Application of Polymerase Chain Reaction to Differentiate HSV– 1 and 2 Serotypes in Culture Negative Intraocular Aspirates From Patients With Viral Retinitis and Keratouveitis

Abstract

Abstract: : Purpose: To standardize and apply a polymerase chain reaction (PCR) on the Glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 and 2 serotypes in culture negative intraocular specimens from presumed intraocular HSV infection. Methods: Seventeen intraocular fluids (13– Aqueous Humor – AH & 4– Vitreous Aspirate – VA) collected from 14 patients (12 viral retinitis and 2 keratouveitis) were subjected to cultures for HSV & PCR followed by DNA hybridization & DNA sequencing of the PCR products to detect DNA polymerase gene of HSV (detects both HSV 1 & 2). In three patients both AH & VA were tested. To differentiate the HSV 1 & 2 serotypes, a semi–nested PCR (snPCR) targeting Glycoprotein D gene using serotype specific primers was standardized and applied onto the 17 intraocular fluids. The specificity of snPCR was verified by application onto five HSV 1 & five HSV 2 clinical isolates & prototype strains. All specimens were also tested for presence of Cytomegalovirus (CMV) and Varicella Zoster Virus (VZV) by nucleic acid amplification methods including a multiplex PCR for three genomes of CMV. Results: Five of 17 intraocular fluids were positive for HSV by PCR and one other was further positive by PCR based DNA hybridization increasing clinical sensitivity by 5.6 %. DNA sequencing of PCR products showed 100% homology with the prototype strains of HSV 1 & HSV 2. Among the six HSV positive intraocular specimens, four were identified as HSV 1 and two as HSV 2 by the semi–nested PCR targeting Glycoprotein D gene. In both AH & VA of two patients with viral retinitis, HSV 1 was detected in one and HSV 2 in another. Among the other patients, CMV DNA was detected in four patients & VZV DNA in five others. Conclusions: The standardized snPCR can be applied directly on to culture negative clinical specimens for rapid differentiation of HSV 1 & 2 serotypes.

Keywords: herpes simplex virus • clinical laboratory testing • retinitis