Yeasts:

Yeasts are unicellular spherical to ellipsoid cells. They reproduce by budding, which result in
blastospore
(blastoconidia) formation. In some cases, as the cells buds the buds fail to detach and
elongate thus forming achain of elongated hyphae like filament called pseudohyphae. This
property is seen in Candia albicans. The same species also have the ability to produce true
hypha, which is seen as germ tube. The difference between the two is that there is a
constriction in psueudohyphae at the point of budding, while the germ tube has no
constriction.
Some yeast such as Cryptococcus and the yeast form of Blastomyces dermatatidis produce
polysaccharide capsule. Capsules can be demonstrated by negative staining methods using
India ink or Nigrosin. The capsule itself can be stained by Meyer Mucicarmine stain.

Reproduction in fungi:
Fungi reproduce by asexual, sexual and parasexual means.
Asexual reproduction is the commonest mode in most fungi with fungi participating in sexual
mode only under certain circumstances. The form of fungus undergoing asexual reproduction
is known as anamorph (or imperfect stage) and when the same fungus is undergoing sexual
reproduction, the form is said to be teleomorph (or perfect stage). The whole fungus,
including both the forms is referred as holomorph. (Taxonomically, the teleomorph or the
holomorph is used, but practically it is more convenient to use the anamorph.)
Asexual reproduction:
Asexual propagules are termed either spores or conidia depending on their mode of
production. Asexual spores are produced following mitosis whereas sexual spores are
produced following meiosis.
The asexual spores of zygomycetes, which are known as sporangiospores form within sac
like structure known as sporangia. The sporangiospores result from the mitotic cleavage of
cytoplasm in the sporangium. The sporangia are borne on special hyphae called

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sporangiophore. This endogenous process of spore formation within a sac is known as
sporogenesis.
Conidia arise either by budding off conidiogenous hyphae or by differentiation of preformed
hyphae. These develop following mitosis of a parent nucleus and are formed in any manner
except involving cytoplasmic cleavage. This exogenous process is known as conidiogenesis,
a process that occurs both in yeasts and moulds. Conidia are borne on specialised structures
called conidiophore.
Conidia production may be blastic or thallic. In blastic development the conidium begins to
enlarge and a septum is formed. Here the conidium originates from part of parent. In thallic
mode of development the conidium is differentiated by a septum before its differentiation.
Thus the conidium results from the conversion of entire parent cell into the conidium.
The cell that gives rise to a conidium is called a conidiogenous cell. Conidiophores are
specialised hyphae that bear conidia or conidiogenous cells. In many cases conidiogenous
cells are referred as phialides.
Sexual Reproduction:
Sexual propagules are produced by the fusion of two nuclei that then generally undergo
meiosis.
The first step in sexual methods of reproduction involves plasmogamy (cytoplasmic fusion of
two cells). The second step is karyogamy (fusion of two compatible nuclei), resulting in
production of diploid or zygote nucleus. This is followed by genetic recombination and
meiosis. The resulting four haploid spores are said to be sexual spores, e.g.
zygospores, ascospores and basidiospores.
If a sexual spore is produced only by fusion of a nucleus of one mating type with a nucleus of
another mating type
(+ and - strains), the fungus is said to be heterothallic. In contrast, homothallic moulds
produce sexual spores following the fusion of two nuclei from the same strain. For sexual
reproduction to occur, two compatible isolates are required.
Zygospores, which are the sexual spores of zygomycetes are round, thick walled reproductive
structures that result from the union of two gamentagia. Ascomycetes produce sexual spores
called ascospores in a special sac like cell known as ascus. In basidiomycetes the
basidiospores are released from basidium, which is the terminal cell of a hyphae.
Parasexual reproduction:
Parasexual reproduction, first seen in Aspergillus is known to occur in basidiomycetes,
ascomycetes and
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deuteromycetes. The process involves genetic recombination without the requirement of
specific sexual structures.
Importance of Spores:
A. Biological
1) Allows for dissemination
2) Allows for reproduction
3) Allows the fungus to move to new food source.
4) Allows fungus to survive periods of adversity.
5) Means of introducing new genetic combinations into a population
B. Practical
1) Rapid identification (also helps with classification)
2) Source of inocula for human infection
3) Source of inocula for contamination

ZYGOMYCETES
Commonly known as bread moulds, these are fast growing, terrestrial, largely saprophytic
fungi. Hyphae are
coenocytic and mostly aseptate. Asexual spores include chlamydoconidia, conidia and
sporangiospores.
Sporangiophores may be simple or branched. Sexual reproduction involves producing a
thick-walled sexual resting spore called a zygospore.
Medically important orders and genera include:
1. Entomophthorales: Conidiobolus and Basidiobolus are involved in subcutaneous
zygomycosis
2. Mucorales: Rhizopus, Mucor, Rhizomucor, Absidia and Cunninghamella are involved in
subcutaneous and systemic zygomycosis (formerly called Mucormycosis)

BASIDIOMYCETES
They exist as saprobes and parasites of plants. Hyphae are dikaryotic and can often be
distinguished by the presence of clamp connections over the septa. Sexual reproduction is by
the formation of exogenous
basidiospores, typically four, on a basidium. Occasional species produce conidia but most are
sterile.
Genera of medical importance include:
1. Teleomorph of Cryptococcus neoformans, which is Filobasidiella neoformans

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2. Agents of basidiomycosis such as Coprinus and Schizophyllium
3. Mushroom poisoning by Aminita, Lepiota, Coprinus and Psilocybe etc.

ASCOMYCETES
They exist as saprophytes and parasites of plants. Hyphae are septate with simple septal
pores. Asexual reproduction is by conidia. Sexual reproduction is by the formation of
endogenous ascospores, typically eight, in an
ascus.
Medically important genera include the:
1. Teleomorphs of known pathogenic fungi e.g. Arthroderma (of Trichophyton and
Microsporum), Ajellomyces
dermatitidis (of Blastomyces dermatitidis), Pseudallescheria boydii (of Scedosporium
apiospermum)
2. Agents of mycetoma, like Leptosphaeria
3. Agents of black piedra, like Piedraia hortae.
DEUTEROMYCETES
Deuteromycetes are also known as Fungi Imperfecti because of absence of sexually
reproducing forms (teleomorph or perfect stage). As their teleomorph continue to be
discovered, they would be classified among the previous categories, until then this remains an
artificial and heterogeneous group.
There are three classes of Fungi Imperfecti.
1. Blastomycetes: These include asexual budding forms of Cryptococcus, Candida,
Torulopsis and Rhodotorula.
Depending on the presence of melanin in their cell walls, they may be non-dematiaceous or
dematiaceous.
2. Hyphomycetes: A class of mycelial moulds which reproduce asexually by conidia on
hyphae. Hyphae are
septate. This class contains the majority of medically important fungi. Dematiaceous
hyphomycetes are those conidial fungi that produce dark brown, green-black, or black
colonies and are the causative agents of
phaeohyphomycosis. Hyaline hyphomycetes include those conidial fungi, which are not
darkly pigmented; colonies may be colourless or brightly coloured. These include the agents
of hyalohyphomycosis, aspergillosis,
dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum.
3. Coelomycetes: These produce acervuli, which are tightly bound mats of hyphae on which
conidia are produced.
Pathogenesis of fungal diseases (Mycoses):

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Most fungi are saprophytic or parasitic to plants and are adapted to their natural environment.
Infection in humans is a chance event, occurring only when conditions are favourable. Except
for few fungi such as the dimorphic fungi that cause systemic mycoses and dermatophytes,
which are primary pathogens, the rest are only opportunistic pathogens.
Human body is a hostile environment and offers great resistance to fungal invasion. Most
fungi are saprophytic and their enzymatic pathways function more efficiently at the redox
potential of non-living substrates than at the relatively more reduced state of living
metabolizing tissue. Some fungi such as Candida and Malasezzia have adapted to human
environment and exist as commensals.
The complex interplay between fungal virulence factors and host defence factors will
determine if a fungal infection will cause a disease. Infection depends on inoculum size and
the general immunity of the host.

Fungal Pathogenicity (virulence factors):

• Ability to adhere to host cells by way of cell wall glycoproteins
• Production capsules allowing them to resist phagocytosis
• Production of a cytokine called GM-CSF by Candida albicans that suppress the production
of complement.
• Ability to acquire iron from red blood cells as in Candida albicans
• Ability to damage host by secreting enzymes such as keratinase, elastase, collagenase
• Ability to resist killing by phagocytes as in dimorphic fungi
• Ability to secrete mycotoxins
• Having a unique enzymatic capacity
• Exhibiting thermal dimorphism
• Ability to block the cell-mediated immune defences of the host.
• Surface hydrophobicity
Host defence factors:
• Physical barriers, such as skin and mucus membranes
• The fatty acid content of the skin
• The pH of the skin, mucosal surfaces and body fluids
• Epithelial cell turnover
• Normal flora
• Chemical barriers, such as secretions, serum factors
• Most fungi are mesophilic and cannot grow at 37oC.
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• Natural Effector Cells (polymorphonuclear leucocytes) and the Professional Phagocytes
(monocytes andmacrophages)
Factors predisposing to fungal infections:
• Prolonged antibiotic therapy
• Underlying disease (HIV infection, cancer, diabetes, etc.)
• Age
• Surgical procedures
• Immunosuppressive drugs
• Irradiation therapy
• Indwelling catheters
• Obesity
• Drug addiction
• Transplants
• Occupation
Immunity to fungal infections:
Mechanism of immunity to fungal infections can be innate or acquired. The non-specific
immunity includes the physical barriers offered by skin and mucus membranes along with
their secretions and normal flora. The pH, body temperature and serum factors along with
phagocytic cells play an important part in providing non-specific immunity. Even though
body mounts both humoral and cell mediated immunity, it is the latter that is the mainstay of
host defence.
Cell mediated immunity:
Immunity is provided non-specifically be effector cells (polymorphonuclear leucocytes) and
professional phagocytes (monocytes and macrophages) and specifically by T lymphocytes.
The phagocytes are very important in defence against Candia, Aspergillus and Zygomycetes
as is evidenced by their severity in granulomatous diseases, myeloperoxidase deficiency and
cytotoxic chemotherapy.
Expression of T-cell-mediated immunity to fungi includes:
• Delayed-type hypersensitivity
• Contact allergy
• Chronic granulomatous reactions
Humoral immunity:

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Even though antibodies are produced against many fungi, their role in protection is not very
clear. However, antibodies help in clearing fungal pathogens through opsonisation, which is
important against Candida and
Cryptococcus. Another component of humoral immunity is the complement, which can act as
opsonins and may even cause damage to their cells through complement activation.
Antibodies are important to fungal serodiagnosis.
Hypersensitivity:
As a result of dermatophyte infection some fungus-free skin lesions of variable morphology
occur elsewhere on the body, which are thought to result from hypersensitivity to the fungus.
These reactions are called "id reaction". These reactions are also seen n Candida infections.
An inflamed boggy lesion of the scalp called the kerion may result from a strong immune
reaction to the dermatophyte. Granulomas due to intracellular fungi represent delayed
hypersensivities. Many fungi are significant allergens to humans, the allergens being spores,
conidia, hyphae and other fungal products. On inhalation they may produce allergic
pulmonary diseases such as allergic
bronchopulmonary aspergillosis, farmer's lung, maple bark stripper's lung, bronchial asthma
etc, which may be Type
I or III hypersensitivity.

Fungal Diseases (Mycoses):

Mycoses can be conveniently studied as:
1. Superficial mycoses
I. Superficial phaeohyphomycosis
II. Tinea versicolor
III. Black piedra
IV. White piedra
I. Dermatophytosis
II. Dermatomycosis

2. Subcutaneous mycoses
I. Chromoblastomycosis
II. Rhinosporidiasis
III. Mycetoma
IV. Sporotrichosis
V. Subcutaneous phaeohyphomycosis
VI. Lobomycosis

3. Systemic (deep) mycoses

I. Blastomycosis
II. Histoplasmosis
III. Coccidioidomycosis
IV. Paracoccidioidomycosis
4. Opportunistic mycoses
I. Candidiasis
II. Cryptococcosis

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III. Aspergillosis

Laboratory diagnosis of mycoses:

Specimen collection: specimen collection depends on the site affected. Different specimens
include hair, skin scrapings, nail clippings, sputum, blood, CSF, urine, corneal scraping,
discharge or pus from lesions and biopsy.
• All specimens must be transported to the laboratory without any delay to prevent bacterial
overgrowth. Incase of delay specimens except skin specimen, blood and CSF may be
refrigerated for a short period.
• Infected hairs may be plucked using forceps. Those hairs that fluoresce under Wood’s lamp
may be selectively plucked. Hairs may be collected in sterilized paper envelopes.
• Surface of the skin must be disinfected with spirit before specimen collection. The
advancing edge of the lesion is scraped with the help of a blunt forceps and collected in
sterilized paper envelopes.
• Discoloured or hyperkeratotic areas of nail may be scraped or diseased nail clipping may be
collected in sterilized paper envelopes.
• Specimens from mucus membranes (oral) must be collected by gentle scraping and
transported to laboratory in sterile tube containing saline. Swabs may be collected from
vagina.
• Corneal scrapings may be collected using a fine needle and inoculated at bedside.
• Pus may be collected by aspiration; use of cotton swabs may give false positive microscopic
results.
• Clean catch urine may be collected in a sterile wide-mouthed container.
• Biopsy specimens must be transported in saline.
In certain cases, pus or exudates must be looked for presence of granules.

Microscopy:
Microscopy is used to observe clinical specimens for the presence of fungal elements or to
identify the fungus following culture. In the latter case, lactophenol cotton blue is stain of
choice, which stains the fungal elements blue. Direct examination of clinical specimens could
be stained or unstained.
• Wet mount: Candida may be observed in urine wet mounts
• 10-20% KOH mount: Several specimens are subjected to KOH mount for direct
examination. The material is mixed with 20% KOH on a slide and a cover slip is placed. The
slide is then gently heated by passing through the flame 2-3 times. The slide is observed on

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cooling. KOH serves to digest the protein debris and clears keratinised tissue and increases
the visibility. Addition of Dimethyl sulphoxide (DMSO) permits rapid clearing in the absence
of heat.
• Calcofluor white: This is a fluorescent dye, which binds selectively to chitin of the fungal
cell wall. The specimen then can be observed under fluorescent microscope.
• India Ink: Capsules of Cryptococcus neoformans can be demonstrated by this negative
staining technique.
• Periodic Acid-Schiff (PAS) stain: On staining by this stain, fungal elements appear bright
magenta coloured while the background stains green. It is useful in staining tissue specimens.
• Giemsa’s stain: It is particularly useful in the detection of Histoplamsa capsulatum in the
bone marrow smears.
• Haematoxylin and Eosin (H&E) stain: Useful for staining tissue sections.
• Gomori’s methenamine silver nitrate (GMS) stain: Outlines of the fungi are black, internal
parts stain pinkblack while the background stains light green. Candida and Aspergillus may
be missed in H&E stained sections, therefore GMS stained sections are essential for tissue
pathology.
• Gridley’s stain: It stains hyphae and yeasts dark blue-pink, tissues deep blue and
background yellow.
• Meyer mucicarmine stain: Capsules of C. neoformans and inner walls of Rhinosporidium
seeberi’s sporangium are stained pink.
• Gram stain: Candida is best demonstrated in clinical specimen by Gram stain.
• Masson-Fontana stain is helpful in staining phaeoid (dematiaceous) fungi in tissue.
• Immunofluorescence: Monoclonal antibody labelled with fluorescent dyes can be used to
detect several fungi in the clinical specimens.

Culture:
One of the most common media used to culture fungi in laboratory is Sabouraud’s Dextrose
Agar (SDA). It consists of peptone, dextrose and agar. High concentration of sugar and a low
pH (4.5-5.5) prevents growth of most bacteria and makes it selective for fungi. Emmon’s
modification of SDA contains 2% dextrose and has pH of 6.8.
Other basal media to grow fungi include Potato Dextrose Agar, Malt Extract Agar etc. Most
fungi are able to grow at room temperature while few pathogenic fungi (e.g, Cryptococcus,
dimorphic fungi) can grow at 37oC. Saprophytic fungi grow much quickly than pathogenic
fungi (e.g, dermatophytes). In such situations the saprophytic fungi can be inhibited by the
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addition of cycloheximide (actidione) to the SDA. Addition of antibiotics such as
Chloramphenicol,
Gentamicin or Streptomycin to SDA serves to inhibit bacterial multiplication. An example of
SDA with cycloheximide and Chloramphenicol is Mycosel agar.
Other specialized media used for different fungi include:
• Brain Heart Infusion Agar general isolation of fungi and conversion of dimorphic fungi.
• Inhibitory Mould Agar, an isolation medium with Chloramphenicol to suppress most
bacteria.
• Caffeic Acid Agar and Birdseed Agar for isolation of Cryptococcus neoformans.
• Corn Meal Agar: Enhances production of chlamydospores in Candida albicans and
formation of conidia in fungi.
• Trichophyton Agars: Used for selective identification of Trichophyton species.
• Dermatophyte Test Medium: Used for recovery of dermatophytes from clinical specimens.
• Sabhi Medium: Isolation of Histoplasma capsulatum.
• ‘CHROM agar Candida’ is useful in identification of Candida species.
Conversion of mould to yeast phase must be demonstrated in vitro for identification of
dimorphic fungi. Since some fungi grow slowly cultures should not be discarded for 4-6
weeks. Fungi are identified on the basis of colony morphology (including pigmentation) and
microscopic observation by tease-mount preparation or slide culture technique.

Serology:
Detection of anti-fungal antibody is helpful in diagnosis of sub-cutaneous and systemic
mycoses, prognosis and response to anti-fungal drugs. Different serologic techniques that are
used include agglutination,
immunodiffusion, counter-immunoelectrophoresis, complement fixation test,
immunofluorescence, RIA and ELISA.
Antigen detection: It is particularly useful in the diagnosis of cryptococcal meningitis from
CSF specimens. The test is performed by Latex Agglutination or immunodiffusion tests. It is
also helpful in the detection of Aspergillus and Candida antigens in systemic infections.
Skin tests: Delayed hypersensitivity reactions to fungal antigens can be demonstrated by skin
tests. A positive skin does not necessarily indicate an active infection; it only indicates
sensitization of the individual. Hence, its value is in epidemiological studies than diagnosis.
These tests may be performed in Histoplasmosis, Candidiasis,

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Sporotrichosis, Coccidioidomycosis, Blastomycosis, Paracoccidiodomycosis and
dermatophytosis.
Molecular techniques:
Newer techniques such as DNA hybridization, PCR are useful in diagnosis of mycoses in a
shorter period as well as detect those fungi that are difficult or dangerous to cultivate in vitro.

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