The Journal of Practical Medicine >

2023 , Vol. 39 >Issue 20: 2664 - 2670

DOI: https://doi.org/10.3969/j.issn.1006-5725.2023.20.019

Medical Examination and Clinical Diagnosis

Clinical application of fluorescence polymerase chain reaction probe melting curve technique in detecting isoniazid drug⁃resistance of Mycobacterium tuberculosis in sputum samples

  • Nenhan WANG ,
  • Yanfeng ZHAO ,
  • Lili TIAN ,
  • Shuangshuang CHEN ,
  • Hao CHEN ,
  • Yixuan REN ,
  • Chuanyou LI ,
  • Xiaowei. DAI
Expand
  • Beijing Center for Disease Prevention and Control，Beijing 100035，China

Received date: 2023-06-28

  Online published: 2023-11-15

Fold

Abstract

Objective To evaluate the application value of the fluorescence PCR probe melting curve（MeltPro） technique in the detection of isoniazid drug- resistance of Mycobacterium tuberculosis （MTB） in sputum samples. Methods In this study， we collected sputum samples of PTB that were identified as MTB by GeneXpert MTB/RIF method（MTB positive） from 2021 to 2022. These sputum samples were detected including smear， Mycobacterium culture， GenoType MTBDRplus，and isoniazid drug?resistance test by MeltPro technique. We did the proportional drug sensitivity test （DST） of isoniazid for culturing positive strains， analyzed the influence of the amount of bacteria in sputum samples on the results of MeltPro technique， compared the difference in the detection rate of isoniazid resistance by proportional DST， GenoType MTBDRplus， and MeltPro technique； The proportional DST results of isoniazid were used as a standard to evaluate the isoniazid drug?resistance detection efficiency of MeltPro technique and GenoType MTBDRplus. Results 157 qualified MTB positive sputum samples were collected， after Mycobacterium culture， we got 130 strains. The rate of success for isoniazid in sputum samples detected by MeltPro technique was84.1%（132/157）. Among different grades of sputum smear results， there is a difference in the successful rate of MeltPro technique （H = 24.169，P = 0.000）， increased with the grade of sputum smear results， the successful rate was increased from64.8% （32/54，the negative sputum）to 100.0%（17/17、6/6，the positive sputum grade 3+、4+）； the successful rate also increased with the increase of MTB grade detected by GeneXpert MTB/RIF， from54.8%（17/31，the very low sputum） to 96.4%（27/28，the high sputum）and the difference between each grade was statistically significant （H = 27.763，P = 0.000）. The detection rate of isoniazid resistance using MeltPro technique， GenoType MTBDRplus and proportional DST were 22.0%（29/132），15.3%（19/124），和14.6%（19/130）， respectively. There was no statistically significant difference between the three methods （χ2 = 2.997，P = 0.223）. Based on the results of proportional DST， the sensitivity， specificity， positive predictive value and negative predictive value of MeltPro technique in the detection of isoniazid were 84.6%（11/13）、91.2%（93/102）、55%（11/20）、97.9%（93/95）， respectively， Kappa is 0.614. The sensitivity， specificity， positive predictive value and negative predictive value of GenoType MTBDRplus in the detection of isoniazid were 55.6%（10/18）、92.3%（84/91）， 58.8%（10/17）， 91.3%（84/92） ，respectively， Kappa is 0.490. Conclusion The MeltPro technique has high sensitivity and specificity in detecting MTB isoniazid resistance in sputum samples of pulmonary tuberculosis patients， and can quickly and accurately screen for isoniazid resistance in patients； To improve the success rate of testing， it is necessary to pay attention to the selection of sputum samples when promoting the application in grassroots laboratories.

Key words： Mycobacterium tuberculosis; fluorescence PCR probe melting curve; drug resistance; phlegm

Cite this article

Nenhan WANG , Yanfeng ZHAO , Lili TIAN , Shuangshuang CHEN , Hao CHEN , Yixuan REN , Chuanyou LI , Xiaowei. DAI . Clinical application of fluorescence polymerase chain reaction probe melting curve technique in detecting isoniazid drug⁃resistance of Mycobacterium tuberculosis in sputum samples[J]. The Journal of Practical Medicine, 2023 , 39(20) : 2664 -2670 . DOI: 10.3969/j.issn.1006-5725.2023.20.019

References

1 中国防痨协会. 耐药结核病化学治疗指南（2019年简版）. 中国防痨杂志， 2019， 41（10）： 1025-1073.
2 SWAIN S S， PAIDESETTY S K， PADHY R N， et al. Isoniazid-phytochemical conjugation： A new approach for potent and less toxic anti-TB drug development［J］. Chem Biol Drug Des， 2020，96（2）：714-730.
3 ZHAO X， YU H， YU S， et al. Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase （KatG） and its S315T mutant［J］. Biochemistry， 2006，45（13）：4131-4140.
4 QIN L， HUANG C H， XU D， et al. Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn（III）： First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location［J］. Free Radic Biol Med， 2019，143：232-239.
5 World Health Organization. WHO treatment guidelines for isoniazid-resistant tuberculosis： supplement to the WHO treatment guidelines for drug-resistant tuberculosis. （2018）
6 World Health Organization. Global tuberculosis report 2020. Geneva：World Health Organization， 2021.
7 SULIS G， PAI M. Isoniazid-resistant tuberculosis： A problem we can no longer ignore［J］. PLoS Med， 2020，17（1）：e1003023.
8 赵雁林，逄宇. 结核病实验室检验规程［M］. 北京：人民卫生出版社， 2015.
9 World Health Organization. Global tuberculosis report 2020. Geneva：World Health Organization， 2021.
10 WHO Expert Group Report Molecular line probe assay more rapid screening of patients at risk of multidrug-resistant tuberculosis（MDR-TB）［R］. Geneva： World Health Organization， 2008.
11 赵国连，崔晓利，康磊，等. 荧光PCR探针熔解曲线技术检测涂阳患者痰标本中结核分枝杆菌耐药性的价值［J］. 中国防痨杂志， 2019，41（2）：149-155.
12 HUO F， MA Y， LIU R，et al. Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using GeneXpert vs Phenotypic Drug Susceptibility Testing［J］. Open Forum Infect Dis， 2020，7（8）：ofaa279.
13 谢芳晖，梁丽，赵霞，等. 肺结核患者痰标本采集的研究进展［J］. 中国防痨杂志， 2022，44（9）：978-982.
14 刘春平，熊德芳，谭耀驹. 两种分子检测技术对结核分枝杆菌临床分离株耐药性诊断价值的评价［J］. 临床肺科杂志， 2021，26（6）：815-819.
15 田丽，周伟，黄星，等. 中国异烟肼耐药结核分枝杆菌基因突变特征分析［J］. 中国防痨杂志， 2022，44（4）：354-361.
16 KHAN A S， PHELAN J E， KHAN M T， et al. Genetic mutations underlying isoniazid-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa， Pakistan［J］. Tuberculosis （Edinb）， 2023，138：102286.
17 CHEN Y， TAKIFF H E， GAO Q. Phenotypic instability of Mycobacterium tuberculosis strains harbouring clinically prevalent drug-resistant mutations［J］. Lancet Microbe， 2023，4（5）：e292.
18 CLSI. Performance standards for susceptibility testing of Mycobacteria， Nocardia spp，and Other Aerobic Actinomycetes［R］. Wayne： Clinical and Laboratory Standards Institute， 2023.
19 《中国防痨杂志》编辑委员会，中国医疗保健国际交流促进会结核病防治分会基础学组和临床学组. 结核分枝杆菌耐药性检测专家共识［J］. 中国防痨杂志， 2019，41（2）：129-137.
20 ZHANG Z， WANG Y， PANG Y，. et al. Comparison of different drug susceptibility test methods to detect rifampin heteroresistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother， 2014，58（9）：5632-5635.
21 龚兰，牛群，王楠，等. 结核分枝杆菌异烟肼耐药基因突变与其他抗结核药物耐药的相关性 ［J］. 实用医学杂志， 2023， 39（11）： 1353-1358.
Options
Outlines

/

〈 〉