Molecular differentiation of multi-drug resistant mycobacterium tuberculosis complex isolated from three provinces in Zambia

Abstract

Multidrug-resistant tuberculosis (MDR-TB) is a disease caused by an acid-fast staining pathogenic bacterium, Mycobacterium tuberculosis that is resistant to at least isoniazid (INH) and rifampicin (RIF), the two most potent TB drugs. Drug-resistant tuberculosis poses a significant problem for successful treatment of tuberculosis (TB). Worldwide, 4.1 percent of new cases and 19 percent of previously treated cases have been estimated to show MDR-TB and in Zambia, 1.8 percent and 2.3 percent MDR TB among new and previously treated cases respectively have been reported. This study was conducted to characterize the isolates of Mycobacterium tuberculosis complex (MTBC) associated with rifampicin (RIF) and isoniazid (INH) drug resistance and to determine the prevalence of mutations attributed to two specific genes (rpoB and katG) and the inhA promoter region which have been linked to resistance against rifampicin and isoniazid. A panel of 40 INH and/or RIF drug resistant clinical isolates of Mycobacterium tuberculosis complex with susceptibility test results from Mycobacteria Growth Indicator Tubes (MGIT) were used. Isolates showing resistance to RIF and INH were analysed by multiplex polymerase chain reaction (PCR) and the amplicons were sequenced using the Big Dye Terminator v3.1 cycle sequencing kit on the ABI Prism 3500xl Genetic Analyser (Life Technologies Corp). All of the analysed isolates were identified to be Mycobacterium tuberculosis. Seventy-five percent (30/40) of these had katG mutations at codon 315, 7.5 percent (3/40) had the katG mutation alone while 2.5 percent (1/40) had mutations in the inhA promoter and rpoB in addition to the katG 315 mutation. KatG S315T mutation and the -18CT inhA mutation accounted for only 2.6 percent (1/39) of the INH-resistant isolates. Of the 40 isolates characterized for rpoB mutations, 39 demonstrated mutations with the most common being S450L (48.7 percent), followed by H445T (20.5 percent). In one phenotypically-RIF resistant isolate, no nucleotide changes were detected in the rpoB gene. Mutations not previously reported were found at codons 535(CAA-GAA, Q535E) and 545(CTG-CCG, L545R). These results demonstrate varying geographical distribution of mutations in the rpoB in distinct regions. One out of 39 (2.6 percent) RIF resistant isolates demonstrated resistance to rifampicin alone which agrees with results from other studies which have reported that rifampicin mono resistance is rare. The results of the current study are consistent with the results from similar studies in China and Mexico which reported that katG S315T and S450L rpoB mutations were the most prevalent mutations in clinical isolates. The high percentage of mutations at codon 315 of the katG gene (75 percent, n=40) demonstrates the importance of this codon in the development of resistance against isoniazid in Mycobacterium tuberculosis species circulating in various provinces of Zambia. Therefore, molecular analysis of these predominant mutations could be useful for the rapid detection of drug resistant tuberculosis.