Journal of Clinical Virology

Mpox poses an ongoing global public health threat, with case numbers rising beyond traditionally endemic regions in Central and Western Africa. Rapid detection of the causative agent, the Monkeypox virus (MPXV), is critical for outbreak control, yet laboratory infrastructure and trained personnel remain scarce in many affected areas. Point-of-care molecular diagnostics offer a practical solution by enabling timely testing without specialized equipment or elaborate nucleic acid extraction. We evaluated the performance of an extraction-free RNase HII-assisted amplification (RHAM) assay for MPXV detection by Pluslife Biotech, a novel isothermal amplification technology providing results in under 30 minutes. The Pluslife RHAM test demonstrated pan-MPXV clade reactivity, detecting all four MPXV clades (Ia, Ib, IIa, IIb) with high analytical sensitivity and no cross-reactivity to other poxviruses or other clinically relevant pathogens. The assay proved compatible with diverse clinical specimen types, including lesion swabs, oropharyngeal swabs, rectal swabs, urine, semen, and wound exudate. As part of routine diagnostics at the German Consultant Laboratory for Poxviruses, in a comprehensive evaluation of 206 clinical specimens against diagnostic real-time PCR, the Pluslife RHAM test achieved a diagnostic sensitivity of 94.2% (95% CI: 85.8-98.4%) and a specificity of 100% (95% CI: 97.3-100%). Notably, samples with higher viral loads (Ct <30) showed 100% sensitivity. Time-to-result correlated significantly with viral load, enabling faster diagnosis in high-viral-load cases. The Pluslife RHAM test represents a practical, sensitive, and rapid point-of-care solution for MPXV detection in resource-limited settings, combining strong analytical performance with operational simplicity to support timely outbreak response and clinical decision-making.

The rapid diagnosis of Campylobacter infections is important for the management of infectious gastroenteritis. Although stool culture is considered the gold standard, its sensitivity is limited and it requires prolonged incubation times. We performed a prospective multicenter study at nine healthcare facilities in Japan to evaluate a Campylobacter rapid antigen test using stool specimens between March 2024 and August 2025. Patients with suspected infectious gastroenteritis were consecutively enrolled and tested using QuickNavi-Campylobacter and compared with the FilmArray Gastrointestinal Panel as the reference method. Discordant results were further evaluated by culturing and additional PCR assays. In total, 410 patients were included in the final analysis. The positive, negative, and total concordance rates between QuickNavi-Campylobacter and FilmArray Gastrointestinal Panel were 79%, 99%, and 93%, respectively. The positive concordance rate decreased in specimens collected [&ge;] 6 days after the onset of symptoms (50%). QuickNavi-Campylobacter demonstrated relatively good concordance with the FilmArray Gastrointestinal Panel in a real-world multicenter setting. These results suggest that this rapid antigen test may be particularly useful for the early diagnosis of suspected campylobacteriosis.

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.

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.

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.

BackgroundClinical metagenomics (CMg) offers high-throughput respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Sequencing cost and the challenges of high host biomass in non-invasive samples are barriers to the use of CMg in high-throughput respiratory pathogen detection. MethodsWe optimised a long-read sequencing workflow to detect RNA viruses in nasopharyngeal swabs, employing pathogen enrichment and ONT sequencing. As a pre-requisite for agnostic pathogen detection, we first derived quality control (QC) criteria and diagnostic thresholds against a gold-standard comprising 23 pathogen targets detected by routine multiplex PCR. We validated this workflow using 344 prospectively collected upper respiratory tract samples submitted for routine testing. FindingsUsing pre-defined QC and positivity criteria, the workflows sensitivity versus PCR was 51% (95%CI: 45%-57%) (133/260 positive targets detected) (ranging from 19%-85% across pathogens with >20 gold-standard detections), and specificity 99.8% (95%CI: 99.6%-99.9%) (3836/3845 negative targets not detected). Sensitivity improved to 58% using post-hoc optimised thresholds, 61% only considering RNA pathogens, 70% excluding rhinovirus/enterovirus and 83% excluding samples with qPCR Ct values [&ge;]35. Read crossover from multiplex sequencing contributed most (7/9) false-positives: only 2 plausible additional pathogens were identified (rhinovirus and coronavirus OC43). 41 respiratory syncytial virus (RSV), 13 influenza A and 10 rhinovirus/enterovirus were successfully sub-typed by sequencing. Multiplexed nanopore sequencing costs were {pound}112/sample. InterpretationAlthough CMg has substantial diagnostic potential, this validation study demonstrates the technical limitations of current metagenomic sequencing methods applied to viral detection in upper respiratory tract samples with high host and low pathogen biomass. Its greater sensitivity at higher viral loads demonstrates the importance of identifying the most appropriate use cases to maximise its utility and value. FundingNational Institute for Health and Care Research Oxford Biomedical Research Centre. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSClinical metagenomics (CMg) provides the potential for rapid respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Searching PubMed for studies published between Jan 1 2018 and December 1 2025 using the terms respiratory tract infection AND metagenomics AND diagnosis, with no language restrictions yielded 734 items. We identified 15 clinical studies assessing the diagnostic performance of a metagenomic workflow for respiratory samples. Only 3 of these included upper respiratory tract samples (nasopharyngeal swabs); the remaining studies exclusively investigated invasive samples from the lower respiratory tract (bronchioalveolar lavage fluid). One of the three relevant studies assessed detection of viral pathogens in 83 specimens from critical care patients as part of pan-microbial nanopore sequencing metagenomic assay; only 2 samples included were nasopharyngeal samples. The second validated a high-depth short-read CMg workflow in 191 samples (85% nasopharyngeal), reporting 93.6% sensitivity and 98.8% specificity across eight respiratory pathogens detected by respiratory PCR. This workflow required substantial equipment and sequencing costs. A third study validated a nanopore sequencing CMg workflow, with lower laboratory footprint and sequencing costs than the second study. In 359 nasopharyngeal samples, they reported 61% sensitivity and 100% specificity against four respiratory viral targets detected by PCR. The costs of CMg mean that routine deployment as a high-throughput test with broad use will require demonstrating good performance across a range of common respiratory pathogens (such as can be assayed with commercially available extended multiplex PCR testing) and in upper respiratory tract samples. Added value of this studyThis prospective study is the first to evaluate nanopore sequencing for detecting a broad range of common respiratory viruses in nasopharyngeal samples. We validated a CMg workflow employing a modernised SISPA step for viral amplification, in 344 prospectively collected non-invasive samples, from which 19 different respiratory pathogens were detected by routine PCR testing. Using pre-defined bioinformatic thresholds overall sensitivity was 51% compared with PCR. This could be improved to 83% by limiting analysis to RNA viruses with Ct <35 and using post-hoc exploratory bioinformatic criteria. Sequencing costs were 45% lower than short-read sequencing. Implications of all the available evidenceOur results demonstrate rigorous bioinformatic thresholds are essential in multiplexed CMg sequencing to reduce false-positive detections, a particular danger with imperfect barcode de-multiplexing. However these thresholds impair true-positive detection in samples with a high ratio of host-to-pathogen biomass. Further research should focus on identifying in which samples and clinical settings CMg can offer greater value in patient care.

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.

BackgroundMpox has spread to 35 countries in Africa, yet many face challenges achieving nationwide PCR-based testing due to cost and limited access in remote and rural areas. Point-of-care antigen-based rapid diagnostic tests (AgRDTs) may help improve diagnostic access. ObjectivesTo evaluate the diagnostic accuracy of five mpox AgRDTs manufactured by Beijing Hotgen Biotech (China), Contipharma (Belgium), Hangzhou Testsea Biotechnology (China), Guangdong Wesail Biotech (China), and NG Biotech (France). MethodsDiagnostic accuracy was assessed using 190 lesion swabs from suspected mpox cases in the Democratic Republic of the Congo, with results compared against the RADI FAST Mpox PCR assay (KH Medical, South Korea). Analytical sensitivity was evaluated using a clade Ib MPXV isolate from the WHO Biohub held by the Geneva University Hospitals, Switzerland. ResultsThe best-performing assay, the Monkeypox Virus Ag Test Kit (Guangdong Wesail Biotech), demonstrated a sensitivity of 77.3% (95% CI: 68.0-84.5) and specificity of 93.5% (95% CI: 86.6-97.0). The Hangzhou Testsea assay showed comparable performance (sensitivity 72.2%, specificity 93.5%). Beijing Hotgen and Contipharma assays exhibited moderate sensitivity (59.8% and 50.5%, respectively) with high specificity (96.8% and 95.7% respectively), while the NG Biotech assay showed the lowest sensitivity (39.2%) but similarly high specificity (96.8%). Analytical testing revealed no major differences across assays, though Guangdong Wesail demonstrated the highest analytical sensitivity, detecting clade Ib virus at a Ct of 28.3. ConclusionSeveral AgRDTs show high positive predictive values for mpox screening in high-prevalence settings, where positive test results may support confirmation but negative results cannot rule out infection. Further multicentre prospective studies are needed to define appropriate use cases as countries transition to sustainable mpox control. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAccess to decentralized mpox diagnostics remains limited in many African countries due to the cost, infrastructure, and turnaround time associated with PCR, particularly in remote settings. Antigen-based rapid diagnostic tests (AgRDTs) could expand access to point-of-care testing, but data supporting their clinical performance has been limited. We searched PubMed and medRxiv on Dec 31, 2025, using the following search string: (mpox OR monkeypox) AND (diagnostic* OR point of care OR lateral flow assay OR rapid antigen test OR AgRDT). Early evaluations of mpox AgRDTs reported very low sensitivity. A large prospective, multicentre field study in Uganda and the Democratic Republic of the Congo (DRC) evaluating a single mpox AgRDT for research use only using lesion swabs reported an overall sensitivity of 70.4%, with substantial variation by country (81.9% in Uganda vs 55.1% in DR Congo), age, and viral load, and specificity of 89.3%. Independent comparative evaluations of multiple commercially available AgRDTs with regulatory approval--particularly in clade I-endemic African settings--have been lacking. Added value of this studyThis study provides the first head-to-head clinical and analytical evaluation of five commercially available mpox AgRDTs using skin or mucosal lesion swab specimens from suspected cases in DRC. By assessing all assays against a molecular reference test under identical conditions and incorporating analytical testing with a clade Ib reference virus isolate, we demonstrate substantial variability in performance between manufacturers tests kits. Two assays achieved sensitivity against PCR of above 70% with moderate specificity (93.5%), comparable to or exceeding results reported in prior field studies, while others showed moderate to poor sensitivity. Analytical testing identified meaningful differences in detection limits, with the best-performing assay detecting clade Ib virus at viral loads corresponding to a Ct value of 28.3. These findings provide critical data to inform further field-based validation projects, assay selection and procurement decisions for use of such AgRDTs in endemic settings. Implications of all the available evidenceAcross studies, mpox AgRDTs consistently demonstrate high specificity but variable and often insufficient sensitivity to function as standalone diagnostic tools. Prior studies have provided evidence that sensitivity is highest for samples with Ct values 25 or lower, and the present comparative evaluation show that positive AgRDT results may be able to reliably confirm mpox infection in high-prevalence settings, whereas negative results cannot safely exclude disease. The heterogeneity in performance between assays underscores the need for field-based independent evaluations of most promising tests before their large-scale deployment, and cautions against treating AgRDTs as a uniform diagnostic test category. As countries transition from emergency response to sustainable mpox control, AgRDTs may have a complementary role in decentralized screening, outbreak confirmation, and triage, provided confirmatory PCR remains available. Further multi-centre prospective studies and continued assay optimization are essential to ensure antigen-based diagnostics can be used to meaningfully strengthen mpox surveillance, preparedness and outbreak response.

Severe neonatal hyperbilirubinaemia represents a considerable cause of mortality and long term-morbidity in neonates born in low resource settings. Early identification of risk factors, such as glucose-6-phosphate dehydrogenase (G6PD) status, has the potential to prevent severe hyperbilirubinaemia and improve the clinical outcomes. The primary aim of the study was to assess equivalency of cord blood and neonatal capillary blood for diagnosis of G6PD deficiency using the quantitative point-of-care "STANDARD G6PDTM" test (SD Biosensor, Korea). Additional secondary aims were to compare the "STANDARD G6PDTM" with gold standard spectrophotometry and to analyse changes in G6PD activity in the first 4 months of life. A total of 75 neonates born in Shoklo Malaria Research Unit (SMRU) clinics were selected based on their G6PD status assessed through routine cord blood screening using the "STANDARD G6PDTM" test. Using activity thresholds established before in this setting, 25 G6PD deficient, 25 G6PD intermediate and 25 G6PD normal neonates were identified and re-tested on capillary blood collected within 24 hours of life and at day 7. They were also followed-up at 1 and 4 months of age to study haematologic and G6PD activity changes over time. The results showed that the "STANDARD G6PDTM" can be used reliably up to one week of life for testing neonates using the same thresholds established in cord blood. Performance of the point-of-care test as compared to the gold standard spectrophotometry remained excellent at all sampling time-points. Nevertheless, G6PD activity assessed longitudinally in the same participants decreased over time, both at 1 month of age and at 4 months of age, and interpretation of results in female infants with intermediate activity might require different thresholds. The study demonstrated that the "STANDARD G6PDTM" can effectively support clinical care in neonates and infants in populations with prevalent G6PD deficiency at the primary care level and especially in low-resource settings.

ObjectivesThe aim of this study was to examine the prevalence of congenital HIV infection at the Neonatal Centre of Excellence Unit (NCOE), Children Division of the University Teaching Hospitals (UTH), and to analyze the characteristics of neonates who tested positive and negative for HIV PCR. Subject and methodsThis study is a pilot investigation that analyzed retrospective cross-sectional data from 757 mother-neonate pairs. The data was collected over a 12-month period from the ward register and file records at the NCOE Unit, UTH - Childrens Division in Lusaka, Zambia. The prevalence and characteristics of HIV among hospitalized neonates were assessed using percentages, Chi-square tests, ANOVA, and binary logistic regression model. The results were reported in terms of p-values, odds ratios, and 95% confidence intervals. ResultsIn 2024, the annual rates of HIV and Syphilis among all neonatal admissions at NCOE were recorded at 4.1% and 5.9%, respectively. The HIV status for 52 neonates (6.9%) was not available. The median age of the neonates at the time of admission was 14 days, with an interquartile range (IQR) of 9 to 21 days. The maternal HIV positivity rate was 11.9%, while the paternal rate was 7.3%. Notably, a greater number and percentage of fathers had unknown HIV status (103, 13.6%) compared to mothers (20, 2.6%). The rate of mother-to-child transmission of HIV was observed to be 51.7%, while the rate of HIV exposure was 48.3%. A total of twenty-nine mothers, accounting for 3.8%, did not attend the antenatal clinic during their pregnancy. Overall, the incidence of teenage pregnancies was 4.9%, and 47 mothers, or 6.2%, delivered outside of healthcare facilities. The rate of cesarean sections was 20.6%, and 57.3% mothers experienced delays in starting breastfeeding. Furthermore, 82.2% of neonates were referred from other healthcare facilities, and 73.6% showed indicators of growth faltering. A significant number of neonates presented at admission with abnormal body temperatures (60.1%), heart rates (66.8%), and respiratory rates (83%). The characteristics of neonates diagnosed with HIV were comparable in all examined aspects to those without HIV. The sole distinctions observed were that mothers of HIV-positive neonates were, on average, significantly older (31.1 years, with a standard deviation of 5.40 years, representing a 3.37-year increase, and a 95% CI of 1.19 to 5.55, with a p-value of 0.003). Furthermore, these HIV-positive neonates had a greater propensity to be born to discordant couples (15.4% vs 1.5%; OR=11.72. 95% CI 3.35, 40.99; p=0.001); mothers with moderate parity (OR = 2.51; 95% CI: 1.05 to 5.88; p=0.032), to be born prematurely (OR=5.83; 95% CI: 1.15 to 29.58; p=0.047), and exhibited significantly impaired postnatal growth (OR= 3.02; 95% CI: 1.26 to 7.24; p=0.010) when contrasted with HIV-negative neonates. Notably, the impairment of postnatal growth manifested earlier than anticipated, with a substantial rate of 74.1% observed among the HIV-positive infants. Neonates whose record of HIV status was missing presented with distinct characteristics compared to those with a known HIV status. Specifically, neonates with unknown HIV status were more frequently born to parents who were either HIV positive or whose HIV status was also unknown, as opposed to parents who were HIV negative (p<0.001). Furthermore, these neonates were less likely to have received antenatal care (p<0.001; OR=4.83, 95% CI 1.96-11.91). They also exhibited delayed initiation of breastfeeding, demonstrated impaired growth, and presented with relatively elevated random blood glucose levels and irregular body temperature at the time of admission. The prevalence of discordant couples was observed to be 20.9% (19 out of 91couples). This rate was notably higher among infants whose HIV status was unknown, at 27.3% (6 out of 22), compared to 20% (10 out of 50) for infants with known HIV status, a statistically significant difference (p<0.001) with an odds ratio of 9.07 (95% confidence interval: 3.16 to 26.00). Among all the 19 identified discordant couples, 18 mothers tested positive for HIV, while only 1 father tested positive. ConclusionThe significant number of mothers lacking antenatal care in this study is a cause for concern and poses a risk to the advancement of the Prevention of Mother-to-Child Transmission (PMTCT) program within the nation. Intensified antenatal care initiatives, encompassing early HIV screening for expectant mothers and their partners, are imperative to enable the timely initiation of Antiretroviral Therapy (ART) and PMTCT services. Particular vigilance is warranted for neonates presenting with an unknown HIV status in healthcare facilities within resource-limited environments. Such infants, especially those born to mothers aged >25years, with inadequate or absent antenatal care, those with moderate parity, premature births, delayed breastfeeding initiation, faltering growth, and abnormal vital signs, should be suspected of HIV positivity and undergo early infant testing to further mitigate infant morbidity and mortality. Regular assessment of these infants feeding, health, and growth shortly after birth is crucial, either through home visits or postnatal clinic appointments. Targeted counseling for mothers (and partners) with unknown HIV status, HIV positive infants, infants with unknown HIV status, women aged >25 years, belonging to discordant couples; and public education are essential for reducing HIV incidence and improving infant health outcomes within the community.

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.

BackgroundChronic hepatitis B virus (HBV) infection (CHB) affects nearly 300 million individuals globally and remains incurable with current antiviral therapies, which suppress viral replication but rarely achieve functional cure defined by sustained loss of hepatitis B surface antigen (HBsAg). CHB is characterized by profound virus-induced immune tolerance that limits the efficacy of conventional therapeutic vaccination strategies. ObjectiveTo evaluate the therapeutic efficacy and immunological mechanisms of HEPLISAV-B, a CpG-1018-adjuvanted HBsAg vaccine, in breaking immune tolerance and inducing functional cure-like responses in a murine model of CHB. DesignUsing the adeno-associated virus-HBV (AAV-HBV) mouse model, mice with high levels of persistent HBV viremia were vaccinated with two doses of HEPLISAV-B. Virological outcomes in the blood and liver, immune responses and mechanisms were assessed. ResultsHEPLISAV-B induced rapid and durable HBsAg clearance, markedly reduced circulating and intrahepatic HBV DNA and RNA, and suppressed viral replication without hepatocellular injury. Vaccination elicited robust, sustained anti-HBs IgG1 and IgA responses, enhanced HBsAg-specific T and B cell immunity, reduced CD4 regulatory T cells, and decreased PD-1 expression on CD4 T cells. Therapeutic efficacy was strictly dependent on CD4 T cells and the CD40/CD40L signaling pathway, but independent of CD8 T cells, indicating a CD4-driven, non-cytolytic antiviral mechanism critical for HEPLISAV-B induced HBV control. ConclusionHEPLISAV-B effectively breaks HBV-induced immune tolerance and restores coordinated antiviral immunity through a CD4 T cell-/CD40L-dependent pathway. The findings support its potential as a therapeutic vaccine in CHB patients. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSChronic HBV infection is marked by profound virus-induced immune tolerance, current antiviral therapies and vaccines fail to reliably induce HBsAg loss or restore effective antiviral immunity, highlighting the need for immune-based therapeutic strategies. What this study addsThis study demonstrates that the clinically approved vaccine HEPLISAV-B can break HBV immune tolerance in a chronic HBV mouse model, inducing durable HBsAg clearance and anti-HBs immunity, non-cytolytic depletion of intrahepatic HBV DNA, through a mechanism strictly dependent on CD4 T cells and CD40/CD40L signaling. How this study might affect research, practice or policyThese findings defined a CD4 T cell-CD40L/CD40 axis that is critical in CHB functional cure, and support testing HEPLISAV-B as a therapeutic vaccine in CHB patients. O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/711721v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1d76f4dorg.highwire.dtl.DTLVardef@cc41cborg.highwire.dtl.DTLVardef@1f39288org.highwire.dtl.DTLVardef@192d0e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Background and AimsHepatitis C virus (HCV) infections were previously treated with interferon (IFN) but today direct acting antivirals (DAAs) with cure rates >95% are available. DAA treatment failure is primarily attributed to resistance associated mutations (RAMs), often imposing a fitness cost. Interferon treatment outcome was shown to be associated with the interferon sensitivity determining region (ISDR), which is part of the replication enhancing domain (ReED) in non-structural protein (NS) 5A. We found that accumulation of mutations in the ReED was indicative of elevated viral genome replication fitness. This study investigates the impact of HCV replication fitness on antiviral treatment outcomes. MethodsWe utilized chimeric HCV subgenomic replicons containing RAMs and ReED sequences from patients after interferon treatment or DAA failure to assess replication fitness of patient isolates in presence and absence of inhibitors. ResultsReplication fitness did not impact on IFN sensitivity in cell culture but resulted in higher remaining antigen levels for highly replicating variants at a given IFN concentration. Furthermore, we identified ReED variants substantially increasing HCV replication in several patients who failed DAA therapy across different genotypes. High replicator ReEDs rescued the fitness loss caused by RAMs like Y93C/H (NS5A) and S282T (NS5B). While high replication fitness did not intrinsically increase drug sensitivity (IC50), it allowed the virus to sustain robust replication despite antiviral pressure. ConclusionsElevated replication fitness might support interferon treatment due to increased antigen presentation, facilitating adaptive immune responses. Furthermore, ReED mediated increase in replication fitness could contribute to DAA treatment failure by preserving higher replication upon treatment and compensating for RAM associated fitness costs. Thus, patients failing DAA treatment should be monitored for RAMs and ReED mutations.

1.Etiological diagnosis of lower respiratory tract infections (LRTIs) relies on sputum or bronchoalveolar lavage (BAL), which may be difficult to obtain or invasive. Exhaled breath aerosol (XBA) sampling offers a non-invasive alternative for pathogen detection. We evaluated the performance of the AveloMask, a face mask-based device designed to capture XBAs for molecular testing. In this prospective paired-sample study, hospitalized adults with pneumonia at three hospitals in Switzerland and Georgia provided an XBA sample using the AveloMask and a lower respiratory tract (LRT) specimen (sputum or BAL). XBA samples were analyzed by multiplex PCR using the Roche LightMix(R) panel and LRT samples were tested using the BioFire(R) FilmArray(R) Pneumonia Panel. Concordance between XBA and LRT samples was assessed using positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA). Ninety-three participants were enrolled and 63 participants provided paired samples. AveloMask sampling identified the dominant pathogen (lowest Ct value in the LRT sample) in 40/47 LRT-positive cases (85.1%). Across all targets, PPA was 61% (95%CI, 50-72%), NPA was 100% (95%CI, 99-100%), and OPA was 95% (95% CI, 92-96%). PPA was higher for bacteria than for viruses and lower PPA was largely driven by reduced detection of low-abundance or co-infecting pathogens. In a subset analysis, AveloMask results showed substantial overlap with standard-of-care testing and could have supported antimicrobial de-escalation. Breath aerosol sampling using the AveloMask enabled non-invasive molecular detection of LRT pathogens in pneumonia cases and may complement conventional standard-of-care testing, particularly when sputum is unavailable.

Background and AimsPropionic acidemia (PA) is a rare autosomal recessive disorder caused by mutations in PCCA or PCCB, which encode the two subunits of propionyl-CoA carboxylase (PCC). PCC deficiency causes toxic metabolite accumulation and multi-organ damage. Current management, including dietary restriction, pharmacological support, and liver transplantation, does not restore enzymatic activity. We developed a dual-gene adeno-associated virus (AAV) therapy that delivers both PCC subunits to treat both PA subtypes. MethodsWe generated a clinically relevant PCCA-R73W knock-in mouse model and administered AAV8 vectors encoding native human PCCA and PCCB under the control of a liver-specific thyroxine-binding globulin promoter (AAV8-TBG-hPCCA-P2A-hPCCB). Metabolite levels and organ safety were longitudinally assessed. ResultsDual-gene therapy produced dose-dependent reductions in plasma C3/C2 ratio, 3-hydroxypropionic acid, 2-methylcitric acid, and propionylglycine, and significantly outperformed single-gene (PCCA-only) therapy. Neonatal facial-vein injection achieved metabolic correction comparable to or better than adult treatment. The longitudinal follow-up revealed sustained efficacy over a 16-week period, with no signs of hepatotoxicity or adverse effects. ConclusionsSingle-dose, dual-gene AAV therapy achieves sustained metabolic correction and demonstrates long-term safety in a clinically relevant PA model, supporting its translational potential for both type I and type II propionic acidemia.

The accurate measurement of Ebola virus (EBOV)-specific antibody responses is crucial to assessing immunity induced by EBOV infection or vaccination. For this purpose, the Filovirus Animal Nonclinical Group (FANG) anti-EBOV glycoprotein (GP1,2) ELISA is considered the "gold-standard". However, it has limitations such as high repeat-rates and variability, and low throughput. Here, we describe two new alternative assays: a Single-Molecule Assay Planar EBOV GP1,2 ELISA and a multiplexed EBOV GP1,2, EBOV nucleoprotein, and EBOV Viral Protein 40 Luminex assay, and compare these with two versions of the FANG ELISA. Samples were selected from participants receiving vaccine or placebo in a randomized, placebo-controlled, double-blinded study of two EBOV vaccines (PREVAIL 1), and a longitudinal cohort study of Ebola virus disease (EVD) survivors and their close contacts (PREVAIL 3). All four assays were concordant in their measurements of anti-EBOV GP1,2-specific immunoglobulin G responses, allowing for the determination of conversion equations for antibody measurements across assays. In addition, all four showed a similar ability to distinguish vaccine recipients from placebo recipients and EVD survivors from their close contacts. Compared to the FANG assays, the Quanterix and Luminex assays had lower variability, lower repeat rates, and higher throughput, making them good alternatives for future studies.

Household transmission of EV-D68 was identified in 35 of 1040 households (3.4%) in the Pacific Northwest between 2022-2024, with an estimated secondary attack rate of 15%. Sequences from within households clustered closely with 0 to 2 pairwise nucleotide differences (median 1) between cases 6-14 days apart (median 7).

BackgroundReliable detection of latent Mycobacterium tuberculosis (Mtb) infection (LTBI) remains challenging, particularly in TB contacts and immunocompromised individuals, where interferon-{gamma} release assays (IGRAs) demonstrate variable sensitivity. IP-10, a chemokine produced at substantially higher concentrations than IFN-{gamma}, represents a promising immune marker. This study aimed to evaluate the diagnostic performance of two IP-10 based assays RIDA(R)QUICK TB (lateral flow) and RIDASCREEN(R) TB (ELISA), by comparison with QuantiFERON-TB Gold Plus (QFT-Plus) assay or a composite reference standard. MethodsA cross-sectional diagnostic accuracy study enrolled 99 adults: 49 with culture-confirmed active pulmonary TB, 30 close TB contacts and 20 individuals with autoimmune disease, in Bucharest, Romania. All participants underwent RIDA Quick, RIDA Screen and QFT-Plus testing. Indeterminate results for all assays were reclassified using a composite reference standard. ResultsAgainst culture in active TB cases, RIDA(R)QUICK TB demonstrated a sensitivity of 85.7% (95% CI: 72.8-94.1) and PPV of 97.7%, while RIDA(R)SCREEN TB achieved 91.8% sensitivity (95% CI: 80.4-97.7) and 97.8% PPV. Specificity and NPV could not be reliably estimated due to the near-absence of true-negative individuals. Agreement with QFT-Plus was moderate to good ({kappa}=0.47-0.93).ROC analysis performed against QFT-Plus as a comparator demonstrated good immunological discrimination for RIDA(R)QUICK TB (AUC = 0.828) and RIDA(R)SCREEN TB (AUC = 0.767), reflecting concordance with QFT-Plus rather than diagnostic accuracy against confirmed infection. ConclusionIP-10 based assays demonstrated higher sensitivity than QFT-Plus and excellent PPV across bacteriological standard, supporting their use as complementary tools for LTBI detection. Larger, more heterogeneous cohorts are needed to accurately define specificity and operational integration.

BACKGROUNDAutomated antimicrobial susceptibility testing (AST) systems are crucial for accurate, timely detection of drug-resistant microbial isolates. This meta-analysis assessed the performance of the BD Phoenix ("Phoenix", BD Diagnostic Solutions), Vitek(R) 2 ("Vitek 2", bioMerieux), and DxM MicroScan WalkAway ("MicroScan", Beckman Coulter, Inc.) AST systems relative to common reference methodology. METHODSA systematic literature search in Ovid (MEDLINE and Embase) yielded 275 unique (not duplicated) records, with 44 additional records retrieved from handsearching; 39 studies met inclusion criteria. Categorical agreement (CA), essential agreement (EA), very major errors (VMEs), and major errors (MEs) for the three instruments were compared to a common reference method. Ratios of proportions were analyzed using random-effect meta-regression. RESULTSThe instruments did not differ significantly in CA, EA, or ME. Vitek 2 showed a higher overall VME rate than Phoenix ([~]44% higher; Vitek 2-to-Phoenix ratio = 1.44; p=0.062 [approaching significance]) and MicroScan (74% higher; ratio = 1.74; p=0.045). No appreciable difference was observed for VME between Phoenix and MicroScan. Subgroup analyses should be interpreted cautiously due to limited overall significance indicating varying performance across systems. Vitek 2 generally had higher relative VMEs for gram-negative organisms and lower relative VMEs for gram-positive organisms, whereas Phoenix showed the opposite pattern. MicroScan had relatively low VMEs when stratified by Clinical and Laboratory Standards Institute (CLSI) criteria; no differences in VMEs were observed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. CONCLUSIONAlthough some VME differences were noted, overall performance of the three systems was comparable. Organism- and drug-specific VME patterns--and updates to CLSI criteria over time--highlight the importance of continued monitoring of current breakpoints for all three instruments.