Two Lancet Infectious Diseases papers highlight the importance of monitoring the failure of dihydroartemisinin-piperaquine treatment for malaria in Cambodia

WWARN Published Date

Two studies, recently published in the Lancet Infectious Diseases, demonstrate that a frontline malaria treatment combining dihydroartemisinin with piperaquine (DHA-PPQ) is rapidly becoming less effective in Cambodia due to the emergence and spread of drug-resistant parasites. Alternative treatment approaches are urgently needed by public health officials and national governments in the region.

Scientists have used whole-genome sequencing and molecular marker analyses to track the spread of multi-drug resistant parasites. Together both analyses are furthering our understanding of the impact of artemisinin resistance on rapid selection of resistance to partner drugs when they are combined, and suggest alternative treatment approaches that could be used to slow the spread of resistant parasites in Cambodia. The research teams were led by experts at the National Institute of Allergy and Infectious Diseases (NIAID), the Wellcome Trust Centre for Human Genetics (WTCHG), the Wellcome Trust Sanger Institute (WTSI), the Wellcome Trust Mahidol-Oxford Tropical Medicine Research Unit (MORU), the Institut Pasteur du Cambodge (IPC) and Paris (IPP), and the National Centre for Human Parasitology, Entomology & Malaria Control in Cambodia.

The first study led by Dr Rick Fairhurst (NIAID, USA) and Dr Roberto Amato (WTSI, Hinxton and WTCHG, Oxford) analysed parasites collected from 2010 to 2013 in three different provinces of Cambodia with notably different levels of ART resistance and DHA-PPQ failure. They carried out whole-genome sequencing of 297 parasites and compared the sequences of the K13 gene that has been associated with the slow clearance of parasites characteristic of artemisinin resistance. They identified many parasites with mutant K13 genes, and confirmed that these mutations were markers of artemisinin resistance. 

Of greater importance, the researchers noted a strong correlation of other genetic changes with parasites from patients who had failed DHA-PPQ treatment. In particular, parasites that carried an exonuclease gene with a particular single nucleotide change on chromosome 13, and those with amplification of two related genes, plasmepsin II and plasmepsin III, on chromosome 14 that encode enzymes involved in parasite digestion of haemoglobin. Both of these genetic changes were strongly associated with the parasites’ ability to survive PPQ exposure in the laboratory. Among 133 patients treated with DHA-PPQ, those who carried parasites with either of these genetic changes failed treatment almost 40% of the time, again suggesting that these changes might be used as markers of PPQ resistance.

DHA-PPQ was recommended in the western provinces in 2008, and nationwide in Cambodia by 2010, following the failure of another ACT called artesunate-mefloquine. The marker of mefloquine resistance had been shown to be an amplification of the pfmdr1 gene, which encodes a transport protein. Should resistance develop to both partner drugs, public health officials may encounter a perilous situation where neither frontline treatment will work effectively to combat malaria in Cambodia.

The absence of viable alternative treatments presents a serious health risk should multi-drug resistant parasites become untreatable and spread to other malaria-endemic regions, including western Asia and Africa. However, the team noticed that the parasites resistant to DHA-PPQ did not carry extra copies of the pfmdr1 marker suggesting that they were not resistant to mefloquine. Perhaps parasites are not able to develop resistance to both PPQ and mefloquine? Based on this idea, the research teams suggest that if true, triple ACTs, an artemisinin plus PPQ AND mefloquine, might be a suitable treatment alternative.

One of the lead authors Dr Roberto Amato, from the WTSI and WTCHG, UK comments: "We hope that that these findings will provide actionable knowledge for public health policy makers in Cambodia and surrounding countries where resistance is likely to continue emerging and spreading. It’s also important that, where needed, alternative treatment approaches are adopted quickly if we’re to keep working towards our goal of saving lives and eliminating malaria for good."

In parallel, a second independent study, led by Dr Didier Ménard, Dr Benoit Witkowski (IPC) and Dr Frédéric Ariey (IPP), developed a new laboratory assay to measure the ability of parasites to survive exposure to PPQ. The presence of extra copies of plasmepsin 2 (now called PfPM2) was assessed to measure the association of this genetic marker with PPQ susceptibility. The researchers tested 134 clinical field isolates, showing a much higher median survival rate in the new assay among parasites with two or more copies of PfPM2 compared with only the usual single copy of the gene. In addition, they studied more than 725 P. falciparum isolates and found that an increased PfPM2 gene copy number was strongly associated with DHA-PPQ treatment failures. 

The team concluded that ‘patients harbouring multi-copy PfPM2 parasites - from studies carried out from 2009 to 2015 in western Cambodia and 2014 to 2015 in eastern Cambodia - had a 20-fold higher risk of recrudescence during the 42-day post-treatment follow-up period. In areas of artemisinin resistance, the clinical efficacy of DHA-PPQ at day 42 fell under 90% when the local proportion of multicopy PfM2 K13 mutant parasites in the population rose above 22%.’

Dr Ménard and colleagues’ combined analysis of K13 polymorphisms and PfPM2 copy number provides the first valuable molecular marker signature for both artemisinin and piperaquine treatment failures. Combining genome sequencing with a molecular toolkit of PfPM2 with K13 and Pfmdr1 monitoring should provide timely information for antimalarial treatment and help track the emergence and further spread of piperaquine resistance in local communities.

Both studies confirm plasmepsin 2–3 amplification as a robust marker for PPQ resistance in the region. They also highlight the urgent need for alternative treatment strategies such as ACT drug rotation, sequential administration or triple ACT combinations to begin addressing the threat of widespread treatment failure, prolong the efficacy of current antimalarials, and urgently support containment and elimination policies in the region.

Philippe Guérin, Director of the WorldWide Antimalarial Resistance Network (WWARN)*, acknowledges: "These findings will support the World Health Organization’s (WHO’s) and national health ministries’ efforts to respond to the ACT drug resistance crisis and monitor the onward march of multi-drug resistant parasites in Cambodia. Large-scale collaborative studies are critical to provide the evidence base to update treatment guidelines and develop urgently needed novel strategies to save the lives of patients, especially vulnerable groups such as pregnant women and infant children.”

View the WWARN pfmdr1 and pfcrt Molecular Surveyor.

Amato et al. Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study. Lancet ID. November 3, 2016.

Witkowski et al. A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study. Lancet ID. November 3, 2016.

* Prof Guérin is Director of WWARN and the Infectious Diseases Data Observatory (IDDO).