Journal of Clinical Microbiology最新文献

{"title":"The potential to improve Lyme disease diagnostics through quantification of immunoglobulin class switching patterns.","authors":"Anna M Schotthoefer","doi":"10.1128/jcm.01020-25","DOIUrl":"https://doi.org/10.1128/jcm.01020-25","url":null,"abstract":"<p><p>N. Nair, A. Marques , E. J. Horn, G. Brown et al., J Clin Microbiol 63:e0034725, 2025, https://doi.org/10.1128/jcm.00347-25 present data to demonstrate that infection by <i>Borrelia burgdorferi</i>, the primary causative agent of Lyme disease in the USA, leads to immunoglobulin class switching in antibodies specifically against the immunodominant antigen, VlsE (variable major protein-like sequence, expressed), in a predictable pattern between the early acute (<1 month illness duration) to late-stage Lyme arthritis stages. Detection of anti-VlsE isotypes in sera was highly specific to Lyme disease patients, and the frequencies and abundances of IgM, IgG, and IgA isotypes progressed in a pattern consistent with the development of an anti-<i>B</i>. <i>burgdorferi</i> antibody response. Applying multivariate and machine learning modeling methods, they found the profile of isotypes quantified performed particularly well at correctly classifying early acute sera samples. They also identified IgG4 as a potential biomarker unique to Lyme arthritis patients. Overall, the findings suggest that improvements in Lyme disease diagnostics may be attained by quantifying specific antibody isotypes against <i>B. burgdorferi</i> antigens.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0102025"},"PeriodicalIF":5.4,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145149148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Off-label evaluation of the BD MAX MDR-TB assay for rapid diagnosis of rifampicin and isoniazid resistance of <i>Mycobacterium tuberculosis</i> clinical isolates in a high-volume reference laboratory.","authors":"Angela Pires Brandao, Fabiane Maria de Almeida Ferreira, Fernanda Cristina Dos Santos Simeao, Lucilaine Ferrazoli, Erica Chimara, Rosângela Siqueira de Oliveira, Juliana Maira Watanabe Pinhata","doi":"10.1128/jcm.00912-25","DOIUrl":"https://doi.org/10.1128/jcm.00912-25","url":null,"abstract":"<p><p>Drug-resistant tuberculosis (TB) remains a primary global health concern. Multidrug-resistant TB is defined by resistance to at least rifampicin (RIF) and isoniazid (INH), the two key drugs used in TB treatment. The BD MAX Multi-Drug Resistant Tuberculosis (BD MAX) assay is a fully automated real-time PCR platform recommended by the World Health Organization for the initial diagnosis of TB and RIF and INH resistance (RIF-R and INH-R) directly from pulmonary clinical samples. This study aimed to assess the off-label performance of BD MAX in clinical <i>M. tuberculosis</i> complex (MTBC) isolates under routine laboratory conditions. The assay was first validated using non-tuberculous mycobacteria (NTM) and MTBC isolates with known mutations. For real-world validation, it was compared to the GenoType MTBDR<i>plus</i> by testing 1,440 clinical isolates prospectively. The BD MAX assay correctly excluded MTBC from all NTM cultures. Among MTBC isolates with known mutations, it identified 19 of 20 RIF-R isolates and 14 of 15 INH-R isolates. In prospective testing, BD MAX achieved 99.6% sensitivity (1,403/1,409), 96.8% specificity (30/31), and 99.5% overall accuracy (1,433/1,440) for MTBC detection. For drug resistance detection, it showed 95.2% (40/42) concordance for RIF, 96.8% (30/31) for INH, and 81.3% (13/16) for MDR when compared to MTBDR<i>plus</i>. Discrepancies between MTBDR<i>plus</i> and BD MAX included heteroresistant cases and unreportable resistance results by BD MAX due to infrequent mutations or low bacterial load. Overall, this study confirms BD MAX as an accurate and reliable tool for MTBC detection and drug resistance profiling in clinical isolates in high-volume TB laboratories.IMPORTANCEThis study highlights the importance of the BD MAX Multi-Drug Resistant Tuberculosis assay (BD MAX) applied in clinical isolates for the detection of multidrug-resistant tuberculosis (MDR-TB), i.e., <i>Mycobacterium tuberculosis</i> resistance to rifampicin and isoniazid. TB is a global health issue, and drug-resistant TB makes treatment more difficult, favoring transmission and disease amplification. The BD MAX platform offers a faster and more automated way to detect TB and drug resistance. The study showed that BD MAX, applied off-label in clinical isolates, accurately identified TB and resistance to rifampicin and isoniazid, with results comparable to those of the widely used line probe assay. This is significant in a high-volume laboratory because it is more straightforward and more rapid than the line probe assay. BD MAX showed some limitations, especially in detecting rare mutations and in cases of low bacterial levels. Overall, this tool could improve TB care, especially in high-volume laboratories.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0091225"},"PeriodicalIF":5.4,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124017","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of <i>Haemophilus influenzae</i>.","authors":"Yaling Cao, Junwen Wang, Zihao Fan, Ling Xu, Zhenzhen Pan, Yinkang Mo, Qiwen Yang, Jing Huang, Feng Ren","doi":"10.1128/jcm.00535-25","DOIUrl":"https://doi.org/10.1128/jcm.00535-25","url":null,"abstract":"<p><p>Timely and accurate diagnosis is essential for the effective treatment of <i>Haemophilus influenzae</i> (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. The aim of this study was to establish a one-pot detection device for HI DNA detection using CRISPR/Cas13a that would be applicable for POCT. We established a two-step polymerase chain reaction (PCR)-CRISPR assay, a two-step recombinase-aided amplification (RAA)-CRISPR assay, and a one-pot RAA-CRISPR assay. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct an extraction-free one-pot detection device, named EFORCA (extraction-free one-pot RAA-CRISPR/Cas13a assay). Validation was performed on 90 simulated samples and 77 clinical samples. The two-step PCR/RAA-CRISPR assay for HI DNA detection was established and optimized, with a detection limit of 1 copy/μL and 100% specificity. The sensitivity and specificity of the two-step PCR-CRISPR assay for the 90 simulated clinical samples were 96.7% and 95%, respectively, and for the 77 clinical samples, 97.5% and 100%, respectively. The one-pot RAA-CRISPR assay and the EFORCA detected 50 and 25 CFU/mL HI in bacterial suspensions within 30 min, respectively. The valuation results for 77 clinical samples demonstrated that the sensitivity of both methods was 95%, which was greater than the sensitivity of quantitative real-time PCR (qPCR) (92.5%) with the same specificity of 100%. We developed an extraction-free one-pot RAA-CRISPR/Cas13a assay for HI detection, which effectively performs a POCT test and is a valuable tool for the early detection and monitoring of HI infection.IMPORTANCETimely and accurate diagnosis is essential for the effective treatment of <i>Haemophilus influenzae</i> (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. In this study, sensitive two-step polymerase chain reaction (PCR)-CRISPR and recombinase-aided amplification (RAA)-CRISPR assays were developed, followed by the establishment of the one-pot RAA-CRISPR assay through integration of RAA, CRISPR, and rapid lysis for HI detection. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct the extraction-free one-pot RAA-CRISPR/Cas13a assay (EFORCA). With its high sensitivity, specificity, rapid turnaround time, and operational simplicity, this assay shows great potential as a practical diagnostic tool for HI infection in small laboratory settings.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0053525"},"PeriodicalIF":5.4,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145124057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Clinical and microbiologic features of <i>Achromobacter</i> species: a 10-year, multicenter experience.","authors":"David R Bayless, Mitchell G Dumais, Jack W McHugh, Nischal Ranganath, Madiha Fida, Supavit Chesdachai, Omar M Abu Saleh","doi":"10.1128/jcm.00724-25","DOIUrl":"https://doi.org/10.1128/jcm.00724-25","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Achromobacter&lt;/i&gt; species are Gram-negative bacilli that predominantly affect individuals with cystic fibrosis or those with immunocompromising conditions. Clinical and microbiologic data on &lt;i&gt;Achromobacter&lt;/i&gt; species are limited due to the rarity of these organisms and challenges with species-level identification. We conducted a 10-year retrospective analysis (1 January 2013 to 14 March 2023) of all bloodstream and non-bloodstream &lt;i&gt;Achromobacter&lt;/i&gt; isolates identified at three tertiary-care Mayo Clinic locations (Minnesota, Arizona, and Florida). Additionally, we examined clinical characteristics of adult patients with &lt;i&gt;Achromobacter&lt;/i&gt; bloodstream isolates. A total of 1,598 &lt;i&gt;Achromobacter&lt;/i&gt; isolates, encompassing 1,545 non-bloodstream isolates and 53 bloodstream isolates, were identified. The most frequently identified species was &lt;i&gt;Achromobacter xylosoxidans&lt;/i&gt;, though species-level identification was not possible for many isolates. Among adult patients with &lt;i&gt;Achromobacter&lt;/i&gt; bloodstream infection, the mean age was 58.3 years (standard deviation ± 16.7), 73.1% had a central venous catheter, and 59.6% were immunocompromised. All-cause mortality was 10.2% at 30 days and 18.4% at 90 days. Bloodstream isolates exhibited greater than 90% susceptibility to meropenem, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The respiratory tract was the most common source of non-bloodstream isolates (57.5%). Non-bloodstream isolates showed high susceptibility to imipenem (94.6%), piperacillin-tazobactam (92.8%), trimethoprim-sulfamethoxazole (92.1%), and meropenem (87.5%) while demonstrating significant resistance to fluoroquinolones and aminoglycosides. Our findings support the use of piperacillin-tazobactam, carbapenems, or trimethoprim-sulfamethoxazole as initial therapy for &lt;i&gt;Achromobacter&lt;/i&gt; infections. Further research is needed to better define the clinical and microbiologic characteristics of &lt;i&gt;Achromobacter&lt;/i&gt; infections.IMPORTANCE&lt;i&gt;Achromobacter&lt;/i&gt; is a rare but important genus of bacteria that tends to cause infection in those with cystic fibrosis, recurrent healthcare exposures, and/or an immunocompromising condition. There is limited data on the clinical profile of &lt;i&gt;Achromobacter&lt;/i&gt; infections as well as optimal antibiotic selection for affected patients. We conducted a retrospective study to improve understanding of the microbiologic and clinical characteristics of &lt;i&gt;Achromobacter&lt;/i&gt;. Our study consists of a 10-year survey of all &lt;i&gt;Achromobacter&lt;/i&gt; isolates processed by three Mayo Clinic tertiary-care centers in Minnesota, Florida, and Arizona from 2013 to 2023. We report multiple findings, including the clinical characteristics of patients with &lt;i&gt;Achromobacter&lt;/i&gt; bloodstream infection, the number of different &lt;i&gt;Achromobacter&lt;/i&gt; species identified, and the sources of isolates not obtained from blood cultures. We additionally present antimicrobial susceptibility results for &lt;i&gt;Achromobacter&lt;/","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0072425"},"PeriodicalIF":5.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086371","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transforming tuberculosis diagnosis with clinical metagenomics: progress and roadblocks.","authors":"Yuqing Chen, Hui Tang, Jieyuan Zheng, Qing Yang, Dongsheng Han","doi":"10.1128/jcm.00537-25","DOIUrl":"https://doi.org/10.1128/jcm.00537-25","url":null,"abstract":"<p><p>Tuberculosis (TB) remains a leading global infectious killer, yet traditional diagnostic methods are inadequate. Acid-fast staining suffers from low sensitivity, and mycobacterial culture requires prolonged incubation because of the slow growth of <i>Mycobacterium tuberculosis</i>. PCR-based molecular assays allow rapid detection, but their capacity for resistance profiling is limited to a narrow set of mutations. Metagenomic next-generation sequencing (mNGS) has emerged as a promising culture-independent tool for TB detection, enabling broad-spectrum pathogen identification and offering added value in complex scenarios including extra-pulmonary disease, mixed infections, and infections in immunocompromised or pediatric populations. Clinical studies indicate that mNGS achieves moderate to high sensitivity and excellent specificity in the diagnosis of tuberculosis. However, its diagnostic performance is often constrained by low mycobacterial read counts, interference from abundant host nucleic acids, and the inability to distinguish active from latent infection. In addition, the accuracy of drug resistance prediction using mNGS remains limited, and the World Health Organization currently endorses targeted NGS as the preferred sequencing-based approach for resistance profiling. Despite these challenges, mNGS has facilitated novel diagnostic strategies that combine pathogen detection with host-response data, thereby broadening its potential clinical utility. Nevertheless, practical barriers such as high cost, complex laboratory workflows, and difficulties in data interpretation continue to restrict widespread adoption in routine practice. Future efforts should prioritize technical optimization, standardized protocols, and integration with conventional diagnostics to establish cost-effective and clinically meaningful roles for mNGS in TB diagnosis and management.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0053725"},"PeriodicalIF":5.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Corynebacterium striatum</i> infections in oncologic patients: clinical spectrum, resistance profiles, and evidence of nosocomial transmission.","authors":"Kenya Yukawa, Sohei Harada, Kohji Komori, Brian Hayama, Daisuke Ohkushi, Koichi Takeda, Taisuke Enokida, Akira Yarimizu, Kazumi Takehana, Kageto Yamada, Michihiko Goto, Kazuhiro Tateda","doi":"10.1128/jcm.00829-25","DOIUrl":"https://doi.org/10.1128/jcm.00829-25","url":null,"abstract":"<p><p>Among <i>Corynebacterium</i> species, <i>Corynebacterium striatum</i> is relatively frequently involved in invasive human infections. In this study, we collected clinical information from patients diagnosed with <i>C. striatum</i> infection at a single cancer center and performed antimicrobial susceptibility testing and whole-genome sequencing of the causative strains. Of the 51 patients with <i>C. striatum</i> infections, 15 (29.4%) had postoperative intra-abdominal infections, eight (15.7%) had postoperative skin and soft tissue infections of the neck, and eight (15.7%) had osteoarticular infections. In 15 patients, <i>C. striatum</i> was detected concomitantly with other bacteria. The median duration of antimicrobial therapy was 25 days, with 43 patients (84.3%) showing clinical improvement by day 14. The crude mortality up to 90 days post-diagnosis was 15.7%. Vancomycin was the most commonly used definitive therapy, and 40 patients (78.4%) received multiple antimicrobial agents. Oral minocycline was often administered in patients requiring long-term treatment. Antimicrobial susceptibility testing of 53 strains, including two strains from follow-up cultures from the same patient, showed that most strains were susceptible to daptomycin and tetracyclines. However, non-susceptibility was noted in two strains (3.8%) for daptomycin and four strains (7.5%) for tetracyclines, each associated with <i>psgA2</i> mutation and <i>tet</i>(W) carriage. Core-genome single-nucleotide polymorphism analysis of the strains and epidemiological reviews of the source patients identified three suspected clusters of nosocomial transmission involving seven patients. This study demonstrated that <i>C. striatum</i> can cause a range of infections in patients with underlying diseases, such as malignancy, and that nosocomial spread of this pathogen may also occur.</p><p><strong>Importance: </strong>The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, employed for bacterial species identification in this study, has enhanced the recognition of <i>Corynebacterium striatum</i> as an important human pathogen in clinical microbiology laboratories. Our study demonstrated that <i>C. striatum</i> is associated with various healthcare-associated infections, including those requiring prolonged antimicrobial therapy, and that nosocomial transmission of this pathogen can result in the development of infections. In addition, several agents other than vancomycin, such as teicoplanin, tetracyclines, and trimethoprim/sulfamethoxazole, have demonstrated favorable activities. The results of this study indicate the need for further research on the mechanisms and modes of nosocomial transmission of <i>C. striatum</i>, as well as the clinical efficacy of alternative agents to vancomycin, particularly those suitable for prolonged treatment, given the potential side effects associated with vancomycin use.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0082925"},"PeriodicalIF":5.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The diagnostic utility of Immunoglobulin G (IgG) avidity in distinguishing between past and acute infection of West Nile Virus (WNV).","authors":"Yaniv Lustig, Vicki Indenbaum, Ravit Koren, Shiri Katz-Likvornik, Osnat Halpern, Ella Mendelson","doi":"10.1128/jcm.00952-25","DOIUrl":"https://doi.org/10.1128/jcm.00952-25","url":null,"abstract":"<p><p>Infection with West Nile Virus (WNV) can cause severe disease; however, due to immunoglobulin M (IgM) persistence and short viremia, confirmatory diagnosis of acute WNV infection often requires two consecutive samples and a laborious neutralization assay. Immunoglobulin G (IgG) avidity assay is being used to differentiate between acute and past infection of several viral diseases but not of WNV. To test whether IgG avidity can be utilized for WNV diagnosis, serum samples from 103 past and 84 acute WNV-infected persons diagnosed in our laboratory according to WHO guidelines were subjected to IgG avidity. ROC curve analysis was applied to measure the diagnostic accuracy of WNV IgG avidity and establish the optimal cut-off. Mean avidity and geometric mean neutralization titers (GMTs) from acute and past infected cases were 26% (95% CI: 21-30) and 183 (95% CI: 144-235) and 63% (95% CI: 59-67) and 110 (95% CI: 82-147), respectively. Optimal avidity cut-off level to distinguish between acute and past infection was 35% with sensitivity, specificity, PPV, and NPV of 81% (95% CI: 72-88), 88% (95% CI: 81-93), 55% (95% CI: 42-67), and 96% (95% CI: 94-98), respectively. Our results suggest that IgG avidity can rule out acute WNV infections in patients with positive WNV IgM and IgG antibodies and replace the neutralization assay as a confirmatory assay in most cases. By using an avidity assay as part of the routine diagnostic protocol of WNV, the diagnosis of WNV will be simplified, rapid, and would substantially reduce the workload and diagnostic difficulty of this flaviviral infection.IMPORTANCEDiagnosing West Nile Virus (WNV) infection typically requires two serum samples collected weeks apart and a labor-intensive confirmatory test using live virus. Here, we evaluated immunoglobulin G (IgG) avidity assay, which measures the binding strength between WNV antigen and IgG antibodies, as a simpler diagnostic approach using only one sample. Our findings showed that IgG avidity reliably identifies past WNV infections but is less effective in confirming the acute phase. Crucially, this method can rule out acute infections and allow for a rapid and definitive diagnosis in most cases. IgG avidity assays offer a streamlined alternative, reducing the need for complex tests and prolonged timelines.</p>","PeriodicalId":15511,"journal":{"name":"Journal of Clinical Microbiology","volume":" ","pages":"e0095225"},"PeriodicalIF":5.4,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":"https://doi.org/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":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"https://doi.org/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":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}