identification of medically important fungi
TRANSCRIPT
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