Sequence analysis of the rifampicin resistance determining region (RRDR) of rpoB gene in multidrug resistance confirmed and newly diagnosed tuberculosis patients of Punjab, Pakistan

Abstract

Molecular screening of new patients suspected for TB could help in the effective control of TB in Pakistan as it is a high TB burden country. It will be informative to understand the prevalence of multi drug resistance for a better drug regimen management in this geographical area. The Rifampicin resistance determining region (RRDR) sequencing was used to identify mutations associated with drug resistance in DNA extracts from 130 known multidrug resistant (MDR) cultured strains and compared with mutations observed in DNA extracts directly from 86 sputum samples from consecutive newly diagnosed cases in Lahore, Pakistan. These newly diagnosed samples were positive for smear microscopy, chest X-ray and presumed sensitive to first line drugs. In the known MDR group the most frequent mutations conferring resistance were found in rpoB531 (n = 51, 39.2%). In the newly diagnosed tuberculosis group with no history of MDR, mutations in rpoB531 were seen in 10 of the samples (11.6%). Collectively, all mutations in the RRDR region studied were observed in 80 (61.5%) of known MDR cases and in 14 (16.3%) of the newly diagnosed cases. Using the RRDR as a surrogate marker for MDR, sequences for the newly diagnosed (presumed sensitive) group indicate much higher levels of MDR than the 3.9% WHO 2015 global estimate and suggests that molecular screening directly from sputum is urgently required to effectively address the detection and treatment gaps to combat MDR in this high burden country.

Drug Resistance and Molecular Characteristics of Mycobacterium tuberculosis: A Single Center Experience

“…Between 85-90% of MTB has been reported that resistance to INH when it has the RIF resistance, and RIF resistance is regarded as a major marker of MDR [37]. The most mutations in rpoB gene concentrated on codon 516, which was inconsistent with relevant studies that codon 531 was more frequently [38][39][40]. However, a study in Brazil showed that codon 516 rpoB mutations exhibited a higher frequency than codon 531 mutations in Haarlen lineage [41].…”

Molecular characterisation of multidrug-resistantMycobacterium tuberculosisisolates from a high-burden tuberculosis state in Brazil

“…The mutation in the codon 531 stands out with higher frequency (61.8%), also found in other studies in the same state [ 22 , 29 ], in other regions of Brazil [ 30 , 31 ] and other countries [ 28 ]. The codon 516 in the rpo B gene was mutated in 16.8% of the isolates and 2.3% of them had single nucleotide exchange, other studies show a bigger mutation rate with SNP in this codon [ 30 , 32 ]. The codon 526 was mutated in 9.9% of the isolates, and similar results were found in MDR isolates from a nearby state [ 31 ].…”

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

“…Pakistan is among the five highest-burden countries for RR cases, the most frequent mutations reported in RRDR regions were Ser450 (64.1%), Asp435 (13.3%) and His445 (12.7%). Earlier studies performed in Pakistan, were on small sample sizes, have also reported similar frequencies of changes at amino acid position 450 (52 and 56.4%), amino acid 435 (4.84% and 15%) and amino acid 445 (7 and 9.68%) [15,16]. Similar frequencies of mutations were reported by other countries in Asia, e.g., Ser450 mutation rates were reported in India (59%), China (60%) Nepal (58.7), and in Russia (66.6%).…”

“…In Swaziland 30% of RR cases, harbored mutations l outside the RRDR [35]. In Uganda, 18% (8/45), in China five mutations were detected outside the RRDR region [15,16]. Similarly, In Syria, one mutation was detected outside the RRDR region [37].…”

“…Pakistan is among the five highest-burden countries for RR cases, having an estimated 15000 cases every year at the rate of 4.2% and 16% in new and previously treated cases, respectively [3]. Only a few small-scale studies, however, have been conducted in the country for the characterization of rpoB mutations among local MTB strains [14][15][16].…”