Journal of Clinical Microbiology最新文献

Lower respiratory tract infections (LRTIs) place a substantial burden on emergency departments (EDs) during winter outbreaks. Their microbiological diagnosis is currently based on either broad-spectrum molecular techniques performed in laboratory, with turnaround times incompatible with ED workflows, or point-of-care systems, restricted to SARS-CoV-2, influenza A/B, and RSV (quadriplex panel). The Spotfire combines rapid result and syndromic approach (10 respiratory viruses and 4 atypical bacteria), but its added value in EDs remains unassessed. During the 2023 winter, trained nurses collected nasopharyngeal swabs from adult patients with LRTI symptoms and operated the Spotfire in the ED of the Poitiers University Hospital. We described the viral epidemiology and evaluated the impact and efficacy of this rapid broad-spectrum diagnosis by comparing the management of patients with or without respiratory pathogen detected. From 15 December 2023 to 15 March 2024, 1,320 samples were analyzed with Spotfire, with a median turnaround time of 37 min and 10 uninterpretable results. A total of 540 (41%) were positive. Influenza A (30.9%), rhinovirus/enterovirus (21.1%), and SARS-CoV-2 (18.7%) were the main viruses detected, while atypical bacteria represented 8% of all pathogens. Overall, a pathogen not represented on the quadriplex panel was detected in 43% samples. Positive test results were associated with faster medical decision (380 ± 234 vs 431 ± 238 min; P < 0.001), fewer hospital admissions (65% vs 78%; P < 0.001), and shorter hospital stay (10 ± 9 vs 12 ± 14 days; P = 0.006) than negative test results. Antibiotic therapy was administered to 42 of 43 of Mycoplasma pneumoniae-positive patients (98%) compared with 110 of 1,265 (9%) negative patients. This study validated the feasibility of this new diagnostic tool in high-volume EDs improving patient flow, antimicrobial decisions, and isolation strategies.IMPORTANCEThis study provides the first real-world evidence supporting the use of a broad multiplex PCR platform for respiratory pathogens directly at the point of care in a high-volume emergency department. By enabling the simultaneous detection of 14 viruses and atypical bacteria within 20 min, this system bridges a critical gap between laboratory diagnostics and bedside clinical decision-making. Its implementation proved feasible and reliable, improving patient flow, antimicrobial stewardship, and infection control measures. Nearly half of the pathogens identified would have been missed by conventional quadriplex assays, highlighting the added diagnostic value of broader syndromic coverage. These findings are of interest to both clinicians and microbiologists, as they provide pragmatic evidence to guide the integration of advanced molecular diagnostics into acute-care workflows and to optimize patient management during respiratory infection surges.

Effective tuberculosis (TB) management relies on prompt diagnosis of Mycobacterium tuberculosis complex (MTBC) and associated drug resistance. The Sanity 2.0 assay is a high-resolution melting assay designed for direct respiratory sample testing, enabling simultaneous detection of MTBC and resistance to rifampicin (RIF), isoniazid (INH), and fluoroquinolones (FQ) in a single step. This study evaluated its diagnostic performance in two registered multicenter trials among bacteriologically confirmed TB patients. Diagnostic performance was evaluated for MTBC detection, as well as for the identification of resistance to RIF, INH, and FQ, using phenotypic drug susceptibility testing, whole-genome sequencing, and a composite reference standard. Agreement analyses were conducted between the Sanity 2.0 assay and Xpert MTB/RIF and Xpert MTB/XDR. Among 611 patients, the Sanity 2.0 assay detected MTBC in 563 patients, exhibiting a sensitivity of 92.1% (95% CI: 89.7-94.0). For detecting resistance to RIF, INH, and FQ, sensitivities exceeded 90%, with specificities of 95.8% (95% CI: 88.5-98.6), 100.0% (95% CI: 96.4-100.0), and 97.8% (95% CI: 93.8-99.3) against the composite reference standard, respectively. The agreement with Xpert MTB/RIF for RIF detection was 98.6% (95% CI: 96.9-99.3). For INH and FQ resistance, the agreement with Xpert MTB/XDR was 92.0% (95% CI: 88.5-94.5) and 94.3% (95% CI: 91.2-96.3), respectively. The Sanity 2.0 assay is a rapid and user-friendly platform capable of detecting both MTBC and key drug resistance. It demonstrated good diagnostic performance and could potentially be an effective alternative to guide individualized anti-TB treatment, especially in resource-limited settings.

Importance: Rapid and accurate detection of both Mycobacterium tuberculosis complex (MTBC) and key drug resistance is critical to improving tuberculosis treatment outcomes and reducing transmission. However, current molecular diagnostic workflows often require sequential testing, which can delay the initiation of effective and individualized therapy. We evaluated the Sanity 2.0 assay, an integrated high-resolution melting test that simultaneously detects MTBC and resistance to rifampicin, isoniazid, and fluoroquinolone resistance directly from respiratory samples in about 2-3 hours. The assay demonstrated excellent performance, with MTBC detection sensitivity of 92.1% and drug resistance sensitivities exceeding 90% and specificities over 95% against a composite reference standard, as well as strong concordance with World Health Organization-endorsed molecular assays. Implementation of the Sanity 2.0 assay could streamline TB diagnostic workflows; enable rapid, single-step resistance profiling; and facilitate timely, individualized treatment-particularly in resource-limited settings where rapid and comprehensive resistance testing remains a critical unmet need.

Although artificial intelligence-particularly large-language models-receives daily attention, the application of AI to image-recognition challenges in clinical microbiology has been under development for several years. In the accompanying article, B. A. Mathison, K. Knight, J. Potts, B. Black, et al. (J Clin Microbiol 63:e01062-25, 2025, https://doi.org/10.1128/jcm.01062-25) (in collaboration with ARUP Laboratories and TechCyte) describe a trained convolutional neural network (CNN) that reviews wet-mount parasitology smears with accuracy and analytical sensitivity exceeding that of a cohort of highly trained medical technologists. The impressive results were enabled by an extensive, globally sourced training set. These findings constitute Part II of the authors' earlier Journal of Clinical Microbiology publication on CNN-based diagnosis of trichrome-stained smears and provide a robust proof-of-concept for integrating AI into clinical microbiology workflows. We comment on the translatability of this technology to routine clinical laboratories.

In 2025, measles cases in the United States have reached their highest level since the disease was officially declared eliminated in 2000. Molecular assays may assist in the early diagnosis of measles and improve contact tracing efforts. In this manuscript, we describe the validation of a real-time PCR assay on the Hologic Panther Fusion Open Access system for the detection of measles virus (MeV) and differentiation between wild-type and vaccine strains. After implementation, we examined 3 months of clinical testing data to understand testing trends and utilization. Over the study period, a total of 525 tests from 491 patients were performed. MeV was detected in 16 specimens with 6 wild-type and 10 vaccine strain identifications. Children less than 10 years old constituted the largest proportion of tested individuals (54.3%) and vaccine strain detections (9/10, median age 1.2 years), while wild-type infections were observed in individuals aged 20-50 (6/6, median age 32.6 years). Those with vaccine strain detected had significantly higher Ct values for the pan-measles target versus wild-type infections (33.6 vs 28.3; P-value < 0.05). Only 4.2% of patients in our cohort received paired serologic and molecular measles testing. When paired data were available, PCR had a positive agreement of 25% and a negative agreement of 98% with IgM results. Molecular testing for MeV with the ability to differentiate wild-type strains from vaccine strains is a helpful tool in the response to measles re-emergence.IMPORTANCEThe 2025 U.S. measles outbreak comes at a challenging time for public health in America. As vaccine hesitancy increases and resources are withdrawn from national and state public health laboratories, historically low incidence diseases develop into nationwide outbreaks that require increased testing capacity. In response to this need, our national reference laboratory developed a measles PCR assay that allows for the detection and separation of vaccine from wild-type strains. The assay was launched on the Hologic Panther fusion system to improve throughput and reduce turnaround times. In this research article, we describe the design of our assay, validation results, and early clinical performance.

Description of new and revised taxa originating from non-domestic animal species continues to be pursued. The majority are bacteria isolated from the alimentary system of a variety of healthy wildlife, adding to our understanding of microbial flora present in the normal state. A few new and revised taxa are associated with disease, including a novel Erysipelothrix species associated with sepsis in albatross and a revised species, Allocoenonia anatina comb. nov., associated with Riemerella anatipestifera-like disease in ducks and geese. Representative bacteria from genera that are additionally under study for the remediation of pollutants and bacteria that are related to significant animal or zoonotic pathogens are additionally discussed.

The World Health Organization (WHO) recommends testing stool with Xpert MTB/RIF Ultra (Xpert-Ultra) as an initial diagnostic test for detection of tuberculosis (TB) and rifampicin resistance in children. The option of testing stool on the Truenat platform could benefit children and their caregivers in remote areas where GeneXpert is not available. We report the results of research to validate a protocol for processing and testing stool on Truenat using an adapted Simple One Step (SOS) stool method in routine settings in Nigeria. Stool specimens from children with presumptive TB were tested with Truenat MTB Plus (Truenat) and Xpert-Ultra using a comparative cross-sectional study design. A total of 510 children were enrolled and submitted a stool specimen. Of these, 482 (94.5%) had valid results on both platforms, 28 (5.8%) with MTB detected and 454 MTB not detected. Of the 28 with MTB detected, eight were detected on both platforms, seven on Truenat only, and 13 on Xpert-Ultra only. The concordance rate between Truenat and Xpert-Ultra was high at 95.8%. Significantly more non-determinate (error/invalid) results were observed with Truenat compared to Xpert-Ultra (4.7% versus 1.2% respectively; P < 0.001). Truenat can accurately detect MTB using 100 mg of stool with an adapted SOS stool protocol. Further optimizing extraction methods could reduce the frequency of non-determinate results. These results hold promise for expansion of childhood TB diagnosis services to hard-to-reach areas with limited infrastructure that are suitable for Truenat placement. Global scale-up of these alternative testing approaches will be necessary to close the gap in childhood TB case finding.IMPORTANCEFollowing WHO recommendations, high TB burden countries have commenced testing stool on GeneXpert to improve TB case finding among children and others who may not easily produce sputum. In previous work, we adapted the Simple One Step (SOS) stool method for GeneXpert for the Truenat platform. In the present study, we validated the revised processing method under routine conditions in health care facilities in Nigeria. We tested stool from 510 children with presumptive TB on both the GeneXpert and Truenat platforms across a variety of health care settings. Concordance between Truenat and Xpert MTB detection was high at 95.8%, confirming stool can be tested on Truenat in routine services. Our study demonstrates that TB diagnostic testing for children can be provided in peripheral health facilities located in remote geographic areas with limited infrastructure where Truenat can be placed. Global scale-up will help further close the gap in childhood TB case finding.

Prion diseases are characterized by misfolding of prion protein (PrP) from correctly folded PrP^C to a disease-associated form, PrP^D. Real-time quaking-induced conversion (RT-QuIC) detects prions by "seeding" reaction mixtures, which contain recombinant PrP, with samples suspected to contain prions, resulting in amplification of misfolded PrP. The assay is sensitive to inhibition by tissue constituents, including blood. Heme, a cofactor of hemoglobin (Hb), has been shown to bind PrP in an isoform-specific manner and to affect the stability of other pathogenic amyloids. Herein, tissue samples from scrapie-positive sheep were used to seed RT-QuIC reactions in the presence of heme-as free hemin, as a cofactor of Hb, and as present in whole blood. At equivalent heme concentrations, the inhibitory action of free heme was the least and that of blood the greatest, suggesting other components of Hb and whole blood have additional inhibitory actions. We also demonstrate that this inhibition of RT-QuIC acts through disruption of the recombinant PrP assay substrate, rather than destruction of PrP^D seeds. Lastly, heme concentrations were measured in several ruminant tissues. Heme levels exceeded inhibitory thresholds in nearly all types of intact tissue but were reduced below inhibitory levels at a 1:1,000 dilution of most tissue types, with whole blood being one of a few notable exceptions. Our results suggest that detection of PrP^D seeding activity is not precluded by exposure to heme in tissue samples, but that the final heme concentration introduced into the RT-QuIC assay mixture is the critical factor that impacts detection sensitivity.

Importance: Real-time quaking-induced conversion (RT-QuIC) is an ultrasensitive amplification assay for the detection of prions. The assay has shown exceptional performance in optimal laboratory conditions, on par with bioassay, and far surpassing current immunoassay diagnostics. However, efforts to apply RT-QuIC as a real-world diagnostic have been hampered by inconsistencies and unexpectedly low sensitivity in some field samples. This study aims to quantify and characterize the mechanism of inhibition from blood and its constituent parts, hemoglobin and heme-omnipresent components of most sample types. Such systematic evaluations of RT-QuIC inhibitory factors represent necessary steps toward the consistent and sensitive performance necessary for a field-applicable diagnostic assay.