An Analytical Method for Assessing Stage-Specific Drug Activity in Plasmodium vivax Malaria: Implications for Ex Vivo Drug Susceptibility Testing.

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC₅₀) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC₅₀ for CQ. Our findings indicate that EC₅₀ values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials. Author Summary: The schizont maturation test (SMT) was developed to monitor drug resistance in malaria parasites. The SMT examines differences in the rate of parasite development when exposed to different drug concentrations, providing an estimate of drug efficacy. While the assay is effective when examining resistance in Plasmodium falciparum, there are concerns regarding its suitability for testing other malaria species, particularly if the drug only targets particular life-cycle stages of the parasite. Blood samples taken from Plasmodium vivax infected individuals exhibit significant heterogeneity in the parasite life-cycle stages present. If a drug targets the early stage parasites, but only late stage parasites are present in the sample, the test will show an erroneously high degree of resistance. In this study, we estimate thresholds which can be used to identify when test results can be considered accurate should the drug being tested only affect specific life stages of the parasites. Chloroquine is used as a case study but the method developed also allows the identification of stage-specific activity in other malarial drugs in P. vivax. For field researchers, this threshold modelling approach will allow for increased confidence in the reliability of P. vivax resistance results and provides a novel means of detecting stage-specific drug activity for new antimalarials. [ABSTRACT FROM AUTHOR]