Detection of Herpes Simplex Virus Genome in Tear Fluid of Patients with Herpetic Keratouveitis and Endotheliitis The authors have no financial interest in the subject matter of this poster Department of Cornea, Cataract & Refractive Surgery and *Ocular Microbiology Dr Rajendra Prasad Centre For Ophthalmic Sciences, AIIMS Radhika Tandon, MD, DNB, FRCS, FRCOphth Dr. Manoj Sharma, MD, Prof.Jeewan.S.Titiyal, MD Dr Tushar Agarwal, MD Dr Namrata Sharma, MD Prof. Gita Satpathy, MD*

Aim  To evaluate the role of the polymerase chain reaction (PCR) for detecting Herpes virus DNA in tear samples of patients with herpetic keratouveitis and endotheliitis.

Patients  Inclusion criteria Clinically diagnosed Keratouveitis and/or endotheliitis  Exclusion criteria Oral acyclovir use within one month

Study Design  Study group: 20 eyes (17 patients) 12 unilateral 3 bilaterally active 2 bilaterally affected with fellow eye quiescent*  Control group: 94 eyes of 54 patients Contra lateral eye of 14 unilateral active disease Both eyes of 40 normal volunteers *59% (10/17) had definite history of recurrent disease

Laboratory Diagnosis  Before starting treatment, tear samples were collected with fire polished microcapillary tubes and subjected to PCR for HSV DNA detection  PCR Protocol 1. DNA extraction: commercial QI Amp DNA blood kit 2. Polymerase chain reaction Primer-111 bp region of HSV 1 thymidine kinase gene (Hofgartner W T et al Clinical chemistry, 1999) Amplification- thermal cycler (Gene Amp PCR system 9700, applied biosystem, USA) 3. Electrophoreses- in 2% agarose gel

Treatment  Tab acyclovir 400 mg (5 times/day) × 7 days followed by Tab acyclovir 400 mg (BD) × 6 months  Topical steroid (1% prednisolone acetate) Adjunct therapy was given as required  Topical antibiotic  Topical mydriatic (2% homatropine)  Topical lubricant (preservative free)  Analgesics (if required)

Clinical typePCRTotal PositiveNegative Viral keratouveitis2911 Endotheliitis279 Control080 Fellow unaffected eyes012 Fellow quiescent eye022 Result  Four of twenty tear samples (20%) from patients yielded PCR positive for Herpes virus DNA, of whom 2 had acute endotheliitis and 2 had keratouveitis.  All four PCR positive cases had recurrent disease.  No HSV DNA was detected in tear samples from both eyes of 40 normal volunteers and fellow eye of unilateral active cases

Figure : PCR result In different clinical categories

At presentation 1 week 1 month (1a, 2a) (1b, 2b) (1c, 2c) 1a 1b 1c 2c 2b 2a 2 cases of Herpetic Keratouveitis who tested PCR positive in tear samples from the affected eye

1a1b1c 2c 2b 2a A patient with bilateral Herpetic Endotheliitis who tested PCR positive in tear samples from both eyes At presentation 1 week 3 weeks (1a, 2a) (1b, 2b) (1c, 2c)

Conclusion  20% cases of active keratouveitis and endotheliitis had positive tear sample PCR test result for HSV DNA  Herpes Virus DNA is detectable in tear samples of such patients and could prove useful in supporting the clinical diagnosis of these cases.

Address for Correspondence Dr Radhika Tandon Professor of Ophthalmology Dr. RP Centre for Ophthalmic Sciences All India Institute of Medical Sciences ( AIIMS) New Delhi India