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Dry season P. falciparum reservoir

Periodic Reporting for period 3 - DrySeasonPf (Dry season P. falciparum reservoir)

Reporting period: 2021-01-01 to 2022-06-30

The mosquito-borne Plasmodium falciparum parasite is responsible for over 200 million malaria cases and nearly half a million deaths each year among African children. Dependent on Anopheles mosquito for transmission, the parasite faces a challenge during the dry season in the regions where rain seasonality limits vector availability for several months. While malaria cases are restricted to the wet season, clinically silent P. falciparum infections can persist through the dry season and are an important reservoir for transmission. Our preliminary data provides unequivocal evidence that P. falciparum modulates its transcription during the dry season, while the host immune response seems to be minimally affected, suggesting that the parasite has the ability to adapt to a vector-free environment for long periods of time. Understanding the mechanisms which allow the parasite to remain undetectable in absence of mosquito vector, and to restart transmission in the ensuing rainy season will reveal complex interactions between P. falciparum and its host. To that end I propose to: (i) Identify the Plasmodium signalling pathway(s) and metabolic profile associated with long-term maintenance of low parasitaemias during the dry season, (ii) Determine which PfEMP1 are expressed by parasites during the dry season and how effectively they are detected by the immune system, and (iii) Investigate the kinetics of P. falciparum gametocytogenesis, its ability to transmit during the dry season, and uncover sensing molecules and mechanisms of the disappearance and return of the mosquito vector.
In the first 18 months we confirmed that indeed a high proportion of children carries P. falciparum parasites throughout the dry season without presenting malaria symptoms, while clinical cases occur only during the transmission season when mosquitoes are available. on the specific aims we have achieved:
regarding Aim i) We sought to profile genetic diversity, gene expression and metabolites of P. falciparum that may be associated with long-term maintenance of low parasitaemias, as well as the parasite’s ability to replicate, and produce sexual forms that may be transmitted to mosquitoes. In work that we are now preparing to submit for publication we show that genetic diversity of P. falciparum during the dry season is not different form that of clinical cases observed in the transmission season; but transcription, however, was found much altered during the dry season. A very large number of differential expressed genes (DEGs) was identified through NGS comparing leucocyte-depleted blood of Malian children with persistent subclinical P. falciparum infections at the end of the dry season, and that of age- and gender- matched Malian children presenting with their first clinical malaria case in the ensuing transmission season. After excluding that host immunity is actively detecting and attacking the infected erythrocytes and leading to the parasites’ DEGs, we followed to investigate how efficiently P. falciparum replication occurs in the dry vs transmission seasons through flow cytometry. In Mali, we cultured parasites immediately after blood collection form subclinical children carrying P. falciparum infections at the beginning, mid and end of the dry season, or at their first clinical malaria episode during the transmission season. Our data shows clearly that parasitaemia in the dry season took less time to increase (~30h in January and ~24h in March and May) compared with the time that parasites from malaria cases needed to increase parasitaemia (>36h in Malaria). Parasites during the dry season were not impaired in their replicative ability, and seemed instead to be reaching the end of a 48h replicative cycle faster than parasites collected from malaria-causing infections in the transmission season. These data could be consistent with a faster-replicating parasite in dry season, which is unlikely given that parasitaemias in this period are maintained low and do not progress to disease; or it could also be that parasites collected in the dry season would be more advanced in the 48h intra-erythrocytic cycle. To test the later hypothesis we measured development of parasites collected straight from the arm of children in the dry season and in malaria cases in the transmission season. We have also performed and are currently analysing the metabolomic analysis of the samples of the dry season vs malaria casaes in the wet season.
on Aim ii) in 2018 blood samples collected in Kalifabougou Mali during the dry and wet seasons started to be analysed to determine expression of var genes. DNA was extracted from an aliquot of each sample to be used in downstream targeted next generation sequencing. Also, for each sample, RNA was extracted using Trizol method then treated the RNA to remove genomic DNA contamination. Reverse transcription of RNA samples into cDNA which was used to quantify parasite material in qPCR reactions. The cDNA together with the genomic DNA samples were used for targeted next generation sequencing of the var genes. For this, the samples were amplified by PCR using degenerate primers designed to target var gene sequences of interest. Amplified products were then sent for sequencing, ad we are currently waiting for the results.
on Aim iii) we have established a 5 color multiplex taqMan qRT-PCR to quantify the proportion of parasites converting in to sexual sates of P. falciparum and the ration between female and male gametocyte production in the dry and wet season.Blood samples collected in Kalifabougou Mali during the dry and wet seasons started to be analysed and first attempts were done to establish mosquito infections from blood of infected individuals from the dry and the wet seasons. LysoPC quantification of donor samples through the dry and the wet season were all determined.
We have move forward in all aims initially planned and we are on track to obtain the expected results by the end of the project. Concretely we are are writing a manuscript covering how dynamic sexual commitment of P. falciparum is over the seasons. which should be ready to submit early in 2021. Plus, we are finalising experiments for a manuscript in preparation regarding the ability of malaria parasites to detect environmental signals. And we are also currently finishing evaluating if P. falciparum remodels its host cell differently in the different seasons. Both of these should be published in the 1st half of 2021. Then we plan to have still another story published prior to the end of the project in dec 2022 concerning the variant gene families expressed on the surface of the infected erythrocytes and how immunity responds to these; and i believe we are n talk with that too, we have sequences DLBalpha Tags of PfEMP1 (the most important variant gene family in P. falciparum) from samples collected in different seasons of the year and we are currently doing that for longitudinal samples of the same children along the year to allow us to identify switching between variants of the family seasonally, and test serology against the different variants.