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Epidemics of keratoconjunctivitis may involve various aetiological agents. Microsporidia are an uncommon difficult-to-diagnose cause of such outbreaks.


During the third quarter of 2022, a keratoconjunctivitis outbreak was reported across Israel, related to common water exposure to the Sea of Galilee. We report a comprehensive diagnostic approach that identified as the aetiology, serving as proof of concept for using real-time metagenomics for outbreak investigation.


Corneal scraping samples from a clinical case were subjected to standard microbiological testing. Samples were tested by calcofluor white staining and metagenomic short-read sequencing. We analysed the metagenome for taxonomical assignment and isolation of metagenome-assembled genome (MAG). Targets for a novel PCR were identified, and the assay was applied to clinical and environmental samples and confirmed by long-read metagenomic sequencing.


Fluorescent microscopy was suggestive of microsporidiosis. The most abundant species (96.5%) on metagenomics analysis was . Annotation of the MAG confirmed the species assignment. A unique PCR target in the microsporidian rRNA gene was identified and validated against the clinical sample. The assay and metagenomic sequencing confirmed in an environmental sludge sample collected at the exposure site.


The real-time utilisation of metagenomics allowed species detection and development of diagnostic tools, which aided in outbreak source tracking and can be applied for future cases. Metagenomics allows a fully culture-independent investigation and is an important modality for public health microbiology.


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