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Background According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). Methods The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Results Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Conclusions Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

Background The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples. Results Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing. Conclusion Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.

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Background The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing. Results Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene. Conclusions The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.

Background Success of India’s TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results. Methods The capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene. Results Out of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene. Conclusion Re-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.

Introduction Childhood tuberculosis (TB) is now a global priority. With the advent of Xpert MTB/RIF, more TB cases in children are being reported. This study was undertaken to evaluate the performance of Xpert in diagnosis of pulmonary and extra-pulmonary TB in children. Methods Specimens from 171 suspected TB cases in children aged

This paper presents an assessment of the existing situation of municipal solid waste management (MSWM) in Bahraich City. The quantity and composition of MSW vary from place to place, and bear a rather consistant correlation with the average standard of living. Field investigations were carried out for quantification, analysis of physico-chemical composition and characterization in disposal site. Studies carried out in these places have revealed that there were many shortcomings in the existing practices used in managing the MSW. These shortcomings pertain mainly to indequate manpower, financial resources, implements and machinery required for effectively carrying out various activities for MSWM. Various adopted treatment technologies for MSW were critically reviewed, alongwith their advantage and limitations. The study was concluded with a few fruitful suggestions, which may be beneficial to encourage the competent authorities/researchers to work towards further improvement in the present system.

Background Diagnosis of tuberculosis (TB) in infants is challenging due to non-specific clinical presentations of the disease in this age-group and low sensitivity of widely available TB diagnostic tools, which in turn delays prompt access to TB treatment. Upfront access to Xpert/MTB RIF (Xpert) testing, a highly sensitive and specific rapid diagnostic tool, could potentially address some of these challenges. Under the current project, we assessed the utility and feasibility of applying upfront Xpert for diagnosis of tuberculosis in infants, including for testing of non-sputum specimens. Methods A high throughput lab was established in each of the four project cities, and linked to various health care providers across the city, through rapid specimen transportation and electronic reporting linkages. Free Xpert testing was offered to all infant (

Background Drug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes. Methods A total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method. Results Of 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours. Conclusion The problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

Background Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. Methods The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Results Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5 %) and 19 (29.2 %) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had “clinical TB” (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed “non-TB” cases. For culture, the sensitivity was low, 79.3 % for pulmonary and 54.3 % for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1 % for pulmonary and 91.4 % for extra-pulmonary TB cases with specificity of 100 % and 93.3 % respectively. Conclusion Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1450-1) contains supplementary material, which is available to authorized users.

Comparison of conventional procedures with single-step & nested multiplex PCR for detection of M. tuberculosis complex in different groups of patients. (DOCX 15 kb)

INTRODUCTION: Tuberculosis (TB) in human immunodeficiency virus (HIV)-infected people remains a major global public health challenge. Cartridge-based nucleic acid amplification test (CBNAAT) has been proved to have a key role in diagnosing TB in patients having HIV infection. METHOD: In this study, we intend to present a retrospective data of HIV-TB co-infected patients during our routine diagnostic algorithm under Revised National TB Control Program (RNTCP). RESULT: A total of 59/207 (28.8%) HIV patients were diagnosed to be positive for TB by CBNAAT in the period January to December 2015.

Lipoprotein lipase (LPL) is a key enzyme for the hydrolysis of triglycerides in the core of chylomicrons and very low-density lipoprotein (Goldberg and Merkel 2001). The various substitutions in the LPL gene have been reported to play roles in the development of dyslipidemia in the general population (Razzaghi et al. 2000; Jap et al. 2003; Yang et al. 2003). The implication of the A1127G gene polymorphism in the pathogenesis of coronary artery disease has been studied extensively in several ethnic groups (Reymer et al. 1995; Gerdes et al. 1997; Ferencak et al. 2003; Hokanson 1999; Wittrup et al. 1999; Arca et al. 2000; Fisher et al. 1995; Minnich et al. 1995; Wittekoek et al. 1998; Hu et al. 2006). In this study, we evaluated for the first time the status in an Indian population, where the prevalence of coronary artery disease has increased drastically in recent years (Mohan et al. 2001).

A surface glycoprotein molecule, E-selectin is involved in adhesion of circulating leukocyte to the activated endothelium and plays a fundamental role in pathogenesis of atherosclerosis. The present study was undertaken to document the status of S128R polymorphism of E-selectin gene in angiographically proven coronary artery disease (CAD) patients from Uttar Pradesh.Genotype of the S128R polymorphism was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 329 angiographically proven CAD patients [n=83 acute myocardial infarction (AMI) and n= 246 AMI-free] and 331 age and sex matched control individuals (angiographically proven not to have CAD).This pilot study revealed a significant association of R allele in coronary artery disease patients in univariate analysis [allele frequency 9.6% in patients vs. 5.6% in control (P = 0.031, OR = 1.57, 95% CI = 1.05 - 2.47)]. However, after binomial logistic regression the significant determinants of CAD were: presence of diabetes (OR: 2.26, P=0.001) hypertension (OR = 2.61, P=0.001), smoking habit (OR=2.038, P=0.001), elevated serum triglycerides (OR=1.967, P=0.001) and low HDL-C (high density lipoprotein cholesterol) (OR=1.107, P=0.001).The interaction of classical risk factors for CAD with S128R polymorphism in our study population showed that the significant determinants of coronary artery disease were presence of diabetes, hypertension, smoking habit, elevated serum triglycerides and low HDL. S128R polymorphism in E-selectin gene was not an independent predictor of CAD in our population.

Hereditary hemochromatosis is an autosomal recessive and most commonly inherited single gene disorder among Caucasians, with a prevalence of 5 per 1,000 and a carrier frequency of 1 in 10. Two point mutations were described and are referred as C282Y and H63D. In the present study, we have analyzed 729 north Indian samples for C282Y and H63D mutations. Of these, no allele of the C282Y mutation was seen, while 3 homozygous and 43 heterozygous for the H63D mutation were seen in the patients of thalassemia group. However, 47 cases were found heterozygous for the H63D mutation among the normal groups (11.16%).