Detection of herpes simplex virus DNA with nested PCR in presumed herpetic keratitis

Purpose. The diagnosis of herpetic keratitis is sometimes difficult due to lack of positive culture result, concurrent mixed infection, or preexisting ocular pathology, therefore we use highly sensitive nested PCR technique for detection of HSV genome in suspected clinical cases. Methods. 23 patients suspected of herpetic keratitis but without characteristic epithelial lesion (dendritic or geographic) were included; viral cultures were done in 10 cases but were all negative. Epithelial strappings (n=21) or aspirated aqueous humor (n=2) were transferred to 2 ml viral trasport medium. DNA in the sample is extracted by QIAamp blood kit, primers (TK1/TK2) amplifying a 327 bp sequence in HSV thymidine kinase region are used for first PCR; the product of first reaction is then subjected to second (nested) PCR using second primers (TK3/TK4) amplifying a 200 bp sequence internal to above-mentioned region. Positive control is done by assaying culture-positive samples; negative control is done by assaying non-HSV viral DNA. Results. Both the results of positive control (sensitivity) and negative control (specificity) approach 100%. The limitation of detection is at least 1 pg viral DNA. For previous culture-negative cases (n=10), 30% (3/10) were positive for first PCR, and additional 30% (3/10) were positive for nested PCR. For those cases without prior culture (n=13), 15.4% (2/13) were positive for first PCR, and additional 61.5% (8/13) were positive for nested PCR. In all, HSV genome was positive in 69.6% (16/23) of all cases. Conclusion. Nested PCR is able to increase the sensitivity of viral genome detection, which is important for clinical diagnosis; as specimen from living cornea is often very tiny in amount.