Laboratory diagnosis of fungal diseases

Name: Purshotam Kumar Sah Kanu
Roll No.: MB 1318/075
Level: M.Sc Microbiology (3rd Sem)
Central Department of Microbiology
Tribhuvan University, Kirtipur
Kathmandu, Nepal
MB 609 Systemic and Diagnostic
Mycology

Laboratory Diagnosis Of Fungal
Diseases- 4 hrs
Cutaneous, Subcutaneous, Systemic,
Opportunistic mycoses, Fungal sinusitis

MEDICAL MYCOLOGY
 Fungi were discovered earlier than bacteria and viruses.
 In the past, most fungi cause skin infections or cosmetic
infections, where bacteria and viruses cause serious fatal
diseases, so there was no interest of studying fungi.
 In 1980, when HIV infection was discovered, increasing
number of immunocompromizing conditions, they found
that fungi produce fatal diseases; from that time, fungi
return to be in focus again.

MEDICAL MYCOLOGY
 It is the science that deals with the study of
pathogenic fungi that produce diseases.

Structure
 Fungi are eukaryoticorganisms have true nuclei with
definite nuclear membrane, nucleolus,
cytoplasmicorganelles.

Structure
 Cell membrane of fungi has sterols, which is the
target of action of antifungal agents.
Ergosteroldominates in contrast to cholesterol in
mammalian membrane.

Structure
•Cell wall of fungi
lacks:
–Peptidoglycan
–Glycerol &
ribitolteichoicacid
–Lipopolysaccharide
•Cell wall composed
of:
–Chitin.
–Glucan(important for
new antifungal agent).
–Mannan.

Source of infection:
Endogenous:
•Normal flora and
it is the main
source in
nosocomial
infection (because
those people in
hospitals are
immunocompromiz
ed).

Source of infection (cont.):
Exogenous:
 This is the main source of fungal infection mainly from
the environment.
 Few fungal infections are communicable between
human or between animals.

Mode of transmission:
Respiratory tract (air
borne infection).
–GIT (food & water borne
infection).
–Blood stream injection.
–Skin = contact.
–Most fungal diseases are
not communicable
between human or
animals.

Most fungi are opportunistic:
–Produce diseases in immunocompromized
patients.
–Little is primary pathogen (cause disease in
person with intact immune system).

Steps of infection:
•I.Adherence:
–By adhesions, e.g. Candida, but filamentous fungi have no
adhesions.
–Fibrinonectin of epithelial cell is the receptors.
–Virulence usually associated with adherence.
ii. Invasion:
–Mechanical trauma to skin or mucosal surface is an
essential step in fungal infection, because most of the
infective element in fungi is the spore and it is non-invasive.
–Some fungi have invasive power like Candida by the
formation of hyphae and pseudo hyphae.

Steps of infection:
iii. Phagocytic interactions:
–Some fungi especially
dimorphic fungi show
resistance to phagocytic
killing.
–Some fungi are
capsulated and can resist
phagocytosis
(Cryptococcus).

Immunity to fungal infections
Innate immunity:
–Non-specific works against all microorganisms.

Immunity to fungal
infections(cont.)
Acquired immunity: The main immunity is cellular
immunity because fungi stay inside the host cell.
Antibodies have limited role in some fungal diseases.

A. Anatomical terminology
(According to the site of
infection):
–Dermatomycosis: Fungal
infection of the skin.
–Pulmonary mycosis:
Fungal infection of the
lung.
–Cardiovascular mycosis:
Fungal infection of the
cardiovascular system.

B. Mycological terminology
(According to the etiology):
–Candidiasis (=
Candidiosis): Fungal
infection by Candida.
–Aspergillosis: Fungal
infection by Aspergillus.
–Cryptococcosis: Fungal
infection by Cryptococcus.
–Histoplasmosis: Fungal
infection by Histoplasma.

Introduction:
 Fungal infections, depending on the tissues that are initially
colonized, can be classified into three major groups as follows:
 Superficial mycoses: These are surface infections of the skin,
affecting the outermost layers of skin, hair, and mucosa.
 Cutaneous mycoses: These are infections of the skin involving
the epidermis and its integuments, the hair, and nails.
 Subcutaneous mycoses: These are infections of the dermis,
subcutaneous tissue, muscle, and fascia.

Superficial mycosis
Infection restricted to upper most
horny layer of skin, hair and nails e.g.
Pitryasisversicolor.

3-Subcutaneous mycosis
(Implantation mycosis):
–Most of fungi are
non invasive.
–Occurs by
implantation of
spores into wounds.
–e.g. Mycetoma
(madura foot), thorn
pricks mycosis.

4-Systemic mycosis:
•Multi organs affected.
Mode of infection:
•Inhalation of spores
of saprophytic fungi.
•Spread of local
mycosis.
Examples:
•Cryptococcosis.
•Histoplasmosis.
•Candidiasis.

5-Opportunistic mycosis:
•Fungal infection by:
–Fungal flora (Candida).
–Saprophytic fungi in the
environment (Aspergillus).
•This infection occur in:
–Immunocompromised host
(Both innate and acquired
immunity).
–Opportunistic conditions like:
–Diabetic patients.
–Cancer patients.
–Corticosteroid & other
immunosuppressive therapy (e.g.
Cytotoxicdrugs).
–Prolonged antibiotic therapy.

A: CUTANEOUS MYCOSES
 Dermatophytoses or cutaneous mycoses are diseases of
the skin, hair, and nail.
 These infections are caused by a homogeneous group of
closely related fungi known as dermatophytes.
 These dermatophytes infect only superficial keratinized
structures, such as skin, hair, and nail, but not deeper
tissues.
 Examples:
a. Infection of skin, hair and nail caused by dermatophytes.
b. Infection of skin, nail and mucous membrane caused by
C. albicans and other candida species.

 The genera Trichophyton, Microsporum, and
Epidermophyton are the principal etiologic agents of
the dermatomycoses.
1. Trichophyton: Trichophyton species infect hair, skin,
or nails.
2. Microsporum: Microsporum species infect only hair
and skin.
3. Epidermophyton: Epidermophyton attacks the skin
and nails but not the hair.
 About 40 species of dermatophytes are known to
cause infection in humans and animals.
Dermatophytes are probably restricted to the
nonviable skin because most are unable to grow at
37°C or in the presence of serum.

Laboratory Diagnosis:
 Introduction:
 Laboratory diagnosis is based on demonstration of
fungal element in clinical specimen by microscopy
and confirmation by culture.
 The specimens include skin scrapings and nail
clippings or hair taken from the areas suspected to be
infected by dermatophytes.
 These entire specimens are treated with alkali
solution to clear epithelial cells and other debris.
 Direct microscopy is useful only for diagnosis, while
culture is always carried out to identify the specific
causative fungal agent.

Cont..
Specimen Collection, Transport, and Processing:
See previous chapter of Specimen
selection,collection, Transportation and processing
A. Specimens:
 Specimens consist of scrapings from both the
skin and the nails plus hairs plucked from
involved areas.
 Microsporum-infecred hairs fluoresce under
Wood’s lighr in a darkened room.

Cont..
B. Microscopic Examination:
 Examination of 10% direct KOH mount may
show fungal
hyphae.
 Three types of hair infections can be
demonstrated in
microscopy of 10% KOH wet mount as follows
(Fig. 72-2):

Cont..
 Specimens are placed on a slide in a drop of 10 to 20
percent potassium hydroxide, with or without
calcofluor white, which is a nonspecilic fungal cell wall
stain viewed with a fluorescent microscope.
 A coverslip is added, and the specimen is examined
immediately and again afrer 20 minutes.
 In skin or nails, regardless of the infecting species,
branched septate hyphae or chains of arthroconidia
(arthrospores) are seen (Fig. 73.6).

Cont..
Fig. 73.6: Dermatophyte in potassium
hydroxide mount of skin or nail scraping
with branched septate hyphae and
arthroconidia

Cont..
 Ectothrix: Ectothrix infection is characterized by
presence
of a layer of arthrospores on the surface of hair shaft
(Color Photo 60)
 It is caused by M. audouinii, M. canis, and
Trichophyton mentagrophytes.
 Endothrix: The clusters of arthrospores are found
entirely
within the hair shaft in endothrix infection (Color Photo
61).
 It is caused by Trichophyton tonsurans, T. violaceum,
and Trichophyton schoenleinii.

 Favus: In favus, there is sparse hyphal growth
and formation
of air spaces within hair shaft.
 It is caused by T. violaceum,T. schoenleinii, and
M. gypseum.

C: Culture
 The clinical specimens are cultured by inoculation
on SDA
containing antibiotics like cycloheximide.
 The media after inoculation are incubated at 25–
30°C for 3 weeks.
 At 25°C most of the pathogenic fungi grow well,
while saprophytic fungi and bacteria are inhibited.
 The cultures are examined at regular intervals,
and dermatophytes are identified based on (a)
colony morphology, ( b) pigment production, and
(c) presence of microconidia and macroconidia.

Cont..
 The LPCB preparation of the colonies shows
microconidia, macroconidia, or both.
 Only macroconidia are present in the Epidermophyton
infection.
 Few macroconidia and more microconidia are
present in
Trichophyton infection.
 Macroconidia are predominantly present in
Microsporum infection.
 The differentiation of three genera is based mainly on
the nature of macroconidia (Table 72-3; Fig. 72-3).

TABLE 72-3 General characteristics of macroconidia
and microconidia of dermatophytes
Microsporum Epidermophyto
n
Trichophyton
Macroconidia Thick-walled,
rough, numerous
Smooth-walled,
numerous
Thin-walled,
smooth, rare
Microconidia Rare Absent Abundant

Key Points
 Trichophyton: Colonies are powdery, velvety, or waxy with pigment
characteristic of different species.
 Microconidia are abundant but macroconidia are very few.
 The microconidia are arranged in clusters along with hyphae and are present on
tip of conidiophores.
 Some species may have special type of hyphae, such as spiral hyphae or racket
hyphae, etc.
 Trichophyton species infect hairs, skin, and nails.
 Microsporum: Infects both skin and hair, but not the nails.
 They produce cottony, velvety, or powdery colonies with white brown pigment.
 Microconidia are relatively scanty but macroconidia are abundant.
 Epidermophyton: Infects skin and nails but not hair.
 It produces powdery and greenish yellow colonies.
 Macroconidia are numerous.
 The macroconidia are club-shaped, multiseptate, and are arranged in groups of
two to three. Chlamydospores are numerous but microconidia are absent.

Other tests:
 Hair perforation test: This test is performed to
differentiate T. rubrum from T. mentagrophytes.
 The test is also used to differentiate M. canis from
Microsporum equinus.
 This test is performed by collecting 5–10 mm
short shaft of human hair and placing it in a Petri
dish with 20 mL of distilled water.
 Then, two to three drops of 10% sterile yeast
extract is added to the Petri dish with hair shafts.
 These hair shafts are inoculated with test fungus
cultured on SDA.

Cont..
 The culture is incubated at 25°C for up to 1
month, during which the hairs are removed and
LPCB mount of the hair is examined
microscopically at weekly intervals for the
demonstration of the perforation of hair.
 T. mentagrophytes (Color Photo 62) shows a
positive hair perforation test characterized by a
wedge-shaped perforation of the hair.
 This test is negative for T. rubrum in which only
surface eruption of hair shaft is demonstrated.

Cont..
 COLOR PHOTO 62. SDA showing colonies of
Trichophyton mentagrophytes.

Cont..
 Urease test: Urease test is carried out to
differentiate T. mentagrophytes from T. rubrum.
 This test is performed by inoculating a tube of
Christensen’s medium with the fungus and
incubating at 25°C for 5 days at room
temperature.
 Most T. mentagrophytes are urease positive
within 5 days.

Cont..
Growth on rice grains: This test is useful to
differentiate
M. canis from M. audouinii.
 In this test, sterile, nonfortified rice is inoculated
with the hyphae of the fungi to be tested.
 The medium is observed for growth after 10 days
of incubation at room temperature.
 M. canis usually grows well and forms many
conidia on rice grain, whereas M. audouinii fails
to grow.

D: Molecular Methods
 Nucleic acid amplification assays for
dermatophytes are not routine.
 Current traditional procedures are more cost
effective for superficial infections.

Treatment
 Treatment of dermatophyte infection is carried out by use
of local antifungal drugs, such as miconazole, clotrimazole,
econazole, etc., or by treatment orally with griseofulvin.
 Topical therapy is satisfactory for most skin infections, but
oral antifungals are required to treat infections of the nail
and scalp, and severe or extensive skin infections.
 Topical agents include azole compounds, terbinafine,
amorol fine and ciclopirox olamine.

Cont..
 Oral griseofulvin is useful for scalp, skin and fingernail
infections, but gives poor results in toenail infections,
even after 18 months’ therapy.
 Terbinafine and itraconazole have largely replaced
griseofulvin for the treatment of nail infections.
 Relatively little has been done to control the spread of
ringworm.
 The prophylactic use of antifungal footpowder after
bathing helps to reduce the spread of infection among
swimmers. Foot-bath containing antiseptic solutions,
which are commonplace in swimming pools, are of no
value.

B: Subcutaneous Mycoses
 Subcutaneous mycosis is defined as fungal infection
associated
with development of characteristic lesion in subcutaneous
tissue and overlying skin with or without extension to bone
and muscle.
 This is caused by a heterogeneous group of fungal
infection of low pathogenic potential introduced in the body
percutaneously from a trivial trauma.
Examples:
 The principal subcutaneous mycoses are mycetoma,
chromomycosis, sporotrichosis and rhinosporidiosis.

TABLE-4 Causative agents of subcutaneous mycoses
Condition Causative agents
Mycetoma Bacteria, fungi
Chromoblastomycosis Fonsecaea pedrosoi
Fonsecaea compactum
Phialophora verrucosa
Cladophialophora carrionii
Rhinocladiella aquaspersa
Phaeohyphomycosis Exophiala jeanselmei
Bipolaris spicifera
Wangiella dermatitidis
Sporotrichosis Sporotrichosis Sporothrix schenckii
Rhinosporidiosis Rhinosporidium seeberi

1. Mycetoma:
 Mycetoma is a slowly progressive, chronic
granulomatous
infection of skin and subcutaneous tissues with
occasional
involvement of underlying fascia and bone
usually affecting
extremities.
 The condition is characterized by a triad of (a)
tumefaction, (b) draining sinuses, and (c) grains
or granules.

Cont..
 The condition was described as early as in
Atharva-Veda:
padmavalmikam means anthill foot
 John Gill first described the condition in Madurai
(India) in 1842.
 Colebrook confirmed it in 1846. Henry Vandyke
Carter, a professor at Grants Medical College,
Mumbai, coined the term mycetoma in 1874.
 Chalmer and Christopherson coined the term
maduramycosis in 1916.

Cont..
 Mycetoma is caused by a number of actinomycetes
and filamentous fungi that enter through penetrating
injuries resulting from thorn pricks, splinters, etc
(Table 72-5). Lower extremities are most commonly
involved.
 Microabscesses are formed in subcutaneous tissues
surrounded by polymorphonuclear inflammatory
reaction.
 The center of the lesion consists of tangled filaments
of these organisms.

Cont..
 During the course of infection, microabscesses burst
open with the formation of chronic multiple sinuses
discharging copious, seropurulent fluid containing
granules.
 The color and consistency of these granules vary
depending on the fungi that cause the disease (Table
72-5).
 The condition is characterized by formation of
painless, localized, swollen lesions on the affected
limbs.
 Multiple discharging sinuses are present.
 Discharge is purulent, mucopurulent, or
serosanguinous and usually contains granules of
varying size, color, and consistency.

Cont..
 Fascia and bone may be affected but underlying
tendons and nerves are never affected.
 The condition during period of time spreads
slowly, but over years by contiguity.
 Sometimes the infection becomes extensive by
involving buttock and trunk.
 Actinomycetes spread faster than eumycetes.
 Hematogenous spread is rare.

TABLE 72-5 Important causative agents of mycetoma
Grain Causative agents
Black grain Madurella mycetomatis
Madurella grisea
Exophiala jeanselmei
Curvularia geniculata
White grain Pseudoallescheria boydii
Acremonium falciforme
Actinomadura madurae
Nocardia brasiliensis
Red grain Actinomadura pelletieri

Cont..
 Mycetoma is documented worldwide but is common in
tropical and subtropical countries.
 Actinomycetoma accounts nearly for 65% of mycetomas
and for 35% of eumycetoma in India. Actinomadura
madurae is the major causative agent.
 Streptomyces somaliensis infection is seen more
frequently in eastern India.
 The condition is more common in rural areas and in males
(3.5:1) than females.
 It affects mostly highly active persons aged 20–40 years.
 It is an occupational hazard affecting farmers, herdsmen,
carpenters, builders, land workers, field workers, etc.

Cont..
 Mycotic mycetomas require surgical treatment by
amputation, while actinomycotic mycetomas are
treated well by sulfonamides and antibiotics.
Key Points:
 High index of clinical suspicion and proper history
is essential for clinical diagnosis of mycetoma.
 Laboratory diagnosis depends on demonstration
of the fungi and fungal filaments in granules, pus,
and biopsy tissue by microscopy.

Cont..
 The granules on naked eye examination may be
hard or soft, round or lobulated, and vary in size
from 0.5 to 3 mm.
 These may be black, white to cream, or red.
 Microscopy of the granules may reveal very thin
fungal filaments, measuring less than 1 m in
diameters in cases of
actinomycotic mycetomas.
 However, the fungal filaments may be broader
and often show septate hyphae and
chlamydospores in mycotic mycetoma

Laboratory Diagnosis
A. Direct Examination:
 The presence of grains in pus collected from draining
sinuses or in biopsy material is diagnostic.
 The grains are visible to the naked eye and their color
may help to identify the causal agent.
 Grains should be crushed in KOH (potassium
hydroxide) and examined microscopically to
differentiate between actinomycetoma and
eumycetoma.
 Material from actinomycetoma grains may be gram-
stained to demonstrate the gram-positive filaments.

B. Culture
 Samples should also be cultured, at both 25 to
30°C and 37°C, on brain-heart infusion agar or
blood agar for actinomycetes and on Sabouraud
agar (without cycloheximide) for fungi.
 The fungi that cause eumycetoma are all septate
moulds that appear in culture within 1 to 4 weeks
 C. Serological Tests:
 Serological precipitin tests are of little value for
diagnosis and are not in routine use.

Treatment:
 The prognosis varies according to the causal agent,
so it is important that the identity is established.
 The management of eumycetoma is difficult involving
surgical debridement or excision and chemotherapy.
 Actinomycetoma responds well to rifampicin in
combination with sulonamides or co-trimoxazole, but
an average of 9 months therapy is required.
Epidemiology and Control of Mycetoma:
 The organisms producing mycetoma occur in soil and
on vegetation.
 Barefoot farm laborers are therefore commonly
exposed. Properly cleaning wounds and wearing
shoes are reasonable control measures.

Chromomycosis
 The term chromomycosis includes
chromoblastomycosis and phaeohyphomycosis
caused by dematiaceous fungi.

2. Chromoblastomycosis
• This disease, also known as chromomycosis, is a
chronic, localized disease of the skin and
subcutaneous tissues, characterized by crusted,
warty lesions usually involving the limbs.
• The disease is mainly encountered in the tropics.
• Like mycetoma, the disease is seen most often
among males in rural areas.
 Chromoblastomycosis is a slowly progressing
granulomatous
 infection caused by several soil fungi.
 These are Fonsecaea pedrosoi, Fonsecaea
compactum, Cladosporium carrionii, and Phialophora
verrucosa.

Cont..
 These fungi are collectively called dematiaceous fungi
because they have brown to black melanin pigment
in their cell wall, and their conidia or hyphae are dark
colored, either gray or black.
 All the fungi causing chromoblastomycosis appear
morphologically similar in tissues stained by
hematoxylin and eosin (H and E) or other stains.
 The infection occurs following introduction of any of
the dematiaceous fungi into the skin through trauma.
 The development of warty nodules that appear at site
of inoculation characterizes chromoblastomycosis.

Cont..
 During the course of infection, these lesions
vegetate and develop to a cauliflowerlike lesion.
 The disease is more common in tropical and
subtropical countries.

Key Points
 Laboratory diagnosis of chromoblastomycosis is
made by demonstration of small clusters of round and
dark brown, thick-walled cells (8–12 m) in tissues.
 The fungi are found in the dermis but are occasionally
seen in the subcutaneous
tissues.
 Direct KOH wet mount of skin is a rapid method for
diagnosis of the condition by demonstrating these
fungi in scales removed from surface of these lesions.
 In addition to these, copper-colored spherical cells in
various stages of cell division are seen.
 These are the tissue forms of the fungus and called
sclerotic or medullar bodies (Fig. 72-4) of the fungus.

Etiological Agents
 The etiological agents are soil inhabiting fungi of the
family Dematiaceae.
 All are dematiaceous fungi, having melaninized cell
walls.
 These include: Phialophora verrucosa, Fonsecaea
pedrosoi, Rhinocladiella aquaspersa, Fonsecaea
compacta and Cladophialophora carrionii.
 They enter the skin by traumatic implantation.
 The lesion develops slowly around the site of
implantation.
 The infection is chronic and characterized by the
slow development of progressive granulomatous
lesions that in time induce hyperplasia of the
epidermal tissue.

A. Microscopic Examination:
 The dark-colored fungal elements are relatively easy
to see on microscopical examination of skin
scrapings, crusts and pus.
 The agents of chromoblastomycosis are identified by
their modes of conidiation.
 Histologically, the lesions show the presence of the
fungus as round or irregular, dark brown, yeastlike
bodies with septae, called sclerotic cells (Fig 73.7).
 Diagnosis can be established by demonstration of
these sclerotic bodies in KOH mounts or in sections
and by culture on Sabouraud’s agar.

Cont..
Fig. 73.7: Chromblastomycosis: KOH
mount of lesion large septate ‘scierotic
bodies’

B. Culture:
 Culture on Sabouraud agar at 25 to 30°C yields
slow-growing, greenish grey to black, compact,
folded colonies.
 Cultures should be incubated for 4 to 6 weeks.
C. Serological Tests: Serological tests are not
used routinely.

3. Phaeohyphomycosis
 Phaeohyphomycosis is a heterogeneous group of
cutaneous
diseases caused by various dematiaceous fungi.
 Phaeohyphomycotic cyst is the most common form
described
in this condition.
 A wide number of dematiaceous fungi have been
associated with various types of phaeohyphomycosis.
 Some of the common causes of phaeohyphomycosis
are
Exophiala jeanselmei, Bipolaris spicifera, and
Wangiella dermatitidis.

Cont..
 The sites of lesions may be cutaneous, subcutaneous,
deeper tissues, or organs like the brain or lung.
 The tissue reactions and morphology of the fungus in
lesions differ from those seen in chromoblastomycosis.
Sclerotic cells or granules are not found.
 Diagnosis of phaeohyphomycosis is made by
demonstration
of darkly pigmented, septate hyphae measuring 5–10 m
diameter in tissues.
 The fungi appear in lesions as distorted hyphal strands.

Cont..
 The drug of choice for treatment of
chromoblastomycosis is 5-fluorocytosine.
 Cautery and surgical removal of early lesion is
also useful.

Clinical Types of
Phaeohyphomycosis
 Phaeohyphomycosis is generally seen in debilitated
or immunodeficient hosts. Some of the clinical types
are:
a. Brain abscess caused by Cladosporium bantianum.
b. Subcutaneous or intramuscular lesions with
abscesses or cysts containing masses of brown
hyphae (formerly known as phaeosporotrichose)
caused by Phiaophora jeanselmei, P.spinifera, P.
dermatitidis or P. richardsiae.

4. Sporotrichosis
 Sporotrichosis is a chronic pyogenic granulomatous
lesion of
the skin and subcutaneous tissue caused by
Sporothrix schenckii.
 S. schenckii is a dimorphic fungus found all over the
world and occurs mainly in Central and South
America, parts of the USA and Africa and Australia.
 It is rare in Europe.
 The fungus is found in soil, decaying woods, thorns,
and on infected animals including rats, cats, dogs,
and horses.

Cont..
 Causative Agent: It is caused by Sporothrix
schenckii, a saprophyte in nature.
 Morphology:
 S. schenckii is a dimorphic fungus.
 In nature and in culture at 25 to 30°C, it develops
as a mould with very thin (1-2 mm) septate
hyphae; spore-bearing hyphae carry clusters of
oval spores.
 The yeast phase is formed in tissue and in
culture at 37°C, and is composed of spherical or
cigar-shaped cells (1-3 × 3-10 mm).

Cont..
 In infected tissues, the fungus is seen as cigar
shaped yeast cells, without mycelia.
 Sometimes ‘asteroid bodies’ are seen in the
lesion, composed of a central fungus cell with
eosinophilic material radiating from it.
 Spore is the infective stage of the fungus.
 It causes infection primarily on the hand or the
forearm through direct contact of the skin by
spores.
 Typically, infection is introduced in skin through a
penetration of thorn.

Cont..
 At the site of thorn injury, it causes a local pustule
or ulcer with the nodules along the draining
lymphatics.
 Frequently, the regional lymph nodes draining the
ulcer enlarge, suppurate, and ulcerate.
 The primary lesion may remain localized or in the
immunocompromised individuals may
disseminate to involve the bones, joints, lung, and
rarely the central nervous system.
 In infected tissue, the yeast appears as round,
oval, or cigarshaped cells with irregular borders.

Cont..
 Periodic acid-Schiff (PAS) or Gomori’s methenamine
silver (GMS) stain is useful to demonstrate these
structures in the stained smears.
 The fungus on SDA at 25°C produces black and
shiny colonies, which become wrinkled and foggy
during course of time.
 The mold contains hyphae bearing flower-like
structures of small conidia on delicate sterigmata.
 Laboratory diagnosis of sporotrichosis is made by
demonstration of asteroid bodies in pus of the
abscesses.

Cont..
 Asteroid bodies consist of a central basophilic
budding yeast cell with eosinophilic material,
which radiates from the center.
FIG. 72-4. Sclerotic bodies.

Pathogenesis
• The fungus is a saprophyte found widely on plants,
thorns and timber.
 Infection is acquired through thorn pricks or other
minor injuries. Rare instances of transmission from
patients and infected horses and rats have been
recorded.
 Sporotrichosis most frequently presents as a nodular,
ulcerating disease of the skin and subcutaneous
tissues, with spread along local lymphatic channels
but seldom extends beyond the regional lymph nodes.
 Most cases occur in the upper limb.

Key Points
 Isolation of the fungi by culture of ulcer exudates or
pus aspirated
from subcutaneous nodule or biopsy material
confirms the diagnosis of sporotrichosis.
 LPCB wet mount of the colony shows thin, delicate
hyphae bearing conidia occurring in a rosette pattern
at the end of delicate conidiophores.
 Conidia are also demonstrated along the sides of the
hyphae.
 Repeated subculture of mycelium of fungi on blood
agar tubes at 37°C induces formation of yeast
colonies.
 The LPCB wet mount of these colonies shows cigar-
shaped yeast-like cells.

 Diagnosis is made by culture as frequently the fungus
may not be demonstrable in pus or tissues.
A. Micrscopic Examination:
Direct microscopy is of little.
B. Culture:
 SDA or blood agar are the media used S. schenckii is
a dimorphic fungus occurring in the yeast phase in
tissues and in cultures at 37°C, and in the mycelial
phase in nature and in cultures at room temperature.

Cont..
 The septate hyphae are very thin (l-2 μm diameter)
and carry flower-like clusters of small conidia borne
on delicate sterigmata (Fig. 73.8)
 Conidia are also produced along the sides of the
hyphae.
C. Serology:
 A latex agglutination test is of value for the diagnosis
of the extracutaneous forms of sporotrichosis.
 The test has poor prognostic value since titres change
little after successful therapy.

Cont..
Fig. 73.8: Sporothrix (Sporotrichum) schenkii:
culture mount showing fine branching hyphae and
pear shaped conidle borne in rosette clusters at tips
of lateral branches and singly along sides of
hyphae

D. Skin Test
 A skin test with sporotrichin antigen is ‘positive in
almost all patients with cutaneous sporotrichosis.
E. Animal Inoculation:
 Rats are highly susceptible and can be infected
by intraperitoneal or intratesticular inoculation.
Treatment: Itraconazole is the drug of choice for
treatment of the condition.

5. Rhinosporidiosis
 Rhinosporidiosis is a chronic granulomatous disease
caused
by Rhinosporidium seeberi.
 More than 90% of cases are reported from India, Sri
Lanka, and South America.
 The cases have been reported from throughout India.
 The endemic foci of infection have been reported from
Tamil Nadu, Andhra Pradesh, Kerala, and Madhya
Pradesh.
Etiology:
 The causative fungus Rhinosporidium seeberi.

Mode of Infection
 The mode of infection of this fungus is not known.
 However, it is suggested that it is transmitted in
dust and
stagnant water or aquatic life.
 Fish is believed to be the natural host of this
fungus. Infection is seen most commonly in
persons taking bath in stagnant pools and in
individuals who dive in streams to collect sand
from river beds.

Cont..
 The disease is characterized by the development
of large
friable polyps or wart-like lesion in the nose,
conjunctiva, or
eye.
 The lesion is also occasionally seen in ears,
larynx, bronchus,
urethra, vagina, rectum, and skin.

 It has not been cultured in cell-free artificial media and
animal inoculation is also not successful. Successful
cultivation of the organism in epithelial cell culture has
been reported.
Demonsration of Sporangia:
 Diagnosis depends on the demonstion of sporangia.
 Direct examination of the surface of the polypoid
growth and histologic examination are the only ways
to make a diagnosis.
 R. seeberi can be identified in hematoxylin and eosin
stained sections, but sometimes one may need
special stains.

 Histologically, the lesion is composed of large
numbers of fungal spherules embedded in a
stroma of connective tissue and capillaries.
 The spherules are 10 to 200 m in diameter and
contain thousands of endospores (6-7 mm in
diameter) (Fig 73.12).
 These spores develop into new sporangia when
released
Fig. 73.9: Rhinosporidiosis:
Sporangium with numerous
endospores

Treatment:
 Treatment of the condition is carried out by
surgery or
cauterization.
 Chemotherapy with dapsone is also useful.

Key Points
 Laboratory diagnosis of rhinosporidiosis depends on
demonstration of sporangia of R. seeberi in tissue sections.
 R. seeberi can be demonstrated in tissue sections stained with
H&E or other special stains, such as GMS stain and PAS stain.
 The stained smear on microscopic examination shows a
hyperplastic
surface epithelium with the presence of chronic inflammatory
exudates.
 The epidermis and stroma are embedded with sporangia (Fig.
72-5).
 The sporangia measure 10–200 m in diameter and contain
hundreds of endospores, each measuring 6–7 m in diameter.
 The spores when released develop into new sporangia.

C. SYSTEMIC MYCOSES
INTRODUCTION:
 Systemic mycoses are caused by fungi of soil,
which are inherently virulent and cause disease in
healthy humans.
 Systemic mycoses-infections that originate
primarily in the lung but that may spread to many
organ systems.

Cont..
 Systemic mycoses are caused by inhalation of
airborne spores produced by the fungi which are
present as saprophytes in soil and on plant material.
 From the lungs the fungus may disseminate to central
nervous system (CNS), bone and other internal
organs.
 The systemic mycoses include coccidioidomycosis,
paracoccidioidomycosis, histoplasmosis,
blastomycosis, and cryptococcosis.

1. Coccidioidomycosis
 This is primarily an infection of the lungs caused by
Coccidioides immitis, a dimorphic fungus which
occurs as mold in the soil and in culture at25˚C and
as a spherule in tissue and in culture at 37˚C of semi-
arid areas, mainly in the south-west USA and northern
Mexico.
 The infection may be inapparent, benign, severe or
even fatal. Agricultural workers with a higher exposure
risk and dark-skinned people are especially prone to
the disease.
 Recovery usually confers lifetime immunity.

Clinical Features
i. Asymptomatic:
 Infection is acquired by inhalation of dust
containing arthrospores of the fungus.
 C. immitis usually causes an asymptomatic or
self-limiting pulmonary illness, but a progressive
and sometimes fatal secondary disease
occasionally develops.

Cont..
ii. Primary Pulmonary Disease:
 The 40 percent of individuals develop a self-limited
influenza-like illness with fever, malaise, cough,
arthralgia, and headache.
 This condition is called Valley fever, San Joaquin
Valley fever, or desert rheumatism.
 iii. Disseminated Disease:
 Less than one percent of infected persons develop
chronic progressive disseminated disease
(coccidioidal granuloma) which is highly fatal.
 It resembles clinically and histologically disseminated
tuberculosis.

Morphology
 The fungus is dimorphic, occurring in the tissue
as a yeast and in culture as the mycelial form.
 The mycelial phase consists of hyphae which
fragment into arthrospores which are highly
infectious.
 In culture and in soil, C. immitis grows as a mold,
producing large numbers of barrel-shaped
arthrospores (4 × 6 μm diameter) which are highly
infectious.
 They characteristically alternate with smaller
intervening empty cells (Figs 74.3 A and B).

Cont..
 C. immitis is probably the most virulent of all human
mycotic agents.
 The inhalation of only a few arthroconidia produces
primary coccidioidomycosis.
 The yeast form is a spherule (15-75 μm diameter)
with a thick, doubly refractile wall and filled with
endospores.
 Endospores are released by rupture of the spherule
wall and develop to form new spherules in adjacent
tissue or elsewhere in the body (Figs 74.3A and B).

Cont..
Figs 74.3A and B: Coccidioides immitis: (A) Arthrospores
formation; (B) Spherule formation with endospores

 Laboratory diagnosis of coccidioidomycosis is
made by demonstration of spherules containing
endospores in
(a) sputum, or smears from the lesion stained by
calcofluor white and
(b) in biopsy material stained by hematoxylin and
eosin, silver, or periodic acid-Schiff stains.

A. Specimens:
 Sputum, pus and biopsy material.
 Diagnosis may be made by microscopic
examination of sputum, pus and biopsy material.
C. Culture:
 Culture is the most definitive method for
diagnosis.
 Specimen is inoculated on SDA medium in the
test tube and incubated at 25-30°C.

Cont..
 The fungus grows well on Sabouraud’s dextrose agar
(SDA) and other media producing white and cottony
colony within 5 days.
 Identification of colonial morphology is not adequate,
because other fungi show similar mycelial forms.
 The arthroconidia are highly infectious and are a
serious danger to laboratory staff.
 Consequently, Petri dishes should never be used for
isolation of the organism and all procedures should be
carried out in Category 3 containment facilities.

Cont..
D. Serological Tests:
 Serological tests such as precipitin test, latex
agglutination test and complement fixation test play
an important part in diagnosis.
 Serodiagnosis of coccidioidomycosis is based on the
demonstration of antibodies to coccidioidal antigens in
patient’s serum.
 Tube-precipitating antigen and the
complementfixation
antigen are the two major antigens used to detect
antibodies.

Cont..
 Enzyme immunoassay (EIA) is the most frequently
used test to detect serum tube-precipitating
antibodies that are IgM antibodies to mycelial phase
antigens.
 These IgM antibodies appear in more than 85% of
patients with primary infection and are found within
the first week after the onset of symptoms.
 In most patients, these antibodies disappear within 6
months.
 IgG antibodies detected by complement fixation
appear later,
with results becoming positive in 85–90% of patients.

Cont..
E. Skin Test:
 Skin tests with coccidioidin, a culture filtrate
antigen from the mycelial phase of C. immitis, or
spherulin, an extract of the spherules are useful.
 The test becomes positive (5 mm diameter of
induration at 48 hours) between 3-21 days of
symptoms.
 About 90 percent of inhabitants in endemic areas
exhibit positive skin test.

Cont..
F. Molecular Method:
 DNA probe is a recent method used for accurate
identification of the fungus.
Treatment:
 Amphotericin B is the drug of choice for treatment of
the
condition.
 Fluconazole can be used for the treatment of mildto
moderate disease and, occasionally, for the treatment
of life-threatening disease in patients in whom
amphotericin B is contraindicated for use.
 It is used as the drug of choice for long-term therapy
of meningeal infection.

Key Points
 Skin test using coccidioidal antigens is used for diagnosis
of coccidioidomycosis.
 It is a delayed-type hypersensitivity reaction that appears
2–21 days after the onset of symptoms and precedes the
appearance of serologic markers.
 Skin test has epidemiologic and prognostic implications.
 Absence of delayed-type hypersensitivity indicates poor
prognosis.
 However, the skin test has limited diagnostic utility
because of its low sensitivity and specificity in endemic
areas.
 Infected individuals may show negative results due to
absence of an immune response and healthy individuals
may show positive results because of previous infection.

2. Paracoccidioidomycosis
• This is a chronic granulomatous disease of the
skin, mucosa, lymph nodes and internal organs.
 It is confined to endemic regions of Central and
South America. As the disease is confined to
South America, it is called ‘South American
blastomycosis’.
Causative Fungus:
 It is caused by Paracoccidioides brasiliensis, a
dimorphic fungus.

Pathogenesis
 It is characterized by primary pulmonary infection that
spreads, by hematogenous route to mucosa of the
nose, mouth and the gastrointestinal tract, skin,
lymphatic system, and the internal organs producing
chronic granulomatous reaction.
 P. brasiliensis is inhaled, and initial lesions occur in
the lung.
 After a period of dormancy that may last for decades,
the pulmonary granulomas may become active,
leading to chronic, progressive pulmonary disease or
dissemination.

Cont..
 In the usual case of chronic
paracoccidioidomycosis, the yeasts spread from
the lung to other organs, particularly the skin and
mucocutaneous tissue, lymph nodes, spleen,
liver, adrenals, and other sites.
 Many patients present with painful sores
involving the oral mucosa.

Laboratory Diagnosis:
1. Specimens:
 Sputum or pus, crusts and biopsies from
granulomatous lesions
2. Direct Microscopy:
 Microscopy of sputum or pus, crusts and biopsies
from granulomatous lesions usually reveals
numerous yeast cells (10-60 mm) with multiple
buds, which is diagnostic. (Figs 74.2A and B).
 Multiple buds attached to the mother cell
(resembling ‘pilot wheel’) by very narrow necks
are the hallmark of this fungus. Tissue sections
should be stained with H & E, PAS and GMS.

Cont..
Figs 74.2A and B: Paracoccidioides brasiliensis (A)
Yeast phase; and (B) Mycelial phase

3. Culture
 P. brasiliensis grows in the mycelial phase in
culture at 25-30°C, and in the yeast phase in
tissue or at 37°C. Blood agar (without
cycloheximide) and incubation at 37°C is
recommended for isolation of the yeast phase
(tissue form).
 Mycelial (mold) phase of the fungus develops on
SDA incubated at 25-30°C.
 Identification depends on conversion from the
mycelial to the yeast phase.

Treatment:
 Itraconazole is the drug of choice for treatment of
paracoccidioidomycosis and is effective in 95% of
patients.
 Imidazole is also an effective agent with a cure
rate of 85–90%.
 Amphotericin B is used as a reserve for treatment
of severe
cases that are refractory to treatment with these
drugs.

Key Points
 Serodiagnosis depends on demonstration of P.
brasiliensis antibodies or antigens in the serum.
 Enzyme-linked immunosorbent assay (ELISA) is
used to detect antibodies to gp43, the main
antigenic determinate of P. brasiliensis with a
reported sensitivity of 95% and a specificity of
93%.
 The test shows cross-reactivity with sera from
patients of histoplasmosis or Lobo disease.

Key Points
 Western blot detects circulating antibodies to gp43 in
the serum in 100% of patients and to gp70, another
antigen of the fungus, in 96% of patients.
 Newer tests that detect Ag7, i.e., antigen found after 7
days of growth, have a sensitivity of 84% and
specificity of 99%.
 This antigen detection test is used to monitor patient
response to therapy and to detect recurrences.
 However, skin tests are rarely helpful.

3. Histoplasmosis
• Histoplasmosis is an intracellular infection of the
reticuloendothelial system caused by the dimorphic fungus
Histoplasma capsulatum.
 The disease was originally described by Darling (1905)
who believed the causative agent to be a protozoon related
to Leishmania donovani.
 Histoplasmosis is also known as Darling’s disease,
reticuloendothelial cytomycosis, cave disease, and
spelunker’s disease.
 H. capsulatum is predominantly found in river valleys in
North and Central America.
 It is endemic in the Ohio, Missouri, and Mississippi River
valleys of the United States.

Cont..
 The colony is characterized by thin, branching,
septate hyphae that produce tuberculate
macroconidia and microconidia.
Macroconidia: are thick-walled, spherical spores
measuring 8–20 m in diameter and have finger-like
projections.
 These are diagnostic form of the fungus.
Microconidia: are smaller and thin-walled spores, and
are
the infectious form of the fungus.

Cont..
 Most infected individuals are asymptomatic.
 Nevertheless, H. capsulatum causes acute
pulmonary histoplasmosis, chronic pulmonary
histoplasmosis, and progressive disseminated
histoplasmosis.
 Majority of patients with acute pulmonary
histoplasmosis
are asymptomatic.
 Incubation period varies from 3 to 14 days.
 Fever, headache, malaise, myalgia, abdominal pain,
and chills are common symptoms.
 Cough, hemoptysis, dyspnea, and/or chest pain may
be present.

Cont..
 Chronic pulmonary histoplasmosis is seen in
patients with underlying pulmonary disease.
 Cough, weight loss, fevers, and malaise are
symptoms.
 Progressive disseminated histoplasmosis is seen
in patients
who are immunocompromised, such as patients
with AIDS.

A. Specimens:
 Blood films, bone marrow slides, and biopsy
specimens may be examined microscopically.
 Specimens for culture include sputum, urine,
scrapings from superficial lesions, bone marrow
aspirates, and buffy coat blood cells.
 In disseminated histoplasmosis, bone marrow
cultures are often positive.

Cont..
 Microscopy of smears of sputum or pus should be
stained by the Wright or Giemsa procedure.
 Liver or lung biopsies stained with PAS or
methenamine-silver may provide a rapid
diagnosis of disseminated histoplasmosis in some
patients.
 H. capsulatum is seen as small, oval yeast cells
(2-4 μm in diameter), typically packed within the
cytoplasm of macrophages or monocytes (Fig.
74.4).

Cont..
Fig. 74.5: H.
capsulatum: Mycelial form
Fig. 74.4: H. capsulatum: Yeast cells in macrophage

Cont..
C. Culture:
 Specimens are cultured in rich media, such as
glucosecysteine blood agar at 37°C and on
Sabouraud’s agar or inhibitory mold agar at 25-30°C.
 On SDA, it forms white to tan fluffy colony with
septate branching hyphae with two types of
unicellular, asexual spores: (1) Large round,
tuberculate macroconidia (8-14 μm) are most
prominent and are diagnostic. (2) Small spores or
microconidia are sessile or stalked, smooth-walled,
round to pyriform, 2-4 μm in diameter (Fig. 74.5).
 On blood agar or other enriched media containing
cysteine, small, round or oval budding yeast cells are
produced.

Cont..
D. Serological Tests:
 Serological tests like latex agglutination,
precipitation and complement fixation become
positive two weeks after infection.
 Serological tests are useful, but cross-reactions
can occur, mainly with C. immitis.
 Antibody tests fail to detect antibodies in up to 50
percent of immunosuppressed individuals.
 Tests for antigen detection by radio-
immunoassay or ELISA are useful, but are not
widely available.

Cont..
E. Histoplasmin Skin Test:
 Delayed hypersensitivity to the fungus can be
demonstrated by histoplasmin skin test.
 The test is similar to tuberculin test but antigen
used is histoplasmin.
 Histoplasmin is a culture filtrate antigen of
mycelial phase of H. capsulatum.

Cont..
 The histoplasmin skin test becomes positive soon
after infection and remains positive for years.
 A positive ‘histoplasmin skin test’ indicates past
or present infection, but does not differentiate
active and past infections.

Treatment:
 Intravenous amphotericin B for up to 3 months is the
treatment of choice for most forms of disseminated
histoplasmosis; this is followed by oral itraconazole
for 6-24 months in immunocompromised patients.
 Ketoconazole and itraconazole give good results in
less ill cases.
 Fluconazole is often recommended for meningitis due
to its better penetration of the cerebrospinal fluid
(CSF).
 Asymptomatic or mild condition needs no treatment.

Key Points
 Detection of specific H. capsulatum antigen in serum and
urine is useful in immunocompromised hosts when
antibody production may be impaired.
 They are usually positive in case of acute progressive
disseminated histoplasmosis.
 Histoplasmin skin test detects a delayed-type
hypersensitivity to histoplasmal antigens that occurs 3–6
weeks after exposure.
 A positive skin test indicates a past or present infection,
but does not differentiate between active and past
infections.
 It is positive in approximately 85–90% immunocompetent
individuals.
 DNA probes and polymerase chain reaction (PCR) are
also used for diagnosis of the condition.

4. Blastomycosis
 Blastomycosis is a chronic infection of the lungs
which may spread to other tissues, particularly skin,
bone and genitourinary tract.
 Blastomycosis is a granulomatous fungal infection
caused
by B. dermatitidis. B. dermatitidis is a dimorphic
fungus, which
occurs in two stages: as mold in soil and as yeast in
tissue.
 The disease has been called North American
blastomycosis because it is endemic and in most
cases occur in the United States and Canada.

Cont..
Fig. 74.1: Blastomyces dermatitidis: yeast and
mycelial forms

 Diagnosis can be made by direct microscopy, culture
of sputum, pus and scrapings from skin lesions and
serology.
A. Specimens:
 Specimens consist of sputum, pus, exudates, urine,
and biopsies from lesions.
 Potassium hydroxide (10%) mount of specimens may
show characteristic thick-walled yeast cells with a
single broadbased bud.
 These may also be apparent in histologic sections.

Cont..
C. Culture:
 Colonies usually develop on Sabouraud’s dextrose
agar or enriched blood agar at 30°C within 2 weeks.
 The identification is confirmed by conversion to the
yeast form after cultivation on a rich medium at 37°C,
or by the exoantigen test or by a specific DNA probe.
D. Serology:
 Antibodies can be measured by the complement
fixation (CF), immunodiffision (ID) tests and enzyme
immunoassay (EIA).
 Overall, serologic tests are not as useful for the
diagnosis of blastomycosis.

Treatment
 Severe cases of blastomycosis are treated with
amphotericin B and itraconazole.
 Uncomplicated pulmonary disease may respond
to fluconazole.

Key Points
 Sputum microscopy is a very useful test for demonstration of
characteristic broad-based budding yeasts of B. dermatitidis.
 KOH wet mount of pus aspirated from skin
microabscesses,fistulae, or subcutaneous abscesses is
useful to
 demonstrate the yeast.
 Sputum culture on SDA medium confirms diagnosis of the
condition.
 However, it may take as early as 5 days or as late as 30 days
for colony formation.
 ELISA is a useful test to detect antibodies against A antigen
of B. dermatitidis in the patient’s serum.
 A titer of greater than 1:32 is suggestive of the condition.
 Radioimmunoassays (RIAs) and Western blot techniques
are the newer tests that use more specific cell wall antigens
of B. dermatitidis for detection of antibodies in the serum.
 Skin test is not reliable for diagnosis.

5. Cryptococcosis
 Cryptococcosis, also called European
blastomycosis, is an acute to chronic disease
caused by an encapsulated yeast,C. neoformans.
 Cryptococcosis is the most common life-threatening
fungal disease in patients with AIDS.
 It is the only systemic mycosis frequently
documented from India.
 Of the 19 species that comprise the genus
Cryptococcus,human disease is associated with
only C. neoformans. C. neoformans was first
described by Busse, a pathologist, in 1984.

Cont..
 Kwon-Chung (1976) have described the perfect
(i.e.,sexual, teleomorphic) form of C. neoformans,
which was named Filobasidiella neoformans.
 Prior to the identification of F. neoformans, which
is mycelial, C. neoformans was considered
monomorphic yeast.

 Laboratory diagnosis of cryptococcal infection is
made by
 demonstration of the yeast in CSF, sputum, pus,
and brain biopsy tissue by smear and culture.
 Methenamine silver or periodic acid-Schiff stains
are used to stain the tissue specimens for
demonstration of the capsule of C. neoformans.
 Fixed tissue may also be stained with
mucicarmine, which preferentially stains C.
neoformans.

Cont..
 India ink preparation is commonly used to detect
budding
 yeast cells in the CSF (Fig. 73-2).
 The capsule appears as a clear halo around the yeast
cells.
 By this method, cryptococci can be demonstrated in
25–50% of patients with cryptococcal meningitis.
 Gram-stained smear of the CSF shows Gram-positive
yeast
cells (Color Photo 63).
 The culture of centrifuged CSF specimens confirms
diagnosis of the condition.

Cont..
 This fungus is identified based on its microscopic
appearance, biochemical test results, and ability
to grow at 37°C (98.6°F).
 Latex agglutination test (LAT) is a frequently used
serological
 test to detect cryptococcal polysaccharide antigen
in the serum or CSF for diagnosis of meningitis.
 LAT is an extremely important adjunct to the
diagnosis.

Treatment:
 Amphotericin B is the drug of choice for initial therapy
in meningitis or other disseminated infections caused
by C. neoformans.
 Amphotericin B may be used alone or in combination
with flucytosine.
 The therapeutic goal for patients with cryptococcal
disease not complicated by HIV infection is to achieve
a permanent cure of the fungal infection.
 The therapeutic goal for patients with concomitant
HIV infection without a CD4 count of greater than 100
cells/L is to control the acute infection, followed by
lifelong suppression of C. neoformans.

D. OPPORTUNISTIC MYCOSES
Introduction:
 The opportunistic fungi usually cause infections in
persons
with impaired host defense, but do not cause disease in
most
of the immunocompetent hosts.
 Since these fungi become pathogens in individuals with
impaired immunity by taking advantage of the host’s
debilitated conditions, they are called opportunistic fungi.
 In recent times, there is an increasing list of exotic and
rare fungi, which have been associated to cause
opportunistic infections.
 But most opportunistic infections are caused by Candida
albicans, Aspergillus spp., Penicillium marneffei, and
various Zygomycetes

Candidiasis
 Candida species are the most common fungal
pathogens that affect humans.
 These species are true opportunistic pathogens that
take advantage of the host’s debilitated condition and
gain access to the circulation and deep tissues.
 The genus Candida includes more than 100 species,
of which only few cause disease in humans.
 C. albicans and occasionally other species cause
candidiasis, a major infection in immunocompromised
hosts.

Candida albicans
 C. albicans is the most common Candida species, which
causes
opportunistic infections in immunocompromised hosts.
 It forms the part of the normal flora of the mucous
membrane of the gastrointestinal, genitourinary, and
respiratory tract.
Properties:
■ C. albicans is ovoid or spherical yeast with a single bud.
■ It forms the part of the normal flora of the mucous
membrane of the gastrointestinal, genitourinary, and
respiratory tract.
 It produces pseudohyphae in the cultures and in tissues.
 Pseudohyphae are elongated yeast that may resemble
hyphae morphologically, but are really not true hyphae.

Cont..
 Candida grows readily on Sabouraud’s dextrose
agar and on bacteriological culture media.
 C. albicans produces creamy white, smooth
colonies with a yeasty odor (Color Photo 64).
■ It can be differentiated from other Candida
species by carbohydrate fermentation reaction and
by characteristic growth properties.
■ Only C. albicans produces chlamydospores on
cornmeal agar culture at 25°C.

Causative Fungal Agents
A. Yeast like fungi (Candida spp., Torulopsis, Cryptococcus),
B. Filamentous fungi (Aspergillus, Mucor, Absidia, Rhizopus,
Cephalosporium, Fusarium, Penicillium, Geotrichum,
Scoulariopsis)
C. Others (Pneumocystis carinii).
Laboratory Tests:
Diagnosis can be established by microscopy and culture.
A. Specimens: Specimens include swabs and scrapings
from superficial lesions, blood, spinal fluid, tissue biopsies,
urine, exudates, and material from removed intravenous
catheters.

B. Direct microscopy:
 Tissue biopsies, centrifuged spinal fluid, and other
specimens may be examined in Gram stained smears
for pseudohyphae and budding cells.
 Wet films or gram-stained smears from lesions or
exudates show budding gram-positive cells.
 As Candida can be seen on normal skin or mucosa
as well, only its abundant presence is of significance.
 Demonstration of mycelial forms indicates
colonisation and tissue invasion and is, therefore, of
greater significance.
 Skin or nail scrapings are first placed in a drop of 10
percent potassium hydroxide (KOH) and calcofluor
white.

C. Culture:
 Cultures are obtained on Sabouraud’s dextrose agar
(SDA) and on ordinary bacteriological culture media,
e.g. blood agar at room temperature or at 37°C.
 Colonies are creamy white, smooth and with a yeast
odour.
 Gram stained smear from colonies shows gram-
positive budding yeast cells (Fig. 75.3).
Fig. 75.3: Gram stained smear of
Candida albiocans

D. Identification
 The following tests are done to differentiate C.
albicans from other species.
 C. albicans is identified by the production of germ
tubes or chlamydospores.
 Other candida isolates are speciated with a battery of
biochemical reactions.
a. Germ tube test: C. albicans has ability to form germ
tubes within two hours when incubated in human serum
at 37°C (Reynolds-Braude phenomenon) (Fig. 75.4 ).
b. Chlamydospores: Chlamydospores develop in a
nutritionally deficient medium such as cornmeal agar at
20°C. They can be seen at the end of pseudohyphae
(Fig. 75.5).

c. Carbohydrate fermentation and carbohydrate
assimilation tests
Fig. 75.4: Candida albicans showing
Germ tubes Fig. 75.5: Formation of
Chlamydospores by Candida
albicans when cultured on
cornmeal agar at 25°C

Cont..
 These are used in identification of C. albicans and
other species of candida.
 Species of candida can be identified depending on
fermentation and assimilation of various
carbohydrates.
 Sugar fermentation and assimilation tests can be
used ro confirm the identification and speciate the
more common candida isolates, such as C tropiealis,
C parapsilosis, C guillier mondii, C kefyr, C krusei,
and C lusitaniae.
 C glabrata is unique among these pathogens
because it produces only yeast cells and no
pseudohyphal forms.

E. Serology:
 Agglutinins appear in the sera of patients but as they
are frequent in normal persons also, they are not
helpful in diagnosis.
 The detection of circulating cell wall mannan, using a
latex agglutination test or an enzyme immunoassay, is
much more specific, but the test lacks sensitivity.
F. Skin Test:
 Delayed hypersensitivity to Candida is so universal
that skin testing with Candida extracts is used as an
indicator of the functional integrity of cell mediated
immunity.

G. Molecular Diagnosis
 DNA probe and polymerase chain reaction (PCR) are
still under evaluation, but appear to be promising.
Treatment: Antifungal therapy forms the mainstay of
treatment of the infections caused by Candida.
 These agents include azoles ( fluconazole, triazole,
ketoconazole), nystatin, and amphotericin B.
 C. glabrata is becoming increasingly important
worldwide
and is intrinsically less susceptible to amphotericin B
and
other azoles (ketoconazole, fluconazole, etc).

Cont..
 Candida krusei is increasingly recognized
because of its resistance to many antifungal
agents.
 It is intrinsically resistant to ketoconazole and
fluconazole.
 It is also less susceptible to all other antifungal
agents including itraconazole and amphotericin B.
 C. lusitaniae is also of clinical significance
because it is resistant to amphotericin B, but it is
susceptible to azoles and echinocandins.

Prevention and Control
 Antifungal prophylaxis is indicated for patients
with invasive
candidiasis who are at high risk of developing
invasive candidiasis.
 There is no vaccine available against candidiasis.

Cryptococcosis
 Cryptococcosis (torulosis, European
blastomycosis, Busse-Buschke disease) is
subacute or chronic infection caused by the
capsulate yeast Cryptococcus neoformans.
 It is most frequently recognized as a disease of
the central nervous system (CNS), although the
primary site of infection is the lungs.
 The disease occurs sporadically throughout the
world but it is now seen most often in patients
with AIDS.

Morphology
 In culture, C. neoformans produces a whitish
mucoid colony in 2-3 days.
 Microscopically, in culture or clinical material, C.
neoformans is a spherical budding yeast (5-10
μm in diameter), surrounded by a thick
polysaccharide capsule (Fig. 75.6).
Fig. 75.6: Cryptococcus neoformans:
India ink preparation of spinal fluid
showing yeast cells surrounded by a
large capsule

A. Specimens:
 Specimens include spinal fluid, tissue, exudates,
sputum, blood, and urine.
 Diagnosis is established by demonstration of capsule,
budding yeast cells in the lesions and by culture.
B. Microscopic Examination—India ink or nigrosine
preparation:
 Specimens are examined in wet mounts, both directly
and after mixing with India ink, which delineates the
capsule. In unstained, wet preparations of CSF mixed
with a drop of India ink or nigrosine, the capsule can
be seen as a clear halo around the yeast cells (Fig.
75.6).

Cont..
 In unstained, wet preparations of CSF mixed with a
drop of India ink or nigrosine, the capsule can be
seen as a clear halo around the yeast cells (Fig.
75.6).
 The yeast cells of C. neoformans are round, 5-10 μm
in diameter, and are surrounded by a
mucopolysaccharide capsule.
 Tissue sections: For examination of tissue sections it
is best to use a specific fungal stain such as PAS.
Alciari blue and mucicarmine stain the capsular
material, enabling the organisms to be differentiated
from H. capsulatum and B. dermatitidis.

C. Culture:
 On Sabouraud agar (without cycloheximide) cultured
at 25-30°C and 37°C, colonies normally appear within
2-3 days.
 Media with cycloheximide inhibit C. neoformans and
should be avoided.In culture, C. neoformans appears
as smooth, mucoid, cream coloured colonies.
 Cultures can be identified by growth at 37°C and
detection of urease.
 Alternatively, on an appropriate. diphenolic sub
strate, the phenol oxidase (or laccase) of C.
neoformans produces melanin in the cell walls and
colonies develop a brown pigment.

Cont..
 Niger seed (bird seed) agar is a differential
medium for presumptive identification of C.
neoformans.
 It produces brown colonies on this medium within
one week when incubated at 30°C.
 C. neoformans produces phenoloxidase, which
oxidizes the caffeic acid in the niger seed into
melanin.

D. Serological tests:
 Cryptococcal capsular polysaccharide antigen
can be detected in CSF and blood by latex
agglutination and ELISA test.
 A whole-cell agglutination test for serum antibody
is positive in less than 50 percent of proven cases
of cyptococcal meningitis.

E. Animal inoculation test:
 Intracerebral or intraperitoneal inoculation into
mice leads to a fatal infection in case of C.
neoformans.
 Capsulated budding yeast cells can be
demonstrated in the brain of the infected mice.

Differentiation of pathogenic (C. neoformans) from other
non-pathogenic cryptococci:
Pathogenic C. neoformans can be differentiated
from nonpathogenic species by its ability to:
1. Grow at 37°C;
2. Hydrolyze urea;
3. Produce phenol oxidase—produce black
colonies on niger seed agar, bird seed agar and
caffeic acid agar, and;
4. Produce disease in mice on intracerebral and
intraperitoneal inoculation (animal inoculation test
positive). Capsulated budding yeast cells can be
demonstrated in the brain of infected mice.

Treatment
 Combination therapy of amphotericin B and
flucyto sine has been considered the standard
treatment for cryptococcal meningitis, though the
benefit from adding flucytosine remains
controversial.
 Fluconazole offers excellent penetration of the
central nervous system.

Aspergillosis:
 A broad spectrum of diseases in humans ranging
from direct
invasion to hypersensitive reactions are caused
by Aspergillus
species.
 Although more than 100 species have been
described, the majority of human diseases are
caused by Aspergillus fumigatus
and Aspergillus niger, and less frequently by
Aspergillus flavus
and Aspergillus clavatus.

A. Specimens:
 Sputum, other respiratory specimens, or lung
biopsy tissue provide good specimens.
 Blood samples are rarely positive.
 On direct examination of sputum with KOH or
calcofluor white or in histologic sections, the
fungus appears as non-pigmented septate
mycelium, 3-5 μm in diameter, with characteristic
dichotomous branching and an irregular outline.
 Rarely the characteristic sporing heads of
Aspergillus species are present.

Cont..
 In tissue sections, Aspergillus species are best seen
after staining with PAS or methenamine-silver.
C. Culture:
 Aspergillus species grow readily on Sabouraud agar
without cycloheximide at 25-37°C. Colonies appear
after 1-2 days.
 Species are identified according to the morphology of
their conidial structures.
 Asexual conidia are arranged in chains, carried on
elongated cells called ‘sterigmata’, borne on the
expanded ends (vesicles) of conidiophores (Fig.
75.7).

Cont..
 As aspergilli are such common contaminants, their
demonstration in exudates and isolation in cultures
have to be interpreted with care.
D. Skin tests:
 Skin tests with A. fumigatus antigen are useful for the
diagnosis of allergic aspergillosis.
E. Serological tests:
 Immunodiffusion, counterimmunoelectrophoresis
(CIE) and ELISA are widely used for the detection of
antibodies in the diagnosis of all forms of
aspergillosis, particularly aspergilloma and allergic
bronchopulmonary aspergillosis.

Cont..
 For diagnosis of invasive aspergillosis, antigen
detection has also been used successfully by
techniques such as ELISA and latex
agglutination.
F. Polymerase chain reaction(PCR):
 This is now increasingly used for diagnosis of
invasive aspergillosis but its precise value is still
being assessed.

Zygomycosis
 Zygomycosis, also known as mucormycosis or
phycomycosis,
is an infection caused by saprophytic molds, such as
Mucor, Rhizopus, and Absidia.
 These fungi are ubiquitous in the environment and
generally saprophytic.
 They rarely cause disease in immunocompetent
hosts, but they are the third most frequent cause of
invasive fungal infection in immunocompromised
patients.
 The fungal agents of zygomycosis have a high degree
of predilection to invade major blood vessels, leading
to ischemia, necrosis, and infarction of adjacent
tissues.

Cont..
 Fever, unilateral facial pain or headache, nasal
congestion, epistaxis, visual disturbance, and
lethargy are the common manifestations.

1. Specimens:
 Recognition of the fungus in tissue by microscopy is
considerably more reliable than culture, but material
such as nasal discharge or sputum seldom contains
much fungal material and examination of a biopsy is
usually necessary for a firm diagnosis.
2. Microscopy:
 Direct examination of curetted or biopsy material in
potassium hydroxide (KOH) may reveal the
characteristic broad, aseptate, branched mycelium
and sometimes distorted hyphae.

Cont..
 However, they are seen much more clearly when
stained with methenamine-silver.
 The hyphae of these fungi do not stain with PAS.
Biopsy is normally the best method of establishing the
diagnosis and should be performed early in the
course of the infection.
3. Culture:
 The fungi are readily isolated on Sabouraud agar
without cycloheximide at 37°C, producing abundant
cottony colonies.
 Isolation is of little diagnostic significance in the
absence of strong supporting clinical evidence of
infection.

4. Identification
 Identification is based on the sporangial
structures (Figs 75.8A to C).
i. Mucor: Shows nonseptate mycelium without
rhizoids (root like structures). Sporangiophores,
which may be branched, terminate in large globose
sporangia containing numerous spores.
ii. Rhizopus: shows nonseptate mycelium with
rhizoids. Unbranched sporangiophores arise in
groups directly above the rhizoids.

Cont..
iii. Absidia: has also rhizoids but sporangiophores
arise from the aerial mycelium inbetween the
rhizoids .
Figs 75.8A to C:
Zygomycetes: (A) Mucor (B)
Rhizopus (C) Absidia

Treatment
 Amphotericin B is the drug of choice for
mucormycosis.
 Surgical removal of necrotic, infected tissues is
frequently useful.
 Many patients survive, but there may be residual
effects such as partial facial paralysis or loss of
an eye.

Pneumocystosis
 Pneumocystis jiroveci, previously known as
Pneumocystis carinii,
is the causative agent of P. carinii pneumonia
(PCP).
 PCP is the most common opportunistic infection
in HIV-infected patients.

Pneumocystis jiroveci
 Pneumocystis is a unicellular fungus found in the
respiratory
tracts of many mammals and humans.
 The genus Pneumocystis was initially mistaken for
trypanosome, then later as a protozoan.
 Biochemical analysis of the nucleic acid composition
of Pneumocystis rRNA and mitochondrial DNA in
1980s established it as a fungus.
 The cyst wall closely resembles that of fungi.
 However, it does not have ergosterol in its membrane
as
do the fungi, but instead has cholesterol.

Clinical Syndromes
 P. jiroveci causes PCP in HIV patients with their
CD4 cells count below 200/L.
 It also causes PCP in other patients with primary
immune deficiencies including
hypogammaglobulinemia and severe combined
immunodeficiency, in organ (e.g., heart, lung, liver
kidney)-transplant recipients’ long-term
immunosuppressive regimens, and in patients
with hematologic and nonhematologic
malignancies.
 Most cases of PCP are asymptomatic.

Cont..
 In symptomatic cases, sudden onset of fever,
nonproductive
cough, dyspnea, and tachypnea are typical
manifestations.
 Bilateral rales and ronchi are present.
 Extrapulmonary manifestations are rare.
 It occurs in AIDS patients during their advanced
stage.

1. Specimens:
 To establish the diagnosis of P. carinii pneumonia,
specimens of bronchoalveolar lavage, lung
biopsy, or induced sputum are stained and
examined for the presence of cysts or
trophozoites.
2. Staining:
 Appropriate stains include Giemsa, toluidine blue,
methenamine silver, and calcofluor white.
 A specific monoclonal antibody is available for
direct fluorescent examination of specimens.

Cont….
 Cyst wall stains black with methenamine silver
staining.
 With the Giemsa stain, the organism appears
round, and the cyst wall is barely visible.
 Intracystic bodies are seen around the interior of
the organism.
 Fluorescent monoclonal antibody staining shows
‘honeycomb’ appearance of the cyst. P. carinii
cannot be cultured.

Cont..
3. Serology:
 While not clinically useful, serology has been
used to establish the prevalence of infection.
 Serological tests can be used for diagnosis in
suspected cases.
 Complement fixation titers of 1:4 or more is
indicative of active disease.
 Latex agglutination test is also used.
4. Polymerase chain reaction (PCR):
 PCR for amplification of P. carinii DNA is a rapid
method for detection of early infection.

Treatment:
 P. jiroveci, although considered a fungus, does not
respond
to treatment with antifungal agents.
 A combination of trimethoprim and sulfamethoxazole
is the drug of choice for treatment of PCP.
 Pentamidine and atovaquone are alternative drugs.
 Acute cases of pneumocystis pneumonia are treated
with trimethoprim-sulfamethoxazole (TMP-SMZ) or
pentamidine isethionate. Prophylaxis can be achieved
with daily TMP-SMZ or aerosolized pentamidine.

Prevention and Control
 Chemoprophylaxis with trimethoprim and
sulfamethoxazole or aerosolized pentamidine is
useful for prevention of infection in patients with
CD4 counts below 200/L.

Penicilliosis
 Penicillium species rarely cause opportunistic
infections in
humans.
 The Penicillium species are identified by their typical
morphology (Fig. 74-6), culture characteristics on the
SDA
medium (Color Photo 69), and microscopy (Color
Photo 70).
 P. marneffei is the only dimorphic fungus in the genus
Penicillium known to cause opportunistic infection.
 There are more than 150 known species of the genus
Penicillium.

Cont..
 Fig. 75.9: Penicillium

Cont..
 P. marneffei causes disseminated infection in
immunocompromised hosts, such as patients with
HIV.
 It develops a necrotizing reaction and involves the
skin, lung, and intestine.
 It causes disseminated infections of
reticuloendothelial system, and also allergic diseases
and mycotoxicoses.
 The disseminated infection is more common in adults
than in children.
 P. marneffei is widely distributed in the nature.
 Its natural habitat is soil.

Cont..
 The fungus infects no mammals other than
humans and bamboo rat.
 Fungus is found in feces of rats and also in
burrows made by the rats in the soil.
 The infection is transmitted by inhalation of
conidia, direct inoculation of the skin, and rarely,
by ingestion of infected rats.
 The condition is more common in rainy season,
in rural areas.

1.Microscopy:
 Demonstration of P. marneffei in the skin and
mucosal
scrapings, sputum, stool, blood, urine, lymph
node, bone
marrow, lung and liver biopsy specimens.
2.Staining:
 Staining of skin, lymph node, and bone marrow
aspirate by hematoxylin and eosin, PAS (periodic
acid-Schiff), Wright’s, and calcofluor white stain
demonstrates yeast cells with transverse septa.

Cont..
3.Histological test:
 Immunohistochemical assay using monoclonal
antibody
against an external wall epitope is used to
identify P. marneffei
in tissues.
 Demonstration of fungal antigen in affected
tissue by using direct immunofluorescence
antibody also helps in diagnosis of the condition.
 Peripheral blood smear shows P. marneffei in
patients with AIDS.

Cont..
4.Culture:
 Culture is the gold standard. P. marneffei is a highly
infectious fungus, hence culture should be done in a
laboratory with a biohazard safety level-2 precautions.
 The fungus on SDA without cycloheximide and at 25°C
produces mycelial grayish white colonies, green center,
white periphery, and bright rose pigmented reverse (Color
Photo 71).
 Microscopy of the colony shows septate hyaline hyphae,
branched conidiophores, and three to five medullae which
produce phialides that bear conidia in chains.
 The fungus on SDA at 37°C produces yeast-like colonies
and cream-colored mucoid with brown red pigment.
 Microscopy of the colony shows pleomorphic ellipsoidal to
rectangular yeast cells with transverse septum

COLOR PHOTO 71. SDA
medium showing colonies of
Penicillium marneffei.
COLOR PHOTO 70. LPCB wet
mount showing Penicillium
(×100).

Cont…
5.Serological test:
 Indirect immunofluorescent antibody test,
immunoblot
assay, and immunodiffusion method using
mycelial phase
culture antigen are used to demonstrate serum
IgG antibodies.
 Immunodiffusion and latex agglutination tests are
used to detect P. marneffei antigen in serum and
urine.

Laboratory diagnosis of fungal diseases

Laboratory diagnosis of fungal diseases

Recomendados

Recomendados

Más contenido relacionado

La actualidad más candente

La actualidad más candente (20)

Similar a Laboratory diagnosis of fungal diseases

Similar a Laboratory diagnosis of fungal diseases (20)

Más de Central Department Of Microbiology, TU

Más de Central Department Of Microbiology, TU (20)

Último

Último (20)

Laboratory diagnosis of fungal diseases