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. 2023 Aug;28(31):2300010.
doi: 10.2807/1560-7917.ES.2023.28.31.2300010.

Metagenomic sequencing for investigation of a national keratoconjunctivitis outbreak, Israel, 2022

Yair Motro  1   2 , Denise Wajnsztajn  3   2 , Ayelet Michael-Gayego  4 , Shubham Mathur  1 , Roberto Bm Marano  1 , Ikram Salah  1 , Chaggai Rosenbluh  5 , Violeta Temper  4 , Jacob Strahilevitz  4 , Jacob Moran-Gilad  1   4
Affiliations

Metagenomic sequencing for investigation of a national keratoconjunctivitis outbreak, Israel, 2022

Yair Motro et al. Euro Surveill. 2023 Aug.

Abstract

BackgroundEpidemics of keratoconjunctivitis may involve various aetiological agents. Microsporidia are an uncommon difficult-to-diagnose cause of such outbreaks.AimDuring 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 Vittaforma corneae as the aetiology, serving as proof of concept for using real-time metagenomics for outbreak investigation.MethodsCorneal 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.ResultsFluorescent microscopy was suggestive of microsporidiosis. The most abundant species (96.5%) on metagenomics analysis was V. corneae. 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 V. corneae in an environmental sludge sample collected at the exposure site.ConclusionsThe 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.

Keywords: Microsporidium; PCR; keratoconjunctivitis; metagenomics; outbreak investigation; public health microbiology; unknown aetiology.

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Conflict of interest statement

Conflict of interest: None declared.

Figures

Figure 1
Figure 1
Slit lamp and fluorescent microscopy findings, keratoconjunctivitis patient, Israel, 2022
Figure 2
Figure 2
Comparison of the isolated MAG contigs with the Vittaforma corneae ATCC 50505 reference genome, keratoconjunctivitis patient, Israel, 2022
Figure 3
Figure 3
Phylogenetic analysis of Vittaforma corneae from a keratoconjunctivitis patient, Israel, 2022
Figure 4
Figure 4
Development and application of real-time PCR for Vittaforma corneae, Israel, 2022
Figure 5
Figure 5
Mapping of clinical metagenomic sample to environmental metagenomic sample and to Vittaforma corneae ATCC 50505, keratoconjunctivitis patient, Israel, 2022
See this image and copyright information in PMC

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