“mykos” meaning mushroom. Mycology is the study of fungi. The fungi possess rigid cell walls: Chitin and ergosterol, mannan and other polysaccharides. Beta-glucan is most important, because it is the target of antifungal drug caspofungin. Fungi are eukaryotic organisms VS bacteria (prokaryotic). The cell membrane of fungus contains ergosterol, unlike human cell membrane which contains cholesterol. Most fungi are obligate aerobes or facultative anaerobes, but none are obligate anaerobes. The natural habitat of most fungi is environment, require a preformed organic source of carbon, association with decaying matter. C. albicans is an exception!!!

1. Medical Microbiology
(Mycology)

2. Outline
• Introduction.
• The Superficial Mycoses.
• The Cutaneous Mycoses.
• The Subcutaneous Mycoses.
• Dimorphic Systemic Mycoses.
• Opportunistic mycosis.

3. Introduction
• “mykos” meaning mushroom.
• Mycology is the study of fungi.
• The fungi possess rigid cell walls:
• Chitin and ergosterol, mannan and other polysaccharides.
• Beta-glucan is most important, because it is the target of antifungal drug
caspofungin.
• Fungi are eukaryotic organisms VS bacteria (prokaryotic).
• The cell membrane of fungus contains ergosterol, unlike human cell membrane which
contains cholesterol.
• Most fungi are obligate aerobes or facultative anaerobes, but none are obligate
anaerobes.
• The natural habitat of most fungi is environment, require a preformed organic source
of carbon, association with decaying matter.
• C. albicans is an exception!!!

5. Classification of fungi
• Taxonomical Classification:
1. Zygomycetes.
2. Ascomycetes.
3. Basidiomycetes.
4. Deuteromycetes or Fungi Imperfecti.
• Morphological Classification:
1. Yeast.
2. Yeast-like form.
3. Molds.
4. Dimorphic fungi.

6. • 1. Yeasts:
• Round or oval unicellular fungi that reproduce by asexual budding.
• On culture medium, such as Sabouraud dextrose agar (SDA), they
produce creamy mucoid colonies.
• Example: Cryptococcus neoformans.
• 2. Yeast-like fungi:
• Pseudohyphae.
• Example: Candida albicans.

7. • 3. Molds:
• Grow as long filaments called hyphae.
• Septate hyphae (transverse walls), whereas others do
not produce walls, hence are called
nonseptate/aseptate (coenocytic) hyphae.
• Nonseptate hyphae are multinucleated.
• The hyphae on their continuous growth form
mycelium.
• The part of the mycelium that projects above the
surface in culture medium is called aerial mycelium,
bear the reproductive spores/conidia. Vegetative
mycelium, penetrate the supporting medium and
absorb nutrients.
• Examples include Aspergillus, Penicillium,
Rhizopus.
• 4. Dimorphic fungi:
• Many of medically important fungi are dimorphic.
• They exist as hyphal/mycelial forms in the soil and
in the cultures at 22–25°C.
• They occur as yeasts or other structures in human
tissue and in the culture at 37°C.
• Examples include Coccidioides immitis,
Paracoccidioides brasiliensis, Histoplasma
capsulatum, Blastomyces dermatitidis, and
Sporothrix schenckii.

9. Reproduction of Fungi
• Sexually: by forming sexual spores and asexually by
forming conidia or asexual spores.
• 1. Sexual spores:
1. Ascospores:
• Formed in a sac called ascus.
2. Basidiospores:
• Formed outside on the basidium.
3. Zygospores:
• Single, large spores with thick wall.
• The fungi that do not produce sexual spores are called
imperfect and are classified as Fungi imperfecti.
2. Asexual spores:
• Produced by mitosis.
• Fungi reproduce asexually by forming conidia.
• The shape, color, and arrangement of the conidia are
helpful for identification of the fungi.
• Asexual spores can be vegetative or aerial spores as
follows:

10. 1. Vegetative spores:
• Arthrospores:
• Formed by fragmentations of the ends of hyphae.
• The arthrospores are the infective stage of C. immitis.
• Chlamydospores:
• Arise by rounding and thickening of hyphal segments. They
are round and thick walled.
• The terminal chlamydospores help in the identification of C.
albicans.
• Blastospores:
• Formed by budding process from parent cells, such as yeast.
• Some yeasts, such as C. albicans can form multiple buds that
do not detach from the parent yeast, thus producing
elongated structures called pseudohyphae.
2. Aerial spores:
• Sporangiospores:
• Formed within a sac called sporangium, which develops at
the ends of the hyphae called sporangiophores (e.g., Mucor
and Rhizopus).
• Conidiospores:
• Called conidia, are spores found externally on the sides or
tips of hyphae. Conidia can be macroconidia or
microconidia.
• Macroconidia are large, aseptate, often multicellular conidia.
• Microconidia are small and single.

12. Lab diagnosis
• Specimens:
• Blood and Bone Marrow.
• Cerebrospinal Fluid (CSF).
• Skin, Hair and Nail Scrapings.
• Sputum, Bronchial Washings and Throat Swabs.
• Tissue Biopsies from Visceral Organs.
• Urine.

13. 1. Direct Microscopy:
• Demonstration of asexual spores, hyphae, or yeast
in various clinical specimens by light microscopy.
• The commonly used clinical specimens are sputum,
lung biopsy material, and skin scrapings.
• The specimen is either treated with 10% KOH or
stained with special fungal stains.
• Use of 10% KOH dissolves tissue material
(clearing), leaving the alkali-resistant fungi
intact.
• Lactophenol cotton blue (LPCB) wet mount.
• Calcofluor-white (CFW) dye is a fluorescent dye
that combines with fungal cell wall and is useful in
identification of fungi in tissue specimens.
• Grocott's Methenamine Silver (GMS) stain and
PAS are useful for demonstration of fungi in tissues.
• India ink preparation of cerebrospinal fluid (CSF)
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.

15. 2. Culture:
• For confirming the diagnosis of fungal infection.
• Sabouraud dextrose agar (SDA) is the most commonly used medium for fungal
culture.
• Other media include CHROM agar, blood agar.
• The media (with low pH) contain chloramphenicol (inhibit the growth of bacteria)
and cycloheximide (inhibit the saprophytic fungi).
• Fungal colony is identified by rapidity of growth, color, and morphology of the
colony.
• 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.

16. 3. Nonculture Methods
• These methods include (a) detection of fungal antigen, (b) detection of
fungal cell wall markers, and (c) detection of fungal metabolites.
• Antigen detection:
• It is useful in immunocompromised hosts where antibody detection is not as
sensitive.
• Detection of fungal antigen in serum, CSF, and urine is increasingly used for
diagnosis of 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.

17. 4. Serology:
• Antibodies in patient’s serum or CSF, especially in systemic fungal infections.
• The complement fixation test, diagnosis of suspected cases of histoplasmosis,
blastomycosis, or coccidiomycosis.
• ELISA (enzyme-linked immunosorbent assay), Western blot, and RIA
(radioimmunoassays).
5. Molecular Diagnosis:
• DNA probes: identify colonies growing in culture at an earlier stage of growth.
• These DNA probes are very useful for rapid diagnosis.
• Detection of Cryptococcus, Histoplasma, Blastomyces, and Coccidioides.
• Mitochondrial DNA has been used for the diagnosis of C. albicans and Aspergillus
species.

18. Fungal infection
• Superficial mycoses:
• These are surface infections of the skin, affecting the outermost layers of
skin, hair, and mucosa. No living tissue is invaded.
• Cutaneous mycoses:
• These are infections of the skin involving the epidermis and its
integuments, the hair, and nails. No living tissue is invaded.
• Subcutaneous mycoses:
• These are infections of the dermis, subcutaneous tissue, muscle, and
fascia.

19. The Superficial Mycoses
• Restricted to the outermost layers of the skin and hair.
• The condition usually causes cosmetic problem, which can be
easily diagnosed and treated.
• It includes four important conditions:
1. Pityriasis versicolor.
2. Tinea nigra.
3. Black Piedra.
4. White Piedra.

20. Malassezia infections
• Malassezia furfur is the causative agent of Pityriasis
versicolor, Pityriasis folliculitis and seborrhoeic dermatitis
and dandruff.
• Lipophilic yeast, normal flora of skin.
• Pityriasis versicolor:
• Chronic, superficial disease of the skin characterized by
well-demarcated white, or brownish lesions on the
trunk, shoulders and arms, rarely on the neck and face,
and fluoresce a pale greenish colour under Wood's
ultra-violet light.
• Young adults are affected most often, but the disease may
occur in childhood and old age.
• Laboratory diagnosis:
• Skin scrapings from patients with superficial lesions,
blood and indwelling catheter tips from patients with
suspected fungaemia.
• Skin scrapings when mounted in 10% KOH, glycerol and
Parker ink solution, show characteristic clusters of
thick-walled round, budding yeast-like cells (spaghetti
& meatballs).
• These microscopic features are diagnostic for
Malassezia furfur and culture preparations are usually
not necessary (only in suspected fungaemia).

21. • M. furfur is a lipophilic yeast, therefore in vitro growth must be stimulated by natural
oils or other fatty substances.
• Sabouraud dextrose agar containing cycloheximide (actidione) with olive oil.
• A more specialized media like Dixon's agar which contains glycerol mono-oleate (a
suitable substrate for growth).
• Optimum temperature for growth is 35-37C; weak growth occurs at 25C.
• Treatment:
• Imidazole.
• Ketoconazole shampoo has proven to be very effective.
• Alternative treatments include zinc pyrithione shampoo or selenium sulfide lotion.

22. Tinea nigra
• Superficial fungal infection of skin.
• Caused by Hortaea (Phaeoannellomyces) werneckii, RG-1,
saprophytic fungus in soil, compost, and on wood in humid tropical
and sub-tropical regions.
• Skin lesions, brown to black macules which usually occur on the
palmar of hands and occasionally the plantar and other surfaces of
the skin.
• Lesions are non-inflammatory and non-scaling.
• Laboratory diagnosis:
• Skin scrapings.
• Direct Microscopy: Skin scrapings should be examined using 10%
KOH and Parker ink or calcofluor white mounts.
• Showing pigmented brown to dark dematiaceous septate hyphal
elements and 2-celled yeast cells producing annelloconidia typical
of Hortaea werneckii.
• Conidia are cylindrical to spindle-shaped.
• Culture: Sabouraud dextrose agar.
• Treatment: topical treatment with Whitfield's ointment (benzoic acid
compound) or an imidazole agent.

23. White piedra
• Superficial fungal infection of the hair shaft caused by Trichosporon
beigelii/ Trichosporon cutaneum.
• Minor component of normal skin flora and is widely distributed in
nature.
• Infected hairs develop soft greyish-white nodules along the hair shaft.
• Infections are usually localized to the axilla or scalp but may also be
seen on facial hairs and sometimes pubic hair. White piedra is common
in young adults.
• The nodules are firmly adhering to the hairs is characteristic of white
Piedra.
• Laboratory diagnosis:
• Epilated hairs with white soft nodules present on the shaft.
• Hairs should be examined using 10% KOH and Parker ink or
calcofluor white mounts. Look for irregular, soft, white or light
brown nodules.
• Culture: Hair fragments should be implanted onto Sabouraud
dextrose agar.
• Dalmau Plate Culture on Cornmeal and Tween 80 Agar: True
mycelium is abundant; arthroconidia.
• Pseudomycelium often occurs with blastoconidia in chains or
clusters.

24. • Hydrolysis of Urea is Positive.
• Treatment:
• Shaving the hairs is the simplest method of treatment, but this is often not
considered acceptable, particularly by women.
• Topical application of an imidazole agent may be used to prevent reinfection.

25. Black piedra
• Superficial fungal infection of the hair shaft caused by
Piedraia hortae.
• An ascomycetous fungus forming hard black nodules on the
shafts of the scalp, beard, moustache and pubic hair.
• Epidemics in families, following the sharing of combs and
hairbrushes.
• Laboratory diagnosis:
• Epilated hairs with hard black nodules present on the
shaft.
• Hairs should be examined using 10% KOH and Parker ink
or calcofluor white.
• Look for darkly pigmented nodules that may partially or
completely surround the hair shaft.
• Culture: Hair fragments should be implanted onto
Sabouraud dextrose agar.
• The usual treatment is to shave or cut the hairs short.
• Piedraia hortae is sensitive to terbinafine.

27. The Cutaneous Mycoses
dermatophytes
Trichophyton species infect hairs, skin, and nails.
Microsporum: Infects both skin and hair, but not the nails.
Epidermophyton: Infects skin and nails but not hair.

28. Dermatophytosis - Ringworm or Tinea
• Scalp, skin and nails infection caused by keratinophilic dermatophytes which
have the ability to utilize keratin as a nutrient source, i.e., they have a unique
enzymatic capacity - keratinase.
• Microsporum, Trichophyton and Epidermophyton.
1. Anthropophilic: isolated from human sources, include (T. rubrum, T.
tonsurans, T. violaceum, M. audouinii and E. floccosum).
2. Zoophilic: Animals are the primary reservoir, (M. canis, M. nanum, T.
mentagrophytes, T. verrucosum).
3. Geophilic: Soil is the primary reservoir of geophilic dermatophytes, (M.
gypseum and T. Terrestre).
• Tinea corporis: AKA ringworm, infect skin only (T. rubrm, T. mentagrophytes,
M. canis).
• Tinea barbae: affect hair of beard, (T. rubrum, T. violaceum, M. canis).
• Tinea pedis: known as athlete foot, infect toe, (T. rubrum and T.
mentagrophytes).
• Tinea unguium: AKA onychomycosis, infect the nails, (T. rubrum and T.
mentagrophytes).
• Tinea cruris: dermatophytosis of the proximal medial thighs, preum and
buttocks, T. rubrum, T. interdigitale and E. floccosum.

29. • Tinea capitis:
• Dermatophytosis of the scalp.
• Three types of in vivo hair invasion are recognized:
• 1. Ectothrix
• Invasion is characterized by the development of arthroconidia on the outside of the
hair shaft.
• The cuticle of the hair is destroyed, and Infected hairs usually fluoresce a bright
greenish yellow colour under Wood's ultraviolet light.
• Common agents include M. canis, M. gypseum, T. equinum and T. verrucosum.
• 2. Endothrix
• Hair invasion is characterized by the development of arthroconidia within the hair
shaft only.
• The cuticle of the hair remains intact and infected hairs do not fluoresce under
Wood’s ultraviolet light.
• All endothrix producing agents are anthropophilic e.g., T. tonsurans and T.
violaceum.
• 3. Favus
• Usually caused by T. schoenleinii, produces favus-like crusts or scutula and
corresponding hair loss.

31. M. canis "Kerion" lesion caused by
M. canis "Kerion" lesion
caused by
T. verrucosum
T. schoenleinii

32. Lab diagnosis
• Skin Scrapings, nail scrapings and epilated hairs.
• The laboratory needs enough specimen to perform both
microscopy and culture.
• In patients with suspected dermatophytosis of skin [tinea or
ringworm] any ointments or other local applications present
should first be removed with an alcowipe.
• Using a blunt scalpel, tweezers, firmly scrape the lesion,
particularly at the advancing border.
• In patients with suspected dermatophytosis of nails
[onychomycosis] the nail should be pared and scraped using
a blunt scalpel until the crumbling white degenerating portion
is reached.
• Any white keratin debris beneath the free edge of the nail
should also be collected.
• Skin Scrapings, nail scrapings and epilated hairs should be
examined using 10% KOH and Parker ink or calcofluor white
mounts.
• Specimens inoculated onto Sabouraud dextrose agar
containing cycloheximide and incubated at 26-28C for 4 weeks.

34. T. Rubrum (Birds on a fence)

35. 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.
• T. mentagrophytes 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.
• Treatment: use of local antifungal drugs, such as
miconazole, clotrimazole, econazole, or by treatment
orally with griseofulvin.

37. The Subcutaneous Mycoses

38. Sporotrichosis
• Chronic mycotic infection of the cutaneous or subcutaneous
tissues and adjacent lymphatics characterized by nodular
lesions which may suppurate and ulcerate.
• Caused by Sporothrix schenckii, RG-2.
• Infections are caused by the traumatic implantation of the
fungus into the skin, or very rarely, by inhalation into the
lungs.
• 1. Fixed cutaneous sporotrichosis: Primary lesions develop at
the site of implantation of the fungus, usually at more
exposed sites mainly the limbs, hands and fingers.
• 2. Lymphocutaneous sporotrichosis: Primary lesions develop
at the site of implantation of the fungus, but secondary
lesions also appear along the lymphangitic channels.
• 3. Pulmonary sporotrichosis: caused by the inhalation of
conidia. Haemoptysis may occur and it can be massive and
fatal.
• 4. Osteoarticular sporotrichosis: stiffness and pain in a
large joint, usually the knee, elbow, ankle or wrist.
Osteomyelitis seldom occurs without arthritis; the lesions
usually confined to the long bones near affected joints.

39. Laboratory-diagnosis:
• A tissue biopsy is the best specimen.
• Tissue sections should be stained using PAS digest,
Grocott's methenamine silver (GMS) or Gram stain.
• Culture: Sabouraud dextrose agar and Brain heart
infusion agar supplemented with 5% sheep blood.
• Conidia are formed in clusters, their arrangement
often suggestive of a flower.
• Conidia are ovoid or elongated, hyaline, one-celled
and smooth-walled.
• Treatment:
• Cutaneous lesions respond well to saturated
potassium iodide, itraconazole and terbinafine.
• Extracutaneous forms of sporotrichosis may need a
combination of antifungal treatment with
Amphotericin B or itraconazole together with
surgical debridement.
Section from a fixed cutaneous lesion showing round Periodic
Acid-Schiff (PAS) positive budding yeast-like cells.
Look for small narrow base budding yeast cells.
Sporothrix schenckii is a dimorphic fungus and this is the
typical yeastlike form seen in tissue.

41. Mycetoma (Madura foot)
• A mycotic infection of humans and animals caused by a
number of different fungi (eumycetoma) and actinomycetes
(actinomycetoma) characterized by draining sinuses,
granules and tumefaction.
• The disease results from the traumatic implantation of the
aetiologic agent and usually involves the cutaneous and
subcutaneous tissue, fascia and bone of the foot or hand.
• Sinuses discharge containing the granules which vary in
size, colour and degree of hardness, depending on the
aetiologic species, and are the hallmark of mycetoma.
• The feet are the most common site for infection.
• Other sites include the lower legs, hands, head, neck, chest,
shoulder and arms. Most cases start out as a small hard
painless nodule which over time begins to soften on the
surface and ulcerate to discharge a viscous, purulent fluid
containing grains.
• Laboratory diagnosis:
• Tissue biopsy or excised sinus, fluid containing the
granules.

42. Causative agents
• Actinomycetoma:
• Actinomadura madurae (white grains).
• Actinomadura pelletieri (red).
• Streptomyces somaliensis (yellow).
• Nocardia spp (white to yellow).
• Eumycetoma:
• Pseudoallescheria boydii (white grains).
• Madurella mycetomatis (black grains).

43. • Serosanguinous fluid containing the granules should
be examined using either 10% KOH and Parker ink or
calcofluor white mounts, and tissue sections should
be stained using H&E, PAS digest, and Grocott's
methenamine silver (GMS).
• Culture: Sabouraud dextrose agar.
• M. mycetomatis have been isolated from soil and are
one of the major causative agents of mycetoma.
• Madurella mycetomatis produce brown diffusible
pigment.
• Treatment:
• Amphotericin B, Itraconazole and Voriconazole.

44. Alyazeed Hussein, BSc, SUST
This has been a presentation of Alyazeed Hussein
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