Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples
7 affiliations
- 1Life Sciences Department, University of Bath, Bath, United Kingdom
- 2Forestry Vocational School, Düzce University, Konuralp Campus, Düzce, Türkiye
- 3Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
- 4Academic Department of Military Medicine, Royal Centre for Defence Medicine, Birmingham, United Kingdom
- 5Department of Evolution, Ecology and Behaviour, University of Liverpool, Liverpool, United Kingdom
- 6Tropical Disease Research Center, Department of Tropical Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
- 7Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Australia
Abstract
Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity (low and standard laboratory doses) and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infection were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author summary: Soil-transmitted helminths (STHs), such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STHs in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in faecal samples from humans infected with STHs, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read (SR) Illumina and long-read (LR) Oxford Nanopore Technologies, but SR outperformed LR shotgun metagenomics. Contamination of bacterial sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.
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BibTeX
@article{obrien2026hunting,
title = {{Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples}},
author = {Katie O’Brien and Ajay Elamaran and Mehmet Dayi and Georgia Keeling and William D. Nevin and Yuchen Liu and Mark Viney and Kieran Reynolds and Charmaine Bishop and Banchob Sripa and Menebere Woubshete and Pamela Sachs Nique and Rhiannon Wright and Jane Younger and Vicky L. Hunt},
journal = {PLOS Neglected Tropical Diseases},
year = {2026},
month = sep,
doi = {10.1371/journal.pntd.0014130},
url = {https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014130},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples
AU - Katie O’Brien
AU - Ajay Elamaran
AU - Mehmet Dayi
AU - Georgia Keeling
AU - William D. Nevin
AU - Yuchen Liu
AU - Mark Viney
AU - Kieran Reynolds
AU - Charmaine Bishop
AU - Banchob Sripa
AU - Menebere Woubshete
AU - Pamela Sachs Nique
AU - Rhiannon Wright
AU - Jane Younger
AU - Vicky L. Hunt
PY - 2026
DA - 2026/09/21
JO - PLOS Neglected Tropical Diseases
DO - 10.1371/journal.pntd.0014130
UR - https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0014130
AB - Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity (low and standard laboratory doses) and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infection were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author summary: Soil-transmitted helminths (STHs), such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STHs in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in faecal samples from humans infected with STHs, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read (SR) Illumina and long-read (LR) Oxford Nanopore Technologies, but SR outperformed LR shotgun metagenomics. Contamination of bacterial sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.
SN - 1935-2735
ER -