Purchase this article with an account.
Nora Cothran, Kacy Ramirez, Rebecca Thompson, Anami Patel, Daniel Fuller, John DeVincenzo; Molecular Quantification of Herpes Simplex Virus (HSV) by Real-Time PCR in Tear Film and Correlation with Human Disease. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2913.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Create a quantitative real-time polymerase chain reaction (qPCR) assay for the molecular detection and quantification of HSV-1 & 2. Quantify the assay’s lower limit of detection (LOD) and dynamic range. Optimize detection from commercially available Schirmer strips, utilizing clinically achievable storage conditions. Quantify HSV-1 & 2 in human tear film and correlate with clinical disease.
A real-time Taqman probe-based qPCR assay, amplifying HSV DNA polymerase, was created with conserved primers for, and melting point discrimination between, HSV-1 & 2. Standard curves of known copy number were made from ultra-purified whole HSV-1 & 2, allowing harmonization of all qPCR HSV assays. LOD was determined by probit statistical analysis of >360 assays, ranging over >9 Log copies/ml. Normal saline, spiked with known quantity of HSV-1, was absorbed onto Schirmer strips, mimicking clinical tear collection. Standard volume tear samples, from the following three groups, were evaluated: A) healthy adults devoid of historical or current ocular surface disease and no history of herpetic eye disease, B) patients presenting to an eye care clinic with symptomatic red eye, C) patients with active dendritic corneal disease (HSV quantity evaluated over time, following therapy initiation).
The LOD for HSV-1 & 2 were 111.24 copies/ml (95% CI, 30.74-250.09) and 453.24 copies/ml (95% CI, 192.13-741.27), respectively. One strip type exhibited qPCR inhibition. Another type was further tested, demonstrating consistent volumes of absorption between strips. Mimicked tear collection and storage of strips were evaluated under various defined conditions including storage of dry strips at room temperature for 7 days before processing. Recovery of HSV-1 from strips was 98.89% of the 4.88 Log copies/ml spiked into the saline. Descriptive statistics, including mean (SD) viral load, were determined for the three study populations. Comparisons of viral loads between groups were made. Sensitivity, specificity, and positive and negative predictive values for dendritic corneal disease, were determined for various viral load thresholds.
A molecular quantification method for HSV-1 & 2 in human tears has been developed, allowing clinical diagnostic trials of herpetic corneal disease and offering potential for improved detection and early therapy.
This PDF is available to Subscribers Only