Journal of Clinical Microbiology最新文献

{"title":"Development and validation of a core genome multilocus sequence typing scheme for <i>Citrobacter freundii</i>: application in outbreak investigations and comparative analysis across the <i>Citrobacter</i> genus.","authors":"Bärbel Kieninger, Gabriel E Wagner, Anca Rath, Anja Eichner, Jürgen Fritsch, Aila Caplunik-Pratsch, Jasmine Alikhani, Parham Heydarzadeh-Ghamsary, Adriana Cabal-Rosel, Werner Ruppitsch, Dag Harmsen, Mohammed R Abdulla, Lena Ulm, Karsten Becker, Wulf Schneider-Brachert, Christian Kohler","doi":"10.1128/jcm.00860-25","DOIUrl":"10.1128/jcm.00860-25","url":null,"abstract":"<p><p><i>Citrobacter freundii</i> is a nosocomial pathogen increasingly associated with multidrug resistance and hospital outbreaks. Despite its growing clinical relevance, no standardized core genome multilocus sequence typing (cgMLST) scheme has been available for high-resolution epidemiological analyses. Here, we developed and validated a robust cgMLST scheme for <i>C. freundii</i> comprising 3,250 target loci based on a curated data set of 825 globally distributed genomes representing extensive sequence type and geographic diversity. Validation against published outbreak data sets from hospitals in Finland and Belgium, as well as environmental and patient isolates from two German university hospitals, proved that the scheme possesses high target gene coverage (median 99.6%) and strong discriminatory power. Additionally, we developed a combined cgMLST scheme for <i>C. freundii</i>, C. <i>portucalensis</i>, C. <i>braakii</i>, and C. <i>europaeus</i>, based on 2,307 shared target loci and target gene coverages of ≥99.7%. This scheme proved suitable for cross-species outbreak analysis. Our analyses revealed environments, such as sinks, shower drains, and toilets, as likely reservoirs where <i>Citrobacter</i> species may persist in hospital settings. These findings suggest that environmental sources could play a significant role in transmission events involving patients, while allele-based cluster analyses indicated that direct patient-to-patient transmission was rare. Given the increasing prevalence of multidrug-resistant (MDR) <i>Citrobacter</i> strains, the level of discrimination achieved by these newly developed cgMLST schemes underscores the importance of accurate species identification and environmental screening in understanding the transmission dynamics of opportunistic healthcare-associated pathogens. Ultimately, this makes them valuable tools for genomic surveillance, outbreak investigation, and infection prevention.</p><p><strong>Importance: </strong>Accurate identification and high-resolution typing of multidrug-resistant bacteria are essential for understanding their transmission dynamics in hospitals, particularly in light of the global spread of resistant strains and the role of environmental reservoirs. The newly developed cgMLST schemes presented here provide standardized, portable tools for both local and global scientific and clinical communities to conduct fine-scale genomic epidemiology across four <i>Citrobacter</i> species. These schemes support detailed outbreak reconstruction, source attribution, and cross-hospital comparisons, capabilities that are critical in an era of increasing antimicrobial resistance and international patient movement. By enabling consistent, species-specific surveillance and comparative analyses, cgMLST enhances infection control and public health responses, facilitating early detection and targeted intervention.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0086025"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12505962/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086276","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 expert rules for carbapenemase class identification in <i>Enterobacterales</i> isolates using the VITEK2 susceptibility testing platform.","authors":"Michaela J Eickhoff, Abigail P Brown, Carol E Muenks, Megan L Porter, Murad Ali, Iftikhar Uddin, Tahir Hussain, Melanie L Yarbrough, Rebekah E Dumm","doi":"10.1128/jcm.00769-25","DOIUrl":"10.1128/jcm.00769-25","url":null,"abstract":"<p><p>Prompt identification of enzyme class in carbapenemase-producing carbapenem-resistant <i>Enterobacterales</i> (CP-CRE) has critical implications for informing appropriate patient treatment, infection prevention, and public health. This study evaluates the performance of prototype bioMérieux Advanced Reporting Tool (bioART) expert rules to rapidly identify <i>Klebsiella pneumoniae</i> carbapenemase (KPC), metallo-β-lactamase (MBL), and OXA-48-like carbapenemase enzymes in CP-CRE. The bioART rules are designed for use with routine cards on the VITEK2 automated antimicrobial susceptibility testing (AST) platform. Results provided by the bioART rules should be interpreted in combination with the instrument Advanced Expert System (AES) to comprehensively evaluate phenotypic resistance phenotypes. Two hundred clinical isolates with varied β-lactam resistance profiles were enrolled, with 196 ultimately analyzed, including 115 CP-CREs. The AES alone detected CP-CRE isolates with a sensitivity of 83% and a specificity of 85%. The combined AES and bioART rules detected CP-CRE with a sensitivity of 96% and a specificity of 83%. Prediction of carbapenemase classes by the bioART rules was analyzed for a subset of 158 isolates, including only the species for which the rules have claimed indications. MBL, KPC, and OXA-48-like enzymes were detected with sensitivities of 97%, 76%, and 47%, respectively. Notably, sensitivity was 90% for single OXA-48-like producers, whereas OXA-48-like enzymes in dual New Delhi metallo-β-lactamase (NDM)/OXA-48-like-producing isolates were undetectable using phenotypic susceptibility patterns, and isolates were reported only as producing NDM enzymes. Overall, laboratories incorporating these tools into carbapenemase screening workflows may consider their utility in prompting confirmatory carbapenemase testing, guiding modifications to AST reporting, and/or prompting additional susceptibility testing.</p><p><strong>Importance: </strong>Carbapenem-resistant bacteria are a major public health concern due to their ability to spread in healthcare settings and cause infections that are difficult to treat with first-line antibiotics. Identification of the enzyme classes responsible for carbapenem resistance plays a crucial role in ensuring that patients receive effective treatments and controlling the spread of these bacteria. In this study, we evaluated the performance of a new approach to identify carbapenemase enzymes without additional hands-on testing. The method is designed for use with the VITEK2 automated susceptibility testing platform to recognize patterns of resistance to antibiotics and make predictions about the possible resistance mechanisms.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0076925"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506011/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086323","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":"Paradoxical carbapenemase activity detected by modified carbapenemase inactivation (mCIM) method in <i>Citrobacter sedlakii</i>.","authors":"D Garrett Brown, Matthew Surette, Sreejana Ray, Kevin Guo, Reagan Chan, Liusheng Huang, Sanchita Das","doi":"10.1128/jcm.00589-25","DOIUrl":"10.1128/jcm.00589-25","url":null,"abstract":"<p><p>Screening for carbapenemase-producing Enterobacterales is a necessity for hospitals serving high-risk patient populations. Detection methods often incorporate the modified carbapenemase inactivation method (mCIM) test to verify carbapenemase production in suspect isolates. Here, we demonstrate the case of a <i>Citrobacter sedlakii</i> isolate recovered from carbapenemase-surveillance culture that was strongly mCIM positive for carbapenemase production, but phenotypically nonresistant to carbapenems. The modified carbapenemase inactivation method with EDTA (eCIM) test, which suggests the presence of a metallo-β-lactamase, was positive as well. Liquid chromatography mass spectrometry additionally confirmed the loss of meropenem during the mCIM test. Whole-genome sequencing detected only the presence of the chromosomal class A serine β-lactamase SED-1 but did not identify any known carbapenemase or additional β-lactamases. Further characterization suggested that the large inoculum utilized in mCIM tests can give the impression of carbapenemase production in non-resistant isolates. Additionally, the large inoculum, combined with weak carbapenemase activity, can mimic the phenotype expected for metallo-β-lactamase in the eCIM test. These results suggest that utilization of the mCIM and eCIM tests without appropriate susceptibility testing can lead to erroneous detection of carbapenemase production.IMPORTANCEPhenotypic tests including the mCIM are often used for detection of carbapenemase production in Enterobacterales. The presence of a carbapenemase gene is confirmed by PCR assays and, while rarely seen, a positive mCIM assay and negative PCR can alert the laboratory to the presence of rare carbapenemases (such as GES and IMI). We report a false-positive mCIM test, which we demonstrate is due to the heavy inoculum of the organism used in the assay. Whole-genome sequencing showed that a class A β-lactamase (<i>bla_SED-1</i>) was present, and mass spectrometry demonstrated degradation of meropenem during the mCIM assay. The inoculum effect should be considered when interpreting mCIM and eCIM assays where a carbapenemase gene is not detected.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0058925"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506007/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956146","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":"Diagnostics and new treatment regimens for TB: can the Xpert MTB/XDR assay fill the gap for fluoroquinolone testing?","authors":"Derek T Armstrong, Emma C E Baird, Logan Pretty, Karen D'Agostino, Matthew Schwartz, Victoria L Campódonico, Nicole Parrish","doi":"10.1128/jcm.00643-25","DOIUrl":"10.1128/jcm.00643-25","url":null,"abstract":"<p><p>Rapid diagnosis of resistance-conferring mutations to antibiotics used for the treatment of tuberculosis (TB) is critical for patient care and public health control efforts. Prior guidelines included the use of fluoroquinolones (FQs) for the treatment of drug-resistant TB, including multidrug-resistant TB, pre-extensively drug-resistant TB, and extensively drug-resistant TB. More recently, a short-course regimen for antibiotic-susceptible TB was introduced, which includes the use of a FQ, a drug class that diagnostic algorithms in the United States (US) typically do not test for if all first-line agents are susceptible. However, FQ mono-resistance has been documented by previous studies, and for this reason, we tested 319 archived <i>Mycobacterium tuberculosis</i> complex (MTBC) strains spanning a 14-year period of time using the Xpert MTB/XDR assay. Resistance to FQs was detected in 4.4% (14/319) of the isolates tested, with mutations predominating in the <i>gyrA</i> region (13/14; 92.9%). A single isolate (1/14; 7.1%) was found to have a <i>gyrB</i> mutation. A broth microdilution assay demonstrated the minimum inhibitory concentrations for resistant strains that ranged from 0.5 µg/mL to 8.0 µg/mL. Importantly, three strains were FQ mono-resistant and would have been completely missed by standard testing algorithms. Although currently unavailable in the US, the GeneXpert XDR assay has the potential to fill the significant diagnostic gap in susceptibility testing of MTBC resistance to FQs and support the use of the currently recommended short-course regimen.IMPORTANCE This study provides insight into the need for additional rapid testing for the detection of drug resistance (specifically to fluoroquinolones) in tuberculosis (TB) cases in the United States (US). The current regimens for TB treatment rely on knowing resistance patterns to optimize treatment, and missed resistance could have a negative impact on the health of the patient, as well as contribute to increased drug-resistance mutations in new TB cases. There are currently limited platforms for expanded rapid drug resistance testing for TB cases in the US, and this study looks at past TB cases that had drug resistance missed by routine testing.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0064325"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12509795/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956100","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":"Analytical evaluation of TriVerity, a rapid diagnostic and prognostic host gene expression test performed on the Myrna instrument using RT-LAMP.","authors":"Claudia Figueiredo-Pereira, Paul Fleming, Mikaela Nicole Alganes, Ran Bi, Ana Mafalda Cavaleiro, Diogo Cruz, Carlota Cunha-Matos, Margarita Davalos-Arias, Dana Farkas, Yehudit Hasin, Kevin Hu, Christos Kampouridis, Ragheb El Khaja, Graciano Leal, Jingyi Lu, Rita Madeira, Michael Mayhew, Breana McBryde, Daniela C Oliveira, Anna Passernig, Davion Pendleton, Elizabeth Popp, Shailee Rasania, Cristina Rebelo, Ana Santiago, Joshua R Shak, Vera P Silva, Ambika Srinath, Timothy E Sweeney, Rodrigo Vieira, Thang Vu, Chris Wilson, Boris Zybin, Natalie N Whitfield, Oliver Liesenfeld, Hjalmar R Bouma, Richard E Rothman, Edward A Michelson, Joao Fonseca","doi":"10.1128/jcm.00352-25","DOIUrl":"10.1128/jcm.00352-25","url":null,"abstract":"<p><p>We evaluated the analytical performance of the TriVerity test system, a benchtop system for rapid measurement and interpretation of host messenger RNAs (mRNAs) measured quantitatively from PAXgene Blood RNA specimens using the TriVerity cartridge on the Myrna instrument. In approximately 30 minutes, 3 scores are generated from 29 host mRNAs for the likelihood of a bacterial infection, a viral infection, and illness severity (7-day need for ICU-level care), each falling into 1 of 5 interpretation bands (very low, low, moderate, high, and very high). Reproducibility, precision, limit of detection, linearity, interference, and sample and cartridge stability were determined per Clinical Laboratory Standards Institute standards. An operator survey assessed TriVerity's ease of use. Reproducibility demonstrated SDs meeting the acceptance criteria of <5.5 score units. The lowest concentration at which 100% of replicates showed measurable amplification was 1 × 10⁶ cp/mL of <i>in vitro</i> RNA transcripts; the limit of quantification and detection were equivalent to blood containing 500 leukocytes/µL. Interference was not observed for 17 potential interferents. The cartridge was stable at room temperature (RT) for 9 and 12 months using accelerated testing at 37°C. TriVerity results were equivalent using fresh and frozen PAXgene Blood RNA samples. All operators agreed or strongly agreed that the TriVerity test system was easy to use. In summary, the analytical performance presented here, along with diagnostic accuracy established in the SEPSIS-SHIELD trial, demonstrates that the TriVerity system is reliable and user friendly, assisting clinicians in managing patients with suspected acute infections and sepsis in acute care settings.</p><p><strong>Importance: </strong>The prompt diagnosis of acute infections and sepsis is critical for better patient outcomes. We introduce a groundbreaking messenger RNA-based test, the first of its kind, designed to diagnose the presence of infection and predict illness severity in adult patients with suspected acute infections or sepsis. Several key findings regarding the accuracy and robustness of the test system are presented, which will be relevant to laboratories or acute care settings implementing the test for patient care. Furthermore, these findings may assist clinical researchers in developing analytical trial protocols aimed at the combined evaluation of RNA-based multi-marker tests with both diagnostic and prognostic test characteristics.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0035225"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144955931","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":"Optimization of a molecularly defined tuberculin formulation: recombinant fusion proteins and epitope surgery.","authors":"Sonya Middleton, Mahavir Singh, Michael Coad, Si Palmer, Tom Holder, Sabine Steinbach, Rebecca Hardiman, H Martin Vordermeier, Gareth J Jones","doi":"10.1128/jcm.00552-25","DOIUrl":"10.1128/jcm.00552-25","url":null,"abstract":"<p><p>Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test that utilizes purified protein derivatives (PPDs) of tuberculin. PPDs suffer from a number of limitations regarding their production, characterization, and performance. Recently, we developed a molecularly defined tuberculin (MDT) that overcame these limitations. The MDT formulation comprises eight antigens, initially presented as seven recombinant proteins (ESAT6, CFP10, Rv3615c, Rv3020c, Rv1789, Rv3478, and Rv3810) and one 20-synthetic peptide pool representing Rv3616c; the recombinant Rv3616c could not be produced. In this paper, we describe the steps taken to simplify the MDT formulation. First, seven of the eight proteins were formulated as three recombinant fusion proteins. Second, the non-immunogenic regions of Rv3616c were identified, using overlapping peptides to probe T-cells from infected cattle. A novel variant, Rv3616c-S4, was designed and then formulated (i) as a stand-alone protein in the MDT-S4 formulation and (ii) fused to Rv1789 for use in the MDT-F formulation. These novel MDT formulations gave comparable signal strength to PPD-B in experimentally infected animals. The MDT-F, comprising only fusion proteins, performed as well as the comparative cervical tuberculin skin test, in terms of relative sensitivity (>2 mm; 96%, 95% CI 80-100, <i>n</i> = 24) and specificity (>2 mm; 100%, 95% CI 89%-100%, <i>n</i> = 30). In conclusion, we have developed a novel, simplified MDT formulation that has significantly advanced our efforts toward licensure and which has the potential to replace the PPDs developed in the 1930s.</p><p><strong>Importance: </strong>Bovine tuberculosis is an infectious livestock disease of global economic and zoonotic importance. Surveillance programs are largely dependent on the skin test, which utilizes tuberculins. These are crude protein extracts of live bacterial cultures, which suffer from a number of limitations regarding their characterization, standardization, production, and performance. We aim to develop a skin test reagent composed of eight defined antigens that could replace the tuberculins. This study describes the refinement of this \"molecularly defined tuberculin\" (MDT) reagent into a fusion protein formulation comprising all eight antigens. The MDT is well defined, easily standardized, and delivers good test performance in experimentally infected, non-infected cattle and cattle sensitized to Johne's disease. The fusion protein formulation is an important step on the developmental pathway to a registered and marketable product.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0055225"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12505890/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956111","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":"Enhancing blood culture volume with ultrathin-wall cannula devices.","authors":"Grant Johnson, Valentin Parvu, Stephane Beauchamp, Mike Cuttler, Arek Zubrzycki, Lauren Cooper","doi":"10.1128/jcm.00208-25","DOIUrl":"10.1128/jcm.00208-25","url":null,"abstract":"<p><p>Suboptimal blood volume collection negatively affects pathogen recovery and time to pathogen detection in clinical blood culture samples. This study evaluated the performance of the ultrathin-wall cannula device of the BD Vacutainer UltraTouch Push Button Blood Collection Set (UltraTouch; BD Life Sciences, Franklin Lakes, NJ) in increasing the blood volume collected compared to regular cannulas. In this retrospective study, the volume of blood collected in negative BD BACTEC Plus Aerobic/F Culture Vials prior to and following blind conversion to UltraTouch was measured from blood background metabolic activity signal acquired during the initial incubation period in the BD BACTEC FX instrument using a custom software application (patent pending). Differences in average fill volume between pre- and post-conversion were evaluated overall and by month. A separate logistic regression was also performed to model the relationship between monthly average blood volume increase (mL) and sample positivity rate. Prevalence rates for identified organisms were also assessed. The blood fill volume of 13,356 pre-conversion and 119,971 post-conversion bottles was compared. The average post-conversion fill volume was significantly higher than that of pre-conversion (7.56 mL [95% CI, 7.54-7.59] vs. 5.68 mL [95% CI, 5.62-5.74], respectively). This 1.88 mL increase in collected blood volume increased sample positivity from 7.0% to 7.7% (<i>P</i> = 0.002; odds ratio [OR], 1.11; 95% CI, 1.04-1.19). This study demonstrated that using an ultrathin-wall cannula blood collection device can significantly increase blood culture volume and sample positivity, supporting improved identification of patients suffering from bloodstream infections.IMPORTANCESubstandard blood fill volume practices in clinical settings negatively affect pathogen recovery which, in turn, may impact patients' care. Whereas professional education regarding the importance of adhering to clinical guidelines has been helpful in partly remediating the issue, the role of blood collection set technologies in optimizing blood culture volume has not been studied as thoroughly. Our study assessed the performance of the BD Vacutainer UltraTouch Push Button Blood Collection Set, which is designed with an ultrathin-wall cannula, to improve blood culture volume.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0020825"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12505989/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145000593","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 extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B <i>Streptococcus</i> by integrating with RPA.","authors":"Chengman Zhao, Ge Li, Chen Shen, Yanjun Xie, Yun Chen, Xiaobo Ying, Yan Chen, Chuanling Zhang","doi":"10.1128/jcm.00819-25","DOIUrl":"10.1128/jcm.00819-25","url":null,"abstract":"<p><p>Group B <i>Streptococcus</i> (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.</p><p><strong>Importance: </strong>This study presents a rapid, convenient, and highly accurate method for Group B <i>Streptococcus</i> (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0081925"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086308","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":"Diagnostic stewardship applied to targeted fungal sequencing.","authors":"Jessica Hudson, Indre Budvytiene, Niaz Banaei","doi":"10.1128/jcm.00896-25","DOIUrl":"10.1128/jcm.00896-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0089625"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506030/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144956082","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":"Photo Quiz: Unexpected yeast in a premature infant-pathogen or passenger.","authors":"Yuan Chao Xue, Lemuel O Aigbivbalu, Katie J Holekamp, Ping Ren","doi":"10.1128/jcm.01111-25","DOIUrl":"10.1128/jcm.01111-25","url":null,"abstract":"","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":"63 10","pages":"e0111125"},"PeriodicalIF":5.4,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12506031/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251476","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}