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Two studies recently published in The Lancet Infectious Diseases identified genic markers of Plasmodium falciparum that are associated with resistance to the antimalarial drug piperaquine.

According to researchers, the classification of these molecular markers will help them track and contain the spread of piperaquine-resistance in Cambodia, where the frontline combination malaria treatment dihydroartemisinin-piperaquine appears to be failing.

"Piperaquine resistance, although currently confined to Cambodia, is a major concern, because patients suffering malaria are almost untreatable," Didier Ménard, PhD, of the Institut Pasteur in Cambodia, said in a press release. "At present, alternative treatments are scarce and the reduced cure rates lead to prolonged parasite carriage, increasing the potential of transmitting the resistant parasites and endangering efforts to control and eliminate malaria."

Plasmepsin 2 and 3 gene amplification linked to piperaquine resistance

Ménard and colleagues reported that dihydroartemisinin-piperaquine failure rates are as high as 60% in Cambodia. The researchers launched an investigation to identify molecular markers associated with drug resistance. They used an in vitro piperaquine survival assay to examine the drug susceptibility profiles of 31 parasite lines collected in Cambodia in 2012 and found an increased number of plasmepsin 2 and plasmepsin 3 genes o­n chromosome 14 in piperaquine-resistant lines.

Plasmepsin 2 amplification was further assessed as a potential signature of resistance in 725 patients treated with dihydroartemisinin-piperaquine between 2009 and 2015. An examination of blood samples collected prior to treatment showed that patients infected with parasites with multiple plasmepsin 2 gene copies were 20.4 (95% CI, 9.1-45.5) times more likely to experience treatment failure within a 42-day follow-up period. The genetic marker predicted piperaquine resistance with 94% sensitivity and 77% specificity.

In an analysis of 1,252 P. falciparum isolates collected across Cambodia from 2002 to 2015, an increase in the proportion of parasites with multiple plasmepsin 2 genes over time strongly correlated with an increase in treatment failure rates, further supporting the link between plasmepsin 2 amplification and resistance (P < .0001). The clinical efficacy of dihydroartemisinin-piperaquine dropped below 90% when the local proportion of parasites with multiple plasmepsin 2 copies rose above 22%.

While their findings showed that plasmepsin 2 and plasmepsin 3 clusters can predict treatment failure, the researchers noted that the association is not proof that plasmepsin gene amplification causes piperaquine resistance.

"We now know that amplification of the plasmepsin 2 and plasmepsin 3 genes is an important factor in determining how well piperaquine kills malaria parasites," Menard said in the release. "A genetic toolkit combining this new marker with markers of artemisinin and mefloquine resistance could be used to track the spread of resistance and provide timely information for containment policies."

Researchers identify additional resistance marker

In a separate study, Rick M. Fairhurst, MD, PhD, of the National Institute of Allergy and Infectious Diseases, Roberto Amato, PhD, of the Wellcome Trust Sanger Institute, Cambridge, U.K., and colleagues observed an association between plasmepsin gene amplification and piperaquine resistance in a larger sample of 297P. falciparum parasites. However, the researchers also identified another marker of resistance - the exo-E415G variant o­n chromosome 13.

For the study, the researchers enrolled 486 patients infected with P. falciparum from three provinces in Cambodia where artemisinin and piperaquine resistance is common (Pursat), emerging (Preah Vihear) or uncommon (Ratanakiri). Before the patients began treatment, the researchers collected parasites from blood samples and exposed them to varying levels of piperaquine to assess the evolution of resistance. The researchers also performed a whole genome sequencing analysis to compare variations in parasitic DNA.

According to the results, parasites were more likely to survive dihydroartemisinin-piperaquine treatment if they had more than o­ne copy of plasmepsin 2 and plasmepsin 3 genes, as well as a mutation o­n chromosome 13 (exo-E415G). In a subset of 133 patients with 63-day clinical efficacy data, both plasmepsin 2 and 3 genes (survival rate = 0.41; 95% CI, 0.28-0.53) and exo-E415G (survival rate = 0.38; 95% CI, 0.25-0.51) were associated with low rates of treatment success.

In other results, the researchers found that the prevalence of these resistance markers substantially increased in Pursat and Preah Vihear where dihydroartemisinin-piperaquine served as the frontline therapy for at least 6 years.

"By surveying the piperaquine resistance marker across Southeast Asia in real-time, we can identify those areas where dihydroartemisinin-piperaquine will not be effective, and this could enable national malaria control programs to immediately recommend alternative therapies such as artesunate-mefloquine," Fairhurst said in a press release. "This approach will be crucial to eliminating multidrug-resistant parasites in Southeast Asia before they spread to sub-Saharan Africa, where most of the world's malaria transmission, illness and death occur."

Mefloquine plus piperaquine may counteract resistance

Arjen M. Dondorp, MD, of the Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine at Mahidol University, Bangkok, and the Center for Tropical Medicine and Global Health at the University of Oxford noted thatmdr1gene amplification was consistently absent in parasites with plasmepsin 2 and plasmepsin 3 amplification. Although this could be due to interrupted use of mefloquine, he suggested that the markers could represent counteracting resistance mechanisms. If so, alternating mefloquine and piperaquine or using the drugs in triple combinations could be "attractive treatment modalities."

"Antimalarial drug resistance threatens to offset the impressive gains made over the past decade in reducing falciparum malaria transmission in the Greater Mekong subregion to the current historically low numbers," Dondorp wrote in an accompanying editorial. "We have to eliminate falciparum malaria from the region now before it becomes close to untreatable."- by Stephanie Viguers

References:

Amato R, et al.Lancet Infect Dis. 2016;doi:10.1016/S1473-3099(16)30409-1.

Dondorp AM.Lancet Infect Dis.2016;doi:10.1016/S1473-3099(16)30414-5.

Witkowski B, et al.Lancet Infect Dis. 2016;doi:10.1016/S1473-3099(16)30415-7.

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