Polymerase Chain Reaction Based Assay for the Diagnosis of Varicella-Zoster Virus in Ocular Surface Disease

Abstract

Purpose: : Our polymerase chain reaction (PCR) based diagnostic assay for Varicella-Zoster Virus (VZV) was originally developed to detect DNA in vitreous samples. When designing this assay the sensitivity was intentionally reduced to limit false positive results in inflamed eyes in patients with AIDS. We have since been using this assay to evaluate aqueous and corneal samples for VZV DNA, even in samples gathered from patients without HIV/AIDS. In the current study we evaluated and optimized our existing assay for the detection of VZV in corneal scrapings.

Methods: : We retested 110 consecutive corneal samples collected over 30 months (June 2007- Dec 2009) that had been previously negative or untested for VZV DNA. Our assay consisted of a polymerase chain reaction amplification of the VZV IE 63 gene using nested primers. The assay was modified to increase its sensitivity from approximately 156 gene copies of VZV DNA/µL to1 gene copy of VZV DNA/µL. All patients were originally tested because either HSV or VZV was being considered as a cause of active corneal disease.

Results: : Of 127 samples originally received during the study period, 12 had tested positive for VZV DNA, 82 had tested negative for VZV DNA, and 2 had contained inhibitors of our assay. Thirty-one samples had not been tested, since the treating physicians had not requested testing for VZV. Sufficient sample volume was present for testing with the updated more sensitive assay in 110 of the clinical specimens that were previously not tested or found to be negative. None of the 31 previously untested samples were positive for VZV DNA. VZV DNA was detected in 5 of 79 samples that had previously tested negative. A review of the medical records of these 5 patients is planned to help us determine whether these cases represent ocular surface disease secondary to VZV or clinically insignificant viral shedding.

Conclusions: : The evaluation of diseases of the cornea may require a more sensitive assay than we currently use. Further studies should clarify the optimal sensitivity of an assay for ocular surface disease without sacrificing specificity.

Keywords: varicella zoster virus • clinical laboratory testing • cornea: clinical science