Persistence of Plasmodium falciparum parasitemia after artemisinin combination therapy: evidence from a randomized trial in Uganda

Abstract

Artemisinin resistance is rapidly spreading in Southeast Asia. The efficacy of artemisinin-combination therapy (ACT) continues to be excellent across Africa. We performed parasite transcriptional profiling and genotyping on samples from an antimalarial treatment trial in Uganda. We used qRT-PCR and genotyping to characterize residual circulating parasite populations after treatment with either ACT or ACT-primaquine. Transcripts suggestive of circulating ring stage parasites were present after treatment at a prevalence of >25% until at least 14 days post initiation of treatment. Greater than 98% of all ring stage parasites were cleared within the first 3 days, but subsequently persisted at low concentrations until day 14 after treatment. Genotyping demonstrated a significant decrease in multiplicity of infection within the first 2 days in both ACT and ACT-primaquine arms. However, multiple clone infections persisted until day 14 post treatment. Our data suggest the presence of genetically diverse persisting parasite populations after ACT treatment. Although we did not demonstrate clinical treatment failures after ACT and the viability and transmissibility of persisting ring stage parasites remain to be shown, these findings are of relevance for the interpretation of parasite clearance transmission dynamics and for monitoring drug effects in Plasmodium falciparum parasites.

Trial registration: ClinicalTrials.gov NCT01365598.

Figure 1. Changes in asexual and sexual stage parasite prevalence over time.

(A) The prevalence of mature gametocytes (MG, PF3D7_1438800) decreases more rapidly in the AL-PQ group (the slope of simple linear regression = −0.0045 (95% CI = [−0.014, 0.0047]) for the AL group and −0.040 [95% CI = [−0.049, −0.032] for the AL + PQ group). (B) The prevalence of rings (PF3D7_0501300) through time is similar in the AL group and the AL + PQ group (the slope of simple linear regression = −0.040 (95% CI = [−0.057, −0.023]) for the AL group and −0.042 [95% CI = [−0.054, −0.029] for the AL + PQ group).

Figure 2. Changes in ring stage and mature gametocyte stages through time.

(A) The number of mature gametocytes after day 3 is significantly smaller in the AL + PQ group (p-value = 0.0057). (B) The number of rings decreases between day 0 and day 3, and the level of the decrease is similar in the AL group and the AL + PQ group (p-value = 0.18). (C) The proportion of the total parasite population that represents mature gametocytes increases during follow-up (Mann-Whitney test, p-values = 7.92 × 10⁻¹⁰ [AL] and 1.01 × 10⁻⁷ [AL + PQ]).

Figure 3. MSP2 genotyping to determine multiplicity of infection.

(A,B) MOI decreases after treatment in both groups (Mann-Whitney test, p-values = 0.0016 [AL] and 0.0081 [AL + PQ]). Instead of having one dominant genotype, multiple genotypes are circulating on later days in some samples. One patient sample has MOI = 11 on day 0 in the AL group and is not shown in the figure. (C) Total number of msp2 types is greater than maximum MOI in some patients, indicating that new genotypes emerge later in the patients.

Figure 4. Molecular barcode analysis to determine multiplicity of infection.

(A,B) Highly polymorphic molecular barcodes suggest that multiple genotypes are circulating both before and after treatment.