Journal of Clinical Virology Plus

BackgroundNeonatal IgM antibodies reflect an in-utero immune response to Treponema pallidum and may offer added diagnostic value. This study evaluated the test performance of treponemal IgM levels measured by the research-use-only (RUO) DPP Syphilis TnT point-of-care (POC) assay for CS risk stratification. MethodsConducted from May 2023 to May 2025, this study tested neonatal serum samples from infants born to mothers with syphilis using the DPP Syphilis TnT RUO POC assay, which reports treponemal and nontreponemal IgM levels as relative light units (RLU). Neonates were classified as Confirmed Proven or Highly Probable CS, Possible CS, or CS Less Likely per guidelines; 23 neonates without maternal syphilis served as controls. Treponemal IgM levels were compared across categories using nonparametric tests and ordinal logistic regression. Diagnostic performance used prespecified cutoffs, with agreement assessed against neonatal RPR. ResultsTwenty-two maternal-neonatal dyads were included. Mean treponemal IgM levels rose with CS severity, peaking in the high-risk group (Possible or Confirmed Proven/Highly Probable CS: 29.9 {+/-} 20.6 RLU) versus CS Less Likely (17.5 {+/-} 20.8 RLU) and controls (3.5 {+/-} 0.8 RLU; p<0.05). Higher IgM levels independently linked to elevated CS risk (OR 1.10 per 1 RLU; p=0.0025). At [&ge;]10 RLU cutoff, treponemal IgM detected 88.9% of high-risk neonates, with 76% agreement to neonatal RPR. ConclusionThe DPP Syphilis TnT RUO POC assays treponemal IgM levels discriminated CS risk categories effectively and may supplement current algorithms to improve neonatal CS stratification.

Influenza H3N2 subclade K (J.2.4.1) is a genetic branch of H3N2 with 11 mutations in hemagglutinin and currently represents the dominant circulating influenza strain. We evaluated antibody responses to H3N2 subclade K before and after influenza vaccination in 46 healthy individuals. Our data show that baseline antibody responses to two H3N2 subclade K variants were lower than to other H1N1 and H3N2 strains and that antibody responses following vaccination were also less robust to the H3N2 subclade K variants. These data indicate that the H3N2 subclade K strain partially evades both prior H3N2 immunity and the current inactivated influenza vaccine.

Individuals with occupational exposure to swine may have disproportionate risk for zoonosis with swine influenza A virus (IAV). To evaluate human antibody responses, sera or plasma from swine veterinarians, swine farm employees, and the general population were tested by hemagglutination inhibition (HI) assays against representative swine and human seasonal influenza vaccine strains. HI data were analyzed by antigenic cartography to assess strain relationships, and reproduction number modeling to evaluate pandemic potential using age-stratified immunity profiles. Occupationally exposed groups had lower human seasonal vaccine uptake (45.5% vs 70%) and significantly lower odds of seropositivity to several H1 and H3 from swine compared to general population cohorts. One swine strain exhibited significant antigenic drift (3.62 AU) from its nearest vaccine strain. Multiple strains required lower R thresholds for pandemic spread (1.09-1.35) than recorded pandemic strains (1.46-1.80). This demonstrates that population immunity gaps heighten zoonotic risk to circulating swine H1 and H3 strains.

BackgroundNeutralising antibody titres are widely used as key immunogenicity endpoints in Ebola virus (EBOV) vaccine and monoclonal antibody clinical trials. However, direct comparison of results across studies remains challenging due to the use of heterogeneous neutralisation platforms, ranging from pseudotyped viruses to live EBOV assays. These limitations restrict assay standardisation, validation, scalability, and compliance with good clinical laboratory practice (GCLP), particularly in outbreak-prone and resource-limited settings. There is an unmet need for neutralisation assays that combine biological authenticity with clinical-trial compatibility. MethodsWe developed and optimised a fluorescence-based microneutralisation assay using a biologically contained EBOV lacking the essential VP30 gene (EBOV{Delta}VP30), enabling multi-cycle viral replication under containment level 2 conditions. Using a defined panel of serum samples from Ebola virus disease survivors and EBOV-negative controls, we benchmarked EBOV{Delta}VP30 neutralisation titres against previously generated data obtained with wild-type EBOV and pseudotyped virus platforms. Assay performance was evaluated in terms of sensitivity, reproducibility, discrimination between positive and negative samples, and correlation with live virus neutralisation. Calibration was performed using the WHO International Standard for anti-EBOV immunoglobulin. FindingsThe EBOV{Delta}VP30 microneutralisation assay robustly distinguished EBOV survivor sera from negative controls (p < 0{middle dot}0001) and demonstrated a strong correlation with live EBOV neutralisation titres (Spearman {rho} = 0{middle dot}8725). This correlation exceeded that observed for HIV-1-based pseudotyped assays and for the vesicular stomatitis virus-based platforms. The fluorescence-based read-out showed comparable sensitivity to conventional immunostaining, supporting its suitability for high-throughput and standardised implementation. Importantly, assay conditions were compatible with BSL-2 laboratories and GCLP-aligned workflows. InterpretationBiologically contained EBOV{Delta}VP30 provides a clinically relevant and scalable alternative to existing neutralisation platforms, bridging the gap between pseudotyped assays and wild-type virus testing. By improving biological relevance while maintaining accessibility and standardisation, this assay has the potential to enhance comparability of immunogenicity data across EBOV vaccine and therapeutic antibody (pre-)clinical trials, aligning with global outbreak preparedness and trial harmonisation objectives. FundingStated in acknowledgement section of manuscript. Research in contextO_ST_ABSEvidence before the studyC_ST_ABSBefore starting this study, we reviewed published work on how neutralising antibodies against Ebola virus are measured in vaccine and monoclonal antibody research. We searched PubMed, Web of Science, and reference lists of key review papers for studies published up to mid-2025, without restricting by language. Search terms included "Ebola virus", "neutralising antibodies", "neutralisation assay", "pseudovirus", "live virus", and "clinical trials". We focused on studies describing neutralisation tests using wild-type Ebola virus as well as commonly used pseudotyped virus systems. From this body of evidence, neutralisation assays using wild-type Ebola virus are considered the most biologically relevant but can only be performed in biosafety level 4 laboratories. This limits their availability, scalability, and use in clinical trials. Pseudotyped virus assays can be performed under lower biosafety conditions and are widely used, but multiple studies have reported variable performance and inconsistent agreement with live virus results. Although biologically contained Ebola viruses have been developed and used in laboratory research, their application as neutralisation assays and their direct comparison with both live virus and pseudotyped systems using the same human serum samples had not been systematically studied. As a result, it remained unclear whether such systems could support reliable immunogenicity assessment in clinical trials. Added value of this studyThis study shows that a biologically contained Ebola virus lacking the VP30 gene can be used to measure neutralising antibodies in a robust and scalable way under biosafety level 2 conditions. By directly comparing this system with wild-type Ebola virus and widely used pseudotyped assays using the same set of human serum samples, we demonstrate that neutralisation results obtained with the biologically contained virus closely align with those of the wild-type virus reference assay. The assay reliably distinguishes samples from Ebola survivors and uninfected individuals and can be read using different detection methods, making it compatible with GCLP-aligned workflows and suitable for further qualification and validation in support of clinical development. This work provides clear evidence that biologically contained Ebola virus can combine biological relevance with practical usability. Implications of all the available evidenceTogether with existing evidence, our findings indicate that biologically contained Ebola virus offers a valuable new option for measuring neutralising antibodies in vaccine and monoclonal antibody clinical trials. By reducing reliance on high-containment laboratories while preserving key features of authentic virus infection, this approach can improve the consistency and comparability of immunogenicity data across studies and sites. Broader use of such assays could support better decision-making during clinical development and strengthen outbreak preparedness. More generally, this work highlights how biologically contained viruses can help advance research licensure of medical countermeasures for high-consequence pathogens in ways that are directly relevant to human health.

A multivariant total subclass analysis has been performed for a control cohort (n=15) and a long COVID patient cohort (n=15) measuring the IgG1, IgG2, IgG3, IgG4 and IgE response to the following 14 variants of SARS-CoV-2: Wuhan, Alpha, Delta, BA.1, BA.2, BA.5, EG.5.1, XBB.1.5, BA.2.75, CH.1.1, BA.2.12.1, BQ.1.1, JN.1, and KP.3. Significant differences (p < 0.05 and p < 0.005) between concentrations of IgG subclasses by variant were found in 24% of variants and in mean-normalised distributions. The medians of the mean-normalised distributions were significantly lower for IgG1 (p < 0.05) in long COVID patients compared with controls, and significantly higher (p < 0.005) for levels of IgG3, IgG4 and IgE for long COVID patients. A preliminary diagnostics classification analysis performed by variation of the mean-normalised upper and lower percentiles symmetrically for IgG3 showed a long COVID diagnostic sensitivity of 80%, and specificity of 80% for the 60th percentile threshold of the control cohort. Three types of long COVID can be identified: patients with at least one variant below the threshold (hypo-immune), patients with at least one variant above the threshold (hyperimmune) and patients with IgG3 levels within the reference range. The multivariant subclass spectrum indicates IgG4 and IgE elevations due to potential chronic antigen exposure from persistent virus or autoimmunity and may indicate potential therapeutic interventions.

Over-the-counter (OTC) lateral flow tests for respiratory viruses have newly emerged since the beginning of the coronavirus disease 2019 (COVID-19) pandemic and are increasingly available to consumers. While all marketed tests have met standards for Emergency Use Authorization (EUA), De Novo classification or 510(k) clearance, limited data are available to inform consumers of their relative performance. We performed a head-to-head benchtop analytical assessment of OTC tests available for purchase from retailers in the United States in the fall of 2025. Contrived specimen dilution panels were prepared from propagated viral stocks of influenza A H1N1pdm09, influenza A H3N2, influenza B (Victoria lineage), and SARS-CoV-2 JN.1 (Omicron variant) in clinical matrix and applied to the swab provided with each kit. Tests were subsequently performed according to the manufacturers instructions for use and results were interpreted by two readers blinded to the starting test material. Lower limits at which 3 of 3 replicates of test material were detected were within a 4-fold dilution for all four viruses and all eight OTC tests evaluated. We found that at low viral concentrations many OTC tests were interpreted as negative at the start of the stated interpretation window but converted to a positive result by the end of the interpretation window. We conclude that eight OTC tests that are currently readily available to consumers perform similarly in a contrived specimen analytical study, but we recommend that users of OTC lateral flow tests allow for the full incubation time before concluding that a test is truly negative.

BackgroundImmunocompromised (IC) individuals are at increased risk for persistent SARS-CoV-2 infections and can develop new viral mutations and lineages not seen in the community. In this case report, a persistent SARS-CoV-2 infection (330 days) in an IC patient is examined for viral mutations and mutations associated with cryptic lineages. Case PresentationThe patient was followed in a longitudinal study examining persistent SARS-CoV-2 in IC patients. The patient provided stool and nasal swab samples biweekly until 28 days post-enrollment, then monthly, and then quarterly after 12 month post enrollment until the participant was no longer positive for SARS-CoV-2. Staff performed RT-qPCR and viral sequencing on the samples. Viral mutations from the XBK lineage were already present in the initial sample. By the end of the infection period, there were 40 fixed consensus changes from XBK of which two mutations were typical for cryptic lineages. Mutations increased steadily over time, with most mutations fixed by day 253, including the cryptic typical mutations. ConclusionThis case demonstrates the potential for persistent SARS-CoV-2 infections to develop mutations and lineages in IC patients and highlights the need for continued SARS-CoV-2 monitoring and treatment in this vulnerable population.

Influenza A, Influenza B, SARS-CoV-2, Respiratory Syncytial Virus and other respiratory pathogens are an ongoing public health concern. The ability to rapidly identify these viruses early in infection is essential for effective treatment and outbreak control. The AMDI Fast PCR Mini Respiratory Panel (MRP) incorporates sample preparation and real-time RT-PCR for detection of Flu A, Flu B, SARS-CoV-2 and RSV from anterior nasal swab (ANS) specimens in less than 10 minutes at the point of care. We established the analytical performance characteristics of the Fast PCR MRP and determined that the limit of detection (LoD) is 250 copies/mL for Flu A and RSV, and 500 copies/mL for Flu B and SARS-CoV-2. In a reproducibility study at 3 clinical sites, there was at least 98.2% positivity for each target for a weak positive (2x LoD) sample, at least 99.6% positivity for a moderate positive (5x LoD) sample and the negative sample returned a negative result for at least 99.3% of the tests. Fast PCR MRP had 100% analytical reactivity to all strains tested (23 Flu A, 6 Flu B, 8 SARS-CoV-2 and 6 RSV) and at least 98% predicted inclusivity from in silico analysis. There was no cross-reactivity to 40 viruses, bacteria and fungi, nor interference for 15 endogenous and exogenous substances in ANS matrix. The Fast PCR MRP delivers excellent analytical performance comparable to high complexity laboratory assays, at the point of care.

IntroductionHemophagocytic lymphohistiocytosis (HLH) is a serious condition induced by Dengue virus which becomes fatal if not detected early and treated appropriately. So objectives of the present study are to observe the different patterns of presentations, clinical features and outcome of HLH induced by Dengue. MethodsIn this observational study, 14 patients admitted and diagnosed HLH as per diagnostic criteria, were included after informed written consent. Study conducted in a period of six months from 01/07/2025 to 31/12/2025. All patients were followed up till discharge. After collection, all data were analyzed by Microsoft Excel 2010. Ethical clearance was taken from Ethical Review Board of the Medical College. ResultsAmong 14 cases, male were more affected then the female (78.6% VS 21.4%) and majority were in between 20 to 50 years age groups. Clinical data showed, all 14 cases had fever for >7 days, joint pain 3(21.4%), headache 11(78.6%), skin rashes 10(71.4%), retro-orbital pain 2(14.3%), vomiting 11(78.6%),bleeding 10(71.4%), cough 4(28.6%), loose motion 9(64.3%), abdominal pain 7(50.0%), anorexia 2(14.3%), Melaena 2(14.3%), jaundice 4(28.6%) and spleenomegaly 9(64.3%). One(7.1%) case had history of Hypertension. Laboratory data showed different level of Bi or Pancytopenia, high ferritin, high TG, low fibrinogen, raised liver enzymes and low sodium. Dengue RT PCR and serology results showed 8(42.9%) cases were both IG M and Ig G dengue antibody positive, 6 cases were RT PCR positive, 2 cases were IgM and another 4 cases were IgG positive. Outcome of patients revealed, among all 14 cases12(85.8%) patients improved uneventfully and 2 were shifted to ICU where one improved and one died. ConclusionDengue is prevailing for long time and different complications are evolving and HLH is a relatively newer incident among the dengue patients. Infection by different serotypes at different time or multiple dengue serotype infection may be related with HLH and it might be a future subject to explore and to evaluate.

Inter-alpha Inhibitor Proteins (IAIP) are serine protease inhibitors and a reliable inflammatory biomarker. We have demonstrated that IAIP levels decrease during sepsis in humans and animal models. Currently, enzyme-linked immunosorbent assay (ELISA) is the standard procedure to measure IAIP levels. We developed a lateral flow immunoassay (LFIA) that allows rapid, point of care detection. We compared IAIP levels in infants with sepsis and/or necrotizing enterocolitis (NEC) by ELISA and LFIA in a multi-center, cross-sectional study. Blood samples were collected from 47 infants with sepsis, 31 sepsis case controls, 52 gestational age (GA)-matched controls and 10 infants with culture-negative sepsis ("clinical sepsis"). We also collected samples from 17 infants with NEC, 7 NEC case controls and 15 GA controls. IAIP levels at presentation of acute events and over the next 72 hours were significantly reduced in infants with sepsis, NEC and culture negative sepsis when compared to controls. IAIP levels did not differ in patients with sepsis or culture negative sepsis. IAIP levels measured by ELISA and LFIA were highly correlated (R2 = 0.9326) and both showed reliable detection of neonatal sepsis, NEC and culture negative sepsis. IAIP levels were 80.0% sensitive and 92.3% specific using LFIA for the detection of neonatal sepsis. For detection of NEC, IAIP levels were 84.6% sensitive and 86.7% specific.

BackgroundAccurate and comparable human papillomavirus (HPV) testing is essential for HPV vaccine research, HPV surveillance, and cervical cancer screening. In 2008, the WHO HPV Laboratory Network initiated global HPV proficiency testing traceable to International Standards (IS), which has been issued regularly since. Here, we summarize recent results and trends over time. MethodsThe HPV genotyping panel, used since 2008, consists of 43 blinded samples, including HPV 6, HPV 11, all oncogenic and vaccine-targeted HPV types, as well as extraction controls. A smaller screening panel, launched in 2022, includes 13 blinded samples designed for cervical screening needs. Laboratories worldwide test the panels using their own methods, and results are centrally evaluated at the International HPV Reference Center. Proficiency has hitherto been defined as absence of false positives, detection of HPV16/18 at 10 IU/{micro}L, and of other oncogenic types at 100 IU/{micro}L (genotyping) or 1000 IU/{micro}L (screening). ResultsParticipation in the genotyping panel increased from 54 laboratories in 2008 to >130 in 2021. Proficiency rose from [~]25% in early years to >80% by 2024, with >99% correct detection for most genotypes. Optional low-copy challenges (e.g., 1 IU/{micro}L HPV16/18) were detected by >95% of laboratories by 2024. Screening panel participation increased from 84 laboratories in 2022 to 132 in 2024, with overall proficiency improving from 77% to 95% and >96% datasets free of false positives. ConclusionsThe global HPV proficiency program enables global laboratory quality assurance. Annual proficiency testing is essential for high-performance HPV testing and evaluation supporting cervical cancer elimination. summaryGlobal HPV proficiency program traceable to international standards shows marked improvement from 2008-2024. Participation expanded worldwide, false positives decreased, and most HPV types reached near-universal detection. Annual proficiency testing supports HPV research, surveillance, screening and evaluation of novel assays.

BackgroundThe epidemiology of suspected pediatric meningoencephalitis has shifted in the era of conjugate vaccines and multiplex PCR diagnostics, with viral pathogens now predominating over bacterial causes. Updated epidemiologic data are essential to adapt diagnostic and therapeutic algorithms to current clinical practice. MethodsThis retrospective single-center study included children and adolescents <18 years who underwent lumbar puncture with cerebrospinal fluid multiplex PCR for suspected central nervous system infection at a tertiary-care pediatric hospital in Germany between 2016 and 2024. Clinical, laboratory, and outcome data were extracted from electronic medical records. Cerebrospinal fluid was analyzed using the BioFire(R) FilmArray(R) Meningitis/Encephalitis Panel. Statistical analyses included descriptive statistics, nonparametric group comparisons, receiver operating characteristic analyses. ResultsAmong 1,198 included children, definite bacterial meningitis was diagnosed in 13 (1.1%), definite viral meningitis in 80 (6.7%), aseptic meningitis of unknown etiology in 131 (11.0%), confirmed/probable encephalitis in 53 (4.4%), and possible encephalitis in 34 (2.8%). Bacterial meningitis accounted for 5.8% of all meningitis cases. A causative pathogen was identified in all bacterial meningitis cases, most commonly Streptococcus pneumoniae (n = 7). Enterovirus (n = 52) and parechovirus (n = 9) predominated in viral meningitis, whereas an infectious etiology was identified in only 13 of 53 confirmed/probable encephalitis cases. The Bacterial Meningitis Score showed a sensitivity of 80.0% and a specificity of 57.6%. The recently published UK-ChiMES-pre- and post-lumbar puncture scores demonstrated sensitivities of 84.6% and 76.9% and specificities of 86.3% and 92.7%, respectively. DiscussionBacterial meningitis was rare in this contemporary cohort, while viral and etiologically unresolved infections predominated despite routine multiplex PCR diagnostics. Clinical prediction scores supported risk stratification, with the UK-ChiMES-pre-lumbar puncture score showing the most favorable balance between sensitivity and specificity and potential to guide diagnostic decisions and antiinfective therapy.

A number of vaccines and long-acting monoclonal antibodies have been shown to be effective in the prevention of respiratory syncytial virus (RSV) disease. However, an immune correlate of protection for RSV has not yet been identified. We conducted a systematic review to identify published reports of immunogenicity and/or efficacy in vaccines and long-acting monoclonal antibodies against RSV and performed a meta-analysis on extracted data to identify any relationship between antibody increase and protection against RSV disease. We identified 130 relevant reports which we classified into an open access evidence map of RSV immunisation products. We found a strong correlation between the immunisation induced rise in neutralising antibody titres and efficacy ({rho}>0.7 for all comparisons, Spearman). For infants, we estimated that each 10-fold increase in neutralising antibody titre rise provides an additional 31% [95% CI 10%-47%], 47% [95% CI 36%-56%] and 57% [95% CI 45%-66%] reduction in the relative risk of symptomatic, moderate and severe disease respectively. For older adults, a 10-fold rise in antibody levels was associated with a 34% [95% CI -2%-57%], 50% [95% CI 22%-67%] and 63% [95% CI 36%-79%] reduction in the relative risk of RSV disease with 1, 2 and 3 symptoms respectively. These results align extremely well with findings from natural history studies and individual-based analysis of correlates of protection studies. This work paves the way for use of neutralising antibodies as a correlate of protection to guide the development, approval, and deployment of RSV vaccines and monoclonal antibodies.

In the past decade, dengue fever has emerged as a major public health on Reunion Island in the Southwest Indian Ocean. During the 2018-2022 outbreak, an unusual increase in ocular complications was reported in some patients. To investigate a potential viral cause, we analysed 447 blood samples from hospitalized patients with and without ophthalmic symptoms. Genetic sequencing revealed the co-circulation of two strains of dengue virus serotype 1, both genetically linked to strains previously identified in Asia. Notably, all patients with ophthalmic symptoms were infected with viruses from a single cluster within genotype I, which harbored several unique mutations. These findings suggest that the rare ocular complications observed during this outbreak may be associated with specific viral cluster. Further laboratory studies are required to confirm this potential link.

Microbial keratitis is a sight-threatening corneal infection with varying etiological agents, primarily bacteria and fungi. Assessing and contrasting the virulence factors of microorganisms isolated from a non-contact lens-associated keratitis (NCLAK) and contact lens-associated keratitis (CLAK) is the goal of the current investigation. Samples were collected from over 60 patients and analysed using standard microbiological techniques, including culture, Gram staining, KOH mount, biochemical tests, antimicrobial susceptibility testing, and biofilm assays. The results demonstrated that CLAK isolates were predominantly bacterial, especially Pseudomonas aeruginosa, known for strong biofilm production and high multidrug resistance. In contrast, NCLAK showed a higher incidence of fungal infections, particularly Candida albicans. The results highlight the significance of early diagnosis, tailored and improved awareness regarding contact lens hygiene to prevent complications associated with keratitis.

BackgroundAntibiotic stewardship during stem cell transplantation (SCT) is challenging.. Procalcitonin (PCT) has been employed successfully in critical care patients to safely guide stewardship. However, procalcitonin guided stewardship has not been robustly assessed in SCT recipients. We sought to evaluate the potential utility of PCT to guide antimicrobial de-escalation during engraftment. Methods100 SCT patients were prospectively enrolled in a "strategy trial" and had infectious complications documented. Lab parameters - CBC, BMP, CRP were obtained daily as standard of care (SOC) while PCT was obtained for research purposes. Providers were blinded to PCT results. We compared duration of antimicrobial escalation between actual events (SOC model) and a proposed PCT model. In this hypothetical PCT model, antibiotic de-escalation would occur if CRP remained <100 mg/dl and PCT <0.25 ng/ml after 3 days of escalation. Escalation events were defined as a substitution or addition of an antimicrobial agent after initiation of prophylactic antimicrobials. Results77 patients had escalation events and of these, 33 had bacterial infections. A total of 136 antimicrobial escalations events were identified, and of these only 39(28.7%) were associated with documented infections. The standard of care model had a mean duration (+SD) of 9.08 (+ 6.08) antibiotic days. If the PCT model were employed, the mean duration (+SD) would be 4.44 (+ 6.16) days (p<0.001). The PCT model, however, would have missed 11 infections\ ConclusionProcalcitonin-guided antimicrobial stewardship during autologous stem cell transplantation is feasible however optimization is necessitated for utilization as a tool to guide antibiotic prophylaxis during SCT.

BackgroundWe previously reported that nasal SARS-CoV-2 viral loads (VL) peaked around the fourth day of symptoms in highly immune adults sampled April 2022 - April 2023, while influenza A VL peaked soon after symptom onset. We hypothesized that SARS-CoV-2 kinetics may have changed due to reduced COVID-19 incidence and altered vaccination patterns. Understanding how viral kinetics evolve over time is essential to inform testing strategies. MethodsParticipants with symptomatic upper respiratory infection were recruited at testing centers in Georgia April 14, 2023-April 11, 2025. Participants reported date of symptom onset and vaccination history. A nasal swab was tested on the Xpert(R) Xpress CoV-2/Flu/RSV-Plus assay and Ct values recorded. Results552 adults ([&ge;]16 years) and 60 children (<16y) were SARS-CoV-2-positive; lowest median Ct values (highest VL) were observed on the 2nd symptomatic day. Adults vaccinated within 12M (n=106) had peak VL on the 3rd day, and those vaccinated [&ge;]12M prior (n=334), on the 2nd. In influenza A-positive adults (n=181) and children (n=147), median VL peaked on the 1st symptomatic day, versus the 4th day in 157 influenza B-positive participants. ConclusionsIn 2023-25, median SARS-CoV-2 VL peaked earlier relative to symptom onset compared to a highly immune 2022-23 cohort. More recent vaccination correlated with delayed peak VL, suggesting more robust immunity correlates with earlier symptom onset relative to peak. Median influenza A VL were highest at symptom onset, versus 4 days into symptoms for influenza B. These findings can inform use of multiplexed antigen tests amidst changing immunity and viral circulation patterns. Summary PointIn 2023-25, median nasal SARS-CoV-2 viral loads peaked around the second day of symptoms, versus the fourth day in a 2022-23 highly immune cohort. Median influenza A viral loads peaked soon after symptom onset, versus on the fourth symptomatic day for influenza B. These observations have implications for multiplexed testing.

Influenza A(H3N2) subclade K virus was detected in Canada early in the 2025/26 influenza season, bearing an antigenic transition in the hemagglutinin (HA) glycoprotein. Analysis of 396 HA sequences from Canada showed antigenic divergence from 2025/26 influenza vaccine strains, consistent with partial mismatch. Phylodynamic analysis revealed sustained pre-vaccine transmission without clear post-vaccine expansion. Phylogenetic and phylogeographic analyses indicated interprovincial mixing within a highly connected metapopulation, highlighting the value of genomic surveillance for real-time epidemiologic inference and public health decision-making.

IntroductionDengue has been prevalent in a regular fashion in Bangladesh and Chattogram for the last 6-7 years and is showing some serotype twisting. So, the objectives of the present study were to explore the burden of dengue serotypes in Chattogram. MethodsIn this study, 223 Dengue RT-PCR positive patients were evaluated for serotyping. Gender and age group, along with cycle threshold (CT) values, were also collected. Data after collection were compiled, analyzed, and plotted in Microsoft Excel and GraphPad Prism 10.4. Ethical clearance was taken to conduct the study. ResultsAmong 223 patients analyzed, males and females were found near equal (113 and 110). Middle-aged patients were more than the extremes of age. The mean {+/-} SD of age was 33.55 {+/-} 13.67 years. Regarding serotype distributions, isolated Den 1, Den 2 and Den 3 were found 1.3%, 73.1% and 6.7%, respectively. Concurrent infections with multiple serotypes were observed in several patients, most notably the Den 2 and Den 3 combination, which accounted for 14.3% (n=32) of the cases. Other co-infections were less frequent: the Den 1 and Den 2 pairing appeared in 3.6% (n=8) of the cohort, while triple-serotype infections (Den 1, 2, and 3) and Den 3/Den 4 pairings were rare, each occurring in only 0.4% of patients. Statistical analysis of CT values revealed no significant sex-based differences for Den 2 and Den 3. However, significant variations in CT values were observed when comparing Den 1 against both Den 2 and Den 3 (p < 0.05). In contrast, the difference between Den 2 and Den 3 Ct values remained statistically insignificant. ConclusionIn the year 2025, Dengue serotypes 2 and 3 were found to be the most prevalent, both in isolated or in combinations and Den 1 and Den 4 were found minimum. Exposure to multiple serotypes and twisting from one serotype to another might influence the dengue outcome in future, which needs further exploration.

BackgroundsFollowing an increase in mpox clade Ib cases in several African countries, the World Health Organization recognized mpox as a public health emergency of international concern, highlighting the need for reliable and accessible diagnostic tools. As several mpox clades are co-circulating in endemic countries, the analytical sensitivity of diagnostic assays should be evaluated for all of them in a comparative manner. MethodsThis study evaluated the analytical sensitivity of three rapid tests (Flowflex Monkeypox virus rapid test from ACON Biotech, Monkeypox antigen rapid test from Assure Tech, and Standard Q Monkeypox Ag Test from SD Biosensor) and two point-of care molecular tests (SD Biosensors M10 and Cepheids Xpert MPOX assays) using serial dilutions of viral stocks from the four clades of Monkeypox virus (MPXV): Ia, Ib, IIa, and IIb. FindingsUpon our analytical comparative benchmarking, the three rapid tests demonstrated comparable analytical sensitivity for all four clades, with a limit of detection of approximately 1,000,000 DNA copies/mL or 1,000 FFU/mL. The two molecular tests demonstrated also comparable analytical sensitivity to an in-house PCR assay for all four clades, detecting concentrations down to 10-100 DNA copies per mL, corresponding to a non-detectable titer of infectious particles. The Xpert assay detected the Clade Ib strain only through its orthopoxvirus target and not its MPXV target, and none of the assays could distinguish between MPXV clades. InterpretationNo differences in sensitivity was observed across MPXV clades neither for Ag-RDTs nor for molecular POCTs. However, the potential of simple, affordable tests, such as Ag-RDTs, is limited by poor sensitivity while the use of highly sensitive POC molecular platforms remains limited by their cost. To date, the lack of accurate, affordable POC MPXV-specific assays with potential to differentiate clades, limits diagnostic capacities as well as our understanding of the virus and its epidemiology. FundingThis work was supported by FIND and internal funds of the Centre for Emerging Viral Diseases. Mpox diagnostic tests were provided by FIND and the World Health Organization (WHO). MOD was supported by Schmidt Science Fellows, in partnership with the Rhodes Trust.