Prevalence and molecular characterization of multi drug resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa

Abstract

Tuberculosis (TB) is a pre-historic disease caused by Mycobacterium tuberculosis (MTB). There are more than 150 species in mycobacteriaceae family however, MTB is the most dominant member causing millions of new infection and deaths each year. Pakistan is on the 5th position among the list of countries having a high prevalence of TB. World health Organization has estimated an incidence of 370 to 650 thousands TB cases in Pakistan in each year. A total of 5759 samples were randomly collected from 25 districts of KPK for MDR prevalence as per criteria used in national TB survey. All the received samples were then subjected to microscopy, genexpert and culture tests for diagnosis of TB at a biosafety level-3 laboratory (BSLIII). Among all TB suspects 1969 (34%) were culture positive and 3121 (54%) were culture negative. The proportion of TB was significantly higher (p =0.001) in female (39%) as compare to male (29%). This might be due to minimal accessibility of femal to the helath care centers. TB is considered as the disease of poverty and is more common in malnurished and old age people. Interestingly, it was observed in the current study that age group 25-34 was more likely infected with TB in the studied region. High incidence of TB in the youg paitents may have socio econoical effect on the country profile thus great attenion must be gvien to early diagnose and treat the young patients who contribute to the country ecomony. A primilnary information of drug resistance was obtained from this study. It was observed that drug resistance was significanly higher in 238(12%) cases due the misuse and non conplaince of antibiotics. This high ration of antibiotic resistance resulted in high incidence of MDR-TB, found in 97 (4.9%) cases. The current study further explored mutation in different genes that confer resistance to antibiotics. DNA sequence of three genes rpoB, inhA and katG genes were analyzed to investigate drug resistance to rifampicin and isoniazid. It was observed that common mutation in rpoB was S531L in 75(77%) isolates followed by D516V in 10(10%). Similarly, S315T was observed the most common mutation in katG gene which was causing resistant in 97 strains. Genotypic analysis was carried out to identify the strain types of each MDR isolate. For this purpose spoligotyping analysis was carried out on all MDR strains along with control group. Results were analyzed in international TB databases for strain identification. Out of total 200 isolates 166 produced valid and interpretable results. (13%) spoligotype patterns were unmatched. These analysis demonstrates that CAS is the dominant genotype in KPK having a proportion of 73% (n=121) of the total isolates. Other genotypes include Beijing 5.4% (n=9), T1 4.2% (n=7) and 4.8% (n=8) were others types. Drug resistance profile was analyzed for all genotypes. Frequency of MDR was higher in Beijing strains (89%) as compared to CAS (56%) and T1 (57%). Statistical analysis showed that the proportion of drug resistance was higher in predominate genotypes (χ (24) = 84., p value= 0.001) as compared to less frequent genotypes. This study reported 13 new spoligotype patterns for the first time in Pakistan and these strains were named as orphan KP. All 13 new spoligotype patterns were analyzed to find out any evolutionary relationship to the previously published genotypes reported from Pakistan. We confirmed that majority of the new spoligotypes are evolved from pre-existed CAS genotypes. The current study provides a preliminary data of MDR-TB prevalence in Khyber Pakhtunkhwa which contributes 13% to the national TB burden in Pakistan. In this first large scale data from Khyber Pakhtunkhwa we found 4.9 % of MDR-TB among the new TB patients. This figure is consistent to the first national drug resistant survey (DRS) of Pakistan that reported 3.7% MDR-TB cases. Molecular profile resulted from this study predicts a high diversity value of MTBC strains in the studied region. Beijing strain is prone to drug resistance but fortunately it is less frequent in Pakistan. It is noteworthy that treatment of MDR-TB is very expensive thus early identification and isolation of patients infected with Beijing strain as diagnosed in this study will be of socio-economical benefit to Pakistan.