Increase in Kelch 13 Polymorphisms in Plasmodium falciparum, Southern Rwanda

Artemisinin resistance in Plasmodium falciparum is associated with nonsynonymous mutations in the Kelch 13 (K13) propeller domain. We found that 12.1% (8/66) of clinical P. falciparum isolates from Huye district, Rwanda, exhibited K13 mutations, including R561H, a validated resistance marker. K13 mutations appear to be increasing in this region.

Rwanda achieved substantial reductions in malaria during 2006-2011, partly due to home-based management using artemether/lumefantrine (6). In 2010, at our study site in Huye district, southern Rwanda, we observed a pattern in the P. falciparum multidrug resistance: 1 gene suggestive of intense artemether/ lumefantrine drug pressure, whereas K13 mutations were absent. However, among P. falciparum isolates, 2.5% in 2014 and 4.5% in 2015 harbored K13 variants, including 2 candidate mutations (7,8). A recent report showed the presence of a validated pfkelch13 mutation, R561H, at 2 sites in Rwanda (9). We conducted a cross-sectional molecular surveillance study to update records of the prevalence of K13 variants in Huye among isolates collected in 2019.

The Study
During September-December 2019, we recruited study patients with uncomplicated malaria seeking treatment at the Sovu Health Centre and Kabutare District Hospital, Huye district, Rwanda. Huye district (population ≈390,000) is located on the central plateau of Rwanda (average altitude 1,700 m, yearly rainfall 1,200 mm, mean temperature 19°C). Malaria transmission peaks in October-November and March-May. In 2010, a total of 11.7% of children had microscopically confirmed Plasmodium infection (8).
We obtained written informed consent from all participants or from the caregivers for children; we also obtained written assent from participants 7-18 years of age. The study was approved by the Rwanda National Ethics Committee. Eligibility criteria for participants included age >1 year; a positive result on a rapid diagnostic test, SD Bioline Malaria Ag Pf/Pan (Abbott Global Point of Care, https://www.globalpointofcare. abbott); and a fever (axillary temperature ≥37.5°C) at the time they sought treatment or within 48 hours beforehand (self-reported). We collected whole blood in S-Monovette EDTA (ethylenediaminetetraacetic

Increase in Kelch 13
Polymorphisms in Plasmodium falciparum, Southern Rwanda acid; Sarstedt, https://www.sarstedt.com) tubes and confirmed malaria diagnosis by microscopy of Giemsa-stained thick blood smears; patients were also seen by a physician. We provided a 3-day regimen of artemether/lumefantrine for treatment, the first dose given under observation. All patients were asked to return after 3 days to evaluate residual parasitemia on Giemsastained thick blood smears.
None of the patients infected with K13 variant parasites tested positive after 3 days of treatment. One pregnant patient sought treatment again. Initially, she had K13 wild-type parasites and was given artemether/ lumefantrine; her day 3 microscopy result was negative. Three weeks later, we detected K13 R561H parasites, possibly due to reinfection, and administered quinine.
The R561H artemisinin resistance mutation is regularly observed across Asia (10). A recent study that reported R561H in 7.4% of isolates collected during 2013-2015 in central Rwanda and 0.7% of isolates in south-central Rwanda (9) suggested that this mutation emerged indigenously and independently from Asia 561H strains. We do not have data in our study to support this. None of the K13 variant parasites showed delayed clearance in our study, which may be due to the partner drug lumefantrine still being effective, similar to observations in Southeast Asia (11). In addition, the absence of delayed parasite clearance despite K13 mutations may reflect partial immunity contributing to parasite elimination (12).
We found other nonsynonymous polymorphisms only once among the isolates tested. C469F and A675V are considered artemisinin resistance candidate

mutations (4) and have previously been seen in East
Africa (7,13,14). G533A and V555A have also been previously reported in Africa but have not yet been evaluated for resistance (7,13). A578S is a common K13 polymorphism across Africa but is not linked to artemisinin resistance (1). Our study has clear limitations. Data from only 2 healthcare facilities, with limited catchment areas, were included. Adherence to treatment was assessed by patient self-report, and drug susceptibility testing was not performed. Future research should include ring-stage susceptibility assays to contribute to understanding the role of K13 mutations in Africa. Separate testing for each drug in a combination for efficacy and continued surveillance for antimicrobial resistance are needed.
Our results show that K13 mutations are present in Rwanda and that their prevalence in P. falciparum malaria patients in the Huye District increased from 0% in 2010 to >12% in 2019. The validated artemisinin resistance mutation R561H occurs in 4.5% of P. falciparum isolates being transmitted in this area. The emergence of artemisinin resistance-related mutations in Rwanda is alarming because it might indicate developing resistance against commonly used antimalarials in this region. Countermeasures need to be considered early, potentially including 3-drug antimalarial combinations (2).