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LABORATORY DIAGNOSIS OF
COMMON FUNGAL DISEASES
Prof. Louella A. Dancel, RMT, MSc.
University of Perpetual Help System DALTA
42nd
PAMET ANNUAL CONVENTION
Manila Hotel – December 2,2006
Clinical specimens submitted for
Fungal Isolation 2004-2006*
Clinical specimen 2004 2005 2006 Total
Respiratory 29 35 34 98
Body fluids 37 17 34 88
Tissues 9 0 23 32
Skin 1 0 0 1
Nails 29 31 20 80
Hair 0 1 0 1
CSF 8 5 8 21
Others 5 20 24 49
TOTAL 118 109 153 380
Fungal Isolates 2004-2006*
Isolate 2004 2005 2006
C.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 isolates
Microsporum 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 compactum
Phialophora verrucosa
Exophiala jeanselmei
Madurella 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 & Culture
SDA
BHIA/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 mycoses
Disease
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 versicolor
KOH of skin scrapings
• clusters of budding
yeast-like cells & short
angular hyphal forms
• “spaghetti and meat
balls”
Superficial mycoses
Pityriasis versicolor
PAS 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 yeast
part 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 piedra
Nodule
• 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 diagnosis
Isolation
• medium - SDA with
chloramphenicol without
cycloheximide
• growth/culture
– rapid
– cream-colored, soft
– membranous, wrinkled radial
furrows, irregular folding
Superficial mycoses
White piedra - lab diagnosis
Isolation
• 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 mycoses
Disease
• Dermatophytosis
Causative organisms
• Dermatophytes
 Microsporum
 Trichophyton
 Epidermophyton
ringworm
Cutaneous mycoses
Disease
• Candidiasis of
skin, mucous
membranes &
nails
Causative organisms
• Candia albicans &
related species
 dermatomycosis  Soil fungi
(Scytalidium, Fusarium, etc.)
 Systemic fungi
(Histoplasma, etc)
Ecological Groups of
Dermatophytes
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
Macroconidia
Rough walled
Microconida
present
Trichophyton
Macroconidia
Smooth walled
Microconidia
present
Epidermophyton
ChlamydoconidiaMacroconidia
Smooth walled
Microconidia
none
Clinical Manifestations of
Dermatophytes
41 laboratorydiagnosisofcommonfungaldiseases-110919091652-phpapp01
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
dermatophyte
Yeast etc.
Laboratory diagnosis
Wipe with water
www.doctorfungus.org
scalpel
Paper / envelope
active edge
Skin scraping specimen
Direct Examination
• Wet mount
KOH
• KOH
– 10% to 30%
– with Parker Superquink
blue-black ink
– gentle warming
p
a
r
k
e
r
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:
• Incubation
Room temperature
At least 2 weeks
Identification
• Gross color & texture
• Microscopic characteristics
• Confirm / compare with
Written descriptions
Drawings
photographs
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 canis
lab diagnosis –
culture
• white cottony growth
Cutaneous
Microsporum canis
• microscopic:
– spindle shaped,
one end pointed,
other end blunt
– thick walled
verrucose
macroconidia
– 6 to 12 cells
www.doctorfungus.org
Cutaneous
Microsporum gypseum
–geophilic
–usually produces a single
inflammatory skin or scalp lesion
• distribution
–worldwide
Cutaneous
Microsporum gypseum
lab diagnosis -
culture
• flat, spreading suede-
like to granular
• cinnamon growth
• yellow brown pigment
on reverse of colony
www.ukneqasmicro.org.uk
Cutaneous
Microsporum gypseum
microscopic:
• 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 mentagrophytes
Microscopic
 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 rubrum
www2.provlab.ab.ca• lab diagnosis
–scanty to moderate
numbers of slender
clavate to pyriform
microconidia
–arranged “en-
thyrse”
Cutaneous
Trichophyton concentricum
Anthropophilic
chronic non-inflammatory tinea
corporis
tinea imbricata – concentric scaling
of skin
Not invade hair
Cutaneous
Trichophyton concentricum
Distribution
Pacific Islands of Oceania
Southeast Asia
Central and South America
Cutaneous
Trichophyton concentricum
Lab diagnosis
Slow growing deeply
folded thallus
Cream to orange
brown in color
Reverse buff to
brown
Cutaneous
Trichophyton concentricum
Microscopic –
“antler tips” hyphae,
chlamydoconidia
Cutaneous
Trichophyton schoenleinii
Anthropophilic
Cause favus
Chronic scarring form
of tinea capitis
Saucer shaped crusted
lesions or scutula
Permanent hair loss
Cutaneous
Trichophyton schoenleinii
Lab diagnosis
Culture
Waxy or glabrous
Deeply folded honeycomb-like
thallus with sub-surface growth
Cutaneous
Trichophyton schoenleinii
Lab diagnosis
Microscopic
Favic chandeliers
No macroconidia
No 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

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41 laboratorydiagnosisofcommonfungaldiseases-110919091652-phpapp01

Editor's Notes

  1. Cutaneous mycoses are fungal infections of the integument and its appendages. They are superficial infections of the skin, hair & nails The fungus localizes only in the cornified layer, stratum corneum, No living tissue is invaded Pathological changes occur in the host because of her reaction to the fungus and its products of metabolism or life. Allergic & inflammatory eczematous host response
  2. Dermatophytosis are caused by a closely related group of organisms, called dermatophytes Which are keratinophilic Very similar & closely related group of fungi that cause a wide variety of clinical conditions A single species may be involved in several disease types, each with its distinctive pathology Most commonest infectious agents of man, the infections are distributed worldwide (geographically & racially) Common term is ringworm
  3. Dermatomycosis are skin infections that are caused neither by dermatophytes nor by the genus candida. They may be caused by soil inhabiting fungi
  4. Tinea capitis is a dermatophyte infection of the scalp, eyebrows and eyelashes caused by species of Microsporum and Trichophyton. type & severity of response is related to the species & strain of dermatophyte causing infection
  5. Tinea capitis is a dermatophyte infection of the scalp, eyebrows and eyelashes caused by species of Microsporum and Trichophyton. type & severity of response is related to the species & strain of dermatophyte causing infection
  6. Favus refers to the similarity of appearance of scutula and honeycombs. Favus is characterized by the occurrence of dense masses of mycelium & epithelial debris which form yellowish, cup-shaped crusts called SCUTULA. The scutulum develops in a hair follicle, with the hair shaft in the center of the raised lesion. Removal of these crusts reveals an oozing, moist red base. After a period of years, atrophy of the skin occurs, leaving a cicatricial alopecia & scarring. Scutula may be formed on the scalp or the glabrous skin.
  7. Tinea corporis is a dermatophyte infection of the glabrous skin most commonly caused by species of the gnera Trichophyton and Microsporum. The infection is generally restricted to the stratum corneum of the epidermis. The clinical symptoms are a result of the fungal metabolites acting as toxins & allergens. Lesions vary from simple scaling, scaling with erythema & vesicles, to deep granulomata. Villous hair in the involved area may be invaded, & the follicle often acts as a reservoir for recrudescence of the disease.
  8. Tinea corporis is a dermatophyte infection of the glabrous skin most commonly caused by species of the gnera Trichophyton and Microsporum. The infection is generally restricted to the stratum corneum of the epidermis. The clinical symptoms are a result of the fungal metabolites acting as toxins & allergens. Lesions vary from simple scaling, scaling with erythema & vesicles, to deep granulomata. Villous hair in the involved area may be invaded, & the follicle often acts as a reservoir for recrudescence of the disease.
  9. Tinea imbricata is a geographically restricted form of tinea corporis caused by Trichophyton concentricum. It is characterized by polycyclic, concentrically arranged rings of papulosquamous patches of scales scattered over and often covering most of the body
  10. Tinea barbae is a dermatophyte infection of the bearded areas of the face and neck, and therefore is restricted to adult males.
  11. Tinea cruris is a dermatophyte infection of the groin, perineum, & perianal region, which is acute or chronic and generally pruritic. The lesion is sharply demarcated, with a raised, erythematous margin & thin, dry epidermal scaling. The disease is found in all parts of the world but is more prevalent in the tropics. It tends to occur when conditions of high humidity lead to maceration of the crural region. A similar condition may involve the axilla or other intertriginous areas. The disease is more common in men, but frequently involves women, usually when it is transmitted by intimate contact or fomite. It may reach epidemic proportions in athletic teams, troops, ship crews, & inmates of institutions. In such cases, it is probably most commonly transmitted by towels, linens, & clothing. E. Floccosum has been isolated from blankets & sheets as well as rugs & other fomites where it can survive for years as infective arthroconidia.
  12. Tinea pedis is a dermatophyte infection of the feet involving particularly the toe webs and soles. The lesions are of several types, varying from mild, chronic, and scaling to acute, exfoliative, pustular, & bullous disease.
  13. Most dermatophyte infections of the hand, particularly of the dorsal aspect, are similar to tinea corporis. Tinea manuum refers to those infections in which the interdigital areas & the palmar surfaces are involved & show characteristic pathologic features. Along with tinea pedis, tinea manuum is one of the commoner types of chronic dermatophytosis in the adult. It has been postulated that this is related to the lack of sebaceous glands & their fungistatic lipids in these two areas.
  14. Tinea unguium is an invasion of the nail plates by a dermatophyte. Onychomycosis is an infection of the nails caused by nondermatophytic fungi and yeasts. Tinea unguium is of at least 2 types: Superficial white onychomycosis – invasion is restricted to patches or pits on the surface of the nail invasive subungual dermatophytosis (ringworm of the nail), in which the lateral or distal edges of the nail are first involved, followed by establishment of the infection beneath the nail plate.
  15. For skin specimen, first clean lesion & periphery with 70% alcohol. Use sterile scalpel or edge of microscope slide, scrape perpendicular to the skin. Scrape around the active edge where the fungus is actively growing. The center of lesion heals first, so the laboratory results are negative using this sample. If the lesion is inflamed or with fissures, clean it with sterile distilled water. Collect skin scrapings in paper envelope or petri dish, or place between 2 slides. Store at room temperature. If patients are young children and are scared of the scalpel, use can use scotch tape to collect specimen for microscopy. Collect moist exudate for candida
  16. These Fungi exhibit characteristic structures in clinical specimens that can be seen microscopically using a brightfield or phase contrast microscope. You can prepare a wet mount, a specimen plus sterile water or NSS, or specimen alone, like exudates. We can use potassium hydroxide, from 10 to 30% depending upon the type of specimen, for skin use 10%, for nails use a stronger concentration, 30%. Potassium hydroxide digests or dissolves proteins, fats & carbohydrates, the tissue clears Making the fungal cell wall, which is resistant to alkali, visible. Addition of Parker blue-black ink, will stain the fungal structures, facilitating microscopic examination, fungi appear bluish green. The preparation is passed 2 or 3 times over an alcohol lamp or bunsen burner, to hasten the reaction. If the result is negative, especially in nail scrapings, you can leave the preparation overnight on the lab table, to give time for digestion to occur. View the preparation again the next day. Question: Can you use Parker permanent blue or black ink? YES
  17. Microscopic examination
  18. Use several media