Fungi. Materials and Methods PART 3: FUNGI 2/28/19. Bedside Diagnos,cs: Beyond the Tzanck. Slide, 15 blade, stain(s), microscope

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1 I do not have any relevant rela,onships with industry. Bedside Diagnos,cs: Beyond the Tzanck PART 3: FUNGI I will be discussing off-label use of medica,ons. *SPECIAL THANKS TO DR KAROLYN WANAT* Travis Vandergriff, MD Director of Dermatopathology UT Southwestern Medical Center Dallas, Texas Fungi 1. Dermatophytes 2. Yeast 3. Subcutaneous mycoses -Chromoblastomycosis -Lobomycosis -Eumycetoma 4. Systemic mycoses -Cryptococcus -Histoplasmosis -Paracoccidiomycosis -Blastomycosis -Coccidiomycosis 5. Angioinvasive -Aspergillus -Fusarium -Zygomyces Materials and Methods Slide, 15 blade, stain(s), microscope 1

2 Step by step procedure for KOH 1) Iden,fy area to obtain sample -leading edge 2) Dislodge scale directly on slide 3) Add 1-2 drops 10-20% KOH or Chlorazol black 4) Coverslip 5) Heat if necessary (match or lighter) 6) EVALUATE Staining Potassium hydroxide 10-20% Chlorazol black: fungus turquoise Swartz Lamkins: fungus blue +/- DMSO (dimethyl sulfoxide): gives more rapid macera,on and clearing of kera,nocytes than potassium hydroxide alone -Prepara,ons do not keep long 62 y/o woman with recurrent rash on her face that has not improved with many creams or lo,ons. KOH was performed 2

3 Which of the following is true? A. This is a posi,ve KOH prep B. No stain was applied to the prep C. The condenser is not in the correct loca,on D. The wrong area was scraped for the prep E. The prep was not heated Power of the condenser Power of the condenser Power of the condenser 3

4 Microscope Set-up To enhance contrast, reduce microscope illumina,on by lowering condenser un,l epithelial cells are clearly visible Scan under 4x to find cells Evaluate at 10x-40x to iden,fy fungus Microscope Set-up To enhance contrast, reduce microscope illumina,on by lowering condenser un,l epithelial cells are clearly visible Diaphragm posi,on rela,vely closed Scan under 4x to find cells Evaluate at 10x-40x to iden,fy fungus DETECTION Potassium hydroxide dissolves kera,naceous debris while chi,n-rich fungi remain intact 42 y/o woman with recalcitrant foot derma,,s and blisters on her heel Chlorazol Black Iden,fy: Branching, septate hyphae Ballooning Going ACROSS cell walls 4

5 Which of the following is true? A. Hyphae turned turquoise because of chi,n in the cell walls B. Hyphae turned turquoise because it is a yeast C. This tes,ng takes a lot longer to perform D. Turquoise color decreases sensi,vity in detec,on DETECTION Chlorazol black Chi,n specific Hyphae will be turquoise Going ACROSS cell walls Look for septa,ons Ballooning 8 y/o boy with itchy scalp Diagnosis 1) Clean off site 2) Wood s lamp 3) Epilate hairs (choose those that fluoresce) 4) Scrape scale using blade ***Use toothbrush, cultureie, coion,p 5) KOH or Chlorazol black 5

6 Tinea capims 28 y/o with recurrent derma,,s on the trunk What is the the most likely diagnosis? A. Candida B. Eczema C. Tinea corporis D. Tinea versicolor Tinea Versicolor KOH: Spaghej and meatballs! Or penne and peas! Easy to find, usually florid! 6

7 Calcofluor white Calcofluor white Binds to cellulose and chi,n Fluoresces and detected under UV light Add one drop of KOH with one drop of calcofluor white to specimen Hyphae fluorescent in nail plate or scale Method Comparison KOH: $<10 -Simplest, inexpensive -Sensi,vity: 80% -Specificity: 72% -PPV 88% -False nega,ve: up to 15% Chlorazol: $<10 -Cost-effec,ve, similar to KOH -Sensi,vity: 94.3% J Am Acad Dermatol 2003;49:193-7 Diagn Microbiol Infect Dis 1990;13:337-9 Mycoses 1993;36:243-5 Calcofluor: $30 Sensi,vity: 92 95% Specificity: 95% PPV: 74% NPV: 99% Fungal culture: $90 Sensi,vity 35 65% Specificity: 82% PPV: 90% PAS: $90 Sensi,vity: 85 >95 (98.8)% Specificity: 72% PPV: 89.7% Fungi 1. Dermatophytes 2. Yeast 3. Subcutaneous mycoses -Chromoblastomycosis -Lobomycosis -Eumycetoma 4. Systemic mycoses -Cryptococcus -Trichosporonosis -Histoplasmosis -Paracoccidiomycosis -Blastomycosis -Coccidiomycosis 5. Angioinvasive -Aspergillus -Fusarium -Zygomyces 7

8 40 y/o with slow growing plaque on his leg What is the best diagnosis? A. Chromomycosis B. Cryptococcosis C. Candidiasis D. Coccidioidomycosis Image courtesy of B. Bustamante Chromomycosis Scrape brown dots Same KOH prep Pigmented bodies Sclero,c bodies Medlar bodies Copper pennies Globe-shaped, cigarcolored, thick-walled structures 4-12 µm in diameter Chromomycosis Slow growing, verrucous plaques secondary to dema,aceous fungi Organisms: Fonsecaea pedrosoi (moist environments) Cladophialophora carrionii (drier environments) Phialophora verrucosa, and Rhinocladiella aquaspersa being less common Treatment: Itraconazole (voriconazole, posaconazole, caspofungin) 8

9 Lobomycosis Tropical climates (La,n America) Lacazia loboi Dimorphic fungi Slow growing (25-30 days) Treatment: Surgery Itraconazole (posaconazole) Clofazimine Image courtesy of B. Bustamante Lobomycosis Cannot be cultured Direct visualiza,on (KOH): Single or mul,ple budding thick walled spherules 9 to 10 microns Thin connec,on 60 y/o man from South American presents with painful sores in his mouth What is the most likely diagnosis? A. Blastomycosis B. Coccidiomycosis C. Cryptococcus D. Histoplasmosis E. Paracoccidiomycosis Image courtesy of B. Bustamante 9

10 Paracoccidiomycosis Paracoccidiomycosis Direct visualiza,on (KOH): Thick-walled round cell 5-50µm Narrow buddings (2-10µm) Mickey Mouse or Mariner s wheel Images courtesy of B. Bustamante ***KOH yields diagnosis in >90% of cases ***Can be more sensi,ve than culture 2/2 contamina,ng organisms Restrepo A, Tobon AM, Agudelo CA. Paracoccidioidomycosis. In: Diagnosis and Treatment of Human Mycoses, 1st edi,on, Hospenthal, DR, Rinaldi, MG (Eds), Humana Press, Totowa, NJ Paracoccidiomycosis Direct visualiza,on (Lactophenol coion blue): --Phenol: kill live organisms --Lac,c acid: preserves fungal structures --Coion blue: stains chi,n in cell walls Add alcohol, then lactophenol/ coion blue stain Vinyl Adhesive Tape Useful in chromoblastomycosis, lobomycosis, paracoccidiomycosis Collect sample (infec,ous agents) in upper layers of epidermis Apply tape to lesions, rub (scaly crust) Put tape on slide with KOH Evaluate! Miranda MFR, Silva AJG. Diagnos,c Microbiology and Infec,ous Disease :

11 24 y/o man from Central American presents with slow growing, fluctuant papulonodules with drainage and grains as shown What is the most likely causamve organism? A. Ac3nomadura pellei3eri B. Acremonium spp C. Madurella spp D. Staphylococcus aureus E. Streptomyces somaliensis Image courtesy of B. Bustamante Mycetoma Madura foot Painless papules and nodules Edema (tumefac,on) Sinus tracts Grains Ac,nomyco,c Eumyco,c Botryomycosis Image courtesy of B. Bustamante 11

12 Mycetoma Eumycetoma: thick filaments, septate Black grains always fungus Boiom image from: Gupta S, Jain K, Parmar C, Shah P, Raval RC. Indian J Sex Transm Dis Jan;31(1): Mycetoma KOH direct examina,on Take grains Evaluate color Apply KOH and press grain Eumycetoma: thick filaments, septate Ac,nomycetoma: thin, fine filaments 67 y/o HIV posi,ve man recently moved from California to Wisconsin and presents with cough and scaly plaques What is the most likely diagnosis? A. Blastomycosis B. Coccidiomycosis C. Cryptococcus D. Histoplasmosis E. Paracoccidiomycosis 12

13 Cryptococcus Tzanck smear Encapsulated round, dark-walled, pleomorphic yeast with clear gela,nous capsule Capsule stains with methylene blue, alcian blue, mucicarmine Cryptococcus Worldwide distribu,on Cryptococcus neoformans 4 th leading cause of opportunis,c infec,on Skin: 10-15% of cases may precede by 2-8 months Treatment: amphotericin B +/- fluconazole Blasto Coccidio Crypto Histo Paracocci 8 to 15 µm round to oval organisms with thick, doublerefractile walls; broad-based, single budding 10 to 80 µm variably sized spherules filled with endospores 5-10 µm round, darkwalled, pleomorphic yeast with clear gelatinous capsules; India Ink stains the background 2-4 µm with a pseudocapsule Parasitized organisms Giemsa or Tzanck best 5-50 µm thick-walled, round cells with narrow (2-10 µm) budding Mariner s wheels & Mickey Mouse 40 year old woman with a history of Gardner syndrome s/p bowel resec,on and small bowel transplant 25 years ago with recurrent episodes of solid organ rejec,on needing treatment with systemic steroids. 13

14 Now with 1 week history of painful plaques on legs. What is the next most appropriate step in management? A. Start amphotericin B. Start cefepime C. Start vancomycin D. Wait for histopathology and tissue culture Touch Prep! Rapid diagnos,c test that can help iden,fy fungi quickly Start empiric therapy earlier Impact pa,ent survival Touch prep KOH: Remove part of specimen (skin biopsy) Take piece of skin and rub base or center of lesion on slide Diagnosis: Thin, septate hyphae, acute angle branching Image courtesy of B. Bustamante 14

15 Aspergillus Scedosporium spp. and Fusarium spp Mucomycosis (Mucor, Rhizopus, Cunninghamella, & Lichtheimia ) KOH PREP PRACTICAL CONSIDERATIONS Septate, thin hyphae Acute angle branching Septate, irregular hyphae with bubbly cytoplasm Branching is 45 to 90 degrees Ribbon-like, aseptate hyphae Wide angle branching CPT code: Medicare reimbursement: approximately $8 CLIA: Moderate complexity, provider performed microscopy (PPM) QUESTIONS? TRAVIS.VANDERGRIFF@UTSOUTHWESTERN.EDU 15