Analysis Of Hsv-1 Genome In Corneal Scrapings Of Patients With Different Hsv Keratitis Forms In Croatia

HSV keratitis has several clinical appearances, sometimes with atypical presentation that can be difficult to distinguish form other causes of keratitis like fungus or Acanthamoeba. Using polymerase chain reaction, we can provide a timely diagnosis for HSV infection and initiate appropriate therapy. Consequently, we hypothesize that levels of HSV-1 replication differ significantly between different subtypes of HSV keratitis, including epithelial, stromal with or without epithelial ulceration and endothelial, which can have a major impact on treatment and selection of antiviral treatment. In addition, we will investigate the appearance of resistance viruses among patients with recurring disease and repeatedly treated with acyclovir.

Department of biotechnology, University of Rijeka

 Our research includes patients within Clinic of Ophthalmology, Clinical hospital Centre Rijeka with different clinical appearance of HSV keratitis. Material was taken via scraping of corneal epithelium from involved eye. Molecular diagnostics includes corneal scrapings analysed by one step PCR, nested PCR and quantitative real time PCR (RTqPCR) and correlated with the type of keratitis. Acyclovir resistance analysis was performed, HSV-1 TK gene was amplified in DNA extracted from the corneal scraping by PCR and sequenced.

We have collected corneal scrapings from 20 patients with diagnosed HSV keratitis. To analyse HSV-1 in corneal scrapings we have tested and optimized different approaches for sensitive and, in a clinical setting, rapid detection of HSV-1. We have tested 4 different primer pairs targeting different genes using purified recombinant HSV-1 genome as the template. The outperforming primers were further validated on different templates, including purified virus, DNA extracted from infected cells in culture and clinical samples. The sensitivity of one step PCR was compared to nested-PCR and RTqPCR. The results of optimization and analysis of 20 clinical samples from patients with different forms of HSV keratitis will be presented.

We have developed highly sensitive PCR and RTqPCR and tested 20 clinical samples. Our initial investigation indicates different levels of DNA HSV in different subtypes of HSV keratitis, which might impact treatment. In addition, we have implemented the PCR/sequencing approach to investigate mutations in the TK gene.