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

Background: The 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.

Methods: The 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).

Results: All 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).

Conclusion: PSA 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.

Fig. 1

In vitro and ex vivo piperaquine survival assays (PSA). Top: synchronization and timing of 200 nM piperaquine exposure for in vitro PSA performed on culture-adapted P. falciparum isolates collected on day 0 from patients subsequently followed-up and presenting or not a late recrudescence. Bottom: timing of 200 nM piperaquine exposure for the ex vivo PSA performed on circulating parasites obtained directly from the blood of patients with falciparum uncomplicated malaria. The ex vivo PSA was performed only on isolates with parasite densities ≥0.1 %. The survival rates were interpretable when the parasite growth rates (parasite density at 72 h/parasite density at 0 h) were >1.5 for the in vitro PSA and >1 for the ex vivo PSA. Dark blue rectangles show culture medium containing 200 nM piperaquine (exposed culture) or 0.5 % lactic acid (non-exposed culture). Light blue rectangles represent complete culture medium without drugs (exposed and non-exposed cultures). PPQ, Piperaquine; PSA, Piperaquine survival assay

Fig. 2

Association between clinical dihydroartemisinin-piperaquine outcome and in vitro and ex vivo piperaquine survival assay (PSA) survival rates. In vitro and ex vivo PSAs were done with 0–3 h post-invasion rings from culture-adapted parasites isolated in 2012–2013 or parasites directly collected from patients with malaria in Rattanakiri, Siem Reap, Stung Treng, and Mondulkiri in 2014, respectively. Results from the in vitro and ex vivo PSAs are expressed as the proportion of viable parasites in the exposed or non-exposed cultures (Fig. 1). Isolates (collected on day 0) are dichotomized according to the clinical outcome of infection in patients enrolled and treated with a 3-day course of dihydroartemisinin-piperaquine (non-recrudescence or recrudescence of P. falciparum infections within 42 days, after PCR-correction). The median of the proportion of viable parasites was significantly higher in isolates from subsequently recrudescent than non-recrudescent patients (in vitro PSAs 51.9 % vs. 34.4 %, respectively, P = 0.04; ex vivo PSA: 39.2 % vs. 0.17 %, respectively, P <1 × 10^–11). Each circle represents a P. falciparum isolate. Red and green colors refer to K13 mutant alleles (C580Y or Y493H) and K13 wild-type alleles, respectively. The black diamonds, the horizontal lines and I bars represent the medians and interquartile ranges. The dotted grey line represents the 10 % survival rate cut-off that distinguishes piperaquine-resistant (≥10 %) from piperaquine-sensitive (<10 %) parasites in PSAs

Fig. 3

Cumulative incidence of clinical failure within 42 days (after PCR-correction) in patients treated with a 3-day dihydroartemisinin-piperaquine course according to K13 allele (wild-type or mutant) and ex vivo piperaquine survival assay (PSA) survival rates of day 0 parasites. The cumulative incidence of clinical failure was significantly higher in patients infected on day 0 by isolates carrying a mutant K13 allele and presenting a PSA survival rate ≥10 % (P <1 × 10^–10, log rank test, Hazard Ratio = 14.3, 95 % CI, 4.6–44.6, Fig. 3). The survival proportion at day 42 for those patients was estimated 25.8 % (SD = 7.9 %)