caused by eating amanita mushroom is the best example of mycotoxicosis. This group of fungi
produces five toxins. Of these, amanitine and phalloidin are the two most potent hepatotoxins.The
toxicity of amanitine is due to its ability to inhibit cellular RNA polymerase, which prevents mRNA
synthesis. Aflatoxin is another fungal toxin produced by Aspergillus flavus that causes disease in
humans. Aflatoxin-B causes a mutation in the P53 tumor-suppressor gene, resulting in a loss of P53
liver, and it induces tumor in liver in animals and is associated with hepatic carcinomas in humans.
Laboratory diagnosis of fungal infections depends on: (a) direct microscopy, (b) culture, (c)
serological tests, (d ) nonculture methods, and (e) molecular methods.
Section IV - Medical Mycology By Dr. Kareem Lilo
Direct microscopic examination depends on demonstration of characteristic asexual spores, hyphae,
specimens by light microscopy. The commonly used clinical specimens are sputum, lung biopsy
The specimen is either treated with 10% KOH or stained
with special fungal stains. Use of 10% KOH dissolves tissue
is a useful method for demonstration of white capsule of C. neoformans in CSF. Gram staining is also
useful to demonstrate Gram-positive Candida species in the specimen.
The disadvantages of microscopy are that it shows low sensitivity and requires an experienced
microscopist for specific identification.
Fungal culture is a frequently used method for confirming
the diagnosis of fungal infection. SDA is the most commonly
used medium for fungal culture. Other media include CHROM
agar, blood agar, etc. The low pH of the medium and addition
of chloramphenicol and cycloheximide to the medium inhibit the growth of bacteria in the specimen
and thereby facilitate the appearance of slow-growing fungi. Fungal colony is identified by rapidity
of growth, color, and morphology of the colony at the obverse and pigmentation at the reverse.
Microscopy of the fungal colony is carried out in lactophenol cotton blue (LPCB) mount to study the
morphology of hyphae, spores, and other structures. The appearance of the mycelium and the nature
of the asexual spores are very much helpful to identify the fungus.
Recently, newer tests like ELISA (enzyme-linked immunosorbent assay), Western blot, and
radioimmunoassays are increasingly used for serodiagnosis of fungal infections.
(c) detection of fungal metabolites.
Section IV - Medical Mycology By Dr. Kareem Lilo
Antigen detection: It is useful in immunocompromised hosts where antibody detection is not as
many fungal infections. Demonstration of antigen indicates recent or active infection. Latex
agglutination test is a frequently used test to demonstrate polysaccharide capsular antigen of C.
neoformans in CSF for diagnosis of cryptococcal meningitis. False-positive reactions due to
Trichosporon beigelli and Capnocytophaga canimorsus are known.
Detection of fungal cell wall markers: Mannan is a highly immunogenic component of the candidal
cell wall. Mannan antigen detection, therefore, is most widely used method in the diagnosis of
candidiasis. Galactomannan is a heat-stable heteropolysaccharide found in the cell walls of all
Aspergillus species. Production of the
galactomannan antigen is proportional to fungal load in tissue,
hence is being used as the prognostic marker for diagnosis
of invasive aspergillosis. A sandwich ELISA using rat monoclonal
antibody EB-A2 against galactomannan antigen is being
currently used in Europe for diagnosis of invasive aspergillosis.
Most pathogenic fungi have 1, 3-beta-D-glucan in their cell walls and minute quantities are secreted
invasive fungal infections. Detection of 1, 3-beta-D-glucan is based on its ability to activate a
coagulation cascade within amebocytes derived from the hemolymph of horseshoe crabs. This uses a
detect certain species, such as C. neoformans and Zygomycetes.
Detection of fungal metabolites: Detection of distinctive fungal metabolites is another approach for
diagnosis of C. albicans infections.
A few drugs are available for therapy of systemic fungal infection, unlike a large number of
They act on the ergosterol of fungal cell membrane that is not found in bacterial or human cell
membrane. Similarly, caspofungin inhibits synthesis of beta-glucan, which is found only in fungal
membrane but not in bacterial or human cell membrane. Table 4-2 summarizes the common
antifungal agents and their primary sites of activity.
Table ( 4-2 )Antifungal agents and primary sites of activity
Section IV - Medical Mycology By Dr. Kareem Lilo
Superficial ,Cutaneous, and Subcutaneous Mycoses
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 caused by different fungi is restricted to the outer most layers of the skin and
hair. The condition usually causes cosmetic problem, which can be easily diagnosed and treated. It
includes four important conditions:
(a) pityriasis versicolor, (b) tinea nigra, (c) black piedra, and
Section IV - Medical Mycology By Dr. Kareem Lilo
Pityriasis versicolor or tinea versicolor is a superficial infection
of the skin caused by Malassezia furfur (Pityrosporum orbiculare).
M. furfur requires fatty acids for growth, hence is cultured on
the Sabouraud’s dextrose agar (SDA) overlayed with a layer of
olive oil. On incubation at 37°C, the fungus produces creamy
colonies within 5–7 days. The fungus is found inparts of the body rich in sebaceous glands. The
lesions of pityriasis versicolor are found most commonly on the upper tissue, arms, and abdomen.
hyphae in KOH preparation of skin scrapings. Characteristic “spaghetti and meatballs” appearance of
fungus is demonstrated in the microscopy of KOH preparation of the skin. Culture is not carried out
routinely for diagnosis. Topical miconazole is treatment of choice.
Tinea nigra is an infection of keratinized layer of skin caused
by Exophiala werneckii or Cladosporium werneckii. C. werneckii is a
dimorphic fungus that produces melanin. The fungus on the SDA grows as yeast with many cells in
various stages of cell division
producing typical two-celled oval structure, on primary isolation from clinical specimen. On
prolonged incubation, elongated hyphae develop and in older cultures, mycelia and conidia are
predominantly found. A well-demarcated brown-black macular lesion, which
appears as brownish spot of the skin, is typical manifestation
of the condition. These brownish to black lesions are most
commonly seen on palms and soles.
Laboratory diagnosis of tinea nigra is made by microscopy of the KOH preparation of skin scrapings
collected from the affected part. Typical darkly pigmented yeast-like cells and hyper fragmented
hyphae are demonstrated. Culture of the skin scraping on the SDA confirms the diagnosis.
Black piedra is a superficial infection of the hair caused by Piedraia
hortae, a dematiaceous fungus. . Culture of specimens
on SDA shows slow-growing brown to reddish black mycelium,
which is considered asexual or anamorphic stage of the fungus.
Section IV - Medical Mycology By Dr. Kareem Lilo
diagnosis of the condition is made by demonstration of nodules containing asci with spindle-shaped
ascospores in 10% KOH mount of the hair.
White piedra is an infection of the hair caused by yeast-like organism Trichosporon beigelli. The
of the colony shows septate hyphae that break rapidly to form arthroconidia. The latter subsequently
become round and develop to Blastoconidia The development of a soft, pasty,
cream-colored growth along infected hair shaft characterizes the condition. The initial growth of T.
epidermis of hair. The infected hair shaft consists of mycelium
that rapidly fragments to arthroconidia.
Laboratory diagnosis of the condition is made by demonstration of fragmented hyphae that develop
into arthroconidia or produce blastoconidia in 10% KOH mount of hair. Culture of the fungus from
clinical specimen confirms the diagnosis.
Features of the organisms causing superficial mycoses are
Table (4-3) Features of the organisms causing superficial 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.
Section IV - Medical Mycology By Dr. Kareem Lilo
The most important dermatophytes that cause infection in
humans are classified into the following three genera:
(i) Trichophyton—causes infection of hair, skin, and nail.
(ii) Microsporum—causes infection of hair and skin.
(iii) Epidermophyton—causes infection of skin and nails, but
The dermatophytes on the basis of their natural habitat and host preferences can be classified into
(i) Anthropophilic species: These dermatophytes are typically adapted to live on human host. They
are transmitted from human to human through fallen hairs, desquamated epithelium, combs, hair
brushes, towels, etc. Examples are Trichophyton rubrum, icrosporum audouinii, and Epidermophyton
their animal host. These zoophilic species are transmitted from infected animals to humans by direct
and indirect contacts with domestic animals (e.g., cat and dog) and occasionally wild animals.
Examples are Trichophyton violaceum and Microsporum canis.
(iii) Geophilic species: These are saprophytic fungi found in
soil or in dead organic substances. They occasionally cause infection in humans and animals.
Examples are Microsporum gypseum and Trichophyton ajelloi. Dermatophytes usually grow only on
fungus antigen may cause secondary
reaction occurs as a result of hypersensitivity response to circulating fungal antigen, and these
lesions do not contain any fungal hyphae.
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
for diagnosis, while culture is always carried out to identify the specific causative fungal agent.
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. 4-1):
hair shaft . It is caused by M. audouinii, M. canis, and Trichophyton mentagrophytes.
Section IV - Medical Mycology By Dr. Kareem Lilo
Endothrix: The clusters of arthrospores are found entirely
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.
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. shows the
classification of subcutaneous mycoses.
Mycetoma is a slowly progressive, chronic granulomatous infection of skin and subcutaneous tissues
with occasional involvement of underlying fascia and bone usually affecting
Figure (4-1) Endothrix and Ectothrix infection
Section IV - Medical Mycology By Dr. Kareem Lilo
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.
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.The condition is characterized by formation of painless, localized, swollen lesions on the
affected limbs.Multiple discharging sinuses are present.
Systemic mycoses are caused by fungi of soil, which are inherently virulent and cause disease in
healthy humans. The systemic ycoses include coccidioidomycosis, paracoccidioidomycosis,
histoplasmosis, blastomycosis, and cryptococcosis.
soil and as yeast at body temperature in mammals. On SDA medium at 37°C, this fungus produces
cottony mycelial growth. The colony is characterized by thin, branching, septate hyphae that produce
tuberculate macroconidia and microconidia.
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.
On culture at 37°C and in tissue, the yeast is a round structure
from the Cryptococcus neoformans yeast, which has a narrow-based bud. On culture at 25°C, the
fungus produces a mycelial growth showing typical pyriform microconidia, which measure 2–4 _m
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 Of the 19
species that comprise the genus Cryptococcus, human disease is associated with only C. neoformans.
Section IV - Medical Mycology By Dr. Kareem Lilo
C. neoformans is a true yeast.
It is an oval and budding cyst and measures 3–6 _m in diameter. The yeast may be single or
may have a single budding daughter cell.
Within the host and in certain culture media, the yeast is urrounded by a wide
polysaccharide capsule. The polysaccharide capsule is composed of mannose, xylose, and
C. neoformans on SDA medium forms smooth, convex, cream-colored colonies at 20–37°C.
Lactophenol cotton blue (LPCB) wet mount of the colony shows budding yeast cells.
C. neoformans has two varieties: C. neoformans var neoformans and C. neoformans var gattii. Based on
antigenic specificity of the capsular polysaccharide, the species has been classified into four
serotypes. These are serotypes A and D (C. neoformans var neoformans) and serotypes B and C (C.
The immune status of the host is the crucial factor in pathogenesis
of cryptococcosis. C. neoformans usually causes most serious infections in patients with
patients undergoing corticosteroid treatment,
patients undergoing organ transplantation,
patients with reticuloendothelial malignancy, and
into the pulmonary alveoli, in which they survive before they are phagocytosed by alveolar
macrophages. Glucosylceramide synthase has been identified as an essential factor in the survival of
C. neoformans in pulmonary alveoli.
Cryptococcal polysaccharide capsule has antiphagocytic properties. Hence, unencapsulated yeast are
readily phagocytosed and destroyed than the encapsulated organisms, which are more resistant to
phagocytosis. The antiphagocytic properties of the capsule prevent recognition of the yeast by
phagocytes and inhibit leukocyte migration into the area of fungal replication.
The host immunity in cryptococcal infection is mediated by both cellular and humoral responses.
the tissues indicates a successful host response against the fungus. Humoral immunity is mediated
Section IV - Medical Mycology By Dr. Kareem Lilo
and the complement play a crucial
role in facilitating the macrophage- and lymphocyte-mediated immune response to the organism.
C.neoformans causes (a) pulmonary cryptococcosis in immunocompetent hosts and in
immunocompromised hosts, (b) CNS
cryptococcosis, and (c) disseminated nonpulmonary non-CNS cryptococcosis.
The clinical manifestations of pulmonary cryptococcosis are widely variable. Pulmonary disease
ffecting immunocompromised hosts. It depends on the immune status of the host
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