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MycologyMycology

Dental / Optometry MicrobiologyDental / Optometry MicrobiologyStephen A. Moser, Ph.D.Stephen A. Moser, Ph.D.

10/10/200810/10/2008

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Reading AssignmentReading Assignment

Medical Microbiology, 4th EditionMurray, PR, et al

Chapters 66 – 71.

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EpidemiologyEpidemiology

GeographyGeography Endemic mycosesEndemic mycoses Worldwide mycosesWorldwide mycoses

Transmission of infectionTransmission of infection Respiratory inhalation (systemic mycoses)Respiratory inhalation (systemic mycoses) Cutaneous inoculation (sporotrichosis)Cutaneous inoculation (sporotrichosis) Systemic invasion by opportunistic normal floraSystemic invasion by opportunistic normal flora

(candidiasis)(candidiasis) Contact with infected hosts (dermatophytoses)Contact with infected hosts (dermatophytoses)

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Epidemiology (Cont.)Epidemiology (Cont.) Risk factors and manifestations of diseaseRisk factors and manifestations of disease

True pathogens versus opportunistsTrue pathogens versus opportunists Environmental risk factors for systemic fungalEnvironmental risk factors for systemic fungal

diseasedisease•• Location and travelLocation and travel•• OccupationOccupation

Host defenses and susceptibility to systemicHost defenses and susceptibility to systemicfungal disease (CMI most important)fungal disease (CMI most important)

•• Congenital and acquired T cell deficiencies (includingCongenital and acquired T cell deficiencies (includingAIDS)AIDS)

•• Immunosuppression (transplants and malignancies)Immunosuppression (transplants and malignancies)•• Diabetes mellitusDiabetes mellitus

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Endemic Distribution for BlastomycosisEndemic Distribution for Blastomycosis

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General CharacteristicsGeneral Characteristics

AerobicAerobic - obligate or facultative - obligate or facultative EukaryoticEukaryotic: membrane bound nucleus: membrane bound nucleus

and cytoplasmic organelles (may beand cytoplasmic organelles (may bemultinucleate)multinucleate)

AchlorophyllousAchlorophyllous MorphologyMorphology (unicellular or (unicellular or multicellularmulticellular)) Saprophytic (heterotrophic)Saprophytic (heterotrophic)

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Characteristics of Fungal CellsCharacteristics of Fungal Cells

Cell wallCell wall: : multilayered polysaccharidemultilayered polysaccharide Cellulose, glucans, mannans, chitin, polypeptidesCellulose, glucans, mannans, chitin, polypeptides Absence of teichoic acids, peptidoglycan, LPSAbsence of teichoic acids, peptidoglycan, LPS

Cell membraneCell membrane PhospholipidPhospholipid bilayerbilayer Ergosterol (relate to chemotherapy)Ergosterol (relate to chemotherapy)

CytoplasmCytoplasm - typical eukaryotic organelles- typical eukaryotic organelles NucleusNucleus - either - either uninucleateuninucleate or multinucleate or multinucleate

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Characteristics of Fungal CellsCharacteristics of Fungal Cells

CapsuleCapsule Present in some species (e.G. Present in some species (e.G. CryptococcusCryptococcus

neoformansneoformans)) Amorphous polysaccharide coatingAmorphous polysaccharide coating FunctionsFunctions and activitiesand activities

AntiphagocyticAntiphagocyticAntigenicAntigenic

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Growth formsGrowth forms Yeast - unicellular fungi which reproduce byYeast - unicellular fungi which reproduce by

budding (budding (CryptococcusCryptococcus)) Mold - hyphae (mycelium)Mold - hyphae (mycelium)

Septate hyphae (Septate hyphae (AspergillusAspergillus))

Non-septate, Non-septate, coenocyticcoenocytic hyphae ( hyphae (MucorMucor)) PseudohyphaePseudohyphae ( (Candida albicansCandida albicans)) Thermal dimorphismThermal dimorphism

Characteristics of Fungal CellsCharacteristics of Fungal Cells

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Differences Between BacteriaDifferences Between Bacteriaand Fungiand Fungi

PROPERTY FUNGI BACTERIA

Cell diameter 5-50 microns 1-5 microns

Nucleus Eukaryotic prokaryotic

Cytoplasmic organelles Present absent

Cell membrane sterols present (ergosterol) absent (exceptMycoplasma)

Cell wall chitin, glucans,mannans, peptides

teichoic acids,peptidoglycan, LPS

Metabolism Mainly aerobes, facultativeanaerobes

obligate and facultativeaerobes and anaerobes

Thermal dimorphism Common in many pathogenicspecies

absent

Differences Between Differences Between Bacteria and FungiBacteria and Fungi

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Examples of Yeast &Examples of Yeast &PseudohyphaePseudohyphae

Pseudohypha

Blastoconidia

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Example of True Septate HyphaeExample of True Septate Hyphae

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Non-septate HyphaeNon-septate Hyphae

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Blastomyces dermatitidisBlastomyces dermatitidis Thermal ThermalDimorphismDimorphism

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Asexual ReproductionAsexual Reproduction

Conidia (spores) Conidia (spores) –– asexual structures asexual structures BlastosporesBlastospores –– formed by budding yeasts formed by budding yeasts

((BlastomycesBlastomyces)) ChlamydosporesChlamydospores –– terminal or intercalary cells terminal or intercalary cells

with thick walls (with thick walls (Candida albicansCandida albicans)) ArthrosporesArthrospores –– formed by fragmentation of formed by fragmentation of

hyphae (hyphae (Coccidioides immitisCoccidioides immitis)) SproangiosporesSproangiospores –– formed in sporangia by formed in sporangia by

cleavage (cleavage (RhizopusRhizopus))

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Classification Based onClassification Based onSexual PhaseSexual Phase

AscomycetesAscomycetes: : Aspergillus,Aspergillus, HistoplasmaHistoplasma,,BlastomycesBlastomyces, Dermatophytes, Dermatophytes

BasidiomycetesBasidiomycetes: : CryptococcusCryptococcus,,MushroomsMushrooms

ZygomycetesZygomycetes: Order Mucorales - : Order Mucorales - MucorMucor,,RhizopusRhizopus

DeuteromycetesDeuteromycetes (Fungi Imperfecti): (Fungi Imperfecti):SporothrixSporothrix, , Coccidioides, CandidaCoccidioides, Candida

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Clinical Types of FungalClinical Types of FungalInfectionsInfections

TYPE DISEASE ORGANISM

1. Superficial Pityriasis versicolor Malassezia furfur

2. Cutaneous Ringworm (Tinea)

Candidiasis

Trichophyton species

Candida albicans and others

3. Subcutaneous Sporotrichosis Sporothrix schenckii

4. Systemic Pathogenic Fungi

Histoplasmosis Histoplasma capsulatum

Blastomycosis Blastomyces dermatitidis

Coccidioidomycosis Coccidioides immitis

Paracoccidioidomycosis

Opportunistic Fungi

Paracoccidioides brasiliensis

Aspergillosis Aspergillus fumigatus and

others

Cryptococcosis Cryptococcus neoformans

Candidiasis Candida albicans and others

Zygomycosis Mucor and Rhizopus species

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Routes of InfectionRoutes of Infection

Inhalation of spores Inhalation of spores –– major factor major factor Inoculation of spores into skinInoculation of spores into skin Disease by normal flora in compromisedDisease by normal flora in compromised

host (Candida)host (Candida) HypersensitivityHypersensitivity Contact with infected hostContact with infected host

(Dermatophytes)(Dermatophytes) MycotoxinsMycotoxins

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Laboratory Diagnosis of FungalLaboratory Diagnosis of FungalInfectionsInfections

Microscopic Examination of tissues and bodyMicroscopic Examination of tissues and bodyfluidsfluids Gram stainGram stain GiemsaGiemsa India InkIndia Ink Potassium hydroxide (KOH) wet prepPotassium hydroxide (KOH) wet prep Hematoxylin and Eosin stainHematoxylin and Eosin stain Periodic-Acid Schiff stain (PAS)Periodic-Acid Schiff stain (PAS) Gomori-Methenamine Silver stain (GMS)Gomori-Methenamine Silver stain (GMS) Mucicarmine or Mucicarmine or AlcianAlcian Blue stain Blue stain

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Budding Yeast - Gram StainBudding Yeast - Gram Stain

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Encapsulated Yeast - India InkEncapsulated Yeast - India Ink

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KOH Prep - Broad-base Budding YeastKOH Prep - Broad-base Budding Yeast

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H&E Stain - Budding YeastsH&E Stain - Budding Yeasts

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PAS Stain - Yeast and HyphaePAS Stain - Yeast and Hyphae

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GMS Stain - Septate HyphaeGMS Stain - Septate Hyphae

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Mucicarmine Stain - Mucicarmine Stain - C. neoformansC. neoformans

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Histopathological Response toHistopathological Response toFungal InfectionFungal Infection

Acute pyogenic abscess (Candida)Acute pyogenic abscess (Candida) Chronic granuloma formationChronic granuloma formation

(Histoplasma)(Histoplasma) Chronic, localized dermal inflammationChronic, localized dermal inflammation

(Dermatophytes)(Dermatophytes) Mixed pyogenic and granulomatousMixed pyogenic and granulomatous

inflammation (Blastomyces)inflammation (Blastomyces) Blood vessel invasion with thrombosis andBlood vessel invasion with thrombosis and

infarction (Mucor, Aspergillus)infarction (Mucor, Aspergillus) Hypersensitivity without tissue reactionHypersensitivity without tissue reaction

(allergic bronchopulmonary aspergillosis)(allergic bronchopulmonary aspergillosis)

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Fungal CulturesFungal Cultures

Utilize Sabouraud agar with antibioticsUtilize Sabouraud agar with antibiotics Identification criteriaIdentification criteria

Temperature of growthTemperature of growth Rate of growthRate of growth Colonial and microscopic morphologyColonial and microscopic morphology Sporulation patternSporulation pattern Biochemical reactions (yeast)Biochemical reactions (yeast)

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Fungal SerologyFungal Serology

Generally poor and not as useful as in otherGenerally poor and not as useful as in otherpathogens such as viruses and bacteria, withpathogens such as viruses and bacteria, withsome exceptions.some exceptions.

Cryptococcal antigen by latex agglutination:Cryptococcal antigen by latex agglutination:serum and CSF.serum and CSF.

CoccidioidesCoccidioides - early IgM response is useful - early IgM response is usefulfor identification of acute primary disease -for identification of acute primary disease -CSF IgG prognostic value.CSF IgG prognostic value.

Skin tests for DTH - problems:Skin tests for DTH - problems: Cross-reactivity.Cross-reactivity. High positive rate in endemic areas.High positive rate in endemic areas.

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CandidiasisCandidiasis Clinical manifestationsClinical manifestations

MucosalMucosal•• VaginitisVaginitis•• EsophagitisEsophagitis•• Oral thrushOral thrush

CutaneousCutaneous Chronic mucocutaneousChronic mucocutaneous SystemicSystemic

•• FungemiaFungemia•• Hepato-spleenicHepato-spleenic•• EndophthalmitisEndophthalmitis•• RenalRenal

Urinary tractUrinary tract

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OralOralCandidiasisCandidiasis

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PseudomembraneousPseudomembraneousCandidiasisCandidiasis

(Thrush)(Thrush)

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Wet Mount - CandidiasisWet Mount - Candidiasis

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CandidiasisCandidiasis

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MucocutaneousMucocutaneousCandidiasisCandidiasis

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Candida sp. Tissue GMS StainCandida sp. Tissue GMS Stain

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AspergillosisAspergillosis

Clinical manifestationsClinical manifestations PneumoniaPneumonia AspergillomaAspergilloma Allergic bronchopulmonaryAllergic bronchopulmonary Disseminated multiorgan involvementDisseminated multiorgan involvement

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AspergillomaAspergilloma

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Aspergilloma Aspergilloma –– Gross Anatomy Gross Anatomy

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Allergic Bronchopulmonary AspergillosisAllergic Bronchopulmonary Aspergillosis

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CNS AspergillosisCNS Aspergillosis

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KOH Prep KOH Prep –– Aspergillus sp. Aspergillus sp.

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AspergillusAspergillus sp GMS Stain sp GMS Stain

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AspergillusAspergillus sp sp –– GMS Stain GMS Stain

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ZygomycosisZygomycosis

Clinical manifestationsClinical manifestations SinusitisSinusitis RhinocerebralRhinocerebral PulmonaryPulmonary RenalRenal

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RhinocerebralRhinocerebralMucormycosisMucormycosis

in Diabeticin DiabeticKetoacidosisKetoacidosis

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Postmortem Postmortem –– Rhinocerebral Mucormycosis Rhinocerebral Mucormycosis

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Non-septate Branching Hyphae (PAS)Non-septate Branching Hyphae (PAS)

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HistoplasmosisHistoplasmosis

Clinical manifestationsClinical manifestations Most cases mild or sub-clinical pulmonaryMost cases mild or sub-clinical pulmonary

diseasedisease•• Dissemination appears to be commonDissemination appears to be common

PneumoniaPneumonia Chronic progressive pulmonary (cavitary)Chronic progressive pulmonary (cavitary) HistoplasmomaHistoplasmoma DisseminatedDisseminated

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HistoplasmosisHistoplasmosis–– Calcified Calcified

LesionsLesions

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DisseminatedDisseminatedHistoplasmosisHistoplasmosis

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Histoplasmosis- GMSHistoplasmosis- GMS

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Histoplasmosis Histoplasmosis –– Bone Marrow Bone Marrow

Histiocyte

H. capsulatum

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Histoplasma capsulatumHistoplasma capsulatumIn vitro In vivo

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Risk factors for endogenous and exogenous endophthalmitisDisease and fungus Risk factors or comments

Endogenous endophthalmitis

C. albicans Central venous lines, neutropenia, abdominal surgery,intravenous drug abuse, broad-spectrum antibiotics

Candida species Central venous lines, neutropenia, abdominal surgery,intravenous drug abuse, broad-spectrum antibiotics

Aspergillus species Neutropenia, endocarditis, intravenous drug abuse, pulmonarydisease being treated with high-dose steroids, organ transplant

Fusarium species Neutropenia, intravenous drug abuse

H. capsulatum, C. immitis, B. dermatitidis, S. schenckii, C. neoformans

May accompany disseminated disease

Exogenous endophthalmitis

Fusarium species Post trauma and post keratitis

Candida species Post surgery, contaminated eye irrigates

P. lilacinus Post surgery, contaminated sterilization solutions

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FUNGAL KERATITISFUNGAL KERATITIS

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ChemotherapyChemotherapy FDA approvedFDA approved

Polyenes (Amphotericin B, lipid encapsulated forms)Polyenes (Amphotericin B, lipid encapsulated forms) Azoles (fluconazole, itraconazole, ketoconazole,Azoles (fluconazole, itraconazole, ketoconazole,

voriconazole)voriconazole) Echinocandin (Caspofungin, Micafungin, Anidulafungin)Echinocandin (Caspofungin, Micafungin, Anidulafungin) Nucleoside derivatives (5-flurocytosine)Nucleoside derivatives (5-flurocytosine) Allyamines (Terbinafine)Allyamines (Terbinafine) Microtubule disruption (Griseofulvin)Microtubule disruption (Griseofulvin)

InvestigationalInvestigational Nikkomycins (chitin synthase inhibitors)Nikkomycins (chitin synthase inhibitors) Echinocandin/pnemocandin/lipopeptide class (inhibit glycanEchinocandin/pnemocandin/lipopeptide class (inhibit glycan

synthesis)synthesis)

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Antifungal Drugs for SystemicAntifungal Drugs for SystemicMycoses - Amphotericin BMycoses - Amphotericin B

Mode of ActionMode of Action Binds to ergosterol, increases membraneBinds to ergosterol, increases membrane

permeability resulting in leakage of cytoplasmicpermeability resulting in leakage of cytoplasmiccomponents and cell death components and cell death –– Fungicidal Fungicidal

Spectrum of ActivitySpectrum of Activity Candida, Crypto, Aspergillus, Candida, Crypto, Aspergillus, HistoHisto, , BlastoBlasto, , CocciCocci,,

etcetc LimitationsLimitations

NephrotoxicityNephrotoxicity

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Proposed Structure ofProposed Structure ofABELCETABELCET

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6161

Mechanism of Action ofMechanism of Action ofABELCETABELCET

6262

Antifungal Drugs for SystemicAntifungal Drugs for SystemicMycoses - 5-flurocytosineMycoses - 5-flurocytosine

Mode of ActionMode of Action Inhibits RNA and DNA biosynthesisInhibits RNA and DNA biosynthesis

Spectrum of ActivitySpectrum of Activity Used in combination with Used in combination with AmphoAmpho B against crypto B against crypto

and Candidaand Candida Good CNS penetrationGood CNS penetration

LimitationsLimitations Narrow spectrum of activityNarrow spectrum of activity Resistance develops if used aloneResistance develops if used alone Can suppress bone marrowCan suppress bone marrow

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Antifungal Drugs for SystemicAntifungal Drugs for SystemicMycoses - FluconazoleMycoses - Fluconazole

Mode of ActionMode of Action Prevents ergosterol synthesis by inhibiting the C-14Prevents ergosterol synthesis by inhibiting the C-14

demethylationdemethylation step (cytochrome P-450 rx) step (cytochrome P-450 rx) FungistaticFungistatic

Spectrum of ActivitySpectrum of Activity Candida, Crypto, Candida, Crypto, TrichsporonosisTrichsporonosis, dermatophytes, dermatophytes

LimitationsLimitations Resistance in some Candida sp Resistance in some Candida sp –– krusei and glabrata krusei and glabrata Not effective for non-dermatophyte moulds.Not effective for non-dermatophyte moulds.

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Antifungal Drugs for SystemicAntifungal Drugs for SystemicMycoses -ItraconazoleMycoses -Itraconazole

Mode of ActionMode of Action Prevents ergosterol synthesis by inhibitingPrevents ergosterol synthesis by inhibiting

the C-14 the C-14 demethylationdemethylation step (cytochrome step (cytochromeP-450 rx)P-450 rx)

FungistaticFungistatic Spectrum of activitySpectrum of activity

Systemic mycoses, yeasts, dematiaceousSystemic mycoses, yeasts, dematiaceousmoulds and dermatophytes.moulds and dermatophytes.

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Early Diagnosis of InvasiveEarly Diagnosis of InvasiveFungal InfectionsFungal Infections

ObstaclesObstacles•• Because of Immunosuppression typical signsBecause of Immunosuppression typical signs

and symptoms of infection are frequently absentand symptoms of infection are frequently absent•• Few clinical features are uniquely specific forFew clinical features are uniquely specific for

systemic fungal infectionsystemic fungal infection•• Sputum and blood cultures are frequentlySputum and blood cultures are frequently

negativenegative•• Invasive proceduresInvasive procedures

–– May be necessary for definitive diagnosisMay be necessary for definitive diagnosis–– Are often complicated in severelyAre often complicated in severely

immunocompromised patientimmunocompromised patient

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Early Diagnosis of InvasiveEarly Diagnosis of InvasiveFungal Infections (Continued)Fungal Infections (Continued)

BenefitsBenefits Early diagnosis permits therapy ofEarly diagnosis permits therapy of

maximal effectivenessmaximal effectiveness Early intervention with antifungalEarly intervention with antifungal

therapy may help decrease the hightherapy may help decrease the highmortality rate associated with seriousmortality rate associated with serioussystemic mycosessystemic mycoses

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DISEASE ETIOLOGIC AGENTS(S) TISSUE APPEARANCE ORGANS INVOLVED

Candidiasis

Candida albicans and others

yeast, hyphae and pseudohyphae

skin, mucous membranes , heart valves

Histoplasmosis

Histoplasma capsulatum

intracellular yeast

lungs and disseminated

Blastomycosis

Blastomyces dermatitidis

thick-walled budding yeast

lungs, skin and bone

Coccidioidomycosis

Coccidioides immitis

spherules with endospores

lungs and disseminated

Aspergillosis

Aspergillus fumigatus and others

mold with septate hyphae

lungs

Cryptococcosis

Cryptococcus neoformans

encapsulated budding yeast

lungs, meninges and skin

Sporotrichosis

Sporothrix schenckii

cigar-shaped yeast (rarely observed)

skin and disseminated

Zygomycosis

Mucor and Rhizopus species

Mold with broad nonseptate hyphae

nasal sinuses, lung, GI tract

Common Deep MycosesCommon Deep Mycoses