Investigating the transmissibility of artemisinin-resistant P. falciparum and the effect of artesunate on gametocyte production

Malaria transmission to mosquitoes relies on the development of a specialised form of the blood stage parasite called gametocyte. Gametocytes are produced in each replicating cycle, and the proportion of young gametocytes (sexual rings) produced per cycle is referred to as the sexual conversion rate (SCR). In P. falciparum infections, gametocytes take approximately 10 days to develop and last in circulation for months. As we move towards malaria eradication, there is an increased interest in using antimalarials to block malaria transmission from humans to mosquitoes by targeting gametocyte stages. However, key tools are lacking to determine the SCR during the course of an infection and the effect of antimalarial drugs on SCR. 

We have developed a malaria volunteer infection study using artemisinin resistant parasites. Briefly, subjects were infected with either artemisinin-resistant or artemisininsensitive parasites and subsequently treated with a single dose of artesunate, an artemisinin-derivate. We will adapt our recently published mathematical model to predict the SCR in samples collected pre-treatment from this volunteer study. Furthermore, we will optimise current molecular assays to determine the SCR and use these estimates to validate the model predictions. Finally, we will assess the effect of artesunate on SCR, by estimating the SCR using molecular assays and comparing the estimates before and after treatment. This project will develop a methodology to determine the effect of antimalarial drugs on gametocyte production, which will help to improve malaria transmission models.

Optimising molecular assays to determine the SCR: Sexual rings from P. falciparum K13-gexp5- GFP in vitro culture will be sorted to prepare three standard curves containing six 10-fold dilutions ranging from 1 x 106 parasites/mL to 1 parasites/mL. Transcript levels of ap2-g or gexp5 from each dilution will be determined by qRT-PCR assays (TaqMan) and digital PCR using the ap2g primers described by Usui et al., and the gexp5, sbp1 and 18SrRNA primers described by Farid et al. A conversion factor from transcripts/mL to sexual rings/mL will be estimated by logistic regression. An identical approach will be followed to estimate the conversion factor from sbp1 and 18SrRNA transcripts to rings stage/mL (asexual and sexual rings parasitaemia). The SCR will be calculated by measuring the ratio between sexual rings/mL and rings stage/mL.

Estimating the SCR in K13-infected subjects before and after treatment: The SCR will be estimated in all 13 K13-infected subjects in pre-treatment samples from Day 4 post-inoculation until Day 9 (six timepoints), and in post-treatment samples collected 48, 56, 60, and 72 hours after artesunate. The effect of the drug on SCR will be assessed by comparing the SCR pre-treatment (Day 9) versus the SCR post-treatment (48, 56, 60 and 72h). The post-treatment timepoints cover the window when sexual rings induced during artesunate-treatment are in circulation5.

Mathematical modelling: The mathematical model described previously will be adopted in this project to estimate the SCR in K13-infected subjects. We will fit the model to all available parasitaemia data from the aforementioned clinical trial.

The data include: i) total parasitemia estimated by qPCR targeting the 18SrRNA and reported as parasites/mL, from 40–50 timepoints post-inoculation per subject, and ii) gametocytemia estimated by qRT-PCR targeting the female pfs25 and the male pfMGET transcripts and reported as gametocytes/mL10, from 9–10 timepoints per subject. The pre-treatment SCR estimated by molecular assays in point 2 will be used to validate the SCR estimates obtained by mathematical modelling.

Drug Resistance
Measurement of Transmission

Nov 2019 — Oct 2023

$15,000

Australia

Australian Centre for Research Excellence in Malaria Elimination (ACREME)