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Singh A.,All India Institute of Medical Sciences | Gopinath K.,All India Institute of Medical Sciences | Gopinath K.,University of Cape Town | Singh N.,Designated Microscopy and Center | And 2 more authors.
International Journal of Mycobacteriology | Year: 2014

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and the disease has remained a major health problem in most of the developing countries, particularly after the emergence of multidrug-resistant TB (MDR-TB). The MDR-TB is an intriguing subject and very little is known about the in vivo processes which take place during the acquisition of MDR. This study describes a unique case of pulmonary TB (PTB) from which four sequential isolates of MTB could be isolated while the patient was on anti-tubercular treatment. The first baseline isolate was sensitive to all drugs, but the subsequent three isolates acquired resistance to multiple drugs and finally the patient died after 27. months post-diagnosis when his fourth isolate became resistant to isoniazid, rifampicin, ethambutol and kanamycin. All sequential cultures were identified as MTB using conventional and molecular methods, including 16s RNA sequencing and the spoligotyping. Spoligotyping followed by comparison with SITVITWEB database revealed that all the isolates belonged to the family of the Central Asian Strain Delhi (CAS1_Delhi, ST26) genotype, and no cross or mixed infections were observed. The drug resistance was further characterized at the molecular level by sequencing the target genes (katG, inhA, rpoB, embB, eis promoter region and rrs). The results revealed mutated alleles associated with resistance to the respective drugs. This unique case indicates that it is possible to isolate MTB during treatment if the strain is acquiring resistance. The data presented from four sequential isolates provides an insight into what sequential genetic and proteomic changes occur in the bacteria during the in vivo acquisition of MDR. © 2013 Asian-African Society for Mycobacteriology.

Singh A.,All India Institute of Medical Sciences | Gopinath K.,All India Institute of Medical Sciences | Gopinath K.,University of Cape Town | Sharma P.,National JALMA Institute for Leprosy and Other Mycobacterial Diseases ICMR | And 4 more authors.
Indian Journal of Medical Research, Supplement | Year: 2015

Background and objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation and conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings. © 2015, Indian Council of Medical Research. All rights reserved.

Kumar P.,All India Institute of Medical Sciences | Pandya D.,AmpliGene India Biotech Pvt. Ltd. | Singh N.,National Institute of Tuberculosis and Respiratory Diseases | Behera D.,National Institute of Tuberculosis and Respiratory Diseases | And 3 more authors.
Journal of Infection | Year: 2014

Objectives: Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples. Materials: Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods. Results: LAMP was able to detect 5fg DNA (one MTB) within 21min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2-100) and 96.1% (95% CI 86.7-99.5) respectively with 93.5% (95% CI 78.5-99.2) of overall specificity. Conclusion: LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective. © 2014 The British Infection Association.

Gandhi K.,National Institute of Tuberculosis and Respiratory Diseases | Gupta S.,National Institute of Tuberculosis and Respiratory Diseases | Singla R.,National Institute of Tuberculosis and Respiratory Diseases
Indian Journal of Tuberculosis | Year: 2016

Background Treatment of pulmonary tuberculosis (PTB) focuses on microbiological cure and radiological improvement. However, many patients develop pulmonary impairment after the completion of anti-tubercular therapy (ATT), which affects their quality of life (QoL). Aim and objective To study the occurrence and severity of pulmonary impairment after tuberculosis (PIAT), risk factors associated with development of PIAT and QoL after development of PIAT. Methodology 146 eligible PTB patients, who completed their ATT during January 2013 to December 2013 at National Institute of TB and Respiratory Diseases (NITRD), New Delhi and peripheral centres were enrolled after informed consent and evaluated. PIAT was graded using spirometric parameters. Severity of dyspnoea was assessed using Borg scale and Medical Research Council (MRC) scale. QoL was assessed using Seattle's Obstructive Lung Diseases Questionnaire (SOLDQ). Results 74% (108) had PIAT. On univariate analysis, smoking, education, body mass index (BMI), duration of illness prior to diagnosis of TB and number of prior ATT courses taken were the significant risk factors associated with the development of PIAT. On multiple logistic regression, patients who had taken ATT more than once was the independent risk factor associated with PIAT. Severity of dyspnoea was increased on both Borg scale and MRC scale with the increase in impairment of lung function. QoL was lower in patients with severe impairment. Conclusion After bacteriological cure of TB after treatment, significant numbers of patients have poor lung function and poor QoL. There is need for prevention and management of such sequelae under national programme. © 2016 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.

Dewan R.K.,National Institute of Tuberculosis and Respiratory Diseases | Saxena R.,National Institute of Tuberculosis and Respiratory Diseases | Gandhi S.,National Institute of Tuberculosis and Respiratory Diseases
Indian Journal of Thoracic and Cardiovascular Surgery | Year: 2016

A 20-year-old male presented to the hospital with discharging pus from anterior chest wall region for 15 days. He had a past history of left-sided pleural effusion for which he was put on ATT by a private practitioner on an empirical basis. In view of an excessive bleeding from the chest wall abscess, the patient was taken up for emergency exploratory thoracotomy. Intra-operatively, it was found that the abscess had ruptured in the thorax eroding the left internal mammary artery which was ligated at its origin, and hemothorax was drained. Post-operatively, the patient had an uneventful recovery. © 2015, Indian Association of Cardiovascular-Thoracic Surgeons.

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