Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions

Abstract

The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections.

Fig. 1

Laboratory strains in mixed clone assays. Results were independent of clonal proportions (A, K1 25% with Dd2 75%; B, K1 50% with Dd2 50%; C, K1 75% with Dd2 25%). Solid lines represent clone growth from mixed cultures. Dotted lines are adjusted values from single-clone controls. Dd2 single-clone controls had a greater overall parasitaemia (not shown) but a lower adjusted parasitaemia than Dd2 in mixture groups. K1 showed the opposite trend. This is explained by a density-dependent growth rate and a shorter life cycle and faster multiplication rate of Dd2 versus K1.

Fig. 2

Native Peruvian strains in competitive assays; comparative growth of clones when alone versus co-cultured with FCR3. FAM fluorescence shows the growth of K1 genotype clones K1, 6390 and MZ1183. Solid line shades illustrate clones in a 50/50 co-culture with FCR3 whereas dotted lines represent single-clone controls.