Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 1)

ACREMONIUM

= Cephalosporium (Corda, 1839).

Pathogenicity

Acremonium

Acremonium has been reported as a rare cause of keratitis and endophthalmitis.

In the literature 17 cases of Acremonium keratitis have been reported between 1965 and 1991. Rosa et al (1994) found 3.2% of Acremonium keratitis in their series and Rodriguez-Areset al reported this as an extremely rare cause of suppurative corneal infection.

Ecology

Cosmopolitan, isolated from soil and plant debris.

MACROSCOPIC MORPHOLOGY

• The growth rate of Acremonium colonies is moderately rapid, maturing within 5 days. The diameter of the colony is 1-3 cm following incubation at 25°C for 7 days on potato glucose agar.

• The texture of the colony is compact, flat or folded, and occasionally raised in the center. It is glabrous, velvety, and membrane-like at the beginning. Powdery texture may also be observed. By aging, the surface of the colony may become cottony due to the overgrowth of loose hyphae.

• The color of the colony is white, pale grey or pale pink on the surface. The reverse side is either uncolored or a pink to rose-colored pigment production is observed (Fig. 8.1).

MICROSCOPIC MORPHOLOGY

• Acremonium spp. possesses hyaline, septate hyphae which are typically very fine and narrow. Vegetative hyphae often form hyphal ropes. Unbranched, solitary, erect phialides are formed directly on the hyphal tips, the hyphal ropes, or both. The phialides are separated from hyphae by a septum and taper towards their apices. At the apices of the phialides is the hyaline conidia 2-3 × 4-8 μm in size. They usually appear in clusters, in balls or rarely as fragile chains.

• The conidia are bound by a gelatinous material. They may be single or multicellular, fusiform with a slight curve or resemble a shallow crescent. These structural properties of conidia vary depending on the species.

• Acremonium falciforme usually produces crescentic, nonseptate conidia. Sometimes, 2 or 3 celled conidia may also be observed. Acremonium kiliense, on the other hand, has short straight conidia and the conidia of Acremonium recifei are usually crescentic and nonseptate (Figs 8.1 and 8.2).

Fig. 8.1: Acremonium species on potato dextrose agar, 25°C, 7 days

Fig 8.2: Acremonium species. Oblong conidia accumulating at the apices of narrow Phialides

ALTERNARIA

Pathogenicity

Occasional agents of keratitis, other infections include onychomycosis, ulcerated cutaneous infection, chronic sinusitis and rare cases of deep infection have also been reported in the immunocompromised patient.

Ecology

Alternaria is a cosmopolitan dematiaceous (phaeoid) fungus commonly isolated from plants, soil, food, and indoor air environment. The production of melanin-like pigment is one of its major characteristics.

MACROSCOPIC MORPHOLOGY

• Alternaria spp. grows rapidly and the colony size reaches a diameter of 3 to 9 cm following incubation at 25°C for 7 days on potato glucose agar (Fig. 8.3).

• The colony is flat, downy to woolly and is covered by grayish, short, aerial hyphae in time (Fig. 8.4).

• The surface is grayish white at the beginning which later darkens and becomes greenish black or olive brown with a light border.

• The reverse side is typically brown to black due to pigment production.

MICROSCOPIC MORPHOLOGY

• Alternaria spp. has septate, brown hyphae.

• Conidiophores are also septate and brown in color, occasionally producing a zigzag appearance. They bear simple or branched large conidia (7-10 × 23-34 μm) which have both transverse and longitudinal septations. These conidia may be observed singly or in acropetal chains and may produce germ tubes. They are ovoid to obclavate, darkly pigmented, muriform, and smooth or roughened.

• The end of the conidium nearest the conidiophores is round while it tapers towards the apex. This gives the typical beak or club-like appearance of the conidia.

Fig. 8.3: Alternaria species. Growth on potato dextrose agar

Fig. 8.4: Alternaria species. Obclavate, muriform conidia in chains

ASPERGILLUS SPECIES

Pathogenicity

At present, some twenty species of Aspergillus have been recognized as opportunistic pathogens. They are the most common cause of mycotic keratitis along with Fusarium spp.

Among all filamentous fungi, Aspergillus is in general the most commonly isolated in invasive infections. It is the second most commonly recovered fungus in opportunistic mycoses following Candida. Almost any organ or system in the human body may be involved.

Onychomycosis, sinusitis, cerebral aspergillosis, meningitis, endocarditis, myocarditis, pulmonary aspergillosis, osteomyelitis, otomycosis, keratitis, endophthalmitis, cutaneous aspergillosis, hepatosplenic aspergillosis, as well as Aspergillus fungemia, and disseminate aspergillosis may develop.

Ecology

Aspergillus spp. abounds 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.

The color of the colony in various Aspergillus species is summarised in Table 8.1

ASPERGILLUS FLAVUS

Macroscopic Morphology

• On Potato dextrose agar, colonies are granular, flat, often with radial grooves, yellow at first but quickly becoming bright to dark yellow-green with age (Fig. 8.5).

Microscopic Morphology

• Conidial heads are typically radiate, mostly 300-400 μm in diameter, later splitting to form loose columns, biseriate but having some heads with phialides borne directly on the vesicle. Conidiophores are hyaline and coarsely roughened, the roughness often being more noticeable near the vesicle (Fig. 8.6).

• Conidia are globose to subglobose (3-6 μm in diameter), pale green and conspicuously echinulate. Some strains produce brownish sclerotia.

Fig. 8.5: Aspergillus flavus growth on potato dextrose agar, 25°C, 5 days

Fig. 8.6: Aspergillus flavus conidial head supported by a rough-walled conidiophore

ASPERGILLUS FUMIGATUS

Macroscopic Morphology

• On potato dextrose agar, colonies show typical blue-green surface pigmentation with a suede-like surface consisting of a dense felt of conidiophores (Figs 8.7A and B).

Microscopic Morphology

• Conidial heads are typically columnar (up to 400 × 50 μm but often much shorter and smaller) and uniseriate. Conidiophores are short, smooth-walled and have conical-shaped terminal vesicles which support a single row of phialides on the upper two thirds of the vesicle (Fig. 8.8). Conidia are produced in basipetal succession forming long chains and are globose to subglobose (2.5-3.0 μm in diameter), green and rough-walled.

• Note, this species is thermotolerant and grows at temperatures up to 55°C.

Figs 8.7A and B: Aspergillus fumigatus growth on Potato dextrose agar, 25°C, 5 days (A—Surface; B—Reverse)

Fig. 8.8: Aspergillus fumigatus uniseriate phialides attached to the upper surface of the vesicle

ASPERGILLUS NIGER

Macroscopic Morphology

• On potato dextrose agar, colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads (Figs 8.9A and B).

Microscopic Morphology

• Conidial heads are large (up to 3 mm × 15-20 μm in diameter), globose, dark brown, becoming radiate and tending to split into several loose columns with age (Fig. 8.10).

• Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle. Conidial heads are biseriate with the phialides borne on brown, often septate metulae.

• Conidia are globose to subglobose (3.5-5.0 μm in diameter), dark brown to black and rough-walled.

Figs 8.9A and B: Aspergillus niger growth on potato dextrose agar, 25°C, 5 days (A—Surface; B—Reverse)

Fig. 8.10: Aspergillus niger with radiate conidial heads

ASPERGILLUS TERREUS

Macroscopic Morphology

• On potato dextrose agar, colonies are typically suede-like and cinnamon-buff to sand brown in color with a yellow to deep dirty brown reverse (Figs 8.11A and B).

Microscopic Morphology

• Conidial heads are compact, columnar (up to 500 × 30-50 μm in diameter) and biseriate (Figs 8.12A and B).

• Conidiophores are hyaline and smooth-walled.

• Conidia are globose to ellipsoidal (1.5-2.5 μm in diameter), hyaline to slightly yellow and smooth-walled.

Figs 8.11A and B: Aspergillus terreus growth on potato dextrose agar, 25°C, 5 days
(A—Pale cinnamon color coloy; B—Dark brown color colony)

Figs 8.12A and B: Aspergillus terreus with biseriate conidial head

ASPERGILLUS NIDULANS

Macroscopic Morphology

On potato dextrose agar, colonies are typically pine green in color with tan to dark red- brown cleistothecia developing within and upon the conidial layer (Figs 8.13A and B). Reverse may be olive to drab-gray or purple-brown. Growth rate is slow to moderate in comparison with other clinically significant Aspergillus species.

Microscopic Morphology

• Conidial heads are short, columnar (up to 70 × 30 μm in diameter) and biseriate (Fig. 8.14).

• Conidiophores are usually short, brownish and smooth-walled.

• Vesicles are hemispherical, small (8-12 μm in diameter), with metulae and phialides occurring on the upper portion.

• Conidia are globose (3.0-3.5 μm in diameter) and rough-walled.

• Aspergillus nidulans is a homothallic species capable of producing the teleomorph (sexual stage) without mating studies. The ascomycetous telemorph (Emericella nidulans) produces brown to black globose cleistothecia (100-250 μm) that are surrounded with globose Hülle cells. Ascospores are reddish brown, lenticular (4 × 5 μm), with two equatorial crests.

Figs 8.13A and B: Aspergillus nidulans growth on potato dextrose agar,
25°C, 7 days (A—Surface and B—Reverse)

Fig. 8.14: Aspergillus nidulans conidial head and round hulle cells (¾)

ASPERGILLUS TAMARII

Ecology

A. tamarii is a member of Aspergillus section Flavi. Thisspecies is widely used in the food industry for the production of soy sauce (known as red Awamori koji) (14) and in the fermentation industry for the production of various enzymes, including amylases, proteases, and xylanolytic enzymes.

Pathogenicity

A. tamarii is able to produce several toxic secondary metabolites, including cyclopiazonic acid and fumigaclavines, it has rarely been encountered as a human pathogen. The only known cases are an eyelid infection, invasive nasosinusal aspergillosis in an immunocompetent patient, and onychomycosis in a 3-year-old boy.

The first cases of A. tamarii fungal keratitis reported by Kredics L et al, 2007 in Coimbatore, South India and they reported as fourth known case worldwide involving this unusual opportunistic human pathogen.

Macroscopic Morphology

• Colonies on malt extract agar at room temperature attained diameters of 6.0 to 7.0 cm in 10 days, producing abundant conidial heads in dull yellowish green shades becoming metallic bronze at maturity (Fig. 8.15).

• These are similar to A. flavus so their molecular identification only to differentate the species level.

Molecular Identification

DNA extraction:

• Masterpure yeast DNA purification kit (Epicenter Biotechnologies, Madison, WI).

DNA amplification:

• Primers 5.8S rRNA gene- ITS 1 and ITS 4

• Primers β-tubulin gene - bT2a and bTsb.

DNA sequencing:

• BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems Inc., foster city CA)

• ABI 3100 DNA Sequencer.

Microscopic Morphology

• The conidiophore stipe was hyaline and rough walled; the conidial heads were radiate; the vesicles were globose to subglobose, 25 to 50 μm in diameter.

• The phialides were borne directly on the vesicle or on metulae (mostly on large heads).

• The conidia were globose to subglobose, 5 to 6.5 μm in diameter, and brownish yellow.

However, in contrast with those of typical wild A. tamarii isolates, some conidia of this isolate were not ornamented with tubercules and warts but were smooth walled and hyaline (Fig. 8.16).

• The isolate grew well at 37°C but was unable to grow at 42°C on malt extract agar medium.

Fig. 8.15: Colony of A.tamarii

Fig. 8.16: Conidial head of A. tamarii

BIPOLARIS

Pathogenicity

Bipolaris is common cause of keratitis and endophthalmitis. Also occasionally the cause of diverse types of phaeohyphomycosis, including sinusitis, peritonitis, endocarditis, osteomyelitis, meningoencephalitis and cutaneous infection; these infections have been recognized in the immunocompromised patient as well as in the normal host.

Ecology

Cosmopolitan, although some species are mainly found in tropical or subtropical areas. Saprobes or pathogens of numerous species of plants.

Macroscopic Morphology

• Bipolaris colonies grow rapidly, reaching a diameter of 3 to 9 cm following incubation at 25°C for 7 days on potato dextrose agar (Figs 8.17A and B).

• The colony becomes mature within 5 days. The texture is velvety to woolly.

• The surface of the colony is initially white to grayish brown and becomes olive green to black with a raised grayish periphery as it matures.

• The reverse is also darkly pigmented and olive to black in color.

Microscopic Morphology

• The hyphae are septate and brown. Conidiophores (4.5-6 μm wide) are brown, simple or branched, geniculate or sympodial, bending at the points where each conidium arises.

• This property leads to the zigzag appearance of the conidiophore. The conidia, which are also called poroconidia, are 3- to 6-celled, fusoid to cylindrical in shape, light to dark brown in color and have sympodial geniculate growth pattern. The poroconidium (30-35 μm × 11-13.5 μm) is distoseptate and has a scarcely protuberant, darkly pigmented hilum (Fig. 8.18).

• This basal scar indicates the point of attachment to the conidiophore. From the terminal cell of the conidium, germ tubes may develop and elongate in the direction of longitudinal axis of the conidium.

• Teleomorph production of Bipolaris is heterothallic. The perithecium is black in color, and round to ellipsoidal in shape. The ascospores are flagelliform or filiform, hyaline in nature and are found in clavate-shaped or cylindrical asci. Each ascus contains eight ascospores.