Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal

Abstract

Background: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance.

Methods: Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1.

Results: Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 - Y184F - was associated with decreased parasite sensitivity to artemisinin.

Conclusions: Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population.

Figure 1

Validation of the DAPI ex vivo drug assay. A. Fluorescence intensity of maximum growth wells versus initial parasitaemia for parasites tested in the DAPI ex vivo drug assay. Pearson ρ = 0.47, linear slope P < 0.0001. B. Box plots showing signal-to-noise ratio (SNR) and Z’-factor for all assays. C-F. Bland-Altman plots showing differences between IC₅₀ values of each technical replicate vs. average IC₅₀ values for amodiaquine (C), artemisinin (D), chloroquine (E), and mefloquine (F). Horizontal lines indicate the mean difference in IC₅₀ values between replicates. Intra-class correlation coefficients (ICC, with corresponding 95% confidence intervals) are displayed on each graph. G-I. Comparison of ex vivo with in vitro IC₅₀ values for artemisinin (G), chloroquine (H), and mefloquine (I), among culture-adapted monoclonal parasites collected in 2009. Mean in vitro IC₅₀ values are plotted with error bars showing the standard error of at least two biological replicates. ρ denotes the Pearson correlation coefficient.

Figure 2

Changes in ex vivo parasite sensitivity over time. IC₅₀ values among parasites collected in Thiès, Senegal and tested against amodiaquine (A), artemisinin (B), chloroquine (C), and mefloquine (D). The number of samples tested each year is indicted in parentheses below each plot. Horizontal lines indicate median IC₅₀ values. The asterisk in panel A indicates an IC₅₀ value off the scale (amodiaquine IC₅₀ = 1140 nM).

Figure 3

Changes in prevalence of known drug resistance-associated mutations over time. Prevalence of resistance-associated mutations in pfcrt(A) and pfmdr1(B) with corresponding 95% point-wise confidence intervals. Mutations were measured by high-resolution melt (HRM) technology and prevalence was calculated by dividing the number of samples containing at least one mutant allele by the total number of samples genotyped each year. Asterisks indicate significant changes over time (P < 0.05 by Fisher-Hamilton exact test).