CONTRIBUTION OF PATHOLOGY TO THE DIAGNOSIS OF FUNGAL INFECTIONS DR GEETIKA KHANNA PROFESSOR OF PATHOLOGY © by CIOauthorLABORATORY VMMC & SAFDARJUNG HOSPITAL NEW DELHI ESCMID Online Lecture Library PATHOLOGY – RELEVANCE IN FUNGAL DISEASE 1. Rapid & cost-effective means of arriving at a presumptive diagnosis …Culture may take wks …Organism may not grow at all. 2. Allows definitive interpretation© by inauthor cases with specific findings - unique fruiting head of Aspergillus spp. - atESCMID least 1 unequivocal, Online intact, endospore Lecture-filled spherule Library of Coccidioides . 3. Provides insight into the diagnostic significance of culture isolates ….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 like Pneumocystis & Lacazia loboi. © by author 5. SometimesESCMID detects Online fungal disease Lecture in clinically Library unsuspected cases in which no culture has been sent. Overview of special stains for fungi 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 © by author 4. Fontana-Masson [black] : Cryptococcus & dematiaceous fungi & some speciesESCMID of Aspergillus, MucoralesOnline & Trichosporon. Lecture Library Cytology (sputum, BAL, CSF, FNA): Calcofluor white, Uvitex, MGG, Pap, PAS, GMS. Reporting of fungal infections in tissue & cytological preparations 1. Describe the fungal elements. Yeast / hyphae / Both/ endosporulating structures. Yeast forms – Diameter, Budding or Fission/Type of budding (narrow based or broad based). Hyphae - Width, septations, branching angle, dilated bizarre forms (Mucorales), Brown pigmentation (Dematiaceous fungi). Also comment on quantity of fungal elements & viability. 2. Clearly state the fungus which is most frequently associated with the described morphology & enlist the fungi that can display a similar morphology. © by author 3. Identify the inflammatory reaction, comment on presence of ESCMIDvascular or tissue Online invasion, necrosis, Lecture or hemorrhage. Library 4. Correlate all pathological findings with clinical features, epidemiology & results of alternative testing if available . Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80. MORPHOLOGICAL CLASSIFICATION OF FUNGAL DISEASES Diseases Where Yeasts or Yeast-Like Structures Are Usually Seen Diseases Where Hyphae Are Usually Seen Diseases caused by Fusarium, Scedosporium, and other hyaline septated molds Diseases caused by Bipolaris/Curvularia and other Dematiaceous fungi Dermatophyte disease Diseases caused by other© bymolds author (coelomycetes) *TheESCMID “clinical spectrum Online & host tissue Lecture response “Library to every fungus is as important as the “morphology of the fungus” & must be kept in mind while making a diagnosis. DISEASES WHERE YEASTS OR YEAST-LIKE STRUCTURES ARE USUALLY SEEN IN TISSUES Blastomycosis Cryptococcosis Histoplasmosis Coccidioidomycosis Candidiasis© by author ESCMID Online Lecture Library Blastomyces dermatitidis Morphology of fungus Clinical spectrum & host tissue D/Ds response •Yeasts meas. 8 -15 μm. • Acute pneumonia: Mixed 1. Larger forms to be suppurative response. differentiated from • Show single, broad-based • Chronic pneumonia: a) Immature spherules budding. Pyogranulomatous inflammation of C. immitis. with numerous multinucleate cells. b) Yeast forms of • Clear space around the • Cutaneous/mucosal lesions: P. Brasiliensis. fungal cell on H&E staining - Marked epithelial hyperplasia. c) Conidia of (corresponds to a thick - Neutrophilic microabscesses in aspergillus. refractile cell wall); stains epithelium. with silver stains. - Chronic inflammation in the dermis/ 2. “Microforms” to submucosal© by tissue. author be differentiated • Larger forms may • Disseminated lesions: Various from the occasionally be accompanied inflammatory responses depending Yeast forms of by the “microESCMID forms”. Onlineon immune status. Lecture LibraryCandida spp., *Tuberculosis or neoplasia can be Histoplasma, present concomitantly Cryptococcus. © by author ESCMID Online Lecture Library Guarner J, Brandt ME. Clin Microbiol Rev 2011;24:247-80. PAS stain. © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Cryptococcus spp. Morphology of fungus Clinical spectrum & host tissue D/Ds response • Spherical-oval yeasts. • Pneumonia & Cryptococcomas: Candida & • Marked variation in size. Granulomatous reaction with fibrosis. Histoplasma - • Narrow-based budding. Occasionally pseudotumour formation. similar in size & can • Thick polysaccharide • Neutrophils unusual – indicate be confused capsule. bacterial superinfection/response to histologically. • Pseudohyphae (with massive necrosis. massive yeast proliferation). • Disseminated disease: Varying infl. Well encapsulated response depending on immune status. organisms easily India ink: Negative stain - • Immunocompetent individuals: Well differentiated using highlights the capsule. formed granulomas with few organisms. capsular stains. Mucicarmine & Alcian blue: • Severe© by T cell author deficiency: Capsule. Minimal inflammation with abundant Organisms with PAS & GMS: Cell wall. intra/extracellular organisms (sheets of poorly formed MasonsESCMID-Fontana: Yeast cell Onlineyeast filled lacunae Lecture- “soap bubble Librarycapsule pose some (permits differentiation from lesion”). problem and need most other pathogenic yeasts to be differentiated except Trichosporon beigelii). by melanin stains. CDC public health image library - Khanna G, Bhattacharya SN, Singhal A,© Singh by N, Sharma author S. Fatal disseminated cryptococcosis in a HIV negative 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 MycolESCMID2007; 45:7, 655-6 Online Lecture Library - Capoor MR, Khanna G, Malhotra R, Verma S, Nair D, Deb M, Aggarwal P. Disseminated cryptococcosis with necrotizing fasciitis in an apparently immunocompetent host: a case report. Medical Mycology 06/2008; 46(3):269-73. © by author ESCMID Online Lecture Library PAS stain. © by author ESCMID Online Lecture Library Mucicarmine stain Histoplasma Morphology of fungus Clinical spectrum & host tissue response D/Ds H. Capsulatum: • Spectrum varies from localized granuloma Include all small • Oval 2-4-μm yeast formation to massive proliferation of MPs sized yeasts which show containing yeast cells. • Narrow-based budding. narrow based • Acute pneumonia: Intra-alveolar aggregates budding & • H&E: Halo corresponding of histiocytes, expand to form nodules of Intracellular to the cell wall. parenchymal/ vascular necrosis. clustering. • Chronic histoplasmosis: Multiple small • GMS & PAS: Highlight the granulomata seen all over, commonly in pleura cell wall. & spleen…May get hyalinized...Rarely residual • Intracellular clustering necrosis© with by yeast author forms may be seen. within macrophages - • Mediastinitis: Massive hilar & mediastinal diagnostic hallmark. LAP; lamellar fibrosis & calcification (fibrosing ESCMID Onlinemediastinitis); organismsLecture rarely found/if Library found • In African histoplasmosis the yeast swollen, distorted, no budding..? non-viable). size is larger (8-15μm) & yeast may be pigmented. Fungus to be Differentiating features differentiated “Micro” forms of B. Presence of larger forms & broad based budding dermatitidis Capsule deficient Show marked size variation & positive melanin staining Cryptococci Endospores of Presence of remnants of a ruptured spherule or an intact spherule. Coccidioides spp. Pneumocystis jirovecii Does not show budding & has a prominent intracystic focus. Penicillium marneffei Fission,© sausage by shape,author transverse septum. Candida glabrata Typically extracellular; no halo, shows greater size variability & pure neutrophilic infiltrate. ESCMID Online Lecture Library Leishmaniasis & H&E stains entire organism; no halo; in leishmaniasis kinetoplast toxoplasmosis seen by the side of the nucleus; in toxoplasmosis infected cells are cardiomyocytes & neurons rather than histiocytes. © by author ESCMID Online Lecture Library CDC public health image library PAS stain PAS stain © by author ESCMID Online Lecture Library GMS stain GMS stain © by author ESCMID Online Lecture Library Coccidioides immitis Morphology of fungus Clinical spectrum & host tissue D/Ds response • Thick walled spherules • Pulmonary & cutaneous lesions R. seeberi: (10-100 μm) which demonstrate a mixed suppurative- • Palatal & nasopharyngeal contain multiple granulomatous response with polyps. endospores (2-5 μm). abundant eosinophils. • Larger sporangia & • Rupture to release the • Eosinophils secrete EBP which endospores. endospores into the induces an intense rim of • 50-100mm, can be seen surrounding tissues. eosinophilic material around fungal with the naked eye as yellow • H&E stains both the elements. “Splendore-Hoeppli pin-head sized dots in a spherules & endospores. phenomenon”. polyp. • GMS & PAS stain the • Immunosuppressed - marked spherule wall & necrosis© without