Performance of shotgun metagenomic sequencing for detection of fungi and parasites in a broad range of clinical samples: a multicentre retrospective study.

To evaluate the analytical performance of clinical shotgun metagenomic sequencing (SMg) for detecting medically relevant fungi and parasites compared with standard of care (SoC), and to define optimal sequencing read thresholds for positivity.We retrospectively analysed 198 clinical samples collected from 187 patients in four university hospitals (2018-2022): blood (n = 37), faeces (n = 63), respiratory fluids (n = 54), other biological fluids (n = 24), and tissue biopsies (n = 20).Total nucleic acids were sequenced (>10 million reads per library) and processed with MetaMIC v2.2.1. Data were normalised as reads per million (RPM).SoC identified microorganisms in 152/198 samples (76.8%). All 46 SoC-negative samples were also negative by SMg. At the genus-level, SoC identified 187 taxa and SMg 175.SMg showed strong concordance with SoC. A uniform 0.1 RPM threshold sustained performance, and type-specific thresholds further improved accuracy.SMg offers benefits for mycology and parasitology, where rare or unexpected pathogens can be found, especially with viruses and bacteria.SMg offers benefits for mycology and parasitology by identifying rare or unexpected pathogens, even in the presence of viral or bacterial co-infections.