Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines.

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Management of early melanoma recurrence despite adjuvant anti-PD-1 antibody therapy☆.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies (PD1) prolong recurrence-free survival in high-risk resected melanoma; however, approximately 25%-30% of patients recur within 1 year. This study describes the pattern of recurrence, management and outcomes of patients who recur with adjuvant PD1 therapy. PATIENTS AND METHODS: Consecutive patients from 16 centres who recurred having received adjuvant PD1 therapy for resected stage III/IV melanoma were studied. Recurrence characteristics, management and outcomes were examined; patients with mucosal melanoma were analysed separately. RESULTS: Melanoma recurrence occurred in 147 (17%) of ∼850 patients treated with adjuvant PD1. In those with cutaneous melanoma (n = 136), median time to recurrence was 4.6 months (range 0.3-35.7); 104 (76%) recurred during (ON) adjuvant PD1 after a median 3.2 months and 32 (24%) following (OFF) treatment cessation after a median 12.5 months, including in 21 (15%) who ceased early for toxicity. Fifty-nine (43%) recurred with locoregional disease only and 77 (57%) with distant disease. Of those who recurred locally, 22/59 (37%) subsequently recurred distantly. Eighty-nine (65%) patients received systemic therapy after recurrence. Of those who recurred ON adjuvant PD1, none (0/6) responded to PD1 alone; 8/33 assessable patients (24%) responded to ipilimumab (alone or in combination with PD1) and 18/23 (78%) responded to BRAF/MEK inhibitors. Of those who recurred OFF adjuvant PD1, two out of five (40%) responded to PD1 monotherapy, two out of five (40%) responded to ipilimumab-based therapy and 9/10 (90%) responded to BRAF/MEK inhibitors. CONCLUSIONS: Most patients who recur early despite adjuvant PD1 develop distant metastases. In those who recur ON adjuvant PD1, there is minimal activity of further PD1 monotherapy, but ipilimumab (alone or in combination with PD1) and BRAF/MEK inhibitors have clinical utility. Retreatment with PD1 may have activity in select patients who recur OFF PD1.

Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

BACKGROUND & OBJECTIVES: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. METHODS: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. RESULTS: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. INTERPRETATION & CONCLUSIONS: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Effect of Cholesterol-loaded Cyclodextrin on Membrane and Acrosome Status of Hariana Bull Sperm during Cryopreservation.

BACKGROUND: The membrane and acrosomal integrity of sperm play a vital role in fertilization process; however they are compromised upon cryopreservation. OBJECTIVE: To study the effect of cholesterol-loaded cyclodextrin (CLC) on membrane and acrosome status of Hariana bull sperm during cryopreservation. MATERIALS AND METHODS: Semen samples collected from Hariana bulls with mass motility ≥ 3+ and individual progressive motility ≥ 70% were utilized in the study. Each ejaculate was split into two parts, one part being evaluated freshly for various seminal attributes and the other part being diluted in Tris diluent (without egg yolk and glycerol) to obtain a final concentration of 120×106 sperm/mL. The diluted semen was divided into four treatments: Group I, without CLC (control); Group II, with CLC at 0.5 mg per 120 million sperm; Group III, at 1.0 mg per 120 million sperm; Group IV, at 2.0 mg per 120 million sperm. All aliquots were incubated for 15 min at 37°C and each sample was diluted with Egg yolk-Tris-Glycerol (EYTG) extender up to 80×106 sperm/mL. The diluted semen samples were packed in French mini straws (0.25 mL), sealed and equilibrated at 4°C for 4 h followed by cryopreservation. The samples at pre-freeze and post-thaw stage were evaluated for membrane and acrosomal integrity, as well as primary, secondary and tertiary acrosomal damages. RESULTS: The membrane and acrosomal integrity was significantly higher in group II as compared to groups I, III, and IV, at pre-freeze and post-thaw stage (P<0.05). The primary and secondary acrosomal damage were significantly reduced in group II compared to other groups (P<0.05). No significant difference in tertiary acrosomal damage was found among different groups. CONCLUSION: CLC improves the membrane and acrosomal integrity, and reduces primary and secondary acrosomal damages during cryopreservation of Hariana bull sperm.

Genetic diversity & drug sensitivity profiles of Mycobacterium tuberculosis isolates from two slums of Jaipur city, Rajasthan, India.

BACKGROUND & OBJECTIVES: Slums are considered as hotspots of tuberculosis (TB). The study of genetic diversity and drug susceptibility profile of Mycobacterium tuberculosis (MTB) will help understand the transmission dynamics and can be used for better prevention and control of the disease. The aim of this study was to determine the drug susceptibility profiles and genetic diversity using the random amplified polymorphic DNA (RAPD) and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU VNTR) of MTB isolates from sputum samples of pulmonary TB patients residing in the two slums of Jaipur city in Rajasthan, India. METHODS: Sputum samples collected from pulmonary TB patients, their contacts and suspects during 2010-2012 were processed for microscopy and mycobacterial culture. Drug susceptibility testing was done by one per cent indirect proportion method on Lowenstein-Jensen medium for first-line anti-TB drugs rifampicin, isoniazid, ethambutol and streptomycin. MTB DNA was extracted by physicochemical method, and DNA fingerprinting was done by RAPD and MIRU VNTR analysis. RESULTS: Among 175 sputum samples collected, 75 were positive (43.8%) for acid-fast bacilli, 83 for MTB culture and four were contaminated. Fifty two isolates (62.7%) were fully sensitive to four drugs, and five (6%) were multidrug resistant (MDR). RAPD analysis of 81 isolates revealed six clusters containing 23 (28.4%) isolates, and 58 (71.6%) were unique. MIRU VNTR analysis clustered 20 (24.7%) isolates, and 61 (75.3%) were unique. INTERPRETATION & CONCLUSIONS: About 62.7 per cent isolates from the sputum samples from slum areas were sensitive to four drugs; six per cent of isolates were MDR. Poly-resistance other than MDR was high (16%). About one-fourth isolates were clustered by either method. RAPD was rapid, less expensive but had low reproducibility. MIRU VNTR analysis could identify to greater extent the epidemiological link in the population studied.

Genetic diversity of Mycobacterium tuberculosis isolates from central India.

BACKGROUND & OBJECTIVES: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. METHODS: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. RESULTS: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS[2]. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. INTERPRETATION & CONCLUSIONS: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis.

THE IMPACT OF DEPRESSION ON MATERNAL RESPONSES TO INFANT FACES IN PREGNANCY.

Research has suggested that prenatal depression may be associated with disrupted maternal responses to infant stimuli, with depressed pregnant women not showing the bias toward distressed infants as that observed in nondepressed pregnant women. The current study examined the effects of depression on self- reported responses to infant stimuli, in early pregnancy. Women with clinical depression (n = 38), and nondepressed women (n = 67) were recruited from a wider cognitive behavioral therapy trial. They completed Maternal Response Scales in which they were presented with images of distressed, neutral, and happy infant faces, with no time limit. The women rated their responses to these images along three dimensions--wanting to comfort, wanting to turn away, and feelings of anxiety--using Likert scales via a computerized task. There was evidence that women with depression in pregnancy showed different responses than did women without depression. Women with depression were substantially more likely to be in the highest quartile for ratings of wanting to turn away, odds (OR) ratio = 4.15, 95% confidence intervals (CIs) = 1.63-10.5, p = .003, and also were substantially less likely to be in the highest quartile for wanting to comfort a distressed infant face, OR = 0.22, 95% CIs = 0.09-0.54, p < .001. Findings are consistent with there being both a heightened avoidant and a reduced comforting response toward distressed infants in depressed pregnant women, providing some support that depression disrupts maternal preparations at a conscious level.

Optimized parametric skin modelling for diagnosis of skin abnormalities by combining light back-scatter and laser speckle imaging.

BACKGROUND/PURPOSE: Optical and parametric skin imaging methods which can efficiently identify invisible sub-skin features or subtle changes in skin layers are very important for accurate optical skin modelling. In this study, a hybrid method is introduced that helps develop a parametric optical skin model by utilizing interdisciplinary techniques including light back-scatter analysis, laser speckle imaging, image-texture analysis and Bayesian inference methods. The model aims to detect subtle skin changes and hence very early signs of skin abnormalities/diseases. METHODS: Light back-scatter and laser speckle image textural analysis are applied onto the normal and abnormal skin regions (lesions) to generate set of attributes/parameters. These are then optimized by Bayesian inference method in order to build an optimized parametric model. RESULTS: The attributes selected by Bayesian inference method in the optimization stage were used to build an optimized model and then successfully verified. It was clearly proven that Bayesian inference based optimization process yields good results to build an optimized skin model. CONCLUSION: The outcome of this study clearly shows the applicability of this hybrid method in the analysis of skin features and is therefore expected to lead development of non-invasive and low-cost instrument for early detection of skin changes.

Evaluation of pectinolytic activities for oenological uses from psychrotrophic yeasts.

UNLABELLED: Of the twenty-three morphotypes of yeasts isolated from soil capable of utilizing pectin as sole carbon source at 6°C, two yeast isolates, one psychrotolerant (PT1) and one psychrophilic (SPY11), were selected according to their ability to secrete pectinolytic enzymes under some oenological conditions (temperature 6 and 12°C and pH 3.5) and ability or inability to grow above 20°C, respectively. As compared to their optimal activity, the three pectinolytic enzymes viz., pectin methyl esterase (PME), endopolygalacturonase (endo-PG) and exopolygalacturonase (exo-PG) isolated and assayed at pH 3.5 from PT1 were found to retain 39, 60 and 60% activity at 12°C and 40, 79 and 74% activity at 28°C, respectively. Likewise, the enzymes PME and endo-PG at pH 3.5 from SPY11 displayed 46 and 86% activity at 12°C and 50 and 60% activity at 28°C, respectively. All these enzymes showed 20-90% of residual activity at pH 3.5 and 6°C. The yeast isolates PT1 and SPY11 were identified as Rhodotorula mucilaginosa and Cystofilobasidium capitatum, respectively, on the basis of morphological, physiological and molecular characteristics. This study presents the first report on pectinolytic activities under major oenological conditions from psychrotolerant isolate R. mucilaginosa PT1 and psychrophilic isolate C. capitatum SPY11. SIGNIFICANCE AND IMPACT OF THE STUDY: The cold-active pectinolytic enzymes (PME, endo-PG and exo-PG) from the newly isolated and identified psychrophilic yeast Cystofilobasidium capitatum SPY11 and psychrotolerant yeast Rhodotorula mucilaginosa PT1that exhibited 50-80% of their optimum activity under some major oenological conditions pH (3.5) and temperatures (6 and 12°C) could be applied to wine production and juice clarification at low temperature. The psychrotrophic yeasts themselves could be applied to cold process for the production of enzymes thus saving cost of energy and protecting process from contamination.

Osmotically controlled oral delivery of ciprofloxacin through asymmetric membrane capsules.

Asymmetric membrane capsules (AMCs) are based on the concept of osmotic pressure but are much simpler to manufacture. Further, they can be suitably optimized by varying the parameters like concentration of pore former, polymer, osmotic agents and solubility enhancers to cater the specific needs of a particular formulation. The main objective of the present work was to exploit the concept of AMCs for the controlled delivery of poorly soluble anti-infective drugs. Ciprofloxacin was chosen as the model drug. Nine AMCs (F1-F9) with varying concentrations of cellulose acetate [CA] (polymer-12% w/v, 16% w/v and 20% w/v) and glycerol (pore former- 50% w/w, 60% w/w and 70% w/w of polymer) were prepared. AMCs F1-F3 were discarded because of poor rigidity. 18 formulations (F4A-F9C) were prepared with the remaining 6 AMCs by varying concentrations of mannitol in the core (osmogen-15% w/w, 25% w/w and 50 % w/w of drug). F6C prepared with 16% CA, 70% glycerol and 50% mannitol gave highest release (57.93 +/- 0.93 %) after 12 h. Scanning electron microscopy revealed asymmetric structure of the membrane and osmotic release (zero order) through pores formed in situ was confirmed. Three concentrations of tartaric acid were used in F6C (T1-5%, T2-15%, T3-20%) for further optimization. T3 gave maximum release after 12 h (82.21 +/- 0.71%) and was selected as final optimized formulation. The study concluded that AMCs containing a suitable osmogen and a solublizer, can successfully deliver poorly soluble anti-infective drugs in a controlled manner.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Omental desmoplastic small round cell tumor with metastasis.

Desmoplastic small round cell tumor (DSRCT) is a very rare diagnosis with about 200 cases reported in literature. DSRCT is a recently described histopathological entity by Gerald and Rosai in 1989. Abdominopelvic cavity especially peritoneum is the most common site. We report a case of a huge omental DSRCT with lymph node metastasis which was initially misdiagnosed as gastrointestinal stromal tumor on radiology. A 26-year-old male presented with complaints of upper abdominal swelling associated with constant dull pain. On examination there was a large 15 × 12 cm intraabdominal mass in the epigastric and umbilical region. Imaging studies were suggestive of neoplastic mesenchymal etiology. Image-guided fine-needle aspiration cytology (FNAC) was suggestive of mesenchymal neoplastic etiology. On laparotomy, there was a huge 20 × 15 cm mass arising from omentum with multiple omental and mesenteric seedlings and mesenteric, peripancreatic and perigastric lymphadenopathy. The patient underwent debulking surgery with uneventful post-operative recovery. Histopathological examination with immunohistochemistry revealed a diagnosis of DSRCT of omentum and small bowel mesentery with lymph node metastasis. Patient then received adjuvant chemotherapy with multiple chemotherapeutic drugs as per P6 protocol and has stable disease at 1 year follow up.

Multi-location evaluation of mungbean (Vigna radiata L.) in Indian climates: Ecophenological dynamics, yield relation, and characterization of locations.

Crop yield varies considerably within agroecology depending on the genetic potential of crop cultivars and various edaphic and climatic variables. Understanding site-specific changes in crop yield and genotype × environment interaction are crucial and needs exceptional consideration in strategic breeding programs. Further, genotypic response to diverse agro-ecologies offers identification of strategic locations for evaluating traits of interest to strengthen and accelerate the national variety release program. In this study, multi-location field trial data have been used to investigate the impact of environmental conditions on crop phenological dynamics and their influence on the yield of mungbean in different agroecological regions of the Indian subcontinent. The present attempt is also intended to identify the strategic location(s) favoring higher yield and distinctiveness within mungbean genotypes. In the field trial, a total of 34 different mungbean genotypes were grown in 39 locations covering the north hill zone (n = 4), northeastern plain zone (n = 6), northwestern plain zone (n = 7), central zone (n = 11) and south zone (n = 11). The results revealed that the effect of the environment was prominent on both the phenological dynamics and productivity of the mungbean. Noticeable variations (expressed as coefficient of variation) were observed for the parameters of days to 50% flowering (13%), days to maturity (12%), reproductive period (21%), grain yield (33%), and 1000-grain weight (14%) across the environments. The genotype, environment, and genotype × environment accounted for 3.0, 54.2, and 29.7% of the total variation in mungbean yield, respectively (p < 0.001), suggesting an oversized significance of site-specific responses of the genotypes. Results demonstrated that a lower ambient temperature extended both flowering time and the crop period. Linear mixed model results revealed that the changes in phenological events (days to 50 % flowering, days to maturity, and reproductive period) with response to contrasting environments had no direct influence on crop yields (p > 0.05) for all the genotypes except PM 14-11. Results revealed that the south zone environment initiated early flowering and an extended reproductive period, thus sustaining yield with good seed size. While in low rainfall areas viz., Sriganganagar, New Delhi, Durgapura, and Sagar, the yield was comparatively low irrespective of genotypes. Correlation results and PCA indicated that rainfall during the crop season and relative humidity significantly and positively influenced grain yield. Hence, the present study suggests that the yield potential of mungbean is independent of crop phenological dynamics; rather, climatic variables like rainfall and relative humidity have considerable influence on yield. Further, HA-GGE biplot analysis identified Sagar, New Delhi, Sriganganagar, Durgapura, Warangal, Srinagar, Kanpur, and Mohanpur as the ideal testing environments, which demonstrated high efficiency in the selection of new genotypes with wider adaptability.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

BACKGROUND & OBJECTIVES: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. METHODS: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. RESULTS: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. INTERPRETATION & CONCLUSIONS: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

BACKGROUND & OBJECTIVES: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. METHODS: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1x107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. RESULTS: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). CONCLUSION: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

BACKGROUND & OBJECTIVES: In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis. METHODS: A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor). RESULTS: The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors. INTERPRETATION & CONCLUSIONS: Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples/isolates.

Does it matter how we measure metatarsal length?

INTRODUCTION: Variations in the relative lengths of the metatarsal bones have been implicated as a cause of forefoot pain. Osteotomies to balance the metatarsal lengths have been described as therapy. Although measurement of metatarsal length is required for these osteotomies, there is no gold standard method to measure it radiologically. We compared the three main radiological methods to measure the second metatarsal length described in the literature. MATERIALS AND METHODS: Standing dorsoplantar radiographs of 81 feet were measured using the methods described by Coughlin, Maestro and Hardy and Clapham. Bland and Altman's method was used to compare the above techniques for reproducibility and agreement between each method. RESULTS: The three methods produced markedly different results. Coughlin's and Maestro's methods showed a relative protrusion of the second metatarsal-relatively longer than the first metatarsal by an average of 4 & 3 mm respectively. Hardy and Clapham's method, however, showed a relative retraction of second metatarsal by an average of 1 mm. Both intra-observer and inter-observer errors were smallest for the Coughlin method (the most reproducible) and largest for the Hardy/Clapham method. Significant lack of agreement was found when we compared the 3 techniques with bias for measuring the mean between methods ranging from 1mm to 5.5mm. CONCLUSIONS: Metatarsal lengths as measured by these three methods can be very variable. Thus their role in planning metatarsal osteotomies and establishing relationship of metatarsal protrusion with metatarsophalangeal joint instability and other similar conditions is questionable and require further studies.

Extraction of Rhododendron arboreum Smith flowers from the forest for the livelihood and rural income in Garhwal Himalaya, India.

Rhododendron arboreum locally known as 'Burans', that bears magnificent flowers is one of the valuable non timber forest produces (NTFPs) in Garhwal Himalaya. These flowers are good source of income for local populace and help them to their subsistence up to some extent. R. arboreum flower can help local population to improve their livelihoods if potential harvesting is carried out sustainably. An attempt has been made to estimate the flower yield, examine extraction techniques, marketing trends and various uses of flowers. Stratified random sampling method was carried out in eight sites varying in altitudes and geographic locations. Flower yield kg/ha for each site was calculated as standard process. Questionnaire based survey was carried out in selected villages for flower extraction and marketing trends. Projections of potential (probable/-could generate) income were made and cost-benefit analysis was also estimated. Tree density of R. arboreum ranked first and Q. leucotrichophora had second rank while 16-25 cm cbh class tree density for R. arboreum was found highest across the sites. Flower yield was significantly (p < 0.001) higher at Khirsu site with 26-35 and 46-55 cm cbh class. There was positively significant correlation (n = 446, p < 0.001, r = 0.53) between flower yield and actual cbh. Flower yield has a direct relation with size of tree whereas yield has been less impacted by the sites. Average yield of flowers across the sites was about 25.3 ton/ha. On average 30% households are engaged in the extraction and trade activities with the extraction rate of 25-350 kg/household/year. A net household income of Rs. 6000-37,000 (89-545 USD) per year was computed from Rhododendron flower extraction and marketing business. The total monetary benefit was significantly higher than the inputs for all value added items on a per day basis. R. arboreum plays important role in ecological and economic sustainability of poor rural people and unemployed youths in Himalayan region. This can reduce unemployment through development of small cottage industry and entrepreneurship at village level by making different food products i.e. juice, squash, sauce and pickle etc.