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DIAGNOSIS OF COMMON FUNGAL DISEASES
1. 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
4. 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
5. 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
6. Data from a Five-year review of
Fungal Isolates at UPM-CPH
Fungal isolates :
Trichophyton mentagrophytes
Trichophyton rubrum
Trichophyton tonsurans
Trichophyton schoenlenii
Trichosporon spp.
7. Data from a Five-year review of
Fungal Isolates at UPM-CPH
• Fungal isolates
Microsporum gypseum
Microsporum canis
Epidermophyton flocossum
Candida albicans
Exophiala werneckii
8. 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
9. Laboratory Methods for
Diagnosis of Mycoses
I. DIRECT EXAMINATION:
*10-30% KOH
*Calcofluor white stain
*Histological stains- H&E, PAS
*India Ink
*Wet mount
10. Laboratory Methods for
Diagnosis of Mycoses
II. Isolation & Culture
SDA
BHIA/BAP
Media with/without antibiotics
• Macroscopic examination of culture
• Microscopic examination using LPCB
11. 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
13. 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
17. 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
24. 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
25. Superficial mycoses
Tinea nigra
• Distribution
– world-wide
– more common in tropical regions of
Central & South America, Africa,
Southeast Asia & Australia
26. Superficial mycoses
Tinea nigra
• KOH
– pigmented brown to dark
olivaceous
(dematiaceous) septate
hyphal elements & 2-
celled yeast cells
27. Superficial mycoses
Tinea nigra
• Etiologic agent
– Exophiala werneckii
saprophyte found in soil,
compost, humus & wood in
humid tropical & sub-tropical
regions
28. Superficial mycoses
Tinea nigra
• Culture on SDA
– initially mucoid,
yeast-like & shiny
black
– with age: aerial
mycelia & dark olive
color
29. 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
32. 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
40. Superficial mycoses
Black piedra - lab diagnosis
• Isolation
-growth very slow
-dark brown to black
-greenish brown,
short aerial
mycelium
Heaped center
Flat periphery
43. Superficial mycoses
White piedra
Nodule
• Soft, white, yellowish,
beige or greenish nodule
• Discrete
• more often coalescent,
forming an irregular
transparent sheath
44. Superficial mycoses
White piedra
• Distribution
– common in S. America & Asia
– sporadic in N. America & Europe
• Etiologic agent
– Trichosporon beigelii or T. cutaneum
84. Cutaneous
Endothrix invasion of hair
• formation of
arthroconidia within
hair shaft
• cuticle of hair remains
intact
• do not fluoresce under
Wood’s UV light
86. Culture:
• Selective media
–SDA with chloramphenicol &
cycloheximide (Mycosel or
Mycobiotic agar)
–Dermatophyte test medium
Non-selective medium
–Sabouraud’s dextrose agar
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
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
Dermatomycosis are skin infections that are caused neither by dermatophytes nor by the genus candida.
They may be caused by soil inhabiting fungi
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
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
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.
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.
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.
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
Tinea barbae is a dermatophyte infection of the bearded areas of the face and neck, and therefore is restricted to adult males.
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.
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.
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.
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.
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
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
