Abstract:

Objective: To investigate the diagnostic value of Nanopore targeted sequencing for detecting non-tuberculous mycobacteria (NTM) in respiratory samples. Methods: Data from 455 patients who underwent Nanopore targeted sequencing of respiratory samples from December 2021 to September 2022 at the Tuberculosis Treatment Center of Hangzhou Red Cross Hospital in Zhejiang Province was retrospectively collected. According to diagnostic criteria, these 455 patients were finally diagnosed as NTM-PD patients (NTM-PD group; 65 patients) and non-NTM-PD patients (non-NTM-PD group; 390 patients), their alveolar lavage fluid or sputum were subjected to simultaneous Nanopore targeted sequencing, BACTEC MGIT 960 liquid cultures (liquid culture) and/or PCR fluorescent probes to compare their efficacy in diagnosing NTM-PD. Results: Using clinical diagnostic results as standard, the sensitivities of Nanopore targeted sequencing, liquid culture, and PCR fluorescent probe for detecting NTM in respiratory samples were 92.3% (60/65), 83.1% (54/65), and 57.1% (36/63), respectively; the specificities were 96.2% (375/390), 98.7% (385/390), and 99.5% (380/382), respectively; and the positive predictive values were 80.0% (60/75), 91.5% (54/59), and 94.7% (36/38); negative predictive values were 98.7% (375/380), 97.2% (385/396), and 93.4% (380/407); Kappa values were 0.831, 0.851 and 0.679, respectively. Using liquid culture as standard, the sensitivities of Nanopore targeted sequencing, and PCR fluorescent probe were 86.4% (51/59), and 73.5% (36/49), respectively; the specificities were 98.0% (388/396) and 94.4% (374/396), respectively; the positive predictive values were 86.4% (51/59) and 62.1% (36/58), respectively; and the negative predictive values were 98.0% (388/396) and 96.6% (374/387), respectively; Kappa values were 0.844 and 0.629, respectively. Among the 65 cases clinically diagnosed as NTM-PD, 50 samples got positive results for both Nanopore targeted sequencing and liquid culture+gene chip, and their strain identification consistency was 96.0% (48/50). Conclusion: Nanopore targeted sequencing demonstrated high sensitivity and specificity in diagnosing NTM-PD and showed strong concordance with conventional identification methods. In terms of strain identification, this method also exhibited high agreement with results obtained from liquid culture combined with gene chips. Consequently, Nanopore targeted sequencing is well-suited for rapid detection of clinical respiratory samples from patients suspected of having NTM-PD, and it can simultaneously provide strain identification results for informing subsequent development of anti-NTM therapeutic regimen.

Key words: Atypical bacterial forms, Mycobacterium infections, Nanopores, Diagnostic techniques and procedures, Sensitivity and specificity