Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea

Abstract

Background: Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments.

Methods: In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria.

Results: We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests.

Conclusions: The emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.).

Fig. 1.. Evidence of delayed parasite clearance associated with the expansion of the mutant Pfkelch13 622I in Eritrea

(A) Proportions of D3+ rate per site and year. D3+ cases were not observed in Akordat (2016, 2017 and 2019), Ghindae (2017, no data were available in 2016 and 2019), Guluj (2016 and 2017), and Tokombia ((2016 and 2017). (B) Allele frequency of P. falciparum kelch13 622I mutant parasites per site and year. Only Pfkelch13 wild-type parasites were observed in Akordat and Ghindae in 2017 (no data were available in 2016 and 2019). (C) Distribution of Pfkelch13 genotypes per site and year. The proportions of each Pfkelch13 allele are shown per year in pie charts (except for Ghindae where only 2017 data were available). The frequency for the Pfkelch13 wild type allele is shown in dark blue, the Pfkelch13 622I allele is in red and the other Pfkelch13 mutants are in yellow. The size of the pie chart is proportional to the sample size. The study sites colored in green correspond to areas where no D3+ case was observed. The study sites where D3+ cases where detected are colored in red (Guluj, Tokombia, and Shambuko). Additional information is presented in Tables S1, S6 and S7.

Fig. 2.. Pfkelch13 R622I mediates low-level artemisinin resistance in P. falciparum parasites.

Results show the percentage of early ring-stage parasites (0–3 hours post-invasion) that survived a 6-hour pulse of 700 nM DHA, relative to DMSO-treated parasites assayed in parallel. Percent survival values are shown as means ± SEM. Results were obtained from two (Dd2^WT) to three (NF54^WT, Dd2^R622I, NF54^R622I and NF54^C580Y) independent experiments, each performed in duplicate (Dd2^WT and Dd2^R622I) or triplicate (NF54^WT, NF54^R622I and NF54^C580Y).

Fig. 3.. Genome-wide phylogenetic tree.

This maximum-likelihood tree is based on 128 P. falciparum Eritrean isolates (red), together with 162 isolates collected worldwide (Africa, Asia and South America) and the 3D7 reference genome from Africa. Labels of Eritrean isolates include the year of collection. The non-Eritrean isolates were sourced from the MalariaGEN P. falciparum Community Project (https://www.malariagen.net/apps/pf/4.0) and are labelled with their accession identifier. Each leaf represents one sample and is colored according to the country of collection (bottom). Eritrean parasites carrying the Pfkelch13 622I mutation are identified by filled red stars at the tip. Eritrean Pfkelch13 622I mutants are closely related to Pfkelch13 wild type parasites originating from East African countries. Scale bar, 0.00005 nucleotide substitutions per character.