|Year : 1969 | Volume
| Issue : 5 | Page : 171-200
Dr. E. V. Srinivasan Oration- Primary keratomycosis
Bangalore Medical College and Minto Ophthalmic Hospital, Bangalore, India
Bangalore Medical College and Minto Ophthalmic Hospital, Bangalore
|How to cite this article:|
Puttanna S. Dr. E. V. Srinivasan Oration- Primary keratomycosis. Indian J Ophthalmol 1969;17:171-200
President, fellow members of the All-India Ophthalmological Society, ladies and gentlemen,
I deem it a great honour to deliver Dr. E. Srinivasan's Oration today and I am highly thankful to the Scientific Body of the All-India Ophthalmological Society for having chosen me to deliver the oration, this year. Dr. E. Srinivasan was a pillar of strength to our Society and was instrumental in the organization of our Society and was one of its founder-members. He was an eminent ophthalmic surgeon and was a constant member at each annual conference, easily recognisable by his colourful turbans. His efforts elevated the status of our Society and steered it from its infancy to its present stature. I had the privilege of knowing him towards the end of his active and busy life. He was interested in several phases of ophthalmology, including detachment surgery. His name has been associated with dystrophies of the cornea and finds a place in Duke Elder's Text Book. He led a busy life, took active part in scientific discussions and his comments were sharp, critical based on his long and rich experience. He served the profession honestly and truthfully and breathed his last just after finishing an operation for detachment of the Retina.
I have selected `Primary Keratomycosis' as the subject of this oration as I have been interested in this field for the past 10 years. Corneal ulcers are of every day occurrence and the communities working in farms, quaries and mining areas, are the major sufferers of corneal blindness in our country. Corneal ulcers pose a challenging problem to the ophthalmologists, as their etiology is of varied origin and their course unpredictable, prognosis uncertain, a specific therapy being still a confused issue, as effective specific antifungal broad-spectrum agents are not still available. Corneal ulcers occur more frequently in elderly debilitated, arteriosclerotic and anaemic persons. They occur among the lower classes exposed to injury and prone to neglect the infection. They occur especially during harvest season and by the time the patient seeks medical aid, the ulcers are much advanced and may recover without perforation but with opacities of varying density or end in perforation and its sequalae leading to blindness.
From January 1967 to December 1967, there were 301 cases of corneal ulcers admitted as inpatients to the Minto Ophthalmic Hospital. [Table - 1] shows the maximum incidence to be in the months of June, July, September and October of 1967. It also indicates the absolute age and sex incidence.
I confine myself to the mycotic aspect of the corneal ulcer which is coming more into light for the past decade, specially, after the advent of anti-biotics and corticosteroids, which are often used indiscriminately.
According to Kronfeld  "our defences against the fungi are still meager and confusing." The known antifungal agents like nystatin and amphotericin B have been tried in some cases with variable results." This means that we are still short of broad-spectrum antifungal agents.
| Incidence of Fungal Infections|| |
Since the report of Leber's  (1870) on fungus keratitis, more than 30 species of saprophytic fungi have been identified as the causative agents of primary exogenous keratomycosis. A review of the literature for the past 13 years shows a definite increase of fungal infections all over the World (Mikami and Stemmermann  ; Chick and Conant  ). Haggerty and Zimmermann  ) reported a fifteenfold statistical increase (1:77) in fungal infections of the eye since the introduction of Cortico-steroids from the incident rate of 1:11329 of previous years. A similar report is by Thygeson, Hogan and Kimura  ).
Surveys reveal that fungi are found in various parts of the human body with incidence as high, as 80 per cent on tongue (Benham and Hopkins  )-, 63.6 per cent in ear canals (Haley  ); and 49 per cent in tonsillar crypts (Bernard, Race and Guy  ). Seligmann  and Vogel, Michael and Timpe  have reported the systemic dissemination of fungus infection, after systemic cortisone therapy. Conant, Smith, Baker, Callaway and Martin  stated that Mycotic Keratitis was mainly due to potentially pathogenic fungi.
As regards corneal infections, the literature is full of clinical case reports since Leber's  in 1879 ,,,, .
As regards Indian authors Sreenivas Rao and Ramakrishna  have reported 13 cases of fungal infection out of 40 cases (33%) of corneal ulcers in his series from Mangalore and Agarwal and Khosla  6 cases isolating Candida Albicans and Aspurg. fumigatus in 2 cases of each and Fusarium and Candida krusic in 1 each. We  have been able to diagnose 34 cases within 10 years.
It is to be noted that in quite a number of studies of un-inflamed eyes fungi have been cultured. [Table - 2] gives an indication of the wide variations in the results obtained by different authors.
[Table - 2],[Table - 3],[Table - 4],[Table - 5],[Table - 6] show frequency of positive cultures from healthy eyes and the reported sites of infection by foreign authors.
Variations in frequency do occur on geographical variations and climatic conditions as in sporotrichum (McGrath and Singer  ). Penicillium predominates in temperate climates and can exist at a lower optimum temperature and Aspergillus thrives in tropics and requires higher optimum temperature but less moisture. Of Fazaka's  258 positive cultures, 71 were of the penicillium species. There appears to be a seasonal variation in the incidence of fungi, the maximum incidence being in the relatively cool and humid months (Sreenivasa Rao et al  ). Thus, ecological differences are responsible for variation in fungal flora of different regions and in our series the maximum number of fungal infections were due to the Aspergillus species. Of course, each genus includes a diversity of organisms, different species vary in pathogenicity and within each species there may be different strains which likewise vary, thus complicating the therapeutic approach. Hammeke and Ellis  report that the incidence of positive cultures below the age of 16 years is lower (4.8 per cent), as compared with adults (10.3 percent). Thus elderly individuals are more prone for fungal infections.
A number of studies indicate that indiscriminate use of antibiotics and corticosteroids potentiate fungal infections of the eye or enhance the pathogenicity, though they may not be the actual cause of the infestation by fungi. Antibiotics and corticosteroids have been variously implicated separately or in combination for this potentiation. ,,,48],,,, .
Even so, a primary fungus keratitis is possible as shown by our own experimental studies in which we successfully produced fungal keratitis in rabbits' cornea by aspergillus fumigatus, penicillium and caphalo-sporium species. (Puttana  ). We have also reported primary aspergillus after the instillation of herbal juices used as native medicines for the eyes. (Puttana  ). Other authors who have reported primary type of mycotic keratitis are Balkrishnan  and Drault-Toufesco  . Wilson, Sexton and Ahern  have reported corneal ulcer from mycotic affection of the skin due o philophore verrucosa.
Non-pathogenic fungi can be converted into pathogenic ones in the presence of mixed infection, as shown experimentally by Agarwal and Khosla  or in the presence of cortico-steroids (Ley  )
In vitro experiments also have proved the unholy effects of indiscriminate use of antibiotics and corticosteroids ,,, Tanaka  has shown, in vitro, the inhibiting effect of chlortetracyclin on C. Albicans in higher concentration (0.25 per cent) and a stimulating effect in a concentration of less than 0.01 per cent.
There is also evidence to show that there has been an increased incidence of systemic fungal infections, along with the ophthalmic, after antibiotic or steroid therapy ,, .
It can be concluded that corticosteroids and antibiotics used indiscriminately or the presence of other nonpathogenic or pathogenic microorganisms can convert a non-pathogenic fungus into a pathogenic one or increase the virulence of pathogenic fungi.
The synergistic effects of these therapeutic preparations or of mixed infections on fungal infections have been attributed variously to different reasons, e.g. the growth and/or the virulence of the pathogens may be directly altered or the protective action of the normal flora is interfered with, or the host cell permeability is altered, permitting the pathogen to become more readily established. (Huppert, McPherson and Cazin  ).
Steroids either in combination with antibiotics or without them are freely prescribed not only by ophthalmologists but also by general practitioners and non-ophthalmic specialists. The abuse of corticosteroids has done more harm than good as in herpetic keratitis and has hastened the progression of the superficial diseases of the cornea to perforation and made the opportunistic fungi like cephalosporium, penicillium and fusarium species to become pathogenic.
Secondly the suppressive, anti-inflammatory effect of corticosteroids may delay the clinical diagnosis of a possible fungal infection which is partly made from the antibiotic resistant characters of fungal infections.
| Fungi and their Classification|| |
Fungi have no chlorophyll in them and are unable to synthesize carbohydrates from the carbon dioxide of the air and hence are heterotrophic, i.e., they depend on prepared food materials supplied to them. They are either parasitic or saprophytic in habit. Reserve carbhydrates in fungi is glycogen. They reproduce by means of specialized cells called spores which on a suitable substrate germinate, producing one or more filamentous processess called germ tubes. These elongate into filaments and each filament is called a hypha. (See diagram)
These hyphae branch, rebranch and intertwine to form a network called mycelium. The mycelium seen above the surface of the medium is aerial mycelium and that below the surface for food collecting is called the vegetative mycelium. The aerial mycelium is reproductive in function and bears spores.
At first, the hyphae are like tubes without any partitions (non-septate). When they divide into chains of cells separated by transverse walls (septa) they are called septate-hyphae. In some fungi, the hyphae remain nonseptate, wherein the protoplasm flows freely uninterrupted through the hollow tubes or cells which contain many nuclei. They are cenositic, i.e., capable of living apart from their host.
It is necessary to be familiar with the following terms while talking of spores of yeasts and fungi.
Spores = Reproductive elements of one of the lower organisms.
Exospores or conidia = Nonsexual spores arising from the end of the hyphae (conidiophores) by budding. These can be micro-(small) or macro(large).
Conidiophore = The specialised branch of the mycelium which bears conidia
Endospores or gonidia = Sexual spores formed in the interior of special hyphae (sporangia).
Zoospores = Endospores that are free and provided with locomotive flagelle. Their cases are known as zoosporangia.
Zygospores = Spores formed by a conjugation between two special hyphae which bear morphologically identical cells and do not show sexual differentiation.
Oospores = spores formed by the conjugation of two sexually differentiated elements.
Chlamydospores = Spores enclosed by two envelopes, the whole forming the reproductive organ.
Sporangiophore = The thread-like stalk which bears at its tip the sporangium of molds.
Sporangium = The encysted structure containing the spores. When it ruptures the spores are scattered about.
Perithecium = A cup or flask-shaped envelope enclosing the fructification of certain fungi and molds.
The classification is based on (1) type of colony, (2) presence or absence of mycelium, (3) type of mycelium, (4) methods of spore development, (5) characteristics of the spores - shape and size of spores.
Fungi are broadly grouped into
I. Pseudomycetes (False fungi)
a) Schizomycetes (bacteria)
b) Myxomycetes or slimy molds (non-pathogenic)
II. Eumycetes (true fungi)
d) Deuteromycetes or fungi imperfecta.
The distinguishing characteristics of the different eumycetes are tabulated in [Table - 8].
Asexual spores on which the classification of fungi imperfecti is based, are of two major types:
(a) Conidiospores : produced by a specialised hypha (conidiophore) arising from septate hyphae and mycelium.
(b) Thallospores : formed by the thallus or congregation of ovoid or round-cells, which represent the simplest form of plant-life (e.g. sea-weeds and algae) are allied to the mycelium of molds. They arise by budding as cells from the parent cells. Sometimes the spores remain encased in a thickwall and form a resting or hybernating stage (chlamydospores). Sometimes the segmentation of the daughter cells result in rectangular or pillow-shaped spores, and assume a septate appearance and are therefore called pseudohyphae (Arthrospores).
From a clinician's point of view, it will suffice to divide fungi into molds and yeast groups. Molds present dry cotton-wooly growths seen on aging foods and have a net work of mycelium. In tissues, molds grow as hyphae beneath the surface and spores are rarely seen unless they reach the surface, where they can fruit off.
Yeasts are round or ovoid-celled organisms and reproduce by budding, as daughter cells. A number of pathogenic fungi are dimorphic, i.e., they resemble molds when growing as saprophytes and assume an yeast form when leading a parasitic life in animal tissues. Some of the yeast-like fungi may form fragile hyphae-like chains called pseudo-hyphae. Fungi and their spores are found everywhere and their number of species run into thousands. Of these, about 50 are pathogenic to man.
True yeasts remain in the Yeast form at both room temperature (20° C) and incubator (body 37°C). They produce no mycelium and reproduce by budding.
Candida species produce soft yeast-like colonies at room and incubator temperatures. The aerial portion is yeast and the vegetative portion contains elongated buds remaining attached to the parent cell which on repeated division produce a chain of attached cells somewhat constricted at their point of division but resembling hyphae-pseudo-hyphae. This colony is not a true yeast but is yeast-like.
Mode of entry : Fungi gain entry into the eye in the following manner.
i. Exogenous by direct invasion of the external eye as in fungal conjunctivitis, fungal keratitis, fungal infection of the lacrimal passages. Infection may extend to deeper tissues, e.g., various strains of aspergillus, candida, nocardia, cephalosporium, actinomyces etc.,
ii. After instillation of herbal drops as a part of native treatment, or through indiscriminate use of antibiotics and cortico-steroids.
iii. Extension from infected neighbouring structures as in fungal dermatitis, nasopharyngitis, sinusitis.
iv. Lack of personal hygiene specially in women suffering from fungal vaginitis and allied fungal disorders.
v. May gain entry into the inner eye by perforating wounds or during operation or postoperatively causing fungus endophthalmitis.
vi. Haemotogenous spread often missed specially when they are from an ocult source.
| Factors Favouring Mycotic Infections of the Cornea|| |
There is evidence to show that even non-pathogenic species under certain conditions assume pathogenic character and proliferate on, and penetrate into tissues producing necrosis. This is specially seen in patients with low resistance as in debilitated individuals, diabetis, leukaemia and cancer.
The contributing role of corticosteroids and antibiotics have already been commented upon. This is due to the fact that the normal balance that exists between bacteria and fungi have been disturbed and the opportunistic fungi gain the upper hand and proliferate readily and rapidly under such favourable conditions. So prolonged therapy with antibiotics and corticosteroids favour fungal infections of the eye.
Any minor trauma of the cornea favours such invasion, because mycotic infections never occur spontaneously upon an intact healthy cornea. Hence trivial injuries to the cornea, especially in the fields caused by a cow's tail while milking, twigs, thorns, infloresence of paddy or ragi or jola and other field crops or dusty grains which may contain these fungal elements can well be the cause of mycotic infections of the cornea.
Women suffering from fungal vaginitis may inadvertently infect their eyes due to lack of personal cleanliness. An investigation of fungal flora of female reproductive tract by Dr. A. P. Rao showed that 8 % among the pregnant group and 4% among the non-pregnant group had no symptoms of vaginitis or cervicites, yet showed positive cultures for fungi and the fungi isolated in their study is shown in [Table - 9]. Hence it is worth our while to investigate the fungal flora of female reproductive tract in case of hypopyon ulcers.
Diagnosis of mycotic ulcers of the cornea : Diagnosis of fungus infection of the cornea presents a problem specially in the initial phase. The diagnostic failure is due to the difficulty of identification by the common methods used for isolation and staining technique, by the slow growth of fungi on culture media and by the absence of pathogenic features to differentiate them from more common bacterial and viral infections. If the diagnosis is delayed or incorrect, the treatment of the ulcer with antibiotics, steroids or both in combination favours the rapid proliferation of the organism resulting in perforation and loss of the eye. Hence the importance of early and correct diagnosis. Proper laboratory technique for isolation and the sensitivity tests will help in the selection of proper anti-fungal agents to combat the infection.
In most cases, a history of trauma with vegetable matter, branches and twigs, etc., or instillation or herbal drops or injury with cow's tail or falling of saw dust should make one suspect the ulcer to be of mycotic origin. Previous treatment with antibiotics and steroids may also be helpful in diagnosis.
A mycotic ulcer presents as a whitish, yellowish or greyish slightly raised plaque or an ulcer with circumscribed edges, surrounded by infiltrative zone associated with hypopyon and runs a slow chronic course. A recalcitrant hypopyon ulcer uncontrolled by the usual antibiotics, should arouse suspicion of the possibility of mycotic infection.
Many species of fungi produce pigment especially on primary isolation and some strains are strong pigment producers and retain this ability under all circumstances. Hence, a pigmented corneal ulcer is presumably of mycotic origin.
Clinically, a fungal ulcer starts as a fluffy white spot with satellitic lesions which coalsce, soon breaking into a shallow ulcer with a surrounding infiltrate and hypopyon soon follows. Distinct branching lines radiating from the margin of the ulcer are seen in the corneal stroma and these precede the satellite lesions. The extension of these hyphate margins beyond the edge of the ulcer helps to differentiate it from the bacterial ulcer which possesses a shaggy necrotic margin which undermines the ulcer. The entire lesion appears elevated well above the surrounding cornea. The ulcer soon extends both in area as well as in depth. Inflammatory signs are proportionately mild and the tension does not rise inspite of massive hypopyon showing that iris and ciliary body are severely involved. The administration of steroids may result in sloughing of the cornea, soon ending in perforation with subsequent loss of the eye. Like bacterial infection, the fungal hyphae may be held back by the resistant Descemet's membrane for a time as they proliferate along the cleavage plane between the stroma and the membrane but unlike bacteria the hyphal elements can penetrate an intact Descemet's membrane and thus gain entry into the inner eye.
Slit lamp examination may help in diagnosing the mycotic ulcer. Folds in Descemet's membrane may be seen with flare in the anterior chamber. A white plaque at the endothelial level is suggestive of mycotic origin.
Hypopyon ulcer occurring in a farmer with a history of trauma by a cow's tail or a twig or cornstalk or inflorescence of rice, ragi or jola or of instillation of any herbal drops, should raise suspicion of fungus origin as it has occurred in agricultural surroundings.
Examinations of corneal scrapings to rule out bacteria as well as fungi should be a routine procedure. If earlier examination of the scrapings does not reveal any pathogenic bacteria, a fungus infection of the cornea should be entertained. Although surface discharge may reveal fungal elements, scrapings from beneath the surface at the edge of the ulcer where the tissue and organisms are not yet quite so necrotic, should be taken and examined both by smear and cultural methods. Gentle wiping or swabs often give a negative culture as mycotic elements tend to invade deeper tissues. Culture is more positive than scrapings. The tissue obtained by debridement or curetting the ulcer is good material for diagnotic purpose. While taking material for culture, it is essential to rinse the conjunctival sac with sterile normal saline after instillation of local anesthesia as anesthetics contain preservatives, which are anti-fungal as well as antibacterial. The Gram stain or Giemsa stain or lactophenol blue and P.A.S. are sufficient to stain the fungi. Histopathological examination with special stains, Gomoris Methenamine, Silver Nitrate technique and Gridley's method used for fungi are more reliable. Where smears are reported negative, culture may be positive and so the culture tubes should not be discharged for at least three weeks as fungi grow slowly. Repeated smears and cultures should be taken in all cases of corneal ulcers presumably caused by unidentified micro-organisms. Artifacts must be distinguished from fungi. The mosaic fungus, oil droplets, vegetable fibres, hydroxide crystals, fat laden macrophages, erythrocytes and amorphous debris may be confused with various forms of fungi and this pitfall should be avoided while examining smears. Positive cultures should not be ignored as merely culture contaminants, specially so when the culture is positive from the cornea of a farmer engaged in agricultural operations. Contaminations are minimal when Litman Oxgall agar is used for primary isolation.
| Types of Keratomycosis|| |
The clinical picture of mycotic ulcer of the cornea presents pleomorophic features, varying from individual to indivial, largely depending on the type of mycotic organisms, the severity of the invading pathogen and its strain, the corneolytic toxin, the resistance of the host tissue and on the age of the patient. It runs a tragic course in debilitated and old individuals and in diabetics. An attempt is made to classify the ulcer according to the etiological agent.
Aspergillus Keratitis : Aspergillus is one of the commonest mycotic organisms that invade the cornea, tearduct and orbit. It belongs to the ascomycetes group of true fungi. Generally, in Aspergillus keratitis superficial layers of the cornea are involved and a typical ulcer is described as a raised circular gray plaque with well demarkated rolled edges. But other types of corneal lessions, like "Cauliflower like elevation" are described (Bothman and Crowe.  ) Corneal ulcers with perforation have been reported (Castroviejo and Munoz Urra  . In our series of mycotic infections of the cornea, aspergillus was responsible for the majority of infections (16 cases). Puttanna, Ramanda Rao, Sirsi and SwamiNathan  reported primary Aspergillus keratitis and have produced experimentally inflammatory response on animal's cornea by injecting culture isolate of the fungus from the clinical cases. Aspergillus flavus oryzae, A. niger and A. nidulans, A. fumigatuswere the strains isolated from the clinical cases and the inflammatory response was not uniform in all cases.
Experimental injection of Aspergillus niger into rat's cornea produced marked keratitis and left a vascularised thin scar in the course of 3 weeks [Figure - 10]. Sections showed giant cells engulfing fungal elements [Figure - 11]. Aspergillus nidulans resulted in mild keratitis resolving in 3 weeks.
I would like to report four cases of Aspergillus keratitis to illustrate the varied inflammatory response seen experimentally on animals.
The first case was of a farmer's wife who gave a history of injury to the left eye by a cotton plant twig while working in the field and had received some native treatment before coming to the hospital. She had a circular corneal ulcer 5 x 5 mm in diameter, hypopyon 5 mm in height and low tension. There was yellowish exudate in the pupillary area. The lacrimal sac was infected which was excised. Corneal scrapings from the ulcer showed hyphae [Figure - 1]. The culture from the corneal scraping and from the fluid from the lacrimal sac was positive for Aspergillus flavus oryzae group on Sabouraud's media at 37° C. The characteristic features of the isolate on Czapek's agar are seen in [Figure - 2],[Figure - 3],[Figure - 4].
The culture was injected into the stroma of rat's cornea at 2 o'clock position at the limbus of the left cornea, the right eye being kept as control.
Signs of marked Keratitis were noticed after 72 hours (Rat 3) [Figure - 5], and the left cornea of rat 8 became infiltrated and ulcerated in 3 days and perforated on the 9th day with bleeding from the ulcer (rat 8-[Figure - 6]). Marked inflammation limited to the anterior segment of the eye was observed on sectioning.
The second case was that of a farmer aged 60 years with a history of injury to the right eye by an inflorescence of jola of 15 days duration. He had an ulcer extending over almost the whole cornea leaving the periphery clear from 10 o'clock to 4 o'clock with a dense pigmented area in the centre, 3 mm. in diameter, surrounded by a less denser area and hypopyon, 4 mm in height [Figure - 7]. Corneal scrapings revealed no bacteria but fungal hyphae were seen. Culture from the scrapings were positive for Aspergillus niger (grown on Saboururaud's media at 37° C. [Figure - 8],[Figure - 9] show the characteristic features of the isolates on Czapek's agar.
Scarification with material from the identified culture on rat's cornea produced fairly marked keratitis. Injections into rat's cornea at 2 o'clock position produced marked keratitis which finally resolved leaving a thin scar which was vascularized from the periphery in the course of six weeks [Figure - 10]. Fungus elements were also demonstrated from the corneal scrapings of the rat. The scrapings from the rat's cornea when cultured was positive to Aspergillus niger. Microscopic examination of a section of the enuclated eyeball shows giant cells engulfing fungal elements. [Figure - 11]
The third case was of a farmer aged 25 years, with a history of injury of 8 days duration from an inflorescence of jola. He had an ulcer cornea L.E. 4 mm in diameter at 6 o'clock position extending towards the centre [Figure - 12]. Scrapings from the cornea showed no bacteria but were positive for fungal hyphae [Figure - 13]. Culture for bacteria was negative but was positive for Aspergillus nidulans on Sabouraud's media at 37°C.
The characteristic features of the isolate on Czapke's agar can be seen in [Figure - 14],[Figure - 15].
Injection of the culture to rat's cornea at 2 o'clock position produced mild keratitis and the keratitis became localised at the centre and resolved finally in about 3 weeks. Fungus elements were demonstrated from the scrapings of the rat's cornea.
The fourth case was of a farmer aged 35 years with a history of foreign body falling into the left eye while working in the field. Eight days later he had a central ulcer 3 mm in diameter with hypopyon 3 mm in height pupilary area filled with yellowish exudate. [Figure - 16]. Corneal scrapings were positive for fungus hyphae and negative for bacteria. Culture was negative for bacteria but fungus grew on Sabouraud's media at 37°C. The colonies on Sabouraud's media were more or less flucose with slight tufted aerial mycel. lium to deep felted or extremely flucose forms, white at first, becoming green with the development of conidial heads. Seen on the reverse, the substratum of Sabouraud's media had changed to yellowish tinge. Conidial heads were columnar compact and the fungus was identified as Aspergillus fumigatus [Figure - 17].
The culture isolate was injected into the centre of the right and left cornea of an albino rat which had previously received cortisone eye drops for one week in the right eye only, the left eye being kept as control. Signs of keratitis were seen in three days in both eyes, the right side being more pronounced than the left. Note reverse nounced than the left. The right cornea became infiltrated and ulcerated in the course of nine days and reached the climax in 22 days.
The culture isolate was also injected into rabbit's cornea which resulted in marked keratitis with engorgement of limbal vessels of both eyes [Figure - 18],[Figure - 19]. Positive signs were noticed on the third day, climax being reached on the 12th day. The left eye [Figure - 19] which did not receive cortisone developed marked keratitis. The scrapings from the rat's cornea revealed hyphae.
The animal experiments showed that the inflammatory response varied widely in the same genera of Aspergillus both in animal cornea and in human lesions. Further the eyes which did not receive cortisone also developed lesions thus proving that primary fungus keratitis could be a clinical entity.
Leber  reported Aspergillus as the causative agent in a hypopyon ulcer. Gingrich  has reported two cases of Aspergillus keratitis due to flavus and A. fumigatus of which ore case ended in perforation and staphyloma formation and the other in Descemetocele followed at a later date by keratoplasty, giving hand motion vision. Castroviejo and Munoz-Urra  have reported 2 cases. In Fazaka's  11 cases of corneal involvement, six were primary and five were secondary. Of the six primary cases three were superficial keratomycosis and three were keratomycosis profunda. The secondary infections were associated with dendritic ulcers, ulcus serpens, eczematous pannus and inclusion catarrah. The same author reported that almost 50 percent of the secondary mycotic invader in 20 primary viral or bacterial infections of the external eye belonged to Aspergillus group.
Agarwal et al  (1965) in their experimental fungal uveitis came to the conclusion that bacteria in combination with fungus produce severe clinical picture which may probably be an additive effect. They are of opinion that Aspergillus being a larger filamentous fungus resists invasion by the cellular defence mechanism and is probably more resitant to bacterial antibiotics.
Histopathological studies show advanced necrosis with a fibrinous network of mycelia. Leucocytes surround the lesion while round cells infiltrate the iris and ciliary body. Hypopyon is due to the chemotactic effect of the corniolytic toxin liberated by the fungus. The perforated ulcer in our experimental series (Rat No. 11) on section showed giant cells engulfing fungal elements as in [Figure - 13].
Candida Keratitis: Candida albicans, the causative agent is an yeast-like organism. Cornmeal agar is used to differentiate the albicans species from the non-pathogenic types. On this medium it appears as treelike branching filaments with large round chlamydospores. This affects lids, conjunctiva, cornea, tear duct and uvea. In our series we had four cases infected with candida species. Mendelblatt  reported the first case of corneal involvement due to candida albicans. Gingrich  reported the two cases of keratomycosis due to candida albicans which responded to Thimerosal ointment I : 5000.
Corneal ulcers due to candida are shallow and indolent with undermined and infiltrated edges (Birge  ) The floor of the ulcer is usually covered with a thin dry membrane which adheres to the corneal tissue. It is associated with iritis and hypopyon. The other type of corneal lesion is superficial punctate keratitis (Mathur  ). Vanwinkle, Melvin, Rehins and Suie  have produced experimental candida corneal infection by scarification method and showed 66 per cent of the infected eyes which received antibiotic treatment developed severe corneal ulceration. Graf  reported a corneal ulcer in a 53 year old man after injury due to candida albicans proved by culture and pathogenicity proved experimentally on rabbits' corneas. The ulcer responded to electro-coagulation and topical application of iodine. Manchester  (1957) has reported corneal ulcer due to candida parapsilosis. Ainley and Smith' reported secondary keratomycosis due to candida parapsilosis which responded to mystatin 25000 units ml. The same species of fungus can produce quite different lesions of varying severity from a benign ulcer to progressive necrotic lesions ending in perforation [Figure - 20],[Figure - 21]. Hanabusa  has shown that candida may elaborate a heat stable toxin capable of producing conjunctivitis.
Fusarium, Keratitis : Fusarium is a soil saprophyte which may cause potato wilt can invade plant and human tissue. It can be isolated from almost any soil or water sample. Septate mycelium is white, conidia are fusiform, conidiophores simple produced on loose mycellium, conidia terminal, single. The infection is said to occur at the time of injury with plant tissue. Sigtenhorst and Gingrich  reported for the first time Fusarium as an etiological factor in fungus keratitis. Puttanna  reported corneal ulcer infected with cephalosporium and fusarium species of fungus. Gingrich  reported 3 cases of keratitis due to fusarium species of fungus, which perforated inspite of treatment. Mikami and Stemmermann  have reported a case of keratomycosis caused by fusarium oxysporium from Hawaii. Anderson and Chick  reported 9 mycotic corneal ulcers of which four were caused by fusarium and responded well to amphotericin B. therapy.
Puttanna  has also reported another case of corneal ulcer due to fusarium species of fungus after instillation of herbal juice used as native treatment. It is believed that the fungus entered the eye through the herbal juice or through the grass leaf used to scratch the ulcer as part of native treatment [Figure - 22],[Figure - 23]. Fusarium keratitis appears to be toxic as the ulcer ended in perforation inspite of treatment. The fungal elements from culture were injected into rat's cornea which showed signs of keratitis 24 hours after injection and a circumscribed ulcer six days after injection. It is not known whether it produces a proteolytic enzyme which dissolves the cornea as does cephalosporium serrae.
Fusarium ulcer though circumscribed was covered with brownish pigmented slough which made us suspect that it could be of mycotic origin.
Cephalosporium Keratitis : Colonies on Czapke's agar spreading felty to fluccose. Pure white consisting of creeping, septate, dichotomously branched hyphae, reverse side colourless. Conidiophores arise as branches of aerial hyphae, short upto 25 µ long. Conidial heads round, conidia elongate, ellipitcal hyaline 9 to 10 µ x 3.5 - 4. Bedell  reported a corneal ulcer from an injury of a cow's tail from which Cephalosporium was isolated. Puttanna  reported a case of corneal ulcer due to cephalo-sporium species. Injection of culture isolate into rat's cornea produced marked keratitis at the site of injection which spread steadily for the first two weeks, finally resolved leaving a thin scar in the course of six weeks. Fungus elements were also demonstrated from the corneal scrapings of the rat. Gingrich  reported a case of cephalosporium keratitis which ended in perforation inspite of treatment with amphoterecin B and sulphacetamide iontophoresis. Burda and Fisher  have extracted a potent proteolytic enzyme from cephalosporium serrae which produced corneal destruction in 2 to 4 hours in rabbits by ulceration and exudative liquifaction and are of opinion that the proteinase may be responsible for the clinical picture of cephalo-sporium keratitis. The exact action of the enzyme is still unestablished and is said to act on the collagen bundles present in the stroma of the cornea. The proteinase activity appears to be active during the initial phases of germination and proliferation of the spores.-Byers, Holland and Allen. 
Penicillium Keratitis : Hyphae creeping, conidiophores erect apically irregularly whorled, penicillately (tufted or brush-like) branched, conidia spherical, hyaline. Early colony white, round, semitranslucent, radiating silk like hyphae. In a course of four days, the colour changes to dirty green and a spore zone appears. These have sterigmata and produce spore chains similar to Aspergillus but lack in vesicle at the end of the conidiophore. In its place the conidophore has numerous branches each with a sterigmata that produce a chain of spores. Thus this will have a brush like (penicillate) appearance [Figure - 24]. Puttanna  reported a case of corneal ulcer due to penicillium species (Ramigena series) and the culture isolate when injected into rat's cornea produced slight keratitis at the site of injection. Scrapings from the rat's cornea showed hyphae. In our series there were 3 corneal ulcers due to the Penicillium species.
Rhizopus Keratitis (phycomycetes group of fungi) phycomycetes are found in tissues as branching nonseptate hyphae measure 5-40 µ in diameter. On artificial media these fungi grow rapidly, forming serial mycelia and characteristic spores of each genera. Puttanna  reported two cases of Rhizopus keratitis after instillation of herbal juice as a part of native treatment [Figure - 25]. The culture isolate was injected into rat's cornea, which showed keratitis 48 hours after injection and there was a haemorrhagic patch on the iris six dys after injection. [Figure - 26]. The cornea became opaque and bulged with prominent keratitis after 7 days.
Lasio diplodia keratifis : Puttanna  reported two cases of Lasiodiplodia l; eratitis after instillation of herbal juice as part of native treatment. White woolly colonies at first, changed in course of time to black resembling the colour of the pigment seen in the centre of the ulcer. Spores were seen after 12 days [Figure - 27]. The culture isolate was injected into rat's cornea and keratitis developed 72 hours after injection which reached its climax after 6 days [Figure - 28]. The corneal scrapings of the rat was positive for fungal elements. Fungus inoculated in the stem of a plant showed luxuriant growth [Figure - 29]. Lasio diplodia appears to be toxic like fusarium clinically as well as in experimental animals.
Cryptococcus Keratitis : Cryptococcus neoformans the causative agent of torulosis does not produce mycelia or spores. In artificial media the colonies are mucoid and appear whitish to tan. Individual cells measure 5 to 20 µ . are round to ovoid and are surrounded by wide, gelatinous, polysacharide capsules. Budding is evident. Fazakas  reported a case of Keratomycosis due to cryptococcus. He describes the corneal involvement as being deep and very extensive which resulted in thin opacity rather transparent.
Prognosis : Prognosis in a mycotic ulcer of the cornea is still unpredictable. It depends on the host resistance, severity of the pathogen, type and strain of the invading fungi and the corneolytic substance produced by it, the available fungi-static or fungicidal agents, early diagnosis, and institution of energetic and immediate treatment. The response to antifungal agents is very varied and no uniformity has been observed. This may be due to poor penetration of the drug and the fact that myceliae have a tendency to burrow deeper into the tissues where the drug does not reach and also due to nonavailability of a universal broad spectrum antifungal agent. Further, the exact nature of the corneolytic toxin is not yet known to us. The known antifungal agents that are in use have side effects as well as toxic to normal tissue. Therapeutic keratoplasty offers an hope but it should be considered before the ulcer spreads in depth as well as in area. Further, lamellar or penetrating keratoplasty depends upon the type of fungus whether mycellial or yeast like type isolated from the culture.
Prophylaxis : The only key to the problem is prevention. Early diagnosis is very essential and many eyes can be saved if the routine repeated smear and cultural examination is followed in every case of ulcer cornea, specially in those engaged in agricultural occupation. Any recalcitrant corneal ulcer which does not respond to the usual line of treatment should arouse suspicion of mycotic ulcer. The fact of secondary micotic infection should also be borne in mind while instituting prolonged corticosteroid therapy for eye conditions. A careful history of trauma to the eye by vegetable agents like corn-stalks, inflorescence, twigs or branches, instillation of herbal juice or native medicine may aid a presumptive diagnosis of mycotic infection.
| Treatment|| |
Therapy of mycotic keratitis can be considered under three sub-divisions (1) Prevention, (2) Medical, (3) Surgical.
Prevention : Farmers should be educated regarding the dangers of mycotic infections and should be advised to wear protective safety glasses to avoid dust and minor trauma to the eyes. Introduction of mechanical devices in agricultural occupations may minimize the incidence of mycotic ulcers, of the cornea. Personal cleanliness in washing hands before touching eyes is essential to avoid fungal infections from outside agencies as in fungal vaginitis. To prevent post-operative infections and possibility of infection during operations, care should be taken to avoid air-borne organisms, spores in glove powder, inadequate sterilisation of instruments by soaking in contaminated solution and drugs and fungi in the ocular and periocular tissues of the patient. Pre-operative prophylaxis with antifungal agents like sodium propionate, argyrol or amphotericin B eye drops is advocated. Routine antibiotic programme and lavish postoperative steroid therapy should be reorientated and should be used with great caution as both are double edged swords. Routine use of fungicidal sprays in the operating room is desirable. Rigid operating room cleanliness by modern methods should be adopted to see that no dust is stirred up during cleaning. Precautions against contamination of fungi from outside agencies like street dust, shoes and other uncovered clothing should be rigidly followed.
Medical: Physical measures suggested in the treatment of mycotic ulcers of the Cornea are (1) Ultraviolet phototherapy as suggested by Chick, Huddell and Sharp  , (2) Thermophore suggested by Allen. 
Various forms of chemo-therapy like 30% Sodium Sulphacetamide solution by iontophoresis, 1: 1000 Merthiolate ophthalmic ointmentt, oral Sodium Iodide with local 0.125% Copper Sulphate and Tincture Iodine, Amphotericin B and nystatin and local cycloheximide have been tried with varying results. The disappointing results are due to poor intra-ocular penetration and local toxicity of the recent anti-fungal drugs. Some fungi are sensitive to some of the sulphonamides and some of the antibiotics. Others seem to flourish in the presence of these agents.
Leber  (1879) was the first to diagnose and report a clinical case of mycotic keratitis, to reproduce the disease in animals and to cure his clinical case he used a combination of curettage and chemical cautery with carbolic acid. The treatment begins with debridement of devitalized tissues which provides material for smear and culture. Debridement helps in the removal of nutrient clinical medium for the fungi and better evaluation of the ulcer and facilitates penetration of the available antifungal drugs used as drops or ointments. Puttanna  has reported good clinical response to instillation of 0.125% copper sulphate and 10%, sodium propionate drops and ointment combined with systemic iodine. Puttanna, Ramananda Rao, Sirsi and Swaminathan  have conducted the antifungal activity of potassium iodide 1%, copper sulphate 1%, salicylic acid 1%, phenol 1%, sodium propionate 20%, Pimaricin 200 u, Mycostatin 200 u. on Aspergillus strains in vitro and showed that copper sulphate, sodium propionate, pimaricin and Mycostatin have antifungal effects. Sodium sulphacetamide has slight fungistatic properties and requires very long term therapy.
Sightenhorst  et al (1957) found sulfacetamide and thimerosal to have general activity against 25 fungi tested in vitro. Thiomersal inhibited all 25 fungi cultures in concentration from 0.0005 to 0.01 mg per ml indicating the greatest sensitivities of all the fungi to all the preparations tested.
Gingrich  advocated 30% sodium sulfacetamide iontophoresis (using the negative electrode and at least 3.0 ml. of solution with a current of three to five ma. for 5-10 minues, every six to eight hours for the first five days. This is followed by Thiomerasal 1 in 1000 ointment at the close of each iontophoresis and repeated three hours later. The active antimycotic factor is heavy metal mercury which like ionic copper or nickel, penetrates the well wall and oxidizes the essential sulphydryl group of enzymes necessary for vital metabolic pathways.
Miller and McCallon  found that silver ions one part per million were effective in releasing cell contents of fungus spores and inhibiting germination of spores. Argyrol is a mild silver protein preparation containing 20% silver and is used preoperatively in Massachussets Eye and Ear Infirmary of Boston and absence of post-operative fungal infections is claimed.
Thiomersal (Merthiolate) is used in a dilution of 1 in 1000 of physiological saline as drops every hour during the day and every two hours at night. It is often combined with oral and topical nystatin.
Allen  recommended oral sodium iodide one gr. to 10 lbs. body weight, three times a day (about 3 gm. per day, for the average male patient). He also advocated focal heat with the Thermophore. for localised or superficial infection. Cautery with tincture of iodine and topical application of 0.125% copper sulphate are also recommended.
A few antifungal agents that have come into recent use are amphotericin B. and nystatin derived from Streptomyces cultures. These have poor penetrating power except by direct introocular injection. These are used either as drops or as ointments. They are effective only on the superficial corneal layers and the fungal elements may persist in deeper layers. Both are highly toxic and have side effects. Mangiarcin and Liebman  have successfully treated a case of hypopyon keratitis due to Aspergillus fumigatus with nystatin. Montana and Serry  showed definite inhibition of candida albicans infection of rabbit's cornea by nystatin and amphotericin B. Sensitivity tests with these agents demonsrated fungistatic activity in fairly low dilutions. Dieckhues  treated keratomycosis with Nystatin and Pimaricin.
Amphotericin B is fungistatic against a wide variety of yeast and fungi including Coccidiodes, Histoplasma. Blastomyces and Candida. It is heat labile and light sensitive. It is used in 5% dextrose and is given intravenously in concentrations of 0.1 mg/cc. as it is not readily absorbed from the gastrointestinal tract.
Chick, Huddell and Sharp  used ultra-violet rays with amphotericin B in the form of eye drops 1.0 mg/ml and felt that higher concentration upto 4 mg /ml could be used. Currie  reported 3 cases of mycotic keratitis which responded to topical application with amphotercin B. In each of these, there was a history of injury to the eye by vegetable matter. Fine  observed that amphotericin B is very irritating to the infected human eye. The drug is too toxic and hence should not be used in vague and undiagnosed conditions. Dosage should be adjusted to minimise toxic effects. Daily dosage should not exceed 1.5 mg/kg of body weight. The initial daily dosage is 0.20 mg/kg to be gradually increased as tolerated. It should be given under close clinical supervision. Side effects like chills, fever, headaches, anorexia and chemical thrombophlebitis have been observed. Kidney function tests and blood urea nitrogen levels should be checked periodically if therapy is prolonged. Sub-conjunctival injection of 125 mg of amphotericin B is tolerated. Drops are used as often as every 15 minutes.
Nystatin : inhibits the growth of a wide variety of fungi, molds and yeasts. It is reasonably well tolerated as a topical application in ointment form containing 100,000 units/gm or by subconjunctival injection of 5000 units suspended in 0.5 cc saline. Dieckhues, Jper and Fegeler  reported a case of corneal fungus infection treated successfully with Nystatin.
Gingrich  found that Nystatin was effective for the early superficial fusarium keratitis but ineffective for the deep infection because of poor penetration. Roberts  used Nystatin in the treatment of Monilia Kerato conjunctivitis.
Casero  reported a case of Aspergillus Keratitis recovered under local and general mycostatin therapy and cephalo-sporium keratitis responding to the instillation of Tricomycin and another case caused by Aspergillus fumigatus recovered under the instillation of tricomycin.
Gricofulvin (Sporostanin) is orally administered and effective in cutaneous fungus infections like athlete's foot. It is of doubtful value in corneal affections though Batista  reports good response by oral administration. Morales  reported a case of mycotic corneal ulcer which cleared up by oral administration of Grisofulvin.
Hamycin : Ahuja Bal, Nath and Nema  tried Hamycin in Glycerine suspension on experimental Keratomycosis and are of opinion that the drug appeared to be effective in candida infections and not with Aspergillus fumigatus.
Thiabindazole : 2-4'-Thiazolyl)benzimidazole. This drug is unusually potent and broad spectrum anti-helimethic. Besides, it possesses in vitro anti-mycotic properties against saprophytic and some pathogenic fungi. Blank and Rebell  found that it is both fungistatic and fungicidal in vitro against the fungi of dermatophytosis, mycetomas and chromomycosis. It is slightly soluble in water and a 0.3% solution was tried in kerato-chromomycosis by Wilson et al  . A 12% suspension in 1% sodium Carboxycellulose might be more effective.
Cycloheximide was used by Lynn  in the treatment of Fusarium keratitis. Cycloheximide is an antifungal antibiotic used in Mycology Laboratories to inhibit the growth of "Saprophytes in pathogenic fungus cultures. Cycloheximide was tolerated by the human eye in aqueous concentration of 0.06%. It has good lipid solubility and unlike Nystatin and Amphotericin B is also soluble in water at concentration of 2%; so one could predict that its ocular penetration would be good.
X 597 C (Hoffman-la-Roche) a new drug is of value in systemic mycosis such as histoplasmosis, blastomycosis and in few cases of systemic aspergillosis.
The curent therapy of Mycotic Keratitis as recommended by Anderson et al begin with Amphotericin B Solution 0.25-0.4% every one to two hours. If this fails, Mycostatin 10,000 µ/gm. is then used as an ointment with the same frequency of application.
The prolonged medical therapy of fungus disease suggests that the biochemical composition of the fungus is a complicated one and demands administration of more potent anti-fungal agent to kill the fungus and excite the host's immunologic reaction, but no such agent has yet been discovered. Lack of response to medical treatment may mean that the therapeutic agent is of low specific activity against the invading pathogen or it may mean that insufficient drug reaches the site where it is needed. Before surgical measures are thought of, the current antifungal armamentarium should be given a fair trial.
Pimaricin, a fungicide isolated from streptomyces natalensis is effective against spores. Puttanna et al  have shown its antifungal activity on different species of aspergillus invitro and appears to be effective. Francois obtained good results with pimaricin, the dose being 50 mg. four times daily in combination with local pimaricin. Kaufman and Wood  used with favourable result pimarcin in a case of Keratomycosis.
Polymyxin : Jawetz (1956) reported that polymyxin B in vitro in high concentrations (20-250 µg/ml) may be fungicidal or fungi-static against several organisms including candida albicans.
Surgical treatment : Physcal and chemical cautery, paracentesis, keratectomy and excision with keratoplasty and conjunctival hooding have been tried with varying results. Medical cure often leaves an ugly corneal scar and if central, will interfere with vision which may need keratoplasty at a later date. Keratoplasty has been advocated in active mycotic infection as a theraputic measure. Successes and failures are on record. Gingrich  has performed successful keratoplasty 56 hours after perforation of the fungal ulcer due to cephalosporium serrae with vision of handmotion 2 months later. Some are of opinion that keratoplasty should be considered only after control of the situation to avoid further spread of the fungi resulting in resistant emdophthalmitis. McLean  (1963) has stated in regard to fungal keratitis "Certainly it would seem preferable to recognise the situation, control the fungal infection if possible and graft the healed scar in the absence of active infection. The type of keratoplasty-Lamellar or penetratitng depends on the depth and area of mycotic infection and the type of fungus, mycelial or yeast type. A penetrating keratoplasty is probably contra-indicated in the presence of an active yeast infection, as the organism may gain entry into the anterior chamber due to lack of mycelial barrier. Berson, Kobayashi and Oglisby  in their experimental fungal keratitis produced by candida albicans confirm this view. The mold type of fungi with their hyphae interwoven prevents the organisms from falling off into the anterior chamber during penetrating keratoplasty. So an early penetrating keratoplasty is recommended in case of central mycotic ulcer due to mold species of fungs. Dhanda  recommends lamellar kerato-plasty in mycotic ulcers in the early phase before the lesion spreads deeply in the corneal stroma. The trephine disc should be located in the healthy recipient bed and the dissection should proceed deeper to the infiltrating zone. The size and thickness of the graft depend on the extent of the ulcer. According to Kaufman and Wood  . lamellar keratoplasty is contraindicated and preserved cornea should never be used if one is driven to do lamallar keratoplasty in mycotic keratitis. However, decision should be taken with careful balancing of the calculated risks and each case deserves balanced judgement.
Conjunctival Hooding : Since our experience has shown that it is very difficult to evaluate the stage of the disease at the time of the treatment and the fungi differ from case to case, it is worthwhile to consider this method as a practcal one as there is difficulty in our country to get suitable eyes for therapeutic keratoplasty. As we are still in darkness regarding the mechanism of pathogenicity in various types of keratomycosis and looking at the confusing results obtained from medical therapy, the conjunctival hood method should be tried at an early phase if medical treatment is unsuccessful. The rationale of this treatment is still not clear but recent studies suggest a serum-fungal inhibiting factor rather than a conjunctival antifungal substance. Kozinn, Caroline and Taschdjian  , Lorincz, Priestly and Jacob.  The flap may not contribute to the strength to the thin cornea but the vessels of the flap may help in rapid healing of the diseased tissue.
Cryotherapy : is also suggested. A slow freeze to the range of 44° c with a thaw might disrupt the hyphal elements and result in destruction of the tissue mycelium.
| Conclusion|| |
The problem of therapy of mycotic ulcers of the cornea is still today an open chapter and the future line of research should be directed in detecting a more potent, less toxic, broad spectrum antifungal agent and to determine the exact nature of the corneolytic substance produced by the pathogen. If the toxin is species specific the therapy of fungal ulcers becomes more confusing and complicated. As there is some degree of anti-biosis between bacteria and fungi, as cultures for bacteria are negative it is worthwhile to observe whether there is any antibiosis between nonpathogenic bacteria and fungi and this may throw a new light in our therapeutic approach to fungal ulcers in the future. This study is still in progress.
To summarise, I would once again stress the early diagnosis clinicallly confirmed by rapid laboratory diagnosis by smear, culture and sensitivity test if possible to select the drug of choice. If the medical treatment fails it is safer to adopt the simple surgical procedure of conjunctival hooding fairly early to save the eye rather than think of therapeutic keratoplasty, whose results are still unpredictable so far in the surgical treatment of mycotic keratitis.
In conclusion, I am deeply thankful to the Scientific Committee of the All India Ophthalmological Society for having nominated me to deliver the oration this year in memory of Dr. E. Sreenivasan and I consider it a great honour to do so and if my remarks have revealed the growing and challenging problem of keratomycosis facing us today and the use of antibiotics and corticosteroids more sparingly and precisely, I think we have honoured the person, as he was cautious and conservative in therapeutic approach inspite of tempting new technique and new drugs coming to light in the advancing field of Medicine.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7], [Figure - 8], [Figure - 9], [Figure - 10], [Figure - 11], [Figure - 12], [Figure - 13], [Figure - 14], [Figure - 15], [Figure - 16], [Figure - 17], [Figure - 18], [Figure - 19], [Figure - 20], [Figure - 21], [Figure - 22], [Figure - 23], [Figure - 24], [Figure - 25], [Figure - 26], [Figure - 27], [Figure - 28], [Figure - 29], [Figure - 30]
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7], [Table - 8], [Table - 9]