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Indian Journal of Medical Microbiology, (2014) 32(3): 310-314
1
Brief Communication
Clinico‑demographical profile of keratomycosis in Delhi, North India A Gupta, *MR Capoor, S Gupta, S Kochhar, A Tomer, V Gupta
Abstract This study was undertaken to evaluate the clinico‑demographical profile of keratomycosis. (January 2004 to January 2012). The corneal scrapings were processed by direct microscopic methods and standard culture techniques. Of 209 cases of keratitis studied, culture yielded growth in 80 cases (38.3%). Out of these 80 cases of growth, fungi were isolated in 77.5% and bacteria in 22.5%. The spectrum of keratomycosis was Aspergillus flavus (22.5%), Fusarium solani (16.1%), A. fumigatus (11.3%), Candida albicans (6.4%), etc., Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent use of un‑warranted anti‑microbial therapy. Key words: Anti‑fungal susceptibility, demography, keratitis, mould, yeast
Introduction Ophthalmic infections are regarded as among the chief causes of ocular morbidity and mortality worldwide. A larger proportion of keratitis is reported from developing countries than in developed countries. The aetiological agents implicated are fungi, bacteria and protozoans. While viral infections are the leading cause of corneal ulcer in developed nations, bacteria, fungi and Acanthamoeba are important aetiological agents in the developing world. It is therefore very important to recognise the prevalence and aetiology of keratitis.[1‑3] In mycotic keratitis, two types have been recognised: Keratitis due to filamentous fungi (especially Fusarium and Aspergillus), which commonly occurs in tropical and subtropical zones, and associated with corneal trauma (and concurrent contamination with plant material); and keratitis due to yeast‑like and related fungi particularly Candida; and mostly associated with corneal disease, local immunosuppression caused by chronic corticosteroid use and systemic disease states that lower host resistance. All the more, the protean appearances *Corresponding author (email: ) Department of Microbiology (AG, SG, AT), Ophthalmology (SK, VG), Microbiology (MRC) Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi ‑ 110 029, India Received: 13-07-2013 Accepted: 16-01-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.136582
of mycotic keratitis might be indistinguishable from a bacterial ulcer. The typical features suggestive of fungal infection are feathery edges or a dry, gray, elevated infiltrate and satellite lesions.[2] Despite advances in diagnosis and medical treatment of keratitis, many patients require surgical intervention such as keratoplasty, enucleation or evisceration because of either failed medical treatment or advanced disease at presentation. Complications of keratitis include secondary glaucoma, corneal scarring, perforation, corneal opacity, corneal thinning, uveitis, severe anterior chamber reaction, severe vision loss and loss of eye. Availability of an antibiogram is necessary to initiate prompt and accurate therapy and would avoid unnecessary and indiscriminate use of steroids or anti‑microbial drugs.[4‑7] There is scarcity of recent data on mycotic keratitis published from north India.[4,8,9] Furthermore, there are limited number of studies from India depicting anti‑fungal susceptibility and therapeutic outcomes.[10] This study attempts to understand the clinico‑demographical profile of keratomycosis from a tertiary care hospital in north India. Materials and Methods Demographic profile of patients in the study The study was undertaken at the Department of Microbiology and Ophthalmology, in a tertiary care in north India, for a period of 8 years (January 2004 to January 2012). Patients with clinical diagnosis of microbial keratitis were included in the study. The corneal scrapings were submitted to the laboratory for microbiological investigations. The patients age, gender, occupation, history of any predisposing factor like trauma, past and current use of topical medicines or use of contact lens use were meticulously recorded. Collection of samples In all suspected cases of keratomycosis, the corneal scrapings were collected for microbiological investigations
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July-September 2014
Gupta, et al.: Keratomycosis in North India
and analysis. The scrapings were obtained aseptically by scraping the base and edge of the ulcers using sterile Kimura’s spatula or No. 15 Baird Parker blade under direct vision through a slit lamp after instillation of anaesthetic eye drops (4% Xylocaine). Inoculation in culture media The material from corneal scrapings was inoculated directly on blood agar (containing 5% sheep blood), chocolate agar, MacConkey’s agar for bacterial culture incubated at 37°C in multiple rows of C‑shaped streaks. Sabouraud’s dextrose agar (Himedia, Mumbai) without cycloheximide was used for fungal culture and was incubated in duplicate at 25°C and 37°C, respectively, for 4 weeks. Direct microscopy Direct microscopy was performed for presumptive identification or evidence of bacterial, fungal or parasitic infection. The mounts of corneal scrapings were prepared in 10% KOH. The smears were also prepared from each sample for Gram’s staining for the demonstration of bacteria, hyphae, pseudohyphae and yeast cells. Giemsa staining for Acanthamoeba was done, wherever indicated. Fungal culture identification The cultures were examined routinely for the growth of colonies. The growth was identified by its cultural characteristics on media and sporulation pattern on lactophenol cotton blue mount. Yeasts The yeasts were identified by their characteristic growth on culture media and microscopic findings. The identification of yeasts was done by growth and microscopic characteristics, germ tube test, HiChrome agar (Himedia, Mumbai), corn meal agar; carbon and nitrogen assimilation tests, and API 20C yeast identification strips (Biomerieux, France).
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Anti‑fungal susceptibility Anti‑fungal susceptibility of Candida and Aspergillus species only were performed by broth microdilution method using RPMI‑1640 medium and E‑strip method using glucose‑methylene blue Mueller-Hinton agar using CLSI guidelines (CLSI 2002). Antifungal susceptibility of Candida and Aspergillus species only were performed by brothmicro dilution method using RPMI-1640 medium and E-strip method using glucose-methylene blue Mueller Hinton agar using CLSI guidelines (Reference method for broth dilution susceptibility testing for yeast, Approved standard, Second Edition, Document M27-A2, CLSI 2002; Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, Approved standard, Document M38-A, CLSI 2004; Method for Antifungal disc diffusion susceptibility testing of yeast, Approved guidelines, Document M44-A, CLSI 2002). It was also decided that the infection with hyaline moulds (non‑Aspergillus moulds) to be treated with 5% natamycin eye drops, infection with Aspergillus to be treated with 0.15% amphotericin and natamycin eye drops; and dematiceous moulds with 5% natamycin eye drops and oral ketoconazole. Bacterial culture identification Blood agar (containing 5% sheep blood), chocolate agar and MacConkey agar were incubated at 37°C and were examined daily and discarded after 7 days if no growth occurred. Bacteria were identified as per the standard protocol. Anti‑bacterial susceptibility Anti‑bacterial susceptibility was performed by Kirby Bauer disc diffusion method as per CLSI guidelines (CLSI 2009. Performance Standards for Anti‑microbial Susceptibility Testing; Nineteenth Informational Supplement. Volume 29 Number 3). The study was approved by ethical committee of the institute.
Moulds
Results
Moulds were identified by characteristic structure and arrangement of conidia on lactophenol cotton blue mount. For non‑sporulating moulds, slide cultures were put up to stimulate sporulation and assist their identification.
The study was conducted in the Department of Microbiology and Ophthalmology for a period of 8 years (January 2004 to January 2012). The parameters that were studied included the age and sex of the patient, the signs and symptoms, the use of contact lenses or any topical medications. A total of 209 corneal scrapings were submitted from patients presenting with complaints of photophobia, lacrimation and reduced vision. All the patients had unilateral eye involvement. There was a male preponderance in this study, with a male: Female ratio of 1.75:1.
Significant growth The cultures were considered positive for growth if at least one of the following criteria was fulfilled • The growth of the same organism is demonstrated on one or more solid media • The growth on one medium is consistent with direct microscopic findings • The same organism is grown from repeated corneal scrapings.
The prevalence of keratitis was found to be more in agriculturists and labourers. A total of 48% of the patients recalled the history of ocular trauma with vegetative or soil.
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A total of 25% of the patients provided the history of previous use of topical medications (corticosteroids or antibiotics). Of all the samples studied, direct microscopic examination revealed microorganisms (bacteria, fungi, Acanthamoeba) in 53 (25.4%) samples as depicted in Table 1. Subsequently on culture, 80 (38.3%) cases exhibited growth. Acanthamoeba was found only on microscopy in a 55‑year‑old female villager and history of bathing in ponds was elicited from her. The microbiological profile in the study was bacteria (22.5%) and fungi (77.5%). Culture on non‑nutrient agar was negative for Acanthamoeba. The distribution of fungal agents in the study was yeasts in 14.3% of cases and filamentous fungi in 85.5% of cases as depicted in Table 2. With regards to the fungal susceptibility, Aspergillus fumigatus, A. flavus and other Aspergillus species were uniformly sensitive to amphotericin B and itraconazole. The MIC of the isolates for all Aspergillus species for amphotericin B ranged from 0.0616 to 1 µg/mL and itraconazole 0.0616 to 0.5 µg/mL, respectively. C. parapsilosis and other non‑albicans Candida were found susceptible to amphotericin B (the MIC ranged from 0.125 to 1 µg/mL) and were found resistant to fluconazole (MIC ≥ 64 µg/mL) and itraconazole (high MIC ≥ 4 µg/mL). One of the unusual pathogens isolated in the study was Aureobasidum pullulans. Table 1: Results of direct microscopy and culture Test Positive cases Percentage Direct microscopy 53 25.4 Culture 80 38.3 Table 2: Distribution of fungal pathogens in the study Filamentous moulds Aspergillus fumigatus Aspergillus flavus Aspergillus niger Aspergillus nidulans Other Aspergillus species Fusarium solanii Penicillium species Rhizopus oryzae Mucor ramosissimus Alternaria alternata Curvularia lunata Aureobasidium pullulans Yeasts Candida albicans Candida tropicalis Candida parapsilosis Other Candida species
7 14 8 1 2 10 3 1 2 3 1 1
11.3% 22.5% 12.9% 1.6% 3.2% 16.1% 4.8% 1.6% 3.2% 4.8% 1.6% 1.6%
4 2 2 1
6.4% 3.2% 3.2% 1.6%
vol. 32, No. 3
All the patients of bacterial keratitis responded to anti‑bacterial therapy. No relapse was observed. Of the 28 patients with mycotic keratitis, 14 were prescribed natamycin 5% eye drops alone and 14 received natamycin 5% eyedrops along with amphotericin B drops. Out of these 14 patients receiving the combination drops, 12 were also prescribed oral itraconazole. In six patients finally therapeutic keratoplasty was done. Therapeutic keratoplasty was done in cases of deep and large ulcers due to Aspergillus species. Complete cure was seen in only 15 patients with mycotic keratitis. Discussion Mycotic keratitis continues to be an important cause of ocular morbidity despite advances in the ocular treatment strategies. In the current study, incidence of fungal ulcers was more in males (63.6%) than in females (36.4%). This study result coincides with the study of other workers.[4] Higher incidence of keratitis in males can be attributed to more outdoor activity of males in field activities related to agriculture and farming. In our study, majority of fungal ulcers were diagnosed in the age group of 21-40 years, whereas a study from north India reported their incidence at 56.67% in the age group of 20-49 years.[4] This is the most common actively working age group.[5] Trauma with vegetative matter and use of topical medications (corticosteroids or antibiotics) were observed to be the predominant predisposing factors in the pathogenesis of corneal ulcer This finding is consistent with the result of other studies.[4,6] Steroids have been proven to alter corneal metabolism there by changing defense mechanism while antibiotics alter normal local flora. Agriculturists were more commonly affected by mycotic keratitis in our study. Similar observations were made in prior studies.[7,8,11] This is attributed to ubiquitous fungal spores and presence of these on senescent plant material. In this study, culture was sterile despite observation of microorganism on direct microscopy. This is attributed to the fact that the patients were already prescribed topical steroids or anti‑fungal agents as prescribed by local doctors before the corneal scrapings were collected in our tertiary‑care referral centre. Microbial pathogens responsible for keratitis though vary geographically, the deciding factor causing keratitis in many cases is the local microbial flora. The fungi isolated from cases of corneal ulcers known to inhabit normal eye and also occur as saprobes in the soil and environmental matter. In this study Aspergillus species was the most frequent aetiological agent of keratitis, followed by F. solanii (16.1%). Other centres from north and western India also reported Aspergillus species as most frequent cause of mycotic keratitis (41%) and (40%), respectively.[9,12,13] However, Fusarium species was found
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Gupta, et al.: Keratomycosis in North India
to be the most common case of fungal keratitis in south India and abroad.[10,14‑16] In a study from Philadelphia, Candida albicans was the most common isolate (46%) reported.[17] One of the unusual pathogens isolated in the study was Aureobasidum pullulans, which is considered a rare entity. The patient responded well to therapy with natamycin eye drops and oral ketoconazole. So far very few reports have appeared about it in the world literature and in most of the cases, keratitis developed following a surgical intervention.[18‑20] Although A. pullulans is regarded as a contaminant, it should be implicated as a pathogen if there exists a clinical and microbiological correlation. Mycotic keratitis is usually treated with a topical anti‑fungal agent sometimes in combination with subconjunctival injections and/or antimycotic agents but therapeutic keratoplasty may be needed for patients whose corneal infection does not resolve as was seen in six cases in this study. The infections caused by yeasts present a more favourable outcome as compared with filamentous fungi. Natamycin, a polyene is considered the drug of choice for filamentous fungi. Nonetheless, poor penetration in non‑severe superficial keratitis and its expense preclude its routine use. In our set of patients, natamycin was used in 14 patients only. Econazole, a triazole has similar effect as that of natamycin against filamentous fungi. Fluconazole works best for Candida species.[2] In patients with C. tropicalis and Aspergillus infection itraconazole was prescribed. Voriconazole works for Candida species, Aspergillus spp., Scedosporium, Fusarium, Paecillomyces species. Amphotericin B covers Candida and Aspergillus spp.[2] Itraconazole and caspofungin are not effective against Fusarium species.[10] In patients with fungal keratitis, angio‑invasive disease, large ulcer size and the presence of other ocular disease were risk factor for treatment failure.[21] Furthermore, it is very important to speciate Aspergillus species and Candida species, as anti‑fungal resistance to polyene and azoles is intrinsic in A. terreus, A. ustus and non‑albicans Candida species (C. kruzei, C. glabrata).[22,23] Though most of the reports of Acanthamoeba as the causative agent of keratitis were in contact lens wearers, the case in this study positive for Acanthamoeba had history of bathing in pond.[5] Corneal trauma, dust and contamination of water are the other reported predisposing factors.[24,25] However, Acanthamoeba could not be isolated on non‑nutrient agar culture may be attributed to use of anti‑acanthamoebic drug by the patient. Low incidence of Acanthamoeba keratitis is attributed to decreased awareness for Acanthamoeba infection. Therefore a high index of suspicion is required especially for Acanthamoeba diagnosis. As even in resource poor settings, KOH, Gram’s, Giemsa stain and culture can be done routinely especially in suspected cases. Though bacteria, fungi and parasites are responsible for keratitis, fungal agents deserve special mention
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due to their opportunistic nature and indolent course of disease. High index of suspicion by ophthalmologists and laboratory confirmation of the case of keratitis could save the eye from blindness. Not a lot many studies have been undertaken so far to understand the demography of such infections. The research in this area needs to delve into the pathogenesis (i.e., adhesion, invasion, toxigenicity) of mycotic agents. In particular, the possible role of fungal extracellular proteinases and fungal morphogenesis in ophthalmic mycoses requires clarification.[26,27] Also, numerous problems are encountered in the timely diagnosis of fungal infections due to inadequate or insufficient quantity of sample, failure of causative pathogen to grow in culture. These can be ameliorated by molecular techniques like DNA amplification, hybridisation assays, etc., the utility of which has already been proven.[28] Furthermore, these are prohibitively expensive for routine use and are available in reference laboratories only. Last but not the least, studies showing susceptibility of the fungal pathogens to antiseptics like polyhexamethyl biguanide can prove helpful to deal with rampant pattern of anti‑fungal resistance. Priorities should be given to develop and undertake trials involving newer anti‑microbials and role of cytokines in keratitis. Proper understanding of microbiological and clinical characteristics of keratomycosis will enable ophthalmologist to avoid unnecessary and indiscriminate use of steroids or anti‑bacterials or anti‑virals and anti‑fungals. Early stage of diagnosis and formulation of an uncompromising management protocol can prevent profound visual morbidity. Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens. References 1. Shokohi T, Nowroozpoor‑Dailami K, Moaddel‑Haghighi T. Fungal keratitis in patients with corneal ulcer in Sari, Northern Iran. Arch Iran Med 2006;9:222‑7. 2. Srinivasan S. Fungal keratitis. Curr Opin Ophthalmol 2004;15:321‑7. 3. Akshaya R, Amrutha P, Nalamada S. A study on the epidemiological and microbiological aspects of keratomycosis, Hyderabad, South India. J Young Invest 2010;20. 4. Chander J, Singla N, Agnihotri N, Arya SK, Deep A. Keratomycosis in and around Chandigarh: A five year study from a north Indian tertiary care hospital. Ind J Pathol Microbiol 2008;51:304‑6. 5. Kotigadde S, Ballal M, Jyothirlatha, Kumar A, Srinivasa R, Shivananda PG. Mycotic keratitis: A study in coastal Karnataka. Ind J Ophthalmol 1992;40:31‑3. 6. Bhartiya P, Daniell M, Constantinou M, Islam FM, Taylor HR. Fungal keratitis in Melbourne. Clin Experiment Ophthalmol 2007;35:124‑30. 7. Venugopal PL, Venugopal TL, Gomathi A, Ramakrishna ES, Ilavarasi S. Mycotic keratitis in Madras. Indian J Pathol Microbiol 1989;32:190‑7. 8. Saha R, Das S. Mycological profile of infectious keratitis from Delhi. Indian J Med Res 2006;123:159‑64. 9. Chowdhary A, Singh K. Spectrum of fungal keratitis in North
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India. Cornea 2005;24:8‑15. 10. Doczi I, Gyetvai T, Kredics L, Nagy E. Involvement of Fusarium spp. in fungal keratitis. Clin Microbiol Infect 2004;10:773‑6. 11. Sathyanarayan MS, Suresh BS, Surekha YA, Mariraj J, Krishna S. Epidemiology and aetiological diagnosis of keratomycosis in a tertiary care hospital in North Karnataka. Int J Curr Res Rev 2013;5:92‑7. 12. 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;81:965‑71. 13. Jadhav SV, Gandham NR, Misra RN, Ujagare MT, Sharma M, Sardar M. Prevalence of fungal keratitis from tertiary care hospital from western part of India. Int J Microbiol Res 2012;4:207‑10. 14. Bharathi MJ, Ramakrishna 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. 15. Lalitha P, Shapiro BL, Srinivasan M, Prajna NV, Acharya NR, Fothergill AW, et al. Antimicrobial susceptibility of Fusarium, Aspergillus, and other filamentous fungi isolated from keratitis. Arch Ophthalmol 2007;125:789‑93. 16. Anusuya D, Ambica R, Nagarathnamma T. The epidemiological features and laboratory diagnosis of keratomycosis. Int J Biol Med Res 2013;4:2879‑83. 17. Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000;19:307‑12. 18. Panda A, Das H, Deb M, Khanal B, Kumar S. Aureobasidium pullulans keratitis. Clin Experiment Ophthalmol 2006;34:260‑4. 19. Chawla B, Sharma N, Titiyal JS, Nayak N, Satpathy G. Aureobasidium pullulans keratitis following automated lamellar therapeutic keratoplasty. Ophthalmic Surg Lasers Imaging 2010;9:1‑3.
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20. Maverick KJ, Conners MS. Aureobasidium pullulans fungal keratitis following LASEK. J Refract Surg 2007;23:727‑9. 21. Lee KH, Chae HT, Yoon KC. Analysis of risk factors for treatment failure in fungal keratitis. J Korean Ophthalmol Soc 2008;49:737‑42. 22. Chandrasekar P. Invasive mold infections: Recent advances in management approaches. Leuk Lymphoma 2009;50:703‑15. 23. Capoor MR, Nair D, Deb M, Verma PK, Srivastava L, Aggarwal P. Emergence of Non‑albicans Candida species and antifungal resistance in a tertiary‑care hospital. Jpn J Infect Dis 2005;58:344‑8. 24. John T, Desai D, Sahm D. Adherence of Acanthamoeba castellanii cysts and trophozoites to unworn soft contact lenses. Am J Ophthalmol 1989;108:658‑64. 25. Girija T, Kumari R, Kamath MG, Ramani R, Mohan R, Shivananda PG. Acanthamoeba keratitis‑‑a report of two cases. Indian J Ophthalmol 1992;40:124‑5. 26. Zhu WS, Wojdyla K, Donlon K Thomas PA, Eberle HI. Extracellular proteases of Aspergillus flavus. Fungal keratitis, proteases, and pathogenesis. Diagn Microbiol Infect Dis 1990;13:491‑7. 27. Thomas PA, Garrison RG, Jansen T. Intrahyphal hyphae in corneal tissue from a case of keratitis due to Lasiodiplodia theobromae. J Med Vet Mycol 1991;29:263‑7. 28. Jaeger EE, Carroll NM, Choudhury S, Dunlop AA, Towler HM, Matheson MM, et al. Rapid detection and identification of Candida, Aspergillus and Fusarium species in ocular samples using nested PCR. J Clin Microbiol 2000;38:2902‑8.
How to cite this article: Gupta A, Capoor MR, Gupta S, Kochhar S, Tomer A, Gupta V. Clinico-demographical profile of keratomycosis in Delhi, North India. Indian J Med Microbiol 2014;32:310-4. Source of Support: Nil, Conflict of Interest: None declared.
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Indian Journal of Medical Microbiology, (2014) 32(3): 310-314
1
Brief Communication
Clinico‑demographical profile of keratomycosis in Delhi, North India A Gupta, *MR Capoor, S Gupta, S Kochhar, A Tomer, V Gupta
Abstract This study was undertaken to evaluate the clinico‑demographical profile of keratomycosis. (January 2004 to January 2012). The corneal scrapings were processed by direct microscopic methods and standard culture techniques. Of 209 cases of keratitis studied, culture yielded growth in 80 cases (38.3%). Out of these 80 cases of growth, fungi were isolated in 77.5% and bacteria in 22.5%. The spectrum of keratomycosis was Aspergillus flavus (22.5%), Fusarium solani (16.1%), A. fumigatus (11.3%), Candida albicans (6.4%), etc., Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent use of un‑warranted anti‑microbial therapy. Key words: Anti‑fungal susceptibility, demography, keratitis, mould, yeast
Introduction Ophthalmic infections are regarded as among the chief causes of ocular morbidity and mortality worldwide. A larger proportion of keratitis is reported from developing countries than in developed countries. The aetiological agents implicated are fungi, bacteria and protozoans. While viral infections are the leading cause of corneal ulcer in developed nations, bacteria, fungi and Acanthamoeba are important aetiological agents in the developing world. It is therefore very important to recognise the prevalence and aetiology of keratitis.[1‑3] In mycotic keratitis, two types have been recognised: Keratitis due to filamentous fungi (especially Fusarium and Aspergillus), which commonly occurs in tropical and subtropical zones, and associated with corneal trauma (and concurrent contamination with plant material); and keratitis due to yeast‑like and related fungi particularly Candida; and mostly associated with corneal disease, local immunosuppression caused by chronic corticosteroid use and systemic disease states that lower host resistance. All the more, the protean appearances *Corresponding author (email: ) Department of Microbiology (AG, SG, AT), Ophthalmology (SK, VG), Microbiology (MRC) Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi ‑ 110 029, India Received: 13-07-2013 Accepted: 16-01-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.136582
of mycotic keratitis might be indistinguishable from a bacterial ulcer. The typical features suggestive of fungal infection are feathery edges or a dry, gray, elevated infiltrate and satellite lesions.[2] Despite advances in diagnosis and medical treatment of keratitis, many patients require surgical intervention such as keratoplasty, enucleation or evisceration because of either failed medical treatment or advanced disease at presentation. Complications of keratitis include secondary glaucoma, corneal scarring, perforation, corneal opacity, corneal thinning, uveitis, severe anterior chamber reaction, severe vision loss and loss of eye. Availability of an antibiogram is necessary to initiate prompt and accurate therapy and would avoid unnecessary and indiscriminate use of steroids or anti‑microbial drugs.[4‑7] There is scarcity of recent data on mycotic keratitis published from north India.[4,8,9] Furthermore, there are limited number of studies from India depicting anti‑fungal susceptibility and therapeutic outcomes.[10] This study attempts to understand the clinico‑demographical profile of keratomycosis from a tertiary care hospital in north India. Materials and Methods Demographic profile of patients in the study The study was undertaken at the Department of Microbiology and Ophthalmology, in a tertiary care in north India, for a period of 8 years (January 2004 to January 2012). Patients with clinical diagnosis of microbial keratitis were included in the study. The corneal scrapings were submitted to the laboratory for microbiological investigations. The patients age, gender, occupation, history of any predisposing factor like trauma, past and current use of topical medicines or use of contact lens use were meticulously recorded. Collection of samples In all suspected cases of keratomycosis, the corneal scrapings were collected for microbiological investigations
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July-September 2014
Gupta, et al.: Keratomycosis in North India
and analysis. The scrapings were obtained aseptically by scraping the base and edge of the ulcers using sterile Kimura’s spatula or No. 15 Baird Parker blade under direct vision through a slit lamp after instillation of anaesthetic eye drops (4% Xylocaine). Inoculation in culture media The material from corneal scrapings was inoculated directly on blood agar (containing 5% sheep blood), chocolate agar, MacConkey’s agar for bacterial culture incubated at 37°C in multiple rows of C‑shaped streaks. Sabouraud’s dextrose agar (Himedia, Mumbai) without cycloheximide was used for fungal culture and was incubated in duplicate at 25°C and 37°C, respectively, for 4 weeks. Direct microscopy Direct microscopy was performed for presumptive identification or evidence of bacterial, fungal or parasitic infection. The mounts of corneal scrapings were prepared in 10% KOH. The smears were also prepared from each sample for Gram’s staining for the demonstration of bacteria, hyphae, pseudohyphae and yeast cells. Giemsa staining for Acanthamoeba was done, wherever indicated. Fungal culture identification The cultures were examined routinely for the growth of colonies. The growth was identified by its cultural characteristics on media and sporulation pattern on lactophenol cotton blue mount. Yeasts The yeasts were identified by their characteristic growth on culture media and microscopic findings. The identification of yeasts was done by growth and microscopic characteristics, germ tube test, HiChrome agar (Himedia, Mumbai), corn meal agar; carbon and nitrogen assimilation tests, and API 20C yeast identification strips (Biomerieux, France).
311
Anti‑fungal susceptibility Anti‑fungal susceptibility of Candida and Aspergillus species only were performed by broth microdilution method using RPMI‑1640 medium and E‑strip method using glucose‑methylene blue Mueller-Hinton agar using CLSI guidelines (CLSI 2002). Antifungal susceptibility of Candida and Aspergillus species only were performed by brothmicro dilution method using RPMI-1640 medium and E-strip method using glucose-methylene blue Mueller Hinton agar using CLSI guidelines (Reference method for broth dilution susceptibility testing for yeast, Approved standard, Second Edition, Document M27-A2, CLSI 2002; Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, Approved standard, Document M38-A, CLSI 2004; Method for Antifungal disc diffusion susceptibility testing of yeast, Approved guidelines, Document M44-A, CLSI 2002). It was also decided that the infection with hyaline moulds (non‑Aspergillus moulds) to be treated with 5% natamycin eye drops, infection with Aspergillus to be treated with 0.15% amphotericin and natamycin eye drops; and dematiceous moulds with 5% natamycin eye drops and oral ketoconazole. Bacterial culture identification Blood agar (containing 5% sheep blood), chocolate agar and MacConkey agar were incubated at 37°C and were examined daily and discarded after 7 days if no growth occurred. Bacteria were identified as per the standard protocol. Anti‑bacterial susceptibility Anti‑bacterial susceptibility was performed by Kirby Bauer disc diffusion method as per CLSI guidelines (CLSI 2009. Performance Standards for Anti‑microbial Susceptibility Testing; Nineteenth Informational Supplement. Volume 29 Number 3). The study was approved by ethical committee of the institute.
Moulds
Results
Moulds were identified by characteristic structure and arrangement of conidia on lactophenol cotton blue mount. For non‑sporulating moulds, slide cultures were put up to stimulate sporulation and assist their identification.
The study was conducted in the Department of Microbiology and Ophthalmology for a period of 8 years (January 2004 to January 2012). The parameters that were studied included the age and sex of the patient, the signs and symptoms, the use of contact lenses or any topical medications. A total of 209 corneal scrapings were submitted from patients presenting with complaints of photophobia, lacrimation and reduced vision. All the patients had unilateral eye involvement. There was a male preponderance in this study, with a male: Female ratio of 1.75:1.
Significant growth The cultures were considered positive for growth if at least one of the following criteria was fulfilled • The growth of the same organism is demonstrated on one or more solid media • The growth on one medium is consistent with direct microscopic findings • The same organism is grown from repeated corneal scrapings.
The prevalence of keratitis was found to be more in agriculturists and labourers. A total of 48% of the patients recalled the history of ocular trauma with vegetative or soil.
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A total of 25% of the patients provided the history of previous use of topical medications (corticosteroids or antibiotics). Of all the samples studied, direct microscopic examination revealed microorganisms (bacteria, fungi, Acanthamoeba) in 53 (25.4%) samples as depicted in Table 1. Subsequently on culture, 80 (38.3%) cases exhibited growth. Acanthamoeba was found only on microscopy in a 55‑year‑old female villager and history of bathing in ponds was elicited from her. The microbiological profile in the study was bacteria (22.5%) and fungi (77.5%). Culture on non‑nutrient agar was negative for Acanthamoeba. The distribution of fungal agents in the study was yeasts in 14.3% of cases and filamentous fungi in 85.5% of cases as depicted in Table 2. With regards to the fungal susceptibility, Aspergillus fumigatus, A. flavus and other Aspergillus species were uniformly sensitive to amphotericin B and itraconazole. The MIC of the isolates for all Aspergillus species for amphotericin B ranged from 0.0616 to 1 µg/mL and itraconazole 0.0616 to 0.5 µg/mL, respectively. C. parapsilosis and other non‑albicans Candida were found susceptible to amphotericin B (the MIC ranged from 0.125 to 1 µg/mL) and were found resistant to fluconazole (MIC ≥ 64 µg/mL) and itraconazole (high MIC ≥ 4 µg/mL). One of the unusual pathogens isolated in the study was Aureobasidum pullulans. Table 1: Results of direct microscopy and culture Test Positive cases Percentage Direct microscopy 53 25.4 Culture 80 38.3 Table 2: Distribution of fungal pathogens in the study Filamentous moulds Aspergillus fumigatus Aspergillus flavus Aspergillus niger Aspergillus nidulans Other Aspergillus species Fusarium solanii Penicillium species Rhizopus oryzae Mucor ramosissimus Alternaria alternata Curvularia lunata Aureobasidium pullulans Yeasts Candida albicans Candida tropicalis Candida parapsilosis Other Candida species
7 14 8 1 2 10 3 1 2 3 1 1
11.3% 22.5% 12.9% 1.6% 3.2% 16.1% 4.8% 1.6% 3.2% 4.8% 1.6% 1.6%
4 2 2 1
6.4% 3.2% 3.2% 1.6%
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All the patients of bacterial keratitis responded to anti‑bacterial therapy. No relapse was observed. Of the 28 patients with mycotic keratitis, 14 were prescribed natamycin 5% eye drops alone and 14 received natamycin 5% eyedrops along with amphotericin B drops. Out of these 14 patients receiving the combination drops, 12 were also prescribed oral itraconazole. In six patients finally therapeutic keratoplasty was done. Therapeutic keratoplasty was done in cases of deep and large ulcers due to Aspergillus species. Complete cure was seen in only 15 patients with mycotic keratitis. Discussion Mycotic keratitis continues to be an important cause of ocular morbidity despite advances in the ocular treatment strategies. In the current study, incidence of fungal ulcers was more in males (63.6%) than in females (36.4%). This study result coincides with the study of other workers.[4] Higher incidence of keratitis in males can be attributed to more outdoor activity of males in field activities related to agriculture and farming. In our study, majority of fungal ulcers were diagnosed in the age group of 21-40 years, whereas a study from north India reported their incidence at 56.67% in the age group of 20-49 years.[4] This is the most common actively working age group.[5] Trauma with vegetative matter and use of topical medications (corticosteroids or antibiotics) were observed to be the predominant predisposing factors in the pathogenesis of corneal ulcer This finding is consistent with the result of other studies.[4,6] Steroids have been proven to alter corneal metabolism there by changing defense mechanism while antibiotics alter normal local flora. Agriculturists were more commonly affected by mycotic keratitis in our study. Similar observations were made in prior studies.[7,8,11] This is attributed to ubiquitous fungal spores and presence of these on senescent plant material. In this study, culture was sterile despite observation of microorganism on direct microscopy. This is attributed to the fact that the patients were already prescribed topical steroids or anti‑fungal agents as prescribed by local doctors before the corneal scrapings were collected in our tertiary‑care referral centre. Microbial pathogens responsible for keratitis though vary geographically, the deciding factor causing keratitis in many cases is the local microbial flora. The fungi isolated from cases of corneal ulcers known to inhabit normal eye and also occur as saprobes in the soil and environmental matter. In this study Aspergillus species was the most frequent aetiological agent of keratitis, followed by F. solanii (16.1%). Other centres from north and western India also reported Aspergillus species as most frequent cause of mycotic keratitis (41%) and (40%), respectively.[9,12,13] However, Fusarium species was found
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July-September 2014
Gupta, et al.: Keratomycosis in North India
to be the most common case of fungal keratitis in south India and abroad.[10,14‑16] In a study from Philadelphia, Candida albicans was the most common isolate (46%) reported.[17] One of the unusual pathogens isolated in the study was Aureobasidum pullulans, which is considered a rare entity. The patient responded well to therapy with natamycin eye drops and oral ketoconazole. So far very few reports have appeared about it in the world literature and in most of the cases, keratitis developed following a surgical intervention.[18‑20] Although A. pullulans is regarded as a contaminant, it should be implicated as a pathogen if there exists a clinical and microbiological correlation. Mycotic keratitis is usually treated with a topical anti‑fungal agent sometimes in combination with subconjunctival injections and/or antimycotic agents but therapeutic keratoplasty may be needed for patients whose corneal infection does not resolve as was seen in six cases in this study. The infections caused by yeasts present a more favourable outcome as compared with filamentous fungi. Natamycin, a polyene is considered the drug of choice for filamentous fungi. Nonetheless, poor penetration in non‑severe superficial keratitis and its expense preclude its routine use. In our set of patients, natamycin was used in 14 patients only. Econazole, a triazole has similar effect as that of natamycin against filamentous fungi. Fluconazole works best for Candida species.[2] In patients with C. tropicalis and Aspergillus infection itraconazole was prescribed. Voriconazole works for Candida species, Aspergillus spp., Scedosporium, Fusarium, Paecillomyces species. Amphotericin B covers Candida and Aspergillus spp.[2] Itraconazole and caspofungin are not effective against Fusarium species.[10] In patients with fungal keratitis, angio‑invasive disease, large ulcer size and the presence of other ocular disease were risk factor for treatment failure.[21] Furthermore, it is very important to speciate Aspergillus species and Candida species, as anti‑fungal resistance to polyene and azoles is intrinsic in A. terreus, A. ustus and non‑albicans Candida species (C. kruzei, C. glabrata).[22,23] Though most of the reports of Acanthamoeba as the causative agent of keratitis were in contact lens wearers, the case in this study positive for Acanthamoeba had history of bathing in pond.[5] Corneal trauma, dust and contamination of water are the other reported predisposing factors.[24,25] However, Acanthamoeba could not be isolated on non‑nutrient agar culture may be attributed to use of anti‑acanthamoebic drug by the patient. Low incidence of Acanthamoeba keratitis is attributed to decreased awareness for Acanthamoeba infection. Therefore a high index of suspicion is required especially for Acanthamoeba diagnosis. As even in resource poor settings, KOH, Gram’s, Giemsa stain and culture can be done routinely especially in suspected cases. Though bacteria, fungi and parasites are responsible for keratitis, fungal agents deserve special mention
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due to their opportunistic nature and indolent course of disease. High index of suspicion by ophthalmologists and laboratory confirmation of the case of keratitis could save the eye from blindness. Not a lot many studies have been undertaken so far to understand the demography of such infections. The research in this area needs to delve into the pathogenesis (i.e., adhesion, invasion, toxigenicity) of mycotic agents. In particular, the possible role of fungal extracellular proteinases and fungal morphogenesis in ophthalmic mycoses requires clarification.[26,27] Also, numerous problems are encountered in the timely diagnosis of fungal infections due to inadequate or insufficient quantity of sample, failure of causative pathogen to grow in culture. These can be ameliorated by molecular techniques like DNA amplification, hybridisation assays, etc., the utility of which has already been proven.[28] Furthermore, these are prohibitively expensive for routine use and are available in reference laboratories only. Last but not the least, studies showing susceptibility of the fungal pathogens to antiseptics like polyhexamethyl biguanide can prove helpful to deal with rampant pattern of anti‑fungal resistance. Priorities should be given to develop and undertake trials involving newer anti‑microbials and role of cytokines in keratitis. Proper understanding of microbiological and clinical characteristics of keratomycosis will enable ophthalmologist to avoid unnecessary and indiscriminate use of steroids or anti‑bacterials or anti‑virals and anti‑fungals. Early stage of diagnosis and formulation of an uncompromising management protocol can prevent profound visual morbidity. Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens. References 1. Shokohi T, Nowroozpoor‑Dailami K, Moaddel‑Haghighi T. Fungal keratitis in patients with corneal ulcer in Sari, Northern Iran. Arch Iran Med 2006;9:222‑7. 2. Srinivasan S. Fungal keratitis. Curr Opin Ophthalmol 2004;15:321‑7. 3. Akshaya R, Amrutha P, Nalamada S. A study on the epidemiological and microbiological aspects of keratomycosis, Hyderabad, South India. J Young Invest 2010;20. 4. Chander J, Singla N, Agnihotri N, Arya SK, Deep A. Keratomycosis in and around Chandigarh: A five year study from a north Indian tertiary care hospital. Ind J Pathol Microbiol 2008;51:304‑6. 5. Kotigadde S, Ballal M, Jyothirlatha, Kumar A, Srinivasa R, Shivananda PG. Mycotic keratitis: A study in coastal Karnataka. Ind J Ophthalmol 1992;40:31‑3. 6. Bhartiya P, Daniell M, Constantinou M, Islam FM, Taylor HR. Fungal keratitis in Melbourne. Clin Experiment Ophthalmol 2007;35:124‑30. 7. Venugopal PL, Venugopal TL, Gomathi A, Ramakrishna ES, Ilavarasi S. Mycotic keratitis in Madras. Indian J Pathol Microbiol 1989;32:190‑7. 8. Saha R, Das S. Mycological profile of infectious keratitis from Delhi. Indian J Med Res 2006;123:159‑64. 9. Chowdhary A, Singh K. Spectrum of fungal keratitis in North
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India. Cornea 2005;24:8‑15. 10. Doczi I, Gyetvai T, Kredics L, Nagy E. Involvement of Fusarium spp. in fungal keratitis. Clin Microbiol Infect 2004;10:773‑6. 11. Sathyanarayan MS, Suresh BS, Surekha YA, Mariraj J, Krishna S. Epidemiology and aetiological diagnosis of keratomycosis in a tertiary care hospital in North Karnataka. Int J Curr Res Rev 2013;5:92‑7. 12. 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;81:965‑71. 13. Jadhav SV, Gandham NR, Misra RN, Ujagare MT, Sharma M, Sardar M. Prevalence of fungal keratitis from tertiary care hospital from western part of India. Int J Microbiol Res 2012;4:207‑10. 14. Bharathi MJ, Ramakrishna 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. 15. Lalitha P, Shapiro BL, Srinivasan M, Prajna NV, Acharya NR, Fothergill AW, et al. Antimicrobial susceptibility of Fusarium, Aspergillus, and other filamentous fungi isolated from keratitis. Arch Ophthalmol 2007;125:789‑93. 16. Anusuya D, Ambica R, Nagarathnamma T. The epidemiological features and laboratory diagnosis of keratomycosis. Int J Biol Med Res 2013;4:2879‑83. 17. Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000;19:307‑12. 18. Panda A, Das H, Deb M, Khanal B, Kumar S. Aureobasidium pullulans keratitis. Clin Experiment Ophthalmol 2006;34:260‑4. 19. Chawla B, Sharma N, Titiyal JS, Nayak N, Satpathy G. Aureobasidium pullulans keratitis following automated lamellar therapeutic keratoplasty. Ophthalmic Surg Lasers Imaging 2010;9:1‑3.
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20. Maverick KJ, Conners MS. Aureobasidium pullulans fungal keratitis following LASEK. J Refract Surg 2007;23:727‑9. 21. Lee KH, Chae HT, Yoon KC. Analysis of risk factors for treatment failure in fungal keratitis. J Korean Ophthalmol Soc 2008;49:737‑42. 22. Chandrasekar P. Invasive mold infections: Recent advances in management approaches. Leuk Lymphoma 2009;50:703‑15. 23. Capoor MR, Nair D, Deb M, Verma PK, Srivastava L, Aggarwal P. Emergence of Non‑albicans Candida species and antifungal resistance in a tertiary‑care hospital. Jpn J Infect Dis 2005;58:344‑8. 24. John T, Desai D, Sahm D. Adherence of Acanthamoeba castellanii cysts and trophozoites to unworn soft contact lenses. Am J Ophthalmol 1989;108:658‑64. 25. Girija T, Kumari R, Kamath MG, Ramani R, Mohan R, Shivananda PG. Acanthamoeba keratitis‑‑a report of two cases. Indian J Ophthalmol 1992;40:124‑5. 26. Zhu WS, Wojdyla K, Donlon K Thomas PA, Eberle HI. Extracellular proteases of Aspergillus flavus. Fungal keratitis, proteases, and pathogenesis. Diagn Microbiol Infect Dis 1990;13:491‑7. 27. Thomas PA, Garrison RG, Jansen T. Intrahyphal hyphae in corneal tissue from a case of keratitis due to Lasiodiplodia theobromae. J Med Vet Mycol 1991;29:263‑7. 28. Jaeger EE, Carroll NM, Choudhury S, Dunlop AA, Towler HM, Matheson MM, et al. Rapid detection and identification of Candida, Aspergillus and Fusarium species in ocular samples using nested PCR. J Clin Microbiol 2000;38:2902‑8.
How to cite this article: Gupta A, Capoor MR, Gupta S, Kochhar S, Tomer A, Gupta V. Clinico-demographical profile of keratomycosis in Delhi, North India. Indian J Med Microbiol 2014;32:310-4. Source of Support: Nil, Conflict of Interest: None declared.
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