PRINCIPLES OF FUNGOUS DISEASE

TOPIC SUMMARY
Types of Fungous Diseases
FUNGOUS ALLERGIES 2. MYCOTOXICOSES Amatoxins Phallotoxins Aflatoxins and other tumorigenic mycotoxins 3. MYCOSES
1.

Mycosis Incidence Portal of entry Classification Pathogenesis Diagnosis Therapy

Types of Fungous Diseases

Fungi are able to cause human disease in 3 generalized ways:
1.

Allergies may follow sensitization to specific fungous antigens

2.

Fungi may elaborate or indirectly generate toxic substances

1. FUNGOUS ALLERGIES
RTs of humans are

constantly exposed to aerosolized conidia and spores containing potent allergens to w/c some individuals are sensitive or hypersensitive
Exposure to spores
Outdoors: 100,000 spores

/m3 Enclosed areas: 1,000,000,000/m3

Depending on the site of

deposition, patients may exhibit:

rhinitis,

Respiratory Allergies
ALLERGY
Cheese Washers lung Maltsters lung Maple-bark strippers lung Sequoiosis Suberosis Wood-pulp workers disease Farmers lung

SOURCE
Cheese Barley malt Maple tree bark Redwood sawdust Cork Wood pulp Stored hay

ETIOLOGY
Penicillium casei Aspergillus clavatus Cryptostroma corticale Aureobasidium pullulans, Graphium Penicillium frequentans Alternaria Faenia rectivirgula, Thermoactinomyces vulgaris Thermoactinomyces saccharii Thermoactinomyces vulgaris, Thermoactinomyces candidus

Bagassosis Humidifier lung

Sugar cane Humidifiers, air conditiners

2. MYCOTOXICOSES
Fungi can generate toxins

(secondary metabolites) secreted directly into the environment

They include a variety of

mycotoxins elaborated by mushrooms

Toxicity is due to ingestion,

resulting to a disease called MYCETISMUS

Most common elaborated toxins: amatoxins and

POISONOUS MUSHROOMS

Amanita mushroom produce amatoxins and phallotoxins;

most potent mycotoxin is

AMATOXIN

Phallotoxins

not absorbed by GIT and are NOT considered as a cause of mycetismus

Other toxins elaborated by Amanita:

Phalloidin

binds to actin in cell membranes disrupting the endoplasmic reticulum

Phalloin ALPHA, BETA and GAMMA AMANITIN

-amanitin binds to a subunit of RNA polymerase II and disrupts protein synthesis

Amanita muscaria

LIVER is the target organ of both

amatoxins and phallotoxins; no antidote for this type of poisoning, treatment is supportive

Human Mycetismus (toxicity due to ingestion)

Site of Involvement
GIT

Etiology
Boletus satanas Lactarius torminosus Lepiota morgani Russula emetica Amanita phalloides Amanita virosa

Mycotoxin
unidentified

Mechanism of Action
-----------------

Symptoms
Nausea and diarrhea (mild to severe)

Prognosis and Treatment

Spontaneous recovery

GIT (cholera type and parasympathetic nervous system)

Amatoxins Phallotoxins

Cholinergic effect on smooth muscles and exocrine glands

(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 Hallucination

2nd phase treated w/ atropine; often FATAL; thioctic acid

Clitocybe species Inocybe species Helvella esculenta

Muscarine

Same

Same

Gyromitrin

GIT toxicity, hemolysis

Self-limiting

CNS

Psliocybe cubensis Psliocybe spp

Psilocybin

Spontaneous recovery

Aflatoxins and Other Tumorigenic Mycotoxins

Aflatoxin produced by

Aspergillus flavus can be mutagenic and carcinogenic

Aflatoxin in corn
Aflatoxin B1 most

potent liver carcinogen; may be present in grains, corn, peanuts, etc..

Aflatoxin in peanut

3. MYCOSES
Fungal infection actual growth of fungi on a

human or animal host.

Fungal infections are named by coupling
etiologic agent - coccidioidomycosis Site of involvement- otomycosis

mycosis to another word that designates the:

Other mycoses are named by adding the

Aspergillossis Candidiasis

suffix sis denoting state/ condition such as:

Establishment of mycosis depends on:

State of host defenses Route of exposure Size of the inoculum Virulence

MYCOSIS
Incidence Portal of entry

Classification Pathogenesis
Diagnosis Therapy

A. Incidence
Not reportable diseases;

prevalence is unknown
However, dermatophytes and

pityriasis versicolor are among the most common infectious diseases globally
Mycoses are always prevalent;

lesions are superficial and historical descriptions of ringworm date from the olden times; e.g. curse of Tutankhamens tomb was actually residual conidia of Aspergillus

B. Portal of Entry
1. SKIN - Abraded,

burned, macerated or integrity has been compromised prone to mycosis

Skins defenses:
amino acids and fats in sebum, 2. hormone-induced changes, salinity, 3. pH, 4. Secretion of specific growth inhibitors
1.

2. RESPIRATORY SYSTEM

RT is exposed daily to a large volume of airborne fungi yet the incidence of respiratory mycoses is low

Factors:

Anatomy of RT determines the depth to w/c particles can be inhaled; size of fungal cells will delimit the extent of penetration:

10 um and above deposited on the tracheal or nasal epithelium 5 10 um in dm penetrate the bronchioles (but may be removed by bronchial secretions) Less than 5 um- inhaled to the alveoli

 In the alveolus, the fungous cell is confronted by surfactant,

humoral serum components, alveolar macrophages, and subsequent inflammatory response leading to inactivation of the fungus

3. Urogenital tract occasionally bridged by Candida albicans

4. GIT may become a source of infection after changes

induced by age, trauma, neoplasm, certain drugs, imbalance in normal 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,

C. Classification of Mycoses
Based on the general body area

predominantly involved:
1. Superficial mycoses 2. Cutaneous mycoses 3. Subcutaneous mycoses 4. Systemic mycoses 5. Opportunistic mycoses

Clinical Classification of Mycoses

Area of Predominant Involvement
Superficial

Mycosis
Pityriasis versicolor Tinea nigra White piedra Black piedra Dermatophytosis Candidiasis of skin, mucosa, or nails Sporotrichosis Chromomycosis Mycetoma Rhinosporidiosis Lobomycosis Subcutaneous phycomycosis Rhinoentorophthoromycosis Primary mycoses Coccidioidomycosis Histoplasmosis Blastomycosis Paracoccidioidomycosis Systemic candidiasis Cryptococcosis Aspergillis Mucormycosis

Etiology
Malassezia furfur Phaeoannellomyces werneckii Trichosporon beigelii Piedra hortae Microsporum species, trichophyton spp, and Epidermphyton floccosum Sporothrix schenckii Philaphora verrucosa; Fonsecaea pedrosoi Pseudallescheria boydii, madurella mycetomatis Rhinosporidium seeberi Loboa loboi Basidiobolus haptosporus Conidiobolus coronatus

Cutaneous

Subcutaneous

Systemic

Coccidioides immitis Histoplasma capsulatum Blastomyces dermatitidis Paracoccidioides brasiliensis Candida albicans, other candida spp Cryptococcus neoformans Aspergillus fumigatus, other aspergillus spp Species of Rizopus, Absidia, Mucor and others

Opportunistic

Tinea nigra

Pityriasis versicolor/ Tinea flava

Black piedra

Dermatophytosis

Tinea capitis

Tinea corporis

Tinea barbae

Tinea unguium

Tinea cruris/jock itch

Tinea pedis/athletes foot

Subcutaneous Mycoses

Sporotrichosis

Chromomycosis

Mycetoma

Rhinosporidiosis

Lobomycosis

Systemic Mycoses

Coccidioidomycosis

Histoplasmosis

Blastomycosis

Paracoccidioidomycosis

Opportunistic Mycoses

Candidiasis

Aspergillis

Cryptococcosis

D. Pathogenesis
Zinnser)

(Table 81 -6, pg 1087

For mycosis to develop, there should be:

1.

Contact b/n the host and fungal pathogen the conditions of exposure: inoculum size, route, host immunity will determine infection
Inherent virulence tissue reactive enzymes, irritants, attachment to host cells, antiphagocytic properties, and inflammatory components In vivo morphology

2.

3.

Hyphae penetrate 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

Some fungi are completely superficial (grow on the host w/o invasion and cause minimal irritation). Two examples:

Piedra formation of nodules on hair Aspergillus spp colonization of external ear or nasal sinuses

E. Diagnosis
1. Direct Examination

some are large enough to be observed in skin scrapings, tissue biopsy material, or body fluids digested with 10% KOH For fluorescent stain calcofluor white for cell wall Hematoxylin and eosin tissue stains PAS, methenamine Ag fungous cell wall

Candida albicans

Cryptococcus neoformans

Histoplasma capsulatum

2. Culture

non-sterile specimens like skin scrapings and sputa are planted on media w/ antibiotic to inhibit bacterial and non-pathogenic fungal growth

Saborauds Agar (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 and

sterile specimens and when presence of H. capsulatum is suspected

Routine cultures are incubated @ 25 30 deg C and must be retained for several weeks before reported as negative
Fungous isolates are identified by appropriate morphologic, physiologic, or antigenic properties

Candida albicans

Histoplasma

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

F. Therapy
Polyenes formation of complexes w/

ergosterol in cell membrane; Amphotericin B

Flucytosine (5-fluorocytosine),

Azoles (imidazoles and triazoles) interfere w/

the synthesis of ergosterol by blocking cytochrome P-450-dependent 14 demethylation of lanosterol (precursor of ergosterol and cholesterol)

Page 1089 Table 81-8 Zinnser

GENERAL CONSIDERATIONS FOR THE LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS

Collection, Transport, and Culturing of Clinical Specimens

A. SPECIMENS Respiratory tract secretions Cerebrospinal fluid Blood Hair, skin, and nail scrapings Urine Tissue, bone marrow, and sterile body fluids B. CULTURE MEDIA and INCUBATION REQUIREMENTS

Respiratory Tract Secretions

Sputum, induced sputum,

Bronchial washings, Bronchoalveolar lavage, Tracheal aspirations most commonly submitted specimens for fungal culture
Cycloheximide

antifungal agent that shd be included in the culture medium to prevent mold overgrowth

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

Blood
Blood cultures provide an accurate method

for determining the etiology of fungal diseases

Automated blood culture systems: BACTEC ESP

Labs w/ high incidence of dimorphic fungi

use the lysis centrifugation system the Isolator. Isolator has been shown to be the optimal recovery medium of H. capsulatum and other filamentous fungi organism are lysed and centrifugation concentrates the organisms before culturing

Isolator rbcs, wbcs w/c may contain the

Optimum temp for culturing is 30 deg C for

21 days

Hair, Skin and Nail Scrapings

For dermatophyte culture; usually

contaminated w/ bacteria
Samples from lesions may be obtained

by scraping the affected area w/ a scalpel blade or glass slide;

Infected hairs are plucked with forceps

Specimens are placed in sterile

container; THEY SHD NOT BE REFRIGERATED

MYCOSEL AGAR w/ chloramphenicol

and cycloheximide is a satisfactory culture medium

Cultures are incubated @ 30 deg C for a

minimum for 21 days

Microscopic Exam of Skin Scrapings

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

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 pressure exerted 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 culture mediums surface; incubated @ 30 deg C for 21 days
Portions of tissue shd be inoculated

Stomacher

on culture medium (not just the broth)

 May be placed directly on the

culture medium w/ similar incubation conditions to that of tissues

sterile body fluids are

centrifuged before culture, and at least 1ml shd be placed on culture medium
Or, place bone marrow and

other body fluids in an Isolar tube and process it as blood culture

All specimens are cultured

CULTURE MEDIA AND INCUBATION REQUIREMENTS

For optimal recovery, a battery of media shd be used: 1. 2.

Media w/ and w/o cycloheximide 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 examined 3x a week during its incubation.
Aside from chloramphenicol and cycloheximide, a combination of 5 ug/ml of gentamicin and 16ug/ml of chloramphenicol are recommended. 5ug ciprofloxacin/ml may 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.

 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 provide humidity.

The macroscopic examination of fungal colonies includes:

1.

Top

Reverse

Rate of growth

2.

General topography best

observed 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; Pigmentation-surface and reverse sides

4.

 Culture media should include:

NITROGEN SOURCE Nitrate Nitrite Amino acids Urea CARBON SOURCE Glucose Vitamins minerals

Fungal Culture Media: Indications for Use

MEDIA INDICATIONS FOR USE

Primary Recovery Media

BHI BHI agar w/ antibiotics BHI biphasic blood culture bottles Dermatophyte test medium Saprobic and pathogenic fungi Dermatophytes Fungi from blood Dermatophytes (screening medium)

Inhibitory Mold Agar

Potato flake agar Mycosel SABHI agar Yeast-Extract PO4 agar

Dermatophytes
Saprobic and pathogenic fungi Dermatophytes Saprobic and pathogenic fungi Dermatophytes

Media Differential Test Media

Ascospore agar Cornmeal agar w/ Tween 80 and trypan blue Cottonseed conversion agar Czapeks Agar Niger seed agar Nitrate reduction medium

Indications for Use

Ascospores in Saccharomyces C. albicans by chlamydospore prodn; C. albicans by microscopic morphology

Conversion of dimorphic fungus B. dermatitidis from mold to yeast form Aspergillus spp. C. neoformans NO3 reduction in confirmation of Cryptococcus

Potato dextrose agar

Rice medium Trichophyton agars 1-7

Pigment production by T. rubrum; preparation of microslide cultures and sporulation of dermatophytes

M. audouinii Trichophyton genus Cryptococcus spp; differentiate T. mentagrophytes from T. rubrum; trichosporon spp. Yeasts by determining fermentation Yeasts by determining CHO assimilation

Urea agar
Yeast fermentation broth Yeast nitrogen base agar

PRIMARY ISOLATION MEDIA

Culture Media
Sabouraud Dextrose Agar (SDA)
SDA w/ cycloheximide and chloramphenicol (SDA CC) Mycosel or Mycobiotic agar

Indication/Purpose
Primary recovery of saprobic and pathogenic fungi

Recovery of pathogenic fungi; bacteria and saprophytic fungi and inhibited Isolation of dermatophytes from hair, skin, and nail specimens; w/ cycloheximide and chloramphenicol Isolation of dermatophytes from hair, skin and nail specimens; w/ antibiotics inhibiting bacteria and saprophytic; fungi produce yellow colonies Isolation of saprophytic and pathogenic fungi from sterile sites; bacterial growth is not inhibited Isolation of dermatophytes; bacteria and saprophytic fungi are inhibited Recovery of fungi from blood or bone marrow

Dermatophyte Test Medium (DTM)

BHI agar

BHI agar w/ cycloheximide and chloramphenicol BHI biphasic blood culture bottles

DIFFERENTIAL TEST MEDIA

Culture Media
Birdseed/ Niger Seed Agar Urea Agar Nitrate Reduction Medium Cornmeal Agar w/ Tween 80

Indication/Purpose
Isolation of Cryptococcus neoformans; black brown colonies due to melanin prodn (phenol oxidase breaks down the medium) Detection of urease prodn by C. neoformans. Differentiation of T. mentagrophytes from T. rubrum Confirmation of NO3 reduction in C. neoformans. Stimulation of conidiation and chlamydospore prodn in Candida spp; differentiation of Candida spp. Stimulation of conidiation; useful in slide cultures; demonstration of pigment prodn of T. rubrum Conversion of mold to yeast phase of Blastomyces dermatitidis Identification of Microsporum audouinii Detection of CHO assimilation through utilization of carbon/nitrogen by yeast in the presence of oxygen Identification of yeasts by fermentation rxns w/ various CHOs Nutritional requirement tests for the differentiation of Trichophyton spp.

Potato Dextrose Agar

Cottonseed Agar Rice Medium Yeast Assimilation Media (carbon or nitrogen) Yeast Fermentation Broth Trichophyton agars

DIRECT MICROSCOPIC EXAM

Calcofluor white stain is

superior than KOH; using fluorescent microscope

1. Saline mount
Quick, simple method to observe fungal

elements like budding yeast, hyphae, and pseudohyphae

The specimen is added to 1 drop of NSS, a

coverslip is applied and examined under LPO and HPO

Major disadvantage is lack of contrast

KOH Preparation
KOH is used to dissolve keratin in

skin, hair, or nail specimens Keratin may obscure the fungal elements in these specimens and KOH facilitates the fungal examination A drop of KOH is added to the specimen and warmed lightly to dissolve keratin; allowed to clear and is examined for hyphae, budding yeast and spherules
Hair can also be examined if the

infection is:

Endothrix fungal invasion w/in the

hair shaft Ectothrix infection outside the hair shaft

Skin scrapings showing the presence of fungal elements in KOH preparation

2. Cellufluor
Brightening agent, can

be added to KOH solution

Binds to chitin and

provides excellent contrast when seen in fluorescent mx

3. India Ink or Nigrosin

Used to demonstrate the capsule

of Cryptococcus neoformans.
CSF can be directly examined for

the presence of C. neoformans: one drop of CSF to one drop of India ink
Preparation is examined under oil

immersion; capsule appears as a clear halo against a dark background

This method, however, has been

replaced by direct Ag testing due to difficulty in interpreting results i.e. wbcs may be mistaken for capsules; cryptococcal infections in AIDS may not be detected in this method

Cryptococcal smear India ink preparation brings out the prominent translucent capsule of the organisms.

4. Lactophenol cotton blue (LPCB)

Imparts blue color to the

cell wall One drop of LCPB is added to the specimen, coverslip is applied, and examined microscopically
Can also be used in the
Aspergillus on LPCB

tease preparation (wet mount) and slide cultures, in which a small portion of an actively growing fungal culture can be examined.

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. neoformans may

appear pale lavender w/ blue inclusions (capsule prevents adequate staining)

6. Giemsa or Wrights Staining

Used for the detection of

intracellular Histoplasma capsulatum in:

Blood smears Lymph nodes Lung, liver, or bone marrow

H. capsulatum appears as a

small, oval yeast, light to dark blue in color

Can also be used in staining

Cryptococcus neoformans. Wrights stain.

C. neoformans

7. Methenamine-Silver Nitrate Staining

Useful for the screening of

clinical specimens Provides good contrast and staining of fungal elements Fungi appear outlined in black against a pale background
Gomori methenamine-silver
Cryptococcus neoformans. Gomori Methenamine silver stain.

nitrate modification used

to detect fungi in histological specimens

8. Periodic acid-Schiff (PAS)

Stains the hyphae of molds

and some yeasts

Principle
PAS oxidizes the hydroxyl

in CHOs of the cell wall to form aldehydes; Aldehydes react w/ basic fuchsin to form PINKPURPLE complex A counterstain of fast green can be used to provide contrast

Malassezia furfur in stratum corneum, periodic acid-Schiff stain

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; they might contain:

H. capsulatum, B. dermatidis, Coccidioides immitis, etc..

Summary of Methods for Direct Examination of Fungi

on Table 50-7 pg 646 Bailey & Scott

MICROSCOPIC EXAMINATION OF FUNGAL GROWTHS

When fungal cultures start to grow, mx

examination is used to observe conidia and spores. Identification methods include:

1. Tease mount 2. Slide culture

3. Cellophane tape mount

1. Tease Mount
Purpose: allows for the rapid examination of conidia, spores

and other microscpic fungal structures.

Procedure: 1. 2. 3.

4.

Remove a small portion of a fungal colony using a bent dissecting needle or wire. Place a drop of lactophenol cotton blue (LPCB) on the slide. Place the culture into the stain. 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. 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. Slide Culture
Purpose: most accurate method to

preserve and observe fungal microstructure; allows preservation of fungi in its original state
Procedure: a small block of agar is inoculated w/ the

suspected 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 microscopically using LPCB.

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