Journal of Clinical Virology

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.

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.

Background SARS-CoV-2 genomic surveillance is vital for public health, but whole-genome sequencing (WGS) remains costly and inaccessible in many resource-limited settings. We developed and validated a multiplex real-time RT-PCR assay for rapid, economical detection of key mutations associated with variants of interest (VOI) and concern (VOC). Methodology Two multiplex mixes (M1, M2) targeting eight mutations in the ORF1a and Spike genes were designed. Analytical validation included sensitivity, specificity, reproducibility, and limit of detection (LoD) using WHO international standards and a respiratory pathogen panel. In parallel, an in silico analysis evaluated oligonucleotide efficacy against 10.4 million SARS-CoV-2 genomes from GISAID/NCBI, assessing inclusivity, target-site secondary structure (RNAalifold), and hybridization energy (Primer3Plus). Results The assay demonstrated 100% clinical sensitivity among samples with valid RT-PCR results (41/42 samples yielded interpretable results, with one inhibited sample excluded from sensitivity calculation), a LoD of 5.7 log10 IU/mL, and 100% analytical specificity against 32 non-SARS-CoV-2 respiratory pathogens. Six out of eight oligonucleotide sets showed >96% inclusivity; two sets exhibited reduced inclusivity (94.03%, 90.14%) and structural features potentially affecting binding against emerging variants. The assay enables direct identification of major VOCs (Alpha, Beta, Gamma, Delta, Omicron) and indirect detection of multiple VOIs (P.2, Epsilon, Kappa, Eta, Iota, Lambda). Conclusion This standardized multiplex assay provides a rapid, sensitive, and low-cost alternative for SARS-CoV-2 variant surveillance in Cuba and similar settings. The integration of experimental and in silico validation offers a robust, adaptable framework to sustain diagnostic accuracy amid viral evolution, optimizing the allocation of scarce sequencing resources.

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.

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.

Identification of acyclovir-resistant herpes simplex virus (HSV) infections is critical for directing appropriate antiviral therapy, particularly for immunocompromised patients where resistance rates can reach 30%. In 2020, the University of Washington Clinical Virology Laboratory launched the first clinical genotypic HSV drug resistance test in the United States. While genotypic testing offers significantly faster turnaround times than traditional phenotypic assays, interpretation depends on established mutational databases and remains challenging when novel variants are identified. Here, we retrospectively reviewed all HSV acyclovir resistance Sanger sequencing tests performed from January 2020 to November 2025 at this primary national reference laboratory. Mutations identified via clinical sequencing were compared against published databases of HSV UL23 mutations to determine their phenotypic effects. Over the nearly six-year study period, 136 samples were sequenced with a median turnaround time of 10.6 days. Among these, 65 samples (47.8%) harbored acyclovir resistance mutations, including 45 frameshift mutations. Notably, across the 100 samples (73.5%) displaying mutations not known to cause acyclovir resistance at the time of clinical testing, we identified 56 distinct mutations, including 23 without prior characterization. Our national experience demonstrates that genotypic testing accelerates actionable results in clinical practice and confirms that frameshift mutations remain a primary driver of acyclovir resistance. Furthermore, by uncovering these 23 novel variants, this work provides critical targets for future biochemical and phenotypic characterization of HSV UL23 mutations.

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.

Hepatitis C virus (HCV) is a hepatotropic virus that causes a spectrum of liver diseases. HCV genetic diversity influences transmission, pathogenesis, prognosis, and response to antiviral therapy. Moreover, resistance associated substitutions (RASs) challenged the existing antiviral treatment. However, data regarding circulating genotypes and RASs are limited in Ethiopia. Therefore, this study aimed to determine HCV genotypes and RASs among apparently healthy blood donors. This study used 97 archived anti-HCV-positive serum samples obtained from apparently healthy blood donors, collected in accordance with national routine blood collection practices. The partial HCV NS5B gene of 45 samples was amplified and sequenced using amplicon-based next-generation sequencing. Genotypes and subgenotypes were determined using NCBI BLAST, Geno2Pheno [hcv], and the Los Alamos HCV database, and a phylogenetic tree as a confirmation. The RASs were determined using Geno2Pheno [hcv]. Of the 97 anti-HCV- positive samples, 45 had detectable HCV RNA, of which 35 yielded high-quality sequences. These strains (n = 35) showed marked homogeneity in NS5B gene sequences: 34 were HCV genotype 4, all subgenotype 4d, and one was HCV genotype 2, subgenotype 2c. Most strains carried D310N, an RAS associated with ribavirin, while no RASs associated with sofosbuvir resistance were detected. There is HCV genotype homogeneity with HCV genotype 4, particularly HCV subgenotype 4d, predominating. No major sofosbuvir RASs were found, but ribavirin RAS D310N was common. Future studies should use broader geographic sampling and whole-genome sequencing to comprehensively characterize RASs, their distributions, clinical significance, and temporal trends, thereby guiding treatment strategies in Ethiopia. ImportanceHepatitis C virus infection remains a major global health concern. Its high genetic diversity influences viral evolution, transmission, and response to antiviral therapy. Despite the significant burden of hepatitis C virus infection in sub-Saharan Africa, molecular data on circulating genotypes, subgenotypes, and resistance associated substitutions remain limited. The significance of our research is in characterizing the genetic diversity and resistance associated substitutions among hepatitis C virus strains circulating in Ethiopia. These findings provide important insights into hepatitis C virus molecular epidemiology and may support genomic surveillance, optimization of treatment strategies, and improved understanding of viral evolution trends and patterns among blood donor populations.

Background: Rapid detection of viral pathogens can be challenging, especially when routine PCR fails. Conventional assays typically detect known viruses which are specifically targeted by the assay, which may result in the failure to identify novel or non-targeted viruses. Broad-range hybrid-capture sequencing enables unbiased detection of viruses, including those that are uncommon or divergent. Methods: We combined the TWIST Comprehensive Viral Research Panel (>3,000 virus species) with Oxford Nanopore Flongle sequencing for easy and quick viral genome detection. The workflow includes random-primed cDNA synthesis, dsDNA conversion, TWIST probe enrichment, and Nanopore sequencing. Performance was evaluated using the QCMD 2024 Viral Metagenomics EQA panel and one clinical sample. Results: All expected targets of the QCMD 2024 Viral Metagenomics EQA panel were detected; eight of thirteen viruses achieved [&ge;]90% genome coverage. The negative control showed no targeted viral reads. Mixed infections of DNA and RNA viruses were resolved accurately. The workflow from nucleic acid extraction to obtaining sequence data was completed within 3 days.

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.

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.

BACKGROUND: Half of positive blood cultures in pediatric emergency departments (PEDs) represent contaminants, driving unnecessary hospitalization, antibiotic exposure, and repeat visits. A clinical decision rule derived at CHU Sainte-Justine showed 99% sensitivity and 60% specificity for distinguishing bacteremia from contaminants but had not been externally validated. We sought to validate this rule in an independent pediatric cohort. METHODS: This retrospective diagnostic study spanned from January 2015 to May 2025 at a tertiary PED in Switzerland, using positive blood cultures from patients younger than 16 years. The four predictors (Gram-negative organisms or Gram-positive cocci in pairs or chains; time to positivity <17 hours; indwelling device; suspected osteoarticular infection) classified each case as low, moderate, or high risk. The primary outcome was bacteremia, adjudicated by two independent reviewers, based on organism identity and infectious disease specialist's assessment. Diagnostic accuracy was assessed with 95% CIs. RESULTS: Of 130 children enrolled (median age 3.8 years [IQR 0.9-9.9]; 61.5% male), 78 (60.0%) had true bacteremia. The rule yielded a sensitivity of 97.4% (95% CI, 91.0-99.7), specificity of 69.2% (95% CI, 54.9-81.3), positive predictive value of 82.6% (95% CI, 73.3-89.7), and negative predictive value of 94.7% (95% CI, 82.3-99.4). Both false-negatives were immunocompetent children with methicillin-susceptible Staphylococcus aureus bacteremia without indwelling devices. Among contaminants, 71% received antibiotics under usual care versus 31% classified as moderate or high risk by the rule. CONCLUSIONS: This first external validation supports the Sainte-Justine rule in a distinct pediatric population, preserving sensitivity with higher specificity. Multicenter validation is warranted before adoption.

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.

Background Human T-cell lymphotropic virus - 1 (HTLV-1) is the causative agent of Adult T-cell Leukaemia/Lymphoma (ATLL), a malignancy of CD4+ cells, and HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), a demyelinating disease. Globally, 10-20 million people are infected, though most remain asymptomatic and about 5% progress to severe disease. Transmission occurs mainly through breastfeeding, sexual contact, contaminated needles, and blood transfusion. In Ghana, evidence on the role of HTLV-1 in haematological malignancies remains scarce. Methods This was a cross-sectional study involving 200 patients with haematological malignancies (Acute Lymphoblastic Leukaemia - 4, Acute Myeloid Leukaemia - 6, Chronic Lymphocytic Leukaemia - 27, Chronic Myeloid Leukaemia - 63, Hodgkin Lymphoma - 21, Multiple Myeloma - 31, Myelodysplasia - 6, Myeloproliferative Neoplasm - 11) at the Haematology Day Care of the Korle-Bu Teaching Hospital. After informed consent was obtained, sera from study participants were tested for anti-HTLV-1 using MP Diagnostics GmbH ELISA immunoassay. Data were analysed using R software version 4.0.2 and SPSS version 31.0.0. Results The study population had a mean age of 49.1{+/-}17.7 years, with majority being females (n=109, 54.5%). Of the 200 samples, 16 (8.0%) were seropositive for HTLV-1, and these were detected in 4 males and 12 females. No statistically significant association was found between HTLV-1 infection and haematological malignancy (exact p = 0.061), sex (p=0.061), and history of blood transfusion (exact p= 1.000). Conclusion The findings show the seroprevalence of HTLV-1 of 8.0% among patients with haematological malignancies. Although there was no probable association between HTLV-1 and haematological malignancies, screening for HTLV-1 in patients with haematological malignancies may help to unravel the exact contribution in these conditions.

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.

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.

Background Current molecular diagnostics for Mycobacterium tuberculosis (MTB) require complex nucleic acid extraction procedures and laboratory infrastructure, limiting their use as point-of-care (POC) tests in resource-limited settings. To address these barriers, we developed an extraction-free one-pot CRISPR assay for rapid detection of MTB directly from minimally processed sputum specimens. Methods The assay integrates ambient-temperature chemical lysis, recombinase polymerase amplification, and CRISPR-Cas12a detection within a single closed-tube workflow. A conserved region of the MTB-specific IS6110 insertion sequence was targeted for detection. Analytical performance was evaluated using serially diluted MTB genomic DNA standards, followed by clinical validation using 100 archived sputum specimens, including 50 MTB-positive samples and 50 MTB-negative controls. Results The extraction-free assay detected MTB genomic DNA within 30 minutes and achieved an analytical limit of detection of 100 copies per reaction. In clinical validation, the assay correctly identified 48 of 50 MTB-positive specimens and 49 of 50 MTB-negative specimens, yielding 96.0% sensitivity and 98.0% specificity. Conclusions This study demonstrates the feasibility of extraction-free one-pot CRISPR-Cas12a detection of MTB directly from sputum specimens. By eliminating conventional nucleic acid extraction while maintaining high analytical sensitivity and diagnostic performance, the platform may facilitate future development of rapid molecular diagnostics for POC and resource-limited settings.

BACKGROUND In line with International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommendations for high-sensitivity cardiac troponin assays, analytical validation and reference limit assessments are required to confirm that an assay meets performance criteria. This study evaluated the analytical performance and established the 99th percentile upper reference limit (URL) for the SPINCHIP High-Sensitivity Cardiac Troponin I (SPINCHIP hs-cTnI) point-of-care assay. METHODS Analytical performance characteristics, including the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), were assessed. Additionally, 1,053 plasma samples and 1,055 whole-blood samples were used to determine the URL. Imprecision around the 99th percentile URL was evaluated as part of the analytical validation. High-sensitivity criteria were assessed by confirming measurable cTnI in [&ge;]50% of healthy individuals (n=432 plasma; n=431 whole blood) and achieving imprecision <10% at the 99th percentile (plasma, n=960; whole blood, n=480). RESULTS SPINCHIP hs-cTnI demonstrated a LoB of 0.3 ng/L; LoDs of 0.8 ng/L (plasma) and 0.9 ng/L (whole blood); and LoQs of 1.1 ng/L (plasma) and 1.4 ng/L (whole blood). The analytical measuring range was 1.1-9,000 ng/L. Imprecision at the common 99th percentile URL (14 ng/L) was 5.8%; for men (URL=16 ng/L) 5.6% and for women (URL=10 ng/L) 6.3%. Greater than 85.2% (94.0% and 76.1% in men and women, respectively) of healthy individuals showed measurable cTnI above the LoD. CONCLUSIONS The SPINCHIP hs-cTnI assay meets the IFCC high-sensitivity requirements, demonstrating <10% imprecision at the 99th percentile, reliable low-concentration precision and cTnI detection in more than half of healthy individuals.