MYCO / VIRO
Transcript of MYCO / VIRO
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FUNGAL DISEASES
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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
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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%
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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
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Data from a Five-year review of Fungal Isolates at UPM-CPH
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
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Data from a Five-year review of Fungal Isolates at UPM-CPH
Fungal isolates :
Trichophyton mentagrophytes
Trichophyton rubrum
Trichophyton tonsurans
Trichophyton schoenlenii
Trichosporon spp.
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Data from a Five-year review of Fungal Isolates at UPM-CPH
• Fungal isolates
Microsporum gypseum
Microsporum canis
Epidermophyton flocossum
Candida albicans
Exophiala werneckii
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Data from a Five-year review of Fungal Isolates at UPM-CPH
Fungal isolates from biopsy materials and exudates (31.2% positive):
Fonsecaea compactum
Phialophora verrucosa
Exophiala jeanselmei
Madurella grisea
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Laboratory diagnosis
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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
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• 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
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• 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
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• 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)
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Wipe with water
www.doctorfungus.org
scalpelPaper / envelope
active edge
Skin scraping specimen
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Direct Examination
• Wet mount
KOH• KOH– 10% to 30%
– with Parker Superquink blue-black ink
– gentle warming
pa
rk
er
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I. DIRECT EXAMINATION:
*10-30% KOH
*Calcofluor white stain
*Histological stains- H&E, PAS
*India Ink
*Wet mount
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II. Isolation & Culture
SDA
BHIA/BAP
Media with/without antibiotics
• Macroscopic examination of culture
• Microscopic examination using LPCB
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Identification
• Gross color & texture• Microscopic characteristics• Confirm / compare with
Written descriptionsDrawingsphotographs
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• III. Biochemical Tests:*Rapid kits for yeasts*Urea test
• IV. Special Tests: *In-vitro hair perforation test
*Germ tube test *Chlamydoconidia formation test
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Mycotic Infections
Superficial
Cutaneous
Subcutaneous
Systemic
Opportunistic
*Mycotoxicosis
*Allergies
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Superficial mycoses
Disease
SKIN
• Pityriasis versicolor
• Tinea nigra
Causative organisms
• Malassezia furfur
• Exophiala werneckii
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Superficial mycoses
Disease
HAIR
• White piedra
• Black piedra
Causative organisms
• Trichosporon beigelii
• Piedraia hortae
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Superficial mycoses
Pityriasis versicolor
• Lesion-An-an”
-Hyperpigmented or hypopigmented macular lesions
www.ethnomed.org
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Superficial mycoses
Pityriasis versicolor
• Lesion– scale readily, giving
a chalky branny appearance
– occurs on the trunk, shoulders & arms, face and neck
Modified from www.columbia.edu
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Superficial mycoses
Pityriasis versicolor
KOH of skin scrapings
• clusters of budding yeast-like cells & short angular hyphal forms
• “spaghetti and meat balls”
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Superficial mycoses
Pityriasis versicolor
PAS of skin scrapings• “spaghetti and meat
balls”
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Superficial mycoses
Pityriasis versicolor
• Culture of skin scrapings– Not necessary– diagnostic microscopic
features– SDA overlaid with peanut
oil, olive oil
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Superficial mycoses
Pityriasis versicolor
• Etiologic Agent– Malassezia globosa
lipophilic yeast
part of skin normal flora
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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
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Superficial mycoses
Tinea nigra
• KOH– pigmented brown to dark
olivaceous (dematiaceous) septate hyphal elements & 2-celled yeast cells
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Superficial mycoses
Tinea nigra
• Etiologic agent– Exophiala werneckii
saprophyte found in soil, compost, humus & wood
in humid tropical & sub-tropical regions
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Superficial mycoses
Tinea nigra
• Culture on SDA– initially mucoid, yeast-
like & shiny black– with age: aerial
mycelia & dark olive color
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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
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Superficial mycoses
Piedra
Two varieties
–White piedra
–Black piedra
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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
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Superficial mycoses
Black piedra - distribution
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Superficial mycoses
Black piedra
• Etiologic agent– Piedraia hortae– source of infection
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Superficial mycoses
Black piedra - lab diagnosis
• Direct microscopy– specimen - hair with nodules
– 25% NaOH or KOH
– dark septate hyphae
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Superficial mycoses
Black piedra - lab diagnosis
• Direct microscopy– round to oval asci;
hyaline, curved to fusiform ascospores
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Superficial mycoses
Black piedra - lab diagnosis
• Isolation – medium
– SDA with chloramphenicol
– SDA cycloheximide
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Superficial mycoses
Black piedra - lab diagnosis
• Isolation
-growth very slow-dark brown to black
-greenish brown, short aerial mycelium
Heaped center
Flat periphery
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Superficial mycoses
White piedra
• Infection of hair shaft– face, axilla, genitals -
common
– scalp, eyebrows, eyelashes - less common
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Superficial mycoses
White piedra
Nodule
• Soft, white, yellowish, beige or greenish nodule
• Discrete• more often coalescent,
forming an irregular transparent sheath
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Superficial mycoses
White piedra
• Distribution
– common in S. America & Asia
– sporadic in N. America & Europe
• Etiologic agent
– Trichosporon beigelii or T. cutaneum
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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
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Superficial mycoses
White piedra - lab diagnosis
Isolation
• medium - SDA with
chloramphenicol without
cycloheximide
• growth/culture– rapid
– cream-colored, soft
– membranous, wrinkled radial
furrows, irregular folding
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Superficial mycoses
White piedra - lab diagnosis
Isolation
• microscopic exam
of culture
– hyaline hyphae
– arthroconidia
– blastoconidia
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Superficial mycoses
White piedra - lab diagnosis
• Physiological studies– does not ferment carbohydrates– assimilate dextrose, lactose, D-xylose,
inositol
– negative KNO3 assimilation
– urease positive– arbutin is split
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Cutaneous mycoses
skin
hair
nails
• No living
tissue
• Host Rxn
to fungus
keratinase
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Cutaneous mycoses
Disease
• Dermatophytosis
Causative organisms
• Dermatophytes
Microsporum
Trichophyton
Epidermophyton
ringworm
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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)
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Clinical Manifestations of Dermatophytes
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Cutaneous
Tinea capitis
www.emedicinehealth.com
Scalp, eyebrow, eyelashes
Microsporum &
Trichophyton
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Cutaneous
Tinea capitis Endothrix
Ectothrix
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Cutaneous
Tinea favosa
• Scutulum
• Mass of mycelia
& epithelial
debris
• Cup shaped
crusts
www.mf.uni-lj.si
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Cutaneous
Tinea corporis
www.cut.ee/
• Non-hairy skin
• Rings with scaly
centers
• Rxn vs fungus
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Cutaneous
Tinea corporis
• E. floccosum
• Trichophyton
• Microsporum
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Cutaneous
Tinea imbricata
Concentric
rings
Trichophyton
concentricum
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Cutaneous
Tinea barbae
• Bearded areas of
face & neck
www.merck.com
www.emedicine.com
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Cutaneous
Tinea cruris
www.dermnetnz.org
Jock itch
Moist groin
area
E. floccosum,
T. rubrum
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Cutaneous
Tinea pedis
www.doctorfungus.org
dermatologie.free.fr
Athlete’s foot
Toe webs & soles,
even nails
Id reaction,
circulating fungal
antigens
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Cutaneous
Tinea manuum
www.dermnetnz.org
• Interdigital areas
& palmar surfaces
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Cutaneous
Tinea unguium
www.dermnetnz.org
Invasion of nail plate by
dermatophytes
Thickened, discolored &
brittle• Onychomycosis- non
dermatophyte
Yeast etc.
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Cutaneous
KOH of skin scrapings
Septate hypha
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Cutaneous
arthrospores
septate hypha
KOH of skin scrapings
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Cutaneous
Ectothrix invasion of hair
• Hair invasion • formation of
arthroconidia on the outside of hair shaft
• cuticle of hair is destroyed
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Cutaneous
Ectothrix invasion of hair
• Hair invasion by a dermatophyte– Microsporum canis
– M. gypseum
– Trichophyton equinum
– T. verrucosum
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Cutaneous
Ectothrix invasion of hair
• Wood’s UV light
• infected hairs fluoresce
• bright greenish yellow under
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Cutaneous
Endothrix invasion of hair
• formation of arthroconidia within hair shaft
• cuticle of hair remains intact
• do not fluoresce under Wood’s UV light
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Cutaneous
Endothrix invasion of hair
• ALL AGENTS ARE ANTHROPOPHILIC
• Trichophyton tonsurans,
• T. violaceum
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Cutaneous
Microsporum canis
netti.nic.fi
• Zoophilic– cats and dogs
• Invades– Hair– skin – rarely nails
• distribution– worldwide
www.vet.ohio-state.edu
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Cutaneous
golden yellow reverse colony
www2.provlab.ab.ca
Microsporum canis
lab diagnosis – culture
• white cottony growth
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Cutaneous
Microsporum canis
• microscopic:
– spindle shaped,
one end pointed,
other end blunt
– thick walled
verrucose
macroconidia
– 6 to 12 cellswww.doctorfungus.org
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Cutaneous
Microsporum gypseum
– geophilic
– usually produces a single inflammatory skin or scalp lesion
• distribution– worldwide
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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
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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
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Cutaneous
Trichophyton mentagrophytes
– zoophilic: mice, cats, horses, sheep, rabbits
– inflammatory skin or scalp lesions in humans
– ectothrix
• distribution– worldwide
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Cutaneous
Trichophyton mentagrophytes
• lab diagnosis - culture
• flat, white to cream color; powdery to granular surface
danival.org
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Cutaneous
Trichophyton mentagrophytes
Microscopic
spherical microconidia forming dense clusters,
“en-grappe”vtpb-www.cvm.tamu.edu
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Cutaneous
Trichophyton mentagrophytes
• spiral hyphae
smooth thin-walled clavate multiseptate macroconidia
Microscopic
www.vet.ohio-state.edu vtpb-www.cvm.tamu.edu
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Cutaneous
Trichophyton mentagrophytes
lab diagnosis
www2.provlab.ab.ca
positive urease production
positive for in-vitro hair perforation
www2.provlab.ab.ca
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Cutaneous
Trichophyton rubrum
• anthropophilic– chronic infections of the skin, nails,
rarely scalp
– ectothrix or endothrix hair infection
• distribution– worldwide
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Cutaneous
Trichophyton rubrum
lab diagnosis – culture
• white, suede-like to downy
• wine red pigment on reverse side
www.pfizer.ch
www4.medfak.lu.se
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Cutaneous
Trichophyton rubrum
www2.provlab.ab.ca • lab diagnosis
– scanty to moderate numbers of slender clavate to pyriform microconidia
– arranged “en-thyrse”
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Cutaneous
Trichophyton concentricum
Anthropophilic
chronic non-inflammatory tinea corporis
tinea imbricata – concentric scaling of skin
Not invade hair
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Cutaneous
Trichophyton concentricum
Distribution
Pacific Islands of Oceania
Southeast Asia
Central and South America
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Cutaneous
Trichophyton concentricum
Lab diagnosis
Slow growing deeply folded thallus
Cream to orange brown in color
Reverse buff to brown
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Cutaneous
Trichophyton concentricum
Microscopic –
“antler tips” hyphae,
chlamydoconidia
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Cutaneous
Trichophyton schoenleinii
Anthropophilic
Cause favus
Chronic scarring form of tinea capitis
Saucer shaped crusted lesions or scutula
Permanent hair loss
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Cutaneous
Trichophyton schoenleinii
Lab diagnosis
Culture
Waxy or glabrous
Deeply folded honeycomb-like thallus with sub-surface growth
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Cutaneous
Trichophyton schoenleinii
Lab diagnosis
Microscopic
Favic chandeliers
No macroconidia
No microconidia
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Cutaneous
Epidermophyton floccosum
• anthrophophilic
• does not invade hair in vivo
• distribution– worldwide
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Cutaneous
Epidermophyton floccosum
Culture• greenish-brown or
“khaki” colored
• suede-like surface
• raised & folded center, with flat periphery
• yellowish brown reverse pigment
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Cutaneous
Epidermophyton floccosum
botit.botany.wisc.edu
Microscopic• smooth thin-walled
macroconidia often in clusters growing directly from hyphae
• no microconidia• numerous
chlamydoconidia
www.fns.uniba.sk
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Subcutaneous mycoses
• Chronic localized infections
• the skin and subcutaneous tissue
epidermis
dermis
subcutaneous
muscle & bone
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Subcutaneous mycoses
• traumatic implantation of the aetiologic agent
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Subcutaneous mycoses
•soil saprophytes of regional epidemiology
•barefooted people
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Subcutaneous mycoses
Disease
• Sporotrichosis
• Chromoblastomycosis
Causative organisms
• Sporothrix schenckii
• Fonsecaea, Phialophora, Cladosporium, etc
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Subcutaneous mycoses
Disease
• Mycotic mycetoma
Causative organisms
• Pseudallescheria, Madurella, Acremonium, Exophiala, etc
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Subcutaneous mycoses
Disease
• Subcutaneous zygomycosis (Entomophthoromycosis)
Causative organisms
• Basidiobolus ranarum,
• Conidiobolus coronatus
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Subcutaneous mycoses
Disease
• Subcutaneous zygomycosis (Mucormycosis)
Causative organisms
• Rhizopus, Mucor, Rhizomucor, Absidia, Saksenaea etc.
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Subcutaneous mycoses
Disease
• Rhinosporidiosis
• Lobomycosis
Causative organisms
• Rhinosporidium seeberi
• Loboa loboi
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Subcutaneous mycoses
Disease
• Phaeohyphomycosis
Causative organisms
• Exophiala bantianum
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subcutaneous
Mycetoma
• Human & animal infection characterized by:– draining sinuses
– granules vary in • Size
• color
• hardness
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subcutaneous
Mycetoma
• infection characterized by:
• tumefaction:
• destruction of bone
• distortion of foot or hand
• hyperplasia at openings of sinus tracts
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subcutaneous
Mycetoma
• etiologic agents
– ACTINOMYCOTIC MYCETOMA
– EUMYCOTIC MYCETOMA
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subcutaneous
Mycetoma
• etiologic agents– ACTINOMYCOTIC MYCETOMA
• Nocardia
• Actinomadura
• Streptomyces
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subcutaneous
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Madurella
info.dom.uab.edu
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subcutaneous
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Fusarium
byebyemold.com
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subcutaneous
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Curvularia
www2.truman.edu
www.dehs.umn.edu
www.mold.ph
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subcutaneous
Mycetoma
www.biologie.uni-halle.de
• etiologic agents• EUMYCOTIC MYCETOMA
– Leptosphaeria
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subcutaneous
www.medicine.cmu.ac.th
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Pseudallescheria
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subcutaneous
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Aspergillus
vtpb-www.cvm.tamu.edu
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subcutaneous
Mycetoma
• etiologic agents– EUMYCOTIC MYCETOMA
• Exophiala
• Acremonium
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subcutaneous
Mycetoma
• Lab diagnosis– collect granules from sinuses, place
sterile gauze overnight
– culture granules to grow etiologic agents
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subcutaneous
Mycetoma
• Lab diagnosis– tissue biopsy; H & E staining
• Madurella mycetomatis - black grains
www.reviberoammicol.com
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subcutaneous
Mycetoma
• Lab diagnosis– tissue biopsy; H & E staining
• Streptomyces pelletierii - red grains• Streptomyces somaliensis -white grains
www.med.sc.edu:85
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subcutaneous
Chromoblastomycosis
• Chronic, slowly
progressive & localized
infection
• Tissue proliferation
around area of inoculation
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subcutaneous
Chromoblastomycosis
• Crusted, verrucose, wart-
like lesions
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Chromoblastomycosis
• Lab diagnosis• KOH of skin scrapings / crusts:
• brown pigmented, planate-
dividing, rounded sclerotic bodies
• Also as copper pennies, brown
fission bodies
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Chromoblastomycosis
• Lab diagnosis• Tissue section
• sclerotic cells
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Chromoblastomycosis
Culture:
• slow growing ,
suede-like, olive
black in color
www.bbges.de
Cladosporium
Fonsecaea sp.
![Page 124: MYCO / VIRO](https://reader038.fdocuments.in/reader038/viewer/2022102602/546e9ba4b4af9f20798b466b/html5/thumbnails/124.jpg)
subcutaneous
Sporothricosis
• Rose gardener’s disease
• Infection of the cutaneous or subcutaneous tissues & adjacent lymphatics
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subcutaneous
Sporothricosis
• Characterized by nodular lesions which may suppurate & ulcerate
dermatology.cdlib.org
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Sporothricosis
• Etiologic agent– Sporothrix schenckii
– Dimorphic fungus
– Found in soil & decaying vegetation
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Sporothricosis
• Lab diagnosis– Stained tissue biopsy
• Yeast-like cells, may bud
• “cigar bodies”
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Sporothricosis- lab dx at 25C
Culture on SDA• moist & glabrous
• wrinkled & folded surface
• color – white to cream to black
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Sporothricosis- lab dx at 25C
Microscopic: • Daisy-like microconidia
• short conidiophores at right angle from thin hyphae
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subcutaneous
Sporothricosis- lab dx at 37C
• Culture on BHI agar:– Yeast colonies
• Microscopic: – Budding yeast cells
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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.
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subcutaneous
Subcutaneous zygomycosis
• Lesions vary considerably in morphology but include plaques, pustules, ulcerations, deep abscesses and ragged necrotic patches.
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subcutaneous
Subcutaneous zygomycosis
• Lab dx– Tissue H & E staining
– infrequently septatethin walled hyphae with
– irregular branching
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Opportunistic Mycoses
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Common Opportunistic Fungicausing systemic mycosis
Aspergillus sppAspergillus spp
Mucor/Rhizopus sppMucor/Rhizopus spp
Cryptococcus neoformansCryptococcus neoformans
Candida albicansCandida albicans
Pneumocystis cariniiPneumocystis carinii
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Aspergillosis
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Etiologic Agents
– Aspergillus fumigatus– Aspergilllus flavus– Aspergillus niger– Aspergillus terreus– Aspergillus clavatus– Aspergillus restrictus– Aspergillus amsteloidami
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• “Aspergillus species produce large quantities of spores which are easily dispersed by wind.”
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Aspergillus fumigatus
• Accounts for over 90% of all infections
• Grows in a wide range of temperature-
can thrive up to 500C
• Inhibited by cycloheximide
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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
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Predisposing factors in aspergillosis
• Immunocompromised host• Presence of other disease, etc
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Modes of Transmissions:
• Respiratory
• Direct Inoculation
• Ingestion
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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
![Page 144: MYCO / VIRO](https://reader038.fdocuments.in/reader038/viewer/2022102602/546e9ba4b4af9f20798b466b/html5/thumbnails/144.jpg)
Other clinical manifestations :
Allergy :•Asthma•Farmer’s lung
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• 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)
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• DIRECT INOCULATION
• Into subcutaneous tissues
( mycetoma)
• Into the blood ( drug abuse,
valve replacement, arterial catheterization etc)
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• Examples of Clinical conditions :
Cornea ( keratitis) Traumatized tissue
DISSEMINATE to other sites
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Ingestion :
Food poisoning
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Disseminated aspergillosis
![Page 150: MYCO / VIRO](https://reader038.fdocuments.in/reader038/viewer/2022102602/546e9ba4b4af9f20798b466b/html5/thumbnails/150.jpg)
• CNS : Multiple hemorrhagic areas of acute necrosis in both cerebral
hemispheres ( Dr. P. Garen)
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• Tracheal ulceration in a patient with AIDS
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Laboratory Diagnosis
•Microscopic Examination :
• Demonstration of organism in the tissues ( biopsy or autopsy materials)
• septate hyphae• dichotomous hyphae• spores
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Histological section of the lungs showing wide hyphe of Aspergillus with acute angle branching . (Dr. Elliot)
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• Histological section showing masses of branching fungal hyphae invading the lung parenchyma and blood vessels. Grocott stain ( Dr. C. Kibbler)
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• Fungal hyphae seen in corneal scrapings (Mr. P. A. Hunter)
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• Aspergillus hyphae with dichotomous branching and septae .( Gomori methenamine
silver stain , Dr. P. Garen)
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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”
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Mucormycosis( Systemic Zygomycosis)
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Includes infections caused by:
• Rhizopus
• Rhizomucor
• Absidia
Non-septate hyphae and sporangium
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Mucormycosis : Clinical forms, probable routes of infection and underlying
conditions
• Rhinocerebral/Rhino-orbital• airborne• Diabetes
• Pulmonary• airborne• Leukemia, lymphoma,• immune suppression
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Mucormycosis : Clinical forms, probable routes of infection and underlying
conditions
• Gastrointestinal• Ingestion• malnutrition, Amebic colitis
• Skin• Traumatic• Diabetes, wounds
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• Rhinocerebral mucormycosis : Bloodstained nasal discharge with left sided ptosis and proptosis ( Dr. J. Snape)
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• Rhinocerebral mucormycosis : View through nasal speculum showing fungal material arising from nasal turbinates
( Prof. R. Y. Cartwright)
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• 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)
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• Rhinocerebral mucormycosis with infarction of the hard palate ( Dr. J. Snape)
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• 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)
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Laboratory Diagnosis
• Microscopic : KOH of tissues ( and with
other stains):
Findings : broad, irregularly shaped,
non-septated hyphae
with right angle branching
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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)
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• Rhinocerebral mucormycosis: Mucor organisms visible in biopsy, showing branching hyphae and sporangia.( Prof. R. Y. Cartwright)
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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
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Cryptococcosis
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“ 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.”
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• 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.
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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
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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
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Cutaneous cryptococcosis
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• Cryptococcosis : Active granulomatous lesion on the nose of a 68 year old woman with Hodgkin’s disease ( Dr. T. F. Sellers Jr.)
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Laboratory Diagnosis
• Microscopic examination• Indian Ink mount ( sputum, CSF sediments)
( 50% sensitivity -less satisfying)
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• Culture of blood from a case of Disseminated cryptococcosis ( Dr. Kibbler)
Laboratory Diagnosis….Laboratory Diagnosis….
• Culture ( 75% sensitivity) : in SAB without cyclohexidine
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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
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Candidemia andDisseminated Candidiasis
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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
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Disseminated candidiasis : Typical skin lesions areDisseminated candidiasis : Typical skin lesions are pinkish –red nodules. Patient with leukemia pinkish –red nodules. Patient with leukemia
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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.
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• “ 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.”
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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
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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
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• “ Candida albicans is the most common species associated with human illness,”
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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
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Laboratory Diagnosis of Candidemia
• Culture : recovery of C. albicans
from culture
• Microscopic:Demonstration of
organism in the biopsy materials
by histological examination
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• Colonies are white and smooth• Presumptive identification is based on
formation of germ tube within 90 minutes of placing the organism in serum
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Characteristics of Candida albicans…
• Gram positive, coccus in shape
Budding cell
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Laboratory Diagnosis…..
• Serology available but is not reliable
because of high false-negative
and false-positive
?
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Pneumocystis carinii
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Pneumocystis carinii cyst
visualized by scanning electron microscope, visualized by scanning electron microscope, platinum coated ( Dr. M. Forte)platinum coated ( Dr. M. Forte)
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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
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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
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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
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• 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……
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• PCP : Fluorescent monoclonal antibody stain
of bronchoalveolar washings ( Dr. K. Nye)
Laboratory Diagnosis……Laboratory Diagnosis……
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• PCP : Transbronchial lung biopsy showing alveolar spaces filled with foamy materials which is composed of a mass of pneumocystis organisms. H & E stain
Laboratory Diagnosis……Laboratory Diagnosis……
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• PCP :Lung section showing densely staining clusters of cysts. ( Methenamine silver stain)
Laboratory Diagnosis……Laboratory Diagnosis……
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Systemic Mycoses
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The Dimorphic Fungi
Blastomyces dermatitidisBlastomyces dermatitidis
Paracoccidiodes brazielensisParacoccidiodes brazielensis
Coccidiodes immitisCoccidiodes immitis
Histoplasma capsulatumHistoplasma capsulatum Histoplasma duboisiiHistoplasma duboisii
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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
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Features of Systemic Mycosis…….
•Mode of transmission :
: Inhalation of large inoculum
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Coccidioidomycosis
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Life Cycle of Coccidiodes immitis
Mycelial at room Temperature
Arthrospores
Inhaled
Spherule in tissue
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Natural habitatThe fungus is found in dry, alkaline dessert soil, like those in San Joaquin Valley in California
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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
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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
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Cutaneous Coccidioidomycosis
Lesions similar to those found on the head of Domingo Escurra
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Provisional diagnosis
• Based on :
• Epidemiologic considerations• Clinical manifestations• Skin response to coccidioidin• Detection of antibody
Serology:
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Laboratory Diagnosis
Specimens :• sputum• biopsy• etc.
Direct Microscopy : KOH/FAT
Culture : media without antibiotics : slants used/bottles
( ( HAZARDOUS)HAZARDOUS)
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• Microscopic Examination : Spherule in the CNS
( from Granulomatous meningitis due to C. immitis ).
Multinucleated giant cellMultinucleated giant cell
spherule containing manyspherule containing many endosporesendospores
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Blastomycosis(North American Blastomycosis)
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• “ Acute or chronic infection of lung, skin, bone or genitourinary tract , characterized by formation of pyogranuloma.”
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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
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Mode of Transmission
• Inhalation of spores ( microconidia)
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Clinical manifestations
•Pulmonary• acute
Symptomatic cases presents like bacterial pneumonia
Clear with/without Treatment
Overwhelming: ARDS ( acute respiratory distress syndrome)
Chronic pulmonary
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Pulmonary Blastomycosis
Differential diagnosis:
• bacterial infections• fungal infections, • lymphomas • neoplasms
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Cutaneous blastomycoses
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Yeast cells are seen in lesions
Other forms of blastomycosis…..Other forms of blastomycosis…..
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Laboratory Diagnosis
• Direct Microscopic examination:
KOH Mount
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Multinucleated giant cell withMultinucleated giant cell with B. dermatitidisB. dermatitidis ( H & E stain)( H & E stain)
Calcofluor white Calcofluor white fluorescent stainingfluorescent staining
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»Culture
Requires 5 days to 4 weeks for growth; can be as short as 2-3 days
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• Serology
•Serum complement fixation•Immunodiffusion test•Radio immunoassay•ELISA
“None have been useful because of low sensitivity and specificity rates.”
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Paracoccidiomycosis( South American Blastomycosis)
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• “ 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.”
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Etiologic agent:Paracoccidiodes brasiliensis
• Dimorphic
Mycelial at room temperature;Indistinguishable from Blastomyces dermatitidis
Yeast at 370 C Multiple-budding cell ( narrow necked buds)
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• Mode of Transmission
Inhalation of spores
10 Pulmonary infection
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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
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Example of Cutaneous paracoccidioidomycosis
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Laboratory Diagnosis
• Demonstration of multiple-budding cell from clinical specimens
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Histoplasmosis( Great “ mimic” in mycosis”
caused by Histoplasma capsulatum )
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•“ 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.”
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Etiologic Agent
•Dimorphic
Mycelial phase at 250C,microconidia andtuberculate macroconidia,
Tissue phase at 370C,Thin-walled,single budding cells inside macrophages
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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
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• Closed environment
Bat caves have high deposits of guano
stable condition
BATS – GIT with ulcer
Cave explorationMay result in exposureTo high levels of organisms
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• Pathogenesis : Mode of transmission
Inhalation of spores
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Pathogenesis…….
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Pathogenesis…….
In 2-4 weeks, specific T cell- mediated immunity develops
Inhibits the growth of the organisms
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Cutaneous Histoplasmosis
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Histoplasmosis in the buccal mucosa
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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
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Laboratory Diagnosis
• Specimens
• Sputum• Buffy coat of blood ( WBC layer)• Sediments of biopsy materials• Gastric lavage• urine
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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
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Laboratory Diagnosis……..
• Culture • Media
» SAB» BAP without antibiotic» Smith and Goodman’s medium –
for heavily contaminated
specimens» Yeast extract with ammonium
hydroxide
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Laboratory Diagnosis………
• Culture
Histoplasma growing Histoplasma growing at room temperatureat room temperature from bone marrowfrom bone marrow specimenspecimen
Yeast colonies at370C
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Yeast colonies of H. capsulatum in blood agar , incubated at 370C
Laboratory Diagnosis…….Laboratory Diagnosis…….
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• Microscopic examination of mycelial form showing micro
and macroconidia of Histoplasma capsulatum
Laboratory Diagnosis…..Laboratory Diagnosis…..
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Laboratory Diagnosis……….
• Complement Fixation Test
• Animal inoculation
• Skin testing » Histoplasmin skin test
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Thank you !