Rapid Detection and Identification of Uveitis Pathogens by Qualitative Multiplex Real-Time PCR

Infectious uveitis is a serious sight-threatening infection commonly caused by herpesviruses and Toxoplasma gondii. Etiologic diagnosis based on the clinical evaluation is often challenging. We developed and validated a multiplex real-time PCR assay coupled with high-resolution melting (HRM) for rap...

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Bibliographic Details
Published in:	Investigative ophthalmology & visual science Vol. 59; no. 1; pp. 582 - 589
Main Authors:	Bispo, Paulo J M, Davoudi, Samaneh, Sahm, Matthew L, Ren, Ai, Miller, John, Romano, John, Sobrin, Lucia, Gilmore, Michael S
Format:	Journal Article
Language:	English
Published:	United States The Association for Research in Vision and Ophthalmology 01-01-2018
Subjects:	Immunology and Microbiology
Online Access:	Get full text
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Summary:	Infectious uveitis is a serious sight-threatening infection commonly caused by herpesviruses and Toxoplasma gondii. Etiologic diagnosis based on the clinical evaluation is often challenging. We developed and validated a multiplex real-time PCR assay coupled with high-resolution melting (HRM) for rapid detection and identification of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), and T. gondii. The assay was designed to target pathogen genome regions that yield products with distinct melting temperatures. Analytical specificity, sensitivity, and precision of HRM identification were determined. Clinical validation was performed by testing 108 intraocular fluids collected from eyes suffering with infectious uveitis (n = 30) and controls (n = 78). A nonoverlapping high-precision profile for each pathogen was generated following HRM (coefficient of variation 0%). The assay was highly sensitive, with a limit of detection of 20 genome copies for herpesviruses and 200 genome copies for T. gondii. The intra- and interassay variability of cycle threshold (Ct) measurement was ≤4% and ≤6%, respectively. Thirteen intraocular specimens collected from suspected cases of infectious uveitis were positive (mean Ct values varied from 19.4 to 27.7). Melting profiles of positive cases were consistent with HSV-2 (n = 5), VZV (n = 5), CMV (n = 2), and T. gondii (n = 1). Amplicon identities were confirmed by sequencing. Control intraocular samples from patients without a clinical diagnosis of infectious uveitis were all negative. This assay allows rapid, sensitive, and reliable detection and identification of the most common known causes of infectious uveitis, making early pathogen information-based intervention possible.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1552-5783 0146-0404 1552-5783
DOI:	10.1167/iovs.17-22597