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Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example <jats:italic>Dirofilaria<\/jats:italic> sp. \u2018hongkongensis\u2019. 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. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies\u2019 (ONT) MinION\u2122 sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott\u2019s test (MKT).<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, <jats:italic>Breinlia<\/jats:italic>, <jats:italic>Brugia<\/jats:italic>, <jats:italic>Cercopithifilaria<\/jats:italic>, <jats:italic>Dipetalonema<\/jats:italic>, <jats:italic>Dirofilaria<\/jats:italic>, <jats:italic>Onchocerca<\/jats:italic>, <jats:italic>Setaria<\/jats:italic>, <jats:italic>Stephanofilaria<\/jats:italic> and <jats:italic>Wuchereria<\/jats:italic>. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids <jats:italic>Acanthocheilonema reconditum<\/jats:italic>, <jats:italic>Brugia<\/jats:italic> sp. Sri Lanka genotype and zoonotic <jats:italic>Dirofilaria<\/jats:italic> sp. \u2018hongkongensis\u2019. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>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\u2019 small and portable MinION\u2122 means that such methods could be deployed for field use.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12866-023-03159-3","type":"journal-article","created":{"date-parts":[[2024,1,20]],"date-time":"2024-01-20T06:01:34Z","timestamp":1705730494000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans"],"prefix":"10.1186","volume":"24","author":[{"given":"Lucas G.","family":"Huggins","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ushani","family":"Atapattu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neil D.","family":"Young","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca J.","family":"Traub","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vito","family":"Colella","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,1,20]]},"reference":[{"issue":"2","key":"3159_CR1","doi-asserted-by":"publisher","first-page":"366","DOI":"10.1128\/CMR.11.2.366","volume":"11","author":"TC Orihel","year":"1998","unstructured":"Orihel TC, Eberhard ML. 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