Journal of Clinical Microbiology最新文献

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.

Lefamulin is a novel pleuromutilin antibiotic with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). While broth microdilution (BMD) is the reference method for susceptibility testing, simpler alternatives such as MIC Test Strip (gradient diffusion strip [GDS]) and disk diffusion (DD) are urgently needed for routine laboratories. We evaluated the accuracy of GDS and DD compared to BMD for lefamulin against S. aureus. A total of 422 non-duplicate clinical isolates of S. aureus (256 methicillin-susceptible S. aureus [MSSA] and 166 MRSA) collected between December 2023 and December 2024 at the Affiliated Tai'an Central Hospital of Qingdao University were tested by BMD, GDS, and DD. Interpretation followed CLSI M100-S34 breakpoints (MIC ≤ 0.25 mg/L or zone diameter ≥ 23 mm = susceptible). BMD served as the reference to calculate categorical agreement (CA), essential agreement (EA), major errors (ME), and very major errors (VME). Lefamulin exhibited potent activity against S. aureus, with 98.6% (416/422) of isolates classified as susceptible by BMD; six isolates (four MSSA and two MRSA) were non-susceptible. Compared with BMD, GDS showed 100% CA and 92.2% EA and yielded no VME and ME, and MICs were 0.49 log_₂ lower than BMD. Disk diffusion with the 20 µg lefamulin disk achieved 99.5% CA (421/422), with a VME rate of 16.7% (1/6) and an ME rate of 0.2% (1/416). Lefamulin GDS fulfilled CLSI criteria but systematically underestimated MICs; BMD confirmation is advised for GDS MIC = 0.25 mg/L. Disk diffusion showed excellent agreement, yet its VME rate surpassed the CLSI threshold owing to scarce non-susceptible isolates; any non-susceptible result by disk should be verified by BMD.

Importance: Lefamulin is a new antibiotic active against methicillin-resistant Staphylococcus aureus, but simple susceptibility tests are lacking. We compared two easy-to-perform methods with reference broth microdilution for 422 S. aureus clinical isolates. Both showed high accuracy after on-site confirmation of borderline results and can be immediately implemented in routine laboratories to guide appropriate lefamulin therapy and help contain resistance.

Blood culture contamination is common, causing diagnostic uncertainty and unnecessary antibiotic use. Analyzing growth patterns within culture sets might offer diagnostic value. We retrospectively analyzed peripheral blood culture sets from 2019 and 2024. Growth pattern (one bottle [discordant] vs both bottles [concordant]) was analyzed according to clinical significance (contaminant vs true pathogen). Overall, 38,216 blood culture sets were analyzed, including 1,491 (3.9%) discordant and 1,938 (5.1%) concordant sets (remaining 34,787 [91.0%] sets were sterile). Discordant sets grew 1,060/1,491 (71.1%) contaminants and 431/1,491 (28.9%) true pathogens. Concordant sets grew 222/1,938 (11.4%) contaminants and 1,716/1,938 (88.5%) true pathogens (P < 0.001). Examining coagulase-negative staphylococci (CoNS) only (2019 data set), 629/642 (98.0%) discordant sets grew contaminants, while 13/642 (2.0%) grew true pathogens. In contrast, Staphylococcus aureus grew in only 82/270 (30.4%) discordant sets. Among 858 first CoNS-positive cultures per patient, 624/636 (98.1%) discordant sets grew contaminants, and 12/636 (1.9%) grew CoNS defined as a true pathogen. The negative predictive value of a discordant first CoNS set to exclude true CoNS bacteremia was 98.1% (95% confidence interval 96.7%-98.9%). Examining aerobic vs anaerobic bottles in 356 discordant sets, contaminants were found more frequently in aerobic bottles (135/356, 37.9% vs 73/356, 20.5%, P = 0.04). The proportion of true pathogens was similar in both (79/356, 22.2% vs 69/356, 19.4%, P = 0.4). Discordant CoNS-positive cultures were strongly associated with contamination. This could assist in interpreting blood culture results and supporting antimicrobial stewardship. Discordance might result from a diversion effect, the aerobic bottle acting as a diversion device for the anaerobic bottle.IMPORTANCERapidly distinguishing blood culture contaminants from true pathogens is essential for optimizing antimicrobial stewardship and avoiding unnecessary antibiotic therapy. In this large, two-period study, we demonstrate that discordant growth of coagulase-negative staphylococci in a two-bottle set has a negative predictive value of 98.1% for true bacteremia. This finding remained robust across both study years and when restricted to first positive cultures, highlighting its reliability. Incorporating simple growth pattern analysis into early blood culture interpretation can provide clinicians with reliable and timely information within 24 hours, supporting more targeted and judicious antibiotic use.

Hepatitis B virus (HBV) DNA testing is essential for the management of HBV infection. Routine HBV DNA tests in central laboratories are expensive and require processed venous blood, limiting accessibility. This study is the first published assessment of the point-of-care Xpert HBV DNA assay performance using fingerstick capillary blood compared with standard-of-care venous blood testing. Participants with chronic HBV infection were enrolled from six hospitals. Fingerstick capillary blood was tested using Xpert HBV Viral Load assay (quantification lower limit: 100 IU/mL). Venipuncture whole blood was tested with COBAS AmpliPrep/COBAS TaqMan HBV DNA Test (gold standard). The sensitivity and specificity of the Xpert were evaluated for identifying HBV DNA ≥100 and >2,000 IU/mL. Agreement between quantitative measurements of assays was assessed. A total of 246 participants were included (median age 45, 46% female, 18% HBeAg positive, 48% on HBV treatment, 6% with cirrhosis). For HBV DNA ≥100 IU/mL, the sensitivity and specificity of the Xpert were 97.0% (95% CI: 94.9, 99.1) and 90.3% (86.6, 94.0), respectively. For HBV DNA >2,000 IU/mL, the sensitivity and specificity were 95.3% (92.7, 98.0) and 95.0% (92.4, 97.8), respectively. Viral load differences in non-concordant samples ranged from 0.1 to 1.1 log IU/mL. Overall, the Xpert viral loads were a mean 0.12 log IU/mL higher than gold standard (95%CI: -0.43, 0.67). In conclusion, minimal differences in HBV DNA levels were identified between the Xpert and gold standard assays, with differences in non-concordant results unlikely to impact clinical decisions. This evidence supports developing a dedicated Xpert HBV DNA fingerstick assay for decentralized care, crucial for remote, resource-limited settings and hard-to-reach populations, including prenatal care for women with HBV.IMPORTANCEThis study represents the first assessment of a point-of-care hepatitis B virus (HBV) DNA assay using fingerstick capillary blood (Xpert HBV Viral Load assay). Our findings demonstrated high sensitivity and specificity for the point-of-care test, with close agreement between the point-of-care and standard-of-care assays across the full quantitative spectrum of HBV viral load measurements. Importantly, the differences between the assays in participants with non-concordant results were not substantial enough to alter clinical management, suggesting that this point-of-care method is both accurate and reliable for clinical use. By highlighting the potential for decentralizing HBV care, our research provides compelling evidence to support the development of a dedicated Xpert HBV DNA point-of-care test. Such a development could greatly benefit patients in remote and resource-limited settings, where access to laboratory-based testing is limited.