Principles of Fungous Disease

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.

3 C ll h T M t


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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

Principles of Fungous Disease

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