The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a costeffective manner.

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IntroductionThere 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.ObjectiveTo 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.MethodologyAdult (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.ResultsOf 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.ConclusionA substantial proportion of new smear positive PTB patients successfully treated with 6 –month thrice-weekly regimen have TB recurrence under program settings.

Tuberculosis (TB) is a major cause of morbidity and mortality, especially in developing countries. Despite significant limitations, microscopy remains the cornerstone of the global TB control strategy. As the TB epidemic escalates, new diagnostic methods that are accurate and also economical and simple to manufacture and deploy are urgently needed. Although several promising antigens have been identified and evaluated in recent years, the reproducible production of high-quality recombinant mycobacterial proteins with minimal batch-to-batch variation is difficult, laborious, and expensive. To determine the feasibility of devising a synthetic peptide-based diagnostic test for TB, we have delineated the immunodominant epitopes of three candidate antigens, Ag85B, BfrB, and TrxC, that were previously identified to be immunogenic in TB patients. The results demonstrate that combinations of carefully selected synthetic peptides derived from highly immunogenic proteins can be the basis for devising an immunodiagnostic test for TB.According to World Health Organization estimates, ϳ9 ϫ 10 6 new cases of tuberculosis (TB) and ϳ2 ϫ 10 6 TB-related deaths occur every year. The major proportion (Ͼ90%) of these cases and deaths occur in low-income developing countries where the diagnosis of TB is based primarily on the examination of sputum smears for acid-fast bacilli. Although microscopy is highly specific, it has some serious limitations, such as the fact that is has a low and variable sensitivity, is time-consuming, and is tedious. The need for multiple visits to provide specimens and obtain results before treatment can be initiated also leads to high rates of dropout of infectious patients (30). A rapid and economical diagnostic test that can provide an accurate means for the identification of sputum smear-positive TB cases is a major diagnostic priority of TB control programs in countries where TB is endemic.Antibody detection-based diagnostic assays have been successfully devised for several infectious diseases (7,15,26,30), but efforts to develop a serodiagnostic test for TB have yielded disappointing results for several decades and no currently available commercial immunodiagnostic tests for TB provide high sensitivity and specificity (24). However, interest in developing these assays for the diagnosis of TB continues due to their adaptability to simple, rapid formats, such as the dipstick and lateral-flow cassette formats, which are extremely useful in the field, where a sophisticated laboratory infrastructure and highly trained personnel are not available (8,30).Our earlier studies of the humoral immune responses elicited at different stages of active TB in humans demonstrated that only a small subset of the Mycobacterium tuberculosis culture filtrate proteins is recognized by antibodies in both patients with early TB and patients with advanced TB and in the presence of human immunodeficiency virus (HIV) coinfection (14,(20)(21)(22)(23). Our earlier studies also identified M. tuberculosis malate synthase (Rv1837c) a...

Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.

GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INHinhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% ( value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR] 400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.KEYWORDS DNA sequencing, high-resolution melt curve analysis, tuberculosis, molecular diagnostics, multidrug resistance, sputum T uberculosis (TB) is one of the world's deadliest transmittable diseases (1). In 2015, worldwide, 10.4 million people were estimated to be infected with TB, with 1.4 million people dying from the disease. Drug-resistant TB is a menace for TB control worldwide, and 60% of reported TB patients estimated to have multidrug-resistant TB (MDR-TB) were not detected in 2015 (1). India holds the dubious distinction of harboring 27% of the global TB burden, with 2.5% of its new TB cases and 16% of previously treated cases having MDR-TB (1). Conventional Mycobacterium tuberculosis culturebased systems for first-line and second-line drug susceptibility testing (DST) are timeconsuming, while commercial liquid culture-based DST reduces turnaround times but

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 4.6days. 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.