Journal of Clinical Virology Plus

Introduction: Herpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap Statement: The comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. Aim: This study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. Methodology: Four hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. Results: HSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. Conclusion: CLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification.

Background: Although the hemagglutination inhibition (HAI) titer remains the gold standard correlate of protection against influenza, it does not fully capture the broader antibody responses that contribute to immunity. Methods: We analyzed immune responses in paired pre-infection and convalescent sera from 306 RT-PCR-confirmed A/H3N2 infections from two household studies (2014-18) in Managua, Nicaragua. Antibody responses were measured by HAI and enzyme-linked immunosorbent assays (ELISAs) against full-length hemagglutinin (HA), the HA stalk, and neuraminidase (NA). Participants were classified as HAI responders ([&ge;]4-fold HAI rise), alternate responders (no HAI rise but [&ge;]4-fold boost in [&ge;]1 ELISA), or no-response individuals (no [&ge;]4-fold rise in any assay). We compared demographic, clinical, and pre-infection antibody characteristics across these groups. We also analyzed predictors of an NA response. Results: Overall, 77% of participants had HAI seroconversion or a 4-fold rise. Among the 23% HAI non-responders, 62% had alternate antibody responses. No-response individuals had the highest pre-infection HAI and full-length HA titers (p < 0.0001), the lowest viral loads, and the fewest fever or influenza like illness (ILI) symptoms (p < 0.01). An NA response was more common among symptomatic individuals (p = 0.0483) and those with low or high baseline NA titers. Conclusions: High baseline HAI titers can limit detectable 4-fold rises and are associated with milder illness. Evaluating additional immune responses may capture a more complete picture of the host response to infection, thereby improving surveillance and informing vaccine development. Keywords: Influenza A/H3N2; Hemagglutination inhibition (HAI); Neuraminidase antibodies; symptomatic vs asymptomatic infection; correlates of protection.

Cetacean pathology is a cornerstone for population and marine ecosystem health monitoring, allowing clear differentiation among natural and anthropogenic threats. Previous studies in the Canary Islands reported natural causes of death in 59.4% (1999-2005) and 81% (2006-2012) of stranded cetaceans, versus anthropogenic causes in 33.3% and 19%, respectively. This study aimed to determine the causes of death (CD), pathologic findings, and epidemiological patterns of 316 cetaceans stranded in the Canary Islands between 2013 and 2018. The CDs were classified in pathologic entities (PEs) emphasizing natural versus anthropic origins. Of 316 animals, 224 (70.9%) from 18 species were suitable for pathological investigations. Among natural PEE, natural pathology associated with good nutritional status (NP-GNS) and natural pathology associated with significant loss of nutritional status (NP-LNS) represented 43/224 (19.2%) and 36/224 (16%) cases, respectively. Natural pathology with undetermined nutritional status (NP-UNS) occurred in 19/224 (8.5%) animals. Intra- and interspecific traumatic interactions (ITI) represented 30/224 (13.4%) cases, followed by neonatal/perinatal pathology (NPN) 19/224 (8.5%) and live-stranding stress and/or capture myopathy (LS-CM) 18/224 (8%). Infectious and parasitic diseases predominated in natural PEs. Anthropogenic PEs included interaction with fishing activities (IFA) in 17/224 (7.6%) cases, vessel collisions (VC) in 9/22 (4%) cases, and foreign body-associated pathology (FBAP) in 3/224 (1.3%) animals. Overall, anthropogenic causes accounted for 12.9% of deaths, natural causes for 73.6%, and the CD could not be established in 30/194 (13.4%) cases. This study reaffirms the trends concerning recognized PEs (NP-GNS, NP-LNS, NP-UNS, ITI, NPN, LS-CM, IFA, VC, and FBAP), expands the body of knowledge on cetacean pathology in the Canary Islands, and reports novel findings including mixed infections, clostridiosis in uncommon species, uremic syndrome secondary to urethral nematodiasis, gas embolism in unusual species, epibiont stomatitis, congenital musculo-skeletal malformations, or neoplastic processes. These findings advance understanding of cetacean mortality patterns and support conservation and management strategies.

Background: How post-COVID-19 condition (PCC) differs from post-acute infection syndromes (PAIS) caused by other respiratory viruses remains uncertain. Comparing these conditions may clarify whether post-acute symptoms reflect specific consequences of SARS-CoV-2 infection or broader post-viral mechanisms. Methods: We conducted a systematic review and meta-analysis of cohort studies comparing persistent symptoms or conditions in adults after SARS-CoV-2 infection with those following other acute respiratory viral infections. PubMed, Embase, and Scopus were searched. Random-effects models were used to estimate pooled risks. Results: Among 9,371 records screened, 22 studies were included and 14 contributed to the meta-analysis. Increased risk after SARS-CoV-2 infection was observed for pulmonary embolism, abnormal breathing, fatigue, hemorrhagic stroke, memory loss/brain fog, and palpitations; heart rate abnormalities showed borderline significance. For most other outcomes pooled estimates were inconclusive. Conclusions: Only a subset of outcomes appears more frequent after SARS-CoV-2 infection, suggesting many symptoms attributed to PCC may reflect broader post-viral syndromes.

BackgroundTo date, accessible diagnostic tools to identify whether a patients pneumonia is a bacterial, or viral infection, are not accurate or timely enough to prevent preemptive antibiotic administration. Relying on single biomarkers or clinical presentations has been insufficient. We aimed to incorporate a wide range of novel biomarkers and clinical presentations in a multivariable model and validate its capacity to differentiate cases of bacterial and viral pneumonia. MethodsData from 457 children aged 2-59 months, admitted to Kilifi County Referral Hospital, Kenya, with bacterial (n = 229) and viral (n = 228) infections, were used to develop and validate a predictive multivariable Poisson regression model to differentiate pneumonia etiology. The Receiver Operating Characteristic curve was used to assess biomarker performance and validate the model internally. ResultsSixty-three percent (63%) of the children presented with severe pneumonia. 72% with viral pneumonia had severe pneumonia, compared to 54% with bacterial pneumonia who had severe pneumonia. In crude analyses, chest-wall indrawing, cough, convulsions, crackles, angiotensinogen, and Serpin Family A Member 1 were significantly associated with pneumonia etiology, controlling for age. However, only chest-wall indrawing remained significant in multivariable analyses after controlling for age. The model demonstrated fair, but inadequate, discrimination, with an Area Under the Curve of 0.61. ConclusionAmong the children admitted to hospital with WHO defined pneumonia, a wide range of biomarkers and clinical presentations still failed to distinguish bacterial from viral pneumonia.

Mpox virus (MPXV) gained increased attention following the declaration of two Public Health Emergencies of International Concern (PHEICs) in 2022 and 2024. The rapid spread of MPXV and the increase in human-to-human transmission highlighted the need for improved diagnostic tools for characterizing infection patterns and transmission dynamics. While PCR is effective for detecting active infections, serological approaches can help identify previous or asymptomatic infections and support retrospective surveillance. However, many serological assays developed during recent outbreaks have not been evaluated in endemic settings such as the Democratic Republic of the Congo (DRC). This study aims to define antigen-specific serological cutoff values to differentiate MPXV-seroreactive individuals from those with other orthopoxvirus (OPXV) exposure or different vaccination histories, specifically for use in the DRC. Here, we analyzed 134 individuals, divided into six distinct cohorts with different exposures. Serum samples were tested using Mesoscale Discovery (MSD) to screen for five MPXV and vaccinia virus (VACV) orthologous antigens: A29L/A27L, A35R/A33R, B6R/B5R, E8L/D8L, and M1R/L1R. Receiver operating characteristic (ROC) analysis identified the best-performing antigens and established seroreactivity cutoff values. A binary composite rule was also evaluated to improve the classification of these results. We identified three MPXV antigens, E8L (cut-off=12.33 AU/mL), A35R (cut-off=5.22 AU/mL), and B6R (cut-off=9.77 AU/mL), that showed the strongest discriminatory performance in the dataset. Collectively, these three antigens form a significant panel that demonstrated clear separation between our mpox survivor cohort and other OPXV-exposed individuals.

There is an ongoing Oropouche Fever (OF) outbreak in Brazil since 2024. There are dengue and chikungunya prediction models available, but none to help discriminate dengue, chikungunya, and OF. Objective: This study aims to develop and validate clinical prediction models for dengue, chikungunya, OF. Methods: This study uses surveillance data from Espirito Santo state / Brazil, from 2023-2025. Epidemiological investigations and biological samples were used to conclude cases as either (a) clinical-epidemiologically confirmed, (b) laboratory confirmed, or (c) discarded. The predictors were all data related to signs, symptoms, and comorbidities available in the notification forms. The analysis was performed using random forest regression models, one for each outcome, in development and validation datasets. Results: A total of 465,280 observations were analyzed, 261,691 dengue cases (56.6%), 18,676 chikungunya cases (4.0%), 12,174 OF cases (2.6%), and 179,115 discarded cases (38.6%). All three models had good discrimination and moderate to good calibration after scaling prediction. The models retained from 26 to 16 predictors each. Leukopenia and vomiting were the most discriminatory predictors for dengue, arthritis, arthralgia, and rash were the most discriminatory for chikungunya, and epidemiological features were the most relevant for OF. The dengue, chikungunya, and OF models had ROC AUC of 0.726, 0.851, and 0.896 in the validation set, respectively. Conclusion: This research identified predictors most discriminative between dengue, chikungunya, and OF. We developed and validated predictive models, one for each condition, with moderate to very good performance available at https://pedrobrasil.shinyapps.io/INDWELL/. One may use them in diagnostic work-up and arbovirus surveillance.

Abstract Introduction: Streptococcus is the second genus involved in bone and joint infections (BJIs) after Staphylococcus. Streptococcus agalactiae is the predominant Streptococcus species implicated in BJIs. However, unlike Staphylococcus-related BJIs, data on S. agalactiae infections remain scarce. Methods: We conducted a retrospective cohort study from the West Region cohort of the CRIOAc registry among six university hospitals including all microbiologically confirmed streptococcal BJI in adults between 2014 and 2023. Results: 1454 patients were included, with a median age of 67 years and 65% male. S. agalactiae was the predominant streptococcal species involved 423/1454(29%). The most prevalent comorbidities identified were obesity (378/1454;26%) and diabetes mellitus (343/1454;24%). Prosthetic joint infections (PJIs) were the most common (653/1454;45%), although diabetic foot osteitis was less prevalent overall, it was significantly more associated with S. agalactiae infections (48/423;11% versus 70/1031;7%, p=0.05). S. agalactiae BJIs were more frequently lower-limb infections and chronic infections (240/423;57% versus 502/1031;49%, p=0.04). Half of the cohort had a polymicrobial infection and were slightly more frequent with S. agalactiae BJIs (235/423;56% versus 498/1031;48%, p=0.1). These results were consistent with a sensitivity analysis excluding diabetic foot related osteitis. Logistic regression analysis identified arteriopathy (OR: 4.16; IC95:1.64-11.24, p=0.003), and obesity (OR: 2.57; IC95: 1.41-4.78, p=0.002) as specific risk factors for S. agalactiae BJIs. Conclusion: S. agalactiae emerges as a prominent and distinct pathogen in complex streptococcal BJIs, with specific risk factors such as arteriopathy, obesity and diabetes mellitus, and more chronic infections.

Background: Spotted fever is caused by spotted fever group rickettsiae (SFGR) belonging to the genus Rickettsia. Transmission to humans is primarily via the bite of infected ticks. Being a vector-borne disease, the occurrence of spotted fever is related to factors that allow the vector to thrive. This spatio-temporal analysis gives an insight into the distribution of cases and correlation with seasonality. Methodology: A suspected AFI patient was considered spotted fever positive if either serology (ELISA/IFA) or molecular assay (Nested PCR/qPCR) was tested positive. Demographic data of confirmed cases were included for the analysis. Results: In the 18-year dataset, a total of 2153 suspected patients were tested for spotted fever, of which 516 (24%) were positive. On spatio-temporal analysis, Vellore district reported 39.9% of cases, Chittoor 38.8%, Tirupattur 12.5%, Ranipet 4.5%, and Tiruvannamalai 4.3%. Maximum spotted fever cases were reported between the months of September to March, with a peak in January. Children below 10 years and housewives were at risk of spotted fever. Conclusion: The findings of this retrospective analysis highlight the importance of considering spotted fever group rickettsioses in patients presenting with acute undifferentiated febrile illness, particularly children aged <10 years, from areas with higher spatial clustering, during or following the monsoon season.

PurposeAntibodies to Epstein-Barr virus (EBV) proteins can predict nasopharyngeal carcinoma (NPC) risk. We previously defined a prototype EBNA1 protein panel and multiplex immunoblot assay that distinguishes NPC risk several years pre-diagnosis. Assay throughput and specificity are critical to effectively implement a population-level screening program. Here, we developed a strip test assay - EBNA1 SeroStrip-HT - with an objective to increase throughput and maximize specificity. Experimental DesignEBNA1 full-length (FL) and glycine-alanine repeat deletion mutants (dGAr) were purified from insect and mammalian cells to screen serum IgA/IgG from prospective cohorts in Singapore and Shanghai, China, with known time intervals to NPC diagnosis. Twenty pre-diagnostic sera within 4 years to diagnosis were compared to 96 healthy controls using a nested case-control study design. ResultsIgA to mammalian-derived EBNA1 dGAr achieved 85.0% sensitivity and 94.8% specificity (AUC, 0.939) for NPC status. IgA to insect-derived EBNA1 dGAr showed the same sensitivity (85.0%) and similar specificity (93.8%) (AUC, 0.941). IgA to insect-derived EBNA1 FL had a higher 90% sensitivity, but lower 91.7% specificity (AUC, 0.940). Combining EBNA1 FL and dGAr results showed that subjects positive for both proteins had a 243.67 odds ratio for NPC incidence compared to double-negative scores. ConclusionThis study demonstrated the efficacy of EBNA1 SeroStrip-HT for NPC risk assessment and stratification in high- and intermediate-risk populations, yielding high accuracy and a 12-fold increased throughput over the prototype. The insect system was appropriate for large-scale production of purified EBNA1. Larger, geographically diverse cohorts are warranted to confirm these results, especially in low-incidence populations.

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

Digital pathology, coupled with advanced image recognition algorithms, represents a transformative frontier in histopathological diagnosis. This sub-Saharan African laboratorys exploratory study investigates the application of a Convolutional Neural Network (CNN) model, specifically leveraging the VGG16 architecture with transfer learning, for automated analysis and classification of selected gastrointestinal (GIT) and liver tissue samples, incorporating both routine and specialized staining protocols. The study utilized a dataset comprising 114 samples (18 liver, 96 GIT images) derived from archival formalin-fixed paraffin-embedded tissue blocks at University College Hospital, Ibadan, Nigeria. Specialized staining techniques included Alcian Yellow for GIT mucin visualization and Massons Trichrome for liver fibrosis assessment, alongside conventional H&E staining. Model performance was evaluated using statistical methodologies including Wilson Score confidence intervals (CI), Bayesian probability assessment, and effect size analysis. Results reveal a striking dichotomy in model performance. The GIT tissue model achieved perfect classification accuracy (100% test accuracy) with exceptional statistical significance (Z=10.0, p<0.0001), Wilson CI [96.29%, 99.99%], Cohens h=1.571, and Bayesian probability >99.99%. Conversely, the liver tissue model demonstrated diagnostic failure (42.86% test accuracy), with Z=-1.428, p=0.9236, Wilson CI [33.59%, 52.65%], Cohens h=-0.144, and Bayesian probability of 7.64%. This performance divergence correlates with training data availability, as the liver dataset fell far below empirically established thresholds (>100-200 samples) for reliable classification. The liver models failure reveals limitations in transfer learning with insufficient data. These findings underscore critical implications for AI-enhanced digital pathology, demonstrating potential deployment of the GIT model as a promising one that supports tissue-specific model development.

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. Methods: A murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a Sandwich-ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. Results: mAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The Sandwich-ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. Conclusion: The FH12-based Sandwich-ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.

Anopheles albimanus (Nyssorhynchus) is featured as the main malaria vector on Hispaniola. However, five other Anopheles species have been reported circulating in the area; four of them belonging to the subgenus Anopheles (An. crucians, An. grabhamii, An. pseudopunctipennis, and An. vestitipennis) and another one to the Nyssorhynchus subgenus (An. argyritarsis). Previous studies on mosquitoes in the genus Anopheles have identified and characterized peptides from immunogenic salivary proteins, with several of these peptides being unique to the Nyssorhynchus and Anopheles subgenera. This underscores their potential use as biomarkers for differentiating exposure to Anopheles mosquitoes in both the Old World and New World. Since both Nyssorhynchus and Anopheles subgenera have been reported in Haiti, a series of ELISAs were conducted to quantify IgG antibody titers against three published antigenic anopheline salivary peptides (gSG6-P1, Peroxi-P3, and Apy-2) in 348 participants registered in Haitis multi-partner/multidisciplinary Malaria Zero Program. This study aimed to evaluate the intensity of human-vector contact with Anopheles from both subgenera in Grand Anse, Haiti. In addition, the study measured antibodies against a panel of Plasmodium falciparum antigens to determine any association between anti-parasite and anti-peptide antibodies. Significantly elevated IgG responses to Peroxi-P3 in comparison to Apy2 and gSG6-P1 in the total study population (p < 0.001) were observed. Additionally, immune responses to Peroxi-P3 and gSG6-P1 differed significantly between [&le;]18-year-olds and >18-year-olds (p = 0.004 and p = 0.002), whereas no sex-based differences were observed for any peptide. Correlation analyses observed a greater number of significant positive associations in immune response between gSG6-P1 and Plasmodium antigens than any other salivary peptide, an occurrence which was more pronounced in [&le;]18-year-olds than >18-year-olds. A marked reduction in IgG responses to Apy2 and Peroxi-P3, but not gSG6-P1, among participants who kept a single household animal species compared with those who owned two or more species or those who did not have household animals was also demonstrated. Spatial analysis revealed heterogenous geographic overlap of high antibody responses among Peroxi-P3, Apy2, and gSG6-P1, alongside geographically overlapping clusters of low antibody responses to Peroxi-P3 and Apy2. These results provide additional data on the utility of anopheline salivary peptides to characterize human-vector-parasite exposure dynamics in low-transmission areas, such as Haiti.

Objectives: Scrub typhus, caused by the bacterium Orientia tsutsugamushi, is frequently underdiagnosed due to its non-specific clinical presentation and the frequent absence of eschar. Most molecular diagnostic assays target single-copy genes of O. tsutsugamushi, which can limit diagnostic sensitivity. We aimed to develop an ultra-sensitive quantitative PCR (qPCR) assay targeting a highly repetitive element in O. tsutsugamushi genome. Methodology: We developed a SYBR Green-based qPCR assay (TranScrub) targeting a multicopy transposase gene of O. tsutsugamushi and compared its performance with assays targeting the 56kDa (single-copy) and traD (multicopy) genes. Diagnostic performance was evaluated using clinical specimens and a panel of blood-borne pathogens. The limit of detection (LOD) was estimated using serial dilutions of quantified template. The assay was further applied to dried blood spot (DBS) samples from patients with acute febrile illness of unknown aetiology, with positives confirmed by Oxford Nanopore amplicon sequencing. Results: Targeting the multicopy transposase gene enabled highly sensitive detection of O. tsutsugamushi, outperforming the conventional 56-kDa assay and matching the traD assay. TranScrub achieved a 91% sensitivity (29/32) and 100% specificity (77/77) using blood-derived DNA, with no cross-reactivity. The LOD was 0.024 genome equivalents/L. Among 81 DBS samples from acute febrile patients of unknown aetiology, 6 (7.5%) tested positive, all confirmed by sequencing. Conclusions: The transposase gene represents a novel target that improves molecular detection of scrub typhus. TranScrub enables sensitive and specific detection from both blood and DBS, supporting its use in clinical diagnosis and field surveillance.

BackgroundDuring 2021-2022, high pathogenicity avian influenza (HPAI) caused mass mortality in wild birds across Europe, with Northern Gannets (Morus bassanus) among the most affected. Following the outbreak, unusual alterations in the species characteristic pale iris were observed in some individuals. MethodsOpportunistically captured gannets on Bass Rock (n=52), selected to represent a range of iris pigmentation, were examined. Slit-lamp biomicroscopy, indirect ophthalmoscopy, rebound tonometry and photography were performed. Iris pigmentation was classified as normal, mottled or black. Eleven birds underwent avian influenza virus (AIV) serology. Histopathology was performed on two eyes. ResultsAbnormal iris pigmentation was found in 74% of adult and immature gannets, with 61% affected bilaterally. Additional signs consistent with uveitis were present in 77% of affected birds. Iris pigmentation abnormalities were positively associated with AIV H5 seropositivity (Fishers exact test, P=0.018). Histopathology from affected eyes showed increased melanin deposition and disorganisation, including loss of a distinct anterior layer of melanocytic cells and hypertrophy of melanocytes within the iris stroma. LimitationsField conditions limited uniform lighting and concurrent serology. ConclusionsIris pigmentation changes were associated with prior HPAI exposure and frequently accompanied by signs of uveitis, suggesting iris alterations may indicate past infection and potential chronic sequelae.

Respiratory syncytial virus (RSV), an approximately 15.2 kb negative sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV/A and RSV/B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 deidentified remnant clinical specimens (102 RSV/A; 112 RSV/B) with RT PCR Ct values <31 were included. RSV genomes were amplified using ARTIC style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV/A (median 53,433x; mean 51,076x) than RSV/B (median 49,699x; mean 46,945x), whereas genomic coverage was slightly lower for RSV/A (median 97.5%; mean 96.6%) than RSV/B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV/A and B.D.E.1 for RSV/B. For RSV/A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra and inter run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0 to 5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. These results demonstrate that the ARTIC-based RSV WGS assay enables near real time surveillance and strengthens data driven public health responses to future outbreaks.

Introduction: Following a large chikungunya outbreak during 2006 to 2008, Sri Lanka did not report any outbreaks for a 16 year period until end of 2008, possibly due to population immunity. Therefore, understanding baseline immunity prior to outbreaks is crucial to inform implementation of vaccine strategies. Methods: We assessed the age stratified seroprevalence for chikungunya in an urban (n=816) and a semi urban (n=380) community in Colombo, Sri Lanka, from September to November 2024, prior to the commencement of the large chikungunya outbreak, in December 2024. Sociodemographic, socioeconomic and clinical data were collected and chikungunya specific IgG measured in serum samples. Results: Of 1196 participants, 410 (34.3%) were chikungunya IgG seropositive. Seroprevalence was significantly higher in urban populations compared with semi urban populations (39.6% vs 22.9%; p<0.001) and increased significantly with age in urban areas but not in semi-urban areas. Living in an urban area was the strongest independent risk factor of chikungunya seropositivity (aOR 7.48, 95% CI 4.05 to 13.81; p<0.001), consistent with the higher population density, poor housing conditions and overcrowding observed in that setting. The use of mosquito nets was independently associated with reduced risk of seropositivity (aOR 0.50, 95% CI 0.27 to 0.93; p=0.029). Almost no individuals aged <16 years had evidence of prior infection (0.55%), indicating minimal transmission in the preceding 16 years. In the urban cohort, seropositivity was significantly associated with diabetes, central obesity, overweight, and hypertension. Conclusions: There appears to have been minimal chikungunya transmission in the 16 years preceding the 2024 outbreak, with a large population susceptible to chikungunya. Higher seroprevalence in urban populations highlights the role of population density, overcrowding, and housing conditions as key drivers of transmission.

The A(H1N1)pdm09 virus remains a major global health threat. This study examined the burden of ICU admission, mortality, and associated predictors among patients with A(H1N1)pdm09 pneumonia in a leading center for infectious diseases in Vietnam. Information on demographic, clinical, and laboratory characteristics, and outcomes was retrieved from medical records of adults admitted with influenza A(H1N1)pdm09 during 2009-2019. Among 729 cases, 21.7% (158/729) developed pneumonia. Among 158 pneumonia cases, 36.7% (58/158) developed moderate-to-severe acute respiratory distress syndrome (ARDS), and 15.2% (24/158) received invasive ventilation. ICU admission and mortality rates were 48.7% (77/158, 95%CI 41.1-56.5%) and 8.2% (13/158, 95%CI 4.9-13.6%), respectively. Predictors of ICU admission included being >60 years old (adjusted OR [AOR] 13.864, 95%CI 2.185-87.956, P=0.005), comorbidities (AOR 6.527, 95%CI 1.710-24.915, P=0.006), AST (AOR 1.013, 95%CI 1.001-1.025, P=0.029), and moderate-to-severe ARDS (AOR 14.027, 95%CI 4.220-46.627, P<0.001). Predictors of mortality were invasive ventilation (AOR 55.355, 95%CI 1.486-2062.375, P=0.030) and double-dose oseltamivir or combination therapy (AOR 32.625, 95%CI 1.594-667.661, P=0.024). In conclusion, mortality is not rare in A(H1N1)pdm09 infection. Monitoring of older patients and those with comorbidities, liver enzyme elevation, or moderate-to-severe ARDS is essential for the timely detection of complications requiring intensive care.

Introduction Improved diagnostics are needed for people at risk of tuberculosis, especially adolescents. Tongue swab (TS) molecular testing has emerged as a promising strategy for tuberculosis diagnosis. We evaluated diagnostic accuracy and acceptability of Xpert MTB/RIF Ultra (Xpert) using TS samples for tuberculosis detection among adolescents. Methods We conducted a cross-sectional diagnostic accuracy study with consecutive recruitment in Vietnam. Adolescents aged 10-19 who were recommended to undergo investigation for tuberculosis and had not received tuberculosis treatment in the past years were eligible. Participants provided TS and sputum samples and completed a structured survey regarding sampling experiences. TS was tested on Xpert, with sputum tested on Xpert and liquid culture. We utilised a composite reference standard of a positive result on sputum Xpert or sputum culture to define disease status. Sensitivity, specificity, and diagnostic yield were calculated for TS Xpert. Results From July to December 2025, we enrolled 225 adolescents from Can Tho and An Giang provinces in southern Vietnam. Fewer than half (96/225, 43%) the participants exhibited a tuberculosis -like symptom, and the majority (157/225, 70%) were close contacts of a person recently diagnosed with tuberculosis. TS were collected from all adolescents, while 116 (52%) could provide mucopurulent sputum. Tuberculosis prevalence was relatively low (12/225, 5.3%). TS Xpert sensitivity (90% CI) and specificity (90% CI) were 58.3% (35.6, 78.0) and 99.5% (97.9, 99.9), respectively. Diagnostic yield among all diagnosed was 58.3% (7/12). TS sampling was highly acceptable to adolescents; the short time and simplicity of collecting TS were considered favourably. Conclusions The sensitivity and diagnostic yield of TS Xpert was relatively low among adolescents recommended for tuberculosis investigation, which includes asymptomatic individuals who may not provide high quality sputum. Specificity was excellent, and everyone could provide a TS. TS high acceptability indicates it remains a promising sample for diagnostic algorithms.