The authors have no financial interest in the subject matter of this poster.
Radhika Tandon, MD, DNB, FRCS, FRCOphth
Department of Cornea, Cataract & Refractive Surgery and *Ocular Microbiology Dr Rajendra Prasad Centre For Ophthalmic Sciences, AIIMS
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 subjectsassessed (percentage of patients with shedding of HSV-1 DNA).
Authors (year) [PCR type]
Subjects, proportion (%)*
Sample method
Total Samplecollected
per subject,no.
Active stromal keratitis Endotheliitis
Yamamoto S et al. (1994) [Conventional PCR]
2/6(33.33%) - Schirmer’s 1
Kudo E et al. (1996) [Nested PCR] 5/15(33.33%) - Schirmer’s 3.3
Pramod NP et al.(2000 ) [(Conventional PCR] 12/40(30%) - Schirmer’s 2
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
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.
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
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
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
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)
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
Figure 1: Distribution of tear samples from various clinical categories
PUK=Peripheral ulcerative keratitisA= active Q= quiescentP= with perforationV= virus
PUK=Peripheral ulcerative keratitisA= active Q= quiescentP= with perforationV= virus
Figure 2: PCR result in different clinical categories
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
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%)
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
Address for Correspondence : Professor Radhika Tandon
([email protected]) Dr. RP Centre for Ophthalmic Sciences, AIIMS, New Delhi 110029
