Defining the In Vivo Phenotype of Artemisinin-Resistant Falciparum Malaria: A Modelling Approach

Background: Artemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment ('day-3'), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions. Methods and Findings: Data from 1,518 patients on the Thai-Myanmar and Thai-Cambodian borders with parasite half-life assessments after artesunate treatment were analysed. Half-lives followed a bimodal distribution. A statistical approach was developed to infer the characteristics of the component distributions and their relative contribution to the composite mixture. Conclusions: Characterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in 'day-3' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the ‘day 3’ threshold to account for initial parasitaemia and sample size. Lisa White and colleagues explore parasite clearance distributions in an area of artemisinin resistance with the aim of refining the phenotypic definition of resistance.Background: Artemisinin and its derivatives are powerful medicines that can quickly reduce the number of Plasmodium parasites in the blood of patients with malaria. Artemisinin combination therapies (ACTs) are recommended by the WHO as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Expanding access to ACTs has been a key contributor to the recent success in reducing the global malaria burden. Several hundred million ACT treatment courses are currently distributed in malaria-endemic countries every year. However, recent findings of artemisinin-resistant malaria parasites have alarmed the global health community. According to the WHO, as of February 2015, artemisinin resistance had been confirmed in five countries in Southeast Asia, and its spread is seen only as a question of time by many experts. Such spread—or independent emergence of artemisinin resistance—in other parts of the world would pose a major health crisis because there are currently no other antimalarial drugs that are as effective and well-tolerated as ACTs. Why Was This Study Done?: Surveillance systems to detect the spread or emergence of artemisinin resistance in malaria-endemic regions are crucial so that control and elimination measures can be undertaken rapidly. Tools for surveillance include clinical studies on parasite clearance from the blood, in vitro parasite testing for artemisinin sensitivity in the laboratory, and assessment of molecular markers. (Tests based on molecular markers of resistance are being developed based on the 2013 discovery of artemisinin-resistance mutations in a parasite gene called Kelch 13 that are associated with delayed parasite clearance after ACT treatment.) The best description of parasite clearance in the individual patient is the parasite half-life, which requires frequent blood sampling. Since this is difficult to do in resource-limited settings, the WHO uses the following working definition: artemisinin resistance in a population is suspected if more than 10% of patients are still carrying parasites three days after the start of ACT treatment. The researchers conducted this study to see how well this working definition matches actual data from individual patients in areas with artemisinin-resistant P. falciparum parasites. What Did the Researchers Do and Find?: The researchers used data on blood samples from 1,518 patients collected along the Thai-Myanmar and Thai-Cambodian borders (where resistance first developed) from 2001 to 2012. All patients had been treated with ACT, and the time it took for the drugs to kill half of the parasites in their blood (the parasite half-life) had been measured. When the researchers used mathematical models to analyse these parasite half-life assessments after ACT treatment, they found that a model that assumed two different parasite subpopulations best explained the actual data. One of these subpopulations—and the only one present in the early years before resistance developed—had a shorter parasite half-life of about 3 hours (equivalent to sensitive parasites that still are killed efficiently by the ACT drugs). The second population began to appear around 2008 and had a longer half-life of about 6.5 hours, equivalent to parasites that had acquired resistance to artemisinin, and consistent with measurements of Kelch 13–mutant parasites. The parasite half-life predicted the likelihood of an artemisinin-resistant infection for individual patients, but this was influenced by how common resistance was in that area: the critical half-life varied between 3.5 hours (in areas where resistance is rare) and 5.5 hours (in areas where resistance is common). This means that there is not a universal cut-off value in parasite half-life to denote an infection as ‘sensitive’ or ‘resistant’ because intermediate half-lives can be part of the lower tail end of the resistant parasite half-life distribution or the top-end of the sensitive one. The model the researchers developed can estimate the background proportion of resistant infections, and this can then be used to calculate the probability for an individual infection to belong to a resistant or sensitive parasite half-life distribution, thereby providing a more accurate assessment of resistance. Taking this into account, the researchers found that the current WHO 'day-3' cut-off value of 10% predicts the potential presence of artemisinin-resistant infections in most but not all scenarios. What Do these Findings Mean?: The findings are relevant to the situation in Southeast Asia, where malaria is less common than in so-called high-transmission areas, including much of Sub-Saharan Africa. Whether the model is useful for resistance surveillance in high-transmission areas will need further study. Nonetheless, the results suggest that a model that analyses data on parasite half-life as distributions of artemisinin-sensitive and artemisinin-resistant populations is a useful tool for surveillance of artemisinin resistance in a geographical area. The model also measures the probability that a parasite strain with a given parasite half-life is resistant to artemisinin, which should help with the evaluation of other surveillance methods, including those based on Kelch 13 mutations and possibly other molecular markers. The WHO—recommended definition of suspected artemisinin resistance in cases where more than 10% of patients still carry parasites on the third day of treatment is useful, but, according to this study, including the initial parasite load (i.e., the number of parasites in a patient’s blood before the start of treatment) would make it more informative. In addition, as the ‘day-3’ cut-off method lacks accuracy in predicting the actual proportion of artemisinin-resistant parasites, a positive result should be followed by a more detailed assessment. Additional Information: This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001823.The WHO issues regular updates about the status of artemisinin-resistant malariaThe WHO provides a list of questions and answers about artemisinin-resistant malariaWikipedia provides information on artemisinin (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)The Bill & Melinda Gates Foundation describes its global strategy to fight and eradicate malaria in the face of artemisinin resistanceThe Mahidol Oxford Research Unit, where several of the study’s authors work, has pages about malaria, including information about treatments and drug-resistance