June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Highly Multiplexed Broad Pathogen Detection Assay for Diagnosis of Ocular Infections
Author Affiliations & Notes
  • Paulo J.M. Bispo
    Harvard Medical School Department of Ophthalmology, Boston, Massachusetts, United States
  • Lawson Ung
    Harvard Medical School Department of Ophthalmology, Boston, Massachusetts, United States
  • James Chodosh
    Harvard Medical School Department of Ophthalmology, Boston, Massachusetts, United States
  • Lucia Sobrin
    Harvard Medical School Department of Ophthalmology, Boston, Massachusetts, United States
  • Michael S Gilmore
    Harvard Medical School Department of Ophthalmology, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Paulo Bispo, None; Lawson Ung, None; James Chodosh, None; Lucia Sobrin, None; Michael Gilmore, None
  • Footnotes
    Support  Tej Kohli Foundation
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 3460. doi:
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    • Get Citation

      Paulo J.M. Bispo, Lawson Ung, James Chodosh, Lucia Sobrin, Michael S Gilmore; Highly Multiplexed Broad Pathogen Detection Assay for Diagnosis of Ocular Infections. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3460.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Eye infections are among the most common causes of blindness worldwide. The sooner effective therapy can be started, the more vision can be saved. However, current diagnostic modalities are time-consuming, lack sensitivity and inclusiveness, and may result in patients being treated with the wrong drug for long periods. We present a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid pathogen identification, with potential to improve treatment choices and at earlier stages of the disease

Methods : Using epidemiological information on the etiologies of ocular infections seen at our hospital and in combination with a literature review, we identified 46 pathogens and 2 resistance/virulence markers that are most commonly detected (>90% of cases). Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains from the same species. Regions of 150 to 300bp in length were selected and a set of primers for pre-enrichment, and two 50mer NanoString compatible probes were designed per target. DNA-DNA hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler

Results : Analytical studies demonstrated highly sensitive detection of a broad spectrum of infectious agents, including bacteria, fungi, viruses and parasites, with limits of detection being as low as 25 femtograms per reaction. We also challenged the diagnostic panel in a pilot clinical study testing samples from infectious keratitis (n=6) and uveitis (n=6) for which the etiologies were confirmed by culture or real-time PCR, and included Gram-positive and -negative bacteria and herpesviruses. The NanoString-based panel correctly identified the causative agent from all clinical specimens. Detection was robust, with probe counts for the targeted pathogen ranging from 5x103 to 4x105. For corneal ulcers, higher probe counts seem to correlate with the severity of presentation

Conclusions : This highly multiplexed panel for detection of ocular pathogens offers a sensitive, comprehensive, and uniform assay run directly on ocular specimens, that could significantly improve diagnostics of sight-threatening infections

This is a 2021 ARVO Annual Meeting abstract.

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