rate of severe leptospirosis can exceed 50%. Early recognition of severe disease can expedite transfer to referral centres for advanced supportive care. The entirely clinical, 3-point SPiRO score can be calculated rapidly at presentation to predict a patients subsequent clinical course. In its derivation study, a SPiRO score of 0 had a negative predictive value (NPV) for intensive care unit (ICU) admission of 98% (95% confidence interval (CI): 96-99). In this validation cohort we sought to confirm the clinical utility of the SPiRO score and to compare its prognostic utility with other leptospirosis-specific and general disease severity scores. Methods: We examined consecutive adults presenting to high-caseload hospitals in tropical Australia with laboratory-confirmed leptospirosis between June 2016 and April 2026. The ability of the SPiRO score to predict requirement for ICU admission before hospital discharge was compared with that of the leptospirosis-specific QuickLepto score and commonly used disease severity scores, namely the SOFA, qSOFA, qSOFA-lactate, NEWS-2, qNEWS, UVA and the SIRS scores. Results: ICU admission was required in 62/309 (20%) episodes of leptospirosis. The SPiRO score performed as well as - or better than - all the other scores in predicting ICU admission. The Area Under the Receiver Operating Characteristic curve for the SPiRO score was 0.83 (95% CI: 0.77-0.89); only the SOFA score had a higher value: 0.84 (0.79-0.90), although the difference was not statistically significant (p=0.08). The SPiRO score had the highest NPV for ICU admission of any of the scores: 95 (95% CI: 91-97)%. Conclusions: The SPiRO score can be calculated easily at the bedside at presentation to expedite the recognition of patients with leptospirosis who are most likely to deteriorate. In resource-limited settings this entirely clinical score can also help reduce unnecessary escalation of care, optimising the use of finite health resources.

Although controversial, limited publications support the notion that HIV-1 can infect CD4-negative cells. The objective of this study was to provide a comprehensive investigation of a universally available CD4-negative cell line model system that can be infected with X4 and R5 HIV-1 to generate integrated proviral DNA and serve to study latent viral reservoirs. The reason that HIV-1 infection of CD4-negative cells has become less investigated is due to a lack of a fully characterized model for the study of this unusual pathway. To address this critical need, human osteosarcoma (HOS) cells, engineered to express either CD4, CCR5 or CXCR4, and easily available from a commercial source were used. CD4 expression was examined using western immunoblot, flow cytometry, anti-CD4 blocking antibody and mRNA expression. Cells were infected with HIV-1 pseudo-enveloped viruses bearing either JR-FL (R5-tropic) or HXB2 (X4-tropic) envelopes, constructed on NL4-3 luciferase/GFP backbone. Infection was monitored by luciferase readout and visualized by GFP immunofluorescence. Raltegravir was used to inhibit integration, and AMD3100 and maraviroc used to block chemokine coreceptors, CXCR4 and CCR5, respectively. Productive versus latent infection was quantified by dual-fluorescence readouts using HI.fate.E. We confirmed that HOS cells lack CD4. HOS cells expressing only CCR5 or CXCR4 supported HIV-1 infection, although infection was significantly lower than in matched CD4-positive controls. Raltegravir treatment blocked proviral integration in all instances. Coreceptor antagonism and envelope-deficient viruses revealed that infection of CD4-negative CXCR4 cells remained CXCR4-dependent, whereas CD4-negative CCR5 cells showed evidence of CCR5-independent infection. Dual-reporter HI.fate.E assays indicated that CD4-negative cells could support both productive and latent infection. These studies establish a universally available cell line model for the study of CD4-negative HIV-1 infection. This cell line model will provide insight into the question of how CD4-negative cells can be infected with HIV-1 and whether CD4-negative cells can provide latent viral reservoirs in HIV/AIDS. Author summarySince the first description of HIV/AIDS in 1981 and the recognition that CD4 was a primary receptor for HIV-1 in 1983, a limited number of reports have suggested that cells lacking CD4 could be infected with HIV-1. These reports continued even when it was shown in 1996 that co-receptors, CXCR4 and CCR5, were also required for HIV-1 infection of CD4 T-helper cells. Indeed, crystallography studies showed that CD4 was required to interact with the HIV-1 envelope gp120 in order to cause conformational changes in the envelope to expose the binding motif for chemokine co-receptor engagement, required for additional conformational changes to expose the gp41 fusion protein, allowing for entry and infection. However, reports continued that cells lacking CD4 could be infected which raised questions as to how this can happen. To address this critical gap, we have identified a cell line, HOS, that is commercially available, having expression of CD4, CXCR4 and/or CCR5. Using these HOS cell lines, we have been able to confirm that HIV-1, either X4 or R5 enveloped viruses, can infect CD4-negative cells. We have also confirmed that infection is productive and allows for latent proviral integration. Our findings provide a system for further studies of the mechanism(s) of HIV-1 infection of CD4-negative cells using a consistent model and may aid in elucidating establishment of viral reservoirs.

Background: Seasonal influenza, commonly known as flu, is an acute respiratory, highly contagious illness caused by influenza viruses. A clear understanding of influenza seasonality is crucial for guiding prevention and treatment strategies, including decisions on vaccination timing to prevent outbreaks. While well documented in temperate regions, data on influenza epidemiology in tropical areas, particularly sub-Saharan Africa, remain limited. We described the types, subtypes and positivity rate of seasonal influenza in Uganda during 2019-2023. Methods: We abstracted data from the National Influenza database on positive seasonal influenza cases confirmed by Polymerase Chain Reaction. The cases were disaggregated by age group, sex, region, month and year of reporting. Using Microsoft excel, we calculated the influenza positivity rate and disaggregated it by strain, sex, age, region and time. Test positivity rate was computed as the number of positive cases as a percentage of the total samples tested. Results: Among 17,957 individuals tested, the overall positivity rate for seasonal influenza was 5% (936 cases). Positivity was higher among males compared to females (7% vs. 4%), with children aged 5-9 years having the highest positivity rate (16%), while individuals aged 50-54 years had the lowest (1%). The median positivity rate was 4%, with a range of 1-16%. Regionally, the central region reported a positivity rate of 5%, with rates across all regions ranging from 5% to 8%. Over time, there was a gradual decline in positivity rates, decreasing from 16.5% in 2019 to 5.3% in 2023. Seasonal influenza exhibited bimodal peaks, with the primary peak occurring between March and May and a secondary peak from October to December. Influenza A was the predominant strain, accounting for 70% of seasonal influenza cases (669/936). Among the Influenza A subtypes, H3N2 was most common, representing 63% of cases (425/669). Conclusions: The declining seasonal influenza positivity rates from 2019 to 2023 and the predominance of Influenza A and H3N2 highlight the need for sustained surveillance in Uganda. Given Influenza A's high genetic variability and potential for novel strain emergence, monitoring circulating strains, informing vaccine development, and implementing targeted interventions for high-risk groups and regions are critical to controlling and preventing outbreaks.

Background. The clinical course of coronavirus disease 2019 (COVID-19), and the ability to anticipate which patients will require intensive care, were poorly characterized in Central Asia during the first pandemic wave. We aimed to describe the clinical features of hospitalized COVID-19 patients at the Tashkent State Medical University, Uzbekistan, and to identify risk factors for intensive care unit (ICU) admission. Methods. In this single-centre cross-sectional study, we reviewed the records of 2500 consecutive patients hospitalized between 11 April and 8 August 2020. Patients were grouped as asymptomatic or symptomatic, and symptomatic patients were compared by ICU versus non-ICU status. Groups were compared with chi-square or Fisher's exact and Mann-Whitney U tests. Univariable and multivariable logistic regression identified risk factors for ICU admission. Results. Of 2500 patients (median age 36 years; 60.9% male), 989 (39.6%) were asymptomatic and 1511 (60.4%) symptomatic. In total, 129 (5.2%) were admitted to the ICU and 38 (1.5%) died. ICU patients were older (median 56 vs 40.5 years) and more often had bilateral pneumonia, oxygen desaturation and cardiometabolic comorbidity. In the multivariable model (AUC 0.82), the independent predictors of ICU admission were ischemic heart disease (aOR 4.20), shortness of breath (aOR 3.22), hypertensive heart disease (aOR 2.93) and male sex (aOR 2.00). Conclusions. Older age, cardiometabolic comorbidity and respiratory compromise identified patients at high ICU risk. As one of the first clinical COVID-19 descriptions from Uzbekistan, these data provide a baseline for preparedness in Central Asia.

Background. Obesity has been associated with higher risk of dengue virus (DENV) infection and disease, yet its influence on antibody responses to DENV remains undefined. Methods. We evaluated whether nutritional status -- based on BMI z-score (BMIz) -- or blood markers of body fat -- leptin and adiponectin --are associated with binding and/or neutralizing antibody responses to DENV in 85 children in the Nicaraguan Pediatric Dengue Cohort Study who experienced a primary DENV infection in 2019. Associations were estimated using linear models adjusting for age, sex, and DENV infection outcome. Results. Compared to children with normal weight, those with obesity had higher quantities of DENV binding antibodies (fold-change [FC] 1.89, 95% confidence interval [CI] 1.02, 3.48) but no difference in neutralizing antibodies. Likewise, leptin concentration was associated with higher quantities of binding antibodies (FC 1.22, 95%CI 1.09, 1.37), while adiponectin was associated with lower quantities (FC 0.79, 95%CI 0.67, 0.94), and neither was associated with neutralizing antibodies. Lower neutralizing efficiency (neutralizing/binding antibodies) was observed in children with obesity (FC 0.67, 95%CI 0.48, 0.93). Conclusions. Our results indicate that obesity is associated with higher antibody quantity (binding) but not higher quality (neutralization) post-primary DENV infection -- implying that antibodies generated by children with obesity have lower neutralization efficiency, requiring greater quantities to reach similar levels of neutralization than children with normal weight. Further, the agreement among the three models using distinct proxies of body fat -- BMIz, leptin, and adiponectin -- demonstrates that adipokines are useful in supplementing BMIz analysis or as independent predictors of immune responses.

Background Sequencing of monkeypox virus (MPXV) from antigen rapid diagnostic tests (Ag-RDTs) could expand genomic surveillance during outbreaks in decentralized settings where sequencing equipment and cold chain transportation are unavailable. We aimed to evaluate the efficacy of MPXV sequencing from MPXV antigen Ag-RDTs. Methods We tested MPXV Ag-RDTs from three different brands using serial dilutions of cultured MPXV subclade Ib. Positive Ag-RDTs with different intensities of the test band were stored for 19 days, either at room temperature or at +4 degree C, after which viral DNA was extracted from the pads of the test cassettes. Metagenomic and tiled amplicon-based Oxford Nanopore technology sequencing methods were then performed. Results Viral DNA extraction from MPXV Ag-RDTs showed a consistent decrease in viral load of 3 logs compared to the initial viral load of the applied viral dilution. Both sequencing methods were able to reach high coverage but the tiled amplicon-based demonstrated more consistent results with a coverage always above 85%. Conclusion This proof-of-concept supports the development of this approach in the field, with the aim of combining genomic surveillance with decentralized testing, including in remote areas.

Background: Blood culture-negative infective endocarditis (BCNIE) poses a significant diagnostic challenge. This study evaluated a multimodal diagnostic algorithm combining serological and molecular methods at the Argentine National Reference Laboratory. Methods: A prospective analysis was conducted on 53 consecutive patients with suspected BCNIE referred between January 2019 and December 2024. The diagnostic workflow included indirect immunofluorescence for Bartonella spp. and Coxiella burnetii, species-specific PCR for Bartonella spp. and Tropheryma whipplei, and broad-range 16S rRNA PCR with Sanger sequencing on available blood and valvular tissue specimens. Results: An etiological diagnosis was established in 17 of 53 patients (32.1%). Bartonella spp. was the predominant pathogen (47.1%; 8/17), followed by T. whipplei (35.3%; 6/17) and Streptococcus spp. (17.6%; 3/17). All Bartonella cases were initially detected via serology, with molecular confirmation achieved exclusively through valvular tissue analysis. Conclusions: Implementing a standardized multimodal diagnostic algorithm significantly enhances etiological yields in BCNIE. The findings emphasize the complementary value of frontline serology and targeted molecular testing, highlighting that simultaneous submission of serum, blood, and valvular tissue is essential for optimal diagnosis.

Background: There are currently no vaccines approved for the prevention or treatment of Orthoebolavirus bundibugyoense (Bundibugyo virus; BDBV). The recombinant vesicular stomatitis virus- Zaire ebolavirus glycoprotein vaccine (rVSV-ZEBOV-GP; ERVEBO) has been widely deployed during Ebola virus disease (EVD) outbreaks caused by Orthoebolavirus zairense (Ebola virus; EBOV). Given the lack of vaccines and medical countermeasures we evaluated development of antibodies to Bundibugyo glycoprotein (GP) following rVSV-ZEBOV-GP vaccination in two EVD outbreak-affected populations in the Democratic Republic of the Congo (DRC). Methods: Between 2018 and 2023, serum samples were collected from vaccine recipients in Mbandaka, Equateur Province (n=482 at baseline), and Beni, North Kivu Province (n=599 at baseline). Antibody reactivity was assessed using a multiplex pan-filovirus immunoassay. We evaluated longitudinal trends in BDBV GP seroreactivity across follow-up visits extending to approximately five years after vaccination. Findings: We collected 2552 samples from 482 participants in Mbandaka and 3297 samples from 599 participants in Beni. BDBV GP responses diverged by location. Baseline BDBV GP seroreactivity differed between sites, with 3.3% of participants reactive in Mbandaka and 10.4% in Beni. In Mbandaka, BDBV GP titers remained unchanged through 6 months post-vaccination but increased markedly between 2.5 and 3.5 years (mean MFI 1,238 to 4,845; p<0.0001), accompanied by a rise in seroreactivity to 35.3%, followed by waning at later visits. In Beni, BDBV GP titers increased rapidly after vaccination, reaching peak seroreactivity at 21 days (52.3%) and 6 months (53.9%), with mean fold increases of 21.9 and 20.1 among baseline-naive participants. Although antibody levels declined after 6 months, EBOV GP and BDBV GP titers remained above baseline, and significant increases in EBOV GP, BDBV GP, and SUDV GP titers were observed between 2.5 and 5 years. Interpretation: We observed detectable BDBV GP seroreactivity in two geographically distinct populations in the DRC. However, there was no consistent evidence showing that rVSV-ZEBOV-GP vaccination was associated with increased seroreactivity to BDBV GP across study populations. Baseline BDBV GP seroreactivity observed prior to vaccination warrants further investigation into the origins and epidemiological significance of pre-existing filovirus-reactive antibodies, including the possibility of prior exposure to related filoviruses, cross-reactive immune responses, high non-specific background reactivity unrelated to filoviruses, or other unrecognized sources of earlier antigenic stimulation. In the absence of licensed BDBV-specific vaccines, these data may inform preparedness planning, evaluation of existing countermeasures, and future vaccine development efforts during current and future BDBV outbreaks. It may further contribute to the study of natural history of filovirus immunity with a direct implication to understanding the possibility of cross-reactive and possible cross-protective responses in humans.

Background: Leptospirosis causes around 1 million cases and 60,000 deaths globally annually, predominantly affecting flood-prone tropical regions. Transmitted through contact with water or soil contaminated by animal urine infected with Leptospira spp., this bacterial zoonosis is classified as a high-risk Group B infectious disease in Vietnam. However, epidemiological and clinical data are scarce, particularly in pediatric populations. This study evaluated clinical and laboratory characteristics of leptospirosis cases at the National Children's Hospital, Hanoi, from January 2023 to August 2025. Methodology : All children admitted with probable or confirmed leptospirosis were enrolled. Clinical and laboratory data were analyzed to characterize disease manifestations and identify risk factors for severe leptospirosis. This was defined by the presence of organ dysfunction, including liver or renal failure, hemorrhage (particularly pulmonary), aseptic meningitis, cardiac arrhythmias, pulmonary insufficiency, or hemodynamic collapse. Principal Findings : Of 84 patients (37 confirmed, 47 probable), the mean age was 9.4 years, with 52.4% male and over half aged [&ge;] 10 years. Fever was the most common presenting symptom (85.7%); gastrointestinal and renal manifestations were frequent, including oliguria in 26.2% of cases. Key laboratory abnormalities included elevated D-dimer (81.8%), elevated C-reactive protein (75.6%), hypoalbuminemia (74.3%), increased liver enzymes (AST 63.3%, ALT 53.2%), and renal impairment ({approx} 46%). Severe disease developed in 48.8% of patients, most frequently as acute kidney injury. Hematuria was independently associated with increased severity (OR = 4.89). Conversely, fever, higher baseline albumin, and longer symptom duration prior to hospitalization were associated with a significantly reduced risk of severe disease. Conclusions : Pediatric leptospirosis in this cohort frequently presented with systemic inflammation and multi-organ involvement, particularly renal impairment. Nearly half the patients developed severe disease. Early recognition of renal signs, especially hematuria, and careful monitoring of albumin levels are critical to identifying children at risk for severe progression and optimizing clinical management.

Purpose: Human Parvovirus B19 (B19V) infection is associated with a broad spectrum of clinical manifestations, including erythema infectiosum, arthropathy, transient red cell aplasia, hepatitis, and adverse fetal outcomes. Due to nonspecific presentations and limited routine testing, B19V infection often remains undiagnosed. Despite recent reports of increased B19V activity worldwide, contemporary data on its presence in Brazil remain scarce. We investigated the circulation of underdiagnosed pathogens in patients with suspected infectious diseases, prioritizing severe and fatal cases. Methods: In this descriptive study, hybrid capture-based whole-genome sequencing was used for a broad-range viral detection and characterization. We analyzed 472 clinical specimens selected for diagnostic investigation between 2024 and 2025, according to surveillance criteria for respiratory and arboviral infections, referred to the Public Health Laboratory of the state of Rio de Janeiro, Brazil. Results: B19V was the second most frequently detected viral pathogens, being identified in 190/472 specimens. Of these, just one sample had been previously tested for this pathogen. Thirty-one cases had higher B19V genomic coverage and were therefore selected for further analysis. Notably, B19V was the only virus detected with substantial genomic coverage in nine individuals, including elderly patients, and meningitis cases with B19V-positive cerebrospinal fluid. Phylogenetic analysis showed that recovered genomes clustered within genotype 1A2. Conclusions: B19V was frequently detected in patients undergoing investigation for infectious diseases and its circulation may represent an important and underrecognized pathogen. These findings support the incorporation of B19V testing into diagnostic algorithms for unexplained infectious diseases, especially in patients presenting atypical symptoms.