myco / viro

FUNGAL DISEASES

Clinical specimens submitted for Fungal Isolation 2004-2006*

Clinical specimen 2004 2005 2006 Total

Respiratory 29 35 34 98

Body fluids 37 17 34 88

Tissues 9 0 23 32

Skin 1 0 0 1

Nails 29 31 20 80

Hair 0 1 0 1

CSF 8 5 8 21

Others 5 20 24 49

TOTAL 118 109 153 380

Fungal Isolates 2004-2006*

Isolate 2004 2005 2006

C.albicans 5.1% 13.8% 11.8%

C.tropicalis 1.8% 3.7% 5.2%

C.parapsilosis 5.1% 6.4% 3.9%

C.glabrata 0.91% 0.91% 3.9%

C.famata 1.8% 4.6% 2.0%

Aspergillus spp. 0.91% 0% 0.65%

Fusarium spp. 0% 0% 0.65%

Data from a Five-year review of Fungal Isolates at UPM-CPH

• Clinical specimens (n=545):

–Skin scrapings

–Nail clippings/scrapings

–Hair

–Exudates

–Biopsy materials


Total clinical specimens tested – 545

Results:

10.8%- (+) for both KOH & culture

59.1%- (-) for both KOH &culture

17.1% - (+)culture, (-) KOH

12.8% - (-) culture, (+) KOH


Fungal isolates :

Trichophyton mentagrophytes

Trichophyton rubrum

Trichophyton tonsurans

Trichophyton schoenlenii

Trichosporon spp.


• Fungal isolates

Microsporum gypseum

Microsporum canis

Epidermophyton flocossum

Candida albicans

Exophiala werneckii


Fungal isolates from biopsy materials and exudates (31.2% positive):

Fonsecaea compactum

Phialophora verrucosa

Exophiala jeanselmei

Madurella grisea

Laboratory diagnosis

Collection, Transport, and Culturing of Clinical specimens

• Respiratory tract secretions (sputum,bronchial washings,BAL & TA)

- culture media with antibacterial antibiotics ; with cycloheximide in

one medium

• CSF – filtered in 0.45m membrane filter

- culture media with no antibiotics

• Blood- automated blood culture systems

(BACTEC, BACT/ALERT,ESP)– adequate for the recovery of yeasts

- lysis- centrifugation system – for dimorphic fungi

Incubation time: 21 daysOptimal temperature: 30C

• Hair, Skin, and Nail Scrapings

-placed in sterile Petri dish or paper envelope before culturing

- Mycosel agar

Incubation time : minimum of 30 days

Incubation temperature: 30C

• Urine - 24-hr urine sample not accepted - centrifuged and sediment used for culture - Culture media with antibacterial agents

Tissue, Bone marrow, and Sterile Body Fluids

- tissues must be processed first (grinding, mincing)

Wipe with water

www.doctorfungus.org

scalpelPaper / envelope

active edge

Skin scraping specimen

Direct Examination

• Wet mount

KOH• KOH– 10% to 30%

– with Parker Superquink blue-black ink

– gentle warming

pa

rk

er

I. DIRECT EXAMINATION:

*10-30% KOH

*Calcofluor white stain

*Histological stains- H&E, PAS

*India Ink

*Wet mount

II. Isolation & Culture

SDA

BHIA/BAP

Media with/without antibiotics

• Macroscopic examination of culture

• Microscopic examination using LPCB

Identification

• Gross color & texture• Microscopic characteristics• Confirm / compare with

Written descriptionsDrawingsphotographs

• III. Biochemical Tests:*Rapid kits for yeasts*Urea test

• IV. Special Tests: *In-vitro hair perforation test

*Germ tube test *Chlamydoconidia formation test

Mycotic Infections

Superficial

Cutaneous

Subcutaneous

Systemic

Opportunistic

*Mycotoxicosis

*Allergies

Superficial mycoses

Disease

SKIN

• Pityriasis versicolor

• Tinea nigra

Causative organisms

• Malassezia furfur

• Exophiala werneckii

Superficial mycoses

Disease

HAIR

• White piedra

• Black piedra

Causative organisms

• Trichosporon beigelii

• Piedraia hortae

Superficial mycoses

Pityriasis versicolor

• Lesion-An-an”

-Hyperpigmented or hypopigmented macular lesions

www.ethnomed.org

Superficial mycoses


• Lesion– scale readily, giving

a chalky branny appearance

– occurs on the trunk, shoulders & arms, face and neck

Modified from www.columbia.edu

Superficial mycoses


KOH of skin scrapings

• clusters of budding yeast-like cells & short angular hyphal forms

• “spaghetti and meat balls”

Superficial mycoses


PAS of skin scrapings• “spaghetti and meat

balls”

Superficial mycoses


• Culture of skin scrapings– Not necessary– diagnostic microscopic

features– SDA overlaid with peanut

oil, olive oil

Superficial mycoses


• Etiologic Agent– Malassezia globosa

lipophilic yeast

part of skin normal flora

Superficial mycoses

Tinea nigra

• Lesion– Gray to black well-

demarcated macular lesions

– most frequently occurring on the palms of the hand

– non-inflammatory & non-scaling lesions

11th.blogspot.com

Superficial mycoses

Tinea nigra

• KOH– pigmented brown to dark

olivaceous (dematiaceous) septate hyphal elements & 2-celled yeast cells

Superficial mycoses

Tinea nigra

• Etiologic agent– Exophiala werneckii

saprophyte found in soil, compost, humus & wood

in humid tropical & sub-tropical regions

Superficial mycoses

Tinea nigra

• Culture on SDA– initially mucoid, yeast-

like & shiny black– with age: aerial

mycelia & dark olive color

Superficial mycoses

Tinea nigra

• Lactophenol cotton blue (LPCB) of culture on SDA– 2-celled, pale brown yeast cells– darkly pigmented septa (annelides)– one cell cylindrical, the other cell is

spindle-shaped– occur in aggregated masses

Superficial mycoses

Piedra

Two varieties

–White piedra

–Black piedra

Superficial mycoses

Black piedra

• Lesion– discrete, hard, gritty,

brown to black concretions / nodules

– infection of hair• scalp hair -common• beard, moustache -

less common• axilla & groin hairs -

rarewww.doctorfungus.org

Superficial mycoses

Black piedra - distribution

Superficial mycoses

Black piedra

• Etiologic agent– Piedraia hortae– source of infection

Superficial mycoses

Black piedra - lab diagnosis

• Direct microscopy– specimen - hair with nodules

– 25% NaOH or KOH

– dark septate hyphae

Superficial mycoses


• Direct microscopy– round to oval asci;

hyaline, curved to fusiform ascospores

Superficial mycoses


• Isolation – medium

– SDA with chloramphenicol

– SDA cycloheximide

Superficial mycoses


• Isolation

-growth very slow-dark brown to black

-greenish brown, short aerial mycelium

Heaped center

Flat periphery

Superficial mycoses

White piedra

• Infection of hair shaft– face, axilla, genitals -

common

– scalp, eyebrows, eyelashes - less common

Superficial mycoses

White piedra

Nodule

• Soft, white, yellowish, beige or greenish nodule

• Discrete• more often coalescent,

forming an irregular transparent sheath

Superficial mycoses

White piedra

• Distribution

– common in S. America & Asia

– sporadic in N. America & Europe

• Etiologic agent

– Trichosporon beigelii or T. cutaneum

Superficial mycoses

White piedra - lab diagnosis

• Microscopic direct examination

– specimen - hair with nodules

– 10% KOH or 25% NaOH + 5% glycerin

– hyaline septate hyphae

– oval or rectangular arthroconidia

– occasional blastoconidia

Superficial mycoses


Isolation

• medium - SDA with

chloramphenicol without

cycloheximide

• growth/culture– rapid

– cream-colored, soft

– membranous, wrinkled radial

furrows, irregular folding

Superficial mycoses


Isolation

• microscopic exam

of culture

– hyaline hyphae

– arthroconidia

– blastoconidia

Superficial mycoses


• Physiological studies– does not ferment carbohydrates– assimilate dextrose, lactose, D-xylose,

inositol

– negative KNO3 assimilation

– urease positive– arbutin is split

Cutaneous mycoses

skin

hair

nails

• No living

tissue

• Host Rxn

to fungus

keratinase

Cutaneous mycoses

Disease

• Dermatophytosis

Causative organisms

• Dermatophytes

Microsporum

Trichophyton

Epidermophyton

ringworm

Cutaneous mycoses

Disease

• Candidiasis of skin, mucous membranes & nails

Causative organisms

• Candia albicans & related species

dermatomycosis Soil fungi (Scytalidium, Fusarium, etc.)

Systemic fungi (Histoplasma, etc)

Clinical Manifestations of Dermatophytes

Cutaneous

Tinea capitis

www.emedicinehealth.com

Scalp, eyebrow, eyelashes

Microsporum &

Trichophyton

Cutaneous

Tinea capitis Endothrix

Ectothrix

Cutaneous

Tinea favosa

• Scutulum

• Mass of mycelia

& epithelial

debris

• Cup shaped

crusts

www.mf.uni-lj.si

Cutaneous

Tinea corporis

www.cut.ee/

• Non-hairy skin

• Rings with scaly

centers

• Rxn vs fungus

Cutaneous

Tinea corporis

• E. floccosum

• Trichophyton

• Microsporum

Cutaneous

Tinea imbricata

Concentric

rings

Trichophyton

concentricum

Cutaneous

Tinea barbae

• Bearded areas of

face & neck

www.merck.com

www.emedicine.com

Cutaneous

Tinea cruris

www.dermnetnz.org

Jock itch

Moist groin

area

E. floccosum,

T. rubrum

Cutaneous

Tinea pedis

www.doctorfungus.org

dermatologie.free.fr

Athlete’s foot

Toe webs & soles,

even nails

Id reaction,

circulating fungal

antigens

Cutaneous

Tinea manuum

www.dermnetnz.org

• Interdigital areas

& palmar surfaces

Cutaneous

Tinea unguium

www.dermnetnz.org

Invasion of nail plate by

dermatophytes

Thickened, discolored &

brittle• Onychomycosis- non

dermatophyte

Yeast etc.

Cutaneous


Septate hypha

Cutaneous

arthrospores

septate hypha


Cutaneous

Ectothrix invasion of hair

• Hair invasion • formation of

arthroconidia on the outside of hair shaft

• cuticle of hair is destroyed

Cutaneous


• Hair invasion by a dermatophyte– Microsporum canis

– M. gypseum

– Trichophyton equinum

– T. verrucosum

Cutaneous


• Wood’s UV light

• infected hairs fluoresce

• bright greenish yellow under

Cutaneous

Endothrix invasion of hair

• formation of arthroconidia within hair shaft

• cuticle of hair remains intact

• do not fluoresce under Wood’s UV light

Cutaneous

Endothrix invasion of hair

• ALL AGENTS ARE ANTHROPOPHILIC

• Trichophyton tonsurans,

• T. violaceum

Cutaneous

Microsporum canis

netti.nic.fi

• Zoophilic– cats and dogs

• Invades– Hair– skin – rarely nails

• distribution– worldwide

www.vet.ohio-state.edu

http://images.google.com.ph/imgres?imgurl=netti.nic.fi/~fajesa/images/nessu2.jpg&imgrefurl=http://netti.nic.fi/~fajesa/males.htm&h=412&w=358&sz=26&tbnid=RdDATdT2TccJ:&tbnh=120&tbnw=105&prev=/images%3Fq%3DMicrosporum%2Bcanis%26start%3D20%26hl%3Den%26lr%3D%26ie%25

Cutaneous

golden yellow reverse colony

www2.provlab.ab.ca

Microsporum canis

lab diagnosis – culture

• white cottony growth

Cutaneous

Microsporum canis

• microscopic:

– spindle shaped,

one end pointed,

other end blunt

– thick walled

verrucose

macroconidia

– 6 to 12 cellswww.doctorfungus.org

http://images.google.com.ph/imgres?imgurl=www.doctorfungus.org/thefungi/img/miccan.jpg&imgrefurl=http://www.doctorfungus.org/thefungi/microsporum_canis.htm&h=391&w=594&sz=41&tbnid=kkCBdqU7uWUJ:&tbnh=87&tbnw=132&prev=/images%3Fq%3DMicrosporum%2Bcanis%26hl%3Den%25

Cutaneous

Microsporum gypseum

– geophilic

– usually produces a single inflammatory skin or scalp lesion


Cutaneous

Microsporum gypseum

lab diagnosis - culture

• flat, spreading suede-like to granular

• cinnamon growth • yellow brown pigment

on reverse of colony

www.ukneqasmicro.org.uk

Cutaneous

Microsporum gypseum

microscopic:

• symmetrical ellipsoidal

• thin walled verrucose

macroconidia

• distal end slightly rounded,

proximal (point of

attachment) is blunt

• 4 to 6 cells

vtpb-www.cvm.tamu.edu

www.medmicro.wisc.edu

Cutaneous


– zoophilic: mice, cats, horses, sheep, rabbits

– inflammatory skin or scalp lesions in humans

– ectothrix


Cutaneous


• lab diagnosis - culture

• flat, white to cream color; powdery to granular surface

danival.org

Cutaneous


Microscopic

spherical microconidia forming dense clusters,

“en-grappe”vtpb-www.cvm.tamu.edu

Cutaneous


• spiral hyphae

smooth thin-walled clavate multiseptate macroconidia

Microscopic

www.vet.ohio-state.edu vtpb-www.cvm.tamu.edu

Cutaneous


lab diagnosis

www2.provlab.ab.ca

positive urease production

positive for in-vitro hair perforation

www2.provlab.ab.ca

Cutaneous

Trichophyton rubrum

• anthropophilic– chronic infections of the skin, nails,

rarely scalp

– ectothrix or endothrix hair infection


Cutaneous

Trichophyton rubrum

lab diagnosis – culture

• white, suede-like to downy

• wine red pigment on reverse side

www.pfizer.ch

www4.medfak.lu.se

Cutaneous

Trichophyton rubrum

www2.provlab.ab.ca • lab diagnosis

– scanty to moderate numbers of slender clavate to pyriform microconidia

– arranged “en-thyrse”

Cutaneous

Trichophyton concentricum

Anthropophilic

chronic non-inflammatory tinea corporis

tinea imbricata – concentric scaling of skin

Not invade hair

Cutaneous


Distribution

Pacific Islands of Oceania

Southeast Asia

Central and South America

Cutaneous


Lab diagnosis

Slow growing deeply folded thallus

Cream to orange brown in color

Reverse buff to brown

Cutaneous


Microscopic –

“antler tips” hyphae,

chlamydoconidia

Cutaneous

Trichophyton schoenleinii

Anthropophilic

Cause favus

Chronic scarring form of tinea capitis

Saucer shaped crusted lesions or scutula

Permanent hair loss

Cutaneous


Lab diagnosis

Culture

Waxy or glabrous

Deeply folded honeycomb-like thallus with sub-surface growth

Cutaneous


Lab diagnosis

Microscopic

Favic chandeliers

No macroconidia

No microconidia

Cutaneous

Epidermophyton floccosum

• anthrophophilic

• does not invade hair in vivo


Cutaneous


Culture• greenish-brown or

“khaki” colored

• suede-like surface

• raised & folded center, with flat periphery

• yellowish brown reverse pigment

Cutaneous


botit.botany.wisc.edu

Microscopic• smooth thin-walled

macroconidia often in clusters growing directly from hyphae

• no microconidia• numerous

chlamydoconidia

www.fns.uniba.sk

Subcutaneous mycoses

• Chronic localized infections

• the skin and subcutaneous tissue

epidermis

dermis

subcutaneous

muscle & bone


• traumatic implantation of the aetiologic agent


•soil saprophytes of regional epidemiology

•barefooted people


Disease

• Sporotrichosis

• Chromoblastomycosis

Causative organisms

• Sporothrix schenckii

• Fonsecaea, Phialophora, Cladosporium, etc


Disease

• Mycotic mycetoma

Causative organisms

• Pseudallescheria, Madurella, Acremonium, Exophiala, etc


Disease

• Subcutaneous zygomycosis (Entomophthoromycosis)

Causative organisms

• Basidiobolus ranarum,

• Conidiobolus coronatus


Disease

• Subcutaneous zygomycosis (Mucormycosis)

Causative organisms

• Rhizopus, Mucor, Rhizomucor, Absidia, Saksenaea etc.


Disease

• Rhinosporidiosis

• Lobomycosis

Causative organisms

• Rhinosporidium seeberi

• Loboa loboi


Disease

• Phaeohyphomycosis

Causative organisms

• Exophiala bantianum

subcutaneous

Mycetoma

• Human & animal infection characterized by:– draining sinuses

– granules vary in • Size

• color

• hardness

subcutaneous

Mycetoma

• infection characterized by:

• tumefaction:

• destruction of bone

• distortion of foot or hand

• hyperplasia at openings of sinus tracts

subcutaneous

Mycetoma

• etiologic agents

– ACTINOMYCOTIC MYCETOMA

– EUMYCOTIC MYCETOMA

subcutaneous

Mycetoma

• etiologic agents– ACTINOMYCOTIC MYCETOMA

• Nocardia

• Actinomadura

• Streptomyces

subcutaneous

Mycetoma

• etiologic agents– EUMYCOTIC MYCETOMA

• Madurella

info.dom.uab.edu

subcutaneous

Mycetoma


• Fusarium

byebyemold.com

subcutaneous

Mycetoma


• Curvularia

www2.truman.edu

www.dehs.umn.edu

www.mold.ph

subcutaneous

Mycetoma

www.biologie.uni-halle.de

• etiologic agents• EUMYCOTIC MYCETOMA

– Leptosphaeria

subcutaneous

www.medicine.cmu.ac.th

Mycetoma


• Pseudallescheria

subcutaneous

Mycetoma


• Aspergillus

vtpb-www.cvm.tamu.edu

subcutaneous

Mycetoma


• Exophiala

• Acremonium

subcutaneous

Mycetoma

• Lab diagnosis– collect granules from sinuses, place

sterile gauze overnight

– culture granules to grow etiologic agents

subcutaneous

Mycetoma

• Lab diagnosis– tissue biopsy; H & E staining

• Madurella mycetomatis - black grains

www.reviberoammicol.com

subcutaneous

Mycetoma

• Lab diagnosis– tissue biopsy; H & E staining

• Streptomyces pelletierii - red grains• Streptomyces somaliensis -white grains

www.med.sc.edu:85

subcutaneous

Chromoblastomycosis

• Chronic, slowly

progressive & localized

infection

• Tissue proliferation

around area of inoculation

subcutaneous

Chromoblastomycosis

• Crusted, verrucose, wart-

like lesions

Chromoblastomycosis

• Lab diagnosis• KOH of skin scrapings / crusts:

• brown pigmented, planate-

dividing, rounded sclerotic bodies

• Also as copper pennies, brown

fission bodies

Chromoblastomycosis

• Lab diagnosis• Tissue section

• sclerotic cells

Chromoblastomycosis

Culture:

• slow growing ,

suede-like, olive

black in color

www.bbges.de

Cladosporium

Fonsecaea sp.

subcutaneous

Sporothricosis

• Rose gardener’s disease

• Infection of the cutaneous or subcutaneous tissues & adjacent lymphatics

subcutaneous

Sporothricosis

• Characterized by nodular lesions which may suppurate & ulcerate

dermatology.cdlib.org

Sporothricosis

• Etiologic agent– Sporothrix schenckii

– Dimorphic fungus

– Found in soil & decaying vegetation

Sporothricosis

• Lab diagnosis– Stained tissue biopsy

• Yeast-like cells, may bud

• “cigar bodies”

Sporothricosis- lab dx at 25C

Culture on SDA• moist & glabrous

• wrinkled & folded surface

• color – white to cream to black


Microscopic: • Daisy-like microconidia

• short conidiophores at right angle from thin hyphae

subcutaneous


• Culture on BHI agar:– Yeast colonies

• Microscopic: – Budding yeast cells

subcutaneous

Subcutaneous zygomycosis

• Mucorales group

• Local traumatic implantation of fungal elements through the skin, especially in patients with extensive burns, diabetes or steroid induced hyperglycemia and trauma.

subcutaneous


• Lesions vary considerably in morphology but include plaques, pustules, ulcerations, deep abscesses and ragged necrotic patches.

subcutaneous


• Lab dx– Tissue H & E staining

– infrequently septatethin walled hyphae with

– irregular branching

Opportunistic Mycoses

Common Opportunistic Fungicausing systemic mycosis

Aspergillus sppAspergillus spp

Mucor/Rhizopus sppMucor/Rhizopus spp

Cryptococcus neoformansCryptococcus neoformans

Candida albicansCandida albicans

Pneumocystis cariniiPneumocystis carinii

Aspergillosis

Etiologic Agents

– Aspergillus fumigatus– Aspergilllus flavus– Aspergillus niger– Aspergillus terreus– Aspergillus clavatus– Aspergillus restrictus– Aspergillus amsteloidami

• “Aspergillus species produce large quantities of spores which are easily dispersed by wind.”

Aspergillus fumigatus

• Accounts for over 90% of all infections

• Grows in a wide range of temperature-

can thrive up to 500C

• Inhibited by cycloheximide

Allergy ( to conidia/or transient growth of organism in body orifices)

Colonization of pre-formed cavities

Invasive, inflammatory, granulomatous, necrotizing diseases of lungs and other tissues Toxicity due to ingestion of contaminated food

RARELY systemic and fatal disseminated diseases

Summary of Clinical syndromes Summary of Clinical syndromes associated with genus associated with genus AspergillusAspergillus

Predisposing factors in aspergillosis

• Immunocompromised host• Presence of other disease, etc

Modes of Transmissions:

• Respiratory

• Direct Inoculation

• Ingestion

Pathogenesis …

Spores disseminated from humidifiers and Air conditioner filters and ducts that have accumulated moistureAnd from environment

Aspergilloma : Tomogram of lung Aspergilloma : Tomogram of lung

cavity containing fungus ball outlinedcavity containing fungus ball outlined

by air spaceby air space

Other clinical manifestations :

Allergy :•Asthma•Farmer’s lung

• Aspergilloma : Fungus ball

within cavity of old tuberculosis

lesion, right upper lobe

Aspergilloma : Macroscopic appearance Aspergilloma : Macroscopic appearance of fungus ball occupying a large of fungus ball occupying a large pulmonary cavity ( Dr. V. E. del Bene)pulmonary cavity ( Dr. V. E. del Bene)

• DIRECT INOCULATION

• Into subcutaneous tissues

( mycetoma)

• Into the blood ( drug abuse,

valve replacement, arterial catheterization etc)

• Examples of Clinical conditions :

Cornea ( keratitis) Traumatized tissue

DISSEMINATE to other sites

Ingestion :

Food poisoning

Disseminated aspergillosis

• CNS : Multiple hemorrhagic areas of acute necrosis in both cerebral

hemispheres ( Dr. P. Garen)

• Tracheal ulceration in a patient with AIDS

Laboratory Diagnosis

•Microscopic Examination :

• Demonstration of organism in the tissues ( biopsy or autopsy materials)

• septate hyphae• dichotomous hyphae• spores

Histological section of the lungs showing wide hyphe of Aspergillus with acute angle branching . (Dr. Elliot)

• Histological section showing masses of branching fungal hyphae invading the lung parenchyma and blood vessels. Grocott stain ( Dr. C. Kibbler)

• Fungal hyphae seen in corneal scrapings (Mr. P. A. Hunter)

• Aspergillus hyphae with dichotomous branching and septae .( Gomori methenamine

silver stain , Dr. P. Garen)

Laboratory Diagnosis…….

• Culture• Isolation of organism from tissues• SDA used• Incubated at room temperature• Identification : Based on the

characteristic spores

produced by the species

“ Fruiting body of A. fumigatus”

Mucormycosis( Systemic Zygomycosis)

Includes infections caused by:

• Rhizopus

• Rhizomucor

• Absidia

Non-septate hyphae and sporangium

Mucormycosis : Clinical forms, probable routes of infection and underlying

conditions

• Rhinocerebral/Rhino-orbital• airborne• Diabetes

• Pulmonary• airborne• Leukemia, lymphoma,• immune suppression

Mucormycosis : Clinical forms, probable routes of infection and underlying

conditions

• Gastrointestinal• Ingestion• malnutrition, Amebic colitis

• Skin• Traumatic• Diabetes, wounds

• Rhinocerebral mucormycosis : Bloodstained nasal discharge with left sided ptosis and proptosis ( Dr. J. Snape)

• Rhinocerebral mucormycosis : View through nasal speculum showing fungal material arising from nasal turbinates

( Prof. R. Y. Cartwright)

• Rhinocerebral mucomycosis : Gross brain specimen viewed from base with acute superficial necrosis of temporal lobe and thrombosis of left internal carotid artery ( Dr. P. Garen)

• Rhinocerebral mucormycosis with infarction of the hard palate ( Dr. J. Snape)

• Rhinocerebral mucormycosis : Advance case with necrosis of nasal and maxillary tissue with black eschar. Note periorbital edema and serosanguinous discharge from eye.

( Prof. R. Y. Cartwright)


• Microscopic : KOH of tissues ( and with

other stains):

Findings : broad, irregularly shaped,

non-septated hyphae

with right angle branching

Histological examination: stained with PASHistological examination: stained with PAS

Rhinocerebral mucomycosis: Brain biopsy from Rhinocerebral mucomycosis: Brain biopsy from a diabetic patient showing the large non-septateda diabetic patient showing the large non-septatedhyphae ( Periodic acid – Schiff stain)hyphae ( Periodic acid – Schiff stain)

• Rhinocerebral mucormycosis: Mucor organisms visible in biopsy, showing branching hyphae and sporangia.( Prof. R. Y. Cartwright)

Laboratory Diagnosis..

• Culture• Care in handling the specimen, since

organism is found in the environment• 3-5 days incubation at room temperature• To identify :LPCB mount of growth

Cryptococcosis

“ A subacute or chronic infection most frequently involving the tissues

of the CNS, but occasionally producing lesions in the skin, bones, lungs or other internal organs.”

• Cryptococcus : found worldwide in the soil. It flourishes in bird guano and is often found in large numbers in pigeon roots, old barns and in soil beneath trees used as bird roosts.

Two varieties of Cryptococcus

C. neoformans C. gattii• Distribution worldwide tropical & subtropical • Reservoir pigeon feces Eucalyptus trees

• Host * AIDS normal host * sarcoid * lymphoma *Corticosteroids * CLL, ALL * Organ transplantation

• Infection in AIDS Yes Rare

Characteristics

• C. neoformans, grows at 370C on SAB or malt agar ( distinguishes it from non-pathogenic species)

• In culture, colonies are visible after 72 hours ( white or tan colored, mucoid, smooth colonies)

• Canavanine-glycol-bromothymol blue agar :• gattii grows to produce color change to cobalt blue• neoformans does not grow, indicator remains yellow

Cutaneous cryptococcosis

• Cryptococcosis : Active granulomatous lesion on the nose of a 68 year old woman with Hodgkin’s disease ( Dr. T. F. Sellers Jr.)


• Microscopic examination• Indian Ink mount ( sputum, CSF sediments)

( 50% sensitivity -less satisfying)

• Culture of blood from a case of Disseminated cryptococcosis ( Dr. Kibbler)

Laboratory Diagnosis….Laboratory Diagnosis….

• Culture ( 75% sensitivity) : in SAB without cyclohexidine

Laboratory Diagnosis….• Demonstration of cryptococcal antigen by

Latex agglutination ( for disseminated infections)

• Serology : Detection of capsular antigen

by antibodies ( use of rabbit’s anti -

C. neoformans capsule antigen) –

95% sensitivity

• PCR

Candidemia andDisseminated Candidiasis

Candidemia and Disseminated candidiasis

• May present as asymptomatic or fulminant sepsis• Must be considered when patient has these risks factors :

• Malignancy• Chemotherapy-induced neutropenia• Organ transplantation• GI surgery• Indwelling catheter• Burns• Exposure to broad spectrum antibiotics

Disseminated candidiasis : Typical skin lesions areDisseminated candidiasis : Typical skin lesions are pinkish –red nodules. Patient with leukemia pinkish –red nodules. Patient with leukemia

Oral candidiasis ,severe infection, plaques of white exudates on the tongue and palate. Severe infection in a patient with AIDS . Earliest clinical sign of HIV infection and may persist for months.

• “ The genus Candida is associated with a number of clinical syndromes usually seen among patients with abrogated immune response, debilitating diseases , as well as on those taking corticosteroids, anti-cancers and on long antibiotic therapy.”

The Genus Candida

• Over a hundred species• Only 10 are commonly associated

with disease• Distribution :

– Saprophytes in human and animal

digestive tract–Candida albicans–Candida albicans var stellatoidea

The Genus Candida• Distribution :

– Saprophytes in soil, air, water, dairy products, fermenting /rotting vegetable products

– Candida tropicalis and pseudotropical

( also found in the respiratory tract)– Candida parapsilosis ( also in the skin)– Candida krusei– Candida guilliermondi– Candida lusitaniae– Candida viswanatii

• “ Candida albicans is the most common species associated with human illness,”

Candida albicans

• Exist primarily in the yeast form

• In tissue:• It may be found in both yeast

and mold form ( pseudohyphae)

• In agar medium:• Surface – oval budding cell or yeast• Lower layer - pseudohyphae

Laboratory Diagnosis of Candidemia

• Culture : recovery of C. albicans

from culture

• Microscopic:Demonstration of

organism in the biopsy materials

by histological examination

• Colonies are white and smooth• Presumptive identification is based on

formation of germ tube within 90 minutes of placing the organism in serum

Characteristics of Candida albicans…

• Gram positive, coccus in shape

Budding cell

Laboratory Diagnosis…..

• Serology available but is not reliable

because of high false-negative

and false-positive

?

Pneumocystis carinii

Pneumocystis carinii cyst

visualized by scanning electron microscope, visualized by scanning electron microscope, platinum coated ( Dr. M. Forte)platinum coated ( Dr. M. Forte)

Pathogenesis….

Organisms found liningThe alveoli: forming thick-Walled cyst with intracysticbodies

Inhaled from another host-Persons has WEAKENED Immune system as in AIDS, PCP

Exchange of gas impaired

Infection in AIDS patients

• They get sick more slowly than other patients and may survive longer without treatment

• They die of the infection

• Signs and symptoms : Triad of:

• Shortness of breath with exertion• Non-productive cough• fever

Laboratory Diagnosis• Imaging

– Chest radiograph – 80% of cases will show diffuse interstitial infiltrates -

PCP : Chest X-ray showing advance disease withPCP : Chest X-ray showing advance disease with

dense infiltrates in both lungsdense infiltrates in both lungs

• Microscopic Examination : using various staining methods

Specimens:– bronchoalveolar-lavage ( BAL) – transbronchial-biopsy

– Basis for diagnosis in > 90% of patients.

– Considered the “ Gold Standard” in the diagnosis

Laboratory Diagnosis……Laboratory Diagnosis……

• PCP : Fluorescent monoclonal antibody stain

of bronchoalveolar washings ( Dr. K. Nye)


• PCP : Transbronchial lung biopsy showing alveolar spaces filled with foamy materials which is composed of a mass of pneumocystis organisms. H & E stain


• PCP :Lung section showing densely staining clusters of cysts. ( Methenamine silver stain)


Systemic Mycoses

The Dimorphic Fungi

Blastomyces dermatitidisBlastomyces dermatitidis

Paracoccidiodes brazielensisParacoccidiodes brazielensis

Coccidiodes immitisCoccidiodes immitis

Histoplasma capsulatumHistoplasma capsulatum Histoplasma duboisiiHistoplasma duboisii

Features of Systemic Mycosis•Host :Host :

: Human host is a dead end in : Human host is a dead end in their life cycletheir life cycle

: Fungi have the ability to elicit: Fungi have the ability to elicit disease process in a disease process in a NORMAL HOSTNORMAL HOST

: In : In immunocompromised immunocompromised host-host- high risk of high risk of disseminated diseasedisseminated disease

Features of Systemic Mycosis…….

•Mode of transmission :

: Inhalation of large inoculum

Coccidioidomycosis

Life Cycle of Coccidiodes immitis

Mycelial at room Temperature

Arthrospores

Inhaled

Spherule in tissue

Natural habitatThe fungus is found in dry, alkaline dessert soil, like those in San Joaquin Valley in California

Clinical manifestations

IP 1-3 weeks

Fever, malaise, cough, chest pain

erythema nodosum in women erythema multiforme in children

Resolution in 10-21 days

Mode of transmissionIs inhalation of

C. immitisC. immitis, , spherule inspherule in the lungs H & E stain the lungs H & E stain ( A.E. Provost)( A.E. Provost)

Right lobe consolidation. The appearance is Right lobe consolidation. The appearance is

non specific and could be produced by other non specific and could be produced by other

pathogenspathogens

Primary Pulmonary coccidioidomycosis

Cutaneous Coccidioidomycosis

Lesions similar to those found on the head of Domingo Escurra

Provisional diagnosis

• Based on :

• Epidemiologic considerations• Clinical manifestations• Skin response to coccidioidin• Detection of antibody

Serology:


Specimens :• sputum• biopsy• etc.

Direct Microscopy : KOH/FAT

Culture : media without antibiotics : slants used/bottles

( ( HAZARDOUS)HAZARDOUS)

• Microscopic Examination : Spherule in the CNS

( from Granulomatous meningitis due to C. immitis ).

Multinucleated giant cellMultinucleated giant cell

spherule containing manyspherule containing many endosporesendospores

Blastomycosis(North American Blastomycosis)

• “ Acute or chronic infection of lung, skin, bone or genitourinary tract , characterized by formation of pyogranuloma.”

Etiologic Agent• Blastomyces dermatitidis

• Dimorphic

Calcofluor white fluorescent Calcofluor white fluorescent staining of staining of B. dermatitidisB. dermatitidisyeastyeast

Mycelial at room temperature;Indistinguishable from P. brazielensis

Mode of Transmission

• Inhalation of spores ( microconidia)

Clinical manifestations

•Pulmonary• acute

Symptomatic cases presents like bacterial pneumonia

Clear with/without Treatment

Overwhelming: ARDS ( acute respiratory distress syndrome)

Chronic pulmonary

Pulmonary Blastomycosis

Differential diagnosis:

• bacterial infections• fungal infections, • lymphomas • neoplasms

Cutaneous blastomycoses

Yeast cells are seen in lesions

Other forms of blastomycosis…..Other forms of blastomycosis…..


• Direct Microscopic examination:

KOH Mount

Multinucleated giant cell withMultinucleated giant cell with B. dermatitidisB. dermatitidis ( H & E stain)( H & E stain)

Calcofluor white Calcofluor white fluorescent stainingfluorescent staining

»Culture

Requires 5 days to 4 weeks for growth; can be as short as 2-3 days

• Serology

•Serum complement fixation•Immunodiffusion test•Radio immunoassay•ELISA

“None have been useful because of low sensitivity and specificity rates.”

Paracoccidiomycosis( South American Blastomycosis)

• “ Chronic granulomatous disease that characteristically produce a primary infection, often inapparent, and then disseminates to form ULCERATIVE granulomata of the buccal, nasal, skin , adrenal glands and occasionally the gastrointestinal tract.”

Etiologic agent:Paracoccidiodes brasiliensis

• Dimorphic

Mycelial at room temperature;Indistinguishable from Blastomyces dermatitidis

Yeast at 370 C Multiple-budding cell ( narrow necked buds)

• Mode of Transmission

Inhalation of spores

10 Pulmonary infection

Clinical Manifestations

• Primary Benign disease:• 10 Pulmonary• Pulmonary : re-infection with allergic

manifestation

• Acute and chronic progressive- disease• Disseminated disease• Acute and chronic progressive pulmonary

disease

Example of Cutaneous paracoccidioidomycosis


• Demonstration of multiple-budding cell from clinical specimens

Histoplasmosis( Great “ mimic” in mycosis”

caused by Histoplasma capsulatum )

•“ Pulmonary infection resembles tuberculosis. It was first recognized as a disease among patients who were X-ray positive, but tuberculin negative.

• Affects the RES cells ; lesions are not confined to the lungs.”

Etiologic Agent

•Dimorphic

Mycelial phase at 250C,microconidia andtuberculate macroconidia,

Tissue phase at 370C,Thin-walled,single budding cells inside macrophages

Two Environments favored byHistoplasma capsulatum

• Open environment

• Nitrogen rich• 22-29ºC• rainfall of 35-50 inches• 67-87% relative humidity• Guano from birds, particularly Sturnus vulgaris or the starlings•Bird not infected but organism can be isolated from its feathers

• Closed environment

Bat caves have high deposits of guano

stable condition

BATS – GIT with ulcer

Cave explorationMay result in exposureTo high levels of organisms

• Pathogenesis : Mode of transmission

Inhalation of spores

Pathogenesis…….

Pathogenesis…….

In 2-4 weeks, specific T cell- mediated immunity develops

Inhibits the growth of the organisms

Cutaneous Histoplasmosis

Histoplasmosis in the buccal mucosa

Histoplasmosis in AIDS

• Causes disseminated disease in 95% of cases with AIDS

• 90% when CD4 count is below 200/ul

• Localized pulmonary disease when CD4 is higher than 300/ul

• CNS involved in 10-20% of cases

• Diagnosis :• High index of suspicion• Detection of antigen by radiommunoassay ( rapid)

• Treatment : amphotericin B and itraconazole


• Specimens

• Sputum• Buffy coat of blood ( WBC layer)• Sediments of biopsy materials• Gastric lavage• urine

Laboratory Diagnosis• Direct Microscopic Examination :

( Wright –Giemsa methods or Grocott silver

methenamine stain)

Cell wall –unstainedMaterial around the cell

Red staining crescent shaped Mass is the cytoplasm

Laboratory Diagnosis……..

• Culture • Media

» SAB» BAP without antibiotic» Smith and Goodman’s medium –

for heavily contaminated

specimens» Yeast extract with ammonium

hydroxide

Laboratory Diagnosis………

• Culture

Histoplasma growing Histoplasma growing at room temperatureat room temperature from bone marrowfrom bone marrow specimenspecimen

Yeast colonies at370C

Yeast colonies of H. capsulatum in blood agar , incubated at 370C

Laboratory Diagnosis…….Laboratory Diagnosis…….

• Microscopic examination of mycelial form showing micro

and macroconidia of Histoplasma capsulatum

Laboratory Diagnosis…..Laboratory Diagnosis…..

Laboratory Diagnosis……….

• Complement Fixation Test

• Animal inoculation

• Skin testing » Histoplasmin skin test

Thank you !

myco / viro

Documents

culture culture media

koh culture

c hair

fungal isolation

koh data

upmcphfungal isolates

albicans c

c urine