Transcript Slide 1

Radhika Tandon, MD, DNB, FRCS, FRCOphth
Dr. Manoj Sharma, MD
Dr. Bhavna Chawla, MS
Dr Namrata Sharma, MD
Prof. Jeewan.S.Titiyal, MD
Prof. Gita Satpathy, MD*
Department of Cornea, Cataract & Refractive Surgery and *Ocular
Microbiology Dr Rajendra Prasad Centre For Ophthalmic Sciences, AIIMS
The authors have no financial interest in the subject matter of this poster.
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
Subjects, proportion (%)*
Sample method
Total Sample
collected
per subject,
no.
-
Schirmer’s
1
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
13/22(59.1%)
0/3
eye rinse
method
1
Authors (year)
[PCR type]
Yamamoto S et al. (1994)
[Conventional PCR]
Kudo E et al. (1996) [Nested
PCR]
Fukuda M, Deai T, et al.
(2008) ) [Real-time PCR]
Active stromal
keratitis
Endotheliitis
2/6
(33.33%)
*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).

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 keratitis
A= active
Q= quiescent
P= with perforation
V= virus
Figure 2: PCR result in different clinical categories
PUK=Peripheral ulcerative keratitis
A= active
Q= quiescent
P= with perforation
V= virus
Figure 3: PCR result in tear samples at presentation and at 3 months
FPCR= PCR at follow-up (at 3 month from initiation of treatment)
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