Mycological profile of infectious Keratitis from Delhi
Background & objectives: Corneal blindness is a major health problem worldwide and infectious keratitis is one of the predominant causes. The incidence of fungal keratitis has increased over the last few years. Though a few studies have been carried out on mycotic keratitis from north and other parts of India, there are none from Delhi. Keeping this in mind, this study was conducted to evaluate the frequency of positive fungal cultures in infectious keratitis and of the various fungal species identified as aetiologic agents in patients attending a tertiary care hospital in East Delhi.
Methods: Corneal scrapings from 346 patients of corneal ulcer with suspected fungal aetiology were subjected to direct examination by 10 per cent KOH mount, Gram stain and culture. The results were examined retrospectively and analyzed.
Results: Of the 346 patients of corneal ulcer investigated, in 77 (22.25%) cases fungal aetiology was identified. Males were more commonly affected and were mostly in the age group of 31-40 yr. It was seen that trauma was the most common predisposing factor especially in the agriculturists and the farmers. Aspergillus flavus was the most common fungus isolated in 31.16 per cent cases, followed by A. fumigatus (16.88%) and Fusarium spp. (7.79%). Yeasts were also isolated in 21.62 per cent cases. Both yeasts and mycelial fungi were isolated in 6.5 per cent of cases.
Interpretation & conclusion: Because of serious consequences of infectious keratitis, it is important to know the exact aetiology of corneal ulcer to institute appropriate therapy in time. Laboratory confirmation should be undertaken and fungal infection should be ruled out before prescribing corticosteroids and antibacterial antibiotics.
Key words Aspergillus * corneal ulcer * fungal keratitis
Mycotic keratitis is an important ophthalmologic problem especially in outdoor workers in tropics. It is an important cause of corneal blindness and usually carries an unfavourable prognosis due to its protracted course and requirement of specific therapy. A report on the causes of blindness worldwide consistently lists corneal ulceration second only to cataract as the major aetiology of blindness and visual disability in many of the developing nations in Asia, Africa, and the Middle East1. The diversity of clinical presentation observed in each case and also new emerging cases each year pose a diagnostic and therapeutic challenge to the ophthalmologists. More than 70 genera of filamentous fungi and yeasts have been identified as the aetiological agents of fungal keratitis2. The prevalence of individual pathogens largely depends on geographical and climatic factors. Keratomycosis occurs mainly in the warm climates and coincides with seasonal increase in temperature and humidity. Trauma, particularly by vegetative or soil matter, seems to be the most common predisposing factor for keratomycosis3. Apart from that, injudicious use of topical corticosteriods and antibacterial agents for external ocular disease enhances the risk further4. It may also occur though rarely among the contact lens wearers5 or by retained intracorneal hair2.
Though studies on mycotic keratitis have been carried out in various parts of India, no report is available from eastern part of Delhi. This study was thus undertaken to evaluate the frequency of positive fungal cultures in infectious keratitis and of the various fungal species identified as aetiologic agents in the patients attending a tertiary care hospital in East Delhi.
Material & Methods
This retrospective study comprised a total of 346 patients of corneal ulcer treated and investigated in the Departments of Ophthalmology and Microbiology of University College of Medical Sciences (UCMS) and Guru Teg Bahadur Hospital (GTBH), Delhi from January 2000 to December 2004. Thirty patients suffering from bacterial, Herpes simplex and Acanthamoeba keratitis were excluded from this study. Data collected included age, sex, information on the history of the infectious process as well as the use of corticosteriods, topical antibiotics or herbal medicine, previous eye surgeries or any condition of immunosuppression, or use of cosmetic or therapeutic contact lens.
In all cases, collection of corneal scrapings for analysis was carried out directly from the base and margin of ulcers aseptically using Kimura’s spatula under direct vision through slit lamp after instillation of anaesthetic eye drops (4% xylocaine). Direct microscopy was performed in 10 per cent KOH wet mount and smears were prepared from each sample for Gram stain also for the demonstration of hyphae, pseudohyphae and yeasts cells. For fungal cultures samples were seeded on the following media: two sets of Sabouraud’s dextrose agar (SDA) with antibiotics chloramphenicol (50 mg/ml) and gentamicin (20 mg/ml) and maintained at 25 and 37°C separately over a period of four weeks; one set of brain heart infusion agar (BHIA) containing chloramphenicol (50 mg/ml) and cycloheximide (actidione) (500 mg/ml) were maintained at 25°C for 4 wk and observed daily. All culture media and antibiotics were obtained from Hi-media Laboratories, Mumbai, India. The characteristics considered for fungus identification were macroscopic aspects of texture, colour, growth rate and microscopic aspects such as mycelium and conidium types, relationship between hyphae and fructification organs by lactophenol cotton blue mount. Micro culture on slides was the technique used for observation of filamentous fungi6. The yeast isolates were identified by standard tests like germ tube, different spore production on corn meal agar (CMA) or rice starch agar (RCA), urease production and sugar fermentation and assimilation tests6.
The results were considered positive when smear results were consistent with culture or growth of the organism was demonstrated on two or more media with negative smear results or repeated appearance in smear with negative culture results.
Statistical analysis: Statistical analysis was done by Modules Exact test of SPSS.
Out of the 346 cases of corneal ulcer investigated, in 77 (22.25%) a fungal aetiology was identified. Of these. 60 (77.9%) were male and 17 (20.07%) female, with a male : female ratio of 3.5:1. The most common age group affected was between 31 to 40 yr in both sexes (Fig.).
Unilateral mycotic infection was observed in 73 patients and bilateral in 4. In cases with bilateral affection of the eye, the same fungus was isolated from both the eyes. Of the 346 cases studied, direct microscopic examination was positive in 67 (19.36%) and in 74 (21.38%) cases, fungi were isolated by culture. Three cases revealed sterile culture inspite of positive direct microscopic findings and in seven cases only cultures were positive. Mycelial fungi were isolated in 58 (78.37%) cases and yeasts in 16 (21.62%) cases. In 5 (6.5%) patients both yeasts and mycelial fungi were isolated. There was an 87.93 per cent (51/58) agreement between the direct examination and culture for filamentous fungi, whereas the same for yeasts was 100 per cent (16/16).
The annual frequency of corneal ulcer and those with a positive culture is shown in Table I.
Among the identified filamentous fungi, most were hyaline. Of the 77 positive specimens, the most frequent agent isolated was Aspergillus flavus in 24 (31.16Cf). followed by Aspergillus furnigatus in 13 (16.88%), Fusarium species and Candida albicans in 6 each (7.79%). The frequency of isolation of both filamentous fungi and yeasts was greater in the year 2004 (Table II).
Occupationally 30 of the 77 cases (38.96%) of mycotic keratitis were agriculturists/farmers, 7 housewives (9.09%), 7 students (9.09%), 12 labourers (15.58%) and the rest were from various walks of life like tradesman, drivers, merchants, factory workers and tailors.
Trauma appeared to be the most common predisposing factors associated with positive fungal isolations (Table II). However, association of filamentous fungi with predisposing factors verses association of yeasts with predisposing factors did not show any significant difference (Modules Exact test of SPSS 13.0; P=0.373).
Corneal blindness is a major public health problem worldwide and infectious keratitis is one of the predominant causes. The incidence of ocular fungal infections has increased in the last few years due to the improvement in microbiologie diagnostic techniques and because of introduction of new therapeutic measures such as widespread use of broad-spectrum antibiotics, immunosuppressive drugs and corticosteriods7.
Prevalence of mycotic keratitis may vary depending upon the country of origin. In India it varies between 6-46.8 per cent in various regions (Table IV). In the present study the occurrence was 22.25 per cent, which was similar to that reported from northern India8,9. However, higher precentage positivity was reported from eastern, western and southern parts of India (Table IV). This regional variation could be because fungal keratitis was expected to be more common in the tropical and subtropical regions than in the temperate regions.
As has been reported worldwide as well as from India, Aspergillus species is the most common isolate in fungal keratitis8. Our results also correlated with this fact. However, Fusarium species was found to be the most common cause of fungal corneal infections in Southern United States, Florida. Brazil. Ghana. Nigeria, Paraguay and Columbia as well as from some parts of south India (Tamil Nadu)1,17,19.
Mycotic keratitis may occur at any age. but highest incidence coincides with the period of maximal activity. In our study also the most affected age group was 31-40 yr, i.e., the active age group and men outnumbered women. Men are involved more commonly than women, and agricultural workers and outdoor manual labourers constituted the largest affected occupational group in our study similar to other studies11,15,18. In contrast, Koiogadde et al from Karnataka13 and Dutta et al from Gauhati12 have reported higher incidences in females as compared to males.
Injury to the eye is an important predisposing factor. We have obtained a definite history of antecedent corneal injury in about one third of our patients, which was in agreement with the findings of other workers from north India9. In our study 3.5 per cent patients gave history of use of topical herbal medicine before ophthalmologic examination. It may be of interest to know that use of topical medicine in the form of breast milk, castor oil, seed oil, onion extract, extract from flower, honey, steam and even dropping of chicken blood have been reported from south India18.
Some patients gave history of receiving topical antibiotics with corticosteroids empirically without any culture being done. It was only in the event of indolent and progressive nature of the ulcer that the patient was referred to a tertiary care hospital where cultures were obtained revealing the fungal aetiology of the infection. A survey carried out in Southern California revealed that about 50 per cent of patients with a clinical diagnosis of infective keratitis were treated with antibiotics without any cultures being obtained20.
In conclusion, the key element in the diagnosis of mycotic keratitis is the clinical suspicion by the ophthalmologists and laboratory confirmation of the fungus before prescribing corticosteroids and antibacterial antibiotics. Therefore, precise identification of the causative fungus and institution of .appropriate treatment strategy could save the eye from this preventable cause of blindness.
Authors thanks the Department of Ophthalmology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi for sending the corneal scraping samples for mycological examination.
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Rumpa Saha & Shukla Das
Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi, India
Received April 6, 2005
Reprint requests: Dr Rumpa Saha, Pool Officer, Department of Microbiology, University College of Medical Sciences &
Guru Teg Bahadur Hospital, Dilshad Garden, Shahdara, Delhi 110095, India
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