Efficient Transmission of Mixed Plasmodium falciparum/vivax Infections From Humans to Mosquitoes

Abstract

Background: In Southeast Asia, people are often coinfected with different species of malaria (Plasmodium falciparum [Pf] and Plasmodium vivax [Pv]) as well as with multiple clones of the same species. Whether particular species or clones within mixed infections are more readily transmitted to mosquitoes remains unknown.

Methods: Laboratory-reared Anopheles dirus were fed on blood from 119 Pf-infected Cambodian adults, with 5950 dissected to evaluate for transmitted infection. Among 12 persons who infected mosquitoes, polymerase chain reaction and amplicon deep sequencing were used to track species and clone-specific transmission to mosquitoes.

Results: Seven of 12 persons that infected mosquitoes harbored mixed Pf/Pv infection. Among these 7 persons, all transmitted Pv with 2 transmitting both Pf and Pv, leading to Pf/Pv coinfection in 21% of infected mosquitoes. Up to 4 clones of each species were detected within persons. Shifts in clone frequency were detected during transmission. However, in general, all parasite clones in humans were transmitted to mosquitoes, with individual mosquitoes frequently carrying multiple transmitted clones.

Conclusions: Malaria diversity in human hosts was maintained in the parasite populations recovered from mosquitoes fed on their blood. However, in persons with mixed Pf/Pv malaria, Pv appears to be transmitted more readily, in association with more prevalent patent gametocytemia.

Keywords: Anopheles dirus; Plasmodium vivax; deep sequencing; gametocyte; malaria transmission.

Figure 1.

Transmission of mixed Plasmodium falciparum (Pf)/Plasmodium vivax (Pv) malaria from 7 Cambodian adults to membrane-fed Anopheles dirus mosquitoes: schematic of species-specific parasite burden. (a) Parasitemia scale. The number of + signs indicates the number of species-specific parasites/μL: 1–999 (+); 1000–9999 (++); 10 000+ (+++). (b) Gametocytemia scale. The number of gametocyte images indicates the number of species-specific gametocytes/μL: 1–49 (1); 50–149 (2); 150+ (3). (c) Oocyst-positive mosquitoes out of 30, as determined by species-specific quantitative polymerase chain reaction (qPCR) at day 9 postfeeding. (d) Sporozoite-positive mosquitoes out of 30, as determined by species-specific qPCR at day 16 postfeeding. Pf, orange; Pv, blue; Pf/Pv coinfection, green.

Figure 2.

Percentage distribution of PfAMA1 and PvMSP1 haplotypes in the blood among mono-Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) infections (A) and mixed Pf/Pv infections (B). Individual haplotypes (CAM_AV numbers) are represented by different colors, with width of bars corresponding to their within-host frequency (%) on the x-axis. Blood from 9 Pf, 5 Pv, and 12 Pf/Pv infections are shown. CAM_AV numbers refer to Cambodia_AnlongVeng, with number indicating the population prevalence of the haplotype (CAM_AV0 is the most common haplotype in the cohort, CAM_AV1 the next most common, etc).

Figure 3.

Minimum spanning trees depicting the relationship of individual PfAMA1 (A) and PvMSP1 (B) haplotypes within persons with mixed Plasmodium falciparum/Plasmodium vivax malaria. For each subject, the minimum spanning tree provides a graphical representation of the in-host frequency of each haplotype (denoted by size of colored orbs) and the number of single-nucleotide polymorphisms and insertion/deletions that separate each haplotype from the next most similar one (denoted by number of pink dots along mutational path between haplotypes).

Figure 4.

Measured genetic diversity in the cohort according to sensitivity of detection of minority haplotypes for Plasmodium falciparum (Pf ) (A) and Plasmodium vivax (Pv ) (B). For infections of each species (n = 21 Pf and 17 Pv), the change in the mean multiplicity of infection ([MOI] left axis) and total number of haplotypes (right axis) in the cohort is shown to decrease as the threshold frequency below which haplotypes are not called in SeekDeep is increased. Although the mean MOI of each species is the same when minority haplotypes at 1% frequency are detected, the number of Pv haplotypes is greater and the measured Pv MOI is more robust to less sensitive genotype detection.

Figure 5.

PfAMA1 haplotypes in patient blood through transmission to mosquitoes. For each subject, the composition of haplotypes and their in-host frequencies are depicted in each stage of the parasite life cycle—blood-stage in human, gametocytes isolated from blood (when available), oocysts in mosquito, and sporozoites in head and thorax of mosquito (A). Haplotypes were detected in pooled mosquito samples, except for subject SN063-M, where individual mosquitoes (Mo2 through Mo20) at oocyst and sporozoite stages were also analyzed (B). On the left, the number of gametocytes/µL of blood detected by microscopy is shown. On the right, the percentage of mosquitoes with oocyst-positive midguts is shown along with the median (range) oocyst count per midgut. The suffix -M depicts a subject with mixed Plasmodium falciparum/Plasmodium vivax infection.

Figure 6.

PvMSP1 haplotypes in patient blood through transmission to mosquitoes. Plasmodium vivax haplotypes within mixed infections are depicted through human-to-mosquito transmission (A). For 4 subjects, data are shown for both pooled and individual mosquitoes, with individual mosquitoes sometimes displaying haplotypes not apparent in pooled samples (B).