Mycology and Virology Midterms PDF

MICRO2 | Mycology and Virology BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE 3 - 2 MIDTERMS: LESSON 5: Agents of Subcutaneous Mycoses SUBCUTANEOUS MYCOSES  Affect subcutaneous tissue  Usually result of traumatic implantation of forei...

MICRO2 | Mycology and Virology BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE 3 - 2 MIDTERMS: LESSON 5: Agents of Subcutaneous Mycoses SUBCUTANEOUS MYCOSES  Affect subcutaneous tissue  Usually result of traumatic implantation of foreign objects into the deep layers of the skin, permitting fungus to gain entry into the host. Any thorn or contaminated objects with the different fungal found in the soul or decaying of organic matter, that would be the entrance of subcutaneous mycoses in to the host o Causative agents—organisms typically found in soil or decaying vegetation  Infections may be grouped by either the  These are the different manifestations of o disease processes they cause chromoblastomycosis o causative agents involved  LETTER A, lower leg. It is a lesion and it has been  Coccidioidomycosis, the most fatal of all the fungal occurring in the past 3 months. This is a lesion of infections, this is common some parts of the US, in chromoblastomycosis Arizona, California, desert areas  LETTER B, knee. There are confluent nodular lesions. CHROMOBLASTOMYCOSIS  LETTER C, tumor like lesion like a cauliflower  Also known as verrucous dermatitidis and  LETTER D, cicatrisation lesion with verruca showing chromomycosis serpiginous and verrucous contours o Nodules become ulcerated and crusted  LETTER E, hyperkertotic, highly keratinzed verrucous  Chronic infection acquired by traumatic inoculation lesion into skin and subcutaneous tissue. After the traumatic  LETTER F, thigh. Purplish soft-flat lesion. introduction the symptoms would not develop LABORATORY DIAGNOSIS instantly, it would take months to years before you  10-40% KOH have the symptoms  Direct mycological examination o Develops over months to years  Histopathology  Cauliflower or tumor-like lesions revealing sclerotic o The presence of muriform cells, this is your bodies and resembling copper pennies sclerotic bodies, it is a confirmatory diagnosis  Cladophialophora spp., Fonsecaea monophora, F. o Fungal elements are most commonly found within pedrosoi and Phialophora verrucosa these agents are dermal macrophage and mainly consists of phaeoids or dematiaceous, it means to say they are sclerotic bodies that are usually 5-12 dark in color micrometers in diameter  Dematiaceous molds o Moderate to slow growth o Velvety to wooly, gray-brown to black due to the presence of melanin in their cell wall o Differentiate by characteristic structures AGENTS OF CHROMOBLASTOMYCOSIS  Cladophialophora o These are the characteristic of muriform cells, o Septate hyphae occur in tissue thick walled and can appear singly or in  Phialophora multiple clusters o Sclerotic bodies and septate hyphae are found o This is a skin biopsy, we have here in subcutaneous tissue hyperkeratosis and pseudoepitheliomatous o Hyphae may be found in joints hyperplasia, the stain used is Hematoxylin-Eosin.  Fonsecaea o Within the dermis, we can see inflammatory cells. o Sclerotic bodies are observed in subcutaneous We have macrophages and lymphocytes as tissue well as plasma cells and neutrophils o Septate hyphae are observed in brain and in o In the arrow, there are brownish color, these are lung tissue the sclerotic bodies.  Immunodiagnosis | CALINDAS, TRISHA FELICITTY S.  Conidia Conidiophore o ELISA, you have to detect the presence of antigens from the agents of chromoblastomycosis o Immunodiffusion  LETTER A, you have to scrape the black dot where it is being pointed. The black dot shows you muriform cells without germination  LETTER B, without germination  LETTER C, with germination  Phialophora verrucosa, colony using Malt Extract  Germination will result in the formation of filaments Agar after 3-4 weeks of contribution at 30 degrees  We can also have a biopsy aside from the black dot celcius. Conidiophores are conidogenous cells scrape. We can perform H&E staining, it would phaeoid, flask shapes and the presence of colorettes. showcase hyperkeratosis and The conidium are oval in shape, single-celled. pseudoepitheliomatous hyperplasia because of the  Fonsecaea pedrosoi, colony using Malt Extract Agar presence of macrophages, giant cells, plasma cells, after 3-4 weeks of contribution at 30 degrees and neutrophils that would contribute to hyperplasia celcius. The conidiophore, sympodial and angular.  LETTER E, violet part is the muriform cells and they They are single-celled. The conidia, there is a are found in the Langerhan cells of your tissue primary and secondary. They are closely similar to Cladophialophora spp., the only difference is that MICROSCOPIC MORPHOLOGY OF FUNGI CAUSING there is more erected and more branching single- CHROMOBLASTOMYCOSIS celled blastoconidium in Cladophialophora spp. it is similar to Rhinocladiella spp. however, there are longer conidiophore, also it is erect and they are also sympodial, however there is a longer conidiophore.  Cladophilophora carrionii, erected conidiophore, single-celled but are branching.  Rhinocladiella aquaspersa, the conidiophore is also erect, they are dark and have conidia. Only found on the upper portion near the tip. The conidoa are elliptical, single-celled, and sympodially arranged. CONIDIA OF PHIALOPHOTA VERSUS CLADOPHIALOPHORA | CALINDAS, TRISHA FELICITTY S.  ADDITIONAL PICTURES  LETTER A, Phialophora  The individual condium could measure 3-5 by 1.5- 3.5 micrometers and they are aggregated and clustered and apices of the phialide.  While the conidia of Cladophialophora, specifically Cladosporum carrionii, the precence of lemoniform to fusiform. They are smooth and slightly verrucous and could be pale olivaceous. PHIALOPHORA VERRUCOSA  LETTER A, flask-shaped  LETTER B, colorette  LETTER C, conidia EUMYCOTIC MYCETOMAS  Uncommon in the United States  Subcutaneous and cutaneous tissue infection o Swelling and exudates (fluids produced from infections) draining to skin surface  Found mostly in tropical and subtropical regions  Infections occur in those who have outdoor occupations  Chronic granulomatous infection usually involving the lower extremities  Dissemination may occur, but it is rare  Most common: o Pseudallescheria boydii (teleomorph) sexual  Cleistotheicia | CALINDAS, TRISHA FELICITTY S.  o Scedosporium boydii (anamorph) asexual  It is your sexual, an individual cleistothecia that have  Single oval conidia ascospores. o Fusarium (Acremonium) falciforme  Mucoid clusters of single or two-celled, PHAEOHYPHOMYCOSIS slightly curved conida borne from phialides  Infections from brownish yeast-like cells, at the tips of long, unbranched, multiseptate pseudohyphae, or hyphae conidiophores  Subcutaneous, localized or systemic infection o Madurella spp.  Cladiophialophora bantiana, Rhinocladiella machenziei,  Produce conidia from the tips of phialides, Verruconic gallopava, Exophiala, Alternaria, but many remain sterile Exsorohilum, Bipolaris, and Culvularia spp. DESCRIPTION OF GRANULES AGENTS OF PHAEOHYPHOMYCOSIS  Alternaria- fluffy and gray to gray-brown or gray- green  Bipolaris- gray-green to dark-brown colonies  Curvularia- similar to Alternaria  Exophiala jeanselmei- gray-green to dark-brown colonies  E. dermatitidis- gray-green to dark-brown colonies  Exserohilum- gray-green to dark-brown ACTINOMYCOTIC MYCETOMA DEMATIACEOUS GENERA INCITING SUBCUTANEOUS PHAEOHYPHOMYCOSIS SCEDOSPORIUM BOYDII ALTERNARIA  Golden-brown hyphae  Large brown conidia resembling a drumstick  Horizontal and longitudinal septa  You could see single, oval conidium CLEISTOTHECIA CONTANING ASCOSPORES  Looks like a drum stick and then it has a horizontal as well as longitudinal septa. It is alternating from dark to light. | CALINDAS, TRISHA FELICITTY S. ADDITIONAL PICTURES ADDITIONAL PICTURE CURVULARIA  Geniculate conidiophores  Conidia o Golden brown o Multi-celled o Curved o Swollen central cell BIPOLARIS  Bent conidiophores  Oblong conidia arranged sympodially  Hilum protruding slightly  Germ tubes at one or both ends  This is a distinguishing characteristic, the swollen central cell, making it like twisted curved conidia ADDITIONAL PICTURES  So bent at sharp angles are called geniculate  They have melanin, so it is brown in color | CALINDAS, TRISHA FELICITTY S.  EXOPHIALA  Exophiala jeanselmei o Slow grower o Shiny brown colonies o Velvety with age o Elongated conidiophore with tapered tip o Uses potassium nitrate o Grows only at 37°C ADDITIONAL PICTURES  Exophiala dermatitidis o Slow grower o Shiny black colonies o Velvety with age o Elongated tubular annellophores o Does not uses potassium nitrate o Grows at 37°C and 40°C to 42°C CONIDIA OF EXOPHIALA SPP. ADDITIONAL PICTURES  Born at the tips of annelide CONIDIA OF EXOPHIALA DERMATITIDIS | CALINDAS, TRISHA FELICITTY S. DIRECT DETECTION o Yeast  Microscopic preparation of tissue during surgery or  2 to 4 μm in diameter; oval or elliptical in autopsy shape, often in a single bud  Calcofluor white or fluorescent microscopy  Cigar-shaped forms measuring to 3 to 10  Fontana-Masson stain μm o Useful to detect melanization that may appear YEAST AND MOLD PHASES OF S. SCHENCKII as a hyaline mold  Mycetoma or Phaeophomycosis o Microscopic granules from lesions o Pigmented hyphae using hematoxylin-eosin or unstained histopathologic sections o Methenamine silver stain or Fontana-Masson stain NUCLEIC-ACID DETECTION  Polymerase Chain Reaction o Normally sterile body fluids  Nucleic acid based sequencing o Ribosomal genes  High-Resolution multi-gene typing systems o Epidemiologic purposes  Cigar-shaped yeast cell CULTIVATION  May represent pathogens, more frequently transient microbiota, inhaled spores or contaminants  Cultured from sterile body sites are significant  Interpretation should be in conjunction with direct detection methods  We cannot use contaminated sampled, in which there are high numbers of indigenous microflora ANTIFUNGAL SUSCEPTIBILITIES  Susceptibilities are known for most relevant species  Interpretative breakpoints have not been established  Amphotericin B and Azoles are generally effective  Arranged as flowers or rosettes, mold form SPOROTHRIX SPP. / “ROSE GARDENER’S” DISEASE SPOROTHRIX SCHENCKII SPECIES COMPLEX  Most common presentation  Causative agent of Rose Gardener’s Disease o Lymphocutaneous sporotrichosis o Nodular and ulcerative lesions  Worldwide distribution o Found in soil and decaying vegetative matter  Particularly plants, wood splinters, and rosebush thorns  Colonies o Gray-white but may turn buff, brown, or brown- black as it matures  Microscopically o Mold  It was first observed from people handling rose  Delicate hyaline, septate hyphae plants  Oval, smooth conidia 2 to 4 μm in diameter supported by a delicate hairlike conidiophore  Arranged laterally along hyphae or in daisy-like clusters | CALINDAS, TRISHA FELICITTY S. ADDITIONAL PICTURES ENTOMOPHTHORALES AND BASIDIOBOLALES  Basidiobolus ranarum  Conidiobolus spp.  Present in soil, decaying vegetable materials, and animal feces  Occur predominantly in immunocompetent individuals DISEASE  Basiodobolomycosis o Subcutaneous tissue of arms, legs, buttocks, trunk, perineum, face, or neck o Woody, hard, painless nodule  Conidiobolus spp. o Infect tissue around nose and on the face o Inhalation of spores or following trauma LABORATORY DIAGNOSIS RHINOSPORIDIUM SEEBERI  Mesomycetozoea protistal eukaryote  Direct detection methods o Morphologically similar to Coccidiodes o Splendore-Hoeppli phenomenon  Organism is found in aquatic environments and  PCR for Basidiobolus entomophthoromycosis resistant spores are present in terrestrial  Culture environments  Route of transmission is unknown o Likely by exposure to spores through breaks in the skin or mucous membranes  Infection results in formation of painless polyps on the mucosa of the nose, eye, larynx, genitalia, and rectum  Rhinosporidiosis o Greater than 300 μm spherical sporangia with endospores and negative culture  Tissue biopsies are the preferred method for diagnosis  Wet mount from polyps demonstrating sporangia and endospores are often present  This is the Splendore-Hoeppli phenomenon TREATMENT OF PARAFUNGALS  The organisms are resistant to antifungals  R. seeberi o Surgical removal of infected tissue and polyps is required LACAZIA LOBOI  Lobomycosis o Rare granulomatous zoonotic fungal infection o Leprosy-like lesions and nodules  Organisms found in soil, on vegetation, and in aquatic animals  Transmission results from traumatic injury or from contaminated water  Asteroid bodies, an in vivo formation of intensely o Dolphin to human and human-to-human eosinophilic material, which is activated due to transmission has been reported allergic reactions. Immune complexes that would try  Diagnosis is by microscopic morphology to surround your fungal elements o Yeast-like cells in chains, connected by tubules IDENTIFICATION  Basidobolus ranarum o Colonies appear slightly yellow with radial folds | CALINDAS, TRISHA FELICITTY S.  o No aerial hyphae present  Conidobolus spp. o Primary conidiophores have swollen apices with o Fast growing fungus globose spores o Produces hyaline, radially folded colonies o Secondary conidia appear pyriform with a o Initially appear waxy and become powdery knob-like adhesive tip o Primary conidia are spherical and have prominent papilla o Zygospores are absent when grown on potato dextrose agar  With a satellite colony  Like villain or like projections  Germinating conidia with zygospores  Colonies showing satellite from forcibly discharged conidia  Swollen sporophore | CALINDAS, TRISHA FELICITTY S. MYCOLOGY AND VIROLOGY AGENTS OF SYSTEMIC MYCOSES Systemic Mycoses Young colonies: As a result of molecular technology, many new tenacious species are being proposed Older colonies: Are produced mostly by dimorphic fungi: Glabrous to ❖ Blastomyces spp., Coccidioides spp., woolly Histoplasma capsulatum, Paracoccidioides spp., Talaromyces Histoplasma Slow growth Microconidia small, capsulatum one-celled, round, marneffei White to dark smooth (2-5 Serologic testing or biopsy detects most tan with age micrometer) infections Patients who are immunocompromised tend to Woolly, cottony, Tuberculated exhibit disseminated infection or granular macroconidia large, round (7-12 General Characteristics micrometer) Hyphalike Dimorphic fungi conidiophores Mold forms in the environment at “room temperature” Alternating ❖ Infective form: inhalation of spores or one-celled, direct traumatic implantation “barrel-shaped” arthroconidia with Yeasts at 35 to 37°C at “body temperature” disjunctor cells Tend to form “deep-seated” infections ❖ Highly virulent in susceptible hosts with Coccidioides Rapid growth deep invasions that can become immitis, systemic (yung may mga cancer or Coccidioides White to tan to autoimmune diseases) posadasii dark gray ❖ This deep-seated can become systemic Young colonies: mycoses through hematogenous route tenacious pero commonly is inhalation Older colonies: Morphology of Systemic Fungi at 22°C cottony Tend to grow in Fungus Macroscopic Microscopic concentric Morphology Morphology riings Blastomyces Slow to Oval, pyriform to Paracoccidioi Slow growth Colonies frequently dermatitidis moderate globose smooth des only produce growth conidia borne on brasiliensis White to beige sterile hyphae short, lateral White to dark hyphalike Colony Fresh isolates may tan conidiophores glabrous, produce conidia Lubiano, Ristian Aaron T. 1  AGENTS OF SYSTEMIC MYCOSES leathery, flat to similar to those of river -Immunode phagocytes wrinkled, B. dermatitidis valleys ficient folded or hosts velvety Bird and prone to bat guano disseminat ed disease Mold to Yeast Conversion of Thermally Dimorphic Alkaline Fungi soil Coccidioide Semi-arid -Primary Spherules Fungus Culture Media Yeast Form s immitis, regions-sou lung (30-60 µm) and Coccidioide thwest -Asymptom Containing Temperature s posadasii United atic endospores States, -Secondary Blastomyces Blood agar, 37 Large yeast Mexico, cavitary dermatitidis dc (8-12 µm) Central and -Progressiv South e Blastoconidia America pulmonary attached by -Multisyste broad base In soil m Histoplasma Pines medium, Small, oval Paracoccidi Central and -Primary Thick-walle capsulatum glucose-cysteine yeast (2-5 µm) oides South lung d yeasts -blood, or BHI brasiliensis America -Granulom (15-30 µm) agar-blood, 37 atous dc In soil -Ulcerative Multiple nasal and buds, Paracoccidioide BHI agar-blood Multiple buccal “mariner’s s brasiliensis agar, 37 dc blastoconidia lesions wheel” budding from -Lymph single, large node yeast (15-30 involvemen µm) t -Adrenals Summary of Systemic Mycoses Fungus Ecology Clinical Tissue Blastomyces dermatitidis Disease Form Clinical Manifestations Blastomyce Mississippi -Primary Large yeast s and Ohio lung (8-12 µm) Most prevalent in middle-aged men dermatitidi river -Chronic Broad-base ❖ Presumably of occupational and s valleys skinbone d bud recreational exposure to the soil -Systemic, ❖ Minsan sa course of work nila sila ang multiorgan laging exposed sa soil Histoplasm Ohio, -Primary Small, oval Patients with primary infection a Missouri, lung yeast (2-5 ❖ They could show flu-like symptoms, capsulatu and -Asymptom µm) in most are asymptomatic m Mississippi atic histiocytes, Lubiano, Ristian Aaron T. 2  AGENTS OF SYSTEMIC MYCOSES When primary disease fails to resolve, ❖ Variety of colony morphologies: white, pulmonary disease may ensue. tan, or brown and may be fluffy to ❖ Cough, weight loss, chest pain, and glabrous fever ❖ Frequently, raised areas known as Progressive pulmonary or invasive disease may spicules are seen in the centers of the follow. colonies. ❖ Results in ulcerative lesions of the skin and bone Yeast Mold In immunocompromised patients may experience ❖ Multiple organ systems involvement (infection) ❖ Rapid death possible Causes blastomycosis ❖ a.k.a. Gilchrist disease, North American blastomycosis, Chicago disease Coccidioides Two responsible indistinguishable species Clinical Manifestations ❖ Blastomyces dermatitidis ❖ Blastomyces gilchristii Considered as the probable most virulent of all Occurs primarily in North America and parts of human mycotic agents Africa Two very similar species infect humans ❖ Endemic in the Mississippi and Ohio ❖ C. immitis and C. posadasii river basins Mode of transmission ❖ Inhalation of only a few arthroconidia Laboratory Diagnosis produces primary coccidioidomycosis Types of clinical infections Specimens of choice ❖ Asymptomatic pulmonary disease and ❖ Tissue or purulent material in allergic manifestations cutaneous skin lesions Direct microscopic examination Allergic Manifestations ❖ Large, spherical, refractile yeasts 8 to 15 μm in diameter with double-contoured Toxic erythema wall and buds connected by a broad Erythema nodosum (desert bumps) base Erythema multiforme (valley fever) ❖ KOH (10%) or calcofluor white may be Arthritis (desert rheumatism) used to enhance yeast cell detection. Primary disease in these cases usually resolve Mold phase without therapy. ❖ Conidia are borne on short lateral ❖ Strong specific immunity to reinfection, branches that are ovoid to detected by the coccidioidin skin test dumbbell-shaped and vary in diameter from 2 to 10 μm. ❖ Microconidia resemble other fungi and are thus not diagnostic. Symptomatic Disease Culture characteristics at 22° C Lubiano, Ristian Aaron T. 3  AGENTS OF SYSTEMIC MYCOSES Primary disease symptoms isotropic growth, remodeling its cell wall into ❖ Fever, respiratory distress, cough, spherule (specialized cell wall) anorexia, headache, malaise, myalgia The spherule will mature(distinct to Disease might progress to secondary coccidioides) and will undergo nuclear division coccidioidomycosis. producing a cellular structure of being 100 µm ❖ Nodules, cavitary lung disease, and/or (nagmature further) and inside they have progressive pulmonary disease endospores (100-300 asexual single-celled ❖ In about 1% of this population, single or propagules “ endospores”) multisystem dissemination follows. When it ruptures, it will release your endospores (continue the life cycle) Epidemiology Inhaled arthroconidia convert to spherules. At maturity, spherules produce endospores via Endemic to Southwestern United States progressive cleavage. ❖ Soil; easily windborne in clouds of dust Rupture of spherule wall releases endospores and penetrate deep into the respiratory into the bloodstream and surrounding tissues. tree, into the alveoli Endospores form new spherules. Endemic areas outside of the United States Direct smear of secretions may reveal spherules ❖ Northern Mexico, Guatemala, containing endospores. Honduras, and parts of South America Geographic locations Spherules of C. immitis ❖ C. immitis—San Joaquin Valley of California ❖ C. posadasii—desert southwest, Mexico, South America Life Cycle Notes Microscopic Examination of Culture Fertile hyphae arising at right angles to the vegetative hyphae ❖ Produces alternating (separated by a disjunctor cell-empty spaces when Both species of coccidioides are dimorphic stained) hyaline arthroconidia fungi that grow saprophytically as hyphae When released, conidia have an annular frill at This process produces single barrel-shaped cells both ends. (approx 3-5 µm) As culture ages, vegetative hyphae also After autolysis, individual barrel-shaped cells fragment into arthroconidia. will be produced (infectious) Variety of mold morphologies at 22° C (Under dry conditions) Arthroconidia are very stable spores remaining viable for up to many Arthroconidia of C. immitis years. When they are inhaled into the lungs up to the terminal bronchiole, the fungus initiates Lubiano, Ristian Aaron T. 4  AGENTS OF SYSTEMIC MYCOSES ❖ Daughter cells are 2 to 5 μm in size are connected by narrow base. ❖ Buds of various sizes can occur. - Some strains may resemble a Mickey Mouse cap in appearance. Mold morphologies ❖ Vary when grown at 22° C - Flat colonies are glabrous to Paracoccidioides brasiliensis leathery, wrinkled to folded, Clinical Manifestations floccose to velvety, pink to beige to brown with yellowish Pulmonary in nature brown reverse. ❖ Causative agent of ❖ Microscopically mold form produces paracoccidioidomycosis - 2 to 10 μm in diameter ❖ Infection is usually unapparent and one-celled conidia asymptomatic. ❖ Subsequent dissemination leads to P. brasiliensis Budding Yeast Resembling a Mariner’s - Ulcerative granulomatous Wheel lesions of the buccal, nasal, and occasionally gastrointestinal mucosa. ❖ Concomitant striking lymph node involvement is evident. ❖ Dissemination to other organs, particularly the adrenals, occurs with compromised host defenses. Modes of transmission ❖ Inhalation of small conidia ❖ Direct inoculation of the skin with Histoplasma capsulatum contaminated dirt Clinical Manifestations ❖ Found primarily in Central America and South America in areas that are typically Infection is usually limited and asymptomatic. humid and experience high- rainfall, ❖ Inhalation of spores with acidic soil conditions ❖ Calcification in the lungs, liver, and spleen Laboratory Diagnosis Heavy exposure may result in acute pulmonary disease. Direct microscopic examination of cutaneous In immunocompromised patients and mucosal lesions ❖ Progressive and potentially fatal ❖ Budding yeast cells 15 to 30 μm in disseminated disease possible diameter Other manifestations ❖ Multipolar budding at the periphery ❖ Mediastinitis, pericarditis, resembling a “mariner’s wheel” mucocutaneous lesions Lubiano, Ristian Aaron T. 5  AGENTS OF SYSTEMIC MYCOSES Chronic pulmonary histoplasmosis in patients with COPD may occur. Histoplasmosis is also known as ❖ Reticuloendothelial cytomycosis ❖ Cave disease ❖ Spelunker’s disease ❖ Darling disease Histoplasma capsulatum: Mycelial form Worldwide distribution ❖ Highest endemicity—Ohio, Missouri, Mississippi river deltas Laboratory Diagnosis Direct microscopic morphology when yeast cells are stained with Giemsa or Wright stain ❖ Small in diameter, with single bud connected by a delicate filament Histoplasma capsulatum: Yeast form ❖ In tissue, yeast cells are clustered in macrophages surrounded by clear space (looks Pseudo encapsulated). ❖ Inflammation reactions cause granulomas that may simulate TB. ❖ Yeast commonly seen within monocytes and macrophages in significant numbers Mold morphology ❖ Mycelium initially white but turn gray or gray-brown Talaromyces marneffei ❖ Young cultures produce Clinical Manifestations ❖ As the colony matures, large echinulate or tuberculate macroconidia emerge. Formerly known as Penicillium marneffei Examples of other available ID methods Only true pathogen in the genus ❖ Direct antigen detection, serologic Common cause of systemic infections in procedures immunocompromised patients who have visited the endemic region of Southeast Asia H. capsulatum in Monocytes (left) and Tissue (right) Infections are usually disseminated with multiple organ involvement and is typically fatal. Laboratory Diagnosis Specimen for fungus isolation Large Tuberculate Macroconidia ❖ Cutaneous lesions or biopsies Lubiano, Ristian Aaron T. 6  AGENTS OF SYSTEMIC MYCOSES ❖ Yeast cells present resembling those of H. capsulatum - Oval to cylindric, measuring 3 to 6 μm long, and may have a cross-wall Mold form ❖ Sparse green aerial and reddish brown vegetative hyphae and production of a red diffusible pigment Talaromyces marneffei Lubiano, Ristian Aaron T. 7  MYCOLOGY AND VIROLOGY LECTURE: AGENTS OF OPPORTUNISTIC MYCOSES & YEAST INFECTIONS INSTRUCTOR: MR. CHARLIE CRUZ, RMT, SM (ASCP) – COLLEGE OF MEDICAL LABORATORY SCIENCE A.Y. 2024-2025 AGENTS OF OPPORTUNISTIC MYCOSES o Sporangiophores may branch GENERAL CHARACTERISTICS o Funnel-like structure of terminal conidiophore Saprobe and saprophyte ▪ Apophysis o Terms that describe free-living microorganisms in the environment that are typically not of o Colony color initially white becoming gray to gray-brown with age concern in humans Mucorales Mucor o The line between these two terms has been increasingly blurred due to the growing number of Clinical significance persons with defects in their immune systems. o Ideal points of saprophyte inoculation o Rhinocerebral zygomycosis o Disseminated disease ▪ Surgical wounds, skin and nail bed infections, severe respiratory infections Isolated from the environment worldwide Members of this group of fungi are found worldwide, often associated with decaying vegetation. Colony characteristics MUCORALES GENERAL CHARACTERISTICS o Grow rapidly Common environmental isolates associated with soil and plants o Form cottony, dirty white colonies They contaminate grains, breads, and fruits that become mousy brown to gray Most often associated with infections of the with age o Sinuses, lungs, and skin of immunocompromised Characteristics Diabetics is a significant risk factor for these infections. o No rhizoids o May be branched or unbranched Mucorales Cunninghamella sporangiophores Figure 1.2 Mucor sp Disseminated disease o Sporangiophores’ ends are slightly bulbous columella extending within a spherical smooth- o Recovered from sinuses or affected walled sporangium. organs o Sporangiophores are spherical or ellipsoid and hyaline or yellow-brown pigmented. Key characteristic o Sporangiola Mucorales Rhizopus ▪ Spherical spores from a large Most common zygomycete that globose columella causes human disease ▪ No encasement in sporangium o Typically involved in diabetic ▪ May be covered with long, fine patients with ketoacidosis spines presenting as rhinocerebral Colony morphology mucormycosis o Cottony, initially white, becoming o May be refractory to treatment gray with age o May be recovered from almost any source Figure 1.0 Cunninghamella LPCB Prep Worldwide distribution Mucorales Lichtheimia o Isolate easily recovered from Clinical significance the environment in decaying o Causes vascular invasion, causing vegetation thrombosis and necrosis of the tissues Characteristics o Usually found in diabetic patients suffering Production of distinct rootlike brown from ketoacidosis rhizoids Figure 1.3 Rhizopus sp. o Infection usually begins in the sinuses. o Nodal Found worldwide ▪ Derived from hyphae immediately adjacent to sporangiophores o Soil, dung, decomposing organic matter o Umbrella-like collapse of postmature sporangium Characteristics Colony morphology o Broad, irregular width, ribbon-like, aseptate o Woolly appearance covering the entire surface of medium hyphae, and sporangia o White becoming gray to brown with age o Rhizoids are similar but more delicate than. Mucorales Syncephalastrum Rhizopus spp. Figure 1.1 Lichtheimia ▪ Internodal rhizoids originating from hyphae between the conidiophores Found in soil and decaying vegetation DAWARA 1  MYCOLOGY AND VIROLOGY LECTURE: AGENTS OF OPPORTUNISTIC MYCOSES & YEAST INFECTIONS INSTRUCTOR: MR. CHARLIE CRUZ, RMT, SM (ASCP) – COLLEGE OF MEDICAL LABORATORY SCIENCE A.Y. 2024-2025 Rare in human disease o Some species have diffusible subsurface pigments on a variety of media. Characteristics o Most known pathogens form green- to tan-colored colonies. o Differ in shape and arrangement of sporangia General microscopic features ▪ Merosporangia o Uniseriate or biseriate Cylindric and arranged in “daisy petals” around a small spherical columella ▪ Uniseriate species have phialides attached directly to the vesicle at the end of the Similar to Aspergillus flavus conidiophore. o Examine hyphae and colony morphology ▪ Biseriate species possess a supporting structure—metula that attach directly to the Rapid growth, colonies cover entire medium vesicle with phialides attached to each metula o Foot cell ▪ Base of origin of the conidiophore resembling a foot Figure 1.4 Syncephalastrums SEPTATE AND HYALINE SAPROPHYTES Aspergillus Figure 1.6 Aspergillus sp. Pathogenic species of Interest o A. fumigatus—species most often OTHER MEMBERS seen Beauveria—a known insect pathogen o A. flavus o Rare isolate o A. terreus ▪ Keratitis o A. niger Chrysosporium Mortality is high, especially in the o Rare isolate immune-compromised. ▪ Skin and nail infections o Particularly neutropenic Fusarium individuals o Mycotic keratitis Infection ▪ Contact lens outbreak from lens solution o Inhalation of spore-contaminated o Banana or boat-shaped macroconidia dust ▪ Sinusitis or bronchopulmonary disease Figure 1.5 Aspergillus sp. ▪ Most frequent cause of disease in the bone marrow of transplant recipients o Disseminates from the lungs homogenously to other organs Fungus balls o Group of fungi in hollow spaces Allergic reactions Colony morphology o Color is derived from the conidia and range from black to white and include yellow, brown, green, gray, pink, beige, tan. DAWARA 2  MYCOLOGY AND VIROLOGY LECTURE: AGENTS OF OPPORTUNISTIC MYCOSES & YEAST INFECTIONS INSTRUCTOR: MR. CHARLIE CRUZ, RMT, SM (ASCP) – COLLEGE OF MEDICAL LABORATORY SCIENCE A.Y. 2024-2025 Figure 1.7 Beauveria sp. Figure 1.8 Chrysosporium sp. o Nail specimens o Implicated in pulmonary disease in immunocompromised o Tan to buff conidia Trichoderma o Emerging pathogen in immunocompromised ▪ Pulmonary and skin infections o Rapid growth, yellow-green to green conidia Figure 1.9 Fusarium sp. Geotrichum o Pulmonary disease in immunocompromised ▪ Abundant arthroconidia Purpureocillium (Paecilomyces) o Potentially serious and difficult to treat ▪ Has been implicated in hospital outbreaks Figure 2.4 Scopulariopsis sp. Figure 2.5 Trichoderma sp. o Flat, velvety, tan, gold, and mauve colonies SEPTATE AND PHAEOID SAPROPHYTES Penicillium Alternaria o Rarely cause disease, fungal sinusitis o Colonies gray to brown or black o Brushlike conidia, green or blue-green colonies o Short conidiophores with conidia that lengthen in acropetal fashion o Angular cross-walls and taper toward distal end Aureobasidium o Rare but found in contaminated dialysis lines o Hyaline conidia that break up into arthroconidia which are responsible for darkening colonies Chaetomium o Infections of the brain of patients with central nervous system (CNS) disease Figure 2.6 Alternaria sp. o Pineapple-shaped perithecia ▪ Curled or straight curled hairs or setae Figure 2.0 Geotrichum sp. Figure 2.1 Paecilomyces sp. Cladosporium o Sinus or traumatic inoculation o Brown to olive to black hyphae and conidia ▪ Branched conidia ▪ Shield appearance Figure 2.2 Penicillium sp. Figure 2.3 Penicillium sp. Scopulariopsis Figure 2.7 Chaetomium sp. Figure 2.8 Cladosporium sp. DAWARA 3  MYCOLOGY AND VIROLOGY LECTURE: AGENTS OF OPPORTUNISTIC MYCOSES & YEAST INFECTIONS INSTRUCTOR: MR. CHARLIE CRUZ, RMT, SM (ASCP) – COLLEGE OF MEDICAL LABORATORY SCIENCE A.Y. 2024-2025 Curvularia AGENTS OF YEAST INFECTIONS o Chronic sinusitis in immunocompromised INCIDENCE AND CLASSIFICATION patients Escalating incidence o Multicelled conidia produced on sympodial o Provides evidence for identifying yeasts a species level conidiophores Classified into two groups based on reproduction characteristics o Frequently seen crescent-shaped conidia with o Those which reproduce sexually by forming ascospores or basidiospores are truly yeasts. three to five cells of unequal size and o Isolates not capable of sexual reproduction are termed yeastlike fungi. enlarged central cell All organisms are referred to here as yeasts. o Rapidly growing phaeoid colony that is cottony and dirty gray to black GENERAL CHARACTERISTICS Macroscopic characteristics Figure 2.9 Curvularia sp. o Colony color—ranges from white to cream or tan, with some species forming a pink- to salmon- Phoma color o Pycnidia appearing as black fruiting bodies lined inside with short conidiophores o Colony texture—varies o Rapidly growing gray to brown colonies ▪ Mucoid and flowing across the culture plate Pithomyces ▪ Butter-like o Barrel shaped singly on short conidiophores ▪ Velvetty to wrinkled o Transverse and longitudinal cross-walls and often echinulate Candida Characteristics Disease ranges from superficial disease to systemic. Premier cause of yeast infection in the world o Candida albicans May be opportunists in immunocompromised people Recovered as normal biota from multiple sites o Skin, oral, mucosa, digestive tract, vagina Candida Species Candida albicans o Thrush o Recognized as an indicator of immunosuppression Figure 3.0 Phoma sp. Figure 3.1 Pithomyces sp. o Can evolve into serious infection in patients receiving long-term antibiotics or other Ulocladium chemotherapeutic agents o Occasional subcutaneous infections Candida glabrata o Dark multicelled conidia on sympodial conidiophores o May account for 21% of all urinary yeast isolates o Angular cross-walls with echinulate surfaces o Tend to be aggressive and difficult to treat with traditional antifungal therapy o Colonies are brown to olivaceous to black Candida auris o An emerging multidrug-resistant yeast linked to high mortality rates in hospital-acquired infections worldwide o In hospital setting, likely transmitted patient to patient o Colonized patients should be placed on contact precautions (recommendation of the CDC). Other notable species o Candida krusei o Candida tropicalis o Candida parapsilosis ▪ Becoming major cause of outbreaks of nosocomial infections Species can be distinguished by differences in their carbohydrate assimilation patterns and other secondary procedures. Figure 3.2 Ulocladium sp. DAWARA 4  MYCOLOGY AND VIROLOGY LECTURE: AGENTS OF OPPORTUNISTIC MYCOSES & YEAST INFECTIONS INSTRUCTOR: MR. CHARLIE CRUZ, RMT, SM (ASCP) – COLLEGE OF MEDICAL LABORATORY SCIENCE A.Y. 2024-2025 DIFFERENTIATING CHARACTERISTICS OF YEAST Rhodotorula Noted for bright salmon-pink color Growth at Corn Meal Agar Resemble Cryptococci 37° 42° 45° Pseudohyp True Arthroconi hae Hyph dia Cycloheximi Ure Nitrat Possess a capsule and are urea-positive C C C ae de a e Some species also nitrate-positive Candida Known to cause opportunistic infections C. albicans + + + + + - R - - C. + - - + + - R - - Pneumocystis dubliniensis P. jirovecii C. glabrata + + + - - - S - - o Formerly considered a protozoa but nucleic acid sequencing showed the organism is a fungus C. + + - + - - R - - o Most infections occur by 2 to 4 years old. guilliermond ▪ Asymptomatic in healthy immunocompetent individuals ii Symptoms in immunocompromised patients C. krusei + + - + - - S V - o Fever, nonproductive cough, and difficulty breathing C. lusitaniae + + + + - - V - - o Chest radiographs may show interstitial infiltrates. C. + - - + - - S - - Life cycle parapsilosis o Nonfilamentous fungus C. + + + + + - S - - o Transmission stellatoidea ▪ Respiratory route C. tropicalis + + + + - - V - - o Three stages Cryptococc ▪ Trophozoite us C. albidus - - - - - - S + + Multiply by binary fission C. + - - - - - S + - ▪ Precyst neoformans ▪ Cyst C. gattii + - - - - - R + - Infective stage Trichosporo + V - + + + R + - Diagnosis n spp. o Biopsy and aspirates of the lung +, Positive; −, negative; R, resistant; S, sensitive; V, variable. ▪ Stains using Giemsa and Grocott’s methenamine silver (GMS) o Calcofluor white Cryptococcus ▪ Blue-white color when stained and viewed under ultraviolet (UV) light Opportunistic infection o Meningitis, pneumonia, and septicemia Stains of Pneumocystis Characteristics o Mucoid appearance due to capsule ▪ India ink test Black background with clear halos around organism Being replaced by cryptococcal antigen tests o Blastoconidia only Figure 3.3 India ink preparation is used primarily to examine cerebrospinal fluid for the presence of the encapsulated yeast Cryptococcus neoformans. This is an India ink preparation from an exudate containing encapsulated budding yeasts Figure 3.4 A, Pneumocystis jirovecii cysts (silver stain). B, P. jirovecii (Giemsa stain). Note the circular arrangement of intracystic bodies within a faint outline of the cyst wall in the center of the field. DAWARA 5 MYCOLOGY AND VIROLOGY INTRODUCTION TO MEDICAL VIROLOGY STUDY NIYO MGA TABLE LIST OF VIRUSES CAUSING DISEASE Characteristics of Viruses Structure Viruses contain a viral genome of RNA or DNA (not both) and a protein coat known as a capsid. Genome + capsid = known as a nucleocapsid Entire virus particle = virion Envelope ❖ Sometimes an envelope surrounds the capsid. ❖ May be more susceptible to environmental changes Other virions do not possess an envelope. Morphologic types of virions ❖ Helical ❖ Icosahedral (a geometric shape with 20 triangular sides) ❖ Complex ❖ When present, the envelope masks the shape of the virion. Size extremes of human viruses ❖ Poxviruses are the largest, poliovirus is the smallest. (eradicated na si poliovirus) Viral Taxonomy Viral Replication Originally, viruses were classified by the Viruses are obligate intracellular parasites. diseases they caused and their host range. ❖ Organisms must be inside a living cell Now, classification is as follows and then use the host machinery to ❖ Orders replicate. ❖ Families based on genome type Steps of infection ❖ Genera (RNA or DNA) ❖ Absorption or attachment ❖ Species - Specific for certain cell ❖ Number of strands in the genome (ds or receptors ss) Receptor distribution will ❖ Morphology determine entry point into the ❖ Presence or absence of an envelope host. ❖ Nucleotide sequences Lubiano, Ristian Aaron T. 1  INTRODUCTION TO VIROLOGY - Most host cell receptors New virions are then released are glycoproteins. by lysis if they are naked - Virus attaches to viruses or by budding if they are specific receptors on enveloped viruses. the susceptible cell by During budding, part of the host means of specialized cell plasma membrane structures on its surface surrounds the viral capsid and known as adhesion becomes the viral envelope. molecules. ❖ Penetration Laboratory Diagnosis of Viral - Naked virions Direct penetration into the cell Infections membrane Examples of Available Viral Testing Methods - Enveloped virions Fusion into the cell Laboratory diagnosis of viral infections Endocytosis (enters cell in a ❖ Varying levels of viral testing are cytoplasmic vacuole) available. - Endocytosis Examples of viral testing methods - Uncoating ❖ Cell cultures Virus loses its protein coat, ❖ Rapid tests releasing the genome. - Viral antigen tests, i.e., IF or Release the genome from enzyme immunoassay (EIA) (for capsid quick diagnostic because it has RNA viruses release their rapid sTAT) genome into the cytoplasm. DNA viruses release their Specimen Selection, Collection, and Transport genome into the host nucleus. - Replication and translation When are viruses most likely to be present? Viral genome then directs the ❖ During early stages of infection host cell to make viral proteins ❖ As early as possible, which may be and replicate the viral genome. before symptoms occur Metabolism of host cell may - May decrease rapidly 3 days be completely stopped. after onset of symptoms Specimens ❖ Assembly or maturation of the virus ❖ Aspirated secretions particles ❖ Swabs: sometimes easier to obtain - Capsid protein subunits - Dacron or rayon aggregate to form capsomers - No calcium alginate swabs and then capsomers combine to ❖ Samples should be kept moist. form the capsid. - Desiccation can destroy fragile - The capsid and genome viruses. associate to form the - Usually use viral transport nucleocapsid. medium. - Release or egress Respiratory, swab, and tissue samples. Lubiano, Ristian Aaron T. 2  INTRODUCTION TO VIROLOGY Buffered isotonic solution with a protein such as albumin, gelatin, or serum, to protect less stable viruses; sometimes with antibiotics to inhibit contamination - Transport media is not indicated for blood, bone marrow (BM), cerebrospinal fluid (CSF) (lumbar puncture), amniotic fluid (amniocentesis), urine, pericardial fluid (pericardiocentesis), and pleural fluid (thoracentesis). ❖ Samples are best cultured immediately. - Refrigerate if necessary at 4° C. Freeze only if longer than 4 days and only at −70° C. Never freeze specimens at −20° Methods in Diagnostic Virology C because this temperature facilitates the formation of ice Four major methods crystals that will disrupt the ❖ Direct detection of virus in clinical host cells and result in loss of specimen viral viability. ❖ Nucleic acid-based detection - Repeated freeze thawing cycles ❖ Isolation of the virus in cell culture is not recommended since it too ❖ Serologic assays to detect viral can result in loss of viral antibodies (plasma cells) viability. - May take 3 to 4 weeks to make antibodies Appropriate Specimens for Maximum Recovery ❖ Direct detection - Microscopy Best if collected from affected site - Cytopathic effect (CPE) from cell Examples of specimen types scrapings ❖ Respiratory specimen - Bright-field light microscopy ❖ Lesion aspirates or surface swabs (for - Direct fluorescent antibody lesions) (DFA) staining ❖ Stool - EIAs ❖ Urine - Nucleic acid-based assays ❖ Blood ❖ Tissue biopsy ❖ CSF A Card Format Rapid Immunochromatographic Membrane Assay Tests Available for Common Viral Pathogens and Specimens for Culture Lubiano, Ristian Aaron T. 3  INTRODUCTION TO VIROLOGY ❖ Finite cell cultures—can divide but passage is limited to about 50 generations - Example is human neonatal lung (HNL) cells. - Diploid cell lines - Greater than 75% normal 2 lines for positive chromosomes 1 line for negative ❖ Heteroploid cell lines (continuous established cell lines) Viral Isolation - Less than 75% have normal chromosomes. Isolating viruses is still the gold standard - Capable of infinite passage against which all other methods are compared. - HEp-2, A549 (human lung ❖ Three methods are used for viral carcinoma cell line), or Vero isolation cells - Cell culture (most commonly used) Cell Cultures Commonly Used in the Clinical Virology - Animal inoculation Laboratory - Embryonated eggs Cell Culture Also known as tissue culture ❖ Growing cells in flask or test tube to detect viral infection Three types CPE on Cell Cultures ❖ Primary cells (primary monkey kidney cells) Herpes simplex virus (HSV) - Obtained from tissue removed ❖ Focal CPE with plaques or clusters of from an animal infected cells - Tissue is finely minced and then ❖ Grows in most cell lines treated with an enzyme i.e., Cytomegalovirus (CMV) trypsin) to disperse individual ❖ HSV-like CPE cells further. ❖ Grows slower but only on diploid - Cells are then seeded on a fibroblasts surface to form monolayer. - Only one cell division occurs. - Viability maintained via periodically removing cells from surface, diluting them, and placing them into a new container (process is known as splitting or passaging) HSV Showing CPE on Rabbit Kidney Cells Lubiano, Ristian Aaron T. 4  INTRODUCTION TO VIROLOGY Centrifugation-Enhanced Shell Vial Culture Cells grown on a round coverslip in a shell vial covered in culture media CMV Showing CPE on Diploid Fibroblast Cells Sample is added, and centrifugation is used to promote viral absorption to cells. ❖ Incubated 24 to 48 hours Test using immunofluorescence Serologic Assays Detection of circulating antibodies to viruses after exposure ❖ Immunoglobulin M (IgM) or CONT.. immunoglobulin G (IgG) Adenovirus Measure host response rather than directly ❖ Produces cell rounding detecting the virus. - Diffuse or focal, appearing like a ❖ Dependent on the ability of host to cluster of grapes respond and produce antibodies Respiratory syncytial virus (RSV) Advantages (quick, nonhazardous) ❖ Syncytial formation ❖ Detects nonculturable viruses - Giant multinucleated cells Determines immune status ❖ Monitors patients who are Adenovirus Showing CPE on HeLa Cells immunosuppressed ❖ Good for epidemiologic or prevalence studies Disadvantages (lack of paired sera, antibodies are not always diagnostic) ❖ Does not work well in severely immunocompromised ❖ Reactivating viruses already have antibodies. CONT.. ❖ Infants with maternal antibody Influenza ❖ Rarely have paired sera (fourfold rise in ❖ CPE generally not detected antibody titer) ❖ Infected cells express a viral ❖ Cross-reactivity of other antibodies hemagglutinin (H) protein on their surface that binds to red blood cells (RBCs). ❖ Hemagglutination or hemadsorption test - Clumps RBCs to infected cells Hemagglutinin from virus Lubiano, Ristian Aaron T. 5