Genetic characterization of zoonotic Dirofilaria asiatica in Cambodian dogs through nanopore metabarcoding

Filarioid nematodes are harmful vector-borne pathogens that afflict humans and other animals, with numerous species transmissible from animals to humans, i.e., zoonotic. In the last decade, one zoonotic filarioid, previously named Dirofilaria sp. Hong Kong genotype has been increasingly found as a cause of disease in people traveling from South Asia and has recently been characterized as a novel species: Dirofilaria asiatica. Nonetheless, the epidemiology of this parasite in Asia remains poorly understood. Therefore, with the aid of a novel pan-filarioid nanopore-based metabarcoding assay, we aimed to elucidate the epidemiology of dirofilariosis in Cambodia. We tested 504 canine blood samples collected from five regions and used the cytochrome c oxidase (cox1) gene sequences obtained to conduct phylogenetic analyzes. We found no evidence of the canine heartworm Dirofilaria immitis nor Dirofilaria repens in the country. Conversely, we detected D. asiatica and genetically characterized it from the eastern district of Tbong Khmum at a local prevalence of 4% (95% CI = 1.6%-9.8%), an area just ~100 km northeast of the country's capital, Phnom Penh. We also detected Acanthocheilonema reconditum from 0.2% of dogs sampled (95% CI = 0%-1%). Phylogenetic and haplotype network analyzes of Cambodian D. asiatica sequences clustered with strong support to those found from canines and humans in Bhutan, Hong Kong, India, and Sri Lanka. This genetic characterization of D. asiatica in Cambodia highlights the need for further epidemiological research into human filariases in Cambodia, given the zoonotic nature of this parasite and that mass drug administration targeting filarioids is no longer conducted in the country.IMPORTANCEParasitic filarioid nematodes are transmitted by blood-sucking arthropods and can cause significant disease in animals and humans (i.e., zoonotic). One such zoonotic filarioid (Dirofilaria asiatica) that has recently been characterized has been found responsible for a growing number of cases of human filariases, particularly in individuals who live in or have traveled to South and Southeast Asia. D. asiatica predominantly infects dogs, and therefore, to better understand this parasite's distribution, we tested 504 dogs from five regions of Cambodia using an advanced diagnostic approach. We found that the local prevalence of D. asiatica in dogs in the eastern district of Tbong Khmum was 4%. Given that mass drug administration programs to control human-infecting filarioid nematodes in Cambodia have stopped, this first detection of D. asiatica in Cambodia is alarming, given its potential to cause disease in vulnerable cohabiting people.