Fungal keratitis in Patients with cornealulcer in Sari, Northern Iran
http://www.100md.com
《伊朗医学》
Department of Medical Mycology and Parasitology, Department of Ophthalmology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Background: Fungal keratitis is a suppurative, ulcerative, and sight-threatening infection of the cornea that sometimes leads to loss of the eye. The objectives of this study were to improve facilities for laboratory diagnosis, to determine the predominant causative microorganisms, and to identify the predisposing factors of mycotic keratitis patients.
Methods: A prospective study of corneal ulcer was conducted in Sari between May 2004 and March 2005. Patients who presented with clinically suspected corneal ulcer to the Ophthalmology Department of Bou-Ali Sina University Hospital in Sari were included in this study. Each patient was examined with slit lamp. Data were collected by examining and questioning the patients. Using standard techniques, corneal scraping was performed by an ophthalmologist. The specimens collected were then smeared on two slides, which were stained with Gram stain (for bacterial keratitis) and 10% potassium hydroxide with or without Calcofluor white stain (for fungal keratitis), and studied under light microscope. The specimens were also inoculated directly on blood agar, Sabouraud dextrose agar, and potato dextrose agar in C-shaped streaks.
Results: A total of 22 patients met the inclusion criteria of this study, among whom 10 (45.5%) were females and 12 (54.5%) were males. The mean ± SD age of patients was 61.5 ± 17.7 (range: 15 – 83) years. In direct microscopy, branching, and septate hyphae were identified in 7 (31.8%) patients. Two (28.6%) fungi (Aspergillus fumigatus and Fusarium spp.) were isolated. Five (31.8%) patients with fungal keratitis were males and 2 (28.6%) were females. The mean ± SD age of patients with fungal keratitis was 60.4 ± 12.1 (range: 39 – 73) years. Three (42.85%) patients with fungal keratitis were farmers. The mean interval between the onset of symptom and diagnosis was 26.4 (range: 1 – 93) days. Trauma with plant debris and straws were noted in two (28.6%) patients with fungal keratitis. Five (71.4%) patients received topical antibiotics. Analyses, using potassium hydroxide with or without Calcofluor white as the gold-standard test, revealed a sensitivity of 71.4% for potassium hydroxide, and 42.9% for Gram stain.
Conclusion: Infections of the cornea due to filamentous fungi are frequent causes of corneal damage and should always be kept in mind. The direct microscopy method is an essential tool in the diagnosis of fungal keratitis. Therefore, wet mount preparation with potassium hydroxide with or without Calcofluor white or only KOH can be relied upon as the single most important screening test for rapid diagnosis of fungal corneal ulcer.
Archives of Iranian Medicine, Volume 9, Number 3, 2006: 222 – 227.
Keywords: Corneal ulceration;diagnosis;fungal keratitis;keratomycosis
Introduction
Fungal keratitis is a suppurative, ulcerative, and sight-threatening infection of the cornea that leads sometimes leads to loss of the eye. Worldwide, the reported incidence of mycotic keratitis is 17% to 36%.1 – 3 In India, it is 44% to 47%.4 – 7 Despite advances in diagnosis and medical treatment of keratomycosis, 15% to 27% of patients require surgical intervention such as keratoplasty, enucleation, or evisceration because of either failed medical treatment or advanced disease at presentation.1 – 11
In general, it has been indicated that although corneal infections have worldwide distribution, the incidence of fungal infections is higher in tropical and semitropical areas and is much more frequent in developing countries. It has also been mentioned that these infections follow trauma, especially with plant debris.1 – 11 The distribution of fungi is not only affected geographically, but also changes with time. It is, therefore, very important to recognize the prevalence and etiology of corneal ulcer.
The following study was conducted at the Ophthalmology Department of Bou-Ali Sina Hospital of Sari, in Mazandaran Province, North of Iran. Mazandaran is located near the Caspian sea and the majority of people are engaged in farming. The objectives of this study were to improve facilities for laboratory diagnosis, to determine the predominant causative microorganisms, and to identify the predisposing factors of mycotic keratitis patients.
Patients and Methods
Patients
A prospective study of corneal ulcer was conducted in Sari between May 2004 and March 2005. Patients who presented with clinically suspected corneal ulcer to the Ophthalmology Department of Bou-Ali Sina University Hospital in Sari, were included in this study. Corneal ulceration was defined as the loss of corneal epithelium with underlying stromal infiltrate and suppuration as-sociated with signs of inflammation, with or without hypopyon.6 Written informed consent was obtained from all patients.
Clinical examination and laboratory investigations
Each patient was examined by slit lamp. The location and size of infiltrate were recorded. Data were then collected by examination and questioning the patients. Using standard techniques,12 corneal scraping was performed by an ophthalmologist, with a sterile surgical blade number 15, or a sterile 21 gauge needle, following the instillation of a local anesthetic (tetracaine hydrochloride 0.5%). Corneal specimen obtained from scraping the ulcer was smeared on two slides, which were stained with Gram stain (for bacterial keratitis) and 10% potassium hydroxide (KOH) with or without Calcofluor white (KOH+CFW) stain (for fungal keratitis), and studied under light microscope. The specimens were also inoculated directly on blood agar, Sabouraud dextrose agar, and potato dextrose agar in C-shaped streaks. Blood agar plates were incubated at 37oC. Sabouraud dextrose agar and potato dextrose agar were incubated at 25oC. The culture was considered significant and reported if the smears demonstrated morphologically similar organism, and/or if the same organism grew in more than one culture media, and/or if there was growth on at least two streaks.
Results
Demographic
A total of 22 patients met the inclusion criteria of this study, of whom 10 (45.5%) were females and 12 (54.5%) were males. The mean ± SD age of patients was 61.5 ± 17.7 (range: 15 – 83) years. Of 22 patients with corneal ulcer surveyed during the period of May 2004 to March 2005 (12 months), fungal keratitis was identified as the principal etiologic agent of corneal ulceration in 7 (31.8%) patients (5 males and 2 females) (Table 1). The mean ± SD age of patients with fungal keratitis was 60.4 ± 12.1 (range: 39 – 73) years. Three (42.9%) patients with fungal keratitis were farmers; one (14.3%) animal husbandman, one (14.3%) laborer, and 2 (28.6%) were housewives. All patients with fungal keratitis lived in rural areas. The mean interval between the onset of symptom and diagnosis was 26.4 (range: 1 – 93) days.
Predisposing factors
Trauma with plant debris and straws were noted in two patients with fungal keratitis. Five patients received topical antibiotics. One patient had diabetes and another had local corneal disease (persistent corneal defect and stromal ulceration). No patient with fungal keratitis had a history of ocular surgery or use of contact lens and topical corticosteroid.
Clinical features
All patients had symptoms of redness and watering. Ocular pain and photophobia were found in 85.7% of patients. Seventy-one point four percent of patients had foreign body sensation; 57.1% had decreased vision. The signs on slit lamp examination are shown in Table 2.
The distinct characteristics of patients with fungal keratitis found in our study—male preponderance (71.4%), outdoor occupa-tion (71.5%), mean age of 60.4 years, and living in rural regions (100%)—are consistent with observations made in the majority of other studies.12 – 14 The incidence is reported to be higher in tropical countries. Apart from a humid atmosphere, agricultural-based live-li-hood in this region and outdoor occupations also make the population more vulnerable to fungal infections.
The cornea may present suppurative keratitis due to viruses, bacteria, parasites, and fungi. Normal conjunctival microbiota usually does not consist of fungi. With some special life or work situation, however, individuals are exposed to corneal trauma and thus become more prone to develop mycotic infection. In our study, 71.4% of patients with fungal keratitis received topical antibiotics before seeking treatment from us. The great number of patients with fungal keratitis using topical antibiotics, who came to the university services, is explained by the fact that this service is a tertiary referral center to which more severe cases that did not respond to initially-applied therapy are referred. Since one of the differential diagnoses of suppurative keratitis is bacterial keratitis, the initial therapy with antibiotics is justified in services without a supporting laboratory. In one report, 56% of patients with fungal keratitis and in another, 55.3% of patients with fungal keratitis had received prior topical antibiotics as a major risk factor.12, 15 However, the various reports have not clearly identified the importance of prior usage of topical antibiotics as a predisposing factor.6, 11, 16
In this study, 28.6% of patients with fungal keratitis had corneal trauma, which is lower than that reported for fungal keratitis in general. Previous corneal trauma is identified as a major predisposing factor among patients with filamentous fungal keratitis.6, 12, 15, 17 A frequency of 33% to 100% has been described in the literature for mycotic keratitis in patients with corneal trauma by things having organic material or foreign body.1, 18, 19 In some other reports, 8.3%17 to 17.6%20 of patients with fungal keratitis had corneal trauma, which is lower than our report. The fewer number of patients with fungal keratitis and corneal trauma could be explained by the fact that trauma might be insensible or as a result of delay existing between the occurrence of trauma and its diagnosis, causing them difficult to recall.
In this study, the most common signs on slit lamp examination were epithelial defect (57.1%) and suppuration (57.1%)—which are nonspecific to fungal keratitis—and feathery pattern which is specific, presented in only one patient. None of our patients had hypopyon on slit lamp examination, whereas Javadi et al noted hypopyon in 52% of their patients with fungal keratitis.21 Rosa et al noted irregular feathery margins (62%), a dry rough texture (47%), and satellite lesions (41%) in their patients.1 During the first week, the signs such as hypopyon and endothelial plaque are not present. In our study, five of seven patients with fungal etiology referred to us between one and ten days of onset of the symptoms. During this phase, specific clinical features were not established. Therefore, the clinical picture may not always be sufficient to establish a diagnosis of fungal keratitis. The importance of establishing a defini-tive diagnosis by microbiologic investigations is unavoidable in such cases.
Aspergillus and Fusarium species, as were also isolated from our patients, are the most common isolated organisms from patients with fungal keratitis in the tropic. Other studies in Iran have reported Aspergillus and Fusarium to be the most common fungal pathogens.21 – 23 Fusarium species have also been found to be the principal fungal pathogen in Ghana, South India, Florida, Paraguay, Nigeria, Tanzania, Hong Kong, China, and Singapore. Aspergillus species predominate in northern India, Nepal, and Bangladesh.24 This phenomenon may be explained by differences in climate and natural environment. Seasonal variations have been observed to be important in the incidence of fungal keratitis and in the predominant genera of fungi isolated from such patients. Such variations have been linked to environmental factors, such as humidity, rainfall, wind, and also to the harvest.1, 19, 25, 26 Environmental conditions including temperature, annual rainfall, windy seasons, and the harvest period have a significant role in increasing the incidence.
There are different diagnostic laboratory methods for diagnosis of fungal keratitis, including direct examination and culture. In the current study, the presence of fungi in corneal scrapings was determined by KOH+CFW stain in 7, KOH stain in 5 (71.4%), and Gram stain in 3 (42.9%) scrapings from patients with keratitis. The smear findings of KOH+CFW as the gold-standard method were compared with the KOH mount, Gram stain, and culture results to calculate the specificity, sensitivity, and predictive values of the other techniques used. Rapid detection of fungal elements in the corneal scraping is of utmost importance in the management of fungal keratitis, especially because the organisms are not amenable to treatment with broad-spectrum, commercially available antibiotics. Fungi can be detected in corneal scraping by a variety of methods including KOH wet preparation,27 acridine orange staining,28 and lactophenol blue staining.29 The sensitivity of these methods varies from 76% to 80%. CFW, with or without KOH, is also a well-established stain for observation of fungal elements in clinical samples with a high sensitivity.27, 30, 31 Sharma et al32 confirmed the superiority of KOH+CFW stain in detection of fungal elements in corneal scraping. It is not only highly specific and sensitive, but also has a high NPV and PPV for both early and advanced keratitis. Since they had seen some false positive results in advanced keratitis, they recom-mended resorting to antifungal therapy whenever a KOH+CFW stain smear is positive for fungus. This is because they believed that the gold-standard of culture has its own limitations and a fungal element is unlikely to be misinterpreted. The false positive results are explained by some observations27, 33 that all samples that show fungal elements in smear may not show growth in the culture. Chander et al30 in their evaluation of CFW staining for diagnosis of fungal corneal ulcer, confirmed the superiority of KOH+CFW in comparison with KOH and culture. In another study,34 KOH wet mount showed fungal elements in 68% of patients with corneal ulcers. Gram stain of corneal scraping did not provide much help. In our series, fungal culture was positive in 26.4% of cases. Vajpayee et al33 noted that cultures were positive in 51.46% of cases, although, the fungus was demonstrated by the KOH preparation in 94.3% of the culture-proven cases. In our study, the culture-positive cases determined in whom the interval between the onset of disease and diagnosis was more than a week and in direct microscopy in which too many fungal elements were seen. The culture-positive patients were in the advance phase of keratitis, whereas the culture-negative cases were in the early phase of keratitis. Having no yield of positive culture could be explained by an insufficient corneal scraping of sample inoculated to culture media and less fungal load in the early stages of keratitis. To the best of our knowledge, the clinical picture may not always be sufficient to establish a diagnosis for fungal keratitis. The present study also showed the effectiveness of KOH+CFW as a rapid and highly reliable method in confirming the diagnosis of fungal keratitis, particularly when empirical antifungal therapy is not justified. Therefore, if KOH+CFW stained smears are negative for fungal filaments and acanthamoeba cysts, the patient must be treated with a broad-spectrum antibiotic, even if the Gram stain is negative for bacteria.
In eras where fungal keratitis is prevalent, a simple, rapid, and reliable KOH+CFW smear exa-mi-nation of corneal scraping early during the disease could result in a significant difference in the course of the disease by helping in early, specific therapy. KOH+CFW stain was significantly more sensitive than KOH wet mount in demonstrating fungal pathogens. However, if UV microscope and CFW are not available, KOH preparation is recommended.
Acknowledgment
We are grateful to the Vice-Chancellor of Research of Mazandaran University of Medical Sciences for financial support. We also thank Dr. A.R. Khalilian for statistical analyses.
References
1 Rosa RH Jr, Miller D, Alfonso EC. The changing spectrum of fungal keratitis in South Florida. Ophthalmology. 1994; 101: 1005 – 1013.
2 Upadhyay MP, Karmacharya PC, Koirala S, Tuladhar NR, Bryan LE, Smolin G, et al. Epidemiologic characteristics, predisposing factors, and etiologic diagnosis of corneal ulceration in Nepal. Am J Ophthalmol. 1991; 111: 92 – 99.
3 Katz NN, Wadud SA, Ayazuddin M. Corneal ulcer disease in Bangladesh. Ann Ophthalmol. 1983; 15: 834 – 836.
4 Dunlop AA, Wright ED, Howlader SA, Nazrul I, Husain R, McClellan K, e al. Suppurative corneal ulceration in Bangladesh. A study of 142 cases examining the microbiological diagnosis, clinical diagnosis, and epidemiological features of bacterial and fungal keratitis. Aust N Z J Ophthalmol. 1994; 22: 105 – 110.
5 Polack FM, Kaufman HE, Newmark E. Keratomycosis. Medical and surgical treatment. Arch Ophthalmol. 1971; 85: 410 – 416.
6 Srinivasan M, Gonzales CA, George C, Cevallos V, Mascarenhas JM, Asokan B, et al. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol. 1997; 81: 965 – 971.
7 Thomas PA. Mycotic keratitis—an underestimated mycosis. J Med Vet Mycol. 1994; 32: 235 – 256.
8 Thomas PA, Geraldine P. Fungal keratitis due to Fusarium and other fungi. J Mycol Med. 1992; 2: 121 – 131.
9 Sharma S, Srinivasan M, George C. The current status of Fusarium species in mycotic keratitis in South India. Indian J Med Microbiol. 1993; 11: 140 – 147.
10 Reddy PS, Satyendran OM, Satapathy M, Kumar HV, Reddy PR. Mycotic keratitis. Indian J Ophthalmol. 1972; 20: 101 – 108.
11 Mahajan VM. Ulcerative keratitis: an analysis of laboratory data in 674 cases. J Ocul Ther Surg. 1985; 4: 138 – 141.
12 Garg P, Gopinathan U, Choudhary K, Rao GN. Keratomycosis: clinical and microbiologic experience with dematiaceous fungi. Ophthalmology. 2000; 107: 574 – 580.
13 Thomas PA. Current perspectives on ophthalmic mycoses. Clin Microbiol Rev. 2003; 16: 730 – 797.
14 Wong TY, Fong KS, Tan DT. Clinical and microbial spectrum of fungal keratitis in Singapore: a 5-year retrospective study. Int Ophthalmol. 1997; 21: 127 – 130.
15 de Andrade AJM, Vieira LA, Hofling-Lima AL, Zorat Yu MC, Fischman Gompertz O, de Freitas D, et al. Laboratorial analysis of fungal keratitis in a university service. Brazilian Arch Ophthalmologia; 2004, 63. Available from: URL: http://www.abonet.com.br/abo/ abo63110.htm (Access date: 15/5/ 2004).
16 Kumari N, Xess A, Shahi SK. A study of keratomycosis: our experience. Indian J Pathol Microbiol. 2002; 45: 299 – 302.
17 Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea. 2000; 19: 307 – 312.
18 Alfonso EC, Rosa RH. Fungal keratitis. In: Krachmer JH, Mannis MJ, Holland EJ, eds. Cornea and External Disease: Clinical Diagnosis and Management. St. Louis: Mosby; 1997: 1253 – 1266.
19 Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol. 1980; 90: 38 – 47.
20 Kunimoto DY, Sharma S, Gary P, Gopinathan U, Miller D, Rao G. Corneal ulceration in the eldery in Hyderabad, South India. Br J Ophthalmol. 2000; 84: 54 – 59.
21 Javadi MA, Hemati R, Mohammadi MM, Farsi A, Karimian F, Einolahi B, et al. Causes of fungal keratitis and its management: review of 23 cases from Labbafinejad Medical Center (LMC) [in Persian]. Bina. 1996; 2: 38 – 54.
22 Mirshahi A, Ojaghi H, Aghashahi D, Jabarvand M. Fungal keratitis in patients at Farabi Hospital, Tehran (1998 – 1999) [in Persian]. Bina. 1999; 5(2): 135 – 143.
23 Berenji F, Elahi SR, Fata AM, Khakshour H, Darakhshan A. Fungal keratitis in patients at Imam Reza Hospital, Mashhad (1982 – 2001) [in Persian]. Med J Mashhad Univ Med Sci. 2003; 45: 49 – 54.
24 Leck AK, Thomas PA, Hagan M, Kaliamurthy J, Ackuaku E, John M, et al. Aetiology of suppurative corneal ulcers in Ghana and South India, and epidemiology of fungal keratitis. Br J Ophthalmol. 2002; 86: 1211 – 1215.
25 Gugnani HC, Gupta S, Talwar RS. Role of opportunistic fungi in ocular infections in Nigeria. Mycopathologia. 1978; 65: 155 – 166.
26 Thomas PA. Fungi in Keratitis: Detection, Susceptibility Patterns, and Pathogenic Mechanisms [PhD thesis]. Tiruchirappalli; India: Bharathidasan University; 1990.
27 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 – 35.
28 Kanungo R, Srinivasan R, Rao RS. Acridine orange stainig in early diagnosis of mycotic keratitis. Acta Ophtalmol. 1991; 69: 750 – 753.
29 Thomas PA, Kuriakos T, Kirupashanker MP, Maharajan VS. Use of lactophenol cotton blue mounts of corneal scrapings as an aid to the diagnosis of mycotic keratitis. Diagn Microbiol Infect Dis. 1991; 14: 219 – 224.
30 Chander J, Chakrabarti A, Sharma A, Saini JS, Panigarhi D. Evaluation of Calcofluor staining in the diagnosis of fungal corneal ulcer. Mycoses. 1993; 36: 243 – 245.
31 Arffa RC, Avni I, Ishibashi Y, Robin J, Kaufman HE. Calcofluor and ink-potassium hydroxide preparations for identifying fungi. Am J Ophthalmol. 1985; 100: 719 – 723.
32 Sharma S, Kunimoto DY, Gopinathan U, Athmanathan S, Grag P, Rao GN. Evaluation of corneal scraping smear examination methods in the diagnosis of bacterial and fungal keratitis. Cornea. 2002; 21: 643 – 647.
33 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.
34 Nag (Roy) D, Chatterjee S, Chatterjee S, Khan M. Role of potassium hydroxide mount in rapid diagnosis of fungal corneal ulcers. JIMA. 2002; 100: 1 – 4.
35 Behboodi H, Mohammadi MJ, Forghanparast K. Epidemiology of infectious keratitis inpatients of Tootoonkaran Hospital, Rasht (1997 – 1998) [in Persian]. Bina. 2001; 7: 3 – 9.
36 Shokohi T. Mycotic Keratitis (case report). J Med Facul Guilan Univ Med Sci. 1999; 13: 61 – 66.(Tahereh Shokohi PhD, Kium)
Background: Fungal keratitis is a suppurative, ulcerative, and sight-threatening infection of the cornea that sometimes leads to loss of the eye. The objectives of this study were to improve facilities for laboratory diagnosis, to determine the predominant causative microorganisms, and to identify the predisposing factors of mycotic keratitis patients.
Methods: A prospective study of corneal ulcer was conducted in Sari between May 2004 and March 2005. Patients who presented with clinically suspected corneal ulcer to the Ophthalmology Department of Bou-Ali Sina University Hospital in Sari were included in this study. Each patient was examined with slit lamp. Data were collected by examining and questioning the patients. Using standard techniques, corneal scraping was performed by an ophthalmologist. The specimens collected were then smeared on two slides, which were stained with Gram stain (for bacterial keratitis) and 10% potassium hydroxide with or without Calcofluor white stain (for fungal keratitis), and studied under light microscope. The specimens were also inoculated directly on blood agar, Sabouraud dextrose agar, and potato dextrose agar in C-shaped streaks.
Results: A total of 22 patients met the inclusion criteria of this study, among whom 10 (45.5%) were females and 12 (54.5%) were males. The mean ± SD age of patients was 61.5 ± 17.7 (range: 15 – 83) years. In direct microscopy, branching, and septate hyphae were identified in 7 (31.8%) patients. Two (28.6%) fungi (Aspergillus fumigatus and Fusarium spp.) were isolated. Five (31.8%) patients with fungal keratitis were males and 2 (28.6%) were females. The mean ± SD age of patients with fungal keratitis was 60.4 ± 12.1 (range: 39 – 73) years. Three (42.85%) patients with fungal keratitis were farmers. The mean interval between the onset of symptom and diagnosis was 26.4 (range: 1 – 93) days. Trauma with plant debris and straws were noted in two (28.6%) patients with fungal keratitis. Five (71.4%) patients received topical antibiotics. Analyses, using potassium hydroxide with or without Calcofluor white as the gold-standard test, revealed a sensitivity of 71.4% for potassium hydroxide, and 42.9% for Gram stain.
Conclusion: Infections of the cornea due to filamentous fungi are frequent causes of corneal damage and should always be kept in mind. The direct microscopy method is an essential tool in the diagnosis of fungal keratitis. Therefore, wet mount preparation with potassium hydroxide with or without Calcofluor white or only KOH can be relied upon as the single most important screening test for rapid diagnosis of fungal corneal ulcer.
Archives of Iranian Medicine, Volume 9, Number 3, 2006: 222 – 227.
Keywords: Corneal ulceration;diagnosis;fungal keratitis;keratomycosis
Introduction
Fungal keratitis is a suppurative, ulcerative, and sight-threatening infection of the cornea that leads sometimes leads to loss of the eye. Worldwide, the reported incidence of mycotic keratitis is 17% to 36%.1 – 3 In India, it is 44% to 47%.4 – 7 Despite advances in diagnosis and medical treatment of keratomycosis, 15% to 27% of patients require surgical intervention such as keratoplasty, enucleation, or evisceration because of either failed medical treatment or advanced disease at presentation.1 – 11
In general, it has been indicated that although corneal infections have worldwide distribution, the incidence of fungal infections is higher in tropical and semitropical areas and is much more frequent in developing countries. It has also been mentioned that these infections follow trauma, especially with plant debris.1 – 11 The distribution of fungi is not only affected geographically, but also changes with time. It is, therefore, very important to recognize the prevalence and etiology of corneal ulcer.
The following study was conducted at the Ophthalmology Department of Bou-Ali Sina Hospital of Sari, in Mazandaran Province, North of Iran. Mazandaran is located near the Caspian sea and the majority of people are engaged in farming. The objectives of this study were to improve facilities for laboratory diagnosis, to determine the predominant causative microorganisms, and to identify the predisposing factors of mycotic keratitis patients.
Patients and Methods
Patients
A prospective study of corneal ulcer was conducted in Sari between May 2004 and March 2005. Patients who presented with clinically suspected corneal ulcer to the Ophthalmology Department of Bou-Ali Sina University Hospital in Sari, were included in this study. Corneal ulceration was defined as the loss of corneal epithelium with underlying stromal infiltrate and suppuration as-sociated with signs of inflammation, with or without hypopyon.6 Written informed consent was obtained from all patients.
Clinical examination and laboratory investigations
Each patient was examined by slit lamp. The location and size of infiltrate were recorded. Data were then collected by examination and questioning the patients. Using standard techniques,12 corneal scraping was performed by an ophthalmologist, with a sterile surgical blade number 15, or a sterile 21 gauge needle, following the instillation of a local anesthetic (tetracaine hydrochloride 0.5%). Corneal specimen obtained from scraping the ulcer was smeared on two slides, which were stained with Gram stain (for bacterial keratitis) and 10% potassium hydroxide (KOH) with or without Calcofluor white (KOH+CFW) stain (for fungal keratitis), and studied under light microscope. The specimens were also inoculated directly on blood agar, Sabouraud dextrose agar, and potato dextrose agar in C-shaped streaks. Blood agar plates were incubated at 37oC. Sabouraud dextrose agar and potato dextrose agar were incubated at 25oC. The culture was considered significant and reported if the smears demonstrated morphologically similar organism, and/or if the same organism grew in more than one culture media, and/or if there was growth on at least two streaks.
Results
Demographic
A total of 22 patients met the inclusion criteria of this study, of whom 10 (45.5%) were females and 12 (54.5%) were males. The mean ± SD age of patients was 61.5 ± 17.7 (range: 15 – 83) years. Of 22 patients with corneal ulcer surveyed during the period of May 2004 to March 2005 (12 months), fungal keratitis was identified as the principal etiologic agent of corneal ulceration in 7 (31.8%) patients (5 males and 2 females) (Table 1). The mean ± SD age of patients with fungal keratitis was 60.4 ± 12.1 (range: 39 – 73) years. Three (42.9%) patients with fungal keratitis were farmers; one (14.3%) animal husbandman, one (14.3%) laborer, and 2 (28.6%) were housewives. All patients with fungal keratitis lived in rural areas. The mean interval between the onset of symptom and diagnosis was 26.4 (range: 1 – 93) days.
Predisposing factors
Trauma with plant debris and straws were noted in two patients with fungal keratitis. Five patients received topical antibiotics. One patient had diabetes and another had local corneal disease (persistent corneal defect and stromal ulceration). No patient with fungal keratitis had a history of ocular surgery or use of contact lens and topical corticosteroid.
Clinical features
All patients had symptoms of redness and watering. Ocular pain and photophobia were found in 85.7% of patients. Seventy-one point four percent of patients had foreign body sensation; 57.1% had decreased vision. The signs on slit lamp examination are shown in Table 2.
The distinct characteristics of patients with fungal keratitis found in our study—male preponderance (71.4%), outdoor occupa-tion (71.5%), mean age of 60.4 years, and living in rural regions (100%)—are consistent with observations made in the majority of other studies.12 – 14 The incidence is reported to be higher in tropical countries. Apart from a humid atmosphere, agricultural-based live-li-hood in this region and outdoor occupations also make the population more vulnerable to fungal infections.
The cornea may present suppurative keratitis due to viruses, bacteria, parasites, and fungi. Normal conjunctival microbiota usually does not consist of fungi. With some special life or work situation, however, individuals are exposed to corneal trauma and thus become more prone to develop mycotic infection. In our study, 71.4% of patients with fungal keratitis received topical antibiotics before seeking treatment from us. The great number of patients with fungal keratitis using topical antibiotics, who came to the university services, is explained by the fact that this service is a tertiary referral center to which more severe cases that did not respond to initially-applied therapy are referred. Since one of the differential diagnoses of suppurative keratitis is bacterial keratitis, the initial therapy with antibiotics is justified in services without a supporting laboratory. In one report, 56% of patients with fungal keratitis and in another, 55.3% of patients with fungal keratitis had received prior topical antibiotics as a major risk factor.12, 15 However, the various reports have not clearly identified the importance of prior usage of topical antibiotics as a predisposing factor.6, 11, 16
In this study, 28.6% of patients with fungal keratitis had corneal trauma, which is lower than that reported for fungal keratitis in general. Previous corneal trauma is identified as a major predisposing factor among patients with filamentous fungal keratitis.6, 12, 15, 17 A frequency of 33% to 100% has been described in the literature for mycotic keratitis in patients with corneal trauma by things having organic material or foreign body.1, 18, 19 In some other reports, 8.3%17 to 17.6%20 of patients with fungal keratitis had corneal trauma, which is lower than our report. The fewer number of patients with fungal keratitis and corneal trauma could be explained by the fact that trauma might be insensible or as a result of delay existing between the occurrence of trauma and its diagnosis, causing them difficult to recall.
In this study, the most common signs on slit lamp examination were epithelial defect (57.1%) and suppuration (57.1%)—which are nonspecific to fungal keratitis—and feathery pattern which is specific, presented in only one patient. None of our patients had hypopyon on slit lamp examination, whereas Javadi et al noted hypopyon in 52% of their patients with fungal keratitis.21 Rosa et al noted irregular feathery margins (62%), a dry rough texture (47%), and satellite lesions (41%) in their patients.1 During the first week, the signs such as hypopyon and endothelial plaque are not present. In our study, five of seven patients with fungal etiology referred to us between one and ten days of onset of the symptoms. During this phase, specific clinical features were not established. Therefore, the clinical picture may not always be sufficient to establish a diagnosis of fungal keratitis. The importance of establishing a defini-tive diagnosis by microbiologic investigations is unavoidable in such cases.
Aspergillus and Fusarium species, as were also isolated from our patients, are the most common isolated organisms from patients with fungal keratitis in the tropic. Other studies in Iran have reported Aspergillus and Fusarium to be the most common fungal pathogens.21 – 23 Fusarium species have also been found to be the principal fungal pathogen in Ghana, South India, Florida, Paraguay, Nigeria, Tanzania, Hong Kong, China, and Singapore. Aspergillus species predominate in northern India, Nepal, and Bangladesh.24 This phenomenon may be explained by differences in climate and natural environment. Seasonal variations have been observed to be important in the incidence of fungal keratitis and in the predominant genera of fungi isolated from such patients. Such variations have been linked to environmental factors, such as humidity, rainfall, wind, and also to the harvest.1, 19, 25, 26 Environmental conditions including temperature, annual rainfall, windy seasons, and the harvest period have a significant role in increasing the incidence.
There are different diagnostic laboratory methods for diagnosis of fungal keratitis, including direct examination and culture. In the current study, the presence of fungi in corneal scrapings was determined by KOH+CFW stain in 7, KOH stain in 5 (71.4%), and Gram stain in 3 (42.9%) scrapings from patients with keratitis. The smear findings of KOH+CFW as the gold-standard method were compared with the KOH mount, Gram stain, and culture results to calculate the specificity, sensitivity, and predictive values of the other techniques used. Rapid detection of fungal elements in the corneal scraping is of utmost importance in the management of fungal keratitis, especially because the organisms are not amenable to treatment with broad-spectrum, commercially available antibiotics. Fungi can be detected in corneal scraping by a variety of methods including KOH wet preparation,27 acridine orange staining,28 and lactophenol blue staining.29 The sensitivity of these methods varies from 76% to 80%. CFW, with or without KOH, is also a well-established stain for observation of fungal elements in clinical samples with a high sensitivity.27, 30, 31 Sharma et al32 confirmed the superiority of KOH+CFW stain in detection of fungal elements in corneal scraping. It is not only highly specific and sensitive, but also has a high NPV and PPV for both early and advanced keratitis. Since they had seen some false positive results in advanced keratitis, they recom-mended resorting to antifungal therapy whenever a KOH+CFW stain smear is positive for fungus. This is because they believed that the gold-standard of culture has its own limitations and a fungal element is unlikely to be misinterpreted. The false positive results are explained by some observations27, 33 that all samples that show fungal elements in smear may not show growth in the culture. Chander et al30 in their evaluation of CFW staining for diagnosis of fungal corneal ulcer, confirmed the superiority of KOH+CFW in comparison with KOH and culture. In another study,34 KOH wet mount showed fungal elements in 68% of patients with corneal ulcers. Gram stain of corneal scraping did not provide much help. In our series, fungal culture was positive in 26.4% of cases. Vajpayee et al33 noted that cultures were positive in 51.46% of cases, although, the fungus was demonstrated by the KOH preparation in 94.3% of the culture-proven cases. In our study, the culture-positive cases determined in whom the interval between the onset of disease and diagnosis was more than a week and in direct microscopy in which too many fungal elements were seen. The culture-positive patients were in the advance phase of keratitis, whereas the culture-negative cases were in the early phase of keratitis. Having no yield of positive culture could be explained by an insufficient corneal scraping of sample inoculated to culture media and less fungal load in the early stages of keratitis. To the best of our knowledge, the clinical picture may not always be sufficient to establish a diagnosis for fungal keratitis. The present study also showed the effectiveness of KOH+CFW as a rapid and highly reliable method in confirming the diagnosis of fungal keratitis, particularly when empirical antifungal therapy is not justified. Therefore, if KOH+CFW stained smears are negative for fungal filaments and acanthamoeba cysts, the patient must be treated with a broad-spectrum antibiotic, even if the Gram stain is negative for bacteria.
In eras where fungal keratitis is prevalent, a simple, rapid, and reliable KOH+CFW smear exa-mi-nation of corneal scraping early during the disease could result in a significant difference in the course of the disease by helping in early, specific therapy. KOH+CFW stain was significantly more sensitive than KOH wet mount in demonstrating fungal pathogens. However, if UV microscope and CFW are not available, KOH preparation is recommended.
Acknowledgment
We are grateful to the Vice-Chancellor of Research of Mazandaran University of Medical Sciences for financial support. We also thank Dr. A.R. Khalilian for statistical analyses.
References
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