Clinical Application of Real–Time Polymerase Chain Reaction in the Diagnosis of Herpetic Eye Diseases

Abstract

Purpose: : To assess clinical values and efficacy of herpes simplex virus (HSV) genome quantification for differential diagnosis of herpetic eye diseases.

Methods: : One hundred and thirty samples of 84 patients with clinically diagnosed herpetic eye diseases or other ocular inflammation were examined for the detection of HSV genome. Total genomic DNA, extracted from corneal epithelial scrapings, aqueous humor, and tear fluid, were assayed for quantification of HSV. Extracted DNA was amplified with primers set in consensus sequence of HSV–1/2 DNA polymerase (92 base pairs), and quantified with real time PCR using SYBR Green incorporation. Identity of the amplified product was confirmed using PCR–based direct sequencing.

Results: : In typical herpetic cases of dendritic or geographic keratitis, HSV genome were more than 10⁴ copies (mean 2.9×10⁵ copies) in tear fluid (200ul of eye wash) and more than 10⁵ copies (mean 9.0×10⁵ copies) in epithelial scrapings. In atypical epithelial keratitis cases, 10^2–3 copies (mean 4.1±1.1×10² copies) of HSV DNA were detected in both of tear fluid and corneal scrapings except one case. In herpetic stromal keratitis cases, mean HSV genome (mean 6.8±3.9×10²copies) was significantly lower than epithelial keratitis cases (student T test, P < 0.05). In 7 eyes of 15 stromal keratitis cases, HSV genome was not detected. In endotheliitis cases, 1 of 5 cases showed presence of HSV twice, however, mean HSV genome(2.9×10² copies/ul) was low. In 3 of 4 herpetic iritis suspected cases, HSV genome was detected in aqueous humor. Interestingly, their mean copy number was 1.1×10⁵/ul, showing significantly higher than endotheliitis cases. In cases submitted for exclusive diagnosis of herpetic diseases, 11 of 65 cases (16.9%) showed positive detection of HSV genome, indicating seemingly unexpected involvement of HSV in various array of ocular diseases.

Conclusions: : Real–time PCR is an informative and simple method to diagnose herpetic disease and evaluate possible viral involvement in various inflammatory ocular diseases. We believe that the quantification of HSV genome by real time PCR, together with evaluation of clinical symptoms and drug responsiveness, can improve the level of diagnosis in HSV–related cases.

Keywords: herpes simplex virus • microbial pathogenesis: clinical studies • clinical laboratory testing