Pseudomonas aeruginosa outbreaks frequently occur in intensive care units (ICUs). In particular, ICU patients requiring mechanical ventilation are vulnerable to P. aeruginosa ventilator-associated pneumonia, which is associated with high morbidity and mortality. Fast and accurate genotyping during the early stage is crucial to document and manage P. aeruginosa outbreaks at the ICU. In this study, we have evaluated the applicability of Oxford Nanopore whole-genome sequencing (WGS) for outbreak investigation and antimicrobial resistance (AMR) prediction. To evaluate whether a Nanopore-only WGS workflow was able to reproduce Illumina-confirmed transmission clusters, 19 P. aeruginosa isolates from ICUs at UZ Brussels (Belgium) that were previously sequenced with Illumina were sequenced using a Nanopore-only workflow based on the latest V14 chemistry, followed by bioinformatic analysis via BugSeq and MBioSEQ Ridom Typer. Although both bioinformatic platforms showed high concordance between Illumina and Nanopore data, MBioSEQ Ridom Typer yielded the lowest allelic distance (maximum one cgMLST allele), confirming all outbreak clusters. When applying the Nanopore-only workflow to longitudinally collected isolates, low genetic heterogeneity (maximum three cgMLST alleles) was observed between isolates from the same patient. WGS and subsequent outbreak analysis of 65 respiratory P. aeruginosa isolates collected from 38 different ICU patients across six Belgian hospitals during a 9-month period showed no intra- or inter-hospital transmission. When the Nanopore-only WGS data were used to predict AMR, there was high categorical agreement (95%) between AMR genotype and phenotype. These findings highlight the potential of Nanopore WGS as a rapid and accurate tool for outbreak investigation of P. aeruginosa.

Global tuberculosis (TB) incidence is slowly decreasing, partly because undiagnosed TB continues to fuel transmission. Point-of-care TB triage tests (TTTs) performed near a patient or at the site of patient care may help bridge this gap, but head-to-head comparisons of different host blood biomarkers within the same cohort are limited. In ambulatory adults in Uganda aged ≥15 years with any cough, fever, weight loss, or night sweats, we evaluated the diagnostic accuracy of C-reactive protein (CRP), monocyte-to-lymphocyte ratio (MLR), interferon-gamma-induced protein 10 (IP-10), and hemoglobin (anemia) head-to-head using a reference standard comprised of culture and/or Xpert MTB/RIF Ultra. Cut-offs of CRP and anemia were from World Health Organization (WHO) recommendations, MLR from the literature, and IP-10 from Youden's index. We included 623 participants (193 people living with HIV [PLWHIV]), of whom 111 (18%) were reference standard-positive. Sensitivity and specificity (95% CI) were 91% (84%-96%) and 78% (74%-81%) for CRP (≥10 mg/L); 72% (63%-78%) and 80% (77%-84%) for MLR (≥0.378); 86% (79%-92%) and 73% (69%-77%) for IP-10 (≥28.75); 69% (60%-78%) and 76% (72%-80%) for anemia (<12 g/dL [female], <13 g/dL [male]), respectively. At 90% sensitivity, specificity was 76% (58%-83%) for CRP; 62% (47%-74%) for MLR; 64% (47%-75%) for IP-10, and 28% (17%-51%) for anemia. There was no significant difference for any of the four TTTs in diagnostic odds ratio between participants with and without a history of previous TB, between smokers and non-smokers, and between males and females. However, MLR performed significantly (P = 0.001) worse in PLWHIV. Combining two index tests in parallel testing improved sensitivity up to 96% at the expense of reduced specificity. The applicable WHO minimum target product profile of 90% sensitivity and 60% specificity was met for CRP irrespective of HIV status, and for IP-10 and MLR when cut-offs were adjusted. Sex, prior history of smoking, and TB did not impact diagnostic performance. Parallel testing may be suitable in high TB prevalence settings, where minimizing missed cases and ensuring timely referral for confirmatory testing are priorities.IMPORTANCEThis study provides strong evidence that host blood protein biomarkers (C-reactive protein [CRP], interferon gamma-induced protein 10 [IP-10], and monocyte-to-lymphocyte ratio [MLR]) can serve as effective point-of-care tools for tuberculosis (TB) triage. CRP reliably meets the World Health Organization (WHO) high-sensitivity target product profile criteria across people living with HIV and HIV-negative populations, while optimized cut-offs enable IP-10 and MLR to achieve comparable performance, expanding feasible options where CRP is unavailable or unaffordable. The minimal influence of sex, smoking history, or prior TB supports broad, unstratified implementation. Evidence that parallel testing reduces missed TB cases is particularly relevant in high-burden settings. Clinically, these findings support integrating biomarker-based triage into routine screening to improve early case detection, although it may require further external validation to ensure cut-offs used are robust and not overly data-driven. For policymakers, the results justify incorporating biomarker-based point-of-care tests into national TB screening algorithms to accelerate diagnosis, improve referral efficiency, and reduce TB transmission.

Ceftiofur is a third-generation cephalosporin approved for veterinary applications and is used to treat Escherichia coli infections in dogs, particularly urinary tract infections (UTIs), in countries including China. Despite this use, there are no approved breakpoints for interpretation of ceftiofur susceptibility testing results. This absence limits the guidance that veterinarians need to effectively prescribe ceftiofur in dogs. It also limits the analysis of data from resistance monitoring and surveillance programs. This study aimed to establish ceftiofur interpretive categories and breakpoints that can be used to assess antimicrobial susceptibility testing results for canine E. coli isolates. Our methods included a definition of the wild-type cutoff (COWT) based on resistance phenotypes from two sources, deriving the pharmacokinetics/pharmacodynamics (PK-PD) cutoff (COPD) through PK-PD analyses and Monte Carlo simulations and determining the clinical cutoff (COCL) from a trial in dogs. Minimal inhibitory concentration (MIC) data were collected from China and the European database (European Committee on Antimicrobial Susceptibility Testing [EUCAST]). COWT was determined as 1 μg/mL. The COPD was 0.25 μg/mL. Through a canine cystitis trial, a COCL of 2 μg/mL was determined. Based on these findings and Clinical and Laboratory Standards Institute (CLSI)-recommended methods, we propose urinary tract infection-specific MIC breakpoints of ≤1 µg/mL (susceptible), 2 μg/mL (intermediate), and ≥4 µg/mL (resistant) and corresponding disk diffusion zone diameter breakpoints, determined by the error rate-bounded (ERB) method, of ≥23 mm (susceptible), 20-22 mm (intermediate), and ≤19 mm (resistant). The systemic breakpoints (non-urine) were determined to be ≤0.125 µg/mL (susceptible), 0.25 μg/mL (intermediate), and ≥0.5 µg/mL (resistant).IMPORTANCECeftiofur can be legally used, but laboratories and veterinarians lack canine-specific breakpoints to determine whether an isolate is susceptible or resistant. Without such criteria, test results can be inconsistent, treatment choices uncertain, and early signs of emerging resistance overlooked. This study fills that gap by defining evidence-based, canine-specific breakpoints for ceftiofur, particularly for urinary tract infections (UTIs). Adoption of these breakpoints will allow more consistent reporting by diagnostic laboratories, improve therapeutic decision-making for veterinarians, and provide a reliable basis for resistance surveillance. These advances strengthen the One-Health antimicrobial stewardship and help preserve the effectiveness of important antimicrobials for companion animals and the wider community.

Staphylococcus lugdunensis is a common skin commensal. However, S. lugdunensis found in multiple blood culture sets is concerning for life-threatening bacteremia. When a single set is positive, the National Healthcare Safety Network classifies S. lugdunensis as a common commensal (contaminant), while multiple publications found bacteremia and suggested an Infectious Diseases (ID) consultation. Here, a retrospective review spanning 9 years at a large health system evaluated the clinical significance of a single-positive S. lugdunensis blood culture when multiple cultures were collected. S. lugdunensis was found in blood cultures from 137 patients, who were stratified into single (n = 91) and multiple (n = 46) set-positive groups. The single-positive group was more likely to be deemed contaminants by clinicians, less likely to have a diagnosis of endocarditis or catheter-related bloodstream infection, and less likely to have an intravascular device (P < 0.01). Treatment occurrence and duration were also statistically different (P < 0.01). Single-positive cultures were statistically more likely to be polymicrobial (P = 0.04). Additionally, each case was reclassified as significant bacteremia, indeterminate bacteremia, or contamination using the Souvenir criteria. In single-positive cultures, significant bacteremia occurred less often (12.1% vs. 43.5%, P < 0.01), while contamination was more common (81.3% vs. 32.6%, P < 0.01). Of note, nearly all significant bacteremia cases involved an intravascular device (96.8%). Taken together, these findings suggest single-positive S. lugdunensis blood cultures are often contaminants, but certain clinical features, including intravascular devices, strongly predict bacteremia.

The co-circulation and rapid expansion of the genus Orthoflavivirus, including dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Yellow fever virus (YFV), and Japanese encephalitis virus (JEV), pose significant global health challenges. Developing inclusive pan-genus molecular diagnostics is hindered by high nucleotide divergence (>25-30%) and the computational limitations of traditional multiple sequence alignment in detecting conserved motifs across large data sets. To overcome these limitations, we developed a systematic alignment-free design pipeline that uses rigorous k-mer analysis and compacted De Bruijn graphs. We analyzed 11,846 RefSeq viral genomes to identify phylogenetically conserved, functionally relevant signatures within the Orthoflavivirus genus as a case study. The pipeline identified a conserved 600 bp region within the non-structural protein 5 gene, facilitating the design of a broad-spectrum TaqMan RT-qPCR assay. Analytical validation demonstrated limits of detection (LODs) of 1-10 copies/µL for DENV1-4, ZIKV, JEV, and YFV, with no cross-reactivity against non-target pathogens; WNV was consistently detected at 1,000 copies/µL in pilot experiments. In a clinical evaluation of archived samples, the assay achieved 97.33% overall accuracy. It demonstrated 100% sensitivity and specificity for DENV serotypes, yielding significantly earlier cycle threshold (Ct) values compared to a standard commercial kit, while ZIKV detection showed 100% specificity with 71.43% sensitivity. This study validates an alignment-free, k-mer-guided strategy for uncovering conserved diagnostic targets in highly variable viral genera, followed by local MSA-assisted primer-probe design. The resulting assay offers a robust tool for broad frontline surveillance, and the computational framework provides a scalable solution for future pandemic preparedness.IMPORTANCEMosquito-borne viruses like dengue, Zika, and West Nile pose a massive threat to global health. However, because these viruses are highly diverse and mutate rapidly, creating a single diagnostic test to detect all of them has been a challenge. Traditional software struggles to find shared genetic targets across such different viruses. To solve this, we developed a new computational approach. By analyzing over 11,000 viral genomes, we successfully identified a shared genetic signature of a group of mosquito-borne viruses. We used this discovery to create a single, highly accurate test capable of detecting multiple dangerous mosquito-borne viruses at once. This breakthrough provides a crucial, ready-to-use tool for frontline clinical diagnosis and global outbreak surveillance. More importantly, our strategy can be quickly adapted to design broad-spectrum tests for other rapidly mutating viruses, significantly strengthening our ability to prepare for and respond to future pandemics.

Whole-genome sequencing (WGS) accelerates drug-susceptibility testing (DST) in Mycobacterium tuberculosis (Mtb). Open-access software tools have become widely available, but the sources of real-world performance variability remain uncharacterized. We performed a systematic review and meta-analysis of the performance of open-access, independently validated WGS-based DST prediction tools. Bivariate random-effects meta-analysis was performed for six maintained tools (TBProfiler, Mykrobe, PhyResSE, MTBseq, GenTB, and SAM-TB). Bivariate meta-regression identified covariates associated with performance variation. Thirty-nine studies comprising 144,623 genomes were included. For the two most extensively validated tools, TBProfiler and Mykrobe, pooled rifampicin sensitivity was 95.4% (95% CI: 93.5-96.7) and 93.7% (92.0-95.1), with a specificity of 97.3% (95.7-98.3) and 97.0% (94.8-98.3), respectively. For isoniazid, the sensitivity was 92.0% (90.4-93.3) and 88.2% (85.5-90.4) and specificity 97.3% (96.0-98.2) and 97.5% (95.8-98.5). For ethambutol, the specificity was heterogeneous across tools (86.5%-95.4%); for pyrazinamide, the sensitivity varied widely (49.9%-80.6%). For fluoroquinolones, both sensitivity and specificity approached 90%, with heterogeneity. For newer agents, data scarcity precluded meaningful assessment. Meta-regression identified rifampicin resistance prevalence as the dominant predictor of decreased specificity across first-line drugs (β -1.5 to -3.6 on logit scale, false discovery rate [FDR] q < 0.05), while lineage composition effects were small and confounded. Current open-access WGS prediction tools achieve clinically useful accuracy as rule-out tests for rifampicin, isoniazid, and fluoroquinolone resistance. Predictive performance for second-line drugs is limited by data scarcity. Methodological limitations, including lineage bias, data leakage, and selective sampling, may undermine the tools' generalizability across diverse global tuberculosis populations.IMPORTANCETuberculosis remains a leading infectious disease killer worldwide. Whole-genome sequencing (WGS) of Mycobacterium tuberculosis offers the potential to rapidly predict drug resistance as a one-stop test, but the accuracy of the software tools used to interpret sequencing results has been inconsistently reported. This meta-analysis leverages the heterogeneity across 39 studies and 144,623 genomes to identify factors that drive inconsistencies in reported performance, providing context-specific guidance for clinical adoption. We show that most tools perform adequately as rule-out tests for resistance to the most important first- and second-line drugs but fall short of specificity targets. Importantly, we identify that the local burden of drug resistance in a study population is the dominant factor driving inconsistencies between reported performance estimates. These findings provide guidance for laboratories considering adopting sequencing-based resistance testing and specify priorities for future tool development and validation.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV) and is characterized by high mortality and non-specific clinical manifestations, which make early and accurate molecular diagnosis essential. This study aims to investigate whether reverse transcription droplet digital PCR (RT-ddPCR) provides improved diagnosis of SFTS compared to existing molecular diagnostic platforms. Detection limits of RT-ddPCR, reverse transcription quantitative PCR (RT-qPCR), and reverse transcription nested PCR (RT-nested PCR) were determined using RNA extracted from metrologically validated SFTSV nucleic acid reference material. Then, diagnostic performance metrics of each PCR method were compared using RNA from 29 SFTS patients and 29 disease controls with scrub typhus. Statistical analyses of key laboratory parameters were performed to identify markers significantly correlated with SFTSV load, as defined by RNA copy number, in early SFTS patients. RT-ddPCR showed the lowest detection limit of 3.82 copies/μL among the three PCR methods. The sensitivity of RT-ddPCR was 82.75% in clinical samples, which was higher than that of RT-qPCR at 72.41%. The specificity of RT-ddPCR was 100% compared to 89.66% for RT-nested PCR. Overall, RT-ddPCR demonstrated the highest diagnostic performance among the three PCR methods, with an accuracy of 91.38% and an area under the curve value of 0.91. A strong correlation between viral RNA loads and laboratory parameters was observed in the early stage of SFTS, particularly with high-sensitivity C-reactive protein, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and activated partial thromboplastin time. Our findings demonstrate that a novel molecular diagnosis method using RT-ddPCR shows improved diagnostic performance for detecting SFTSV RNA from SFTS patients and potentially contributes to accurate early diagnosis and enhanced clinical diagnosis.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is a high-mortality tick-borne infection caused by SFTS virus (SFTSV), and reliable molecular diagnosis is essential for patient care in endemic regions. Existing PCR-based assays for SFTS show limited sensitivity and specificity, particularly in patients with suspected or early-stage disease. Here, we describe the development and analytical validation of a reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection and quantification of SFTSV RNA from patient sera. By using metrologically validated viral nucleic acid reference materials and clinical samples, this assay demonstrates significantly improved sensitivity and overall diagnostic performance compared with conventional reverse transcription quantitative PCR and reverse transcription nested PCR. RT-ddPCR can provide clinical microbiology laboratories with a useful tool to enhance early and reliable molecular diagnosis of SFTS.

Helicobacter pylori infection is linked to gastritis, peptic ulcers, and gastric cancer. The urea breath test (UBT), while gold standard for active infection diagnosis, poses radioactive risks and equipment dependency. Fecal molecular detection offers a non-invasive alternative but requires validation. This multicenter study evaluates a novel fecal PCR assay (Cellrun PCR assay, 16S rRNA-targeted) against UBT and a commercial kit (comparison reagents). A multicenter trial enrolled 866 patients (May 2023-Sep 2024) from four hospitals. Of 777 valid samples, positive detection rates, diagnostic consistency (kappa), and clinical performance (sensitivity/specificity) were compared across UBT, Cellrun PCR assay, and comparison reagents. Positive rates were 44.02% (UBT), 43.89% (Cellrun PCR assay), and 44.14% (comparison reagents), with no significant differences (P > 0.05). Cellrun PCR assay exhibited near-perfect agreement with UBT (Kappa = 0.9713, P < 0.001) and matched comparison reagents' consistency (Kappa = 0.966, P < 0.001). Sensitivity and specificity for Cellrun PCR assay were 98.2% (AUC = 0.985), comparable to comparison reagents (AUC = 0.983; P = 0.614). Weak-positive agreement was 85.7%, while post-treatment sensitivity/specificity reached 94.74%/97.06% (AUC = 0.985). Cellrun PCR assay achieved 100% accuracy in interference samples (Kappa = 1.0). False negatives correlated with low bacterial loads or processing variability; false positives arose near detection thresholds. Cellrun PCR assay demonstrates UBT-comparable sensitivity and specificity, validating its equivalence to established molecular diagnostics for Helicobacter pylori detection.IMPORTANCEHelicobacter pylori infection is a major global health burden, linked to gastritis, peptic ulcers, and gastric cancer. Current gold-standard urea breath tests (UBTs) carry radioactive risks and require specialized equipment, limiting accessibility. Fecal molecular detection offers a non-invasive alternative, but existing assays need robust validation. Our study validates the novel Cellrun PCR assay, showing it matches UBT's accuracy and established commercial kits. This user-friendly, non-radioactive tool enhances diagnostic accessibility, especially in resource-limited settings, enabling early H. pylori detection and targeted treatment-critical for reducing associated morbidity and mortality. Its reliability supports broader clinical adoption, advancing global efforts to manage this prevalent infection.

Assessment of cytomegalovirus (CMV) cell-mediated immunity (CMV-CMI) using standardized, commercially available interferon-gamma release assays (IGRAs) may improve the clinical management of CMV infection in allogeneic hematopoietic stem cell transplant recipients (allo-HCT). Studies comparing the performance of CMV IGRA assays have been conducted in this setting, revealing frequent discrepancies between assays. Here, we compared the performance of a newly released VIDAS CMV IGRA (bioMérieux) and the T-SPOT. CMV (Oxford Immunotec) was evaluated in a cohort of 85 allo-HCT recipients who received letermovir prophylaxis. A total of 107 whole blood samples were collected at either day +100 (n = 53) or day +180 (n = 54) after allo-HCT and analyzed in parallel. A moderate qualitative agreement between assays was observed (Cohen's kappa coefficient, 0.47; 95% CI, 0.30-0.63). Discrepant results were obtained in 29 specimens, most of which were collected at day +100. A moderate correlation (rho, 0.54; P < 0.001) was observed between T-SPOT pp65-SPC and IFN-γ levels measured by the VIDAS CMV assay, whereas the correlation was weaker for T-SPOT IE-1 SPC and VIDAS IFN-γ levels (rho, 0.32; P < 0.001). The previously described booster effect of CMV DNAemia on the magnitude of CMV-CMI was captured by the VIDAS CMV assay. Neither the qualitative nor the quantitative results obtained with either IGRA predicted protection from CMV DNAemia following LMV discontinuation. While our data support the validity of the VIDAS CMV assay for the assessment of CMV-CMI in allo-HCT, they reinforce the idea that results from commercially available IGRA assays are not interchangeable, particularly in allo-HCT recipients with impaired CMV-CMI reconstitution.

Molecular syndromic panels such as the BioFire FilmArray Gastrointestinal Panel (BF-GIP) have been widely adopted for gastrointestinal illness diagnosis due to their fast turnaround times and broad pathogen coverage. Recently, the BF-GIP demonstrated increased rates of norovirus false-positive detections, prompting a Class II recall of more than two million tests in February 2024 and resulting in an estimated burden of more than a quarter-million false-positive norovirus results reported over the last 5 years. We examined the prevalence of BF-GIP norovirus false positives across four hospitals from December 2024 to June 2025. Among 185 BF-GIP norovirus-positive results confirmed with the BD MAX Enteric Viral Panel, the false discovery rate ranged from 31% to 74% across sites, with the highest rate seen at a specialized cancer care hospital. Deep sequencing of BF-GIP pouches (n = 42) confirmed the Noro-1 assay as the primary source of off-target amplification, identifying 78 off-target species-predominantly commensal stool bacteria-compared to only two species for the Noro-2 assay. Off-target species amplified by the Noro-1 assay were recovered from both false-positive and true-negative pouches, suggesting no single species accounted for the false-positive results. Partial primer complementarity at off-target loci and amplicon Tm values within the acceptable range support mispriming of gut microbiota as the underlying cause. False-positive pouches exhibited significantly higher crossing point values than true-positive pouches for both assays (Noro-1: 26.6 vs 11.1, P = 0.013; Noro-2: 30.0 vs 13.1, P < 0.001), consistent with low-level off-target amplification. These findings highlight the high false discovery rate of the Noro-1 assay, identify bacterial species involved in mispriming, and demonstrate the need to redesign this assay to ensure reliable testing and improved patient care.

Antibiotic-resistant Helicobacter pylori (H. pylori) represents a significant global challenge to the efficacy of eradication therapies, underscoring the urgent need for rapid and precise pretreatment resistance profiling. Traditional genotyping assays frequently neglect synonymous mutations, resulting in false-negative results and reduced diagnostic accuracy. In this study, we developed a single-tube, synonymous codon-optimized multiplex real-time PCR (qPCR) assay for the qualitative detection of H. pylori and the simultaneous identification of mutations associated with resistance to clarithromycin (23S rRNA gene) and levofloxacin (gyrA gene). The probe design was informed by clinical mutation data and synonymous codon usage patterns from 453 isolates, achieving over 98% coverage of naturally occurring variants. The assay exhibited high sensitivity (limit of detection: 2-20 copies/μL), excellent repeatability (coefficient of variation <5%), and robust specificity. Clinical validation using 316 gastric mucosal specimens demonstrated 97.47% concordance with a commercial infection detection kit and 100% and 98.08% agreement with Sanger sequencing for 23S rRNA and gyrA resistance genotyping, respectively. This assay effectively addresses the critical limitation posed by synonymous mutations, providing a rapid, reliable, and clinically accessible tool to guide personalized H. pylori eradication therapy and promote antimicrobial stewardship.

Overuse of respiratory pathogen profiles (RPPs) in immunocompetent patients offers limited clinical benefit and may contribute to unnecessary hospital costs. Evidence supports RPP use primarily in severely ill, immunosuppressed, or pediatric patients, while smaller, targeted tests are more appropriate for routine cases . The goal of this study was to evaluate the impact of a clinical decision support (CDS) intervention on RPP utilization and associated reagent costs. We reviewed RPP utilization across the health system and implemented a CDS tool with pre-approved clinical indications to guide appropriate RPP ordering or redirect testing to a smaller seasonal respiratory viral profile (SRVP). Initiation of an RPP order through the CDS tool presented the SRVP as the default, pre-selected option, with users prompted with specific built-in clinical indications before selecting whether to proceed with an RPP or SRVP. Data from inpatient and emergency department settings from January 2022 to January 2026 were used to assess RPP ordering practices pre- and post-CDS implementation. The impact of the CDS implementation on RPP orders was assessed using an interrupted time series analysis. Retrospective analysis revealed a year-to-year increase in RPP orders from 2022 to 2024. After CDS implementation, there was an immediate and significant decrease in RPPs compared to the pre-CDS period (-946.70, 95% CI: -1,320.2 to -573.2). This corresponded to $2.26 million in reagent cost savings. Overall, the introduction of the CDS successfully reduced the overutilization of large respiratory pathogen syndromic panels by encouraging the use of a SRVP when clinically appropriate.

Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging clustered regularly interspaced short palindromic repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes, unique markers of the S. flexneri 2a serotype, were designed to ensure specificity. Recombinase polymerase amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/µL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within 1 h. Both readouts allowed visual interpretation without specialized equipment. Clinical validation of the CRISPR-Cas12a assay demonstrated a diagnostic sensitivity of 95% and specificity of 98%, comparable to PCR when evaluated using the same clinical specimens. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assay's simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings.IMPORTANCERapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method's simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.

Malassezia pachydermatis is a zoophilic yeast found on the skin and in the outer ear canal of many mammals. It normally maintains a commensal lifestyle, but can cause dermatitis and otitis in predisposed hosts, particularly in atopic dogs. M. pachydermatis is genetically diverse, with strains clustering into at least three phylogroups based on molecular typing, a pattern we now confirm through whole-genome sequencing (WGS). Accurate species and strain-level identification is essential for understanding its epidemiology, pathogenic potential, and response to treatment. In this study, we established Fourier transform infrared (FTIR) spectroscopy as a rapid, cost-effective method for distinguishing M. pachydermatis from other Malassezia species, including M. globosa, M. furfur, M. restricta, and M. sympodialis. Within M. pachydermatis, FTIR spectroscopy resolved even closely related strains with high accuracy, producing clusters congruent with multilocus sequence typing (MLST)- and WGS-based phylogeny. The incorporation of an artificial neural network classifier further enhanced the discriminatory power, enabling robust and automated strain assignment. These findings demonstrate the potential of FTIR spectroscopy as a practical tool for large-scale epidemiological surveillance of M. pachydermatis and for clinical and veterinary applications where strain-level identification could inform treatment and management of Malassezia-associated diseases.IMPORTANCEMalassezia pachydermatis is a yeast that commonly inhabits the skin and ear canals of mammals but can cause dermatitis and otitis in predisposed hosts, especially dogs with allergies or immunosuppression. This species displays substantial genetic diversity, with strains falling into distinct phylogroups that may differ in their biology and clinical significance. Determining these differences has typically required advanced molecular or genomic methods, which can be costly and time-consuming. In this study, we demonstrate that Fourier transform infrared spectroscopy can rapidly and accurately distinguish M. pachydermatis from other Malassezia species and resolve genetic groups within the species in a way that reflects whole-genome relationships. This capability offers a practical tool for investigating the epidemiology and inter-/intraspecies diversity of M. pachydermatis and for guiding targeted management of Malassezia-associated diseases in both veterinary and, potentially, human medicine.

Whenever next generation sequencing is used in clinical and public health laboratories, rigorous quality control and quality assurance are key components of a quality management system. However, both the development and maintenance of a quality management system encompassing next generation sequencing present significant challenges, such as selection of appropriate sequencing platforms, complex workflows, allocation of time and resources, kit and reagent management, potential discontinuation of technical support, specialized bioinformatic workflows, compliance with regulatory and/or accreditation bodies, and the necessity of highly trained personnel. In an effort to address such challenges, the Centers for Disease Control and Prevention collaborated with public health partners to establish the Next Generation Sequencing Quality Initiative, which focuses on the development of a sequencing-focused quality management system. Here, we describe the ongoing efforts of the Next Generation Sequencing Quality Initiative to develop a foundational quality management system specific to next generation sequencing, as well as collaborations aimed to assist partners in addressing ongoing and emerging quality-related challenges associated with assay implementation in laboratories for surveillance and diagnostic applications.