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2020 OMIG Abstract

Evaluation of Metagenomic Deep Sequencing as a Diagnostic Test for Infectious Keratitis

Prajna Lalitha, MD1, N. Venkatesh Prajna1, Misra Sikha MD1, Rameshkumar Gunasekaran MD1,
Armin Hinterwirth, PhD2, Lee Worden, PhD2, Cindi Chen, MS2, Lina Zhong, BS2, Zijun Liu MS2,
Thomas M. Lietman, MD2,3, Gerami D. Seitzman2,3, Thuy Doan, MD, PhD2,3

1Aravind Eye Hospital, Madurai, Tamil Nadu, India; 2Francis I. Proctor Foundation, UCSF, San Francisco, CA, USA; 3Department of Ophthalmology, University of California, San Francisco, CA, USA

Purpose: To compare the diagnostic capability of RNA and DNA deep sequencing to smears and culture in the diagnosis of infectious keratitis.

Design: Prospective diagnostic test study, using latent class analysis (LCA) in the absence of a gold standard.

Methods:: Corneal scrapings from 46 consecutive patients presenting to the Aravind Eye Hospital in Madurai, India, with acute infectious keratitis were obtained. Diagnostic KOH and Gram stain smears, RNA sequencing (RNA-seq) and DNA sequencing (DNA-seq) was performed. For genomic sequencing processing and analysis, clinical samples were de-identified and laboratory personnel were masked to any prior diagnostic result. The sensitivity and specificity of each test for an unobservable gold standard was estimated using latent class analysis (LCA).

Results: 46 corneal ulcers were evaluated. Microbial smears for KOH and Gram stain, were positive in 32 samples (70%). Nine were identified as bacterial and 23 as fungal organisms. Corneal cultures were positive in 24 samples (52%). Seven grew bacterial organisms and 17 cultured fungal organisms. RNA-seq detected pathogens in 33 samples (72%), with 13 samples positive for bacteria and 20 samples positive for fungal. DNA-seq detected pathogens in 21 samples (46%) of which eight were bacterial and 13 were fungal. LCA estimated the sensitivities and specificities for conventional diagnostics, RNA-seq, and DNA-seq. For bacterial ulcers, the performance of conventional diagnostics is estimated at 61% sensitivity and 94% specificity, RNA-seq had a 100% sensitivity and 97% specificity, and DNA-seq showed a 62% sensitivity and 97% specificity. For fungal ulcers, the respective estimated sensitivities and specificities of conventional diagnostics were 95% and 81% for RNA-seq were 100% and 100% , and for DNA-seq were 65% and 100%.

Conclusion: This comparative study demonstrates MDS is an effective tool for the identification of organisms responsible for corneal ulcers. In this population of severe, central, predominately fungal corneal ulcers, RNA-seq outperforms DNA-seq as a diagnostic test. MDS may be a less useful tool when the infectious burden is so high and in environments where fungal keratitis is common, as conventional diagnostics, in particular KOH preparation and Gram stain, perform very well in this population.

Disclosure: C (G Seitzman)

Support: Supported by the Career Developmental Award and the unrestricted departmental funding provided by Research to Prevent Blindness and the K08EY026986.



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