Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia.

BACKGROUND: The accurate monitoring and evaluation of malaria vectors requires efficient sampling. The objective of this study was to compare methods for sampling outdoor-biting Anopheles mosquitoes in Cambodia. METHODS: In the Cambodian provinces of Pursat, Preah Vihear, and Ratanakiri, six different mosquito trapping methods were evaluated: human landing collection (HLC), human-baited tent (HBT), cow-baited tent (CBT), CDC miniature light trap (LT), CDC miniature light trap baited with molasses and yeast (LT-M), and barrier fence (F) in a Latin square design during four or six consecutive nights at the height of the malaria transmission season. RESULTS: Using all traps, a total of 507, 1175, and 615 anophelines were collected in Pursat, Preah Vihear, and Ratanakiri, respectively. CBTs captured 10- to 20-fold more anophelines per night than the other five sampling methods. All 2297 Anopheles mosquitoes were morphologically identified and molecularly typed using standard morphological keys and sequencing the rDNA ITS2 region to distinguish cryptic species, respectively. Overall, an extremely diverse set of 27 known Anopheles species was sampled. CBTs captured the same molecular species that HLCs and the other four traps did, as well as additional species. Nine specimens representing five Anopheles species (Anopheles hyrcanus, Anopheles barbirostris sensu stricto, Anopheles barbirostris clade III, Anopheles nivipes, and Anopheles peditaeniatus) were infected with Plasmodium falciparum and were exclusively captured in CBTs. CONCLUSIONS: These data indicate that cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia. This sampling method captured high numbers of anophelines with limited sampling effort and greatly reduced human exposure to mosquito bites compared to the gold-standard human landing collection.

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies.

BACKGROUND: When rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed. METHODS: Several species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented. RESULTS: Anopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner. CONCLUSIONS: Anopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.

Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae.

Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

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

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.