Research priorities in Maternal, Newborn, & Child Health & Nutrition for India: An Indian Council of Medical Research-INCLEN Initiative.

In India, research prioritization in Maternal, Newborn, and Child Health and Nutrition (MNCHN) themes has traditionally involved only a handful of experts mostly from major cities. The Indian Council of Medical Research (ICMR)-INCLEN collaboration undertook a nationwide exercise engaging faculty from 256 institutions to identify top research priorities in the MNCHN themes for 2016-2025. The Child Health and Nutrition Research Initiative method of priority setting was adapted. The context of the exercise was defined by a National Steering Group (NSG) and guided by four Thematic Research Subcommittees. Research ideas were pooled from 498 experts located in different parts of India, iteratively consolidated into research options, scored by 893 experts against five pre-defined criteria (answerability, relevance, equity, investment and innovation) and weighed by a larger reference group. Ranked lists of priorities were generated for each of the four themes at national and three subnational (regional) levels [Empowered Action Group & North-Eastern States, Southern and Western States, & Northern States (including West Bengal)]. Research priorities differed between regions and from overall national priorities. Delivery domain of research which included implementation research constituted about 70 per cent of the top ten research options under all four themes. The results were endorsed in the NSG meeting. There was unanimity that the research priorities should be considered by different governmental and non-governmental agencies for investment with prioritization on implementation research and issues cutting across themes.

Cross-sectional Serosurvey of Crimean-Congo Hemorrhagic Fever Virus IgG in Livestock, India, 2013-2014.

We conducted a cross-sectional serosurvey of Crimean-Congo hemorrhagic fever (CCHF) among livestock in 22 states and 1 union territory of India. A total of 5,636 samples from bovines, sheep, and goats were screened for CCHF virus IgG. IgG was detected in 354 samples, indicating that this virus is widespread in this country.

Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model.

BACKGROUND & OBJECTIVES: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. METHODS: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. RESULTS: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. INTERPRETATION & CONCLUSIONS: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection.

Evaluation of gidB alterations responsible for streptomycin resistance in Mycobacterium tuberculosis.

OBJECTIVES: To evaluate gidB alterations for possible impact on the cumulative mechanism underlying the acquisition of high-level streptomycin resistance in Mycobacterium tuberculosis. METHODS: Fifty-two isolates with high streptomycin resistance and 23 isolates with low streptomycin resistance were sequenced for mutational analysis in the rpsL, rrs and gidB region. As the gidB protein has a complex substrate and no activity assay has yet been formulated, mutants of interest were subjected to in silico modelling and were structurally mapped together with active-site amino acid residues for assessment of the relevance to activity of the mutations found. RESULTS: Eight novel sense mutations and four novel mis-sense mutations in gidB were identified. Findings showed that active-site morphology is not only greatly affected by mutants lying in close proximity to the active-site pocket, but also by other mutations altering secondary-structure motifs and having an overall effect on protein structure. CONCLUSIONS: We conclude that gidB mutations address many unanswered questions and explain the whole story behind phenotypic streptomycin-resistant strains exhibiting no mutation in rpsL or rrs. They also validate the hypothesis of sequential progression of resistance from low to high due to the existence of gidB alterations in the genetic background.

Perceptions of State Government stakeholders & researchers regarding public health research priorities in India: an exploratory survey.

Public health research has several stakeholders that should be involved in identifying public health research agenda. A survey was conducted prior to a national consultation organized by the Department of Health Research with the objective to identify the key public health research priorities as perceived by the State health officials and public health researchers. A cross-sectional survey was done for the State health officials involved in public health programmes and public health researchers in various States of India. A self-administered semi-structured questionnaire was used for data collection. Overall, 35 State officials from 15 States and 17 public health researchers participated in the study. Five leading public health research priorities identified in the open ended query were maternal and child health (24%), non-communicable diseases (22%), vector borne diseases (6%), tuberculosis (6%) and HIV/AIDS/STI (5%). Maternal and child health research was the leading priority; however, researchers also gave emphasis on the need for research in the emerging public health challenges such as non-communicable diseases. Structured initiatives are needed to promote interactions between policymakers and researchers at all stages of research starting from defining problems to the use of research to achieve the health goals as envisaged in the 12th Plan over next five years.

Exploring the Role of C-C Motif Chemokine Ligand-2 Single Nucleotide Polymorphism in Pulmonary Tuberculosis: A Genetic Association Study from North India.

The C-C motif chemokine ligand-2 (CCL2) was evidenced to be associated with tuberculosis susceptibility in some ethnic groups. In the present study, effort was made to find out the association of CCL2-2518 A>G and -362 G>C variants with susceptibility to TB in a population from North India. The genotyping was carried out in 373 participants with pulmonary TB (PTB) and 248 healthy controls (HCs) for CCL2-2518 A>G and -362 G>C polymorphisms by PCR-RFLP and by melting curve analysis using fluorescence-labeled hybridization fluorescent resonance energy transfer (FRET) probes, respectively, followed by DNA sequencing in a few representative samples. Genotype and allele frequencies were compared by the chi-squared test and crude and Mantel-Haenszel (M-H) odds ratio (OR). OR was calculated using STATA/MP16.1 software. Further, CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß levels were measured in serum samples of these participants using commercially available kits. Our analysis indicated that the homozygous mutant in both -2518 GG (OR = 2.07, p = 0.02) and -362 CC (OR = 1.92, p = 0.03) genotypes was associated with susceptibility to pulmonary TB. Further, heterozygous genotypes -2518AG (OR = 0.60, p = 0.003) and -362GC (OR = 0.64, p = 0.013) provide resistance from PTB disease. Haplotype analysis revealed AC haplotype (p = 0.006) to be a risk factor associated with PTB susceptibility. The serum CCL2 level was significantly elevated among participants with -2518 AA genotype compared to -2518 GG genotype. CCL2 level was observed to be positively correlated with IL12p70, IFN-γ and TNF-α, thus suggesting the immunological regulatory role of CCL2 against pulmonary tuberculosis. CCL2-2518 GG and -362 CC genotypes were found to be associated with susceptibility to pulmonary tuberculosis and CCL2-2518AG and CCL2-362GC with resistance from PTB. AC haplotype was found to be a risk factor for PTB in the present study. It may be hypothesized from the findings that -2518G allele could be responsible for lower production of CCL2 which leads to defective Th1 response and makes a host susceptible for pulmonary tuberculosis.

Setting priorities in child health research in India for 2016-2025: a CHNRI exercise undertaken by the Indian Council for Medical Research and INCLEN Trust.

BACKGROUND: Millennium Development Goal 4 (MDGs) mobilised countries to reduce child mortality by two thirds the 1990 rate in 2015. While India did not reach MDG 4, it considerably reduced child mortality in the MDG-era. Efficient and targeted interventions and adequate monitoring are necessary to further progress in improvements to child health. Looking forward to the Sustainable Development Goal (SDG)-era, the Indian Council of Medical Research and The INCLEN Trust International conducted a national research priority setting exercise for maternal, child, newborn health, and maternal and child nutrition. Here, results are reported for child health. METHODS: The Child Health and Nutrition Research Initiative (CHNRI) method for research priority setting was employed. Research ideas were crowd-sourced from a network of child health experts from across India; these were refined and consolidated into research options (ROs) which were scored against five weighted criteria to arrive weighted Research Priority Scores (wRPS). National and regional priority lists were prepared. RESULTS: 90 experts contributed 596 ideas that were consolidated into 101 research options (ROs). These were scored by 233 experts nationwide. National wRPS for ROs ranged between 0.92 and 0.51. The majority of the top research priorities related to development of cost-effective interventions and their implementation, and impact evaluations, improving data quality; and monitoring of existing programs, or improving the management of morbidities. The research priorities varied between regions, the Economic Action Group and North-Eastern states prioritised questions relating to delivering interventions at community- or household-level, whereas the North-Eastern states and Union Territories prioritised research questions involving managing and measuring malaria, and the Southern and Western states prioritised research questions involving pharmacovigilance of vaccines, impact of newly introduced vaccines, and delivery of vaccines to hard-to-reach populations. CONCLUSIONS: Research priorities varied geographically, according the stage of development of the area and mostly pertained to implementation sciences, which was expected given diversity in epidemiological profiles. Priority setting should help guide investment decisions by national and international agencies, therefore encouraging researchers to focus on priority areas. The ICMR has launched a grants programme for implementation research on maternal and child health to pursue research priorities identified by this exercise.

Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

Molecular modelling and docking analysis of katG and rpoB gene in MDR-TB isolates from North Central Indian population.

Tuberculosis caused by Mycobacterium tuberculosis, requires multi drug therapy approach. Drug resistance in M. tuberculosis is caused by mutations in specific regions in drug target genes. The study aimed to identify mutations in katG and rpoB genes and investigate the drug-drug target interactions. A total of 27 MDR-TB isolates were sequenced for katG and rpoB genes and docking and MIC analysis were performed. Three types of mutations for katG gene (Arg463Leu in all isolates of Sahariya and non-tribes; Asp529Thr and Asp529His, each in two isolates only, in Sahariya) were observed. In rpoB gene, the Ser531Leu change was observed in 17/21 isolates in Sahariya and 3/6 isolates in non-tribes. The docking analysis revealed that the drugs isoniazid and rifampicin bind to different residues in mutant forms than their proposed active sites, making active binding sites rigid and causing resistance. The MIC for isoniazid was found to range from 0.2 to 5µg/ml in Sahariya tribe, whereas, in non-tribes, it is 0.2µg/ml and 1µg/ml. The MIC for rifampicin was observed at 64µg/ml in both the population groups. The study explored the possible functional variation in isoniazid and rifampicin resistance with respect to the identified mutations. The present results indicate that these mutations affect the drug binding affinity and are causing resistance.

Prevalence of Mycobacterium tuberculosis Beijing genotype and its association with drug resistance in North India.

The global presence and rapid dissemination of Beijing genotype of Mycobacterium tuberculosis, makes it an important issue of public health. Its presence and association with multi-drug resistance has been shown in many settings. In present study we tried to find its prevalence and association with drug resistance in North India. One hundred and twenty four M. tuberculosis isolates were analyzed with spoligotyping, further drug susceptibility testing was done by 1% proportional method. Out of these, 11 (8.9%) M. tuberculosis isolates were identified as Beijing and 113 (91.1%) as non-Beijing genotypes. While looking at their drug susceptibility patterns, 6 (54.5%) & 22 (19.5%) were found to be multi drug resistant (MDR) among Beijing and non-Beijing isolates respectively. Our study concluded that the Beijing strains were not so common in north India and these strains do not fully associate with MDR.

Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis.

Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.

Status of multidrug resistant tuberculosis (MDR-TB) among the Sahariya tribe of North Central India.

BACKGROUND: The incidence/prevalence of tuberculosis (TB) is reported to be high in the Sahariya tribe of North Central India. The outbreaks of different drug-resistant isolates of Mycobacterium tuberculosis emphasized the need for continuous monitoring of resistance to anti-tuberculosis drugs. This study aimed to assess the profile of multidrug resistant TB among the Sahariya tribe and their non-tribal neighbors for first line drugs through field-based investigations. METHODOLOGY: A total of 274 sputum positive pulmonary TB individuals were enrolled and studied for their drug susceptibility profile by the proportion method. RESULTS: A total of 21 cases from Sahariya and 6 from non-tribes were identified with MDR-TB. Thus Sahariya tribe showed a 1.95-fold increased risk of developing drug resistance than non-tribes. Significant differences were observed for developing drug sensitivity between Sahariya males and females when analyzed for resistance developed to any drug and overall drug resistance vs. sensitive isolates, respectively. A 4.46-fold risk was found for MDR-TB among the smokers of Sahariya tribe, whereas, the non-tribes did not show any significant association. CONCLUSION: The drug susceptibility profile developed in the present study indicates that drug-resistant tuberculosis is emerging as a serious public health concern in Sahariya tribe. Urgent and effective control measures and better management policies are needed for the prevention of MDR-TB in the tribe.

Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: preliminary findings of the study of 161 new cases from a new endemic focus in himachal pradesh, India.

Localized cutaneous leishmaniasis (LCL) in India is due mostly to Leishmania tropica. It is mainly endemic in the deserts of Rajasthan. Recently, Himachal Pradesh has been identified as a new endemic focus for the disease. In the last few years, the number of new cases has been increasing almost to epidemic proportions. This report presents the preliminary findings of clinico-epidemiologic and investigative results of 161 new localized cases of LCL seen between May 2001 and December 2003. The study populaton was composed of 80 males and 81 females between 10 months and 75 years of age. All were indigenous to the sub-alpine valley along the Satluj River in the mountainous region of the Kinnaur District (altitude = 700-2,900 meters). Most patients were seen from April to September and had 1-8 lesions (duration = 1-6 months) that involved mainly the face. Tissue smears were positive for amastigotes in 37% and histopathology showed non-caseating epitheloid cell granuloma in 77% of the cases. Analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ribosomal gene region of 10 biopsy specimens showed amplicons indistinguishable from L. donovani in eight cases and L. tropica in two cases. Leishmania was cultured on modified Nicole-Novy-McNeal (NNN) medium containing RPMI 1640 medium and heat-inactivated fetal bovine serum from 13 of 38 biopsy samples. Three of these isolated strains were identified as L. donovani while a fourth was L. tropica by PCR-RFLP of the ribosomal internal transcribed spacer region. One strain had a gp63 sequence identical to that of east African strains. Another strain had a unique gp63 sequence that has not been found in L. donovani complex strains. Sand flies trapped in the cattle sheds of a few patients were identified as Phlebotomus longiductus (Parrot 1928). Treatment with intralesional sodium stibogluconate was effective in all patients without any major side effects. One patient developed lupoid leishmaniasis that responded to higher dose of sodium stibogluconate. Though rarely reported as a cause of LCL, L. donovani seems to be the predominant pathogen in this new focus of cutaneous leishmaniasis. Phlebotomus longiductus is a possible vector, albeit based on circumstantial evidence.

Advances in the diagnosis and treatment of leprosy.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Over recent years, many important advances have been made in developing molecular diagnostics, in identifying highly effective drugs and designing multidrug regimens for treatment, and in unravelling the genomic structure and functions of the leprosy bacillus. Using the new information about specific sequences of M. leprae, several gene probes and gene amplification systems for confirming diagnosis and monitoring treatment have been developed. Among these, polymerase chain reaction (PCR)-based methods have been useful in confirming the diagnosis in paucibacillary leprosy (where few bacilli are present). RNA-targeting systems for monitoring the progress of treatment, in situ hybridisation techniques for analysing specimens with nonspecific histological features, and molecular methods for direct detection of rifampicin/dapsone resistance are other major technological advances with immense applied value. Several effective regimens for the treatment of leprosy have been developed, which include rifampicin, clofazimine and dapsone as core drugs. Although these regimens are generally satisfactory, limitations in terms of persisting activity and late reactions/relapses in paucibacillary leprosy, and persistence of dead and/or live organisms in multibacillary forms of the disease, have been observed.

Toll-like Receptor 1 743 A>G, 1805 T>G & Toll-like Receptor 6 745 C>T gene polymorphism and tuberculosis: a case control study of north Indian population from Agra (India).

Infection with Mycobacteriumtuberculosis possibly depends on host genetic factors and is thought to be the major cause of differential susceptibility to the disease. In the present study, 205 pulmonary tuberculosis cases and 127 healthy controls were studied for the association of Toll-like Receptor (TLR) variants (TLR1 variants 743A>G and 1805T>G, and TLR6 variant 745 C>T) in north Indian population. The frequency of heterozygous genotypes (AG) in TB cases (0.47) and HCs (0.61), differed significantly (p value = 0.02). The association of AG genotypes in HCs was adjusted for gender as gender was observed to be a confounder and M-H OR was found to be 0.62 (p = 0.044). On categorizing the cases basing on AFB smear positivity, the heterozygous genotypes (AG) was found to be associated with low bacillary load (scanty and 1+) (P = 0.002). No association was observed for either TLR1 1805 T>G or TLR6 745 C>T polymorphism. Level of serum IL6 was found to be significantly higher among healthy controls with TLR1 GG genotype compared to healthy controls with AA (p = 0.035) and AG (p = 0.005) genotypes. Thus, it may be suggested that the heterozygous condition for TLR1 743 A>G provide resistance from the disease. However, in depth study is required to understand the mechanism for possible protective responses.

A toolbox for tuberculosis (TB) diagnosis: an Indian multi-centric study (2006-2008); evaluation of serological assays based on PGL-Tb1 and ESAT-6/CFP10 antigens for TB diagnosis.

BACKGROUND: The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status. METHODS: Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data. RESULTS: Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results. CONCLUSIONS: In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients.

A toolbox for tuberculosis (TB) diagnosis: an Indian multicentric study (2006-2008). Evaluation of QuantiFERON-TB gold in tube for TB diagnosis.

BACKGROUND: The aim of this multicentric prospective study in India was to assess the performance of the QuantiFERON TB-Gold in tube (QFT-GIT), Tuberculin Skin Test (TST) and microbiological results as additional tools for diagnosing active tuberculosis (TB) and latent infection (LTBI) according to Human Immunodeficiency Virus (HIV) status. METHODS: Individuals with and without active TB and HIV infection were enrolled between 2006-2008. QFT-GIT and TST results were analyzed per se and in combination with microbiological data. RESULTS: Among the 276 individuals (96 active pulmonary TB and 180 no active TB) tested by QFT-GIT, 18 indeterminate results (6.5%) were found, more significantly numerous in the HIV-infected (15/92; 16.3%) than the HIV-uninfected (3/184; 1.6%)(p<0.0001). QFT-GIT sensitivity for active TB was 82.3% and 92.9% respectively after including or excluding indeterminate results. Clinical sensitivity was significantly lower in the HIV-infected (68.4%) than the HIV-uninfected (91.4%) patients (p = 0.0059). LTBI was detected in 49.3% of subjects without active TB but varied according to TB exposure. When the TST and QFT-GIT were concomitantly performed, the respective sensitivity for active TB diagnosis was 95.0% and 85.0% in the HIV-uninfected (p = 0.60), and 66.7% and 51.5% in the HIV-infected patients (p = 0.32). QFT-GIT and TST respective specificity for active TB in the HIV-uninfected was 25.0% and 57.1% (p = 0.028), and 64.8% and 83.3% in the HIV-infected (p = 0.047). In those with active TB, QFT-GIT results were not associated with microbiological parameters (smear grade, liquid culture status, time-to-positivity of culture) or clinical suspicion of active TB score (provided by the clinicians at enrollment). Combining microbiological tests with both immunological tests significantly increased sensitivity for active TB diagnosis (p = 0.0002), especially in the HIV-infected individuals (p = 0.0016). CONCLUSION: QFT-GIT and TST have similar diagnostic value for active TB diagnosis. In HIV-infected patients, combining microbiological tests with both immunological tests significantly increases the sensitivity for active TB diagnosis.

A toolbox for tuberculosis diagnosis: an Indian multicentric study (2006-2008): microbiological results.

BACKGROUND: The aim of this multicentric prospective study in India was to assess the value of several microbiological tools that contribute to the diagnosis of tuberculosis (TB) according to HIV status. METHODS: Standard microbiological tools on individual specimens were analyzed. RESULTS: Among the 807 patients with active TB, 131 were HIV-infected, 316 HIV-uninfected and 360 had HIV-unknown status. Among the 980 non-active TB subjects, 559 were at low risk and 421 were at high risk of M. tuberculosis (Mtb) exposure. Sensitivity of smear microscopy (SM) was significantly lower in HIV-infected (42.2%) than HIV-uninfected (75.9%) (p = 0.0001) and HIV-unknown pulmonary TB patients (61.4%) (p = 0.004). Specificity was 94.5% in non-TB patients and 100% in health care workers (HCW) and healthy family contacts. Automated liquid culture has significantly higher diagnostic performances than solid culture, measured by sensitivity (74.7% vs. 55.9%) (p = 0.0001) and shorter median time to detection (TTD) (12.0 vs. 34.0 days) (p = 0.0001). Specificity was 100% in HCW and cured-TB patients, but was lower in non-TB patients (89%) due to isolation of Mycobacteria other than tuberculosis (MOTT). TTD by both methods was related to AFB score. Contamination rate was low (1.4%). AccuProbe hybridization technique detected Mtb in almost all culture-positive specimens, but MOTT were found in 4.7% with a significantly higher frequency in HIV-infected (15%) than HIV-uninfected TB patients (0.5%) (p = 0.0007). Pre-test classification significantly increased the diagnostic value of all microbiological tests in pulmonary TB patients (p<0.0001) but to a lesser degree in extrapulmonary TB patients. CONCLUSIONS: Conventional microbiological tools led to results similar to those already described in India special features for HIV-infected TB patients included lower detection by SM and culture. New microbiological assays, such as the automated liquid culture system, showed increased accuracy and speed of detection.

Assessment of the N-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem.

BACKGROUND: The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. METHODOLOGY/PRINCIPAL FINDINGS: Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M. tuberculosis and two as M. fortuitum and M. chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.

Mycobacterial antigen(s) induce anergy by altering TCR- and TCR/CD28-induced signalling events: insights into T-cell unresponsiveness in leprosy.

Present study investigates the role of Mycobacterium leprae (M. leprae) antigens on TCR- and TCR/CD28-induced signalling leading to T-cell activation and further correlates these early biochemical events with T-cell anergy, as prevailed in advanced stages of leprosy. We observed that both whole cell lystae (WCL) and soluble fraction of M. leprae sonicate (MLSA) not only inhibited TCR, thapsigargin and ionomycin induced calcium fluxes by diminishing the opening of calcium channels, but also TCR- or TCR/CD28-induced proximal signalling events like phosphorylation of Zap-70 and protein kinase-C (PKC) activity. Study of TCR- and TCR/CD28-induced downstream signals revealed that M. leprae antigens curtail phosphorylation of both Erk1/2 and p38MAPK, consequently altering terminal signalling events like reduced binding of NFAT on IL-2 promoter and transcription of IL-2 gene, diminished expression of activation markers (CD25 and CD69). Furthermore, M. leprae fractions significantly inhibited IL-2 secretion and T-cell blastogenesis in healthy individuals. Altogether, results suggest that M. leprae interferes with TCR/CD28-induced upstream as well as downstream signalling events resulting in reduced IL-2 production and thus inhibition in T-cell proliferation, which might be responsible for T-cell unresponsiveness leading to stage of immunosuppression and consequently, for the progression of disease.