Kelch13 mutations in Plasmodium falciparum and risk of spreading in Amazon basin countries

Abstract

Background: The first potential focus for artemisinin resistance in South America was recently confirmed with the presence of the C580Y mutation in the Plasmodium falciparum kelch 13 gene (pfk13) in Guyana.

Objectives: This study aimed to strengthen pfk13 monitoring in the Amazon basin countries, to compile the available data and to evaluate the risk of spreading of mutations.

Methods: Sanger sequencing was done on 862 samples collected between 1998 and 2019, and a global map of pfk13 genotypes available for this region was constructed. Then, the risk of spreading of mutations based on P. falciparum case importation between 2015 and 2018 within countries of the Amazon basin was evaluated.

Results: No additional pfk13 C580Y foci were identified. Few mutations (0.5%, 95% CI = 0.3%-0.8%) in the propeller domain were observed in the general parasite population of this region despite a high proportion of K189T mutations (49.1%, 95% CI = 46.2%-52.0%) in the non-propeller domain. Case information revealed two patterns of intense human migration: Venezuela, Guyana and the Roraima State in Brazil; and French Guiana, Suriname and the Amapá State in Brazil.

Conclusions: There are few pfk13 mutant foci, but a high risk of dispersion in the Amazon basin, mainly from the Guiana Shield, proportionate to mining activities. Therefore, access to prompt diagnosis and treatment, and continuous molecular monitoring is essential in these geographical areas.

Figure 1.

Distribution of pfk13 mutations in the Amazon basin between 1983 and 2019. Pie charts represent pfk13 allele frequencies per country. The total number of analysed samples is shown with the percentage and the 95% CI (in brackets) when a mutation was identified. Results are shown in the map of the Annual Falciparum Incidence (AFI) in the Americas region in 2017 (Annual Country Reports to PAHO/CDENT/Malaria). For Brazil, results are regrouped by closely located states. Notably, most Colombian and Ecuadorian samples (mutant included) represented here were collected on the Pacific coast (99.8% and 93.7%, respectively) and not in the Amazon region of the country. Brazilian states: AC, Acre; AM, Amazonas; AP, Amapá; MT, Mato Grosso; PA, Pará; RO, Rondônia; and RR, Roraima. Countries: COL, Colombia; ECU, Ecuador; GUF, French Guiana; GUY, Guyana; PER, Peru; SUR, Suriname; and VEN, Venezuela.

Figure 2.

Ring-stage survival assays for parasites from French Guiana. The survival rate of ring-stage parasites pfk13 WT (n = 115), pfk13 K189T (n = 24), pfk13 K189N (n = 1) and pfk13 R/K255 (mixed genotype, n = 1) after a 6 h pulse of 700 nM dihydroartemisinin as measured by microscopy 66 h later. Horizontal bars represent mean±SEM percentage survival.

Figure 3.

Map of P. falciparum infections imported between countries in the Amazon basin, 2015–18. Number of P. falciparum mono and mixed infections imported by country/state of diagnosis and putative country of infection. Each country/state is represented by a standardized location not linked with the putative contamination place. Lines in the map represent the cumulative numbers of infections recorded during the 2015–18 period. Lines between countries include bidirectional data, while lines from countries to Brazilian states are unidirectional (imported in Brazil). The width and colour of the lines are proportional to the number of imported cases. The grey P. falciparum transmission layer is based on the Annual Falciparum Incidence (AFI) reported during the 2015–18 period. Unstable corresponds to AFI <1 for ≥1 year and endemic (stable) corresponds to AFI >1 for all years. Countries: BOL, Bolivia; COL, Colombia; ECU, Ecuador; GUF, French Guiana; GUY, Guyana; PER, Peru; SUR, Suriname; and VEN, Venezuela. Brazilian states: AC, Acre; AM, Amazonas; AP, Amapá; BA, Bahia; CE, Ceará; GO, Goiás; MA, Maranhão; MG, Minas Gerais; MT, Mato Grosso; PA, Pará; PI, Piauí; PE, Pernambuco; RO, Rondônia; RR, Roraima; and TO, Tocantins.

Figure 4.

Number of P. falciparum mono and mixed infection cases by place of diagnosis and putative place of infection 2 weeks prior to diagnosis in Guyana, 2015–18. Lines represent a set of localities wherein one is where a patient’s smear was taken for diagnosis and the second is the place where the patient self-reported they were 2 weeks prior to diagnosis. The width and colour of the lines represent the number of cases of P. falciparum mono and mixed infections. GUF, French Guiana.