Journal of Clinical Microbiology最新文献

Several nucleic acid and antigen assays have been developed to detect HIV in plasma at levels below the detection limit of standard clinical assays; however, comparative assessment of performance of these ultrasensitive assays on identical panels has been limited. Here, we report the relative performance characteristics of three manual (ultracentrifugation concentration and laboratory-developed PCR) and two automated commercial assays for single-copy detection of HIV RNA, as well as two ultrasensitive HIV p24 assays, using blinded panels of plasma samples containing HIV at low concentrations. Independent sample sets were studied in two phases: in the first phase, qualification panels consisted of analytic standards in serial dilution and clinical plasma samples from virologically suppressed people with HIV (PWH). HIV detection in clinical samples was infrequent using the ultrasensitive p24 assays (mean 11%). In contrast, a higher proportion of the same samples were detected using single-copy RNA assays (mean 61%). In the second phase, evaluation panels of clinical plasma samples (n = 80) from virally suppressed PWH and low-copy analytic standards assessed performance of one automated and two manual single-copy RNA assays with the highest qualification phase detection frequencies. The automated assay performed comparably in three separate laboratories and consistently detected HIV RNA in over half (mean 57%) of samples from virally suppressed PWH, whereas the manual assays detected HIV in ≤40% of the same samples. An automated single-copy assay provides a scalable method for measuring residual HIV viremia that may be broadly useful in pathogenesis and cure-directed studies.

Importance: Most people with HIV (PWH) on antiretroviral therapy have viral loads below the detection limit of clinical assays, yet virus is often present and detectable at very low levels using ultrasensitive research assays. Clinical trials evaluating curative interventions and interpreting outcomes of analytical treatment interruptions depend on reliable assays to assess and quantify changes in HIV persistence often at very low levels. We conducted a two-stage head-to-head, blinded comparison of multiple ultrasensitive HIV RNA and p24 assays, first using 50 low viral load plasma samples, then further evaluating the top-performing assays on a 144-member blinded panel composed of duplicate contrived and clinical specimens from well-suppressed PWH. Single-copy RNA methods performed better than p24 assays, and a fully automated, 9-replicate commercial RNA assay demonstrated high sensitivity, reproducibility across laboratories, and practical scalability that can be applied to measure the impact of interventions in HIV cure trials.

Tuberculosis (TB) remains a leading global infectious killer, yet traditional diagnostic methods are inadequate. Acid-fast staining suffers from low sensitivity, and mycobacterial culture requires prolonged incubation because of the slow growth of Mycobacterium tuberculosis. PCR-based molecular assays allow rapid detection, but their capacity for resistance profiling is limited to a narrow set of mutations. Metagenomic next-generation sequencing (mNGS) has emerged as a promising culture-independent tool for TB detection, enabling broad-spectrum pathogen identification and offering added value in complex scenarios including extra-pulmonary disease, mixed infections, and infections in immunocompromised or pediatric populations. Clinical studies indicate that mNGS achieves moderate to high sensitivity and excellent specificity in the diagnosis of tuberculosis. However, its diagnostic performance is often constrained by low mycobacterial read counts, interference from abundant host nucleic acids, and the inability to distinguish active from latent infection. In addition, the accuracy of drug resistance prediction using mNGS remains limited, and the World Health Organization currently endorses targeted NGS as the preferred sequencing-based approach for resistance profiling. Despite these challenges, mNGS has facilitated novel diagnostic strategies that combine pathogen detection with host-response data, thereby broadening its potential clinical utility. Nevertheless, practical barriers such as high cost, complex laboratory workflows, and difficulties in data interpretation continue to restrict widespread adoption in routine practice. Future efforts should prioritize technical optimization, standardized protocols, and integration with conventional diagnostics to establish cost-effective and clinically meaningful roles for mNGS in TB diagnosis and management.

Historic detection methods for diarrheal pathogens are time-consuming, laborious, and may lack specificity and sensitivity. Multiplexed molecular panels for enteric pathogens are widely used for rapid and accurate results. We evaluated the performance of the investigational use only (IUO) Hologic Panther Fusion GI Bacterial and Expanded Bacterial assays (Fusion GI Bac), the Verigene Enteric Pathogens (EP), and BioFire FilmArray Gastrointestinal (BioFire GI) panels for the detection of primary bacterial causes of gastrointestinal infection. We conducted a multi-platform evaluation for the detection of Salmonella, Shigella/Enteroinvasive Escherichia coli, Campylobacter, Shiga toxin-producing E. coli, Vibrio, and Yersinia enterocolitica. Limited analysis of Plesiomonas shigelloides and E. coli O157 was also performed. A total of 591 stool specimens underwent complete analysis: 263 frozen positive specimens and 328 freshly collected samples. We used a 2-out-of-3 consensus as the reference for assay performance. Positive and negative percent agreement (PPA and NPA) were high for most pathogen/assay pairs. Individual false-positive (FP) or false-negative (FN) results were seen for all pathogens and assays. PPAs below 95% in the retrospective samples were seen for Shiga toxin (Verigene EP, 89.3%) and Y. enterocolitica (Verigene EP, 88.9% and Fusion GI Bac, 72.2%). There were 12 FP results for Vibrio affecting all assays. Factors contributing to erroneous results, including freeze/thaw effects, are discussed. Overall, the IUO Panther Fusion GI Bacterial and Expanded Bacterial assays were comparable to two commercially available GI panels with a straightforward workflow using a high-throughput instrument.

Importance: Bacterial causes of diarrhea lead to significant morbidity and mortality around the world. Historic testing methods, for example, antigens and culture. For these reasons, molecular testing for enteric bacterial pathogens has become widely used, but there are limited numbers of commercially available tests on the market, especially those suitable for higher-throughput testing. We show that the high-throughput, random- access investigational use only Hologic Panther Fusion Gastrointestinal (GI) Bacterial assay and Panther Fusion GI Expanded Bacterial assay perform comparably to existing assays.

Biofilms are structured communities of microorganisms encased in a self-produced polymeric matrix that typically adhere to surfaces. Recent research, however, has revealed that non-attached aggregates share many common traits with the surface-dependent biofilms. This mode of bacterial growth provides enhanced protection against antibiotics and resistance to host immune defenses. Biofilms require higher antibiotic concentrations than those needed to inhibit planktonic bacteria, necessitating prolonged high-dose and combination therapies to achieve effective eradication. This increased resistance is attributed to multiple factors, including the protective extracellular matrix, reduced metabolic activity of bacteria within the biofilm, and also the ability of bacterial genomes to rapidly adjust in response to environmental changes. Diagnostic modalities such as sonication, tissue culture, and polymerase chain reaction-based assays currently dominate clinical diagnostics of biofilm infections due to their practicality, cost-effectiveness, and proven reliability. Recent research has led to innovative treatment strategies that target biofilm structure, enhance drug delivery, and modulate host-pathogen interactions. This review summarizes our current knowledge of biofilm formation, explores the current techniques for detecting microbial biofilms, and discusses future perspectives for advancing diagnostic and therapeutic strategies.