LABORATORY DIAGNOSIS OF COMMON FUNGAL DISEASES

Prof. Louella A. Dancel, RMT, MSc.

University of Perpetual Help System DALTA

42nd PAMET ANNUAL CONVENTIONManila Hotel – December 2,2006

Clinical specimens submitted for Fungal Isolation 2004-2006*

Clinical specimen 2004 2005 2006 Total

Respiratory 29 35 34 98Body fluids 37 17 34 88Tissues 9 0 23 32Skin 1 0 0 1Nails 29 31 20 80Hair 0 1 0 1CSF 8 5 8 21Others 5 20 24 49TOTAL 118 109 153 380

Fungal Isolates 2004-2006*Isolate 2004 2005 2006C.albicans 5.1% 13.8% 11.8%C.tropicalis 1.8% 3.7% 5.2%C.parapsilosis 5.1% 6.4% 3.9%C.glabrata 0.91% 0.91% 3.9%C.famata 1.8% 4.6% 2.0%Aspergillus spp. 0.91% 0% 0.65%Fusarium spp. 0% 0% 0.65%

Data from a Five-year review of Fungal Isolates at UPM-CPH

• Clinical specimens (n=545):–Skin scrapings–Nail clippings/scrapings–Hair–Exudates–Biopsy materials

Data from a Five-year review of Fungal Isolates at UPM-CPH

Total clinical specimens tested – 545 Results: 10.8%- (+) for both KOH & culture 59.1%- (-) for both KOH &culture 17.1% - (+)culture, (-) KOH 12.8% - (-) culture, (+) KOH

Data from a Five-year review of Fungal Isolates at UPM-CPH

Fungal isolates : Trichophyton mentagrophytes Trichophyton rubrum Trichophyton tonsurans Trichophyton schoenlenii Trichosporon spp.

Data from a Five-year review of Fungal Isolates at UPM-CPH

• Fungal isolatesMicrosporum gypseum

Microsporum canis Epidermophyton flocossum Candida albicans Exophiala werneckii

Data from a Five-year review of Fungal Isolates at UPM-CPH

Fungal isolates from biopsy materials and exudates (31.2% positive):

Fonsecaea compactumPhialophora verrucosa

Exophiala jeanselmeiMadurella grisea

Laboratory Methods for Diagnosis of Mycoses

I. DIRECT EXAMINATION: *10-30% KOH

*Calcofluor white stain *Histological stains- H&E, PAS *India Ink *Wet mount

Laboratory Methods for Diagnosis of Mycoses

II. Isolation & CultureSDABHIA/BAP

Media with/without antibiotics

• Macroscopic examination of culture• Microscopic examination using LPCB

Laboratory Methods for Diagnosis of Mycoses

• III. Biochemical Tests:*Rapid kits for yeasts*Urea test

• IV. Special Tests: *In-vitro hair perforation test

*Germ tube test *Chlamydoconidia formation test

Mycotic Infections

Superficial

Cutaneous

Subcutaneous

Systemic

Opportunistic

*Mycotoxicosis

*Allergies

Superficial mycoses• superficial cosmetic

fungal infections of the skin or hair shaft

• no living tissue is invaded

• no cellular response from the host

• no pathological changes

• patients unaware of infection

Superficial mycosesDisease

SKIN• Pityriasis

versicolor

• Tinea nigra

Causative organisms

• Malassezia furfur

• Exophiala werneckii

Superficial mycoses

Disease

HAIR• White piedra

• Black piedra

Causative organisms

• Trichosporon beigelii

• Piedraia hortae

Superficial mycoses

Pityriasis versicolor• Lesion

-An-an”-Hyperpigmented or

hypopigmented macular lesions

www.ethnomed.org

Superficial mycoses

Pityriasis versicolor• Lesion

– scale readily, giving a chalky branny appearance

– occurs on the trunk, shoulders & arms, face and neck

Modified from www.columbia.edu

Superficial mycoses

Pityriasis versicolor

• Lesion– fluoresce pale greenish under Wood’s

lamp• Distribution

– worldwide– more common in tropical than

temperate climates

Superficial mycoses

Pityriasis versicolorKOH of skin scrapings

• clusters of budding yeast-like cells & short angular hyphal forms

• “spaghetti and meat balls”

Superficial mycoses

Pityriasis versicolorPAS of skin scrapings• “spaghetti and meat

balls”

Superficial mycoses

Pityriasis versicolor• Culture of skin

scrapings– Not necessary– diagnostic microscopic

features– SDA overlaid with

peanut oil, olive oil

Superficial mycoses

Pityriasis versicolor

• Etiologic Agent– Malassezia globosa

lipophilic yeastpart of skin normal flora

Superficial mycoses

Pityriasis versicolor

• Treatment– keratinolytic agents applied locally– Mild fungicides– Miconazole– Selenium sulfide (1%) shampoo

Superficial mycoses

Tinea nigra• Lesion

– Gray to black well-demarcated macular lesions

– most frequently occurring on the palms of the hand

– non-inflammatory & non-scaling lesions

11th.blogspot.com

Superficial mycoses

Tinea nigra

• Distribution– world-wide– more common in tropical regions of

Central & South America, Africa, Southeast Asia & Australia

Superficial mycoses

Tinea nigra

• KOH– pigmented brown to dark

olivaceous (dematiaceous) septate hyphal elements & 2-celled yeast cells

Superficial mycoses

Tinea nigra

• Etiologic agent– Exophiala werneckii

saprophyte found in soil, compost, humus &

wood in humid tropical & sub-tropical regions

Superficial mycoses

Tinea nigra

• Culture on SDA– initially mucoid,

yeast-like & shiny black

– with age: aerial mycelia & dark olive color

Superficial mycoses

Tinea nigra

• Lactophenol cotton blue (LPCB) of culture on SDA– 2-celled, pale brown yeast cells– darkly pigmented septa (annelides)– one cell cylindrical, the other cell is

spindle-shaped– occur in aggregated masses

Superficial mycoses

Tinea nigra• Treatment

– keratinolytic agents (Whitfield’s ointment)

– tincture of iodine, 2% salicylic acid, 3% sulfur

– miconazole nitrate, imidazoles, triazoles

Superficial mycoses

Tinea nigra

• Prevention– avoid exposure to sources,

contaminated material

Superficial mycoses

Piedra

• Fungus infection of the hair shaft

• presence of firm, irregular nodules

• Nodules - fungal elements cemented together along the hair shaft

• Multiple infections of the same strand

Superficial mycoses

Piedra

Two varieties–White piedra–Black piedra

Superficial mycoses

Black piedra• Lesion

– discrete, hard, gritty, brown to black concretions / nodules

– infection of hair• scalp hair -common• beard, moustache -

less common• axilla & groin hairs -

rare

www.doctorfungus.org

Superficial mycoses

Black piedra - distribution

Superficial mycoses

Black piedra

• Etiologic agent– Piedraia hortae– source of infection

Superficial mycoses

Black piedra - lab diagnosis

• Direct microscopy– specimen - hair with nodules– 25% NaOH or KOH– dark septate hyphae

Superficial mycoses

Black piedra - lab diagnosis

• Direct microscopy– round to oval asci;

hyaline, curved to fusiform ascospores

Superficial mycoses

Black piedra - lab diagnosis

• Isolation – medium

– SDA with chloramphenicol

– SDA cycloheximide

Superficial mycoses

Black piedra - lab diagnosis• Isolation -growth very slow

-dark brown to black-greenish brown,

short aerial mycelium

Heaped center

Flat periphery

Superficial mycoses

White piedra• Infection of hair

shaft– face, axilla, genitals -

common– scalp, eyebrows,

eyelashes - less common

Superficial mycoses

White piedra• Infection of hair

shaft– less common

scalp

eyebrow

eyelashes

Superficial mycoses

White piedraNodule

• Soft, white, yellowish, beige or greenish nodule

• Discrete• more often coalescent,

forming an irregular transparent sheath

Superficial mycoses

White piedra

• Distribution– common in S. America & Asia– sporadic in N. America & Europe

• Etiologic agent– Trichosporon beigelii or T. cutaneum

Superficial mycoses

White piedra

• Ecology– soil, stagnant water– decaying fruit– spoiled food– sputum & body surfaces– horses

Superficial mycoses

White piedra - lab diagnosis

• Microscopic direct examination– specimen - hair with nodules

– 10% KOH or 25% NaOH + 5% glycerin

– hyaline septate hyphae

– oval or rectangular arthroconidia

– occasional blastoconidia

Superficial mycoses

White piedra - lab diagnosisIsolation• medium - SDA with

chloramphenicol without cycloheximide

• growth/culture– rapid– cream-colored, soft– membranous, wrinkled radial

furrows, irregular folding

Superficial mycoses

White piedra - lab diagnosisIsolation• microscopic exam

of culture– hyaline hyphae

– arthroconidia

– blastoconidia

Superficial mycoses

White piedra - lab diagnosis

• Physiological studies– does not ferment carbohydrates– assimilate dextrose, lactose, D-xylose,

inositol– negative KNO3 assimilation– urease positive– arbutin is split

Superficial mycoses

Piedra - Treatment

• Shaving or cutting infected hair• Topical fungicides

– 1:200 bichloride of mercury– benzoic acid & salicylic acid combinations– 3% sulfur ointment– 2% formalin

Cutaneous mycoses

skin

hair

nails

• No living tissue

• Host Rxn to fungus

keratinase

Cutaneous mycosesDisease

• Dermatophytosis

Causative organisms

• Dermatophytes Microsporum

Trichophyton

Epidermophyton

ringworm

Cutaneous mycosesDisease

• Candidiasis of skin, mucous membranes & nails

Causative organisms

• Candia albicans & related species

dermatomycosis Soil fungi (Scytalidium, Fusarium, etc.)

Systemic fungi (Histoplasma, etc)

Ecological Groups ofDermatophytes

Geophilic

• inhabit soil where they decompose keratinaceous debris

• Dead animals

Zoophilic

• parasitic on animals

www.saanendoah.com www.kolumbus.fi

Anthropophilic fungi

• primarily parasitic to man• man as exclusive host• for maintenance & dissemination of

species

Anthropophilic fungi• Anthropophilic

fungi:– Examples:

• M. audonii• T. rubrum• T. schoenleinii• T. tonsurans• T. violaceum

Classification of Dermatophytes

Microsporum

MacroconidiaRough walled

Microconidapresent

Trichophyton

MacroconidiaSmooth walled

Microconidiapresent

Epidermophyton

ChlamydoconidiaMacroconidiaSmooth walled

Microconidianone

Clinical Manifestations of Dermatophytes

Tinea capitis

www.emedicinehealth.com

Scalp, eyebrow, eyelashes Microsporum &

Trichophyton

Tinea capitis Endothrix Ectothrix

Tinea favosa

• Scutulum• Mass of mycelia

& epithelial debris

• Cup shaped crusts

www.mf.uni-lj.si

Tinea corporis

www.cut.ee/

•Non-hairy skin•Rings with scaly

centers•Rxn vs fungus

Tinea corporis

• E. floccosum • Trichophyton

• Microsporum

Cutaneous

Tinea imbricata

Concentric rings

Trichophyton concentricum

Cutaneous

Tinea barbae

• Bearded areas of face & neck

www.merck.com

www.emedicine.com

Cutaneous

Tinea cruris

www.dermnetnz.org

Jock itch

Moist groin

area

E. floccosum,

T. rubrum

Cutaneous

Tinea pedis

www.doctorfungus.org

dermatologie.free.fr

Athlete’s foot

Toe webs & soles,

even nails

Id reaction,

circulating fungal

antigens

Cutaneous

Tinea manuum

www.dermnetnz.org

• Interdigital areas & palmar surfaces

Cutaneous

Tinea unguium

www.dermnetnz.org

Invasion of nail plate by

dermatophytes

Thickened, discolored &

brittle• Onychomycosis- non

dermatophyteYeast etc.

Laboratory diagnosis

Wipe with water

www.doctorfungus.org

scalpelPaper / envelope

active edge

Skin scraping specimen

Direct Examination

• Wet mountKOH• KOH

– 10% to 30%– with Parker Superquink

blue-black ink– gentle warming

pa

rk

er

Cutaneous

KOH of skin scrapings

Septate hypha

Cutaneous

arthrospores

septate hypha

KOH of skin scrapings

Cutaneous

Ectothrix invasion of hair

• Hair invasion • formation of

arthroconidia on the outside of hair shaft

• cuticle of hair is destroyed

Cutaneous

Ectothrix invasion of hair

• Hair invasion by a dermatophyte– Microsporum canis– M. gypseum– Trichophyton equinum– T. verrucosum

Cutaneous

Ectothrix invasion of hair

• Wood’s UV light• infected hairs

fluoresce• bright greenish

yellow under

Cutaneous

Endothrix invasion of hair

• formation of arthroconidia within hair shaft

• cuticle of hair remains intact

• do not fluoresce under Wood’s UV light

Cutaneous

Endothrix invasion of hair

• ALL AGENTS ARE ANTHROPOPHILIC

• Trichophyton tonsurans,

• T. violaceum

Culture:

• Selective media– SDA with chloramphenicol &

cycloheximide (Mycosel or Mycobiotic agar)

– Dermatophyte test medium

Non-selective medium– Sabouraud’s dextrose agar

Culture:

• IncubationRoom temperatureAt least 2 weeks

Identification

• Gross color & texture• Microscopic characteristics• Confirm / compare with

Written descriptionsDrawingsphotographs

Mycology

Cutaneous

Microsporum canis

netti.nic.fi

• Zoophilic– cats and dogs

• Invades– Hair– skin – rarely nails

• distribution– worldwide

www.vet.ohio-state.edu

Cutaneous

golden yellow reverse colony

www2.provlab.ab.ca

Microsporum canislab diagnosis –

culture• white cottony growth

Cutaneous

Microsporum canis• microscopic:

– spindle shaped, one end pointed, other end blunt

– thick walled verrucose macroconidia

– 6 to 12 cellswww.doctorfungus.org

Cutaneous

Microsporum gypseum

– geophilic– usually produces a single

inflammatory skin or scalp lesion• distribution

– worldwide

Cutaneous

Microsporum gypseumlab diagnosis -

culture• flat, spreading suede-

like to granular • cinnamon growth • yellow brown pigment

on reverse of colony

www.ukneqasmicro.org.uk

Cutaneous

Microsporum gypseummicroscopic: • symmetrical ellipsoidal• thin walled verrucose

macroconidia• distal end slightly rounded,

proximal (point of attachment) is blunt

• 4 to 6 cells

vtpb-www.cvm.tamu.edu

www.medmicro.wisc.edu

Cutaneous

Trichophyton mentagrophytes

– zoophilic: mice, cats, horses, sheep, rabbits

– inflammatory skin or scalp lesions in humans

– ectothrix• distribution

– worldwide

Cutaneous

Trichophyton mentagrophytes

• lab diagnosis - culture

• flat, white to cream color; powdery to granular surface

danival.org

Cutaneous

Trichophyton mentagrophytesMicroscopic

spherical microconidia forming dense clusters,

“en-grappe”vtpb-www.cvm.tamu.edu

Cutaneous

Trichophyton mentagrophytes

• spiral hyphae

smooth thin-walled clavate multiseptate macroconidia

Microscopic

www.vet.ohio-state.edu vtpb-www.cvm.tamu.edu

Cutaneous

Trichophyton mentagrophytes

lab diagnosis

www2.provlab.ab.ca

positive urease production

positive for in-vitro hair perforation

www2.provlab.ab.ca

Cutaneous

Trichophyton rubrum• anthropophilic

– chronic infections of the skin, nails, rarely scalp

– ectothrix or endothrix hair infection

• distribution– worldwide

Cutaneous

Trichophyton rubrum

lab diagnosis – culture

• white, suede-like to downy

• wine red pigment on reverse side

www.pfizer.ch

www4.medfak.lu.se

Cutaneous

Trichophyton rubrumwww2.provlab.ab.ca • lab diagnosis

– scanty to moderate numbers of slender clavate to pyriform microconidia

– arranged “en-thyrse”

Cutaneous

Trichophyton concentricumAnthropophilicchronic non-inflammatory tinea corporistinea imbricata – concentric scaling of skinNot invade hair

Cutaneous

Trichophyton concentricum

DistributionPacific Islands of OceaniaSoutheast AsiaCentral and South America

Cutaneous

Trichophyton concentricumLab diagnosisSlow growing deeply folded thallusCream to orange brown in color Reverse buff to brown

Cutaneous

Trichophyton concentricum

Microscopic –

“antler tips” hyphae,

chlamydoconidia

Cutaneous

Trichophyton schoenleiniiAnthropophilicCause favusChronic scarring form of tinea capitisSaucer shaped crusted lesions or scutulaPermanent hair loss

Cutaneous

Trichophyton schoenleiniiLab diagnosisCultureWaxy or glabrousDeeply folded honeycomb-like thallus with sub-surface growth

Cutaneous

Trichophyton schoenleiniiLab diagnosisMicroscopic

Favic chandeliersNo macroconidiaNo microconidia

Cutaneous

Epidermophyton floccosum

• anthrophophilic• does not invade

hair in vivo

• distribution– worldwide

Cutaneous

Epidermophyton floccosum

Culture• greenish-brown or

“khaki” colored• suede-like surface• raised & folded center,

with flat periphery• yellowish brown reverse

pigment

Cutaneous

Epidermophyton floccosum

botit.botany.wisc.edu

Microscopic• smooth thin-walled

macroconidia often in clusters growing directly from hyphae

• no microconidia• numerous

chlamydoconidia

www.fns.uniba.sk