Prevalence and Genetic Variation among Drug Resistance Genes of Mycobacterium tuberculosis

Tuberculosis (TB) is a widespread and habitually deadly infectious disease caused by Mycobacterium species in humans, mainly by Mycobacterium tuberculosis (MTB). Drug resistant of TB increases day by day and there are few drugs effective against MTB, so this is a significant threat to TB controls. Tuberculosis and drug resistance TB (DR-TB) are big problem so early diagnosis of tuberculosis and DR-TB is essential for clinical management and public health control measures. For the first time, this study used wellestablished methods particularized somewhere else such as polymerase chain reaction (PCR), multiplex allele-specific PCR (MAS-PCR) and DNA sequencing for the diagnosis of tuberculosis and drugs resistant genes in Pakistan. A total of 6006 TB suspects specimens collected from March, 2009 to August, 2013 from 11 centers allover Punjab were included in the present study. The mean ages of patients were 39.7+ 18.5 years. These specimens were subjected to spot and morning smear microscopy. The morning specimens were subjected to culture and PCR. The culture positive specimens were then analyzed for drug susceptibility testing (DST). Randomly selected 123 drug resistant and 37 drug susceptible specimens were further analyzed by MAS-PCR. Those specimens for which MAS-PCR failed to detect known mutation were further analyzed by Sanger sequencing for mutation, if any, in the genes (rpoB, katG and embB genes). Among them 3196 (53%) were females and 1358 (22.6%) were previously treated patients. Microscopy of spot specimens of 2136 (35.6 %) were found positive for Mycobacterium species. Almost similar percentage of morning specimens (37%) was found positive on microscopy. Out of 6006 suspects specimens cultured, 2445 (40.7%) culture were positive for Mycobacterium species. Among 2445 positive culture 47 (1.9%) were Mycobacterium Other Than Tuberculosis (MOTT). When culture was used as a gold standard, specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of morning specimen microscopy was 97.1% , 83.2%, 95.4% and 88.9% respectively, whereas specificity, sensitivity, PPV and NPV of PCR was 98%, 93.8%, 97.2% and 95.6% respectively. Upon univariate analysis, statistically significant weak positive association (P<0.001, ?=0.120) was observed between culture positive for MTB and male gender, while moderate positive association (P<0.001, ?=0.228) was observed between culture positive for MTB and age group (10-25 years) and history of TB treatment (P<0.001, ?=0.221). Two hundred and seventy three (11.5%) of the studied patients showed resistance to one or more first line anti-TB drugs. Among drug resistant TB patients, 221 (81%) were multi-drug resistant, 41 (15%) were mono-resistant, while the remaining 11 (4%) were poly drug resistant TB patients. Upon univariate analysis, prevalence of MDR-TB had statistically significant negligible positive association (P<0.008, ?=0.07) and moderate positive association (P <0.001, ?=0.25) with age category (10-25 years) and positive TB treatment history respectively. When DST was used as a gold standard, sensitivity, specificity, PPV and NPV of MAS-PCR was 91.8%, 100%, 100% and 82.2% respectively for rpoB gene of rifampicin, whereas sensitivity, specificity, PPV and NPV of MAS- PCR was 80.5%, 100%, 100% and 60.7% respectively for katG gene of isoniazid. The sensitivity, specificity, PPV and NPV of MAS- PCR was 5.7%, 100%, 100% and 24.2% for mabA gene of isoniazid respectively, whereas sensitivity, specificity, PPV and NPV of MAS-PCR was 72.7%, 100%, 100% and 71.1% respectively for embB gene of ethembutol when DST was used as gold standard. In the present study, 33 specimens resistant to rifampicin by DST method and failed to determine by MAS-PCR were bidirectionally sequenced for unknown mutations. In twelve (12) specimens, a rare mutation in worldwide and novel mutation in Pakistan at codon 515 (Met515Iso) and a common variant at codon 516 (Met516Tyr) in each sample was detected in rpoB gene. In 4 specimens, a rare mutation in worldwide and novel mutation in Pakistan at codon 513 (Gln513Pro) was identified in rpoB gene. Similarly, in additional 4 specimens, M515V and H526W were identified in rpoB gene. Moreover, a rare mutation in worldwide and novel mutation in Pakistan (Leu533Pro) was found in 5 specimens in rpoB gene. In 8 specimens undetermined by MAS-PCR, no mutation was detected in rpoB gene. In fifteen (15) specimens resistant to isoniazid by DST method and failed to determine by MAS-PCR were bidirectionally sequenced for unknown mutation. In these 15 specimens, a common mutation at codon 315 (S315T) was detected in each specimens in katG gene. Fourteen (14) specimens resistant to at least ethambutol by DST method and failed to give result by MAS-PCR were sequenced bidirectionally for unknown mutation. In nine (9) specimens, a common mutation at codon 306 (Met306Iso) was determined in embB gene. In five (5) specimens, sequence matched completely with wild type embB gene sequence. This study concludes that Tuberculosis (TB) is more prevalent in males than in females. </BR>\ud Moreover it is advisable to not just rely on spot smears for detection of tuberculosis but \ud both spot and morning smears will lead to more accurate diagnosis as the later is more \ud effective. Careful design of treatment regimen from the available arsenal of anti\ud tubercular drugs will help in better resolution of the infection in the first encounter \ud minimizing risks of development of resistance in future. Novel mutations in different \ud genes of MTB leading to altered response to anti-tubercular medicines in Pakistan are not \ud out of question and should be closely monitored to adopt effective treatment plans for TB \ud in future