Journal of Clinical Microbiology最新文献

Sandfly-borne phleboviruses cause febrile illness and neuroinvasive disease in humans. While infections are reported in the Mediterranean region, the discovery of previously unknown phleboviruses in sandflies from Kenya suggests a wider geographic distribution. Detection and characterization of novel phleboviruses are often hindered by low-quality and low-viral-load samples. We developed a capture-based target enrichment next-generation sequencing approach that showed a 99%-100% fold enrichment of viral genomes from primary material and provides a robust tool for generating complete genomes of both known and previously unknown viruses. From a collection of 15,652 sandflies in Kenya, we recovered seven complete coding sequences of Embossos, Bogoria, and Kiborgoch viruses, and of two previously unknown phleboviruses, which were named Sosoik and Shable viruses. Sosoik virus shared 83% amino acid identity in its RdRp gene with that of Bogoria virus, while Shable virus shared ca. 88% amino acid identity with viruses of the Salehabad serocomplex. Additionally, a reassortant of Shable virus was detected that possessed an M segment from an undescribed Ponticelli-like virus. DNA barcoding of blood-fed sandflies revealed several potentially novel Sergentomyia species and evidence of host-feeding on humans, livestock, and reptiles, suggesting possibilities for zoonotic transmission. Overall, our findings increase the known genetic diversity of Old World sandfly-borne phlebovirus species from 18 to 25 (by 38.9%), including the detection of viruses from all pathogenic sandfly-borne phlebovirus serocomplexes in East Africa, opening new horizons in disease ecology research.IMPORTANCEKnowledge of the genetic diversity of circulating pathogens is crucial for providing appropriate diagnostics and disease management. This study established a novel capture-based target enrichment next-generation sequencing approach that enabled the near-complete viral genome recovery from primary samples, while native NGS yielded negative or poor-quality results. In addition to the five recently discovered sandfly-borne phleboviruses in Kenya, two previously unknown phleboviruses were detected in sandflies from the same region. The viruses were detected in several sandfly species, which showed diverse host-feeding behaviors, including mixed feeding on humans and chickens. The study significantly advances the understanding of sandfly-borne phleboviruses by uncovering their broader geographic distribution and genetic diversity, particularly in East Africa, highlighting the importance of expanding surveillance efforts beyond traditionally studied regions.

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.

Plasma microbial cell-free DNA (mcfDNA) sequencing is a novel diagnostic tool for pediatric complicated community-acquired pneumonia (cCAP). However, rigorous evaluation of real-world mcfDNA performance is lacking. We compared mcfDNA sequencing to a composite reference standard consisting of blood cultures, pleural fluid (PF) cultures, and targeted PF PCRs in children with cCAP requiring pleural effusion or empyema drainage at Children's Hospital Colorado from 2022 to 2024. We calculated positive/negative percent agreement (PPA/NPA), Jaccard similarity index, and theoretical time to pathogen detection. We investigated mcfDNA positivity in pediatric controls without bacterial infections. Receiver operating characteristic (ROC) analysis explored potential mcfDNA detection cutoff values to define "true clinical positivity." Across 45 cCAP cases, mcfDNA sequencing detected a probable pathogen in 86.7% versus 8.9% by blood culture, 20.0% by PF culture, and 71.1% by PF PCRs (P < 0.001). Against the culture+PCR composite reference standard, mcfDNA had a PPA of 91.9% (83.1%-100.0%), NPA of 35.7% (10.6%-60.8%), and a Jaccard index of 0.74. Bacterial cfDNA was detected in 52% of controls. ROC analysis yielded an area under the curve of 0.9, with potential optimal detection cutoffs ranging between 158 and 491 mcfDNA molecules per microliter. Theoretical median time to pathogen detection was 3.0 days with mcfDNA versus 4.5 days for pleural fluid PCRs, driven primarily by PF sampling time. Plasma mcfDNA sequencing had a significantly higher diagnostic yield than cultures and a similar yield to PF PCRs; however, over half of non-bacterial controls had low-level mcfDNA detected, potentially complicating interpretation. mcfDNA detection level cutoffs may help elucidate the clinical significance of detected pathogens.IMPORTANCEMore sensitive diagnostic tests, particularly non-invasive options, are needed to better identify the causative organism(s) in children with complicated community-acquired pneumonia and help inform pathogen-directed therapy. A novel, potentially powerful diagnostic tool for pneumonia is plasma microbial cell-free DNA sequencing, available commercially as Karius Spectrum. However, unknowns regarding its real-world performance and proper role in clinical practice remain. This study aims to address two ongoing concerns: first, the lack of robust comparisons of microbial cell-free DNA (mcfDNA) sequencing performance against validated conventional and state-of-the-art diagnostic modalities (i.e., pleural fluid testing and blood cultures); and second, the unknown baseline positivity rates of mcfDNA in children without bacterial infections. Results from this study may help inform clinical practice decisions and testing implementation strategies.

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.