Enhanced Targeted NGS Assay for Comprehensive Diagnosis in Tuberculosis and Drug-Resistant Tuberculosis Patients.

Background: Accurate and timely diagnosis of tuberculosis (TB) and drug-resistant TB is crucial; however, current methods have limitations in sensitivity and scope, especially for detecting drug resistance and differentiating other respiratory pathogens.

Methods: We developed a multiplex PCR-based targeted next-generation sequencing (tNGS) assay and validated its analytical performance, including limit of detection (LoD), precision, and resistance to interference. A single-centre prospective study was conducted with 181 suspected TB patients to evaluate the assay's clinical performance using bronchoalveolar lavage or sputum samples, compared against microbiological culture and Xpert MTB/RIF.

Results: The tNGS assay demonstrated a low LoD of 10 copies/mL for TB and other respiratory pathogens, with high precision and resistance to interference. In clinical validation, it achieved 94.94% sensitivity in confirmed TB cases and showed a 92.86% positive percent agreement with Xpert MTB/RIF for rifampicin resistance, while also identifying additional mutations. The assay accurately detected nontuberculous mycobacteria (NTM) and other respiratory pathogens, aiding differential diagnosis.

Conclusion: The tNGS assay provides reliable detection of TB, drug-resistant mutations, and respiratory pathogens, including NTM, thereby enhancing differential diagnosis and supporting effective treatment strategies in the management of patients with suspected TB.