Identification of Medically Important Fungi

Dr.Kedar Karki

Introduction

Separation of taxa is still primarily based on the method of spore production of the perfect or sexual state (teleomorph) and three major subdivisions, the Zygomycetes, Ascomycetes and Basidiomycetes are generally recognized. 

Introduction

However for medical mycologists two other groups are of particular importance; the yeasts and the hyphomycetes or conidial moulds. 

the following groups: Dermatophytes Yeasts Dimorphic Pathogens Hyphomycetes (hyaline moulds) Hyphomycetes (dematiaceous moulds) Coelomycetes Zygomycetes Oomycota  

Dermatophytes

Microscopic morphology of the micro and/or macroconidia is the most reliable identification character, but you need a good slide preparation and you may need to stimulate sporulation in some strains. 

Dermatophytes

Culture characteristics such as surface texture, topography and pigmentation are variable and are therefore the least reliable criteria for identification. 

Dermatophytes

Clinical information such as the site, appearance of the lesion, geographic location, travel history, animal contacts and race is also important, especially in identifying rare non-sporulation species like M. audouini, T. concentricum and T schoenleinii etc.  Note: mating experiments are not practical for the clinical mycology laboratory.

Three genera are recognised:

Epidermophyton: Smooth thin-walled Macroconidia

only present, no microconidia, colonies a green-brown to khaki colour.

Epidermophyton floccosum On Sabouraud's dextrose agar colonies are

usually slow growing, greenish-brown or khaki coloured with a suede-like surface, raised and folded in the centre, with a flat periphery and submerged fringe of growth. Older cultures may develop white pleomorphic tufts of mycelium. A deep yellowish-brown reverse pigment is usually present. Microscopic morphology shows characteristic smooth, thin-walled macroconidia which are often produced in clusters growing directly from the hyphae. Numerous chlamydoconidia are formed in older cultures. No microconidia are formed.

Culture of Epidermophyton floccosum.

Macroconidia of E. floccosum

Chlamydoconidia of E. floccosum.

Clinical significance:

Epidermophyton floccosum is an anthropophilic dermatophyte with a world-wide distribution which often causes tinea pedis, tinea cruris, tinea corporis and onychomycosis. It is not known to invade hair in vivo and no specific growth requirements have been reported.

Clinical significance:

E. floccosum infections may become epidemic among personnel using common shower or gym facilities, e.g. athletic teams, troops, ship crews and inmates of institutions.

Microsporum:

Macroconidia with rough walls present, microconidia may also be present.

Microsporum sp.Teleomorph: Arthroderma sp.

Microsporum species form both macro- and microconidia on short conidiophores. Macroconidia are hyaline, multiseptate, variable in form, fusiform, spindle-shaped to obovate, ranging from 7 to 20 by 30 to 160 um in size, with thin- or thick- echinulate to verrucose cell walls. Their shape, size and cell wall features are important characteristics for species identification. Microconidia are hyaline, single-celled, pyriform to clavate, smooth-walled, 2.5 to 3.5 by 4 to 7 um in size and are not diagnostic for any one species.

Microsporum sp.Teleomorph: Arthroderma sp.

The separation of this genus from Trichophyton is essentially based on the roughness of the macroconidial cell wall, although in practice this may sometimes be difficult to observe. Seventeen species of Microsporum have been described, however only the more common species are included in these descriptions.

Microsporum spp

Microsporum audouinii Microsporum canis Microsorum canis var. distortum Microsporum cookei Microsporum equinum Microsporum ferrugineum

Microsporum canis Teleomorph: Arthroderma otae

On Sabouraud's dextrose agar, colonies are flat, spreading, white to cream-coloured, with a dense cottony surface which may show some radial grooves. Colonies usually have a bright golden yellow to brownish yellow reverse pigment, but non-pigmented strains may also occur.

Culture of Microsporum canis

Macroconidia of M. canis

M. canis growing on boiled polished rice grains

Macroconidia

Macroconidia are typically spindle-shaped with 5-15 cells, verrucose, thick-walled and often have a terminal knob. A few pyriform to clavate microconidia are also present.

Macroconidia

Macroconidia and/or microconidia are often not produced on primary isolation media and it is recommended that sub-cultures be made onto boiled polished rice grains to stimulate sporulation.

Clinical significance:

Microsporum canis is a zoophilic dermatophyte of world-wide distribution which is a frequent cause of ringworm in humans, especially children. Invades hair, skin and rarely nails.

Cats and dogs are the main sources of infection. Invaded hairs show an ectothrix infection and fluoresce a bright greenish-yellow under Wood's ultra-violet light.

Microsporum spp

Microsporum fulvum Microsporum gallinae Microsporum gypseum Microsporum nanum Microsporum persicolor

Clinical significance:

The genus Microsporum contains a number of important species that are the principle causative agents of animal and human dermatophytoses [tinea and ringworm].

Trichophyton:

Microconidia present, smooth-walled macroconidia may or may not be present.

Trichophyton sp.Teleomorph: Arthroderma sp.

The genus Trichophyton is characterized by the development of both smooth-walled macro- and microconidia.

Macroconidia are mostly borne laterally directly on the hyphae or on short pedicels, and are thin- or thick-walled, clavate to fusiform, and range from 4 to 8 by 8 to 50 um in size.

Macroconidia are few or absent in many species. Microconidia are spherical, pyriform to clavate or of irregular shape and range from 2 to 3 by 2 to 4 um in size.

Microconidia, macroconidia and nodular organs of T. equinum

Horse infection and culture of Trichophyton equinum.

Dimorphic Fungal Pathogens

These are fungal infections of the body caused by fungal pathogens which can overcome the physiological and cellular defences of the normal human host by changing their morphological form.  They are geographically restricted and the primary site of infection is usually pulmonary, following the inhalation of conidia. 

Descriptions for the following fungi are provided.

Blastomyces dermatitidis Coccidioides immitis Histoplasma capsulatum Paracoccidioides brasiliensis

Coccidioides immitis

On Sabouraud's dextrose agar at 25C, colonies are initially moist and glabrous, but rapidly become suede-like to downy, greyish white in color with a tan to brown reverse. However, considerable variation in both growth rate and culture morphology has been noted (Rippon, 1988). Microscopic morphology shows typical single-celled, hyaline, rectangular to barrel-shaped, alternate arthroconidia, 2.5-4.0 x 3.0-6.0 um in size, separated from each other by a disjunctor cell. This arthroconidial state has been classified in the genus Malbranchea and is similar to that produced by many non-pathogenic soil fungi such as Gymnoascus species.

Culture of Coccidioides immitis.

Arthroconidia of C. immitis.

Culture of Coccidioides immitis.

In the past microscopic morphology, conversion from the mould form to the yeast or spherule form, and animal pathogenicity have all been used for identification; however exoantigen tests are now the method of choice, at least for Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum. 

Hyaline Hyphomycetes

Hyaline Hyphomycetes include those conidial fungi which are not darkly pigmented, colonies may be colorless or brightly colored.  These include the agents of hyalohyphomycosis, Aspergillosis, dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum.  Descriptions of the following genera are avaiable:

genera AcremoniumAspergillusBeauveria

ChrysosporiumFusariumGeotrichumGliocladiumGraphiumMadurella

genera Malbranchea

PaecilomycesPenicilliumScedosporiumScopulariopsisSepedoniumTrichodermaTrichotheciumVerticillium

Identification:

Identification of the hyphomycetes is primarily based on microscopic morphology including; (a) conidial morphology, especially septation, shape, size, color and cell wall texture;

Identification:

(b) the arrangement of conidia as they are borne on the conidiogenous cells, for example are they solitary, arthrocatenate, blastocatenate, basocatenate or gloiosporae etc.,

Identification:

(c) the type conidiogenous cell, for example non-specialized or hypha-like, phialide, annellide or sympodial etc., and (d) other additional features such as the presence of sporodochia or synnemata.

Culture characteristics, although less reliable may also be

useful.  These include surface texture, topography and pigmentation, reverse pigmentation and growth at 37C.  For identification, potato dextrose agar and cornmeal agar are two of the most suitable media to use and exposure to daylight is recommended to maximize culture color characteristics.

  Culture Characteristics.  . Least reliable as the media and growth

conditions play an important part.  Examine the following characteristics:

Surface texture [glabrous, suede-like, powdery, granular, fluffy, downy, cottony etc].

Surface topography [flat, raised, heaped, folded, domed, radial grooved].

  Culture Characteristics.  Surface pigmentation [white, cream,

yellow, brown, green, grey, black etc]. Reverse pigmentation [none, yellow,

brown, red etc]. Growth rate [eg colonies growing less

than 5 mm in 14 days etc]. Growth temperature studies are also

often very useful [37C, 40C & 45C].

Penicillium sp.

Penicillium sp.

Colonies are usually fast growing, in shades of green, sometimes white, mostly consisting of a dense felt of conidiophores. Microscopically, chains of single-celled conidia (ameroconidia) are produced in basipetal succession from a specialized conidiogenous cell called a phialide. The term basocatenate is often used to describe such chains of conidia where the youngest conidium is at the basal or proximal end of the chain. In Penicillium, phialides may be produced singly, in groups or from branched metulae, giving a brush-like appearance known as a penicillus. The penicillus may contain both branches and metulae (penultimate branches which bear a whorl of phialides). All cells between the metulae and the stipes of the conidiophores are referred to as branches.

Penicillium sp.

The branching pattern may be either simple (non-branched or monoverticillate), one-stage branched (biverticillate-symmetrical), two-stage branched (biverticillate-asymmetrical) or three- to more-staged branched. Conidiophores are hyaline and may be smooth- or rough-walled. Phialides are usually flask-shaped, consisting of a cylindrical basal part and a distinct neck, or lanceolate (with a narrow basal part tapering to a somewhat pointed apex). Conidia are globose, ellipsoidal, cylindrical or fusiform, hyaline or greenish, smooth- or rough- walled. Sclerotia may be produced by some species.

Conidiophores of P. verrucosum var. cyclopium showingtwo-stage branching. Simple conidiophore of P. cheresanum showing long chains of single-celled

phialoconidia.

Morphological structures and types of conidiophore branchingin Penicillium. a. simple; b. one-stage branched;

c. two-stage branched; d. three-stage branched

Clinical significance:

Many species of Penicillium are common contaminants on various substrates and are known as potential mycotoxin producers.

Fusarium sp.

Culture of Fusarium sp.

Culture of Fusarium sp.

Colonies are usually fast growing, pale or brightly colored (depending on the species) and may or may not have a cottony aerial mycelium. The color of the thallus varies from whitish to yellow, brownish, pink, reddish or lilac shades. Species of Fusarium typically produce both macro- and microconidia from slender phialides.

Macroconidia are hyaline, two- to several-celled, fusiform- to sickle-shaped, mostly with an elongated apical cell and pedicellate basal cell. Microconidia are 1- to 2-celled, hyaline, pyriform, fusiform to ovoid, straight or curved. Chlamydoconidia may be present or absent.

Microconidia of Fusarium solani are usually abundant, cylindrical to oval, 1- to 2-celled and formed from long lateral phialides.

. Macroconidia Fusarium solani are formed after 4-7 days from short multi-branched conidiophores which may form sporodochia

Chlamydoconidia Fusarium solani are hyaline, globose,smooth- to rough-walled, borne singly or in pairs on short lateral hyphal branches, or they are intercalary.

Clinical manifestations linical manifestations Clinical manifestations of

hyalohyphomycosis caused by Fusarium; include cutaneous and subcutaneous infections, endophthalmitis, osteomyelitis, and arthritis following traumatic implantation. Peritonitis has also been reported in patients on continuous ambulatory peritoneal dialysis (CAPD).

Clinical manifestations

Disseminated infections are similar to disseminated aspergillosis, however fungemia and ulcerated skin lesions are often more pronounced. The typical patient is granulocytopenic and receiving broad-spectrum antibiotics for unexplained fever.

Penicillium sp. colonizing an orange showing typical green surface pigmentation with a suede-like surface consisting

of a dense felt of conidiophores.

Culture of Penicillium sp. on Sabouraud’s dextrose agar.

Penicillium sp.

Penicillium sp. showing chains of single celled phialoconidia (ameroconidia) produced in basipetal succession from a specialized conidiogenous cell called a phialide.  The term basocatenate is often used to described such chains of conidia where the youngest conidium is at the basal or proximal end of the chain.  In Penicillium phialides may be produced singly or in groups or from branched metulae, giving a brush-like appearance, i.e. a penicillus

Penicillium sp.

Aspergillus fumigatus

Aspergillus fumigatus

On Czapek dox agar showing typical blue-green surface pigmentation with a suede-like surface consisting of a dense felt of conidiophores.

Aspergillus fumigatus

Aspergillus fumigatus

Microscopic morphology of Aspergillus fumigatus showing typical columnar, uniseriate conidial heads.  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.  Conidia are produced in basipetal succession forming long chains   Conidia are globose to subglobose, green and rough-walled to echinulate

Aspergillus fumigatus

Aspergillus niger

Aspergillus flavus

Aspergillus flavus on Czapek dox agar.  Colonies are granular, flat, often with radial grooves, yellow

Mucor sp.

Mucor sp.

Microscopic morphology of a Mucor sp. showing erect, simple sporangiophores forming a terminal, globose sporangium, packed with sporangiospores and with a well developed subtending columella visible. 

Culture of Candida albicans

Bubbing yeast cells of Candida albicans