Fitness Consequences of Plasmodium falciparum pfmdr1 Polymorphisms Inferred from Ex Vivo Culture of Ugandan Parasites

Abstract

bPolymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene impact sensitivity to multiple antimalarials. In Africa, polymorphisms at N86Y and D1246Y are common and have various impacts on sensitivity to different drugs. To gain insight into the fitness consequences of these polymorphisms, we cultured parasites isolated from children with malaria in Tororo, Uganda, where the multiplicity of infection is high, and used pyrosequencing to follow polymorphism prevalences in culture over time. Of 71 cultures, parasites in 69 were successfully analyzed at N86Y and parasites in 68 were successfully analyzed at D1246Y over 3 to 36 days of culture. For position 86, the sequences of 39/69 (56.5%) parasites remained stable (>90% prevalence over 2 to 17 time points), with 82.1% of these being stable for the 86Y mutation. For position 1246, the sequences of 31/68 (45.6%) parasites remained stable, with 64.5% of these being stable for the wild-type D1246 sequence (P ‫؍‬ 0.0002 for comparison of stable mutant genotypes for the two alleles). Defining allele selection as a >15% change in prevalence between the first and last samples assessed, for position 86, 11 samples showed selection, with selection toward 86Y occurring in 72.7% of alleles; for position 1246, 14 samples showed selection, with selection toward D1246 occurring in 64.3% of alleles (P ‫؍‬ 0.11 for comparison of selection of mutations at the two alleles). Among the 7 samples with selection at both alleles, 5 showed selection for both 86Y and D1246. Overall, consistent trends in the direction of selection were seen, although differences were not statistically significant. Our results suggest fitness advantages for parasites with the pfmdr1 86Y mutation and wild-type D1246, highlighting the complex interplay between drug resistance and fitness in malaria parasites. (This study has been registered at ClinicalTrials-.gov under registration no. NCT00948896 and NCT00993031.)

Competition between Plasmodium falciparum strains in clinical infections during in vitro culture adaptation

“…We evaluated the complexity of infections using the msp1 block 2 allele types and several polymorphic sites of genes associated with drug resistance. Consistent with findings from other endemic areas (Nsobya et al, 2008; Ochong et al, 2013), we observed a trend of gradual decrease of parasite populations during culture, suggesting that some parasites are less fit. A large proportion of the parasites essentially became monoclonal during in vitro culture for 3–4 weeks.…”

“…Most studies on in vitro parasite competition performed thus far focused on genetically defined laboratory strains (Hayward et al, 2005; Peters et al, 2002) and clinical isolates from high-transmission areas where complexity of infection is high (Nsobya et al, 2008; Ochong et al, 2013). In this study, we assessed the competition between parasite populations in mixed clinical infections from a hypoendemic area near the China-Myanmar border during in vitro culture adaption.…”

“…In co-culture experiments, the 184F, 1034C, 1042D, and 1246Y mutations all incur a fitness cost relative to the wild-type (Hayward et al, 2005). During ex vivo culture of African clinical samples, parasites carrying the mutant 86Y and the wild-type D1246 appeared to have fitness advantages (Ochong et al, 2013). Parasites with increased copy number of the pfmdr1 gene such as those selected for resistance to mefloquine and artemisinin family drugs are less fit (Chen et al, 2010; Cui et al, 2012; Preechapornkul et al, 2009).…”

“…In nature, competition between parasite strains in clinical isolates is definitely much more complex. During culture adaptation of clinical isolates, some parasite clones are often lost and parasites with certain mutations in genes conferring drug resistance may be selected due to fitness advantages (Nsobya et al, 2008; Ochong et al, 2013). In this study, we collected P. falciparum clinical isolates in a malaria hypoendemic area and followed the dynamics of the parasite populations in the mixed infections during in vitro culture adaptation.…”

The interplay between drug resistance and fitness in malaria parasites

“…Considering impacts of pfmdr1 polymorphisms on the fitness of clinical isolates, P. falciparum was cultured from children with malaria in a region of Uganda with high multiplicity of infection (and thus high likelihood of mixed infections), and changes in the prevalence of the pfmdr1 N86Y and D1246Y alleles were followed over time (Ochong et al ., ). Most cultures did not undergo changes in culture, but for those that did show selection 8/11 selected towards mutant 86Y, 9/14 selected towards wild‐type D1246, and 5/7 with selection at both alleles selected towards 86Y and D1246.…”