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MYCOLOGY REVIEW Fungi vs.

Bacteria: Criteria Size Cellular differentiation Nuclear & cell structure Chemical Composition Cell Wall Cell Membrane Reproduction Spore Life Cycle Bacteria 3 microns Unicellular Prokaryotic Peptidoglycan (-) Sterols (except Mycoplasma) Asexual (Binary Fission) For Adverse conditions Thermoresistant Simple Fungi Yeast 3 microns at least (like bacteria) Unicellular Eukaryotic Chitin, Glucan & Mannans (+) Sterols Ergosterols Asexual (B.F.) and Sexual For Reproduction Not Thermoresistant Simple complex Mold 3 feet Multicellular Capsule: Some fungi have capsules. Cryptococcus neiformans the only pathogenic yeast which has a capsule

Morphology: Dimorphism the ability to exist as two morphologic forms Thermally dimorphic dimorphism is induced by change in temperature YEASTS/SPIRULES Body Temp. Animal Habitat Parasitic Facultative anaerobes Reproduce via Budding or Sporulation Yeast phase Exist at conditions: Temp O2 Nutrients MOLDS Room Temp. Natural Habitat Saprobic Aerobes Sporulation Hyphal/Mycelial phase Temp O2 Nutrients

Thermally dimorphic fungi in our mycology module: Sporothrix schenckii Histolasma capsulatum Blastomyces dermatidis Coccidioides immitis Paracoccidioides braziliensis Penicillium marneffei

Hyphae of mold can be classified: 1. Septate/Non-septate a. Septate with crosswalls; unicellular per septation; can still reproduce when it breaks b. Non-septate coenocytic/aseptate; continuous; multicellular; cannot survive when it breaks 2. Reproductive/Vegetative a. Reproductive aerial; sprout from vegetative hyphae vertically; produce sexual spores b. Vegetative - anchors fungus to substrate; absorbs nutrients and water 3. Hyaline/Dematiaceous a. Hyaline non-pigmented b. Dematiaceous pigmented (black-brown) Tissue forms: what we see in histopathologic examination (so, kapag may microscope sa prax, ito lang ang madalas na nasa pointer) YEAST CELLS Organism Disease Type of Mycoses Intracellular Histoplasmosis capsulatum Histoplasmosis Endemic Respiratory Broad-based Blastomyces dermatitidis Blastomycosis Endemic Respiratory Multiple buds (like a mariners Paracoccidioides braziliensis Paracoccidioidomycosis Endemic Respiratory wheel) Encapsulated Cryptococcus neoformans Cryptococcosis Opportunistic Cryptococcus gatti With Transverse septum Penicillium marneffei Penicilliosis marneffei Endemic Respiratory Cigar-shaped Sporothrix schenckii Sporotrichosis Subcutaneous HYPHAE Coenocytic Zygomycetes Zygomycosis Opportunistic Dichotomously branched Aspergillus spp. Aspergillosis Opportunistic A.fumigatus (most common) Hyaline, septate Dermatophytes Dermatophytosis Cutaneous Demetiaceous, septate Several dematiaceous fungi Phaeohyphomycosis Subcutaneous Phaeoanneliomyces wernekii Tinea nigra Superficial YEAST CELLS &PSEUDOHYPHAE Candida spp. Candidiasis Opportunistic C. albicans (most common) GRANULES Several dematiaceous and nonMycetoma Subcutaneous dematiaceous fungi SPHERULES Coccidiodes immitis Coccidioidomycosis Endemic Respiratory FISSION/SCLEROTIC BODIES Several dematiaceous fungi Chromomycosis Subcutaneous The next part is a compilation of all the diseases we tackled in our mycology module. Dr.Padla said that our prax will include the laboratory diagnosis-part of our lectures.

I. SUPERFICIAL MYCOSES limited to the stratum corneum, the dead layer of the skin affect only nonliving tissues also called tineas (larva, worm) infections are usually cosmetic problems, easily diagnosed and treated Ptyriasis versicolor Etiology: caused by Malassezia furfur, Tissue form: short hyphae and clusters of yeast-like cells Infectious form: short hyphae and clusters of yeast-like cells Laboratory Diagnosis: o KOH examination: skin scrapings o Spaghetti and meatball appearance (short hyphae and clusters of yeast-like cells) o o Woods lamp: lesions fluoresce golden yellow Cannot be cultured unless olive oil and fatty acids are incorporated

Tinea Nigra Etiology: caused by Phaeoanneliomyces wernekii Tissue form: dematiacious hyphae Infectious form: dematiacious hyphae Piedra Black Piedra Etiology: Piedra hortae Hair shaft: perfect (teleomorphic) state spindle-shaped ascospores w/in asci develop Culture: asexual (anamorphic) state slow growing brown to reddish hyphae with many chlamydoconidium-like cells KOH: (crushed nodules) - asci containing fusiform ascospores with whiplike extensions White piedra Trichosporon beigelii yeast-like, hyphae that fragmnet into arthroconidia Laboratory Diagnosis: direct microscopic examn: KOH treated skin scrapings: pigmented budding cells, budding fragments, blanching hyphae

Laboratory Diagnosis

Direct KOH: septate hyphae that fragment into arthroconidia wc rapidly round up Blastoconidia may be present along hyphae Confirmed by culture

II. CUTANEOUS MYCOSES a.k.a. Dermatophytoses; normally called tinea and more commonly ringworms result of the host reaction to the enzymes released by the fungus during its digestive process involve infections that extent into the (epidermis/outer keratinized layer SKIN, HAIR, and NAILS Living tissue not invaded; systemic spread is rare Most common human mycoses CAUSE: Dermatophytes o keratinophilic fungi; able to utilize keratin as nitrogen source o Dermatophytes classified according to: Ecological niche or habitat: 1. Anthropophilic exclusively in association with humans 2. 3. Zoophilic found in association with domesticated and wild animals and birds Geophilic species frequently isolated from the soil

3 Genera: These genera, in the anamorphic (asexual) state, are classified according to sporulation patterns, morphologic features and nutritional requirements: 1. 2. 3. Trichophyton infects skin, hair and nails (tip: three=Tri) Microsporum infects skin and hair (tip: MSH) Epidermophyton (E. flocossum) infect skin and nail (tip: ESN)

Trichophyton 1. T. rubrum

Microsporum 1. M. cruisis

Epidermophyton 1. E. floccosum

2. T. mentagrophytes 3. T. verrucosum 4. T. tonsurans 5. T. suodanese 6. T. violaceum 7. T. schoenleinii 8. T. concentricum

2. M. gypseum 3. M. audoinii 4. M. ferrugineum 5. M. canis

GENERAL CHARACTERISTIC OF THREE GENERA: GENUS MACROCONIDIA Trichophyton Usually rare, smooth, pencilshaped, thin-walled, normally contain 3-8 cells Microsporum Numerous, rough, spindleshaped, echinulate, thickwalled, singly, usually contains 3-7 cells Epidermophyton Numerous, smooth-walled, club-shaped, formed singly, in pairs or clusters, contain 2-4 cells in groups TRICHOPHYTONS SPECIES T. rubrum T. mentagrophytes T. verrucosum T. tonsurans T. suodanese T. violaceum T. schoenleinii T. concentricum MICROSPORUMS SPECIES M. cruisis M. gypseum M. audoinii M. ferrugineum M. canis MACROCONIDIA Variable number Not always present Absent Rare and irregular form Absent Rare/Absent Absent Absent/Rare

MICROCONIDIA Few to numerous, round or oval, borne singly or in grapelike clusters Few club-shaped, borne singly, usually present

Absent

MICROCONIDIA Oval shape, periform, singly along hyphae Numerous, borne in clusters Absent Numerous, tear/club shape Tear shape along hyphae Rare/Absent Rare Absent/Rare; balloon shape

MACROCONIDIA Numerous, long, rough knob like ends, spiky, with many cells Numerous, symmetric, with round ends, smooth walled Distorted if present Rare, resemble those of M. canis Smooth edge, round ends, knob-like

MICROCONIDIA

EPIDERMOPHYTON SPECIES E. floccosum

MACROCONIDIA Smooth, club-shaped with rounded ends, numerous

MICROCONIDIA Absent

Trichophyton Infects: skin, hair & nails Transmission: Human to human Animal to human

Microsporum Infects: skin & hair Transmission: Human to human Animal to human Soil to human

Epidermophyton Infects: skin & nails Transmission: Human to human

TISSUE FORMS AND INFECTIOUS PARTICLES: o hyaline septate hyphae SELECTIVE MEDIUM USED: o Mycobiotic Agar containing Cyclohexamide (inhibits saprophytic fungi growth) Chloramphenicol and Gentamycin (inhibits bacterial growth to prevent contamination)

LABORATORY DIAGNOSIS: o KOH exam of skin scrapings, hair stubs/roots, nail clippings shows hyaline, septate hyphae o Woods Lamp Examination Fluorescence in cases of tinea capitis cause by Tinea schoenleinii, Microsporum canis and Microsporum audouinii

Culture on Mycobiotic Agar Cultural and morphological; characteristics used in species identification CONFIRMATORY TESTS: Hair Penetration Test distinguishes Tinea mentagrophyte from Tinea rubrum Urease Test - distinguishes Tinea mentagrophyte from Tinea rubrum NOTE: In annular lesions with central clearing and advancing border sample is obtained from the edge to yield a more positive result and prevent false negative Dermatophytes have no blastoconidia because they are not a budding yeast They are seldom cultured

7 DIFFERENT TYPES OF DERMATOPHYTOSES: TINEA CAPITIS CAUSES: Microsporum species and Trichophyton species Types of Tinea capitis with different causative agents: o Microsporum audouinii infections grey patches covered with scales develop with broken stumps of the infected hair hair stumps are surrounded by spores hence ECTOTHRIX type of hair infection o Kerion o Greater inflammatory reaction caused by Microsporum canis Caused by spores of animal origin A severe inflammatory type characterized by boggy, tumid, suppurating mass studded with pustules and broken hair. When the condition resolved, there may be areas of permanent hair loss.

black dot tinea capitis produced by Tinea violaceum and Tinea tonsurans hairs are invaded within the shaft hence ENDOTHRIX type of hair infection it breaks off at the surface of the hair shaft leaving stumps that look like black dots in the follicles NOTE: An endothrix infection will always indicate that the pathogen is anthrophilic in origin Favus Caused by Tinea schoenleinii Affects mainly the scalp and is characterized by scutula (cup-shaped disc around the hair follicle with a peculiar moussy odor) and cicatricial alopecia ENDOTHRIX

LABORATORY DIAGNOSIS: o Woods light suspected hair infection should always be examined under Woods light (UV >365 nm) as lesion of M. canis and M. auduoinii show a diagnostic brilliant yellow-green fluorescence. Those due to T. schoenleinii may cause a grey-green fluorescence. o Direct microscopic examination o Culture

TINEA BARBAE CAUSES: Microsporum species and Trichophyton species acquired from animals LABORATORY DIAGNOSIS: o Direct microscopic examination o Culture TREATMENT: o Systemic griseofulvin o Clipping or shaving the beard is recommended

TINEA CORPORIS CAUSES: Microsporum species, Trichophyton species and Epidermophyton floccosum o Microsporum species are the predominant cause of tinea corporis in children

TINEA IMBRICATA CAUSE: Trichophyton concentricum LABORATORY DIAGNOSIS: o Direct microscopic examination of scrapings in 10%-20% KOH o Culture

TINEA CRURIS

CAUSES: T. mentagrophyte or T. rubrum, E. floccosum LABORATORY DIAGNOSIS: o Direct microscopic examination o Culture

TINEA PEDIS CAUSES: T. mentagrophyte or T. rubrum, E. floccosum o T. mentagrophyte commonly produces an inflammatory type of tinea pedis whereas T. rubrum produces a chronic type LABORATORY DIAGNOSIS: o Direct microscopic examination o Culture

TINEA MANUUM CAUSES: T. mentagrophyte or T. rubrum, E. floccosum LABORATORY DIAGNOSIS: o Direct microscopic examination o Culture

TINEA UNGIUM

CAUSES: Trichophyton species and E. floccosum LABORATORY DIAGNOSIS: o Direct microscopic examination of the nail o Culture TREATMENT: o All nail infections are extremely resistant to treatment o Systemic griseofulvin after identification of the agent

Dermatophytoses Tinea capitis Tinea barbae Tinea corporis >Tinea imbricata Tinea cruris Tinea pedis Tinea manuum Tinea ungium

Location Scalp hair Beard hair Non-hairy smooth skin Groin Interdigital spaces of feet Hand Nail

III. Subcutaneous Mycoses It is characterized by deep ulcerating lesions usually developing at the site of the trauma where the fungus is implanted. It involves the deeper layer of the dermis, subcutaneous tissues and bone systemic spread. Mimics some bacterial infections, with the exception of sporotrichosis Causative organisms are ubiquitous in nature, cultural isolation wouldnt be enough. Tissue invasion must be demonstrated by KOH and tissue preparations. Sporotrichosis A. Etiology Sporothrix schenckii Yeast forms inside infected tissues At 250, the fungus is a mold B. Laboratory Diagnosis Clinical materials use in laboratory examination include pus from abscesses, biopsy materials of subcutaneous or organ lesions or sputum in cases of pulmonary infection KOH mount is not diagnostically useful since organisms are sparse Round to oval to cigar-shaped yeast cells can be demonstrated better with GMS stain rather that tissue smears Asteroid bodies represents the host immune response (basophilic yeast surrounded by eosinophilic rays) Culture is necessary to confirm diagnosis; colonies are initially white and membranous becoming black and leathery by age

Mycetoma (Madura Foot)

A.

It is characterized by swelling, abscess formation and draining Etiology Eumycetoma (true fungi) Exophiala jeanselmei, Pseudallescheria boydii, Madurella mycetomatis and Madurella grisea Actinomycetoma(bacteria) Actinomadura madurae, Actinomadura pelletieri, Streptomycis somaliensis, Nocardia braziliensis, and Nocardia asteroids

B.

Epidemiology Saprophytes from the soil and is acquired by direct inoculation Laboratory diagnosis So that you can distinguish between a bacteria and a fungal infection KOH examination (Direct microscopic examination) presents granules that shows the etiologic agents; color is species specific Culture is also use to identify the species

C.

Chromomycosis A. Dark brown nodules at site of infection Etiology Dematiaceous (black) fungi Fonsecaea pedrosoi, Foncecaeae compactum, Phialophora verrucosa and Cladosporum carrionii Laboratory Diagnosis KOH in skin scrapings or histopathological infection; crushed black dots appear as thick walleddark brown, 5-12 mm in diameter, round and sclerotic bodies with single or multiple cells formed by crosswalls Brown, blanched distorted hyphae may also be present Culture techniques are also done for taxonomic identification; slow growing, black colonies

B.

Phaeohyphomycosis Localized subcutaneous infection forming granulomas, cysts and abscesses. A. Etiology Several dematiaceous fungi Laboratory Diagnosis Histopathological examination; Organisms are seen as pigmented, septate hyphal fragments Culture for taxonomic identification; dark colored colonies develops

B.

IV. Opportunistic Mycoses Candidiasis A. Etiology Candida albicans, C. tropicalis, C. krusei, C. parapsilosis and C. glabrata Clinical disease 1. Superficial Candidiasis The most common diseases involves the skin, nails and mucosal surface of the vagina, mouth esophagus and the bronchial tree; Skin and nail infections usually mimics dermatophytoses C. Cutaneous mycoses as seen in diabetics Candidiasis of the nail usually causing hardening and thickening of the nail Vulvovaginal candidiasis is also seen in diabetics and can be transferred sexually Thrush (oral) usually presents white, creamy patches on the mucus membranes and corners of the mouth

B.

Laboratory Diagnosis KOH examination of the infected nail, skin, mucosal scrapings; in most instances, blastoconidia are seen singly or in clusters at the constrictions of their pseudohyphae Histopathological examination of C. albicans are demonstrated as budding yeast of pseudohyphae or both Culture; in isolation media, C albicans grows as opaque cream colored colonies with pasty consistency while in microscopic examination, pseudohyphae with clusters of round blastoconidia are seen Germ tubes at 370 in serum identifies C. albicans

SYSTEMIC MYCOSES Most serious of all mycoses Originate primarily in the LUNGS Major types: Endemic Respiratory Mycoses A. Histoplasmosis B. Blastomycosis C. Coccidioidomycosis D. Paracoccidioidomycosis E. Penicilliosis marneffei Opportunistic Mycoses A. Candidiasis B. Aspergillosis C. Cryptococcosis D. Zygomycosis Endemic Respiratory Mycoses Portal of entry: Respiratory Tract Mode of Transmission: Inhalation of infectious conidia Infectious form: conidia (asexual form) Agents: have restricted geographic distribution (area where the fungi thrive) exhibit thermal dimorphisms with 2 forms: Yeast seen at 37 degrees Celsius Mold seen at room temperature, natural habitat primary site of infection = lungs Microconidia infectious form; the small the size, the better. MYCOSES OCCUR AS CAUSED BY INFECTIOUS PARTICLE DIAGNOSIS (KOH/ histopath exam) TISSUE FORM Small round to oval intracellular yeast cells often with histiocytes finding yeast cell in tissues does not diagnose Histoplasmosis, others are histoplasma CULTURE AT ROOM TEMPERATURE (Saborauds Agar) 27C fine septate hyphae (hyaline) tuberculate macronidia (means spikes around the structure) and small micronidia (both seen in Lactophenol Cotton Blue Mount) which is diagnostic of H. capsulatum (infectious particle) non-capsulated At 37 Celsius (BHI)

Histoplasmosis / Darlings/ Cavers/ Spelunkers disease

Pulmonary (PTB like) 60% asymptomatic and 40% primary pulmonary histoplasmosis Disseminated infection with (RES) Reticuloendotheli al System involvement and muco-cutaneous infection

Histoplasm a capsulatu m Ascomycet e -Thermally dimorphic facultative intracellula r -grows in soil contaminat e by bat or bird excreta Blastomyc es dermatitidi s Ascomycet e -Thermally dimorphic - soil saprophyte

Microconidia (can easily go through the lungs and settle in the alveoli)

creamy, pasty yeast colonies small oval yeast cells (cluster self yeast cells) non-infectious, patient cant transmit Importance of observation at Body Temp: Demonstrate dimorphism: Hyphae (from culture) yeast cell (seen microscopically, budding not intracellularly) Double-walled yeast cells with single broad-based bud seen also in KOH/histopath

Blastomycosis aka North American Blastomycosis

PTB like but with lesser calcifications Cutaneous result of infection of the skin or sometimes a manifestation of disseminated form; microabscesses; crusty, elevated lesions Disseminated with lower percentage

conidia / microconidia (No macroconidia )

Round, double-walled yeast often with single bud connected by a broad base (bowling pin appearance)

Pyriform conidia borne singly on septate hyphae (on Lactophenol Cotton Blue mount)

MYCOSES

OCCUR AS

CAUSED BY

INFECTIOUS PARTICLE

Coccidioido mycoses/ San Joaquin Valley fever (found in California, an endemic area)

PTB like with egg shell cavity on x-ray Disseminated Meningitis: common cause of death

Coccidiode s immitis Deuteromy cete (asexual only) -Thermally dimorphic -soil saprophyte

Arthoconidia - rectangular shaped, thallic conidiogeny (conidia formed inside hyphae) -an asexual spore which can be disseminated by fragmentation of septate hyphae and blastic yeast cells Hyphal fragments produce arthroconidia Conidia

DIAGNOSIS (KOH/ histopath exam) TISSUE FORM Spherules containing endospores (not infectious)

CULTURE AT ROOM TEMPERATURE (Saborauds Agar) Hyphae bearing cylindrical, barrelshaped arthrocondia separated by dysjunctor cells (empty degenerate cells)

At 37 Celsius (BHI)

White floccose colonies No yeast-like colonies on routine mycology agar

Paracoccidi oidomycosi s South American Blastomyco sis/ LutzSplendorAleidas disease a new disease

PTB-like Disseminated (oral and nasal mucosa): hepatosplenomegaly is prominent chronic granulamatous lesion or mulberry lesion sometimes with cervical adenitis skin of face-most common site of infection dimorphic and pigmented Pulmonary disseminated (RES)

Paracoccid ioides braziliensis Deuteromy cete (asexual only) -Thermally dimorphic - soil saprophyte

Large, thinwalled yeast cells, with multiple narrow based buds

Septate hyphae with no typical pattern sporulation

Yeast cells with ships or mariner;s wheel appearance.

Penicilliosis marneffei produces pigment colonies

Penicillium marneffei -only Penicillium species that is thermally dimorphic soil saprophyte

Conidia

Small, ovoid yeast cells with transverse septum Yeast cell reproduction is by tranverse fission/ schizogony

restricted to SEA (Southeast Asia) Thailand (most common in Chang Mai areas) Endemic due to mode of transmission Opportunistic they affect immunocompromised px naturally occurring infections in bamboo rats new discovery

Conidiophore branch into metullae which support the phialides that bear chains of conidia Colonies produce diffusible red pigment distinct character of P. marneffei (other penicillium have yellow pigment) Penicillus paintbrush/fingerlik e appearance

Round to oval cells with crosswalls.

Opportunistic Mycoses MYCOSES OCCUR AS Candidiasis

INFECTIOUS FORM Yeast cells, pseudohypha e

CAUSED BY Candida spp. C. albicans (most common)

DIAGNOSIS TISSUE FORM Gram Stain/KOH/ histopath exam: small budding yeast cells and pseudohyphae Germ Tube test: Specific for C. albicans

CULTURE AT ROOM TEMPERATURE Pseudohyphae with clusters of round blastoconidia Chlamydosores, yeast cells and pseudohyphae roduced on chlasmydospore/cornme al agar

MYCOSES Aspergillosis

OCCUR AS Allergic form most important allergic bronchopulmonary aspergilliosis Asthmatic attack or hay fever Aspergilloma fungus ball formation of intertwining hyphal elements in previous TB cavities. Disseminated invasive aspergillosis Pulmonary (cryptococcoma) -a solitary pulmonary nodule in the mediastinum mimicking a carcinoma. Disseminated -have a tendency for CNS infection causing meningitis.

INFECTIOUS FORM Conidia

CAUSED BY Aspergillus spp. A.fumigatus (most common) -soil saprophyte -Monomorphic mold; has a different sporing head than penicillium

DIAGNOSIS TISSUE FORM KOH/histopath exam Wide, septated, dichotomously branced (acute angle) hyphae - distinct characteristics. Conidiophore supports swollen vesicle covered by phialides bearing radial chains of conidia

CULTURE AT ROOM TEMPERATURE Sporing head with rows of phialides bearing conidiospores Conidiospores arise from foot cell

Cryptococcos is also called BusseBuschkes disease or torulosis

Noncapsulated yeast cells which is smaller because it loses its capsule. Monomorphi c yeast.

1. Cryptococcus neoformans (Filobasidiella neoformans) -in pigeon excreta -affect the immunocompromised -Clinical disease: Meningoencephatlitis 2 biotypes: a. Cryptococcus neoformans var. neoformans. Serotype D b. Cryptococcus neoformans var. grubii Serotype A 2.Cryptococcus gatti (Filobasidiella bacillispora) -soil under Eucalyptus tree Serotype: B,C -affect the immunocompetent -Clinical disease: pneumonia Filamentous fungi belonging to Phylum Zygomycota asexual sporangiospores Class Zygomycetes (asexual and sexual form) Asexual conidia Sexual zygospore Genera: 1.Rhizopus 2.Mucor 3. Absidia 1st 2 genera more common

India ink/histopath exam India ink-negative stain (stains background) Yeast cells with wide capsule.

Creamy, mucoid yeast colonies. Encapsulated yeast cells Bird Seed agar (Niger) brown to black pigment colonies due to melanin protein due to phenyloxidase produced by the organism. Chemical test: Urease Test (+) pink color Caffeic Acid Test: (+) phenoloxidase production L-canavarine-glycinebromthymol blue (CBG medium) C. neoformans (yellow) C. gatti (blue) Serologic test: antigen detection - latex agglutination & ELISA Morphologic features: 1. Rhizopus spp. sporangia around sporangiosphores unbranched, nodal columellae hemispherical no collarette remains when sporangia ruptures rhizoids well develop. 2. Mucor spp. sporangia round sporangiospores branched, internodal no rhizoids columellae round to oval 3. Absidia spp. sporangia pyriform apophysis with colarettes remain when sporangia ruptures rhizoids rudimentary.

Zygomycosis / Mucormycosi s

- Rhinocerebral - Pulmonary - Cutaneous - GIT - Disseminated

Sporangiospo re born inside the sporangium Conidia not contained in a sporangium; produced by septate.

KOH/histopath exam Aseptate, wide hyphae branching at right angle. Wide, ribbon like.