Nanopore-based targeted sequencing (NTS) for drug-resistant tuberculosis: an integrated tool for personalized treatment strategies and guidance for new drug development

Abstract

Background: Drug-resistant tuberculosis has emerged as a major public health issue that requires immediate attention. NTS is an innovative method that allows for the direct detection of clinical samples without the need for culture. It could provide more accurate, reliable, and comprehensive information on drug resistance.

Methods: We collected clinical data retrospectively from patients suspected of having drug-resistant tuberculosis who visited the tuberculosis department at the Second Hospital of Nanjing in Jiangsu Province, China, from December 2023 to December 2024. The diagnostic efficiency of NTS for different types of drug-resistant tuberculosis and antimicrobial resistance was calculated. The relationship between resistance genes, mutated amino acids, and mutation sites was demonstrated.

Results: In this study, a total of 107 patients with drug-resistant tuberculosis were included, comprising 43 cases of mono-drug resistant tuberculosis, 20 patients with poly-drug resistant tuberculosis, 22 cases of multidrug-resistant tuberculosis, 21 cases of pre-extensively drug-resistant tuberculosis and 1 case of extensively drug-resistant tuberculosis. The accuracy of NTS in diagnosing drug-resistant tuberculosis ranged from 42.9 to 93.0%. Except for second-line injectable drugs, NTS achieved a sensitivity of over 70% for other anti-tuberculosis drugs. Serine was identified as the most frequently mutated amino acid in both the rpoB gene (66.2%, 49/74) and the katG gene (86.3%, 44/51). Additionally, the most frequently mutated amino acids in the embB gene, rpsL gene, and gyrA gene were methionine (94.7%, 44/51), lysine (100%, 28/28), and aspartic acid (66.7%, 20/30), respectively.

Conclusion: NTS could effectively and precisely deliver comprehensive drug resistance results, assisting medical professionals to create more personalized treatment plans. Besides, it would encourage the development of new anti-tuberculosis drugs to broaden clinical treatment options for drug-resistant tuberculosis.

Keywords: Amino acids; Drug-resistant genes; Drug-resistant tuberculosis(DR-TB); Nanopore-based targeted sequencing(NTS); Targeted next-generation sequencing(tNGS).

Fig. 1

Flowchart illustrating the classification of participants in the study

Fig. 2

(a) Bar chart illustrating accurate diagnoses of drug-resistant tuberculosis identified by NTS; (b) Upset diagram showing the positive intersection of the three detection methods for rifampicin-resistant tuberculosis patients

Fig. 3

(a) Sankey diagram illustrating the relationships between genes associated with rifampicin resistance, the mutated amino acids, and the specific locations of resistance mutations; (b) Sankey diagram displaying the relationships between genes associated with isoniazid resistance, the mutated amino acids, and the specific locations of resistance mutations; (c) Sankey diagram presenting the relationships between genes associated with ethambutol resistance, the mutated amino acids, and the specific locations of resistance mutations; (d) Sankey diagram showing the relationships between genes associated with streptomycin resistance, the mutated amino acids, and the specific locations of resistance mutations; (e) Sankey diagram revealing the relationships between genes associated with fluoroquinolone resistance, the mutated amino acids, and the specific locations of resistance mutations; (f) Radial bar chart of pyrazinamide resistance related mutations

Fig. 4

(a) Half violin plot showing differences in tuberculosis sequence numbers in clinical specimens with different Xpert MTB/RIF detection results; (b) Half violin plot illustrating variations in tuberculosis sequence numbers in clinical specimens with different NTS test results; (c) Radar chart of diagnostic performance of NTS for different types of low bacterial load drug-resistant tuberculosis; (d) Radar chart of diagnostic manifestations of anti-tuberculosis drug resistance in clinical specimens with low bacterial load by NTS RIF: rifampicin; INH: isoniazide; EMB: ethambutol; SM: streptomycin; AMK + KM + CM: amikacin + kanamycin + capreomycin; FQ: fluoroquinolones