An exploratory study on the application of nanopore sequencing for detecting Mycobacterium tuberculosis drug resistance in respiratory specimens

Abstract

Background

This study aimed to evaluate the diagnostic efficacy of nanopore sequencing for Mycobacterium tuberculosis (MTB) drug resistance in respiratory specimens from pulmonary tuberculosis (PTB) patients. It compared it to the Xpert MTB/RIF and fluorescent polymerase chain reaction (PCR) melting curve to explore the validity and feasibility of detecting MTB drug resistance in respiratory specimens.

Methods

This study retrospectively analyzed 52 respiratory specimens. The proportional method applied the phenotypic drug susceptibility test (pDST) to respiratory specimens. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), consistency statistic (kappa) with phenotypic drug susceptibility testing (pDST), and the area under the curve (AUC) from the receiver operating characteristic (ROC) curve were calculated for nanopore sequencing, Xpert MTB/RIF, and fluorescent PCR melting curve. These calculations used the pDST results as the reference standard.

Results

Among the resistance mutation genes detected by nanopore sequencing, rpoB, and katG were the most frequent, followed by embB, rpsL, gyrA, inhA, ahpC, gyrB, gid, and rrs. In bronchoalveolar lavage fluid (BALF) specimens, nanopore sequencing showed high sensitivity (100.00%,90.32%,82.35%,82.35%,100.00%,76.92%), specificity (70.00%,81.82%,88.00%,96.00%93.75%,93.10%0.100.00%), and AUC values (0.85,0.86,0.85, 0.89,0.97,0.85) for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), levofloxacin (LFX), moxifloxacin (MFX). Nanopore sequencing exhibited good detection efficacy (kappa value ≥ 0.70) and perfect diagnostic resistance value (AUC value ≥ 0.85). For RIF, nanopore sequencing showed Kappa values of 0.01 and 0.38 and AUC values of 0.02 and 0.18 higher than the Xpert MTB/RIF and fluorescent PCR melting curve, respectively; for INH, nanopore sequencing had a higher Kappa value of 0.65 and a higher AUC value of 0.32 than the fluorescent PCR melting curve. Nanopore sequencing provided superior overall performance.

Conclusion

Nanopore sequencing has significant technical advantages and clinical application potential in detecting MTB drug resistance. Its rapid and highly accurate detection capabilities support early diagnosis and personalized treatment of drug-resistant MTB. As the technology continues to mature and the cost is further reduced, it is expected that nanopore sequencing technology will play a more important role in MTB resistance detection.