Détermination des facteurs associés à l'engagement sexuel chez Plasmodium falciparum

Malaria parasite can encounter heterogenous hosts and different type of host cells. In particular during the intra-erythrocytic cycle the parasite replicates inside an erythrocyte and can encounter different erythrocytes in term of maturation stage or in host carrying haemoglobin variants modifying the red blood cell environment. The impact of this heterogeneity on mosquito-transmissible stages is particularly unexplored. Characterising their effects on both sexual commitment and transmission to mosquitoes is of paramount importance in the fight against malaria. Here we leveraged new technologies including single cell transcriptomics as well as samples collected in naturally infected carriers to understand the impact of RBC heterogeneity on transmission phenotypes.Firstly, we find that sickle cell trait carrier has an enhanced mosquito transmission through both an increase in prevalence of transmission stages and are more infectious to mosquitoes, suggesting they excessively contribute to malaria transmission and should be taken into account in our understanding of the spread of malaria.Secondly, we find that P. falciparum preferentially invades younger erythrocytes and that the biomass of infected reticulocyte is important within an infection. We further show that parasites preferentially commit to sex within younger erythrocytes, suggesting that the host cell is one of the key determinants in environmentally-induced sexual fate determination.Our data show cases the power of combining cutting edge methods with parasites in their natural environment as a source for identifying new and exciting parasite biology. This work has important implication for our understanding of how parasites maximise their transmission in response to a carrying environment and therefore on the spread of malaria and how it may be targeted in transmission blocking interventionsKey words: Sexual commitment, sickle cell trait, host cell preferenceAu cours de son cycle intra-érythrocytaire chez l’Homme, Plasmodium falciparum, l’agent pathogène responsable du paludisme se réplique dans les érythrocytes. Le parasite peut envahir des érythrocytes à différentes étapes de leur maturation et il peut aussi se retrouver chez des hôtes porteurs d’anomalies de l’hémoglobine qui modifient les propriétés de l’érythrocyte. L’impact de cette hétérogénéité des globules rouges sur le développement des stades sexués, seuls stades infectieux pour le vecteur, est particulièrement inexploré.Dans cette thèse, nous avons cherché à caractériser l’effet de l’hétérogénéité de l’environnement érythrocytaire sur l’engagement sexuel du parasite et sur la transmission chez le moustique. Nous avons développé des approches basées sur de nouvelles technologies, dont la transcriptomique en cellule unique, pour rechercher des facteurs associés à l’engagement sexuel à partir d’échantillons de terrain prélevés au Cameroun chez des porteurs asymptomatiques. Au Cameroun, nous avons réalisé des gorgements de moustiques sur sang contenant des gamétocytes issus de porteurs du trait drépanocytaire ou d’une hémoglobine normale pour la mesure de l’infection selon le génotype de l’hôte.Dans un premier temps, nous avons montré que les gamétocytes circulant chez des porteurs du trait drépanocytaire sont plus infectieux pour le vecteur. La prévalence et la charge oocystique des moustiques femelles gorgés sur sang d’individus HbAS sont significativement plus élevées que celles obtenues pour des femelles nourries sur sujets HbAA. Les sujets drépanocytaires contribueraient alors de manière accrue à la transmission du paludisme et ces résultats doivent être pris en compte dans les interventions de prévention afin de mieux protéger ces populations plus à risque de propager le parasite.Dans un second temps, nous avons observé que P. falciparum envahit préférentiellement les érythrocytes jeunes et que la biomasse parasitaire est plus élevée dans les réticulocytes infectés. Nous avons aussi montré que les parasites évoluent plus souvent vers des stades sexuellement engagés dans des érythrocytes plus jeunes, ce qui suggère que la cellule hôte est un des déterminants clés de l’engagement sexuel.Ce travail de thèse apporte des résultats importants pour notre compréhension de la transmission et particulièrement sur les mécanismes mis en jeu par le parasite pour optimiser sa transmission selon l’environnement dans lequel il se développe. Nos résultats vont apporter de nouvelles voies pour le développement de stratégies bloquant la transmission.Mots clés : Engagement sexuel, trait drépanocytaire, préférence des cellules hôtes

Determination of factors associated with sexual commitment rings

Au cours de son cycle intra-érythrocytaire chez l’Homme, Plasmodium falciparum, l’agent pathogène responsable du paludisme se réplique dans les érythrocytes. Le parasite peut envahir des érythrocytes à différentes étapes de leur maturation et il peut aussi se retrouver chez des hôtes porteurs d’anomalies de l’hémoglobine qui modifient les propriétés de l’érythrocyte. L’impact de cette hétérogénéité des globules rouges sur le développement des stades sexués, seuls stades infectieux pour le vecteur, est particulièrement inexploré.Dans cette thèse, nous avons cherché à caractériser l’effet de l’hétérogénéité de l’environnement érythrocytaire sur l’engagement sexuel du parasite et sur la transmission chez le moustique. Nous avons développé des approches basées sur de nouvelles technologies, dont la transcriptomique en cellule unique, pour rechercher des facteurs associés à l’engagement sexuel à partir d’échantillons de terrain prélevés au Cameroun chez des porteurs asymptomatiques. Au Cameroun, nous avons réalisé des gorgements de moustiques sur sang contenant des gamétocytes issus de porteurs du trait drépanocytaire ou d’une hémoglobine normale pour la mesure de l’infection selon le génotype de l’hôte.Dans un premier temps, nous avons montré que les gamétocytes circulant chez des porteurs du trait drépanocytaire sont plus infectieux pour le vecteur. La prévalence et la charge oocystique des moustiques femelles gorgés sur sang d’individus HbAS sont significativement plus élevées que celles obtenues pour des femelles nourries sur sujets HbAA. Les sujets drépanocytaires contribueraient alors de manière accrue à la transmission du paludisme et ces résultats doivent être pris en compte dans les interventions de prévention afin de mieux protéger ces populations plus à risque de propager le parasite.Dans un second temps, nous avons observé que P. falciparum envahit préférentiellement les érythrocytes jeunes et que la biomasse parasitaire est plus élevée dans les réticulocytes infectés. Nous avons aussi montré que les parasites évoluent plus souvent vers des stades sexuellement engagés dans des érythrocytes plus jeunes, ce qui suggère que la cellule hôte est un des déterminants clés de l’engagement sexuel.Ce travail de thèse apporte des résultats importants pour notre compréhension de la transmission et particulièrement sur les mécanismes mis en jeu par le parasite pour optimiser sa transmission selon l’environnement dans lequel il se développe. Nos résultats vont apporter de nouvelles voies pour le développement de stratégies bloquant la transmission.Mots clés : Engagement sexuel, trait drépanocytaire, préférence des cellules hôtes.Malaria parasite can encounter heterogenous hosts and different type of host cells. In particular during the intra-erythrocytic cycle the parasite replicates inside an erythrocyte and can encounter different erythrocytes in term of maturation stage or in host carrying haemoglobin variants modifying the red blood cell environment. The impact of this heterogeneity on mosquito-transmissible stages is particularly unexplored. Characterising their effects on both sexual commitment and transmission to mosquitoes is of paramount importance in the fight against malaria. Here we leveraged new technologies including single cell transcriptomics as well as samples collected in naturally infected carriers to understand the impact of RBC heterogeneity on transmission phenotypes.Firstly, we find that sickle cell trait carrier has an enhanced mosquito transmission through both an increase in prevalence of transmission stages and are more infectious to mosquitoes, suggesting they excessively contribute to malaria transmission and should be taken into account in our understanding of the spread of malaria.Secondly, we find that P. falciparum preferentially invades younger erythrocytes and that the biomass of infected reticulocyte is important within an infection. We further show that parasites preferentially commit to sex within younger erythrocytes, suggesting that the host cell is one of the key determinants in environmentally-induced sexual fate determination.Our data show cases the power of combining cutting edge methods with parasites in their natural environment as a source for identifying new and exciting parasite biology. This work has important implication for our understanding of how parasites maximise their transmission in response to a carrying environment and therefore on the spread of malaria and how it may be targeted in transmission blocking interventionsKey words: Sexual commitment, sickle cell trait, host cell preferenc

Détermination des facteurs associés à l'engagement sexuel chez Plasmodium falciparum

Malaria parasite can encounter heterogenous hosts and different type of host cells. In particular during the intra-erythrocytic cycle the parasite replicates inside an erythrocyte and can encounter different erythrocytes in term of maturation stage or in host carrying haemoglobin variants modifying the red blood cell environment. The impact of this heterogeneity on mosquito-transmissible stages is particularly unexplored. Characterising their effects on both sexual commitment and transmission to mosquitoes is of paramount importance in the fight against malaria. Here we leveraged new technologies including single cell transcriptomics as well as samples collected in naturally infected carriers to understand the impact of RBC heterogeneity on transmission phenotypes.Firstly, we find that sickle cell trait carrier has an enhanced mosquito transmission through both an increase in prevalence of transmission stages and are more infectious to mosquitoes, suggesting they excessively contribute to malaria transmission and should be taken into account in our understanding of the spread of malaria.Secondly, we find that P. falciparum preferentially invades younger erythrocytes and that the biomass of infected reticulocyte is important within an infection. We further show that parasites preferentially commit to sex within younger erythrocytes, suggesting that the host cell is one of the key determinants in environmentally-induced sexual fate determination.Our data show cases the power of combining cutting edge methods with parasites in their natural environment as a source for identifying new and exciting parasite biology. This work has important implication for our understanding of how parasites maximise their transmission in response to a carrying environment and therefore on the spread of malaria and how it may be targeted in transmission blocking interventionsKey words: Sexual commitment, sickle cell trait, host cell preferenceAu cours de son cycle intra-érythrocytaire chez l’Homme, Plasmodium falciparum, l’agent pathogène responsable du paludisme se réplique dans les érythrocytes. Le parasite peut envahir des érythrocytes à différentes étapes de leur maturation et il peut aussi se retrouver chez des hôtes porteurs d’anomalies de l’hémoglobine qui modifient les propriétés de l’érythrocyte. L’impact de cette hétérogénéité des globules rouges sur le développement des stades sexués, seuls stades infectieux pour le vecteur, est particulièrement inexploré.Dans cette thèse, nous avons cherché à caractériser l’effet de l’hétérogénéité de l’environnement érythrocytaire sur l’engagement sexuel du parasite et sur la transmission chez le moustique. Nous avons développé des approches basées sur de nouvelles technologies, dont la transcriptomique en cellule unique, pour rechercher des facteurs associés à l’engagement sexuel à partir d’échantillons de terrain prélevés au Cameroun chez des porteurs asymptomatiques. Au Cameroun, nous avons réalisé des gorgements de moustiques sur sang contenant des gamétocytes issus de porteurs du trait drépanocytaire ou d’une hémoglobine normale pour la mesure de l’infection selon le génotype de l’hôte.Dans un premier temps, nous avons montré que les gamétocytes circulant chez des porteurs du trait drépanocytaire sont plus infectieux pour le vecteur. La prévalence et la charge oocystique des moustiques femelles gorgés sur sang d’individus HbAS sont significativement plus élevées que celles obtenues pour des femelles nourries sur sujets HbAA. Les sujets drépanocytaires contribueraient alors de manière accrue à la transmission du paludisme et ces résultats doivent être pris en compte dans les interventions de prévention afin de mieux protéger ces populations plus à risque de propager le parasite.Dans un second temps, nous avons observé que P. falciparum envahit préférentiellement les érythrocytes jeunes et que la biomasse parasitaire est plus élevée dans les réticulocytes infectés. Nous avons aussi montré que les parasites évoluent plus souvent vers des stades sexuellement engagés dans des érythrocytes plus jeunes, ce qui suggère que la cellule hôte est un des déterminants clés de l’engagement sexuel.Ce travail de thèse apporte des résultats importants pour notre compréhension de la transmission et particulièrement sur les mécanismes mis en jeu par le parasite pour optimiser sa transmission selon l’environnement dans lequel il se développe. Nos résultats vont apporter de nouvelles voies pour le développement de stratégies bloquant la transmission.Mots clés : Engagement sexuel, trait drépanocytaire, préférence des cellules hôtes

Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates

International audienceBackground.The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT).Methods. Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2.Results. None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0–1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases.Conclusion. This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa

Efficacy of dihydroartemisinin/piperaquine in patients with noncomplicated Plasmodium falciparum malaria in Yaoundé, Cameroon

International audienceBackground: Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. Objectives: This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. Patients and methods: Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. Results: The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmep-sin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine. Conclusions: Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin-and piperaquine-resistant parasites, unlike Southeast Asia

Antimalarial drug resistance in the Central and Adamawa regions of Cameroon: Prevalence of mutations in P. falciparum crt, Pfmdr1, Pfdhfr and Pfdhps genes

International audienceThe spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt , Pfmdr1 , Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher’s exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P 96%), however no SNP was detected at codon 164. In Pfdhps , the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype ( Pfdhfr 51I/59R/108N+ Pfdhp 437G) was found almost 90% of the samples. The wild-type genotype ( Pfdhfr N51/C59/S108/164I+ Pfdhps A437/K540/A581) was never identified and the sextuple mutant ( Pfdhfr 51I/59R/108N+ Pfdhp 437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P . falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers

Abstract Background Sickle cell trait (SCT) refers to the carriage of one abnormal copy of the β-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas. Methods The study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source. Results Among the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29–1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype. Conclusion Plasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite’s enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells