Host and parasite factors of Plasmodium falciparum asymptomatic persistence during the dry season

Malaria is a mosquito-borne disease caused by Plasmodium parasites and transmitted by female mosquitoes of the genus Anopheles. The majority of deaths occurs in the African continent and in 2019 Plasmodium falciparum killed nearly 400,000 people. In many areas of the globe, dry season periods limit Anopheles mosquito availability reducing malaria transmission to virtually zero. In Mali, during the 5- to 6-month dry season P. falciparum persists in a fraction of the human population in clinically silent infections that serve as reservoir to restart transmission when mosquitoes return in the ensuing wet season. However, the mechanism by which P. falciparum bridges rainy seasons without promoting malaria symptoms or being cleared by host immunity remained elusive. In this work, we used samples from a village in Mali where individuals are exposed to an alternating six-month dry and rainy season. We show that P. falciparum persists in the blood of asymptomatic individuals during the dry season at low parasite densities and very seldom causing disease. By comparing immune system markers in individuals that carried or not infections during the dry season, we found that asymptomatic infections only minimally affect the host immune response during this period. We compared the transcription profile in parasites collected at the end of the dry season or during the first clinical malaria case in the ensuing transmission season and we found that the parasites segregated by season, where parasites from the dry season have a transcription profile reflecting a longer circulation within each 48-hour intra-erythrocytic cycle when compared to malaria causing P. falciparum. In agreement with a different transcriptional profile that indicates a longer circulation of P. falciparum in the dry season, we found more developed parasites in the dry season by microscopy and flow cytometry in comparison with parasites isolated from malaria patients in the transmission season. And we further show that circulation of older parasites in the dry season increases splenic clearance, promoting the low parasite densities found in the dry season. All together we describe how seasonally altering the competence of adhesion to the endothelium by parasites, which is a hallmark of P. falciparum malaria, affects parasitaemia growth and disease presentation. To understand how P. falciparum achieves this less adhesive phenotype during the dry season, we investigated if parasites could respond to seasonal serologic factors. We cultured P. falciparum parasites in the presence of plasmas from different times of the year anddetermined parasite development, replication and host cell remodelling in vitro, which could seasonally favour asymptomatic infections. We did not observe any differences in parasite development, replication, or host cell remodelling when parasites were grown in different season plasmas. Although the in vitro experiments we were able to do could not disprove the existence of a sensing mechanism of the season by P. falciparum, because it may be that we were unable to provide the condition exactly replicating what occurs in vivo, it seems unlikely since we also observed older parasites in circulation in asymptomatic infections during transmission season. This all suggesting that the longer circulation phenotype observed is not unique to the dry season, but likely linked to low parasitaemias that remain below the clinical radar. Finally, we questioned when the P. falciparum dry season asymptomatic infections were transmitted during the preceding transmission season. We followed retrospectively children that were infected at the end of one dry season until when they had first become parasite positive in the preceding transmission season, capturing fortnightly longitudinal data on parasite density fluctuations, and measuring the parasite genetic diversity by ama1 amplicon sequencing. We found that clonal infections that reached the end of the dry season had been mainly transmitted towards the end of the rainy season, while clones transmitted early in the transmission season were unlikely to persist until the end of the dry season. These data suggest that infection length is tailored to bridge the length of the dry season, and favours a model where early transmitted infections that are not treated will present a “dry season” phenotype still during the transmission season (as observed in asymptomatic infections during the transmission season), while later transmitted infections are more likely reach the end of the dry season and resume transmission as the wet season ensues. The results from this work begin to elucidate on the mechanisms that allow P. falciparum to survive the dry season, bridging two transmission seasons. The knowledge acquired in this work about the complex interaction between P. falciparum, the human host and the environment, might inform strategies that will aid to the ultimate goal of malaria eradication by targeting the silent reservoirs of P. falciparum