Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens infecting animals and humans

Abstract

Background: Filarial worms are important vector-borne pathogens of a large range of mammalian hosts, including humans and are responsible for some of the most pervasive, and pernicious diseases within the tropics. In humans, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa are all categorized as neglected tropical diseases. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. ‘hongkongensis’. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Methods: Here we present a novel long-read metabarcoding assay for deep-sequencing the filarial worm cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies’ (ONT) MinIONTM sequencer. We assessed the overall performance of our assay against commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott’s test (MKT) Results: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. ‘hongkongensis’. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified additional filarioid species and numerous additional mono- and coinfections. Conclusions: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT’ small and portable MinIONTM means that such methods could be deployed for field use.