Dematophytes

Dermatophytes
(name based on the Greek for 'skin plants') are a
common label for a group of three types of fungus
that commonly causes skin disease in animals and
humans.
These anamorphic (asexual or imperfect fungi)
genera are: Microsporum, Epidermophyton and
Trichophyton. There are about 40 species in these
three genera.
Species capable of reproducing sexually belong in the
teleomorphic genus Arthroderma, of the Ascomycota
The organisms are transmitted by either direct
contact with infected host (human or animal) or
by direct or indirect contact with infected
exfoliated skin or hair in combs, hair brushes,
clothing, furniture, theatre seats, caps, bed linens,
towels, hotel rugs, and locker room floors.
Depending on the species the organism may be
viable in the environment for up to 15 months.
 There is an increased susceptibility to
infection when there is a preexisting injury to the
skin such as scares, burns, marching, excessive
temperature and humidity.
Dermatophytes are classified as anthropophilic,
zoophilic or geophilic according to their normal
habitat.
Geophilic species
species are usually recovered from the soil but
occasionally infect humans and animals.
They cause a marked inflammatory reaction,
which limits the spread of the infection and
may lead to a spontaneous cure but may also
leave scars.
Anthropophilic dermatophytes
are restricted to human hosts and produce a mild, chronic
inflammation.
Zoophilic organisms
are found primarily in animals and cause marked
inflammatory reactions in humans who have contact with
infected cats, dogs, cattle, horses, birds, or other animals.
This is followed by a rapid termination of the infection.
Dermatophytes cause infections of the skin, hair and nails due
to their ability to obtain nutrients from keratinized material.
The organisms colonize the keratin tissues and
inflammation is caused by host response to metabolic
by-products.
are usually restricted to the nonliving cornified layer of the
epidermis because of their inability to penetrate viable
tissue of an immunocompetent host.
Invasion does elicit a host response ranging from mild to
severe.
The development of cell-mediated immunity
correlated with delayed hypersensitivity and
an inflammatory response is associated with
clinical cure.
Whereas the lack of or a defective cell-mediated
immunity predisposes the host to chronic or
recurrent dermatophyte infection.
Some of these infections are known as ringworm or
tinea.
Toe- and fingernail infection are referred to as
onychomycosis.
Dermatophytes usually do not invade living tissues,
but colonize the outer layer of the skin.
 Occasionally the organisms do invade subcutaneous
tissues, resulting in kerion development.
 Types of Dermatophyte Infections

Athlete's foot or tinea pedis.

 Jock itch or tinea cruris.
Ringworm of the body or tinea corpora.
Facial ringworm or tinea faciei.
Blackdot ringworm or tinea capitis.
Ringworm of the hands or tinea manuum.
Ringworm of the nail, Onychomycosis, or tinea unguium.
Anthropophilic

Epidermophyton floccosum
Microsporum audouinii
Trichophyton mentagrophytes
(cottony and velvety)
Trichophyton rubrum
Trichophyton schoenleinii
 Trichophyton soudanense
Trichophyton tonsurans
Trichophyton violaceum
Zoophilic

Microsporum canis (cats, dogs, etc.)

Microsporum equinum (horses)
 Microsporum nanum
Trichophyton mentagrophytes(granular)
(rodents, rabbits, hedgehogs, etc.)
Trichophyton verrucosum (cattle)
Geophilic

Microsporum gypseum
The dermatophytes consist of three genera:
Epidermophyton
produces only macroconidia, no microconidia and
consists of 2 species, one of which is a pathogen.
Microsporum
Both microconidia and rough-walled macroconidia
characterize Microsporum species.
There are 19 described species but only 9 are
involved in human or animal infections.
Trichophyton
When produced the macroconidia of Trichophyton
species are smooth-walled.
There are 22 species, most causing infections in
humans or animals.
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.
Culture characteristics such as surface texture,
topography and pigmentation are variable and are
therefore the least reliable criteria for identification.
The asexual spores may be large (macroconidia,
chlamydospores) or small (microconidia,
blastospores, arthroconidia)
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.
Tinea capitis
Tinea capitis refers to dermatophytosis of the scalp. Three
types of in vivo hair invasion are recognised:
1. Ectothrix invasion is characterised by the development
of arthroconidia on the outside of the hair shaft.
The cuticle of the hair is destroyed and infected hairs
usually fluoresce a bright greenish yellow colour under
Wood's ultraviolet light.
Common agents include M. canis, M. gypseum, T.
equinum and T. verrucosum.
2. Endothrix hair invasion is characterised by the
development of arthroconidia within the hair shaft
only.
The cuticle of the hair remains intact and infected
hairs do not fluoresce under Wood's ultraviolet light.
All endothrix producing agents are anthropophilic eg
T. tonsurans and T. violaceum.
3. Favus usually caused by T. schoenleinii, produces
favus-like crusts or scutula and corresponding hair
loss.
Endothrix
Ectothrix
Blackdot ringworm or tinea capitis
Infected hair shafts are broken off just at the base,
leaving a black dot just under the surface of the skin.
Scraping these residual black dot will yield the best
diagnostic scrapings for microscopic exam.
Numerous green arthrospores will be seen under the
microscope inside the stubbles of broken hair shafts
at 400x.
Tinea capitis can not be treated topically, and must
be treated systemically with antifungals
 Laboratory Identification of
Dermatophytes
Specimen Collection:
Skin should be scraped from the margin of the lesion.
Hair should be plucked, not cut, from the edge of the
lesion.
Choose hairs that fluoresce under a Wood's lamp or, if
none fluoresce, choose broken or scaly ones.
Nails scrapings are obtained from the nail bed or from
infected areas after the outer layers are discarded.
A Wood's lamp is a diagnostic tool used in
dermatology by which ultraviolet light is shone (at a
wavelength of approximately 365 nanometers) onto
the skin of the patient; a technician then observes
any subsequent fluorescence. For example,
porphyrins — associated with some skin diseases —
will fluoresce pink.
Direct Examination:
A small sample of the specimen is selected for direct
microscopic examination and investigated for the presence of
fungal elements.
The specimen is mounted in a small amount of potassium
hydroxide or calcofluor white.
The KOH slides are gently heated and allowed to clear for 30 to
60 minutes before examining on a light or phase contrast
microscope.
Calcofluor white slides are examined on a fluorescent
microscope.
When present in the direct examination dermatophytes appear as
hyaline (non-pigmented), septated elements.
 Hyphae rounding up into arthroconidia are diagnostic of
dermatophyte involvement. Without the presence of arthroconidia
the elements could also be due to a non-dermatophyte agent of
onycomycosis or a small segment of a contaminating organism.
When hair is involved the arthroconidia may be found on the
periphery of the hair shaft (ectothrix) or within the shaft
(endothrix).
Culture
Nails are scraped or minced into small pieces
Hair is cut into short segments
Each specimen is divided between at least two types of
culture media
The use of antibiotics will inhibit the overgrowth of bacteria
and incorporation of cycloheximide will prevent the
overgrowth of the rapidly growing saprophytic fungi
The cultures are incubated at 30°C and examined frequently
for 4 weeks.
Potato dextrose agar is a media useful for the
production of pigment.
Sabouraud dextrose agar (Emmon's modification) is
a non-selective media which supports the growth of
most fungi.
A special media called Dermatophyte Test Medium
(DTM) has been formulated to grow and identify
dermatophytes.
 Without having to look at the colony, the hyphae, or
macroconidia - one can identify the dermatophyte by
a simple color test.
The specimen (scraping from skin, nail, or hair) is
embedded in the DTM culture medium
It is incubated at room temperature for 10 to 14 days.
If the fungus is a dermatophyte, the medium will
turn bright red.
If the fungus is not a dermatophyte, no color change
will be noted.
If kept beyond 14 days, false positive can result even
with non-dermatophytes.
Specimen from the DTM can be sent for species
identification if desired.
Hair perforation
Many dermatophytes have the ability to degrade
hair. The hair perforation test determines whether
the organism simply erodes the hair shaft or
produces an enzyme that will penetrate and invade
the shaft resulting in perforating bodies or cones.
To distinguish between isolates of
dermatophytes, particularly Trichophyton
mentagrophytes and its variants.
Ingredients:
Autoclaved blonde pre-pubital hair cut into short
pieces (1cm)
Sterile distilled water 5 ml in a suitable vial.
Method:
1. Place hair in water in vial.
2. Inoculate with small fragments of the test fungus.
3. Incubate at room temperature.
4. Individual hairs are removed at intervals up to 4 weeks and
examined microscopically in lactophenol cotton blue.
Isolates of T. mentagrophytes produce marked localised
areas of pitting and marked erosion whereas those of T.
rubrum do not.
Slide culture
The slide culture is a method of examining the
microscopic structures of a fungus.
 The organism is grown on a glass coverslip placed
on a block of agar.
When sufficient growth has occurred the coverslip is
placed on a drop of mounting media on a microscope
slide and examined by phase contrast microscopy.
Urea
Christensen's urea broth indicates the presence of
the enzyme urease, which splits urea into ammonia,
resulting in an alkaline environment. The phenol red
indicator turns the media from a straw yellow to
fuschia at pH 8.4.
Vitamin requirements
Certain species of dermatophytes have distinctive
nutritional requirements that may be beneficial to
differentiate from similar species.
The agar base is vitamin free and various vitamins
are added to the basal media.
The growth on the vitamin-enriched media is
compared to the vitamin free media to determine
enhancement of aerial mycelium.
 Trichophyton sp.

Description and Natural Habitats

Trichophyton is a dermatophyte which inhabits the
soil, humans or animals. Related to its natural
habitats, the genus includes anthropophilic,
zoophilic, and geophilic species.
Some species are cosmopolitan. Others have a
restricted geographic distribution.
The genus Trichophyton has several species. Most
common are Trichophyton mentagrophytes,
Trichophyton rubrum, Trichophyton schoenleinii,
Trichophyton tonsurans, Trichophyton verrucosum,
and Trichophyton violaceum.
Trichophyton rubrum is the commonest causative
agent of dermatophytoses worldwide
. Trichophyton species may cause invasive infections
in immunocompromised hosts .
Several morphological and physiological
characteristics are used in differentiation and
identification of Trichophyton species.
Trichophyton differs from Microsporum and
Epidermophyton by having cylindrical, clavate to
cigar-shaped, thin-walled or thick-walled, smooth
macroconidia.
Macroscopic Features

The growth rate of Trichophyton colonies is slow to

moderately rapid. The texture is waxy, glabrous to
cottony. From the front, the color is white to bright
yellowish beige or red violet. Reverse is pale,
yellowish, brown, or reddish-brown
T. rubrum
Colonies flat, woolly, or granular to cottony, white to
cream with a blood red or olivaceous reverse;
 The red pigment production is best seen on potato
dextrose agar or cornmeal dextrose agar.
Microscopic morphology
The downy type is characterized by the production of
scanty to moderate numbers of slender clavate
microconidia and no macroconidia.
The granular type is characterized by the production
of moderate to abundant numbers of clavate to pyriform
microconidia and moderate to abundant numbers of
thinwalled, cigar-shaped macroconidia.
 Microconidia are characteristically tear-shaped and
form singly all along the sides of the hyphae.
Some species may be sterile and the use of specific
media is required to induce sporulation
T.rubrum
Lab identification
++++++ pigment production on specific media
(PDA, cornmeal agar).
- ve in-vitro hair perforation test.
 T. violaceum

Trichophyton violaceum is an anthropophilic fungus

causing inflammatory or chronic non-inflammatory
finely scaling lesions of skin, nails, beard and scalp,
producing the so-called "black dot" tinea capitis.
Distribution is world-wide, particularly in the Near
East, Eastern Europe, USSR and North Africa.
Invaded hairs show an endothrix infection and do
not fluoresce under Wood's ultra-violet light.
On Sabouraud's dextrose agar, colonies are very slow
growing, glabrous or waxy, heaped and folded and a
deep violet in colour.
Cultures often become pleomorphic, forming white
sectors and occasional non-pigmented strains may
occur.
Hyphae are relatively broad, tortuous, much
branched and distorted.
Young hyphae usually stain well in lactophenol
cotton blue, whereas older hyphae stain poorly and
show small central fat globules and granules.
 No conidia are usually seen, although occasional
pyriform microconidia have been observed on
enriched media. Numerous chlamydoconidia are
usually present, especially in older cultures.
Nutritional requirements:
T. violaceum has a partial nutrient requirement for
thiamine. There is minimal growth on casein
vitamin-free agar.
The partial requirement for thiamine separates this
organism from T. gourvillii, T. rubrum, and other
species that may produce purple pigmented colonies.
 T. mentagrophytes

T. mentagrophytes is the zoophilic form of T.

mentagrophytes with a world-wide distribution and
a wide range of animal hosts including mice, guinea-
pigs, kangaroos, cats, horses, sheep and rabbits.
Produces inflammatory skin or scalp lesions in
humans, particularly in rural workers.
 Kerion of the scalp and beard may occur.
Invaded hairs show an ectothrix infection but do not
fluoresce under Wood's ultra-violet light
On Sabouraud's dextrose agar, colonies are generally
flat, white to cream in colour, with a powdery to
granular surface.
Some cultures show central folding or develop raised
central tufts or pleomorphic suede-like to downy
areas.
 Reverse pigmentation is usually a yellow-brown to
reddish-brown colour.
Numerous single-celled microconidia are formed,
often in dense clusters.
Microconidia are hyaline, smooth-walled, and are
predominantly spherical to subspherical in shape,
however occasional clavate to pyriform forms may
occur.
 Varying numbers of spherical chlamydoconidia,
spiral hyphae and smooth, thin-walled, clavate
shaped, multicelled macroconidia may also be
present.
Hydrolysis of Urea: Positive within 7 days
(usually 3 to 5 days).
Hair Perforation Test ("in vitro"): Positive
within 14 days.
Positive hair perforation test
T. verrucosum
thiamine and inositol (not all isolates require
inositol) required for growth; growth enhanced at
37.°
 Microsporum sp.

Microsporum species form both macro- and

microconidia on short conidiophores.
 Macroconidia are hyaline, multiseptate, variable in
form, fusiform, spindle-shaped to obovate, 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-walledand are not diagnostic for any
one species.
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.
Microsporum canis
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.
Colonies (SDA) 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.
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.
Macroconidia are typically spindle-shaped with 5-15
cells, verrucose, thick-walled and often have a
terminal knob, 35-110 x 12-25 µm
A few pyriform to clavate microconidia are also
present.
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.
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.
 Identification

Growth on Rice Grains: good growth of white

aerial mycelium with production of yellow pigment.
Microscopy reveals numerous characteristic
macroconidia and microconidia.
Reverse Pigment on Potato Dextrose Agar:
Bright yellow (both M. audouinii and M. canis var.
equinum are salmon to pinkish-brown).
Vitamin Free Agar (Trichophyton Agar No.1):
Good growth indicating no special nutritional
requirements.
Hair Perforation Test: Positive at 14 days.
Key Features:
distinctive macroconidia and culture characteristics.
Abundant growth and sporulation on polished rice
grains and in vitro perforation of hair.
Dysgonic strains of M. canis are rare but may also
occur.
 Cultures are typically heaped and folded and yellow-
brown in colour. Macroconidia are usually absent in
these strains.
 However, typical colonies and macroconidia of M.
canis are usually produced by this variant when
subcultured onto polished rice grains.
 Epidermophyton floccosum

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.
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.
Macroconidia of E. floccosum
Chlamydoconidia of E. floccosum.
 Treatment of dermatophytosis

Dermatophytes are located in the stratum corneum

within the keratinocytes. The signs and symptoms
that appear in infected individuals are due to acute
and chronic inflammatory changes that appear in the
dermis.
For these reasons, antifungal agents should have the
ability to penetrate the stratum corneum cells to be
efficient when applied topically.
The vast majority of antifungals are fungistatic with
the concentrations achieved in the skin when applied
topically; the growth of dermatophytes is delayed
and these are shed with the skin renewal and healing
is achieved.
The antifungal agents and the components
incorporated on the vehicle should be non-irritant
and well tolerated.
Skin lesions located on face, trunk and limbs usually
require two or three weeks of treatment.
Inflammatory dermatophyte infections of the feet
should be treated for four or six weeks and
hyperkeratotic lesions of palms and soles are best
treated with oral antifungals since they are usually
unresponsive to topical antifungals.
Oral treatment is indicated in widespread skin
lesions, tinea capitis, tinea barbae, tinea unguium,
in skin lesions with folliculitis, and when either there
is no response to topical treatment or tolerance is not
adequate.
Griseofulvin is still currently the gold standard for
treatment of dermatophytosis (excluding tinea
unguium).
No new oral antifungal agents appeared until
ketoconazole was introduced in 1980. This drug was
an advance in the treatment of mycoses, but
nowadays there are many concerns about its use,
basically due to a significant incidence of
idiosyncratic hepatic toxicity
Itraconazole is a triazole agent, poorly water-soluble
and whose bioavailability improves when the drug is
taken with a fatty meal.
Accumulation in skin is slow and antifungal
therapeutically active high concentrations persist up
to a month after the end of treatment.
Fluconazole is a triazole that is water-soluble and
extremely well absorbed.
Most of the drug is excreted unchanged in the urine
since it undergoes no hepatic metabolism. It is
eliminated more slowly from skin, and therefore
clinical cures may be achieved after the withdrawal
of treatment.
Voriconazole is a third generation triazole under
preclinical development
From a dermatological point of view it is interesting
to note that voriconazole is active in vitro against
dermatophytes and Malassezia spp
Terbinafine is a fungicidal allylamine that is
absorbed from the gastrointestinal tract.
When treatment with terbinafine ceases the
concentration in stratum corneum remains high (0,1
μg/ml) for 8 weeks and enables the use of short
courses of treatment.
The incidence of adverse effects with terbinafine
therapy is approximately 10%.
 Malassezia

Malassezia (formerly known as Pityrosporum)

is a genus of related fungi, classified as yeasts,
naturally found on the skin surfaces of many animals
including humans. It can cause hypopigmentation on
the trunk and other locations in humans if it
becomes an opportunistic infection
Malassezia were originally identified by the French
scientist Louis-Charles Malassez in the late 19th
century.
In the mid 20th century, it was reclassified into two
species:
Pityrosporum (Malassezia) ovale which is lipid
dependent and found only on humans. P. ovale was
later divided into two species, P. ovale and P.
orbiculare, but current sources consider these terms
to refer to a single species of fungus, with M. furfur
the preferred name.
Pityrosporum (Malassezia) pachydermatis, which is
lipophilic but not lipid dependent and found on the
skin of most animals.
In the mid 1990s, scientists at the Pasteur Institute
in Paris, France discovered additional species,
Currently there are 10 recognized species:
M. furfur, M. pachydermatis , M. globosa ,
M. restricta , M. slooffiae , M. sympodialis[8]
M. nana[ ,M. yamatoensis ,M. dermatis
M. obtusa
As the fungus requires fat to grow, it is most
common in areas with many sebaceous glands: on
the scalp, face, and upper part of the body. When the
fungus grows too rapidly, the natural renewal of cells
is disturbed and dandruff appears with itching (a
similar process may also occur with other fungi or
bacteria).
Pityriasis versicolor:
 This is a chronic, superficial fungal disease of the
skin characterised by well-demarcated white, pink,
fawn, or brownish lesions, often coalescing, and
covered with thin furfuraceous scales.
The colour varies according to the normal
pigmentation of the patient, exposure of the area to
sunlight, and the severity of the disease.
Lesions occur on the trunk, shoulders and arms,
rarely on the neck and face, and fluoresce a pale
greenish colour under Wood's ultra-violet light.
Young adults are affected most often, but the disease
may occur in childhood and old age.
Laboratory diagnosis:

Clinical material:
Skin scrapings from patients with superficial lesions,
blood and indwelling catheter tips from patients with
suspected fungaemia.
Direct Microscopy:
 Skin scrapings taken from patients with Pityriasis
versicolor stain rapidly when mounted in 10% KOH,
glycerol and Parker ink solution and show
characteristic clusters of thick-walled round,
budding yeast-like cells and short angular hyphal
forms up to 8um in diameter.
 These microscopic features are diagnostic for
Malassezia furfur and culture preparations are
usually not necessary.
Culture:
 Culture is only necessary in cases of suspected
fumgaemia.
M. furfur is a lipophilic yeast, therefore in vitro
growth must be stimulated by natural oils or other
fatty substances.
The most common method used is to overlay
Sabouraud's dextrose agar containing cycloheximide
(actidione) with olive oil or alternatively to use a
more specialized media like Dixon's agar.
Symptomatic scalp infections are often treated with
selenium disulfide or ketoconazole containing
shampoos. Other treatments include coal tar,
zinc pyrithione (ZPT), miconazole, or tea tree oil
medicated shampoos.
 Hydrogen peroxide is also used to manage
symptoms of itching.