Lysis of pathogenic and nonpathogenic Entamoeba histolytica by human complement: methodological analysis

The effect of nonimmune human serum on Entamoeba histolytica trophozoites was studied: (a) using whole serum in the presence of Ca and Mg ions allowing complement activation via both the alternative and classical pathways or in the presence of MgEGTA permitting alternative pathway activation only; (b) using different E. histolytica isolates; (c) varying serum and trophozoite concentrations and the time of incubation; and (d) using three different methods to quantify lysis, i.e., microscopic inspection, flow cytometry and 111In release. All three methods yielded similar results, with flow cytometry being most sensitive in identifying membrane damage and 111In release being most valid in determining cell death. Microscopic analysis was reliable only when a chamber was used to calculate the number of complement treated cells in relation to the initial cell count. E. histolytica isolates were classified into three groups according to their susceptibility to lysis by complement: (i) pathogenic isolates after long term cultivation in vitro were susceptible; (ii) pathogenic isolates after recent in vivo passage were less susceptible; and (iii) nonpathogenic isolates were nearly unaffected by exposure to the alternative pathway alone. The extent of lysis of the various isolates correlated with the degree of complement consumption in the serum samples, suggesting that unlysed isolates did not activate complement under the conditions employed. In general, lysis of susceptible trophozoites increased with the serum concentration and with the time of incubation. However, when the trophozoite concentration was 10(6)/ml or higher, lysis no longer reflected complement susceptibility because of exhaustion of the complement supply.