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Clinical Microbiology Reviews, October 2003, p. 730-797, Vol. 16, No. 4
0893-8512/03/$08.00+0 DOI: 10.1128/CMR.16.4.730-797.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Ocular Microbiology, Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli 620001, India
SUMMARY INTRODUCTION ETIOLOGICAL AGENTS AND LABORATORY DIAGNOSIS OF OPHTHALMIC MYCOSES Etiological Agents Hyaline filamentous fungi. Dematiaceous (phaeoid) fungi. Yeasts and zygomycetous fungi. Thermally dimorphic fungi. Organisms of uncertain taxonomic classification. Laboratory Diagnosis Direct microscopic detection of fungi in ocular samples. Culture. Sensitivity testing of fungi isolated from ophthalmic lesions. PCR. PATHOGENESIS Putative Agent Factors in the Pathogenesis of Mycotic Keratitis Invasiveness. Toxigenicity. Putative Host Factors in the Pathogenesis of Mycotic Keratitis ANTIFUNGAL AGENTS USED TO TREAT OPHTHALMIC MYCOSES General Considerations Polyenes Natamycin. Amphotericin B. Azoles Miconazole. Ketoconazole. Itraconazole. Fluconazole. Miscellaneous Compounds Polyhexamethylene biguanide. Chlorhexidine. Silver sulfadiazine. CLINICAL FEATURES, PREDISPOSING FACTORS, AND MANAGEMENT OF SPECIFIC OPHTHALMIC MYCOSES Fungal Infections of the Orbit Acute rhinocerebral (rhino-orbito-cerebral) zygomycosis. (i) Surgical debridement and restoration of sinus drainage. (ii) Intravenous amphotericin B. (iii) Other therapeutic options. Chronic rhinocerebral zygomycosis. Treatment of fulminant infections caused by non-Mucorales fungi. Orbital aspergillosis. Mycotic Infections of the Eyelids Mycotic Dacryocanaliculitis Mycotic Dacryocystitis Mycotic Dacryoadenitis Mycotic Conjunctivitis Conjunctival rhinosporidiosis. Mycotic Keratitis (Keratomycosis) Risk factors. Fungi causing mycotic keratitis. Diagnosis. (i) History and clinical features. (ii) Noninvasive techniques. (iii) Microbiological investigations. Management. (i) Specific antifungal therapy. (ii) Measures to suppress corneal damage due to microbe- or host tissue-derived factors. (iii) Therapeutic surgery. Mycotic Scleritis Intraocular Mycoses (Excluding Endophthalmitis) FUNGAL OCULAR INFECTIONS AFTER OPHTHALMIC SURGICAL PROCEDURES OPHTHALMIC MYCOSES ASSOCIATED WITH AIDS OPHTHALMIC MYCOSES ASSOCIATED WITH OCULAR BIOMATERIALS FUTURE RESEARCH IN OPHTHALMIC MYCOSES Diagnostic Methods New Antifungal Compounds Pathogenesis of Ophthalmic Mycoses ACKNOWLEDGMENTS REFERENCES
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A third problem is in assessing the accuracy of the genus or species identification of a fungal strain isolated in culture. For example, a fungal strain isolated from a patient with keratitis was initially identified as Arthrobotrys oligospora but later reidentified as Cephaliophora irregularis (128); C. irregularis was subsequently isolated from another patient with keratitis as well (235). Similarly, a filamentous fungus isolated from an intraocular lesion arising out of a retained contact lens was identified as Scedosporium prolificans (19); it now appears that this identification may have been erroneous (J. Guarro and J. Gené, Letter, J. Clin Microbiol. 40:3544, 2002).
To overcome these limitations, reports of single cases or small numbers of patients were considered acceptable for this review if they satisfied criteria similar to those described earlier (237): when an adequate clinical history was presented that suggested a mycotic infection; when the fungus was seen in the clinical specimens; and when the morphology of the fungus in the clinical specimens was consistent with the reported etiologic agent. Papers describing a series of patients with keratitis (120, 334) or other ophthalmic infection (313), many of which were based on retrospective analysis of patient records, were assessed differently since such publications rarely provided detailed descriptions of the fungi isolated from individual patients or of the appearance of the fungi in the specimens or tissues. The observations made in these papers were considered valid if definite criteria had been used to assess the significance of the fungi isolated; for example, the presence of clinical features suggesting a fungal infection, growth of the same fungus from repeated samples, growth of the same fungus on two or more solid media, or confluent growth at the site of inoculation in one solid medium with direct microscopic demonstration of fungal hyphae or yeast cells in the sample (85, 120, 208, 216, 364, 377).
A recent review of fungal infections of the eye (194) listed exceptions to the rule requiring isolation of the fungus from ocular tissue. The exceptions listed included entities such as endogenous endophthalmitis, in which fungi known to cause this disease had been isolated from blood culture and the clinical presentation was compatible with vascular dissemination of the fungus; histoplasmosis and coccidioidomycosis, which are commonly associated with characteristic chorioretinal lesions and in which isolation of the fungus from another anatomical site or measurement of titers of antibody to the fungus is usually deemed sufficient evidence to establish one of these fungi as the cause of the eye disease; and ophthalmic infections due to Cryptococcus neoformans, which usually occur in conjunction with meningoencephalitis and in which isolation of cryptococci from blood and/or cerebrospinal fluid is usually sufficient to explain the associated eye findings. Most of these exceptions pertain to reports of intraocular mycoses, whereas the present review highlights external ophthalmic infections.
In this review, fungal genera and species are cited as they have been reported in the literature. Unfortunately, in the majority of published reports, the strains have not been deposited in recognized culture collections to permit others to confirm the validity of the identifications; moreover, there is a need to apply modern molecular biological and other methods to the process of identification of fungi in the future (129; J. Guarro and J. Gené, Letter, J. Clin. Microbiol. 40: 3544, 2002). Hence, at present, only an uncritical compilation of the fungal genera and species as reported is possible.
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An overwhelming number of fungal genera and species have been implicated as causes of ophthalmic mycoses, and this number is steadily increasing. Species and genera of fungi implicated as genuine ophthalmic pathogens in the past 5 years include Chrysosporium parvum (415), Metarhizium anisopliae var. anisopliae (76), Phaeoisaria clematidis (131), and Sarcopodium oculorum (132). In this review, no attempt has been made to list every single fungal genus or species implicated in ophthalmic infection, given the limitations listed above. Instead, the salient features of the most important genera and species are highlighted, since it appears that only a relatively small number are repeatedly isolated in ophthalmic mycoses or have been isolated from more than one ocular site (Tables 1 to 5). For purposes of simplicity, the fungal genera and species have been grouped as hyaline filamentous fungi (Table 1), dematiaceous fungi (Table 2), yeasts and zygomycetes (Table 3), thermally dimorphic fungi (Table 4), and organisms of uncertain classification, namely, Pythium insidiosum, Rhinosporidium seeberi, and Pneumocystis carinii (Table 5). In Tables 1 to 5, brief descriptions and line drawings are included to highlight the salient microscopic morphological features of some ocular fungal pathogens which may be unfamiliar to most clinical microbiologists; more intricate details are provided in other papers and specialist mycology texts (50, 237, 238, 325, 329, 373).
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Aspergillus spp. abound in the environment worldwide, thriving on a variety of substrates such as corn, decaying vegetation, and soil. These fungi are also common contaminants in hospital air (367) and have been implicated in a recent outbreak of endophthalmitis following cataract surgery that was traced to ongoing hospital construction (375); they are also implicated in other types of ophthalmic mycoses.
Scedosporium apiospermum (teleomorph Pseudallescheria boydii) (Fig. 2) has been isolated from soil, sewage, and polluted water and from the manure of farm animals (373). It has been reported to cause severe ocular infection following trauma by plant material, contact with polluted water, and immunosuppression (211, 325, 379, 430). The fungus Scedosporium prolificans, which was first described as a human pathogen in 1984, has been reported as a cause of sclerokeratitis (202, 370).
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Species of Acremonium (Fig. 2) are widespread, occurring in soil, decaying plant material, and the air (129). Several cases of keratitis (93, 237, 315, 317) and occasional cases of endophthalmitis (93) due to Acremonium spp. have been reported in the literature.
Dematiaceous (phaeoid) fungi. The primary factor unifying the dematiaceous fungi (Table 2; Fig. 3) is the dark pigmentation of their hyphae (238). At least 20 species of fungi belonging to 11 different genera have been implicated as causes of keratitis (the most frequently reported ones are listed in Table 2). Dematiaceous fungi have been reported to be the third most frequent cause of mycotic keratitis (behind Aspergillus and Fusarium) (111, 120, 208, 288, 364, 383) and may also cause infections of the orbit (164, 167, 233 W. J. Chang, C. L. Shields, J. A. Shields, P. V. De Potter, R. Schiffman, R. C. Eagle, Jr., and L. B. Nelson, Letter, Arch. Ophthalmol. 114: 767-768, 1996) or intraocular infections (182). These fungi exhibit a brown-to-olive-to-black color in the cell walls of their vegetative cells, conidia or both, colonies thus appear olive to black.
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Ocular infections by the zygomycetes (Table 3; Fig. 5) include rhino-orbitocerebral zygomycosis (435) and keratitis (231). Although Rhizopus spp., especially Rhizopus arrhizus, are most frequently involved, other genera of the order Mucorales may also cause ocular disease (87, 323, 435). The detection of fungi belonging to the Mucorales by direct microscopy in clinical material or tissue sections (Table 3) is more significant than their isolation in culture (323, 324).
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Blastomyces dermatitidis (Fig. 6), which has been isolated from moist soil with high organic content, is known to cause pulmonary, cutaneous, osteoarticular, and genitourinary disease (373). Ocular infections include eyelid lesions (26, 355; G. C. Barr and J. W. Gamel, Letter, Arch. Ophthalmol. 104:96-97, 1986), orbital disease (215, 409), keratitis (332), and endophthalmitis (215, 338).
Histoplasmosis is classically caused by Histoplasma capsulatum var. capsulatum, while a variant form, known as African histoplasmosis or large-celled histoplasmosis, is caused by H. capsulatum var. duboisii. The disease is most prevalent in the central region of North America, in Central and South America, in the tropics, and in certain river valleys in temperate regions (373). H. capsulatum var. capsulatum has been implicated in the "presumed ocular histoplasmosis syndrome" and in several other ophthalmic infections, mostly of intraocular structures (118, 180, 224, 303, 424); H. capsulatum var. duboisii has been reported to cause orbital disease (5).
Sporothrix schenckii (Fig. 6), which has been isolated from soil and decaying plant material worldwide, generally causes nodular lesions in the cutaneous and subcutaneous tissues, which ultimately suppurate, ulcerate, and drain. This fungus has been reported to cause lesions of the orbit (369), sclera (I. Brunette and R. D. Stulting, Letter, Am. J. Ophthalmol 114:370-371, 1992), and intraocular structures (205).
Organisms of uncertain taxonomic classification. Pythium insidiosum (Table 5), a cosmopolitan fungus-like aquatic organism, is found predominantly in swampy environments, where water lilies, various vegetables, and especially certain grasses support the asexual phase of its life cycle; motile zoospores, which appear to be chemotactically attracted to plant leaves or human and horse hairs, are the likely infective particles (244). This organism, originally considered to be an oomycete in the kingdom Fungi and later a member of the kingdom Protoctista (244, 373), is now placed in the kingdom Stramenopila, containing organisms that are related to algae (373). P. insidiosum has been implicated in diseases of plants and animals (horses, cattle, dogs, cats, or fish), particularly in tropical and subtropical parts of the world (22, 155, 260, 381). In Thailand, this organism causes subcutaneous lesions and chronic inflammation and occlusion of blood vessels (especially of the lower extremities) in thalassemic and nonthalassemic patients (381). Keratitis due to P. insidiosum has been noted in tropical (22, 155, 244, 411) and temperate (260) regions. Two particularly aggressive cases of orbital cellulitis with deep facial tissue involvement have occurred in the United States (244).
Rhinosporidium seeberi (Table 5; Fig. 7) an endosporulating microorganism which causesrhinosporidiosis, has traditionally been considered a fungus but is now of uncertain taxonomic classification (295). Lesions of rhinosporidiosis manifest as polypoid or papillomatous, very friable, proliferative outgrowths principally in the nasal cavity; ocular lesions may account for 13% of all lesions, with the ratio of nasal to ocular lesions being 1.4:1 (284).
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The ability to detect and differentiate gram-positive and gram-negative bacteria within 3 min in an ocular sample is the most important function of the Gram stain (329) (Table 7); an additional advantage is that fungi (Fig. 9), filamentous bacteria, and cysts of the protozoon Acanthamoeba can also be detected (314, 329). Identification of the fungal genus by direct examination is generally not possible (175, 271). Direct microscopy of corneal scrapes stained by a fluorescent Gram stain technique permitted a rapid presumptive diagnosis of mycotic keratitis in five patients (335); culture confirmed the diagnosis in all five (three infections were due to F. solani, and one each was due to A. flavus and C. albicans). This stain also detected fungi in the vitreous biopsy specimen of one patient with culture-proven endophthalmitis due to A. flavus (335). Advantages of this fluorescence technique over the conventional method need to be assessed by experiments with samples from more patients.
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Lactophenol cotton blue is a mounting medium commonly used in microbiology laboratories for preparing mounts of fungal cultures. This mounting medium has been recommended for the preparation of clinical samples, including corneal scrapes and aqueous and vitreous aspirates, for direct microscopic examination (24). Although lactophenol cotton blue mounts of ocular samples can be stored for long periods, they must be sealed properly to prevent dehydration.
The Gomori methenamine silver (GMS) and the periodic acid-Schiff (PAS) stains are special stains for detection of fungi in tissue. A modified GMS staining technique has been used for this purpose in corneal scrapes (216), in paraffin-embedded tissue sections (406), and in other ocular samples (Table 7). The entire procedure comprises nine steps and takes about 1 h. This stain can also detect filamentous bacteria such as Nocardia and cysts of Acanthamoeba (175). Although widely available, the PAS technique has been infrequently used as a stain for smears from ophthalmic specimens; the reason for this is not known. PAS stains fungal elements well, and hyphae and yeast cells can be readily distinguished; fungal structures were detected in 91% of the PAS-stained sections of corneal buttons which were positive by culture (431).
In recent years, nonspecific fluorochromatic stains have become popular for the detection of fungi in ocular samples. Calcofluor white appears to be the most widely used of these stains (56, 120, 351, 372) since it can detect fungi in 50% of smears previously considered negative by Gram and Giemsa staining methods (372). Calcofluor white is more sensitive than KOH wet mounts in detecting the common ocular fungi F. solani, A. fumigatus, and C. albicans in corneal scrapes (55, 120, 351). A fluorescence microscope fitted with appropriate filters is needed to view mounts of ocular samples that have been stained with calcofluor white. Blankophor and Uvitex 2B, while similar to calcofluor white in many respects, have certain other advantages for detecting fungi in specimens (337, 414) but have apparently not been used widely for the diagnosis of ophthalmic mycoses; the reasons for this are not known.
Several recent studies of small numbers of patients (126, 179) have confirmed that the acridine orange stain is useful to detect fungal hyphae in corneal scrapes. However, the sensitivity of this method in diagnosing culture-proven mycotic keratitis and its specificity when used for patients with ulcerative keratitis need to be assessed in a large series of patients. A fluorescence microscope fitted with appropriate filters is needed for this technique.
Lectins are ubiquitous proteins, which are particularly common in plant seeds that bind specifically to carbohydrates. Fluorescein-conjugated concanavalin A was found to provide consistently bright staining of the fungal structures in corneal scrapes from 18 patients with culture-proven mycotic keratitis (330) and was thought to be a promising first-line fluorochromatic stain to visualize fungi in ocular samples. Again, this technique does not appear to be used as widely as calcofluor white, perhaps because of the cost involved in preparing the necessary reagents.
Garcia et al. (110) have recently described a peroxidase-labeled wheat germ agglutinin staining technique for diagnosis of experimental mycotic keratitis due to C. albicans, A. fumigatus, and F. solani. In addition to excellent sensitivities and specificities for detecting these infections, there was a high degree of test-retest and inter-rater concordance between two independent observers for all three fungi tested. This technique needs to be assessed in the clinical setting, since the use of the peroxidase label for the lectins would eliminate the need for expensive fluorescence microscopes fitted with appropriate filters. One potential disadvantage of this technique is that tissue sections of corneal biopsy material are required, whereas ophthalmologists and patients would probably feel more comfortable if corneal scrapes could be used as the samples.
When fungi such as Candida or Aspergillus are stained with eosin, they fluoresce under UV illumination; this facilitates their detection. Mucin and vegetable fibers do not interfere with this fluorescence (314). Fluorescence microscopy of a tissue section stained with hematoxylin-eosin revealed the presence of yeast cells of B. dermatitidis in periocular cutaneous lesions that had initially been misdiagnosed as squamous cell carcinoma (229).
Because of their size, polysaccharide content, and morphologic diversity, most mycotic agents can be satisfactorily stained and studied in tissue sections by light microscopy. Sections stained with hematoxylin-eosin have many advantages (Table 7), but species of Fusarium or Candida may not be stained at all. Similarly, fungal structures can be easily detected in sections of corneal tissue stained with the GMS or PAS stains (406), but little else can be visualized. Hence, a replicate tissue section stained with hematoxylin-eosin should always be examined before special stains for fungi are used; alternatively, a section stained with GMS can be counterstained with hematoxylin-eosin for simultaneous demonstration of a mycotic agent and the evoked tissue response (57).
Direct immunofluorescence of fungi in formalin-fixed, paraffin-embedded ocular tissue sections has been used to confirm presumptive histologic diagnoses of ocular infection due to B. dermatitidis, H. capsulatum var. capsulatum, S. schenckii, P. insidiosum, and a zygomycete (98, 224, 244, 283, 409). Other dimorphic fungi and hyaline filamentous fungi can also be detected by this technique (57). Factors that have possibly prevented the routine use of immunofluorescence for diagnosis of ophthalmic mycoses include the need for a fluorescence microscope fitted with appropriate filters, antibodies of good quality, and the standardization of reagents and procedures. This technique is especially helpful when atypical forms of an agent are encountered or when infectious elements are sparse. Moreover, for retrospective studies, tissue sections previously stained by the hematoxylin-eosin, Giemsa, and modified Gram procedures can be decolorized in acid-alcohol and then restained with the specific reagents used for immunofluorescence; however, this is not possible with sections previously stained with GMS or PAS (57).
Culture. Even with the advent of many new techniques, culture remains the cornerstone of the diagnosis of most ophthalmic mycoses, except for rhinosporidiosis (since Rhinosporidium seeberi cannot be cultivated) and perhaps rhino-orbito-cerebral zygomycosis, where direct microscopic examination of necrotic material or biopsy samples yields more reliable results (324). Commonly used culture media include Sabouraud glucose neopeptone agar (Emmons' modification, neutral pH) incubated at 25°C, blood agar (preferably sheep blood agar) incubated at 25 and37°C, brain heart infusion broth incubated at 25°C, and thioglycolate broth incubated at 25 to 30°C (271). These media were found to be sufficient to permit the isolation of different types of ocular fungi (216, 334). Using these different media, growth of fungi was identified within 2 days in 54%, within 3 days in 83%, and within 1 week in 97% of patients with mycotic keratitis; a positive initial culture was observed in 90% of scrapings (334).
Other media that have been found useful for primary isolation of ocular fungi include chocolate agar (334), cystine tryptone agar (384) and rose bengal agar (P. A. Thomas, unpublished observations). Since many of these media also support bacterial growth, antibacterial antibiotics, such as chloramphenicol (40 µg/ml) or a penicillin-streptomycin combination, are usually incorporated to suppress bacterial growth and permit the isolation of fungi alone. However, cycloheximide must never be used in culture media meant for the isolation of ocular fungi, since most of the fungi implicated in ocular infections are suppressed by this chemical (271). Wherever possible, it is best to use more than one medium, preferably a combination of appropriate solid and liquid media, and to incubate these at 37°C and at 25 to 30°C for the optimal recovery of ocular fungi; the use of liquid-shake cultures may facilitate the recovery of ocular fungi (398). However, some workers feel that since liquid cultures are prone to contamination by environmental fungi, they should not be used in the microbiological workup of patients with mycotic keratitis, to avoid erroneous results (364, 398). Uninoculated culture media should be incubated for a long period to ensure the sterility of the media used; frequent sterility checks are needed.
Sensitivity testing of fungi isolated from ophthalmic lesions. The clinical relevance of antifungal susceptibility testing is thought to lie in guiding the clinician in the selection of an appropriate antifungal compound. Such tests have been reported to help in the selection of the appropriate antifungal in different ophthalmic mycoses (161, 173, 233, 234). Unfortunately, many of these reports have not provided details of the test procedures used, the criteria by which MICs were deemed significant, details of the severity of the clinical lesions, or the criteria used for authentic diagnosis of mycotic infection. The use of reproducible tests conforming to rigorous standards, such as the approved document (M27A) of the National Committee for Clinical Laboratory Standards (NCCLS) for sensitivity testing of yeasts (261), and a standard method for susceptibility testing of filamentous fungi, especially Aspergillus spp., may clarify in the future whether antifungal susceptibility testing is at all useful in guiding the therapy of ophthalmic mycoses. Interestingly, when the in vitro antifungal susceptibilities of nine isolates of filamentous fungi were determined by the NCCLS method in 11 different laboratories and compared to antifungal treatment outcomes in animal infection models, only a limited association between MIC and treatment outcome was seen, due to drawbacks in the models used (278). Curvularia senegalensis was isolated from a patient with mycotic keratitis, and the MIC of itraconazole for this isolate was found (by a broth microdilution method performed as described by NCCLS guidelines for filamentous fungi) to be 0.25 µg/ml; however, the patient did not respond to antifungal therapy with natamycin or itraconazole (130). Above all, the relationship between in vitro susceptibility data and clinical response to topical antifungal medication needs to be clarified; hitherto, no studies have been performed in this important area.
PCR. Since the revolutionary molecular biology technique of PCR involves enzymatic amplification of even minute quantities of a specific sequence of DNA (Table 8), it is of great benefit in rapidly detecting the presence of organisms which are difficult to culture. Ocular samples which can be submitted for PCR include intraocular fluid (aqueous or vitreous), tears, any fresh ocular tissue, formalin-fixed or paraffin-embedded tissue, and even stained or unstained cytology slides or tissue sections from which DNA can be extracted. Minute samples (1 to 10 µl) of aqueous, vitreous, or tear fluids generally suffice (311). Table 8 summarizes the salient observations of studies employing PCR in the diagnosis of ophthalmic mycoses. The results of all these studies suggest that PCR is more sensitive than culture as a diagnostic aid in ophthalmic mycoses. However, concern persists regarding the specificity of this technique and the problems that may arise from the production of false-positive results. In most of these studies, insufficient detail has been provided to permit an independent assessment of the adequacy of the techniques used for culture. In the diagnosis of ophthalmic mycoses, PCR would probably be most valuable in providing a positive result in a shorter period than that required for culture (91, 92) and in identification of a fungal isolate which does not sporulate (22). Although PCR is more advantageous than the estimation of antibodies in serum or ocular fluids because of its extreme sensitivity and specificity, it cannot be used (unlike serological tests, for which serial antibody titers can be studied) to monitor the patient's response to treatment. PCR does not distinguish viable from nonviable organisms; it may therefore be difficult to assess the relevance of a positive PCR result in a healing corneal ulcer, where culture is negative (7), or in locations such as the conjunctival sac, where fungi may be found as transient commensals (112). A few culture media will suffice to detect and grow the common ocular pathogens, but PCR must be multiplexed for each microorganism that is suspected; the use of panfungal primers may alleviate this problem. Finally, PCR can detect only fungi for which the DNA sequence is known and primers are available; it also does not provide details of cellular morphology or localization (311).
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The key agent factors thought to be involved in pathogenesis of mycotic infections include adherence, invasiveness, morphogenesis, and toxigenicity (Table 9). There is a paucity of data relating to the role of fungal adhesins in pathogenesis of mycotic keratitis.
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Morphogenesis and phenotypic switching permit fungi to adapt to live in different microenvironments and to survive in the infected host (341). The presence of "intrahyphal hyphae" or "hypha-in-hypha," and thickened fungal cell walls (Table 9) may reflect such morphogenesis occurring in fungi invading corneal tissue; these morphological alterations may constitute a barrier against antifungal drugs or host defenses (392, 393) or may be a virulence factor for fungi in corneas where the defense mechanisms have been compromised by the application of corticosteroids (190). Rigorous experimental and other studies are required to elucidate these aspects. Interestingly, in the study referred to earlier (406), there was no mention of the occurrence of such morphological changes in the fungi seen in corneal tissue.
Toxigenicity. Fusarium spp. are known to cause myelosuppression through toxin production (263), but little is known about whether Fusarium toxins such as nivalenol, T-2 toxin, deoxynivalenol, diacetoxyscirpenol and fusaric acid contribute to the pathogenesis of mycotic keratitis (Table 9). The results of two studies (316, 383) suggest that these factors do not make any such contribution, but further investigation is required.
Some other studies have examined the possible role of fungal proteinases in the pathogenesis of mycotic keratitis (Table 9). Clearly, isolates of F. solani and A. flavus from patients with keratitis possess the ability to secrete proteinases (71, 119, 438). What is not clear, however, is whether these fungi actually secrete these proteinases when infecting corneal tissue and whether such proteinases appreciably influence the outcome of such infections. A recent study attempted to correlate the presence of fungal proteinases in vitro and in an experimental animal system (119). When corneal isolates of A. flavus and F. solani were grown in vitro, the fungal cultures were found to contain predominantly serine proteinase activity, and, to a lesser extent, metalloproteinase activity. However, homogenates of rabbit corneas that had been infected with the same strains of A. flavus and F. solani exhibited metalloproteinase activity alone, and no serine proteinase activity; this suggests that although the fungal strains could secrete proteinases in vitro, they did not do so while infecting corneal tissue. None of the available evidence conclusively establishes or refutes the contribution of fungal proteinases to the pathogenesis of mycotic keratitis. This requires the demonstration of fungal toxins and enzymes in situ in fungus-infected tissues (320) in humans. Similarly, the disease produced in experimental animals by fungal strains secreting a particular proteinase or toxin should be more severe than that produced by a mutant not secreting these products. With the rapid strides made in molecular biological techniques, it should be possible, in the coming years, to investigate these aspects.
A transient commensal fungal flora is present in a variable percentage of healthy eyes (363). Fungal conidia from the environment which colonize the conjunctival sac as innocuous commensals possibly turn pathogenic after ocular trauma or corticosteroid use, after which they invade corneal tissue through minute breaks in the corneal epithelium (268). This hypothesis needs to be tested in a suitable experimental model.
In some cases of mycotic keratitis which are responding well to antifungal therapy, a sudden deterioration accompanied by renewed tissue destruction (in the absence of a demonstrable microbial cause) has been noted; this phenomenon is thought to occur because dying fungal hyphae may elicit a type of hypersensitivity reaction (100). This hypothesis also needs testing in a suitable experimental model; if substantiated, it may result in modifications to conventional therapeutic protocols for mycotic keratitis.
Polymorphonuclear leukocytes are known to be pivotal in preventing fungal infections since they phagocytize and subsequently destroy fungal structures by oxygen-dependent mechanisms; the presence of disease or the use of corticosteroids, tetracycline, doxycycline, or certain other drugs may interfere with these mechanisms and hence lower the host resistance to fungal infection (366). Polymorphonuclear leukocytes, other acute inflammatory cells, the corneal epithelium, and keratocytes appear to also play a key role in sterile corneal ulceration (184); however, their role in stromal matrix degradation is not clear. When amidated glucose oxidase was inoculated into rabbit corneas, an initial corneal opacification and a later corneal melting were observed; the initial lesions were thought to arise due to the effects of hydroxyl radicals derived from hydrogen peroxide-generated glucose oxidase, with the later lesions occurring after the release of collagenases and lysosomal hydrolases from invading phagocytic cells (53). In another study, the basal proteolytic activity (65 kDa) detected in uninfected rabbit corneas was shown to reside in matrix metalloproteinase 2 (MMP-2) (119). When rabbit corneas were experimentally infected with A. flavus or F. solani, additional proteolytic activity (92 and 200 kDa) was detected, with the 92-kDa activity being identified as MMP-9. The expression of 92- and 200-kDa gelatinases correlated positively with the number of polymorphonuclear leukocytes in infected corneas. These authors contended that activated corneal cells or inflammatory cells (polymorphonuclear leukocytes) were responsible for the increased proteolytic activities seen in fungus-infected corneas.
Lesions simulating keratitis were produced in rabbit eyes by applying lipid mediators, such as prostaglandins, leukotriene, and platelet-activating factor (395). The urokinase-plasminogen activator system plays an important role in the regulation of collagen synthesis, secretion, and activation during wound remodeling and stromal ulceration (30). MMP-2 and MMP-9, derived from corneal stromal keratocytes, have also been shown to contribute to the degradation of corneal stroma and epithelial basement membrane, respectively (94). It is not known to what extent these various factors contribute to the progression of stromal ulceration in a case of mycotic keratitis, but they certainly need to be considered when dealing with a patient whose keratitis is refractory to antifungal therapy alone.
There is compelling experimental (276) and clinical (366, 394, 428) evidence to suggest that the administration of corticosteroids may predispose humans to mycotic keratitis. This may occur because corticosteroids suppress ocular immune mechanisms by inhibiting chemotaxis and ingestion by phagocytes, by blocking degranulation, and by reducing the production of phagocytes (366). They may also cause changes in the infecting fungal strain itself, the reasons for which are not clear (394).
Traditional eye remedies are routinely used for the "therapy" of eye ailments in many agricultural communities in the developing world. In India, traditional remedies described include extracts of green leaves, the juice of the banyan tree, coconut and castor oil, goat and human breast milk, and chicken blood (120, 388). Fungi contaminating such concoctions could conceivably be carried into the deeper corneal layers when applied to a traumatized cornea. The use of certain oils may be associated with excessive corneal irritation, thus predisposing to mycotic keratitis. Experimental studies may help to clarify the validity of such hypotheses.
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The concentration of the drug applied to the eye may be increased by the preparation of fortified eye drops (107), but this is not generally done for antifungals. Frequent topical application of drops is a useful means of achieving therapeutic levels in the eye, but this is laborious and may cause irritation. Ointments and subconjunctival injections may prolong the contact time between the antifungal and the corneal and conjunctival tissue. Only amphotericin B and miconazole are available as ophthalmic ointments. Subconjunctival injections can be painful for the patient and inconvenient for the physician (107) and can cause damage to the ocular tissue at the site of injection (236).
Collagen shields, iontophoresis, and pumps have all been used in an attempt to enhance drug delivery to the eye. The use of iontophoresis and pumps has not gained acceptance, and these techniques should now probably be considered obsolete. However, the collagen shield, which is shaped like a contact lens and is packaged in a dehydrated form and rehydrated before use, has been used to promote corneal epithelial healing and deliver drugs. The source of the collagen may be porcine sclera or bovine corium (107). The collagen shield has been found useful to deliver drugs to the eye since therapeutic levels of medication are delivered reliably with a minimum number of applications. Drug delivery depends on absorption and subsequent release of the medication by the shield. When a solution containing a water-soluble drug is used for rehydration, the drug becomes trapped in the interstices of the collagen matrix; the drug is released as the shield dissolves (107). Shields soaked in water-soluble drugs have been found to produce corneal and aqueous levels comparable to those obtained with frequent topical therapy. The prolonged exposure time of medication to the cornea provided by a presoaked shield may produce higher levels in tissue than a single drop that is rapidly carried away by the tears (107). Currently, the only antifungal to be used in collagen shields is amphotericin B (267, 299, 347). Clearly, the potential of this technique for delivery of antifungals to the eye should be explored further.
Natamycin. Natamycin was the first antifungal specifically developed for topical ophthalmic use (Table 10) and is currently the only topical ophthalmic antifungal compound approved by the Food and Drug Administration of the United States (267). It is reported to have a broad spectrum of activity against various fungi, including species of Fusarium, Aspergillus, Acremonium, Penicillium, Lasiodiplodia, and Candida (236, 267, 271), but the validity of the methods used to derive these data, as well as the relevance of these data to the clinical use of natamycin, which is given only topically, is a contentious issue.
Natamycin is poorly soluble in water. It is stable in a 5% suspension and, in this form, adheres well to the cornea for clinically useful periods (236). The 5% topical ophthalmic suspension, although viscous, is well tolerated and causes no pain or secondary corneal damage (236). Punctate keratitis is sometimes encountered (170). It was initially thought that natamycin penetrated the cornea and conjunctiva poorly after topical application, that effective drug levels were not achieved in either the cornea or aqueous, and that it was therefore useful only in the treatment of superficial mycotic keratitis (236). However, radiolabeling studies suggest that it actually penetrates the cornea well after topical application (274). Thirteen topical applications every 5 min resulted in a drug concentration of approximately 2.5 mg/g cornea in rabbit corneas debrided of epithelium; levels peaked at approximately 10 min after administration. Far lower levels (7.0 µg/g) were attained in corneas where the epithelium was left intact (274). It is unclear whether these levels are actually achieved during therapy of clinical mycotic keratitis.
Natamycin is the drug of choice for therapy of mycotic keratitis in many countries (235, 288, 328, 334), particularly for keratitis due to filamentous fungi. It has also been used in association with other treatment modalities for therapy of mycotic scleritis (370), conjunctivitis, and endophthalmitis (267); controlled clinical trials are needed to confirm the efficacy of natamycin for these indications.
Amphotericin B. Amphotericin B (Table 10) is variably fungistatic and occasionally fungicidal, depending on the concentration achieved in serum (187) and the susceptibility of the pathogens; maximum activity is seen at a pH range from 6.0 to 7.5. Amphotericin B has been administered by the intravenous, topical, intravitreal, and intracameral routes for therapy of ophthalmic mycoses (236, 267).
For intravenous infusion of amphotericin B, a solution of 0.1 mg/ml in a 5% solution of dextrose is used (saline cannot be used since the drug may precipitate out). Unused solutions should be discarded after 24 h. Amphotericin B is both heat labile and light sensitive; hence, the dry powder should be refrigerated and protected from light (236). The recommended dosage is usually 1 mg/kg of body weight/day; smaller doses may be relatively ineffective (236). However, since tolerance to amphotericin B varies greatly among patients, the dosage must be individually adjusted; the safest approach is to initially give low test doses and to gradually increase the dose (236). Treatment needs to be given only once daily, or on alternate days once clinical improvement is noted; alternate-day therapy is advised for at least 2 months for many infections, with administration of a total dose of at least 3.0 g of amphotericin B (236). Renal toxicity is estimated to occur in almost 80% of patients receiving intravenous amphotericin B (187); this should be zealously guarded against by frequent monitoring of the blood urea nitrogen and other tests of kidney function. Headaches, chills, fever, and anorexia are common with systemic use; other adverse side- effects include moderate anaemia, nausea, vomiting, gastrointestinal cramps and diarrhea, and local thrombophlebitis at the infusion site (236). In view of these toxic effects, treatment should be reserved for patients in whom a diagnosis of mycotic infection is reasonably well
