Intra-host dynamics of co-infecting parasite genotypes in asymptomatic malaria patients

Malaria-infected individuals often harbor mixtures of genetically distinct parasite genotypes. We studied intra-host dynamics of parasite genotypes co-infecting asymptomatic adults in an area of intense malaria transmission in Chikhwawa, Malawi. Serial blood samples (5 ml) were collected over seven consecutive days from 25 adults with asymptomatic Plasmodium falciparum malaria and analyzed to determine whether a single peripheral blood sample accurately captures within-host parasite diversity. Blood samples from three of the participants were also analyzed by limiting dilution cloning and SNP genotyping of the parasite clones isolated to examine both the number and relatedness of co-infecting parasite haplotypes. We observed rapid turnover of co-infecting parasite genotypes in 88% of the individuals sampled (n = 22) such that the genetic composition of parasites infecting these individuals changed dramatically over the course of seven days of follow up. Nineteen of the 25 individuals sampled (76%) carried multiple parasite genotypes at baseline. Analysis of serial blood samples from three of the individuals revealed that they harbored 6, 12 and 17 distinct parasite haplotypes respectively. Approximately 70% of parasite haplotypes recovered from the three extensively sampled individuals were unrelated (proportion of shared alleles <83.3%) and were deemed to have primarily arisen from superinfection (inoculation of unrelated parasite haplotypes through multiple mosquito bites). The rest were related at the half-sib level or greater and were deemed to have been inoculated into individual human hosts via parasite co-transmission from single mosquito bites. These findings add further to the growing weight of evidence indicating that a single blood sample poorly captures within-host parasite diversity and underscore the importance of repeated blood sampling to accurately capture within-host parasite ecology. Our data also demonstrate a more pronounced role for parasite co-transmission in generating within-host parasite diversity in high transmission settings than previously assumed. Taken together, these findings have important implications for understanding the evolution of drug resistance, malaria transmission, parasite virulence, allocation of gametocyte sex ratios and acquisition of malaria immunity.