15 Causative Agents Mycoses
15 Causative Agents Mycoses
15 Causative Agents Mycoses
Zaporizhzhya
2015
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УДК 579.28 : 61:616.992-092(075.8)=111
ББК 62.64 я 73
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AUTHORS:
Yeryomina A.K., senior lecturer of the chair of microbiology, virology and
immunology, candidate of Biological Sciences.
Kamyshny A.M., the heat of the chairof microbiology, virology, and immunology,
doctor of medicine.
Voitovich A.V., assistant of the chair of microbiology, virology and immunology,
candidate of Biological Sciences.
Sukhomlinova I. E., senior lecturer of the chair of normal physiology, candidate of
Medicine.
Kirsanova E.V., assistant professor of the chair gygien and ecology, candidate of
Medicine.
The independent practical work of students is an important part of the syllabus in the
course of microbiology, virology, immunology. It helps students to study this fundamental
subject.
The systematic independent work enables to reach the final goal in the students’
education. It is also important while preparing the students for their future clinic work with
patients. These theoretical material, questions and tests help students to get ready for
examination.
The methodical manual for practical lessons on microbiology, virology, immunology
for the medical students of 2 -3 year of the study are approved by the Central Methods Board
of ZSMU as a methodical manual on practical lessons for students of the medical faculty.
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CONTENS
Opportunistic mycoses…………………………………………………………………………….31
Antifungal antibiotics……………………………………………………………………………...44
Quizzes……………………………………………………………………………………………..52
Correct answers……………………………………………………………………………………65
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FUNGI. FUNGAL INFECTIONS
They have rigid chetinous cell walls but lack differentiation of root, stem
and leaves. Morphologically the fungi may be either simple oval cells or long
tubular septate hyphae showing true lateral branching.
The yeasts and fungi need organic compounds as nutrients. Their role in
nature appears to be as scavanger i.e., breaki ng down the complex carbohydrates
and proteins of dead bodies of other organisms. Needless to mention that only a
few of them are pathogenic. In many ways, fungi have been of service to man, as
in the making of bread, fermented drinks, cheese, antibiotics etc.
Fungi can be divided into four groups each of which have some human
pathogenic species.
The part of the mycelium which grow on and penetrates into the substrate,
absorbing nutrients for growth is called vegetative mycelium.
On artificial medium they are seen as filamentous mould colony which may
be dry and powdery. The pathogenic members are trichophyton,
microsporurn and epidermophyton.
2. Yeasts: They are unicellular occurring as spherical or ellipsoidal cells.
They reproduce by budding. On solid media they form moist, Hum.
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The part of mycelium which protrudes into the air is called aerial compact,
creamy, mucoid colonies resembling to those of staphylococci. Cryptococcus
neoforman is the only important pathogens.
3. Yeast like fungi: They grow partly as yeasts and partly as long filamentous
cells joined end to end forming a pseudomycelium. On solid medium moist
creamy colored colonies are produced. Candida albicans is the example.
4. Dimorphic fungi: They grow in mycelial form at low temperature 22 °C and
in soil whereas growth at 37°C and in animal body occurs in yeast form. The
pathogenic members are Histoplasma capsulatum, sporotrichum, blastomyces
and Coccidioides immitis.
SYSTEMIC CLASSIFICATION
Based on sexual spore formation fungi are kept in 4 classes as described below:
Phycomycetes: They are fungi having non-septate hyphae. They form endogenous
asexual spore (sporangio spore) contained within sac like structure called
sporangia.
Sexual spores are also found & are of two varieties oospore and zygospore.
1. Ascomycetes: They form sexual spores (ascospores) within a sac. This sac is
called ascus. They include both yeasts and filamentous fungi. They form septate
hyphae.
2. Basidiomycetes: They reproduce by means of sexual reproduction.
Basidiospores are borne at the tip of bisidium. These basidia are sometime quite
large leaf like structure as in mushroom. They form septate hyphae.
3. Fungi imperfecti: They consist of group of fungi whose sexual phases
have not been identified. Fungi of medical importance belong to this group
e.g. Sporothrix schenkii.
polysaccharides.
2. The cytoplasmic membrane contains sterols.
3. The cytoplasm contains true nuclei with nuclear membrane, mitochondria and
endoplasmic reticulum.
4. Fungi may be unicellular or multicellular.
5. They divide asexually, sexually or by both process.
Taxonomical Classification
Fungi are placed in the phylum Thallophyta. There are four classes of fmgi. Their
characteristics are described in flow chart' shown below.
(Irregular plant masses lacking definite root, stem and leaf structures)
4 Classes
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Fig.1. Vegetative forms of fungi
1. Yeasts
(i) Round to oval unicellular fungi.
(ii) Reproduce by budding.
(iii) Form creamy mucoid colonies on culture media.
(iv) The important pathogenic yeast is Cryptococcus neoformans.
These yeasts grow partly as yeasts and partly as chains of elongated budding cells
joined end to end forming pseudohypha. Example is С. albicans.
3. Moulds
(i) They grow as branching filaments called hyphae. Hyphae may be septate or
non-septate.
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(ii) The hyphae continue to grow and branch to form tangled mass of growth
known as mycelium.
4. Dimorphic F ungi
(i) Dimorphic fungi exist as yeasts in the host tissue and in the cultures at 37°C and
as hyphal (mycelial) forms in the soil and in the cultures at 22-25°C.
(ii) Blastomyces dermatitidis, Para-coccidioides brasiliensis, Coccidioides
immitis, Histoplasma capsulatum and Sporothrix schenekii are examples of
dimorphic fungi.
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Fig.4. Aerial spor es
V. LABORATORY DIAGNOSIS
Dir ect Micr oscopy
1. Potassium Hydroxide (KOH) Preparation
Specimen is placed in a drop of 10% KOH on a microscopic slide and covered
with a coverslip. It is heated gently and examined under microscope. Yeasts cells and
hyphae may be observed (Fig. 5).
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Hyphal diameter, presence or absence of septa and of special structures help in
diagnosis. Special hyphal structures frequently found are spring like helical coils
(spiral hyphae), resembling tennis racquets (racquet hyphae) and numerous short
branches appearing at the ends of hyphae (favic chandelier) (Fig. 6).
3. Gram Staining
It is done to observe Gram positive yeasts as in case of Candida species.
Culture
1. Sabouraud's dextrose agar (SDA) and SDA medium with antibiotics are
inoculated and incubated at 25°C and 37°C for three weeks.
Chloramphenicol is added in the culture medium to suppress the growth of
contaminating bacteria while cycloheximide (actidione) is incorporated to
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suppress the contaminating fungi.
Brain heart infusion (BHI) agar with blood and antibiotics is another medium
used for primary isolation of fungi.
2. Microscopy is performed from fungal colony (in teased mounts or slide
cultures) to study the morphology of hyphae, spores and other structures. Teased
mounts are prepared in lactophenol cotton blue (LCB) which contains lactic
acid, phenol and cotton blue.
3. Slide culture is done for studying the exact morphology of the fungus.
Tissue Sections
Fungal elements in tissue can be identified by methenamine silver stain and
Periodic" Acid Schiff (PAS) stain.
A. Superficial Mycoses
1. These are strictly surface infections involving skin, hair, nail and mucosa.
B. Subcutaneous Mycoses
1. Saprophytic fungi of soil or decaying vegetation are usually introduced into
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2. subcutaneous tissue and produce a progressive local disease.
3. Subcutaneous mycoses include mycetoma, chromoblastomycosis, sporotrichosis
and rhinosporidiosis.
С. Systemic Mycoses
1. These are caused by fungi of soil and is acquired by inhalation.
Superficial mycoses
Dermatophytes
(i) Dermatophytes are a group of fungi that infect only superficial keratinised tissue
(skin, hair and nails) without involving the living tissue.
(ii) They break down and utilise keratin.
(iii) They are incapable of penetrating subcutaneous tissue.
(iv) They cause dermatophytoses, also known as tinea or ringworm.
Classification
Dermatophytes are classified into three genera as follows :
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Genus Infection of
1. Trichophyton — Hair, skin, nail
2. Microsporum — Hair, skin
3. Epidermophyton — Skin, nail
Clinical types
• In favus, there is sparse hyphal growth and formation of air spaces within
the hair shaft.
• Two types of hair infection may be present, ectothrix and endothrix. In ectothrix,
a sheath of arthospores is present on the surface of hair shaft, while the arthospore
formation occurs entirely within the hair shaft in endothrix (Fig. 7).
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Laboratory diagnosis
Specimens: Skin, hair and nail
Direct microscopy
• Direct 10% KOH mount may show fungal hyphae
Culture
* SDA and SDA with antibiotics are used.
* Culture media are incubated at 25-30°C for three weeks.
* Identification of dermatophytes is based on
— colony morphology
— pigment production
— microconidia and macroconidia
* Colony characters
— white to creamy, cottony growth
— reverse of media is red in T. rubrum.
* Microscopy
— lactophenol cotton blue preparation from colony reveals microconidia,
macroconidia or both.
The following are the characterstics of three genera :
Genus Trichophyton — More microconidia, very few macroconidia
Genus Microsporum — Predominant macroconidia
Genus Epidermophyton — Macroconidia
Treatment of dermatophytoses
Topical antifungal agents are generally used for treatment. Oral griseofulvin
is the drug of choice.
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Subcutaneous Mycoses
1. Mycetoma
(i) Mycetoma is a chronic granulomatous infection of the subcutaneous tissue,
usually affects the foot and rarely the other parts of body.
(ii) The disease was first described from Madurai, South India. It is therefore
commonly referred to as Madura foot or Maduramycosis.
(iii) It is seen mainly in the tropical countries.
Aaetiology
* Mycetoma is caused by a number of actinomycetes and filamentous fungi (Table 1).
Eumycetoma Actinomycetoma
Madurella grisea
Nocardia brasiliensis
Pathogenesis
• The causative organism is believed to enter the body through minor trauma.
• The disease begins as a subcutaneous swelling usually of foot, which enlarges and
burrows into the deeper tissues producing characteristic abscess. The abscess
bursts with the formation of chronic multiple sinuses discharging viscid,
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seropurulent fluid containing granules.
• The colour and consistency of the granules vary with the different
causative agents.
Laboratory diagnosis
• Diagnosis is made from examination of granules.
• The granules are composed of very thin filaments in actinomycotic mycetoma
while they are broader and often show septae and chlamydospores in mycotic
mycetoma.
Treatment
Actinomycotic mycetoma usually respond well to sulphonamides and antibiotics, but
mycotic mycetoma may require amputation.
2. Chromomycosis
(i) It is a chronic, localised infection of skin and subcutaneous tissue.
(ii) The fungus enters the body through a wound.
Aetiological agents
• It is caused by several darkly pigmented fungi of the family Dematiaceae.
These include:
Fonsecaea pedrosoi
Fonsecaea compactum
Cladosporium carrionii
Phialophora verrucosa
* The term chromomycosis includes chromoblastomycosis and other infections
caused by dematiaceou fungi (Phaeohyphomycosis). Chromoblasto mycosis is
usually confined to the subcutaneous tissue of the feet and lower legs.
Phaeohyphomycosis may affect cutaneous, subcutaneous, deeper tissues or organs
like brain or lung.
Laboratory diagnosis
Chromoblastomycosis
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• Histologically, in H and E staining they appear as yeast like bodies with
septae, called sclerotic bodies.
• These sclerotic bodies can be demonstrated in КОН mounts and
by culture on SDA.
Phaeohyphomycosis
• The fungi appear in lesions as distorted hyphal strands.
Sclerotic bodies are absent.
Treatment
• Amphotericin В and 5-fluorocytosine have been found useful.
3. Sporotrichosis
Causative agent
• Sporothrix schenckii — a dimorphic fungus.
Oral manifestations
Laboratory diagnosis
• Diagnosis is made by culture as the fungus may not be demonstrable in
pus or tissues.
• S. schenckii occurs in the yeast phase n the tissues and in cultures at 37°C,
and in the mycelial phase in cultures at 22°C-25°C (dimorphic fungus).
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• Yeast phase appears as cigar-shaped ells and mould form contains hyphae
carrying flower like clusters of small conidia borne on delicate sterigmata
(Fig. 8).
4. Rhinosporidiosis
(i) Rhinosporidiosis is a chronic granulomatous disease characterised by
formation of friable polyps, usually confined to the nose, mouth or eye.
(ii) Causative agent is Rhinosporidium seeberi.
(iii) More than 80% cases are reported in India and Sri Lanka.
(iv) The mode of infection is not known but most infections occur in males who
have frequent contact with stagnant water or aquatic life.
Oral manifestations
Oronasopharyngeal lesions appear as soft red polypoid growth which spread
to pharynx and larynx. These lesions often contain mucoid discharge and are
vascular.
Laboratory diagnosis
• The fungus has not been cultivated.
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• Diagnosis depends on the demonstration of sporangia.
Systemic mycoses
1. Histoplasmosis
(i) Causative fungus: Histoplasma capsulatum, a dimorphic fungus
(ii) It is primarily a disease of reticuloendothelial system
(iii) Histoplasma capsulatum is an intracellular parasite.
Source of infection
• The fungus is present in the soil enriched with excreta of birds or bats.
• Human infection results from inhalation of spores.
Clinical features
The large majority of infections are asymptomatic.
• Some individuals develop pulmonary disease which resembles tuberculosis.
• Disseminated histoplasmosis develops only in a minority of infected
individuals.
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• Involvement of reticuloendothelial system results in lymphadenopathy,
fever, hepatosplenomegaly, anaemia and a high rate of fatality.
• Granulomatous and ulcerative lesions may develop on the skin or
mucosa.
Oral manifestations
Thе nodular, ulcerative or vegetative oral lesions may be present on the
buccal lucosa, lips, gingiva, tongue to palate. The ulcerative areas are
indurated and are usually covered by a grey membrane.
Laboratory diagnosis
Specimens
* Sputum
* Bone-marrow aspirate
* Peripheral blood
* Scrapings from dermal or mucosal ulcers
* Biopsies of lymph nodes and other organs.
Direct examination
• Smears of sputum or pus are stained with Giemsa or Wright stains. On
microscopy, H. capsulatum appears as small oval yeast cell, packed within
the cytoplasm of macrophages or monocytes (Fig. 10).
Culture
• SDA or brain heart infusion (BHI) agar with cycloheximide and
chloramphenicol are inoculated.
• The yeast phase is formed in cultures at 37°C. White cottony mycelial growth
containing large thick walled, pherical spores with tubercles or finger like
projections (Fig. 11) appears at 25°C.
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Fig. 10. Histoplasma capsulatum: yeast cells in macrophage
Hyphae
Microconidies
Treatment
• Amphotericin В has been found useful in treatment of histoplasmosis.
2. Blastomycosis
(i) Causative fungus: Blastomyces dermatitidis, a dimorphic fungus.
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(ii) It is a chronic infection of the lungs which may spread to other tissues,
particularly skin, bone and genitourinary tract.
(iii) As the infection is confined to the North American Continent, it is also known as
North American blastomycosis.
Source of an infection
• Inhalation of conidia of fungus growing as sanrophytes in the soil.
Clinical features
• Asymptomatic.
• Mild primary pulmonary disease.
• Disseminated lesions have been found in immunocompromised patients including
AIDS.
• Cutaneous blastomycosis.
Oral manifestation
The oral lesions may be primary in origin or secondary to lesions of some other
sites in the body. These lesions may resemble to those of actinomycosis.
However, abscess formation is not as prominent as that in actinomycosis. Tiny
ulcers may be the only main clinical feature.
Morphology
B. dermatitidis is a dimorphic fungus. In tissues and in cultures at 37°C, the
fungus appears as spherical or oval budding yeast cells with thick, double
contoured walls.
Each cell has only a single broad based bud. At 25°C, the culture is
filamentous with septate hyphae and many round or oval conidia (Fig. 12).
Laboratory diagnosis
Specimens
• Sputum
• Pus
• Scrapings from skin lesions
Direct examination
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• 10% KOH mount shows thick walled yeast cells with a single broad based bud.
• H and E and PAS stains also show yeast cells in sections.
Culture
• SDA or blood agar.
• Mycelial phase occurs slowly incubation at 25°C. Yeast phase seen in cultures
incubated at 37°C.
Treatment
Amphotericin B is the drug of choice.
3. Paracoccidioidomycosis
(i) Causative fungus: Paracoccidioides brasiliensis, a dimorphic
fungus.
(ii) It is a chronic granulomatous disease involving lungs, mucosa, skin
and lymphatic system.
(iii) As the disease is confined to South America, it was formerly called
South American blastomycosis.
Source of an infection
By inhalation of spores from environmental sources.
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Clinical features
* Primary pulmonary infection that spreads by haematogenous
route to mucous membranes of mouth, nose, lymph node and adjacent
skin, producing chronic granulomatous reaction.
* Ulcerative granulomas of the buccal and nasal mucosa are a
prominent feature.
Oral manifestations
The fungus may enter through the periodontal tissues. It
produces a severe regional lymphadenopathy. When the fungus
penetrates the tissues, it may produce pappillary lesions of the oral
mucosa. Extensive oral ulceration is also a common feature.
Morphology
• As a dimorphic fungus, it grows as yeast cells with multiple buds in
tissues and in cultures at 37°C and in the mycelial phase at 25°C
(Fig.13).
Laboratory diagnosis
Specimens
• Sputum
• Pus
• Biopsies from granulomatous lesior
Direct microscopy
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• Microscopical examination in КOH mount of the specimen shows
numerous yeast cells with multiple buds.
• Tissue sections should be stained with H and E and PAS stains.
Culture
• Mycelial phase of the fungus develops on SDA incubated at 25°C.
• Yeast phase may be obtained by inoculating specimen on enriched media
such as BHI agar and incubating it at 37°C.
Treatment
Amphotericin B is the drug of choice.
4. Coccidioidomycosis
Clinical features
• Asymptomatic.
• Primary pulmonary disease — mild influenza-like fever to severe
pneumonia.
• Disseminated disease.
Oral manifestation
Proliferative granulomatous ana ulcerative lesions appear on the oral
mucosa and skin. These lesions tend to heal by scar formation. Lesions
are often chronic in nature. Sometimes it may occur as lytic lesions of the
jaws.
Morphology
• Being a dimorphic fungus, it occurs in the tissue as a yeast and in
culture (both at 37°C and at 25°C) as the mycelial form.
• The yeast form is a spherule with a thick doubly retractile wall and
filled with endospores (Fig. 14). Each endospore gives rise to a new
spherule.
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• The mycelial phase contains pseudohyphae which fragment into
arthrospores (Fig. 14) which are highly infectious.
Specimens
• Sputum
• Pus
• Biopsy material
Direct microscopy
• Microscopic examination of the specimen detects large number of
mature spherules.
Culture
• Specimen is inoculated on SDA medium in the test tube.
• Inoculated media are incubated at 25°C for 3 weeks.
• Septate hyphae which fragment into thick walled arthrospores in
chains are found.
Skin test
• It is an intradermal skin test using 'coccidioidin', an antigen from
the fungus.
5. Cryptococcosis
(i) Causative fungus: Cryptococcus neoformans, a capsulated yeast.
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(ii) It is a soil saprophyte and is particularly abundant in the feces of
Morphology
• Spherical budding cell having a prominent polysaccharide capsule (Fig. 15).
• It is a true yeast and is Gram positive.
• Capsule may be demonstrated by India-ink or nigrosin staining.
pigeons.
(iii) Several cases of cryptococcosis have been identified in India.
Antigeniсity
• On the basis of cryptococcal polysaccharide antigen, C. neoformans has four
serotypes (А, В, С and D).
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India-ink staining Romanowsky-Giemsa staining
Source of infection
• Infection is usually acquired by inhalation of dust containing yeast cells.
Pathogenesis
• C. neoformans is pathogenic in humans and animals.
• The disease is usually seen in immunocompromised host.
• Most infections are asymptomatic.
• Pulmonary cryptococcosis may lead to a mild pneumonitis.
• Cryptococcal meningitis occurs by haematogenous spread. It is often seen in
AIDS patients.
• Skin, lymph nodes, bones and other organs may be involved when dissemination
of infection occurs. Cutaneous cryptococcosis varies from small ulcers to large
granulomas.
Laboratory diagnosis
Specimens
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• CSF
• Sputum
• Pus
• Brain tissue
Direct microscopy
• Specimen mixed with a drop of India ink or nigrosin shows round budding yeast
cells. In India ink, capsule appears as a clear halo around the yeast cells.
• Gram staining shows Gram positive yeast cells.
• The histopathological examination of tissue can be done by staining with H
and E, PAS and mucicarmine stains.
Сulture
• Sediment from a centrifuged CSF is inoculated on SDA and incubated at 37°C.
Other specimens may also be inoculated on SDA.
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VII. OPPORTUNISTIC MYCOSES
Some saprophytic fungi usually do not infection under special conditions such
as immunocompromised individuals and in terminal stages of chronic
disease.
The incidence of these fungal infections has increased with widespread use of
antibiotics, corticosteroids and immunosuppressive drugs.
These are called opportunistic fungi.
• Some of these are common laboratory contaminants in culture media and
grow on virtually anything.
Aspergillus and Penicillium grow on damp bread and other organic matter.
• These fungi can produce serious and even fatal infections in persons who are
otherwise debilitated.
Candidiasis
1. Causative fungus: Candida albicans (80-90% of cases).
2. Causative fungus: Candida albicans (80-90% of cases).
3. Candidiasis is an infection of skin, mucosa and internal organs, caused
by yeast like fungus Candida albicans, and occasionally by other
Candida species.
4. Candida albicans is the normal inhabitant of skin, gastrointestinal tract,
oral and vaginal cavities.
Morphology
C. albicans is an ovoid or spherical budding yeast cell, 3-5 um in diameter.
(Fig. 16).
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Chlamydiaspore
Pseudohyphae
Blastospores
Pathogenesis
(i) Candidias is an opportunistic endogenous infection.
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(b) Skin and nail infections
• Skin—Infections of axillae, groin, perineum and submammary folds.
• Nails—Infections of finger webs, nail folds and nails may occur.
Paronychia and onychia are seen in those persons who frequently
immerse their hands in water.
• In infants it may lead to napkin dermatitis
(c) Systemic candidiasis
* Urinary tract infection
* Intestinal candidiasis: It is a frequent sequel to oral antibiotic
therapy and present as diarrhoea.
* Pulmonary candidiasis.
* Endocarditis.
* Meningitis.
* Septicaemia.
(d) Oral
manifestations
Various manifestations of oral candidiasis are as follows :
• Thrush
• Chronic oral candidiasis
• Cronic mucocutaneous candidiasis
• Angular stomatitis (angular cheilitis)
• Circumoral candidal dermatitis.
Thrush (Pseudomembranous candidiasis): The lesions are soft, white, slightly
elevated plaques frequently occuring on the buccal mucosa of tongue, but may
also be seen on the gingiva, palate and floor of the mouth. In severe cases,
the entire oral cavity may be affected.
Laboratory Diagnosis
(i) Direct microscopy
Gram stained smears and KOH mounts from lesions of skin, nail or
mucous membranes show budding Gram positive yeast cells.
(ii) Culture
(a) Candida species grow well on SDA and ordinary bacteriological culture
media e.g. blood agar.
(b) They grow at 25-37°C within 24 hours.
(c) Cream coloured, smooth, pasty colonies appear.
(d) Gram stained smear from colonies shows Gram positive budding
yeast cells.
(iii) Identification
To differentiate C. albicans from other species, the following tests are
done.
(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).
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(b) Сhlamydospores: Сhlamydospores develop in a nutritionally deficient
medium such as cornmeal agar at 20°C. They can be seen at the end of
pseudohyphae.
Treatment
(i) Predisposing factors are to be removed in all cases.
(ii) Topical application of polyene (nystatin) or imidazole (miconazole,
clotrimazole) is effective in superficial infections.
(iii) Amphotericin В is administered alongwith 5 fluorocytosine in systemic
infections.
Aspergillosis
1. Aspergilli are ubiquitous in nature.
2. Aspergillus fumigatus is the main opportunistic pathogen.
3. Other important species are A. niger and A. flavus. (Fig. 17).
Laboratory Diagnosis
Specimens
• Sputum
• Bronchoalveolar lavage
• Biopsy
Direct microscopy
• KOH preparation of the specimen reveals non-pigmented septate hyphae with
characteristic dichotomous branching (at an angle of approximately 45°C).
• Biopsy sections can be stained with H and E and PAS staining and examined for
the characteristic hyphae.
Culture
• The clinical specimen is inoculated on SDA without cycloheximide and
incubated at 25°C.
• Colonies appear within 1-2 days and show a velvety to powdery surface.
Colonies are coloured. A fumigatus—green coloured colonies, A niger—black
colonies and A flavus— golden-yellow coloured colonies.
• Lactophenol cotton blue preparation from colonies shows branching and septate
hyphae.
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Asexual conidia are arranged in chains, carried on sterigmata, borne
on the expanded ends (vesicles) of conidiophores.
(Fig. 18).
Penicillosis
Etiology: Penicillium
Culture:
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Conidia
Phialide
Conidiophore
Hyphae
Septate
Clinical picture:
Penicillium species are implicated in otomycosjs and mycotic
keratitis. In otomycosis there is inflammation, puritis, exfoliation of
epithelium and often, by deafness when ear canal is occluded by a plug of
hyphae. In mycotic keratitis there may be corneal ulcer or hypopyon or
both. However corneal trauma, corneal disease glaucoma predis pose to
mycotic. There may be pulmonary and cerebral penicillosis.
Treatment:
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Zygomycosis
1. Zygomycosis includes mucormycosis and entomophthoramycosis. The latter
is an infection of the subcutaneous tissue or paranasal sinuses.
2. Three genera of class Zygomycetes, Mucor, Rhizopus and Absidia are
associated with zygomycosis. (Fig. 20, 21).
Laboratory Diagnosis
Specimens
* Scrapings from the lesions, skin lesion scraping
* Pus
* Tissue biopsy
* Sputum
Direct microscopy
• KOH wet mounts of specimens show non-septate hyphae.
• Histological sections stained with H and E stain reveal the presence of
hyphae.
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Sporangiospores Sporangium
Sporangiospores
Sporangiophore
Hypha
Hypha
Rhizoids
Fig. 20. Fungi: genus Mucor Fig. 21. Fungi: genus Rhizopus
Culture
• Fungi can be readily grown on SDA without cycloheximide at 37°C.
Lactophenol cotton blue preparation of colonies shows branched
sporangiophores arising randomly along aerial mycelium. In the case of mucor
rhizoids are absent. Rhizopus has rhizoids, and sporangiospore arise in groups
directly above the rhizoids. Absidia has also rhizoids but sporangiophores arise
from the aerial mycelium in between the rhizoids.
Histological sections:
Microscopic examination shows broad, non septate, irregular hyphae in
thrombosed vessels or sinuses surrounded with leukocytes and giant cells.
Clinical picture:
Zygomycosis and phycomycosis (mucormycosis) is a systemic disease which may
involve internal organs with predilection for blood vessels. Sometime phycomycosis
may be seen as a chronic infection of subcutaneous tissue.
Treatment:
In some cases amphotericin В may be useful.
Pneumocystis carinii
1. Pneumocystis carinii causes pneumonia in immunocompromised patients. Until
recently, it was thought to be a protozoan, but now it has been included in
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fungi. Prior to the introduction of chemoprophylaxis, it was a major cause of
death among AIDS patients.
2. P. carinii has two stages: thin walled trophozoites and thick walled spherical
cysts. Cyst contains 4 to 8 nuclei.
3. Bronchoalveolar lavage, lung biopsy and induced sputum are the specimens
used for diagnosis.
Pneumocystis
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4. Direct demonstration of trophozoites and cysts can be done by Giemsa,
toludine blue, methenamine silver and calcofluor white stains.
Cyst wall stains black with methenamine silver staining. Fluorescent
monoclonal antibody staining shows honeycomb appearance of the cyst.
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Antibiotics produced by fungi.
ANTIFUNGAL ANTIBIOTICS
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of the cell membranes, causing cell lysis.
medically important fungi but must be given intravenously (not absorbed orally)
and causes many side effects. It may also be given intrathecally (into the
cerebrospinal fluid).
Itraconazole, given orally, has now proven useful for many of these infections.
2) Antifungal agents that are used for less serious systemic infections:
The oral azole drugs. The prototype is ketoconazole. There are now new agents
Griseofulvin (taken orally) and the many topical antifungal agents such as nystatin
Amphotericin B
The classic antifungal antibiotic is amphotericin B. Most species of fungi are
susceptible to it, and although it has many side effects, it remains the drug of
choice for most serious systemic fungal infections: Systemic Candida infections.
Cryptococcal meningitis: used in combination with flucytosine. Severe
pneumonia and extrapulmonary Blastomycosis, Histoplasmosis, and
Coccidioidomycosis. Invasive Aspergillosis. Invasive Sporotrichosis.
Mucormycosis.
Adverse Effects
On the wards this drug is referred to as amphoterrible and Awfultericin
because of its numerous adverse effects:
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1) Renal toxicity: (Poor Mr. Kidney!) There is adose-dependent azotemia
(increase in BUN and creatinine reflecting kidney damage) in most patients taking
this drug. This is reversible if the drug is stopped. The creatinine level must be
followed closely, and if it becomes too high (creatinine > 3), the dosage may have
to be lowered, terminated, or switched to alternate day regimens.
2) Acute febrile reaction: A shaking chill (rigors) with fever occurs in some
people after IV infusion. This is a common side effect.
3) Anemia.
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Amphoterrible.
Amphotericin B colloidal dispersion (ABCD: Amphocil): Ampho B +
cholesterol sulfate. Rigors still occur but nephrotoxicity is reduced. Amphotericin
B lipid complex (ABLC): Ampho B + dimyristoylphosphatidylglycerols and
dimyristoylphosphatidylcholines.
Rigors still occur but there is less nephrotoxicity.
Liposomal amphotericin B (Ambisome): A unilamellar liposome containing a
mixture of 1 molecule of amphotericin B surrounded by a coating of nine
molecules of lipid (soy lecithin, cholesterol, and distearoylphosphatidylglycerol),
like a coated jaw breaker. There is little nephrotoxicity or rigors.
Some hospitals make their own concoction by adding amphotericin B
deoxycholate to Intralipid (parenteral fat for intravenous feeding) in a mixture of 1-
2 mg amphotericin B per ml lipid.
Less nephotoxicity is seen, but once again we do not yet know enough about
antifungal efficacy.
Flucytosine is rarely used alone because of rapid development of resistance.
Think of it as the tag team wrestling partner of amphotericin B.
Amphotericin B busts holes in the cell membranes and flucytosine enters and
inhibits DNA/RNA synthesis.
Most fungi are resistant to flucytosine, but Cryptococcus,and Candida are the
exceptions. Flucytosine use mostly limited to the treatment of
cryptococcalmeningitis, in conjunction with Amphotericin B.
Adverse Effects
1) Bone marrow depression, resulting in leucopenia and thrombocytopenia.
Remember that most antimetabolite type drugs will do this (methotrexate,
sulfadrugs, 5-fluorouracil, etc.).
2) Nausea, vomiting, diarrhea. This again is common with the antimetabolites,
such as the chemotherapeutic drugs.
The reason for these adverse effects is that the drugs damage DNA during its
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formation in rapidly dividing cells such as bone marrow and GI epithelial cells.
Clotrimazole and miconazole are too toxic for systemic use and for this
reason, are primarily used for topical fungal infections, including pityriasis
versicolor, cutaneous candidiasis, and the dermatophytosis (tinea pedis, corporis,
etc.).
Clotrimazole troches (like candies) are sucked to treat oral Candida (thrush),
and clotrimazole vaginal suppositories treat Candida vaginitis.
Ketoconazole, fluconazole, and itraconazole are tolerated orally and have many
important uses for systemic fungal infections.
Ketoconazole
Ketoconazole, one of the imidazoles, is the drug of choice for chronic
mucocutaneous candidiasis ( Candida on every surface). It is NOT used for
systemic candidiasis (amphotericin B, remember?).
Ketoconazole is currently not used for the treatment of systemic fungal
infections. The safer, more efficacious, oral itraconazole and old faithful,
amphotericin B, are the first line drugs.
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Adverse Effects
1) GI: Nausea, vomiting, and anorexia, all common.
2) Hepatotoxicity: This is usually seen as a temporary rise of hepatic enzymes but
on rare occasions can lead tohepatic necrosis. Follow enzymes when on this drug.
3) Inhibition of testosterone synthesis: Ketoconazole inhibits the cytochrome P-
450 system, which is
important in testosterone synthesis. The result is gynecomastia, impotence,
decreased sex drive (libido), and decreased sperm production.
4) Adrenal suppression.
Fluconazole
Fluconazole is one of the triazoles; it is less toxic and has broader antifungal
coverage than ketoconazole.
Like ketoconazole it is used for cutaneous Candida infections but it is also
used as a second-line agent behind amphotericin B for systemic candidiasis and
cryptococcal meningitis.
In AIDS patients who have had cryptococcal meningitis, maintenance with
fluconazole will prevent relapses.
The big picture with fluconazole is that it kills Candida albicans very well:
1) Studies comparing it to amphotericin B in the treatment of systemic Candida
albicans infection (in
non-neutropenic patients) demonstrated equivalent effcancy.
2) A single dose of fluconazole very effectively clears candida vaginitis.
Itraconazole
This triazole is becoming the next amphotericin B but in an oral formulation
without the many amphoterrible side effects!!!
Itraconazole is now used as first-line treatment for chromoblastomycosis,
histoplasmosis, coccidioidomycosis, blastomycosis, and possibly for invasive
aspergillosis.
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The main problem with this drug is poor oral absorption. Taking it with acid
drinks such as orange juice or colas enhances absorption (need low pH). A new IV
Nystatin
Griseofulvin
keratin precursor cells in the skin, hair, and nails, where it inhibits the growth of
fungi in those cells.
Note that it does not kill the fungi; it just inhibits their growth (static rather
thancidal). The uninfected drug-infiltrated keratin precursor cells mature and move
outward toward the keratinized layer.
As the older, infected cells fall off with normal cell turnover, this translates
into a slow cure of skin fungus.
Adverse effects of griseofulvin are uncommon. They include headache,
nausea, vomiting, photosensitivity, and mental confusion, in addition to bone
marrow suppression (leukopenia and neutropenia).
Potassium Iodide
Potassium iodide is used to treat sporotrichosis. Remember that you get
sporotrichosis from pricking your finger in the garden. "You get Sporotrichosis
while Potting plants." If the infection becomes systemic, amphotericin B or
itraconazole is better.Summary of the anti-fungal drugs.
Terbinafine
Terbinafine is a new oral fungicidal agent that blocks fungal cell wall
synthesis. It blocks ergosterol synthesis by inhibiting the formation of squalene
epoxide from squalene.
Terbinafine tends to accumulate in nails, and is therefore useful for tinea
unguium (onychomycosis). It also appears useful in the treatment of tinea pedis,
tinea capitis, and tinea corporis.
Since it is not metabolized by the cytochrome p450 system (as do the azole
antifungals), there is little potential or drug-drug interactions.
52
Quizzes
6. A sporangium contains…
53
A. Spherules
B. Sporangiospores
C. Chlamydospores
D. Oidia
E. Arthrospores
9. Which is a eukaryote?
A. Mycoplasma
B. Bacteria
C. Chlamydia
D. Fungi
E. Viruses
56
D. Endosporulating spherules
57
A. Candidiasis
B. Aspergillosis
C. Histoplasmosis
D. Penicillosis
E. Chlamydiasis
30. Some saprophytic fungi usually do not produce disease but may cause
infection under special conditions such as immunocompromised individuals
and in terminal stages of chronic disease. Name the species of Candida.
A. C. albicans
B. C. septicum
C. C. novyi
D. C. fallax
E. C. carnis
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B. Mucor
C. Penicillus
D. Candida
E. Histoplasma
34. Candida species grow well on Sabouraud dextrose agar. What colonies
grow on this medium?
A. Red with metal shine
B. Colorless
C. White and cream
D. Black and grey
E. Pink
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D. Vaginal cavity
E. All of the above
37. Many fungi produce poisonous substances that can cause acute or chronic
intoxication. Name this toxin.
A. Endotoxin
B. Enterotoxin
C. Anatoxin
D. Antitoxin
E. Cytotoxin
38. Fungi had been recognized as causative agents of human disease carlier
that bacteria. What is the name these diseases?
A. Chlamidiosis
B. Salmonellosis
C. Mycoplasmosis
D. Shigellosis
E. Mycosis
39. Fungi can be divided into four classes each of which have one human
pathogenic speacies. Which one of the following groups is not a classification
of fungi?
A. Yeast
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B. Moulds
C. Fungi imperfecti
D. Yeast-like fungi
E. Dimorphic fungi
61
D. Buchin medium
E. Blood agar
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following?
A. Budding yeast cells with pseudohyphae
B. Yeast-like forms with very large capsules
C. Arthrospores
D. Endosporulating spherules
50. Many fungi produce poisonous substances that can cause acute or chronic
intoxication. Name this toxin.
A. Endotoxin
B. Enterotoxin
C. Anatoxin
D. Antitoxin
E. Cytotoxin
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C. Ergosterol
D. Peptidoglican
E. Keratin
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57. Which of the following stain is used for cryptococcus?
A. Negative India ink
B. Giemsa
C. Gram
D. Albort
E. Neisser
60. Many fungi produce poisonous substances that can cause acute or chronic
intoxication. Name this toxin.
A. Endotoxin
B. Enterotoxin
C. Anatoxin
D. Antitoxin
E. Cytotoxin
61. The fungal nucleous…
A. Contains true chromosomes
B. Contains DNA fragments
65
C. Differs from the bacterial nucleous
D. All of the above
CORRECT ANSWERS
Test Answ e r s
1 A B C *D E
2 A B C *D E
3 A B C *D E
4 A B C *D E
5 *A B C D E
6 A *B C D E
7 A *B C D E
8 A B *C D E
9 A B C *D E
10 A *B C D E
11 A B C D *E
12 A B *C D E
13 A B *C D E
14 A B C *D E
15 A B *C D E
16 A B C *D E
17 A *B C D E
18 *A B C D E
19 *A B C D E
20 A B C *D E
21 *A B C D E
22 A B C *D E
23 *A B C D E
24 A B C D *E
25 *A B C D E
26 A *B C D E
27 A B C D *E
28 A B C D *E
29 A B C *D E
30 *A B C D E
31 A *B C D E
32 *A B C D E
33 A B *C D E
34 A B *C D E
35 A B C D *E
36 A *B C D E
37 *A B C D E
38 A B C D *E
66
39 A B *C D E
40 A *B C D E
41 A B C *D E
42 A B C *D E
43 *A B C D E
44 A B *C D E
45 *A B C D E
46 A B C *D E
47 *A B C D E
48 A B C *D E
49 A B *C D E
50 *A B C D E
51 A *B C D E
52 A *B C D E
53 *A B C D E
54 A B *C D E
55 *A B C D E
56 A B C *D E
57 *A B C D E
58 *A B C D E
59 A B C *D E
60 A B *C D E
61 A B C *D E
PRACTICAL LESSON № 1
THEME: Morphology of fungi.
CONTROL GUESTIONS:
2. Aspergillus
3. Pernicillium
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PRACTICAL LESSON № 2
THEME: Laboratory diagnostics of mycosis. Yeast – like fungus Candida.
CONTROL GUESTIONS:
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TASK 1. Mark the methods of laboratory diagnostics of diseases caused by
fungi.
Methods Mycosis
Microscopic
Microbiological
Serological
Allergical
PRACTICAL LESSON № 3
70
1. Name the taxonomy of the causative agents of the systemic mycoses.
Genus
Species
_________________________________________ ________________________________________
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2. Fill in the table.
Blastomycosis
Histoplasmosis
Coccidioido-
mycosis
Cryptococcosis
4. Name the preparations which are used for treatment of the systemic mycoses.
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
______________________________________________________________________________
5. Name the preparations which are used for prophylaxis of the systemic
mycoses.
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
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5.Name the causative agent of the systemic mycoses and indicate their tissue
and mycelia forms.
____________________________________________
____________________________________________
____________________________________________
____________________________________________
____________________________________________
1 2
____________________________________________
____________________________________________
____________________________________________
____________________________________________
____________________________________________
1 2
____________________________________________
____________________________________________
____________________________________________
____________________________________________
____________________________________________
_________________________________________
1 2
____________________________________________
____________________________________________
____________________________________________
____________________________________________
____________________________________________
73