Development and application of genomic tools for characterising genetic variation in soil-transmitted helminths

Abstract

Soil-transmitted helminths (STHs) are intestinal parasitic worms that represent a significant global health challenge, perpetuating poverty and disease. For decades, the diagnosis of STHs has depended on the microscopic detection and identification of eggs or larvae in faecal samples. However, these techniques can be labour-intensive, and their diagnostic performance and sensitivity rely heavily on sample preparation and the prevalence of infections. Molecular tests, such as quantitative polymerase chain reactions (qPCRs), are increasingly being used due to their sensitivity, especially in low-prevalence settings. These tests target species-specific ribosomal RNA genes or tandem repetitive regions found throughout the helminth genome. Currently employed qPCR assays were primarily developed and validated using a limited number of geographically restricted parasite isolates. However, it is increasingly recognised that parasitic worms in humans and animals are genetically diverse and distributed within structured populations both locally and globally. This genetic variation can occur in the sequences targeted by molecular methods. Therefore, differences within a single species that characterise STH populations may also affect the sensitivity and specificity of diagnostic tests in various settings. To date, there have been only a few studies on the population genetics of STHs, which limits our understanding of the extent of genetic variation that could interfere with molecular diagnostic methods. Additionally, such studies assessing variation in helminths have mainly relied on the availability of adult worm material. This reliance depends on the organisation of worm expulsion studies following anthelmintic treatment of the affected individuals, which can be logistically and ethically challenging. Meanwhile, faecal samples iv from infected individuals offer an untapped source of helminth egg material for genetic analysis. The aims of the thesis were to: (i) evaluate the feasibility of low-depth shotgun metagenomic analysis, also known as ‘genome skimming,’ using faecal samples to diagnose helminth infections and recover genetic information from helminths; (ii) examine the genetic variation of nuclear and mitochondrial DNA targets associated with STHs and assess their impact on quantitative PCR (qPCR) diagnostics; and (iii) enhance the recovery and enrichment of low-abundance targets, such as STHs, in faecal samples. This thesis demonstrates that low-depth shotgun metagenomics, when applied to faecal samples, can effectively confirm helminth infections, with reduced sensitivity compared to qPCR, but adds value in recovering complete mitochondrial genomes. Additionally, data from this work highlight the issue of bacterial and host contamination in current helminth analyses, which presents significant challenges for data interpretation when dealing with metagenomic samples derived from faeces. Bioinformatics analysis of the largest STH cohort to date offered insight into the population structure of STHs and revealed that genetic variations exist in diagnostic targets, which can affect qPCR amplification and potentially lead to false-negative results. Finally, given the limited amount of parasitic DNA present in faecal samples and the cost involved in metagenomic application, this thesis employs a highly efficient targeted enrichment method to extract helminth DNA from these samples. This marks the first instance of such an approach being used for helminths. The data presented in this work lay a strong foundation for future research aimed at enhancing our understanding of genetic variation in helminths, without requiring adult worm specimens. Additionally, it highlights the necessity of thoroughly assessing current and future diagnostics before implementation to effectively inform control measures and considerations regarding the cessation of deworming for STHs. Collectively, these findings form a foundational basis for the genomic epidemiology and sustainable control of STHs as a public health concern.