Application of real-time PCR in pathogenic detection of necrotizing herpes stromal keratitis

Background

Herpes stromal keratitis (HSK) is a common infectious ocular surface disease, with a higher recurrent rate, especially in necrotizing HSK.The diagnosis of HSK primarily depends on signs and symptoms, and specific laboratory diagnostic is lack.

Objective

This study was to clarify the expression of herpes simplex virus (HSV) in the corneal epithelium scrapings and tears of necrotizing HSK patients.

Methods

Thirty eyes of 30 patients with necrotizing HSK were enrolled in Nanjing Hospital of Nanjing Medical Hospital from September 2012 to September 2013 under the patient’s informed consent.The eyes were examined by slit lamp microscope and scored.HSK patients received local and systemic therapy for 8 weeks and then an oral maintenance dose for 6 months.Corneal epithelial scrapings and tears samples were collected for HSV DNA detection by real-time PCR before and the 1st, 2nd, 4th, 6th and 8th week after therapy respectively.The difference of HSV positive rate was compared between corneal epithelium scrapings and tears samples using Chi-square test.Multilevel mixed effective model was employed to evaluate HSV concentration change in the samples at various time points in the HSV-positive patients of initial visit.The correlation between HSV concentration and clinical score was analyzed by Spearman rank correlation.

Results

HSV-positive rate was 46.4% (13/30) in the corneal epithelial scrapings and 13.3% (4/30) in the tear samples, showing a significant difference between them (P=0.006). HSV-positive rate was significantly lower in the corneal epithelial scrapings 1 week, 2 weeks and 4 weeks after treatment than before (P=0.001, 0.003, 0.004), and no HSV was detected 6 weeks and 8 weeks after treatment.No significant change in HSV-positive rate in tear samples in 1 week and 2 weeks after treatment in comparison with before treatment (P=1.000, 0.583), and no HSV was detected after 4 weeks following treatment.The HSV concentration was 2 460(2 165-636 500)/ml in initial 13 HSV-positive eyes of corneal epithelial scrapings and 0(0-1 150)/ml in initial 13 HSV-positive eyes of tear samples.Multilevel mixed effective model determined that HSV concentration was significantly lower in corneal epithelial scraping than that in tear (P=0.005), and HSV concentration was reduced with the lapse of time (P=0.001), with the faster rate of decline in the corneal epithelial scrapings (P=0.049). A positive correlation was found between initial HSV concentration and clinical scores (r_s=0.844, P=0.000).

Conclusions

Real-time PCR appears to be a powerful molecular tool for the detection of HSV in the HSK, especially in corneal epithelial scrapings of lesion.The initial positive outcome of viral DNA in corneal epithelial scrapings predicts a severe clinical procedure.

Herpes simplex/diagnosis; Corneal stroma; Keratitis; Necrosis; DNA, viral/analysis; Epithelium/virology; Tears/virology; Real-time PCR