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Chakravarty J.,Banaras Hindu University | Sundar S.,Banaras Hindu University | Chourasia A.,Banaras Hindu University | Singh P.N.,Banaras Hindu University | And 7 more authors.
BMC Infectious Diseases | Year: 2015

Background: The National AIDS Control Organization of India has been providing free second line antiretroviral therapy (ART) since 2008. This observational study reports the survival and virologic suppression of patients on second-line ART under programmatic condition and type of mutations acquired by those failing therapy. Methods: 170 patients initiated on second-line therapy between 2008 and 2012 were followed up till 2013. Viral Load (VL) was repeated at 6 months for all patients and at 12 months for those with VL > 400 copies/ml at 6 months. Adequate virological response was defined as plasma HIV-1 VL < 400 copies/ml and virological failure was defined as VL > 1000 copies/ml. Genotyping was done in 16 patients with virological failure. Results: Out of 170 patients, 110 (64.7 %) were alive and on therapy and 35 (20.5 %) expired. In the first year the occurrence of death was 13.7/100 person years while between1 and 5 year it was 3.88/100 person years. In the first year, duration of immunological failure > 12 months, weight < 45 kg, WHO clinical stage 3 and 4 and WHO criteria CD4 count less than pretherapy baseline [hazard ratio HR 4.2. 15.8, 11.9 & 4.1 respectively] and beyond first year poor first and second line adherence and first line CD4 count < 200/μL [HR 5.2,15.8, 3.3 respectively] had high risk of death. 119/152 (78.2 %) had adequate virological response and 27/152 (17.7 %) had virological failure. High viral load at baseline and poor second line adherence (Odds Ratio 3.4 & 2.8 respectively) had increased risk of virological failure. Among those genotyped, 50 % had major Protease Inhibitor mutation (M46I commonest) however 87.5 % were still susceptible to darunavir. Conclusions: Second line therapy has shown high early mortality but good virological suppression under programmatic conditions. © 2015 Chakravarty et al. Source

Kumar P.,All India Institute of Medical Sciences | Sen M.K.,Vardhman Mahavir Medical College VMMC and Safdarjung Hospital | Chauhan D.S.,National JALMA Institute of Leprosy and Other Mycobacterial Diseases | Katoch V.M.,Dep Of Health Research Ministry Of Health And Family Welfare | And 2 more authors.
PLoS ONE | Year: 2010

Background: The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. Methodology/Principal Findings: Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. Conclusions/Significance: To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis. © 2010 Kumar et al. Source

Chaturbhuj D.N.,National Dairy Research Institute | Nirmalkar A.P.,National Dairy Research Institute | Paranjape R.S.,National Dairy Research Institute | Tripathy S.P.,National JALMA Institute of Leprosy and Other Mycobacterial Diseases
PLoS ONE | Year: 2014

Objectives: Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples. Design: The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity. Methods: The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000-1.8 million RNA copies/ml. Results: Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load <1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective. Conclusions: The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients. © 2014 Chaturbhuj et al. Source

Husain S.,National JALMA Institute of Leprosy and Other Mycobacterial Diseases
Indian Journal of Leprosy | Year: 2014

Peripheral nerve involvement results in deformities formation in leprosy. High doses of (40-60 mg) steroids along with the anti-leprosy drugs is preferred even though the 70-75% cases develop deformity with the above treatment. 772 ulnar nerves, 120 median nerves and 108 posterior tibial nerves not responding to above medical treatment in 12 weeks, were undertaken for external and internal nerve trunk decompression. These cases were followed-up for 5-20 years at various intervals. The pain in nerves (neuritis) recovered in all cases of ulnar, median and posterior tibial nerves. Full sensory recovery with pin prick/feather or cotton wool touch was seen in 50% cases of all the three nerves. 20% cases maintain the pre-operative levels of sensory status. Plantar ulcers healed within 6 months after decompression of posterior tibial nerve. Only 6 cases showed reoccurrence. Overall motor recovery in ulnar nerve was seen 89% and 70% in median nerve. The sensory recovery restores protective sensation which prevents secondary injuries. The improvement of motor power gave better functional hands and improved the appearance which in absence of surgical intervention was not possible. © Hind Kust Nivaran Sangh, New Delhi. Source

Singh G.,Panjab University | Arya S.,Panjab University | Narang D.,Indian Institute of Science | Jadeja D.,Panjab University | And 3 more authors.
Molecular Biology Reports | Year: 2014

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif 'GXSXG'. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K m and Vmax of enzyme was determined to be 21.29 U mg -1 protein, 714.28 μM and 62.5 μmol ml-1 min -1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser50, Asp 180 and His203 residues by site-directed mutagenesis. © 2013 Springer Science+Business Media Dordrecht. Source

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