Molecular Detection of Drug-Resistant Mycobacterium tuberculosis in Sputum Specimens from the New and Previously Treated Tuberculosis Cases at the National Reference Chest Diseases Laboratory in Lusaka, Zambia

Abstract

Background: Drug-Resistant Tuberculosis (DR-TB) is one of the major public health issues globally. Zambia is highly burdened by TB and multi-drug resistant TB. In this study, sputum samples obtained from the new and previously treated cases of TB were examined for drug-resistant Mycobacterium tuberculosis (MTB). Methods: Sputum specimens were processed using the N-acetyl-L-cysteine-sodium hydroxide method, stained and examined using fluorescent technique and microscopy respectively. Mycobacterial DNA was extracted using the Genolyse kit, then subjected to multiplex polymerase chain reaction amplification and reverse hybridization. Drug-resistance and mutations in MTB genes were detected using the Genotype MTBDRplus VER 2.0 and MTBDRsl VER 2.0 assays. Results: A total of 329 MTB-positive sputum specimens, 102 from the new TB cases and 227 from previously treated TB cases, were analysed for drug-resistance. Among the new TB cases, 3.9% had Rifampicin (RIF) mono-resistance, 12.8% Isoniazid (INH) mono-resistance, and 17.7% had Multi-Drug Resistance (MDR). For the previously treated TB cases, 10.1% had RIF mono-resistance, 6.6% INH mono-resistance, 33.0% MDR, 1.8% poly-drug resistance, and 0.8% had pre-Extensively Drug-Resistance (pre-XDR). Mutations identified were rpoB (Ser531Leu, His526Asp, Asp516Val, His526Tyr, and Glu510His), katG (Ser315Thr 1 and Ser315Thr 2), InhA (Cys15Thr), gyrA (Ala90Val and Asp94Gly), and eis (Cys14Thr), each with a varying frequency. Conclusion: DR-TB is prevalent, especially MDR-TB, which is currently the most worrisome form of DR-TB and an emerging threat hampering efforts in the control of TB in Zambia. The early detection and effective treatment of TB cases are key in the control of DR-TB.

Drug-target genes and their spontaneous mutations associated with resistance to first-line, second-line, third-line, novel and repurposed anti-tuberculosis drugs in Mycobacterium tuberculosis resistant strains

“…Isoniazid resistance is brought about by mutations in several genes of M. tuberculosis such as the katG, inhA, ahpC, kasA, oxyR, furA, fabG1-inhA, and ndh genes. 3,34,37 However, Current research has shown that resistance to isoniazid can also be caused by an upregulation of efflux pumps or isoniazid inactivators. 38,39 Mutations in the katG, inhA and its promoter, and the oxyR-ahpC intergenic region frequently confer resistance to INH.…”

“…1,2 It causes high mortality and morbidity rates, it kills one person every 21 seconds. 3,4 TB is caused by species of the Mycobacterium tuberculosis complex (MTBC), some of which are adapted to humans (Mycobacterium tuberculosis [MTB] and Mycobacterium africanum), while others to animals (Mycobacterium bovis, Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium mungi, and Mycobacterium caprae), and others are smooth bacilli (Mycobacterium canettii). [5][6][7] MTB is the major causative pathogen for human TB among the species of MTBC, as it causes 97-99% of all TB cases globally.…”

“…[5][6][7] MTB is the major causative pathogen for human TB among the species of MTBC, as it causes 97-99% of all TB cases globally. 3 TB is the second leading cause of death from a single infectious agent, after coronavirus disease 2019 (COVID-19), a highly infectious disease caused by a virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 8,9 The current TB morality is severely impacted by COVID-19 and not HIV/AIDS.…”

“…2 DR-TB can be transmitted through the air from one person to another or can develop when MTB strains become resistant to anti-TB drugs due to a number of factors which include: poor adherence, poor compliance, poor selection of regimens, low efficacy anti-TB drugs, late diagnosis, interrupted supply, stock-outs, spontaneous mutations, and chromosomal replication errors. 3,10,[17][18][19][20][21] Different categories of DR-TB have been defined and they include; multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant tuberculosis (Pre-XDR-TB), extensively drug-resistant tuberculosis (XDR-TB), extremely drug-resistant tuberculosis (XXDR-TB), and totally drug-resistant tuberculosis (TDR-TB). 4,5,22 MDR-TB is TB resistant to at least rifampicin (RIF) and isoniazid (INH).…”

“…23,24 Pre-XDR TB is TB resistant to RIF, INH, plus any one fluoroquinolone (FLQ) (levofloxacin, moxifloxacin, ofloxacin, or gatifloxacin), or any one of the second-line injectable drugs (SLIDs) (kanamycin, capreomycin, or amikacin). 3 XDR-TB is TB resistant to RIF, INH, plus any FLQs, plus any SLIDs. 10 XXDR-TB is a type of TB characterized by the presence of MTB isolates that show in-vitro resistance to all first-and second-line anti-TB drugs tested.…”