Journal of Clinical Microbiology最新文献

{"title":"CDC dengue typing kit fails to detect dengue virus 2 sylvatic genotype.","authors":"Diamilatou Balde, Mignane Ndiaye, Agathe Shella Efire, Ousmane Faye, Amadou Alpha Sall, Manfred Weidmann, Oumar Faye, Idrissa Dieng","doi":"10.1128/jcm.01741-24","DOIUrl":"10.1128/jcm.01741-24","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0174124"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345212/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659364","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":"Evaluation of the MIC test strips for antifungal susceptibility testing of <i>Candidozyma auris</i> (<i>Candida auris</i>) using a representative international collection of isolates.","authors":"Maria Siopi, Sevasti Leventaki, Ioannis Pachoulis, Bram Spruijtenburg, Jacques F Meis, Spyros Pournaras, Georgia Vrioni, Athanasios Tsakris, Joseph Meletiadis","doi":"10.1128/jcm.00399-25","DOIUrl":"10.1128/jcm.00399-25","url":null,"abstract":"<p><p>We compared MIC test strips (MTS) with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international panel of 100 <i>Candidozyma auris</i> (<i>Candida auris</i>) isolates belonging to different clades. The agreement (±1 twofold dilution) between the methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and MTS-specific wild-type upper limit values (WT-ULVs) were determined. The MTS-CLSI agreement was poor to weak for posaconazole (3%), itraconazole (20%), voriconazole (31%), and 5-flucytosine (37%), and moderate to strong for isavuconazole (58%), anidulafungin (68%), caspofungin (72%), micafungin (77%), and amphotericin B (85%). Most fluconazole MICs were off-scale, precluding a corresponding estimation. Significant interpretation discrepancies were recorded using the CDC's breakpoints for amphotericin B (66% CA, 34% major errors; MaEs), but not for fluconazole (98% CA, 1% MaEs, 1% very major errors; VmEs), anidulafungin (97% CA, 3% MaEs, 0% VmEs), micafungin (99% CA, 1% MaEs, 0% VmEs), and caspofungin (95% CA, 5% MaEs, 0% VmEs). Discrepancies were minimized using the amphotericin B method-specific WT-ULV of 4 mg/L (98% CA, 2% MaEs). The MTS-specific WT-ULVs of echinocandins could help to detect 100% of <i>FKS1</i> mutants. MTS generated higher MICs than the CLSI for azoles and 5-flucytosine. MTS could accurately detect fluconazole and echinocandin resistance among <i>C. auris</i> isolates. Nevertheless, it overestimated amphotericin B resistance as per the CDC's breakpoint of 2 mg/L. This can be improved by using the MTS-specific WT-ULV of 4 mg/L.IMPORTANCE<i>Candidozyma auris</i> (<i>Candida auris</i>) may exhibit resistance to multiple and sometimes even all currently available classes of antifungals. Hence, antifungal susceptibility testing (AFST) is of key importance to guide the clinician in therapeutic decision-making and to detect novel patterns of resistance. Gradient diffusion strips, referred to both Etest and MIC test strip (MTS), are broadly used in laboratory routine for AFST of yeasts. We therefore compared MTS with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international panel of 100 <i>C</i>. <i>auris</i> isolates belonging to different clades. Significant interpretation discrepancies were recorded for amphotericin B (66% categorical agreement, 34% major errors), which could be minimized using the amphotericin B method-specific wild-type upper limit value of 4 mg/L. MTS generated higher MICs than the CLSI for azoles and 5-flucytosine. MTS could accurately detect fluconazole and echinocandin resistance.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0039925"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345157/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553626","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":"Sulbactam-durlobactam in combination with aztreonam and carbapenems against carbapenem-resistant <i>Acinetobacter baumannii</i>: an assessment using the MIC-based broth disk elution.","authors":"C Koenig, D P Nicolau, T E Asempa","doi":"10.1128/jcm.00709-25","DOIUrl":"10.1128/jcm.00709-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0070925"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345227/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698697","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":"Genomic-enabled classification of three <i>Mycobacteroides abscessus</i> subspecies and an effective subspecies-specific identification method.","authors":"Zelin Yu, Ruibai Wang","doi":"10.1128/jcm.00697-25","DOIUrl":"10.1128/jcm.00697-25","url":null,"abstract":"<p><p><i>Mycobacteroides abscessus</i> (Mab) is a clinically significant non-tuberculous mycobacterium. It comprises three distinct subspecies being considered to have different macrolide susceptibilities, transmission patterns, and treatment outcomes. In this study, systematic analysis was conducted on 2,006 Mab genomes deposited in the National Center for Biotechnology Information genome database, and the taxonomic classification of their subspecies was revised accordingly. The findings revealed that: (i) in terms of three distinct subspecies classification, the analysis based on core genes and average nucleotide identity (ANI) values was completely consistent; (ii) ANI was a reliable criterion for Mab species and subspecies classification, with defined thresholds of 95% ANI for species-level and 98% ANI for subspecies-level differentiations; and (iii) the integrity of the <i>erm</i>(41) gene or the similarity of the <i>rpoB</i> gene was an unreliable characteristic for Mab subspecies, and the assertions that subspecies <i>massiliense</i> lack inducible resistance to macrolides also cannot be sustained. Moreover, through a subspecies re-classification of genomes and pangenome analysis, Mab subspecies-specific genes were successfully identified, and a novel single-gene test with enhanced clinical applicability was developed. Additionally, the impact of reference genome selection on taxonomic classification highlighted the importance of adopting a standardized set of reference genomes in species/subspecies identification to significantly enhance the comparability across different studies.IMPORTANCE<i>Mycobacteroides abscessus</i> (Mab) is a clinically challenging non-tuberculous mycobacteria species. The accurate identification of subspecies is of utmost importance for clinical diagnosis and treatment, as well as for research on pathogenicity, drug resistance, and other related aspects. This study provided a clear average nucleotide identity threshold for Mab subspecies classification, as well as revised options of the three Mab subspecies, new and accurate Mab subspecies-special biomarker, and a detection technique with practical clinical application.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0069725"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345259/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698696","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":"Advanced specificity and sensitivity studies relative to a research use only transcription-mediated amplification-based assay for <i>Treponema pallidum</i> RNA detection.","authors":"Trinity Krueger, Grace Kadonsky, Elizabeth Thelen, Amanda Zapp, Josephine Moore, Ahmet Muslu, Allan Pillay, Irene A Stafford, Erik Munson","doi":"10.1128/jcm.00388-25","DOIUrl":"10.1128/jcm.00388-25","url":null,"abstract":"<p><p>Preliminary experimentation has suggested that a research-use-only real-time transcription-mediated amplification assay for <i>Treponema pallidum</i> (RUO <i>T. pallidum</i> TMA) yields instances of <i>T. pallidum</i> nucleic acid detection that coincide with non-treponemal serology in men who have sex with men (MSM) at increased risk for sexually transmitted infection. To further characterize the specificity of RUO <i>T. pallidum</i> TMA testing, 3,586 rectal swab specimens reported as \"not detected\" by the assay generated a mean endpoint FAM fluorescence of 637.5 units. Introduction of <i>Treponema denticola</i>, <i>Treponema phagedenis</i>, and <i>Treponema refringens</i> nucleic acid into matrices generated mean endpoint FAM fluorescence ranging from 620.7 to 633.5 units (<i>P</i> ≥ 0.15 versus control). Introduction of lubricant to a pooled rectal swab matrix did not result in elevated FAM fluorescence (mean endpoint value 628.3 units [95% CI 612.0, 644.6]; <i>P</i> = 0.67). Introduction of talcum powder, urine, seminal fluid, and blood also failed to generate increased FAM fluorescence (<i>P</i> ≥ 0.20). Analytic sensitivity assessment was measured by serial 10-fold dilution of <i>T. pallidum</i> whole organism or <i>in vitro</i> 23S rRNA transcript in specimen transport medium or pooled rectal swab matrix and interrogation by the assay. Probit analysis estimated sensitivity (95% detection) of RUO <i>T. pallidum</i> TMA at 421-5,707 <i>in vitro</i> transcript copies/mL and 9-48 <i>T. pallidum</i> cells/mL, depending on dilution matrix. These results support RUO <i>T. pallidum</i> TMA as a highly sensitive method for <i>T. pallidum</i> detection that is not impacted by potentially cross-reactive organisms or interfering substances and may have adjunctive diagnosis capability for syphilis in MSM.</p><p><strong>Importance: </strong>Research-use-only <i>Treponema pallidum</i> transcription-mediated amplification (RUO <i>T. pallidum</i> TMA) has the potential to improve laboratory diagnosis of syphilis, particularly in patients at increased risk for sexually transmitted infection. The high analytic sensitivity and lack of cross-reactivity of the assay can facilitate other laboratories exploring the use of the test in a research setting.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0038825"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345250/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144333165","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 Clinical and Laboratory Standards Institute M45 antimicrobial susceptibility testing methods and breakpoints for <i>Pseudomonas</i> other than <i>Pseudomonas aeruginosa</i>.","authors":"Patricia J Simner, Harley Harris, Emily Jacobs, Haley Stambaugh, Amira Bhalodi, Mark Fisher, Tsigereda Tekle, Jennifer Lu, Romney Humphries","doi":"10.1128/jcm.00368-25","DOIUrl":"10.1128/jcm.00368-25","url":null,"abstract":"<p><p>The purpose of this study was to establish tentative Clinical and Laboratory Standards Institute (CLSI) M45 MIC and disk diffusion (DD) breakpoints (BPs) for <i>Pseudomonas</i> other than <i>Pseudomonas aeruginosa</i> (POPA). Mechanisms of antimicrobial resistance (AMR) and the modified carbapenem inactivation method (mCIM) to detect carbapenemase production were also evaluated. MIC data from <i>P. aeruginosa</i> and POPA from 2013 to 2022 were evaluated to compare the MIC distributions and modal MICs relative to the CLSI M100 <i>P. aeruginosa</i> BPs. A disk-to-MIC correlation study with 83 isolates was completed by testing reference broth microdilution and DD from the same inoculum, and the error-rate bounded method was used to establish DD BPs. For most antimicrobials, the modal MICs between <i>P. aeruginosa</i> and POPA were within 1-doubling dilution and lower than the M100 <i>P. aeruginosa-</i>susceptible BP. For amikacin, the modal MIC for POPA was 2-doubling dilutions lower than <i>P. aeruginosa</i> and was evaluated relative to the Enterobacterales BP. For aztreonam and trimethoprim-sulfamethoxazole, the modal MICs were elevated, and no BPs were set. New DD correlates were established for most antimicrobial agents, except for fluoroquinolones, where the <i>P. aeruginosa</i> correlates were suitable. AMR genes conferring resistance to multiple antimicrobial classes were identified by WGS. Beta-lactamase genes were identified in 30 (36.1%) isolates, with metallo-beta-lactamases (90.6%) predominating. The mCIM had a sensitivity and specificity of 100%. Upon review, the CLSI M45 committee proposed tentative MIC and DD BPs for expanded-spectrum cephalosporins (ceftazidime and cefepime), carbapenems (meropenem and imipenem), fluoroquinolones (ciprofloxacin and levofloxacin), and the aminoglycosides (amikacin and tobramycin).<b>IMPORTANCE</b><i>Pseudomonas</i> species other than <i>Pseudomonas aeruginosa</i> (POPA) can cause opportunistic infections which may be difficult to treat due to a variety of antimicrobial resistance mechanisms. Antimicrobial susceptibility testing is a critical component of patient management for these infections. Currently, the Clinical and Laboratory Standards Institute (CLSI) M100 non-Enterobacterales breakpoints and methodology are utilized for POPA by US clinical laboratories and likely do not accurately predict susceptibility results. The purpose of this study was to establish tentative CLSI M45 MIC and disk diffusion breakpoints for POPA. Mechanisms of antimicrobial resistance and the modified carbapenem inactivation method to detect carbapenemase production were also evaluated. We present the data used by the volunteers tasked by CLSI to develop POPA breakpoints in the M45 guidelines.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0036825"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345185/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528215","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":"PCR sensitivity for <i>Mycoplasma pneumoniae</i> detection in nasopharyngeal and oropharyngeal swabs: a comparative study.","authors":"Daisuke Kitagawa, Shin Nishihara, Masayuki Murata, Mai Onishi, Takahiro Mori, Soshi Hachisuka, Tenshin Okubo, Naohiro Yamamoto, Hiroki Nishikawa, Masayuki Onaka, Rika Suzuki, Soma Suzuki, Ayu Yamamoto, Ritsuki Uejima, Fumihiko Nakamura, Sayaka Yoshida, Taito Kitano","doi":"10.1128/jcm.00458-25","DOIUrl":"10.1128/jcm.00458-25","url":null,"abstract":"<p><p>The differential impact of sample type on polymerase chain reaction (PCR) detection of <i>Mycoplasma pneumoniae</i> (MP) has rarely been investigated. The study aimed to evaluate the diagnostic performance of PCR for the detection of MP and to measure MP DNA load between nasopharyngeal and oropharyngeal swabs. Nasopharyngeal and oropharyngeal samples were obtained simultaneously to evaluate their diagnostic performance in children with suspected MP. Two commercially available PCR tests, multiplex PCR and Smart Gene Myco, were used to analyze the nasopharyngeal and oropharyngeal samples, respectively. Furthermore, real-time PCR (RT-PCR) tests were conducted on both sample residues to validate the results. In total, 422 participants underwent simultaneous PCR testing using nasopharyngeal and oropharyngeal swabs; 139 samples (32.9%) from nasopharyngeal swabs and 176 samples (41.7%) from oropharyngeal samples that tested positive using commercially available tests. RT-PCR tests were positive for 136 (32.2%) nasopharyngeal and 183 (43.4%) oropharyngeal residual samples. With the RT-PCR test of the residual extract from oropharyngeal swabs as a reference, the sensitivity and specificity of detecting MP were 74.9% (95% confidence interval 67.9%-81.0%) and 99.2% (97.0%-99.9%) with the multiplex PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) and 100.0% (98.5%-100.0%) with the Smart Gene Myco on oropharyngeal samples. A negative correlation was observed between fluoroquinolone use and oropharyngeal DNA loads (<i>P</i> = 0.004). The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study indicates that oropharyngeal samples should be used to detect MP rather than nasopharyngeal samples.IMPORTANCEObtaining the best sample is crucial for the accurate diagnosis of <i>Mycoplasma pneumoniae</i> (MP) and timely and appropriate treatment. This study aimed to assess the diagnostic performance of MP detection using polymerase chain reaction (PCR) tests between nasopharyngeal and oropharyngeal samples. This study showed that the sensitivity of detecting MP was 74.9% (95% confidence interval 67.9%-81.0%) with a commercially available PCR test on nasopharyngeal swabs, and 96.2% (92.3%-98.4%) with a commercially available PCR test on oropharyngeal samples. The sensitivity of MP detection was significantly better in oropharyngeal samples than in nasopharyngeal samples. This study supports the idea that oropharyngeal samples should be used to detect MP. The results contribute to guidance in the recommendation regarding sampling methods to detect MP. Accurate identification of MP is crucial not only for timely and appropriate antimicrobial treatment but also for efficient epidemiological surveillance.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0045825"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345179/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553627","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":"Comparative performance of cobas 4800 HPV Test and Anyplex II HPV HR for high-risk human papillomavirus detection.","authors":"Luani R Godoy, Mariam El-Zein, Elizaveta Padalko, Bo Verberckmoes, Bodine Van Eenooghe, Heleen Vermandere, Sónia Dias, Ana Gama, Bernardo Vega Crespo, Vivian Alejandra Neira, Eduardo L Franco, Adhemar Longatto-Filho","doi":"10.1128/jcm.00200-25","DOIUrl":"10.1128/jcm.00200-25","url":null,"abstract":"<p><p>Numerous molecular tests are available to detect human papillomavirus (HPV). We compared the analytical performance of cobas and Anyplex for detection of high-risk (HR) carcinogenic HPV genotypes, assessed the composition of HPV types (other than 16 and 18) that influenced cobas performance, and considered the impact of viral load on test performance. We used data from the Early Detection of Cervical Cancer in Hard-to-Reach Populations of Women Through Portable and Point-of-Care HPV Testing project, which involved collection (2019-2022) of cervicovaginal samples from 1,042 women aged 21-74 years in Belgium (<i>n</i> = 244), Portugal (<i>n</i> = 309), Brazil (<i>n</i> = 244), and Ecuador (<i>n</i> = 245). Samples were tested by cobas (provides individual results for HPV16 and HPV18 and a pooled result for 12 other HR-HPV types) and Anyplex (provides separate results for 14 HR-HPVs). We calculated HPV positivity by each test and compared performance between tests by calculating Cohen's kappa statistics. Based on 938 samples with complete data from both tests, positivity rates by cobas were 13.4%, 3.6%, 34.3%, and 45.3% for HPV16, HPV18, 12 pooled HR-HPVs, and any HR-HPV, respectively. Corresponding HPV positivity rates by Anyplex were 14.9%, 3.7%, 37.9%, and 50.0% for the same categories, respectively, with high concordance; kappa statistics were 0.90, 0.87, 0.82, and 0.85, respectively. Based on 355 samples that tested positive for at least 1 of the 12 pooled HR-HPVs, most types showed high agreement (80.9%-100.0%) between individual-Anyplex and pooled-cobas HPV results, except for HPV68 (61.3% agreement). Our findings suggest that the two commercial tests may have different performances, depending on the specific HPV types detected, emphasizing the need for continued research on conditions that may affect these tests, especially for less common or less studied HPV types.IMPORTANCEThis study compared two commercial tests-cobas and Anyplex-for detecting high-risk HPV types in women undergoing routine cervical cancer screening or referred for colposcopy. Both tests provide separate results for HPV16 and HPV18, but Anyplex also identifies the remaining 12 high-risk HPV types individually, while cobas groups them together. Overall, we found a high level of agreement between the two tests, supporting their use in clinical practice. However, differences in detecting certain HPV types, particularly those that are less common or less studied, emphasize the importance of choosing the right test. As more countries switch to HPV-based cervical cancer screening, using tests that provide detailed results could help improve risk assessment and optimize patient care.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0020025"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345211/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591365","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":"An open-source nanopore-only sequencing workflow for analysis of clonal outbreaks delivers short-read level accuracy.","authors":"Nick Vereecke, Thomas B Yoon, Ting L Luo, Brendan W Corey, Francois Lebreton, Patrick T Mc Gann, John P Dekker","doi":"10.1128/jcm.00664-25","DOIUrl":"10.1128/jcm.00664-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;In this work, we present an optimized nanopore long-read only sequencing workflow for epidemiologic analysis of clonal outbreaks built with open-source tools. A set of unrelated clinical &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; isolates (&lt;i&gt;n&lt;/i&gt; = 10) was chosen for workflow optimization, and sequencing libraries were prepared using a modified rapid barcoding strategy that incorporates temperature ramps to improve performance for high-GC content genomes. Sequencing data were used to benchmark the performance of the dorado suite (v0.9.1), including its basecaller, pre-assembly read error correction, and post-assembly polishing algorithms. All long-read assemblies and core genome multilocus sequence typing (cgMLST) were performed with Flye and pyMLST, respectively. Results were compared with a standard reference Illumina short-read approach, and discordant positions were determined at the core and whole-genome levels. Optimal performance was found with dorado sup@v5.0.0 basecalling with the inclusion of dorado error correction and dorado polish with its bacterial model. This workflow was then validated with four retrospective hospital outbreak isolate sets, including &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; (&lt;i&gt;n&lt;/i&gt; = 12), &lt;i&gt;P. aeruginosa&lt;/i&gt; (&lt;i&gt;n&lt;/i&gt; = 11), &lt;i&gt;Enterococcus faecium&lt;/i&gt; (&lt;i&gt;n&lt;/i&gt; = 10), and &lt;i&gt;Staphylococcus aureus&lt;/i&gt; (&lt;i&gt;n&lt;/i&gt; = 10). The nanopore-only assemblies obtained from the optimized pipeline demonstrated fully concordant cgMLST-based minimum spanning trees compared to the Illumina short-read reference. At the whole-genome level, high concordance was also observed, with as few as two discordant positions per genome compared to short-read assemblies. This optimized library preparation and open-source computational workflow enables nanopore-only clonality and outbreak analysis with performance comparable to that of Illumina short-read sequencing and will contribute critically to hospital infection control.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;For the past decade, bacterial whole-genome sequencing has been performed using high-accuracy short-read sequencing. More recently, long-read sequencing with Oxford Nanopore Technologies (ONT) instruments has emerged as a potential alternative based on multiple advantages, including lower costs, portability, and speed. However, this platform has suffered from basecall error rates that were too high for many applications in clinical microbiology, including outbreak tracing. With the release of new flow cell chemistries and basecall algorithms, the accuracy has improved dramatically, making this approach feasible for outbreak investigations. In this work, we optimize a streamlined nanopore-only workflow for epidemiologic analysis of bacterial pathogens. The workflow was validated with isolates from four previously identified clinical outbreaks with varying GC content and demonstrated fully concordant cgMLST clustering as compared to short-read references. This workflow will facilitate the broader implemen","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0066425"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345217/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144659363","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":"Waste to worth: diagnostic accuracy of Xpert MTB/XDR on contaminated liquid cultures to salvage the detection of drug-resistant tuberculosis.","authors":"Yonas Ghebrekristos, Erick Auma, Zama Mahlobo, Rouxjeane Venter, Natalie Beylis, Jay Achar, Brigitta Derendinger, Sarishna Singh, Megan Burger, Christoffel Opperman, Robin Warren, Grant Theron","doi":"10.1128/jcm.00580-25","DOIUrl":"10.1128/jcm.00580-25","url":null,"abstract":"<p><p>Mycobacterium Growth Indicator Tube (MGIT) 960 culture is critical for tuberculosis (TB) drug susceptibility testing (DST) but is vulnerable to contamination. We evaluated the accuracy of Xpert MTB/XDR, a molecular DST for isoniazid, fluoroquinolone, amikacin, and ethionamide, on to-be-discarded contaminated growth. Xpert MTB/XDR was applied to acid-fast-bacilli-negative, contaminated cultures from sputum from people with rifampicin-resistant TB when Xpert MTB/XDR on sputum was unsuccessful (not resistant or susceptible for all drugs), either at diagnosis (Cohort A) or during treatment monitoring (Cohort B). Future DSTs within 3 months served as a reference standard. We determined potential care cascade improvements. In Cohort A, 10% (66/650) of people had a contaminated culture; 89% (59/66) of contaminated growths were Xpert MTB/XDR TB-positive. Sensitivity and specificity for isoniazid, fluoroquinolone, amikacin, and ethionamide resistance were 100% (95% confidence interval [CI] 85, 100) and 100% (79, 100); 100% (59, 100) and 100% (89, 100); 100% (16, 100) and 100% (91, 100); and 100% (72, 100) and 96% (78, 100), respectively. In Cohort B, 22% (28/129) of people with a contaminated culture were Xpert MTB/XDR TB-positive. Of these, 57% (16/28), 7% (2/28), and 43% (12/28) were isoniazid-, fluoroquinolone-, and ethionamide-resistant (in two, one, and four people, respectively, this would be the first resistant result). In both cohorts, time-to-DST could improve by a median (IQR) of 22 (12-42) days. Xpert MTB/XDR on contaminated MGIT960 cultures had high sensitivity and specificity for DST. This approach could mitigate culture contamination's negative effects and improve gaps in the drug-resistant TB diagnostic cascade.</p><p><strong>Importance: </strong>Culture contamination is a common impediment to drug susceptibility testing for tuberculosis, the single biggest infectious cause of death globally. Xpert MTB/XDR is a World Health Organization-recommended rapid molecular test for second-line drug resistance. We evaluated Xpert MTB/XDR on contaminated liquid culture growth that would otherwise be discarded, with the people who provided these specimens potentially lost from care cascades. By applying Xpert MTB/XDR to contaminated growth in a high-volume programmatic laboratory, we found the number of people who had second-line DST improved, as did the number of resistant cases diagnosed and time to diagnosis. Furthermore, DST information was generated in people who otherwise would have had none. This approach can therefore reduce the effect of culture contamination on tuberculosis DST, permitting earlier diagnosis and effective treatment initiation and potentially ultimately contributing to improving clinical outcomes and reducing transmission of drug-resistant TB.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0058025"},"PeriodicalIF":5.4,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12345176/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144540423","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}