Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa

Abstract

Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread.

Figure 1. Infection of different Anopheles species by Cambodian P. falciparum clinical isolates.

Three ART-sensitive parasites carrying WT K13 alleles (KH-C/WT) and six ART-resistant parasites from three Western Cambodian founder populations carrying different mutant K13 alleles (WKH-F02/R539T, WKH-F04/Y493H and WKH-F03/C580Y) were used to infect malaria vectors of Africa (An. coluzzii) and Southeast Asia (An. dirus and An. minimus) in parallel. Infection intensity was measured by counting the number of parasite oocysts per mosquito midgut in individual mosquitoes 8 days after they were fed gametocyte-infected erythrocytes. Each dot represents the oocyst count in a single mosquito midgut; black bars indicate the mean number of oocysts per midgut for all mosquitoes dissected. At least four independent feeds were performed for each parasite–mosquito combination. n, number of fed mosquitoes checked for oocysts; % Inf., proportion of fed mosquitoes with ≥1 oocyst; Spz., sporozoites were found in the salivary glands of a subset of mosquitoes dissected on day 14 after feeding (+), or their presence could not be determined (ND), typically owing to low infection rates, or were not examined (NE).

Figure 2. Relative infectivity of P. falciparum clinical isolates to An. dirus, An. minimus and An. coluzzii.

Estimates (points) and 95% confidence intervals (lines) for odds ratios of any oocyst infection, done by a separate logistic regression for each isolate that controls for replicate feeds. The top panel shows the odds for infection of An. dirus over the odds for An. coluzzii, the middle panel shows the odds for An. minimus over the odds for An. coluzzii and the bottom panel shows the odds for An. minimus over the odds for An. dirus. Isolates are colored according to population: KH-C/WT (green), WKH-F02/R539T (blue), WKH-F04/Y493H (yellow) and WKH-F03/C580Y (red).

Figure 3. Distribution of Pfs47 haplotypes among Cambodian P. falciparum clinical isolates.

The Pfs47 alleles of 516 parasite isolates were genotyped against the 3D7 reference genome (ver. 3). This procedure identified 22 different haplotypes, which are listed in order of decreasing prevalence in our sample collection (Supplementary Tables 1 and 2). The Pfs47 haplotypes of parasites from the core population of Eastern Cambodia (KH-C) and multiple founder populations of Western (WKH), Northern (NKH) and Northeastern (NEKH) Cambodia are indicated. The six ART-resistant isolates we used in our study belong to the WKH-F02, WKH-F04 and WKH-F03 founder populations (*), all of which possess the same Pfs47 haplotype 1 (Supplementary Tables 1 and 2). The three ART-sensitive isolates we used belong to the KH-C/WT population (**) and have the same Pfs47 haplotype 3 (Supplementary Tables 1 and 2). It is noteworthy that Pfs47 haplotype 1 is present in four of five ART-resistant founder populations, whereas all 22 Pfs47 haplotypes are present in ART-sensitive isolates (Supplementary Table 2).