Quantification of multiple infections of Plasmodium falciparum in vitro (Malaria Journal)

Background: Human malaria infections caused by the parasite Plasmodium falciparum often contain morethan one genetically distinct parasite. Despite this fact, nearly all studies of multiple strain P.falciparum infections have been limited to determining relative densities of each parasitewithin an infection. In light of this, new methods are needed that can quantify the absolutenumber of parasites within a single infection. Methods: A quantitative PCR (qPCR) method was developed to track the dynamic interaction of P.falciparum infections containing genetically distinct parasite clones in cultured red bloodcells. Allele-specific primers were used to generate a standard curve and to quantify theabsolute concentration of parasite DNA within multi-clonal infections. Effects on dynamicgrowth relationships between parasites under drug pressure were examined by treating mixedcultures of drug sensitive and drug resistant parasites with the anti-malarial drug chloroquineat different dosing schedules. Results: An absolute quantification method was developed to monitor the dynamics of P. falciparumcultures in vitro. This method allowed for the observation of competitive suppression, thereduction of parasites numbers due to the presence of another parasite, and competitiverelease, the improved performance of a parasite after the removal of a competitor. Thesestudies demonstrated that the presence of two parasites led to the reduction in density of atleast one parasite. containing both a drug resistant and drug sensitive parasites resulted in anincreased proportion of the drug resistant parasite. Moreover, following drug treatment, theresistant parasite experienced competitive release by exhibiting a fitness benefit greater thansimply surviving drug treatment, due to the removal of competitive suppression by thesensitive parasite. Conclusions: The newly developed assay allowed for the examination of the dynamics of two distinctclones in vitro; both competitive suppression and release were observed. A deeperunderstanding of the dynamic growth responses of multiple strain P. falciparum infections,with and without drug pressure, can improve the understanding of the role of parasiteinteractions in the spread of drug resistant parasites, perhaps suggesting different treatmentstrategies.