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.