Journal of Clinical Virology

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.

The 2026 Bundibugyo ebolavirus (BDBV) outbreak underscores the need for rapidly deployable molecular diagnostics. We developed and analytically validated reverse-transcription quantitative PCR assays detecting BDBV, Zaire ebolavirus, and Sudan ebolavirus. The platform includes a BDBV singleplex assay, a duplex assay with a human internal control, a four-target multiplex assay for ebolavirus differentiation, and a probe-free SYBR Green assay. We adapted the assays to a portable qPCR instrument, reducing runtime from 65 to 35 minutes, and validated lyophilized reagents to reduce cold-chain requirements. All TaqMan formats achieved a 95% limit of detection of 5 copies per reaction across instruments and reagent types; the SYBR Green assay achieved 50 copies per reaction. The assays detected viral RNA in contrived clinical samples without cross-reactivity among ebolavirus species tested. We shared the protocols in real time through Ampliphi (https://www.ampliphi.bio), a new open-access platform for rapidly disseminating diagnostic assays, and through protocol.io.

Nucleic acid extraction remains the principal infrastructure barrier to molecular influenza testing outside centralised laboratories, since bead-based purification is normally tied to mains-powered extractors and trained operators. We evaluated SmartLid, a centrifugation-free format in which a removable magnetic key shuttles paramagnetic beads through pre-aliquoted lysis/binding, wash, and elution buffers without pipetting or powered instrumentation, against an automated magnetic-bead extractor (Nextractor NX-48S) on 311 nasopharyngeal specimens from the 2024-2025 influenza season at a National Influenza Centre. Paired eluates were amplified under identical monoplex RT-qPCR conditions for influenza A(H1N1)pdm09, A(H3), and B/Victoria. Both methods gave 100% specificity (47/47 negatives; no false positives). Subtyping succeeded in 263/264 reference-positive specimens after SmartLid extraction versus 241/264 after automated extraction (99.62% versus 91.29%; difference 8.33 percentage points; discordant pairs 23 versus 1; McNemar P < 0.001). Across 240 complete pairs, cycle threshold (Ct) values were lower after SmartLid extraction (median paired difference -2.78 cycles; estimated location shift -2.60 cycles, 95% CI -2.82 to -2.37; P < 0.001) with rank-ordering of specimens conserved between methods (Spearman rho = 0.84). The advantage was preserved across all three subtypes and in both fresh and frozen specimens (adjusted P < 0.001). Specimens recovered only after SmartLid extraction had higher Ct values than dual-detected specimens (median 34.37 versus 28.54; P < 0.001), locating the gain near the assay detection limit. An instrument-free manual format can therefore exceed the extraction efficiency of an automated reference workflow, extending quality-assured influenza subtyping beyond centralised laboratories.

Hybrid capture approaches can detect a broad range of pathogens directly from clinical samples with greater sensitivity than untargeted approaches. Clinical adoption has been limited by long turnaround times, complex workflows, and perceived high costs. We iteratively developed and validated a rapid hybrid capture workflow based on Illumina's RPIP protocol (standard workflow). Rapid-RPIP v1 reduced wet-lab time from ~11.4 to ~7.3 h, for a turnaround time (TAT) of <15 h. Rapid-RPIP v2 further reduced wet-lab time by ~0.7 h and used Illumina Turbo Sequencing shortening the sequencing time from ~5.5 to ~2.5 h thus achieving a TAT of <10 h without compromising sensitivity and yielding higher RPKMs for RNA viruses than the standard workflow. This approach could be readily adapted to a broad range of clinical and public health applications.

Background: Reactivation of herpes simplex viruses (HSV) can occur in the early post-allogeneic hematopoietic cell transplant (aHCT) period despite antiviral prophylaxis. Few studies have assessed HSV infection in the modern era, in which acyclovir/valacyclovir is recommended for up to 1 year post aHCT. We evaluated the incidence and management of breakthrough HSV during the first 100 days post-aHCT over two decades. Methods: Patients who received their first aHCT at Fred Hutchinson Cancer Center between 2002-2022 were reviewed for breakthrough HSV infection within the first 100 days on prophylaxis (acyclovir 800 mg or valacyclovir 500 mg twice daily). Cases were identified via culture, polymerase chain reaction, and/or direct fluorescent antibody testing; clinical records were reviewed for symptoms, outcomes, and prophylaxis/treatment regimens. Refractory/resistant (R/R) infections were defined according to consensus guidelines. Results: We reviewed data from 4,357 aHCT recipients aged [&ge;]18 years, among whom 3,749 (86%) were HSV seropositive and 23 developed breakthrough HSV infection (observed probability = 0.6%). Among those who had an infection, the median time from transplant to first positive test was 46 days (IQR: 24.0-69.5). Oral and genital mucosa were the most common sites of infection. In total, 11 of 23 (47.8%) patients with breakthrough HSV developed R/R infection. Conclusions: Breakthrough HSV infections are rare in the first 100 days after aHCT among patients receiving antiviral prophylaxis. Refractory/resistant infections were uncommon but represented almost half of breakthrough cases. Our findings highlight the sustained effectiveness of universal prophylaxis in the early post-transplant period.

Objectives: Capturing adequate blood volume for blood cultures is critical for accurate detection of bloodstream infections. Pediatric volume targets vary by age and weight, whereas adult targets are standardized. The BD BACTEC FXI Culture System (FXI) contains an integrated calibrated load cell capable of automatically reporting blood volume measurements for each vial loaded onto the system. This study evaluated the accuracy of the FXI's blood volume measurements in simulated pediatric and adult patients. Methods: Mock pediatric and adult blood draws were performed, using bagged whole blood, to replicate real-world collection protocols. Syringe-collected blood volumes ranged from 2.0 to 15.0 mL for pediatric patients, depending on mock patient weight, and were fixed at 40.0 mL for adults. Samples were inoculated into BD BACTEC Peds Plus/F, Plus Aerobic/F, and Lytic/10 Anaerobic/F Culture Vials, with a target volume of 2.0 to 10.0 mL per bottle. Reference blood volumes were determined gravimetrically using manually obtained pre- and post-inoculation weights with a blood-specific gravity of 1.055 g/mL and were compared to the automatically measured, gravimetric-based blood volumes reported by the BACTEC FXI Culture System. Results: Automated volume estimates were accurate to a mean error of -0.03 mL per bottle (SD, 0.40 mL; n=168; 95% CI, -0.09 mL, 0.03 mL) and -0.08 mL (SD, 0.79 mL; n=72; 95% CI, -0.26 mL, 0.10 mL) when assessing total volume collected per patient. Conclusions: Our findings demonstrate that the automated system can quantify blood volumes in BACTEC culture vials and support blood volume monitoring for pediatric and adult collections. The gravimetric approach is also amenable to full automation for efficient and accurate blood volume determination.

Background: HBV coinfection is more common among people with HIV than in the general population. Nucleotide reverse transcriptase inhibitors (NRTIs) in antiretroviral therapy (ART) are also active against HBV. Growing use of NRTI-sparing regimens raises concern about HBV reactivation. Objectives: To determine the rate, cumulative incidence, and factors associated with HBsAg clearance in HIV/HBV-coinfected patients, and to characterize the genetic/structural basis of occult HBV infection (OBI) in the same cohort. Study design: We retrospectively reviewed 256 people with HIV at Kumamoto University Hospital in Japan (1986-2025), stratified by HBsAg clearance status; clinical factors were compared and cumulative incidence estimated by Kaplan-Meier analysis. OBI cases underwent HBsAg-region sequencing. Results: Fifteen patients were analyzed (12 HBsAg-positive, 3 OBI). HBsAg clearance occurred in 7/12 (58%). The Kaplan-Meier estimated cumulative incidence reached 25% by 2 years and 64.3% at approximately 4.4 years, after which no additional clearance events were observed during follow-up. Clearance was associated with higher CD4 ([&ge;]200 cells/L: 71% vs 0%) and lower HIV-RNA (<105 copies/mL: 86% vs 20%); HBV-DNA was significantly higher in the clearance group than OBI (adjusted p=0.007). All OBI sequences matched genotype C2; one also carried a rare genotype A2 'a' determinant substitution, N131K (0.23% of genotype A sequences in HBVdb). Conclusions: HBsAg clearance was frequent, concentrated within 3-4 years of ART, and associated with preserved immune status and HBV replicative activity. A rare 'a' determinant substitution may cause false-negative HBsAg in OBI, informing NRTI-sparing ART selection.

Background: Distinguishing Zika virus (ZIKV) from dengue virus (DENV) infection remains a major diagnostic challenge in co-endemic regions due to antigenic cross-reactivity. We evaluated a multiplex electrochemiluminescence Meso Scale Discovery System (MSD) NS1 assay to define ZIKV infection with high specificity across diverse immune backgrounds. Methods: We analyzed longitudinal samples from PCR-confirmed ZIKV and DENV infections across multiple cohorts, assessed correlations between MSD NS1 signal intensities and PRNT50 titres, and performed ROC analyses to derive seroconversion thresholds. We then applied these criteria to identify ZIKV seroconversions in Brazil (2015-2024) and Thailand (2015-2025). Findings: ZIKV infections produced sustained NS1 responses, whereas primary DENV infections showed minimal ZIKV cross-reactivity and secondary DENV infections exhibited only transient, low-level increases. MSD NS1 signal intensities correlated with PRNT50 titres for ZIKV and all four DENV serotypes (rho = 0.54-0.84). ROC analyses identified a fold-change of at least 10.96 as the optimal ZIKV seroconversion cutoff, with an AUC of 100% among DENV-naive and 90.7% among DENV-exposed participants. Adding a second criterion - ZIKV/DENV fold-change ratio > 1 - further increased specificity. Application of this dual-threshold framework revealed high ZIKV incidence during the 2015-2016 epidemic in Brazil and very low incidence in the post-epidemic period. In the Thailand cohort, synchronous seroconversion among household members suggested potential intra-household transmission. Interpretation: The MSD NS1 multiplex assay provides a scalable, quantitative, and highly specific approach for identifying ZIKV infection and estimating seroincidence in co-endemic settings. The dual-threshold framework (fold-change of at least 10.96 and ZIKV/DENV ratio > 1) enhances diagnostic precision, offering a robust tool for surveillance and transmission studies in flavivirus-endemic regions.

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

Access to accurate cost-effective technologies for typing high-risk human papillomaviruses (hrHPV) is critical to expand cervical cancer screening and inform vaccination strategies. Compared with clinical-standard quantitative polymerase chain reaction (qPCR) assays, HPV genotyping by next-generation sequencing (NGS) provides greater flexibility, scalability, and genotype specificity. We have developed a method for HPV genotyping, HPV Phased Amplicon Multiplex Sequencing (PhAM-Seq), that uses combinatorial barcoding of amplicons with short, variable-length inline sequences to enable higher throughput and lower per-sample costs than conventional amplicon sequencing approaches. We evaluated HPV PhAM-Seq using degenerate and type-specific primers targeting the L1 and E6-E7 gene loci in a blinded cohort of 170 cervical samples previously typed by the Seegene Anyplex II HPV28 Detection qPCR assay. Across eight common hrHPV types (HPV16, 18, 31, 33, 35, 45, 52, and 58), HPV PhAM-Seq demonstrated >80% overall agreement with qPCR using degenerate L1-targeting primers, with the highest sensitivity for HPV16, 31, 33, and 58. Sensitivity for HPV35, 45, and 52 improved to 85% or greater with type-specific primers targeting genes E6/E7. Parallel processing and sequencing enables a single technician to assay hundreds of samples per week at a reagent cost of around $10 USD per sample, with laboratory automation and sequencing on higher-output platforms enabling further scaling and cost reduction to a scale amenable to population-level surveillance. We include a detailed SOP; tools for primer design, sequencing library construction, and sample tracking; and all scripts needed for data analysis to ensure HPV PhAM-Seq can be readily implemented for scalable, cost-effective hrHPV genotyping or extended to other similar applications.

Background: Amplicon-based next-generation sequencing is a cornerstone of viral genomic surveillance, but it often produces datasets with highly uneven read coverage due to PCR amplification bias. This leads to excessive data generation and substantial computational overhead for downstream bioinformatic analysis. Results: We evaluated the efficacy of two data reduction strategies on 220 SARS-CoV-2 clinical specimens and 12 wastewater samples: random subsampling with SeqKit and read depth normalization with BBNorm. FASTQ files were reduced to target depths from 10X to 10,000X to compare genome coverage, lineage assignment concordance, and computational resource usage. Both strategies markedly reduced total processing time. For clinical isolates, read depth normalization corrected for amplification bias resulting in highly concordant lineage assignments at a target depth of just 200X. Random subsampling preserved inherent bias but produced reliable assignments at depths over 500X. Reducing target depths below 500X in complex wastewater mixtures severely degraded variant deconvolution accuracy and heavily skewed lineage abundance profiles for both methods. Conclusion: Both random subsampling and read depth normalization successfully reduce computational overhead while preserving key clinical sequencing metrics at appropriate reduction settings. The optimal method depends on the analytical goal, ranging from computationally lightweight random subsampling for rapid lineage assignment, to read depth normalization for generating high-quality consensus sequences. However, aggressive data reduction should be avoided in environmental surveillance, where maintaining a high baseline sequencing depth is critical for accurate mixed-variant deconvolution.

In resource-limited settings plasma is usually transported to centralized laboratories for HIV RNA quantification; often by motor vehicles using dry ice. Any decay in plasma HIV RNA when near the threshold used to define virologic failure could impact clinical management. However, maintaining a cold chain during transport can be costly and limit the frequency of shipments, which can delay clinical assessments and also negatively impact patient care. Transporting plasma at ambient temperatures could facilitate timely measurements of plasma HIV RNA viral loads (VL). We conducted a stability study of HIV RNA in plasma held for 1, 3, 5, and 7 days at a range of temperatures (4{degrees}C, 30{degrees}C, and 37{degrees}C) to simulate the durations and temperatures specimens could experience during transport. Thirteen large volume plasma specimens with VL ranging from 2.7-5.53 log10 copies/mL were evaluated. Mean changes in VL between Day-0 to Day-1, Day-3, Day-5 or Day-7 were calculated, with decays >0.5 log10 c/mL considered clinically relevant. Compared to Day 0, the VL of plasma stored at 4{degrees}C and 30{degrees}C remained stable across the Days tested. The VL in plasma stored at 37{degrees}C had a progressive decline at Day-1, Day-3, Day-5 and Day-7 (means of -0.04, -0.19, -0.33. -0.34 log10 c/mL, respectively), with a decay of >0.5 log10 observed in 3/13 (23%) specimens at Day-7. A linear regression analysis showed that plasma specimens stored at 37{degrees}C significantly decayed over time (slope -0.054, 95% CI -0.079, -0.029; p=0.01). Any decay observed among specimens stored at 4{degrees}C and 30{degrees}C was not clinically relevant nor statistically significant. Our observations suggest that HIV RNA levels in plasma should remain stable if transported to centralized laboratories at ambient temperatures of [&le;]30{degrees}C or chilled by cold packs over a period [&le;]7 days. Transporting specimens with cold packs instead of dry ice could reduce costs and facilitate the timely monitoring of VL used to assess and guide antiretroviral therapy.

Point-of-care Hepatitis C Virus (HCV) RNA assays reduce diagnostic turnaround time but depend on benchtop instrumentation and continuous electricity, limiting their deployment in the harm reduction and community settings where confirmatory testing is most needed, as people who use drugs (PWUD) carry a disproportionate share of the HCV burden in the United States. This is a systemic problem in diagnostic development, where decision-making and design requirements overlook point-of-use stakeholders. Closing the gap requires integrating real-world constraints throughout design rather than validating against user needs once a product already exists. Here, we apply a human-centered design (HCD) approach to inform rigorous, stakeholder-derived design requirements, implementation considerations, and early value proposition for a novel point-of-need HCV RNA test intended for deployment in harm reduction and community settings in Indiana. To determine design specifications grounded in real-world context, our objectives were (1) identifying and characterizing context-specific experiences and barriers to HCV testing among higher-risk populations; (2) assessing the perceived benefits and acceptability of the proposed test within real-world settings across direct and indirect user groups; and (3) translating the user needs and contextual constraints into design requirements and implementation considerations that support the test's clinical, operational, and user-centered value. We conducted 18 semi-structured interviews with frontline staff and HCV testing/treatment pipeline experts (n=11) and people who get tested (n=7) across harm reduction organizations, syringe service programs, and community testing settings, analyzed using Rapid Qualitative Analysis guided by the PARRQA framework. Stakeholders responded positively to a single-encounter point-of-need RNA test, and implementation considerations, including funding restrictions, staffing structures, and diverse deployment settings, directly shaped design requirements spanning turnaround time, sample type and volume, portability, result output, target operator, and ease of use. Benchmarking these stakeholder-derived specifications against the FIND Dx HCV target product profile (TPP) showed that stakeholder input confirmed, modified, or extended several TPP criteria and introduced requirements the TPP does not address. Together, these objectives constitute an upstream, evidence-driven design process that translates contextual and stakeholder knowledge into actionable engineering requirements, highlighting the need for diverse stakeholder engagement at all stages of the design process for closing the translation gap between laboratory-validated diagnostic tools and effective point-of-need deployment.

Healthcare workers are occupationally exposed to blood and body fluids and require effective protection against hepatitis B virus infection. Evidence on hepatitis B surface antibody (anti-HBs) seroprotection among healthcare workers in Namibia remains limited. We estimated the prevalence of anti-HBs seroprotection and examined associated factors among healthcare workers tested at Eastgate Clinic in Windhoek, Namibia. We conducted a cross-sectional analysis of routinely collected anti-HBs test records from 1 January to 31 December 2025. The first chronological test per individual was included. Anti-HBs concentrations [&ge;]10 mIU/mL were classified as seroprotective. Results outside the assay reporting range were treated as censored but remained classifiable for the binary outcome. Prevalence estimates were reported with Wilson 95% confidence intervals (CIs). Modified Poisson regression with robust standard errors was used to estimate adjusted prevalence ratios (aPRs). The register contained 304 test records representing 299 unique individuals. After excluding three individuals with missing anti-HBs results, 296 were analysed; 163 (55.1%) were female. Overall, 148 individuals were seroprotected, corresponding to a prevalence of 50.0% (95% CI 44.3-55.7%). Seroprotection was lowest among participants aged 18-29 years (35.3%) and highest among those aged 50-59 years (71.1%). Each 10-year increase in age was associated with a higher prevalence of seroprotection after adjustment for sex (aPR 1.14, 95% CI 1.06-1.24; p=0.001), whereas sex was not independently associated with seroprotection (aPR 0.97, 95% CI 0.78-1.22; p=0.797). The age association remained consistent across sensitivity analyses. Half of the healthcare workers tested had anti-HBs concentrations below the operational seroprotection threshold. These findings support systematic vaccination verification, appropriately timed post-vaccination testing, and documented follow-up of non-protective results. The absence of vaccination history, occupational characteristics, testing indications, and additional hepatitis B serological markers limited causal and clinical interpretation.

Background The COVID-19 pandemic substantially altered respiratory pathogen circulation worldwide. However, longitudinal analyses of changes in respiratory pathogen ecology across the pandemic and post-pandemic periods remain limited. Methods We analyzed 19,968 respiratory samples tested with the BioFire(R) FilmArray(R) Respiratory Panel at a hospital in Tokyo, Japan, between January 2020 and March 2026. We evaluated temporal changes in pathogen circulation, age-specific epidemiology, co-detection patterns, pairwise pathogen associations, and clinical parameters. Results At least one respiratory pathogen was detected in 27.8% of tests. Respiratory pathogens resurged asynchronously following the relaxation of COVID-19-related public health measures. Influenza virus circulation remained markedly suppressed until late 2022 before re-emerging in successive large seasonal epidemics, whereas other pathogens, including RSV, human metapneumovirus, and Mycoplasma pneumoniae, exhibited distinct resurgence patterns. Pathogen distributions also varied by age. Human rhinovirus/enterovirus remained predominant among young children, whereas SARS-CoV-2 predominated among older adults. Co-detection occurred in 14.0% of positive specimens and was significantly more frequent in younger patients. Pairwise analysis identified both positive and negative pathogen associations; however, the patterns varied across age groups and study periods. Conclusions Respiratory pathogen circulation changed substantially during the transition from the COVID-19 pandemic to the post-pandemic period, with pathogen-specific, age- and period-dependent patterns. Continued surveillance is warranted to determine how respiratory pathogen circulation will evolve and to inform infection control strategies in the post-pandemic era.

BackgroundHuman Mastadenovirus B (HAdV-B) causes ocular and respiratory infections and is capable of causing outbreaks in healthcare settings. HAdV-B114, a recombinant genotype only formally recognised in 2023, has been reported predominantly from Germany, the USA, China and Japan; genomic data from UK clinical settings remain limited. This study analysed 68 HAdV-B whole genomes from ocular and respiratory-associated samples collected at a Nottingham hospital across the pre- and post-COVID-19 restriction periods (2017-2023). MethodsClinical samples typed positive for HAdV-B by partial hexon Sanger sequencing were selected for whole-genome sequencing using a novel overlapping PCR amplicon strategy, sequenced on the Oxford Nanopore Technologies PromethION platform. Phylogenetic and pairwise difference analyses were used to characterise genotype composition and genomic relatedness. SimPlot and amino-acid comparison characterised divergence between sub-lineages. ResultsWhole-genome phylogenetics confirmed that two species B genotypes were detected in Nottingham University Hospitals between 2017-23: HAdV-B114 (n=56) and HAdV-B7 (n=12). HAdV-B114 was detected before and after COVID-19 restrictions, with an increase in 2022-2023, consistent with post-restriction expansion of a pre-existing lineage. Pairwise difference analysis identified 24 genomically identical B114 sequences; associated metadata argued against a single nosocomial transmission chain interpretation, with the pattern more consistent with repeated detection of a widely circulating community strain, with the possibility of localised onward transmission. Within the B114 clade, multiple monophyletic groups were identified by whole-genome phylogenetics. ConclusionsThis study adds whole-genome resolution to the existing UK HAdV-B114 data, demonstrates the utility of WGS for clinical adenovirus surveillance and shows the benefits of combining epidemiological data with genomic analysis.

Sample preparation remains a barrier for decentralized, swab-based molecular testing of tuberculosis (TB). Extraction-free workflows offer a simpler alternative, but systematic benchmarking against standard methods is lacking. We evaluated five novel lysis devices, BLINK Shaker Prototype, nPOC-BB, SPS-1, Truelyse, and Thermolyse, against a heat- and bead-beating reference method using contrived M. tuberculosis-spiked tongue and sputum swabs. The primary outcome was lysis efficiency, measured as the relative DNA recovery compared with the reference workflow. Secondary outcomes included nuclease inactivation, biosafety, and usability. In the reference buffer, lysis efficiencies ranged from 51-63% to 95-154% on tongue swabs and 12-54% to 280-644% on sputum swabs across the five devices. In proprietary buffers, performance varied more widely, with lysis efficiencies of 2-4% to 64-80% on tongue swabs and 1% to 94-398% on sputum swabs. Complete biosafety inactivation was achieved by three devices; two showed residual growth (<0.02%). Lysis efficiency of several devices met or exceeded the reference, supporting the feasibility of extraction-free workflows for TB diagnosis, with further optimization of buffer compatibility and biosafety profiles expected to enhance performance.

Macrolides remain the first-line antimicrobial treatment for pertussis, primarily caused by Bordetella pertussis. Emergence of macrolide-resistant B. pertussis in Australia and globally presents a growing concern for clinical management. As molecular diagnosis predominates for pertussis, limited numbers of cultures are available for antimicrobial susceptibility testing. The A2037G mutation in the 23S rRNA gene is most commonly associated with macrolide resistance in B. pertussis, yet no commercial molecular assay is available for its detection. In this study, we adapted and validated a previously published real-time PCR assay as a laboratory developed test (LDT) on the Panther Fusion(R) system using Open Access functionality to detect the macrolide resistance-associated mutation in B. pertussis from culture isolates and clinical specimens. Total turnaround time was ~2.5 h for the first result and 2-3 min for subsequent results in the same batch, with an estimated cost of AUD 18 per sample, excluding labour. A total of 30 samples were included in the validation panel to assess assay performance. All isolates and clinical specimens in the validation panel produced results concordant with the expected mutation status, demonstrating 100% accuracy, sensitivity and specificity. The assay demonstrated a limit of detection of 1.5 x 103 CFU/mL (22 CFU/reaction). This Panther Fusion(R) LDT provides a rapid and fully automated approach for molecular detection of the targeted mutation associated with macrolide resistance in B. pertussis. Its integration into routine molecular workflows as a reflex test following identification of B. pertussis may facilitate timely identification of resistant infections.

Broad-range RT-qPCR assays are essential for detecting highly diverse viruses within virus families, but designing suitable primers remains challenging because of extensive sequence diversity and biases in public sequence databases. Here, we present a semi-automated workflow that combines sequence clustering and varVAMP to streamline pan-RT-qPCR primer design. The workflow reduces sequence redundancy, improves the representation of viral diversity, and facilitates the identification of broadly reactive primer candidates. We demonstrate the workflow using the arboviral genera Orthoflavivirus and Alphavirus as proof-of-concept datasets, showing its applicability to large and genetically diverse viral sequence collections. The workflow provides a practical, scalable, and reproducible framework for developing broad-range primer candidates and may support surveillance, diagnostics, and preparedness for emerging or divergent viruses.

BACKGROUND Diagnosis of Tuberculosis (TB) from stool specimen using the Xpert MTB/RIF Ultra assay (Xpert-Ultra assay) is important to confirm diagnosis for presumptive TB patients who are unable to produce sputum. We evaluated diagnostic performance of the Xpert-Ultra assay in stool specimen among adult migrant population living in generalized HIV epidemic situation. METHODS A prospective, cross-sectional study was conducted at outpatient and inpatient departments of the Shoklo Malaria Research Unit (SMRU) clinics and Mae Tao Clinic (MTC) located in Thailand-Myanmar border area. Presumptive TB patients of any age who were registered between November 14, 2022, and May 23, 2023, were eligible for inclusion based on reported signs and symptoms and/or radiological findings. Using liquid MTB culture in sputum as reference standard, evaluation of diagnostic performance of the Xpert-Ultra assay in stool was performed, and it was also compared with performance of smear microscopy and Xpert-Ultra assay in sputum specimen. RESULTS Total 113 participants were included in the analysis; 9 (7.96 %) had human immunodeficiency virus (HIV) infection, and 31 (27.43%) had confirmed TB on culture results. Among these culture-confirmed TB cases, the sensitivity of Xpert-Ultra assay in stool specimen was 90.32 % (95% confidence interval [CI], 74.25% to 97.96%). Although the absolute difference in sensitivity of Xpert-Ultra assay in stool was 3.23 % lower than sputum (95% CI: -9.46 % to 3.00 %), there was no statistically significant difference between the two sample types. The specificity of Xpert-Ultra assay in stool specimen was 98.78% (95% CI, 93.39% to 99.97%) against culture-negative TB cases, giving an absolute difference of 1.22 % (95% CI, -1.16% to 3.59%) compared to sputum Xpert-Ultra assay. This method demonstrated that diagnostic performance was consistent with World Health Organization (WHO) target product profiles on low-complexity assays for detecting Mycobacterium tuberculosis (MTB). CONCLUSIONS The Xpert-Ultra assay in stool specimen can be considered as a potential, alternative method in diagnosis of presumptive pulmonary TB in adults when respiratory sample is difficult to collect.