Children develop Immunity to cryptosporidiosis in a high transmission intensity area

Abstract

Background: Cryptosporidium is one of the top causes of diarrhea in Bangladesh infants. Cryptosporidium infections lead to the production of antibody immune responses, which were associated with a decrease in parasite burden and decreased disease severity in subsequent infections.

Methods: We conducted a longitudinal study of cryptosporidiosis from birth to five years of age in an urban slum of Dhaka Bangladesh. We then retrospectively tested the concentration of anti-Cryptosporidium Cp17 or Cp23 IgA in surveillance stool samples collected from 54 children during their first 3 years of life by enzyme-linked immunosorbent assay (ELISA). We also assessed the concentration of both IgA and IgG antibodies specific to Cryptosporidium Cp17 and Cp23 in the concentration of anti-Cryptosporidium Cp17 or Cp23 IgA and IgG antibodies in the children's plasma (1- 5 years).

Results: The seroprevalence of both anti- Cp23 and Cp17 antibodies was high at ≤ one year of age and reflected the exposure of these children in this community to cryptosporidiosis. In Bangladesh, the prevalence of cryptosporidiosis is high during the rainy season (June to October) but decreases during the dry season. In younger infants' plasma anti-Cp17 and Cp23 IgG and anti-Cp17 IgA levels were markedly increased during the rainy season in line with the higher initial exposure to the parasite at this time. Both anti-Cp17, anti-Cp23 fecal IgA and the parasite burden declined during repeat infections.

Conclusions: We found that anti-Cryptosporidium plasma and fecal antibody levels in children could contribute to the decrease in new infections in this study population.

Keywords: Cp17; Cp23; Cryptosporidium; Cryptosporidium hominis.

Figure 1. Flow diagram of Birth COHORT Study

Figure 2

Incidence of Diarrheal Cryptosporidiosis in the Mirpur cohort. Data shown is only from the 286 children who remained in the study for the entire 5 years; Diarrheal cryptosporidiosis phenotype is defined as an infection in which symptoms were coincident with the qPCR detection of Cryptosporidium in fecal DNA. Positive samples were classified as a separate infection only if occurring greater than 65 days after the preceding positive sample. Infections occurring within each 6-month interval were grouped and the number shown on the Y axis. X axis indicates child age in days. Although cryptosporidiosis was known to decrease in frequency diarrheal stool collection and analysis took place > 3 years of age although there was a decrease in surveillance. All of the children in the group of 286 children shown in this graph had reached 4 years of age prior to the COVID-19 pandemic-related hiatus in surveillance sample collection (Supplemental Table 1).

Figure 3:

Decline in the frequency of sub-clinical cryptosporidiosis in the Mirpur cohort. A) Sub-clinical cryptosporidiosis phenotype is defined as an infection in which no symptoms were observed at the time of qPCR detection of Cryptosporidium in fecal DNA. Stool surveillance was decreased after the children reached 3 years of age with monthly stool samples being collected from only a third of the infants, all other collections occurred bi-annually. Y axis indicates the number of fecal samples collected during each 30.4-day window (year end is indicated by the filled bar). The graph clearly shows the transition at year 3 from continuing monthly surveillance to a proportion of the cohort switching to bi-annual cross-sectional surveillance B) Sub-clinical cryptosporidiosis is expressed as a percentage of the number of samples collected to account for the differences in collection frequency. Y-axis indicates the percentage of infections binned into 6-month intervals as shown on the X axis which indicates the child’s age in years. The dotted lines indicate the 95% CI of the non-linear curve fit line.

Figure 3:

Decline in the frequency of sub-clinical cryptosporidiosis in the Mirpur cohort. A) Sub-clinical cryptosporidiosis phenotype is defined as an infection in which no symptoms were observed at the time of qPCR detection of Cryptosporidium in fecal DNA. Stool surveillance was decreased after the children reached 3 years of age with monthly stool samples being collected from only a third of the infants, all other collections occurred bi-annually. Y axis indicates the number of fecal samples collected during each 30.4-day window (year end is indicated by the filled bar). The graph clearly shows the transition at year 3 from continuing monthly surveillance to a proportion of the cohort switching to bi-annual cross-sectional surveillance B) Sub-clinical cryptosporidiosis is expressed as a percentage of the number of samples collected to account for the differences in collection frequency. Y-axis indicates the percentage of infections binned into 6-month intervals as shown on the X axis which indicates the child’s age in years. The dotted lines indicate the 95% CI of the non-linear curve fit line.

Figure 4

Infections and samples collected in a subset of infants selected for additional analysis A) Infections Child Within 5 Years of Birth Y axis indicates the number of infections X axis the individual infants selected for this analysis B) On the X-axis is the child’s age in years. On the Y-axis is the Child’s unique ID. Green lines represent Plasma Collection time points, the red dots represent stool samples that tested positive for Crypto, and the blue dots represent stool samples that tested positive for both Cp17 and Cp23 IgA & IgG.

Figure 5

Analysis of the seasonal IgG ( A & B) and IgA antibodies (C-F) to the Cp17 (A,C,E) and Cp23 (B,D,F) antigens in plasma (A-D) and fecal material (E&F). Relative antibody levels are shown on the Y-axis with the children’s age in days shown on the X-axis. Blue symbols are samples collected during the Bangladesh rainy season (June to October); red symbols dry season. Linear regression P-values and R² values are shown for IgG and IgA, as well as a line (colored per season) and confidence intervals (gray bands) fit to each.

Figure 6

Parasite burden and anti-Cp17 and Cp23 fecal IgA decline in repeat infections. Analysis was restricted to samples in which both fecal IgA was able to be measured and the sample had a Cryptosporidium positive Cq value. X values: The number of repeat Cryptosporidium infections per child was defined using the entire data set as previously defined (Kabir et al 2021). A) In line with previous observations the parasite burden was decreased during repeat infections. Fecal IgA B) anti-Cp17 and C) anti-Cp23 also declined during repeat infections. (*) p value is less than 0.05, (**) p value less than 0.01, (***) p value is less than 0.00. Analysis was restricted to paired 1^st and 2^nd infections (n=19) and repeated using the mixed-effect model (Supplemental Figure 2) p=0.0041