A combination of new screening assays for prioritization of transmission-blocking antimalarials reveals distinct dynamics of marketed and experimental drugs.

OBJECTIVES: The development of drugs to reduce malaria transmission is an important part of malaria eradication plans. We set out to develop and validate a combination of new screening assays for prioritization of transmission-blocking molecules. METHODS: We developed high-throughput assays for screening compounds against gametocytes, the parasite stages responsible for onward transmission to mosquitoes. An existing gametocyte parasitic lactate dehydrogenase (pLDH) assay was adapted for use in 384-well plates, and a novel homogeneous immunoassay to monitor the functional transition of female gametocytes into gametes was developed. A collection of 48 marketed and experimental antimalarials was screened and subsequently tested for impact on sporogony in Anopheles mosquitoes, to directly quantify the transmission-blocking properties of antimalarials in relation to their effects on gametocyte pLDH activity or gametogenesis. RESULTS AND CONCLUSIONS: The novel screening assays revealed distinct stage-specific kinetics and dynamics of drug effects. Peroxides showed the most potent transmission-blocking effects, with an intermediate speed of action and IC50 values that were 20-40-fold higher than the IC50s against the asexual stages causing clinical malaria. Finally, the novel synthetic peroxide OZ439 appeared to be a promising drug candidate as it exerted gametocytocidal and transmission-blocking effects at clinically relevant concentrations.

Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi.

We previously selected a line of the malaria vector mosquito Anopheles stephensi refractory (resistant) to the human malaria parasite Plasmodium falciparum, using in vitro infections with P. falciparum gametocytes. This report presents data on the genetic background of refractoriness. The results of F1-crosses and backcrosses show that refractoriness to P. falciparum in our A. stephensi line is autosomal and semi-dominant to susceptibility. The expression of refractoriness is apparently affected by a cytoplasmic factor. Interpretation of data from the crosses by quantitative trait locus analysis shows that one gene or two unlinked interacting autosomal genes, or groups of closely linked genes, are involved.

Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development.

The Plasmodium falciparum zygote/ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development. Anti-25 kD transmission blocking antibody, given to infected Anopheles stephensi or A. gambiae mosquitoes in an additional bloodmeal, 3-6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it. This reaction caused a significant reduction in the number of developing sporozoites. Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.

Feeding behaviour and sporozoite ejection by infected Anopheles stephensi.

Anopheles stephensi mosquitoes infected with Plasmodium falciparum sporozoites were allowed to feed individually through fresh whole thickness mouse skin. More sporozoites were ejected into the skin in clusters than into the blood. Deposition of sporozoites in the blood was an infrequent occurrence and always coincided with ejection of these stages into the skin--perhaps a spill-over effect. The number of probes before feeding (median 4.5) was not correlated with the sporozoite inoculum (median 8), nor was the number of sporozoites in the glands (median 14,500). However, the number of sporozoite clusters in the skin (median 1) was positively correlated with the inoculum size. The median value of the sporozoite inoculum was 22, when only those mosquitoes that ejected sporozoites were included. When feeding was interrupted and recommended on a new membrane, sporozoite ejection occurred with equal frequency on both occasions. Sporozoites disappeared from the site of bites in living mice within 2 h of feeding. The epidemiological significance of these observations is discussed.