1. The authors have no financial interest in the subject matter of this poster. Dr. Manoj Sharma, MD Dr. Bhavna Chawla, MS Dr Namrata Sharma, MD Prof. Jeewan.S.Titiyal, MD Prof. Gita Satpathy, MD* Radhika Tandon, MD, DNB, FRCS, FRCOphth Department of Cornea, Cataract & Refractive Surgery and *Ocular Microbiology Dr Rajendra Prasad Centre For Ophthalmic Sciences, AIIMS

2. Table 1- Shedding of herpes simplex virus type 1 (HSV-1) DNA in active stromal keratitis and / or endotheliitis, as detected by polymerase chain reaction analysis of tear samples obtained from different method *Data are the total no. of subjects with shedding of HSV-1 DNA/total no. of subjects assessed (percentage of patients with shedding of HSV-1 DNA). Authors (year) [PCR type] Subjects, proportion (%)* Sample method Total Sample collected per subject, no. Active stromal keratitis Endotheliitis Yamamoto S et al. (1994) [Conventional PCR] 2/6 (33.33%) -Schirmer’s1 Kudo E et al. (1996) [Nested PCR] 5/15(33.33%)-Schirmer’s3.3 Pramod NP et al.(2000 ) [(Conventional PCR] 12/40(30%)-Schirmer’s2 Fukuda M et. al (2003) [Real-time PCR] 8/14(57%)0/3 eye rinse method 1 Fukuda M, Deai T, et al. (2008) ) [Real-time PCR] 13/22(59.1%)0/3 eye rinse method 1

3.  To evaluate the role of Polymerase Chain Reaction (PCR) in confirmation of diagnosis of clinically suspected Herpetic stromal keratitis or endotheliitis in tear samples  To evaluate the effect of antiviral therapy on test result.

4.  Inclusion criteria › Clinically diagnosed cases of active stromal keratitis and endotheliitis  Exclusion criteria › Pure epithelial keratitis with no stromal involvement › H/o previous oral acyclovir use within one month

5.  Study group: 66 eyes( 59 patients) › 52 Unilateral & 7 Bilateral affected  Control group: 130 eyes of 90 patients › Contra lateral eye of 50* unilateral affected patients › Both eyes of 40 normal volunteers *2 patients had contralateral phthisis bulbi and sample from phthisical eye was not taken

6.  Before starting treatment, tear samples from both eyes of patients were collected by fire polished microcapillary tube and subjected to PCR for HSV DNA detection  PCR Protocol › DNA extraction: commercial QI Amp DNA blood kit › 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) › Electrophoreses- in 2% agarose gel

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

8.  Follow up examination was done Day 2, day 3-6, day-12, day 13-17, day 18-22 & at 3 mths  Repeat PCR was done 3 months after initiating treatment

9. Figure 1: Distribution of tear samples from various clinical categories PUK=Peripheral ulcerative keratitis A= active Q= quiescent P= with perforation V= virus

10. PUK=Peripheral ulcerative keratitis A= active Q= quiescent P= with perforation V= virus Figure 2: PCR result in different clinical categories

11. FPCR= PCR at follow-up (at 3 month from initiation of treatment ) Figure 3: PCR result in tear samples at presentation and at 3 months

12. PCR Negative Necrotizing keratitis with perforation (0/6) Recurrence in Graft (0/1) Quiescent stromal keratitis (0/3) Control sample Positive 13 (20%) (Active) Stromal keratitis 8/30(26%) Necrotizing keratitis 1/3 (33%) Keratouveitis 2/11 (22%) Endotheliitis 2/9 (22%)

13.  20% cases of active Herpetic stromal keratitis and endotheliitis had positive tear sample PCR test result. All positive cases showed good response to treatment and no HSV DNA was detected after antiviral therapy  Positive PCR test result can therefore be used as a marker of active viral replication in cases of active stromal keratitis & endotheliitis

14. Address for Correspondence :  Professor Radhika Tandon (radhika_tan@yahoo.com)  Dr. RP Centre for Ophthalmic Sciences, AIIMS, New Delhi 110029