Journal of Clinical Microbiology最新文献

{"title":"Evaluation of fully automated chemiluminescent enzyme immunoassays for hepatitis B core-related antigen components, phosphorylated and non-phosphorylated hepatitis B core antigens: clinical significance and dynamics during hepatitis B e antigen seroconversion.","authors":"Takanori Suzuki, Chiharu Ohue, Osamu Arai, Yuka Inose, Katsuya Nagaoka, Shintaro Ogawa, Takako Inoue, Kentaro Matsuura, Katsumi Aoyagi, Shintaro Yagi, Yasuhito Tanaka","doi":"10.1128/jcm.00385-25","DOIUrl":"10.1128/jcm.00385-25","url":null,"abstract":"<p><p>To assess the performance and clinical relevance of three assays-hepatitis B core-related antigen (HBcrAg), hepatitis B core antigen (HBcAg), and phosphorylated HBcAg (pHBcAg)-in quantifying HBcrAg, a critical biomarker of hepatitis B virus (HBV) infection, fully automated chemiluminescent enzyme immunoassays (CLEIA) for two HBcAg variants were developed. Cutoff values for HBcAg and pHBcAg assays were established at 2.50 and 2.10 LogU/mL, respectively. The strong correlations among components (<i>r</i> = 0.896-0.970) were observed by using purchased 100 plasma samples. When the dynamics of HBcrAg components were analyzed by using a series of hepatitis B e antigen (HBeAg) seroconversion panel, alongside correlations with HBV markers, distinct dynamics and molecular profiles were observed. For the assessment of the clinical relevance of these assays, serum samples were obtained from 102 HBV-infected patients treated with nucleos(t)ide analog (NA). Both HBcAg and pHBcAg levels were significantly reduced during NA therapy, but in different patterns, suggesting HBV activity independent of HBV DNA and persistent HBcrAg components derived from pc-mRNA. OptiPrep density gradient ultracentrifugation analysis identified pHBcAg as the dominant component in high-density fractions. Immunoprecipitation and Western blotting confirmed that HBcAg components were predominantly enveloped by hepatitis B surface antigen (HBsAg), with pHBcAg identified as the major component of empty viral particles. Although the limitation in sample size in this study, the revealed distinct dynamics of HBcAg, pHBcAg, and HBcrAg during seroconversion and treatment suggest these assays could serve as independent biomarkers for monitoring intrahepatic HBV activity and treatment efficacy.IMPORTANCEThis study evaluates novel fully automated chemiluminescent enzyme immunoassay (CLEIA) systems for hepatitis B core antigen (HBcAg) and phosphorylated HBcAg (pHBcAg) and identifies pHBcAg as the predominant component of hepatitis B virus (HBV)-derived empty viral particles, challenging previous assumptions and providing new insights into HBV biomarkers. HBcAg and pHBcAg show distinct dynamics in hepatitis B e seroconversion and nucleos(t)ide treatment from the other biomarkers including hepatitis B core-related antigen (HBcrAg), HBV RNA, and HBV DNA. The developed CLEIA systems for HBcAg and pHBcAg show promise as tools for monitoring intrahepatic HBV activity, immune clearance, and noninfectious viral replication. Incorporating these biomarkers into clinical practice could refine HBV management strategies, improve reactivation risk prediction, and advance precision medicine approaches.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0038525"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421862/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873439","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 Brown-Elliott et al., \"Utility of Sequencing the <i>erm</i>(41) Gene in Isolates of <i>Mycobacterium abscessus</i> subsp. <i>abscessus</i> with Low and Intermediate Clarithromycin MICs\".","authors":"Barbara A Brown-Elliott, Sruthi Vasireddy, Ravikiran Vasireddy, Elena Iakhiaeva, Susan T Howard, Kevin Nash, Nicholas Parodi, Anita Strong, Martha Gee, Terry Smith, Richard J Wallace","doi":"10.1128/jcm.00887-25","DOIUrl":"10.1128/jcm.00887-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0088725"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421867/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956114","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":"A novel single-tier serologic test to diagnose all stages of Lyme disease.","authors":"Andrew E Levin, Gary P Wormser, Elizabeth J Horn, Nadezhda Karaseva, Drew Miller, Hunter Kellogg","doi":"10.1128/jcm.00483-25","DOIUrl":"10.1128/jcm.00483-25","url":null,"abstract":"<p><p>Lyme disease, a bacterial zoonosis, is the most commonly reported vector-borne disease in the United States. Laboratory diagnosis has relied on a two-tier serologic approach, originally comprising an ELISA, or another first-tier assay, followed by separate IgG and IgM immunoblots to confirm a positive first-tier result. This standard two-tier testing (STTT) approach provides high specificity, but at the cost of low sensitivity in early Lyme disease. Recent studies have shown that a modified two-tier (MTTT) testing approach, in which a second ELISA replaces the immunoblot, can provide an increase in test sensitivity without a loss of specificity. Nevertheless, neither STTT nor MTTT is considered sensitive enough for diagnosing patients with erythema migrans, the most common clinical manifestation of early Lyme disease. We have developed a novel ELISA methodology termed \"Hybrid Lyme ELISA\" for single-tier Lyme antibody detection, which relies on the simultaneous binding of individual antibody molecules to the <i>Borrelia burgdorferi</i> surface protein VlsE and to the C6 peptide derived from it. This dual binding requirement builds exceptionally high specificity into the assay, eliminating the majority of non-specific antibody interactions. In this study, the single-tier Hybrid Lyme ELISA was shown to provide greater sensitivity, but with equivalent specificity, to both STTT and MTTT. In addition, given the >90% sensitivity of the Hybrid Lyme ELISA in patients with erythema migrans, this assay may not only transform serologic testing from two-step to single-step testing, but may also provide a means for the first time to diagnose patients with erythema migrans.IMPORTANCEThe diagnosis of Lyme disease, a tick-borne spirochetal infection caused by <i>Borrelia burgdorferi</i> sensu lato, is subject to two major limitations: the need for a two-tier serologic testing algorithm to provide adequate specificity, and the low sensitivity of this algorithm in practice for detection of early Lyme disease manifesting with the erythema migrans skin lesion, the most common clinical manifestation. This study presents the first description of a new assay, the Hybrid Lyme ELISA, which demonstrates sensitivity high enough to potentially diagnose over 90% of patients with erythema migrans, and specificity high enough to preclude the need for a second-tier test. These test characteristics suggest the potential for the Hybrid Lyme ELISA to be the first single-tier serologic test suitable for laboratory diagnosis of all stages of Lyme disease.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0048325"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421848/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955951","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":"Establishing the reference broth microdilution MIC method for cefepime-taniborbactam.","authors":"Adam Belley, Susan M Cusick, Dan C Pevear, Laura Koeth, Jeanna DiFranco-Fisher, Nimmi Kothari, Stephen Hawser, Greg Moeck","doi":"10.1128/jcm.00661-25","DOIUrl":"10.1128/jcm.00661-25","url":null,"abstract":"<p><p>The investigational β-lactam/β-lactamase inhibitor combination cefepime-taniborbactam is intended as therapy for serious infections caused by Gram-negative pathogens resistant to third-generation cephalosporins and carbapenems. Establishing a susceptibility testing reference method for cefepime-taniborbactam that conforms to the Clinical Laboratory Standards Institute (CLSI) M07 and International Standards Organization 20776-1:2019 standards is necessary to inform patient care. This study describes the reference broth microdilution MIC method for cefepime-taniborbactam (taniborbactam fixed at 4 µg/mL). In a CLSI M23 Tier 2 study that included nine clinical microbiology laboratories, the CTX-M-15-producer <i>Escherichia coli</i> NCTCC 13353 was determined to be appropriate for routine quality control (QC) as ranges for cefepime (≥64 µg/mL) and cefepime-taniborbactam (0.12 to 1 µg/mL) were non-overlapping, thereby simultaneously controlling for cefepime antibacterial activity and taniborbactam β-lactamase inhibition. Of the cefepime-taniborbactam MIC results obtained, 99.6% (269/270) were within the identified QC range; similarly, 100% (79/79) and 98.0% (98/100) of the QC values from a surveillance study and from the two central microbiology laboratories of the CERTAIN-1 Phase 3 clinical study (clintrials.gov identifier NCT03840148) were in range, respectively. Modifications to the standard medium (pH, cation content, or supplementation with human serum, albumin, polysorbate-80, or pulmonary surfactant) or assay parameters (inoculum density, incubation duration, and atmosphere) revealed that only inoculum titers (e.g., ≥5 × 10⁶ CFU/mL) exceeding the CLSI M07 standard (2-8 × 10⁵ CFU/mL) increased MIC values above the QC range. These results demonstrate the robustness and reliability of the cefepime-taniborbactam broth microdilution MIC reference method when performed following the approved standards.</p><p><strong>Importance: </strong>This study focuses on a new antibiotic combination called cefepime-taniborbactam that is being developed to treat serious infections caused by bacteria that are often resistant to current treatments. To make sure this new antibiotic combination can be used safely and effectively once it has been approved for clinical use, we developed a standardized laboratory method to measure its activity against certain bacteria that are widely used during quality control testing. The method was assessed in multiple labs and proved to be reliable, accurate, and consistent. It also held up well under different testing conditions, showing that it is a dependable tool for guiding treatment decisions. This is an important step in meeting the challenge of antibiotic-resistant infections since it will help clinicians evaluate cefepime-taniborbactam as a potential treatment option as they strive to improve the care of patients suffering from serious infections.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0066125"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421830/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144873438","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":"Will we miss specific IgA detection for the diagnosis of congenital toxoplasmosis? A French retrospective monocenter study on 483 infants.","authors":"Lya Hamet, Hélène Guegan, Sorya Belaz, Jean-Pierre Gangneux, Florence Robert-Gangneux","doi":"10.1128/jcm.00379-25","DOIUrl":"10.1128/jcm.00379-25","url":null,"abstract":"<p><p>To estimate the performance of anti-<i>Toxoplasma</i> IgA assay for the diagnosis of congenital toxoplasmosis, a retrospective monocenter study was conducted comparing serological results obtained in the framework of routine diagnosis workup. All infants born to mothers infected with <i>Toxoplasma gondii</i> during pregnancy from 2010 to 2023 with at least 6 months of serological follow-up were included. Four hundred and eighty-three cases (1,171 sera) were included, of which 56 infants (11.6%) were infected. Twenty out of 60 infants (33.3%) with positive IgA were not infected. The sensitivity to detect IgA antibodies in infected neonates was 71.4%. The specificity was 97.3%. The mean time to detect IgA was 7.7 ± 13.0 days in infected neonates. Anti-<i>Toxoplasma</i> IgA was the earliest positive serological test in only two cases (5.0%) but turned negative at 1 month in the absence of specific treatment, suggesting non-specific detection. IgA was associated with anti-<i>Toxoplasma</i> IgM, neosynthetized IgG or IgM on comparative Western blotting (WB), or both IgM and neosynthetized IgG or IgM on WB, in 12 cases (30.0%), 2 cases (5.0%), and 19 cases (47.5%), respectively. Sixteen infants with no IgA after birth were diagnosed via neosynthetized IgG or IgM on comparative WB and/or IgM (<i>n</i> = 10), or via PCR on amniotic fluid (<i>n</i> = 5), or persistent IgG (<i>n</i> = 1). Our study suggests that anti-<i>Toxoplasma</i> IgA is not a critical serological parameter for the diagnosis of congenital toxoplasmosis, and the recent withdrawal of commercialized reference anti-<i>Toxoplasma</i> IgA assays should not affect patient care.IMPORTANCEThis study will help clinical microbiologists estimate the impact of the withdrawal of anti-<i>Toxoplasma</i> IgA reference assays for the diagnosis of congenital toxoplasmosis and will contribute to actualize the recommendations for laboratory diagnosis.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0037925"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421870/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816829","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":"Overview of changes in the Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing: M100 32nd and 33rd editions.","authors":"Audrey N Schuetz, Andrea Ferrell, Janet A Hindler, Romney Humphries, April M Bobenchik","doi":"10.1128/jcm.01623-23","DOIUrl":"10.1128/jcm.01623-23","url":null,"abstract":"<p><p>The Clinical and Laboratory Standards Institute Subcommittee on Antimicrobial Susceptibility Testing (AST) publishes annual updates to the M100 <i>Performance Standards for Antimicrobial Susceptibility Testing</i>. This important document contains key information critical to the laboratory's performance of accurate and current AST. This minireview will highlight and provide supporting data for major changes in the M100 32nd and 33rd editions published in February 2022 and March 2023, respectively. New and revised breakpoints for gram-negative and gram-positive organisms are explained. Proper use of the restructured M100 Table 1 guidance for antimicrobial agents to be considered for testing and reporting is outlined. Reporting guidance for multidrug-resistant gram-negative bacilli is discussed. Other topics within this minireview include additional agents to test and report for direct blood disk diffusion of gram-negative bacilli and quality control range changes and troubleshooting. The minireview concludes with other issues under consideration by the AST Subcommittee.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0162323"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421849/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144794553","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 evaluation of plazomicin susceptibility testing of Enterobacterales on VITEK 2 and VITEK 2 Compact Systems.","authors":"Edith Csiki-Fejer, Maria Traczewski, Gary W Procop, Thomas E Davis, Meredith Hackel, Gilles Zambardi","doi":"10.1128/jcm.00449-25","DOIUrl":"10.1128/jcm.00449-25","url":null,"abstract":"<p><p>The objective of the present study was to evaluate the VITEK 2 antimicrobial susceptibility test (AST) for Gram-negative (GN) plazomicin performance using the VITEK 2 and VITEK 2 Compact Systems in clinical settings and to demonstrate its equivalence to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) technique. The clinical study conducted at four external sites followed the requirements detailed in the US Food and Drug Administration (FDA) and International Standards Organization (ISO) guidance documents and included clinical, challenge, quality, and reproducibility studies. In this multisite study, a total of 979 Enterobacterales isolates were tested. The VITEK 2 card minimum inhibitory concentration (MIC) results were compared with the reference BMD MIC results. The performance following ISO criteria indicated Essential Agreement (EA) = 97.8% and showed that VITEK 2 plazomicin MIC results for Enterobacterales tend to be in exact agreement when compared with the CLSI BMD reference method, except for <i>Escherichia coli,</i> when MICs tend to be in exact agreement or at least one doubling dilution lower. The analysis was also performed following the FDA criteria using the breakpoints defined by the FDA: ≤2 susceptible (S), 4 intermediate (I), and ≥8 resistant (R) for Enterobacterales. The analysis showed the following performance: EA = 98.7%, category agreement (CA) = 99.4%, and major errors (ME) = 0.1%, with no very major errors (VME) present. There are three species, <i>Klebsiella pneumoniae</i>, <i>Escherichia coli,</i> and <i>Serratia marcescens,</i> for which the trend is ≥ 30% and therefore addressed as a note in the US label. The plazomicin test met the ISO and FDA criteria of ≥95% reproducibility and ≥95% quality control (QC) results within acceptable ranges for QC organisms.IMPORTANCEThe VITEK 2 AST-GN plazomicin test is a new, automated alternative to the BMD reference method for determining minimum inhibitory concentrations (MIC) of Enterobacterales, expanding the range of automatic AST testing.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0044925"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144760210","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":"All you need to know about equipment validation for sterility testing.","authors":"James E T Gebo, Kayla M Feehely, Chase E Lattimore, Dylan P Mogavero, Anna F Lau","doi":"10.1128/jcm.01477-24","DOIUrl":"10.1128/jcm.01477-24","url":null,"abstract":"<p><p>Product sterility testing requests are becoming increasingly common in clinical microbiology laboratories due to the rapid development of advanced therapies. Most laboratory directors are hesitant to bring on such tests because they fall under current Good Manufacturing Practices (cGMP) regulated by the United States Food and Drug Administration (FDA), where expectations differ from clinical requirements (regulated by the Centers for Medicare and Medicaid Services). When considerations are made for cGMP testing in the clinical lab, most focus is placed on analytical test validation. In this mini-review, we provide an overview of one critical element within the cGMP quality system-validation of equipment, software, and systems through installation, operational, and performance qualification (IQ, OQ, and PQ or IOPQ). This terminology is not common in clinical laboratories, and the IQ and OQ portions are often overlooked, not performed, and/or not documented, although phase II CAP requirements exist (COM.30550 and COM.30575). This mini-review will provide an overview of the IOPQ framework, what is included and how it differs from CAP requirements, important considerations for an IOPQ, and a summary of FDA citations relating to equipment validation. We provide examples using blood culture systems, controlled temperature units (CTUs; e.g., incubator), and the laboratory information management system, given their likelihood for use in cGMP activities in the clinical lab. Importantly, the IOPQ requirements summarized here would have also been relevant to laboratory-developed tests (LDTs) classified as \"devices\" prior to the March 31, 2025, annulment of the ruling.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0147724"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421869/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144816828","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":"Luciferase reporter mycobacteriophage (TM4::<i>GeNL</i>) enables rapid assessment of drug susceptibilities and inducible macrolide resistance in <i>Mycobacterium abscessus</i> complex.","authors":"Saranathan Rajagopalan, Lahari Das, Donna J Kohlerschmidt, Amy K Rourke, Salika M Shakir, Michelle H Larsen, Max R O'Donnell, Wendy A Szymczak, Vincent E Escuyer, Phyu M Thwe, William R Jacobs","doi":"10.1128/jcm.00841-25","DOIUrl":"10.1128/jcm.00841-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Mycobacterium abscessus&lt;/i&gt; (MAB) infections are challenging to treat due to high-level resistance to anti-tuberculosis drugs, carbapenems, fluoroquinolones, and tetracyclines. Clarithromycin and amikacin are considered cornerstone drugs for MAB treatment due to their superior efficacy; however, assessing clarithromycin susceptibility typically takes 7 to 14 days because of inducible macrolide resistance. We developed a 48 h luciferase reporter mycobacteriophage drug susceptibility testing (LRM-DST) assay using TM4::&lt;i&gt;GeNL&lt;/i&gt; to evaluate MAB drug susceptibility. For this proof-of-principle study, we performed DST on 26 MAB clinical isolates using TM4::&lt;i&gt;GeNL&lt;/i&gt; and Sensititre RAPMYCO2 plates. Genome sequencing was performed to identify the MAB sub-species and for understanding intrinsic and acquired drug resistance mechanisms. We detected clarithromycin resistance in 12/26 (46%) by both Sensititre and LRM-DST. All clarithromycin-resistant isolates carried full-length inducible erythromycin ribosomal methylase-41 [&lt;i&gt;erm(41&lt;/i&gt;)] gene, and none had &lt;i&gt;rrl&lt;/i&gt; (23S rRNA) mutations. Amikacin resistance or intermediate amikacin resistance was detected in 6/26 (23%) isolates. Mutations in the &lt;i&gt;rrs&lt;/i&gt; (16S rRNA) gene at positions 1375A &gt; G (&lt;i&gt;E. coli&lt;/i&gt; 1408A &gt; G) conferred amikacin resistance in four isolates; however, no mutations were identified in two of the amikacin intermediate-resistant isolates. LRM-DST results were in 100% and 92.3% concordance with Sensititre DST results of clarithromycin (kappa coefficient, 1.0; 95% confidence interval [CI], 0.84 to 1.0) and amikacin (kappa coefficient, 0.752; 95% confidence interval [CI], 0.43 to 1.0). In addition, LRM-DST results for bedaquiline, moxifloxacin, imipenem, linezolid, and cefoxitin correlated well with Sensititre DST. Our findings suggest that LRM-DST can provide reliable phenotypic MAB-DST information in 48 h for prompt clinical management.IMPORTANCE&lt;i&gt;Mycobacterium abscessus&lt;/i&gt; (MAB) is a notorious human pathogen causing severe infections in individuals with cystic fibrosis and immunocompromised patients. Treatment options for MAB are very limited as they are resistant to multiple drugs through intrinsic and acquired resistance mechanisms. While macrolides and amikacin are the key drugs in the fight against MAB infections, emerging drug resistance is compromising their efficacy. Current growth-based drug susceptibility testing (DST) method takes 7 to 14 days to identify clarithromycin susceptibility due to the presence of inducible macrolide resistance and 3 to 5 days for other drugs. We developed a novel luciferase reporter mycobacteriophage (LRM) based phenotypic DST method using TM4::&lt;i&gt;GeNL&lt;/i&gt; to assess MAB drug susceptibility based on metabolic inhibition rather than growth. LRM-DST significantly reduces the DST turnaround time to 48 h and provides rapid assessment of MAB susceptibility to drugs, including inducible macrolide resistance, thereby accelerating the trea","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0084125"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421814/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956133","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":"Type-specific EV-D68 real-time RT-PCR assay for the detection of all extant enterovirus D68 strains.","authors":"Terry Fei Fan Ng, W Allan Nix, Shannon L Rogers, Brian Emery, Shur-Wern Chern, Kiantra Butler, M Steven Oberste","doi":"10.1128/jcm.01492-22","DOIUrl":"10.1128/jcm.01492-22","url":null,"abstract":"<p><p>We developed a type-specific Enterovirus D68 (EV-D68) real-time RT-PCR (rRT-PCR) assay termed CDC2022, which targets sequences encoding conserved amino acid regions of all extant EV-D68 strains. We targeted three motifs conserved among all strains in the last 60 years. The assay achieved 100% (281/281) sensitivity and 100% (344/344) specificity when tested with a collection of 625 respiratory specimens, compared to the gold-standard EV semi-nested VP1 PCR and sequencing assay (snPCR/Seq). CDC2022 gave negative results with 289/289 non-target viruses, including 104 EV A-D isolates, 165 rhinovirus (RV) isolates or clinical specimens, and 14 other common respiratory viruses. The limit of detection (LOD) of the CDC2022 assay is 361 copies per reaction, as determined using serially diluted RNA transcripts. It can detect as few as 0.28 CCID₅₀ per reaction with titrated isolates. An <i>in silico</i> \"phylo-primer-mismatch\" analysis was performed to visualize primer/probe mismatches and to compare CDC2022 with other EV-D68 rRT-PCR assays. It showed that CDC2022 has the fewest primer/probe mismatches among all assays analyzed and is suitable for all clades. As a type-specific assay targeting conserved amino acids, the CDC2022 assay allowed detection of newer strains from 2024. The CDC 2022 assay could provide a critical tool for molecular surveillance of EV-D68.IMPORTANCEEV-D68 has caused recurring respiratory disease outbreaks in the United States since 2014. As recurrent outbreaks and continued virus evolution are expected for EV-D68, the CDC2022 rRT-PCR provides a robust test that detects known strains as well as potential emerging strains. This type-specific assay approach is critical for national EV-D68 surveillance and clinical diagnostics. An <i>in silico</i> \"phylo-primer-mismatch\" approach is invented to show EV-D68 assay robustness, but it has utility in new molecular tests for pathogen detection.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0149222"},"PeriodicalIF":5.4,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12421836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956103","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}