Journal of Clinical Microbiology最新文献

The diagnosis of mosquito-borne viral infectious diseases can be challenging, in part due to the complexity of antibody cross-reactivity between many of these viruses. This is further complicated by the unpredictable nature of climatic variability affecting disease transmission, exotic virus incursion, and the potential emergence of new strains of viruses with increased virulence. A thorough understanding of virus biology, locally relevant transmission patterns, and principles of diagnostic tests is required for the investigation of suspected clinical cases. This review provides guidance for veterinarians, researchers, and policy-makers on the diagnosis and management of alphavirus and orthoflavivirus infections in animals with a One Health perspective, including interpretation of laboratory results. Biosecurity and biosafety considerations and the zoonotic potential of mosquito-borne infections are also discussed.

Ceftobiprole is a new-generation cephalosporin with activity against Staphylococcus aureus, but a comprehensive evaluation of MIC Test Strip (MTS) and disk diffusion (DD) methods using EUCAST and FDA breakpoint is lacking. Using broth microdilution (BMD) as the reference standard, we assessed the performance of MTS and DD for determining ceftobiprole susceptibility among 422 clinical S. aureus isolates. Ceftobiprole retained potent activity, with MIC₅₀/MIC₉₀ of 0.5/1 mg/L and an overall susceptibility rate of 98.8%; methicillin-resistant Staphylococcus aureus (MRSA) showed 97.6% susceptibility. MTS demonstrated high agreement with BMD, achieving 98.8% categorical agreement (CA), 1.2% major error (ME), and zero very major error (VME) under European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, meeting the acceptability limits of ISO 20776-2:2021 and CLSI M52. DD performance was breakpoint-dependent: under EUCAST criteria, the overall CA was 89.6%, with 10.6% ME and 0 VME. The 40.3% of MRSA that fell into the EUCAST-defined Area of Technical Uncertainty (ATU) zone were all shown to be susceptible by the reference BMD method. Under Food and Drug Administration (FDA) breakpoints, the CA rose to 94.1% and ME fell to 1.7%. Consequently, MTS offers a reliable routine option; DD is suitable for routine testing under FDA breakpoints, while for EUCAST users, MRSA isolates in the ATU zone should be confirmed by BMD or MTS. VME was not determined under FDA breakpoints due to the absence of resistant isolates.

Importance: Ceftobiprole offers a β-lactam option for Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA); however, its susceptibility testing remains insufficiently examined across both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Food and Drug Administration (FDA) interpretive criteria. We systematically compared MIC Test Strip (MTS) and disk diffusion (DD) with broth microdilution (BMD), demonstrating breakpoint-specific performance for methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA and providing practical recommendations that allow laboratories to report accurate results regardless of the guidelines they follow.

Respiratory tract infections remain a major cause of global morbidity, highlighting the need for rapid and accurate diagnostic testing. This multicenter study evaluated the clinical and analytical performance of the updated NxTAG Respiratory Pathogen Panel v2 (RPPv2), a high-throughput multiplex molecular assay designed to detect 21 viral and bacterial respiratory pathogens. A total of 2,145 nasopharyngeal swab specimens, including both prospectively enrolled and archived specimens, were analyzed across five U.S. clinical sites. The NxTAG RPPv2 was assessed against FDA-cleared molecular assays and bi-directional sequencing, with contrived specimens used to supplement low-prevalence targets. Valid results were obtained for 99.7% of specimens after a single retest, with minimal performance differences between fresh and frozen specimens. Among viral targets, 15 of 18 (one viral target did not have clinical positive specimens for analysis) achieved ≥95% positive percent agreement (PPA) across the prospective and pre-selected arms. For bacterial targets, Mycoplasma pneumoniae and Chlamydia pneumoniae showed PPA values of 92.3% and 100%, respectively. Lower PPA values were observed for parainfluenza 4 (90.3%). Negative percent agreement (NPA) was consistently high across targets, with all viral and bacterial analytes achieving ≥99.2%. Reproducibility across sites and operators was 99.9%. Overall, the NxTAG RPPv2 demonstrated high accuracy, reproducibility, and operational efficiency, supporting its clinical utility for rapid and reliable diagnosis of respiratory tract infections.

Importance: Respiratory infections remain a major global health challenge, particularly during seasonal epidemics and pandemics. Rapid and accurate pathogen detection is essential to guide treatment decisions, limit transmission, and reduce unnecessary antibiotic use. This multicenter study evaluated the updated NxTAG Respiratory Pathogen Panel v2 (RPPv2), a high-throughput molecular assay capable of detecting 22 common viral and bacterial respiratory pathogens from a single sample. The panel demonstrated high accuracy, reproducibility, and reliability across multiple clinical sites and sample types, including both fresh and frozen specimens. These findings support the NxTAG RPPv2 as a comprehensive, efficient diagnostic tool that can support respiratory infection diagnosis and contribute to multifaceted antimicrobial stewardship efforts.

Laboratory-developed tests (LDTs) play a critical role in meeting unmet diagnostic needs, particularly for rare infections and high-acuity or immunocompromised patient populations. However, current US regulatory and reimbursement frameworks have constrained innovation and delayed implementation of many essential laboratory diagnostic tests. Here, we propose a pragmatic, collaborative model focusing on infectious disease molecular diagnostics that maintains analytical rigor while allowing clinical validity to be supported by evidence from the medical literature and clinical judgment. To address the resource constraints faced by hospital laboratories, we envision a voluntary, national repository where laboratories and manufacturers contribute standardized analytical and, where appropriate, clinical validation data for both newly developed and modified Food and Drug Administration (FDA)-cleared assays. Data elements such as accuracy, limit of detection, analytical measurement range, analytical specificity, and inclusivity compared with reference determinations would be aggregated within groups representing technically identical assays, enabling cumulative validation and shared use of high-quality evidence. By leveraging these shared data sets, hospital laboratories could implement LDTs in-house with minimal redundant validation, enabling broader access to testing near the point of patient care and faster turnaround times. Manufacturers could use the same data to support applications for expanded intended use of existing FDA-cleared and -approved tests. This framework would strengthen hospital laboratory diagnostic capacity, accelerate test implementation, and improve patient outcomes and healthcare system efficiency.

Isoniazid (INH) is a critical antibiotic used worldwide for the treatment and prophylaxis of tuberculosis. Drug resistance (DR) to INH is the single most common type of DR, mediated by multiple genes/loci, including katG, inhA, mabA, mabA-inhA, and the oxyR-ahpC intergenic region. Over the course of 6 years, we performed a two-phase study of 3,696 Mycobacterium tuberculosis complex (MTBC) strains, aiming to determine the molecular basis of INH resistance and assess whole-genome sequencing (WGS) for predicting resistance. In phase 1, we performed a side-by-side study, including 1,767 strains with paired phenotypic drug susceptibility testing (DST) and genotypic DST. We found WGS capable of accurately predicting INH resistance with a sensitivity of 90.3% and a specificity of 99.8%. The negative predictive value of WGS for INH susceptibility was 98.8%. Based on these findings, we developed a molecular testing algorithm where phenotypic DST (pDST) was reduced and applied this new testing algorithm in phase 2 to 1,929 MTBC strains, resulting in streamlined testing, reduced cost, and decreased turnaround time (TAT). The prevalence of INH resistance among MTBC strains in New York was found to be 10.2%. Of the 3,696 isolates tested, 337 were predicted INH-resistant by WGS. Of 41 additional strains exhibiting phenotypic INH resistance, 38 were found to have mutations in genes known to be associated with INH resistance. This study demonstrates the utility of WGS as a molecular tool for predicting INH DR and shows that the vast majority of INH resistance in MTBC has a molecular basis in known resistance loci.

Importance: Isoniazid (INH) is one of the two most critical antibiotics used as part of standard treatment of tuberculosis and is also used as preventative therapy for contacts of tuberculosis patients, despite having a higher rate of drug resistance than all other antibiotics used in standard therapy. Furthermore, isoniazid resistance typically precedes rifampin resistance in the development of multidrug-resistant TB. As such, the reliable detection of INH resistance is crucial for case management and to limit the acquisition of additional drug resistance. The present study describes a whole-genome sequencing approach to predicting INH resistance from clinical isolates and models how this technology can be used within a reduced phenotypic drug susceptibility testing algorithm to limit duplicate testing, saving resources and time while maintaining the sensitivity of resistance detection.

Diagnosing pulmonary diseases caused by non-fumigatus Aspergillus species remains challenging. We conducted a single-center, retrospective observational study in a French Respiratory Medicine Department. Patients with at least one respiratory sample positive for a non-fumigatus Aspergillus species, without concurrent A. fumigatus isolation over a 12-month period, were included. The primary objective was to determine the prevalence of pulmonary events (colonization or pulmonary diseases) associated with non-fumigatus Aspergillus species. Secondary objectives included species characterization and assessment of positive results for available diagnostic tests, including direct examination and fungal culture from respiratory samples, galactomannan in bronchoalveolar lavage, and serum A. fumigatus-specific IgG. Between April 2017 and January 2022, 497 patients (39.6%) had cultures positive for non-fumigatus Aspergillus species. Among them, 52 (10.5%) experienced pulmonary events: 36 were colonized, and 16 had a documented pulmonary disease. Aspergillus niger was the most frequently isolated species (41%), followed by Aspergillus flavus (27%) and Aspergillus nidulans (10%). Positive results were observed in 10/437 (2.3%) samples for direct examination, 75/808 (9.3%) for fungal culture, 7/94 (7.4%) for galactomannan in bronchoalveolar lavage, and 12/49 (24.5%) for serum Aspergillus fumigatus-specific IgG. Among patients with non-fumigatus Aspergillus positive respiratory samples, most were colonized, while nearly one-third had clinically significant pulmonary diseases, underscoring the clinical relevance of these species. Low positivity rates across diagnostic tests underscore the need for repeated respiratory sampling and fungal culture and suggest that assays primarily designed for A. fumigatus may under-detect these pulmonary events.

Importance: Non-fumigatus Aspergillus species are recognized as pulmonary pathogens, but their diagnosis is poorly documented. In this 5-year, single-center study, 497 of 1,256 patients had at least one positive respiratory culture for a non-fumigatus species, with 36 considered colonized and 16 with documented lung disease. A. niger, A. flavus, and A. nidulans accounted for almost four-fifths of the isolates. Routine tests produced poor results, with positivity rates of 2.3% for microscopy, 9.3% for repeat culture, 7.4% for bronchoalveolar galactomannan, and 24.5% for Aspergillus fumigatus serum IgG. Overall, the study shows that non-fumigatus species can cause treatable chronic lung disease, but that current diagnostics miss most cases. Until more sensitive tests are available, clinicians must rely on repeated respiratory sampling and culture to identify these infections.

Carbapenem-resistant organisms (CROs) pose a major threat to global health due to limited therapeutic options and their capacity for rapid dissemination. Among these, carbapenemase-producing (CP) strains are of greatest concern, as they hydrolyze most β-lactams, and carbapenemase genes are readily spread via mobile genetic elements. Historically, clinical laboratories relied solely on minimal inhibitory concentration (MIC) results and interpretive criteria to guide therapy, with carbapenemase testing performed mainly for epidemiologic purposes. However, changing carbapenemase epidemiology, the introduction of enzyme-specific novel β-lactam combination agents for treatment, and updated Clinical and Laboratory Standards Institute (CLSI) guidance have renewed the importance of carbapenemase testing among carbapenem-resistant Enterobacterales. The 2025 CLSI M100 update now recommends carbapenemase testing for most Enterobacterales resistant to at least one carbapenem, emphasizing differentiation of key enzymes, such as KPC, NDM, and OXA-48-like, to inform therapeutic decisions and support antimicrobial stewardship. This minireview summarizes the evolution of CLSI guidance from early breakpoint establishment through the "MIC-only" era to the current antimicrobial resistance mechanism-driven framework. Key issues addressed include the clinical limitations of prior clinical breakpoints, challenges in balancing sensitivity and specificity of screening criteria to guide carbapenemase testing in different settings, and the expanding role of rapid phenotypic and molecular detection methods. Revisions to the EDTA-modified carbapenem inactivation method (eCIM) are discussed in light of increasing co-production of metallo-beta-lactamase and serine-carbapenemases. Reintegration of carbapenemase testing into clinical workflows highlights the role of the clinical microbiology laboratory as a critical component of antimicrobial stewardship.