Serum IgG responses and seroconversion patterns to Cryptosporidium gp15 among children in a birth cohort in south India.
ABSTRACT: The correlates of protective immunity to cryptosporidiosis are not well understood. This study was conducted to assess the effect of maternal serum IgG against Cryptosporidium gp15 on responses to this antigen in children with (cases) and without (controls) PCR-confirmed cryptosporidial diarrhea. Maternal sera (n = 129) and sera from cases (n = 39) and controls (n = 90) collected at 3.5, 9, and 24 months of age were tested for serum IgG against Cryptosporidium gp15 by enzyme-linked immunosorbent assay (ELISA). Seroconversion patterns were evaluated by estimating probabilities of seroconversion along three time points based on the transition pathways by using a first-order Markov chain process and empirical Bayesian estimates. There was no difference in serum IgG levels or seropositivity rates to gp15 between cases and controls across all time points in children or in IgG levels to this antigen between mothers of cases and controls. The most common transition pathway can be described as a seronegative child at 3.5 months who seroconverts at 9 months and remains seropositive at 24 months. This pattern remained stable irrespective of the serological status of the mother or the case or control status of the child. Children were most likely to be exposed to Cryptosporidium for the first time between the ages of 3 and 9 months, and most of the children seroconverted by 24 months. The high degree of seroconversion among control children is suggestive of high rates of asymptomatic transmission in this region.
Project description:Although Cryptospridium hominis is the dominant Cryptosporidium species infecting humans, immune responses to cognate antigens in C. hominis-infected persons have not been reported. We investigated antibody responses to the immunodominant gp15 antigen from C. hominis and C. parvum, in C. hominis-infected Bangladeshi children less than five years of age with diarrhea (cases) and uninfected children with diarrhea (controls). We also investigated polymorphisms in the C. hominis gp15 sequence from cases. Serum IgG responses to gp15 from both species were significantly greater in cases than controls. In spite of polymorphisms in the gp15 sequence, there was a significant correlation between antibody levels to gp15 from both species, indicating cross-reactivity to conserved epitopes. Cases with acute diarrhea had a significantly greater serum IgA response to gp15 compared with those with persistent diarrhea, suggesting that this response may be associated with protection from prolonged disease. These findings support further investigation of gp15 as a vaccine candidate.
Project description:Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention.Children (N = 497) were recruited at birth in semiurban slums in Vellore, India, and followed for 3 years with twice-weekly home visits. Stool samples were collected every 2 weeks and during diarrheal episodes were tested for Cryptosporidium species by polymerase chain reaction (PCR). Serum samples obtained every 6 months were evaluated for seroconversion, defined as a 4-fold increase in immunoglobulin G directed against Cryptosporidium gp15 and/or Cp23 antigens between consecutive sera.Of 410 children completing follow-up, 397 (97%) acquired cryptosporidiosis by 3 years of age. PCR identified 1053 episodes of cryptosporidiosis, with an overall incidence of 0.86 infections per child-year by stool and serology. The median age for the first infection was 9 (interquartile range, 4-17) months, indicating early exposure. Although infections were mainly asymptomatic (693 [66%]), Cryptosporidium was identified in 9.4% of diarrheal episodes. The proportion of reinfected children was high (81%) and there was clustering of asymptomatic and symptomatic infections (P < .0001 for both). Protection against infection increased with the order of infection but was only 69% after 4 infections. Cryptosporidium hominis (73.3%) was the predominant Cryptosporidium species, and there was no species-specific protection.There is a high burden of endemic cryptosporidiosis in southern India. Clustering of infection is suggestive of host susceptibility. Multiple reinfections conferred some protection against subsequent infection.
Project description:Cryptosporidium is a major cause of diarrhea in children in developing countries. However, there is no vaccine available and little is known about immune responses to protective antigens. We investigated antibody responses to p23, a putative vaccine candidate, in children in Bangladesh with cryptosporidiosis and diarrhea (cases) and uninfected children with diarrhea (controls), and p23 gene polymorphisms in infecting species. Serum IgM, IgG, and IgA responses to p23 were significantly greater in cases than controls after three weeks of follow-up. Cases with acute diarrhea had significantly greater serum IgA and IgM responses than those with persistent diarrhea, which suggested an association with protection from prolonged disease. The p23 sequences were relatively conserved among infecting species and subtype families. Although most children were infected with Cryptosporidium hominis, there was a cross-reactive antibody response to C. parvum antigen. These results support further development of p23 as a vaccine candidate.
Project description:BACKGROUND AND AIMS: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P). METHODS: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease. RESULTS: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease. CONCLUSIONS: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.
Project description:BACKGROUND & AIMS:Early nutrition may affect the risk of celiac disease. We investigated whether amount of gluten in diet until 2 years of age increases risk for celiac disease. METHODS:We performed a 1-to-3 nested case-control study of 146 cases, resulting in 436 case-control pairs matched for sex, birth year, and HLA genotype generated from Swedish children at genetic risk for celiac disease. Newborns were annually screened for tissue transglutaminase autoantibodies (tTGA). If tested tTGA positive, time point of seroconversion was determined from frozen serum samples taken every 3 months. Celiac disease was confirmed by intestinal biopsies. Gluten intake was calculated from 3-day food records collected at ages 9, 12, 18 and 24 months. Odds ratios (OR) were calculated through conditional logistic regression. RESULTS:Breastfeeding duration (median, 32 wk) and age at first introduction to gluten (median, 22 wk) did not differ between cases and tTGA-negative controls. At the visit before tTGA seroconversion, cases reported a larger intake of gluten than controls (OR, 1.28; 95% confidence interval [CI], 1.13-1.46; P = .0002). More cases than controls were found in the upper third tertile (ie, >5.0 g/d) before they tested positive for tTGA seroconversion than controls (OR, 2.65; 95% CI, 1.70-4.13; P < .0001). This finding was similar in children homozygous for DR3-DQ2 (OR, 3.19; 95% CI, 1.61-6.30; P = .001), heterozygous for DR3-DQ2 (OR, 2.24; 95% CI, 1.08-4.62; P = .030), and for children not carrying DR3-DQ2 (OR, 2.43; 95% CI, 0.90-6.54; P = .079). CONCLUSIONS:The amount of gluten consumed until 2 years of age increases the risk of celiac disease at least 2-fold in genetically susceptible children. These findings may be taken into account for future infant feeding recommendations.
Project description:Cryptosporidium parvum and Cryptosporidium hominis isolates from sporadic, drinking water-associated, and intrafamilial human cases together with C. parvum isolates from sporadic cases in livestock were collected in the United Kingdom between 1995 and 1999. The isolates were characterized by analysis of three microsatellite markers (ML1, GP15, and MS5) using PCR amplification. Within C. hominis, four alleles were detected within the GP15 and MS5 loci, and a single type was detected with ML1. C. parvum was more polymorphic; 12 alleles were detected with GP15, 6 were detected with MS5, and 3 were detected with ML1. Multilocus analysis of polymorphisms within the three microsatellite loci was combined with those reported previously for an extrachromosomal small double-stranded RNA. Forty multilocus types were detected within these two species: 9 were detected in C. hominis, and 31 were detected in C. parvum. In C. hominis, heterogeneity was almost exclusively found in samples from sporadic cases. Similarity analysis identified three main groups within C. parvum, and the group that predominated in human infection was also found in livestock. Multilocus types of C. parvum previously identified only in humans were not detected in livestock. Isolates of both C. hominis and C. parvum from separate waterborne outbreaks were genetically homogeneous, suggesting preferential or point source transmission of certain types of these two species of parasites.
Project description:BACKGROUND:The importance of Cryptosporidium as a pediatric enteropathogen in developing countries is recognized. METHODS:Data from the Global Enteric Multicenter Study (GEMS), a 3-year, 7-site, case-control study of moderate-to-severe diarrhea (MSD) and GEMS-1A (1-year study of MSD and less-severe diarrhea [LSD]) were analyzed. Stools from 12,110 MSD and 3,174 LSD cases among children aged <60 months and from 21,527 randomly-selected controls matched by age, sex and community were immunoassay-tested for Cryptosporidium. Species of a subset of Cryptosporidium-positive specimens were identified by PCR; GP60 sequencing identified anthroponotic C. parvum. Combined annual Cryptosporidium-attributable diarrhea incidences among children aged <24 months for African and Asian GEMS sites were extrapolated to sub-Saharan Africa and South Asian regions to estimate region-wide MSD and LSD burdens. Attributable and excess mortality due to Cryptosporidium diarrhea were estimated. FINDINGS:Cryptosporidium was significantly associated with MSD and LSD below age 24 months. Among Cryptosporidium-positive MSD cases, C. hominis was detected in 77.8% (95% CI, 73.0%-81.9%) and C. parvum in 9.9% (95% CI, 7.1%-13.6%); 92% of C. parvum tested were anthroponotic genotypes. Annual Cryptosporidium-attributable MSD incidence was 3.48 (95% CI, 2.27-4.67) and 3.18 (95% CI, 1.85-4.52) per 100 child-years in African and Asian infants, respectively, and 1.41 (95% CI, 0.73-2.08) and 1.36 (95% CI, 0.66-2.05) per 100 child-years in toddlers. Corresponding Cryptosporidium-attributable LSD incidences per 100 child-years were 2.52 (95% CI, 0.33-5.01) and 4.88 (95% CI, 0.82-8.92) in infants and 4.04 (95% CI, 0.56-7.51) and 4.71 (95% CI, 0.24-9.18) in toddlers. We estimate 2.9 and 4.7 million Cryptosporidium-attributable cases annually in children aged <24 months in the sub-Saharan Africa and India/Pakistan/Bangladesh/Nepal/Afghanistan regions, respectively, and ~202,000 Cryptosporidium-attributable deaths (regions combined). ~59,000 excess deaths occurred among Cryptosporidium-attributable diarrhea cases over expected if cases had been Cryptosporidium-negative. CONCLUSIONS:The enormous African/Asian Cryptosporidium disease burden warrants investments to develop vaccines, diagnostics and therapies.
Project description:Diarrhea is a leading contributor to childhood morbidity and mortality in sub-Saharan Africa. Given the challenge of blinding most water, sanitation, and hygiene (WASH) interventions, diarrheal disease outcome measures in WASH intervention trials are subject to potential bias and misclassification. Using the platform of a cluster-randomized controlled trial of a household-based drinking water filter in western province, Rwanda, we assessed the impact of the drinking water filter on enteric seroconversion in young children as a health outcome and examined the association between serologic responses and caregiver-reported diarrhea. Among the 2,179 children enrolled in the trial, 189 children 6-12 months of age were enrolled in a nested serology study. These children had their blood drawn at baseline and 6-12 months after the intervention was distributed. Multiplex serologic assays for Giardia, Cryptosporidium, Entamoeba histolytica, norovirus, Campylobacter, enterotoxigenic Escherichia coli and Vibrio cholerae were performed. Despite imperfect uptake, receipt of the water filter was associated with a significant decrease in seroprevalence of IgG directed against Cryptosporidium parvum Cp17 and Cp23 (relative risk [RR]: 0.62, 95% confidence interval [CI]: 0.44-0.89). Serologic responses were positively associated with reported diarrhea in the previous 7 days for both Giardia intestinalis (RR: 1.94, 95% CI: 1.04-3.63) and C. parvum (RR: 2.21, 95% CI: 1.09-4.50). Serologic responses for all antigens generally increased in the follow-up round, rising sharply after 12 months of age. The water filter is associated with reduced serologic responses against C. parvum, a proxy for exposure and infection; therefore, serologic responses against protozoa may be a suitable health outcome measure for WASH trials among children with diarrhea.
Project description:The CRM197-conjugated 7-valent pneumococcal vaccine (PCV7) is protective against vaccine serotype disease and nasopharyngeal carriage. Data on PCV7-induced mucosal antibodies in relation to systemic or natural anticapsular antibodies are scarce.In a randomized controlled setting, children received PCV7 at age 2 and 4 months (2-dose group), at age 2, 4 and 11 months (2+1-dose group) or no PCV7 (control group). From 188 children paired saliva samples were collected at 12 and 24 months of age. From a subgroup of 15 immunized children also serum samples were collected. IgG and IgA antibody-levels were measured by multiplex immunoassay.At 12 months, both vaccine groups showed higher serum and saliva IgG-levels against vaccine serotypes compared with controls which sustained until 24 months for most serotypes. Salivary IgG-levels were 10-20-fold lower compared to serum IgG, however, serum and saliva IgG-levels were highly correlated. Serum and salivary IgA-levels were higher in both vaccine groups at 12 months compared with controls, except for serotype 19F. Higher salivary IgA levels remained present for most serotypes in the 2+1-dose group until 24 months, but not in the 2-dose group. Salivary IgA more than IgG, increased after documented carriage of serotypes 6B, 19F and 23F In contrast to IgG, salivary IgA-levels were comparable with serum, suggesting local IgA-production.PCV7 vaccination results in significant increases in salivary IgG and IgA-levels, which are more pronounced for IgG when compared to controls. In contrast, salivary anticapsular IgA-levels seemed to respond more to natural boosting. Salivary IgG and IgA-levels correlate well with systemic antibodies, suggesting saliva might be useful as potential future surveillance tool.
Project description:Probiotics have a possible role in the treatment of pediatric acute gastroenteritis. We report the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on intestinal function, immune response, and clinical outcomes in Indian children with cryptosporidial or rotavirus diarrhea.Children with gastroenteritis aged 6 months to 5 years, testing positive for either rotavirus or Cryptosporidium species in stool (coinfections were excluded), were randomized to LGG (ATCC 53103) or placebo, once daily for 4 weeks. Baseline demographic and clinical details were obtained. Sera were tested for immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies to Cryptosporidium and rotavirus, and the lactulose to mannitol ratio for intestinal permeability was determined at baseline and at the end of follow-up.Of the 124 children enrolled, 82 and 42 had rotavirus and cryptosporidial diarrhea, respectively. Median diarrheal duration was 4 days; one-third of the children had severe diarrhea. Baseline and clinical parameters were comparable between children receiving LGG and placebo. At the end of follow-up, fewer children with rotavirus diarrhea on LGG had repeated diarrheal episodes (25% vs 46%; P = .048) and impaired intestinal function (48% vs 72%; P = .027). Significant increase in IgG levels postintervention (456 vs 2215 EU; P = .003) was observed in children with rotavirus diarrhea receiving LGG. Among children with cryptosporidial diarrhea, those receiving LGG showed significant improvement in intestinal permeability.LGG has a positive immunomodulatory effect and may be useful in decreasing repeated episodes of rotavirus diarrhea. Improvement in intestinal function in children with rotavirus and cryptosporidial gastroenteritis emphasizes the role of probiotics in treating intestinal impairment after infection.CTRI/2010/091/000339.