Mycology and Virology Lecture PDF

MYCOLOGY AND VIROLOGY O3B OVERVIEW OF FUNGAL IDENTIFACCTION REPRODUCTIVE FORMS METHODS AND STRATEGIES...

MYCOLOGY AND VIROLOGY O3B OVERVIEW OF FUNGAL IDENTIFACCTION REPRODUCTIVE FORMS METHODS AND STRATEGIES Teleomorph MYCOLOGY Sexual form Anamorph The study of fungi Asexual form Taxonomy Synanomorph Environmental impact Different asexual forms in the same fungus. Genetic and biochemical properties CLINICAL CLASSIFICATION OF THE FUNGI PREVENTED OTHER LABORATORIES FROM OFFERING MYCOLOGY SERVICES SUPERFICIAL (CUTANEOUS) MYCOSES Economic constraints Infections involve the hair, skin, or nails without direct Diagnostic clinical laboratory is performed by invasion of deep tissue reference laboratory Infect keratinized tissue Lack of experience Dermatophytes Shortage of trained individuals ▪ Ringworm Training and continuing education ▪ Athlete’s foot Tinea infection GENERAL FEATURES SUBCUTANEOUS MYCOSES Most fungi are saprophytic (they live on dead organic matter. Infections are confined to subcutaneous tissue without Fungi are yeast (moist) and/or molds (filamentous) dissemination Organisms that display both yeast and mold forms are Sporothrix schenckii dimorphic. They are thermally dimorphic when dependent on SYSTEMIC MYCOSES temperature Fungi that have more than one form or stage are Infections often involve the lungs but can disseminate polymorphic to any organ Paracoccidioidomycosis. TAXONOMY OF FUNGI OPPORTUNISTIC MYCOSES Chitin Cell wall Infections occur in patients who are Ergosterol immunocompromised Cell membrane Infections can become systemic Reproduction Candidiasis Sexual and asexual Fungal infection caused by candida Lack chlorophyll Lack of susceptibility to antibacterial antibiotics Saprophytic nature PHYLA Zygomycota Asexual reproduction by sporangiospores Sexual reproduction by zygospores Ascomycota Asexual reproduction by conidia Sexual reproduction by ascospores Basidiomycota Sexual reproduction via basidiospores (on basidia) Deuteromycota Asexual reproduction by conidia JEMALENE BARION 1  WORKING SCHEMA Septate hyphae VIRULENCE FACTORS OF MEDICALLY IMPORTANT FUNGI Coenocytic hyphae PATHOGENESIS AND SPECTRUM OF DISEASE CLINICAL SPECIMENS Types of pathogens Respiratory secretions Primary (immunocompetent host) Antibiotic and antifungal should be in culture Opportunistic (immunocompromised host) media Common virulence factors Cerebrospinal fluid Size of organism Should be filtered before culturing Ability to grow at 37⁰C at neutral pH Blood Conversion from mycelial form to yeast or spherule form Eye Toxin production Corneal scrapings or vitreous humor Hair, skin, and nail scrapings Mycosel agar should be used Vaginal Selective and inhibitory plates should be used Urine JEMALENE BARION 2  Tissue, bone marrow, and sterile body fluids All tissues should be minced before culturing CULTURING RECOMMENDED MEDIA With and without cycloheximide With and without an antibacterial agent Inhibitory agar (Chloramphenicol) Brain-Heart Infusion (BHI) containing antibiotics Esculin-based media with chloramphenicol and gentamicin AGAR PLATES Are preferred over screw-capped agar Better aeration Larger surface area Greater ease of handling DIRECT DETECTION METHODS INCUBATION Potassium hydroxide (KOH) preparation Room temperature (30⁰C) Calcofluor white stain Minimum of 21 to 30 days Superior to KOH Humidity range of 40% to 50% India ink stain Examined at least three times a week Lactophenol cotton blue mounts Grocott’s methenamine silver (GMS) stain Periodic acid-Schiff (PAS) stain JEMALENE BARION 3  FUNGAL STAINS PARACOCCIDIOIDES BRASILIENSIS Mariner’s wheel SPHERULES HISTOPLASMA CAPSULATUM Small Oval to round budding cells COCCIDIODES IMMITIS Often found clustered in histiocytes Spherules vary in size Some may contain endospores, other may be empty SPOROTHRIX SCHENCKII Small Oval to round to cigar shaped RHINOSPORIDIUM SEEBERI Single or multiple buds present Large, thick-walled sporangia containing sporangiospores are present CRYPTOCOCCUS NEOFORMANS Cells exhibit great variation in size Usually spherical but may be football shaped Buds single or multiple and “pinched off” Capsule may or may not be evident YEAST AND PSEUDOPHYPHAE OR HYPHAE MALASSEZIA FURFUR (IN FUNGEMIA) Small Bottle-shaped cells Buds separated from parent cell by a septum Emerge from a small collar CANDIDA SPP. BLASTOMYCES DERMATITIDIS Single budding Pseudohyphae Broad base bud Are constricted at the ends and remain attached like links of sausage Hyphae Are septate JEMALENE BARION 4  HYALINE MOLDS, ZYGOMYCETES, SPECIMEN COLLECTION, TRANSPORT, AND DERMATOPHYTES, AND OPPORTUNISTIC AND PROCESSING SYSTEMIC MYCOSES Deep lesions or tissues and sterile sites should be MUCORALES (ZYGOMYCETES) collected rapidly and aseptically. Sufficient quantity is essential to improve the Rhizopus identification and recovery of the fungal isolate. Mucor Rhinocerebral forms of infection Actinomucor elegans Nasal discharge or scrapings, sinus aspirate, Cokeromyces recuryatus or a tissue specimen from a vascularized Lichtheimia tissue. Rhizomucor pusillus Respiratory samples Saksenaea spp. Sputum and bronchoalveolar lavage fluids Syneciphalastrum racemosum If Respiratory samples in negative results Transbronchial or percutaneous computed Cunninghamella spp. tomography – guided biopsy of pulmonary lesions GENERAL CHARACTERISTICS Collected for the microbiology laboratory and the histology laboratory. Preservatives used for histologic Large, ribbon-like hyphae processes, such as formalin, are inhibitory to fungal Occasional septa growth. Sporangia – sac-like fruiting structures Transported in sterile containers. Tissue (biopsy Sporangiospores – produce internally spherical, yellow specimens) should be moistened by adding a few or brown spores drops of sterile saline to the container. Specimens Sporangiophore – each sporangium is formed at the tip should be transported to the laboratory within 2 hours of a supporting structure of collection and processed immediately. Mucorales Stolons – septate hyphae that connect are extremely sensitive to environmental changes. sporangiophores Rhizoids – root-like structures that anchor organism to DIRECT DETECTION METHODS agar surface Stains EPIDEMIOLOGIC FACTORS A mucormycosis may be diagnosed rapidly by examining tissue specimens or exudate from Has worldwide distribution infected lesions in a calcofluor white and Is found on decaying organic matter, bread, or in the potassium hydroxide preparation. soil If the sample is too thick, a false negative Infection is acquired through inhalation, percutaneous result may occur because of insufficient routes, or the ingestion of spores. dissociation of tissues. It is recommended that the negative slide be SPECTRUM OF DISEASE maintained overnight and reviewed again the next day. Mucormycosis Branching, broad-diameter, predominantly Patients who are immunocompromised are at non-septate hyphae are observed. risk It is important that the laboratory notify the Uncontrolled diabetes clinician of these findings, because Mucorales Those with prolonged drug therapy grow rapidly, and vascular invasion occurs at Infection caused by Mucorales a rapid rate Marked propensity for vascular invasion and Antigen-Protein rapidly produce thrombosis and necrosis of Antigen-protein–based assays are not used tissue. for the diagnosis of mucormycosis. In Rhinocerebral Form addition, beta-D-glucan testing is not useful The nasal mucosa, palate, sinuses, orbit, for diagnosis. face, and brain are involved Nucleic Acid-Based Testing Massive necrosis with vascular invasion and Nucleic acid testing may be performed on infraction formalin-fixed, paraffin-embedded, fresh or Other infections frozen tissue samples. Perineural invasion Polymerase chain reaction (PCR) Lung infections amplification of the internal transcribed Gastrointestinal (GI) infections spacer, as well as seminested PCR of the Diagnosis 18S ribosomal ribonucleic acid Based on direct examination, nucleic acid- (RNA)/deoxyribonucleic acid (DNA) based testing, or recover pf fungus sequence, has been used to confirm identification in samples that have been identified as histopathology positive. JEMALENE BARION 5  CULTIVATION area (apophysis) at the junction of the sporangium and the sporangiophore. Growth media containing high concentrations of DERMATOPHYTOSES carbohydrates inhibit the production of asexual fruiting bodies that are required for the proper identification of Refers to the infection of the skin, nails or hair that are the Mucorales species. caused by fungi classified as dermatophytes. Potato Dextrose Media Tinea capitis – scalp 2% malt, and cherry decoction (acidic) agars Tinea pedis – foot be used for cultivation. Growth and Tinea corporis – trunk of the body development of the mycelium in the Tinea barbae – face and neck Mucorales occurs within 24 to 48 hours. Tinea unguium – fingernails and toenails Subcultures should be incubated at 27°C to Tinea cruris – groin area 30°C Ringworms Colonies characteristically produce a fluffy, Athlete’s foot white to gray or brown hyphal growth that Jock itch resembles cotton candy and that diffusely Dermatomycosis covers the surface of the agar within 24 to 96 hours. BACKGROUND The hyphae can grow very fast and may lift the lid of the agar plate (also known as a “lid Dermatologist described these diseases by the body lifter”). The hyphae appear to be coarse. The area entire culture dish or tube rapidly fills with Tinea capitis – scalp loose, grayish hyphae dotted with brown or Mycologist felt more comfortable referring to the black sporangia scientific name of etiologic agent, paying little attention to the clinical picture and site of infection. APPROACH TO IDENTIFICATION To compromise over these different forms of terminology is to name the disease by the body area Mucorales are characterized by the production of and to designate the specific etiologic agent branched, non-septate, wide mycelia (10 to 20 μm). Tinea barbae due to Microsporum gypseum Sexual reproduction The formation of a thick walled zygospore; EPIDEMIOLOGY however, morphology of the zygospores is not generally useful for routine identification Contained in 3 major genera unless the species is homozygous. Microsporum Asexual reproduction occurs Epidermophyton The formation of sporangiospores in saclike Trichophyton structures termed sporangiophores. Organisms occur worldwide, mainly on soil and on Columella (singular) animals, including man. The central axis of the sporangia (multispored An outstanding feature of these organisms that is they structure) are all keratinophilic. Apophysis Hair baiting – isolating dermatophyte fungi from soil. a swelling of the sporangiophore below the columellae (plural). Some species also CLINICAL FORMS produce rhizoids that hold the sporangiophore within the soil or growth substrate. The Tissues they invade rhizoids are then connected to a branching root, or stolon Rhizopus spp. HAIR mostly unbranched sporangiophores with rhizoids that appear opposite the point where Ectothrix Infection – grow only on the outside of the hair the stolon arises, at the base of the shaft sporangiophore. Endothrix Infection – invade only on the inside of the Mucor spp. hair characterized by sporangiophores that are Wood’s or black light singularly produced or branched and have a UV light with max. emission at round sporangium at the tip filled with 3,660angstroms sporangiospores. They do not generally have Preliminary clue regarding the etiologic agent; rhizoids or stolons, which distinguishes this infecting fungi fluoresce when the hair is genus from the other genera of the examined Mucorales. This type of examination should be done Lichtheimia spp. routinely in suspected cases of tinea capitis characterized by the presence of rhizoids that If fluorescing hairs are observed, they should originate between branched or whorled be removed for direct examination sporangiophores along the stolons between preparations and culturing the rhizoids. The sporangia of Lichtheimia spp. are pyriform and have a funnel-shaped JEMALENE BARION 6  NAILS Causes sporadic outbreak hair and skin infections Infected hair looks chalky and dull. worldwide Nail is raised, with debris and keratinized cell Zoophilic underneath it. Erythema is common in the lesion caused by M. canis Nail infections are most often caused by organism Colony: white and fluffy, sometimes rich canary yellow belong to the genus Trichophyton, some investigators color on the bottom of the colony shows through the believe that Candida albicans, causes a relatively high white, fluffy mycelium on the top percentage of the nail infections that are thought to be Microscopically: spindle-shape macroconidia (8-12 caused by dermatophytes. septa) Regardless of the etiologic agent, one only observes hyaline, septate hyphae in nail tissue. MICROSPORUM FERRUGINEUM It is impossible to know the specific etiologic agent without first obtaining pure culture Common cause of tinea capitis Nail infection caused by dermatophytes evolve very Anthropophilic and causes infected hair to fluoresce slowly; several months to years may pass as the Ectothrix infection in hair disease develops Colony (2 colonial forms) Very slow-growing, wrinkled, flat, leathery and SKIN white Rapid growing colony, flat, leathery and white Small isolated lesions may appear on any part of the Microscopic: few structures of any diagnostic body value. Occasionally septate hyphae appear to Regardless of the etiologic agent, one observes have exceptionally thick septa (bamboo hyaline, septate hyphae ONLY in skin. hyphae) IDENTIFICATION MICROSPORUM GYPSEUM Before therapy is initiated, one need only be certain Geophilic (resides on soil) that a fungus is the source of a given patient’s problem Isolated from hair baiting It is important to identify a specific etiologic agent Sporadically, it has been reported to cause hair and because in some instances the therapeutic regimen is skin infections (tinea barbae) affected and information of this type is of value Ectothrix infections epidemiologically. Infected hairs do not fluoresce Colony IDENTIFICATION PARAMETERS Flat, light brown (cinnamon color) and powdery appearance Clinical picture Microscopic Wood’s light Thin-walled, spindle shaped pores (4-6 septa) Direct examination Infected hair (endothrix/ectothrix) EPIDERMOPHYTON Skin or nail (look for hyaline, septate hyphae) Culture This genus contains only a single species Mycosel or Mycobiotic medium (1-3 weeks This fungus attacks skin and nails incubation) EPIDERMOPHYTON FLOCCOSUM GENERA OF DERMATOPHYTES Infects only man and anthrophilic Microsporum, Epidermophyton, Trichophyton Colony All of these organisms should be culture on Sabouraud Yellow to green (olive drab) color and is quite Dextrose Agar (SDA) with antibiotics (Mycosel or wrinkled or folded Mycobiotic) and cultured for several weeks at room Microscopic temperature Club-shaped spores None of these organisms are dimorphic Spores usually formed single or in clumps Very few biochemical procedures are available Spores contains 2-5 septa and the end of it is Identification is based on the gross and microscopic quite blunt or rounded morphology of the colony Spores on dermatophytes fungi may be called conidia. TRICHOPHYTON MICROSPORUM May attack skin, hair, and nails Infected hair does not fluoresce Species of the genus Microsporum infect hair and skin Infect man and other animals only. Difficult to cure Hair – usually ectothrix infection. There organisms rarely produce macroconidia, making identification difficult MICROSPORUM CANIS JEMALENE BARION 7  Microscopic Underside of the colonies is rich red to brown May form macroconidia and produced hyphae color in spiral forms and other strange structures Microscopic Forms abundance of microconidia which are TRICHOPHYTON CONCENTRICUM usually club-shaped and borne directly on the hyphae Anthrophilic Infects the skins and induces the formation of TRICHOPHYTON VERRUCOSUM characteristics circular skin lesions This type of clinical picture is called tinea imbricata Usually found in the cattle, thus very buffalo must also Colony be suspect Slow growing, velvety in appearance and Transmitted directly to man via contaminated fomites white to orange brown in color Human tinea corporis Microscopic; few distinguishing features Large ectothrix infection Some strains are stimulated by the addition of thymine Grows better at 35⁰C than room temperature TRICHOPHYTON MENTAGROPHYTES AIR CONTAMINANTS PENICILLIUM Worldwide cause of athlete’s foot. Also cause tinea capitis, tinea corporis, tinea barbae, Rate of growth and tinea cruris. Rapid, mature within 4 days Several variants of organism (anthrophilic, zoophilic) Colony Ectothrix infection Surface at first white then becoming powdery, Colonial forms: bluish green, with a white border Downy – culture is white, very fluffy (anthrophilic) ASPERGILLUS Granular – colony is very flat (zoophilic) Underside of the colonies is pale yellow Septate hyphae Difficult to differentiate with T. rubrum Conidiospore is Microscopic enlarge at the tip, Numerous round microconidia forming a swollen ▪ Grape-like clusters (“en grappe”) vesicle Some strains produced so called “spiral hyphae” Rate of growth Differentiation test for T. rubrum Rapid, ▪ Hair penetration test mature within 3 days ▪ T. rubrum – grow only on the outside Colony of the hair Surface at first white, then any shade of ▪ T. mentagrophytes – burrow inside yellow, green brown or black depending on the hair; producing V-shaped shaft the species TRICHOPHYTON RUBRUM ALTERNARIA Caused long-established foot and toenail infections Hyaline, septate and dark May also cause tinea corporis, skin lesions with red Conidiospores are septate of variable length margin Hand grenade shaped Rare occasion, cause tinea capitis, infected hair does Rate of growth not fluoresce (endothrix); anthrophilic Rapid, mature within 5 days Cultures are quite variable, identification can be Colony frustrating Surface at first grayish white becoming Microscopically greenish black or brown with white border Very small microconidia which are described as being pyriform to peg shaped. CURVALARIA TRICHOPHYTON TONSURANS Hyaline, septate hyphae Conidia large usually contain 4 cells and may appear Anthrophilic and worldwide curve due to swelling of a central cell Endothrix infection which inside of the entire hair shaft Rate of growth seems to be filled with spores Rapid, mature within 5 days Hair infection causes the hair either to burst open or to Colony grow in a coil in the stratum corneum (“Black dot” tiena Dark olive green to brown or black capitis Reverse is black Refer to as “adult ringworm infection” Cultures Powdery and are yellow to reddish brown JEMALENE BARION 8  GEOTRICHUM With age, black area of conidiation appear. Reverse is black Coarse true hyphae that segment into rectangular arthroconidia varying in size and in roundness of their ACREMONIUM ends Hyphae break up into individual cells Extremely delicate, septate hyphae; erect, Rate of growth unbranched, tapering phialides form directly on the Rapid, mature in 4 days fine, narrow hyphae Colony Rate of growth White, moist, yeast-like, and easily picked up Rapid, mature within 5 days Short white, cottony, aerial, mycelium Colony At first compact, folded and feltlike, the SCOPULARIOPSIS becomes overgrown with loose, white cottony hyphae Septate, hyphae Conidia are larger, often thick walled, round to lemon OPPORTUNISTIC FUNGUS DISEASE shaped Long chain of spores 2 categories Rate of growth Pathogens Rapid, mature within 5 days Laboratory contaminants (saprophytes) Colony Those organisms which under normal conditions do Surface at first white, usually powdery light not cause disease. brown with a light tan periphery Predisposing factors Therapeutic regimen RHIZOPUS Surgery Coenocytic hyphae CANDIDIASIS Formation of woot like structure called Acute or chronic, superficial or disseminated mycosis rhizoids caused by species of the genus Candida Dark spherical Most versatile of all fungal pathogens structure known as Can mimic anything from dermatophytes infection of sporangium the skin to tuberculosis Rate of growth Synonyms Rapid, mature Moniliasis within 4 days Mycotic vulvovaginitis Colony Thrush Quickly covers agar surface with dense Candidiasis growth that is cotton candy-like; at first white, Candida endocarditis then gray to yellowish brown. TMG SMEAR SYNCEPHALASTRUM Trichomonas Hyphae broad an almost non-septate Monilia Sporangium formation is different Gonorrhea Numerous small, almost clear projections Each of these individual structures is THRUSH sporangium and each contain 3-5 sporangiospores Disease of oral mucous membrane Rate of growth Formulation of white, creamy patches seen most often Rapid, mature within 3 days on the tongue Colony morphology Quickly fills petri dish with white, cotton candy NAIL INFECTIONS fluff, then turns dark gray to almost white. Reverse is white. Simulate ringworm infections Nails become hardened and thickened (but not brittle) NIGROSPORA assume brownish color Last for years “Black spore” Septate hyphae MYCOTIC VULVOVAGINITIS Rate of growth Rapid, mature within 4 days Rather common disease in diabetics, pregnant women Colony and those on birth control pills. Compact, wolly, at first white, then gray JEMALENE BARION 9  Lesions resemble a simple eczematoid dermatitis or DIRECT MICROSCOPIC EXAM may show vesicular pustules or form a gray white pseudo membrane Sputum Skin scrapings MACROSCOPIC AND MICROSCOPIC APPEARANCE Vaginal swabs OF CANDIDA SPP. Biopsy material from any type of organ; even blood Regardless of the material, it should be placed immediately on a microscope slide Place 1-2 drops of 10-20% KOH If material is solid tissue, tease it apart Add a coverslip, heat gently, press down coverslip Look for yeast cells and pseudo hyphae PSEUDOHYPAL STRUCTURES IN CANDIDA SYSTEMIC CANDIDIASIS Occurs in patients who carry more yeast in mouth, and GI system POTASSIUM HYDROXIDE (KOH) Predisposed with individual with On antibiotic or/and steroid therapy Pulmonary Candidiasis Immunosuppressed Few yeast cells, and form almost exclusively Recipients with organ transplantation pseudo hyphae Infancy – old age – pregnancy Normal floral (oral flora) ▪ On antibiotic therapy Form mostly yeasts cells Indisposed with trauma occluding lesions Many cases of AIDS are suspected by Immunosuppression, major event in AIDS observation of Oral Cavity patients Diabetes mellitus CULTURE Zinc and iron deficiencies SDA TISSUE PHASE AND HISTOPATHOLOGY 2-3 days as white, typical yeast colonies Regardless of the spp. most of the colonies All Candida spp. appear the same when growing in appear the same vivo. Monomorphic PAS Stain – best for visualizing Candida spp. in ▪ Cryptococcus neoformans histopathology slides Capsule All Candida spp. appear pink. Positive urea agar ▪ Candida spp. SUPERFICIAL FORMS Negative urea agar except Candida krusei Budding yeast cells and few pseudo hyphae MICROSCOPIC PREPARATION FROM CULTURE INTERNAL ORGANS Non-encapsulated budding yeast cells Few pseudo hyphae Very few yeast cells, reproducing mostly with pseudo First clue that Candida spp. has been culture hyphae CORNMEAL/CHLAMYDOSPORE AGAR Old standby identification method JEMALENE BARION 10  Only members of the genus Candida form yeast cells ASPERGILLOSIS and pseudohyphae. Results Granulomatous, necrotizing disease of the lungs which Yeast cell only – not Candida spp. often disseminates hematogenous to various organs Yeast cells and pseudohyphae – Candida Fatal spp. Fungus ball Yeast cells, pseudohyphae, and In this form of the disease, the fungus takes chlamydospores – Candida albicans up residence in an old lung cavity Result of old tuberculosis lesions RAPIS IDENTIFICATION ETIOLOGY Incubate unknown yeast in serum at 35-37⁰C After 1-2 hours, only Candida albicans (and many C. Aspergillus – largest fungal genera stelleroidea) forms germ tubes 20-30 Aspergillus spp. – human disease Aspergillus fumigatus Aspergillus flavus Aspergillus niger Epidemiology Worldwide Saprophytes in soil PREDISPOSING FACTORS Normal man can develop effective defense mechanism Individual who develops systemic aspergillosis have compromised immunologic responsiveness TISSUE FORM AND HISTOPATHOLOGY PAS recommended for visualizing the fungus Invade internal organs; characterized by the ff. Hyaline (non-dematiaceous), septate hyphae Dichotomously branched hyphae Under no condition, does not observe yeast THERAPY cells In rare instances, in alveoli, one may observe Candidiasis w/o systemic involvement he spore bearing heads of an Aspergillus spp. Effective against thrush, mycotic vulvovaginitis, and superficial skin lesions DIRECT MICROSCPIC OBSERVATION ▪ Econazole ▪ Isoconazole Sputum or any type of tissue ▪ Nystatin 1-2 drops of 10-20% KOH ▪ Miconazole If material is tissue – tease apart Nail infection Result Griseofulvin – little value Hyaline dichotomously branched septate Nystatin – involving paronychia hyphae Ketoconazole – topical-oral Sputum – very small, rough-walled spores (3- Gentian violet – Candida vaginitis 4 microns diameter) Sodium caprylate and sodium or calcium propionate 1% Thymol in chloroform – Candida onychia CULTURE Amphotericin B Drugs available to treat systemic Candidiasis (1990) SDA with or without antibiotics Amphotericin B Monomorphic 5-fluorocytosine Can be incubated at room temperature and 35-37⁰C Ketoconazole Grow better upon initial isolation at 35-37⁰C, rather than at room temperature ORIENTAL CONSIDERATIONS Aspergillus fumigatus – grows at 45⁰C Endogenous in origin ASPERGILLUS FUMIGATUS STD in origin – in some forms Transmitted via IV needles and other medical Gross morphology of colonies paraphernalia Black, green, yellow, orange, and white Most common cause of disseminated aspergillosis Light green colony after 3-4 days JEMALENE BARION 11  Dark green as colonies matures Pulmonary form – maybe transitory and may pass Blue in color – after 1-2 weeks unrecognized. Other laboratory test SSynonys Few laboratories test exist Torulosis Serologic procedure – experimental stage European blastomycosis Oriental consideration Infectious particle of the etiologic are ETIOLOGY everywhere Cryptococcus neoformans THERAPY Crypto ▪ Greek for hidden Prognosis for pulmonary and disseminated ▪ Organism is surrounded by capsule aspergillosis has been grave Only encapsulated yeast which is pathogenic Amphotericin B – used for many years with to man disappointing results Filobasidella – perfect (sexual) phase of this Itraconazole (1990) – used orally and has minimal side organism effects; recommended even lone term therapy for immuno-suppressed patients EPIDEMIOLOGY Fluconazole – disappointing Worldwide; in soils, especially those containing pigeon PHYCOMYCOSIS (ZYGOMYCOSIS) feces Disseminated by wind currents, enters the lungs Mucormycosis (old term) Zygomycosis PREDILECTIONS Systemic disease caused by a number of closely related fungi belonging to the class of fungi known as Patients with some defect in their cell mediated Phycomycetes immune system Involve almost any internal organ, however, the E.A. AIDS has a predilection for blood vessels Lymphoma Chronic infection of subcutaneous tissues Tuberculosis Certain form of surgery (organ transplant) ETIOLOGY Prolonged therapy with steroids Belong to the genus Mucor DIRECT MICROSCOPIC EXAMINATION Species belonging to the following genera can cause this disease Spinal fluid – material commonly used for direct Absidia examination and culture Basidiobolus Sputum or biopsy material – if lung involvement Conidiobolus Rhizopus INDIA INK EPIDEMIOLOGY China ink Place the material to be examined directly on the slide Ubiquitous 1-2 drops of slightly diluted India ink Soil; all types of plants parts, including fruits Place coverslip Look for yeast cells PREDILECTIONS TISSUE FORM AND HISTOPATHOLOGY Systemic form – most common for patient with: Uncontrolled diabetes mellitus PAS Leukemia Mucicarmine Lymphoma Look for encapsulated yeast cells Prolonged therapy with antibacterial antibiotics, steroids, and anticancer drugs CULTURE DIRECT EXAMINATION Monomorphic SDA with/without antibiotics KOH 1-3 weeks of incubation Large coenocytic hyphae Shiny, light tan yeast colonies Rarely forms hyphae, always produce capsule and CRYTOCOCCOSIS Urease (+) Acute or chronic, pulmonary or systemic or meningeal mycosis JEMALENE BARION 12  OTHER LABORATORY TESTS Worldwide, most common in Central and South America, Africa, India occurs near or south of the Serological (Latex agglutination) equator Intraperitoneal or intracerebral inoculation of C. neoformans, into mice, fata cryptococcosis results. CLINICAL FORMS THERAPY Foot is most common infected part Deformed Amphotericin B and 5-fluorocytosine (synergistic Predilections effect) Puncture wound in the foot Isolated cryptococcomas in the lungs may be removed surgically and follow up with non-toxic fluconazole TISSUE FORM AND HISTOPATHOLOGY Fluconazole – treatment of choice Stains: H&E, PAS, Brown, and Brenn (Tissue), Gram ORIENTAL CONSIDERATIONS Methenamine Silve (GMS) LPO: numerous, discrete granules Diagnosed throughout the Orient HPO: Septate hyphae and numerous chlamydospores Cryptococcus neoformans var. gattii in the granules of eumycotic mycetoma Natural habitat has never been found In 1990, Dr. David Ellis, published the occurrence of DIRECT MICROSCOPIC EXAMINATION this variety in association with the tree Eucalyptus camaldulensis Pus, or other exudate – observe for granules 10-20% KOH MISCELLANEOUS MYCOSES CULTURE To present a group a several diseases that are not frequently reported from oriental countries Not necessary for general diagnosis Mycetoma BHI/SDA – for suspicious diagnosis Chromoblastomycosis (chromomycosis) Room temp. incubation for 3-6 weeks is recommended Phaeohyphomycosis Penicilliosis OTHER LABORATORY TESTS Rhinosporidiosis Animal inoculation – not necessary MYCETOMA Serological test – available but are rarely needed Chronic granulomatous infection, which produced THERAPY tumor-like lesions and sinus tract formation with the presence of granules, esp. foot. Actinomycotic Skin and subcutaneous tissue involved originally High doses and prolonged treatment with Fascia and bone may be infected as the disease antibacterial antibiotics progresses Eumycotic mycetoma Other term Itraconazole Madura foot Regardless of the types of mycetoma, some surgical Maduromycosis procedures, esp. debridement, are usually very helpful ETIOLOGY (2 DISTINCT GROUP) CHROMOBLASTOMYCOSIS (CHROMOMYCOSIS) Actinomycotic Verrucous dermatitidis Actinomycetales Chronic, localized disease of the skin and Actinomadura subcutaneous tissues Nocardia Large, rough, firm, verrucous lesions appear on Streptomyces extremities, especially leds and feet Bacteria Often called chromoblastomycosis; implies that it Granules: very fine delicate filaments contains buds, but is not true Eumycotic True fungi ETIOLOGIC AGENT Large, coarse septate hyphae ▪ Allescheria Major causes of chromomycosis ▪ Madurella Cladosporium carriionii ▪ Phialophora Fonsecaea compactum Fonsecaea pedrosoi EPIDEMIOLOGY Phialophora verrucosa Grow on soil as saprophytes JEMALENE BARION 13  EPIDEMIOLOGY The classic term “systemic mycoses” used to refer to the dimorphic fungi, is somewhat misleading, because Worldwide other fungi, including complex and Candida spp. and Temperate region or topical areas Aspergillus spp., may also caused disseminated Predilections systemic infections. Not spread by man to man Trauma and contact with soil BLASTOMYCES SPP. Tissue form and histopathology Fission bodies Is found in the Ohio and Mississippi River Valley Direct microscopic exam Regions, as well as Ontario, Wisconsin, and Minnesota Round, thick dematiaceous cells Blastomycosis occurs in dogs and humans No hyphae found Produces acute or chronic suppurative and granulomatous infections THERAPY Identifications Large, spherical, thick-walled yeast (8-15µm) Itraconazole Single bud connected to the parent cell Advanced or generalized cases PCR Surgical removal Serology for Blastomyces dermatitidis Early stages Other terms Gilchrist’s disease PHAEOHYPHOMYCOSIS Chicago disease North American blastomycosis Synonyms Chronic infection of the lungs Cerebral chromoblastomycosis Suppurative and granulomatous lesions Dematiaceous infection Confused w/ other systemic mycosis or neoplasm Cladosoriosis Phaesporotrichosis ETIOLOGY Chromomycetoma Blastomyces dermatitidis CLINICAL FORMS Ajellomyces dermatitidis Perfect (sexual age) Subcutaneous cysts – present as single, firm, discrete, well defined subcutaneous nodules EPIDEMIOLOGY Ethmoid Invasion – massive invasion of the ethmoid bone occurred Mississippi River Valley Basin (US) Deep tissue invasion – most serious disease form and Organism is thought to grow in soil prognosis is not favorable CLINICAL FORMS ETIOLOGY Pulmonary Wangiella spp. Mild respiratory symptoms: fever, cough, and Exophiala spp. hoarseness Cladosporium spp. Productive cough and weight loss Hormodendrum spp. Systemic Drechslera spp. – most common cause of frequently Involvement of liver and spleen “fatal deep tissue invasion” form Granulomatous lesions are present, abscess All are dematiaceous fungi occur Cutaneous SYSTEMIC MYCOSES Skin lesions may result inoculation from soil Are produced mostly by dimorphic fungi: PREDILECTIONS Blastomyces spp. Coccidioides spp. Age ranges from 6 to 80 years old Histoplasma capsulatum 9x as many men as women Paracoccidioides brasiliensis No occupational predilection Talaromyces marneffei Sporothrix spp. TISSUE FORM AND HISTOPATHOLOGY Serologic testing or biopsy detects most infections Patients who are immunocompromised tend to exhibit Grows as yeast in tissue disseminated infection 8-15 microns in diameter * Is mostly found in North America “Broad-based bud” These infections may be detectable only by serology or Infectious particle after histopathologic review of tissues removed because of lesions found during a roentgenographic examination. JEMALENE BARION 14  ▪ Test requires 2-3 months after the onset of disease to develop detectable antibody Enzyme immunoassay (EIA) ▪ The latter test has met with mixed ▪ Acceptance by mycologist THERAPY Amphotericin B Drug of choice (DOC) Although it is very toxic and must be administered intravenously for several weeks Itraconazole Use in mild cases Less toxic ORIENTAL CONSIDERATION Not disease of the Orient North America (including Ontario and Quebec in Canada) COCCIDIOIDES SPP. Is found in the deserts of southwest of the United States is acquired by inhalation of arthroconidia Only yeast tat reproduces in animal with broad-based causes respiratory tract infections bud identification nonbudding, thick-walled spherule (20- DIRECT MICROSCOPY EXAMINATION 200µm) spherules containing nonbudding endospores Sputum PCR Pulmonary form Serology Skin scrapings Cutaneous form COCCIDIODES IMMITIS 10-20% KOH Quick and simplest test Cocci Culture (Dimorphic fungi) Desert fever SDA, Mycosel or Mycobiotic Valley fever Fluffy, white to brownish white fungus with Desert rheumatism pyriform form “The bumps” San Joaquin Valley Fever CLINICAL FORMS Primary Pulmonary Occurs 7-28 days after inhalation of single spore (infectious agent) Positive skin test – hallmark of disease Rash called erythema nodosum or erythema multiforme OTHER LABORATORY TESTS Benign Form Precipitin and complement fixation (+) Animal inoculation Hallmark of the disease (well-defined lung Impractical to do routinely cavitation) Skin test May exist for years Blastomycin Disseminated Form Little value because of cross reactivity Spread internal organs (brain) – meningitis Serological test Prognosis is grave Immunodiffusion test (precipitin) Precipitin titer disappear ▪ Requires 2-3 weeks to become ▪ CF–rise positive ▪ skin test from positive to negative Complement fixation (CF) Test called anergy JEMALENE BARION 15  Serological test HISTOPLASMA CAPSULATUM Is seen in the Midwestern and Southern US Is associated with the inhalation of aerosolized conidia: Chicken houses Roosting places for birds (e.g., starlings) Bat guano (in caves) Produces chronic granulomatous infection Identification May be detected in bone marrow or blood using Wright’s stain Is intracellularly found in cells as small, round- to-oval yeast cells (2-5µm) Demonstrates tuberculate macroconidia (mycelial form) PCR Serology EPIDEMIOLOGY Southwestern part of US Lower Sonoran Life Zone Little rainfall “Histo” – fungus is frequently found in histiocytes Extreme high summer temp “plasma” – early, erroneous concept that the E.A was Little vegetation other than cacti protozoan. Few inhabitants other than rattlesnakes and lizards “capsulatum” – erroneous conclusion that the organism has a capsule in vivo Great Desert Area of southwestern US and Mexico Emmonsiella capsulata – perfect (sexual stage) TISSUE FORM AND HISTOPATHOLOGY Darling’s disease Dimorphic CLINICAL FORMS Arthrospores enter human body, covert into completely different forms thick-walled structure called sporangia Primary acute or spherules Inhalation of infectious particle (microconidia) Flu-like, chest pain, shortness of breath, spherules hoarseness Sputum specimen positive skin test – outstanding feature Chronic cavitary Large pulmonary lesion occurs Mistaken for TB Severe disseminated Fatal disease of reticuloendothelial system Arthrospores Barrel-shaped appearance of alternate cells Infectious agent EPIDEMIOLOGY Grow in soil or lab cultures SDA Mississippi River Valley Basin Feces of starlings, chicken and bats Lives in soil in mycelial form and produce microconidia (infectious particle) TISSUE FORM AND HISTOPATHOLOGY Produces septate hyphae Smallest yeast cells of the major H&E DIRECT MICROSCOPIC EXAM PAS – best method Sputum, tissue or skin is available KOH (10-20%) Look for spherules containing endospores JEMALENE BARION 16  heal. Additional bumps or nodules may appear later near the original lesions. Cording effect SPOROTRICHOSIS Florist Plant nursery workers who have handled sphagnum moss Rose gardeners Children who have played on bales of hay Greenhouse workers who have handled thorns contaminated by the fungus CULTURE SDA / Mycobiotic Agar/Mycosel Agar Grow 5-10 days Colony brown w/ greasy appearance Sporothrix schenckii (SDA – room temp.) ▪ Flower-like clusters of spores (conidia) ▪ Septate hyphae ▪ Spores – infectious particles BHI Tan yeast colored yeast Gram stain black Sporothrix schenckii (BHI) ▪ Slender yeast cells ▪ Spherical yeast cells SPOROTHRIX SPP. TISSUE FORM AND HISTOPATHOLOGY Referred to as “rose gardener’s” disease Not seen in human tissue Acquired Laboratory test through No other lab test needed trauma Serological test (disseminated form) (thorns) Direct microscopic exam Appears as Rarely helpful round-to-oval cigar-shaped THERAPY yeast cells Demonstrates Potassium Iodide (oral) ovoid Primary case microconidia in mycelial form Amphotericin B Chronic, subcutaneous mycoses w/ lymphatic Systematic case involvement Itraconazole Common inhabitant of soil and epidermis of many Good response plants Systematic case Avenue of infections Scratch from rose bush (thorny plants) SERODIAGNOSIS Forcing fungus to subcutaneous tissue Fungal serologies are rapid and useful tests that may CLINICAL FORM aid the diagnosis of systemic fungal infections These tests have also been useful to study the The first symptom is usually a small painless nodule epidemiology of these fungal infections, because even (bump) resembling an insect bite. individuals with historically distant, asymptomatic, or The first nodule may appear any time from 1-2 weeks subclinical infections often have developed an antibody after exposure to the fungus response to the infecting pathogen The nodule can be red, pink, or purple in color, and it Two assays, complement fixation and usually appears on the finger, hand, or arm where the immunodiffusion, should be used together to detect fungus has entered through a break in the skin antibodies directed toward Blastomyces dermatitidis, The nodule will eventually become larger in size and Histoplasma capsulatum, and Coccidioides immitis may look like an open sore or ulcer that is very slow to JEMALENE BARION 17  COMPLEMENT FIXATION ASSAY TITERS EPIDEMIOLOGY AND PATHOGENESIS DISEASE 1:8 to 1:16 Suggest active infection with Blastomyces Superficial infections dermatitidis and Histoplasma capsulatum and Mycetoma 1:32 or greater indicate active disease. Chromoblastomycosis Titers as low as 1:2 to 1:4 have been identified in Phaeohyphomycosis patients with coccidioidomycosis. Titers greater than 1:16 usually indicate active disease. SUPERFICIAL INFECTION TINEA NIGRA IMMUNODIFFUSION TEST Skin infection caused by Hortaea werneckii One or two bands of identify, the H and M bands, may It is manifested by blackish brown, macular patches on occur in patients with histoplasmosis the palm of the hand or the sole of the foot The presence of both bands indicates active infection Lesions have been compared with silver nitrate The presence of an M band may indicate early or staining of the skin chronic infection BLACK PIEDRA DEMATIACEOUS (MELANIZED) MOLDS A fungal infection of the hair, scalp, and occasionally Agents of superficial and subcutaneous mycoses the axillary and pubic hair Skin and subcutaneous tissue involvement Hair and scalp infection caused by Piedraia hortae Ubiquitous in nature Humans and animals – accidental host AGENTS Classification Slow growing in 7 to 10 days Hortaea werneckii Rapid growing in less than 7 days Infects skin Tissue contains hyphal fragments and GROWTH RATE budding yeast Piedraia hortae Slow growers Infects hair Cladosporium spp. Tissue contains asci-containing nodules Verruconis gallopava cemented to hair shafts Exophiala dermatitidis Hortaea jeanselmei MYCETOMA Hortaea werneckii Fonsecaea spp. Chronic granulomatous infection involving lower Phialophora spp. extremities Piedraia hortae The infection is characterized by swelling, purplish Madurella mycetomatis discoloration, tumorlike deformities of the Rapid growers subcutaneous tissue, and multiple sinus tracts that Alternaria spp. drain pus containing yellow, white, red, or black Bipolaris spp. granules Curvularia spp. Bacterial (actinomycotic) or fungal (eumycotic) Exserohilum spp. Pseudallescheria boydii TWO TYPES OF MYCETOMA Actinomycotic (bacterial) mycetomas are caused by the aerobic actinomycetes, including Nocardia, Actinomadura, and Streptomyces spp. Eumycotic (fungal) mycetomas are caused by a heterogeneous group of fungi that have septate hyphae. AGENTS Are found mostly in tropical and subtropical regions Infections occur in those who have outdoor occupations Two fungal mycetomas: White grain ▪ Pseudallescheria boydii ▪ Acremonium JEMALENE BARION 18  ▪ Fusarium spp. PHAEOHYPHOMYCOSIS Black grain ▪ Madurella spp. Infections from brownish yeast-like cells, ▪ Exophiala jeanselmei pseudohyphae, or hyphae ▪ Curvularia spp. AGENTS PSEUDALLESCHERIA BOYDII Alternaria Fluffy and gray to gray-brown or gray-green. Grows rapidly (5-10 days) Golden-brown hyphae Is initially white and fluffy but becomes brownish gray Large brown conidia resembling a drumstick Reverse surface is tan to dark brown Horizontal and longitudinal septa Reproduction: Asexual ▪ Scedosporium apiospermum ▪ Two anamorph morphologies: culture-based and Graphium stage Sexual ▪ Teleomorphic form, producing brown-black cleistothecia Bipolaris CURVULARIA SPP. Gray-green to dark-brown colonies Bent conidiophores Oblong conidia arranged sympodial Hilum protruding slightly Germ tubes at one or both ends CHROMOBLASTOMYCOSIS Chronic infection acquired by traumatic inoculation into skin Curvularia Lesions revealing sclerotic bodies and resembling Similar to Alternaria copper pennies Geniculate conidiophores Conidia AGENTS ▪ Golden brown ▪ Multicelled Cladophialophora ▪ Curved Septate hyphae occur in tissue ▪ Swollen central cell Phialophora Sclerotic bodies and septate hyphae are found in subcutaneous tissue Hyphae may be found in joints Phialophora verrucosa Exophiala jeanselmei Slow grower Shiny brown colonies Velvety with age Elongated conidiophore with tapered tip Uses potassium nitrate Fonsecaea Grows only at 37°C Sclerotic bodies are observed in Exophiala dermatitidis subcutaneous tissue. Slow grower Septate hyphae are observed in brain and Shiny black colonies lung tissue. Velvety with age