Longitudinal analysis of cryptosporidium species-specific immunoglobulin G antibody responses in Peruvian children

Abstract

Cryptosporidium species are ubiquitous in the environment and are frequently detected in the stools of children who live where sanitation conditions are poor. To better characterize the immune response to these parasites, we monitored immunoglobulin G (IgG) antibody levels in a cohort of children from Lima, Peru. Two new enzyme-linked immunosorbent assays based on the C. parvum (bovine, subtype IIa) Iowa strain 17-kDa and 27-kDa antigens were used to measure IgG antibody levels in longitudinal serum samples. Antibody responses were detected during infections with C. parvum, C. felis, and C. meleagridis and with four different subtypes of C. hominis. We also noted that the magnitude of the antibody response was related to the number of previous infections and that older children generally had higher levels of antibodies to the two C. parvum antigens. Antibody responses were not associated with infections with either Cyclospora sp. or Giardia sp. We believe the antibody assays will be important tools for monitoring the success of future public health interventions.

FIG. 1.

Representative profiles showing the diagnosis of Cryptosporidium episodes by stool assay and by serologic antibody assay. Longitudinal serum samples from four children (A to D) were analyzed by ELISA for antibodies to the 17-kDa (dashed line with closed square) and 27-kDa (solid line with closed circle) antigens as described in Materials and Methods. Responses are presented in arbitrary units based upon a standard curve serum with a 6,400 maximum value. An asterisk indicates a serum sample that meets the definition for a positive antibody response as described in Materials and Methods. Regularly collected stool samples from the children were screened for the presence of Cryptosporidium oocysts by acid-fast microscopy, and the first day of each identified infection episode is indicated by an arrow. Microscopy-positive stool samples were subjected to PCR and RFLP analysis (46) in order to identify the genotype of Cryptosporidium parasite: C. parvum of the bovine genotype (B), C. felis (F), C. hominis or human genotype (H), and C. meleagridis (M) were detected. Stool samples that were unavailable for analysis are indicated by a U.

FIG. 2.

The magnitude of the antibody response is correlated with age. The first available sample collected from each child (n = 61, 71, 63, and 28) in each age group (<12 months, 12 to 23 months, 24 to 35 months, and 36 to 47 months, respectively) was analyzed for antibodies to the 27-kDa (A) and 17-kDa (B) antigens by ELISA as previously described. Boxes enclose the 25th to 75th percentiles, whiskers indicate the 10th to 90th percentiles, and closed circles indicate the 5th and 95th percentiles for each age group. The medians (indicated by bars) are 120, 162, 250, and 359 arbitrary units for the 27-kDa antigen assay and 35, 35, 63, and 116 arbitrary units for the 17-kDa antigen assay, respectively. The first available sample collected from each child in each age group was also used to calculate a prevalence rate (C). A child was considered to be positive at a given time point for antibodies to Cryptosporidium if the antibody levels to both the 27-kDa and 17-kDa antigens were above the cutoff values (>160 and >57 arbitrary units, respectively).

FIG. 3.

The magnitude of the antibody response increases with experience of infection. A total of 108 Cryptosporidium infection episodes were diagnosed by serologic assay using the definitions described in Materials and Methods. Antibody levels to the 27-kDa (A) and 17-kDa (B) antigens were grouped according to the number of previous infections (0, n = 39; 1, n = 36; >1, n = 33). Boxes enclose the 25th to 75th percentiles, whiskers indicate the 10th to 90th percentiles, and closed circles indicate the 5th and 95th percentiles for each age group. The medians (indicated by bars) are 464, 1,380, and 1,414 arbitrary units, respectively, for the 27-kDa assay and 166, 397, and 435 arbitrary units, respectively, for the 17-kDa assay.