Medical MycologyCandida

Candidiasis: fungal infection due to any of the Candida species Candidemia: the presence of Candida spp in the blood Invasive Candidiasis/disseminated Candidiasis: presence of Candida spp in the tissues or organs as a result of dissemination on the blood Normal commensals Found in the skin Opportunistic pathogen of GI tract, sputum, female genital tract and urinary tract Intact skin is important in defense against Candida infection Immunosuppression: DM and Iatrogenic: antibiotics, indwelling catheters, prosthetic implants and abdominal surgery Thrush: Oral candidiasis with creamy white, curdlike patches on the tongue and other mucosal surfaces Vulvovaginitis: DM, antibiotic theraphy, pregnancy: scanty to thick curdlike discharge, may also have edema and intense pruritus Erosio Digitalis Blastomycetica: Candida infection occuring between the fingers and toes Candida Folliculitis: infection at the hair follicles Candida Balanitis: penile vesicles to patches resembling thrush, severe pruritis and burning Paronychia/Onchomycosis: candidiasis on nails: associated with frequent immersion of hands in water Diaper rash CNS Candidiasis: affects both parenchyma and meninges: hydrocephalus Respiratory tract candidiasis: pneumonia Cardiac candidiasis: endocarditis, myocarditis UTI: cystitis The genus Candida is characterised by globose to elongate yeast-like cells or blastoconidia that reproduce by multilateral budding, polar

budding if present on a narrow base, pseudohyphae and occasionally true hyphae may also be present. Identification: Ensure that you start with a fresh growing pure culture; streak for single colony isolation if necessary. Chromogenic agars are now being used for primary isolation for both the detection of mixed flora and rapid species identification, especially

from non-sterile sites. Germ Tube Test. A rapid screening test for Candida albicans and Candida dubliniensis.

0.5 mL of serum, containing 0.5% glucose, is lightly inoculated with the test organism and incubated at 35OC for 2-3 hours. On microscopy, the production of germ tubes by the cells is diagnostic for Candida albicans.

YEAST C. trpicalisC. albicansC. parapsilosis GERM TUBE POSITIVEC. glabrata

CHROM agar Candida plate showing chromogenic colour change for C. albicans (green),C. tropicalis (blue), C. parapsilosis (white) and C. glabrata (pink).

Candida albicans on Sabouraud’s dextrose agar showing typical cream coloured, smooth surfaced, waxy colonies

Direct smear of urine from a patient with candidiasis of the kidney showing C. albicans in mycelial or tissue phase with blastoconidia budding from the pseudohyphae.

Candida albicans Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Spherical to subspherical budding blastoconidia, 2-7 x 3-8 μm in size. India Ink Preparation: Negative - No capsules present. Dalmau Plate Culture on Cornmeal and Tween 80 Agar: Branched pseudohyphae with dense verticils of blastoconidia. Spherical

chlamydospores, mostly terminal, often on a slightly swollen subtending cell are formed near the edge of the cover slip.

Key Features: germ tube positive, production of chlamydospores on Dalmau plate culture, fermentation of glucose, sugar assimilation profile and a distinctive green colour on CHROMagar.

Note: germ tube negative variants, known as C. claussenii, and sucrose-negative variants described as C. stellatoidea have proven to be synonymous with C. albicans. C. albicans is a commensal of mucous membranes and the gastrointestinal tract.

Candida dubliniensis Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Spherical to subspherical budding blastoconidia, 3-8 x 2-7 μm in size. India Ink Preparation: Negative - No capsules present. Key Features: germ tube positive, similar to C. albicans, except for absence of growth at 45oC; glycerol (mostly +), methyl-a-D-glucoside (-),

trehalose (-), and D-xylose (-). Initial colonies dark green colour on CHROMagar and producing rough colonies on bird seed agar. C. dubliniensis is an uncommon cause of

candidemia and mucosal infection, especially in HIV patients. Candida glabrata

Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Ovoid to ellipsoidal budding blastoconidia, 3.4 x 2.0 μm in size. No pseudohyphae or chlamydospores produced. India Ink Preparation: Negative - No capsules present. Key Features: germ tube negative yeast and sugar assimilation pattern. Candida glabrata is one of the most common yeast species to be found on the body surface and is often isolated as an incidental finding from skin and urine.

It has been implicated as an “opportunistic” cause of both superficial and systemic infections, especially in immunocompromised patients, and it has been isolated from patients with septicemia, pyelonephritis, pulmonary infections, endocarditis and hyperalimentation.

Candida krusei Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Predominantly small, elongated to ovoid budding blastoconidia, 2.0-5.5 x 4.0-15.0 μm. India Ink Preparation: Negative - No capsules present. Key Features: germ tube negative yeast and sugar assimilation pattern. Candida krusei is regularly associated with some forms of infant

diarrhoea and occasionally with systemic disease. It has also been reported to colonise the gastrointestinal, respiratory and urinary tracts of patients with granulocytopenia

Candida parapsilosis Complex Recently Candida parapsilosis has been recognised as 3 species: C. parapsilosis, C. orthopsilosis and C. metapsilosis Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Predominantly small, globose to ovoid budding blastoconidia, 2.0-3.5 x 3.0-4.5 μm, with some larger elongated forms present. India Ink Preparation: Negative - No capsules present. Key Features: germ tube negative yeast and sugar assimilation pattern. Candida parapsilosis is an opportunistic human pathogen which may

cause cutaneous infections, especially of the nail and systemic disease, especially endocarditis. Other clinical manifestations include endophthalmitis and fungemia.

Candida tropicalis

Culture: Colonies (SDA) white to cream-coloured smooth, glabrous yeast-like. Microscopy: Spherical to subspherical budding yeast-like cells or blastoconidia, 3-5.5 x 4-9 μm. India Ink Preparation: Negative - No capsules present. Key Features: germ tube negative yeast and sugar assimilation pattern. Candida tropicalis is a major cause of septicemia and disseminated

candidiasis

Fungi Aerobic, nucleated which reproduce sexually and/or asexually Filamentous branched somatic structures surrounded by a true cell wall Grow as a mass of branching, interlacing filaments Asexual: via mitosis Sexual: via meiosis Mycology - the study of fungi Fungi - molds and yeasts Molds - exhibit filamentous type of growth Yeasts - pasty or mucoid form of fungal growth General considerations Fungi stain gram positive, and require oxygen to survive Fungi are eukaryotic, containing a nucleus bound by a membrane, endoplasmic reticulum, and mitochondria. (Bacteria are prokaryotes and

do not contain these structures.) Fungi are dependent upon enzymes systems to derive energy from organic substrates - saprophytes - live on dead organic matter - parasites - live on living organisms

Sexual Germinate by means of spores which produces one or more filamentous germ tubes Each germ tube elongates by growth of the distal end to become long filaments Each filament is called a hypha which eventually branch and intertwine to form a mass called a mycelium

Parts of a mycelium Aerial mycelium: above the agar. Develops reproductive spores Vegetative mycelium: beneath the agar. Grows into the substrate and absorbs food for further growth

Types of Hypha Nonseptate or coenocytic: no transverse walls or septa divide the hyphae. Septate: transverse walls or septa are formed at regular intervals along the filament

Colony type at different temperatures

Monomorphic or monophasic: produce one type of colony at both room and incubation temperature Ex: Cryptococcus neoformans: yeast only. Aspergillus spp: filamentous only. Candida albicans: yeast only. Dimorphic or diphasic: produce a yeastlike colony at incubation temp (yeast phase) and a filamentous type of colony at room temp (mycelial

phase) Ex: Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis

Spores Sexual spores: produced by the fission of two nuclei which may or may not belong to the same strain within a species Zygospores: produced by the tips of approximating hyphae conjugating and resullting in large thick-walled bodies from fusion of contents of

the terminal portion

Basidiospores: sexual spores typicallt 4 in number produced at the ends of club shaped structures called a basidium

Ascospores: produced within an enlarged cell by nuclear fusion enclosed in a sac called an Ascus or Ascocarp. Typically, there may be 4-8 spores within an ascus

Asexual spores: arise by the differentiation of spore bearing hyphae without nuclear fission Sporangiospores: produced inside a sporangium.

Conidiospores: produced on a specialized hypha and are freed by abstriction at the point of attachment. Microconidia: small and single celled

Macroconidia: large and multicellular

Colonies, hyphae, microconidia and macroconidia of Trichophyton tonsurans Conidia according to shape: Fusiform: spindle shaped Clavate: club shaped Muriform: multiseptate, both transverse and longitudinally

Typical spindle-shaped macroconidia of Microsporum canis

chlamydospores of E. floccosum.

Alternaria alternata showing branched acropetal chains and multi-celled,

obclavate to muriform conidia with short conical beaks.

Thallospores: Athrospores: frragmentation or segmentation of mycelium results in the production of rectangular thick walled spores

Arthroconidium formation in G. candidum. Hyphal elements are progressively compartmentalised by fragmentation of septa. Conidial secession is by the centripetal separation (schizolysis) of a so called double septum and concomitant rupture of the original outer hyphal wall layer

Blastospores: formed by a simple budding process from cells of the mycelium. Budding may be in one cell to one spore basis (Blastomyces dermatitidis) or multispore (Paracoccidioides brasiliensis) basis

Blastomyces dermatitidis Multiple, narrow base budding yeast cells “steering wheels” of P. brasiliensis.

Infectious Mycotic DiseasesSuperficial:

Dermatomycosis: dermatophytes: Microsporum, Epidermophyton, Trichophyton

Pityriasis versicolor: Malazzezia furfur Tinia nigra: Cladosporium werneckii Piedra: white: Trichosporum cutaneum

black: Piedra hortai Otomycosis: Aspergillus, Mucor, Penicillium, Rhizopus Keratitis: Fusarium, Aspergillus, Curvularia, Penicillium,

Cephalosporium, Candida species Subcutaneous :

Sporotrichosis: Sporothrix schenckii Chromomycosis: Phialophora verrucosa, Fonsecae pedrosoi,

Cladosporium carrioni

Mycetoma: Nocardia, Streptomyces

Deep seated: Coccidioidomycosis Histoplasmosis Blastomycosis

Opportunistic: Candidiasis Cryptococcosis Aspergillosis Zygomycosis Geotrichosis Penicillosis

Identification of molds

The genus Rhizopus is characterised by the presence of stolons and pigmented rhizoids, the formation of sporangiophores, singly or in groups from nodes directly above the rhizoids, and apophysate, columellate, multispored, generally globose sporangia.

After spore release the apophyses and columella often collapse to form an umbrellalike structure. Sporangiospores are globose to ovoid, one-celled, hyaline to brown and striate in many species. Colonies are fast growing and cover an agar surface with a dense cottony growth that is at first white becoming grey or yellowish brown with sporulation.

Culture, sporangia, sporangiophores and rhizoids of R.oryzae.

Cladosporium species have a world-wide distribution and are amongst the most common of air-borne fungi. Some 500 species have been described.

Conidiophores and conidia of Cladosporium cladosporioides

Isolates of Cladosporium are frequently isolated as contaminants. RG-1 organisms. The pathogenic species have now been transferred to the genus Cladophialophora.

Key Features: dematiaceous hyphomycete forming branched acropetal chains of conidia, each with a distinct hilum.

Fusarium Colonies are usually fast growing, pale or bright coloured (depending on the species) with or without a cottony aerial mycelium. The colour of the thallus varies from whitish to yellow, pink, red or purple shades. Species of Fusarium typically produce both macro- and

microconidia from slender phialides.

Microconidia on short phialides and macroconidia of F. oxysporum

Scopulariopsis: Colonies are fast growing, varying in colour from white, cream, grey, buff to brown, black, but are predominantly light brown. Microscopic

morphology shows chains of single-celled conidia produced in basipetal succession from by a specialised conidiogenous cell called an annellide.

Conidiophores (annellides) and conidia of Scopulariopsis brevicaulis

Aspergillus Colonies are usually fast growing, white, yellow, yellow-brown, brown to black or shades of green, mostly consisting of a dense felt of erect

conidiophores. Conidiophores terminate in a vesicle covered with either a single palisade-like layer of phialides (uniseriate) or a layer of subtending cells (metulae) which bear small whorls of phialides (the so called biseriate structure). The vesicle, phialides, metulae (if present) and conidia form the conidial head. Conidia are one-celled, smooth or rough walled, hyaline or pigmented are produced in long dry chains which may be divergent (radiate) or aggregated in compact columns (columnar). Some species may produce Hülle cells or sclerotia

Diagnostic test for Aspergillus

Wet smear Fungal stains: PAS; silver stain Fluorescent techniques Immunohistochemistry SDA

Penicillium Colonies are usually fast growing, in shades of green, sometimes white, mostly consisting of a dense felt of conidiophores. In Penicillium, phialides may be produced singly in groups or from branched metulae, giving a brush-like appearance (a penicillus).

Conidiophores of P. verrucosum var. cyclopium showing two-stage branching. Simple

Chromoblastomycosis Chronic mycosis of the skin and subcutaneous tissue Sclerotic bodies/muriform bodies in histopath These are globose to oval cells with thick brown walls that reproduce by septation

Chromoblastomycosis Phialophora verrucosa Fonsecaea pedrosi Cladosporium carionnii Rhinocladiella aquaspersa Botryomyces caespitosus

Mucormycosis (Phycomycosis, Zygomycosis) Rhizopus Cunninghamella Mucor Absidia Rhizomucor Syncephalastrum

Clinical syndromes Sinusitis Pneumonia Skin and wound infections Cerebral infections GIT lesions Cardiac

Sporangia, columella with a conspicuous Culture, sporangia, sporangiophores and rhizoids of R.oryzae.

collarette (arrow)

and sporangiospores of Mucor.