A Prospective Surveillance Study to Detect Antimalarial Drug Resistance, Gene Deletions of Diagnostic Relevance and Genetic Diversity of Plasmodium Falciparum in Mozambique

Genomic data constitutes a valuable adjunct to routine surveillance that can guide programmatic decisions to reduce the burden of infectious diseases. However, genomic capacities remain low in Africa. This study aims to operationalize a functional malaria molecular surveillance system in Mozambique that generates reliable and reproducible genomic data over time for malaria control and elimination. This prospective surveillance study seeks to generate P. falciparum genetic data to 1) monitor molecular markers of drug resistance and deletions in rapid diagnostic test targets; 2) characterize transmission sources (imported/introduced or indigenous) in low transmission settings; and 3) quantify transmission levels and the effectiveness of antimalarial interventions.

Nine provinces were identified through consultation with the NMCP for inclusion in the study: Maputo, Maputo City, Gaza, Inhambane, Niassa, Manica, Nampula, Zambezia and Sofala. Selection of study sites will be stratified by transmission intensity into two major strata: A) low transmission (Maputo province and Maputo City, where individual case notification is in effect for the purpose of a complete interruption of transmission), and B) medium-to-high transmission areas (Gaza, Inhambane, Niassa, Manica, Nampula, Zambezia and Sofala provinces, targeted by control (non-elimination) strategies). The provinces were selected to provide a range of low and medium-to-high transmission intensity, geographies, climatic conditions, malaria interventions mixes and populations (symptomatic, household contacts and sentinel groups). In the low transmission areas, districts and locations will be sampled based on historic surveillance data. Additionally, in Maputo province, the Ponta d'Ouro border post will be included to capture potential imported cases of malaria.

Dried blood spot samples and rapid diagnostic tests will be collected across the study districts through a combination of dense (all malaria cases) and targeted (a selection of malaria cases) sampling of malaria symptomatic cases in low malaria transmission and medium-to-high malaria transmission areas, respectively. In medium-to-high malaria transmission areas a second, non-HRP2 based test will be carried out and discrepant results suggestive of pfhrp2/3 deletions will be recorded and further analysed. Pregnant women attending their first antenatal care visit will be also included to assess their value as sentinel populations for molecular surveillance. Written informed consent will be sought from all survey participants before surveys and blood sample collections are conducted. In that case, information about the purpose of the sample collection will be explained, any questions will be clarified, and written informed consent will be obtained. The information sheet and consent form will also include text explaining informed consent for future use of biological specimens. In case of minors, consent will be sought from parents or guardians. The field worker or nurse will read the informed consent form to adults or the child's parent/guardian and will ask them if he/she agree his/her child to participate in the study. A witness will be required to sign the informed consent in case parent/guardian of the participant is illiterate. The consent will be available in Portuguese and translated into local language spoken at the study areas. The information sheet includes a brief description of the study, the data collection procedure, the blood sample collection procedure, the potential harm to participants, the expected benefits and the voluntary nature of participation at all stages of the interview and blood sampling. Two copies will be signed by the participant and the study team member obtaining the consent. One copy will be given to the participant and the other copy will be kept in the study file at the CISM office.

Sample size per sampling domain has been estimated taking into account antimalarial and diagnostic resistance as a primary use case, considering the negligible carriage of molecular markers of artemisinin resistance and pfhrp2/3 deletions in Mozambique, and setting 5% as the warning threshold. Assuming a 10% of loss of samples or uninterpretable analysis, a sample size of up to 500 per sampling domain would be adequate to: a) estimate a proportion of 0.05 (markers of drug resistance or pfhrp2 deletion) with 0.026 absolute precision and 95% confidence and b) achieve a power of 80% for detecting an increase of genetic marker (resistance or deletion) from 0 ‰ to 5% at a two-sided p-value of 0.01. It is estimated that up to 500 participants will be recruited among all ages for the study in each sampling unit (i.e. province) across those accessing APEs and health facilities (except Maputo city and Maputo province where continuous collections may exceed this number and household contact sample collection may increase this further). We will target up 500 RDT-positive pregnant women attending ANC clinics in all medium-to-high transmission districts and in low transmission areas each year for a period of three years. A flexible and adaptive sampling scheme will be followed, where a) estimates generated during the first half of the project will inform subsequent sampling schemes and b) not all the samples collected will be analysed (some of them will be stored as reference materials or for confirmation of findings).

The project will also leverage from intervention studies and surveillance activities being conducted in Mozambique between 2021 and 2024, namely: the Malaria Indicator Survey (2022-2023) in southern Mozambique; the Therapeutic Efficacy Survey (2022) in sentinel sites in the country (Cabo Delgado, Tete, Sofala, Zambézia and Inhambane); reactive surveillance activities (Maputo province); a Phase III cluster-randomized, open-label, clinical trial to study the safety and efficacy of ivermectin mass drug administration to reduce malaria transmission (Zambezia; 2022); a large-scale implementation development project aiming at maximising the delivery and uptake of intermittent preventive treatment in infancy (IPTi) (Inhambane; 2022-2024); a hybrid effectiveness-implementation study to evaluate the feasibility and effectiveness of seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine and amodiaquine (Nampula; 2022); and a programmatic delivery of a population-based mass drug administration with dihydroartemisinin-piperaquine (Gaza Province; 2022-2023).