Quantitative analysis of herpes simplex virus-1 transcript in suspected viral keratitis corneal buttons and its clinical significance

Purpose: The evaluation of Herpes Simplex virus-1 (HSV-1) transcript by different investigative methods (qPCR, PCR and IHC) in corneal buttons from suspected viral keratitis patients and the comparison of results with histopathological findings and clinical diagnosis. Methods: Sixty corneal buttons, 30 suspected viral keratitis, and 30 controls (keratoconus and bullous keratopathy) obtained after primary penetrating keratoplasty, were included in the study. All the corneal buttons were subjected to reverse transcriptase quantitative PCR (qPCR) for the detection of latency-associated transcript (LAT) gene, conventional PCR for polymerase (pol) gene, and immunohistochemistry (IHC) for HSV-1 antigen respectively. After obtaining baseline preoperative clinical data, all the patients were followed up for three years. The results obtained were correlated with clinicopathological features and follow-up data. Results: Of the 30 suspected viral keratitis patients there were 6 females and 24 males with mean age 46.5 ± 24.62 years (3-80 yrs). There was a marked male preponderance (80%). HSV-1 LAT transcript was detected in 23% (7/30) corneal buttons by qPCR, HSV-1 DNA in 6.7% (2/30) and HSV-1 antigen in 30% (9/30) cases by conventional PCR and IHC respectively. A statistically significant association was found between qPCR and DNA PCR (P = 0.04). All the 30 control corneas were negative for HSV-1 LAT gene, DNA and antigen. Conclusion: Detection of HSV-1 LAT transcript by qPCR may be superior to HSV-1 DNA PCR (conventional) and IHC, which has low sensitivity. However, the utility of HSV-1 LAT mRNA analysis as a diagnostic modality by qPCR needs to be validated on a larger patient cohort.