Applications, complexities, and opportunities of metagenomics for clinical diagnostics and viral discovery

Metagenomic next-generation sequencing (mNGS) has transformed infectious disease research by enabling broad-range detection of microbial nucleic acids from clinical and environmental samples. However, despite its growing adoption in diagnostic and surveillance settings, substantial variability in analytical workflows and interpretive criteria continues to limit reproducibility and clinical implementation. The absence of standardised frameworks for defining positive identifications creates uncertainty in distinguishing clinically relevant pathogens from background signal, particularly in low-biomass samples such as cerebrospinal fluid (CSF). This thesis examined the analytical, clinical, and ecological applications of mNGS across human and animal health to improve diagnostic precision and interpretive consistency. A systematic review of 195 published clinical metagenomic studies identified marked heterogeneity in thresholds used to define positive detections, with nine distinct criteria reported, including genome coverage, read counts, and relative abundance. Over 70% of studies highlighted interpretive ambiguity due to the lack of standardised frameworks, and more than half cited inconsistent thresholding as a barrier to reproducibility. These findings underscore the need for harmonised bioinformatic pipelines and validated reference databases, such as FDA-ARGOS. Building on these findings, a heuristic thresholding strategy was applied to 660 CSF metagenomic datasets from adult and paediatric cohorts to evaluate diagnostic performance in central nervous system infections. A ≥30% genome coverage threshold reduced background signal and improved interpretive clarity. However, no single threshold eliminated all ambiguity, as pathogen abundance varied across individuals and clinical contexts. Extending beyond human diagnostics, metagenomic surveillance of 98 wildlife samples from Kruger National Park identified picobirnavirus sequences in multiple herbivore hosts. Phylogenetic analyses placed these sequences within the proposed Betapicobirnavirus lineage and suggested limited cross-species transmission with possible host adaptation. Collectively, this work demonstrates the versatility of mNGS as both a diagnostic and ecological tool while emphasising that analytical standardisation, contextual interpretation, and multidisciplinary evaluation are essential for responsible implementation. By integrating human and wildlife perspectives, this thesis advances efforts to standardise metagenomic interpretation and strengthen One Health-aligned genomic surveillance of emerging infectious diseases.

Grantor dc:publisher

Stellenbosch : Stellenbosch University

Year dc:date.issued

2026

Author dc:creator

• Maharaj, Akhil

Advisors dc:contributor.advisor

• De Oliveira, Tulio
• Tegally, Houriiyah
• Moir, Monika
• Hofstra, Marije

Language dc:language.iso

en

Repository record dc:identifier.uri

https://scholar.sun.ac.za/handle/10019.1/136268

OAI identifier oai:identifier

oai:scholar.sun.ac.za:10019.1/136268