The risk of recurrent venous thromboembolism (VTE) approaches 40 per cent of all patients after 10 yr of follow up. This risk is higher in patients with permanent risk factors of thrombosis such as active cancer, prolonged immobilization from medical diseases, and antiphospholipid syndrome; in carriers of several thrombophilic abnormalities, including deficiencies of natural anticoagulants; and in patients with unprovoked presentation. Patients with permanent risk factors of thrombosis should receive indefinite anticoagulation, consisting of subtherapeutic doses of low molecular weight heparin in cancer patients, and oral anticoagulants in all other conditions. Patients whose VTE is triggered by major surgery or trauma should be offered three months of anticoagulation. Patients with unprovoked VTE, including carriers of thrombophilia, and those whose thrombotic event is associated with minor risk factors (such as hormonal treatment, minor injuries, long travel) should receive at least three months of anticoagulation. The decision as to go on or discontinue anticoagulation after this period should be individually tailored and balanced against the haemorrhagic risk. Post-baseline variables, such as the D-dimer determination and the ultrasound assessment of residual thrombosis can help identify those patients in whom anticoagulation can be safely discontinued. As a few emerging anti-Xa and anti-IIa compounds seem to induce fewer haemorrhagic complications than conventional anticoagulation, while preserving at least the same effectiveness, these have the potential to open new scenarios for decisions regarding the duration of anticoagulation in patients with VTE.

There has been an increased influx of probiotic products in the Indian market during the last decade. However, there has been no systematic approach for evaluation of probiotics in food to ensure their safety and efficacy. An initiative was, therefore, taken by the Indian Council of Medical Research (ICMR) along with the Department of Biotechnology (DBT) to formulate guidelines for regulation of probiotic products in the country. These guidelines define a set of parameters required for a product/strain to be termed as 'probiotic'. These include identification of the strain, in vitro screening for probiotic characteristics, animal studies to establish safety and in vivo animal and human studies to establish efficacy. The guidelines also include requirements for labeling of the probiotic products with strain specification, viable numbers at the end of shelf life, storage conditions, etc., which would be helpful to the consumers to safeguard their own interest.

Background & objectives : It was hypothesized that both thrombogenic and atherogenic factors may be responsible for premature coronary heart disease (CHD) in young Indians. A case-control study was performed to determine cardiovascular risk factors in young patients with CHD in India. Methods: Successive consenting patients <55 yr with an acute coronary event or recent diagnosis of CHD were enrolled (cases, n=165). Age- and gender-matched subjects with no clinical evidence of CHD were recruited as controls (n=199). Demographic, anthropometric, clinical, haematological, and biochemical data were obtained in both groups. Univariate and multivariate logistic regression were performed to identify important risk factors. Results: In cases vs. controls mean systolic BP, diastolic BP, platelet counts, LDL cholesterol, non-HDL cholesterol, triglycerides, and fibrinogen were higher and HDL cholesterol lower (P<0.001). The presence of current smoking, low fruit and vegetables intake, high fat intake, hypertension, diabetes, low HDL cholesterol, and high LDL cholesterol, total:HDL ratio, fibrinogen and homocysteine was significantly higher in cases (P<0.05). Multivariate logistic regression analysis (age adjusted odds ratio, 95% confidence intervals) revealed that smoking (19.41, 6.82-55.25), high fat intake (1.66, 1.08-2.56), low fruit and vegetables intake (1.99, 1.11-3.59), hypertension (8.95, 5.42-14.79), high LDL cholesterol [2.49 (1.62-3.84)], low HDL cholesterol (10.32, 6.30-16.91), high triglycerides (3.62, 2.35-5.59) high total:HDL cholesterol (3.87, 2.35-5.59), high fibrinogen (2.87, 1.81-4.55) and high homocysteine (10.54, 3.11-35.78) were significant. Interpretation & conclusions : Our results showed that thrombotic (smoking, low fruit/vegetables intake, fibrinogen, homocysteine) as well as atherosclerotic (hypertension, high fat diet, dyslipidaemia) risk factors were important in premature CHD. Multipronged prevention strategies are needed in young Indian subjects.

Background & objectives : Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions : There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples.

Background & objectives : Genital tuberculosis (GTB) is one of the major causes for severe tubal disease leading to infertility. Unlike pulmonary tuberculosis, the clinical diagnosis of GTB is difficult because in majority of cases the disease is either asymptomatic or has varied clinical presentation. Routine laboratory values are of little value in the diagnosis. An absolute diagnosis cannot be made from characteristic features in hysterosalpingogram (HSG) or laparoscopy. Due to the paucibacillary nature of GTB, diagnosis by mycobacterial culture and histopathological examination (HPE) have limitations and low detection rate. The objective of this study was to evaluate the efficacy of PCR technique, culture and histopathological examination in the diagnosis of GTB in female infertility. Methods: This study included 72 infertile women who met the inclusion and exclusion criteria. After a detailed history and clinical examination all patients were subjected to investigations including pelvic sonogram, HSG and laparoscopy. Endometrial samples from were allocated for AFB smear, culture and HPE examination. Only 49 samples were available for PCR using IS 6110 and TRC ₄ primers. In seven patients peritoneal fluid was also taken for culture and PCR. Based on the clinical profile and laparoscopic findings, a diagnostic criteria was derived to suspect GTB. Specific diagnostic tests were evaluated against this diagnostic criterion. Results: Laparoscopy was suggestive of tuberculosis in 59.7 per cent of cases, AFB smear was positive in 8.3 per cent, culture was positive in 5.6 per cent, HPE positive in 6.9 per cent and PCR was positive in 36.7 per cent of cases. Based on the diagnostic criteria, GTB was suspected in 28 of the 49 cases. On evaluating against the diagnostic criteria, the sensitivity of PCR, HPE and culture were 57.1, 10.7, 7.14 per cent respectively. The concordance of results between the clinical criteria and specific diagnostic tests were analysed by Kappa measure of agreement. The culture and HPE showed mild agreement with the clinical criteria, whereas PCR showed a moderate agreement. PCR was positive in Two of the 21 cases in whom GTB was not suspected. False positive PCR in these two cases were ruled out by multiple areas of sampling and re-sampling in one case. The PCR results were negative in 12 of the 28 cases. PCR using TRC ₄ primers had a higher sensitivity (46.4%) than IS 6110 primers (25%) in detecting clinically suspected GTB. Interpretation & conclusions : Our results showed that conventional methods of diagnosis namely, HPE, AFB smear and culture have low sensitivity. PCR was found to be useful in diagnosing early disease as well as confirming diagnosis in clinically suspected cases. False negative PCR was an important limitation in this study.

Background & objectives : This study attempts to compare the pattern of growth of Indian children as assessed by weight for age, height for age and BMI for age with the WHO standards for growth (2006) and to explore the implications of differences in undernutrition rates in the 0-59 months of age group as assessed by these three indices. Methods: From the National Family Health Survey -3 database, growth curves for height, weight and BMI for age in Indian preschool children were computed using LMS software and compared with the WHO (2006) standards. Using the WHO (2006) standards, trends in prevalence of undernutrition as assessed by height, weight and BMI for age in the 0-59 month age group were computed. Results: During the first three months there was no increase in underweight and stunting rates. There was progressive increase in underweight and stunting rates between 3-23 months of age. Low BMI for age and wasting rates were highest at birth. Interpretation & conclusions :Poor growth is an adaptation to chronic low energy intake and stunting is a measure of cumulative impact of chronic energy deficiency on linear growth. It is important to prevent stunting because it is not readily reversible. Low BMI is an indictor of current energy deficit. Early detection of energy deficit using BMI for age and expeditious interventions to correct the deficit might be effective in prevention of stunting.

Background & objectives : Fas receptor and Fas Ligand (FasL) system has been implicated in the resistance to apoptosis, insensitivity to chemotherapy and in providing immune privileged status to most of the tumours. However, no reports are available on Fas and FasL expression in patients with tobacco-related oral carcinoma. Therefore, the present study was undertaken to observe Fas and FasL expression and their correlation with clinicopathological features as well as cell cycle parameters. Methods: Immunohistochemistry for Fas, FasL and DNA flow cytometry for cell cycle parameters was successfully done on 41 paraffin embedded tumour and 10 normal samples. The results were evaluated for possible association of Fas and FasL with clinicopathological features and cell cycle parameters. Results: Weak Fas expression was observed on the cell membrane only in 2 of 41 (5%) oral tumours while FasL immunoreactivity was seen in 26 of 41 (63.4%) tumours. In contrast, all ten normal oral tissues exhibited strong cytoplasmic and membrane Fas receptor immunoreactivity but absence of FasL staining. Older patients, greater tumour size and lymph node positivity were found to be associated with high expression of FasL. Significantly higher (P<0.01) expression of FasL was observed in oral tumours with aggressive DNA pattern like aneuploidy and high S-phase fraction. Interpretation & conclusions: Downregulation of Fas receptor and up-regulation of Fas ligand appear to be an important feature of tobacco-related intraoral carcinoma. Association of FasL expression with advanced clinical stage and aggressive DNA pattern suggests that the Fas and FasL system may be used as an important prognostic variable in patients with tobacco-related intraoral squamous cell carcinoma.

Background & objectives : Psychosocial stressors leading to allostatic load need to be explored further as these have great scope for early intervention. Stress studies done in India are mostly based on sources of stress and objective measures of stress. Therefore, the objective of the present study was to assess stress appraisal among students (16-17 yr) and to identify institution-specific differences (Private vs. Government) in stress appraisal and coping. Methods: The study was carried out among 16-17 yr old apparently normal students. Eighty students were recruited from six schools ensuring equal representation from gender/category of schools (Government/ Private). Validated and culturally adaptable behavioural scales for perceived stress (PSS), stressful life events (LES) and coping were administered. Psychological morbidity was assessed using GHQ-12. Data on 75 students were available for statistical analysis. Results: The students of both Government and Private schools showed similar stress perception, though the former tend to have a higher mean score. The scores were significantly higher on avoidance coping (P<0.05). The stepwise regression model showed coping as the independent predictor of perceived stress (R ² = 10%). Interpretation & conclusions : Students from Government schools had significantly higher scores on avoidance coping and therefore, suitable for a systematic study on chronic stress for early intervention.

Background & objectives : We evaluated pro- and anti-oxidant disturbances in sepsis and non-sepsis burn patients with systemic inflammatory response syndrome (SIRS). Adhesion molecules and inflammation markers on leukocytes were also analyzed. We hypothesized that oxidative stress and leukocyte activation markers can lead to the severity of sepsis. Methods: In 28 severe sepsis and 27 acute burn injury patients blood samples were collected at admission and 4 days consecutively. Oxidative stress markers: production of reactive oxygen species (ROS), myeloperoxidase, malondialdehyde and endogenous antioxidants: plasma protein sulphydryl groups, reduced glutathione, superoxide dismutase and catalase were measured. Flow cytometry was used to determine CD11a, CD14, CD18, CD49d and CD97 adhesion molecules on leukocytes. Procalcitonin, C-reactive protein, fibrinogen, platelet count and lactate were also analyzed. Results: Pro-oxidant parameters were significantly elevated in sepsis patients at admission, ROS intensity increased in burn patients until the 5 ^th day. Endogenous antioxidant levels except catalase showed increased levels after burn trauma compared to sepsis. Elevated granulocyte activation and suppressed lymphocyte function were found at admission and early activation of granulocytes caused by increasing activation/migration markers in sepsis. Leukocyte adhesion molecule expression confirmed the suppressed lymphocyte and monocyte function in sepsis. Interpretation & conclusions : Severe sepsis is accompanied by oxidative stress and pathological leukocyte endothelial cell interactions. The laboratory parameters used for the evaluation of sepsis and several markers of pro- and antioxidant status were different between sepsis and non-sepsis burn patients. The tendency of changes in these parameters may refer to major oxidative stress in sepsis and developing SIRS in burns.

Background & objectives : Observation of an increased frequency of an intermediate deficiency of serum alpha1-antitrypsin (α1-AT) in patients with Tropical Pulmonary Eosinophilia (TPE) was earlier reported. Though the possibility of existence of an acquired deficiency was suggested, without phenotyping a hereditary α1-AT deficiency in TPE could not totally be ruled out. In this study, we have done Pi (Protease inhibitor) phenotyping to investigate the possibility of association of any heterozygous (or homozygous) α1-AT deficiency in patients with TPE. Methods: Serum a1antitrypsin (α1-AT) was measured in 103 patients (Group A) with TPE, 99 patients with pulmonary eosinophilia who had associated intestinal worm infestation (Group B) and 43 healthy volunteers who served as controls. In 19 α1-AT deficient patients (9 of Group A and 10 of Group B), α1-AT level was measured before and after treatment. In 58 patients with TPE and in 5 controls, phenotyping was done. Results : Fifteen patients of Group A and 16 from Group B showed intermediate α1-AT deficiency (150 mg % or less. None of the control subjects had α1-AT deficiency (<200 mg%). After treatment with DEC and/or deworming, in 19 patients there was a significant (P < 0.001) rise in α1-AT levels. Results of phenotyping showed that all had M₁ or M ₂ allele and none had S or Z variant (either homozygous or heterozygous) thus ruling out any underlying genetic cause for the observed α1-AT deficiency. Interpretation & conclusions : The observed α1-AT deficiency may be due to the chronic inflammation in TPE and associated oxidative stress. However, in such α1-AT deficient patients with TPE and those with worm infested pulmonary eosinophilia, faecal α1-AT concentration and faecal α1-AT clearance should be routinely estimated to rule out the possibility of any intestinal protein loss.

Background & objectives: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales' disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. Methods: The TAC assay was done spectrophotometrically in the serum of Eales' disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. Results: TAC was found to be significantly lower in Eales' disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales' disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). Interpretation & conclusions: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Background & objectives : Chronic cough and chronic phlegm are important indicators of respiratory morbidity, accelerated lung function decline, increased hospitalization and mortality. This study was planned to estimate the prevalence of chronic cough and phlegm in the absence of dyspneoa and wheezing and to study its associated factors in a representative population of Mysore district. Methods: A cross-sectional survey was planned in a representative population of Mysore taluk. Eight villages were randomly selected based on the list of villages from census 2001. Trained field workers using the Burden of Obstructive Diseases questionnaire carried out a house-to-house survey. Results: A total of 4333 adult subjects were enrolled in the study with 2333 males and 2000 females. The prevalence of chronic cough in the community was 2.5 per cent and that of chronic phlegm was 1.2 per cent. A significant association was observed between chronic cough and age, gender, occupation and smoking and chronic phlegm with age, gender, occupation, indoor animals and smoking. A multivariate analysis confirmed independent association of age, occupation and smoking for chronic cough and age and smoking for chronic phlegm. On sub-group analysis of males, heavy smokers had higher prevalence of chronic cough and chronic phlegm as compared to light smokers and non smokers. Interpretation & conclusions: The prevalence of chronic cough was 2.5 per cent and chronic phlegm was 1.2 per cent in the general population in Mysore which is lower than that observed in other studies. Heavy smoking was an important preventable risk factor identified in this study and efforts towards smoking cessation are crucial to achieve good respiratory health in the community.

Background & objectives: Mosquitoes transmit serious human diseases, causing millions of deaths every year and the development of resistance to chemical insecticides resulting in rebounding vectorial capacity. Plants may be alternative sources of mosquito control agents. The present study assessed the role of larvicidal activities of hexane, chloroform, ethyl acetate, acetone, and methanol dried leaf and bark extracts of Annona squamosa L., Chrysanthemum indicum L., and Tridax procumbens L. against the fourth instar larvae of malaria vector, Anopheles subpictus Grassi and Japanese encephalitis vector, Culex tritaeniorhynchus Giles (Diptera: Culicidae). Methods: Larvicidal activities of three medicinal plant extracts were studied in the range of 4.69 to 1000 mg/l in the laboratory bioassays against early 4 ^th instar larvae of An. subpictus and Cx. tritaeniorhynchus. The mortality data were subjected to probit analysis to determine the lethal concentrations (LC ₅₀ and LC ₉₀ ) to kill 50 and 90 per cent of the treated larvae of the respective species. Results: All plant extracts showed moderate effects after 24 h of exposure; however, the highest toxic effect of bark methanol extract of A. squamosa, leaf ethyl acetate extract of C. indicum and leaf acetone extract of T. procumbens against the larvae of An. subpictus (LC ₅₀ = 93.80, 39.98 and 51.57 mg/l) and bark methanol extract of A. squamosa, leaf methanol extract of C. indicum and leaf ethyl acetate extract of T. procumbens against the larvae of Cx. tritaeniorhynchus (LC ₅₀ =104.94, 42.29 and 69.16 mg/l) respectively. Interpretation & Conclusions: Our data suggest that the bark ethyl acetate and methanol extract of A. squamosa, leaf ethyl acetate and methanol extract of C. indicum, acetone and ethyl acetate extract of T. procumbens have the potential to be used as an ecofriendly approach for the control of the An. subpictus, and Cx. tritaeniorhynchus.

Background & objectives: The greater tendency to diabetes in Indians may be due to genetic factors in addition to environment and diet. CD36, a class B scavenger cell surface receptor mediates internalization of oxidized low density lipoprotein (Ox-LDL) leading to the formation of macrophage foam cells. CD36 deficiency is related to phenotypic expression of the metabolic syndrome, frequently associated with atherosclerotic cardiovascular diseases resulting in raised levels of glucose thereby contributing to type 2 diabetes (T2DM). Therefore, the association of human CD36 gene mutation to T2DM needs investigation. We undertook this study to investigate CD36 gene status in north Indian subjects by screening for the deletion of exons 3, 4 and 5 and certain polymorphisms. Methods: Clinical characteristics were compared between 300 T2DM patients and 100 healthy controls. Deletion analysis was carried out for exons 3, 4 and 5 of CD36 gene in 300 T2DM patients using PCR and agarose gel electrophoresis. Genotype analysis for two polymorphisms 478C>T and delAC in exons 4 and 5 respectively was carried out using PCR-RFLP method. Results: Biochemical parameters such as fasting and post-prandial glucose levels, total cholesterol, LDL-cholesterol and blood pressure were slightly raised in the T2DM patients when compared with controls with lowered HDL-cholesterol. No exonic deletion was observed in the 300 patients and 100 controls screened. All individuals were found to be homozygous (CC and -/-) for the two polymorphisms studied. Interpretation & conclusions: Although no exonic deletion was found in T2DM patients, our study suggests that all 15 exons need to be screened for mutations which lead to CD36 deficiency. Genotyping studies of the two SNPs in the CD36 gene confirmed the absence of exons 4 and 5 deletion. This is perhaps the first report from India suggesting that CD36 is one of the several important genes that need to be explored in relation to T2DM.

Background & objectives: The P300 wave is an event related potential (ERP) elicited by infrequent, task-relevant stimuli and appeared at about 300 ms, represents higher cognitive function of information processing, working memory or stimulus categorization. Hypobaric hypoxia deteriorates the cognitive function during the short term stay (days to few weeks) at high altitude. The present study was carried out to evaluate the P300 responses during long duration stay (1 month and 6 months) at high altitude (HA, 4115 m) in a sample of Indian lowlanders. Methods: The study was carried out on 18 healthy male volunteers at sea level (SL). The volunteers were stage inducted to 4115 m altitude in the Eastern Himalayas. The P300 was recorded after 1 and 6 months of their stay at HA. Results: The latencies of peaks N100, P200 and N200 waves did not show any significant changes after 1 and 6 months of stay at HA as compared to SL. The P300 latency was significantly delayed after 1 month and further delayed after 6 month of residence at 4115 m. The P200 and P300 amplitudes did not show any changes. Interpretation & conclusions: The increase in P300 latency indicated that long duration of stay at high altitude slows the stimulus evaluation processes. The observations suggest that hypoxia causes slowing of the signal processing at HA. The magnitude of the effects of hypobaric hypoxia may be dependent upon the duration of residence at high altitude.

Background & objectives: Hyponatremia is a common problem encountered in patients presenting with nonspecific symptoms. We undertook this study to investigate the clinical profile of patients with hyponatremia, the precipitating factors, the response to therapy and to compare, using these parameters, hyponatremia at presentation to that developing in the hospital. Methods: Seventy consecutive patients with serum sodium less than or equal to 125 mmol/l at presentation or at any time during hospital admission were identified and studied using a proforma. The severity of hyponatremia, therapy given and time taken for recovery were analysed. Results: The mean age of patients was 48.1 ± 16.1 yr. The mean serum sodium was 117.8 ± 6.4 mmol/l. Confusion, headache and malaise were the most common symptoms, two patients had seizures, and 20.0 per cent patients showed no clinical manifestations. Nausea was significantly (P<0.05) more common in patients presenting with hyponatremia. 22 patients (31.4%) developed hyponatremia during their stay in the hospital. 3 patients (4.3%) presented with hyponatremia which got worse during the admission period. Most had multiple precipitating factors, decreased intake being the most common (82.9%), followed by increased losses (65.7%) and miscellaneous factors (70.0%). Drugs, fluid overload and inappropriate Ryle's tube feeds more commonly precipitated hyponatremia in in-hospital patients. Time taken for recovery showed negative correlation with the serum sodium. Patients with in-hospital hyponatremia took significantly longer time to recover (P<0.05). Interpretation & conclusions: Decreased intake was found to be the commonest cause of hyponatremia, thus, ensuring adequate oral intake, especially in patients on liquid diet and in manual labourers, and correction of hyponatremia as soon as an abnormality is detected is important.