Host Immune and Metabolic Determinants of Sexual Conversion in Plasmodium Parasites IMMETASEX

Study Overview
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Brief Summary
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Understanding the sexual conversion of the malaria parasite is essential to interrupt malaria transmission. A new tool is developed that, based on expression analysis of sexual stage biomarkers, will estimate sexual conversion rates in natural infections.

Detailed Description
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Understanding the sexual conversion of the malaria parasite is essential to interrupt malaria transmission. At each replicating cycle within erythrocytes, a proportion of asexual parasites converts into non-replicative sexual stages, which are the only forms able to infect mosquitos. The rate at which sexual stages are produced, is known as basal sexual conversion rate. Changes in the host immune and metabolic environment associated with the development of malaria disease, such as depletion of lysophosphatidylcholine in plasma, have been associated with increased sexual conversion rates in vitro. It is hypothesised that immune and metabolite factors that are altered during malaria infection induce sexual conversion in Plasmodium falciparum parasites. In this project, a new tool is developed that, based on expression analysis of sexual stage biomarkers, will estimate sexual conversion rates in natural infections. The aim is to identify immune factors and metabolites that induce sexual conversion using in-house developed sexual conversion assays, and experimental mosquito infections. Finally, transcriptional mechanisms are explored driving parasite sexual conversion in the host environment during disease using single-cell RNA-sequencing approaches. This research will provide essential knowledge on the factors that affect sexual conversion in the host and potentially inform novel strategies to interrupt transmission.

Official Title
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Host Immune and Metabolic Determinants of Sexual Conversion in Plasmodium Parasites IMMETASEX

Conditions
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Severe Malaria, Uncomplicated Malaria, Asyptomatic Plasmodium Infection

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age: ≥ 1 year Willing and able to provide written informed consent (or assent for minors with written informed consent by parent(s) and/or guardian(s). Pilot: -symptomatic for P. falciparum -/Travel to P. falciparum endemic area within the last month WP1: Resident in Nanoro district non-symptomatic individuals WP2: Positive for P. falciparum infection via Rapid Diagnostic Tests (RDT) Age: ≥ 1 and ≤ 12 years Patients are included when suspected of the following conditions: I. Severe malaria by infection with P. falciparum is defined in the presence of P. falciparum asexual parasitemia, and as one or more of the following: Impaired consciousness: A Blantyre coma score < 3 (when patients are ≤ 6 years) or Glasgow coma score < 10 (when patients are ≥ 6 years). Prostration: Generalized weakness so that the person is unable to sit, stand or walk without assistance. Multiple convulsions: More than two episodes within 24 hours. Clinical manifestation of respiratory distress (e.g., rapid, deep and labored breathing). Diagnosis through exclusion: absence of an identified alternative cause. II. Uncomplicated malaria by infection with P. falciparum is defined as a patient who presents with lethargic profile (e.g. fever) and a positive parasitological test for P. falciparum, but with no features of severe malaria. Exclusion Criteria: Delayed developmental status or history of chronic illness Participation in another study Previous malaria treatment or prophylaxis in the last week Inability or unwillingness of the parents or guardians to provide informed consent WP1: Symptoms of malaria, as defined by presence of fever (body temperature >37.5 °C or history of fever during the past 48 hours) with a positive RDT (RDT are performed always when there is presence of fever) Any plans to leave the study are in the coming 10 days WP2: Severe anemia (will be determined via clinical examination), since blood samples can hardly be withdrawn, co-morbidities. A questionnaire will be used during the clinical assessment that addresses following exclusion criteria: x Antimalarial drug treatment or other medication during the past week x If the patient had a meal within 4 hours before admission x Patients with acute meningitis (as clinically evaluated according to the local guidelines) x Patients with developmental delay or history of chronic illness x Vaccination during the past week

Ages Eligible for Study
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Minimum Age: 1 Year

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Pilot study Belgium Patients<br>Patients (P. falciparum-infected) No intervention 6 ml of venous blood sampled at one time point | |
| Pilot study Belgium Controls<br>Control non-infected individuals No intervention 6 ml of venous blood sampled at one time point | |
| Work package 1 Burkina Faso Asymptomatic<br>Asymptomatic (P. falciparum-infected) No intervention Venous blood sample (maximum of 8 ml) at 1 time point. 300µl of finger prick blood at four follow-up visits 24, and 48 and 72h and day 10 after the enrollment. | |
| Work package 1 Burkina Faso uncomplicated patients<br>uncomplicated patients (P. falciparum-infected) No intervention Venous blood sample (maximum of 8 ml) at 1 time point. | |
| Work package 1 Burkina Faso Controls<br>Control non-infected individuals No intervention Venous blood sample (maximum of 8 ml) at 1 time point. | |
| Work package 2 Mozambique Uncomplicated malaria patients<br>Uncomplicated malaria patients No intervention Venous blood sample (maximum of 6 ml) at 1 time point. | |
| Work package 2 Mozambique Severe malaria patients<br>Severe malaria patients No intervention Venous blood sample (maximum of 6 ml) at 1 time point. | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| To validate a Sexual Conversion Estimator tool | To validate a Sexual Conversion Estimator tool to accurately estimate SC rates and future transmission potential in epidemiological samples. First, the investigators will measure expression levels of SRBs and directly determine SC rates in samples from malaria asymptomatic patients. Second, the investigators will use a machine learning classifier to determine the combination of SRBs that best predicts SC rates; and third, the investigators will measure the predictive value of the Estimator tool for future transmission potential | 2023-2025 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| To investigate in malaria patients associations between host immune and metabolic factors and P. falciparum sexual conversion and infection potential | In patients with uncomplicated and severe malaria, the investigators will investigate associations between host inflammatory molecules, parasite-specific Ab and metabolic factors, with parasite SC rates. The investigators hypothesize that SC rates will be higher in severe malaria patients and associated with specific molecules and metabolites. | 2023-2025 | 
| To validate associations between sexual conversion and host immune and metabolic factors in vitro | An in vitro assay, based on recently described gametocyte-reporter parasite lines 16 will be used to validate candidate associations identified in Objective 2. First, the effect of patient serum samples and specific metabolites and inflammatory factors via in vitro SC assays is tested. Next, in vitro cultures with significantly induced SC will be fed to Anopheles stephensi mosquitoes to assess infection potential via standard membrane feeding assays. It is hypothesized that plasma/factors identified in Objective 2, induce SC in vitro and increase mosquito infections. | 2024-2026 | 
| To explore transcriptional mechanisms driving parasite sexual conversion in the host environment during uncomplicated and severe malaria disease | Use of a single cell RNA-sequencing (scRNA-seq) approaches to generate the first in-depth characterization of gene expression patterns of very early sexual stages in the host circulation during natural infections in uncomplicated and severe clinical presentations (samples collected in Objective 2). | 2024-2026 |