Journal of Clinical Virology

Several mpox outbreaks, caused by subclades Ia and Ib monkeypox virus (MPXV), are ongoing in the Democratic Republic of the Congo. The World Health Organization declared the ongoing mpox outbreak a Public Health Emergency of International Concern due to the geographic expansion of subclade Ib. We assessed the performance of a novel multiplex real-time PCR assay designed for MPXV detection and simultaneous subclade Ib identification. This assay demonstrates high accuracy and specificity, underscoring its use for the current mpox outbreak.

We evaluated the BOSCH Vivalytic MPXV assay using serial dilutions of monkeypox virus clade Ib, a new offshoot of clade I identified in 2023. The assay detected viral DNA down to [~]100 copies/mL demonstrating comparable analytical sensitivity to our in-house reference PCR and to other commercial platform-based mpox molecular assays.

Prior to the international spread of Monkeypox in May 2022, PCR kits for the detection of Orthopoxviruses, and specifically for monkeypox virus, were rarely available. Here we describe the evaluation of eleven recently-developed commercially available PCR kits for the detection of Monkeypox virus DNA.

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.

BackgroundMolecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. MethodsClinical performance of XpertXpress(R) SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. ResultsThe three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1-2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97% and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. ConclusionTP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhancediagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections. IMPORTANCEViral respiratory infections represent a major burden globally, weighed down by the COVID-19 pandemic, and threatened by spillover of novel zoonotic influenza viruses. Since respiratory infections share clinical presentations, identification of the causing agent for patient care and public health measures requires laboratory testing for several pathogens, including potential zoonotic spillovers. Simultaneous detection of SARS-CoV-2, influenza, and RSV in a single RT-PCR accelerates time from sampling to diagnosis, preserve consumables, and streamline human resources to respond to other endemic or emerging pathogens. Multiplex assays have the potential to sustain and even expand surveillance systems, can utilize capacity/capability developed during the COVID-19 pandemic worldwide, thereby strengthening epidemic/pandemic preparedness, prevention, and response.

Deep sequencing of the whole hepatitis B virus genome increases the analytical resolution and has the potential to improve molecular epidemiology investigations. The aim of this work was to develop and evaluate the performance of such deep sequencing using the Nanopore technology. The method includes an initial PCR step to generate two overlapping amplicons that cover the whole relaxed circular HBV genome found in circulating viral particles and covalently closed circular DNA in infected hepatocytes, followed by sequencing using the Nanopore rapid barcoding kit that allows parallel analysis of several samples in one reaction. The libraries can be sequenced with the standard Nanopore flow cell on MiniIon or GridIon devices, as well as the Flongle. The performance of the method was evaluated by comparing Nanopore and Sanger sequences or qPCR results from 64 clinical samples. The Nanopore-derived consensus sequences were, on average, 99.9% similar to those from Sanger sequencing and the full HBV genome was determined in samples with HBV DNA levels of approximately 3 log10 IU/mL with MagNA pure 96 extraction and < 2 log10 IU/mL using a high-volume manual extraction protocol on a subset of samples from patients with very low viral load (1.62-3.74 IU/mL). A perfect agreement with Sanger/qPCR-derived genotype was seen. The cost of sequencing per genome using the Nanopore method is low, ranging 6-37euros. We conclude that whole-genome sequencing of HBV with Nanopore is well suited for genomic characterization, antiviral resistance mutation analysis and genotyping of HBV in a routine laboratory setting.

Influenza A viruses (IAV) are a global health concern, infecting a wide range of hosts, including humans, birds, and pigs. Whole genome sequencing (WGS) is crucial for genomic surveillance under a One Health framework, providing insights into IAV evolution and transmission. We evaluated several laboratory workflows for next generation sequencing (NGS), aiming to optimise and unify the WGS process across IAV samples of human, swine, and avian origins. Multiple combinations of RNA extraction methods, one-tube RT-PCR protocols and primer sets were systematically tested for their efficacy in generating high-quality PCR and NGS products across all viral segments. We assessed performance based on sequencing quality, including read counts, segment coverage and amplification bias across various host types, subtypes, and viral loads. High-quality RNA extraction was critical for achieving reliable sequencing results, particularly in low viral load samples. Uniform amplification across all IAV genome segments was best achieved using an optimised RT-PCR protocol that reduced amplification bias for shorter fragments, including defective interfering particles (DIPs). Although variability in performance remained across host types and sample qualities, the refined workflow showed consistent improvements in sequencing outcomes, offering a promising foundation for a unified genomic surveillance. While further refinements may be needed for specific contexts, this study provides an improved, broadly applicable workflow that enhances genomic surveillance of influenza A viruses under a One Health framework, facilitating better understanding of viral evolution and transmission across species.

ObjectivesThis multicentre study investigated the utility of next-generation sequencing (NGS) to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load (from 0.2 to 6207 IU/ml). Methods23 HBV DNA positive plasma samples of genotypes A-E and one HBV-negative control sample were assayed blindly via 9 established NGS methods from 6 European laboratories. Methods included untargeted metagenomics, pre-enrichment by probe-capture followed by Illumina sequencing, and HBV-specific PCR pre-amplification followed by sequencing with Nanopore or Illumina. ResultsFull HBV genomes were obtained only from samples with viral loads >1000 IU/ml using probe-capture methods, >200 IU/ml using PCR-Illumina methods, >10 IU/ml using PCR-Nanopore methods, and in no samples using metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads in all PCR-based methods. Probe-capture and metagenomic methods detected additional viruses not routinely screened in blood donations, including polyomaviruses and herpesviruses; positive results were confirmed by PCR. ConclusionsNGS may delineate whole-genome sequences at low viral loads if supported by a PCR pre-amplification step. Probe-capture methods also reliably detect HBV without pre-amplification but achieve limited genome characterisation at low viral loads; they may additionally detect a wide range of blood-borne viruses.

BackgroundAccurate and accessible diagnostics for hepatitis E virus (HEV) are essential for outbreak preparedness and surveillance, particularly in low-resource settings. Dried blood spots (DBS) offer a simple, scalable alternative to serum, but their diagnostic performance for HEV remains poorly characterised. MethodsPaired DBS and serum samples were collected from suspected HEV cases during an outbreak in Bentiu, South Sudan. HEV RNA was detected and quantified by real-time RT-PCR, and anti-HEV IgM and IgG antibodies were measured by ELISA. DBS performance was compared to serum across assays, and associations between DBS positivity and serum viral load were analysed using logistic regression. ResultsAmong 100 serum RT-PCR positive samples, 83 were positive by DBS (sensitivity 83.0%, 95% CI: 74.5-89.1). Ct values from DBS and serum were strongly correlated ({rho} = 0.74), and most discordances at low viral loads (Ct > 30). DBS achieved 88.3% sensitivity (95% CI: 77.8-94.2) and 100% specificity (95% CI: 91.2-100) for IgM, while IgG sensitivity and specificity reached 95.0% (95% CI: 86.3-98.6) and 97.5-100%, respectively. Results were consistent across DBS card types and age groups. ConclusionsDBS provide a reliable, practical alternative to serum for HEV diagnostics. Despite slightly reduced sensitivity at low viral loads, DBS maintained high specificity and strong correlation with serum results. Their ease of collection, storage, and transport without cold chain requirements supports their application for HEV surveillance and outbreak response in resource-limited settings. Further optimisation of elution methods to increase analyte concentration, along with improved storage conditions, could enhance diagnostic sensitivity.

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

Human Cytomegalovirus (HCMV) infection represents a life-threating pathogen for immunocompromised patients. Molecular quantitative testing on whole blood or plasma represents the gold standard for diagnosis of invasive HCMV infection and for monitoring antiviral treatment in individuals at risk of CMV disease. For these reasons, accurate standardization towards the 1st WHO International standard between different centres and diagnostic kits represent an effort for a better clinical management of CMV-positive patients. Herein, we evaluate for the first time the performance of a new TMA (Transcription Mediated Amplification) kit towards qPCR chemistry, used as routine method, on whole blood samples. 755 clinical whole blood specimens were collected and simultaneously tested with TMA and qPCR assays. Data showed 99.27% agreement for positive quantified samples and 89.39% agreement for those not detected between two tested methods. Evaluation of viraemia in positive samples highlighted a good correlation for TMA and qPCR chemistries in terms of International Units ({Delta}Log10 IU/ml: - 0.29 {+/-} 0.40). TMA assay showed a significant correlation with qPCR also in monitored patients until three months, thus allowing accurate evaluation of viraemia in transplanted patients. Moreover, preliminary data about analytical sensitivity of TMA chemistry onto DBS samples showed 86.54% correlation with whole blood specimens. Thus, TMA chemistry showed a good agreement with qPCR assay, used as current diagnostic routine, and offers important advantages: FDA and IVD approval on plasma and whole blood, automated workflow with minimal hands-on time, random access loading, thus enabling a rapid and reliable diagnostics in HCMV-infected patients.

Hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV) are transfusion transmissible infections (TTIs) agents that threaten the safety of the blood supply. Surveillance of the variance of those viruses is an important way to monitor their diversity and evolution to improve safety in blood transfusion. In this study, we characterized the specimens of blood donors from 13 blood centers located in 5 Chinese regions. Samples collected between 2014 and 2017 were screened with serological and molecular tests conducted on Abbott ARCHITECT and m2000 platforms. Sequencing was used to determine the classifications. The HBV immune escape mutations were also analyzed for assessing vaccine breakthrough risks and challenges for diagnostic tests. For HIV, 11 genotypes or recombinants were identified. The predominant genotype was C, which accounts for 42%. For HBV, the genotypes of B, C and D were identified, with B and C predominating. The major subgenotype was B2, comprising 84.1% of all infections. 79 out of 113 (69.9%) samples carried escape mutations in the "a" determinant region with 69 (87.3%) multiple mutants and 15 (19%) escape mutants which will affect HBsAg detection. For HCV, 7 genotypes or subtypes were identified. The major genotype was 1b (48%), followed by 6a (16.7%) and 2a & 3a (10%). This study provides the information of diversity of HBV, HCV and HIV strains circulating in blood centers from 5 regions in China. These data can also be scientific basis for development of detection assays that mitigate the impact of viral diversity on performance. ImportanceThe prevalence of TTIs in blood donations is important for evaluating blood safety and it can also reflect the burden of these disease among populations. Virus variance is threat to blood safety due to it may affect assays detection by nucleic acid, antigen and antibody-based methods in blood donors. HIV, HBV and HCV exhibit high degrees of genetic diversity, with different strains predominating in different geographic locations. The aim of this study is to assess the diversity of HBV, HCV and HIV among blood donors in China. In this study, 13 blood centers located in 5 Chinese regions were involved and the most informative phylogenetic regions of each virus had been sequenced. This will benefit for viral monitoring by subtype/genotype analyses to determine whether the distributions of variants are changing over time and geographically, and to speculate whether previously rare subtypes are becoming established in blood donors in China.

This study presents and evaluates a rapid and all-in-one SARS-CoV-2 RT-LAMP based molecular detection system, including RNA extraction or not, for point-of-care or massive testing of naso-pharyngeal swabs. The point-of-care format uses LoopX(C), a small portative device ensuring optimal LAMP reaction and automated reading with 95.2% and 95.5% sensitivity and specificity respectively. This system might also be useful for testing other sample types such as saliva.

H5N1 is a highly pathogenic avian Influenza A subtype that has been known to also infect mammalian hosts and presents a potential public health risk. To address and mitigate the affects of new SNPs, found in recent Canadian outbreaks, on diagnostic detection we developed two qRT-PCR assays by modifying current probes to match sequences detected in the east and west coast of Canada. These assays were multiplexed with a third qRT-PCR assay targeting the M segment, allowing streamlined detection of Influenza A and subtyping for H5. This three-plex qRT-PCR was validated by assessing analytical specificity, limit-of-detection, precision, and accuracy.

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.

The Chikungunya virus, a global arbovirus, is currently causing a major outbreak in the Western African region, with the highest cases reported in Senegal and Burkina Faso. Recent molecular evolution analyses reveal that the strain responsible for the epidemic belongs to the West African genotype, with new mutations potentially impacting viral replication, antigenicity, and host adaptation. Real-time genomic monitoring is needed to track the viruss spread in new regions. A scalable West African genotype amplicon-based Whole Genome Sequencing for multiple Next Generation Sequencing platforms has been developed to support genomic investigations and identify epidemiological links during the viruss ongoing spread. This technology will help identify potential threats and support real-time genomic investigations in the ongoing spread of the virus.

Influenza (Flu) and respiratory syncytial virus (RSV) are responsible for lower respiratory tract infections (LRTIs) associated with significant hospitalization among young children. In the present study, the performances of a triplex PCR assay detecting Flu A/B and RSV were compared with our in-house single-plex assays using 160 stored respiratory specimens previously tested using a panel of laboratory-developed real-time RT-PCR. Of them, 61 were positive for FluA, 41 for FluB, and 58 for RSV. All samples were retrospectively quantified with Respiratory Viral (RV) ELITe MGB(R) Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe InGenius(R) system. Overall, the total percentage agreement observed was 93.4% (57/61) for FluA, 92.7% (38/41) for FluB, and 86.2% (50/58) for RSV. A significant correlation of VL values was observed between the two methods for FluA and RSV ({rho}= 0.91 and 0.84). This finding was supported by the strength of agreement between the two methods, as showed by the linear regression analysis (R2 =0.84 and 0.80). FluB viral load values measured by RV Panel were less significantly correlated ({rho}= 0.77 and R2 =0.56). The bland-Altman analysis showed how 84.2% (48/57) of FluA and 86.0% of RSV (43/50) samples fell within {+/-}1.0 Log10 variation from our laboratory results, while only 21.1% (8/38) of FluB results fell within this range. The great majority of FluB samples (29/30) outside range had values higher than +1.0 Log10 (median +2.1 Log10 range +1.0 to +3.5 Log10). In conclusion, RV ELITe MGB(R) Panel constitutes a valid and robust system for simultaneous detection and quantification of Flu A/B and RSV.

Background. West Nile virus (WNV) genomic surveillance in the United States relies largely on mosquito and avian sequencing, while human-derived genomes remain scarce. Nucleic acid testing (NAT)-reactive blood donations provide a standardized source of acute human-phase virus, but low donor viremia complicates genome recovery. This study evaluated a sequencing strategy for WNV surveillance using these samples. Study Design and Methods. Amplicon sequencing, hybridization capture, and shotgun RNA-seq were evaluated for WNV lineage 1a recovery from donor plasma. Amplicon performance was characterized using a WHO International Standard dilution panel quantified by RT-dPCR, contemporary 2025 donations, archival 2010-2011 donations, and technical replicates. Two donations were processed by all three methods from matched plasma to compare performance metrics and consensus concordance. Results. Amplicon sequencing recovered near-complete genomes across the full dilution panel, including the lowest measured input, and across the viral-load range represented by the selected donor samples. Recovery from the two archival plasma samples was similar to that observed among contemporary donations. In the two matched donations, all three methods generated identical consensus sequences across shared callable positions. At lower input, amplicon and capture maintained near-complete recovery, whereas shotgun RNA-seq decreased to 87.2% coverage at 10X. For libraries achieving near-complete recovery, WNV-mapped-read requirements were similar, but amplicon sequencing required substantially fewer total reads. Discussion. NAT-reactive blood donations can support WNV genomic surveillance. Amplicon sequencing is an efficient first-pass approach for expected lineage 1a WNV, with capture and shotgun RNA-seq serving as escalation strategies for divergent lineages or unbiased pathogen detection.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

Unbiased long read sequencing approaches for clinical metagenomic sample analysis holds enormous potential for pathogen detection, including improved detection of unknown, novel or emerging viruses. However, the rapid rate of development in nanopore sequencing and library preparation methods complicates the process of selecting a standardized method for unbiased RNA virus detection. Here, we evaluate multiple sequencing approaches to identify a workflow with sufficient sensitivity, limits of detection, and throughput for potential utilization in a clinical laboratory setting. Four separate library preparation methods for the Oxford Nanopore Technologies MinION sequencer are compared, including direct RNA, direct cDNA, rapid cDNA, and double stranded cDNA. We also establish that depletion of host RNA is not required and can be deleterious for viral RNA detection in some instances when using samples in viral transport media (VTM) or plasma. Using unbiased whole genome amplification following reverse transcription, we achieve limits of detection on the order of 1.95E03 GE/mL of Venezuelan Equine Encephalitis Virus (VEEV) spiked in human plasma. We also report initial detection of 5.43E06 GE/mL of coronavirus 229E spiked into VTM samples containing human background RNA which are expected to decrease significantly during upcoming testing. These metrics were achieved within a 6-plex multiplex reaction, illustrating the potential to increase throughput and decrease costs for relevant sample analysis. Data analysis was performed using EPI2ME Labs framework and open access tools that are readily accessible to most clinical laboratories. Taken together, this work describes an optimized method for unbiased nanopore sequencing and analysis of RNA viruses present in two common clinical matrices.