Nanopore long-read metabarcoding for comprehensive characterisation of parasitic nematodes in humans and animals: a development and diagnostic validation study

BACKGROUND: Parasitic nematodes of vertebrates impose a substantial burden on human and animal health; however, advanced diagnostic tools for the accurate characterisation of these parasites are scarce. One promising but nascent approach is metabarcoding, which enables the simultaneous characterisation of all parasitic nematodes in a sample, termed the nemabiome. However, earlier iterations of this approach could not detect socioeconomically important nematodes such as those in the genera Strongyloides and Trichuris. Therefore, we aimed to develop a novel nemabiome method capable of detecting all parasitic nematodes to support their accurate control in people and animals. METHODS: In this development and diagnostic validation study, we used a comparative molecular target approach to identify optimal barcoding genes to create a novel nemabiome assay capable of detecting all vertebrate-infecting parasitic nematodes in a sample. DNA for developing the assay was extracted from 154 positive control samples sourced from ten different countries. For diagnostic validation, samples were chosen from a biobank of field samples collected as part of two projects in the Asia-Pacific, specifically 190 human (n=95, collected from Vanuatu) and canine (n=95, collected from Cambodia) faecal samples were used. We leveraged the long-read sequencing capabilities of the Oxford Nanopore Technologies platform to ensure high taxonomic resolution. The diagnostic performance of the assay was benchmarked against that of well-established quantitative PCR (qPCR) assays, using Cohen's kappa (κ) statistic and assessment of diagnostic sensitivity and specificity values. FINDINGS: We identified suitable barcoding genes for nematode species in clades I and IV (18S ribosomal DNA) and clades III and V (internal transcribed spacers). Validation using positive controls confirmed the assay's ability to detect parasites representing 24 different genera across all parasitic nematode clades. Through testing on field samples, the nemabiome assay identified 72 parasitic nematode-positive dogs (76%) and 57 positive people (60%), including zoonotic taxa identified in both hosts. Benchmarking using field samples showed a diagnostic specificity of 99·6% (95% CI 98·8-99·9) and diagnostic sensitivity of 86·0% (81·4-89·9), with diagnostic agreement categorised as substantial (κ≥0·69) or high (κ≥0·81) between the nemabiome assay and qPCR for comparable parasites. Moreover, the nemabiome assay revealed a greater diversity of parasitic nematode species (n=11) than those detected by conventional molecular techniques. INTERPRETATION: This nemabiome assay offers a comprehensive approach for the precise characterisation of parasite communities (co-infections) affecting humans and vertebrates. Unlike target-specific molecular methods, such as qPCR, our approach facilitates the detection of previously unidentified parasites, including zoonotic and cryptic species, and also elucidates interhost transmission pathways and animal reservoirs. Using this advanced method in additional host species and locations could strengthen parasite control programmes, including the management of emerging and re-emerging parasitic threats. FUNDING: The University of Melbourne, National Health and Medical Research Council, and Bridges to Development.