Abstract:

Objective To analyze the consistency between fluorescence PCR probe melting curve and Micropore-plate drug sensitivity tests (MicroDST) in determining the drug resistance of Mycobacterium tuberculosis (MTB), as well as the correlation between MTB gene mutation and drug resistance, in order to provide reference for optimization of clinical diagnosis and treatment. Methods From January to December, 2019, a total of 343 MTB clinical isolates from patients in Xi’an Chest Hospital were collected, all the specimens were tested using fluorescence PCR probe melting curve and MicroDST. Based on the results of MicroDST, the detection efficiency of fluorescence PCR probe melting curve in MTB resistance of isoniazid, rifampin, streptomycin, ethambutol, moxifloxacin and levofloxacin were evaluated. And the correlation between the gene mutation of MTB detected by fluorescence PCR probe melting curve and the minimum inhibitory concentration (MIC) of MicroDST was analyzed. Results With reference of the results of MicroDST, the sensitivity, specificity, and Kappa value of fluorescence PCR probe melting curve for isoniazid, rifampin, streptomycin, ethambutol, moxifloxacin and levofloxacin were 96.20% (76/79), 95.28% (242/254), 0.88; 93.62% (44/47), 94.58% (279/295), 0.79; 96.88% (62/64), 94.96% (264/278), 0.86; 93.33% (14/15), 95.37% (309/324), 0.61; 92.31% (24/26), 97.16% (308/317), 0.80; 91.18% (31/34), 99.35% (307/309), 0.92. Among the isolates, results of which by the fluorescence PCR probe melting curve were inconsistent with the phenotypic drug sensitivity, the lower phenotype drug sensitivity rates were found in those with the mutation of AhpC promoter region (-44--30 and -15-3) of isoniazid, the rpoB 507-512 of rifampin, the rrs 905-908 of streptomycin and the embB 406 of ethambutol, and the rates were 1/5, 1/5, 1/10 and 1/5, respectively. However, the phenotype drug sensitivity rates were higher in the isolates with mutation of the KatG 315 of isoniazid, the rpoB 529-533 of rifampin and the rpsL 43 of streptomycin, the rates were 92.42% (61/66), 92.31% (36/39) and 95.74% (45/47), respectively. Conclusion It has showed a good consistency in detection of drug resistance of MTB by fluorescence PCR probe melting curve and MicroDST. The drug resistance of MTB to isoniazid, rifampin and streptomycin was correlated with some gene mutations.

Key words: Mycobacterium tuberculosis, Polymerase chain reaction, Molecular probe techniques, Drug resistance,bacterial, Comparative Study