MYCOLOGY-VIROLOGY

MIDTERM LECTURE NOTES

ENABLING OBJECTIVES: At the end of the period, the students will be able to.
1. Discuss each medically important fungus as to: Morphology and Physiology methods of transmission pathogenesis and clinical manifestations methods of diagnosis prevention and control 2. Perform slide preparation of fungal cultures 3. Identify a fungus based on gross and microscopic appearance

CONTENTS
Subcutaneous Mycoses Sporothrix schenkii Agents of Chromomycosis Agents of Mycetoma Rhinosporidium seeberi Loboa loboi Basidioboulus spp. Conidiobolus spp.

Systemic Mycoses Histoplasma spp. Cocciciodes immitis

Opportunistic Mycoses Candida spp.

Blastomyces dermatitidis Paracoccidiodes braziliensis

SUBCUTANEOUS MYCOSES
Caused by exogenous fungi that normally reside in nature, mostly in soil and vegetations Portal of entry Chronic infections Sporotrichosis Mycetoma Chromoblastomycosis Phaeohyphomycosis

Sporotrichosis
Rose gardeners disease Chronic infection of the subcutaneous tissues and lymphatics trauma (thorns or splinters) hand, arm or leg Occupational hazard

Sporotrichosis
Clinical manifestations
Fixed cutaneous sporotrichosis Lymphocutaneous sporotrichosis Pulmonary sporotrichosis Osteoarticular sporotrichosis

Sporotrichosis
Fixed cutaneous sporothricosis Primary lesion begins as a small, non-healing ulcer, commonly in the index finger or the back of the hand Lymphocutaneous sporotrichosis nodular lesions lymphatic vessels and lymph nodes draining the region

Sporotrichosis
Sporothrix schenckii
Dimorphic fungi aspirated pus from nodules, swabs, scrapings, biopsy tissue Macroscopic Rapidly growing, white, pasty, moist colony that later becomes brown, black, wrinkled or leathery Microscopic Mycelial form: narrow, septate hyphae with pyriform conidia arranged singly or in a flowerette

Mycetoma
Madura foot or Maduromycosis Traumatic inoculation with several saprophytic fungi lower extremities but may occur in any part of the body

Mycetoma: clinical manifestation

Chronic infection characterized by swelling, purplish discoloration, tumor-like deformities of subcutaneous tissue Multiple sinus tracts that drain pus containing yellow, white, red or black granules

Mycetoma: Types
1. Actinomycotic (bacterial)
Actinomycetes

Actinomyces Nocardia Streptomyces

MYCETOMA
2. Eumycotic (fungal)
Pseudallescheria boydii most common Acremonium falciforme Exophiala jeanselmei Curvularia Madurella mycetomatis Madurella grisea
White grain mycetoma Black grain mycetoma

Pseudallescheria boydii
Ascomycota group Soil, standing water and sewage Clinical specimens: granules from the lesions
Pseudoallescheriasis Meningitis Arthritis Endocarditis Brain abscess

Pseudallescheria boydii
Macroscopic
Rapidly growing (5-10 days), initial growth as a white fluffy colony after several wks to brownish-gray colony Reverse tan to dark brown

Microscopic
Asexual form : Scedosporium apiospermum golden brown elliptic, single-celled conidia borne singly from the tips of conidiophores Sexual form : brown sac-like cleistothecia containing asci and ascospores

Chromoblastomycosis
(Chromomycosis)
Traumatic inoculation Chronic infection producing warty or cauliflower-like or tumor-like lesions mostly in the lower extremities Epidermis hyperplasia

Chromoblastomycosis
(Chromomycosis)
Etiologic agents
Cladosporium (Cladophialophora carrionii) Phialophora (Phialophora verrucosa) Fonsecaea (F. pedrosoi, F. compacta) Rhinocladiella aquaspersa

Chromoblastomycosis
Macroscopic
All grow slowly and produce heaped-up and slightly folded, darkly pigmented colonies with a gray to olive to black velvety colonies; reverse side of colonies is jet black

Microscopic
Cladosporium: chains of budding blastoconidia borne from branching conidiophores Phialophora: short flask-shaped phialides with collarette Fonsecaea: conidial heads with sympodial arrangement of conidia, primary conidia giving rise to secondary or tertiary conidia

Fonsecaea
F. pedrosoi F. compacta Polymorphic Spherical w/ broad base connecting the conidia 1. Phialides 2. Chains of blastoconidia Smaller and more compact than pedrosoi 3. sympodial

 Rhinocladiella
Produces lateral or terminal condia from conidiogenous cell ( sympodial) Conidia are elliptical to clavate

Sclerotic Bodies
Characteristic histologic findings in tissues with chromoblastomycosis Copper-colored, septate cells that appear to be dividing

Phaeohyphomycosis
Caused by dematiaceous fungi other than those causing chromomycosis Tissue morphology is mycelial Subcutaneous and systemic infection

Clinical manifestation
Subcutaneous phaeohyphomycosis
Cystic lesion, abscess

Paranasal sinus phaeohyphomycosis

sinusitis

Cerebral phaeohyphomycosis
Immunosuppressed

Phaeohyphomycosis
Etiologic Agents
Exophiala jeanselmei Wangiella dermatitidis Phialophora richardsiae Alternaria spp Bipolaris spicifera Curvularia spp

subQ: exophiala and wangiella paranasal sinusitis ( allergic rhinitis or immunosuppression) : Bipolaris, Exserohilum, Curvularia and Alternaria

Exophiala
Macroscopic
Grow slowly (7-21 days) and initially grows black yeast-like colonies; as colonies age they become filamentous, velvety, gray to black

Microscopic
Pale brown conidiophores that form cylindrical annellids, hyaline conidia gather at its tip

Wangiella dermatitidis
Subcutaneous phaeohyphomycosis

cystic lesions occur most often in adults : subcutaneous phaeomycotic cyst

Alternaria
Macroscopic Grow rapidly and appear fluffy, gray to gray brown or gray green colonies Microscopic Hyphae: septated and golden brown, Conidiophores: simple sometimes branched which bear a chain of large brown conidia resembling a drumstick

Bipolaris
Macroscopic Rapid grower, gray-green to dark brown powdery colonies Microscopic Hyphae are dematiaceous and septate; conidiophores are twisted at the ends where conidia are attached; conidia are oblong and multicelled

Curvularia
Macroscopic Rapid growing, most are fluffy, gray to black colonies

Microscopic Hyphae are dematiaceous and septate conidiophores are twisted at the ends where conidia are attached conidia are multicelled, curved with a central swollen cell

SYSTEMIC & OPPORTUNISTIC MYCOSES

Primary Systemic Mycoses
Coccidioidomycosis Histoplasmosis Blastomycosis Paracoccidioidomycosis

Opportunistic Mycoses
Candidiasis, systemic Cryptococcosis Aspergillosis Mucormycosis

Primary Systemic Mycoses

Caused by dimorphic fungi Dimorphic fungi
Yeast phase
When grown on enriched media usually supplemented with blood at 35-37C Is observed in vivo and is also known as the tissue or invasive phase

Mycelial phase
Observed on SDA at 25-30C Saprophytic, observed in vitro

Dimorphic Fungi
Identified by
Growth characteristics Colonial morphology Microscopic characteristics of conidia and hyphae Conversion of mycelial to yeast phase Antigen testing Nucleic acid probes

Transmission
Inhalation of fungal spores Lead initially to pulmonary infection which may be symptomatic or asymptomatic Dissemination to other body sites can occur

Coccidioidomycosis
Acquired through inhalation of the infective arthroconidia Approximately 60% are asymptomatic and self-limited respiratory tract infections Infection may become disseminated to visceral organs, meninges, bone, skin, lymph nodes and subcutaneous tissue

Coccidioidomycosis
Etiologic agent: Coccidioides immitis Clinical specimens: Sputum, tissues or body fluids Direct microscopic examination from clinical specimens Non-budding, thick-walled spherule, 20-200 um in diameter containing either granular material or numerous small non-budding spores

Coccidioides immitis
Macroscopic
Colonies appear after 3-21 days, delicate fluffy white which turn tan or brown with age

Microscopic
Mycelial phase: septate, branched hyphae that produce thick-walled barrel-shaped, rectangular arthroconidia that alternate with empty alternate cells Yeast phase: large, round, thick-walled spherules with endospores observed in tissues and direct examination

Coccidioides immitis
Other nonvirulent fungi that resemble C. immitis microscopically may be found in the environment and may produce hyphae that may dissociate into arthroconidia Considered as the most infectious of all fungi Extreme caution should be observed in handling cultures of this organism If culture plates are used, they should be handled only in a biological safety cabinet Cultures should be sealed in tape if the specimen is suspected of containing C. immitis

Safety Precautions in Handling C. immitis Cultures

cotton-plugged tubes is discouraged and screw-capped tubes are preferred All microscopic preparations for examination should be performed inside a BSC Cultures should be autoclaved as soon as the final identification of C. immitis is made

Histoplasmosis
A chronic granulomatous infection that is primary and begins in the lungs, produce cavitary lesions disseminate to the lymph node, liver, spleen, bone marrow, kidneys, meninges Heart infxn in immunocompromised indls

Histoplasmosis
inhalation of conidia or small hyphal fragments 95% are asymptomatic and self-limited most prevalent pulmonary mycosis of humans and animals

Histoplasma capsulatum
Direct microscopic examination
Difficult to visualize in the sputum and other tissues bone marrow smear: Wright or Giemsa-stained Rarely in peripheral blood Intracellular yeast in macrophages

Histoplasma capsulatum
Macroscopic Slow growing SDA: white to brown mold with fine fluffy texture reverse side: white, yellow or tan BHI moist, white to cream heaped colony Microscopic Mycelial phase: septate hyphae with large spherical or pyriform tuberculate macroconidia; some produce small round smooth microconidia

Blastomycosis
Chronic suppurative and granulomatous infection which involve the lungs and spread to the long bones, soft tissue and skin inhalation of the conidia and hyphal fragments

Blastomyces dermatitidis
Direct microscopic examination of tissues or body fluids
large, spherical, thick-walled yeast cells 8-15 u usually with a single bud that is connected to its parent cell by a broad base Mycelial phase: delicate, septate hyphae with round or pyriform conidia borne singly on conidiophores resembling lollipops

Blastomyces dermatitidis
Macroscopic
Growth rate is 7-21 days SDA: colony at first white, waxy, yeast-like and later becoming cottony with white aerial mycelium; turns tan to brown with age BHI with blood: cream to tan, waxy. Wrinkled colonies

Paracoccidioidomycosis
Chronic granulomatous infection that begins as a primary pulmonary infection asymptomatic but may disseminate to produce ulcerative lesions

Paracoccidioides brasiliensis
Direct microscopic examination of sputum, mucosal biopsy, exudates
Large, round or oval yeast cells, 8- 40 um, producing multiple buds and each is attached to the parent cell by a narrow base

Paracoccidioides brasiliensis
Macroscopic: SDA: white, glabrous, leathery colony which turns tan-brown with age BA: cream to tan, moist, wrinkled colony which turns waxy with age Microscopic Mycelial phase small, septate, branched hyphae with intercalary and terminal chlamydospores few pyriform microconidia

Paracoccidioides brasiliensis
Yeast phase large, round to oval, thick-walled yeast cells (8-40 u) with multiple buds with a narrow base mariners wheel

Opportunistic Mycoses
Candidiasis
Most frequently encountered opportunistic fungal infection Etiologic agents
Candida albicans C. tropicalis C. parapsilosis C. glabrata

Candidiasis
are part of the normal flora, seen in the oropharynx, GIT, GUT, skin Infections are believed to be endogenous in origin or nosocomial

Candida Infections In Normal And Immunocompromised Hosts

Intertriginous candidiasis (skin folds) Onychomychosis and paronychia Perleche Oral thrush Vulvovaginitis Pulmonary infection Eye infections Endocarditis Meningitis

Candida Infections In Normal And Immunocompromised Hosts

Fungemia and disseminated infection
Most commonly seen in immunocompromised patients Onychomycosis and esophagitis caused by Candida albicans are very common in AIDS patients

Predisposing Factors For Candidiasis

Alteration in the normal skin and mucous membrane barriers Prolonged antibiotic administration Use of immunosuppressive drugs Diseases of the immune system

Candida
Direct microscopic examination of clinical specimens
Budding yeast cells Pseudohyphae Definitely identified microscopically by production of germ tubes and chlamydospores

Candida
Macroscopic
Candida species cannot be differentiated based on colonial appearance Colonies are produced within 24-48 hours Raised, cream-colored, opaque, 1-2 mm; after several days on agar medium hyphae may be observed

Germ Tube Test

A hypha-like extension of the yeast cells with no constriction at the point of origin Candida albicans will form germ tubes when incubated with serum at 37C for a few hours

Cornmeal Agar with Tween 80

Conidiation ID of Candida spp and other yeasts through examination of
hyphae, blastoconidia, chlamydospores. and arthroconidia

Tween 80
reduce the surface tension to allow conidiation

Cornmeal Agar with Tween 80

Procedure
colony from the 1 culture media Inoculate a plate of CMA with 1% T80 and trypan blue by making 3 parallel cuts about inch apart at a 45 angle to the culture medium RT for 48 hours After 48 hours, remove and examine the areas where cuts into the agar were made

Commonly encountered yeast in CMA-T80 Agar

Organism Arthroconidia Blastoconidia Pseudohyphae or Hyphae Chlamydoconidia on hyphae

C. albicans -

Spherical clusters at regular intervals on pseudohyphae Small, spherical, tightly compact Elongated, clusterered at septae of pseudohyphae Present but not characteristic

C. glabrata

None

C. krusei -

Branched pseudohyphae

C. parapsilosis -

Sagebrush like, Giant hyphae

Commonly encountered yeast in CMA-T80 Agar

Organism

Arthro- conidia

Blastoconidia

Pseudohyphae or Hyphae PH present, not characteristic PH present, not characteristic Rare, usually not seen Rudimentary H sometimes present Septated hyphae is present

C. kefyr (pseudotropicalis) C. tropicalis

Elongated, parallel to pseudohyphae Randomly appear on PH &H

C. neoformans

Round to oval separated by capsule Large and spherical

Saccharomyces

Trichosporon

Numerous, resemble Geotrichum

Maybe present but difficult to find

Cryptococcosis
An acute, subacute or chronic fungal infection that has several manifestations
Disseminated disease
with or without meningitis in immunocompromised patients Meningitis occur 2/3 of patients very common in patients with AIDS

Cryptococcus neoformans
Saprophyte pigeon, bat, or bird droppings, decaying vegetations, fruit, plants Inhalation lungs then disseminate to meninges and other sites

Cryptococcus neoformans
Direct microscopic examination
Spherical, single or multiple budding, thick-walled yeast cell (2 to 15 um) surrounded by a wide, refractile polysaccharide capsule

Macroscopic
Colonies appear in 1-5 days smooth, white to tan, mucoid, gelatin-like colonies (soap-bubble) Brown-black colonies on Niger seed agar

Organism

Capsule

Germ Tube + -

Blastoconidia + +

Arthroconidia -

Chlamydospore + V

C. albicans C. tropicalis

C. parapsilosis C. glabrata C. neoformans Geotrichum

+ -

+ + + -

T. beigilii

Organism G
C. albicans C. tropicalis + +

FERMENTATION M
+ +

Urease L
-

Nitrate Reduction

S
+

C. parapsilosis C. glabrata C. neoformans Geotrichum T. beigilii

+ + -

+ +

Aspergillosis
disseminated infection in IC patients Other infection
invasive lung infection Pulmonary or sinus fungus ball (tangled mass of hyphae) Mycotic keratitis allergic pulmonary aspergillosis External otomycosis Onychomycosis Sinusitis, endocarditis, CNS infxn

inhalation

Aspergillus fumigatus
Direct microscopic examination
Septate hyphae that usually show dichotomous branching (45 angle branching)

Microscopic
Branching septate hyphae that terminate in conidiophore

Macroscopic
Rapidly growing mold (2-6 days) fluffy to granular, white to blue green colonies
MOST COMMONLY RECOVERED SPP FROM IC PATIENT A. FLAVUS SOMETIMES RECOVERED Botany repeated branching into two equal parts. Methenamine silver stained tissue section showing dichotomously branched, septate hyphae.

Aspergillus fumigatus
expands into a large DOME-SHAPED vesicle with BOTTLE-SHAPED phialides from which chains of conidia arise

Zygomycosis (Mucormycosis)
Decaying vegetable matter, old bread or in soil Acquired by inhalation Less common cause of infection as compared to Aspergillus Rhinocerebral infection involving nasal mucosa, palate, sinuses and brain Perineural invasion Retro-orbital spread (brain) Lungs, GIT

Zygomycetes
Direct microscopic examination of tissue specimens or exudates
Branching non-septate hyphae

Macroscopic
Fluffy, white to gray to brown colonies covering the surface of the agar within 24-95 hours, grayish hyphae with brown to black sporangia

Zygomycetes
Microscopic
large ribbon-like hyphae
irregular in diameter non-septate

Sporangia
Sac-like sporangiospores at the tip of sporangiophore

Stolons
Connects sporangiphore Rhizoids are attached

Mucor
Sporangiophores the tip of which have sporangia filled with sporangiospores No rhizoids and stolons

Rhizopus
unbranched sporangiophores with rhizoids that appear at the point at which the stolon arises

Penicillium
When clinically significant, clinical manifestations include bronchopulmonary, endocarditis, cutaneous ulcers of extremities

Penicillium marneffei
Macroscopic
Colonies with shades of green, blue-green, white, pink , other colors, Surface velvety to powdery colonies

Fusarium
Infections becoming more common esp in IC patients (Hyalohyphomycosis) Common environmental flora
mycotic keratitis after traumatic implantation into the cornea

Other infections: sinusitis, wound (burn) infections, allergic fungal sinusitis, respiratory tract secretions

Fusarium
Macroscopic
Grow rapidly (2-5 days) fluffy to cottony and may appear pink, purple, yellow, or green

Microscopic Hyphae are small and septated Large sickle- or boat-shaped macroconidia; microconidia are produced

Pneumocystis jiroveci (carinii)

Opportunistic atypical fungus causing pneumonia in immunocompromised hosts Ideal specimen broncho-alveolar lavage fluid or lung biopsy Does not grow in routine culture methods

Pneumocystis jiroveci
Two forms
Trophozoite forms
pleomorphic GIEMSA

Cystic forms
4 to 7 um, do not bud, with intra-cystic bodies Methenamine silver Calcofluor white IF stain

 ADDITIONALS FOR ANTIFUNGAL AGENTS

Anti-fungal agents
I. II. III. IV. Polyene macrolide antifungals Antimetabolite Azole antifungal drugs Echinocandins

Polyene macrolide antifungals

Agent Amphotericin B (liposomal prep) Source Streptomyces nodosus Function Binds ergosterol and alter selective permeability Treatment for IV:Aspergilossis Candida spp. Cryptococcus Zygomycetes R: P.boydii, A. terreus, Trischosporon, Fusarium Oral or vulvovaginal candidiasis Oral tx: dermatophytes non responsive to azole

Nystatin

S. noursei

Not absorbed by GIT, not given parenterally (TOXIC) Binds microtubular protein (mitosis)

Griseofulvin

Penicillium

Antimetabolite
5- Fluorocytosine (Flucytosine)
5-fluorouracil
incorporated to fungal RNA and inhibit protein synthesis

Fluorodeoxyuridine monophosphate
Inhibitor of DNA synthesis

Combination therapy w/ AmB

Candida spp. and C. neoformans Side effect & resistance when used alone

Azole antifungal drugs

Agent Clotrimazole & Miconazole Fluconazole Application Topical or intravaginal Oral or IV Treatment Mild dermatophytosis (T.versicolor) Candida and Cryptococcus (CNS) Adverse rxn Burning, itching, skin irritation S or R (C. glabrata) R: C.krusei & Rhodotorula spp) Elevated liver enzymes, nausea, dose relatedgynecomastia; Decreased libido; oligospermia

Ketoconazole

Topical or oral

Mild ParacoccidioidomycosisBla stomyces & Histoplasmosis Chronic mucocutaneous candidiasis P. boydii

Azole antifungal drugs

Agent Application Treatment Adverse rxn

Itraconazole

Expanded activity w/ ketoconazole

Voriconazole (new triazole)

Expanded activity compared w/itraconazole

Aspergillosis Sporothricosis Cryptococcosis Onchymycosis Blastomycosis Fusarium C.krusei & C. glabrata

GIT & vestibular disturbances, edema, skin irritation

R: Zygomycetes; Elev.liver enzymes; visual disturbances

Echinocandins
Agent Application Treatment Adverse rxn

Caspofungin Micafungin Anidulafungin

Fungicidal
Fungistatic

Candida spp (krusei,glabrata) Aspergillus

R: C.neoformans Trichosporon, Rhodotorula Zygomycetes

Selenium sulfide

Shampoo Sporicidal

Malasezzia furfur T. tonsurans

Potassium iodide

oral

Cutaneous/lymp Bitter taste, hatic allergic rash sporothricosis and anorexia