Analysis of the application of a gene chip method for detecting Mycobacterium tuberculosis drug resistance in clinical specimens: a retrospective study

Abstract

Most Mycobacterium tuberculosis (Mtb) resistant to rifampicin (RIF) has mutations in the rpoB gene, while most Mtb resistant to isoniazid (INH) has mutations in the katG gene or inhA promoter. We used gene chip technology to detect mutations in these genes to determine the resistance of Mtb to RIF and INH. A total of 4148 clinical specimens with sputum smear positivity for acid-fast bacilli (AFB) were detected. Then, taking the results of the drug sensitivity test (DST) as the reference standard, the detection efficiency of sputum samples from different grades of positive smears was compared in detail. We found that the sensitivity of the gene chip method for detecting sputum samples with a grade ≥ AFB 2 + was higher than that of sputum samples with a grade ≤ AFB 1 + (P < 0.05). When the grade of the sample was ≤ AFB 1 +, the sensitivity of the gene chip method was 72.6% for RIF, 67.3% for INH, and 60.0% for MDR-TB. When the grade of the sample was ≥ AFB 2 +, the sensitivity of the gene chip method was 84.5% for RIF, 78.2% for INH, and 73.9% for MDR-TB. The results show that gene chip technology can be directly used to diagnose drug-resistant tuberculosis in clinical specimens, and the diagnostic efficiency for the detection of sputum specimens with a grade ≥ AFB 2 + is better than that of other sputum specimens.

Figure 1

The layout of the DNA microarray method module; each detection panel includes 4 detection modules, which can detect two specimens at the same time. Modules 1 and 3 are used to detect mutations in the rpoB gene, and modules 2 and 4 are used to detect mutations in the katG gene and inhA promoter. QC quality control probe; EC external control probe; BC blank control; NC negative control probe; IC internal control probe; WT wild-type.

Figure 2

The percentage of major mutation sites in the rpoB gene from 2011 to 2020.

Figure 3

Specimen processing procedure: A total of 5,911 sputum smear-positive specimens were collected. After experimental processing, 4148 specimens that were positive with the DNA microarray method and DST were finally included in the study. NTM, nontuberculous mycobacteria; DST, drug sensitivity test.

Figure 4

Pattern diagrams of several common drug-resistant gene mutations detected by the DNA microarray method. The white box is the detection site of the wild-type codon, and the red box is the site of the detected mutant codon. (a) rpoB gene Leu511Pro (CTG → CCG); (b) rpoB gene Asp516Tyr (GAC → TAC); (c) rpoB gene His526Tyr (CAC → TAC); (d) rpoB gene Ser531Trp (TCG → TGG); (e) rpoB gene Ser531Leu (TCG → TTG); (f) katG gene Ser315Thr (AGC → ACC); (g) katG gene Ser315Asn (AGC → AAC); (h) inhA gene promoter-15 (C → T).