Mycology (MTAP) PDF

MTAP 07/08/23 T...

MTAP 07/08/23 Trans # 4 MYCOVIROLOGY (MYCOLOGY) OUTLINE Spores → FUNGI have spores for REPRODUCTION. I. Mycology D. Chromomycosis ▪ Sexual spore II. Fungi E. Rhinosporidiosis ▪ Asexual spore A. Generalities of fungi VII. Systemic mycosis → BACTERIA have endospores but it is usually for PROTECTION. III. Phases of Fungi A. Blastomycosis Metabolism A. Yeast B. Paracocci → Heterotrophic = source of carbon/nutrients is from complex B. Mold C. Paracoccidioidomycosis substances. C. Asexual reproduction D. Histoplasmosis D. Sexual reproduction VIII. Opportunistic mycosis → Photoautotrophs = make their own energy using light and CO2 IV. Superficial mycoses A. Candidiasis → Chemoautotrophs = source of energy is from chemical reaction A. Tinea versicolor B. Cryptococcosis Oxygen Requirement B. Piedra C. Aspergillosis → FUNGI can either be aerobic or facultative anaerobic depending on C. Tinea nigra D. Talaromycosis the phase of the Fungi. V. Cutaneous mycoses E. Pneumocystis ▪ Mold (Aerobic) A. Microsporum pneumonia ▪ Yeast (Facultative Anaerobe) B. Epidermophyton F. Zygomycosis III. PHASES OF FUNGI C. Trichophyton IX. Laboratory diagnosis of VI. Subcutaneous mycoses fungal diseases Polymorphic Fungi A. Rose Gardener’s A. Microscopic examination → Having multiple forms and structures B. Lobomycosis B. Culture Dimorphic Fungi C. Mycetoma C. test → Having two (2) phases which are dependent on the temperature ▪ Yeast-like at body temperature (37°C), then transforms into; I. MYCOLOGY − Recovered through biopsy Study of fungi ▪ Mold-like at room temperature (25°C) Fungi also known as “Thallophytes”. − Cultured in lab → Eukaryotic organisms → Usually pathogenic fungi → Plant-like but lack stems and roots. ▪ ex. Sporothrix, Paracoccidioides, Blastomyces, Histoplasma, Talaromyces (Penicillium) marneffei, Coccidioides → Heterotrophs − Talaromyces marneffei was formerly known as Penicillium II. FUNGI marneffei A. GENERALITIES OF FUNGI Fungus In vitro (25°C) In vivo (37°C) Characteristic Fungi Bacteria Blastomyces Type of Cell Eukaryotic Prokaryotic No sterols except Cell Ergosterols are Present in Mycoplasma, Membrane Coccidioides Ureaplasma Composed of complex Peptidoglycan Cell Wall carbohydrates such as chitin, (NAG and NAM, glucans, and mannans peptides) Histoplasma Endospores (not Sexual and Asexual (for for Reproduction; Spores reproduction) some asexual Paracoccidioide spores s Heterotrophic Metabolism Heterotrophic/Chemoheterotrophic Photoautotrophic Chemoautotrophic Sporothrix Aerobic, Oxygen Aerobic (Molds) Facultative Table 2. Forms of Fungi in Room and Body temp. Requirement Facultative Anaerobe (Yeasts) Anaerobe, Anaerobe A. YEAST pH Unicellular; round; non-filamentous pH 5 (acidic) Neutral pH Requirement Some can produce: Penicillin, → Pseudohyphae – daughter cells (blastoconidia) that fail to detach from Antibiotic Polyenes, Imidazole, Griseofulvin Tetracyclines, mother cell. Sensitivity Aminoglycosides ▪ Majority of Candida spp. Table 1. Generalities of Fungi On additional information: Table 1 Type of Cell → Eukaryotic – True nucleus → Prokaryotic – No true nucleus Cell Membrane → Both have cell membrane. However, Bacteria does not have sterols EXCEPT: ▪ Mycoplasma ▪ Ureaplasma Figure 1. Yeast Cell Wall → Most important carbohydrate (CHO) in the cell walls of Fungi is the Reproduction CHITIN Budding MTAP – MYCOVIROLOGY MLS 4-4 1 of 12  Binary Fission NODULAR Microsporum enlarged knots of canis Temperature and Oxygen Requirement closely twisted hyphae Grows at 35 – 37 C (body temperature) Facultative anaerobe FAVIC CHANDELIER Tricophyton Morphology antler like appearance scheonleinii Moist, creamy, or buttery colonies with an alcohol-like odor → Bacterial-like RHIZOIDS Rhizopus Can be identified by biochemical test root like processes Larger colonies than bacterial colonies B. MOLD Multicellular; filamentous; elongated PECTINATE BODIES Microsporum looks like a tooth of a auduoinii Capable of forming hyphae/thallus comb → Thallus – Basic functional unit Table 3. Hyphae. According to presence/absence of cross walls (septum) → Aseptate / Coenocytic / Mucoraceous / Pauciseptate Hypha C. ASEXUAL REPRODUCTION ▪ Absen or sparse crosswalls All conidia are ASEXUAL spores. ▪ Example: Conidia, Microconidia, Macroconidia − Zygomycetes o Rhizopus Conidia o Mucor → Forms from a conidiophore o Absidia → Arises from the side of hypha → Septate hypha Microconidia ▪ With crosswalls → Small unicellular According to pigmentation (melanin) → May appear as: → Phaeoid/Dematiaceous ▪ Teardrop ▪ Pigmented hypha - agents of subcutaneous mycoses ▪ Pyriform ▪ Example Macroconidia − Alternaria, Bipolaris, Exophiala, Phialophora, Cladosporium → Large multicellular; spindle shaped → Hyaline/Moniliaceous → May appear as: ▪ Non-pigmented ▪ Echinulate ▪ Example − Spiny − Aspergillus, Rhizopus, Acremonium ▪ Smooth, rough ▪ Thick or thin − Microsporum tends to have thick macroconidia − Trichophyton tends to have thin macroconidia ▪ Tuberculate − Tuberculate has protrusion ▪ Club Shaped Figure 2. Aseptate hyphae and Septate hyphae ▪ Muriform – hand grenade (Alternaria) − Muriform is usually seen in cultures of Alternaria Mycelium ▪ Sickle shape → mass/aggregate of hypha − Fusarium can exhibit sickle shape or boat shaped Macroconidia → contains the ff: ▪ Thallus – submerged/vegetative hyphae ▪ Aerial – visible or found on the surface ▪ Fertile hyphae – contains the spores (for reproduction) Figure 4. Muriform On additional information: Table 1 Red Circle – conidiophore Figure 3. Mycelium Green Circle – hypha Reproduction Fragmentation Spore formation Temperature & Oxygen Requirements Grows at room temperature Aerobic Figure 5. Tuberculate (left), Sickle shape (middle), Microconidia (right) Morphology Blastoconidia Dry, cottony with a velvety surface Arise from simple budding Can be identified/classified by appearance or type of spores Parent cell>>daughter cell Vegetative Hypha Seen In Picture RACQUET HYPHAE Epidermophyton resemble tennis floccosum racquet SPIRAL Trichophyton corkscrew like mentagrophytes MTAP – MYCOVIROLOGY 2 of 12  Figure 6. Blastoconidia Derived from fusion from 2 identical cells from the same hypha Chlamydoconidia Single large spores with thick walls Thick-walled spores; resistant; formed during unfavorable conditions Oospores → Terminal = found on the tip Derived from fusion from 2 non-identical separate hypha ▪ Example. Candida albicans → Intercalary = found in between the hyphal element → Sessile = found on the side of the hypha Fungi that are able to form this type of spore are the Candida albicans. → One way to identify them is their ability to form terminal chlamydoconidia Figure 11. Basidiospores (upper left), Ascospores (upper right), Zygospores Figure 7. Chlamydoconidia (lower left), Oospores (lower right). Arthroconidia Barrel shaped or rectangular spores MYCOSES – FUNGAL INFECTIONS Produced from fragmentation IV. SUPERFICIAL MYCOSES Seen in Coccidioides → Possess dysjunctor cells “checkered appearance” Noninvasive; involve the horny nonliving layer of the skin and extrafollicular parts (hair) of the skin. Geotrichum Usually, patients are asymptomatic → “Hockey stick” appearance Commonly acquired through close contact or direct contact with the infected A. TINEA VERSICOLOR Also known as Pityriasis versicolor Asymptomatic; non-inflammatory Variation in pigmentation Only affects the skin and hair usually, they do not spread or disseminate. Etiologic Agent and MOT Figure 8. Coccidioides with dysjunctor cells (left) and Geotrichum (right) MOT: → Direct Skin contact → May disseminate parental nutrition-neonates, elderly, immunocompromised Causative agent: Malassezia furfur → It is a yeast like organism. Figure 9. Fragmentation of hypha resulting to arthroconidia Sporangiospores Enclosed spores (in a sporangium) → Inside the sporangium are the sporangiospores Arise from a sporangiosphore Example: Zygomycetes → Rhizopus → Absidia Figure 12. Hyperpigmentation (left) and Hypopigmentation (right) → Mucor On additional information: Hypopigmentation in dark skin individual. Hyperpigmentation in fair skin individuals. Malassezia furfur may find its way in the blood and disseminate. Lab Diagnosis and Diagnostic Feature Direct KOH Mount → Examined skin scrapings from suspected patient. → Look for the presence of spaghetti and meat balls appearance. Medium: SDA with olive oil → Microscopic Examination of colonies ▪ appears as bowling pin or medicine bottles → Olive oil is used because the organism is a lipophilic organism that prefers growing in lipid rich substances. Figure 10. Sporangiospores D. SEXUAL REPRODUCTION Mnemonic: BAZO Basidiospores Enclosed in a clubshaped structure (basidium) Ascospores Enclosed in a sac (ascus) Ascocarps – fruiting bodies composed of asci Figure 13. Direct KOH Mount (left) and SDA w/ Olive oil (right) Zygospores On additional information: SDA - Sabouraud Dextrose Agar MTAP – MYCOVIROLOGY 3 of 12  Lab Diagnosis and Diagnostic Feature B. PIEDRA Mistaken as melanoma or skin cancer Also known as: “Stained appearance” or blackening on palms, foot, forearms → Tinea nodosa Direct Microscopic Examination: → Beigel’s disease → Branching septate hypha, dematiaceous → Chignon’s disease Figure 17. Stained appearance (left) and Branch septate hypha (Right) Figure 14. Piedra → On additional information: Fungal elements of Hortaea werneckii tend to be pigmented or On additional information: dematiaceous Piedra means stone → It is called stone because you can find this nodule attached to the hair shaft. (Figure 14) V. CUTANEOUS MYCOSES Piedra infection does not affect the whole hair but only on where the Targets keratinized tissues nodule is present. → Have enzymes to breakdown/degrade and utilize keratin Etiologic Agent and MOT Affects: → Hair Black piedra → Skin → Caused by fungus called Piedraia hortae → Nails → Affected areas Infections are called tinea/ringworm infections ▪ Scalp hair ▪ Eyebrows, Dermatophytes ▪ Eyelashes → Fungal agents that cause cutaneous mycoses ▪ Pubic hair → 3 important dermatophytes: White piedra ▪ Microsporum → Caused by Trichosporon beigeli ▪ Epidermophyton ▪ Trichophyton → Affected areas ▪ usually on facial & genital hair Grouping of Select Dermatophytes based on the Host Preference and Natural Habitat Anthropophilic Zoophilic Geophilic Epidermophyton Microsporum canis Microsporum floccosum gypseum Microsporum audouinii Trichophyton equinum Trichophyton Trichophyton concentricum mentagrophytes (granular isolates) Trichophyton Trichophyton verrucosum Figure 15. White Piedra mentagrophytes (velvety and cottony isolates) Nodules Trichophyton rubrum → Black piedra Trichophyton schoenleinii ▪ Pigmented Trichophyton tonsurans ▪ Hard Trichophyton violaceum ▪ Firmly attached Table 4. Dermatophytes. → White piedra ▪ Less/ none pigmented On additional information: ▪ The nodule is slightly soft or mucilaginous Anthropophilic – source of infection is from humans ▪ Not firmly attached Zoophilic – source of infection is from animals Lab Diagnosis and Diagnostic Feature Geophilic – source of infection is from environment (soil) Direct Microscopic Exam Tinea Description Picture → Black Piedra – asci with ascospores; chlamydoconidia → White Piedra - blastoconidia and arthroconidia Fungal infections affecting the T. capitis scalp Severe type of T. capitis T. favosa Permanent alopecia Figure 16. Black Piedra (left) and White Piedra (Right) C. TINEA NIGRA Also known as Keratomycosis nigricans palmaristhe palms. Fungal infection affecting the Etiologic Agent and MOT T. barbae facial hair Etiologic agent: Hortaea werneckii (Exophiala werneckii) MTAP – MYCOVIROLOGY 4 of 12  Fungal infection affecting T. corporis the body Circular lesions on the body Figure 19. M. canis echinulate macroconidia with curved end (left); Colonies of M. canis (right) Lesion on the “singit” or T. cruris inguinal area M. gypseum Also called as “jock itch” 3-9 called broadly spindle shaped rough- walled macroconidia with rounded terminal ends Microconidia → Few, single or in small clusters Wood’s lamp: Negative Also called as “athlete’s foot” Colonies: Tan/buff; cinnamon T. pedis Most common fungal infection in the world T. unguium Ringworm of the nails Figure 20. M. gypseum macroconidia with round end (left); Colonies of M. gypseum (right) M. audouinii Rare, bizarre distorted macroconidia T. mannum Vegetative Hypha: Lesions on the hand → Pectinate hypha T. manuum ▪ clue for looking for M. audouinii → Presence of chlamydoconidia Rice agar: Negative → No growth Presence of concentric Colonies: Cottony white, salmon lesions in the body Associated with tinea capitis T. imbricata Causative agent: Trichophyton concentricum Table 5. Tinea infections (Ringworm). A. MICROSPORUM Infects SKIN and HAIR Figure 21. Pectinate hypha of M. audouinii (left) M. audouinii colony (right) → Macroconidia: B. EPIDERMOPHYTON ▪ Large ▪ Spindle Shaped Infects skin and nails ▪ thick walled Macroconidia: smooth walled ▪ multiseptate (>6 cells) NO MICROCONDIA → Microconidia: few or absent Wood’s Lamp: Positive → Ectothrix infection: mosaic pattern Culture Wood’s Lamp test (UV lamp): (+) Fluorescence except M.gypseum (-) → Smooth walled club shaped macroconidia E. floccosum Macroconidia: → Snow paddle → Club shaped → Smooth walled Colonies → Khaki green → Velvety Associated with → Jock itch Figure 18. Wood’s Lamp positive (left) & Macroconidia (right) → Athlete’s foot M. canis → Tinea unguium → Tinea mannum or Tinea manuum Zoophilic Large, spindle shaped rough/echinulate macroconidia, terminal ends are curved (beaked apex) Few or no microconidia Colonies: Bright yellow (reverse) Associated with tinea capitis Figure 22. Club shaped macroconidia (left); Khaki green colonies (right) MTAP – MYCOVIROLOGY 5 of 12  C. TRICHOPHYTON 2nd most common cause of epidemic tinea capitis Infects skin, hair, and nails → Very common agent of Cutaneous mycoses, since it can affect the skin, the hair, and the nails. Macroconidia: Smooth, club shaped, thin walled Microconidia: Numerous, spherical, tear shaped Endothrix: arthroconidia in row; favus variety (in T. schoenleinii) Wood’s Lamp: (-) → Trichophyton VS Microsporum ▪ Trichophyton is negative for Wood’s Lamp Figure 25. Balloon Forms Macroconidia T. schoenleinii Slow growing fungi Conidia ABSENT Vegetative Hypha: Favic Chandelier → We don’t look for spores but the Hypha that are Favic Chandelier and antler-like Causes: Tinea favosa Figure 23. Trichophyton → Tinea favosa causes permanent alopecia Colonies: white, wrinkled T. rubrum T. mentagrophytes Few, cigar shaped Macroconidia Pencil shaped, smooth smooth walled Tear shaped Spherical “Birds on a fence” Grape-like Microconidia arrangement (single and clusters lateral along hypha) → Also called “en → Also called “en thryse” grappe” Cornmeal Intense Red Agar with 1% Figure 26. Favic Chanderlier glucose Scanty red (red pigment T. verrucosum production) Slow growing fungi similar to T. schoenleinii Urease Rat tail macroconidia Negative Positive Production Distinct feature: Chlamydoconidia in chains Positive (V shaped perforation) Hair Baiting Test/Hair Negative Perforation Test Tinea cruris Tinea unguium Associated Tinea corporis Tinea barbae Diseases Tinea capitis Tinea pedis Table 6. T. rubrum and T. mentagrophytes. Figure 27. Rat tail macroconidia T. violaceum Violet purple waxy colonies Conidia absent; swollen hyphae with cytoplasmic granules Can also cause tinea favosa Figure 24. T. rubrum (left and middle) and T. mentagrophytes (right). VI. SUBCUTANEOUS MYCOSES Also known as Inoculation mycoses On additional information: Chronic fungal diseases Figure 24. Affects subcutaneous and deeper layers of tissue (e.g., muscles, bone) → T. rubrum (left and middle picture) ▪ Left picture: Pencil shape appearance → Lesions appear on the extremities ▪ Middle picture: “birds on a fence” arrangement MOT: Traumatic skin puncture → T. mentagrophytes (right picture) A. ROSE GARDENER’S ▪ Red: Grape like clusters / en grappe Also known as ▪ Yellow: Cigar shape → Sporotrichosis ▪ Green: Spiral hyphae → Rose Handler’s Disease Hair Perforation Test Most commonly manifested as Lymphocutaneous sporotrichosis → Culture suspected to be T. mentagrophyte is added to sterile hair, then will be allowed to grow Etiologic Agent and MOT → Incubation: 2-3 weeks Causative agent: Sporothrix schenckii → Positive Result – hair shaft has V shaped perforation MOT: T. tonsurans → Skin Puncture → Inhalation (in other references) Macroconidia: Rare Microconidia: Balloon forms (aged microconidia) Lab Diagnosis and Diagnostic Feature Colonies: Dimorphic fungi — expect a mold form and yeast form → Suede like → Mold Form (Room temp) → No red pigment in cornmeal dextrose agard ▪ Flowerette microconidia or Daisy Head conidia MTAP – MYCOVIROLOGY 6 of 12  → Yeast Form → Sinus tracts ▪ Cigar shape → bone involvement ▪ Normally found in tissue biopsy of affected patients Dx: Presence of granules/ grains (recovered from the lesions) Asteroid Bodies → Granules can be pigmented or not pigmented depending on the type of → Additional findings in tissue biopsy fungi that causes it.. → Are composed of eosinophilic (pinkish) structures that are remnants of an immune complex reaction Figure 28. Daisy head conidia (left); Asteroid bodies (right) Figure 30. Granulomas lesion in the foot (left); Granules/grains (right) B. LOBOMYCOSIS Also known as “Keloidal Blastomycosis” On additional information: → Name originated from the presence of keloid-like lesions on the In mycetoma, there is a presence of granulomas or granulomatous extremities lesions in the foot, wherein the lesions would have sinus tracts. Etiologic Agent and MOT D. CHROMOMYCOSIS CA: Lacazia loboi Also known as → Formerly known as Loboa loboi → Chromoblastomycosis Lab Diagnosis and Diagnostic Feature → Mossy Foot Disease → Dermatis Verrucosa Chronic, localized, subepidermal infection → Hematomycosis Presence of keloidal/keloid-like, verrucoid nodular lesions on the extremities. Caused by wide variety of fungi (pigmented) Tissue biopsy from lesions → Microscopic examination using GMS (Grocott methenamine silver) stain → Large yeast cells (lemon shaped) with multiple buds & characteristically form short chains Figure 31. Chromoblastomycosis foot Etiologic Agent and MOT Figure 29. Keloidal blastomycosis (left); Lemon shaped yeast cells (right) Fonsecaea compacta On additional information: Fonsecaea pedrosoi (mixed sporulation) GMS stain (Grocott methenamine silver) Phialophora verrucosa → Preferred stain for tissue specimens with fungal elements or fungi Cladophialophora carrionii C. MYCETOMA Rhinocladiella aquaspersa Also known as: On additional information: → Mycetoma/Madura foot These fungi are polymorphic which means it has varying structures → Maduromycosis/Watering Can Foot → Fonsecaea – asterisk like This type of chronic disease may be caused by fungi or bacteria → Rhinocladiella – conidia on the side → Fungal: Eumycotic → Phialophora- conidia from a flask shaped structure ▪ Fungi that cause mycetoma → Cladosporium – conidia in chains ▪ More common in Africa → Bacterial: Actinomycotic ▪ Bacteria that cause mycetoma ▪ This includes Actinomycosis israelii and Nocardia may also be associated ▪ More commonly encountered in South and Central America Botyromycotic → Lesions that look like mycetoma → Caused by bacterial infection → Usually caused by S. aureus Lab Diagnosis and Diagnostic Feature Etiologic Agent and MOT Dematiaceous fungi Fungal Wartlike, cauliflower like lesions Pseudoallescheria boydii There is no bone involvement → Most common cause of white grain mycetoma in US Dx: Sclerotic Bodies Madurella mycetomatis → Also known as Medlar or Muriform Bodies → Most common agent associated with mycetomas → Morphology is similar to copper pennies which have a round structure. → Most common worldwide Leptosphaeria Lab Diagnosis and Diagnostic Feature Granulomatous tumor/lesions of Subcutaneous Tissue Chronic Skin and Subcutaneous Skin Infection S/S: → Weeping lesions ▪ Weeping meaning “nagtutubig” Figure 32. Sclerotic bodies MTAP – MYCOVIROLOGY 7 of 12  E. RHINOSPORIDIOSIS Etiologic Agent and MOT Etiologic Agent and MOT Causative agent: Paracoccidioides brasiliensis Rhinospordium seeberi → dimorphic → Considered as a protozoan Lab Diagnosis and Diagnostic Feature Lab Diagnosis and Diagnostic Feature Room temperature (Mold) Lesions/Masses in Nose & Pharynx → Chlamydoconidia Causes: → Microconidia → Swelling Body Temperature (Yeast) → Difficulty in breathing especially in the nose → Multiple buds or “Mariner’s wheel appearance” Dx: Sporangium → “Mickey mouse cap” → filled with endospores Exoantigen Test: Bands 1,2,3 → Sporangium is found is present when biopsy is performed Manifestation: → TB like pulmonary infection → Suppurative and → Granulomatous Ulcers of mucosal areas (nasopharynx ulcer) Figure 32. Sporangium Figure 34. Paracoccidioides brasiliensis VII. SYSTEMIC MYCOSIS C. COCCIDIOIDOMYCOSIS Primarily caused by dimorphic fungi Also known as: Acquired thru Inhalation of spores (airborne) → San Joaquin Valley Fever Mold forms of B. dermatitidis, C. immitis, H. capsulatum = BSL 3 → Desert fever Specimens: respiratory samples; biopsy specimens → Desert rheumatism Serologic Test: Exoantigen Testing → Cocci On additional information: → The bumps Healthy individuals/ immunocompromised patients can be affected. Endemic area: San Joaquin Valley area in California. It is geographically limited. There are only certain parts of the world Etiologic Agent and MOT where this is endemic Coccidioides immitis – most virulent of all human mycotic infections. Serologic Test: Exoantigen Testing → Other species: C.posadasii → classic serologic test for systemic → used to detect fungal antigens On additional information: Coccidioides immitis found primarily in San Joaquin Valley area in A. BLASTOMYCOSIS California. Mycotic Infection: → There are also reports in nearby states such as Texas and Arizona. → Blastomycosis C.posadasii → Gilchrist’s disease → It is found outside the US or outside the endemic area. → North American blastomycosis Lab Diagnosis and Diagnostic Feature → Chicago disease Room temperature (Mold) – Arthroconidia Etiologic Agent and MOT Body temperature (Not a yeast) Blastomyces dermatitidis → Look for the presence of spherule. Mode of transmission: Inhalation of spores (airborne) ▪ Spherule Lab Diagnosis and Diagnostic Feature − A thick-walled structure containing endospores. − Hallmark of Coccidioides in the biopsy Endemic in River Valley of Southeast, MidWest and Southern MAJOR BIOHAZARD TO LAB PERSONNEL Room Temperature (Mold) → Process it inside the BSL 3 laboratory → lollipop microconidia Exoantigens: Body Temperature (Yeast) → IDCF → Yeast cells with broad based budding → HL Exoantigen Test: SPECIFIC A BAND → TP → Positive for A Band High risk for dissemination: Filipino and African Races. Manifestation: → Chronic, Suppurative Granulomatous Skin Disease → Pulmonary Disease → Affects bone, skin, prostate On additional information: Respiratory infection that spreads. Figure 35. Coccidiodomycosis The patient can get granuloma in the bones, skin, and prostate. On additional information: A unique characteristic for Coccidioides in body temperature is you need to look for the presence of the spherule not a yeast. D. HISTOPLASMOSIS Also known as: Figure 33. Mold (lollipop microconidia) and Yeast Blastomycosis. → Darling’s Disease → Reticuloendothelial Cytomycosis B. PARACOCCIDIOIDOMYCOSIS → Cave disease Mycotic infection → Spelunker disease → South American blastomycosis Other species: H. duboisii (causes African Histoplasmosis) → Para Etiologic Agent and MOT → Paracocci Endemic area: South America, particularly in Brazil Histoplasma capsulatum MTAP – MYCOVIROLOGY 8 of 12 Lab Diagnosis and Diagnostic Feature Lab Diagnosis and Diagnostic Feature Endemic Area: Major cause of fungemia (fungi in blood) → Central Mississippi Able to form germ tubes, blastoconidia, chlamydospores (terminal) → Missouri Screen with: Germ Tube Test to differentiate with C. tropicalis → Ohio River Valleys → Presumptive test Reservoir: → C. albicans (+) → Bats Confirm with: Corn Meal Agar → Starlings (bird) → look for chlamydospore, hyphae, and blastoconidia Intracellular Mycotic Infection of the RES (Reticuloendothelial System) Able to grow at 42°C to differentiate with C. dubliniensis Room Temperature: (Mold) → C. dubliniensis: (+) Germ tube test; (-) at 42°C → Tuberculate Macroconidia Other species of Candida: → Pyriform Microconidia → Candida kefyr: “logs on a stream” (appearance on culture) Body Temperature: (Yeast) → Candida parapsilosis: fungemia in NICU; sagebrush colonies → Small budding yeast cells within monocytes/macrophages ▪ Especially among neonates in the Neonatal Intensive Care Unit → Yeast: can be mistaken for amastigotes of Leishmania (NICU) Exoantigen: H and M → Candida glabrata: no pseudohypha Stain: Note: Update on Candida auris → Giemsa → Emerging species of Candida which is associated with multi-drug → PAS resistance (MDR) → GMS Additional information: ▪ visualize yeast In the presumptive diagnosis of Candida albicans, we perform Germ Tube Test → It is considered as the most cost-effective method in the diagnosis C. albicans → In real practice, C. albicans is the only organism that is Germ Tube positive → In some cases, although we only encounter C. albicans as Germ Figure 36. Yeast cell inside the macrophage or monocyte (left), Tube positive, in some rare cases, we may also encounter other Tuberculate (middle). species of Candida that may form Germ Tube which is C. dubliniensis. Additional information: ▪ C. dubliniensis is also Germ Tube (+) If it affects the RES, it can also affect the mononuclear cells – monocytes, One way of differentiating C. albicans from C. dubliniensis is through macrophages, and tissues in the bone marrow. incubating them at 42C For room temperature → POSITIVE RESULT → Tuberculate Macroconidia. ▪ Grows at 42C = C. albicans For body temperature look for small budding yeast cells. → NEGATIVE RESULT → It is intracellular. ▪ Do not grow at 42C = C. dubliniensis ▪ inside monocyte or macrophages To determine if it is a germ tube, there should be no constriction at the base VIII. OPPORTUNISTIC MYCOSIS → However, if it has a constriction, it is not considered as a germ Fungal infections usually seen in immunocompromised or debilitated tube patients (elderly). → Immunocompromised patients like cancer and AIDS patients Etiologic agents are usually saprophytic fungi which are usually not virulent (non-pathogenic). → The ordinary fungi that we encounter every day is the one who caused opportunistic mycosis Neutropenia (low neutrophil or defect in neutrophil) → Important risk factor in increasing the chance to develop opportunistic mycosis. A. CANDIDIASIS Figure 38. Chlamydospores, Pseudohyphae, and Blastospore of C. albicans Associated with → Moniliasis → Thrush → Mycotic Vulvovaginitis → Candida perionychia → Candida endocarditis Additional information: Candida can grow in an ordinary culture media, even in BAP → Candida colonies on BAP have “foot-like” or pedicle appearance on the side that arise from the colony. Figure 39. Germ Tube Test B. CRYPTOCOCCOSIS Associated with: → Torulosis → European blastomycosis → Busse-Buschke disease Etiologic Agent and MOT Cryptococcus neoformans → Capsulated Yeast Figure 37. Candida albicans on BAP → Associated with: Etiologic Agent and MOT ▪ Fungal Meningitis – Common among patients with AIDS Candida albicans ▪ Cutaneous lesions MTAP – MYCOVIROLOGY 9 of 12  Animal Reservoir: Pigeons → From the feces (guano) of the Pigeons Figure 43. Yeast Form (left); Mold Form (right) Figure 40. Cryptococcus neoformans. E. PNEUMOCYSTIS PNEUMONIA Lab Diagnosis and Diagnostic Feature Etiologic Agent and MOT Culture Medium: SDA (Saboraud Dextrose Agar) w/o cycloheximide Causative agent: Pneumocystis jirovecii/P. carinii (antibiotic) → Formerly classified as a protozoa Selective Medium: Bird Seed/Niger Seed Agar (Staib’s Medium) → Colony Morphology: Brown Black colonies Lab Diagnosis and Diagnostic Feature Other Tests: Highly associated with pneumonia especially among AIDS patients → Urease → Phenol Oxidase → Detect GCM Ag – glucuronoxylomannan Other Spp of Cryptococcus: C. gatti → Can affect healthy individuals and immunocompromised Figure 44. Honeycomb exudates On additional information: Honeycomb exudates (pinkish structures) can be found on lung tissue biopsy. Figure 41. Bird Seed Agar (Cryptococcus neoformans) → Fluid inside the spaces or the alveoli of the lungs ▪ Patients have difficulty in breathing because of the exudates C. ASPERGILLOSIS accumulating in the air sacs. Disease caused by Aspergillus species: → Aspergillosis → Fungus Ball → Aspergilloma → Farmer’s Lung Etiologic Agent and MOT Aspergillus species: → A. niger → A. fumigatus – Most common Figure 45. Broncho Alveolar Lavage biopsy → A. flavus On additional information: Lab Diagnosis and Diagnostic Feature Broncho Alveolar Lavage (BAL) Presence of Dichotomous Septate Hypha in tissue biopsy → preferred specimen to detect the organism → Diochotomous – means branching Hypha Diagnostic stage: Swelling vesicle → Presence of oocyst and trophozoites. ▪ Oocyst – “Punched ping pong ball” appearance F. ZYGOMYCOSIS Etiologic Agent and MOT Members of Zygomycetes: → Rhizopus - w/ rhizoids → Absidia → Mucor Figure 42. Diochotomous Septate Hypha (Aspergillus) Rapid growers; “Lid lifters” → Rapidly fatal D. TALAROMYCOSIS → Those infected with this kind of fungi would die a few days. Etiologic Agent and MOT → Causes obstruction in the brain and other tissues. Talaromyces marneffei (Formerly known as Penicillum marneffei) Lab Diagnosis and Diagnostic Feature Lab Diagnosis and Diagnostic Feature Biopsy: Presence of Aseptate/Coenocytic Hypha Talaromyces marneffei Culture: SDA w/o cycloheximide → Dimorphic: → Cycloheximide inhibit or kills Zygomycetes. ▪ BT: Yeast form (Sausage like shape) ▪ RT: Mold form (Paint brush like appearance/ Flask shaped phialides) Animal Reservoir: Bamboo Rat TB like symptoms: → Affects HIV positive patients in Southeast Asia ▪ Take note of Travel history from Southeast Asia → Chronic cough Figure 46. Zygomycetes Culture: IX. LABORATORY DIAGNOSIS OF FUNGAL DISEASES → Yellow pink colonies with diffusible red pigment A. MICROSCOPIC EXAMINATION MTAP – MYCOVIROLOGY 10 of 12  Microscopic Description Cycloheximide: Examination prevents growth of 1. Saline Mount To observe hyphal elements contaminating Rapid method for detection of fungi on hair, skin, fungi (saphrophytic nails fungi) Serves as clearing reagent to remove debris Chloramphenicol: 2. 10% KOH → Clearing time: 10-15 mins (depending on the anti-bacterial lab) Allows growth of ▪ Heat increases rate of clearing dermatophytes and to ▪ Use 40% DMSO separate saprophytes Also known as Aman Medium Dermatophyte and bacteria 3. Lactophenol Excellent mounting medium for most fungi Test Medium Cotton Blue Most widely used stain pH indicator: Phenol red Contains lactic acid, phenol, and cotton blue (stain) Dermatophytes: red A fluorescence stain color (+) → Exhibits bluish-white or apple-green For Trichophyton rubrum fluorescence depending on filter used and T. mentagrophytes; Stains chitin chlamydospore 4. Calcofluor production Cornmeal Agar White Stain with 1% For pigment production glucose Test results: Trichophyton rubrum (+) 1 min. T. mentagrophytes (-) 5. India Ink Demonstration of encapsulated yeast Cornmeal Agar Periodic Acid Schiff with Tween 80 Presumptive ID of For differentiation of fungi in tissue scrapings and Tryphan Candida → Stains fungi magenta blue 6. PAS Isolate and maintains BHI Blood yeast phase of systemic Agar fungi (incubate at 37C) 3 mins. Hucker’s Modification 7. Gram Stain Use of crystal violet and ammonium oxalate Fungi is always gram-positive Czapeck’s For Aspergillus 8. Giemsa Stain Agar Grocott Methenamine Silver → Best fungal stain for tissues ▪ Fungi is stained black 9. GMS Birdseed Agar For Cryptococcus 10. Acridine For tinea versicolor Cottonseed Orange For Blastomyces Papanicolaou’s stain Medium 12. Pap’s → Used when suspecting Candida albicans in cervicovaginal smear 13. Masson For demonstration of melanin pigment or pigmented Fontana fungi Table 7. Microscopic Examination B. CULTURE Culture Media Purpose Picture ID of M. auduoinii (no growth); negative result Saboraud General purpose culture Rice Medium M. auduoinii = (-) Dextrose Agar medium (pH 5.6) Other Microsporum spp. = (+) Commercially available SDA with cycloheximide Mycosel or and chloramphenicol Mycobiotic *The antibiotics SABHI Recovery of mentioned are (Saboraud Blastomyces and inhibitors* Brain Heart Histoplasma; Yeast Infusion) conversion MTAP – MYCOVIROLOGY 11 of 12  For differentiation of T. Urease Test rubrum and T. Medium mentagrophytes 1-5 = contains casein Trichophyton Agars 6-7 = contains ammonium nitrate Table 8. Culture Incubation Temperature: Room temperature Culture held for 30 days before reporting as negative. → Or may be held for 4 weeks Plates tend to dehydrate during prolonged incubation, prepare plated media at least 40 mL of agar medium. → Since incubation takes long, we must increase the amount of volume of culture media or plate media → Not the usual 20-25 mL for Bacteriology C. TESTS Tests Description Use of plasma/serum Cost effective test to presumptively identify C. 1. Germ Tube albicans Test Incubation for 3-4 hrs. Observe for elongated structure (3-4x the length of yeast) 2. L-DOPA Ferric Rapid ID of C. neoformans Citrate Test 3. Urease Test (+) C. neoformans, T. mentagrophytes 4. Hair Baiting (+) T. mentagrophytes Test Serologic test for systemic mycoses (dimorphic fungi) Principle: Microscopic Immunodiffusion using 5. Exoantigen commercial antisera Test Used to confirm identity of fungal isolates Serologic confirmation → Detection of fungal antigens 6. Complement Fixation Test 7. Antigen Beta 1,3 glucan for yeast Detection CAD test for C. neoformans 8. MALDI-TOF 9. PCR 10. CHO For definitive ID of yeasts Utilization Test Table 8. Biochemical tests. AST (Antifungal Susceptibility Testing) Methods Disk Diffusion → CLSI M44-A2 = for Candida Broth Dilution → CLSI M27-A3 = for yeast → CLSI M38-A2 = for filamentous fungi E-Test MTAP – MYCOVIROLOGY 12 of 12