Over the last decade, there has been a remarkable progress in controlling P. falciparum malaria in the Greater Mekong subregion [1]. However, this achievement is threatened by the rising resistance to antimalarials. In 2008-2009, slow parasite clearance after artemisinin therapy of falciparum malaria was first documented in western Cambodia, and presumed to reflect emerging artemisinin resistance [2, 3]. Since then, artemisinin resistance has spread or emerged independently throughout the Greater Mekong subregion [4, 10, 19]. As a result of the falling efficacy of artemisinin-mefloquine in Thailand, DHA-PPQ was introduced as the first-line treatment in 2015 [8, 20], but treatment failure soon appeared in neighbouring Cambodia and subsequently the eastern border region of Thailand, likely due to the emergence of PPQ resistance [11, 13, 21-23]. At present, markers of resistance to artemisinin and PPQ have been identified [5, 9, 12, 16, 17, 24].
Kelch13 mutations are the most well-established markers of artemisinin resistance [19, 25]. It has been shown that whereas C580Y was the main mutation in the eastern GMS, F446I was more common in Myanmar [10]. C580Y has now been found in many parts of Thailand but become near fixation along the Cambodian border [4, 10, 15, 26]. The distributions of other kelch13 alleles in the country vary geographically - with Y493H and R539T found mainly in the east, P574L in the west and R561H in northwest [26]. In this study, a new mutation, G533S, was detected in 2014 and became the most common resistance mutation in the study site. This mutation was not detected in a previous study from the area in 2012-2014 [26]. Despite this, the timing of its first detection in 2014 and the study site proximity to Myanmar and China were in agreement with the G533S appearance on the China-Myanmar border at around the same time [18]. Importantly, the frequency of G533S rose to 100% in 2019. Although this mutation has not been formally associated with clinical resistance, parasites carrying this mutation were recently shown to have elevated ring-stage survival compared to the wild-type parasite [18].
Amplification of plasmepsin-2 is widely used as a marker to trace PPQ resistance [9, 12-15]. Previous studies suggested that this genotype was restricted to eastern GMS [10, 14, 15]. Consistent with these reports, none of the P. falciparum isolates in Tak province from 2013-2019 had plasmepsin-2 amplification.
The second molecular marker of PPQ resistance is Pfcrt [16, 27]. Several mutations including T93S, H97Y, and F145I were implicated in treatment failure [13, 24]. The frequencies of these mutations have risen in eastern GMS [14, 15], but remained much less prevalent in the Thai-Myanmar border region [28]. In the present study, no parasite had T93S and H97Y and only one parasite had F145I. Because the F145I mutation has been shown to confer a high level of resistance of PPQ through transfection [16] and be associated with a five-fold increased risk of DHA-PPQ treatment failure [24], the detection of this mutation raises a concern over its potential future establishment in the area.
In summary, this study detected for the first time the kelch13 G533S mutation in Tak province of western Thailand. This mutation has been associated with artemisinin resistance in vitro [18] and its increase in frequencies was observed during 2014-2019. In addition, although no plasmepsin-2 amplification was observed, the PPQ resistance Pfcrt F145I mutation was detected in a parasite that also carried the G533S mutation. Given the threat of drug resistance, a close monitoring of resistance markers and treatment failure in the area is well warranted.
Limitations
The study involved a small number of samples - 55 for kelch13 mutation, 51 for plasmepsin-2 amplification, and 54 for Pfcrt mutation - from one district of Tak Province, Thailand. As such, it has limited power to detect rare mutations and the results may not be representative of the broader area of the western Thailand. In addition, although the kelch13 G533S was first detected in 2014, its first emergence in the study area may have been earlier.