Journal of Clinical Microbiology最新文献

Intestinal parasitic infections (IPIs) have a worldwide distribution and have a major impact on health, work capacity, and economy in many countries. Light microscopy is still considered the reference method for IPI diagnosis but is labor-intensive. KU-F40, an automated feces analyzer, combines automated microscopic examination of stool samples and deep learning artificial intelligence. The aim of this study is to evaluate the performance of KU-F40 for the diagnosis of IPI. A random collection of stool samples prescribed for IPI investigation was retrospectively collected from six clinical laboratories in Belgium along with external quality controls. All samples were analyzed in our laboratory by wet mount preparation using classic light microscopy as reference. We assessed the sensitivity and specificity for parasite detection/identification. Finally, we studied the improvement in parasite detection rate when increasing the number of pictures taken to 150% and 200% of the standard settings. A total of 267 clinical stool samples were included. Using standard settings, overall sensitivity and specificity were 86% and 45%, respectively. When considering only clinically relevant parasites, sensitivity was 95%. Increasing the number of pictures allowed to improve detection rate, but it remained under 90% for several targets. KU-F40 offers an innovative approach and provides welcome automation in the diagnosis of IPI. Currently, its performance does not allow it to be used as a screening tool with automatic validation of negative results. Critical missing features could enhance its performance, including the addition of a 10x magnification objective and additional parasites currently absent from the database.IMPORTANCEIntestinal parasitic infections have a worldwide distribution and are a global health concern in many countries. Light microscopy is still considered the reference method for diagnosis but is labor-intensive, time-consuming, and requires highly skilled and motivated technologists. In this paper, we evaluate the KU-F40, an automated feces analyzer designed to diagnose intestinal parasitic infections by combining automated light microscopy and deep learning artificial intelligence for detection and presumptive identification of several protozoans and helminths. As it relies on microscopy, this method enables the detection and identification of a predefined panel of parasites, whose morphology is known to the system and included in the database, without requiring prior diagnostic suspicion, similarly to multiplex PCR assays. The automation could improve the quality, standardization, and turnaround time of stool parasitology. This study is the first to evaluate the performance of the KU-F40 on a wide range of parasites, collected from six Belgian hospitals, including our two national reference centers.

Calodium hepaticum (syn. Capillaria hepatica) is a zoonotic parasite of murid rodents and other mammals, while also being an important agent of hepatic capillariasis in humans. In a cross-sectional survey of schoolchildren across three provinces (Huambo, Uíge, and Zaire) in Angola, we found 39 fecal specimens that were considered positive for Trichuris trichiura eggs using the Kato-Katz thick smear but were negative by quantitative polymerase chain reaction (qPCR). Further morphological examination of these putative Trichuris eggs identified them as having an asymmetrical bipolar plug morphology, resembling capillariid eggs. Molecular characterization through Sanger sequencing and nanopore metabarcoding confirmed these eggs as C. hepaticum, indicating spurious human passage. Once passed in human feces, these eggs may embryonate and become infective in the environment, with the potential to cause hepatic capillariasis in humans. Future research should assess the prevalence of both spurious passage and hepatic capillariasis in humans in Angola, as well as evaluate the impact of misidentification of these nematodes with Trichuris eggs on soil-transmitted helminths surveys.IMPORTANCECalodium hepaticum causes hepatic capillariasis in humans living in low- to upper-middle income countries. This disease is mainly caused by ingestion of embryonated eggs, whereas consumption of eggs that are unembryonated, such as those found in the livers of infected animals, leads to spurious passage. Notably, unembryonated eggs are morphologically similar to those of T. trichiura, leading to potential misdiagnosis in routine fecal examinations and inaccurate estimates of soil-transmitted helminth prevalence. In this study, we detected C. hepaticum eggs in fecal specimens initially identified as positive for T. trichiura eggs by the Kato-Katz thick smear but tested negative by qPCR. Our findings highlight diagnostic challenges posed by such morphological similarities. Additionally, the detection of C. hepaticum eggs in fecal specimens from school-aged children suggests a potential risk of hepatic capillariasis in this population, underscoring the importance for local health authorities and medical practitioners in Angola to consider this parasite as a potential differential diagnosis in cases of unexplained hepatic dysfunction.

Whole-genome sequencing of Mycobacterium tuberculosis can be a valuable tool for TB surveillance and treatment, providing insights into transmission patterns and comprehensive drug susceptibility testing. However, the slow growth of M. tuberculosis means traditional culture-based sequencing methods can take weeks to return results, which has limited the widespread adoption of these techniques and limited their use in clinical decision-making. Tiled amplicon sequencing is a fast, reliable, and cost-effective method of whole-genome sequencing that can be done directly on clinical specimens and has been implemented at scale in academic and public health laboratories across the world; it was the cornerstone of SARS-CoV-2 sequencing and has been adapted for a wide range of viral pathogens. However, similar methods are not yet available for far larger bacterial genomes. Extending this approach to M. tuberculosis would significantly reduce the cost, labor, and turnaround time for whole-genome sequencing. We designed a tiled amplicon panel consisting of 5,128 primers that covers the entire M. tuberculosis genome, the largest tiled amplicon sequencing panel we are aware of to date. Applying our amplicon panels to clinical samples of sputum, we show the ability to recover whole-genome bacterial sequences without the need for culture. The resulting sequence data can be used to determine M. tuberculosis lineage and reliably identify markers of drug resistance. Using this approach in clinical settings could reduce the time needed for comprehensive drug susceptibility testing from weeks to days and enable genomic epidemiology to be performed at scale, even in resource-limited settings.IMPORTANCEWe have developed and tested an amplicon panel, TB-seq, for the priority pathogen Mycobacterium tuberculosis, demonstrating recovery of near-full genomes directly from patient sputum, including mixed and low-concentration samples. This approach significantly reduces the turnaround time for this slow-growing bacterium while maintaining high accuracy in detecting clinically relevant mutations, including those associated with drug resistance. Given the global burden of tuberculosis and the critical need for faster diagnostic solutions, we believe our method has the potential to improve clinical decision-making and public health strategies.

Mycobacterium xenopi causes non-tuberculous mycobacterial pulmonary disease (NTM-PD) that is difficult to treat. However, data on the genomic population structure, antimicrobial susceptibility, and the clinical significance of this pathogen remain scarce. We analyzed 76 clinical M. xenopi isolates from 70 patients collected between 1995 and 2020 in Frankfurt am Main, Germany. All isolates underwent phenotypic drug susceptibility testing and whole-genome sequencing. Cluster analysis, including isolates from this study and all hitherto available high-quality M. xenopi genome data sets in the Sequence Read Archive (n = 11), was performed by core genome multilocus sequence typing. In our cohort, only 26.5% of patients met criteria for clinically relevant NTM-PD. Phylogenetic analysis identified three large hospital-associated clusters (≤10 allelic difference), each involving between 7 and 20 patients and persisting for over 18 years, suggesting prolonged transmission chains or a common environmental source. We also defined three major clades (≤50 allelic difference), two of which contained isolates from the United Kingdom. Clofazimine and guideline-recommended antimycobacterial agents showed good in vitro efficacy, except rifampicin, with 23.6% resistance. This study represents a major expansion of M. xenopi genomic resources and provides insights into the genomic population structure, phenotypic susceptibility, and clinical characteristics of M. xenopi. Guideline-recommended antimycobacterials show good in vitro activity, while clofazimine may be a valuable addition to M. xenopi therapy. The identified clusters underscore the need for further investigation into transmission dynamics and globally successful clones.IMPORTANCEMycobacterium xenopi is an increasingly recognized opportunistic lung pathogen that is difficult to treat. Infections often occur in patients with pre-existing health conditions and can present substantial diagnostic and therapeutic challenges. A deeper understanding of its genetic diversity and resistance mechanisms is essential for optimal patient management and for clarifying potential transmission routes. By analyzing 76 whole-genome sequences together with detailed clinical information and phenotypic drug-susceptibility data, this study substantially expands the available genomic repertoire for M. xenopi. While clinical relevance was limited in our cohort, most guideline-recommended antimicrobial agents showed good efficacy in vitro. The detection of closely related strains might point toward a common environmental source of infection. These findings highlight the need for continued surveillance and provide a comprehensive foundation that supports more accurate monitoring, improved understanding of disease behavior, and future investigations into M. xenopi pathogenicity.

Invasive fungal diseases (IFDs) are common and often fatal in severe burn patients due to skin barrier loss and immune dysfunction. However, current definitions of invasive mold infections are poorly adapted to this population. This study evaluated the characteristics of various diagnostic criteria and their combinations in relation to clinical outcomes in burn patients. We conducted a retrospective cohort study of all patients admitted to the Burn ICU from 2014 to 2023 with ≥15% total burn surface area and at least one sample sent to the mycology lab. Criteria included direct microscopy, culture (respiratory, skin, or tissue), species-specific quantitative PCR (qPCR) (Aspergillus, Mucorales, and Fusarium) on plasma/tissue/bronchoalveolar lavage fluid, and serum galactomannan. Among 276 patients, 489/6,184 samples were positive, including 281 skin biopsies (direct examination and conventional culture) and 132 plasma specimens (qPCR). Positive diagnostic criteria ≥1 was found in 93 patients (33.7%): Aspergillus (25.7%), Mucorales (10.9%), and Fusarium (9.8%). Twenty-seven patients (9.8%) had ≥2 criteria involving ≥2 mold types. Mortality rose with the number of positive criteria: 12.7% (0), 10.7% (1, 2), 27.3% (3, 4), and 46.7% (≥5) (P < 0.001). Plasma qPCR was positive in 81.3% of Mucorales, 40% of Aspergillus, and 15.4% of Fusarium cases with skin involvement. Skin biopsies (direct examination and conventional culture) combined with species-specific plasma qPCR enhance timely and reliable IFD diagnosis in burn patients. Mortality correlated with the number of positive criteria and coexistence of multiple mold species, underscoring the need for broad antifungal coverage and the value of multi-criteria diagnostics to guide treatment.IMPORTANCEInvasive mold infections are frequent and often fatal complications in patients with severe burns, occurring in up to 20% of cases with a total burn surface area exceeding 15%. Despite their severity, no standardized case definition currently exists to guide research or clinical management in this population. The performance of existing mycological diagnostic criteria remains unknown in burn patients. In this 10-year retrospective study, we evaluated the diagnostic performance of individual and combined mold-related criteria in relation to patient outcomes, analyzing more than 6,000 clinical samples. These findings provide a first comprehensive assessment of mold diagnostic markers in the burn population.

Systemic shifts in antimicrobial resistance rates can be due to epidemiologic shifts in microbial susceptibility patterns or artifactual shifts introduced by technical biases in antimicrobial susceptibility testing (AST)-both ultimately leading to changes in antimicrobial prescribing. To reduce technical variability, quality control (QC) criteria for AST are published by manufacturers and standards organizations. However, traditional QC metrics, in isolation, are fallible. In this study, we describe a systematic shift in daptomycin AST results between 2022 and 2025 in isolates tested in two independent health systems. Comprehensive analysis of clinical isolate AST results and retrospective mining of QC data from this period revealed a subtle shift that led to a 5%-22% decrease in overall susceptibility rates for certain organisms, most notably Enterococcus faecium. As daptomycin is a key treatment option for these difficult-to-treat infections, this increase in resistance rates paralleled a decrease in prescribing daptomycin for infections with these organisms. Importantly, this trend was undetectable through routine QC processes and only became apparent through systematic review of patient data. Our findings highlight the opportunity to integrate routine patient data analysis into microbiology QC practices to enhance detection of subtle but clinically relevant changes in AST performance.

Importance: In this study, we report a critical incident of technical variability using daptomycin gradient diffusion methodology that was undetectable using routine quality control metrics. More broadly, this study underscores the opportunity to incorporate additional modalities, such as clinical patient results, into a comprehensive quality assurance plan to ensure high-quality antimicrobial susceptibility testing results. Given the dynamic spread of multidrug resistance in bacteria, accurate susceptibility testing results are critical to identify and respond to shifts in local epidemiology.

Bovine tuberculosis, a zoonotic disease caused primarily by Mycobacterium bovis, poses a significant threat to cattle health and farming livelihoods within the United Kingdom (UK). Disease control in cattle is complicated by the persistence of M. bovis in European badgers, the UK's principal wildlife reservoir. Accurate diagnostic tools for both species are essential for effective surveillance and disease control. Many existing badger serodiagnostic tests, which include MPB70, MPB83, and ESAT6-CFP10 antigens, have relatively modest sensitivities (~50%-60%), limiting their utility in surveillance. To address this issue, we used an unbiased and comprehensive antigen discovery approach to identify new diagnostic targets. This strategy identified Rv3616c as a novel antigen with promising diagnostic test potential for M. bovis infection in badgers. Overlapping peptides spanning the full Rv3616c amino acid sequence were screened to identify the most diagnostically informative epitopes. A pool of four Rv3616c peptides, used in an indirect enzyme-linked immunosorbent assay (ELISA), had a sensitivity of 85.71% (95% CI: 77.19-91.96), a specificity of 94.80% (95% CI: 90.35-97.59), and a diagnostic accuracy of 91.51% (95% CI: 87.54-94.54). The existing validated Badger M. bovis Ab Test, when used alone, had a sensitivity of 73.47% (95% CI: 63.59-81.88); however, parallel interpretation with the Rv3616c ELISA could increase overall sensitivity to 91.84% (95% CI: 84.55-96.41), with minimal loss of specificity. These findings support the use of Rv3616c-derived peptides in serodiagnostic tests to improve the detection of M. bovis infection in badgers and enhance tuberculosis surveillance in this wildlife reservoir.IMPORTANCEAccurate diagnosis of Mycobacterium bovis infection in wildlife reservoirs is essential for controlling bovine tuberculosis (bTB), a zoonotic disease that threatens human health, animal welfare, and farming livelihoods. In the United Kingdom, European badgers are the principal wildlife reservoir, complicating efforts to eradicate bTB in cattle. Existing serodiagnostic tests for badgers have moderate sensitivity, limiting effectiveness in surveillance. To address this, this study used an unbiased, comprehensive antigen discovery approach and identified several new diagnostic targets, including the Rv3616c protein. A test based on specific Rv3616c-derived peptides had a high diagnostic accuracy (91.51%) and, when used in parallel with a validated test, improved test sensitivity while maintaining specificity. These synthetic peptides are scalable, cost-effective, and adaptable to different diagnostic platforms. The findings reveal an antigen with diagnostic potential that could inform the development of new tests for bTB surveillance in wildlife, supporting One Health principles and global tuberculosis elimination strategies.