Home > Characterization of Drug Resistance-Associated Mutations and Genetic Diversity in Mycobacterium Tuberculosis Isolates from Khyber Pakhtunkhwa, Pakistan
Characterization of Drug Resistance-Associated Mutations and Genetic Diversity in Mycobacterium Tuberculosis Isolates from Khyber Pakhtunkhwa, Pakistan
The Khyber Pakhtunkhwa region of Pakistan has been known for a high burden of tuberculosis (TB). However, there is little information about the molecular characteristics of Mycobacterium tuberculosis (M. tuberculosis) strains predominant in the province. Therefore, this study was planned to study the genetic diversity and molecular patterns responsible for the extensive drug resistance in the M. tuberculosis isolates isolated from clinical samples. In the current study total 794 patients were tested for suspected tuberculosis by fluorescence microscopy and GeneXpert system at the Provincial TB Reference Laboratory, Khyber Pakhtunkhwa, Pakistan. Among the 794 patients studied, 170 samples gave positive results and were further tested for drug susceptibility, out of which 163 samples gave results of drug susceptibility test. Among these 163 isolates, 79 (48.46%) were resistant to isoniazid, 47 (28.83%) were resistant to streptomycin, 56 (34.35%) were resistant to rifampicin, 31 (19.01%) were resistant to ethambutol, 16 (9.81%) were resistant to pyrazinamide, 59 (36.1%) were resistant to ofloxacin, 12 (7.36%) were resistant to capreomycin and 11 (6.74%) were resistant to amikacin. Further, these resistant isolates were selected for mutation analysis in the ‘hot spot regions’ of katG, rpoB, inhA, rpsL, embB, pncA, gyrA and gyrB genes. For the mutation analysis of the hotspot regions of katG and inhA genes, 30 resistant isolates were selected randomly. Among these 30 isolates, 21 (70%) had a mutation in katG gene and two (6.6%) had the most common C15T mutation in inhA promoter region. In 21 katG mutant isolates, 15 showed Ser315Thr, three showed Gly316Ser, two showed Ser315Arg mutations while one isolate was positive for a double mutation of Ser303Trp and Lys274Arg. Fourteen isolates were analyzed for the rpoB gene mutation. The mutations detected in rpoB gene in the selected isolates were Ser450Leu, Asp435Gly, Ser450Gln, Gly455Asp and Pro454His. Sixteen isolates were analyzed for streptomycin resistant-mutation in the rpsL gene. The rpsL gene mutations detected in six isolates were Lys43Arg, Lys88Arg, and Lys111Ile. Sixteen isolates were analyzed for ethambutol resistant-mutations of the embB gene. Out of these, 10 isolates were positive for Ala281val, Met306Leu, and Met306Val mutations. Eight isolates were analyzed for pyrazinamide resistant mutations of the pncA gene. Among them, 5 isolates showed a mutation in pncA gene, which included Gln141Pro, Ser65Ser*, Gly132Ser and Cys138stop mutations. The insertion of G at position 392 was also detected in pncA gene. Twenty isolates were analyzed for ofloxacin resistant mutations in gyrA and gyrB genes. Asp94Gly mutation in gyrA gene was detected in nine isolates while no mutation for the gyrB gene was detected in the selected isolates. These findings provide insights into the genetic variability existing against various drugs in M. tuberculosis circulating in Khyber Pakhtunkhwa, Pakistan. The results showed some novel mutations in the genes, katG, rpoB, rpsL and pncA. Small molecule inhibitors of the proteins encoded by the drug resistant genes, i.e., KatG, gyrA, pncA and rpoB of M. tuberculosis, were identified using computational methods. On the basis of the docking score and binding interactions, ten small molecule compounds were identified as novel inhibitors of each protein. These compounds showed significant interaction with the active site of the protein. The genetic diversity of M. tuberculosis isolates was estimated using RAPD and PCR restriction enzyme analysis.Fifty-two MTBC isolates were randomly selected for RAPD-PCR and PCR-restriction enzyme analysis. M. Bovis and M. tuberculosis H37Rv were used as reference strains. RAPD-PCR differentiated the strains and authenticated the high molecular diversity.PCR restriction enzyme analysis (PRA) profiles of all isolates and reference strains were found to be the same. High diversity values were found in the studied area implying high population diversity of M. tuberculosis that’s being prevalent in Khyber Pakhtunkhwa, Pakistan. The development of M. tuberculosis resistant strains is a major challenge severely affecting public health worldwide. New drugs obtained from natural resources could be a viable option to avoid serious drug resistance issues. On this quest, Aspergillus and Penicillium spp. were isolated from the surface soil. The secondary metabolites of Aspergillus and Penicillium were screened for anti-tuberculosis activity. The secondary metabolites of Aspergillus were active against M. tuberculosis whereas those of Penicillium showed no significant inhibitory activity. The results provide an overview of the existing nature of Multi drug resistance in M. tuberculosis circulating in Khyber Pakhtunkhwa province of Pakistan.
Gastric substances that potentially increase the esophageal mucosal damage are: gastric acid, pepsin, bile salts, and pancreatic enzymes. From all of these substances, the highest potential for reflux damage is gastric acid. Although the main cause of clinical symptoms of GERD is acid reflux, it has been known that there are subgroups with typical reflux symptoms that do not provide sufficient response or not responsive to PPI treatment. Despite the improvement of esophagitis, there is no clinical improvements in reflux symptoms of 30% respondents. Therefore, this study was designed to determine fasting gastric acidity with endoscopic findings in patients with GERD. A comparative-analysis study, which determine the fasting gastric acidity from endoscopic findings in patients with GERD. Samples recruited using consecutives sampling technique and divided into groups of esophagitis and non-esophagitis reflux. A total of 40 samples involved in this study. The Mann-Whitney test, was used for analyzing the difference between fasting gastric acidity from endoscopic findings of esophagitis lesions in patient with GERD. The median value for fasting gastric acidity in the esophagitis reflux group was 1.88 (0.82-4.84), whereas the median value for fasting gastric acidity in the non-esophagitis reflux group was 2.49 (0.68-5.97). The Mann-Whitney test result was p=0.298 (p>0.05). This study shows that there is no significant difference of fasting gastric acidity from endoscopic findings between esophagitis and non esophagitis reflux groups in patients with gastroesophageal reflux disease (GERD). This study shows that esophagitis lesions are not affected by gastric acidity.
The study was designed to find out the leadership behaviours of head teachers and its impact on school discipline at secondary level in Punjab. The major objectives of the study were to identify the leadership behaviours of head teachers at secondary level, to explore the existing condition of school discipline, to find out the impact of leadership behaviours of head teachers on school discipline and to compare the leadership behaviours of head teachers gender and locality wise. Three separate and self-constructed instruments were developed for the stud. The interview protocol was designed for head teachers to find out the leadership behaviours of head teachers and school discipline. One questionnaire was designed for teachers to explore the leadership behaviours of head teachers and other questionnaire for students to examine the existing condition of school discipline. Simple random sampling and convenient sampling techniques were used to collect data for the study. The province of Punjab was divided into three zones. Two districts were selected randomly from each zone and 144 secondary schools were chosen from six randomly selected districts of the Punjab. The selected districts were Attock, Sargodha, Shaiwal, Gujrat, Bahawalpur and Layyah. Thirty-six head teachers, 432 secondary school teachers and 2304 secondary school students were selected as a sample of the study. The collected data was entered into SPSS files. To interpret the quantative data, frequency, mean score, standard deviation t-test of independent sample and regression analysis techniques were applied. To analyze qualitative data, the researcher transcribed the interviews, find sub themes and themes of the data to generalize the results. The findings revealed that democratic leadership behaviour received highest rating, while autocratic leadership at second rating. Autocratic and democratic leadership behaviours of head teachers had a positive impact on school discipline. Laissez-faire leadership behaviour of head teachers had not a significant impact on school discipline. Gender wise comparison indicates that there was no significant difference in the opinions of male and female teachers about leadership behaviours. Locality wise comparision also shows that there was no significant difference in the opinions of rural and urban teachers about leadership behaviours of head teachers. Discipline condition of schools was satisfactory at secondary level. It is recommended that head teachers should adopt democratic leadership behaviour to maintain schools discipline in a constructive and positive way.