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Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr, pfdhps, pfmdr1, pfcrt, and pfK13, and genetic diversity was established from two known AmpSeq markers, cpmp and csp. Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P

Experimental studies on the biology of malaria parasites have been mostly based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of somePlasmodium falciparumclinical isolates, in analyses that focused on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points during several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and timepoints. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity although proportions of genotypes varied over time. Drug-resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (includingAP2-HS, EPACandSRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identifiedde novovariants not detected in the bulk isolate sequences. Interestingly, most of these were nonsense mutants and frameshifts disrupting the coding sequence ofEPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of Identity-By-Descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within parasite populations.

Background In areas where Plasmodium falciparum malaria is seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season. Methods In The Gambia, a cohort of 42 individuals with quantitative polymerase chain reaction-positive P falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate infection persistence. The influence of human host and parasitological factors was investigated. Results A large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16 of 40). At the start of the dry season, the majority of these persistent infections (82%) had parasite densities >10 p/µL compared to only 5.9% of short-lived infections. Persistent infections (59%) were also more likely to be multiclonal than short-lived infections (5.9%) and were associated with individuals having higher levels of P falciparum-specific antibodies (P = .02). Conclusions Asymptomatic persistent infections were multiclonal with higher parasite densities at the beginning of the dry season. Screening and treating asymptomatic infections during the dry season may reduce the human reservoir of malaria responsible for initiating transmission in the wet season.