Journal of Clinical Microbiology最新文献

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.

Non-tuberculous mycobacterial (NTM) skin infections present diagnostic and therapeutic challenges in resource-limited primary care settings. This retrospective study evaluated the diagnosis and treatment of NTM skin infections at a primary care hospital in Shouguang City, a coastal region of China with high-risk occupational exposure to aquatic environments, from 2021 to 2022. We analyzed 144 patients, using real-time PCR on formalin-fixed paraffin-embedded (FFPE) tissue samples to validate empirical diagnoses based on clinical symptoms, exposure history, and histopathology. Our results demonstrated a high concordance (79.17%) between empirical and molecular diagnoses, confirming the reliability of the primary care hospital's diagnostic approach. Real-time PCR successfully identified specific NTM species in a subset of cases, with Mycobacterium marinum and Mycobacteroides abscessus being the most prevalent. Empirical treatment with combination antibiotics (primarily clarithromycin and rifampicin) was highly effective, achieving a cure rate of 90.97%. This study highlights the effectiveness of integrated clinical-histopathological assessment for managing NTM infections in resource-limited settings and underscores the value of molecular techniques such as PCR in enhancing diagnostic precision and facilitating retrospective research using archived FFPE samples.

Importance: This study addresses a critical gap in managing non-tuberculous mycobacterial (NTM) skin infections in resource-limited primary care settings, with particular relevance to coastal regions like Shouguang, where occupational exposure to marine environments elevates infection risk. By validating empirical diagnosis through molecular testing, we demonstrate that integrated clinical-histopathological assessment is a reliable method for identifying NTM infections where advanced diagnostics are unavailable. Our adaptation of real-time PCR to archived FFPE tissues provides a feasible model for retrospective species identification and research, overcoming key resource constraints. The high documented cure rate with clarithromycin-rifampicin regimens provides practical treatment benchmarks for regions with similar epidemiological profiles. These findings offer immediate clinical value by: (i) reinforcing the reliability of empirical diagnosis in resource-limited contexts, (ii) establishing a practical pathway for molecular confirmation using stored samples, and (iii) validating effective treatment protocols for high-risk occupational groups. As NTM incidence increases globally, this work equips primary care systems with evidence-based strategies to improve diagnostic accuracy and patient outcomes in settings with high marine exposure while simultaneously enabling future research through the utilization of existing biospecimen repositories.

Population-based HIV Impact Assessment surveys were conducted in 14 countries to measure HIV prevalence, incidence, and viral load suppression in children aged 0-14 years and adults aged ≥15 years. We examined the performance of rapid HIV testing algorithms in multiple countries. Survey participants received an HIV diagnosis based on two serial tests in Cameroon, Côte d'Ivoire, Eswatini, Haiti, Kenya, Lesotho, Malawi, Mozambique, Tanzania, and Zambia. Whereas in Ethiopia, Kenya, Namibia, Uganda, and Zimbabwe, diagnosis was based on a tie-breaker algorithm. Testers received intensive training on specimen collection, testing, and results reporting. HIV-positive and indeterminate results were confirmed by Geenius HIV-1/2 test (Bio-Rad). Pooled test 1 and 2 positive concordance, test 2 and 3 negative concordance, and the positive predictive value (PPV) of the algorithm were calculated. A total of 312,069 participants aged ≥18 months (except for Eswatini, Mozambique, and Uganda, where only adults aged 15+ years were included) were tested by either the two-test (n = 243,311) or tie-breaker (n = 98,757) algorithm for diagnosis. The combined test 1 and 2 positive concordance and PPV were 93.8% and 99.4%, respectively, for countries with a two-test algorithm. Similarly, test 1 and 2 positive concordance, test 2 and 3 negative concordance, and PPV were 87.5%, 84.4%, and 98.8%, respectively, for the tie-breaker algorithm. The PPV ranged from 94.2% to 99.9%, with higher PPV in high burden countries. The HIV testing algorithms performed with high accuracy, with PPV reaching 99% or higher. Adoption of continuous quality improvement is essential to ensure accuracy of HIV diagnosis in service delivery settings.

Importance: HIV diagnostic testing in most African countries follows national algorithms that typically use two tests, with or without a tie-breaker. We assessed the accuracy of these algorithms using data from population-based surveys in 14 sub-Saharan African countries, where all HIV-positive results were further confirmed with the Geenius HIV-1/2 supplemental assay. Our findings show that inter-test concordance and positive predictive values (PPVs) varied by HIV prevalence, with higher PPVs observed in higher-prevalence settings. Overall, the PPV of HIV diagnosis was close to 99%, indicating that two-test algorithms can provide highly accurate results when testing is performed with strict adherence to quality standards and tester competency. These results underscore the importance of quality assurance (QA) and suggest that countries with lower HIV prevalence may benefit from adopting a three-test algorithm. However, such changes should be accompanied by careful attention to logistics, procurement, training, record keeping, and other QA measures.

The incidence of syphilis has risen dramatically in many regions despite the availability of affordable facility-based testing and curative treatment. The recent approval of over-the-counter syphilis self-tests (SSTs) represents an important advance for expanding diagnostic access and disease control and prevention. Evidence demonstrates that SSTs are accurate, usable, and acceptable. However, as with HIV and COVID-19 self-testing, implementation challenges remain, including ensuring equitable access, supporting vulnerable groups, securing linkage to care, and maintaining quality assurance.