Artemisinin-Resistant Plasmodium falciparum K13 Mutant Alleles, Thailand–Myanmar Border

To the Editor: Artemisinin resistance (ART-R) in Plasmodium falciparum phenotypes may have evolved independently in various areas of the Greater Mekong Sub-region (1,2), prompting the World Health Organization to change its regional policy from containment to elimination (3). Risks associated with ART-R include compromised use of artemisinin combined therapy, partner drug resistance selection, total ART-R resistance, and geographic extension to other malaria-endemic regions (2,3). Characterization of ART-R in each setting and rapid update of listed phenotypes classified as in vitro resistant to this antimalarial drug are needed.


Artemisinin-Resistant
To the Editor: Artemisinin resistance (ART-R) in Plasmodium falciparum phenotypes may have evolved independently in various areas of the Greater Mekong Subregion (1,2), prompting the World Health Organization to change its regional policy from containment to elimination (3). Risks associated with ART-R include compromised use of artemisinin combined therapy, partner drug resistance selection, total ART-R resistance, and geographic extension to other malaria-endemic regions (2,3). Characterization of ART-R in each setting and rapid update of listed phenotypes classified as in vitro resistant to this antimalarial drug are needed.
Detected in western Cambodia in 2008, ART-R has been observed in neighboring countries, notably at the Thailand-Myanmar border (4,5). Resistance is partial and manifests by an increased parasite clearance halflife (PCHL) of >5 hours in patients receiving artemisinin monotherapy or artemisinin combined therapy (6). In vitro, ART-R phenotype has been characterized by the ring-stage survival assay (RSA 0-3h , cutoff 1%) (7) and mutations in the propeller domain of a kelch gene on chromosome 13 (K13) (8,9). However, tremendous K13 variability in different genetic backgrounds requires confirmation of specific alleles as ART-R markers (2,3); even statistically significant clinical associations are rarely unequivocal (5-9).
On the Thailand-Myanmar border where ART-R has been documented (4), we investigated K13 mutations in clinical and in vitro phenotypes. Patients with nonsevere P. falciparum hyperparasitemia infections treated during 2011-2013 at the Shoklo Malaria Research Unit (Mae Sot, Thailand) were treated with artesunate, then combined artesunate/mefloquine (5). We retrospectively selected 33 case-patients on the basis of PCHL outcome to analyze a broad parasite clearance distribution with available cryopreserved isolates. Full written consent from all patients was obtained. PCHL was calculated on the basis of initial and repeated parasitemia measurement every 6 hours until undetectable asexual parasitemia (6) was achieved. Venous blood samples were cryopreserved before drug administration (day 0).
Short-term, culture-adapted parasites (3% hematocrit; RPMI-1640 supplemented with 10% human serum, 0.05 mg/mL hypoxanthine, 2 mg/mL sodium bicarbonate, 2 mg/mL glucose, 0.04 mg/mL gentamicin, 0.3 mg/mL L-glutamine in a 37°C candle-jar atmosphere) were split for blinded RSA 0-3h and K13 genotyping. We performed RSA 0-3h in duplicate by selecting early rings (0-3 h) in a combination of percoll gradient and sorbitol lysis, followed by a 6-h exposure to 700 nmol/L dihydroartemisinin (7). RSA survival rate was measured microscopically 66 hours after drug removal and calculated as the quotient of parasitemia upon DHA exposure over control parasitemia with dimethyl sulfoxide. Only 25 isolates that were successfully culture-adapted provided RSA survival rates.
In detail (Figure), C580Y and N458Y mutants were consistently associated with PHCL >5h and RSA values >1%. The C580Y allele has been repeatedly confirmed as a molecular marker of ART-R (5,7-9). Previous reports have inconsistently associated the N458Y mutation with ART-R; 7 case-patients with PCHL >5 h were reported by Ashley et al. (5), and 1 artemisinin sensitive case was reported at the China-Myanmar border (10). Nevertheless, this mutation has not been confirmed in vitro (3). We confirmed the mutation in vitro, and in vivo, according to the World Health Organization definition (3), this K13 allele as a molecular marker of ART-R.
Conflicting data observed between PCHL and RSA values for 4 mutant alleles (E252Q, P441L, G538V, and  A675V) require further targeted approaches to relate them to previous reports. In a study in which only PCHL were reported (5), the proportion of slowly clearing infections were 69%, 0%, 30%, and 61% for the P441L, E252Q, G538V, and A675V alleles, respectively. Discrepancies can result from confounding pharmacologic (drug level, partner drug), immunologic, and parasitologic (genetic background, parasitic stage at treatment initiation) factors.
RSA results and K13 genotypes were associated with delayed parasite clearance, emphasizing the pertinence of each method to define ART-R. In this area, N458Y is a marker of ART-R. To solve conflicts about specific mutations, more detailed characterization in vitro and in vivo is needed.