Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations

Abstract

BackgroundThe declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies.MethodsThe piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594).ResultsAll parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10 %, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2 % vs. 0.17 %, P <1 × 10−7). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10 % were associated with 32-fold higher risk of recrudescence (95 % CI, 4.5–224; P = 0.0005).ConclusionPSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0539-5) contains supplementary material, which is available to authorized users.

Plasmodium falciparum Resistance to Artemisinin Derivatives and Piperaquine: A Major Challenge for Malaria Elimination in Cambodia

“…These anomalous curves presented a paradoxical increase in [ 3 H]-hypoxanthine incorporation at PPQ concentrations above 100-200 nM, the physiological concentration of PPQ observed in the blood of patients treated with a standard 3-day course of DHA-PPQ 82 To overcome the limitations of current assays and provide a more robust assessment of PPQ resistance, a new in vitro assay (named "Piperaquine Survival Assay" PSA) based on the same principle as the RSA was developed. 82 This assay involves exposing tightly synchronized culture-adapted parasites (in vitro format) or parasites directly collected from patients (ex vivo format) to a pharmacologically relevant dose of PPQ (200 nM for 48 hours), to mimic in vivo conditions.…”

“…The most recent clinical studies conducted in Cambodia between 2012 and 2015 have reported an alarming increase in the proportion of late clinical treatment failure following a standard 3-day course of DHA-PPQ. Treatment failure rates increased from 25% in 2010 to 46% in 2014 20,[82][83][84][85] in western Cambodia, suggesting that DHA-PPQ is becoming ineffective within only a few years of its nationwide deployment. The high treatment failure rates suggest that parasites with reduced susceptibility to both artemisinin and PPQ are now prevalent in western/northern Cambodia.…”

“…82 This assay involves exposing tightly synchronized culture-adapted parasites (in vitro format) or parasites directly collected from patients (ex vivo format) to a pharmacologically relevant dose of PPQ (200 nM for 48 hours), to mimic in vivo conditions. The in vitro PSA was found to discriminate efficiently between PPQ-resistant and sensitive parasites, with resistance defined as a survival rate in the PSA ≥ 10% (defined as the cutoff for resistance on the basis of results for the isolates of patients with recrudescent infection).…”

“…20,82,84,86 For instance, it has been observed that all in vitro PPQ-resistant isolates had a single Pfmdr-1 copy but the converse was not true, as not all isolates with a single copy of Pfmdr-1 were in vitro PPQ resistant. This finding strongly supports recent recommendations for the use of artesunate plus MQ as an alternative first-line treatment in provinces in which DHA-PPQ is failing.…”

“…This finding strongly supports recent recommendations for the use of artesunate plus MQ as an alternative first-line treatment in provinces in which DHA-PPQ is failing. 20,82,84,86 However, further investigations are required to determine whether the elimination of parasites with multiple copies of Pfmdr-1 has resulted from the removal of MQ pressure or the selective pressure exerted by PPQ. This last finding also suggests that PfMDR1, a pump involved in the intraparasitic influx/efflux of metabolites or drugs, may play a significant role in parasite susceptibility to PPQ.…”