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

Frequency of multi-drug resistance and mutations in Mycobacterium A tuberculosis isolates from Punjab state of India

Data regarding prevalence of multi-drug resistant tuberculosis [MDR-TB] and associated common mutations is scarce from Punjab region. The study was designed to determine rate of MDR-TB among presumptive MDR-TB from Punjab and mutation patterns using GenoType MTBDRplus assay. Total of 812 consecutive sputum samples were received from January 2012 to July 2013, from 14 districts of Punjab at the National Reference Laboratory at New Delhi for diagnosis of MDR-TB as hand holding activity. Presumptive MDR-TB patients were identified on basis of criterion B defined by the programme. Smear positive and negatives patients were found to be 636/798 [79.7%] and 162/ 798 [20.3%] respectively. Total of 606 GenoType MTBDRplus tests were conducted and mutations in rpoB, kat G and inhA genes analyzed. Total of 94/606 [15.5%], 43/606 [7.1%] and 40/606 [6.6%] were found to be RIF and INH resistant, mono-RIF resistant and 40/606 [6.6%] mono-INH resistant respectively

Commonest known mutation for RIF in rpoB gene and INH in kat G gene was S531L [80/ 137; 58.4%] and S315T1 [119/134; 88.8%] respectively. Mutations in inhA were found in 21/134 ]15.7%] strains. Average turn-around time [TAT] for dispatch of result toPunjab was 4.6 days. Prevalence of RIF resistance in Punjab was found to be 22.6%. Common mutations for RIF and INH were similar to that in other regions of country. GenoType MTBDRplus was found to be useful assay for rapid detection of MDR-TB, responsible for determining better management of MDR-TB patients under the programme

First and second line drug resistance among treatment naive 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 naive 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 naive 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 naive 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

Phenol-ammonium sulfate microscopy method for diagnosis of pulmonary tuberculosis

Collection and processing of sputum samples for the detection of acid fast bacilli [AFB] is hazardous for health-workers in developing countries with limited facilities. The phenol ammonium sulfate [PhAS] method involves smear microscopy and Ziehl-Neelson [ZN] staining of precipitates/ floccules formed in sputum samples when PhAS is added. The present study has been designed to assess the performance and safety of this method. The study was conducted from January 2011 to March 2011 at the Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi. A total of 1038 sputum samples were subjected to ZN staining before and after treatment with PhAS. The smear microscopy results of the PhAS treated and untreated samples were compared. In addition, 200 representative samples were inoculated after processing by petroff's method directly for culture and after treatment with PhAS. The sensitivity, specificity, positive predictive value and negative predictive value of the PhAS solution treated ZN smear microscopy method were found to be 98.8%, 88.5%, 98.0% and 92.7% respectively in comparison with direct smear microscopy. The overall correlation between the two methods was found to be 97.3%. None of the PhAS treated samples grew Mycobacterium tuberculosis on culture. Sputum microscopy with PhAS solution is a safe, reliable and inexpensive alternative for direct microscopy. This method can be conveniently applied for usage in microscopy centers with limited bio-safety facilities

Predominance of Beijing genotype in extensively drug resistant Mycobacterium tuberculosis isolates from a tertiary care hospital in New Delhi, India

Out of a total of 311 Mycobacterium tuberculosis isolates from sputum specimens subjected to first- and second-line drug-susceptibility testing [DST] at a hospital serving as a referral center for chronic tuberculosis [TB] cases in New Delhi, 232/311 [74.6%] isolates were found to be resistant to isoniazid and rifampicin. Among multidrug-resistant [MDR] isolates, 119/232 [51.3%] were resistant to four first-line drugs [streptomycin, isoniazid, rifampicin and ethambutol]. Mono-resistance to isoniazid was observed in 18 [5.7%] isolates, while none of the isolates tested showed mono-resistance to rifampicin. 50/232 [21.5%] isolates met the definition of extensively drug resistant [XDR] TB, i.e., additional resistance to a fluoroquinolone and at least one of the three injectable second-line drugs: kanamycin, capreomycin, or amikacin. Spoligotyping of the XDR-TB isolates revealed 14 patterns; 39/50 [78%] isolates being grouped in three clusters vs. 11/50 [22%] isolates being unique. SIT1/Beijing represented the largest cluster [n = 21, 42%], followed by SIT26/CAS1-Delhi [n = 10, 20%] and SIT 53/T1 [n = 8 isolates; 16%]. This study corroborates recent observations from North India suggesting that both Beijing and CAS1-Delhi lineages constitute the bulk of XDR-TB isolates that are disseminating rapidly across a large geographical region in and around the capital city of India