Integrating local malaria molecular monitoring into regular malaria indicator surveys on Bioko Island: high association between urban communities and low density infections
Authors: Stabler Thomas C. , Hosch Salome , Nyakarungu Elizabeth , Giger Johanna Nouria , Elonga Monserrat Kobe , Bibang Rosalia Nzang , Ndong Victoria Mangue , Mometolo Ilda Elobe , Bolopa Alejandro Lopelo , Bedoung Rosa Maria Oko , Bijeri Jose Raso , Roka Esperanza Willy , Nguema Ana Gertrudis , Ichinda Victoria Palacios , Bakale Salvador , Esokolete Cristina Nka , Rivas Matilde Riloha , Galick David S. , Phiri Wonder Philip , Guerra Carlos A. , García Guillermo A. , Montemartini Luca , Schindler Tobias , Silva Joana C. , Daubenberger Claudia 
Published: November 2024

Background:
Effective malaria control requires accurate identification of Plasmodium infections to tailor interventions appropriately. Rapid diagnostic tests (RDTs) are crucial tools for this purpose due to their small size and ease-of-use functionality. These tests typically target the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen. However, some strains of P. falciparum have deletions in the hrp2 and hrp3 genes, which may result in a false negative diagnosis using HRP2-based RDTs. Additionally, RDTs have a detection limit of less than 100 parasites per microliter, insufficient for identifying low density infections that sustain malaria transmission. This study explores integrating molecular monitoring using a novel cartridge-based PCR test, PlasmoPod, using samples from a malaria indicator surveys (MIS) on Bioko Island, Equatorial Guinea to enhance detection of low density infections and inform targeted malaria control strategies.
Methods:
The study utilized a combination of RDTs and the DiaxxoPCR device for molecular monitoring. The PlasmoPod employs qPCR targeting 18S rDNA/rRNA, capable of detecting low parasite density infections and is significantly more sensitive than HRP2-based RDTs. Samples from the 2023 MIS were extracted from dried blood spots (DBS), qPCR run in duplicate on the PlasmoPod. Epidemiological data from the MIS were merged with molecular data and the association between various risk factors to malaria infection by qPCR, and risk factors to low density infections were measured.
Results:
The integration of molecular monitoring revealed a proportion of low density infections that circumvented RDTs diagnosis. Notably, individuals in urban communities and those reporting recent fever were more likely to harbor low density, asymptomatic malaria infections. Findings suggest that urban residents, although less associated to malaria infection than rural residents, may be serving as a transmission reservoir. The relationship between low density infections and individuals who recently reported fever may reflect recent antimalarial treatment or natural clearance, and thus have lingering parasites in their blood.
Conclusion:
The study highlights the limitations of HRP2-based RDTs in detecting low density infections and underscores the potential of molecular tools like PlasmoPod in malaria surveillance. By identifying elusive transmission reservoirs and tracking parasite importation, molecular monitoring can play a crucial role in achieving malaria elimination. The findings advocate for the broader implementation of molecular diagnostics in malaria programs, especially in areas with low transmission, to enhance the detection and targeting of hidden reservoirs of infection.