Improving the performance of enteric vaccines in the developing world.

Vaccines against important enteric pathogens such as rotavirus and poliovirus have shown lower efficacy in some populations. The application of new technologies and diverse scientific disciplines are needed to realize the promise of truly universal and effective solutions to combat those and other enteric diseases.

Research priorities in Maternal, Newborn, & Child Health & Nutrition for India: An Indian Council of Medical Research-INCLEN Initiative.

In India, research prioritization in Maternal, Newborn, and Child Health and Nutrition (MNCHN) themes has traditionally involved only a handful of experts mostly from major cities. The Indian Council of Medical Research (ICMR)-INCLEN collaboration undertook a nationwide exercise engaging faculty from 256 institutions to identify top research priorities in the MNCHN themes for 2016-2025. The Child Health and Nutrition Research Initiative method of priority setting was adapted. The context of the exercise was defined by a National Steering Group (NSG) and guided by four Thematic Research Subcommittees. Research ideas were pooled from 498 experts located in different parts of India, iteratively consolidated into research options, scored by 893 experts against five pre-defined criteria (answerability, relevance, equity, investment and innovation) and weighed by a larger reference group. Ranked lists of priorities were generated for each of the four themes at national and three subnational (regional) levels [Empowered Action Group & North-Eastern States, Southern and Western States, & Northern States (including West Bengal)]. Research priorities differed between regions and from overall national priorities. Delivery domain of research which included implementation research constituted about 70 per cent of the top ten research options under all four themes. The results were endorsed in the NSG meeting. There was unanimity that the research priorities should be considered by different governmental and non-governmental agencies for investment with prioritization on implementation research and issues cutting across themes.

Convalescent phase sera from children infected with G12 rotavirus cross-neutralize rotavirus strains belonging to the Wa genogroup.

The emergence of G12 rotaviruses raises questions about the ability of candidate vaccines in providing protection against such emerging genotypes. Therefore, we assessed cross-neutralization against four reference rotavirus strains namely Wa (G1P[8]), DS-1 (G2P[4]), 116E (G9P[11]) and RV024 (G12P[6]) using paired sera from 28 children infected with G1P[8], G2P[4], G9P[6/8] or G12P[6] genotypes. Convalescent sera of G12P[6]-infected children demonstrated heterotypic response against 116E and Wa strains (50 and 33.3 %). In contrast, none of the four G2P[4]-infected children seroconverted against Wa or RV024 rotaviruses. The geometric mean neutralizing antibody titre in convalescent sera of G12P[6]-infected children was eightfold higher against strains belonging to the Wa genogroup (i.e. G1, G9 and G12 rotavirus) than against strains belonging to the DS-1 genogroup (G2 rotavirus). In conclusion, this study demonstrates that neutralization in part may be genogroup specific, and thus a monovalent vaccine based on the Wa genogroup is likely to protect against the G12 rotaviruses.

Population-based incidence of intussusception and a case-control study to examine the association of intussusception with natural rotavirus infection among indian children.

BACKGROUND: A rotavirus vaccine previously licensed in the United States was withdrawn because it caused intussusception. Data on background intussusception rates in developing countries are required to plan pre- and postlicensure safety studies for new rotavirus vaccines. Also, it is unclear whether natural rotavirus infection is associated with intussusception. METHODS: Passive surveillance for intussusception in a large, well-defined, poor, urban population in Delhi, India, was conducted in 2 phases. Intussusception was confirmed by ultrasonography or surgery. Fecal samples obtained from patients with intussusception at study hospitals (irrespective of their residence in study areas) and healthy control subjects were tested for rotavirus with use of enzyme immunoassay. If available, resected intestinal tissue samples were tested for rotavirus with use of immunohistochemistical analysis and reverse-transcription polymerase chain reaction. RESULTS: The incidence of intussusception requiring hospitalization was 17.7 cases per 100,000 infant-years of follow-up (95% confidence interval, 5.9-41.4 cases per 100,000 infant-years). Detection rates of rotavirus in stool samples did not differ significantly between case patients and control subjects (4 of 42 case patients vs 6 of 92 control subjects), and no evidence of rotavirus was detected in any of the 22 patients with intussusception for whom intestinal tissue samples were available. CONCLUSIONS: The incidence of intussusception among Indian infants appears to be lower than that reported in other middle- and high-income countries. Natural rotavirus infection does not appear to be a major cause of intussusception in Indian infants.

Genomic characterization of nontypeable rotaviruses and detection of a rare G8 strain in Delhi, India.

In the present investigation we molecularly characterized nontypeable rotavirus strains previously identified during surveillance in New Delhi, India. The majority of strains were demonstrated to belong to genotype G1 (54.5%) or P[8] (77.8%) on the basis of nucleotide sequencing of fragments from their VP7 and VP4 genes. The other genotypes detected included G2, G8, G9, G12, and P[4]. A G8P[6] strain, strain DS108, was detected for the first time in northern India. The VP7 gene of DS108 was most homologous with the VP7 gene of a bovine G8 strain, strain A5 (98.9%), indicating its bovine parentage. In contrast, the VP4 gene had a high degree of nucleotide sequence homology (92.9% to 99.1%) with the VP4 genes of human P[6] strains. The VP6 gene and nonstructural genes (NSP1 to NSP3 and NSP5) were most homologous with the VP6 gene and nonstructural genes of human rotaviruses belonging to the DS1 genogroup. Interestingly, the NSP4 gene of DS108 clustered within genotype E6 that until now had only two representative strains, both with G12P[6] specificity (strains RV176-00 and N26-02). Together, these results indicate that G8 strain DS108 belongs to the DS1 genogroup and could be the result of the acquisition of the VP7, VP4, and NSP4 genes by a human G2P[4] strain from more than one donor, similar to the evolution of G12P[6] strain RV176-00. The present study highlights the importance of characterizing multiple genes of nontypeable rotavirus strains to detect novel strains and get a more complete picture of rotavirus evolution.

Research priorities to reduce global mortality from newborn infections by 2015.

BACKGROUND: Newborn infections are responsible for approximately one-third of the estimated 4.0 million neonatal deaths that occur globally every year. Appropriately targeted research is required to guide investment in effective interventions, especially in low resource settings. Setting global priorities for research to address neonatal infections is essential and urgent. METHODS: The Department of Child and Adolescent Health and Development of the World Health Organization (WHO/CAH) applied the Child Health and Nutrition Research Initiative (CHNRI) priority-setting methodology to identify and stimulate research most likely to reduce global newborn infection-related mortality by 2015. Technical experts were invited by WHO/CAH to systematically list and then use standard methods to score research questions according to their likelihood to (i) be answered in an ethical way, (ii) lead to (or improve) effective interventions, (iii) be deliverable, affordable, and sustainable, (iv) maximize death burden reduction, and (v) have an equitable effect in the population. The scores were then weighted according to the values provided by a wide group of stakeholders from the global research priority-setting network. FINDINGS: On a 100-point scale, the final priority scores for 69 research questions ranged from 39 to 83. Most of the 15 research questions that received the highest scores were in the domain of health systems and policy research to address barriers affecting existing cost-effective interventions. The priority questions focused on promotion of home care practices to prevent newborn infections and approaches to increase coverage and quality of management of newborn infections in health facilities as well as in the community. While community-based intervention research is receiving some current investment, rigorous evaluation and cost analysis is almost entirely lacking for research on facility-based interventions and quality improvement. INTERPRETATION: Given the lack of progress in improving newborn survival despite the existence of effective interventions, it is not surprising that of the top ranked research priorities in this article the majority are in the domain of health systems and policy research. We urge funding agencies and investigators to invest in these research priorities to accelerate reduction of neonatal deaths, particularly those due to infections.

Designing primary healthcare systems for future in India.

Changing epidemiology, rapid urbanization, and rising expectations of populations are creating new challenges and opportunities for India's primary healthcare system. A group of primary care experts, practitioners, and researchers got together to design key elements of primary healthcare models for the future that would address these challenges and make use of emergent opportunities in rural and urban India. Based on experiences and evidence from India and across the globe shared in the consultation, the article lays out a vision and components of India's primary healthcare for future. It provides answers to questions such as how will healthcare be financed and organized, what mechanisms will assure quality of services, who will provide primary healthcare, and what role will technology have. Finally, it provides an agenda for primary healthcare practitioners and researchers to translate this vision into action.

Evaluation of IFN-gamma response to rotavirus and non-structural protein NSP4 of rotavirus in children following severe rotavirus diarrhea.

BACKGROUND: Rotavirus (RV) is the commonest cause of severe gastroenteritis in young children worldwide. However the natural immune mechanisms controlling and preventing rotavirus disease in humans are not fully understood. OBJECTIVE: To examine cellular immune responses to whole rotavirus (vaccine strain, 116E) and non-structural protein-4 (116E-NSP4) in children during natural rotavirus-infection. STUDY DESIGN: Gamma-interferon (IFN-gamma) responses were evaluated by enzyme-linked immunospot assay in peripheral blood mononuclear cells from children with RV (n=26) or non-RV (n=10) gastroenteritis and from RV-exposed adults (n=10). Additionally, IL-4 responses were assessed in 5 of the 10 adults and 6 of 26 RV-infected children. RESULTS: IFN-gamma secreting cells specific to whole RV were detected in 68% of RV-positive children and to NSP4 in 43% of these children between 4 and 30 days of illness onset. IFN-gamma responses were transient and were found higher in RV-exposed adults than in children (P<0.05). Within the RV-positive group, IFN-gamma responses in children with prior RV-exposure were higher than children without prior exposure (P<0.05). The response to whole RV and NSP4 were positively correlated (P<0.01, r(s)=0.66). CONCLUSIONS: Significant IFN-gamma responses to rotavirus candidate vaccine strain 116E were detected in children during natural RV-infection and in RV-exposed adults. Significant IFN-gamma responses to NSP4 were also observed in these study groups.

Natural immunity to rotavirus infection in children.

Annual deaths in infants and young children due to rotavirus (RV) infection are around 100,000 in India and about 600,000 globally. Development of a vaccine for this disease is a high priority. The protective mechanisms for RV diarrhea in human are not fully understood, but it is known that children develop natural immunity against RV. Early exposure to RV results in most severe episode of diarrhea and subsequent infections are milder or asymptomatic. Of the immune responses measured during natural infection, RV-specific antibodies have been well documented, whereas data on cellular immunity in humans are sparse. It is generally thought that two outer capsid proteins VP4 and VP7 play a critical role in protective immunity by stimulating production of neutralizing antibodies. While serotype- specific protection mediated by antibodies directed against the outer capsid proteins may be a mechanism of protection, such a correlate for protection has been difficult to demonstrate in humans during clinical trials. Increasing evidences suggest that viral proteins that lack a capacity of eliciting neutralizing antibody response also induce protective immunity. Limited efforts have focused on the role of non-structural proteins in protective immunity. This review describes current understanding of antibody responses in children with focus on responses specific to viral antigens with their possible role in protective immunity. We have also briefly reviewed the responses elicited to non-antibody effectors during RV infection in human subjects.

Folate, but not vitamin B-12 status, predicts respiratory morbidity in north Indian children.

BACKGROUND: Vitamin deficiencies are often part of malnutrition, which predisposes to acute lower respiratory tract infections. OBJECTIVE: The objective was to measure the association between cobalamin and folate status and subsequent respiratory morbidity. DESIGN: A prospective cohort study was conducted in 2482 children aged 6-30 mo nested in a zinc supplementation trial. We measured plasma concentrations of folate, cobalamin, methylmalonic acid, and total homocysteine (tHcy) and followed the children for 4 mo. RESULTS: We observed 1176 episodes of acute lower respiratory tract infections. Children with folate concentrations in the lowest quartile (interquartile range: 6.4-20.0 nmol/L) had a 44% higher incidence [adjusted incidence rate ratio (IRR): 1.44; 95% CI: 1.23, 1.70] of acute lower respiratory tract infections than did children in the other 3 quartiles. For tHcy, the IRR was 1.24 (1.07, 1.40) in a comparison of those in the highest quartile with those in the other quartiles. Breastfeeding was associated with high folate concentrations and protection against subsequent respiratory tract infections. This protection was significantly and substantially reduced after adjustment for plasma folate concentrations at baseline. Compared with the children in the other 3 quartiles, the IRR for being in the lowest quartile of cobalamin was 1.13 (0.76, 1.03) and for being in the highest quartile of methylmalonic acid was 1.12 (0.96, 1.31). CONCLUSIONS: Poor folate status appears to be an independent risk factor for lower respiratory tract infections in young children. This study also suggests that the protective effect of breastfeeding is partly mediated by folate provided through breast milk.

Effect of zinc added to multi-vitamin supplementation containing low-dose vitamin A on plasma retinol level in children--a double-blind randomized, controlled trial.

In a community-based double-blind randomized trial in children aged 6-35 months, both intervention and control groups received a multi-vitamin syrup containing vitamin A, while the intervention group had zinc gluconate (equivalent to 10 mg of elemental zinc) additional in the syrup. There was a significant decrease in diarrhoea and pneumonia in the intervention group. This study was undertaken to investigate if addition of zinc to vitamin A had improved plasma retinol levels, which, in turn, was responsible for the effects observed in the intervention group. In a randomly-selected subsample of 200 children--100 each from the intervention and the control group, plasma retinol levels after 120 days of supplementation were measured. There was no difference in the mean plasma retinol levels [the difference in the mean 0.46 microg/dL (95% confidence interval -1.42-2.36)] between the two groups following supplementation. No difference in plasma retinol levels was observed in the subgroups based on base-line nutritional status and plasma zinc levels. Addition of zinc to low-dose vitamin A in this study did not improve the vitamin A status of children and cannot explain morbidity effects of the intervention.

Pathways to infant mortality in urban slums of Delhi, India: implications for improving the quality of community- and hospital-based programmes.

The study aimed at obtaining insights into the processes underlying infant deaths to help identify preventive interventions which may bring down infant mortality rates further. Verbal autopsies were performed on 162 deaths of liveborn infants that occurred in a birth cohort in two urban slums of Delhi, India, between February 1995 and August 1996. A structured verbal autopsy form was used for ascertaining the cause of death. The narratives of caretakers on seeking of care and treatment received for illness were reviewed to identify the actions and behaviours that might have contributed to death. Seeking of care was less common (57%) for illnesses that led to death in the first week of life than at later ages. The first-week deaths commonly (61%) occurred within 24 hours of recognition of illness which might have been too a short time for effective interventions by care providers. Only six of 45 neonates who had features of sepsis, pneumonia or meningitis, major congenital malformations, birth asphyxia, or prematurity were advised by primary care providers for hospitalization. Similarly, only 25 (41%) of 61 older infants who had severe malnutrition and sepsis or meningitis, diarrhoea or pneumonia, or other illnesses were referred to hospital. Parenteral antibiotics were prescribed less often than warranted. Only two of 16 neonates with serious bacterial infections and eight of 19 postneonates with features of sepsis or meningitis received parenteral antibiotics. Inappropriate healthcare practices were common among the practitioners of modern and indigenous systems of medicine and registered medical practitioners. Forty percent of the neonates and a little over half of the older infants, advised for hospitalization, were taken to hospital. Fifteen percent of the infants taken to hospital were refused admission. Of 21 hospitalized infants discharged alive, five (23%) died within 48 hours and 13 (62%) within a week of returning home. A major effort is required to improve skills of healthcare providers of the biomedical and indigenous systems of medicine in caring for neonates and infants. Development of home-based treatment regimens for young infants and objective criteria for their hospitalization and discharge should receive a high priority.

A cost-benefit analysis of typhoid fever immunization programmes in an Indian urban slum community.

Many economic analyses of immunization programmes focus on the benefits in terms of public-sector cost savings, but do not incorporate estimates of the private cost savings that individuals receive from vaccination. This paper considers the implications of Bahl et al.'s cost-of-illness estimates for typhoid immunization policy by examining how community-level incidence estimates and information on distribution of costs of illness among patients and the public-health sector can be used in the economic analysis of vaccination-programme options. The findings illustrate why typhoid vaccination programmes may often appear to be unattractive to public-health officials who adopt a public budgetary perspective. Under many plausible sets of assumptions, public-sector expenditure on typhoid vaccination does not yield comparable public-sector cost savings. If public-health officials adopt a societal perspective on the economic benefits of vaccination, there are many situations in which different vaccination programmes will make economic sense. The findings show that this is especially true when public decision-makers recognize that (a) the incidence of typhoid fever is underestimated by blood culture-positive cases and (b) avoided costs of illness represent a significant underestimate of the actual economic benefits to individuals of vaccination.

Costs of illness due to typhoid fever in an Indian urban slum community: implications for vaccination policy.

Data on the burden of disease, costs of illness, and cost-effectiveness of vaccines are needed to facilitate the use of available anti-typhoid vaccines in developing countries. This one-year prospective surveillance was carried out in an urban slum community in Delhi, India, to estimate the costs of illness for cases of typhoid fever. Ninety-eight culture-positive typhoid, 31 culture-positive paratyphoid, and 94 culture-negative cases with clinical typhoid syndrome were identified during the surveillance. Estimates of costs of illness were based on data collected through weekly interviews conducted at home for three months following diagnosis. Private costs included the sum of direct medical, direct non-medical, and indirect costs. Non-patient (public) costs included costs of outpatient visits, hospitalizations, laboratory tests, and medicines provided free of charge to the families. The mean cost per episode of blood culture-confirmed typhoid fever was 3,597 Indian Rupees (US$ 1=INR 35.5) (SD 5,833); hospitalization increased the costs by several folds (INR 18,131, SD 11,218, p<0.0001). The private and non-patient costs of illness were similar (INR 1,732, SD 1,589, and INR 1,865, SD 5,154 respectively, p=0.8095). The total private and non-patient ex-ante costs, i.e. expected annual losses for each individual, were higher for children aged 2-5 years (INR 154) than for those aged 5-19 years (INR 32), 0-2 year(s) (INR 25), and 19-40 years (INR 2). The study highlights the need for affordable typhoid vaccines efficacious at 2-5 years of age. Currently-available Vi vaccine is affordable but is unlikely to be efficacious in the first two years of life. Ways must be found to make Vi-conjugate vaccine, which is efficacious at this age, available to children of developing-countries.

Implementation of the WHO Multicentre Growth Reference Study in India.

The World Health Organization (WHO) Multicentre Growth Reference Study (MGRS) Asian site was New Delhi, India. Its sample was drawn from 58 affluent neighborhoods in South Delhi. This community was selected to facilitate the recruitment of children who had at least one parent with 17 or more years of education, a key factor associated with unconstrained child growth in this setting. A door-to-door survey was conducted to identify pregnant women whose newborns were subsequently screened for eligibility for the longitudinal study, and children aged 18 to 71 months for the cross-sectional component of the study. A total of 111,084 households were visited over an 18-month period. Newborns were screened at birth at 73 sites. The large number of birthing facilities used by this community, the geographically extensive study area, and difficulties in securing support of pediatricians and obstetricians for the feeding recommendations of the study were among the unique challenges faced by the implementation of the MGRS protocol at this site.

Modifications in flow cytometric estimation of t cell subsets and b cells in peripheral blood to reduce the cost of investigation.

The development of monoclonal antibodies combined with flow cytometry has revolutionized the analysis of lymphocyte subsets. These newer methods using the Q-prep leucocyte preparation system require only 1-2 ml of blood as compared to 10 ml required traditionally. One of the main impediments in the use of this superior technology in Indian laboratories has been the high cost of reagents. This study evaluated methods to reduce the cost of assays. In the first experiment from 26 healthy subjects, 2ml venous blood samples in EDTA (ethylenediamine tetra-acetate) were obtained. Each sample was divided into two equal portions, one portion was stained using diluted monoclonal antibody, whereas the other portion was stained using standard concentrations of antibodies. In the second experiment, blood samples from 12 subjects were again divided into 2 portions; one portion of each pair was processed using commercial Q-prep reagents while the other portion was processed using our own reagents. In the first experiment, which evaluated use of a diluted antibody against the standard recommended concentrations, a 5-tube panel that estimated CD3, CD4, CD8, CD20 was used. In the second experiment CD3, CD4 and CD8 were estimated. The total cost per sample for a 5-panel estimation was however reduced from $39.11 to $1.10.Given the proven advantages of using a whole blood stain-lyse method for T cell subset estimations, its use should be encouraged in developing country settings. With the suggested methods the whole blood Q-prep could be performed at appreciably reduced costs, without loss in precision.

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting.

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38-45°C). In the second experiment, blood samples were stained within 1-2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38-45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.

Rotavirus gastroenteritis in India, 2011-2013: revised estimates of disease burden and potential impact of vaccines.

While improvements in oral rehydration use and access to healthcare have contributed to impressive gains in child survival, diarrheal diseases remain the second most important cause of child mortality in India. Pathogen specific disease rates, while key to deciding on the utility of specific public health interventions such as vaccines, are extremely difficult to obtain in developing country settings with less than optimal health access, diagnostic services and information systems. This study combined disease burden within five cohorts of infants followed up for diarrheal morbidity with data from the nationally representative Indian Rotavirus Surveillance Network and applies rates of rotavirus related events to UNICEF birth and mortality estimates for India. These estimates, while limited by the lack of data from nationally representative population based studies, use methods consistent with those employed by the World Health Organization Child Health Epidemiology Reference Group. We estimate that 11.37 million episodes of rotavirus gastroenteritis occur each year in India, requiring 3.27 million outpatient visits and 872,000 inpatient admissions when health access is unconstrained, resulting in a need for Rs. 10.37 billion each year in direct costs. An estimated 78,000 rotavirus-associated deaths occur annually of which 59,000 occur in the first 2 years of life. Introduction of a rotavirus vaccine of similar efficacy to the Rotavac in the national immunization program would result in 686,277 fewer outpatient visits, 291,756 fewer hospitalizations and 26,985 fewer deaths each year in India, assuming no indirect effects for the vaccine.