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Characteristics of Fungi Non-motile eukaryotes lacking chlorophyll

– Contain nucleus, mitochondria, 80S ribosomes– Cell wall is composed of polysaccharides ,

polypeptides and chitin and the cell membrane contains sterol which prevent many antibacterial antibiotics being effective against fungi.

– Larger than bacteria– Relatively simple nutritional requirements, wide range

of growth rates• Form visible colonies in days to weeks

Unicellular or multicellular depending on the species

Fungi can be divided into: Yeast, Mould (filamentous fungi) and Dimporphic fungi

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Molds Multicellular, tubular structures (hyphae) Hyphae can be septate (regular crosswalls) or

nonseptate (coenocytic) depending on the species (grow by apical extension)

– Vegetative hyphae grow on or in media (absorb nutrients); form seen in tissue, few distinguishing features

– Aerial hyphae contain structures for production of spores (asexual propagules); usually only seen in culture

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Molds - identification Identification based on colony morphology

(pigment, texture) and morphology of reproductive structures– Conidia - spores formed by budding (blastoconidia) or

disarticulation of existing hypha (arthroconidia)

– Sporangiospores - produced by free-cell formation within sporangium in nonseptate molds

1. sporangium2. sporangiophore3. Endospores4. Nonseptate hyphae5. rhizoids

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1. Conidiopspores2. Phialides3. Vesicle4. Conidiophore5. Septate hyphae

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Colonial Morphology of Fungi

Cryptococcus neoformans

Candida albicans

Wangiella dermatitidis

Aspergillus fumigatus

T. menta-grophytes

Trichophyton tonsurans

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Yeasts Unicellular, 3-5 µm, reproduce by budding

(blastoconidia formation) or fission Identified by microscopic morphology (grow

on cornmeal agar) and biochemical tests (sugar assimilation, enzymatic activity).

Molds and yeast are not exclusive forms, some species may exist in both yeast and mold forms (dimorphism).

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Classification of Fungi Taxonomy is based on structural features of the

teleomorph (sexual phase).

– Zygomycota - includes all fungi with nonseptate hyphae

– Ascomycota - includes most human pathogens

– Basidiomycota - mainly plant pathogens

– Deuteromycota (fungi imperfecti)

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Isolation of fungi Direct examination:-

Wet specimen preparations

– e.g. Aspergillus hyphae in sputum or Cryptococcus neoformans in CSF (mixed with india ink)

– Potassium hydroxide (KOH) cleared specimens , e.g. dermatophyes (ringworm fungi) in skin scrapings, nails or hair

– Stained preparation: e.g. Candida albicans in Gram stained smears of vaginal discharge or pneumocystis carinii in Giemsa or other stained preparations of broncho-alveolar lavage or sputum.

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Isolation of fungi Culture Media

– General purpose media• BHI + blood, inhibitory mold agar• Sabouraud dextrose agar

– Mycosel• Sabouraud + chloramphenicol and cycloheximide• For isolation of dermatophytes (some pathogenic

fungi are inhibited by cycloheximide)

Incubation– 30°C for 30 days

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Molds• Dermatophyte • Fungi causing mycetoma الفطري القدم داء• Fungi causing chromomycosis ملون فطر• Aspergillus species• Zygomycetes: Mucor فطري عفن

Yeast• Candida albicans• Cryptococcus neoformans• Malassezia furfur

Dimorphic • Histoplasma species• Blastomyces dermatitidis• Paracoccidioides brasiliensis• Coccidioides immitis

Medically important fungi

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Fungal Infections Fungi generally have low pathogenic potential

– Only a few true pathogens; many opportunists– Most are acquired from exog/environ sources – Pathology caused by tissue invasion and/or host

inflammatory response. Many fungal infections can be characterized by extent of invasion

Type of infection Example

superficial Malasezzia furfurcutaneous dermatophytessubcutaneous sporotrichosis

deep/systemic histoplasmosisopportunistic candidiasis

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Dermatophytes Fungi that cause superficial/cutaneous infections

– Confined to keratinized tissue (hair, skin, nails) – Cause of ringworm (eg tinea capitis, tinea pedis); inflammation greatest at

advancing edge.– All are molds. Many species produce macroconidia

• Epidermophyton (2 spp), Microsporum (16 spp), Trichophyton (24 spp)

– Transmitted indirectly via desquamated skin and hair (combs االمشاط hatsقبعه , showers, etc).

Diagnosis based on direct examof scrapings, culture on selective media.

M. canis

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Subcutaneous Mycoses Caused by direct inoculation of organisms from

soil or decaying plants– Generally cause localized infection– Hands and feet are most common sites

Sporotrichosis المبوغة الشعيرات داء– Caused by dimorphic fungus Sporothrix schenkii

• Mold in environment, yeast in infected tissue– Commonly affects hands trauma from thorns causes lympho-cutaneous ;(eg rose gardens)شوكهinfection

• Extracutaneous (pulmonary) and disseminated disease uncommon

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Subcutaneous Mycoses (continued)

Chromoblastomycosis البرعمي الفطرالملون– Slow, progressive, granulomatous

infection.– Skin lesions contain dark brown sclerotic

bodies. – Caused by dematiaceous (black) molds,

eg Cladosporium, Phialophora. Mycetoma

– Swollen lesion, granules (containing organisms) draining from sinuses.

– Can be caused by fungi eg Pseudoallescheria or actinomycetes (prokaryotes)

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Candidiasis Genus Candida - diverse group of yeasts

– Budding yeast, stain Gram-positive– ID based on biochemical tests and morphology (corn

meal agar) C. albicans - most important pathogen

– Multiple forms: budding yeast, pseudohyphae, true hyphae. Forms germ tubes (in presence of serum).

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Pathogenesis of Candida infections

Most infections are endogenous– Candida is component of normal oral, GI, vaginal flora

Pathogenic factors– Essential role of mucosal adherence– Alterations in micro-environment and/or microbial flora

predispose to symptomatic infection. – Germ tube formation, proteinases, phospholipases

may contribute to local invasion by C. albicans

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Candida infections

Cutaneous infections - nails, diaper rash,

Mucosal infections– thrush (tongue, oral mucosa), pseudo-membrane;

seen with inhaled steroids, cancer, HIV– esophagitis in suppressed pts (mucosal invasion)– vulvovaginitis (discharge containing epithelial cells,

pseudohyphae, hyphae)

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Candida infections Candidemia/disseminated candidiasis

– Candida spp are 4th leading cause of nosocomial bloodstream infections

– Antibiotics, iv catheters increase risk• Antibiotics eliminate normal GI flora, permit

overgrowth of Candida and entry across damaged mucosa

• IVs provide entry through skin– Dissemination to kidney, brain, myocardium, eye

is common.• Ocular candidiasis - white cotton ball-like

lesions of retina; can cause blindness

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Candida infections (continued)

Urinary tract candidiasis – Usually seen in pts with urinary catheter.

Hepatosplenic candidiasis– Occurs in severely compromised (neutropenic)

pts. Multifocal abscesses

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Candidiasis - Diagnosis Direct microscopic examination

– Important to demonstrate tissue invasion in mucosal infection; positive culture alone may be due to colonization

Culture– Candida spp grow well on standard media. – Candidemia readily detected with commercial

blood culture systems.

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Encapsulated yeast – Identification based on presence of capsule, urease,

growth at 37°C, melanin synthesis, and sugar assimilation.

Major virulence factors – Polysaccharide capsule

• Antiphagocytic• Immunosuppressive

– Melanin synthesis

Cryptococcus neoformans

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C. neoformans - Clinical features Acquired by inhalation

– Most infections are asymptomatic. May present as isolated pulmonary nodule (r/o carcinoma)

Cryptococcal meningitis– Dissemination from lung. Life-threatening– Major opportunistic infection in HIV pts with low CD4

counts

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Cryptococcocal meningitis Diagnosis

– CSF WBC count may not be elevated• Poor prognostic sign

– Direct detection of capsular antigen in CSF• Latex agglutination or EIA• High sensitivity and specificity; has displaced

India ink (lacks sensitivity)– Culture - gold standard

Treatment– Amphotericin B + 5-fluorocytosine– In HIV patients, C. neoformans cannot be

eradicated, requires suppressive therapy.

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Histoplasma capsulatum - Dimorphism Filamentous mold in environment

– Thin septate hyphae, microconidia, and tuberculate macroconidia (8-14 µm)

Budding yeast (2-4 µm) in tissue– Dimorphic transition is thermally dependent and

reversible (25°C 37°C).

Hyphae, micro- and macroconidia Yeast within histiocyte

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H. capsulatum - Epidemiology– H. capsulatum can be cultured from soil in endemic

areas. Abundant growth in soil containing bird feces (starling roosts, chicken houses) or bat guano (caves).

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H. capsulatum - Pathogenesis Conidia or hyphal fragments are inhaled,

ingested by macrophages. Organisms convert to yeast phase,

proliferate in nonimmune macrophages, and spread through RES.

Dissemination is common and occurs early, but is usually asymptomatic.

CMI response results in macrophage activation, increased fungicidal activity. Infection is contained but not necessarily eradicated.

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Histoplasmosis - Self-limited Syndromes Acute pulmonary histoplasmosis accounts

for most cases of symptomatic infection– Fever, chills, headache, myalgia, anorexia,

nonproductive cough, pleuritic chest pain.– Enlarged lymph nodes, patchy infiltrates. – Patients usually improve in several weeks.

5-10% symptomatic cases develop inflammatory syndromes (arthritis, erythema nodosum, or pericarditis)

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Chronic pulmonary histoplasmosis Slowly progressive pulmonary disease. Usually associated with preexisting lung

disease.– Cough, dyspnea, chest pain, fatigue, fever, night

sweats, and weight loss

Usually progresses if untreated– AmB or itraconazole (depending on disease

severity) reduces symptoms, improves radiographic findings, eliminates H. capsulatum from sputum

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H. capsulatum - Culture

Histoplasma produces mycelial growth with characteristic warty الثألول conidia whenمثلcultured at room temperature.

Small microconidia and characteristic large , round , spiny macroconidia are produced.

At 37 C on certain media it is possible to induce the yeast phase of this dimorphic fungus– Sputum best for pulmonary histoplasmosis– Bone marrow or blood best for disseminated

histoplasmosis

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H. capsulatum – Direct examination

Examination of infected tissue (eg bone marrow, liver, skin, GI mucosa) can provide rapid presumptive evidence of disseminated histoplasmosis

Intracellular yeasts can be seen on peripheral smears in severe disseminated disease

NEJM 342:28

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C. immitis - Dimorphism Grows as hyphae in environment. Forms thick-

walled arthroconidia alternating with thin-walled cells.

Spherule كرية filled with endospores in infected tissue.

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C. immitis - Pathogenesis Arthroconidia are inhaled and convert to

spherules that grow to 20-150 µm. Partially resistant to killing by phagocytic cells.

Spherules undergo multiple nuclear divisions and segmentation of cytoplasm to produce hundreds of endospores (2-5 µm). The spherule ruptures, releasing endospores that form new spherules.

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C. immitis - Epidemiology

Can be cultured from soil in areas where disease is endemic. Expected number of infections is 100,000 annually

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C. immitis - Respiratory Infections 40% of pulmonary infections are symptomatic

– Most are self-limited– Fatigue, cough, chest pain. May also have fever,

dyspnea, myalgia, and headache

Pulmonary nodules– 4% of infections give rise to solitary وحيدة nodule (

5 cm)– Can form cavities, infreq rupture into pleural space

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C. immitis - Disseminated infections Occurs in ~ 0.5% of infections. Increased risk:

– HIV, organ transplants, steroids, Hodgkin’s

Skin is most common site of dissemination Joints and Bones

– Prominent synovitis, effusion; knee most common– Vertebrae (multiple) > long bones– culture (50%) and histopathology for diagnosis

Meningitis– Headache, vomiting, alt mental status. WBC

(mono), prot, gluc in CSF. Culture usually neg.

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C. immitis - Culture and Histopathology Culture - white fluffy وبريmold at 25-30ºC.

– Arthrospores are suggestive but not diagnostic

Histopathology– Acute inflammation (PMNs and Eos) assoc with

active infection and ruptured spherules– Granulomas assoc with chronic infection,

unruptured spherules

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Blastomyces dermatitidisMicrobiology

– Hyphae with microconidia at room temp– Convert to broad-based budding yeast at

37ºC. pulmonary infection (asymptomatic or

pneumonia); chronic pulmonary disease common

Disseminated disease: skin (common) and bones

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B. dermatitidis - Diagnosis Culture

– White light tan mold at room temp, not diagnostic– Identification based on conversion to yeast at 37ºC,

Histopathology– Thick walled broad based

budding yeast– suppurative and/or

granulomatous inflammation

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Paracoccidiodes brasiliensis Microbiology

– Hyphae at room temp. – Converts to yeast with

multiple buds at 37ºC.

– Probably acquired by inhalation. Pulmonary infection can be asymptomatic, acute, or chronic

– Extrapulmonary disease in adults > 30 usually involves oropharyngeal mucosa and regional lymph nodes.

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Penicillium marneffei Only dimorphic species in genus Penicillium

• Infection probably occurs through inhalation

• Chronic illness, low-grade fever, wt loss, skin lesions, disseminated infection

– Intracellular forms resemble H. capsulatum; extracellular forms exhibit septa (cells divide by fission, not budding)

– Grows as mold at 25-30ºC• Produces soluble red pigment• Converts to yeast phase at 37ºC

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Aspergillus spp. More than 100 species of Aspergillus. Septate

hyphae branching at 45 angle. Omnipresent مكان بكل .in environment. Aموجودة

fumigatus is thermotolerant (up to 55oC) and is found in high concentrations in compost اوراق.sitesالشجر

Most human disease caused by A. fumigatus, A. flavus, and A. niger.

Opportunistic pathogen, airborne spread

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Aspergillus Infections Allergic bronchopulmonary aspergillosis Aspergilloma (fungus ball)

– Colonization of preexisting lung cavity (TB, abscess, etc(.

Invasive pulmonary aspergillosis – Occurs in pts with immunosuppression and

neutropenia. – Vascular invasion, infarction, cavitation– Hematogenous dissemination common (ocular,

cerebral, cutaneous involvement)

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Aspergillosis - Diagnosis Direct examination

– Difficult to distinguish branching septate hyphae of Aspergillus spp from other opportunistic fungi, eg Pseudallescheria, Fusarium.

Culture– Aspergillus spp grow well on standard media.

Airborne contaminants are a problem. – Need to see several colonies from one specimen

or same organism in multiple specimens.

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Mucormycosis Infections caused by Mucoraceae

– Mucor, Rhizopus, Absidia

– Broad nonseptate hyphae, sporangia.• Widespread in environment; found in

decaying vegetables and fruits, soil, old bread; grow and sporulate on materials containing carbohydrates. R. oryzae is most common clinical isolate.

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Mucormycosis - Clinical features Rhinocerebral/craniofacial mucormycosis

– Infection of paranasal sinuses with extension from ethmoid into orbit or frontal lobe (also cavernous sinus thrombosis).

– Prompt diagnosis essential; direct exam of turbinate scrapings and/or sinus aspirate.

Pneumonia – Resembles invasive pulmonary aspergillosis

Cutaneous infection– Associated with localized trauma– Cellulitis with central necrotic area.

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Antifungal Drugs Polyenes (Amphotericin B, nystatin) Azoles (fluconazole, itraconazole) Echinocandins (caspofungin) 5-Fluorocytosine