A Comparison of the Clinical and Molecular Diagnosis of Herpes Simplex Keratitis


Purpose: To compare the clinical and molecular diagnoses of Herpes Simplex Keratitis (HSK). Materials and Methods: Conjunctival swabs (after fluorescein and anaesthetic wash out) and detailed questionnaire data were obtained from 146 participants. Corneal rims and conjunctival epithelial cells were infected with Herpes Simplex Virus (HSV) type 1 or HSV2 and supernatant collected. HSV1; HSV2; Varicella Zoster Virus (VZV) and Adenovirus (ADV) DNA was assessed using two real time Polymerase Chain Reaction (PCR) methods. Results: Of the 146 participants recruited, 54 were clinically diagnosed with typical epithelial lesions and 38 with atypical epithelial lesions, 17 with old inactive HSK and 37 healthy volunteers. HSV1 DNA was detected in 28 (30%) of the 92 participants with clinically suspect HSK. Patients who presented with typical epithelial lesions had a higher positive rate (46%) than those who presented with atypical type lesions (8%), when using primers against the Glycoprotein (Gp) G region of the virus. When the same samples were retested with primers against the GpB region, the positive rate for the typical and atypical cases increased to 52% and 11% respectively. Antiviral use at the time of sampling reduced the rate of PCR positivity by 20% (p < 0.05). ADV DNA was detected in 6% of the typical cases and 8% of the atypical cases. All control participants with no history of HSK were negative for HSV1 DNA. Sample quantity was confirmed by testing for housekeeping control genes, beta-actin and beta-2 macroglobulin. PCR results from in vitro control investigations of HSV1 and 2 infected corneal rims and conjunctival epithelial cells were 100% positive for infected and 100% negative for uninfected samples when assessed using both PCR methods. Conclusions: Clinical diagnosis of typical HSK is not always confirmed by PCR. Concomitant use of an antiviral reduces levels of PCR positivity. Given this and the findings that other ocular surface pathogens may mimic HSK pathology, and that choice of gene amplification region can also affect accurate detection of HSV1 by PCR, we propose the use of a multiplex assay. This would perform PCR using primers spanning a number of different regions within one gene and would also target a number of different viral genes to ensure potentially different HSV1 viral strains or other viruses do not affect the test and lead to disagreements between the clinical and molecular diagnosis of HSK. From these findings, this paper proposes a clinical supportive algorithmic guide to manage such disagreements.

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McGilligan, V. , Moore, J. , Tallouzi, M. , Atkinson, S. , O’Neill, H. , Feeney, S. , Novitskaya, E. , Sharma, A. , Shah, S. , Jackson, J. , Frazer, D. and Moore, T. (2014) A Comparison of the Clinical and Molecular Diagnosis of Herpes Simplex Keratitis. Open Journal of Ophthalmology, 4, 65-74. doi: 10.4236/ojoph.2014.43011.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Kaye, S. and Choudhary, A. (2006) Herpes Simplex Keratitis. Progress in Retinal and Eye Research, 25, 355-380.
[2] Farooq, A.V. and Shukla, D. (2012) Herpes Simplex Epithelial and Stromal Keratitis: An Epidemiologic Update. Survey of Ophthalmology, 57, 448-462.
[3] Al-Yousuf, N., Mavrikakis, I., Mavrikakis, E. and Daya, S.M. (2004) Penetrating Keratoplasty: Indications over a 10-Year Period. British Journal of Ophthalmology, 88, 998-1001.
[4] Halberstadt, M., Machens, M., Gahlenbek, K.A., Böhnke, M. and Garweg J.G. (2002) The Outcome of Corneal Grafting in Patients with Stromal Keratitis of Herpetic and Non-Herpetic Origin. British Journal of Ophthalmology, 86, 646-652.
[5] Marangon, F.B., Miller, D. and Alfonso, E. (2007) Laboratory Results in Ocular Viral Diseases: Implications in Clinical-Laboratory Correlation. Abo-ArquivosBrasileiros De Ofthalmologia, 70, 189-194.
[6] Rübben, A., Baron, J.M. and Grussendorf-Conen, E.I. (1997) Routine Detection of Herpes Simplex Virus and Varicella Zoster Virus by Polymerase Chain Reaction Reveals that Initial Herpes Zoster Is Frequently Misdiagnosed as Herpes Simplex. British Journal of Ophthalmology, 137, 259-261.
[7] Fukuda, M., Deai, T., Higaki, S., Hayashi, K. and Shimomura, Y. (2008) Presence of a Large Amount of Herpes Simplex Virus Genome in Tear Fluid of Herpetic Stromal Keratitis and Persistent Epithelial Defect Patients. Seminars in Ophthalmology, 23, 217-220.
[8] Khodadoost, M.A., Sabahi, F., Behroz, M.J., Roustai, M.H., Saderi, H., Amini-Bavil-Olyaee, S. and Arzenani, M.K. (2004) Study of a Polymerase Chain Reaction Based Method for Detection of Herpes Simplex Virus 1 DNA among Iranian Patients with Ocular Herpetic Keratitis Infection. Japanese Journal of Ophthalmology, 48, 328-332.
[9] Lee, S.Y., Kim, M.J., Kim, M.K., Wee and W.R. (2013) Comparative Analysis of Polymerase Chain Reaction Assay for Herpes Simplex Virus 1 Detection in Tear. Korean Journal of Ophthalmology, 27, 316-321.
[10] Jackson, R., Morris, D.J., Cooper, R.J., Bailey, A.S., Klapper, P.E., Cleator, G.M. and Tullo, A.B. (1996) MultiplexPolymerase Chain Reaction for Adenovirus and Herpes Simplex Virus in Eye Swabs. Journal of Virological methods, 56, 41-48.
[11] Van Doornum, G.J., Guldemeester, J., Osterhaus, A.D. and Niesters, H.G. (2003)
Diagnosing Herpesvirus Infections by Real-Time Amplification and Rapid Culture. Journal of Clinical Microbiology, 41, 576-580.
[12] Weidmann, M., Armbruster K. and Hufert, F.T. (2008) Challenges in Designing a Taqman-based Multiplex Assay for the Simultaneous Detection of Herpes Simplex Virus Types 1 and 2 and Varicella-Zoster Virus. Journal of Clinical Virology, 42, 326-334. http://dx.doi.org/10.1016/j.jcv.2008.03.005
[13] Heim, A., Ebnet, C., Harste, G. and Pring-Akerblom, P. (2003)
Rapid and Quantitative Detection of Human Adenovirus DNA by Real-Time PCR. Journal of Medical Virology, 70, 228-239. http://dx.doi.org/10.1002/jmv.10382
[14] Sivadon, V., Lebon, P. and Rozenberg, F. (1998) Variations of HSV-1 Glycoprotein B in Human Herpes Simplex Encephalitis. Journal of Neurovirology, 4, 106-114.
[15] Rekabdar, E., Tunbäck, P., Liljeqvist, J.A. and Bergström, T. (1999) Variability of the Glycoprotein G Gene in Clinical Isolates of Herpes Simplex Virus Type 1. Clinical and Diagnostic Laboratory Immunology, 6, 826-831.
[16] Fan, Q., Lin, E., Satoh, T., Arase, H. and Spear, P.G. (2009) Differential Effects on Cell Fusion Activity of Mutations in Herpes SimplexVirus 1 Glycoprotein B (gB) Dependent on Whether a gD Receptor or a gB Receptor Is Overexpressed. Journal of Virology, 83, 7384-7390.
[17] Norberg, P., Bergström, T., Rekabdar, E., Lindh, M. and Liljeqvist, J.A. (2004) Phylogenetic Analysis of Clinical Herpes Simplex Virus Type 1 Isolates Identified Three Genetic Groups and Recombinant Viruses. Journal of Virology, 78, 10755-10764.
[18] Kolb, A.W., Ané, C. and Brandt, C.R. (2013) Using HSV-1 Genome Phylogenetics to Track Past Human Migrations. PLoS ONE, 8, e76267.
[19] Haarr, L. and Skulstad, S. (1994) The Herpes Simplex Virus Type 1 Particle: Structure and Molecular Functions. Review article. Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 102, 321-346.
[20] Wald, A., Corey, L., Cone, R., Hobson, A., Davis, G. and Zeh, J. (1997) Frequent Genital Herpes Simplex Virus 2 Shedding in Immunocompetent Women. Effect of Acyclovir Treatment. Journal of Clinical Investigation, 99, 1092-1097.
[21] Besada, E. and Reynolds, S. (2005) Incidental Corneal Pseudodendrite Recurrence upon Reinitiation of Latanoprost. Clinical & Refractive Optometry, 16, 233-236.
[22] Mariko, T., Yasuhisa, S., Hiroshi, S. and Kimiyo, F. (1999) Keratomcosis:Clinical diagnosis, Medical and Surgical Treatment. Journal of Medical Education and Research, 5, 3-10.
[23] Pavan-Langston, D. (1990) Herpes Simplex Virus Ocular Infections: Current Concepts of Acute, Latent and Reactivated Disease. Transactions of the American Ophthalmology Society, 88, 727-796.
[24] Goldschmidt, P., Rostane, H., Saint-Jean, C., Batellier, L., Alouch, C., Zito, E., Bourcier, T., Laroche, L. and Chaumeil, C. (2006) Effects of Topical Anaesthetics and Fluorescein on the Real-Time PCR Used for the Diagnosis of Herpesviruses and Acanthamoeba Keratitis. British Journal of Ophthalmology, 90, 1354-1356.
[25] Koizumi, N., Nishida, K., Adachi, W., Tei, M., Honma, Y., Dota, A., Sotozonao, C., Yokoi, N., Yamamotoo, S. and Kinoshita, S. (1999) Detection of Herpes Simplex Virus DNA in Atypical Epithelial Keratitis Using Polymerase Chain Reaction. British Journal of Ophthalmology, 83, 957-960.
[26] Athmanathan, S., Pranesh, V.M., Pasricha, G., Garg, P., Vemuganti, G.K. and Sharma, S. (2001) Atypical Herpes Simplex Keratitis (HSK) Presenting as a Perforated Corneal Ulcer with a Large Infiltrate in a Contact Lens Wearer: Multinucleated Giant Cells in the Giemsa Smear Offered a Clue to the Diagnosis. BMC Ophthalmology, 1, 1.
[27] Polcicova, K., Biswas, P.S., Banerjee, K., Wisner, T.W., Rouse, B.T. and Johnson, D.C. (2005) Herpes Keratitis in the Absence of Anterograde Transport of Virus from Sensory Ganglia to the Cornea. Proceedings of the National Academy of Science of the United States of America, 102, 11462-11467.

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