Rohit Sarin

Impact of Introducing the Line Probe Assay on Time to Treatment Initiation of MDR-TB in Delhi, India

Setting National Institute of Tuberculosis and Respiratory Diseases (erstwhile Lala Ram Sarup Institute) in Delhi, India. Objectives To evaluate before and after the introduction of the line Probe Assay (LPA) a) the overall time to MDR-TB diagnosis and treatment initiation; b) the step-by-step time lapse at each stage of patient management; and c) the lost to follow-up rates. Methods A retrospective cohort analysis was done using data on MDR-TB patients diagnosed during 2009–2012 under Revised National Tuberculosis Control Programme at the institute. Results Following the introduction of the LPA in 2011, the overall median time from identification of patients suspected for MDR-TB to the initiation of treatment was reduced from 157 days (IQR 127–200) to 38 days (IQR 30–79). This reduction was attributed mainly to a lower diagnosis time at the laboratory. Lost to follow-up rates were also significantly reduced after introduction of the LPA (12% versus 39% pre-PLA). Conclusion Introduction of the LPA was associated with a major reduction in the delay between identification of patients suspected for MDR-TB and initiation of treatment, attributed mainly to a reduction in diagnostic time in the laboratory.

Efficacy and Safety of Mycobacterium indicus pranii as an adjunct therapy in Category II pulmonary tuberculosis in a randomized trial

Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.

First and second line drug resistance among treatment naïve pulmonary tuberculosis patients in a district under Revised National Tuberculosis Control Programme (RNTCP) in New Delhi.

There is limited information of level of drug resistance to first-line and second line anti-tuberculosis agents in treatment naïve pulmonary tuberculosis (PTB) patients from the Indian region. Therefore, the present prospective study was conducted to determine the antimicrobial susceptibility to first-line and second line anti-TB drug resistance in such patients. Sputum samples from consecutive treatment naïve PTB cases registered in Lala Ram Sarup (LRS) district, under RNTCP containing 12 Directly Observed Treatment Centre’s (DOTS), were enrolled using cluster sampling technology. A total of 453 samples were received from July 2011 to June 2012. All samples were cultured on solid medium followed by drug susceptibility to first and second line anti-tubercular drugs as per RNTCP guidelines. Primary multi-drug resistance (MDR) was found to be 18/453; (4.0%). Extensively drug resistance (XDR) was found in one strain (0.2%), which was found to be resistant to other antibiotics. Data of drug resistant tuberculosis among treatment naïve TB patients are lacking in India. The presence of XDR-TB and high MDR-TB in small population studied, calls for conducting systematic multi-centric surveillance across the country.

Index-TB guidelines: Guidelines on extrapulmonary tuberculosis for India.

Extrapulmonary tuberculosis (EPTB) is frequently a diagnostic and therapeutic challenge. It is a common opportunistic infection in people living with HIV/AIDS and other immunocompromised states such as diabetes mellitus and malnutrition. There is a paucity of data from clinical trials in EPTB and most of the information regarding diagnosis and management is extrapolated from pulmonary TB. Further, there are no formal national or international guidelines on EPTB. To address these concerns, Indian EPTB guidelines were developed under the auspices of Central TB Division and Directorate of Health Services, Ministry of Health and Family Welfare, Government of India. The objective was to provide guidance on uniform, evidence-informed practices for suspecting, diagnosing and managing EPTB at all levels of healthcare delivery. The guidelines describe agreed principles relevant to 10 key areas of EPTB which are complementary to the existing country standards of TB care and technical operational guidelines for pulmonary TB. These guidelines provide recommendations on three priority areas for EPTB: (i) use of Xpert MTB/RIF in diagnosis, (ii) use of adjunct corticosteroids in treatment, and (iii) duration of treatment. The guidelines were developed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria, which were evidence based, and due consideration was given to various healthcare settings across India. Further, for those forms of EPTB in which evidence regarding best practice was lacking, clinical practice points were developed by consensus on accumulated knowledge and experience of specialists who participated in the working groups. This would also reflect the needs of healthcare providers and develop a platform for future research.

Sequence Analysis of Fluoroquinolone Resistance-Associated Genes gyrA and gyrB in Clinical Mycobacterium tuberculosis Isolates from Patients Suspected of Having Multidrug-Resistant Tuberculosis in New Delhi, India

ABSTRACT Fluoroquinolones (FQs) are broad-spectrum antibiotics recommended for the treatment of multidrug-resistant tuberculosis (MDR-TB) patients. FQ resistance, caused by mutations in the gyrA and gyrB genes of Mycobacterium tuberculosis, is increasingly reported worldwide; however, information on mutations occurring in strains from the Indian subcontinent is scarce. Hence, in this study, we aimed to characterize mutations in the gyrA and gyrB genes of acid-fast bacillus (AFB) smear-positive sediments or of M. tuberculosis isolates from AFB smear-negative samples from patients in India suspected of having MDR-TB. A total of 152 samples from patients suspected of having MDR-TB were included in the study. One hundred forty-six strains detected in these samples were characterized by sequencing of the gyrA and gyrB genes. The extracted DNA was subjected to successive amplifications using a nested PCR protocol, followed by sequencing. A total of 27 mutations were observed in the gyrA genes of 25 strains, while no mutations were observed in the gyrB genes. The most common mutations occurred at amino acid position 94 (13/27 [48.1%]); of these, the D94G mutation was the most prevalent. The gyrA mutations were significantly associated with patients with rifampin (RIF)-resistant TB. Heterozygosity was seen in 4/27 (14.8%) mutations, suggesting the occurrence of mixed populations with different antimicrobial susceptibilities. A high rate of FQ-resistant mutations (17.1%) was obtained among the isolates of TB patients suspected of having MDR-TB. These observations emphasize the need for accurate and rapid molecular tests for the detection of FQ-resistant mutations at the time of MDR-TB diagnosis.

Drug resistance detection and mutation patterns of multidrug resistant tuberculosis strains from children in Delhi

A total of 312 sputum samples from pediatric patients presumptive of multidrug resistant tuberculosis were tested for the detection of drug resistance using the GenoTypeMTBDRplus assay. A total of 193 (61.8%) patients were smear positive and 119 (38.1%) were smear negative by Ziehl–Neelsen staining. Line probe assay (LPA) was performed for 208 samples/cultures (193 smear positive samples and 15 cultures from smear negative samples). Valid results were obtained from 198 tests. Of these, 125/198 (63.1%) were sensitive to both rifampicin (RIF) and isoniazid (INH). 73/198 (36.9%) were resistant to at least INH/RIF, out of which 49 (24.7%) were resistant to both INH and RIF (multidrug resistant). Children with tuberculosis are often infected by someone close to them, so strengthening of contact tracing in the program may help in early diagnosis to identify additional cases within the household. There is a need to evaluate newer diagnostic assays which have a high sensitivity in the case of smear negative samples, additional samples other than sputum among young children not able to expectorate, and also to fill the gap between estimated and reported cases under the program.

Recurrence of tuberculosis among newly diagnosed sputum positive pulmonary tuberculosis patients treated under the Revised National Tuberculosis Control Programme, India: A multi-centric prospective study

Introduction There is lack of information on the proportion of new smear—positive pulmonary tuberculosis (PTB) patients treated with a 6-month thrice-weekly regimen under Revised National Tuberculosis Control Programme (RNTCP) who develop recurrent TB after successful treatment outcome. Objective To estimate TB recurrence among newly diagnosed PTB patients who have successfully completed treatment and to document endogenous reactivation or re-infection. Risk factors for unfavourable outcomes to treatment and TB recurrence were determined. Methodology Adult (aged ≥ 18 yrs) new smear positive PTB patients initiated on treatment under RNTCP were enrolled from sites in Tamil Nadu, Karnataka, Delhi, Maharashtra, Madhya Pradesh and Kerala. Those declared “treatment success” at the end of treatment were followed up with 2 sputum examinations each at 3, 6 and 12 months after treatment completion. MIRU-VNTR genotyping was done to identify endogenous re-activation or exogenous re-infection at TB recurrence. TB recurrence was expressed as rate per 100 person-years (with 95% confidence interval [95%CI]). Regression models were used to identify the risk factors for unfavourable response to treatment and TB recurrence. Results Of the1577 new smear positive PTB patients enrolled, 1565 were analysed. The overall cure rate was 77% (1207/1565) and treatment success was 77% (1210 /1565). The cure rate varied from 65% to 86%. There were 158 of 1210 patients who had TB recurrence after treatment success. The pooled TB recurrence estimate was 10.9% [95%CI: 0.2–21.6] and TB recurrence rate per 100 person–years was 12.7 [95% CI: 0.4–25]. TB recurrence per 100 person–years varied from 5.4 to 30.5. Endogenous reactivation was observed in 56 (93%) of 60 patients for whom genotyping was done. Male gender was associated with TB recurrence. Conclusion A substantial proportion of new smear positive PTB patients successfully treated with 6 –month thrice-weekly regimen have TB recurrence under program settings.

Diabetes Prevalence and its Impact on Health-Related Quality of Life in Tuberculosis Patients

To determine the prevalence of diabetes mellitus (DM), assess its influence on health‐related quality of life (HRQoL) among patients with TB.

Multidrug-resistant tuberculosis among different types of suspected cases: Study from New Delhi

There are limited data of multidrug-resistant tuberculosis (MDR-TB) diagnosed in various patient categories by implementing Programmatic Management of Drug Resistant TB (PMDT) using line probe assay (LPA) from our country. Samples from presumptive MDR-TB from five districts of New Delhi were subjected to LPA from 1st October 2011 to 31st December 2014. The MDR-TB diagnosed in 4th & 5th month follow-up positives were significantly higher than other categories of the patients. Only 3/232 (2.2%) RIF resistants were diagnosed among smear negative re-treatment cases. The data suggest interim cost-benefit analysis of the program especially among smear negatives retreatment cases.

Amplification of Hsp 65 gene and usage of restriction endonuclease for identification of non tuberculous rapid grower mycobacterium

BACKGROUND The rapid grower mycobacteria have emerged as significant group of human pathogen amongst the Runyon group IV organisms that are capable of causing infection in both the healthy and immunocompromised hosts. Study aimed to identification of species amongst rapid grower non tuberculous mycobacterial isolates by polymerase chain reaction - restriction enzyme analysis (PRA). Analysis and comparison of results with standard biochemical tests. METHODS Rapid grower non tuberculous mycobacteria had been collected from liquid culture section during the study period. All isolates were identified by conventional biochemical tests. A 441bp fragment of hsp65 genes was amplified and digested by two restriction enzymes, BstEII and HaeIII. Digested products were analyzed using polyacrilamid gel electrophoresis (PAGE). RESULTS During study, 121 rapid grower mycobacterial isolates were subjected for species identification. Isolates were obtained from pulmonary samples (72) and extrapulmonary samples (49). In the PRA test 8 different types of rapid grower mycobacteria were identified after analyzing the fragments generated through restriction enzymes. Mycobacterium chelonae (57/121) was the most common isolate in pulmonary and extrapulmonary samples. Mycobacterium fortuitum (42), Mycobacterium abscessus (11), Mycobacterium immunogen (06), Mycobacterium peregrinum (02), Mycobacterium smegmatis (01), Mycobacterium wolinskyi (01), Mycobacterium goodii (01) were identified as other species of rapid grower non tuberculous mycobacteria. CONCLUSION PRA is a rapid and accurate system for the identification of species of non tuberculous mycobacteria. Results of PRA and biochemical tests are concordant up to 98%.