Optimisation and validation of capture mNGS for predicting antimicrobial resistance

Antibiotic resistance critically compromises bacterial infection treatment. While antimicrobial susceptibility testing (AST) remains the standard for resistance assessment, its culture dependence is time-consuming. Clinical metagenomic next-generation sequencing (mNGS) offers rapid pathogen detection and antibiotic resistance gene (ARG) profiling. However, low ARG detection sensitivity and unclear genotype-phenotype correlations limit its clinical utility.

We developed capture mNGS approach with probe-based ARG enrichment and a host-attribution algorithm for precise ARG-bacteria linkage. Its ARG detection sensitivity was comparatively analysed against standard mNGS. Using phenotypic AST as reference, we then evaluated the clinical predictive value of capture mNGS-detected ARGs in a retrospective cohort from Sun Yat-sen Memorial Hospital (SYSMH) and an external cohort from Liuzhou Worker's Hospital (LWH). In addition, a prospective cohort from SYSMH was used to explore the clinical utility of ARG detection by mNGS.

Compared to standard mNGS, capture mNGS significantly enhanced ARG detection sensitivity, achieving a 44-fold increase in sequencing depth. In our retrospective cohort, key resistance genes detected by capture mNGS accurately predicted phenotypic resistance: blaCTX-M achieved a sensitivity of 1.00 (95% CI: 0.86, 1.00) and specificity of 1.00 (95% CI: 0.59, 1.00) for ceftriaxone resistance prediction, with an area under the receiver operating characteristic curve (AUC) of 0.93 (95% CI: 0.87, 0.99). BlaKPC demonstrated a sensitivity of 0.94 (95% CI: 0.73, 1.00) and specificity of 1.00 (95% CI: 0.95, 1.00) for carbapenem resistance (AUC = 0.97, 95% CI: 0.92, 1.00). Similarly, blaOXA-23 exhibited a sensitivity of 0.95 (95% CI: 0.82, 0.99) and specificity of 1.00 (95% CI: 0.69, 1.00) for carbapenem resistance (AUC = 0.97, 95% CI: 0.94, 1.00), which was externally validated in the LWH cohort. In addition, mecA showed a sensitivity of 0.94 (95% CI: 0.71, 1.00) and specificity of 0.94 (95% CI: 0.81, 0.99) for oxacillin resistance (AUC = 0.94, 95% CI: 0.87, 1.00). Whereas blaTEM/blaSHV showed higher false-positive rates for cephalosporin resistance and ErmB/ErmC showed lower sensitivity (0.6, 95% CI: 0.32, 0.84) for macrolide-lincosamide-streptogramin (MLS) resistance. Capture mNGS reported results (median turnaround time (TAT): 24.71 h (IQR 22.74-41.00)) were shorter than AST (median TAT: 73.16 h (IQR 54.19-93.42)). In a prospective cohort, the time to guide antibiotic therapy based on reported positive ARGs was significantly shorter than that based on reported resistant phenotypes from AST.

These results highlight that ARGs can be leveraged to rapidly and accurately predict bacterial resistance phenotypes with high sensitivity and specificity, thereby guiding antibiotic management in clinical practice.

The National Natural Science Foundation of China, the Guangdong Science and Technology Department, Science and Technology Projects in Guangzhou.