contribution of pathology to escmid onlin lctr lirary y

CONTRIBUTION OF PATHOLOGY TOCONTRIBUTION OF PATHOLOGY TOTHE DIAGNOSIS OF FUNGALTHE DIAGNOSIS OF FUNGAL

INFECTIONSINFECTIONS

DRDR GEETIKAGEETIKA KHANNAKHANNAPROFESSORPROFESSOR OFOF PATHOLOGYPATHOLOGYCIOCIO LABORATORYLABORATORYVMMCVMMC && SAFDARJUNGSAFDARJUNG HOSPITALHOSPITALNEWNEW DELHIDELHI

DRDR GEETIKAGEETIKA KHANNAKHANNAPROFESSORPROFESSOR OFOF PATHOLOGYPATHOLOGYCIOCIO LABORATORYLABORATORYVMMCVMMC && SAFDARJUNGSAFDARJUNG HOSPITALHOSPITALNEWNEW DELHIDELHIESCMID Online Lectu

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1.1. Rapid & costRapid & cost--effective means of arriving at a presumptiveeffective means of arriving at a presumptivediagnosisdiagnosis…Culture may take wks…Culture may take wks…Organism may not grow at all.…Organism may not grow at all.

22. Allows. Allows definitive interpretation in cases with specific findingsdefinitive interpretation in cases with specific findings-- unique fruiting head ofunique fruiting head of AspergillusAspergillus spp.spp.-- at least 1 unequivocal, intact, endosporeat least 1 unequivocal, intact, endospore--filled spherule offilled spherule of CoccidioidesCoccidioides..

PATHOLOGYPATHOLOGY –– RELEVANCE IN FUNGAL DISEASERELEVANCE IN FUNGAL DISEASE

1.1. Rapid & costRapid & cost--effective means of arriving at a presumptiveeffective means of arriving at a presumptivediagnosisdiagnosis…Culture may take wks…Culture may take wks…Organism may not grow at all.…Organism may not grow at all.

22. Allows. Allows definitive interpretation in cases with specific findingsdefinitive interpretation in cases with specific findings-- unique fruiting head ofunique fruiting head of AspergillusAspergillus spp.spp.-- at least 1 unequivocal, intact, endosporeat least 1 unequivocal, intact, endospore--filled spherule offilled spherule of CoccidioidesCoccidioides..ESCMID Online Lectu

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3. Provides insight into the diagnostic significance of cultureisolates….gives information on vascular/tissue invasion & host reaction….address the frequently encountered problems of contamination &

infection vs colonization.

4. May be the only method of diagnosis for fungal pathogens likePneumocystis & Lacazia loboi.

5. Sometimes detects fungal disease in clinically unsuspectedcases in which no culture has been sent.

3. Provides insight into the diagnostic significance of cultureisolates….gives information on vascular/tissue invasion & host reaction….address the frequently encountered problems of contamination &

infection vs colonization.

4. May be the only method of diagnosis for fungal pathogens likePneumocystis & Lacazia loboi.

5. Sometimes detects fungal disease in clinically unsuspectedcases in which no culture has been sent.ESCMID Online Lectu

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Overview of special stains for fungiOverview of special stains for fungiHPE:1. PAS [red] & GMS [black]:• Both highlight the cell wall - Used as screening tools.• GMS more sensitive than PAS…signal to noise issue..stains

lysosomes, inflammatory cells & tissue reticulin.• PAS gives a better visualization of surrounding tissue compared to GMS.• GMS with H&E counterstain - best combination for fungus & host reaction.

3. Mucicarmine & Alcian Blue [red & blue]: Capsule of Cryptococcus

4. Fontana-Masson [black] : Cryptococcus & dematiaceous fungi & somespecies of Aspergillus, Mucorales & Trichosporon.

Cytology (sputum, BAL, CSF, FNA):Calcofluor white, Uvitex, MGG, Pap, PAS, GMS.

HPE:1. PAS [red] & GMS [black]:• Both highlight the cell wall - Used as screening tools.• GMS more sensitive than PAS…signal to noise issue..stains

lysosomes, inflammatory cells & tissue reticulin.• PAS gives a better visualization of surrounding tissue compared to GMS.• GMS with H&E counterstain - best combination for fungus & host reaction.

3. Mucicarmine & Alcian Blue [red & blue]: Capsule of Cryptococcus

4. Fontana-Masson [black] : Cryptococcus & dematiaceous fungi & somespecies of Aspergillus, Mucorales & Trichosporon.

Cytology (sputum, BAL, CSF, FNA):Calcofluor white, Uvitex, MGG, Pap, PAS, GMS.

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1. Describe the fungal elements.1. Describe the fungal elements. Yeast / hyphae / Both/ endosporulating structures.Yeast / hyphae / Both/ endosporulating structures. Yeast formsYeast forms –– Diameter, Budding or Fission/Type of budding (narrowDiameter, Budding or Fission/Type of budding (narrow

based or broad based).based or broad based). HyphaeHyphae -- Width, septations, branching angle, dilated bizarre formsWidth, septations, branching angle, dilated bizarre forms

(Mucorales), Brown pigmentation (Dematiaceous fungi).(Mucorales), Brown pigmentation (Dematiaceous fungi). Also comment on quantity of fungal elements & viability.Also comment on quantity of fungal elements & viability.

2. Clearly state the fungus which is most frequently associated with2. Clearly state the fungus which is most frequently associated withthe described morphology & enlist the fungi that can display athe described morphology & enlist the fungi that can display asimilar morphology.similar morphology.

3. Identify the inflammatory reaction, comment on presence of3. Identify the inflammatory reaction, comment on presence ofvascular or tissue invasion, necrosis, or hemorrhage.vascular or tissue invasion, necrosis, or hemorrhage.

4. Correlate all pathological findings with clinical features,4. Correlate all pathological findings with clinical features,epidemiology & results of alternative testing if available .epidemiology & results of alternative testing if available .

RReporting of fungal infections in tissue & cytologicaleporting of fungal infections in tissue & cytologicalpreparationspreparations

1. Describe the fungal elements.1. Describe the fungal elements. Yeast / hyphae / Both/ endosporulating structures.Yeast / hyphae / Both/ endosporulating structures. Yeast formsYeast forms –– Diameter, Budding or Fission/Type of budding (narrowDiameter, Budding or Fission/Type of budding (narrow

based or broad based).based or broad based). HyphaeHyphae -- Width, septations, branching angle, dilated bizarre formsWidth, septations, branching angle, dilated bizarre forms

(Mucorales), Brown pigmentation (Dematiaceous fungi).(Mucorales), Brown pigmentation (Dematiaceous fungi). Also comment on quantity of fungal elements & viability.Also comment on quantity of fungal elements & viability.

2. Clearly state the fungus which is most frequently associated with2. Clearly state the fungus which is most frequently associated withthe described morphology & enlist the fungi that can display athe described morphology & enlist the fungi that can display asimilar morphology.similar morphology.

3. Identify the inflammatory reaction, comment on presence of3. Identify the inflammatory reaction, comment on presence ofvascular or tissue invasion, necrosis, or hemorrhage.vascular or tissue invasion, necrosis, or hemorrhage.

4. Correlate all pathological findings with clinical features,4. Correlate all pathological findings with clinical features,epidemiology & results of alternative testing if available .epidemiology & results of alternative testing if available .

Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80.


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Diseases Where Yeasts or YeastDiseases Where Yeasts or Yeast--Like Structures Are Usually SeenLike Structures Are Usually Seen Diseases Where Hyphae Are Usually SeenDiseases Where Hyphae Are Usually Seen Diseases caused byDiseases caused by Fusarium, Scedosporium,Fusarium, Scedosporium, and other hyalineand other hyaline

septated moldsseptated molds Diseases caused byDiseases caused by Bipolaris/CurvulariaBipolaris/Curvularia and otherand other DematiaceousDematiaceous

fungifungi Dermatophyte diseaseDermatophyte disease Diseases caused by other molds (coelomycetes)Diseases caused by other molds (coelomycetes)

**The “clinical spectrum & host tissue response “ to every fungus isThe “clinical spectrum & host tissue response “ to every fungus isas important as the “morphology of the fungus” & must be kept inas important as the “morphology of the fungus” & must be kept inmind while making a diagnosis.mind while making a diagnosis.

MORPHOLOGICAL CLASSIFICATION OF FUNGAL DISEASESMORPHOLOGICAL CLASSIFICATION OF FUNGAL DISEASES Diseases Where Yeasts or YeastDiseases Where Yeasts or Yeast--Like Structures Are Usually SeenLike Structures Are Usually Seen Diseases Where Hyphae Are Usually SeenDiseases Where Hyphae Are Usually Seen Diseases caused byDiseases caused by Fusarium, Scedosporium,Fusarium, Scedosporium, and other hyalineand other hyaline

septated moldsseptated molds Diseases caused byDiseases caused by Bipolaris/CurvulariaBipolaris/Curvularia and otherand other DematiaceousDematiaceous

fungifungi Dermatophyte diseaseDermatophyte disease Diseases caused by other molds (coelomycetes)Diseases caused by other molds (coelomycetes)

**The “clinical spectrum & host tissue response “ to every fungus isThe “clinical spectrum & host tissue response “ to every fungus isas important as the “morphology of the fungus” & must be kept inas important as the “morphology of the fungus” & must be kept inmind while making a diagnosis.mind while making a diagnosis.


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BlastomycosisBlastomycosis CryptococcosisCryptococcosis HistoplasmosisHistoplasmosis CoccidioidomycosisCoccidioidomycosis CandidiasisCandidiasis

DISEASES WHERE YEASTS OR YEASTDISEASES WHERE YEASTS OR YEAST--LIKELIKESTRUCTURES ARE USUALLY SEEN IN TISSUESSTRUCTURES ARE USUALLY SEEN IN TISSUES

BlastomycosisBlastomycosis CryptococcosisCryptococcosis HistoplasmosisHistoplasmosis CoccidioidomycosisCoccidioidomycosis CandidiasisCandidiasis


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Blastomyces dermatitidisMorphology of fungus Clinical spectrum & host tissue

responseD/Ds

•Yeasts meas. 8 -15 μm.

• Show single, broad-basedbudding.

• Clear space around thefungal cell on H&E staining(corresponds to a thickrefractile cell wall); stainswith silver stains.

• Larger forms mayoccasionally be accompaniedby the “micro forms”.

• Acute pneumonia: Mixedsuppurative response.• Chronic pneumonia:Pyogranulomatous inflammationwith numerous multinucleate cells.• Cutaneous/mucosal lesions:- Marked epithelial hyperplasia.- Neutrophilic microabscesses inepithelium.- Chronic inflammation in the dermis/submucosal tissue.• Disseminated lesions: Variousinflammatory responses dependingon immune status.*Tuberculosis or neoplasia can bepresent concomitantly

1. Larger forms to bedifferentiated froma) Immature spherulesof C. immitis.b) Yeast forms ofP. Brasiliensis.c) Conidia ofaspergillus.

2. “Microforms” tobe differentiatedfrom theYeast forms ofCandida spp.,Histoplasma,Cryptococcus.

•Yeasts meas. 8 -15 μm.

• Show single, broad-basedbudding.

• Clear space around thefungal cell on H&E staining(corresponds to a thickrefractile cell wall); stainswith silver stains.

• Larger forms mayoccasionally be accompaniedby the “micro forms”.

• Acute pneumonia: Mixedsuppurative response.• Chronic pneumonia:Pyogranulomatous inflammationwith numerous multinucleate cells.• Cutaneous/mucosal lesions:- Marked epithelial hyperplasia.- Neutrophilic microabscesses inepithelium.- Chronic inflammation in the dermis/submucosal tissue.• Disseminated lesions: Variousinflammatory responses dependingon immune status.*Tuberculosis or neoplasia can bepresent concomitantly

1. Larger forms to bedifferentiated froma) Immature spherulesof C. immitis.b) Yeast forms ofP. Brasiliensis.c) Conidia ofaspergillus.

2. “Microforms” tobe differentiatedfrom theYeast forms ofCandida spp.,Histoplasma,Cryptococcus.


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Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80.


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PAS stain.


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Cryptococcus spp.

Morphology of fungus Clinical spectrum & host tissueresponse

D/Ds

• Spherical-oval yeasts.• Marked variation in size.• Narrow-based budding.• Thick polysaccharidecapsule.• Pseudohyphae (withmassive yeast proliferation).

India ink: Negative stain -highlights the capsule.Mucicarmine & Alcian blue:Capsule.PAS & GMS: Cell wall.Masons-Fontana: Yeast cell(permits differentiation frommost other pathogenic yeastsexcept Trichosporon beigelii).

• Pneumonia & Cryptococcomas:Granulomatous reaction with fibrosis.Occasionally pseudotumour formation.• Neutrophils unusual – indicatebacterial superinfection/response tomassive necrosis.• Disseminated disease: Varying infl.response depending on immune status.• Immunocompetent individuals: Wellformed granulomas with few organisms.• Severe T cell deficiency:Minimal inflammation with abundantintra/extracellular organisms (sheets ofyeast filled lacunae - “soap bubblelesion”).

Candida &Histoplasma -similar in size & canbe confusedhistologically.

Well encapsulatedorganisms easilydifferentiated usingcapsular stains.

Organisms withpoorly formedcapsule pose someproblem and needto be differentiatedby melanin stains.

• Spherical-oval yeasts.• Marked variation in size.• Narrow-based budding.• Thick polysaccharidecapsule.• Pseudohyphae (withmassive yeast proliferation).

India ink: Negative stain -highlights the capsule.Mucicarmine & Alcian blue:Capsule.PAS & GMS: Cell wall.Masons-Fontana: Yeast cell(permits differentiation frommost other pathogenic yeastsexcept Trichosporon beigelii).

• Pneumonia & Cryptococcomas:Granulomatous reaction with fibrosis.Occasionally pseudotumour formation.• Neutrophils unusual – indicatebacterial superinfection/response tomassive necrosis.• Disseminated disease: Varying infl.response depending on immune status.• Immunocompetent individuals: Wellformed granulomas with few organisms.• Severe T cell deficiency:Minimal inflammation with abundantintra/extracellular organisms (sheets ofyeast filled lacunae - “soap bubblelesion”).

Candida &Histoplasma -similar in size & canbe confusedhistologically.

Well encapsulatedorganisms easilydifferentiated usingcapsular stains.

Organisms withpoorly formedcapsule pose someproblem and needto be differentiatedby melanin stains.


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CDC public health image library

- Khanna G, Bhattacharya SN, Singhal A, Singh N, Sharma S. Fatal disseminated cryptococcosis in a HIVnegative elderly Indian woman - early diagnosis by polarizing microscopy. Indian J Pathol Microbiol 2004;47(4): 542-44.- Randhawa HS, Chowdhari A, Khanna G. Comment on Singh et al’s “Cryptococcosis in a bandicoot rat.”Med Mycol 2007; 45:7, 655-6- Capoor MR, Khanna G, Malhotra R, Verma S, Nair D, Deb M, Aggarwal P. Disseminated cryptococcosiswith necrotizing fasciitis in an apparently immunocompetent host: a case report. Medical Mycology 06/2008;46(3):269-73.


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PAS stain.


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Mucicarmine stain


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HistoplasmaMorphology of fungus Clinical spectrum & host tissue response D/DsH. Capsulatum:• Oval 2-4-μm yeast

• Narrow-based budding.

• H&E: Halo correspondingto the cell wall.

• GMS & PAS: Highlight thecell wall.

• Intracellular clusteringwithin macrophages -diagnostic hallmark.

• In Africanhistoplasmosis the yeastsize is larger (8-15μm) &yeast may be pigmented.

• Spectrum varies from localized granulomaformation to massive proliferation of MPscontaining yeast cells.• Acute pneumonia: Intra-alveolar aggregatesof histiocytes, expand to form nodules ofparenchymal/ vascular necrosis.• Chronic histoplasmosis: Multiple smallgranulomata seen all over, commonly in pleura& spleen…May get hyalinized...Rarely residualnecrosis with yeast forms may be seen.• Mediastinitis: Massive hilar & mediastinalLAP; lamellar fibrosis & calcification (fibrosingmediastinitis); organisms rarely found/if foundswollen, distorted, no budding..? non-viable).

Include all smallsized yeastswhich shownarrow basedbudding &Intracellularclustering.

H. Capsulatum:• Oval 2-4-μm yeast

• Narrow-based budding.

• H&E: Halo correspondingto the cell wall.

• GMS & PAS: Highlight thecell wall.

• Intracellular clusteringwithin macrophages -diagnostic hallmark.

• In Africanhistoplasmosis the yeastsize is larger (8-15μm) &yeast may be pigmented.

• Spectrum varies from localized granulomaformation to massive proliferation of MPscontaining yeast cells.• Acute pneumonia: Intra-alveolar aggregatesof histiocytes, expand to form nodules ofparenchymal/ vascular necrosis.• Chronic histoplasmosis: Multiple smallgranulomata seen all over, commonly in pleura& spleen…May get hyalinized...Rarely residualnecrosis with yeast forms may be seen.• Mediastinitis: Massive hilar & mediastinalLAP; lamellar fibrosis & calcification (fibrosingmediastinitis); organisms rarely found/if foundswollen, distorted, no budding..? non-viable).

Include all smallsized yeastswhich shownarrow basedbudding &Intracellularclustering.


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Fungus to bedifferentiated

Differentiating features

“Micro” forms of B.dermatitidis

Presence of larger forms & broad based budding

Capsule deficientCryptococci

Show marked size variation & positive melanin staining

Endospores ofCoccidioides spp.

Presence of remnants of a ruptured spherule or an intact spherule.

Pneumocystis jirovecii Does not show budding & has a prominent intracystic focus.Pneumocystis jirovecii Does not show budding & has a prominent intracystic focus.

Penicillium marneffei Fission, sausage shape, transverse septum.Candida glabrata Typically extracellular; no halo, shows greater size variability &

pure neutrophilic infiltrate.

Leishmaniasis &toxoplasmosis

H&E stains entire organism; no halo; in leishmaniasis kinetoplastseen by the side of the nucleus; in toxoplasmosis infected cellsare cardiomyocytes & neurons rather than histiocytes.


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CDC public health image library


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PAS stain

PAS stain


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GMS stain

GMS stain


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Coccidioides immitisMorphology of fungus Clinical spectrum & host tissue

responseD/Ds

• Thick walled spherules(10-100 μm) whichcontain multipleendospores (2-5 μm).• Rupture to release theendospores into thesurrounding tissues.• H&E stains both thespherules & endospores.• GMS & PAS stain thespherule wall &endospores.• PAS staining fades withthe maturity of theorganism.

• Pulmonary & cutaneous lesionsdemonstrate a mixed suppurative-granulomatous response withabundant eosinophils.• Eosinophils secrete EBP whichinduces an intense rim ofeosinophilic material around fungalelements. “Splendore-Hoeppliphenomenon”.• Immunosuppressed - markednecrosis without granulomasformation.• Occ. mycelia may be seen in thecavitary or skin lesions.

R. seeberi:• Palatal & nasopharyngealpolyps.• Larger sporangia &endospores.• 50-100mm, can be seenwith the naked eye as yellowpin-head sized dots in apolyp.

Endospores/Youngspherules withoutendospores can be confusedwith Blastomyces,Emmonsia, Candida &Histoplasma.*Pneumocytis &Coccidioides can co-exist.

• Thick walled spherules(10-100 μm) whichcontain multipleendospores (2-5 μm).• Rupture to release theendospores into thesurrounding tissues.• H&E stains both thespherules & endospores.• GMS & PAS stain thespherule wall &endospores.• PAS staining fades withthe maturity of theorganism.

• Pulmonary & cutaneous lesionsdemonstrate a mixed suppurative-granulomatous response withabundant eosinophils.• Eosinophils secrete EBP whichinduces an intense rim ofeosinophilic material around fungalelements. “Splendore-Hoeppliphenomenon”.• Immunosuppressed - markednecrosis without granulomasformation.• Occ. mycelia may be seen in thecavitary or skin lesions.

R. seeberi:• Palatal & nasopharyngealpolyps.• Larger sporangia &endospores.• 50-100mm, can be seenwith the naked eye as yellowpin-head sized dots in apolyp.

Endospores/Youngspherules withoutendospores can be confusedwith Blastomyces,Emmonsia, Candida &Histoplasma.*Pneumocytis &Coccidioides can co-exist.


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PAS stain

CDC public health image library collection


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H&E stain

Capoor MR, Khanna G, Rajni, Batra K, Nair D, Venkatchalam VP, Aggarwal P. Rhinosporidiosis in Delhi,North India: Case Series from a Non-endemic area and Mini-review. Mycopathologica 2009 Apr 5.168(2):89-94.


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Candida spp.Morphology of fungus Clinical spectrum & host tissue

responseD/Ds

• C. albicans: Mats ofyeasts measuring 3-5 μmin dm intermingled withpseudohyphae(filaments), which showperiodic constrictions butnot true septations.

• C. glabrata: Does notproduce pseudohyphae.

•.Superficial & invasive disease.• HPE very important to defineinvasion.• Neutrophilic inflammation withsome lymphocytes andmacrophages, fibrin, andcoagulative necrosis. Rarely fewgiant cells and granulomas.• Invasion of BVs may causemycotic aneurysms &thrombophlebitis.• Necrotizing vasculitis is seenbut no demonstrable organismindicating that Candida solublefraction causes the necrotizinglesions.

• C. albicans - to bedifferentiated from Aspergillusspp. (presence of trueseptations). & Trichosporonspp. (presence of aconstriction between the baseof the blastospore & the germtube).

• C. glabrata - to bedifferentiated from similar sizedyeast forms like Histoplasma.

• C. albicans: Mats ofyeasts measuring 3-5 μmin dm intermingled withpseudohyphae(filaments), which showperiodic constrictions butnot true septations.

• C. glabrata: Does notproduce pseudohyphae.

•.Superficial & invasive disease.• HPE very important to defineinvasion.• Neutrophilic inflammation withsome lymphocytes andmacrophages, fibrin, andcoagulative necrosis. Rarely fewgiant cells and granulomas.• Invasion of BVs may causemycotic aneurysms &thrombophlebitis.• Necrotizing vasculitis is seenbut no demonstrable organismindicating that Candida solublefraction causes the necrotizinglesions.

• C. albicans - to bedifferentiated from Aspergillusspp. (presence of trueseptations). & Trichosporonspp. (presence of aconstriction between the baseof the blastospore & the germtube).

• C. glabrata - to bedifferentiated from similar sizedyeast forms like Histoplasma.


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PAS stain

GMS stainGMS stain


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DISEASES CAUSED BY OTHER FUNGI &ORGANISMS RESEMBLING FUNGI THAT DISPLAYYEASTS OR YEAST-LIKE STRUCTURES INTISSUES • Pneumocystis

• Sporothrix• Penicillium• Paracoccidioides brasiliensis• R. seeberi.• Emmonsia crescens

• Pneumocystis• Sporothrix• Penicillium• Paracoccidioides brasiliensis• R. seeberi.• Emmonsia crescens

- Capoor MR, Ramesh V, Khanna G, Singh A, Agarwal P. Sporotrichosis in Delhi among the migrantpopulation from Uttarakhand, India.Tropical Doctor 01/2011; 41(1):46-8.- Capoor M, Nair D, Deb M, Ramesh V, Khanna G, Rajni, Aggarwal P, Chand R, Chowdhary A,Mussa AY, Randhawa HS. Endemic occurrence of sporotrichosis in Uttaranchal, India – report of twoautochthonous cases. Indian J Dermatology 2007; 50(4 suppl11): S10-S13.


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Fungus Morphology & staining Pathological D/Ds

Pneumocystisjirovecii

Cysts 2-6μm in size with an intracysticthickening called “capsular dot”.Stained with GMS, Gram Weigert & Giemsa

Small yeasts with narrow basedbudding & intracellular clustering.

Penicilliummarneffei

• Dimorphic fungus –Yeast 2-6µm in size.• Divides by fission (no budding); resemble asausage with transverse septum.

• Small yeasts with narrow basedbudding & intracellular clustering.• Most striking clinico-pathologicalsimilarity with histoplasmosis.

Sporothrixschenckii

• Round-oval-cigar shaped yeast measuring 2-6µm• Narrow based or tube like budding.• Can be intra & extracellular.• Splendor-Hoeppli phenomenon (40-92%cases).

1. Histoplasmosis (Intracellularclustering)2. Candida (pseudohyphae)3. Leishmania (Kinetoplast)

• Round-oval-cigar shaped yeast measuring 2-6µm• Narrow based or tube like budding.• Can be intra & extracellular.• Splendor-Hoeppli phenomenon (40-92%cases).

Paracoccidioidesbrasiliensis

• Spherical yeasts; double contoured wall;size varying between 4-60µm.• Shows budding with multiple tear drop budssurrounding the parent cell (pilot wheelappearance) better seen with GMS.

Yeast forms showing 1-3 buds withabsence the “pilot wheelappearance” need to bedifferentiated from C.neoformans,S. schenckii & Lacazia loboi.

Emmonsiacrescens

• Dimorphic fungus, inhaled or inoculated inthe skin.• Called “Adiaspiromycosis or Haplomycosis”due to presence of adiaspores which are large,double walled, empty structures measuring20-400μm. GMS stains the wall to demonstratefenestrations.

Adiaspores need to bedifferentiated from Coccidioidesspherules which containendospores while adiaspores areempty.


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CDC public health image library collection


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PAS stain

GMS stainGMS stain


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H&EGMS

H&E


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Diseases Where Hyphae Are Usually SeenDiseases Where Hyphae Are Usually Seenin Tissuein Tissue

1. Small yeasts intermingled with pseudohyphae & hyphae(morphology consistent with Candida; D/D Aspergillosis, Hyalineseptated hyphae).2. Non-pigmented (hyaline) septated hyphae with acute angledbranching (morphology consistent with Aspergillosis; D/D Hyalineseptated hyphae)..3. Non-pigmented (hyaline) pauciseptate ribbon like hyphaewith right angled branching (morphology consistent withMucorales; however, Aspergillus spp. & Hyaline septated hyphaecan sometimes be confused).4. Pigmented irregular hyphae & yeast like structures both withseptations (morphology consistent with Dematiaceous fungi).

1. Small yeasts intermingled with pseudohyphae & hyphae(morphology consistent with Candida; D/D Aspergillosis, Hyalineseptated hyphae).2. Non-pigmented (hyaline) septated hyphae with acute angledbranching (morphology consistent with Aspergillosis; D/D Hyalineseptated hyphae)..3. Non-pigmented (hyaline) pauciseptate ribbon like hyphaewith right angled branching (morphology consistent withMucorales; however, Aspergillus spp. & Hyaline septated hyphaecan sometimes be confused).4. Pigmented irregular hyphae & yeast like structures both withseptations (morphology consistent with Dematiaceous fungi).


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Aspergillus spp.Morphology &staining

Clinical spectrum & host tissue response D/Ds

• Thin hyphae (3-12μm).• Septate.• Show acute-angle(45o) ordichotomousbranching.• Vesicles withconidia (when thefungi are present incavitary lesions orskin).

• A. Niger infectionusually shows calciumoxalate crystals in theHPE specimen.

1. Allergic aspergillosis(ABPA/AFRS):AM with non-invasive hyphae; bronchial wallmay show E, N, MPs, granulomas, vasculitis &interstitial fibrosis.2. Chronic colonizing aspergillosis/aspergilloma: Two types-a)Thin walled aspergillomas (fungal ballsurrounded by fibrosis).b)Chronic cavitary/necrotizing aspergilloma (anecrotic layer with abundant hyphae surroundedby granulation tissue & an outer layer offibrosis).3. Invasive aspergillosis:Angioinvasion may lead to hemorrhage,infarction & septic embolization to other organs.

1. Other Hyalineseptated molds(Hyalino-hyphomycetes):1. Fusarium spp.2. Scedosporiumspp.3. Trichoderma spp.4. Paecilomycesspp.

2. Sometimesmucorales.

• Thin hyphae (3-12μm).• Septate.• Show acute-angle(45o) ordichotomousbranching.• Vesicles withconidia (when thefungi are present incavitary lesions orskin).

• A. Niger infectionusually shows calciumoxalate crystals in theHPE specimen.

1. Allergic aspergillosis(ABPA/AFRS):AM with non-invasive hyphae; bronchial wallmay show E, N, MPs, granulomas, vasculitis &interstitial fibrosis.2. Chronic colonizing aspergillosis/aspergilloma: Two types-a)Thin walled aspergillomas (fungal ballsurrounded by fibrosis).b)Chronic cavitary/necrotizing aspergilloma (anecrotic layer with abundant hyphae surroundedby granulation tissue & an outer layer offibrosis).3. Invasive aspergillosis:Angioinvasion may lead to hemorrhage,infarction & septic embolization to other organs.

1. Other Hyalineseptated molds(Hyalino-hyphomycetes):1. Fusarium spp.2. Scedosporiumspp.3. Trichoderma spp.4. Paecilomycesspp.

2. Sometimesmucorales.ESCMID Online Lectu

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PASChowdhary A, Randhawa H.S., Khanna G, Chakravati A, Naglot A, Roy P. Occurrence and Etiology ofFungal-Rhinosinusitis in a New Delhi Teaching Hospital. International Journal of Infectious Diseases12/2008; 12.


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Mucormycosis(zygomycosis)

Morphology & staining Clinical spectrum & hosttissue response

D/Ds

• Nonpigmented• Broad (5- to 20-µm)• Ribbon-like• Pauciseptate hyphae showingright-angled branching(Mucorales).• The hyphae may vary in width,appear folded or crinkled, andbe sparse or fragmented.• H&E, GMS & PAS demonstrateonly the cell wall with no structureinside.• Important to demonstrateinvasion in the vessel wall or inthe lumen.

• Mucorales causeangioinvasive disease inimmunosuppressed hosts,wherein the hyphal elementswill be found amidst necrosis,hemorrhage, & vascularthrombosis.• May present as rhinocerebral, pulmonary orcutaneous disease, any ofwhich may disseminate.

Molds which producenonpigmented hyphae intissues

1. Aspergillus spp.2. Other hyaline

septate molds likeFusarium orScedosporium.

• Nonpigmented• Broad (5- to 20-µm)• Ribbon-like• Pauciseptate hyphae showingright-angled branching(Mucorales).• The hyphae may vary in width,appear folded or crinkled, andbe sparse or fragmented.• H&E, GMS & PAS demonstrateonly the cell wall with no structureinside.• Important to demonstrateinvasion in the vessel wall or inthe lumen.

• Mucorales causeangioinvasive disease inimmunosuppressed hosts,wherein the hyphal elementswill be found amidst necrosis,hemorrhage, & vascularthrombosis.• May present as rhinocerebral, pulmonary orcutaneous disease, any ofwhich may disseminate.

Molds which producenonpigmented hyphae intissues

1. Aspergillus spp.2. Other hyaline

septate molds likeFusarium orScedosporium.


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H&E

Diwakar A, Dewan RK, Chowdhary A, Randhawa HS, Khanna G, Gaur SN. Zygomycosis- a case reportand overview of the disease in India. Mycosis 2007; 49:1-8.


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Naturally pigmented molds whose hyphae and conidia contain melanin.Naturally pigmented molds whose hyphae and conidia contain melanin. Cause skin & S/T infections preceded by trauma.Cause skin & S/T infections preceded by trauma. Three associated clinical entities:Three associated clinical entities: Eumycetoma,Eumycetoma,

Chromoblastomycosis & Phaeohyphomycosis.Chromoblastomycosis & Phaeohyphomycosis. Other associated clinical syndromesOther associated clinical syndromes -- onychomycosis, keratitis,onychomycosis, keratitis,

allergic disease, pneumonia, brain abscesses, and disseminatedallergic disease, pneumonia, brain abscesses, and disseminateddisease.disease.

Respiratory disease by inhalation of conidia.Respiratory disease by inhalation of conidia. Dematiaceous fungi most frequently associated with eosinophilia &Dematiaceous fungi most frequently associated with eosinophilia &

AFRS/ABPA are Bipolaris & Curvularia (clinicopathological featuresAFRS/ABPA are Bipolaris & Curvularia (clinicopathological featuressimilar to Aspergillosis).similar to Aspergillosis).

Diseases caused by Bipolaris/ Curvularia & otherDiseases caused by Bipolaris/ Curvularia & otherDematiaceous fungiDematiaceous fungi

Naturally pigmented molds whose hyphae and conidia contain melanin.Naturally pigmented molds whose hyphae and conidia contain melanin. Cause skin & S/T infections preceded by trauma.Cause skin & S/T infections preceded by trauma. Three associated clinical entities:Three associated clinical entities: Eumycetoma,Eumycetoma,

Chromoblastomycosis & Phaeohyphomycosis.Chromoblastomycosis & Phaeohyphomycosis. Other associated clinical syndromesOther associated clinical syndromes -- onychomycosis, keratitis,onychomycosis, keratitis,

allergic disease, pneumonia, brain abscesses, and disseminatedallergic disease, pneumonia, brain abscesses, and disseminateddisease.disease.

Respiratory disease by inhalation of conidia.Respiratory disease by inhalation of conidia. Dematiaceous fungi most frequently associated with eosinophilia &Dematiaceous fungi most frequently associated with eosinophilia &

AFRS/ABPA are Bipolaris & Curvularia (clinicopathological featuresAFRS/ABPA are Bipolaris & Curvularia (clinicopathological featuressimilar to Aspergillosis).similar to Aspergillosis).ESCMID Online Lectu

re Library

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Pathology:• Biopsy specimens should be obtained from areas with pigment.

• HPE shows pigmented , thin, 2-6 μm, hyphae, some irregularlyswollen (toruloid or moniliform) with prominent septations &constrictions & terminal or intercalated vesicular swellings.

• In cases where no pigmentation on H&E , FM staining fordemonstration of melanin pigment.

• Pigmented yeast like cells showing septations & budding.

- S. Agarwal, M.R. Capoor, V. Ramesh, Rajni, G. Khanna. First case of Acremoniumkiliense mycetoma in a New Delhi resident: A brief review. Journal De Mycologie Médicale.2011; 1.1016- Azad K, Khanna G, Capoor MR, Gupta S. Cladophialophora carrionii: an etiological agentof cutaneous chromoblastomycosis from a non-endemic area, North India. Mycoses. 2009Dec 17.

Pathology:• Biopsy specimens should be obtained from areas with pigment.

• HPE shows pigmented , thin, 2-6 μm, hyphae, some irregularlyswollen (toruloid or moniliform) with prominent septations &constrictions & terminal or intercalated vesicular swellings.

• In cases where no pigmentation on H&E , FM staining fordemonstration of melanin pigment.

• Pigmented yeast like cells showing septations & budding.

- S. Agarwal, M.R. Capoor, V. Ramesh, Rajni, G. Khanna. First case of Acremoniumkiliense mycetoma in a New Delhi resident: A brief review. Journal De Mycologie Médicale.2011; 1.1016- Azad K, Khanna G, Capoor MR, Gupta S. Cladophialophora carrionii: an etiological agentof cutaneous chromoblastomycosis from a non-endemic area, North India. Mycoses. 2009Dec 17.


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Fusarium species insubcutaneous tissue

showing an irregular dilatedhyphae

Dematiaceousfungus, cerebral

phaeohyphomycosisESCMID Online Lectu

re Library

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Fungus HPE

Eumycetoma • Grains are interwoven mycelia lined by intensely eosinophilicmaterial (Sp. Hoeppli phenomenon).• Dematiaceous fungi produce black grains while Scedosporium,Acremonium produce white grains.• Eumycotic mycetoma shows septate hyphae, 2-6 μm in dm,staining positive with GMS & PAS.• Actinomycotic mycetoma shows delicate, branched, gram positivefilaments sometimes showing beading & meas. <1μm..

Chromoblastomycosis Pigmented round structures (“copper penny lesions” / “scleroticbodies” / “muriform cells” ) with internal septations in multipleplanes.Intense epidermal HP & HK & pyogranulomatous response.

Phaeohyphomycosis • Minimal changes in the epidermis.• Cyst wall comprised of dense collagen with granulomatousinflammation.• Center has geographic necrosis with FB giant cells & fungalelements (yeasts & septated hyphae).


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Hyaline fungi difficult to observe in theHyaline fungi difficult to observe in thekeratin layer using H&E.keratin layer using H&E.

GMS or PAS required for identification.GMS or PAS required for identification. HPE findings:HPE findings:

-- HK with focal PK.HK with focal PK.-- Spongiosis & neutrophilic microSpongiosis & neutrophilic microabscesses in acute cases.abscesses in acute cases.-- Varying degrees of PV lymphocytes &Varying degrees of PV lymphocytes &plasma cells with prominent papillaryplasma cells with prominent papillarydermal edema.dermal edema.-- Severe inflammation of hair folliclesSevere inflammation of hair folliclesand shafts (neutrophilicand shafts (neutrophilic –– kerion;kerion;mononuclearmononuclear-- Majocchi’s granulomaMajocchi’s granuloma ))

Dermatophyte diseaseDermatophyte disease

Hyaline fungi difficult to observe in theHyaline fungi difficult to observe in thekeratin layer using H&E.keratin layer using H&E.

GMS or PAS required for identification.GMS or PAS required for identification. HPE findings:HPE findings:

-- HK with focal PK.HK with focal PK.-- Spongiosis & neutrophilic microSpongiosis & neutrophilic microabscesses in acute cases.abscesses in acute cases.-- Varying degrees of PV lymphocytes &Varying degrees of PV lymphocytes &plasma cells with prominent papillaryplasma cells with prominent papillarydermal edema.dermal edema.-- Severe inflammation of hair folliclesSevere inflammation of hair folliclesand shafts (neutrophilicand shafts (neutrophilic –– kerion;kerion;mononuclearmononuclear-- Majocchi’s granulomaMajocchi’s granuloma ))

PAS


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Reported sensitivity of pathological studies is more than 80%,Reported sensitivity of pathological studies is more than 80%,however, it has some inadequacies:however, it has some inadequacies:

1.On special staining1.On special staininga) Yeasts can be confused with neurosecretory granules & melanin.a) Yeasts can be confused with neurosecretory granules & melanin.b) Hyphae can be confused with collagen fibers & basementb) Hyphae can be confused with collagen fibers & basementmembrane material.membrane material.

2. Some transversally cut hyphae can look like yeasts that may even2. Some transversally cut hyphae can look like yeasts that may evenappear to be budding.appear to be budding.

3. Histopathology usually cannot provide the fungal genus and3. Histopathology usually cannot provide the fungal genus andspecies.species.

4. Multiple infections may be difficult to interpret.4. Multiple infections may be difficult to interpret.

Inadequacies of HPEInadequacies of HPEReported sensitivity of pathological studies is more than 80%,Reported sensitivity of pathological studies is more than 80%,however, it has some inadequacies:however, it has some inadequacies:

1.On special staining1.On special staininga) Yeasts can be confused with neurosecretory granules & melanin.a) Yeasts can be confused with neurosecretory granules & melanin.b) Hyphae can be confused with collagen fibers & basementb) Hyphae can be confused with collagen fibers & basementmembrane material.membrane material.

2. Some transversally cut hyphae can look like yeasts that may even2. Some transversally cut hyphae can look like yeasts that may evenappear to be budding.appear to be budding.

3. Histopathology usually cannot provide the fungal genus and3. Histopathology usually cannot provide the fungal genus andspecies.species.

4. Multiple infections may be difficult to interpret.4. Multiple infections may be difficult to interpret.ESCMID Online Lecture Library

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1.1. The fungus present in the cultures is a colonizer or a contaminant.The fungus present in the cultures is a colonizer or a contaminant.

2. The tissue is sampled from two different areas, and different samples2. The tissue is sampled from two different areas, and different samplesare sent to microbiology and pathology.are sent to microbiology and pathology.

3. The pathologic specimen has not been extensively studied.3. The pathologic specimen has not been extensively studied.

Causes forCauses for Positive Cultures but Negative HPEPositive Cultures but Negative HPE

1.1. The fungus present in the cultures is a colonizer or a contaminant.The fungus present in the cultures is a colonizer or a contaminant.

2. The tissue is sampled from two different areas, and different samples2. The tissue is sampled from two different areas, and different samplesare sent to microbiology and pathology.are sent to microbiology and pathology.

3. The pathologic specimen has not been extensively studied.3. The pathologic specimen has not been extensively studied.


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1.1. When the tissue in the microbiology laboratory is ground tooWhen the tissue in the microbiology laboratory is ground tooaggressively and the fungal cells are destroyed.aggressively and the fungal cells are destroyed.

2. When the fungus in the tissue is not viable.2. When the fungus in the tissue is not viable.

3. When the tissue is sampled from two different areas &3. When the tissue is sampled from two different areas &different samples are sent for HPE & culture.different samples are sent for HPE & culture.

Causes for Positive HPE but Negative CulturesCauses for Positive HPE but Negative Cultures

1.1. When the tissue in the microbiology laboratory is ground tooWhen the tissue in the microbiology laboratory is ground tooaggressively and the fungal cells are destroyed.aggressively and the fungal cells are destroyed.

2. When the fungus in the tissue is not viable.2. When the fungus in the tissue is not viable.

3. When the tissue is sampled from two different areas &3. When the tissue is sampled from two different areas &different samples are sent for HPE & culture.different samples are sent for HPE & culture.


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In developing countriesIn developing countries -- major role, despite limitations, asmajor role, despite limitations, asimproved culture facilities & molecular profiling of fungi, thoughimproved culture facilities & molecular profiling of fungi, thoughideal, are not widely available for routine diagnostic use.ideal, are not widely available for routine diagnostic use.

Microbiologists, Pathologists, and Clinicians need to be aware ofMicrobiologists, Pathologists, and Clinicians need to be aware ofthe pitfalls of morphological diagnosis, and the alternative teststhe pitfalls of morphological diagnosis, and the alternative teststhat can be performed to make organismthat can be performed to make organism--specific diagnoses.specific diagnoses.

To Conclude….To Conclude….

In developing countriesIn developing countries -- major role, despite limitations, asmajor role, despite limitations, asimproved culture facilities & molecular profiling of fungi, thoughimproved culture facilities & molecular profiling of fungi, thoughideal, are not widely available for routine diagnostic use.ideal, are not widely available for routine diagnostic use.

Microbiologists, Pathologists, and Clinicians need to be aware ofMicrobiologists, Pathologists, and Clinicians need to be aware ofthe pitfalls of morphological diagnosis, and the alternative teststhe pitfalls of morphological diagnosis, and the alternative teststhat can be performed to make organismthat can be performed to make organism--specific diagnoses.specific diagnoses.


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References & Resources

1. Schwarz J. Hum Pathol. 1982;13:519-533.2. Watts JC. Am J Clin Pathol. 1994;102:711-712.3. Renshaw AA. Am J Clin Pathol. 1994;102:736-740.8.4. Tarrand JJ et al. Am J Clin Pathol. 2003;119:854-858.5. Weydert JA et al. Arch Pathol Lab Med. 2007;131:780-783.6. Watts JC et al. Am J Clin Pathol. 1998;109:1-2.7. Guarner J, Brandt ME. Clin Microbiol Rev. 2011;24:247-80.8. Das A et al. Histopathology. 2009;54:854-9.9. Challa S et al. Eur Arch Otorhinolaryngol. 2010;267:1239-45.10. Sangoi AR et al. Am J Clin Pathol. 2009;131:364-75.11. Lee S et al. Med Mycol. 2010;48:886-8.

References & Resources

1. Schwarz J. Hum Pathol. 1982;13:519-533.2. Watts JC. Am J Clin Pathol. 1994;102:711-712.3. Renshaw AA. Am J Clin Pathol. 1994;102:736-740.8.4. Tarrand JJ et al. Am J Clin Pathol. 2003;119:854-858.5. Weydert JA et al. Arch Pathol Lab Med. 2007;131:780-783.6. Watts JC et al. Am J Clin Pathol. 1998;109:1-2.7. Guarner J, Brandt ME. Clin Microbiol Rev. 2011;24:247-80.8. Das A et al. Histopathology. 2009;54:854-9.9. Challa S et al. Eur Arch Otorhinolaryngol. 2010;267:1239-45.10. Sangoi AR et al. Am J Clin Pathol. 2009;131:364-75.11. Lee S et al. Med Mycol. 2010;48:886-8.


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