Introduction An important cause of ocular morbidity as well as blindness in developing countries, including India, is microbial keratitis. The incidence of microbial keratitis worldwide, ranges from 6.3 to 710 cases per 100,000 population per year. 1 Microorganisms, like bacteria, viruses, fungi and parasites trigger an infective process, leading to severe corneal inflammation, ulceration, scarring or even perforation, with devastating consequences. Fungal or mycotic keratitis is a major cause of corneal blindness in tropical countries. Fungi are usually taken as opportunistic agents of infection. They rarely infect the healthy, intact cornea but in a compromised or immunosuppressed cornea, almost any fungal species is capable of inducing infection. Moreover, widespread use of corticosteroids and broad-spectrum antimicrobials has led to an increased incidence of fungal infections. In India, half of all infectious corneal ulcers are fungal. 2 In tropical countries, filamentous fungi are more common, whereas yeast such as Candida is more prevalent in temperate climates. In recent years, however, the rate of infection by filamentous fungi has increased. The clinical presentation of fungal keratitis is non-specific and indolent and patients are initially treated for bacterial, viral or even amoebic infections. Lack of appropriate therapy can lead to the infection developing into an endophthalmitis and the eye can 1
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Introduction
An important cause of ocular morbidity as well as blindness in developing countries,
including India, is microbial keratitis. The incidence of microbial keratitis worldwide, ranges
from 6.3 to 710 cases per 100,000 population per year.1 Microorganisms, like bacteria,
viruses, fungi and parasites trigger an infective process, leading to severe corneal
inflammation, ulceration, scarring or even perforation, with devastating consequences.
Fungal or mycotic keratitis is a major cause of corneal blindness in tropical countries.
Fungi are usually taken as opportunistic agents of infection. They rarely infect the healthy,
intact cornea but in a compromised or immunosuppressed cornea, almost any fungal species
is capable of inducing infection. Moreover, widespread use of corticosteroids and broad-
spectrum antimicrobials has led to an increased incidence of fungal infections. In India, half
of all infectious corneal ulcers are fungal.2 In tropical countries, filamentous fungi are more
common, whereas yeast such as Candida is more prevalent in temperate climates. In recent
years, however, the rate of infection by filamentous fungi has increased. The clinical
presentation of fungal keratitis is non-specific and indolent and patients are initially treated
for bacterial, viral or even amoebic infections. Lack of appropriate therapy can lead to the
infection developing into an endophthalmitis and the eye can be lost forever. Alternatively,
the permanent scarring associated with delay in treatment can lead to significant loss of
vision.
Therefore, the major challenge in the management of fungal keratitis is the difficulty in
clinical and microbiological diagnosis. Although laboratory testing of corneal scrape
specimens remains the gold standard in the diagnosis of infectious keratitis, some organisms
are difficult to detect or culture in vitro and others are slow growing. Also corneal cultures are
positive only in 52.5-67% of cases.3 Further, the false-negative rate is another important
limitation in the diagnostic ability of corneal cultures.4 In such cases, delays in appropriate
treatment may occur.
Confocal microscopy is a non-invasive diagnostic modality for imaging the living
human cornea thereby it can provide its qualitative and quantitative analysis in healthy and
pathological states. Thus in vivo confocal microscopy (IVCM) can play a complementary role
in the early diagnosis of corneal infections and in monitoring the prognosis of the disease
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process. Many published studies have shown confocal microscopy to be effective in the
diagnosis of Acanthamoeba5-7, fungal8-10, Nocardia11 and Microsporidia12 keratitis. A recent
study by Kanavi, et al in 133 microbial keratitis cases demonstrated that confocal microscopy
had sensitivity of 94% and specificity of 78% among patients with fungal keratitis.13
The rapidity of diagnosis, relative ease of use, and noninvasive nature of confocal
microscopy are clearly advantageous when compared to traditional laboratory techniques in
suspected cases of fungal keratitis. Hence the present study is being planned to determine if
confocal microscopy can provide a better and rapid method for diagnosing fungal keratitis
and thereby help in initiating appropriate and timely medical or surgical treatment.
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Review of Literature
Fungi are a significant cause of microbial keratitis in warm and temperate climates (42-
46.8%).14,15 The most common microorganisms that cause fungal keratitis in tropical climates
are filamentous fungi i.e. Fusarium solani and other Fusarium species and Aspergillus
species. Whereas in temperate climates yeast such as Candida is more prevalent. Risk factors
for fungal keratitis include prior ocular injury with vegetative matter, long-term steroid use,
Gel, CIBA Vision, Atlanta, GA) will be used as the coupling gel. In all cases, the scan will be
done before corneal scrapings are taken for microbiologic examination. The confocal
scanning will be performed in 4 quadrants and the images will be analyzed to identify cellular
details and evidence of fungal filaments. A diagnosis of either fungal keratitis or no
organisms will be documented. All cases will be subjected to corneal scrapings after confocal
microscopy. The microbiologist evaluating the smears and cultures will be masked from the
confocal findings.
All cases will undergo corneal scrapings from base and margins of the ulcer with a
disposable no. 15 Bard Parker blade under topical anaesthesia (4% xylocaine drops) under
aseptic conditions. The material will then be plated on two glass slides for Gram’s staining
and KOH wet mount and would be examined under microscope using high power
magnification. The material from the corneal scrapings will also be directly inoculated on
blood agar and brain heart infusion (BHI) agar and incubated at 25ºC and 37ºC. Two sets of
Sabouraud dextrose agar containing antibiotics but without actidione will also be inoculated
and incubated at 25ºC and 37ºC, separately. The corneal scrapings will be inoculated on agar
plates in a ‘C’ or ‘S’ -shaped streak. All media would be incubated for 4 weeks and would be
checked everyday during first week and twice a week during next three weeks.17 All the
smears and cultures will be evaluated by a microbiologist. Cases will be defined as all those
in which fungal filaments will be identified on any one or more smear examination methods
(KOH/Gram) or showed significant growth in culture.
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Statistical Analysis
The normality of the measurable data like age, visual acuity, size of ulcer, etc. will be assessed using Kolmogorov-Smirnov Test. Comparisons will be performed using Mann-Whitney U Test and Student t-test for normally distributed data and skewed data respectively. The association of various categorical/classified data will be analyzed using Chi-square Test or Fisher’s exact test whichever is applicable. The normally distributed data will be expressed as mean , standard deviation, range etc. whereas skewed data will be expressed as median and inter quartile range. The categorical data will be expressed as frequencies, percentages etc.
The data will also be depicted graphically using histograms, bar diagrams and pie charts.
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Ethical Considerations
All the procedures, which will be carried out in the present study, are routinely required
and performed in the Department of Ophthalmology, GMCH, Chandigarh. This study will
not render the subject to additional risk. It will be conducted on the ethical guidelines for
biomedical research on human subjects as given by Central Ethics Committee on Human
Research (CEHER ), ICMR, New Delhi,2006.and in the tenets of “Declaration of
Helsinki”,2008.
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Information to the patient
Purpose of Research
Fungal keratitis is an important cause of ocular morbidity in developing countries. The
purpose of this study is to see if confocal microscope can provide a means for early and
accurate diagnosis of fungal keratitis and thereby prevent delay in starting of appropriate
treatment.
Study Procedure
A detailed ophthalmological examination will be done including visual acuity and confocal
microscopy. Corneal scrapings will be sent for microbiological examination.
Confidentiality
Your medical records will be treated with utmost confidentiality and will be revealed only to
other doctors/scientists involved with this study. The results of this study would be published
in scientific journals/thesis but you will not be identified by name.
Your participation and rights
Your participation in the study is absolutely voluntary and you may withdraw from the study
any time without having to give reasons for the same. In any case, you will receive
appropriate treatment for your condition under routine services at GMCH, Chandigarh.
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Patient Informed Consent Form
Patient identification number for this trial : __________________
Title of project : Confocal microscopy versus microbiological evaluation in diagnosing suspected fungal keratitis
Name of Principal Investigator : _______________________ Tel. No. _______________
The contents of the information sheet dated ____________ (version) ________that was provided have been read carefully by me / explained in detail to me, in a language that I comprehend, and I have fully understood the contents. I confirm that I have had the opportunity to ask questions.
The nature and purpose of the study and its potential risks/ benefits and expected duration of the study, and other relevant details of the study have been explained to me in detail. I understand that my participation is voluntary and that I am free to withdraw at any time, without giving any reason, without my medical care or legal right being affected.
I understand that the information collected about me from my participation in this research and sections of any of my medical notes may be looked at by responsible individuals. I give permission to these individuals to have access to my records.
I agree to take part in the above study.
Date:
………………………………………………….. Place:
(Signature/Left Thumb impression)
Name of the participant : ____________________________
Son/Daughter/Spouse of : ____________________________
Any other complaints : _______________________________________________________History of any trauma : ______________________________History of contact lens use : _____________________________ History of any ocular surgery : ___________________________
2) PAIN : Grade 0 - No pain Grade 1 - Occassional mild pain Grade 2 - Constant mild pain Grade 3 - Moderate to severe pain Grade 4 - Constant severe pain
3) REDNESS : Grade 0 - No redness Grade 1 - redness in 1 quadrant Grade 2 - redness in 2 quadrants Grade 3 - redness in 4 quadrants Grade 4 - redness all around
4) PHOTOPHOBIA : Grade 0 - No photophobia Grade 1 - Photophobia only in bright light Grade 2 - Photophobia in day light Grade 3 - Photophobia in dim light Grade 4 - Photophobia without light
7) CORNEAL OEDEMA : Grade 0 - Only in ulcer area Grade 1 - Beyond ulcer area Grade 2 - Involving ½ of cornea Grade 3 - Involving whole of cornea but iris can be seen Grade 4 - Whole of cornea involved and no iris detail seen
10) DEPTH OF ULCER : Grade 0 - Epithelium normal Grade 1- Only Epithelium involved Grade 2 - <20% of stroma involved Grade 3 - 20-50 % of stroma involved Grade 4 - > 50 % of stroma involved
11) INFILTRATE : Grade 0 - No infiltrateGrade 1 - Only upto epithelial surfaceGrade 2 - Infiltate may be dense but superficial and limited to ulcer baseGrade 3 - Infiltrate dense extending upto mid stromaGrade 4 - Infiltrate dense extending deeper than mid stroma or into Sclera
12) HYPOPYON : Grade 0 - No hypopyon Grade 1 - Upto 1 mm Grade 2 - > 1-2 mm Grade 3 - > 2-3 mm Grade 4 - > 3mm