This study evaluated the transmissibility of a new P. falciparum 3D7 blood-stage parasite bank (MBE-008) to determine its suitability for evaluating transmission blocking interventions in future malaria volunteer infection studies (VIS). We demonstrated that transmissibility of MBE-008 parasites was high, with gametocytes infective to mosquitoes in all four participants on day 25 following inoculation. Furthermore, intensity of transmission was high, with a median 94% (range, 12–100%) of mosquitoes positive for oocysts, and 76% (range, 8–94%) positive for sporozoites. This study has therefore demonstrated that this parasite bank is suitable for evaluating transmission blocking interventions in malaria VIS.

The transmissibility of this new P. falciparum 3D7-MBE-008 parasite bank is comparable to that demonstrated for the P. falciparum 3D7-V2 bank, which has been utilized for numerous malaria VIS including several studies evaluating transmission blocking interventions^17,18,34. In the most recent transmission VIS utilising the 3D7-V2 bank and conducted using the same study design as the current study, transmission to mosquitoes on day 25 post-inoculation was demonstrated in 6/6 participants, with transmission intensity also being high (median 86% [range, 22–98%] of mosquitoes positive for oocysts and 57% [range, 4–92%] positive for sporozoites)¹⁸. Taken together, with this study design (albeit with a different P. falciparum 3D7 bank), transmission has therefore been demonstrated in 10 of 10 participants, with high intensity transmission (median 89% [interquartile range, 61–98%] of mosquitoes positive for oocysts and 70% [interquartile range, 32–91%] positive for sporozoites across these 2 studies). The current study has therefore further confirmed the utility of the malaria VIS for evaluating transmission blocking interventions. Blocking malaria transmission is a critical component of malaria control programs, particularly given the recent emergence of artemisinin resistant parasites in Africa³⁵. Evaluating transmission blocking activity is therefore a key step in the clinical development of new antimalarial agents, and malaria VIS offer a valuable platform to expedite the development of agents with transmission blocking activity.

Primaquine is currently the only antimalarial recommended for the purpose of transmission blocking³⁶. Therefore, we took the opportunity to observe the effect of primaquine on gametocyte density and transmission to mosquitoes, as well as the duration of gametocytemia and transmission to mosquitoes in the absence of any transmission blocking intervention. Given the small sample size of this pilot study, we did not intend to make definitive comparisons between treatment groups. For the 2 participants administered primaquine (0.25 mg/kg), the decline in gametocytemia was relatively modest, with gametocytes persisting at a high level 4 days post-primaquine in one participant. This delay in clearance of gametocytes is consistent with previous studies in malaria-endemic regions involving supplementation of antimalarial regimens with a single low dose of 0.25 mg/kg primaquine as a transmission blocking agent^4,5,7. In these studies, gametocyte densities reduced significantly between 2 and 7 days of treatment, but persisted up to 28^4,7 or 42 days⁵. Importantly, the continued presence of gametocytes after primaquine treatment has not been shown to correlate with mosquito transmission, suggesting sterilisation of gametocytes preceding their clearance^37,38.

In both participants administered primaquine, substantial reductions in transmission were observed by day 29 (4 days post-primaquine). These results are again generally consistent with two Phase 2 studies in patients in malaria-endemic regions, where the same dose of primaquine administered alongside antimalarial treatment resulted in complete block in transmission within 2 days^5,7. The low level of transmission demonstrated 4 days post-primaquine in one participant in the current study likely reflects the fact that transmission intensity in this participant was particularly high at baseline (98% of mosquitoes positive for oocysts), in comparison to the substantially lower baseline transmission intensities reported in the Phase 2 studies (medians of 11–27% and 23–24% reported by Dicko et al.⁵ and Stone et al.⁷, respectively). The high levels of transmission observed in our study were aided by our gametocyte enrichment process. In a separate Phase 2 study, transmission intensity between non-enriched and enriched participant samples was compared before and after antimalarial treatment supplemented with low dose primaquine (0.25 mg/kg)³⁹. Following treatment, 2 participants who were initially non-infectious were able to infect mosquitoes following gametocyte enrichment. However, while the high intensity of transmission observed in malaria VIS may not reflect real-world scenarios and may result in more prolonged transmission following a transmission blocking intervention than observed in Phase 2 studies, the relative reductions in transmission are comparable. Furthermore, the high intensity of transmission at baseline in malaria VIS increases the power to evaluate transmission blocking interventions in these studies, enabling interventions to be evaluated with relatively low samples sizes.

We also evaluated gametocytemia and transmission to mosquitoes in 2 participants who were not administered any transmission blocking intervention. The duration of transmission in these 2 participants was long, with a high level of transmission demonstrated 22 days following the first dose of piperaquine, and one participant still infectious to mosquitoes 31 days following the first dose of piperaquine. These findings demonstrate the longevity of gametocytes in the absence of gametocytocidal treatment, consistent with the major contribution of asymptomatic parasitemia/gametocytemia to ongoing transmission in malaria-endemic regions^40,41,42,43, and highlight the critical importance of incorporating transmission blocking interventions into malaria control programs.

An important observation in this study was that 2 participants experienced recrudescence of asexual parasites following administration of 2 doses of piperaquine, confounding the analyses of gametocyte dynamics and infectivity to mosquitoes between and within participants. We therefore performed simulations to estimate a piperaquine dosing strategy that would prevent parasite recrudescence in future studies. These simulations determined that a three-dose regimen, with 960 mg administered 2 days and 9 days following the initial 480 mg dose, will have a high likelihood of preventing recurrence of asexual parasitemia for a sufficient duration to assess transmission blocking interventions. This analysis will inform the design of future malaria VIS evaluating transmission blocking interventions.

In addition to the recrudescence of asexual parasitemia that occurred in 2 participants, this study had other limitations. First, transmission was not assessed as planned on days 26 and 27 (1 and 2 days following administration of primaquine), limiting our ability to assess the early transmission blocking activity of primaquine. Second, as stated above, the small sample size of this pilot study meant that there was insufficient statistical power to definitively assess the impact of primaquine on gametocytemia or transmission to mosquitoes. Third, the fact that we did not observe gametocyte exflagellation in any of the samples in this study limited our ability to further delineate this aspect of the transmission blocking mechanism of primaquine.

In summary, this study demonstrated the transmissibility of a new P. falciparum 3D7 parasite bank (MBE-008), confirming its suitability for use in future malaria VIS evaluating transmission blocking interventions. Together with another recent study using the same study design with a different P. falciparum 3D7 bank, transmission to mosquitoes has now been demonstrated in 10 of 10 participants, with a high level of transmission intensity. Taken together these studies have confirmed the utility of malaria volunteer infection studies for evaluating transmission blocking interventions.