Abstract:

Objective: To evaluate the efficacy of PCR fluorescent probe method for detecting Mycobacterium tuberculosis complex (MTBC) and rifampicin resistance in sputum samples, and to provide feasibility data for further development of a multi-center clinical trial of MTBC and rifampicin resistance nucleic acid detection kit. Methods: Using prospective research method, three sputum specimens (immediate sputum, night sputum and morning sputum) from each of 205 patients with suspected pulmonary tuberculosis who met the enrollment criteria and were initially diagnosed at the pulmonary outpatient clinics of three designated tuberculosis hospitals in Shanghai from December 2021 to August 2022 were collected, and sputum smears, BACTEC MGIT 960 (MGIT 960), GeneXpert MTB/RIF (Xpert) and PCR fluorescent probe method were performed separately to evaluate the efficacy of PCR fluorescence probe method for detecting MTBC and rifampicin resistance in sputum samples. In addition, 96 clinical strains identified as MTBC (47 rifampicin-resistant strains and 49 rifampicin-sensitive strains) stored in the tuberculosis laboratory of Shanghai Center for Disease Control and Prevention were randomly selected to be tested for rifampicin resistance by PCR fluorescent probe method and Xpert, then rifampicin resistance-related gene mutations identified by the two methods were analyzed. Results: Among 205 patients with suspected pulmonary tuberculosis, the MTBC detection rate by PCR fluorescent probe assay was 22.44% (46/205), which was not statistically different from MGIT 960 (21.95% (45/205); χ²=0.014, P=0.904), Xpert (20.98% (43/205); χ²=0.129, P=0.719), and sputum smear (16.10% (33/205); χ²=2.650, P=0.104). Based on clinical diagnosis, the sensitivities (95%CI) of PCR fluorescent probe method, Xpert, MGIT 960 and sputum smear for the detection of MTBC were 48.10% (36.93%-59.56%), 48.10% (36.93%-59.56%), 50.63% (39.23%-61.97%) and 40.51% (29.79%-52.15%), the specificities (95%CI) were 93.65% (87.47%-97.12%), 96.03% (90.52%-98.53%), 96.03% (90.52%-98.53%) and 99.21% (95.01%-99.96%), the coincidence rates were 76.10% (156/205), 77.56% (159/205), 78.54% (161/205) and 76.59% (157/205), and the Kappa values were 0.453, 0.482, 0.507 and 0.446, respectively. There was no significant difference between MTBC detection rate of PCR fluorescent probe method (10.73% (19/177)) and Xpert (8.47% (15/177); χ²=0.793, P=0.373) on testing smear-negative and extremely low-grade positive samples. Six rifampicin resistance gene mutations out of 46 MTBC were detected by PCR fluorescence probe method, and the mutation rate was 13.04% (6/46), while only 1 gene mutation out of 43 MTBC was detected by Xpert, and the mutation rate was 2.33% (1/43). Among 96 clinical strains, 56 strains were found to have rifampicin resistance gene mutation by both methods. Except for mutation types of two probes, the mutation types of all other probes were consistent, and most of them occurred in probe E (529-533) and FAM channel (531/533 mutation). Conclusion: Based on clinical diagnosis, the efficacy of PCR fluorescent probe method in detecting MTBC is basically equivalent to MGIT 960, Xpert and sputum smear, and its efficacy is consistent with Xpert in detecting rifampicin resistance of MTBC clinical strains. It is considered that PCR fluorescent probe method is not only a new technology equivalent to Xpert detection function, but also has the advantages of operation simplicity, lower price, real one-piece and fully enclosed testing platform. It is suggested that PCR fluorescent probe method should be further optimized in the process of clinical popularization to improve detection rate of MTBC, and multi-center clinical trials should be carried out to verify the study.

Key words: Mycobacterium tuberculosis, Drug resistance, bacterial, Rifampin, Microbial sensitivity tests, Molecular diagnostic techniques