Journal of Clinical Microbiology最新文献

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 As vaccine hesitancy increases and resources are withdrawn from national and state public health laboratories, historically low incidence diseases develop into nationwide outbreaks that require increased testing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.

Clinical laboratories are increasingly using diagnostic tests directly on positive blood cultures, which may lead to fewer attempts to recover bacterial isolates. Consequently, public health laboratories can benefit from assays that directly process blood culture samples without requiring submission of clinical isolates to determine additional pathogen features not identified by clinical tests, such as vaccine serotype and bacterial genomic relatedness, for surveillance and outbreak response purposes. In partnership with the Minnesota Active Bacterial Core surveillance (ABCs) site, we identified blood culture samples positive for ABCs streptococcal pathogens and characterized them by a direct whole-genome sequencing from blood culture (dWGS) assay. The dWGS results were compared with the results of a reference method (WGS of isolates from the same cultures) to evaluate concordance in pathogen features and genome assemblies. Of the 97 eligible blood culture samples, 83 (86%) passed dWGS quality control criteria and were subjected to a total of 655 dWGS-based tests, which yielded 651 (99.3%) evaluable results. The percent agreement with reference results was 100% (83/83) for M protein gene (emm)/capsular types and 100% (81/81) for multilocus sequencing types. For genotypic antimicrobial susceptibility testing prediction, the percent prediction agreement was 100% (487/487), false resistant prediction rate was 0% (0/417), and the false susceptible prediction rate was 0% (0/66). Assemblies of pathogen genomes from the same patient differed by 1.08 ± 1.68 (mean ± SD) sites per genome. The dWGS assay can extract high-quality, important streptococcal strain characteristics directly from positive blood culture samples to support evolving public health needs.IMPORTANCEWhole-genome sequencing (WGS) technologies have emerged as a transformative toolkit used by public health microbiology laboratories to detect and characterize pathogens. The surveillance of bacterial diseases often relies on clinical laboratories to submit pathogen isolates to regional or national public health laboratories, which have the capacity to routinely conduct WGS-based strain characterization. Clinical laboratories are increasingly using diagnostic tests directly on positive blood cultures, which may lead to fewer attempts to recover bacterial isolates. The study evaluated a direct whole-genome sequencing from blood culture (dWGS) assay that directly processes blood culture samples. The dWGS assay recovered high quality, important streptococcal strain characteristics, including vaccine serotypes and whole-genome assemblies, without requiring submission of clinical isolates. Thus, the dWGS assay represents a promising tool for addressing the evolving needs of public health laboratories in the metagenomics era.

Mycoplasma pneumoniae (MP) is a major cause of acute respiratory diseases. While macrolide resistance in MP has been recognized for decades, the recent surge in its prevalence has emerged as a growing clinical and public health concern. Rapid, sensitive, and specific detection of MP and macrolide-resistant Mycoplasma pneumoniae (MRMP) are crucial for preventing the spread of the disease. By leveraging the precise programmable recognition and cleavage capabilities of Pyrococcus furiosus Argonaute nuclease (PfAgo), we have established a short-PCR- based method that targets both the RepMP1 gene and the A2063G mutation in the 23S rRNA, enabling efficient detection of MP and MRMP. The high GC content around the 2063 site in the 23S rRNA complicates primer and PfAgo guide DNA (gDNA) design, posing challenges to the differentiation between mutant and wild-type strains. Through meticulous design and screening of PfAgo gDNA, along with optimization of reaction conditions, we achieved a high sensitivity, where the method detects as few as 0.5 copies per reaction for MP, 5 copies per reaction for the A2063G mutation, and 14 copies per reaction for wild-type MP. The entire detection procedure can be completed in 53 min using either extracted DNA or extraction-free oropharyngeal swab samples, with no cross-reaction with the tested eight common pathogens. This methodology has been successfully applied to the detection of MP and MRMP in 58 oropharyngeal swabs, providing robust support for epidemic control of MP and offering critical guidance for the appropriate treatment of MRMP cases.IMPORTANCEThe PCR-PfAgo system developed in this study establishes a rapid, sensitive, and specific detection platform that is crucial for early identification of Mycoplasma pneumoniae and the macrolide resistance-associated A2063G mutation, thereby guiding clinical treatment and controlling the spread of resistant strains. With advantages including enhanced sensitivity and superior specificity, the system accurately discriminates between resistant and sensitive strains, providing critical guidance for the timely and appropriate use of antibiotics in respiratory infections. This work demonstrates the feasibility of Argonaute protein-based systems for clinical diagnostics and provides a scalable framework for detecting other pathogens and resistance markers, laying the groundwork for future developments in multiplex detection, instrument-free readout, and prospective clinical validation.

Human papillomavirus (HPV) is a prevalent sexually transmitted infection linked to genital warts and various cancers. While effective screening methods exist for women, detecting HPV in men remains challenging due to asymptomatic infections and the lack of standardized, non-invasive diagnostic tools. This study aimed to assess the correlation between HPV DNA detection in urine and semen samples from men attending a fertility clinic in Ecuador. This cross-sectional study included 106 sexually active male patients referred for semen analysis at the Fertility Unit of Hospital del Río, Cuenca, Ecuador, between June 2024 and March 2025. Each participant provided paired semen and urine samples, which were analyzed for HPV genotyping. Sociodemographic and behavioral data were also collected. HPV DNA was detected in 15% of semen samples and 9.4% of urine samples. Both low- and high-risk genotypes were identified in semen and urine. In semen samples, high-risk HPV was detected in 9.4% and low-risk HPV in 5.7%. In urine, high-risk HPV was detected in 8.5% and low-risk HPV in 0.9% of samples. Among high-risk genotypes, HPV 58 was the most frequent in urine (20%), while HPV 81 was the most frequent in semen (19%). Genotypes such as HPV 16, 18, 39, 52, and 66 were detected exclusively in semen. Compared with semen sampling, urine sampling showed a sensitivity of 37.5% and a specificity of 95%. Agreement between the two sample types was fair (kappa = 0.39). Although urine sampling offers a non-invasive option, its limited sensitivity restricts its utility as an HPV detection method in asymptomatic men. Semen analysis provides higher detection rates and may be more reliable for HPV screening in male fertility populations.

Importance: This study addresses a critical knowledge gap regarding human papillomavirus (HPV) detection in men, a topic insufficiently covered in current control and prevention strategies. While screening and vaccination efforts in women have advanced considerably, the absence of standardized, well-accepted, and non-invasive methods for HPV detection in men limits the recognition of their roles as reservoirs and transmitters in the HPV transmission pathway. Evaluating alternative solutions for HPV testing, such as urine and semen sampling, not only contributes valuable scientific knowledge to improve detection in this population but also establishes the foundation for public health policies on male HPV testing. Ultimately, this work supports the goal of reducing global HPV prevalence and its associated diseases.

The VITEK REVEAL antimicrobial susceptibility testing (AST) system is a fast in vitro diagnostic device that determines the antimicrobial susceptibility profile of gram-negative organisms from positive blood culture after independent organism identification. The objectives of this study were to (i) compare the performance of VITEK REVEAL to disk diffusion (DD) and (ii) evaluate implementation of VITEK REVEAL into a laboratory workflow that employs matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as the primary method for organism identification. A total of 150 monomicrobial, GN-positive blood cultures were enrolled, of which 47.3% (71/150) contained Escherichia coli, 17.3% (26/150) Klebsiella pneumoniae, and 15.3% (23/150) Pseudomonas aeruginosa, with the remaining comprising other Enterobacterales and one Acinetobacter spp. The time to result (TTR) from blood culture positivity to AST results was compared for DD with tiered testing for multidrug-resistant organisms and VITEK REVEAL. Overall, the standard-of-care workflow had a combined TTR for AST of 37.2-54.7 h, while the workflow of MALDI-TOF MS + VITEK REVEAL had a TTR of 14.0 h due to the time necessary to identify the organism using MALDI-TOF MS. The categorical agreement (CA) for claimed organism-antibiotic combinations was 97.0%, with a very major discrepancy (VMD) of 2.9% (8/279) and a major discrepancy (MD) of 0.3% (5/1957), following adjudication of discrepant results with broth microdilution. The CA for organisms with an extended-spectrum beta-lactamase phenotype (n = 29) was 93.2%, with VMD of 2.0%, and MD of 0.7%. In conclusion, the VITEK REVEAL reduced the TTR for AST by at least 23 h compared to standard-of-care DD, with a CA of 97%.IMPORTANCEBloodstream infections caused by gram-negative bacteria require effective and timely treatment. Laboratory testing informs the choice of antibiotics, but traditional methods can require multiple days for results. The VITEK REVEAL system determines antibiotic susceptibility directly from a positive blood culture within 8 h, though it requires an independent method for organism identification, such as mass spectrometry or nucleic acid amplification testing. In this study, we examined VITEK REVEAL as part of a workflow incorporating mass spectrometry for organism identification. This combined approach provided susceptibility results approximately 1 day earlier than standard disk diffusion while maintaining high accuracy for most organism-antibiotic combinations, including those active against multidrug-resistant organisms. Incorporating VITEK REVEAL into routine laboratory workflows has the potential to accelerate targeted therapy and limit unnecessary broad-spectrum use in patients with gram-negative bloodstream infections.