Enhancing infectious intestinal disease diagnosis through metagenomic and metatranscriptomic sequencing of 1000 human diarrhoeal samples.

Background: Current surveillance of diarrhoeal disease is hindered by limitations of traditional diagnostic approaches, which often fail to identify the causative organism, particularly for novel or hard-to-culture bacterial pathogens. Sequencing nucleic acids directly from stool can overcome such constraints, but such approaches need to reliably detect pathogens identifiable by conventional methods.

Methods: As part of the INTEGRATE study, we analysed stool microbiomes from 1067 patients with gastroenteritis symptoms using direct sequencing, and compared findings with standard diagnostic techniques (culture, immunoassay, microscopy, and single-target PCR) and molecular assays (Luminex xTAG GPP) for detection of bacterial and viral pathogens in the UK.

Results: We found strong positive correlations between metatranscriptomic reads and traditional diagnostics for six out of 15 pathogens. The metatranscriptomic data were highly correlated with the Luminex assay for eight out of 14 pathogens. In contrast, metagenomic sequencing only showed a strong positive correlation with traditional diagnostics for three of 15 pathogens, and with Luminex for four of 14 pathogens. Compared with metagenomics, metatranscriptomics had increased sensitivity of detection for four pathogens, while metagenomics was more effective for detecting five pathogens. Metatranscriptomics gave near-complete transcriptome coverage for Human mastadenovirus F and detected Cryptosporidium via identification of Cryptosporidium parvum virus (CSpV1). A comprehensive transcriptomic profile of Salmonella enterica serovar Enteritidis was recovered from the stool of a patient with a laboratory-confirmed Salmonella infection. Furthermore, comparison of RNA/DNA ratios between pathogen-positive and pathogen-negative samples demonstrated that metatranscriptomics can distinguish pathogen-positive/negative samples and provide insights into pathogen biology. Higher RNA/DNA ratios were observed in samples that tested positive via gold-standard diagnostics.

Conclusions: This study highlights the power of directly sequencing nucleic acids from human samples to augment gastrointestinal pathogen surveillance and clinical diagnostics. Metatranscriptomics was most effective for identifying a wide range of pathogens and showed superior sensitivity. We propose that metatranscriptomics should be considered for future diagnosis and surveillance of gastrointestinal pathogens. We assembled a rich data resource of paired metagenomic and metatranscriptomic datasets, direct from patient stool samples, and have made these data publicly available to enhance the understanding of pathogens associated with infectious intestinal diseases.