Current control strategies for soil-transmitted helminths (STH) emphasize morbidity control through mass drug administration (MDA) targeting preschool- and school-age children, women of childbearing age and adults in certain high-risk occupations such as agricultural laborers or miners. This strategy is effective at reducing morbidity in those treated but, without massive economic development, it is unlikely it will interrupt transmission. MDA will therefore need to continue indefinitely to maintain benefit. Mathematical models suggest that transmission interruption may be achievable through MDA alone, provided that all age groups are targeted with high coverage. The DeWorm3 Project will test the feasibility of interrupting STH transmission using biannual MDA targeting all age groups. Study sites (population ≥80,000) have been identified in Benin, Malawi and India. Each site will be divided into 40 clusters, to be randomized 1:1 to three years of twice-annual community-wide MDA or standard-of-care MDA, typically annual school-based deworming. Community-wide MDA will be delivered door-to-door, while standard-of-care MDA will be delivered according to national guidelines. The primary outcome is transmission interruption of the STH species present at each site, defined as weighted cluster-level prevalence ≤2% by quantitative polymerase chain reaction (qPCR), 24 months after the final round of MDA. Secondary outcomes include the endline prevalence of STH, overall and by species, and the endline prevalence of STH among children under five as an indicator of incident infections. Secondary analyses will identify cluster-level factors associated with transmission interruption. Prevalence will be assessed using qPCR of stool samples collected from a random sample of cluster residents at baseline, six months after the final round of MDA and 24 months post-MDA. A smaller number of individuals in each cluster will be followed with annual sampling to monitor trends in prevalence and reinfection throughout the trial.Trial registrationClinicalTrials.gov NCT03014167

BackgroundHuman immunology is a growing field of research in which experimental, clinical, and analytical methods of many life science disciplines are utilized. Classic epidemiological study designs, including observational longitudinal birth cohort studies, offer strong potential for gaining new knowledge and insights into immune response to pathogens in humans. However, rigorous discussion of methodological issues related to designs and statistical analysis that are appropriate for longitudinal studies is lacking.MethodsIn this communication we address key questions of quality and validity of traditional and recently developed statistical tools applied to measures of immune responses. For this purpose we use data on humoral immune response (IR) associated with the first cryptosporidial diarrhea in a birth cohort of children residing in an urban slum in south India. The main objective is to detect the difference and derive inferences for a change in IR measured at two time points, before (pre) and after (post) an event of interest. We illustrate the use and interpretation of analytical and data visualization techniques including generalized linear and additive models, data-driven smoothing, and combinations of box-, scatter-, and needle-plots.ResultsWe provide step-by-step instructions for conducting a thorough and relatively simple analytical investigation, describe the challenges and pitfalls, and offer practical solutions for comprehensive examination of data. We illustrate how the assumption of time irrelevance can be handled in a study with a pre-post design. We demonstrate how one can study the dynamics of IR in humans by considering the timing of response following an event of interest and seasonal fluctuation of exposure by proper alignment of time of measurements. This alignment of calendar time of measurements and a child's age at the event of interest allows us to explore interactions between IR, seasonal exposures and age at first infection.ConclusionsThe use of traditional statistical techniques to analyze immunological data derived from observational human studies can result in loss of important information. Detailed analysis using well-tailored techniques allows the depiction of new features of immune response to a pathogen in longitudinal studies in humans. The proposed staged approach has prominent implications for future study designs and analyses.

Stunting or reduced linear growth is very prevalent in low-income countries. Recent studies have demonstrated a causal relationship between alterations in the gut microbiome and moderate or severe acute malnutrition in children in these countries. However, there have been no primary longitudinal studies comparing the intestinal microbiota of persistently stunted children to that of non-stunted children in the same community. In this pilot study, we characterized gut microbial community composition and diversity of the fecal microbiota of 10 children with low birth weight and persistent stunting (cases) and 10 children with normal birth weight and no stunting (controls) from a birth cohort every 3 months up to 2 years of age in a slum community in south India. There was an increase in diversity indices (P <0.0001) with increasing age in all children. However, there were no differences in diversity indices or in the rates of their increase with increasing age between cases and controls. The percent relative abundance of the Bacteroidetes phylum was higher in stunted compared to control children at 12 months of age (P = 0.043). There was an increase in the relative abundance of this phylum with increasing age in all children (P = 0.0380) with no difference in the rate of increase between cases and controls. There was a decrease in the relative abundance of Proteobacteria (P = 0.0004) and Actinobacteria (P = 0.0489) with increasing age in cases. The microbiota of control children was enriched in probiotic species Bifidobacterium longum and Lactobacillus mucosae, whereas that of stunted children was enriched in inflammogenic taxa including those in the Desulfovibrio genus and Campylobacterales order. Larger, longitudinal studies on the compositional and functional maturation of the microbiome in children are needed.

BackgroundRotavirus infection causes a significant proportion of diarrhea in infants and young children worldwide leading to dehydration, hospitalization, and in some cases death. Rotavirus infection represents a significant burden of disease in developing countries, such as those in South Asia.MethodsWe conducted a meta-analysis to examine how patterns of rotavirus infection relate to temperature and precipitation in South Asia. Monthly rotavirus data were abstracted from 39 published epidemiological studies and related to monthly aggregated ambient temperature and cumulative precipitation for each study location using linear mixed-effects models. We also considered associations with vegetation index, gathered from remote sensing data. Finally, we assessed whether the relationship varied in tropical climates and humid mid-latitude climates.ResultsOverall, as well as in tropical and humid mid-latitude climates, low temperature and precipitation levels are significant predictors of an increased rate of rotaviral diarrhea. A 1°C decrease in monthly ambient temperature and a decrease of 10 mm in precipitation are associated with 1.3% and 0.3% increase above the annual level in rotavirus infections, respectively. When assessing lagged relationships, temperature and precipitation in the previous month remained significant predictors and the association with temperature was stronger in the tropical climate. The same association was seen for vegetation index; a seasonal decline of 0.1 units results in a 3.8% increase in rate of rotavirus.ConclusionsIn South Asia the highest rate of rotavirus was seen in the colder, drier months. Meteorological characteristics can be used to better focus and target public health prevention programs.

IntroductionRotavirus vaccine efficacy ranges from >90% in high socio-economic settings (SES) to 50% in low SES. With the imminent introduction of rotavirus vaccine in low SES countries, understanding reasons for reduced efficacy in these settings could identify strategies to improve vaccine performance.MethodsWe developed a mathematical model to predict rotavirus vaccine efficacy in high, middle and low SES based on data specific for each setting on incidence, protection conferred by natural infection and immune response to vaccination. We then examined factors affecting efficacy.ResultsVaccination was predicted to prevent 93%, 86% and 51% of severe rotavirus gastroenteritis in high, middle and low SES, respectively. Also predicted was that vaccines are most effective against severe disease and efficacy declines with age in low but not high SES. Reduced immunogenicity of vaccination and reduced protection conferred by natural infection are the main factors that compromise efficacy in low SES.DiscussionThe continued risk of severe disease in non-primary natural infections in low SES is a key factor underpinning reduced efficacy of rotavirus vaccines. Predicted efficacy was remarkably consistent with observed clinical trial results from different SES, validating the model. The phenomenon of reduced vaccine efficacy can be predicted by intrinsic immunological and epidemiological factors of low SES populations. Modifying aspects of the vaccine (e.g. improving immunogenicity in low SES) and vaccination program (e.g. additional doses) may bring improvements.

Cryptosporidium spp., a common cause of diarrhea in children, were investigated in the first multisite study in India. Diarrheal stools from hospitalized children aged <5 years from Delhi, Trichy, and Vellore were analyzed by microscopy, PCR-restriction fragment length polymorphism (RFLP), and/or sequencing at the small-subunit (SSU) rRNA and Cpgp40/15 loci for species determination and subgenotyping, respectively. Seventy of 2,579 (2.7%) children, 75% of whom were <2 years old, had cryptosporidial diarrhea as determined by microscopy. Genotyping and subgenotyping showed that Cryptosporidium hominis was the most commonly identified species (59/67 children), and subgenotypes Ie, Ia, Ib, and Id were common in all centers. A novel C. parvum subgenotype, IIn, was identified in Vellore. Meteorological analysis revealed a higher rate of cryptosporidial positivity during hotter and drier weather in Delhi.Cryptosporidium spp. are an important cause of endemic parasitic diarrhea in children in developing countries. In addition to causing symptoms associated with watery diarrhea, vomiting, and weight loss, early childhood cryptosporidiosis has been shown by studies to be associated with subsequent faltering of growth (reviewed in reference 11). Cryptosporidium hominis and C. parvum cause the majority of infections in children in developing countries, with C. hominis predominating and occasional reports of infection with zoonotic species such as C. felis, C. canis, C. meleagridis, and C. muris (30). C. hominis infection has been found to be associated with greater levels of oocyst shedding (4) and longer durations of oocyst shedding (31) and diarrhea (15) than C. parvum infection. In a recent community-based study in Vellore, we found increased levels of severity of diarrhea in C. hominis-infected children compared to the levels observed in children infected with other species (1).Cryptosporidium spp. have been classified into several distinct subgenotypes based on extensive polymorphisms in the Cpgp40/15 (also referred to as GP60) locus by use of PCRrestriction fragment length polymorphism (RFLP) or sequencing of PCR products (reviewed in reference 30).A number of studies from India have reported Cryptosporidium spp. in diarrheal stool samples from children, with positivity rates of up to nearly 20% (17) and asymptomatic infection rates of up to 10% (19), using stool microscopy for detection. However, only three studies have used molecular techniques for identification of cryptosporidiosis in children in India (9,13,22), suggesting that the actual infection rates may be significantly higher. In a previous hospital-based study in Vellore, we that found that PCR (15.2%) identified more than 3 times the number of cases of cryptosporidial diarrhea than microscopy (4.4%) (2). The aim of the present study was to identify the Cryptosporidium species and Cpgp40/15 subgenotypes associated with cryptosporidial diarrhea in hospitalized children from 3 centers in the country, since no studies have examined cryptosporidiosis using the...

Diarrhoea and water-borne diseases are leading causes of mortality in developing countries. To understand the socio-cultural factors impacting on water safety, we documented knowledge, attitudes and practices of water handling and usage, sanitation and defecation in rural Tamilnadu, India, using questionnaires and focus group discussions, in a village divided into an upper caste Main village and a lower caste Harijan colony. Our survey showed that all households stored drinking water in wide-mouthed containers. The quantity of water supplied was less in the Harijan colony, than in the Main village (P<0.001). Residents did not associate unsafe water with diarrhoea, attributing it to 'heat', spicy food, ingesting hair, mud or mosquitoes. Among 97 households interviewed, 30 (30.9%) had toilets but only 25 (83.3%) used them. Seventy-two (74.2%) of respondents defecated in fields, and there was no stigma associated with this traditional practice. Hand washing with soap after defecation and before meals was common only in children under 15 years (86.4%). After adjusting for other factors, perception of quantity of water received (P<0.001), stated causation of diarrhoea (P=0.02) and low socio-economic status (P<0.001) were significantly different between the Main village and the Harijan colony. Traditional practices may pose a significant challenge to programmes aimed at toilet usage and better sanitation.

CTRI/2010/091/000339.