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.

High prevalence of Plasmodium falciparum gametocyte infections in school-age children using molecular detection: patterns and predictors of risk from a cross-sectional study in southern Malawi

“…qRT-PCR was conducted on the preserved RNA of all PCR-positive and ~1% of PCR-negative individuals to test for stage-specific P. falciparum mRNA. qRT-PCR used a multiplex assay developed by Joice et al [46] and validated in a clinical trial in Uganda by Chang et al [47] that distinguishes mature (stage IV–V) gametocytes from other stages.…”

“…The density of circulating gametocytes was estimated using the mean threshold cycle (Ct) values of the two or three positive wells from qRT-PCR [47]. Briefly, the relationship between Ct values and gametocyte density was estimated using linear regression of samples that had microscopically-quantified gametocyte densities.…”

Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial

“…An important caveat is that our markers were selected based on expression in a single laboratory-adapted parasite line, NF54. Although we previously assessed SBP1 transcript levels in 3D7 4 and Pfs25 transcript levels in NF54, NF135, and NF166 parasites 38 and found highly comparable levels between parasite isolates, this does not mean that in vitro expression can be directly extrapolated to expression in in vivo samples. Our quantitative approach may thus be affected by possible differences between in vitro-cultured parasites and patient samples.…”

“…It is currently unclear whether these are true indications of persisting asexual parasites and, until now, artemisinin resistance has not been demonstrated in Africa 30 . Before the current study, the stage specificity of these markers had not been rigorously examined with, for example, conflicting evidence on the stage specificity of SBP1 4 . At present, there is no consensus on the value of molecular tools to detect asexual parasites with high sensitivity and specificity or interpretation of RNA transcripts after antimalarial treatment 4 .…”

“…Precise quantification of circulating parasite stages is necessary to accurately assess stage-specific clearance 3 by antimalarials and could support the evaluation of treatment efficacy 4 in clinical trials. Indeed, artemisinin-based combination therapy (ACT), the first-line treatment against P. falciparum malaria, does not equally affect all malaria parasite developmental stages: although it is highly active against asexual blood stages responsible for malaria morbidity and mortality, it does not clear mature stage gametocytes, which may persist after treatment, 5 , 6 often at densities below the range that is detectable by microscopy, and may allow posttreatment malaria transmission to mosquitoes 7 , 8 .…”