Uncovering the Diversity of Pathogenic Free - Living Amoebae in Freshwater Environments of Ghana: A Combined Culture Enrichment and Metabarcoding Approach

ABSTRACTFree-living amoebae (FLA) such asNaegleriaandAcanthamoebaare opportunistic pathogens increasingly linked tofatal andsevere human infections, particularly in settings with limited hygiene,water sanitation and diagnostic infrastructure.In this pilot study, we explored the diversity of potentially pathogenic FLA in sectors of the Kakum River Basin, near Cape Coast, Ghana using a combined approach of filtered, pelleted, and culture-enriched metabarcoding. Our results revealed a wide range ofFLAfromAmoebozoaandHeteroloboseaclades, including several of clinical relevance,such asAcanthamoeba, Vermamoeba. Balamuthiaand Paravahlkampfia. Importantly,various Naegleriaspecies were also detected and morphologicallyconfirmed, raising public health concerns given the favorable environmental conditions forNaegleriafowleriproliferationin the sampling sites. The use of culture-enriched metabarcoding was particularly valuable in recovering organisms that may encyst and be missed by direct methods. This study underscores the importance of integrative and sensitive molecular approaches for detecting neglected pathogens in vulnerable communities. Our findings provide a foundation for larger epidemiological studies that incorporate clinical data and support the development of cost-effective monitoring tools forFLA-associated public health threats in sub-Saharan Africa