Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 2)

CANDIDA 

Ecology 

Candida is a genus of yeasts. Clinically, the most significant member of the genus is Candida albicans, which can cause numerous infections (called candidiasis or thrush) in humans and other animals, especially in immunocompromised patients. (Ryan KJ et al, 2004). Various Candida species are members of gut flora in animals, including C. albicans. 

Pathogenicity 

C. albicans is the most common cause of both superficial and systemic candidosis. It is also often present as part of the commensal flora of the mouth, vaginal mucosa and gastrointestinal tract, and may be isolated from these sites in the absence of disease. 

Most episodes of yeast infections in corneal ulcers and other ocular infections are due to various Candida species, predominantly Candida albicans and usually occur in the presence of systemic illness (diabetes mellitus or immunocompromise) or ocular diseases like lid abnormalities or dry eyes) or endogenous endophthalmitis and in patients receiving prolonged topical medications or topical corticosteroids. 

Macroscopic Morphology 

• Candida albicans grows well on potato dextrose agar (Fig. 8.19), Sabouraud’s agar and most routinely used bacteriological media. 

• Convex, entire margin, non-mucoid, smooth texture of cream colored pasty colonies usually appear after 24 - 48 hours incubation at 35-37°C. 

• The colonies have a distinctive yeast smell. 

Microscopic Morphology 

• The round—oval shaped budding cells can be easily seen by direct microscopy in stained or unstained preparations. 

• Candida albicans produces true germ tubes when incubated in serum for 2-3 hours at 37°C (Fig. 8.20). 

• These are parallel-sided tubes which are formed at right angles to the parent cell and are at least twice as long as the parent cell before cross walls are formed.

Fig. 8.19: Cream colored pasty colonies on potato dextrose agar (2 days)

Fig. 8.20: C.albicans produces true germ tubes

CEPHALIOPHORA IRREGULARIS 

Ecology

The genus cephaliophora contains coprophilous fungi. In India, they are generally found growing on the dung of rats. They have also been reported from moist wood. There are two species known to be prevalent in India: Cephaliophora tropica and Cephaliophora irregularis. The genus Cephaliophora was established by Thaxter in 1903 as a new genus of Deuteromycetes consists of two species of dung inhabiting fungi. One species, C.irregularis Thaxter was isolated from mouse dung. C.tropica was isolated from the dung of various animals from Jamaica, Liberia, Java and China.

Pathogenicity

In 1990 Thomas et al reported a first case of human infection of keratitis caused by C. irregularis from Tiruchirapalli, South India. The second case of mycotic keratitis due to C.irregularis reported from Christian Medical College, Vellore (Mathews 1995).

Macroscopic Morphology (Fig. 8.21)

• Rapid growth

• Hyaline mycelial growth

• Later became cottony without a pigment being formed

• Colonies on blood agar had a similar appearance.

Microscopic Morphology (Figs 8.22A to C)

• Revealed hyaline, short, sessile, septate and solitary conidiophores

• Bearing clusters of pale brown conidia

• The conidia were mostly two-celled, pyriform or triangular.

Fig. 8.21: Cottony white colony of C.irregularis on potato dextrose agar (7 days)

Figs 8.22A to C: Two conidium (A) and Cluster of conidia borne on short swollen conidiogenous cells B and C (400 X magnification)

CLADOSPORIUM 

Pathogenicity 

One of the most common dematiaceous fungi to cause keratitis and endophthalmitis. Generally nonpathogenic, with the exception of Cladosporium carrionii an agent of chromoblastomycosis. This is a chronic subcutaneous infection characterized by verrucous lesion and the formation of brown, sclerotic fission cells (copper pennies) in tissue. It is often confined to a single limb. 

Ecology 

Many species are cosmopolitan fungi or soil, plant debris and leaf surfaces. Cladosporium is very frequently isolated from air, especially during seasons in which humidity is elevated. 

Macroscopic Morphology 

• The growth rate of Cladosporium colonies is moderate on potato dextrose agar at 25°C and the texture is velvety to powdery (Figs 8.23A and B). Similar to the other dematiaceous fungi, the color is olivaceous green to black from the front and black from the reverse. 

• Most of the Cladosporium spp. do not grow at temperatures above 35°C. 

Microscopic Morphology 

• Cladosporium spp. produces septate brown hyphae, erect and pigmented conidiophores, and conidia. 

• Conidia of Cladosporium spp. in general are elliptical to cylindrical in shape, pale to dark brown in color and have dark hila (Fig. 8.24). They occur in branching chains that readily disarticulate. Conidial wall is smooth or occasionally echinulate. 

Figs 8.23A and B: Cladosporium species growth on potato dextrose agar, 25°C, 5 days

Fig. 8.24: Cladosporium species Shield-shaped conidia with scars (¾) at points of attachment, bicellular conidium (→)

COLLETOTRICHUM SPP 

Pathogenicity 

Colletotrichum generally associated with some form of trauma. They manifest as keratitis or subcutaneous lesions, although a case of invasive infections has been reported. Five species of Colletotrichum have been reported to cause infections in humans. They are Colletotrichum coccodes, C.crassipes, C. dematium, C. gloeosporioides, and C. graminicola. The predominant infection is keratitis following traumatic implantation, but subcutaneous and systemic infections among immunosuppressed patients have also been reported. 

Ecology 

The genus Colletotrichum is one of the most important genera of plant pathogens. It has a worldwide distribution but is found mainly in subtropical and tropical regions. Colletotrichum species cause economically significant diseases of plants (generally referred to as anthracnoses) that affect cereals and grasses, legumes, vegetables, and perennial crops, including fruit trees. 

Macroscopic Morphology (Figs 8.25, 8.26A and B) 

Colonies usually darkly pigmented with white aerial mycelium, consisting of numerous black sclerotia and light brown colored conidial masses, reverse is dark brown.

Fig. 8.25: Colletotrichum species growth on potato dextrose agar, 25°C, 7days

Figs 8.26A and B:  5 days old culture of C. coccodes (A—Surface; B—Reverse)

Microscopic Morphology (Figs 8.27 and 8.28) 

• Sclerotia are usually abundant, setose, spherical and are often confluent. 

• Conidia are straight, fusiform, attenuated at the ends, 16-22 × 3-4 μm. Appressoria are common, clavate, brown, 11-16.5 × 6-9.5 μm. 

• The key morphological features which identify the genus are its acervular conidiomata, often with setae (dark-pigmented, unbranched, thick-walled sterile hyphae usually pointed  at the tip), producing elongated slimy conidia, and the presence of appressoria (thick-walled swellings at the end of a hypha or germ tube useful for attaching the fungus to the host surface before penetration of the tissue).

Fig. 8.27: Few conidia (¾) with setae (→) of Colletotrichum

Fig. 8.28: Cylindrical conidia of Colletotrichum coccodes

CURVULARIA 

Pathogenicity 

Infection of the cornea, reported in 1959, was the first human disease proved to be caused by Curvularia. It is the most common dematiaceous fungus to cause corneal ulcer. Other ocular infections consist of conjunctivitis, dacryocytitis, sino-orbital cellulites, and endophthalmitis. But the cornea is the most commonly infected site. 

Several Curvularia species are zoo pathogenic. Wound infection is the most common disease caused by Curvularia and ranges from onychomycosis to skin ulceration and subcutaneous mycetoma. Other human Curvularia infections are invasive and allergic sinusitis and bronchopulmonary disease. Abscesses of the lung, brain, liver, and connective tissue have occurred. Nosocomial infections include dialysis-related peritonitis and post surgical endocarditis. 

Ecology 

Most species are facultative pathogens of tropical or subtropical plants, but a few are commonly isolated in temperate agricultural areas. 

Macroscopic Morphology (Fig. 8.29) 

• Curvularia produces rapidly growing, woolly colonies on potato dextrose agar at 25°C. 

• From the front, the color of the colony is white to pinkish gray initially and turns to olive brown or black as the colony matures. 

• From the reverse, it is dark brown to black. 

Microscopic Morphology 

• Septate, brown hyphae, brown conidiophores, and conidia are visualized. Conidiophores are simple or branched and are bent at the points where the conidia originate. This bending pattern is called sympodial geniculate growth. 

• The conidia (8-14 × 21-35 μm), which are also called the poroconidia, are straight to pyriform, brown, multiseptate, and have dark basal protuberant hila (Fig. 8.30). 

• The septa are transverse and divide each conidium into multiple cells. The central cell is typically darker and enlarged compared to the end cells in the conidium. 

• The central septum may also appear darker than the others. The swelling of the central cell usually gives the conidium a curved appearance. 

• The number of the septa, shape (straight or curved),color of the conidia (dark vs pale brown), as well as the existence of dark median septum, and the prominence of geniculate growth pattern are the major microscopic features that help in differentiation of Curvularia spp. among each other. 

• For instance, the conidia of Curvularia lunata have 3 septa and 4 cells, while those of Curvularia geniculata mostly have 4 septa and 5 cells.

Fig. 8.29: Curvularia species growth on Potato dextrose agar, 25°C, 5 days

Figs 8.30A and B: Curvularia species growth curved poroconidia with larger and darker central cell (→)

EXSEROHILUM 

Pathogenicity 

One of the common dematiaceous fungus to cause keratitis. Exserohilum species cause phaehyphomycosis affecting skin, sub cutaneous tissue, nose and paranasal sinuses, rarely endocarditis and osteomyelitis. Some cases of subcutaneous or deep phaehyphomycosis have been reported in humans and animals. 

Ecology 

Cosmopolitan, facultative plant pathogens, also occasionally isolated from soil. 

Macroscopic Morphology (Fig. 8.31) 

• Moderately rapid growth 

• Texture velvety 

• Color dark olive to black on the surface and reverse. 

Microscopic Morphology (Fig. 8.32) 

• Hyphae septate, pale brown 

• Conidiophores brown, geniculate at the apex 

• Proconidia cylindrical to ellipsoidal, multicellular, distoseptate with a protuberant hilum at the base 

• Germ tubes developing in the direction of the conidial long axis or on the side.

Fig. 8.31: Exserohilum species growth on potato dextrose agar, 25°C, 7 days

Fig. 8.32: Exserohilum species geniculate conidiophore, (→) poroconidium with protuberant hilum and darker septa at the ends (¾)

FUSARIUM 

Pathogenicity 

Species of Fusarium are widespread saprobic fungi that cause important diseases of plants, particularly major crop plants and of humans. They have long been regarded as important pathogens in eye infections, especially keratitis. 

As well as being common plant pathogens, Fusarium spp are causative agents of superficial and systemic infections in humans. Infections due to Fusarium spp. are collectively referred to as fusariosis. The most virulent Fusarium spp is Fusarium solani. Trauma is the major predisposing factor for development of cutaneous infections due to Fusarium strains. Disseminated opportunistic infections, on the other hand, develop in immunosuppressed hosts, particularly in neutropenic and transplant patients. Fusarium infections following solid organ transplantation tend to remain local and have a better outcome compared to those that develop in patients with hematological malignancies and bone marrow transplantation patients. 

Keratitis, endophthalmitis, otitis media, onychomycosis, cutaneous infections particularly of burn wounds, mycetoma, sinusitis, pulmonary infections, endocarditis, peritonitis, central venous catheter infections, septic arthritis, disseminated infections, and fungemia due to Fusarium spp. have been reported. 

Outbreaks of nosocomial fusariosis have also been reported. Existence of Fusarium in hospital water distribution systems may result in disseminated fusariosis in immuno- suppressed patients. Fusarium may also exist in soil of potted plants in hospitals. These plants constitute a hazardous mycotic reservoir for nosocomial fusariosis. 

Clusters of Fusarium keratitis were reported among contact lens users in Asia beginning in February 2006. 

Ecology 

Cosmopolitan, frequently isolated from soil. Certain species are important plant pathogens, other produce toxins in grains or stored animal feed. 

• Fusarium solani 

• F. oxysporum 

• F. dimerum. 

Macroscopic Morphology (Figs 8.33 and 8.34) 

• Fusarium spp. grow rapidly on Sabouraud’s dextrose agar at 25°C and produce woolly to cottony, flat, spreading colonies. The only slow-growing species is Fusarium dimerum (Figs 8.35A and B). 

• From the front, the color of the colony may be white, cream, tan, salmon, cinnamon, yellow, red, violet, pink, or purple. From the reverse, it may be colorless, tan, red, dark purple or brown.

Figs 8.33A and B: Fusarium species growth on potato dextrose agar, 25°C, 5 days (A—Surface; B—Reverse)

Figs 8.34A and B:  F. semitectum (A—Surface; B—Reverse)

Figs 8.35A and B: F. dimerum (A—Surface; B—Reverse)

• A sclerotium, which is the organized mass of hyphae that remains dormant during unfavorable conditions, may be observed macroscopically and is usually dark blue in color. 

• On the other hand the sporodochium a cushion-like, moist mat of hyphae bearing conidiophores over its surface, is usually absent in culture. When present, it may be observed in cream to tan or orange color, except for, which gives rise to blue-green or blue sporodochia. 

Microscopic Morphology 

• Hyaline septate hyphae, conidiophores, phialides, macroconidia, and microconidia are observed microscopically. In addition to these basic elements, chlamydospores are also produced by Fusarium chamydosporum, Fusarium napiforme, Fusarium oxysporum, Fusarium solani (Figs 8.36A and B), Fusarium semitectum (Fig. 8.37) and Fusarium sporotrichoides. 

• Phialides are cylindrical, with a small collarette, solitary or produced as a component of a complex branching system. Monophialides and polyphialides (in heads or in chains) may be observed. Macroconidia (3-8 × 11-70 μm) are produced from phialides on unbranched or branched conidiophores. They are 2- or more celled, thick-walled, smooth, and cylindrical or sickle-shaped. 

• Macroconidia have a distinct basal foot cell and pointed distal ends. They tend to accumulate in balls or rafts. Microconidia (2-4 × 4-8 μm), on the other hand, are formed on long or short simple conidiophores. They are 1-celled (occasionally 2- or 3-celled), smooth, hyaline, ovoid to cylindrical, and arranged in balls (occasionally occurring in chains). 

• Chlamydospores sometimes present. They are thick-walled, hyaline, intercalary or terminal. 

• Macroscopic and microscopic features, such as color of the colony; length and shape of the macroconidia, the number, shape and arrangement of microconidia, and presence or absence of chlamydospores are key features for the differentiation of Fusarium species.

Figs 8.36A and B: Macroconidium with foot cell of F. solani (¾macroconidium; → microconidia; → foot cell)

Fig. 8.37: Macroconidium of F. semitectum