Artemisinin combination therapy—the use of a potent, short-acting artemisinin and a less-potent, long-acting partner drug—is recommended worldwide for the treatment of Plasmodium falciparum malaria.1 Dihydroartemisinin–piperaquine, one of the few artemisinin combination therapies still effective against multidrug-resistant P falciparum in southeast Asia, was adopted as the first-line antimalarial treatment in Cambodia in 2008. Several earlier studies2, 3, 4 documented the excellent safety and tolerability of dihydroartemisinin–piperaquine in Cambodia, as well as efficacy of 96–98% after 28 days or 63 days in the Cambodian provinces of Oddar Meancheay, Siem Reap, Pursat, and Kratie.3, 4, 5, 6 However, the rapid spread of artemisinin resistance in Cambodia7, 8, 9, 10, 11 and throughout mainland southeast Asia10, 11, 12 threatens the efficacy of dihydroartemisinin–piperaquine and all other artemisinin combination therapies.13 This danger arises because as more parasites become resistant to artemisinin, more parasites need to be eliminated by the lone partner drug; therefore, they are more likely to spontaneously develop genetic resistance to piperaquine and other partner drugs.
Preliminary evidence for this development has been provided by three studies that show declining efficacy of dihydroartemisinin–piperaquine shortly after its widespread deployment in western Cambodia. In a 2008–10 study,14 the efficacy of dihydroartemisinin–piperaquine after 42 days was 75% in Pailin and 89% in Pursat, but 100% in Preah Vihear and Ratanakiri in northern and eastern Cambodia. Because dihydroartemisinin–piperaquine failures were found not to be associated with piperaquine 50% inhibitory concentration (IC50) in this study, and piperaquine plasma concentrations at 7 days were not measured, piperaquine resistance in Pailin and Pursat could not be confirmed. The emergence of piperaquine resistance is also difficult to reconcile with concomitant decreases in piperaquine IC50 values in Pailin and Pursat.14 In a 2013 study,15, 16 the efficacy of dihydroartemisinin–piperaquine after 42 days in Oddar Meancheay was 46%. Although patients with recrudescence or cure had similar exposures to piperaquine in this study, the piperaquine IC50 values for recrudescent parasites were not higher than those for non-recrudescent parasites. Given this result, piperaquine resistance in this province also could not be confirmed. In a 2011–13 study,17 the proportion of recrudescent infections by 42 days after dihydroartemisinin–piperaquine treatment was higher in western Cambodia (15%) than in eastern Cambodia (3%). Patients with recrudescence or cure in this study had similar exposures to piperaquine and carried parasites with similar piperaquine IC50 values. In view of these findings and the lack of a genetic marker, piperaquine resistance in western Cambodia has not been confirmed, although increasing piperaquine IC50 values in northern Cambodia suggest that it may be emerging.18
Research in context
Evidence before this study
We searched PubMed using the terms “dihydroartemisinin”, “piperaquine”, “efficacy”, and “Cambodia” without any date or language restrictions on June 5, 2015. We identified 13 articles, six of which were original clinical trials of the efficacy of dihydroartemisinin–piperaquine for treatment of uncomplicated Plasmodium falciparum malaria in Cambodia. Three studies from 2001–05 showed that efficacy was 96–98% before dihydroartemisinin–piperaquine was widely used. Three later studies reported reduced efficacy (46–89%) in 2008–13, after dihydroartemisinin–piperaquine became widely used. Treatment failure has been linked to parasite kelch13 mutations, which are associated with artemisinin resistance. All three of the later studies found no association between treatment failures and high piperaquine in-vitro IC50 values (a measure of parasite susceptibility to piperaquine). The role of in-vivo piperaquine resistance in treatment failures has not been adequately assessed.
Added value of this study
Our findings suggest that dihydroartemisinin–piperaquine treatment is failing in Pursat and Preah Vihear, where artemisinin resistance is prevalent, but remains highly efficacious in Ratanakiri where artemisinin resistance is uncommon. Treatment failures were not associated with older patient age, higher initial parasite density, or high piperaquine plasma concentration at 7 days. Instead, recrudescent parasites had more kelch13 mutations and high piperaquine IC50 values, indicating that dihydroartemisinin–piperaquine failures are due to both artemisinin and piperaquine resistance. These recrudescent parasites also have reduced mefloquine IC50 values and lack multiple copies of pfmdr1, a genetic marker for mefloquine resistance.
Implications of all the available evidence
Dihydroartemisinin–piperaquine is failing quickly in four western Cambodian provinces (Pailin, Pursat, Oddar Meanchey, and Preah Vihear), and is associated with parasite resistance to both artemisinin derivatives and piperaquine. Evidence of piperaquine resistance in P falciparum should prompt efforts to map this phenotype in Cambodia and other southeast Asian countries, to elucidate its molecular mechanism, and to discover new drugs that circumvent piperaquine resistance. Artesunate plus mefloquine should be tested as a first-line therapy where dihydroartemisinin–piperaquine failures have been documented, and also as a salvage treatment for dihydroartemisinin–piperaquine failures in Cambodia. Clinical trials should be done of a triple-drug regimen of dihydroartemisinin–piperaquine plus mefloquine.
The lack of clear evidence of piperaquine resistance in Cambodia hinders efforts to define its role in dihydroartemisinin–piperaquine failures, identify and validate genetic markers for use in large surveillance programmes, and study its molecular mechanism. We did a cohort study to identify piperaquine-resistant P falciparum infections in Cambodia. We postulated that such infections would be associated with artemisinin resistance,19 dihydroartemisinin–piperaquine failures, adequate piperaquine exposure, and decreased susceptibility of P falciparum isolates to piperaquine in vitro. We also postulated that dihydroartemisinin–piperaquine would fail more often in areas where artemisinin resistance is prevalent than where it is emerging. We therefore compared the efficacy of dihydroartemisinin–piperaquine for the treatment of uncomplicated P falciparum malaria in Pursat, Preah Vihear, and Ratanakiri, where the prevalences of kelch13 mutations—a genetic marker for artemisinin resistance in Cambodia and elsewhere in southeast Asia9, 10—were 76%, 21%, and 4%, respectively, in 2011–12.10 We also compared the prevalence of kelch13 mutations, plasma piperaquine concentrations after 7 days, and in-vitro piperaquine IC50 values between non-recrudescent and recrudescent infections to investigate the presence of piperaquine-resistant parasites.