Planning action for strengthening the ecosystem for Ayurvedic research.

The establishment of AYUSH independent ministry has a commitment towards strengthening the AYUSH ecosystem. Health problems involving chronic diseases, non communicable diseases etc, there is a scope for complementary as well as preferential choice of any system including Ayurveda. There are a large number of clinical setups in national/state Ayurveda institutions, but the quality publication outcome is low. In the present article, being a scientist and science manager, I have just summarized my opinion to strengthen and growth of Ayurveda. Development of special validation methods for Ayurveda research based on Ayurveda principle is an hour of need. Randomized controlled trials on the use of new innovations are needed in Ayurveda. To strengthen Ayurveda, interdisciplinary approach may prove beneficial. Value addition based synergy and partnerships with ICMR/DBT/DST/ICAR would be beneficial for the growth of Ayurveda.

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method.

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein-Jenson (L-J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L-J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L-J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Expression profile of CXCL12 chemokine during M. tuberculosis infection with different therapeutic interventions in guinea pig.

Mycobacterium indicus pranii (MIP) already established as an immune-modulator in mycobacterial infections generates immune response by acting on CXC chemokines. In the present study, the immunomodulatory effect of MIP in conjunction with chemotherapy against M.tb infection was evaluated by colony forming units (CFUs) following aerosol infection to guinea pig and by measuring CXCL12 chemokine expression using q-PCR and in situ RT-PCR. Different experimental groups included, infection (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis+chemotherapy (RvChMw) group and normal healthy (NH) group. In the combination of immunoprophylaxis+chemotherapy (RvChMw) group, the CFU counts reduced significantly (p<0.001) at 4th week of infection as compared to other treated groups (RvMw and RvCh group). The expression of CXCL12 was recorded in all the treated groups of animals. The study demonstrated suppressed expression of CXCL 12 in both immunoprophylaxis as well as chemotherapy groups (6th and 8th week) that become elevated in immunoprophylaxis plus chemotherapy group (10th week), at which time point no CFUs were detected in RvCh and RvChMw group. The findings indicate that the expression of CXCL12 is associated with good response to anti - tubercular treatment. Thus, prior immunization with MIP appears to show good immunomodulatory effect to release CXCL12 chemokine during infection and also correlates with enhanced effect to chemotherapy.

Predominance of Central Asian and European families among Mycobacterium tuberculosis isolates in Kashmir Valley, India.

BACKGROUND: As there are no data available regarding the strains of Mycobacterium tuberculosis circulating in Kashmir Valley, India, the current study aimed at describing the genetic diversity of M. tuberculosis strains in this region, by spoligotyping and 12-locus-based MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat). METHODS: Sputa from 207 smear positive cases with newly diagnosed pulmonary tuberculosis were subjected to culture for M. tuberculosis. Eighty-five isolates confirmed as M. tuberculosis were subjected to drug susceptibility testing and molecular typing by spoligotyping and MIRU-VNTRs. RESULTS: Drug susceptibility results of 72 isolates revealed 76.3% as fully sensitive while 5.5% as multidrug resistant (MDR). Spoligotyping of 85 isolates detected 42 spoligotypes with 50 isolates (58.8%) clustered into seven spoligotypes. SIT26/CAS1_Del was the major spoligotype (23, 27%) followed by SIT127/H4 (12, 14.1%); CAS lineage (37.6%) was predominant, followed by Haarlem (25.8%) and ill-defined T clade (23.5%). MIRU-VNTR analysis displayed 82 MIRU patterns from 85 strains, including 3 small clusters and 79 unique. MIRU 26 was found to be the most discriminatory locus. CONCLUSIONS: Kashmir Valley has CAS as the predominant lineage of M. tuberculosis similar to the rest of the Indian sub-continent, while it is peculiar in having Euro American lineages such as Haarlem and ill-defined T clade.

Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases.

BACKGROUND & OBJECTIVES: Mycobacterium tuberculosis (M. tuberculosis) has four homologous mammalian cell entry (mce) operons (mce1-4) that encode exported proteins and have a possible role in the virulence mechanism of this pathogen. The expression of mce operon is considered to be complex and not completely understood. Although expression of mce operon at different in vitro growth phases has been studied earlier, its expression in different M. tuberculosis isolates under different growth phases is not yet studied. The present preliminary study was conducted on a limited number of isolates to know the trend of expression pattern of mce operon genes in different M. tuberculosis isolates under different growth stages. METHODS: In this study, we monitored the transcriptional profile of selected mce operon genes (mce1A, mce1D, mce2A, mce2D, mce3A, mce3C) in different M.tuberculosis isolates (MDR1, MDR2, and sensitive isolate) at early exponential and stationary phases using real-time quantitative PCR. RESULTS: The expression ratio of all selected mce operon genes in all M. tuberculosis isolates was reduced at the initial phase and increased substantially at a later phase of growth. Higher expression of mce1 operon genes was found in all M. tuberculosis isolates as compared to other mce operon genes (mce2 and mce3 operons) at stationary growth phase. INTERPRETATION & CONCLUSIONS: the higher expression of mce operon genes at stationary phase (as compared to early exponential phase) suggested growth phase dependent expression of mce operon genes. This indicated that the mce operon genes might have a role in M. tuberculosis survival and adaptation on the onset of adverse condition like stationary phase. Identification of differentially expressed genes will add to our understanding of the bacilli involved in adaptation to different growth conditions.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India.

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Aflatoxin contamination in stored rice variety PAU 201 collected from Punjab, India.

BACKGROUND & OBJECTIVES: The present study was carried out on stored rice variety PAU 201 in Punjab that was not permitted for milling and public distribution due to the presence of damaged grains at levels exceeding the regulatory limits of 4.75 per cent. The aim of the study was to determine fungal and aflatoxin contamination in the rice samples to assess hazard from the presence of damaged grains. Presence of iron in discoloured rice grains was also assessed. METHODS: Stored samples of paddy of PAU 201 rice variety were collected from six districts of Punjab, milled and analysed for presence of fungal and aflatoxin contamination. Scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis and Prussian blue staining was used to determine fungal spores and presence of iron, respectively. RESULTS: Aflatoxin analysis of rice samples indicated that none exceeded the Food Safety and Standards (Contaminants, Toxins and Residues) Regulations, 2011 tolerance limit of 30 µg/kg and majority of the samples had levels <15 µg/kg. The proportion of damaged grains exceeding the limit of 5 per cent was observed in 85.7 per cent of the samples. SEM and Prussian blue staining and EDX analysis of black tipped and pin point damaged rice grains did not show presence of fungal structures and presence of iron. INTERPRETATION & CONCLUSIONS: The results of the study indicated that the stored rice samples did not pose any health concern with respect to aflatoxin contamination as per the criteria laid down by the Food Safety and Standards Authority of India.

Protective efficacy of Mycobacterium indicus pranii against tuberculosis and underlying local lung immune responses in guinea pig model.

Tuberculosis kills two million people each year. As the current vaccine BCG fails to prevent adult cases of TB, an improved vaccine and/or vaccination strategy is urgently needed to combat TB. Previously we reported the higher protective efficacy of Mycobacterium indicus pranii (MIP), formerly known as Mycobacterium w (M.w) as compared to BCG in murine model of TB. In this study we further evaluated the protective efficacy of MIP in guinea pig model of TB. Modulation of post infection immune response was analyzed in the lungs of MIP immunized and control groups. We found reduced bacterial loads, improved pathology and organized granulomatous response at different post infection time points in the MIP-immunized group as compared to the BCG-immunized group. Combined results suggest that MIP-immunization results in heightened protective Th1 response as compared to BCG group, early after infection with M.tb and a balanced Th1 versus immunosuppressive response at late chronic stage of infection. The study demonstrates the higher antigen presenting cells function both inside the granuloma as well as in the single cell suspension of the lung in the MIP-immunized group. We further demonstrate that live MIP is safe to use in vivo as we observed quick clearance of MIP from the body and no untoward reaction was found. Aerosol route of immunization provided higher protection. Further this study provides evidence that MIP-immunization gives significantly better long term protection as compared to BCG against TB.

Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens.

BACKGROUND: The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples. METHODS: Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques. RESULTS: It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%. CONCLUSIONS: This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.

Absence of nucleotide-binding oligomerization domain-containing protein 2 variants in patients with leprosy and tuberculosis.

Crohn's disease-associated NOD 2 variants (Arg702Trp and 3020insC) were found to be monomorphic (wild), and 7 subjects were heterozygous for Gly908Arg SNP in 263 patients with tuberculosis, 260 patients with leprosy and 270 healthy controls residing in northern Indian states. This is the first report to suggest the minimal role of these variants in susceptibility/resistance to TB and leprosy in this population.

Potential of Mycobacterium vanbaalenii as a model organism to study drug transporters of Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium ulcerans: homology analysis of M. tuberculosis drug transporters among mycobacterial species.

Drug efflux pumps have been one of the important mechanisms of drug resistance in Mycobacterium tuberculosis. There is a prerequisite to study the behavior and mechanisms of these drug efflux pumps in detail for being considered in future anti-TB drug designing. The need of a rapid grower non-pathogenic mycobacterium with significant genomic homology for such type of studies is often being felt. During microarray and Real-Time PCR analysis of drug efflux pump genes of M. tuberculosis, we found 10 genes to be over-expressed during stress induced by common anti-TB drugs. In the present study homology analysis of these genes was done in order to know its phylogenetic relationship among other bacteria/mycobacteria. It was found that amino acid sequences of 7 out of 10 genes were significantly (>40%) identical to a non-pathogenic rapid grower environmental mycobacterium, Mycobacterium vanbaalenii. The protein sequences of M. vanbaalenii share important sequence motifs with M. tuberculosis useful for drug efflux mechanism based study across species. Like Mycobacterium smegmatis, it can be used as a model organism to study drug efflux pumps of M. tuberculosis and also other pathogenic mycobacteria such as Mycobacterium ulcerans and Mycobacterium marinum.

Diagnostic value of interferon- gamma release assays (QuantiFERON-TB Gold® In Tube) in childhood tuberculosis.

OBJECTIVE: To evaluate the efficacy and diagnostic potential of interferon- gamma release assays QuantiFERON-TB Gold® In Tube (QFT-TB-IT) for the detection of gamma interferon against the antigen ESAT-6, CFP-10, TB 7.7(p4) of Mycobacterium tuberculosis and compare the reactivity with other methods (smear, culture and IS6110 targeting PCR). METHODS: The study was carried out on children with pulmonary and extra pulmonary tuberculosis along with matching and PPD+ controls (82 cases, 48 matching controls and 20 PPD+ controls). RESULTS: QFT-TB-IT showed a significantly higher sensitivity (51.2%) as compared with LJ medium culture method (11.0%; p < 0.001), BacT/Alert 3D system (12.1%; p < 0.001), ZN staining (19.5%; p < 0.001) and PCR (45.1%; p < 0.05). Specificity of QFT-TB-IT was 48.0%. CONCLUSIONS: Less specificity of this assay may be due to high endemicity of tuberculosis. However, further studies are recommended.

Genetic polymorphisms among Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in northern India.

Restriction fragment length polymorphism (RFLP) based on IS6110 is considered the gold standard for Mycobacterium tuberculosis molecular typing. It is useful to discriminate among M. tuberculosis strains, investigate outbreaks and distinguish between reactivation and re-infection. We studied polymorphisms among M. tuberculosis isolates from northern India using RFLP to determine the presence of a correlation between IS6110 based fingerprints and drug resistance and to look for relapse and transmission among patients and their contacts. RFLP patterns of PvuII digested genomic DNA of 100 M. tuberculosis isolates were analyzed using southern blotting with a 245 bp IS6110 probe. Drug sensitivity testing (DST) was conducted for rifampicin (40 microg/ml), isoniazid (1 microg/ml), ethambutol (2 microg/ml) and streptomycin (4 microg/ml) using the proportion method. A high degree of polymorphism was seen among the M. tuberculosis isolates and the number of IS6110 copies varied from 0 to 14, with a predominance of isolates with 11 bands. Seventy-five isolates had a high number of bands, 9 had an intermediate number, 6 isolates had a low number and 10 isolates had no bands. No correlation between IS6110 band numbers and RFLP banding patterns was found with drug resistance or for any particular geographical area, although clustering was seen amongst MDR-TB cases. No cases of relapses or transmissions were seen.