Principles of Ocular Mycology
John Brinser
The Kingdom Fungi is composed of five classes of fungi that are medically important: the Zygomycetes; the Ascomycetes; the Basidiomycetes; the Deutermycetes [Fungi Imperfecti]; and the Oomycetes (Table 1). Zygomycetes have a saclike cell, a sporangium, in which the entire internal contents are cleaved into spores. Some Zygomycetes form vegetative hyphae similar to roots called rhizoids and vegetative hyphae similar to runners called stolons. The stolons are useful in identifying several genera by their location to the origin of the sporangiophore and the location of the rhizoids. Ascomycetes are characterized by the development of asci, saclike cells that usually contain eight ascospores. Asci may form within a specialized fruiting body termed an ascocarp. The Basidiomycetes have vegetative cells that are dikaryotic (n + n). This dikaryotic state is maintained by the use of clamp connection and dolipore septa that prevent the nuclei moving from cell to cell. Deutermycetes [Fungi Imperfecti] include yeasts and filamentous fungi in which the perfect state or telomorph are not known. The Deutermycetes can be separated into three subclasses: the Blastomycetes comprised of yeast and two subclasses of filamentous fungi based upon the presence or absence of fruiting structures, namely, Coelomycetes and Hypomycetes. The majority of the filamentous fungi isolated in the clinical microbiology laboratory belong to the subclass Hypomycetes. Members of the Class Oomycetes produce biflagellated oospores within a large cell, the oogonium, which has been fertilized by a smaller cell, the anthidium. Oomycetes do not grow on ordinary mycologic media and are not isolated in clinical microbiology laboratories. Filamentous fungi that remain sterile despite attempts to induce the formation of conidia or spore producing structures are placed in a group called the Mycelia-Sterilia. Reasons for the lack of formation of reproductive structures may be due to nutritional needs, appropriate environment, or the lack of a compatible mating strain.
TABLE ONE. Simplified Taxonomic Scheme of Medically Important Fungi | |||||||||||||||||||||||||||||||||||||||||||||||
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Approximately 20 genera of fungi are capable of causing systemic infections in humans, 20 genera cause cutaneous infections, and 12 genera are capable of causing severe localized subcutaneous disease.1 However, there is an ever-increasing number of opportunistic fungal pathogens that may produce disease in the debilitated or immunocompromised host and in the nonimmunocompromised host.
Human fungal infections occur when an individual is exposed to the fungus in its natural environment by either direct inhalation of the spores, by ingestion of the spores, or by trauma.
When an opportunistic fungal pathogen is isolated from a normally sterile site and is capable of growing at 35 °C, it must be considered a possible pathogen.
FUNGAL CHARACTERISTICS
Fungi are a heterogeneous, heterotrophic, and ubiquitous group of eukaryotic organisms that require complex organic compounds for growth. They are capable of living as either saprophytes, parasites, or as symbiots. Characteristics that separate them from bacteria include having a nucleus with a nucleolus, mitochondria, 80S ribosomes, centrioles, and a flagellum which has a 9 + 2 fibril configuration when the fungus is motile. Fungal cell walls are composed of chitin, chitosan, glucan, mannan, and occasionally cellulose. When a preformed organic compound is supplied as a carbon source, they are capable of synthesizing proteins and most of the amino acids and vitmains necessary for their growth. The capability of a fungus to produce enzymes governs its ability to utilize substrates. Excess food that is produced can be stored as either glycogen or as oil. The temperature range of fungal growth is between 0 to 35 °C, with an optimum range between 20 to 30 °C. Fungi, unlike bacteria, prefer growth in an acid environment with an optimum pH of 6.
Fungi exist primarily in either a filamentous or yeast form. Dimorphism occurs when certain fungiexist either as a filamentous fungus at 25 °C or as a yeast at 35 °C. Filamentous fungi are characterized by the development of tubular structures termed hyphae which branch and intertwine to form the mycelium. The hyphal walls are parallel because they grow by linear elongation. Hyphae may be either septated or nonseptated. The crosswalls in septated hyphae may be close together or widely spaced depending on the characteristic of the genus. The septations may be either complete or incomplete. An incomplete septum has a central pore which allows the protoplasm to extend from one cell to the next.
Certain yeasts are capable of producing pseudohyphae which can be confused with true hyphae. True hyphae can be distinguished from pseudohyphae because true hyphal cell walls are parallel to each other without constrictions of the cell wall at the location of each septum and the septations are straight and easily seen. When fungi are exposed to an unfavorable environment, specialized hyphal structures such as chlamydospores, vesicles, or sclerotia may be produced.
Reproduction in fungi occurs either asexually, sexually, or by a combination of both methods. Sexual reproduction involves the union of nuclei or gametes. Asexual reproduction occurs by either fragmentation, fission, budding, or by the formation of spores. In fragmentation, the hyphae break into individual cells called arthrospores. Fission occurs when the yeast cell splits into two daughter cells. Budding is found in the majority of yeasts and occurs when the parent cell produces an outgrowth termed a daughter cell. The daughter cell increases in size and then detaches, forming a second organism. If multiple daughter cells are formed, they may exist in various stages of development.
The most common type of asexual reproduction occurs with the formation of spores. These spores are consistent in their size, shape, color, arrangement, and number of cells in each spore; therefore, these characteristics are very important in the identification of the organism. Certain fungi are capable of producing more than one type of spore, for example the macroconidia and microconidia of Fusarium species and the dermatophytes.
OCULAR FUNGAL INFECTIONS
Fungi are part of the normal eyelid flora in up to 17% of the normal population. The species that are present are representative of the geographic area of residence and the occupation of the individual. Individuals who work outside, such as farmers and construction workers, have a higher incidence of fungi growing on their eyelid margins than does someone who works in an air-conditioned office building. Fungi, especially yeasts, are capable of growing in eye makeup; therefore, women may have an increased incidence of yeast growing on the eyelids.
Fungal infections of the eyelids are caused primarily by yeasts and the dermatophytes, rarely by the dimorphic fungi and may be accompanied by the loss of eyelashes.
Conjunctival fungal infections present either as an oculoglandular syndrome or as an inflammatory mass. The oculoglandular syndrome can be caused by Blastomyces dermatitidis, Paracoccidioides brasiliensis or Sporothrix schenckii. Rhinosporidium seeberi produces a cystic pedunculated mass in the conjunctiva.
The incidence of fungal keratitis, worldwide, has increased because of a greater awareness of fungi causing corneal ulcers and the incorporation of fungal media into the routine workup for all corneal ulcers. In addition, there is a true increase in the number of fungal corneal ulcers because of an increase in outdoor activities and outdoor occupations, the enhanced survival of patients with altered host defenses, and the use of topical corticosteroids.
There are two distinct disease entities involved in fungal keratitis. The first is caused by filamentous fungi, normally present in soil and vegetative matter, and occurs primarily in healthy males. These ulcers are caused by direct inoculation of the fungus following trauma to the cornea. The second entity is caused by yeast and yeastlike fungi and occurs in patients who have pre-existing corneal disease, have had corneal surgery, or are on long-term immunosuppressive drugs including corticosteroids. The exception to this second entity is those corneal ulcers that occur following trauma while applying eye makeup.
Fungal endophthalmitis can be either exogenous or endogenous in origin. Exogenous fungal endophthalmitis occurs in healthy individuals after a penetrating injury to the globe in which the fungus is deposited into either the anterior chamber, the vitreous, or both. The fungus is usually filamentous in type and is present in the soil or vegetativematter and accompanies the foreign body into the eye. Progression of the infection is dependent on size of the inoculum at the time of injury, the growth rate of the fungus, and the status of the host’s immunologic system.
Endogenous fungal endophthalmitis occurs primarily in patients who are in a compromised state of health. Risk factors include the use of antibiotics, corticosteroids and cytotoxic agents, the use of indwelling intravenous catheters and hyperalimentation, increased survival of patients with debilitating diseases, and the use of intravenous narcotic drugs. The spread of the fungus to the eye characteristically occurs following a fungemia, usually due to a yeast. Growth of the yeast occurs slowly and, unlike bacteria, the organisms do not diffuse rapidly through the vitreous. Yeast and yeastlike fungi grow by budding or extending pseudohyphal and hyphal elements into the vitreous, forming colonies within the vitreous body (“fluff balls”). The most common agents are Candida species (primarily C. albicans), Cryptococcus species, and Pneumocytis carinii. The presence of budding yeast cells or pseudohyphal elements in a urine microscopic study performed on a patient with suspected endophthalmitis and a recent history of intravenous catheter use is highly suggestive of an endophthalmitis due to a yeast. The major filamentous fungus seen in endogenous endophthalmitis is Aspergillus species, which is primarily seen in patients with a history of intravenous drug use.
CULTURE MEDIA
Media necessary for the primary isolation of ocular fungal pathogens include blood agar containing 10% sheep blood, Sabouraud dextrose agar, brain heart infusion agar, and SABHI (a combination of Sabouraud dextrose agar and brain heart infusion agar).2 There are two different types of Sabouraud dextrose agar available for use in the clinical mycology laboratory. The first has a pH 5.6, contains 4% dextrose and is used for the isolation and identification of dermatophytes. The second (Emmons modification) has a pH of 6.5, contains 2% dextrose, and is used for the isolation of opportunistic and dimorphic fungi from clinical specimens. If the Emmons modification is available, it is the medium of choice. Media containing cycloheximide (Mycosel [BioQuest, Cockeysville, MD] and Mycobiotic agar [Difco, Detroit, MI]) should not be used because cycloheximide inhibits the growth of mycelium-producing fungi which constitute the majority of ocular pathogens. In its place, either Sabouraud dextrose agar containing 100 μg/mL of gentamicin or Snyder medium (BioQuest, Cockeysville, MD) should be used to isolate fungi from ocular specimens contaminated with bacteria. Broths that can be used include Sabouraud dextrose broth, brain heart infusion broth, and Czapek’s-Dox broth. However, the majority of media used for the isolation of bacteria are capable of supporting fungal growth, such as blood agar, chocolate agar, mannitol salts agar, eosin methylene blue agar, and thioglycolate medium.
Two additional media which can be used in ocular mycology as a primary and as a differential medium are potato dextrose agar and Czapek’s agar. Potato dextrose agar is used to increase sporulation and pigment production in fungi. Its major use in ocular mycology is for the production of macroconidia necessary for the identification of Fusarium species. Czapek’s agar is used for the primary isolation and pigment production necessary for the identification of Aspergillus species.
The majority of microbiology laboratories utilize screw-cap tube media for the isolation of fungi. Ideally, culture media in petri dishes should be used rather than tube media because they provide a larger area for inoculation, especially for “C” streaks. They also provide better aeration for growth and afford easier preparation of microscopic slides for identification when growth appears. The major drawback to using petri dishes is that the media will dry out during the extended incubation period. To reduce this problem, petri dishes should be poured with at least 40 mL of agar per 100 × 15 mm petri dish and the relative humidity of the incubator should be 40% to 50%. This can be accomplished by placing a pan of water in the bottom of the incubator. The petri dishes can be sealed with Parafilm (American National Can Company) to further prevent drying.
Fungal cultures should be incubated at 25 to 30 °C for 4 to 6 weeks and examined three times a week for growth. If infection by a dimorphic fungus is suspected (e.g., Histoplasma capsulatum), the cultures should be held for 8 weeks. The petri dishes should be opened only in a biologic safety cabinet.
DIRECT MICROSCOPIC EXAMINATION
Routine microbiologic stains for fungi include the Gram stain, Giemsa stain, and the fluorochrome stains: acridine orange and calcofluor white.2,3 The cell walls and septations of fungi do not stain with either Gram or Giemsa stain. With Gram stain, the internal contents of the filamentous fungal hyphal elements stain either gram positive, gram negative, gram variable, or do not stain and remain hyaline. Yeast and pseudohyphal elements usually stain gram positive. With the Giemsa stain, the internal contents of yeast and filamentous fungi stain dark blue, while the cell walls and septations remain clear or colorless. Potassium hydroxide (KOH)(20%) with 40% dimethyl sulfoxide (DMSO) is used to examine skin scrapings for the presence of fungi. The KOH clears the epithelial cells so that the hyphae can be more easily seen. Because corneal epithelial cells are already clear, the KOH preparation should be reserved for specimens from the ocular adnexa.
The KOH preparation has been replaced by the use of direct fluorescent staining such as the acridine orange and the calcofluor white stains. Specimens of the cornea, aqueous and vitreous fluids can be examined using either a drop of acridine orange stain or calcofluor white stain. The main drawback to the use of fluorochrome stains is that they require the use of a fluorescent microscope; however, because the stained organisms are viewed against a black background, they are easier to detect. The three most common stains are the acridine orange stain, the calcofluor white stain, and fluorescein-conjugated lectins.
The acridine orange stain [AO], buffered at a pH of 3.8 to 4, stains RNA reddish orange and DNA light green. Bacteria stain reddish orange, filamentous fungi stain bright green, and yeast stain reddish orange cytoplasm with a green nucleus. The background is either black or a light green. Erythrocytes and pigment granules do not stain, which makes the AO stain useful for examining anterior chamber and vitreous fluids.
Calcofluor white binds to both chitin and cellulose resulting in yeast, filamentous fungi, and cysts of Acanthamoeba staining bright green.
Lectins are plant glycoproteins that bind to specific carbohydrates of fungi, mycobacteria, and Acanthamoeba species. Identification is achieved by using a standard panel and comparing the fluorescence pattern of the unknown.
HISTOPATHOLOGICAL STAINS
The hematoxylin and eosin (H & E) stain is useful for demonstrating the type of tissue reaction that is present, but it is not an acceptable stain for demonstrating yeast and fungi. They either stain poorly or are difficult to distinguish from the tissue. The exception to this is the excellent staining of the hyphae of Zygomycetes.
The periodic acid-Schiff (PAS) stain is specific for fungal polysaccharides and can be used for both direct staining and for tissue sections. The fungal polysaccharides are oxidized to aldehyde groups at the 1,2-glycol positions by periodic acid. These aldehyde groups react with Schiff’s leucofuchsin reagent to produce a deep reddish color.
Gomori’s methenamine silver (GMS) stain is specific for fungal cells which stain brown-black against a light green background. Chromic acid oxidizes the fungal polysaccharides to aldehyde groups which then reacts with the methenamine-silver nitrate to form a brown-black complex. The GMS stained slide can be counterstained with hematoxylin and eosin rather than the light green counterstain to allow visualization of the tissue reaction.
The major drawback to using these stains as a primary means of establishing a fungal etiology in keratitis and endophthalmitis is that they require technical methods that may not be available at night and on the weekends. Slides can be prepared and held until they can be stained by the histology laboratory personnel.
IDENTIFICATION CHARACTERISTICS
Unlike bacteria which are identified using biochemical testing, fungi are identified by using the gross colonial morphology and, more importantly, the microscopic morphology.4,5,6 Exceptions to this are the yeasts and the nutritional testing of the dermatophytes. Use of the microscopic morphologic characteristics is the most definitive method to identify fungi because these characteristics are stable. Microscopic identification is based on the spore shape, the method of spore production, and the arrangement of the spores. The size and color of the hyphae provide additional information. There are times when spore production on primary isolation is absent or very sparse and sporulation media such as potato dextrose agar or cornmeal agar must be used to induce the fungus to sporulate. It is important when attempting to identify an unknown isolate that the medium used be the same as was used to create the identification scheme.
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