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Year : 2007  |  Volume : 55  |  Issue : 1  |  Page : 33-36

Evaluation of impression smear in the diagnosis of fungal keratitis

1 Department of Ophthalmology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Cytology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission28-Aug-2005
Date of Acceptance27-Jul-2006

Correspondence Address:
Reema Bansal
Department of Ophthalmology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0301-4738.29492

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Purpose: To evaluate and compare impression smear with the conventional mechanical corneal scraping by potassium hydroxide (KOH) method in the diagnosis of fungal keratitis.
Materials and Methods: Fifty consecutive cases of clinically suspected fungal keratitis were included. Under topical anesthesia, impression smear was obtained from the lesion, followed by the conventional mechanical scraping and both were transferred to sterile glass slides. The slides were examined under direct microscopy with KOH 10% wet mount. Impression smear KOH was compared with the scraping KOH and culture.
Results: The KOH smear was positive in 36 (72%) patients with impression smear technique and positive in 35 (70%) patients from corneal scrapings. Sensitivity of impression smear technique as compared to scraping was 97.14%, specificity 92.86%, positive predictive value 94.4%, negative predictive value 92.86%, false positive rate 5.56%, false negative rate 7.14% and accuracy was 94%. As compared to culture, the sensitivity was 100%, specificity 46.67%, positive predictive value 55.56%, negative predictive value 100%, false positive rate 53.33%, false negative rate 0% and accuracy was 68%.
Conclusion: Impression smear KOH examination is comparable to conventional mechanical corneal scraping KOH examination in making a tentative diagnosis of fungal keratitis and can be accurately relied upon for initiating anti-fungal therapy.

Keywords: Culture, diagnosis, fungal, impression, keratitis, potassium hydroxide, smear

How to cite this article:
Jain AK, Bansal R, Felcida V, Rajwanshi A. Evaluation of impression smear in the diagnosis of fungal keratitis. Indian J Ophthalmol 2007;55:33-6

How to cite this URL:
Jain AK, Bansal R, Felcida V, Rajwanshi A. Evaluation of impression smear in the diagnosis of fungal keratitis. Indian J Ophthalmol [serial online] 2007 [cited 2019 Nov 12];55:33-6. Available from:

Impression smear KOH and culture

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Impression smear KOH and culture

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KOH preparation of impression smear and
conventional scraping

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KOH preparation of impression smear and
conventional scraping

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Features of ulcer

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Features of ulcer

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In India, fungal keratitis results in a significant degree of ocular morbidity.[1] Although the specificity of cultures makes them indispensable for the confirmation of a diagnosis, direct smear examination of the specimen is of immense help in early diagnosis and treatment.[2] Potassium hydroxide (KOH) 10% wet-mount is one of the oldest and principle modalities for demonstration of fungi in corneal scrapings.[3],[4] There are many studies establishing the efficacy of KOH smear of mechanical scraping over culture as gold standard for the diagnosis of fungal keratitis.[5],[6],[7] Chowdhary and Singh have highlighted the sensitivity of KOH examination as a simple and rapid means of making a tentative diagnosis of fungal keratitis.[8] Impression cytology was first introduced in ophthalmology by Egbert et al . who reported the possibility of removing the cells from the surface of the epithelium by pressing a cellulose acetate filter paper on the bulbar conjunctiva.[9] Impression cytology has been used to diagnose dry eye conditions such as vitamin A deficiency,[10] contact lens wear,[11] chemical/thermal burns, limbitis,[12] herpes simplex keratitis[13] and acanthamoeba keratitis.[14] To the best of our knowledge, there is no study reported regarding the use of impression smear in the diagnosis of fungal keratitis. The present study was undertaken to evaluate the accuracy of impression smear KOH examination in the diagnosis of fungal keratitis and to compare it with the conventional KOH method following scraping.

  Materials and Methods Top

Our study included 50 consecutive patients of clinically suspected fungal keratitis who visited the outpatient ophthalmic department of the Post Graduate Institute of Medical Education and Research, Chandigarh, from June 2001 to December 2002.

A detailed history was taken and a thorough slit-lamp examination was done for all the patients. The size, depth and margins of the infiltrate were noted. The presence of satellite lesions and hypopyon height was noted. The presence of an epithelial defect and pigmentation on the surface was noted.

Inclusion criteria

Clinically suspected cases of fungal keratitis, on the basis of at least two of the following criteria:

a) Patients with a history of trauma to the eye with vegetable or organic matter

b) Patients with clinical signs more than symptoms

c) Ulcers with irregular and feathery margins

d) Ulcers with satellite lesions

e) Presence of an endothelial plaque, fibrinoid aqueous reaction and hypopyon formation

f) Dry-looking ulcers

g) Pigmented ulcers

Exclusion criteria

  1. Patients suffering from bacterial keratitis
  2. Patients already on treatment with antifungal drugs.

Formal informed consent was obtained from all patients. Xylocaine (4%) eye drops were instilled in the conjunctival sac. A 3 x 5 mm precut and autoclaved strip of cellulose acetate filter paper (GSWP 02500 Millipore, 0.22 m pore size) was applied to the lesion [Figure - 1]. The filter paper strip was gently pressed for five seconds with a blunt end of a glass rod [Figure - 2]. Using a blunt forceps the filter paper was gently peeled off and immediately transferred on the surface of an autoclaved glass slide, leaving the impression on the slide. One drop of 10% KOH was added on the slide, a cover slip applied and then the impression was observed under a standard light microscope [Figure - 3].

Corneal scrapings were taken from the base and edge of the ulcers aseptically, with a sterile Bard-Parker blade (No 15), using local anesthetic solution (4% xylocaine) and were seen under direct microscopy with 10% KOH for evidence of hyphae, pseudohyphae and yeast cells. The scraped specimen was sent for inoculation in Sabouraud's dextrose agar medium. All the corneal scrapings were also sent for routine Gram's stain and bacterial culture in blood agar, chocolate agar and Mac Conkey agar. Cases were diagnosed to be having fungal keratitis if any one of the following was present:

  1. KOH smear was positive
  2. Fungal culture was positive
  3. Patients responded to anti-fungal treatment

In the statistical analysis, the sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of culture and KOH staining of impression smear were calculated.

  Results Top

Our study included 50 patients. Thirty-eight (76%) were males and 12 (24%) were females. The patients ranged in age from 15 to 78 years. Twelve patients gave a definite history of trauma with vegetative matter and 16 patients gave history of fall of dust particle in the eye. The various features of the ulcers have been highlighted in [Table - 1]. Forty-two patients belonged to a rural background and eight were from an urban background. In six of our patients, impression smear proved to be a therapeutic adjunct as the lesion came out in toto when the impression smear was taken.

In this series, KOH smear from impression smear technique was positive in 36 (72%) patients. The KOH smear from direct corneal scrapings was positive in 35 patients (70%). Culture reports were positive in only 20 eyes (40%). Thirteen out of 50 cases were negative for both KOH (impression smear or scraping) and fungal culture [Table - 2][Table - 3], as well as Gram stain and bacterial culture. Eleven of these eyes responded satisfactorily to anti-fungal therapy. The remaining two eyes, however, developed corneal melting and required penetrating keratoplasty. The fungal hyphae are known to penetrate deep in the corneal stroma where the organisms are inaccessible to the usual diagnostic and therapeutic measures. The histopathology report of the corneal buttons of these two eyes revealed fungal hyphae along with necrosis and inflammation.

The sensitivity of the impression smear technique in comparison to conventional mechanical corneal scraping was 97.14%. The specificity of the technique was 92.86%. The positive predictive value of impression smear was 94.44%. The negative predictive value was 92.86%. The false positive rate was 7.14%. The false negative rate was 2.86% and accuracy of the technique was 94%.

The sensitivity of KOH preparation of the impression smear technique in comparison to culture was 100%. The specificity was 46.67%. The positive predictive value of impression smear was 55.56%. The negative predictive value was 100%. The false positive rate was 53.33%. The false negative rate was 0% and accuracy of the technique was 68%.

  Discussion Top

A KOH wet mount preparation of the corneal scrapings is a simple and sensitive method for diagnosis.[15] In a study by Arora et al . impression debridement of corneal lesions has been done for various types of corneal ulcers, removal of superficial foreign bodies, filaments of filamentary keratitis and superficial infiltration around cataract-related exposed corneo-scleral sutures.[16] In a prospective study of 171 cases of clinically suspected fungal corneal ulcers, although cultures were positive in 88 eyes (51.46%), the fungus could be demonstrated by KOH preparation in 94.3% (83 of 88) of the culture-proved cases and 93.6% (160 of 171) of the overall eyes.[6] Gopinathan et al . in their large series (1354 eyes) of fungal keratitis, have reported the diagnostic utility of smears of corneal scrapings using KOH preparation, Calcofluor White (CFW), Gram and Giemsa-stains.[17] The smears established the fungal cause in 95.4% (1277 out of 1354) eyes. The KOH preparation alone revealed fungus in 91.0% (1219) eyes. Sharma et al .[7] in their series of 114 patients of early keratitis and 363 cases of late keratitis, found that in early keratitis, KOH with CFW stain had a sensitivity of 61.1% and a specificity of 99.0%. The KOH with CFW stain in advanced keratitis had a sensitivity of 87.1% and a specificity of 83.7%.

These studies[6],[7],[17] clearly establish the superiority of the KOH smear of direct corneal scrapings over culture as the gold standard for the diagnosis of fungal keratitis though culture is required for species identification. Sharma et al . have recommended resorting to anti-fungal therapy whenever a KOH+CFW-stained smear is positive for fungus because they believed that the gold standard of culture has its own limitations and a fungal element is unlikely to be misinterpreted.[7] The false positives have been explained by earlier observations that all samples that show fungal filaments in smears may not show growth in culture.[5],[6] Corneal cultures and sensitivity testing provide the basis for understanding the epidemiology of ulcerative keratitis. E test is a simple and reproducible method of determining drug susceptibilities of organisms causing fungal keratitis.[18] But sensitivity testing for fungi is not routinely done due to its availability only in research settings.

In the light of the above-specified studies, the results of impression smear with KOH preparation were compared with corneal scrapings and culture in the present study. Keeping KOH positivity from mechanical corneal scraping as the gold standard, the sensitivity of impression smear technique was 97.14%, the specificity was 92.86% and the accuracy of the technique was 94%. When compared with culture, the sensitivity of impression smear KOH was 100%, the specificity was 46.67% and accuracy was 68%. The high false positive rate of 53.33% is due to nongrowth of the fungus on culture, as seen in earlier studies also.[5],[6],[7]

To the best of our knowledge, the use of impression cytology in fungal keratitis has not been evaluated. In the present study, an attempt has been made to establish the use of impression technique in the diagnosis of fungal keratitis. It is a safe, simple, repeatable and atraumatic procedure. It gives quick results and does not require expensive equipment like a slit-lamp or an operating microscope. It is helpful in obtaining specimen materials from small lesions, which is otherwise difficult by the conventional techniques of mechanical scraping. With mechanical scraping, the infected cells as well as some surrounding normal cells are inadvertently removed and there is risk of corneal perforation, especially in impending perforations. There is risk of injury to the basement membrane, Bowman's layer and superficial stroma[7] in superficial ulcers and risk of precipitation of a perforation in impending perforation.

The impression smear technique relies on cellular adherence to the filter paper rather than mechanical abrasion of the subepithelial areas. Therefore there is little chance of damage to these structures or to the adjacent epithelium. When the filter paper is applied and peeled off from the normal cornea, the cohesive forces of epithelial cells to the underlying structures counteracts the adhesive force to the filter paper, which results in removing only the most superficial epithelial layers. In contrast, when the filter paper is applied to the corneal lesion, only the affected cells are removed.[7] The cohesive force of degenerated cells to the underlying structures is reduced. It has a valuable place as an adjunct therapy because it removes some lesions in toto, removes necrotic material, inflammatory cells, organisms and degenerating epithelium from most corneal ulcers.[7] For conventional techniques greater magnification is needed to stay within the confines of the lesion and to avoid areas of imminent perforation. The material obtained may not spread well on slides and cells may get distorted and crushed with loss of spatial relation. With impression smear technique all of these disadvantages are overcome, especially if the sampling is done in community practice setting or in cases of impending perforation. Nevertheless, mechanical debridement should still be preferred in all cases of fungal keratitis in the hands of an ophthalmologist as it maximally reduces fungal load and enhances drug penetration

  Conclusion Top

We conclude that impression smear and mechanical scraping are equally sensitive and specific in making tentative diagnosis of fungal keratitis. However, further studies are needed to substantiate the safety of this newer procedure.

  References Top

Srinivasan M, Gonzales CA, George C, Cevallos V, Mascarenhas JM, Asokan B, et al . Epidemiology and etiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol 1997;81:965-71.  Back to cited text no. 1
Rao NA. A laboratory approach to rapid diagnosis of ocular infections and prospects for the future. Am J Ophthalmol 1989;107:283-91.   Back to cited text no. 2
Sandhu DK, Randhawa IS, Singh D. The correlation between environmental and ocular fungi. Indian J Ophthalmol 1981;29:177-82.  Back to cited text no. 3
Laverde S, Moncada LH, Restrepo A, Vera CL. Mycotic keratitis: 5 cases caused by unusual fungi. Sabouraudia 1973;11:119-23.  Back to cited text no. 4
Sharma S, Silverberg M, Mehta P, Gopinathan U, Agrawal V, Naduvilath TJ. Early diagnosis of mycotic keratitis: Predictive value of potassium hydroxide preparation. Indian J Ophthalmol 1998;46:31-5.  Back to cited text no. 5
Vajpayee RB, Angra SK, Sandramouli S, Honavar SG, Chhabra VK. Laboratory diagnosis of keratomycosis: Comparative evaluation of direct microscopy and culture results. Ann Ophthalmol 1993;25:68-71.  Back to cited text no. 6
Sharma S, Garg P, Gopinathan U, Athmanathan S, Garg P, Rao GN. Evaluation of corneal scraping smear methods in the diagnosis of bacterial and fungal keratitis: A survey of eight years of laboratory experience. Cornea 2002;21:643-7.  Back to cited text no. 7
Chowdhary A, Singh K. Spectrum of fungal keratitis in North India. Cornea 2005;24:8-15.  Back to cited text no. 8
Egbert PR, Lauber S, Maurice DM. A simple conjunctival biopsy. Am J Ophthalmol 1977;84:798-801.  Back to cited text no. 9
Natadisastra G, Wittpenn JR, West KP Jr, Muhilal, Sommer A. Impression cytology for detection of vitamin A deficiency. Arch Ophthalmol 1987;105:1224-8.  Back to cited text no. 10
Saini JS, Rajwanshi A, Dhar S. Clinicopathological correlation of hard contact lens related changes in tarsal conjunctiva by impression cytology. Acta Ophthalmol (Copenh) 1990;68:65-70.  Back to cited text no. 11
Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:1476-85.  Back to cited text no. 12
Nakagawa H, Uchida Y, Takamura E, Nakagawa Y, Araki H, Watanabe M. Diagnostic impression cytology for herpes simplex keratitis. Jpn J Ophthalmol 1993;37:505-13.  Back to cited text no. 13
Florakis GJ, Folberg R, Krachmer JH, Tse DT, Roussel TJ, Vrabec MP. Elevated corneal epithelial lines in Acanthamoeba keratitis. Arch Ophthalmol 1988;106:1202-6.  Back to cited text no. 14
Bharathi MJ, Ramakrishnan R, Vasu S, Meenakshi R, Palaniappan R. Epidemiological characteristics and laboratory diagnosis of fungal keratitis. A three-year study. Indian J Ophthalmol 2003;51:315-21.  Back to cited text no. 15
Arora I, Singhvi S. Impression debridement of corneal lesions. Ophthalmology 1994;101:1935-40.  Back to cited text no. 16
Gopinathan U, Garg P, Fernandez M, Sharma S, Athmanathan S, Rao GN. The epidemiological features and laboratory results of fungal keratitis: A 10-year review at a referral eye care center in South India. Cornea 2002;21:555-9.  Back to cited text no. 17
Inoue T, Inoue Y, Asari S, Sunada A, Inoue Y, Yamamoto S, et al . Utility of Etest in choosing appropriate agents to treat fungal keratitis. Cornea 2001;20:607-9.  Back to cited text no. 18


  [Figure - 1], [Figure - 2], [Figure - 3]

  [Table - 1], [Table - 2], [Table - 3]

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