Plasmodium falciparum Kelch 13 mutations and treatment response in patients in Hpa-Pun District, Northern Kayin State, Myanmar

Resistance to the artemisinin derivatives in Plasmodium falciparum, characterized by delayed parasite clearance in patients treated with artesunate, emerged in Western Cambodia in 2007 [1‐3]. This phenotypic trait was also documented in a large study on the Thai–Myanmar border in 2012 and was [1] found to be heritable [4] and associated with strong selective sweeps in the plasmodial genome [5]. In 2014, non-synonymous single nucleotide polymorphisms in the Kelch propeller domain on chromosome 13 (k13) were found to be strongly associated with resistance to artemisinin derivatives [6]. The link between certain k13 mutations and the clinical resistance phenotype (delayed parasite clearance) has been demonstrated by a number of clinical [7‐10], in vitro [11] and transfection studies [12]. In the areas where k13 mutations have been found, the distribution of different alleles has been variable. For example, the C580Y polymorphism is the most common mutation in Cambodia [13, 14], Laos [15, 16] and Vietnam [16], while the F446I mutants are most common on the Myanmar–China border [9, 17, 18]. On the Thailand–Myanmar border near Mae Sot, the E252Q allele was the most abundant before 2008, but it has since been replaced by C580Y, an allele now close to fixation in this area [19]. The C580Y mutant parasites have emerged at least twice at different locations [20, 21]. Isolates from Cambodia and Vietnam appear to have a common ancestor and they differ from resistant parasites in Myanmar [13, 21]. The C580Y allele found on the Myanmar–China border appears to have originated from the Thailand–Myanmar border [17]; indicating that genetic mutations (at least C580Y) associated with artemisinin resistance in P. falciparum are not only emerging “de novo” in different locations but also spreading contiguously.

In 2011, the World Health Organization (WHO) proposed a strategy of containment of artemisinin resistant malaria in the region [22], but this has not contained the spread of resistance; parasites carrying k13 mutations have now spread to the China–Myanmar [23] and India–Myanmar borders [7, 21, 23, 24]. The C580Y genotype has increased in frequency replacing other genotypes in most of Southeast Asia, and a single C580Y parasite lineage has swept across the Eastern Mekong subregion spreading from Western Cambodia to Southern Vietnam indicating that a “hard” selective sweep has now replaced initial “soft” selective sweeps [19].

More importantly, k13 alleles under strong selective pressure confer (or are associated with) slow parasite clearance after artemisinin treatment leaving a larger number of surviving (artemisinin resistant) parasites exposed to the partner drugs [25]. The loss in efficacy of DHA-piperaquine in Cambodia and Viet Nam [26, 27] and mefloquine-artesunate on the Thai–Myanmar border [28] have demonstrated how slow parasite clearance due to artemisinin resistance unavoidably leads to the failure of ACT.

This report presents data collected in 2013 from a remote region along the Myanmar–Thai border in Hpa-pun District, Myanmar (Fig. 1). While artemisinin resistance was well established by 2010 [4] in the border district of Mae Sot (Thailand), 200 km further south, which is a well-connected area encompassing the Trans-Asia highway, data from elsewhere in the remote, hilly and forested parts of eastern Myanmar are few [10, 24]. This study provided an opportunity for examining the phenotype and genetic diversity of k13 of parasites from a secluded relatively inaccessible region with limited access to ACT (Fig. 1). Parasite clearance half-life and k13 genotyping results from 35 patients treated with 3 days artesunate monotherapy followed by a full course of ACT are presented.

Throughout South East Asia parasite clearance rates are decreasing among patients with uncomplicated P. falciparum malaria, a pattern that is strongly associated with mutations in the k13 gene of P. falciparum and accompanying partner drug resistance. Until now, 108 non-synonymous polymorphisms of k13 gene have been found in all malarious regions of the world. Most of the mutations in the propeller region of the gene are associated with the slow parasite clearance phenotype, and in Asia show evidence of recent selection [16, 19]. Historical data from the Thai–Myanmar border (Mae Sot area) show that the prevalence of k13 mutations has increased significantly, and among them the C580Y allele has out-competed others in recent years signifying that a “hard” selective sweep has replaced an initially “soft” one [19]. The C580Y mutation also dominates in the Eastern Greater Mekong sub-region although this successful lineage had a different genetic origin to the lineage in Eastern Myanmar. In this study in a relatively inaccessible region 200 km away but days travel from Mae Sot, the most frequent kelch mutant allele was F446I. This is the predominant allele in the North of Myanmar which was significantly associated with slow parasite clearance.

The main limitation of this study is the small sample size, which is related to both the remoteness of the study site, logistical difficulties related to working in this remote area, and the relatively high parasitaemia threshold for inclusion (i.e. ≥ 10,000/µL). The parasitaemia threshold was chosen in order to obtain reliable parasite clearance curves.