Study on the detection of rifampicin resistance of Mycobacterium tuberculosis in sputum samples using fluorescence PCR probe melting curve technique

doi:10.19982/j.issn.1000-6621.20230086

Abstract

Abstract:

Objective: To evaluate the application value of the fluorescence PCR probe melting curve (MeltPro) technique in the detection of rifampicin-resistance of Mycobacterium tuberculosis (MTB) in sputum samples. Methods: Sputum samples of primary treated pulmonary tuberculosis (PTB) with positive GeneXpert MTB/RIF from Beijing Center for Disease Control and Prevention between May 2021 and May 2022 were collected. These sputum samples were tested using smear, mycobacterium isolation, and rifampicin-resistance by MeltPro technique. Drug sensitivity test (DST) of rifampicin was used for culturing positive strains, the differences in the detection rate of rifampicin resistance using DST, GeneXpert MTB/RIF, and MeltPro technique were compared. The proportional DST results of rifampicin were used as a standard to evaluate the drug-resistance detection efficiency of MeltPro technique. Results: A total of 158 qualified positive MTB sputum samples were collected, after mycobacterium culture, 130 strains were got. The success rate of the detection by MeltPro technique for rifampicin in sputum samples was 79.1% (125/158). As the grade of sputum smear results increased, the success rate of MeltPro technique increased from 59.3% (32/54, negative) to 100.0% (18/18, positive, grade +++); the success rate also increased with the increase of MTB grade detected by GeneXpert MTB/RIF, from 64.5% (20/31, extremely low) to 92.9% (26/28, high), the differences between each grade were statistically significant (H=22.271, P=0.001; H=15.637, P=0.001). The detection rates of rifampicin resistance using MeltPro technique, GeneXpert MTB/RIF method and proportional DST were 17.6% (22/125), 12.7% (20/158) and 13.1% (17/130), respectively. There was no statistically significant difference between the three methods (χ²=1.537, P=0.464). Based on the results of proportional DST, the sensitivity, specificity, positive predictive value, negative predictive value, Kappa of MeltPro technique in the detection of rifampicin were 100.0% (17/17), 94.6% (88/93), 77.3% (17/22), 100.0% (88/88), 0.845, respectively, and were 100.0% (17/17), 97.3% (110/113), 85.0% (17/20), 100.0% (110/110), 0.906, respectively of GeneXpert MTB/RIF in the detection of rifampicin. Conclusion: The MeltPro technique has high sensitivity and specificity in detecting rifampicin resistance of MTB in sputum samples of PTB, and can quickly and accurately screen the rifampicin resistance; attention should be paid to the quality of sputum samples when promoting the application in grassroots laboratories.

Key words: Mycobacterium tuberculosis, Polymerase chain reaction, Drug resistance, Sensitivity and specificity

CLC Number:

R521
R446.5

Wang Nenhan, Tian Lili, Chen Shuangshuang, Zhao Yanfeng, Fan Ruifang, Chen Hao, Ren Yixuan, Zhang Jie, Yi Junli, Yang Xinyu, Yu Lan, Ding Beichuan, Li Chuanyou, Dai Xiaowei. Study on the detection of rifampicin resistance of Mycobacterium tuberculosis in sputum samples using fluorescence PCR probe melting curve technique[J]. Chinese Journal of Antituberculosis, 2023, 45(6): 583-588. doi: 10.19982/j.issn.1000-6621.20230086

Figures/Tables 3

References 12

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检测方法	比例法药敏		敏感度 [%(95%CI)]	特异度 [%(95%CI)]	阳性预测值 [%(95%CI)]	阴性预测值 [%(95%CI)]	Kappa值 (95%CI)
检测方法	耐药(份)	敏感(份)	敏感度 [%(95%CI)]	特异度 [%(95%CI)]	阳性预测值 [%(95%CI)]	阴性预测值 [%(95%CI)]	Kappa值 (95%CI)
MeltPro技术^a			100.0 (77.8~100.0	94.6 (87.3~98.0)	77.3 (54.2~91.3)	100.0 (94.8~100.0)	0.845 (0.714~0.976)
耐药	17	5
敏感	0	88
GeneXpert MTB/RIF^a			100.0 (77.8~100.0)	97.3 (91.9~99.3)	85.0 (61.1~96.0)	100.0 (95.8~100.0)	0.906 (0.800~1.012)
耐药	17	3
敏感	0	110

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