Journal of Clinical Microbiology最新文献

{"title":"Enhanced genomic surveillance of enteroviruses reveals a surge in enterovirus D68 cases, the Johns Hopkins health system, Maryland, 2024.","authors":"Amary Fall, Julie M Norton, Omar Abdullah, Andrew Pekosz, Eili Klein, Heba H Mostafa","doi":"10.1128/jcm.00469-25","DOIUrl":"10.1128/jcm.00469-25","url":null,"abstract":"<p><p>This study reports increased Enterovirus D68 (EV-D68) circulation in 2024, re-establishing its biennial circulation cycle after its interruption during the COVID-19 pandemic. A total of 1,395 respiratory and cerebrospinal fluid (CSF) samples, positive for rhinovirus/enterovirus, collected from January to November 2024 were screened. EV-D68 was the predominant enterovirus detected (72.6% of EV-positive samples), with cases peaking in October, consistent with historical seasonal patterns. Demographically, children under 5 years were predominantly infected with EV-D68 (41.6% of cases). Phylogenetic analysis revealed the co-circulation of two EV-D68 subclades: B3 (71%) and A2 (29%). Subclade B3 was primarily associated with pediatric infections (median age: 5 years), while A2 was more common in adults (median age: 42 years). Comparative genomic analysis of the 2024 B3 genomes, along with genomes from 2018 and 2022, identified the emergence of four amino acid substitutions, including three in nonstructural proteins (3C: I597V; 3D: I950V, T2173A) and one in the structural protein VP2 (T145S). The six positive enterovirus CSF samples diagnosed in 2024 included six different types: EV-D68, E9, E30, E18, CV-A9, and CV-B1. Notably, the 2024 EV-D68 outbreak did not coincide with a reported increase in acute flaccid myelitis (AFM) cases. This study highlights the importance of EV-D68 genomic surveillance for monitoring EV-D68 evolution, given its association with severe respiratory disease and neurological complications. Enhanced surveillance is also critical for the early detection of the emergence of enteroviruses, such as EV-C105, identified in this study.IMPORTANCEEnteroviruses (EVs), a genus within the <i>Picornaviridae</i> family, are small, single-stranded RNA viruses linked to a wide spectrum of diseases, including neurological conditions. Despite their prevalence, they remain understudied. EV-D68 and EV-A71 have raised global public health concerns due to outbreaks of acute flaccid myelitis (AFM) and encephalomyelitis in North America and Europe. EVs exhibit high genetic variability, and viral evolution has been associated with changes in neurovirulence. Notably, EV-D68 epidemics in 2014, 2016, and 2018 coincided with spikes in AFM cases. However, AFM reports from 2019 to 2022 were low, even with a significant increase in EV-D68 infections in 2022. We developed an EV-D68 genomic surveillance workflow to investigate genotype associations with severe disease. Our previous work linked amino acid substitutions in 2018 strains to increased disease severity. This study analyzes EV-D68 evolution in 2024 and documents the return of its biennial circulation pattern following disruption during the COVID-19 pandemic.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0046925"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239729/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144258126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The utility of syndromic respiratory pathogen panels: the premise of flexible and customizable approaches.","authors":"Julie M Norton, Gaby Dashler, Eili Klein, Heba H Mostafa","doi":"10.1128/jcm.00313-25","DOIUrl":"10.1128/jcm.00313-25","url":null,"abstract":"<p><p>Extended respiratory panels have been limited to specific patient populations due to cost and inconclusive clinical utility. Customizing syndromic panels offers a way to balance clinical utility and available resources. In this study, we evaluated strategies and assessed the value of flexible, customized respiratory panels. A total of 200 specimens from symptomatic patients (December 2023 to September 2024), negative for SARS-CoV-2/Flu/RSV, were tested with the LIAISON PLEX Respiratory Flex Assay-an extended respiratory panel that offers flexibility in target selection. The study assessed additional diagnoses, correlations with institutional and state-wide pathogen prevalence, and whether customizable panels could optimize diagnostic yield. Sixty-two samples (31%) negative for SARS-CoV-2/Flu/RSV tested positive for other targets, primarily rhinovirus/enterovirus (60%), correlating with local and state prevalence. Weighted estimates for 18,373 symptomatic patients during the study period modeled a prevalence of 14.3% for rhinovirus/enterovirus, followed by HPIV-3, adenovirus, and coronavirus. During the study period, 6% of patients received the standard of care extended respiratory panel order after a negative SARS-CoV-2/Flu/RSV result, duplicating SARS-CoV-2/Flu/RSV testing. Leveraging a flexible feature could have resulted in an estimated staff time reduction of 5,545 minutes for a second swab collection and running a second test, in addition to the cost of running two different panels during a single encounter. Local respiratory pathogen prevalence data can guide target selection in customized panels. The inclusion of high-prevalence targets can increase the likelihood of diagnosis from 12% to nearly 30%. Flexibility in customizing targeted pathogen panels could enhance diagnostic value while conserving institutional resources.IMPORTANCERapid and accurate identification of pathogens causing respiratory tract infections can aid in guiding treatment decisions, reducing healthcare costs, and supporting real-time surveillance of infectious diseases within a community. Limitations of clinical utility beyond SARS-CoV-2/Flu/RSV are primarily driven by cost and the lack of specific treatment options. There is a need to balance clinical gaps with testing cost and diagnostic stewardship. In this study, we evaluated the utility of flexible, customized respiratory viral panels and reportable targets within a broader set of available targets in an extended respiratory panel.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0031325"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239722/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144258128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mismatch-corrected CHIKV CDC Trioplex assay oligos restore sensitive pangenotype viral detection.","authors":"Lika Aminata Diouf, Mignane Ndiaye, Diamilatou Balde, Agathe Shella Efire, Moussa Dia, Fatou Thiam, Manfred Weidmann, Oumar Faye, Idrissa Dieng","doi":"10.1128/jcm.00490-25","DOIUrl":"10.1128/jcm.00490-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0049025"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239721/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144266387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of <i>Mucorales</i> antigen in bronchoalveolar lavage samples using a newly developed lateral-flow device.","authors":"Julie Rousselot, Laurence Millon, Emeline Scherer, Nathalie Bourgeois, Sebastien Imbert, Damien Dupont, Anne Debourgogne, Danièle Maubon, Anne Pauline Bellanger, Christopher R Thornton","doi":"10.1128/jcm.00226-25","DOIUrl":"10.1128/jcm.00226-25","url":null,"abstract":"<p><p>A murine IgG2b monoclonal antibody, named TG11, binding to an extracellular polysaccharide antigen secreted by all <i>Mucorales</i> fungi has been recently developed and integrated into a lateral-flow device (TG11-LFD). The aim of this study was to establish the clinical performance of TG11-LFD on bronchoalveolar lavage (BAL) fluids for the diagnosis of mucormycosis. Thirteen BAL samples from 13 patients with mucormycosis, all of which tested positive for <i>Mucorales</i> qPCR (<i>Mucor/Rhizopus</i> [<i>n</i> = 5], <i>Lichtheimia</i> [<i>n</i> = 2], <i>Rhizomucor</i> [<i>n</i> = 5], and <i>Cunninghamella</i> [<i>n</i> = 1]), were used to assess the TG11-LFD. We also selected 49 BAL samples from 25 patients with other invasive fungal infections (IFI) (aspergillosis, <i>Pneumocystis</i> infection, candidiasis, and possible IFI) and from 20 patients without IFI for use as negative controls. The intensities of the test and control lines were recorded using a Cube reader. The diagnostic performance was assessed by analyzing the receiver operating characteristics (ROC) curve with the Jamovi software package (version 2.6.13). The area under the curve of the ROC curve was 0.739. Using a threshold value positivity ≤531 artificial units, the TG11-LFD test has a sensitivity and specificity of 76.92% and 75.51%, respectively, a positive predictive value of 45.45%, and a negative predictive value of 92.5%. In this study, we evaluated the performance of TG11-LFD on clinical samples for the first time and demonstrated its significant potential for enhancing the rapid detection of mucormycosis. Combining antigen detection with qPCR, as successfully applied in the diagnosis of aspergillosis, is likely to yield the most reliable diagnostic approach.IMPORTANCEMucormycosis is a severe emerging, invasive fungal disease caused by fungi in the order <i>Mucorales</i>. The mortality rate remains high at approximately 50%. Rapid diagnosis and prompt initiation of targeted treatment are associated with an improved prognosis. Gold standard diagnostic procedures have poor sensitivity and long turnaround times. <i>Mucorales</i> polymerase chain reaction in blood and respiratory samples has improved diagnosis, but this technique is not widely available due to high costs and the need for specialist equipment. A prototype lateral-flow device (TG11-LFD) incorporating a mouse monoclonal antibody, which binds to an extracellular polysaccharide antigen specific to <i>Mucorales</i> fungi, has been recently developed. In this study, we evaluated for the first time the performance of the TG11-LFD test on clinical bronchoalveolar lavage fluids for diagnosing mucormycosis. With 76.92% sensitivity and 75.51% specificity, this innovative, simple, and affordable approach shows great potential for improving the rapid diagnosis of mucormycosis.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0022625"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239717/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144325905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Performance of liquid Amies transport medium for the recovery of <i>Salmonella</i> and <i>Shigella</i> from stool compared to molecular testing.","authors":"Laura Collier, Morgan A Pence","doi":"10.1128/jcm.00453-25","DOIUrl":"10.1128/jcm.00453-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0045325"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239730/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144215991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated identification of <i>Salmonella</i> serotype using MALDI-TOF mass spectrometry and machine learning techniques.","authors":"Jun Ren, Jintao Xia, Mengyu Zhang, Chunhong Liu, Yuanyuan Xu, Jianing Wu, Yingzhu Li, Mingming Zhou, Shengjie Li, Wenjun Cao","doi":"10.1128/jcm.00037-25","DOIUrl":"10.1128/jcm.00037-25","url":null,"abstract":"<p><p><i>Salmonella</i> serotyping is essential for epidemiological studies and clinical treatment guidance. However, traditional serological agglutination methods are time-consuming, technically complex, and difficult to adopt at scale. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and cost-effective microbial identification technique, but it cannot be used to differentiate <i>Salmonella</i> serotypes. This study aims to integrate MALDI-TOF MS with machine learning algorithms to develop and validate a model for <i>Salmonella</i> serotype identification, improving efficiency and simplifying workflows. A total of 692 <i>Salmonella</i> isolates from Children's Hospital, Zhejiang University School of Medicine (ZUCH) and Wanbei Coal-Electricity Group General Hospital (WCGH) were analyzed using MALDI-TOF MS, generating 2,048 spectra. The ZUCH data were randomly divided into training and internal validation sets. The WCGH data were used as an external validation set. Ten machine learning algorithms were evaluated for their ability to identify eight <i>Salmonella</i> serotypes (B, C1, C2/3, D, E, Not A-F, <i>Salmonella</i> Typhimurium, and <i>Salmonella</i> Enteritidis). From 192 initial features, 16 features were selected for the final model construction. XGBoost demonstrated the best discriminative ability (area under the receiver operating characteristic curve [AUC] = 0.9898, sensitivity = 0.88, and specificity = 0.98) for the training set. The streamlined XGBoost model achieved AUCs of 0.9662 and 0.9778 for the internal and external validation sets, respectively, accurately identifying <i>Salmonella</i> serotypes. To enhance usability, the model was deployed as a Streamlit-based application, facilitating interaction and broader application. MALDI-TOF MS combined with XGBoost provides a fast and accurate method for <i>Salmonella</i> serotype identification, offering an efficient solution for laboratory diagnostics and epidemiological studies.</p><p><strong>Importance: </strong><i>Salmonella</i> serotyping is vital for outbreak tracking and clinical guidance, but traditional methods are slow and laborious. This study combines matrix-assisted laser desorption ionization-time of flight mass spectrometry with machine learning (XGBoost) to enable rapid, accurate, and cost-effective serotyping. The streamlined model performed excellently in validation and was deployed as a user-friendly Streamlit app, enhancing usability. This innovation simplifies workflows, reduces diagnostic time, and supports scalable use in clinical and public health settings, improving outbreak response and epidemiological research.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0003725"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239726/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144266386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Point-Counterpoint: Cascade reporting-useful tool to support antimicrobial stewardship, or dangerously misleading.","authors":"Joseph L Kuti, Joseph D Lutgring, Patricia J Simner, Samia N Naccache, Emily L Heil","doi":"10.1128/jcm.01708-24","DOIUrl":"10.1128/jcm.01708-24","url":null,"abstract":"<p><p>Addressing antimicrobial resistance requires multi-disciplinary action, including from the clinical laboratory. Cascade reporting of the antimicrobial susceptibility test (AST) is a strategy used by some laboratories to nudge clinicians toward the use of more narrow-spectrum antimicrobials. Cascade reporting involves suppression of broader-spectrum antimicrobials if the narrower-spectrum first-line antimicrobials show in-vitro susceptibility. Studies have shown that cascade reporting can reduce use of broad-spectrum antimicrobials and reduce antimicrobial resistance rates. However, implementing cascade reporting can be complex, and some question the effectiveness on impacting long-term prescribing behaviors. Furthermore, there are concerns surrounding compliance and the possible negative impact on public health surveillance for antimicrobial resistance. In this article, experts weigh in on the benefits and risks associated with implementing AST cascade reporting. General consensus is that cascade reporting is a benefit to antimicrobial stewardship, but protocols need careful implementation to minimize risk to patients and public health.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0170824"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12279290/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144317049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction for Weyand et al., \"Fatal interactions: pneumonia in bighorn lambs following experimental exposure to carriers of <i>Mycoplasma ovipneumoniae</i>\".","authors":"Logan K Weyand, Brandi L Felts, E Frances Cassirer, Jonathan A Jenks, Daniel P Walsh, Thomas E Besser","doi":"10.1128/jcm.00426-25","DOIUrl":"10.1128/jcm.00426-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0042625"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144258125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inter-laboratory variability in cytomegalovirus DNA quantification: implications for standardization and clinical monitoring.","authors":"D Boutolleau, A-S L'Honneur, R Germi, B Chanzy, C Archimbaud-Jallat, C Rzadkowolski, J B Raimbourg, D Gauthier, V Thibault","doi":"10.1128/jcm.01911-24","DOIUrl":"10.1128/jcm.01911-24","url":null,"abstract":"<p><p>Cytomegalovirus (CMV) infection monitoring is a key element in the management of immunocompromised patients. CMV DNA quantification in plasma or whole blood is the best indicator for clinicians to adjust immunosuppressive or antiviral therapies. Despite the availability of internationally standardized material, the commutability of CMV quantification results across laboratories remains inadequate. To assess inter-laboratory variability in CMV DNA quantification, we conducted a blinded study in seven independent laboratories. Each participant received a panel of 92 specimens for CMV quantification using their routinely used standard platform. While quantifications were highly correlated and reproducible, large discrepancies were observed with differences up to 1.45 log₁₀ IU/mL between techniques for identical specimens. However, quantification scattering was lower for the World Health Organization (WHO) international standard or a commercially tested control (interquartile range = 0.129) than for clinical specimens (0.469; <i>P</i> = 0.0142). Blind quantification of the WHO or the commercial standard indicated that all techniques, except for fully integrated platforms, did not align well with the expected values, and most platforms tended to quantify specimens and standards differently. Recalibration of all platforms against the same standard improved the spread of results, but differences of up to 1.19 log₁₀ IU/mL remained for the same specimens. Achieving commutability in CMV quantification remains an elusive goal. Efforts should focus on improving both the assay calibrators and the run controls, which currently do not appear to simulate the unique characteristics of circulating CMV in patients. Until this is resolved, each transplanted patient should be consistently monitored by the same laboratory on the same platform.IMPORTANCEOur conclusions support previous work on this topic describing the diversity of circulating cytomegalovirus (CMV) DNA forms and the difficulties in standardizing CMV viral load (VL) measurement. The inter-assay reproducibility of CMV VL measurement is primarily influenced by the extraction procedure and the amplicon size generated by the technique. Viral standards generated from cell culture supernatant do not reflect circulating CMV forms from patient samples. We highlight the need to develop a new international standard that better reflects the circulating forms of CMV and demonstrate the risk of tracking a patient's CMV VL in different laboratories.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0191124"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239724/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144225624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification and quantification of <i>gyrA</i> variants in fluoroquinolone-resistant <i>Mycobacterium tuberculosis</i> in a MeltArray reaction.","authors":"Chunxia Tu, Biyi Su, Yunfan Xiong, Zihan Xia, Chunlin Xiang, Yaoju Tan, Ye Xu, Qingge Li","doi":"10.1128/jcm.00146-25","DOIUrl":"10.1128/jcm.00146-25","url":null,"abstract":"<p><p>Fluoroquinolones (FQs) resistance in <i>Mycobacterium tuberculosis</i> (MTB), primarily driven by mutations in the quinolone resistance-determining region (QRDR) of <i>gyrA</i>, poses a major concern in treating for multidrug-resistant tuberculosis (MDR-TB). These QRDR mutations are known to confer varying levels of resistance, leading to differences in treatment outcomes. Here, we introduced the MeltArray MTB/FQs assay, a multiplex PCR method that detected 11 <i>gyrA</i>-QRDR mutations and quantified their fractions via a polynomial regression algorithm-based formula, enabling mutation identification and quantification within 3 h in one reaction. This assay, with a limit of detection (LOD) of 50 copies/reaction, could identify mixtures containing 5% mutant gDNA across 50 to 50,000 copies/reaction. Clinical evaluation of 442 culture samples displayed 95.23% sensitivity and 99.32% specificity compared with phenotypic antimicrobial susceptibility testing (pAST). Evaluation of 121 paired sputum-culture samples revealed sensitivities of 90.32% in sputum samples and 95.24% in culture samples, both with specificities of 100%, when compared with pAST. Further evaluation of 285 sputum samples showed 93.75% positive percent agreement (PPA) and 98.10% negative percent agreement (NPA) in comparison with the MeltPro MTB/FQs kit (Zeesan Biotech, China). All mutant samples identified by MeltArray MTB/FQs but classified as susceptible by pAST or as wild type by MeltPro MTB/FQs were confirmed through Sanger sequencing and droplet digital PCR (ddPCR). The formula for predicting mutation fraction (MUT%) showed accuracy rates of 88.00%, 88.89%, and 83.33% in the training, validation, and test data sets, respectively, when compared with ddPCR results. Overall, MeltArray MTB/FQs assay offers an upgraded alternative for routine FQs resistance monitoring.IMPORTANCERising FQs resistance has driven the spread of pre-extensively drug-resistant tuberculosis (pre-XDR-TB), challenging global tuberculosis (TB) control efforts. Conventional molecular assays for FQs resistance often cannot distinguish between low-level and high-level resistance mutations or detect low-fraction heteroresistant populations. In this study, we established a MeltArray MTB/FQs assay that can identify all the 11 critical mutations in the <i>gyrA</i>-QRDR with a LOD of 50 copies/reaction, enabling direct, culture-independent analysis of sputum samples. By using an algorithm to quantify mutations at levels as low as 5% in mixtures, MeltArray achieved both mutation identification and quantification within 3 h in a reaction, thus providing a powerful tool for early detection and precise management of pre-XDR-TB.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0014625"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12239727/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}