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8/13/2019 Principles of Fungous Disease
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PRINCIPLES OF
FUNGOUS DISEASE
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TOPIC SUMMARY
Types of Fungous
Diseases
1. FUNGOUS
ALLERGIES
2. MYCOTOXICOSES
Amatoxins
Phallotoxins
Aflatoxins and other
tumorigenicmycotoxins
3. MYCOSES
Mycosis
Incidence
Portal of entry
Classification
Pathogenesis
Diagnosis
Therapy
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Types of Fungous Diseases
Fung i are able to cause human
dis ease in
3 generalized ways:
1. Allergies may follow
sensitization to specific
fungous antigens
2. Fungi may elaborate or
indirectly generate toxic
substances
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1 FUNGOUS ALLERGIES RTs of humans are
constantly exposed toaerosolized conidia andspores containing potentallergens to w/c someindividuals are sensitive orhypersensitive
Exposure to spores Outdoors: 100,000 spores/m3
Enclosed areas:1,000,000,000/m3
Depending on the site ofdeposition, patients mayexhibit: rhinitis,
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Respiratory AllergiesALLERGY SOURCE ETIOLOGY
Cheese Washers lung Cheese Penici l l ium casei
Maltsterslung Barley malt Asp ergi l lus clavatus
Maple-bark strippers lung Maple tree bark Cryptostroma cort icale
Sequoiosis Redwood sawdust Aureobasid ium pu l lu lans, Graphium
Suberosis Cork Penici l l ium frequentans
Wood-pulp workers disease Wood pulp Alternar ia
Farmers lung Stored hay Faenia rectiv irgu la,Thermoact inom yces vulgar is
Bagassosis Sugar cane Thermoact inom yces sacchari i
Humidifier lung Humidifiers, air conditiners Thermoact inom yces vulgar is,
Thermoact inomyces cand idus
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2 MYCOTOXICOSES
Fungi can generate toxins(secondary metabolites)
secreted directly into the
environment
They include a variety ofmycotoxins elaborated by
mushrooms
Toxicity is due to ingestion,resulting to a disease calledMYCETISMUS
Most common elaborated
toxins: amatoxinsand
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POISONOUS MUS ROOMS
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Amanita mushroom produce
amatoxins and phallotoxins;
most potent mycotoxin is
AMATOXIN
Phallotoxinsnot absorbed by GIT and
are NOT considered as a cause of
mycetismus
Other toxins elaborated by Amanita:
Phalloidinbinds to actin in cellmembranes disrupting the endoplasmic
reticulum
Phalloin
ALPHA, BETA and GAMMA AMANITIN
-amanitin binds to a subunit of
RNA polymerase II and disrupts
protein synthesis
LIVER is the target organ of both
amatoxins and phallotoxins; no antidote
for this type of poisoning, treatment is
supportive
Amanita muscaria
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Human Mycetismus (toxicity due to ingestion)
Site of
Involvement
Etiology Mycotoxin Mechanism of
Action
Symptoms Prognosis and
Treatment
GIT Boletus satanasLactarius torminosus
Lepiota morgani
Russula emetica
unidentified ----------------- Nausea and diarrhea(mild to severe)
Spontaneousrecovery
GIT (cholera type
and parasympathetic
nervous system)
Amanita phalloides
Amanita virosa
Clitocybe species
Inocybe species
Helvella esculenta
Amatoxins
Phallotoxins
Muscarine
Gyromitrin
Cholinergic effect on
smooth muscles and
exocrine glands
Same
GIT toxicity,
hemolysis
(1)Violent vomiting,
diarrhea,
dehydration, muscle
cramps
(2)Renal & hepatic
failure, lacrimation,
salivation twitching,
jaundice, coma
Violent intestinal
upset, perspiration,
salivation
Nausea, vomiting,
diarrhea,
hemoglobinuria,
jaundice
2ndphase treated w/
atropine; often
FATAL; thioctic acid
Same
Self-limiting
CNS Psliocybe cubensis
Psliocybe spp
Psilocybin Hallucination Spontaneous
recovery
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Aflatoxins and Other
Tumorigenic Mycotoxins
Aflatoxin produced by
Asperg i llus f lavus can be
mutagenic and
carcinogenic
Aflatoxin B1most
potent liver carcinogen;
may be present in grains,corn, peanuts, etc..
Aflatox in inpeanut
Af latox in in corn
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3. MYCOSES Fungal infectionactual growth of fungi on a
human or animal host.
Fungal infections are named by couplingmycosisto another word that designates the:
etiologic agent - coccidioidomycosis
Site of involvement- otomycosis
Other mycoses are named by adding thesuffix sis denoting state/ condition such as:
Aspergillossis
Candidiasis
Establishment of mycosis depends on:
State of host defenses
Route of exposure
Size of the inoculum
Virulence
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MYCOSIS
Incidence
Portal of entry
Classification
Pathogenesis
Diagnosis
Therapy
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A. Incidence Not reportable diseases;
prevalence is unknown
However, dermatophytes and
pityriasis versicolor are among
the most common infectiousdiseases globally
Mycoses are always prevalent;
lesions are superficial and
historical descriptions ofringworm date from the olden
times; e.g. curse of
Tutankhamens tomb was
actually residual conidia ofAs er illus
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B. Portal of Entry
1. SKIN - Abraded,burned, macerated orintegrity has beencompromisedprone tomycosis
Skins defenses:1. amino acids and fats in
sebum,
2. hormone-inducedchanges, salinity,
3. pH,
4. Secretion of specificgrowth inhibitors
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2. RESPIRATORY SYSTEM RT is exposed daily to a large
volume of airborne fungi yetthe incidence of respiratorymycoses is low
Factors:
Anatomy of RTdetermines the depth tow/c particles can beinhaled;
size of fungal cells willdelimit the extent ofpenetration:
10 um and abovedeposited on the trachealor nasal epithelium
510 um in dmpenetrate the bronchioles(but may be removed bybronchial secretions)
Less than 5 um- inhaledto the alveoli
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In the alveolus, the fungous cell is confronted by surfactant,humoral serum components, alveolar macrophages, andsubsequent inflammatory response leading to inactivation of thefungus
3. Urogenital tract occasionally bridged by Candidaalbicans
4. GITmay become a source of infection after changes
induced by age, trauma, neoplasm, certain drugs, imbalance innormal flora
5. Iatrogenic inoculation through:
contaminated indwelling catheters,
during surgery, after antibacterial or immunosuppressive
chemotherapy,
administration of steroids,
radiation treatment
Fungi are introduced to the host directly,
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C. Classification of Mycoses
Based on the general body areapredom inant ly involved:
1. Superficial mycoses
2. Cutaneous mycoses
3. Subcutaneous mycoses
4. Systemic mycoses
5. Opportunistic mycoses
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Clinical Classification of Mycoses
Area of
Predominant
Involvement
Mycosis Etiology
Superficial Pityriasis versicolor
Tinea nigra
White piedra
Black piedra
Malassezia furfur
Phaeoannellomyces werneckii
Trichosporon beigelii
Piedra hortae
Cutaneous Dermatophytosis
Candidiasis of skin, mucosa, or nails
Microsporum species, trichophyton spp, and
Epidermphyton floccosum
Subcutaneous Sporotrichosis
Chromomycosis
Mycetoma
Rhinosporidiosis
Lobomycosis
Subcutaneous phycomycosis
Rhinoentorophthoromycosis
Sporothrix schenckii
Philaphora verrucosa; Fonsecaea pedrosoi
Pseudallescheria boydii, madurella mycetomatis
Rhinosporidium seeberi
Loboa loboi
Basidiobolus haptosporus
Conidiobolus coronatus
Systemic Primary mycosesCoccidioidomycosis
Histoplasmosis
Blastomycosis
Paracoccidioidomycosis
Coccidioides immitis
Histoplasma capsulatum
Blastomyces dermatitidis
Paracoccidioides brasiliensis
Opportunistic Systemic candidiasis
Cryptococcosis
Aspergillis
Mucormycosis
Candida albicans, other candida spp
Cryptococcus neoformans
Aspergillus fumigatus, other aspergillus spp
Species of Rizopus, Absidia, Mucor and others
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Pityriasis versicolor/ Tinea flava
Tinea nigra
Black piedra
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Dermatophytosis
Tinea corporis
Tinea capitis
Tinea barbae
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Tinea cruris/jock itch
Tinea unguium
Tinea pedis/athletes foot
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Subcutaneous Mycoses
Sporotrichosis Chromomycosis Mycetoma
Rhinosporidiosis
Lobomycosis
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Systemic Mycoses
Coccidioidomycosis BlastomycosisHistoplasmosis
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Paracoccidioidomycosis
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Opportunistic Mycoses
Candidiasis Aspergillis Cryptococcosis
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D. Pathogenesis (Table 81 -6, pg 1087Zinnser)
For mycos is to develop , there should be:
1. Contact b/n the host and fungal pathogenthe conditions
of exposure: inoculum size, route, host immunity will determine
infection
2. Inherent virulencetissue reactive enzymes, irritants,
attachment to host cells, antiphagocytic properties, and
inflammatory components
3. In vivo morphology Hyphaepenetrate lumina of vessels and lymphatics
Spherical structures (yeasts, sporangia, sclerotic cells)less
confined and can be transported through circulation
4. Attachment of the fungus to host tissue C. albicans et al have
surface ligands and receptors that facilitate binding
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Some fungi are
completely superficial
(grow on the host w/o
invasion and cause
minimal irritation). Two
examples: Piedraformation of
nodules on hair
Aspergillus spp
colonization of external ear
or nasal sinuses
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E. Diagnosis1. Direct Examination
some are large enough to
be observed in skin
scrapings, tissue biopsy
material, or body fluidsdigested with 10% KOH
For fluorescent stain
calcofluor white for cell
wall Hematoxylin and eosin
tissue stains
PAS, methenamine Ag
fungous cell wall
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Candida
albicans
Histoplasma
capsulatumCryptococcusneoformans
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2. Culture non-sterile specimens like skin scrapings and sputa are planted on
media w/ antibiotic to inhibit bacterial and non-pathogenic fungal
growth
SaboraudsAgar (routine agar for fungal culture)
2-4% glucose, 1% neopeptone, 2% agar
Inhibitory Mold Agar complex medium containing
chloramphenicol and gentamicin to inhibit bacteria
BHI w/ sheep blood isolation of fungi from normally andsterile specimens and when presence of H. capsulatum is
suspected
Routine cultures are incubated @ 2530 deg C and must be
retained for several weeks before reported as negative
Fungous isolates are identified by appropriate morphologic,
physiologic, or antigenic properties
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Candida
albicans
Histoplasma
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3. Serology Mycoserologic techniques include
measurement of specific antibodies, antigens,and delayed and immediate hypersensitivity
Evaluation of the number and functional T cell
subpopulations, immunoglobulin classes, and
lymphokine production
Polymerase chain reaction (PCR)rapid
direct amplification of fungous DNA
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F. Therapy
Polyenesformation of complexes w/ergosterol in cell membrane; Amphotericin B
Flucytosine(5-fluorocytosine),
Azoles(imidazoles and triazoles)interfere w/the synthesis of ergosterol by blockingcytochrome P-450-dependent 14 -demethylation of lanosterol (precursor ofergosterol and cholesterol)
Page 1089 Table 81 8 Zinnser
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R i t T t
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Respiratory Tract
Secretions
Sputum, induced sputum,Bronchial washings,
Bronchoalveolar lavage,
Tracheal aspirationsmost
commonly subm i ttedspecimens for fungal
cul ture
Cycloheximideantifungal agent that shd be
included in the culture
medium to prevent mold
overgrowth
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Cerebrospinal Fluid
CSF for fungal culture shd be filtered
through a 0.45 membrane filter attached to a
sterile syringe
Cultures shd be examined and moved to
another location daily
If less than 1ml, it shd be centrifuged and 1
drop aliquots of the sediment shd be placed
onto several areas on the agar surface
Media used shd not contain antibacterial or
antifungal agents
CSF shd be examined immediately; if not
possible, it shd be kept @ RT or placed in a
30 deg C incubator
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Blood Blood cultures provide an accurate method
for determining the etiology of fungaldiseases
Automated blood culture systems: BACTEC
ESP
Labs w/ high incidence of dimorphic fungiuse the lysis centrifugation systemtheIsolator.Isolator has been shown to bethe optimal recovery medium of H.capsulatum and other filamentous fungi
Isolatorrbcs, wbcs w/c may contain theorganism are lysed and centrifugationconcentrates the organisms beforeculturing
Optimum temp for culturing is 30 deg C for
21 days
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Hair, Skin and Nail Scrapings For dermatophyte culture; usually
contaminated w/ bacteria
Samples from lesions may be obtainedby scraping the affected area w/ ascalpel blade or glass slide;
Infected hairs are plucked with forceps
Specimens are placed in sterilecontainer; THEY SHD NOT BEREFRIGERATED
MYCOSEL AGAR w/ chloramphenicoland cycloheximide is a satisfactoryculture medium
Cultures are incubated @ 30 deg C for aminimum for 21 days
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Microscopic Exam of Skin Scrapings
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Urine
Shd be processed ASAP
24-hr urine sample is
unacceptable for culture
All urine samples shd be
centrifuged and the sediment is
cultured for adequate isolation
of colonies
Use culture media w/
antibacterial agents to eliminate
Gram (-) bacteria
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Tissue, Bone Marrow, and Sterile
Body Fluids All tissues shd be processed before
culturing by mincing, grinding or
placement in a Stomacher.
The Stomacher expresses
cytoplasmic contents by pressureexerted from the action of rapidly
moving metal paddles against the
tissue in a broth suspension; after
processing, at least 1ml of specimen
shd be spread on the culturemediums surface; incubated @ 30
deg C for 21 days
Portions of tissue shd be inoculated
on culture medium (not just thebroth
Stomacher
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May be placed directly on the
culture medium w/ similar
incubation conditions to that oftissues
sterile body fluids are
centrifuged before culture, andat least 1ml shd be placed on
culture medium
Or, place bone marrow andother body fluids in an Isolar
tube and process it as blood
culture
All specimens are cultured
CULTURE MEDIA AND INCUBATION
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CULTURE MEDIA AND INCUBATION
REQUIREMENTS
For optimal recovery, a battery of media shd be used:1. Media w/ and w/o cycloheximide
2. Media w/ and w/o an antibacterial agent (for sterile fluids)
Plates are preferred than tubes since they provide better
aeration of cultures
Fungal cultures shd be incubated for 2 to 4 wks and examined3x a week during its incubation.
Aside from chloramphenicol and cycloheximide, a
combination of 5 ug/ml of gentamicin and 16ug/ml ofchloramphenicol are recommended. 5ug ciprofloxacin/mlmay also be used.
Most fungi grow best and more rapidly on 30degC. RT(25degC) is also acceptable if a 30deg C incubator is not
available.
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Fungi prefer moisture and increased
humidity for growth. All plates shd be
sealed w/ parafilm or oxygen
permeable tape. A pan of water is
placed in the incubator to providehumidity.
The macroscopic examination of
fungal colonies includes:
1. Rate of growth
2. General topographybestobserved on the reverse side. Flat,
heaped, folded, rugose, umbonate,
wrinkled/verrucose
3. Texture-best observed in a cross
section; related to the length of
aerial hyphae;
4. Pigmentation-surface and
reverse sides
Top Reverse
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Culture media should include:
NITROGEN SOURCE CARBON SOURCE
Nitrate Glucose
Nitrite Vitamins
Amino acids minerals
Urea
C f
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Fungal Culture Media: Indications for Use
MEDIA INDICATIONS FOR USEPrimary Recovery MediaBHI Saprobic and pathogenic fungi
BHI agar w/ antibiotics Dermatophytes
BHI biphasic blood culture bottles Fungi from blood
Dermatophyte test medium Dermatophytes (screening medium)
Inhibitory Mold Agar Dermatophytes
Potato flake agar Saprobic and pathogenic fungi
Mycosel Dermatophytes
SABHI agar Saprobic and pathogenic fungi
Yeast-Extract PO4 agar Dermatophytes
Media Indications for Use
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Media Indications for Use
Differential Test Media
Ascospore agar Ascospores in Saccharomyces
Cornmeal agar w/ Tween 80 and
trypan blue
C. albicans by chlamydospore prodn; C. albicans by
microscopic morphology
Cottonseed conversion agar Conversion of dimorphic fungus B. dermatitidis frommold to yeast form
CzapeksAgar Aspergillus spp.
Niger seed agar C. neoformans
Nitrate reduction medium NO3 reduction in confirmation of Cryptococcus
Potato dextrose agar Pigment production by T. rubrum; preparation ofmicroslide cultures and sporulation of dermatophytes
Rice medium M. audouinii
Trichophyton agars 1-7 Trichophyton genus
Urea agar Cryptococcus spp; differentiate T. mentagrophytes from T.rubrum; trichosporon spp.
Yeast fermentation broth Yeasts by determining fermentation
Yeast nitrogen base agar Yeasts by determining CHO assimilation
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PRIMARY ISOLATION MEDIACulture Media Indication/Purpose
Sabouraud Dextrose Agar
(SDA)
Primary recovery of saprobic and pathogenic fungi
SDA w/ cycloheximide and
chloramphenicol (SDA CC)
Recovery of pathogenic fungi; bacteria and saprophytic fungi
and inhibited
Mycosel or Mycobiotic agar Isolation of dermatophytes from hair, skin, and nail specimens;
w/ cycloheximide and chloramphenicol
Dermatophyte Test Medium
(DTM)
Isolation of dermatophytes from hair, skin and nail specimens;
w/ antibiotics inhibiting bacteria and saprophytic; fungi produce
yellow colonies
BHI agar Isolation of saprophytic and pathogenic fungi from sterile sites;
bacterial growth is not inhibited
BHI agar w/ cycloheximide
and chloramphenicol
Isolation of dermatophytes; bacteria and saprophytic fungi are
inhibited
BHI biphasic blood culture
bottles
Recovery of fungi from blood or bone marrow
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DIFFERENTIAL TEST MEDIACulture Media Indication/Purpose
Birdseed/ Niger Seed Agar Isolation of Cryptococcus neoformans; black brown colonies due to
melanin prodn (phenol oxidase breaks down the medium)Urea Agar Detection of urease prodn by C. neoformans.
Differentiation of T. mentagrophytes from T. rubrum
Nitrate Reduction Medium Confirmation of NO3 reduction in C. neoformans.
Cornmeal Agar w/ Tween 80 Stimulation of conidiation and chlamydospore prodn in Candida spp;
differentiation of Candida spp.
Potato Dextrose Agar Stimulation of conidiation; useful in slide cultures; demonstration of
pigment prodn of T. rubrum
Cottonseed Agar Conversion of mold to yeast phase of Blastomyces dermatitidis
Rice Medium Identification of Microsporum audouinii
Yeast Assimilation Media
(carbon or nitrogen)
Detection of CHO assimilation through utilization of carbon/nitrogen by
yeast in the presence of oxygen
Yeast Fermentation Broth Identification of yeasts by fermentation rxns w/ various CHOs
Trichophyton agars Nutritional requirement tests for the differentiation of Trichophyton spp.
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DIRECT MICROSCOPIC EXAM
Calcofluor white stain is
superior than KOH; using
fluorescent microscope
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Skin scrapings showing the
presence of fungal elements inKOH preparation
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2. Cellufluor
Brightening agent, canbe added to KOH
solution
Binds to chitin andprovides excellent
contrast when seen in
fluorescent mx
3 I di I k Ni i
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3. India Ink or Nigrosin Used to demonstrate the capsule
of Cryptococcu s neoformans.
CSF can be directly examined forthe presence of C. neoformans:one drop of CSF to one drop ofIndia ink
Preparation is examined under oilimmersion; capsule appears as aclear halo against a darkbackground
This method, however, has beenreplaced by direct Ag testing dueto difficulty in interpreting resultsi.e. wbcs may be mistaken forcapsules; cryptococcal infections
in AIDS may not be detected inthis method
Cryptoc occ al smear Ind ia
ink p reparat ion b r ings
out the prominenttranslucent capsule of
the organisms.
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5 H k M difi ti f G St i
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5. Hucker Modification of Gram Stain
Fungi may appear Gram
positive (but very poorly);so Hucker stain is used as
the primary stain, then
follow Gram stain
procedure
C. neofo rmans may
appear pale lavender w/
blue inclusions (capsule
prevents adequate
staining)
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6. Giemsa or Wrights Staining
Used for the detection of
intracellular Histoplasmacapsulatum in:
Blood smears
Lymph nodes
Lung, liver, or bone marrow
H. capsu latum appears as asmall, oval yeast, light to dark
blue in color
Can also be used in stainingC. neoformans
Cryptococcus neoformans.Wrights stain.
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7. Methenamine-Silver Nitrate Staining
Useful for the screening ofclinical specimens
Provides good contrast and
staining of fungal elements
Fungi appear outlined inblack against a pale
background
Gomori methenamine-silvernitrate modificationused
to detect fungi in histological
specimens
Cryptococcus neoformans.
Gomori Methenamine silver
stain.
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8. Periodic acid-Schiff (PAS)
Stains the hyphae of moldsand some yeasts
Principle
PAS oxidizes the hydroxylin CHOs of the cell wall toform aldehydes;
Aldehydes react w/ basicfuchsin to form PINK-
PURPLEcomplex A counterstain of fast
green can be used toprovide contrast
Malassezia furfu r in stratum
corneum, periodic acid-Schiff
stain
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Extent of Identification of Fungi
recovered from clinical
specimens
All yeasts shd be screened for the presence of C.neoformans
All respiratory secretions shd be cultured; theymight contain: H. capsulatum,
B. dermatidis,
Coccidioides immitis, etc..
Summary of Methods for Direct Examination of Fungion Table 50-7 pg 646 Bailey & Scott
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MICROSCOPIC EXAMINATION OFFUNGAL GROWTHS When fungal cul tures s tar t to g row, mxexaminat ion is used to observe con id ia and
spores. Ident i f ication m ethods inc lude:
1. Tease mount
2. Slide culture3. Cellophane tape mount
1 T M t
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1. Tease Mount Purpose: allows for the rapid examination of conidia, spores
and other microscpic fungal structures.
Procedure:
1. Remove a small portion of a fungal colony using a bent dissecting
needle or wire.
2. Place a drop of lactophenol cotton blue (LPCB) on the slide. Placethe culture into the stain.
3. Place a coverslip over the culture and using a pencil eraser, press
down gently to disperse the mycelium. OR, using two dissecting
needles, gently tease apart the mycelium and then add the
coverslip.
4. Observe slide under low and high dry magnification for fungal
characteristics.
Disadvantage: disrupts arrangement of spores due to teasing or
pressure in applying the coverslip
2 Slid C lt
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2. Slide Culture Purpose: most accurate method to
preserve and observe fungalmicrostructure; allows preservation of
fungi in its original state
Procedure:
a small block of agar is inoculated w/ thesuspected fungi and placed on a slide.
This slide is laid on a bent glass rod in a
sterile petri plate w/ a piece of filter
paper.
Growth is examined microscopicallyusing LPCB.
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3. Cellophane Tape Mount Purpose: examination of fungal
morphology
Procedure:
Application of double-sticky tape or
cellophane tape looped back on itself to
the surface of the fungal colony Aerial hyphae will adhere to the tape and
examined w/ LPCB