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.
Highlights
IntroductionResistance to two first line predominant anti-TB drugs i.e., isoniazid (INH) and rifampicin (RIF), is termed as ‘multidrug resistance tuberculosis’ [4]
Pakistan is among the top 20 countries with a high TB and multidrug resistant (MDR)-TB burden [1]
To gain further information about the prevalence of MDR in newly diagnosed patients in this area of Lahore, Pakistan, the rifampicin resistance determining region (RRDR) region of the rpoB gene from two groups was sequenced; the first group comprised extracted DNA from130 MDR strains from patients diagnosed previously with TB, and with a history of resistance to first line antituberculosis drugs; the second group comprised 86, DNA extracts directly from sputum samples from consecutive newly diagnosed patients, with clinical symptoms of TB, positive in sputum smear microscopy acid fast bacilli (AFB), chest X-ray positive and presumed drug susceptible with no history of resistance to any first line tuberculosis drugs
Summary
Resistance to two first line predominant anti-TB drugs i.e., isoniazid (INH) and rifampicin (RIF), is termed as ‘multidrug resistance tuberculosis’ [4] Molecular methods for this are diverse and each method has its benefits and drawbacks; for example PCR-RFLP [5] and allele-specific PCR [6]. Several molecular techniques have been evolved to detect the gene mutation related to resistance These include hybridization methods; single strand polymorphism, DNA sequencing and other PCR based methods [5, 7, 8]. Drug resistance prediction from the whole genome sequence is possible using publically available software which rapidly analyses all known gene targets and identifies mutations associated with resistance enabling targeted treatment [11] but requires culture and is currently prohibitively expensive for high burden countries. In May 2016, WHO issued guidance that “people with TB resistant to rifampicin, with or without resistance to other drugs, should be treated with an MDR-TB treatment regimen.” Together with MDR-TB, these are referred to as MDR/RR-TB
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