Impact of schistosome infection on Plasmodium falciparum Malariometric indices and immune correlates in school age children in Burma Valley, Zimbabwe

Abstract

A group of children aged 6-17 years was recruited and followed up for 12 months to study the impact of schistosome infection on malaria parasite prevalence, density, distribution and anemia. Levels of cytokines, malaria specific antibodies in plasma and parasite growth inhibition capacities were assessed. Baseline results suggested an increased prevalence of malaria parasites in children co-infected with schistosomiasis (31%) compared to children infected with malaria only (25%) (p = 0.064). Moreover, children co-infected with schistosomes and malaria had higher sexual stage geometric mean malaria parasite density (189 gametocytes/µl) than children infected with malaria only (73/µl gametocytes) (p = 0.043). In addition, a larger percentage of co-infected children (57%) had gametocytes as observed by microscopy compared to the malaria only infected children (36%) (p = 0.06). There was no difference between the two groups in terms of the prevalence of anemia, which was approximately 64% in both groups (p = 0.9). Plasma from malaria-infected children exhibited higher malaria antibody activity compared to the controls (p = 0.001) but was not different between malaria and schistosome plus malaria infected groups (p = 0.44) and malaria parasite growth inhibition activity at baseline was higher in the malaria-only infected group of children than in the co-infected group though not reaching statistical significance (p = 0.5). Higher prevalence and higher mean gametocyte density in the peripheral blood may have implications in malaria transmission dynamics during co-infection with helminths.

Figure 1. Study Design.

The arrows indicate the different times at which urine, blood and stool samples were obtained from study participants for diagnosis. The malaria seasons at baseline and follow up times are indicated as well as treatment of schistosome infected children with praziquantel (PZQ).

Figure 2. Numbers of Recruited Participants and Cohort.

Recruitment, loss to follow up and the follow up of cohort after grouping by diagnosis at baseline survey (T0) and the malaria season status are shown. The numbers of subjects grouped into various diagnostic groups were 485 at T0, 321 at T6 and 281 at T12. At T6 and T12 the numbers of children that tested positive for malaria only were 9 and 14, respectively and those tested positive for malaria and schistosomes were 33 and 21, respectively. A subset of subjects from each of the 4 categories based on the baseline diagnosis was maintained at subsequent follow-up time points T6 and T12. These subjects (numbers in parentheses) were chosen based on the availability of adequate serum volumes for follow-up laboratory studies. Only a representative fraction from the uninfected (N) and schistosome only (S) infected children were included in analyses due to limited reagent quantities, especially recombinant antigens used in ELISA.

Figure 3. Effect of schistosome infection on malaria prevalence.

Total number of children testing positive for malaria at T0, T6 and T12 among schistosome positive (SP) and negative (SN) are shown on x-axis. The bars show the percentage values for each survey time point. A higher proportion of schistosome positive children (black bars) had malaria parasites at T0 (p = 0.064) and T6 (p = 0.053) compared to the schistosome negative children (white bars). At T12 the malaria prevalence was not different for the two groups.

Figure 4. Percentage distributions and geometric mean parasite densities of sexual (gametocyte) and asexual (ring) parasites in malaria-infected children.

Numbers inside bars represent geometric mean parasite density values for sexual (white bars) and asexual (black bars) in the M (n=14) and S+M (n=28) groups for a subset of subjects for which these data were available. Co-infected children had a higher proportion of gametocyte infection (p=0.06) and higher mean gametocyte density in blood (p=0.043) compared to malaria only infected children (M).

Figure 5. Mean hemoglobin levels and prevalence of anemic children for each diagnostic group at different sampling times.

At T0, differences in the hemoglobin levels (Figure 5a) in groups M and S+M were statistically significant (p<0.001 by t-test) as compared to group N. A comparison between M and S+M and groups N and S revealed non-significant p values, 0.743 and 0.183, respectively. Figure 5b shows results of proportion of anemic children in various groups, p values at T0 for differences between groups N and M, groups N and S+M and groups N and S were 0.01, 0.001, and 0.273, respectively. The numbers in each bar represent ‘N’ for each group and the error bars represent SEM.

Figure 6. Comparisons of mean OD ratio of IgG to crude and specific P. falciparum antigens at different time points.

The antibody levels were determined by ELISA from serum samples from children in various diagnostic groups. Generally antibody activity increased with time from T0 to T12 with higher activity in children who had malaria at baseline. Total number of serum samples for data on entire population in Figure 6a were T0 (n = 446), T6 (n = 342), and T12 (n = 310). Figure 6b shows data on 82 children in the cohort at all three time points (T0, T6, T12). Figure 6c shows antibody reactivity results of population sera to MSP-1 (n = 193) and AMA-1 (n = 198) at T0. Figure 6d shows results for cohort children to MSP-1 (n = 79) and AMA-1 (n = 81) at survey T0. Comparison of OD ratios between groups M and S+M or groups N and M for AMA-1 revealed p values of 0.006 and 0.001, respectively by Mann-Whitney test. Error bars represent SEM.

Figure 7. Comparisons of IgG2 and IgG3 isotype-specific antibody activity in serum from subjects in different groups.

Cohort sera samples from three time points (T0, T6 and T12) were analyzed by ELISA to detect IgG2 and IgG3 isotype-specific antibody activity to MSP-1 (Figures 7a and 7c, respectively) and AMA-1 (Figures 7b and 7d, respectively). Error bars represent SEM.

Figure 8. Mean growth inhibition activity of different groups at different malaria seasons and survey times.

Ability of subjects' plasma samples to inhibit P. falciparum growth from each group at T0 (Fig. 8a), T6 (Fig. 8b) and T12 (Fig. 8c) are shown. The box plots display in vitro percentage growth inhibition activities of P. falciparum by participants' plasma samples (at 10% concentration) after 48 h growth of synchronized W2 culture. The middle horizontal line in each box indicates the median percentage growth inhibition for each diagnostic group, and the box indicates the 25^th and 75^th percentiles. The whisker caps extending from each box indicate the minimum and maximum values. Individual marked points represent a few outlier values. Figure 8d represents mean growth inhibition data for the entire cohort at three transmission seasons (T0, T6, T12).