Yi-Wei Tang

The laboratory diagnosis of microbial infections has undergone a profound transformation over the past century, evolving from classical phenotypic methods to molecular genetic approaches and now toward ultra-sensitive molecular phenotyping. While this progression is often described as linear, a closer examination reveals a cyclical pattern: an initial reliance on observable biological function, followed by a shift toward genetic abstraction, and, more recently, a return to phenotypic measurement at unprecedented molecular resolution. This “diagnostic circle” reflects a deeper epistemological trajectory shared across multiple scientific disciplines, wherein knowledge alternates between direct observation and abstraction before converging into integrative, high-resolution frameworks. In this Perspective, we trace the evolution of diagnostic microbiology through three major phases - classical phenotypic methods, molecular/genetic diagnostics, and emerging ultra-sensitive phenotypic technologies - and argue that the field is entering a new era in which functional biology can be measured directly with molecular precision. We propose that this transition represents not merely technological progress but a conceptual shift toward restoring biological context, with significant implications for clinical decision-making, antimicrobial stewardship, and the future of precision infectious disease diagnostics.

Given the current global challenges posed by antimicrobial resistance, the early and accurate identification of carbapenem-resistant Gram-negative bacilli (CR-GNB) is crucial for patient outcomes. This study evaluates the combined diagnostic performance of Xpert Carba-R and Loop-mediated isothermal amplification (LAMP) in the early detection of CR-GNB in patients with hospital-acquired pneumonia (HAP) or ventilator-associated pneumonia (VAP) and investigates the effect of early treatment based on detection results on the prognosis of these patients. In this single-center prospective study, 250 HAP/VAP patients were enrolled. Respiratory specimens underwent bacterial culture, antimicrobial susceptibility testing, Xpert Carba-R, and LAMP. Diagnostic performance (sensitivity, specificity, positive predictive value [PPV], negative predictive value [NPV]) of the combined test to detect CR-GNB was assessed. Clinical utility in guiding early antibiotic therapy was evaluated. The combined method demonstrated 64.87% sensitivity (95% CI: 52.89%–75.61%) and 94.32% specificity (95% CI: 89.80%–97.24%), with perfect 100% sensitivity and specificity for detecting CR-GNB in Klebsiella pneumoniae . Patients testing positive were randomized to early or non-early treatment groups. For severe patients, the early treatment group showed significantly improved clinical outcomes, including lower acute physiology and chronic health evaluation-II (APACHE-II) scores ( P < 0.05) and sequential organ failure assessment (SOFA) scores ( P < 0.01) at 15 days post-treatment, along with reduced 30-day hospitalization costs ( P < 0.05). Notably, among the critically ill patients, compared with the non-early treatment group, the early treatment group achieved a clinical improvement rate of 81.25% and a microbiological eradication rate of 56.25%, showing a clear advantage. IMPORTANCE: An urgent clinical need exists for rapid detection of carbapenem-resistant Gram-negative bacilli (CR-GNB) in respiratory specimens to facilitate early and precise antibiotic therapy. To address this gap, we developed an innovative dual-method assay: Xpert Carba-R to detect five major carbapenemases, and loop-mediated isothermal amplification (LAMP) to identify common Gram-negative respiratory pathogens. Integration of these methods enables simultaneous and early determination of bacterial species and their resistance profiles. We validated this assay against conventional culture-based antimicrobial susceptibility testing (AST) and assessed its clinical impact on patient outcomes. Results indicate that this integrated approach provides clinically actionable data, serving as a viable alternative to conventional diagnostic workflows. This study is registered with the Chinese Clinical Trial Registry as ChiCTR2400090694 .

Background: Rapid and accurate detection and identification of viral pathogens have an impact on physician decision‐making for patients with acute respiratory illness (ARI). We aimed to evaluate the Xpert Xpress Flu/RSV test for the management of antibiotic prescribing in pediatric outpatients with ARI. Methods: We performed a prospective, quasi‐randomized, controlled study in Beijing Children's Hospital between December 1, 2021 and April 28, 2022. Outpatients with ARI aged 28 days to 18 years were enrolled and randomly assigned to the Xpert Xpress Flu/RSV test (Xpert) group or the influenza (Flu) antigen test (control) group. Both tests were performed on site. Results: A total of 771 patients were enrolled and assigned randomly to the Xpert ( n = 398) and the control ( n = 373) groups. There was no statistically significant difference in antibiotic prescriptions between the two groups, whereas a significant difference was observed for the prescriptions of oseltamivir ( p < 0.001). In Flu B‐positive patients, a statistically significant decrease in antibiotic use and increase in antiviral use were observed in both Xpert and control groups. Cephalosporin use was significantly decreased in respiratory syncytial virus (RSV)‐positive patients in the Xpert group before ( n = 8, 17.4%) and after ( n = 1, 2.2%)visit ( p = 0.035). Among clinical and laboratory parameters, shorter fever length (OR = 0.366) and positive Flu B or RSV (OR = 3.99) were two independent factors for antibiotic withdrawal by logistic regression analysis. There was no significant difference in duration of fever, clinical outcomes, and expenditure between the two groups at the 7‐day and 30‐day follow‐up. Conclusions: Use of Xpert Xpress Flu/RSV at point‐of‐care in pediatric outpatients with ARI reduced antibiotic prescription, which has the potential to improve antibiotic stewardship.

Background: Respiratory tract infections remain the main sites of carbapenem-resistant Enterobacteriaceae (CRE) infections in China and limited information on enzyme genotypes and subtypes in China is available. In this study, we used whole-genome sequencing (WGS) and the Xpert Carba-R assay for the detection, genotyping and subsubtyping of CRE genes from respiratory specimens collected from Southeast (Zhejiang, Jiangxi and Fujian Provinces) and Northwest (Gansu Province) regions of China. Methods: Respiratory specimens from hospitalized patients clinically diagnosed with pneumonia from seven tertiary hospitals in China from October 2022 to November 2023 were included. The CRE enzyme genes in specimens were directly detected and genotyped by using the Carba-R assay. Carbapenem resistance and genotypes were compared to a referee in combination of phenotypical and genotypical analyses of pure cultures recovered from the respiratory specimen. The CRE enzyme subtypes in isolates were determined by the WGS. Results: A total of 433 respiratory specimens were collected and tested in this study. The Carba-R assay detected 180 bla KPC (41.3%), 64 blaNDM (14.8%), 5 blaIMP (1.2%), 4 blaVIM (0.9%), and 4 blaOXA-48 (0.9%) genes. In comparison to the above referee, the sensitivity and specificity of the Carba-R assay for CRE detection were 100 and 90.4%. While there were no genotype distribution differences between the Southeast and Northwest regions, more KPC subtypes were identified in the Northwest ( n = 5) than Southeast ( n = 2) region. Conclusion: The Carba-R assay satisfactorily detected and identified carbapenemase genes directly in the respiratory specimens. A variety of CRE enzyme genotypes and subtypes were identified in respiratory specimens especially the Northwest region of China.

Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

Cox proportional hazards model results for PFS and OS. Each row reflects a model with that variable; models across all patients are stratified on treatment arm. Baseline HCV only includes those with pretreatment HCV titer data available (n = 43), and first sample HCV includes baseline HCV titer data for those with it available and cycle 2 data for those missing baseline (n = 53)