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Joveeta Joseph Ruben, Sai Jeevana Madhuri Guda, Bhavani Sontam, Bupesh Bagga, Ranjit Konduri, Savitri Sharma; Evaluation of multiplex real-time polymerase chain reaction for the detection of HSV-1 & 2 and VZV in the diagnosis of viral keratitis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3604.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the role of multiplex real time PCR in the diagnosis of viral keratitis by quantification of the herpes simplex virus and varicella zoster virus (HSV 1 & 2, VZV) DNA in the corneal scrapings of patients with clinical viral keratitis and normal controls.
After appropriate ethical approval, 50 corneal scrapings from 50 patients with suspected HSV keratitis were included in the study. Corneal epithelial cells from 33 eyes of 21 patients undergoing photorefractive keratectomy surgery in essentially normal eyes served as controls. DNA was extracted and analysed for the presence of HSV-1 (Glycoprotein D gene) by conventional PCR in all samples and for the presence of HSV 1 & 2 and/or VZV by real-time PCR (R-gene® kit, bioMérieux) in all samples. Corneal scraping of patients with clinical viral keratitis had also been tested for HSV-1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records of all patients were reviewed.
Very low copy numbers of HSV1 and VZV DNA were detected by real time PCR in 8/15 controls (HSV1, mean-14.3 ±7.96, range: 3 - 29.1 copies/mL) and 2/15 controls (VZV, mean-10.7 ±10.9, range 3 - 18.5 copies/ml). HSV2 however was not detected in any of the control samples. Copy numbers above the mean +1 SD of controls were considered significant in patient samples for HSV1 and VZV. Evaluation of corneal scrapings for HSV 1 from patients showed that 39/50 (78%) were positive (copy number mean-1.18 x 106 ± 3.77 x 106 copies/ml, range: 44.6 - 1.5 x 107 /mL) by real time PCR, 11 out of 48 (23%) by IFA and 20/50 (40%) by conventional PCR. The mean copy number of HSV1 DNA was significantly higher in corneal samples from patients compared to controls (p<0.0001). Double infection with HSV-1 (1.5 x 107 copies/ml) and HSV-2 (3.57 x 104 copies/ml) in one case and VZV infection (1.03 x 102 copies/ml) in another was detected by real-time PCR.
Multiplex real-time PCR reliably detects high copy numbers of HSV1/2 and VZV DNA in patients with clinical viral keratitis and the DNA level is significantly higher than normal individuals who may harbor latent virus. Although expensive, real time PCR is easy to perform, is superior to conventional uniplex PCR and IFA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory, either alone or in combination with IFA.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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