Genotypic Characterization of Drug-Resistant Mycobacterium Tuberculosis among the New and Previously Treated Tuberculosis Cases, from Health Facilities Across Seven Provinces of Zambia

Abstract

Drug-resistant tuberculosis (DR-TB) is one of the major public health problems globally. The emergence of Mycobacterium tuberculosis (MTB) resistant strains is a serious public health challenge to the prevention and control of TB globally. Zambia is ranked among countries with a high-burden of TB, TB/HIV and multidrug-resistant/rifampicin resistant tuberculosis (MDR/RR-TB) in the world. Drug-resistant TB is causing high mortality and morbidity rates in Zambia. Drug-resistant TB is mainly caused by mutations in the target genes of MTB. In this study, sputum samples obtained from the new and previously treated cases of TB were examined for DR-MTB. Sputum specimens were processed using the N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method. Mycobacterial DNA was extracted from the processed sputum using the Genolyse technique. Extracted DNA was subjected to multiplex polymerase chain reaction (PCR) amplification and reverse hybridization. Drug-resistance and mutations in MTB were detected using the Hain genotype MTBDRplus ver 2.0 and MTBDRsl ver 2.0 assays for first- and second-line anti-TB drugs, respectively. A total of 329 positive sputum specimens were analysed for drug-resistant TB. One hundred and two (102) specimens came from the new TB cases and 227 from the previously treated TB cases. Among the new TB cases, 3.9% had rifampicin mono-resistance (RIFr), 12.8% isoniazid mono-resistance (INHr), and 17.7% had MDR. While among the previously treated TB cases 10.1% had RIFr, 6.6% INHr, 33.0% MDR, 1.8% polydrug-resistance, and 0.8% had pre-extensively drug-resistance (pre-XDR). The rpoB MUT 3 (Ser531Leu) mutation was the most frequent (8.6%), conferring resistance to RIF, among the new TB cases. The same mutation was the most frequently detected (10.9%), conferring resistance to RIF, among the previously treated TB cases. The InhA MUT 1 (Cys15Thr) mutation was the most frequent (28.6%), conferring resistance to INH, among the new TB cases, while the katG MUT 1 (Ser315Thr 1) mutation was the most frequent (6.7%), conferring resistance to INH, among the previously treated TB cases. The rpoB MUT 3 (Ser531Leu) and the katG MUT 1 (Ser315Thr 1) mutations were the most frequent (14.3%), conferring resistance to both RIF and INH, among the new TB cases. The same mutations were the most frequently detected (18.5%), conferring resistance to both RIF and INH, among the previously treated TB cases. The rpoB MUT 2A (His526Tyr) and gyrA MUT 1 (Ala90Val) mutations were the most frequent (1.7%), conferring resistance to both RIF and fluoroquinolones (FLQs), among the previously treated TB cases. Two MTB isolates had pre-XDR among the previously treated TB cases. The first isolate had a mutation profile; rpoB MUT 2A (His526Tyr), katG MUT 1 (Ser315Thr), and gyrA MUT 3C (Asp94Gly). The second isolate had the profile; rpoB MUT 2B (His526Asp), katG MUT 2 (Ser315Thr 2), and eis MUT 1 (Cys14Thr). These two combination mutation profiles had the same frequency of 0.8%. Drug-resistant TB is prevalent in Zambia, especially among the previously treated TB cases. This calls for intensified drug-resistance surveillance. It is recommended to diagnose DR-TB early using the Hain genotype MTBDR ver 2.0 assays. There is also an urgent need to use anti-microbials appropriately to prevent or minimize antimicrobial resistance.