THESIS PROTOCOL FOR M.S.

DEGREE (OPHTHALMOLOGY)

TOPIC : UTILITY OF MULTIPLEX POLYMERASE CHAIN

REACTION FOR DETECTION OF HERPES SIMPLEX
VIRUS-1 AND 2 IN PATIENTS WITH VIRAL KERATITIS
NAME OF CANDIDATE : Dr. PREETY REKHA DAS
POST–GRADUATE STUDENT

DEPARTMENT OF OPHTHALMOLOGY,

ASSAM MEDICAL COLLEGE & HOSPITAL, DIBRUGARH

NAME OF GUIDE : Dr. MAMONI BARUAH

PROFESSOR

DEPARTMENT OF OPHTHALMOLOGY,

ASSAM MEDICAL COLLEGE & HOSPITAL, DIBRUGARH

NAME OF CO–GUIDE : Dr. REEMA NATH

PROFESSOR AND HOD

DEPARTMENT OF MICROBIOLOGY,

ASSAM MEDICAL COLLEGE & HOSPITAL, DIBRUGARH

NAME OF CO–GUIDE : Dr. RAJENDRA NATH GOGOI

ASSISTANT PROFESSOR

DEPARTMENT OF OPHTHALMOLOGY,

ASSAM MEDICAL COLLEGE &HOSPITAL, DIBRUGARH

PLACE OF STUDY : ASSAM MEDICAL COLLEGE & HOSPITAL, DIBRUGARH

TYPE OF STUDY : HOSPITAL BASED OBSERVATIONAL STUDY

DURATION OF STUDY : ONE YEAR

UNIVERSITY : SRIMANTA SANKARADEVA UNIVERSITY OF HEALTH

SCIENCES, GUWAHATI, ASSAM

SIGNATURE SIGNATURE SIGNATURE SIGNATURE SIGNATURE SIGNATURE

OF THE OF THE OF THE OF THE OF THE HEAD OF THE
CANDIDATE GUIDE CO–GUIDE CO–GUIDE OF DEPTT. PRINCIPAL
 INTRODUCTION

Infectious keratitis is the infection of cornea associated with epithelial defects

and signs of inflammation. It is one of the leading causes of ocular morbidity and blindness

worldwide. In most cases these infections are preventable and treatable; therefore a thorough

understanding of epidemiology, diagnosis and treatment is essential. A large number of

bacteria, fungi, virus and protozoa have been identified and implicated as infectious agents in

infectious keratitis. Infectious keratitis caused by virus result in a broad spectrum of eye

diseases and can lead to corneal opacities, ulcers, scarring, vascularisation, perforation and

even visual impairment. The virus can infect individual layers of cornea or may involve all

the layers of cornea in severe forms. The viruses most frequently causing keratitis are Herpes

Simplex Virus,1 Adenovirus2,3 and Varicella Zoster Virus (VZV).4 Herpes Simplex Virus

(HSV) is the major cause of viral keratitis, of which HSV 1 accounts for around 90% and

HSV2 accounts for around 10% of all Herpes Simplex Keratitis.5

Primary infections due to Herpes Simplex Virus usually occur in childhood and

are spread by droplet transmission or less frequently by direct inoculation. Primary HSV

infection of the eye usually manifests as blepharoconjunctivitis. Primary HSV keratitis is rare

and occurs in only 3% -5% cases.6 Recurrent infections due to reactivation occur in 27% of

patients at 1 year and in 60% patients at 20 years of primary infection. 6 Although it can affect

all parts of the eye it most commonly causes keratitis. The risk of subsequent recurrent

infection increases with the number of recurrences.

Herpes Simplex Keratitis may present as epithelial keratitis, stromal keratitis

or endotheliitis. Diagnosis is usually made based on the clinical presentations. Giemsa

staining can be done to look for the presence of multinucleated giant cells, lymphocytes and
intranuclear eosinophillic inclusion bodies. HSV DNA can be detected in corneal scrapings

or conjunctival swabs by PCR. Immunofluorescence assay to detect antibodies against HSV

and viral culture for isolation of the organism can also be done.

Polymerase chain reaction (PCR) is an investigative technique in molecular

biology. Here an organism is identified by amplifying a single or few copies of its DNA

across several magnitudes to produce millions of DNA copies and thus helps in identification

of organism.

The purpose of this study is to evaluate the role of multiplex PCR for rapid

diagnosis of Herpes Simplex Keratitis which will help in early management of the disease

and thus reduce ocular morbidity.

The timely and rapid diagnosis of Herpes Simplex Keratitis has become urgent

given the availability of specific antiviral drugs and appropriate patient management

strategies in addition to the traditional diagnosis based on clinical evaluation. Although the

conventional cell culture technique is considered as gold standard for the diagnosis of Herpes

Simplex virus, it is costly and its major drawback is the long isolation period. Moreover cell

culture cannot be established in all routine diagnostic laboratories.7 PCR is highly sensitive in

identifying infectious organisms especially viruses with the help of specific primers and

probes. For HSV detection in specimens, multiplex PCR detects HSV 1 and 2 genome targets

in a single PCR reaction along with a housekeeping gene as internal quality control. 8

Conventional PCR is increasingly being replaced by Real time PCR which is more rapid and

sensitive.
RESEARCH QUESTION:

Is multiplex PCR useful in detection of HSV-1 and 2 in corneal scrapings and

conjunctival swabs of Viral Keratitis patients?

HYPOTHESIS:

Multiplex PCR is supposed to be useful in detecting HSV-1 and 2 in the

corneal scrapings and conjunctival swabs for rapid diagnosis of Viral Keratitis.
 AIM

1. To evaluate the use of multiplex PCR in the detection of HSV1 and HSV2 in patients

with Viral Keratitis.

OBJECTIVE

1. To detect the presence of HSV-1 and HSV-2 in corneal scrapings or conjunctival

swabs of patients with clinically diagnosed Viral Keratitis.

REVIEW OF LITERATURE
Corneal infections are the second among the commonest cause of monocular blindness in the

developing countries.9 In India, there are currently almost 6.8 million people who have

Snellen vision of less than 6/60 because of corneal involvement in one eye out of which

approximately one million have bilateral blindness.10 Corneal ulcer is a silent epidemic. An

annual incidence of 113 cases per 100000 people was reported from a tertiary Centre in South

India which is as high as 10 times the number isolated from a study conducted in the United

states.

It was already reported that 90% of corneal blindness occur in the developing nations and the

prevalence of corneal blindness in India is 0.45 % that translates to approximately 5.4 million

people.11

The distinction between bacterial, fungal or viral etiology is essential for management.

Currently, the gold standard for diagnosing and characterizing microbial (bacterial and

fungal) keratitis is culture.11 Viral ulcers are diagnosed based on clinical findings. Culture

results are highly specific but have suboptimal sensitivity ranging from 35 -70 %

worldwide.12

A population study from central China shows that the prevalence of infectious suppurative

keratitis was 0.148%; the prevalence of viral, bacterial, and fungal keratitis being 0.065%,

0.068%, and 0.015%, respectively. There were no statistically significant differences found

between the prevalence of viral and bacterial corneal ulcers.13

A prospective study in France was conducted and a systematic screening on corneal samples

for bacterial, fungal, amoebic and viral organisms was performed which concluded the
following - 78.5% were caused by bacteria (Pseudomonas aeruginosa was the most frequent),

3 % by mycobacteria, 10.5%, 6% and 2% by virus, fungus and by protozoa respectively.14

HSV keratitis is a leading cause of corneal blindness in the United States and of unilateral

infectious blindness in the world. The ocular prevalence of Herpes infection has been

estimated at 150 cases among 10,000 individuals in developed nations, with an incidence of

5–20 cases per 10,000 individuals per year.15

Pathogenesis of Herpes Simplex Keratitis: - Herpes Simplex Virus is a member of the

Herpesviridae family. It is a linear double stranded DNA virus with an icosahedral capsid

surrounded by poorly defined tegument enclosed in a host cell derived envelope with viral

derived glycoproteins projections. Humans are the only natural reservoirs of Herpes Simplex

Virus. The sources of infection are by direct contact of mucous membrane with infected

lesions, by infected secretions or by fomites from children and adults with active disease and

also asymptomatic carriers shedding the virus in their secretions.16

Primary infection without previous viral exposure usually occurs in childhood. Due to

maternal antibodies it is uncommon during the first 6 months of life. Most primary infections

are subclinical or cause mild fever, malaise and upper respiratory tract symptoms. In the eyes

there may be blepharitis and follicular conjunctivitis but are usually mild and self limiting.

The virus enters the epithelial cells on contact, replicates, enters the sensory nerve endings

and travel in a retrograde fashion via the sensory neurons to the trigeminal ganglion where it

remains latent. The cornea may also be a site where HSV remains latent or replicates.16

Recurrent infections occur when the latent virus is reactivated by various triggers such as

fever, hormonal change, ultraviolet irradiation, emotional stress, prostaglandin analogues,

immunosuppression, trauma and trigeminal injury. Although it can affect all parts of the eye

it most commonly causes keratitis.16

Clinical presentation of Herpes Simplex Keratitis: - HSV can affect different layers of the

cornea with varying clinical presentations causing respective morbidities—namely, the

epithelium, stroma, or endothelium. Epithelial keratitis usually presents as superficial

punctuate keratitis but can also present as dendritic ulcer in severe cases or as geographical

ulcer. Stromal keratitis usually occurs as an immune-mediated response to non-replication. It

can affect all the layers of the cornea. In endotheliitis / Disciform keratitis the inflammation is

limited to the corneal endothelium with keratic precipitates and corneal edema and

Descemet’s folds. It may be the result of active HSV infection of endothelium or a

hypersensitivity reaction to viral antigen in the cornea.17

In India a retrospective case series study detected 212 (169 new) cases of viral keratitis over a

period of 4 years, based on clinical examination. It is a predominantly unilateral disease, but

1.3–12% of cases are reported as bilateral.18

A retrospective case series in Aravind eye hospital Madurai detected 212 (169 new) cases of

viral keratitis over a four-year duration. The diagnosis was purely based on clinical findings.

The various types of clinical presentations in this series was as follows: dendritic ulcers

(15.91%), geographic ulcers (4.09%), stromal keratitis (53.64%), both epithelial and stromal

keratitis (17.73%) and endotheliitis (8.64%).19

The utility of PCR analysis was assessed against other diagnostic markers: HSV isolation on

cell culture, HSV antigen detection by indirect IF, detection of anti-HSV IgG by ELISA and

detection of HSV-specific tear secretory IgA by ELISA. These tests showed overall

sensitivity values of 22.4%, 39.8%, 30.4% and 20.3% respectively. PCR gave a positive

result in 82.1%.20

Polymerase chain reaction (PCR) is a widely used investigative technique in molecular

biology where a selected region of a DNA molecule is amplified by several magnitudes to

produce millions of DNA copies. PCR was invented by Kary Mullis in 1983 for which he
received the Nobel Prize. PCR is used for rapid and highly specific diagnosis of a number

infectious disease including those caused by bacteria and viruses.21

The process of PCR consists of three major steps:21

1) Denaturation: the hydrogen bonds between complementary bases of double stranded

DNA is broken to yield two single stranded DNA.

2) Annealing: two different primers are attached to each single stranded DNA template

containing the target region.

3) Elongation: DNA polymerase is used to synthesize new DNA strand complementary

to the DNA template strands.

These steps are repeated at appropriate temperature and chemical environment for

necessary amplification.

In Conventional PCR the amplified PCR products are run on a gel electrophoresis to analyze

them. It gives the result only at the end of the reaction and the results are in the form of bands

in the gel and thus more time consuming.20

In Quantitative or Real time PCR the quantity of a target sequence is measured i.e. it

determines whether a DNA sequence is present in a sample and the number of its copies in

the sample. Here a special dye is used that helps in production of signal with every cycle and

the signal strength increases as the number of copies of gene increase. In contrast to

conventional PCR, it can detect amplifications during the early phases of reaction and thus is

less time consuming.22

Multiplex PCR consists of multiple primer sets within a single PCR mixture to produce

amplicons of varying sizes which are specific to different DNA sequences. In multiplex PCR

multiple genes are targeted at once and additional information is gained from a single test

run.22
Satpathy et. al. evaluated the role of PCR in suspected viral keratitis patients in corneal

scrapings and tear fluid.23 They compared the results with virus isolation and

Immunofluorescence assay. PCR was found to be much more sensitive than the other two

modalities and the detection rate with corneal scraping was significantly higher than tear

fluid.

Ma et al. reported the results of RT-PCR in diagnosing viral necrotizing keratitis. 24 They

found a viral positivity rate of 46.4% in corneal epithelial scrapings.

Fukuda et al. studied RT-PCR in tear fluid in all variants of HSV keratitis. They reported

highest number of copies of HSV-DNA in herpetic epithelial keratitis followed by active

stromal keratitis and persistent epithelial defect. Their detection rate was higher at 88.1% for

epithelial keratitis and 59.1% for stromal keratitis.

Guda et al. the authors have reported significantly higher positivity with the multiplex RT-

PCR as compared to immunofluorescence assay (IFA) and conventional PCR. 25 The study

emphasizes the role of multiplex RT-PCR for the detection of HSV and varicella zoster

(VZV) DNA in corneal scrapings of suspected herpes keratitis in an ocular microbiology

laboratory.

There are studies supporting the isolation of HSV from corneal scrapings using PCR and also

monitoring drug response using qualitative real time PCR.

 MATERIALS AND METHODS

PLACE OF STUDY: Department of Ophthalmology, Assam Medical College and Hospital,

Dibrugarh, Assam.

STUDY DESIGN: Hospital based observational study

DURATION OF STUDY: 1 year

STUDY POPULATION: Patients attending the Out-patient department of Ophthalmology in

Assam Medical College and Hospital clinically diagnosed with Viral Keratitis.

SAMPLE SIZE: All patients attending the Out- patient department of Ophthalmology in

Assam Medical College and Hospital clinically diagnosed with Viral Keratitis fulfilling the

inclusion criteria during the study period.

INCLUSION CRITERIA:

 Patients above 15 years of age clinically diagnosed with Viral

Keratitis.

EXCLUSION CRITERIA:

 Patients with combined bacterial or fungal infections.

 Patients with other corneal degenerative or immune related keratitis.

 Patients not willing to provide consent.

METHODOLOGY:

In this hospital based, observational study, patients above 15 years of age attending the Out-

patient department of Ophthalmology, Assam Medical College and Hospital with clinically
diagnosed Viral keratitis will be taken up for study. A complete general, medical and

Ophthalmological examination will be peformed as per structured proforma (Annexure-II). A

written informed consent (AnnexureIII) would be obtained from every participant after

providing them with the detailed information about the study. They will also be given the

liberty to withdraw their consent at any given point of time. The instruments to be used for

examination of the patients will include:

 Snellen’s Visual Acuity Chart.

 Fluorescein sodium ophthalmic strips.

 Wisp of cotton.

 Anterior segment Slit-lamp Bio-microscopy.

 Goldman Applanation tonometry.

The corneal scrapings and conjunctival swabs of these patients will be collected

under topical anesthesia and slit lamp magnification with sterile blade no.15, in a TPP tube

with VTM and then stored at -80 degree centigrade until processed for PCR. By PCR the

viral DNA for HSV-1 and 2 will be detected. The test will be conducted in the Multi-

disciplinary Research Laboratory of Assam Medical College and Hospital.

ETHICAL CONSIDERATION:

The study proposal will be submitted to the Institutional Ethics Committee (H) of Assam

Medical College and Hospital, Dibrugarh for review and appraisal and study will be

commenced after approval.

STATISTICAL ANALYSIS:

The results and observations made in the study will be presented in terms of

counts, percentage, mean±SD. Statistical analysis will be done using student ‘t’ test or chi-

square test and p-value of <0.05 will be considered as statistically significant.

 RESULTS AND OBSERVATIONS

Table 1: Age distribution

Age No. Of No. of patients No. of patients No. of patients

group Patients positive for positive for negative for
(in years) HSV 1 by PCR HSV 2 by PCR HSV By PCR

15 – 30
31 – 45
46 – 60
>60
Total

Table 2: Sex distribution

Sex No. of No. of patients No. of patients No. of patients

patients positive for positive for negative for
HSV 1 by PCR HSV 2 by PCR HSV by PCR

Male
Female
Total
Table 3: Clinical presentation and PCR result for HSV 1

Symptoms No. Of Patients with PCR Percentage (%)

Patients positive for HSV 1

Pain

Redness

Watering

Diminution of
vision
Intolerance to
light
Total

Table 4: Clinical presentation and PCR result for HSV 2

Symptoms No. Of Patients with PCR Percentage (%)

Patients positive for HSV 2

Pain

Redness

Watering

Diminution of
vision
Intolerance to
light
Total

Table 5: Symptom duration and PCR result for HSV 1

 Duration No. of Patients with PCR Percentage
Patients positive for HSV 1 (%)

< 1 week

1-2 weeks

2-4 weeks

>4 weeks

Total

Table 6: Symptom duration and PCR result for HSV 2

Duration No. of Patients with PCR Percentage

Patients positive for HSV 2 (%)

< 1 week

1-2 weeks

2-4 weeks

>4 weeks

Total

Table 7: Clinical diagnosis and PCR result for HSV 1

 Clinical No. of patients Patients with Percentage (%)
diagnosis PCR positive for
HSV 1
Epithelial
Stromal
Endothelial
Total

Table 8: Clinical diagnosis and PCR result for HSV 2

Clinical No. of patients Patients with Percentage (%)

diagnosis PCR positive for
HSV 2
Epithelial
Stromal
Endothelial
Total

More tables may be added during the course of study if needed

DISCUSSION

The results and observations made in the study will be discussed in details with relevant

study on the subject by other authors statistically analysed.

CONCLUSION

The results of the study will be summarized and a conclusion will be derived from the

statistical analysis of the observations made during the study.

 REFERENCES

1. Nahmias AJ, Visintine AM, Caldwell DR, Wilson LA. Eye infections with herpes

simplex viruses in neonates. Survey of ophthalmology. 1976 Jan 1;21(2):100-5.

2. Keenlyside RA, Hierholzer JC, D'Angelo LJ. Keratoconjunctivitis associated with

adenovirus type 37: an extended outbreak in an ophthalmologist's office. Journal of

Infectious Diseases. 1983 Feb 1;147(2):191-8.

3. Sprague JB, Hierholzer JC, Currier RW, Hattwick MA, Smith MD. Epidemic

keratoconjunctivitis: a severe industrial outbreak due to adenovirus type 8. New

England Journal of Medicine. 1973 Dec 20;289(25):1341-6.

4. Mondino BJ, Brown SI, Mondzelewski JP. Peripheral corneal ulcers with herpes

zoster ophthalmicus. American journal of ophthalmology. 1978 Nov 1;86(5):611-4.

5. Biney EE, Orrett FA. Screening of human corneas for herpes simplex virus by tissue

culture and polymerase chain reaction. Japanese Journal of Medical Science and

Biology. 1997;50(4-5):151-60.

6. Beck RW,Asbell PA, Cohen EJ, Dawson CR, Hyndiuk RA, Jones DB, et.al. Oral

acyclovir for herpes simlex virus eye disease- Effect on prevention of epithelial

keratitis and stromal keratitis. Archives of ophthalmology. 2000 Aug 1;118(8):1030-

6.

7. Khodadoost MA, Sabahi F, Behroz MJ, Roustai MH, Saderi H, Amini-Bavil-Olyaee

S, Arzenani MK. Study of a polymerase chain reaction-based method for detection of

herpes simplex virus type 1 DNA among Iranian patients with ocular herpetic keratitis

infection. Japanese journal of ophthalmology. 2004 Jul 1;48(4):328-32.

8. Watzinger F, Suda M, Preuner S, Baumgartinger R, Ebner K, Baskova L, et.al. Real-

time quantitative PCR assays for detection and monitoring of pathogenic human

viruses in immunosuppressed pediatric patients. Journal of clinical microbiology.

2004 Nov 1;42(11):5189-98.

9. Rathore AS, Gogate P, Murthy GV, Nirmalan PK, Rao GV, Shamanna BR, et.al.

Present status of the National Programme for Control of Blindness in India.

Community Eye Health Journal. 2008 Mar;21(65).

10. Shah A, Sachdev A, Coggon D, Hossain P. Geographic variations in microbial

keratitis: an analysis of the peer-reviewed literature. British Journal of

Ophthalmology. 2011 Jun 1;95(6):762-7.

11. Srinivasan M. Infective keratitis: A challenge to Indian ophthalmologists. Indian

journal of ophthalmology. 2007 Jan 1;55(1):5.

12. Leck AK, Thomas PA, Hagan M, Kaliamurthy J, Ackuaku E, John M, Newman MJ,

Codjoe FS, Opintan JA, Kalavathy CM, Essuman V. Aetiology of suppurative corneal

ulcers in Ghana and south India, and epidemiology of fungal keratitis. British Journal

of Ophthalmology. 2002 Nov 1;86(11):1211-5.

13. Cao J, Yang Y, Yang W, Wu R, Xiao X, Yuan J, et.al. Prevalence of infectious

keratitis in Central China. BMC ophthalmology. 2014 Dec;14(1):43.

14. Hoffart L, Dornadin A, Drancourt M. Epidemiology of microbial keratitis: A review

of 508 cases. Acta Ophthalmologica. 2012 Sep;90.

15. Inoue T, Ohashi Y. Utility of real-time PCR analysis for appropriate diagnosis for

keratitis. Cornea. 2013 Nov 1;32:S71-6.

16. Farooq AV, Shukla D. Herpes simplex epithelial and stromal keratitis: an

epidemiological update. Survey of ophthalmology. 2012 Sep 1;57(5):448-62.

17. Rowe AM, Leger AS, Jeon S, Dhaliwal DK, Knickelbein JE, Hendricks RL. Herpes

keratitis. Progress in retinal and eye research. 2013 Jan 1;32:88-101.

18. Pramod NP, Rajendran P, Kannan KA, Thyagarajan SP. Herpes simplex keratitis in

South India: clinico-virological correlation. Japanese journal of ophthalmology. 1999

Jul 1;43(4):303-7.

19. Kabra A, Lalitha P, Mahadevan K, Prajna NV, Srinivasan M. Herpes simplex keratitis

and visual impairment: a case series. Indian journal of ophthalmology. 2006 Jan

1;54(1):23.

20. Farhatullah S, Kaza S, Athmanathan S, Garg P, Reddy SB, Sharma S. Diagnosis of

herpes simplex virus-1 keratitis using Giemsa stain, immunofluorescence assay, and

polymerase chain reaction assay on corneal scrapings. British journal of

ophthalmology. 2004 Jan 1;88(1):142-4.

21. Bartlett JM, Stirling D, editors. PCR protocols. Totowa, NJ: Humana Press; 2003

Aug.

22. Bustin SA. Developments in real-time PCR research and molecular diagnostics.

Expert review of molecular diagnostics. 2010 Sep 1;10(6):713-5.

23. Satpathy G, Mishra AK, Tandon R, Sharma MK, Sharma A, Nayak N, Titiyal JS,

Sharma N. Evaluation of tear samples for Herpes Simplex Virus 1 (HSV) detection in

suspected cases of viral keratitis using PCR assay and conventional laboratory

diagnostic tools. British journal of ophthalmology. 2011 Mar 1;95(3):415-8.

24. Ma JX, Wang LN, Zhou RX, Yu Y, Du TX. Real-time polymerase chain reaction for

the diagnosis of necrotizing herpes stromal keratitis. International journal of

ophthalmology. 2016;9(5):682.

25. Guda SJ, Sontam B, Bagga B, Ranjith K, Sharma S, Joseph J. Evaluation of

multiplex real-time polymerase chain reaction for the detection of herpes simplex
virus-1 and 2 and varicella-zoster virus in corneal cells from normal subjects and

patients with keratitis in India. Indian journal of ophthalmology. 2019 Jul;67(7):1040.

 ANNEXURE- I

LIST OF ABBREVIATIONS

HSV Herpes Simplex Virus

VZV Varicella Zoster Virus

DNA Deoxyribonucleic acid

PCR Polymerase chain reaction

IF Immunofluorescence

IgG Immunoglobulin G

IgA Immunoglobulin A

ELISA Enzyme linked immunosorbent assay

RT-PCR Real time polymerase chain reaction

IFA Immunofluorescence assay

SD Standard deviation
 ANNEXURE –II

PROFORMA

Case No. :

Hospital No. :

Name :

Age :

Sex :

Religion :

Occupation :

Address :

Date of Admission :

Date of Examination :

HISTORY:

Chief Complaints:

 H/o Pain in eye : Yes/No

 H/o redness : Yes/No

 H/o watering : Yes/No

 H/o intolerance to light : Yes/No

 H/o diminution of vision : Yes/No

 Any other relevant complaints:

History of Presenting Illness:

 Onset

 Duration

 Discharge

 Use of any medication

 Any other relevant history

History of Past Illness:

 History of similar illness

 History of ocular injury

 History of Ocular Surgery/Past Treatment

 History of any systemic illness, like Diabetes mellitus, Hypertension

Family History:

 Any Similar Complaints:

Physical Examination

General Examination

 Pulse :

 Blood pressure :

 Temperature :

 Respiratory Rate :

Systemic Examination

 Respiratory System :

 Cardiovascular System :

 Gastrointestinal System :

 Central Nervous System:

Ocular Examination

Right eye Left eye

(1) Visual Acuity (Distant/Near) :

(Snellen’s Chart)

(2) Colour vision :

(3) Symmetry of the Orbit :

(4) Alignment :

(5) Forehead :

(6) Eyebrow :

(7) Eyelid :

(8) Movements of the eyeball :

(9) Conjunctiva :

(10)Cornea :

(11)Anterior Chamber :

(12)Iris (Colour/Pattern/Atrophy) :

(13)Pupil (Size/Shape/Reaction) :

(14)Lens (Clear/Pigments on Anterior Capsule) :

(15)NLD Patency :

INVESTIGATIONS

1) R/E Blood

2) RBS

3) Corneal scrapings for detection HSV DNA by PCR

 ANNEXURE-III

PATIENT CONSENT FORM

Title of the Study: “UTILITY OF MULTIPLEX POLYMERASE CHAIN REACTION FOR

DETECTION OF HSV 1 AND 2 IN PATIENTS WITH VIRAL

KERATITIS”

Name of the Principal/Co-investigator ____________________________________

Name of the Institution : ____________________________________

Documentation of the Informed Consent:

I, … … … … … … … … … … … … … … … … have read the information in this form (or

it has been read to me). I was free to ask any questions and they have been answered. I am

over 18 years of age and, exercising my free power of choice, hereby give my consent to

include my child as a participant in the study “UTILITY OF MULTIPLEX POLYMERASE

CHAIN REACTION FOR DETECTION OF HSV-1 AND 2 IN PATIENTS WITH VIRAL

KERATITIS.” I have read and understood this consent form

and the information provided to me.

1. I have had the consent document explained to me.

2. I have been explained about the nature of the study.

3. My rights and responsibilities have been explained to me by the investigator.

4. I have been advised about the risks associated with participation in the study.
5. I have informed the investigator of all the treatments my child is taking or have taken

in the past … … … … months including any desi (alternative) treatments.

6. My child have not participated in any research study within the past … … … …

month(s).

7. I am aware of the fact that I can opt out my child of the study at any time without

having to give any reason and this will not affect my future treatment in the hospital.

8. I am also aware that the investigators may terminate participation of my child in the

study at any time, for any reason, without my consent.

9. I hereby give permission to the investigators to release the information obtained from

me as result of participation in this study to the sponsors, regulatory authorities,

Government agencies, and ethics committee. I understand that they may inspect my

original records.

10. Identity of my child will be kept confidential if my data are publicly presented.

11. If, despite following the instructions, my child is physically harmed because of any

substance or any procedure as stipulated in the study plan, [Treatment will be carried

out free at the investigational site / the sponsor will bear all the expenses], if they are

not covered by my insurance agency or by a Government program or any third party.

12. I have had my questions answered to my satisfaction.

13. I have decided to enlist my child be in the research study. I am aware, that if I have

any questions during this study, I should contact at one of the addresses listed above.
Name and signature/thumb impression of the parents or guardian or legal representative

if participant incompetent:

… … … … … … … … … … (Name) … … … … … … … … (Signature)

Date: … … … … … … Time: … … … … …

Name and signature of impartial witness (required for illiterate parents or guardian):

… … … … … … … … … … (Name) … … … … … … … … (Signature)

Date: … … … … … … Time: … … … … …

Address and contact number of the impartial witness: ___________________

… … … … … … … … … … (Name) … … … … … … … … (Signature)

Date: … … … … … … Time: … … … … …

Name and signature of the Principal/Co-investigator obtaining consent:

… … … … … … … … … … (Name) … … … … … … … … (Signature)

Date: … … … … … … Time: … … … … …
 ANNEXURE-III (B)

“UTILITY OF MULTIPLEX POLYMERASE CHAIN REACTION FOR

DETECTION OF HSV 1 AND HSV 2 IN PATIENTS WITH VIRAL
KERATITIS”

Dr. Preety Rekha Das