Anamika Gupta

Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS-PCR

There is a growing need to develop rapid laboratory research methods to counter the menace of drug resistant tuberculosis (MDR‐TB) cases worldwide especially in developing countries. The present study was undertaken to investigate the type and frequency of rpoB and katG mutations in rifampicin (RIF) and isoniazid (INH) resistant strains respectively of Mycobacterium tuberculosis (MTB) circulating in Northern India and to explore the utility of multiplex‐allele‐specific (MAS)‐PCR assay for detection of drug‐resistant MTB isolates in low resource set up. J. Clin. Lab. Anal. 27:31–37, 2013.

Studies on the hypoglycemic effects of Murraya paniculata Linn. extract on alloxan-induced oxidative stress in diabetic and non-diabetic models

Abstract Objective To evaluate the effect of extract of Murraya paniculata Linn. (Family – Rutaceae) on blood glucose, cholesterol, triglyceride and lipid level and antioxidant status in alloxan induced diabetic and non-diabetic rats. Methods Hydro-alcoholic extract of M. paniculata leaves (100, 200 and 400 mg/kg) was administered orally for 14 days and its effect on blood glucose, cholesterol, triglycerides and lipid level were estimated in serum. Liver free radical (lipid peroxidation, LPO) and antioxidant (Super oxide dismutase, SOD; catalase, CAT; and reduced glutathione peroxidase, GPx) were also measured after 14 days treatment with extract. Glucose level in non-diabetic rats was estimated after 21 days treatment with M. paniculata extract. Results Oral administrations of M. paniculata extract (100, 200 and 400 mg/kg) for 14 days significantly reduced the levels of blood glucose, cholesterol, and triglyceride and lipid level. Liver free radical (LPO) significantly reduced and antioxidants (SOD, CAT and GPx) status significantly increase after 14 days treatment of extract in diabetic rats. M. paniculata 200 and 400 mg/kg significantly decrease glucose level in non-diabetic rats after 21 day and caused hypoglycemia in normal rats. Conclusions M. paniculata leaves extract posses hypoglycemic effect in oxidative stress condition and also in non-diabetic condition. Hypoglycemic action may be by potentiating of the insulin effect by increasing either the pancreatic secretion of insulin from beta cells of islets of langerhans or its release from the bound form. M. paniculata could be a potential source of hypoglycemic agent with antioxidant properties.

PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis

BackgroundThe Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India.MethodsA total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations.ResultsThe sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding.ConclusionsHence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein.

Detection of drug resistance in Mycobacterium tuberculosis: Methods, principles and applications.

The growing emergence of multidrug resistant tuberculosis (MDR-TB) strains is obstructing efforts for the control and management of TB. Proper management of MDR-TB relies on early recognition of drug resistance followed by timely treatment initiation. Several diagnostic methods, both phenotypic and molecular, have been developed in last few years for rapid identification of drug resistant (DR)-TB. Revised national tuberculosis control programmes (RNTPs) may find it tough to choose from the puzzling variety of rapid tests. Here, we present an outline of the available methods, discussing their basis, advantages and deficiencies.

Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates

Purpose: The presence of embB306 mutation in ethambutol (EMB)-susceptible (EMBs) clinical isolates questions the significance of these mutations in conferring resistance to EMB. The present study was carried out to determine the occurrence of embB306 mutation in EMB-resistant (EMBr) and EMBs strains of M. tuberculosis. One hundred and four multidrug-resistant tuberculosis (MDR-TB) strains were also included to establish the relevance of excessive use of rifampicin (RIF) and isoniazid (INH) in occurrence of embB306 mutations in EMBs M. tuberculosis isolates. Materials and Methods: Deoxyribonucleic acid (DNA) from M. tuberculosis clinical strains was isolated by cetyltrimethylammonium bromide (CTAB) method. Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 M. tuberculosis isolates by using standard proportion method and multiplex-allele-specific polymerase chain reaction assay, respectively. Results: The overall frequency of embB306 mutations in EMBr isolates was found to be five times higher than its occurrence in EMB-susceptible isolates (50% vs 10%). Further, the association between embB306 mutation and EMB-resistance was observed to be statistically significant (P = 0.000). Conclusion: The embB306 is not only the main causative mutation of EMB resistance, but is a sensitive applicant marker for EMB-resistance study.