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