Genetic polymorphisms in plasmodium falciparum associated with resistance to artemisinin combination therapy: complexity of infection during short-term culture and characterization of in vitro sensitivity in Kampala Uganda

Abstract

Background Artemisinin-based combination therapy (ACT) is currently advocated in Africa as a means of improving treatment efficacy and slowing the development of drug resistance. However, the selection of resistant parasites, particularly to artemisinin partner drugs, remains a concern. We describe a series of studies focusing on molecular determinants of Plasmodium falciparum resistance to antimalarial drugs and their correlation with the in vitro susceptibility to key antimalarials in practice. This work was also extended to address the important questions about the polyclonal diversity of patient isolates as they were adapted to in vitro culture.

Objectives i). Assessed changes in occurrence of various polymorphisms of a drug resistant allele’s of pfmdr-1 and pfcrt between baseline and new infections during therapy with artesunateamodiaquine (AS/AQ). ii). Evaluated changes in complexity of infection (COI) during culture. iii). Assessed the impact of various amodiaquine containing regimens on the sensitivity of recurrent P. falciparum isolates. iv). Determined in vitro senstivity patterns of P. falciparum isolates in Uganda against various antimalarial drugs. v). Evaluated associations between parasite genetic polymorphisms, in vitro drug sensitivity, and clinical outcomes after antimalarial combination therapy.

Methods Polymorphisms of the pfmdr-1 and pfcrt genes known to play a role in altered drug sensitivity to some antimalarials were analysed by nested PCR and restriction fragment length polymorphism techniques. Complexity of infection Plasmodium falciparum isolates were determined based on msp-2 polymorphisms using nested PCR. For the in vitro sensitivity studies, IC50’s for P. falciparum isolates were measured using an HRP-2-based ELISA.

Results a).Treatment with the combination of (AS/AQ) resulted in significant enrichment of isolates harboring the N86Y and D1246Y mutations, and the wild type residueY184, in the pfmdr1gene. b). Using the msp-2 gene marker, isolates were found to lose certain genotypes in vitro during culture adaptation, leading to a reduced complexity of infection (COI). Some isolates were also found to harbor new genotypes that appeared after day 0, suggesting that in some cases parasites classified as new infections post-treatment were actually present at the time of treatment initiation and would therefore be misclassified. c). Antimalarial regimens containing amodiaquine were associated with subsequent infections displaying a reduced in vitro susceptibility (although this was not reflected in higher rates of treatment failure). d). Patterns of cross resistance, or inverse susceptibility, were observed between several antimalarial drugs.

Conclusion Novelty of this study is the analysis of changes of complexity of infection during isolate adaptation to in vitro culture, and evidence for decreased susceptibility to amodiaquine pending treatment with regimens containing this drug (though these changes did not lead to reduced clinical efficacy).The study also provides important baseline data to further evaluate the in vitro susceptibility of parasite strains in Kampala Uganda and assess any signs of emerging resistance. Lastly, provides useful information on the prevalence of mutations in pfcrt and pfmdrI and their association with elevated drugsIC50 values in vitro.