Primer of Epidemiology IV. Study designs II: Interventional or experimental designs

In this article, we describe experimental study designs and focus on randomized controlled trials. Experimental studies are intervention studies in which the investigator tests a new treatment on a selected group of patients. In a controlled design, the effects of an intervention (new treatment) are measured by comparing the outcome in the experimental group with that in a control group. Experimental studies are similar to cohort studies except that the exposure is a deliberate change (intervention) made by the researcher in one group of participants and it overcomes confounding because the treatment is assigned randomly. Further, we discuss various types of randomization (random sequence allocation) and importance of allocation concealment and blinding for proper assessment of outcomes in randomized trials.

Addressing Pediatric Anemia in India: Can the Lack of Operational Evidence be Holding Back Action.

Objectives: In India, 79% of children less than three years are anemic. Intermittent iron supplementation, home-fortification with multiple micronutrient powders (MMP), and delayed cord clamping are scientifically recommended interventions to prevent pediatric anemia. Little is known about the extent to which these interventions are integrated into programs in India. Examine to what extent anemia control programs in India incorporate scientific recommendations and analyze their operational evidence-base. Methodology: We critically reviewed published (2000-2012) and grey literature, and government and non-government program documentation. Results: Published literature on interventions to reduce pediatric anemia in India is scant; there are six efficacy studies (1-delayed cord clamping; 1-use of MMP; 3- iron supplementation; 1- fortified milk) and no effectiveness studies. Intermittent iron supplementation delivered by frontline workers is the only intervention included in government programs. Only 2 of the 22 NGO programs reviewed provided iron supplements. Little is known about the operational successes and challenges on the demand and supply-side of the programs and of effectiveness of these programs. Neither national nor NGO programs include newer interventions such as MNPs or delayed cord-clamping. Conclusions: A poor evidence base on challenges and solutions to delivering iron supplementation in India precludes from strengthening operational strategies for iron supplementation. Lack of evidence on the efficacy and effectiveness of other recommended interventions prevents identification of alternate strategies for addressing pediatric anemia in India. A research agenda that focuses on closing these operational and effectiveness knowledge gaps is essential to make progress towards reducing the burden of anemia in India.

Suppression of Eis and expression of Wag31 and GroES in Mycobacterium tuberculosis cytosol under anaerobic culture conditions.

A major impediment in chemotherapy of Tuberculosis (TB) is the persistence of M. tuberculosis in a latent or dormant state, possibly perpetuated by paucity of oxygen within the lung granuloma. Proteome analysis of the anaerobically persisting microbe could therefore provide novel targets for drugs against latent TB infection (LTBI). An Indian clinical isolate of M. tuberculosis was cultured under aerobic and anaerobic conditions following Wayne’s hypoxia model and its cytosolic proteins were resolved by two-dimensional gel electrophoresis (2DE). Peptide mass fingerprinting of 32 differentially expressed spots using MALDI TOF-TOF MS-MS resulted in identification of 23 proteins. Under the anaerobic culture conditions, expression of 12 of these proteins was highly suppressed (>2 fold reduction in spot volumes), with 4 of them (GrpE, CanB, MoxR1 and Eis) appearing as completely suppressed since corresponding spots were not detectable in the anaerobic sample. On the other hand, 4 proteins were highly expressed, with two of them (Wag31 and GroES) being uniquely expressed under anaerobic conditions. Suppression of Eis could make the anaerobically persisting bacilli susceptible to the aminoglycoside antibiotics which are known to be acetylated and inactivated by Eis. Although all 4 over-expressed proteins can be considered as putative drug targets for LTBI, Wag31 appears particularly interesting in view of its role in the cell wall biogenesis.

Prevalence of Household-level Food Insecurity and Its Determinants in an Urban Resettlement Colony in North India.

An adequate food intake, in terms of quantity and quality, is a key to healthy life. Malnutrition is the most serious consequence of food insecurity and has a multitude of health and economic implications. India has the world’s largest population living in slums, and these have largely been underserved areas. The State of Food Insecurity in the World (2012) estimates that India is home to more than 217 million undernourished people. Various studies have been conducted to assess food insecurity at the global level; however, the literature is limited as far as India is concerned. The present study was conducted with the objective of documenting the prevalence of food insecurity at the household level and the factors determining its existence in an urban slum population of northern India. This cross-sectional study was conducted in an urban resettlement colony of South Delhi, India. A pre-designed, pre-tested, semi-structured questionnaire was used for collecting socioeconomic details and information regarding dietary practices. Food insecurity was assessed using Household Food Insecurity Access Scale (HFIAS). Logistic regression analysis was performed to determine the factors associated with food insecurity. A total of 250 women were interviewed through house-to-house survey. Majority of the households were having a nuclear family (61.6%), with mean familysize being 5.5 (SD±2.5) and the mean monthly household income being INR 9,784 (SD±631). Nearly half (53.3%) of the mean monthly household income was spent on food. The study found that a total of 77.2% households were food-insecure, with 49.2% households being mildly food-insecure, 18.8% of the households being moderately food-insecure, and 9.2% of the households being severely food-insecure. Higher education of the women handling food (OR 0.37, 95% CI 0.15-0.92; p≤0.03) and number of earning members in the household (OR 0.68, 95% CI 0.48-0.98; p≤0.04) were associated with lesser chance/odds of being food-insecure. The study demonstrated a high prevalence of food insecurity in the marginalized section of the urban society. The Government of India needs to adopt urgent measures to combat this problem.

Differential microglial and astrocytic response to bacterial and viral infection in the developing hippocampus of neonatal rats.

Polyinosinic:polycytidylic acid (Poly I:C; 5 mg/kg body weight, ip) and lipopolysaccharide (LPS; 0.3 mg/kg body weight, ip) induced microglial and astrocytic activation in Sprague Dawley rats. Higher microglial and astrocytic activities were noticed in Poly I:C infused rats throughout the hippocampus till postnatal day 21 with a comparatively weaker response in LPS group. However, LPS induced inflammation persisted even after postnatal day 21, indicating thereby, that the Poly I:C (viral mimic) produces an acute inflammation, while LPS (bacterial endotoxin) produces chronic inflammation when exposed during early neonatal life.

Possible role of CYP2C9 & CYP2C19 single nucleotide polymorphisms in drug refractory epilepsy.

Background & objectives Multiple drug resistance in epilepsy is a common problem and one third of epilepsy patients remain non responsive to antiepileptic drug (AED) therapy. In this study we aimed to investigate the relationship between the genetic polymorphism of cytochrome P450 genes, namely CYP2C9 and CYP2C19 with multiple drug resistance in epilepsy patients. Methods: A total of 402 patients with epilepsy were enrolled in this study; 128 were drug resistant and 274 were drug responsive. The peripheral blood samples of the patients with epilepsy were collected. Drug compliance was confirmed in 20 per cent patient population using HPLC. Genotyping of CYP2C9 (*2 and *3), and CYP2C19 (*2 and *3) was carried out by PCR-RFLP. Results: The genotype frequencies of CYP2C9 430 C>T (*2 variant) and CYP2C9 1075 A>C (*3 variant) did not differ significantly in drug resistant versus responsive patients. After combining CYP2C9 *2 and CYP2C9 *3, the frequency of CYP2C9*1/*3 was significantly lower in drug resistant as compared to drug responsive epilepsy patients (P=0.03, OR=0.53, 95%CI=0.30-0.95). Similarly, combined frequency of all the slow and poor metabolizer variants (2C9 *1/*2, *1/*3 and *2/*3) was also lower as compared to drug resistant group (P=0.03, OR=0.60, 95% CI 0.38-0.96). There was no significant differences in genotypic or allelic distribution of CYP2C19*2 while CYP2C19*3 was monomorphic in northern Indian population. Interpretation & conclusions: Our results demonstrated significant involvement of CYP2C9 genetic variants in the modulation of epilepsy pharmacotherapy confirming the important role of CYP2C9 mutants preventing epilepsy patients from developing drug resistance.

Effect of nickel-stresses on uptake, pigments and antioxidative responses of water lettuce, Pistia stratiotes L.

Water lettuce plants were exposed to various concentrations (0, 0.01, 0.1, 1.0 and 10.0 ppm) of nickel as nickel sulphate in nutrient medium. The effect of graded nickel (Ni+2) concentrations on visible symptoms of toxicity, pigments (chlorophyll a, b and total) and antioxidative attributes were evaluated. Plants exposed to high nickel (1.0 and 10.0 ppm) showed visible toxicity symptoms, such as wilting, chlorosis in young leaves, browning of root tips and broken off roots, observed at 6 days after treatment. Nickel was accumulated more in root (863.3 3g g-1 dry weight) than leaves (116.2 3g g-1 dry weight) at 6 days of treatment. Nickel exposure decreased chlorophyll a, b and total chlorophyll contents. Relative water content decreased at high nickel (1.0 and 10.0 ppm). Antioxidants, such as proline content and peroxidase activity increased with increase in nickel concentrations, whereas, other carotenoids and protein contents at 1.0 ppm and activity of catalase at 10 ppm of nickel were decreased. The low level of nickel stimulates photosynthetic pigments and antioxidative attributes. The study may be helpful in phytoremedial strategies and biological indication of nickel toxicity in aquatic plants.

Indigenous recombinant streptokinase vs natural streptokinase in acute myocardial infarction patients: Phase III multicentric randomized double blind trial.

BACKGROUND: Streptokinase is the most widely used thrombolytic agent and can now be made using recombinant DNA technology. The present trial was initiated to assess an indigenous recombinant streptokinase (Shankinase, r-SK). AIM: To compare the efficacy and safety of indigenous recombinant streptokinase (Shankinase, r-SK) and natural streptokinase (Streptase, n-SK). SETTINGS AND DESIGN: Double blind, randomized, non-inferiority, multicentric, parallel study. MATERIALS AND METHODS: Patients of AMI < 6 hours of chest pain and 2 mm ST elevation in 2 contiguous chest leads V(1)-V(6) or 1 mm in limb leads were randomized to receive 1.5 miu of either r-SK or n-SK. CK Peaking and decrease of > or = 50% ST segment were used to assess reperfusion. STATISTICAL ANALYSIS: Difference in the groups was assessed by chi-square or paired t test as required. Probability value < 0.05 was considered significant with 95% confidence interval. RESULTS: Overall 150 patients were recruited (96 r-SK group and 54 in n-SK group) and demographic and clinical profile of the groups was comparable. Reperfusion was seen in 68.2% (58) and 69.4% (34) patients in r-SK and n-SK groups respectively. Commonly seen adverse events were fever in 7 (8.5%), hypotension in 3 (3.6%), nausea in 2 (2.4%) patients. Minor bleeding were seen in 4 (4.8%) of patients. CONCLUSION: Indigenous recombinant Streptokinase (r-SK) is as efficacious as natural streptokinase (n-SK) in establishing reperfusion as assessed by non-invasive parameters with comparable side effect profile.

Safety and efficacy of an indigenous human recombinant interferon alpha 2b (Shanferon) in patients with chronic hepatitis B.

Interferon treatment is the established option for the treatment of patients with chronic hepatitis B without decompensated liver disease. However, such treatment is expensive. We report here our data of a multi-center, open-label trial of the use of an indigenously produced interferon in the treatment of chronic HBeAg-positive chronic hepatitis B. Adult patients with chronic HBeAg-positive hepatitis B with elevated serum transaminase activity and positive serum HBV DNA test were treated with 5 MU/day of an indigenously produced interferon (Shanferon; Shantha Biotechnics, Hyderabad, India) for 4 months, and were then followed up for 6 months. Of the 39 patients enrolled, 36 completed the treatment and 33 completed the post-treatment follow-up. Of the 33 patients who completed the study, end-of-treatment biochemical and virological responses were observed in 10 (30%) and 5 (15%) respectively. Sustained biochemical and virological responses were observed in 15 (45%) and 7 (21%), patients respectively. Adverse effects led to the discontinuation of treatment in only one patient. Our data suggest that safety and efficacy of the indigenously produced interferon were similar to those previously reported results with interferon from other sources.

Safety and efficacy of an indigenous recombinant interferon-alpha-2b in patients with chronic myelogenous leukaemia: results of a multicentre trial from India.

BACKGROUND: Compared to hydroxyurea, treatment with interferon-alpha (IFN-alpha) is known to prolong survival in patients with chronic phase of chronic myelogenous leukaemia (CML) and was considered as first-line therapy till recently. We conducted a multicentre trial using an indigenous recombinant IFN-alpha-2b to evaluate its efficacy and toxicity in chronic phase CML. METHODS: Between September 2000 and August 2001, patients with chronic phase CML were recruited within 8 weeks of diagnosis at 7 centres in India. The study was approved by the Ethics Committee of each participating Institute and Informed, written consent was obtained from all patients. All patients were given the study drug in a dose of 5 million units daily subcutaneously. Response and survival analyses were done with intent-to-treat analysis. RESULTS: One hundred and fourteen patients (75 men and 39 women) were included in the study. Their ages ranged from 18 to 62 years (median 37 years). Fifty-seven per cent of patients had a haematological response; complete response in 31.6% and partial response in 25.4%. The median time to achieve complete haematological response was 6 months (range 3-12 months). Cytogenetic response was seen in 39.4% of patients; complete in 1.8%, partial in 28% and minimal in 9.6%. The median time to achieve partial and complete cytogenetic response was 6 and 12 months, respectively. Nineteen patients had progression (blast crisis n=15, accelerated phase n=4) while on treatment. Two patients refused further treatment after the initial 4 weeks due to IFN-a toxicity, mainly bone pains and fever. The major toxic effects of treatment were fever (78%), fatigue (25.4%) and myalgia (52%). No patient died of toxicity. Currently, 95 patients are alive, 91 in the chronic phase and 4 in the accelerated phase. Four patients were lost to follow up and all 15 patients with blast crisis died of progressive disease at a median Interval of 6.5 months (range 1-15 months). The Kaplan-Meier probability of survival at 36 months was 76%. CONCLUSION: This study confirms the efficacy of the indigenous recombinant IFN-alpha-2b in chronic phase CML. The drug has a toxicity profile similar to that of other preparations.