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INTRODUCTION TO MYCOLOGY
The term "mycology" is derived from Greek word "mykes" meaning mushroom
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The ability of fungi to invade plant and animal tissue was observed in early 19th century but the first documented
animal infection by any fungus was made by Bassi, who in 1835 studied the muscardine disease of silkworm and
proved the that the infection was caused by a fungus Beauveria bassiana
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He is also regarded as father of medical mycology
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Beneficial Effects of Fungi:
1
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2
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The fermentation property is used for the industrial production of alcohols, fats, citric,
oxalic and gluconic acids
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Important sources of antibiotics, such as Penicillin
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Model organisms for biochemical and genetic studies
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Saccharomyces cerviciae is extensively used in recombinant DNA technology, which includes the Hepatitis
B Vaccine
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Some fungi are edible (mushrooms)
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Yeasts provide nutritional supplements such as vitamins and cofactors
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Penicillium is used to flavour Roquefort and Camembert cheeses
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Ergot produced by Claviceps purpurea contains medically important alkaloids that help in inducing uterine
contractions, controlling bleeding and treating migraine
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Fungi (Leptolegnia caudate and Aphanomyces laevis) are used to trap mosquito larvae in paddy fields and
thus help in malaria control
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Destruction of food, lumber, paper, and cloth
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Animal and human diseases, including allergies
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Toxins produced by poisonous mushrooms and within food (Mycetism and Mycotoxicosis)
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Plant diseases
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Spoilage of agriculture produce such as vegetables and cereals in the godown
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Damage the products such as magnetic tapes and disks, glass lenses, marble statues, bones and wax
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They are eukaryotic; cells contain membrane bound cell organelles including nuclei, mitochondria, golgi
apparatus, endoplasmic reticulum, lysosomes etc
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2
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3
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All fungi
possess cell wall made of chitin
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Are chemoheterotrophs (require organic compounds for both carbon and energy sources) and fungi lack
chlorophyll and are therefore not autotrophic
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Fungi are osmiotrophic; they obtain their nutrients by absorption
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They obtain nutrients as saprophytes (live off of decaying matter) or as parasites (live off of living matter)
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All fungi require water and oxygen and there are no obligate anaerobes
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Typically reproduce asexually and/or sexually by producing spores
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They grow either reproductively by budding or non-reproductively by hyphal tip elongation
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Food storage is generally in the form of lipids and glycogen
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In 1969 R
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Traditionally the classification proceeds in this fashion:
Kingdom - Subkingdom - Phyla/phylum - Subphyla - Class - Order - Family - Genus- Species
This classification is too complicated to be dealt here
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Based on Sexual reproduction:
1
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2
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3
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4
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This is a heterogeneous group of fungi where no sexual reproduction has yet been
demonstrated
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Moulds (Molds): Filamentous fungi Eg: Aspergillus sps, Trichophyton rubrum
2
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Yeast like: Similar to yeasts but produce pseudohyphae Eg: Candida albicans
4
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They exist as yeasts in tissue and in vitro at 37oC and as moulds in their natural habitat and in vitro at room
temperature
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5 million species of fungi
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But, for the
classification sake they are studied as moulds, yeasts, yeast like and dimorphic fungi
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They have rigid cell wall composed of chitin, which may be layered
with mannans, glucans and other polysaccharides in association with polypeptides
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Some fungi such as Cryptococcus and yeast form of Histoplasma capsulatum possess
polysaccharide capsules that help them to evade phagocytosis
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Fungal membranes possess ergosterol in contrast to cholesterol found in mammalian cells
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A unique property of nuclear membrane is that it
persists throughout the metaphase of mitosis unlike in plant and animal cells where it dissolves and re-forms
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Moulds:
The thallus of mould is made of hyphae, which are cylindrical tube like structures that elongates by growth at tips
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It is the hypha that is responsible for the filamentous nature of mould
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They may be septate or aseptate
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These cross walls, called septa have small pores through which cytoplasm is
continuous throughout the hyphae
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With exception
of zygomycetes (Rhizopus, Mucor), all moulds are septate
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When a septate hyphal strand is damaged, the pores
between adjacent compartments can be plugged, thus preventing death of the whole hyphal strand
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Vegetative mycelium are those that penetrates the surface of the medium and absorbs nutrients
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Aerial mycelium are those that grow above the agar surface
3
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Since hypha is the structural unit of mould, the mycelium
imparts colour, texture and topography to the colony
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Examples are
species of Bipolaris, Cladosporium, Exophiala,
Fonsecaea, Phialophora and Wangiella Those hyphae
that don't possess any pigment in their cell wall are
called hyaline
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Some of these are:
a) Spiral hyphae: These are spirally coiled hyphae commonly seen in Trichophyton mentagrophytes
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Commonly seen in Microsporum audouinii
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They occur in Trichophyton schoenleinii and Trichophyton violaceum
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Often seen
in Trichophyton mentagrophytes and Microsporum canis
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Seen in Epidermophyton floccosum, Trichophyton mentagrophytes
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g) There are structures in the hyphae, which arise out of modification of a single cell and transform into thick
walled resting cells
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In some fungi such as Trichosporon beigeilli and Coccidioides immitis some alternating cells
become thick walled and subsequently the intervening cells disintegrate leaving behind arthrospores (or
arthroconidia)
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They reproduce by budding, which result in blastospore
(blastoconidia) formation
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This property is seen in Candia albicans
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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
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Capsules can be demonstrated by negative staining methods using India ink or Nigrosin
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Some yeasts are pigmented
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True yeasts such as Saccharomyces cerviciae don't produce pseudohyphae
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Reproduction in fungi:
Fungi reproduce by asexual, sexual and parasexual means
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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)
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(Taxonomically, the teleomorph or the
holomorph is used, but practically it is more convenient to use the anamorph
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Asexual spores are
produced following mitosis where as sexual spores are produced following meiosis
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The sporangiospores result from the mitotic cleavage of cytoplasm in the sporangium
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This endogenous process of spore formation within a sac is known
as sporogenesis
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These develop
following mitosis of a parent nucleus and are formed in any manner except involving cytoplasmic cleavage
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Conidia are
borne on specialised structures called conidiophore
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In blastic development the conidium begins to enlarge and a septum is
formed
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In thallic mode of development the conidium is
differentiated by a septum before its differentiation
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The cell that gives rise to a conidium is called a conidiogenous cell
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In many cases conidiogenous cells are referred as phialides
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The first step in sexual methods of reproduction involves plasmogamy (cytoplasmic fusion of two cells)
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This is
followed by genetic recombination and meiosis
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g
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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
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For sexual reproduction to occur, two compatible isolates are
required
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Ascomycetes produce sexual spores called ascospores in a special sac like cell
known as ascus
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Parasexual reproduction:
Parasexual reproduction, first seen in Aspergillus is known to occur in basidiomycetes, ascomycetes and
deuteromycetes
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Importance of Spores:
A
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4) Allows fungus to survive periods of adversity
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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
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Asexual spores include chlamydoconidia, conidia and sporangiospores
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Sexual reproduction involves producing a thick-walled sexual resting
spore called a zygospore
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Entomophthorales: Conidiobolus and Basidiobolus are involved in subcutaneous zygomycosis
2
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Hyphae are dikaryotic and can often be distinguished by the
presence of clamp connections over the septa
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Occasional species produce conidia but most are sterile
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Teleomorph of Cryptococcus neoformans, which is Filobasidiella neoformans
2
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Mushroom poisoning by Aminita, Lepiota, Coprinus and Psilocybe etc
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Hyphae are septate with simple septal pores
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Sexual reproduction is by the formation of endogenous ascospores, typically eight, in an
ascus
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Teleomorphs of known pathogenic fungi e
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Arthroderma (of Trichophyton and Microsporum), Ajellomyces
dermatitidis (of Blastomyces dermatitidis), Pseudallescheria boydii (of Scedosporium apiospermum)
2
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Agents of black piedra, like Piedraia hortae
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As their teleomorph continue to be discovered, they would be classified among the previous
categories, until then this remains an artificial and heterogeneous group
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1
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Depending on the presence of melanin in their cell walls, they may be non-dematiaceous or dematiaceous
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Hyphomycetes: A class of mycelial moulds which reproduce asexually by conidia on hyphae
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This class contains the majority of medically important fungi
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Hyaline hyphomycetes include those conidial fungi, which are not darkly pigmented; colonies
may be colourless or brightly coloured
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3
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Pathogenesis of fungal diseases (Mycoses):
Most fungi are saprophytic or parasitic to plants and are adapted to their natural environment
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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
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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
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The complex interplay between fungal virulence factors and host defence factors will determine if a fungal infection
will cause a disease
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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
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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

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Chemical barriers, such as secretions, serum factors
Most fungi are mesophilic and cannot grow at 37oC
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)
• 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
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The pH, body
temperature and serum factors along with phagocytic cells play an important part in providing non-specific
immunity
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Cell mediated immunity:
Immunity is provided non-specifically be effector cells (polymorphonuclear leucocytes) and professional phagocytes
(monocytes and macrophages) and specifically by T lymphocytes
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Expression of T-cell-mediated immunity to fungi includes:
• delayed-type hypersensitivity
• contact allergy
• chronic granulomatous reactions
Humoral immunity:
Even though antibodies are produced against many fungi, their role in protection is not very clear
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Another component of humoral immunity is the complement, which can act as opsonins and may
even cause damage to their cells through complement activation
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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
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These
reactions are also seen n Candida infections
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Granulomas due to intracellular fungi represent delayed
hypersensivities
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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
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Superficial mycoses
I
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Tinea versicolor
III
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White piedra

2
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Dermatophytosis
II
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Subcutaneous mycoses
I
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Rhinosporidiasis
III
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Sporotrichosis
V
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Lobomycosis
4
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Blastomycosis
II
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Coccidioidomycosis
IV
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Opportunistic mycoses
I
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Cryptococcosis
III
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Other mycoses
I
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Occulomycosis
7
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Mycetism and mycotoxicosis

Laboratory diagnosis of mycoses:
Specimen collection: specimen collection depends on the site affected
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• All specimens must be transported to the laboratory without any delay to prevent bacterial overgrowth
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• Infected hairs may be plucked using forceps
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Hairs may be collected in sterilized paper envelopes
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The advancing edge of the
lesion is scraped with the help of a blunt forceps and collected in sterilized paper envelopes
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• Specimens from mucus membranes (oral) must be collected by gentle scraping and transported to
laboratory in sterile tube containing saline
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• Corneal scrapings may be collected using a fine needle and inoculated at bedside
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• Clean catch urine may be collected in a sterile wide-mouthed container
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In certain cases, pus or exudates must be looked for presence of granules
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In the latter case, lactophenol cotton blue is stain of choice, which stains the fungal
elements blue
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• Wet mount: Candida may be observed in urine wet mounts
• 10-20% KOH mount: Several specimens are subjected to KOH mount for direct examination
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The slide is then gently heated by passing
through the flame 2-3 times
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KOH serves to digest the protein debris and
clears keratinised tissue and increases the visibility
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• Calcofluor white: This is a fluorescent dye, which binds selectively to chitin of the fungal cell wall
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• India Ink: Capsules of Cryptococcus neoformans can be demonstrated by this negative staining technique
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It is useful in staining tissue specimens
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Haematoxylin and Eosin (H&E) stain: Useful for staining tissue sections
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Candida and Aspergillus may be missed in H&E stained
sections, therefore GMS stained sections are essential for tissue pathology
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Meyer mucicarmine stain: Capsules of C
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Gram stain: Candida is best demonstrated in clinical specimen by Gram stain
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Immunofluorescence: Monoclonal antibody labelled with fluorescent dyes can be used to detect several
fungi in the clinical specimens
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It
consists of peptone, dextrose and agar
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5-5
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Emmon’s modification of SDA contains 2% dextrose and has pH of 6
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Other basal media to grow fungi include Potato Dextrose Agar, Malt Extract Agar etc
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g, Cryptococcus, dimorphic fungi) can grow at 37oC
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g, dermatophytes)
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Addition of antibiotics such as Chloramphenicol,
Gentamicin or Streptomycin to SDA serves to inhibit bacterial multiplication
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Other specialized media used for different fungi include:
• Brain Heart Infusion Agar general isolation of fungi and conversion of dimorphic fungi
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• Caffeic Acid Agar and Birdseed Agar for isolation of Cryptococcus neoformans
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• Trichophyton Agars: Used for selective identification of Trichophyton species
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• Sabhi Medium: Isolation of Histoplasma capsulatum
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Conversion of mould to yeast phase must be demonstrated in vitro for identification of dimorphic fungi
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Fungi are identified on the basis of colony
morphology (including pigmentation) and microscopic observation by tease-mount preparation or slide culture
technique
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Different serologic techniques that are used include agglutination,
immunodiffusion, counter-immunoelectrophoresis, complement fixation test, immunofluorescence, RIA and ELISA
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The
test is performed by Latex Agglutination or immunodiffusion tests
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Skin tests: Delayed hypersensitivity reactions to fungal antigens can be demonstrated by skin tests
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Hence, its value is in
epidemiological studies than diagnosis
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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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