The pncA gene mutations of Mycobacterium tuberculosis in multidrug-resistant tuberculosis.

The pncA gene encodes pyrazinamidase enzyme which converts drug pyrazinamide to active form pyrazinoic acid, but mutations in this gene can prevent enzyme activity which leads to pyrazinamide resistance. The cross-sectional study was carried out during 2016-2017 for 12 months. The purpose of the study was to detect mutation at codon 12 and codon 85 in the pncA gene in local multidrug-resistant tuberculosis (MDR-TB) patients by developing a simple molecular test so that disease could be detected timely in the local population. DNA extracted from sputum-cultured samples from MDR-TB patients and subjected to semi-multiplex allele-specific PCR by using self-designed primers against the pncA gene. Among 75 samples, 53 samples were subjected to molecular analysis based on purified DNA quantity and quality. The primers produced 250 and 480 bp fragments, indicating the mutations at codon 12 (aspartate to alanine) and codon 85 (leucine to proline) respectively. MDR-TB was more common in the age group 21-40 years. Fifty-seven percent of samples (n = 30) were found positive for pncA mutations, whereas 43% of samples (n = 23) showed negative results. Thirteen percent of samples (n = 4) had mutations at codon 12 in which aspartate was converted to alanine, and they produced an amplified product of 480 bp. Eighty-seven percent of samples (n = 26) had mutations at codon 85 in which leucine was converted to proline and amplified product size was 250 bp. The mutations were simple nucleotide substitutions. The prevalence of mutations in which leucine was substituted by proline was higher than the mutations in which aspartate was substituted by alanine. A high prevalence of substitution mutation (CTG → CCG; leucine to proline) was detected in MDR-TB cases. Earlier detection of MDR-TB via an effective molecular diagnostic method can control the MDR tuberculosis spread in the population.

Molecular detection of hupB gene of Mycobacterium tuberculosis in young patients with acute lymphoblastic leukemia (ALL) on Chemotherapy.

Cancer chemotherapy can lead to the mycobacterial infections. Tuberculosis has been reported a serious complication in leukemia patients who undergo chemotherapy. The study was focused to find mutations in hupB gene of M. tuberculosis in 50 acute lymphoblastic leukemia (ALL) patients through semi multi complex PCR. A column based DNA isolation method was adopted for DNA isolation. The gene for histone-like protein (hupB [Rv2986c]) of M tuberculosis was amplified to detect two closely related mycobacterial species. Primers M and S (histone like protein HupB) were utilized to generate amplicons of 318 bp and 291 bp for M. tuberculosis and M. bovis, respectively. Out of fifty ALL patients, 21 (42%) were females and 29 (58%) were males. The prevalence of ALL was found higher in males as compared to females. The prevalence of ALL was higher in patients of age group 5-10 years. The results of the amplification showed that, the 318 bp fragment specific for M. tuberculosis was observed in seven samples (14%), while 291 bp fragment specific for M. bovis was not observed in any sample. Children with ALL were found at higher risk for tuberculosis. A risk evaluation of tuberculosis infection must be conducted before managing chemotherapy.

NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.