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

ABSTRACT Free-living amoebae (FLA) such as Naegleria and Acanthamoeba are opportunistic pathogens increasingly linked to fatal and severe 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 of FLA from Amoebozoa and Heterolobosea clades, including several of clinical relevance, such as Acanthamoeba, Vermamoeba. Balamuthia and Paravahlkampfia . Importantly, various Naegleria species were also detected and morphologically confirmed, raising public health concerns given the favorable environmental conditions for Naegleria fowleri proliferation in 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 for FLA-associated public health threats in sub-Saharan Africa.