Clinical Microbiology - Mycology Chapters 58-63

Urease test

Heavy inoculum placed on Christensen's urea agar, incubated at 35-37ºC​ Rapid if positive within several hours​ Crypotococcus neoformans is rapid positive​ Not rapid if positive in 1-2 days​ Rhodotorula, some Candida species, and Trichosporon are not rapidly positive​

Cryptococcus neoformans - Approach to ID

India Ink​ Birdseed agar - shows substrate utilization patterns and pigment production​ Serologic testing​ ELISA​ Plasma and urine to find cryptococcal antigen​ Rapid urease test and failure to utilize inorganic nitrate - presumptive ID​

Geotrichum candidum: Approach to ID

Initially appears as a white to creamcolored, yeastlike colony Some isolates may appear as white, powdery molds. Hyphae are septate and produce numerous rectangular to cylindrical to barrel-shaped arthroconidia Arthroconidia do not alternate but are contiguous

Sporothrix spp.

. has been shown to be a complex of numerous species. Those involved in human infection include S. schenckii, Sporothrix brasiliensis, Sporothrix globosa, and Sporothrix luriei. Sporothrix spp. have a worldwide distribution, and their natural habitat is living or dead vegetation. Humans acquire the infection (sporotrichosis) through trauma (thorns, splinters, bites, or scratches), usually to the hand, arm, or leg. The infection is an occupational hazard for farmers, nursery workers, gardeners, florists, and miners; it is commonly known as rose gardener's disease. Infections with S. brasiliensis have been transmitted from the bites or scratches of stray cats. Pulmonary sporotrichosis rarely occurs as a result of inhalation of spores. Pathogenesis: Sporothrix spp., also dimorphic fungi, are often associated with chronic subcutaneous infections. The primary lesion begins as a small, nonhealing ulcer, often of the index finger or the back of the hand. The infection is characterized by the development of nodular lesions of the skin or subcutaneous tissues at the point of contact and later involves the lymphatic channels and lymph nodes that drain the region. Pulmonary infection may be seen in patients who inhale the spores of Sporothrix spp

Laboratory Safety

A common sense approach to the handling of specimens protects the laboratory from contamination and workers from becoming infected.​ Mold cultures and clinical specimens must be handled in a class II BSC​ Yeast cultures may be handled on the bench top.​ An electric incinerator or a gas flame is suitable for decontaminating a loop used to transfer yeast cultures. ​ Cultures of organisms suspected of being pathogens should be sealed with tape to prevent laboratory contamination and should be autoclaved as soon as the definitive identification is made. ​

Cryptococcus neoformans - Pathology & Spectrum of disease

Acute, subacute, or chronic fungal infections - differences depend on yeast variant and host immune status​ Can cause pulmonary infections, cryptococcal meningitis, disseminated disease, endocarditis, hepatitis, renal infection, pleural effusion, may colonize respiratory tract​ May have a characteristic polysaccharide capsule which contains compounds not recognized by phagocytes​ Melanin production by phenoloxidase, which may decrease immune system response​ Appears as a single- or multiple-budding thick-walled yeast 2-15 µm surrounded by a wide, refractile polysaccharide capsule (Figure 62-6), extreme variation in the size of the yeast cells​ CAD antigen test used for CSF or serum, but may cross-react with Trichosporon​

Laboratory Diagnosis: Tissue, bone marrow, sterile body fluids

All tissues should be processed before culturing by mincing; not grinding is critical​ Tissue pieces should be pressed into the appropriate culture media so that they are partially embedded, and the cultures should be incubated at 30°C for 21 days (incubation may be extended if clinical suspicion of a mycotic disease is high)​. Bone marrow may be collected in a heparinized syringe. The sample should be placed directly onto the surface of appropriate culture media and incubated​. Sterile body fluids should be concentrated by centrifugation before culturing, and at least 1 mL of specimen should be placed on the surface of appropriate culture media. An alternative is to place bone marrow and other body fluids in an Isolator tube (Pediatric Isolators are recommended) and process it as a blood culture​

Polyene Macrolide Antifungals

Amphotericin B ​ Produced by Streptomyces nodosus​ Commonly used intravenously to treat invasive Aspergillosis, Candida, Cryptococcus, and Mucorales​ Binds ergosterol, can cause renal insufficiency​ Resistant organisms: Pseudoallescheria boydii, Scedosporium prolificans, Aspergillus terreus, Trichosporon species, and most Fusarium species​ Nystatin​ Produced by Streptomyces noursei​ Not absorbed by GI tract​ Used to treat oral or vulvovaginal candidiasis​ Parenteral use is toxic​ Griseoflulvin​ Produced by Penicillium​ Binds microtubular proteins for mitosis​ Taken orally to treat dermatophytoses​ 5-fluorocytosine​ Acts synergistically with amphotericin B​.

Yeast- Laboratory Diagnosis

Antigen detection​ all systemic fungal infections can be detected using fungal cell wall antigen Beta-1,3-glucan​ Molecular methods​ PNA FISH for C. albicans, C. glabrata, and C. tropicalis​ Real-time PCR​ Multiplex PCR​

Phaeohyphomycosis

Any infection caused by a dematiaceious organism​ Molds, brownish, yeastlike cells, pseudohyphae, and hyphae​ Subcutaneous, localized, or systemic​ "phaeohyphomycotic cysts, progressive soft tissue infection, ​ brain abscess, sinusitis, endocarditis, mycotic keratitis, ​ pulmonary infection, and systemic infection"​ Neurological infections associated with: Cladophialophora bantiana, Rhinoladiella mackenziei, Verruonis gallopava, Exophiala dermatitidis, ​ Also associated with phaeoyphomycosis: Alternaria, Exsrohilum, Bipolaris, Exophiala jeanslmei, Exophiala dermatitidis, Exophila spinifera, Cladophialophora bantiana, and Curvularia species ​

Mucorales: Approach to ID

Approach to identification- Mucorales are characterized by the production of branched, nonseptate, wide mycelia (10-20 mm). Rhizopus spp. have unbranched sporangiophores with rhizoids that appear opposite the point where the stolon arises, at the base of the sporangiophore. Mucor spp. are characterized by sporangiophores that are singularly produced or branched and have a round sporangium at the tip filled with sporangiospores. They do not have rhizoids or stolons, which distinguishes this genus from the other genera of the Mucorales. Lichtheimia spp. (Absidia spp.) are characterized by the presence of rhizoids that originate between sporangiophores. The sporangia of Lichtheimia spp. are pyriform and have a funnelshaped area (apophysis) at the junction of the sporangium and the sporangiophore Lichtheimia produce white, fast-growing wooly colonies that become grayish brown

Systemic Mycoses

Blastomyces species Coccidiodes species Emmonsia species Histoplasma capsulatum Paracoccidiodes brasiliensis Talaromyces marneffei Sporothrix species Epidemiology Pathogenesis and spectrum of disease Specimen collection, transport, and processing Laboratory diagnosis: Direct detection methods Antigen-protein Molecular methods Cultivation Serologic testing

Candida spp. - Laboratory ID

Budding yeast cells 2-4 µm and/or pseudohyphae with regular points of constriction resembling sausage links​ True septate hyphae produced by C. albicans and C. dubliniensis​ Presence of large numbers of blastoconidia, hyphae, and pseudohyphae forms may be clinically significant​ C. glabrata are smaller at 1-4 µm​ Colony morphology not useful for differentiating Candida species although Candida albicans can have "feet"​.

Candida spp. - Approach to ID

CHROMagar Candida​ Germ tube test - 3 hours to complete​ C. albicans will produce germ tubes or chlamydoconidia​ C. dublineiensis germ tube positive also​ C. tropicalis makes pseudo-germ tubes​ Non-albicans Candida ID'd by fermentation of assimilation of certain carbohydrates​ C. glabrata ferments glucose and trehalose​ C. tropicalis ferments sucrose and maltose​ C. albicans has chlamydoconidia on cornmeal agar containing 1% Tween 80 and trypan blue after incubation at room temperature for 24-48 hours​ C. albicans have "feet" on BAP​ C. glabrata - rapid trehalose test, failure to produce a germ tube, maltose hydrolysis in 30-second dipstick assay​.

Pneumocystis jiroveci

Causes pneumocystis pneumonia (PCP) in immunocompromised patients Opportunistic, atypical fungus Responds to antiprotozoal drugs but not to antifungal drugs in patients with PCP Cultivation and propagation is difficult Three life cycles - trophic form (similar to a trophozoite), sporozoite (precyst), and ascus (cyst, diagnostic form) Cell membrane contains cholesterol instead of ergosterol, cell wall is flexible and susceptible to osmotic disturbances, sequencing shows greater homology with fungi than with protozoa

Malassezia spp.

Causes tinea versicolor - superficial, brownish, scaly areas on light-skinned people and lighter areas on dark-skinned people​ Can cause disseminated infections in young infants or adults receiving lipid replacement therapy​ Detect through direct microscopic examination of skin scrapings - oval or bottle shaped cells with monopolar budding (Figure 62-7) commonly describes as "spaghetti and meatballs"​ Recovery not required for diagnosis, to culture agar must be overlaid with long-chain fatty acid such as olive oil​ Growth in presence of long-chain fatty acid with "bowling pin" or "pop bottle" morphology = sufficient ID​ ​

Laboratory Diagnosis: CSF

Cerebrospinal fluid collected for culture should be filtered through a 0.45-mm membrane filter attached to a sterile syringe. After filtration, the filter is removed and placed on the surface of an appropriate culture medium with the inoculum side down. ​ Alternatively, the specimen may be centrifuged and the concentrated sediment used to inoculate the culture medium. ​ If plated using a filter, the filter should be moved to another location every other day​ If less than 1 mL of specimen is submitted for culture, it should be centrifuged, and 1-drop aliquots of the sediment should be placed on several areas on the agar surface.​ If prompt processing is not possible, samples should be kept at room temperature or placed in a 30°C incubator. CSF specimens should never be refrigerated​

Dematiaceous: Laboratory Diagnosis

Chromoblastomycoses​ Scrapings from crusted lesions with 10% KOH "show musiform cells (aggregation of dark brown cells that resemble stones in a stone wall) or sclerotic bodies" 4-10 µm with fission planes that resemble copper pennies​ Mycetoma and phaeohyphomycosis​ Slides show "yellowish brown, septate to moniliform hyphae (string of beads), with or without budding yeast cells"​ Exophiala species show dematiacious yeasts​ Smears of granules from Pseudoallescheria boydii are white to yellow, 0.2-2 mm in diameter, "loosely arranged, intertwined septate hyaline hyphae cemented together"​ Hematoxylin-eosin or unstained histology slides - pigmented hyphae​ Methenamine silver - stains fungal elements black, impossible to differentiate hyaline from dematiaceous molds​ Fontana-Masson stain - confirms presence of pigmented hyphae​ Serologic testing - useful for allergy testing​ Molecular methods ​ not routinely used but can be useful in normally sterile specimens​ Sequencing of ribosomal genes can be helpful​ MALDI-TOF MS - rapid identification​.

Dermatiaceious Fungi: Mycetoma

Chronic granulomatous infection usually in lower extremities​: Symptoms - "swelling, purplish discoloration, tumorlike deformities of subcutaneous tissue, and multiple sinus tracts that drain purulent material containing yellow, white, red, or black granules"​. Granule color varies by causative organism​ Progresses to the bone, muscle, or other tissues and can require amputation​. Tropical and subtropical regions​. Bacterial mycetomas - Aerobic actinomycetes including Nocardia, Actinomadura, and Streptomyces​. Fungal mycetomas - variety of septated fungi with hyphae, "white grain" or "black grain" mycetomas​ Hyaline septate molds (not dematiaceious) ​ White grain mycetomas - Pseudoallescheria boydii and Acremonium species ​ Black grain mycetomas - Exophiala jeanselmei, Curvularia species, Cladophialophora bantiana, Trematosphaeeria grisea (AKA Madurella grisea), and other Madurella species​ Madurella mycetomatis is most common​ Pseudoallescheria boydii most common in US​ Organisms found in soil, standing water, sewage, acquired through traumatic inoculation​

Azole antifungals

Clotrimazole and miconazole​ Synthetic imidazoles for topical or intravaginal application​ Fluconazole​ Exceptionally soluble in water oral or intravenous administration​ Excellent activity against Candida species and Cryptococcus​ Therapeutic levels reachable in CNS​ Candida krusei and Rhodotorula are resistant​ No activity against Aspergillus, Fusarium, or Mucorales​ Ketoconazole​ Oral or topical imidazole, an alternative for amphotericin B​. Is fungistatic in vivo​ Itraconazole​ Similar in spectrum to ketoconazole​ Voriconazole​ Expanded spectrum compared to itraconazole​ Mucorales are resistant​ Posaconazole ​ Oral, effective against dermatophytes but less effective against Fusarium species and Pseudoallescheria boydii​

Acremonium spp.: Approach to ID

Colonies of Acremonium spp. are rapid-growing and also may appear yeastlike when initial growth is observed. Mature colonies become white to gray to rose or reddishorange. Microscopically, small septate hyphae that produce single, unbranched, tubelike phialides are observed.

Fusarium: Approach to ID

Colonies of Fusarium spp. grow rapidly, within 2 to 5 days, and are fluffy to cottony and may be pink, purple, yellow, green, or other colors, depending on the species. Microscopically, the hyphae are small and septate and give rise to phialides producing either single-celled microconidia, usually borne in gelatinous heads similar to those seen in Acremonium spp. or large, multicelled macroconidia that are sickle- or boat-shaped and contain numerous septations The most common medium used to induce sporulation is cornmeal agar. The keys to identification of Fusarium spp. are based on growth on potato dextrose agar.

Paecilomyces spp.

Colonies of Paecilomyces spp. are often velvety, tan to olive brown, and somewhat powdery. Microscopically, Paecilomyces spp. resemble Penicillium spp. in that a penicillus is formed. The phialides of Paecilomyces spp. are long, delicate, and in contrast to the more blunted phialides of Penicillium spp. The penicillus produces numerous chains of small, oval conidia that are easily dislodged

Penicillium: Approach to ID

Colonies of Penicillium spp. are most commonly shades of green or blue-green, but pink, white, or other colors may be seen. The surface of the colonies may be velvety to powdery because of the presence of conidia. Microscopically, hyphae are hyaline and septate and produce brushlike conidiophores (i.e., penicilli). Conidiophores produce metulae from which flask-shaped phialides producing chains of conidia arise

Scopulariopsis spp.

Colonies of Scopulariopsis spp. initially appear white but later become light brown and powdery. Conidia are large, have a flat base, and are rough-walled Scopulariopsis brumptii has been reported to have caused a brain abscess in a liver transplant patient and invasive infection in bone marrow recipients. S. candida and S. acremonium have been identified in association with invasive sinusitis.

Laboratory Diagnosis: Eye (corneal scraping or vitreous humor)

Corneal scrapings collected by a physician should be placed directly onto microscopic slides and inoculated onto noninhibitory media in either a X- or C-shaped pattern​. Vitreous humor aspiration should be concentrated by centrifugation, and the sediment should be used for smears and culture. ​ Samples should be processed as soon as possible and stored at room temperature.​ Media containing cycloheximide should be avoided to prevent inhibition of potential isolates.​

Cryptococcus neoformans

Cryotococcus neoformans-Cryptococcus gattii complex​ 2 species, 5 serotypes​ Canavanine-glycine-bromothymol blue (CCB) agar used to differentiate serotypes - color change from yellow-green to cobalt blue​ Cryptococcus associated with avian (pigeon) excretia, aerosolization is an important mode of transmission​ India Ink to detect capsule in Cryptococcus neoformans​ Cultured on routine fungal media without cycloheximide at 25-30ºC​ To recover C. neoformans from CSF - 0.45 mm filter with sterile syringe or plating pellet after centrifugation​ Grow in culture in 1-5 days, smooth white mucoid to creamy colony but on inhibitory mold agar appears as golden yellow, nonmucoid​.

Phaeohyphomycosis - Approach to ID

Curvularia - multicellular conidia with septation in the horizontal axis, hyphae are dematiaceous and septate, conidiophores are bent (genticulate) where conidia are attached, conidia are golden-brown, multicelled, and curved with a central swollen cell, the ends are lighter in color than the swollen cell, (see Figure 60-11)​ Exophiala - dematiaceous, yeastlike cells (Figure 60-12) but daughter cells are produced by annelids instead of true buds, feltlike filamentous colonies have cylindrical conidiophores with tapered tips​ E. jeanselmei - utilized potassium nitrate, can grow at 37ºC​ E. dermatitidis - does not utilize potassium nitrate, can grow at 37ºC and also at 40-42ºC​

General characteristics of Dermatiaceious Fungi

Dematiaceious = dark coloration due to production of melanin​ Superficial and subcutaneous mycoses, sometimes invasive or disseminated disease​ Found everywhere in nature - saprophytes, plant pathogens​ Humans and animals are accidental hosts usually due to traumatic inoculation​ Slow growing (7-10 days) or rapid growing (less than 7 days)​ In nonsterile sources the significance can be questionable​ Can be contaminants on plates​ sometimes found growing in unopened packages of media from company​ Sometimes grow on plates if dropped on the floor (picking up contaminant) and allowing to grow further​ If not growing on streak line/inoculation zone it's likely a contaminant (Ex: edge of plate, not on streak line)​. Epidemiology: Superficial infections​ Mycetoma​ Chromoblastomycosis​ Phaeohyphomycosis ​

Opportunistic Molds: Laboratory Diagnosis

Direct detection methods Stains- demonstrate septate hyphae that usually show evidence of dichotomous branching, often of 45 degrees Some hyphae may have rounded, thick-walled cells. Antigen-protein: the galactomannan assay, targets antigens of Aspergillus spp. the assay may yield false-positive results because of cross-reactivity with other nonAspergillus molds, including Talaromyces marneffei, Histoplasma capsulatum, Fusarium oxysporum, Paecilomyces spp., and Alternaria spp The beta-D-glucan assay is designed to detect antigens common to all clinically important fungi. Molecular methods- Nucleic acid amplification assays are not commonly performed to detect or identify these fungi. Multiplex amplification and real-time PCR assays have been developed for the detection of systemic aspergillosis in respiratory specimens, blood, tissue, and CSF. MALDI-TOF MS- Has potential to provide quick and accurate species identification. Cultivation - Correlation with biopsy results is the best means of establishing the significance of an isolate. Most Aspergillus spp. are susceptible to cycloheximide. Therefore specimens submitted for recovery or subculture of these species should be inoculated onto media that lack this ingredient Serologic testing has no value for diagnosis

Pneumocystis jiroveci: Epidemiology

Epidemiology: Found worldwide Transmitted person-person via airborne particles Immunocompetent people are reservoirs for transmission to immunocompromised Most common opportunistic infection in HIV and AIDS patients HAART therapy decreases incidence, but many patients do not respond to therapy or comply

Dermatophytes: Laboratory Diagnosis

Direct detection methods- Calcofluor white or potassium hydroxide preparations reveal the presence of hyaline septate hyphae or arthroconidia. Microscopic examination of infected hairs may reveal the hair shaft to be filled with masses of large arthroconidia (4-7 mm) in chains, characteristic of an endothrix type of invasion. The hair may show external masses of spores that ensheath the hair shaft; this is characteristic of the ectothrix type of hair invasion. Hairs infected with Trichophyton schoenleinii reveal hyphae and air spaces within the shaft Antigen-protein- Antigen-protein-based assays are not useful for the detection . Molecular methods- not routine Cultivation- Because the dermatophytes generally present a similar microscopic appearance in infected hair, skin, or nails, final identification typically is made by culture.

Entomophthorales: Laboratory Diagnosis

Direct detection methods- The Splendore-Hoeppli phenomenon (formation of asteroid bodies), the formation of eosinophilic crystals that appear radiate, starlike, asteroid, or club-shaped around a fungal infection, are associated with hyphae in tissue sections stained with hematoxylin-eosin. This is highly indicative of entomophthoromycosis. Antigen-protein- No antigen tests Molecular methods- A single polymerase chain amplification assay has been developed for the diagnosis of Basidiobolus entomophthoromycosis Cultivation- Tissue samples should be sliced or minced and cultivated on potato dextrose agar or Sabouraud agar. cultures should be incubated at 37°C (Conidiobolus spp.) with a second culture incubated at 25°C to 30°C (Basidiobolus sp.) Approach to identification- B. ranarum colonies appear slightly yellow pigmented with radial folds. No aerial hyphae is present. After 7 to 10 days of growth the culture will produce aseptate mycelia with free uninucleated hyphal elements. Conidiobolus spp. is a fast-growing fungus that produces hyaline, radially folded colonies that initially appear waxy and become powdery when mycelia begin to develop. The primary conidia are spherical and have prominent papilla (small bumps). Villose (hairlike spines) conidia appear as the colony ages. C. coronatus can be differentiated from the other species based on the absence of zygospores when grown on potato dextrose agar. Serologic testing- No serologic tests are available

Mucorales: Laboratory Diagnosis Molecular Methods

Direct detection methods-The diagnosis of mucormycosis is primarily based on direct examination. Stains- calcofluor white and potassium hydroxide preparation. If the sample is too thick, a false negative result may occur because of insufficient dissociation of tissues. Branching, broad-diameter, predominantly nonseptate hyphae are observed. Antigen-protein-Antigen-protein-based assays are not used for the diagnosis of mucormycosis. Molecular methods- Nucleic acid testing may be performed on formalin-fixed, paraffin-embedded, fresh or frozen tissue sample. PCR amplification of the internal transcribed spacer, as well as seminested PCR of the 18S ribosomal ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) sequence, has been used to confirm identification in samples that have been identified as histopathology positive. A real-time PCR assay has also been developed that amplifies the cytochrome b gene. Fluorescent in situ hybridization that does not require DNA extraction or amplification has the potential to improve the identification of the fungi. The technique uses synthetic oligonucleotides specific to the 5.8S and 18S ribosomal ribonucleic acid (rRNA) of the fungi.

Epidermophyton floccosum

E. floccosum, the only member of the genus Epidermophyton, is a common cause of tinea cruris and tinea pedis. Specimens submitted for dermatophyte culture should not be refrigerated before culture, and cultures should not be stored at 4°C Direct examination of skin scrapings using the calcofluor white or potassium hydroxide preparation, the fungus is seen as fine branching hyphae. E. floccosum grows slowly; the growth appears olive green to khaki, with the periphery surrounded by a dull orange-brown. After several weeks, colonies develop a cottony white aerial mycelium that completely overgrows the colony; the mycelium is sterile and remains so even after subculture. Microscopically, numerous smooth, thin-walled, club-shaped, multiseptate (2-4 µm) macroconidia are seen They are rounded at the tip and are borne singly on a conidiophore or in groups of two or three. Microconidia are absent, spiral hyphae are rare, and chlamydoconidia are usually numerous The absence of microconidia is useful for differentiating this organism from Trichophyton spp.; the morphology of the macroconidia (smooth, thin walled) is useful for differentiating it from Microsporum spp

Mucorales: Epidemiology & Pathogenesis, Spectrum of Disease

Epidemiology and pathogenesis -The Mucorales are an important cause of morbidity and mortality in immunocompromised patients, particularly patients with diabetes mellitus. The organisms involved have a worldwide distribution and are commonly found on decaying vegetable matter or old bread (bread mold) or in soil. The organism is generally acquired by inhalation or ingestion of spores or through percutaneous routes. Other immunocompromised patients who are susceptible to infection with Mucorales include patients with hematologic malignancies such as acute leukemia and stem cell, kidney, and liver transplant patients. Immunocompetent individuals may acquire skin infections with these fungi after traumatic injection with contaminated material. Spectrum of disease- uncontrolled diabetes mellitus and transplant patients who are undergoing prolonged corticosteroid, antibiotic, or cytotoxic therapy. The organisms that cause mucormycosis (an infection caused by Mucorales) have a marked propensity for vascular invasion and rapidly produce thrombosis and necrosis of tissue. One of the most common presentations is the rhinocerebral form Perineural invasion also occurs in mucormycoses and is a potential means of retroorbital spread Other types of infection involve the lungs and gastrointestinal tract. Localized skin infections in immunocompetent patients with severe burns and infections of subcutaneous tissue in patients who have undergone surgery.

Dermatophytes: Epidemiology & Pathogenesis, Spectrum of Disease

Epidemiology and pathogenesis- The dermatophytes break down and utilize keratin as a source of nitrogen. They usually are incapable of penetrating the subcutaneous tissue, unless the host is immunocompromised. Species of the genus Trichophyton are capable of invading the hair, skin, and nails. Microsporum spp. involve only the hair and skin Epidermophyton spp. involves the skin and nails. Spectrum of disease: Cutaneous mycoses Usually referred to as tinea (Latin for "worm" or "ringworm") are perhaps the most common fungal infections of humans The gross appearance of the lesion is an outer ring of the active, progressing infection, with central healing within the ring tinea corporis (ringworm of the body); tinea cruris (ringworm of the groin, or "jock itch"); tinea capitis (ringworm of the scalp and hair); tinea barbae (ringworm of the beard); tinea unguium (ringworm of the nail); and tinea pedis (ringworm of the feet, or "athlete's foot").

Entomophthorales: Epidemiology and Pathogenesis; Spectrum of Disease

Epidemiology: The organisms are primarily present in the soil, decaying vegetable material, and animal feces. Infections are more commonly identified in warm climates. Infections associated with Conidiobolus spp. have been identified in Africa, Madagascar, Mayotte, India, China, Japan, and South America. B. ranarum has been associated with infections in India, Myanmar, and Africa. Recent gastrointestinal infections with B. ranarum have been identified in the United States. Spectrum of Disease: Infections from B. ranarum, basidiobolomycosis are primarily localized to subcutaneous tissue of the arms, legs, buttocks, trunk, perineum, face, or neck The infection presents as a woody, hard, painless nodule. Gastrointestinal infections have been noted. Conidiobolus spp. primarily infect the tissue around the nose and on the face. Infection is believed to be through inhalation of the spores in the nasal cavity or inoculation after trauma. After infection, swelling occurs that extends to the nose, cheeks, eyebrows, upper lip, palate, and pharynx.

Coccidioides spp.

Found primarily in the desert portion of the southwestern United States and in the semiarid regions of Mexico and Central and South America The infection (coccidioidomycosis) is acquired by inhalation of the infective arthroconidia. Pathogenesis: Approximately 60% of patients with coccidioidomycosis are asymptomatic and ha The infection may become disseminated, with extension to visceral organs, meninges, bone, skin, lymph nodes, and subcutaneous tissue. self-limited respiratory tract infections. Dissemination occurs most commonly in individuals of dark-skin. Pregnancy also appears to predispose females to disseminated infection

General Features of Fungi

Generally can be categorized into two groups based on the appearance of the colonies formed:​ 1. The yeasts produce moist, creamy, opaque or pasty colonies on media​ 2. Filamentous fungi or molds produce fluffy, cottony, woolly, or powdery colonies​ Several systemic fungal pathogens that exhibit either a yeast or yeastlike phase and filamentous forms are referred to as dimorphic.​ Thermally dimorphic- When dimorphism is temperature-dependent. These fungi produce a mold form at 25°C to 30°C and a yeast form at 35°C to 37°C under certain circumstances.​ Fungi that have more than one independent form or spore stage in their life cycle are called polymorphic fungi. The polymorphic features of this group of organisms are not temperature-dependent.​

Conventional yeast ID methods

Germ tube test usually first step to ID​ Cornmeal agar morphology​ Used to determine whether it produces blastoconidia, arthroconidia, pseudohyphae, true hyphae, and/or chlamydoconidia​ Not useful for uncommon yeasts​ Carbohydrate utilization​ Most commonly used conventional method​ Results compared to charts in lab manuals definitive ID​ Phenoloxidase detection using niger seed agar​ Cryptococcus neoformans can be positive​.

Mucorales: Cultivation

Growth media containing high concentrations of carbohydrates inhibits the production of asexual fruiting bodies that are required for the proper identification of the Mucorales species. It is recommended that media such as potato dextrose, 2% malt, and cherry decoction (acidic) agars be used for cultivation. Growth and development of the mycelium in the Mucorales occurs within 24 to 48 hours. Subcultures should be incubated at 27°C to 30°C. Colonies characteristically produce a fluffy, white to gray or brown hyphal growth that resembles cotton candy and that diffusely covers the surface of the agar within 24 to 96 hours. The hyphae can grow very fast and may lift the lid of the agar plate (also known as a "lid lifter"). The entire culture dish or tube rapidly fills with loose, grayish hyphae dotted with brown or black sporangia.

Microsporum audouinii

M. audouinii was once the most important cause of epidemic tinea capitis among school children in the United States. The organism is anthropophilic and is spread directly by means of infected hairs on hats, caps, upholstery, combs, or barber clippers Infected hair shafts fluoresce yellow-green under a Woods lamp. Colonies of M. audouinii generally grow more slowly than other members of the genus Microsporum (10-21 days), and they produce a velvety aerial mycelium that is colorless to light gray to tan. The reverse side often appears salmon-pink to reddish brown. Atypical vegetative forms, such as terminal chlamydoconidia and antler and racquet hyphae, are the only clues to the identification of this organism.

Microsporum canis

M. canis is primarily a pathogen of animals (zoophilic); it is the most common cause of ringworm infection in dogs and cats in the United States. Children and adults acquire the disease through contact with infected animals. Human-to-human transfer has been reported. Hairs infected with M. canis fluoresce a bright yellow-green under a Woods lamp Direct examination of a calcofluor white or potassium hydroxide preparation of infected hairs reveals small spores (2-3 mm) outside the hair Colonies of M. canis grow rapidly, are granular or fluffy with a feathery border, white to buff, and characteristically have a lemon-yellow or yellow-orange fringe at the periphery. On aging, the colony becomes dense and cottony and a deeper brownish-yellow or orange and frequently shows an area of heavy growth in the center. The reverse side of the colony is bright yellow, becoming orange or reddish-brown with age Microscopically, M. canis shows an abundance of large (15-20 µm X 60-125 µm), spindle-shaped, multisegmented (four to eight) macroconidia with curved ends.These are thick-walled with spiny (echinulate) projections on their surfaces. Microconidia are usually few in number

Dermatophytes: Approach to ID

Microscopically, Trichophyton organisms are characterized by smooth, club-shaped, thin-walled macroconidia with three to eight septa ranging from 4 X8 µm to 8 X 15 µm The macroconidia are borne singly at the terminal ends of hyphae or on short conidiophores; the microconidia (which may be described as "birds on a fence") predominate and are usually spherical, pyriform (teardrop-shaped), or clavate (clubshaped) and 2 to 4 µm

Microsporum gypseum

Microsporum gypseum, a free-living organism of the soil (geophilic) that only rarely causes human or animal infection Infected hairs generally do not fluoresce under a Wood lamp. However, microscopic examination of the infected hairs shows them to be irregularly covered with clusters of spores (5-8 µm), some in chains M. gypseum grows rapidly as a flat, irregularly fringed colony with a coarse, powdery surface that appears to be buff or cinnamon color. The underside of the colony is conspicuously orange to brownish. Microscopically, macroconidia are seen in large numbers and are characteristically large, are ellipsoidal, have rounded ends, and are multisegmented (three to nine) with echinulated surfaces

Laboratory Diagnosis: Molecular Methods

Molecular methods- no molecular method has been accepted as a routine diagnostic tool in clinical mycology​. Reports in the literature deal predominantly with select organisms such as the Advan Dx PNA FISH for the identification of Candida spp. from blood cultures (Advan Dx, Woburn, MA)​. MALDI- Matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF MS) is a biophysical method that significantly reduces the time required to specifically identify fungal organisms​

Non-albicans Candida

Most commonly encountered opportunistic fungal infections​ Candida glabrata ​ resistant to fluconazole and echinocandins​ isolated from endocarditis, meningitis, and disseminated disease​ Candida tropicalis​ Prevalent in patients with hematologic malignancies​ May invade GI, particularly in oncology patients due to secretion of aspartyl proteases and tropiase​ Candida krusei​ inherently resistant to azole antifungals​ Candida parapsilosis​ Fungemia in neonatal ICU's​ selective growth in hyperalimentation solutions and ability to grow on intravascular catheters.

Paracoccidiodes brasillensis

Most commonly found in South America, with the highest prevalences in Brazil, Venezuela, and Colombia. It also has been seen in many other areas, including Mexico, Central America, and Africa The exact mechanism by which paracoccidioidomycosis is acquired is unclear; It has a pulmonary origin and is acquired by inhalation of the organism from the environment. Because mucosal lesions are an integral part of the disease process, it also is speculated that the infection may be acquired through trauma to the oropharynx. Pathogenesis: P. brasiliensis produces a chronic granulomatous infection (paracoccidioidomycosis) that begins as a primary pulmonary infection. It often is asymptomatic and then disseminates to produce ulcerative lesions of the mucous membranes. Ulcerative lesions are commonly present in the nasal and oral mucosa, gingivae, and less commonly the conjunctivae. Lesions occur commonly on the face in association with oral mucous membrane infection. The lesions are characteristically ulcerative, with a serpiginous (snakelike) active border and a crusted surface. Lymph node involvement in the cervical area is common. Pulmonary infection is common, and progressive chronic pulmonary infection is found in approximately 50% of cases. In some patients dissemination occurs to other anatomic sites, including the lymphatic system, spleen, intestines, liver, brain, meninges, and adrenal glands.

Candida albicans

Most commonly isolated yeast​ Oroesophageal candidiasis, intertriginous candidiasis, paronchyia, onychomycosis, respiratory infections, vulvovaginitis, thrush, pulmonary infection, eye infection, endocarditis, meningitis, fungemia, or disseminated infection​ Thrush found in newborns or HIV/immunocompromised patients​ Significance in respiratory tract is difficult to determine​ Pathogenesis is complex, involves adhesion to epithelium, commonly colonize mucosal surfaces but can invade and cause infection​ Can form biofilms with Staphylococcus aureus​ Can undergo phenotypic switching to produce pseudohyphae and hyphae to become filamentous, plays a role in pathogenesis​.

Shared characteristics of fungi

Most fungi share the following characteristics:​ • Chitin in the cell wall ​ • Ergosterol in the cell membrane ​ • Reproduction by means of spores, produced asexually or sexually ​ • Lack of chlorophyll ​ • Lack of susceptibility to antibacterial antibiotics​ • Saprophytic nature (derive nutrition from organic materials)​

Commercially available yeast ID systems

Multiple species identification systems - API-20C AUX​ Microscan Rapid YS​ Vitek biochemical cards​ Others​ CHROMagar Candida​ MALDI-TOF MS​

Rhodotorula spp.

Normal microbiota of skin​ Can resemble Cryptococcus as round, oval-shaped budding yeast with capsules​ Shower curtains, toothbrushes, bathtub grout​ Can cause septicemia, meningitis, peritonitis, and infections of peritoneal dialysis​.

Dematiaceous molds - cultivation

Often represent transient microbiota, inhaled spores, or contaminants, therefore interpret culture results in conjunction with direct examination for fungal elements and histology results​ Superficial infections​ Hortaea werneckii - tinea nigra, ​ Grows very slowly (2-3 weeks), olive to black, shiny, yeast-like​ As culture ages velvety gray aerial hyphae appears​ Piedra hortae - black Piedra​ Grows on media lacking cycloheximide​ Slow-growing, brown to black, produce aerial mycelium​ Cyphellophora - slender, curved, 1-3 septate conidia on collarettes, melanized cultures without budding cells​ Exophiala - a black yeast, morphologically variable, moist at first then become wooly or velvety​ Can grow at 40ºC​ Phialophora - produce phialides and have no budding cells​ Neoscytalidium dimidiatum - rapidly growing black arthroconidia​. Mycetoma​ White grain:​ Pseudoallescheria boydii - grows rapidly in 5-10 days​ White fluffy colony that changes to mousy brownish gray, reverse is tan to dark brown​ Acremonium - grow slowly and make gray to brown colonies​ Black grain:​ Madurella - slow growing, white when young, olive-brown when aging with brown diffusible pigment​ Exophiala jeanselmei - slow growing, yeastlike, darkly pigmented olive to black, as ages develops a velvety appearance with aerial hyphae​ Curvularia - fast growing, fluffy or downy, olive-gray to black colony​ Tinea grisea - slow growing, velvety colonies smooth or radially furrowed, dark gray or olive brown to black, reverse is black, hyphae are septate and nonsporulating​. Chromoblastomycosis​ Cladophialophora, Phialophora species, and Fonsecaea species​ All dematiaceious and slow growing​ Heaped-up slightly folded, darkly pigmented colonies with gray to olive to black and velvety or suedelike appearance, reverse is jet black​ Phaeohyphomycosis​ Rapidly growing dematiaceious fungal colonies appear the same, microscopic exam necessary to speciate​ Alternaria - fluffy, gray to gray-brown or gray-green​ Curvularia - appear as Alternaria​ Bipolaris and Exserohilum - gray-green to dark brown and slightly powdery​ Exophiala jeanselmei and Exophiala dermatitidis - slow growing (7-21 days), shiny, black, yeastlike colonies that become filamentous and velvety with age​ Exophiala spinifera - large stiff conidiophores​ Cladophialophora bantiana - long, poorly-branched conidial chains, can grow at 40ºC​ Rhinocladiella mackenziei - brown conidiophores with elongated conidia on denticles​ Verrucosa gallopava - rusty brown to olive colony with 1-3 septate conidia on small denticles​.

Aspergillus spp.

Pathogenesis and spectrum of disease Aspergillus spp. are capable of causing disseminated infection, as is seen in immunocompromised patients, but also of causing a wide variety of other types of infections, including a pulmonary or sinus fungus ball, allergic bronchopulmonary aspergillosis, external otomycosis (a fungus ball of the external auditory canal), mycotic keratitis, onychomycosis (infection of the nail and surrounding tissue), sinusitis, endocarditis, and central nervous system (CNS) infection

Fusarium and other Hyaline septate opportunistic molds

Pathogenesis and spectrum of disease contd... Acremonium spp. are also recognized as important pathogens in immunocompromised hosts; these have been associated with disseminated infection, fungemia, subcutaneous lesions, and esophagitis. Penicillium spp. are among the most common organisms recovered by the clinical laboratory. In North America, they are rarely associated with invasive fungal disease. However, they may be a cause of allergic bronchopulmonary penicilliosis or chronic allergic sinusitis. Purpureocillium lilacinum (previously Paecilomyces lilacinus) appears to be the most pathogenic species and has been associated with endophthalmitis, cutaneous infections, and arthritis Paecilomyces variotii has also been shown to be an important pathogen, causing endocarditis, fungemia, and invasive disease

Pseudozyma spp.

Plant pathogen, beige to tan, moist, wrinkled colonies ​ 6 species isolated from blood disseminated infections​ Typically in immunocompromised, thrombocytopenic, neutropenic​.

Trichosporon spp. - Approach to ID

Presence of predominating contiguous arthroconidia that are rectangular with rounded ends with septate hyaline hyphae​ Positive for urease production (Blastoschizomyces and Geotrichum are negative)​

Purpureocillium

Previously Paecilomyces lilacinus Purpureocillium lilacinum exhibits colonies that are considered lilac in color exhibiting shades of lavender to pink. Chlamydospores are absent. Optimal growth temperature is 25°C to 33°C.

Sporobolomyces spp.

Salmon to pink on routine media​ Ubiquitous in environment​ Can cause skin, respiratory, eye, or CNS infections​ S. holsatiscus, S. roseus, and S. salmonicolor associated with human infections​.

Echinocandins

Semisynthetic lipopeptide antifungal agents that target the fungal cell wall ​ Anidulagungin, caspofungin, and micafungin​ Effective against fluconazole-resistant Candida species​ Used for prophylaxis​ Only available as IV medications​.

Aspergillus: Approach to ID

Serologic testing- The use of serology for Aspergillus spp. is limited to assistance in the diagnosis of bronchopulmonary aspergillosis and fungus ball. Serology currently has no value for the diagnosis of disseminated aspergillosis.

Laboratory Diagnosis: Serologic testing

Serologic testing- no commercially available procedures exist for serologic testing of most fungi.​ Serology testing may be a useful tool with select organisms, such as Cryptococcus, Blastomyces, Histoplasma, and Aspergillus spp. ​ Acute and convalescent titers need to be monitored during treatment of the fungal infection.​ Complement fixation (CF) is a sensitive method that is difficult to perform and interpret. Cross reactions with other fungal antibodies can also be a problem.​ Immunodiffusion testing is a simple, cost-effective procedure. Although it is 100% specific, it is relatively insensitive and is not used as a screening tool. This test also requires 2 to 3 weeks to exhibit a positive result.​ Enzyme immunoassays for both antibody and antigen have been used.​

Microsporum spp.: Laboratory Diagnosis

Species of the genus Microsporum are immediately recognized by the presence of large (8-15 µm X 35-150 µm), spindle-shaped, echinulate (covered with small spines), rough-walled macroconidia with thick walls (up to 4 mm) containing four or more septa The exception is Microsporum nanum, which characteristically produces macroconidia with two cells Microconidia, when present, are small (3-7 mm) and club-shaped and are borne on the hyphae, either laterally or on short conidiophores. Cultures of Microsporum spp. develop either rapidly or slowly (5-14 days) and produce aerial hyphae that may be velvety, powdery, glabrous, or cottony, varying in color from whitish, to buff, to a cinnamon brown, with varying shades on the reverse side of the colony

Pneumocystis jiroveci - Laboratory Diagnosis

Specimen collection and transport - BAL is the best specimen; induced sputum, tracheal aspirates, pleural fluid, transbronchial biopsy, and cellular material from bronchial brushings are acceptable Extrapulmonary infections require biopsy Stains Giemsa somewhat visible, but difficult to identify, stains nuclei reddish-purple with light blue cytoplasm, cysts more easily recognized than trophic forms Calcofluor white, methenamine silver, and immunofluorescent stains help visualize cysts - spherical to concave, 4-7 µm, do not bud, contain distinctive intracystic bodies. Direct detection of (1-3) beta-D-glucan A component of the ascus (cyst) cell wall Other fungi secrete this but in lower amounts Uses patient serum Confirmatory testing is needed to rule out other fungi if positive Molecular methods Real-time PCR results must be correlated with history and clinical presentation due to potential for colonization Cultivation - difficult to cultivate outside the lung Serologic testing is not useful

Mucorales: Specimen collection, transport, and processing

Specimens from deep lesions or tissues and sterile sites should be collected rapidly and aseptically. Samples collected for the diagnosis of rhinocerebral forms of infection should include nasal discharge or scrapings, sinus aspirate, or a tissue specimen from a vascularized tissue. Specimens should be transported in sterile containers. Tissue (biopsy specimens) should be moistened by adding a few drops of sterile saline to the container. Specimens should be transported to the laboratory within 2 hours of collection and processed immediately

Blastomyces spp.

The disease (blastomycosis) is most commonly found in North America and extends southward from Canada to the Mississippi, Ohio, and Missouri river valleys; Mexico; and Central America Patients with blastomycosis often have a history of exposure to soil or wood, particularly near waterways Blastomycosis is more common in males than in females and seems to be associated with outdoor occupations or activities. The disease also occurs in dogs. Pathogenesis: Commonly produce an acute or chronic suppurative and granulomatous infection. Blastomycosis begins as a respiratory infection and is probably acquired by inhalation of the conidia or hyphal fragments of the organism. The infection may spread and involve secondary sites of infection in the lungs, long bones, soft tissue, and skin.

Direct Microscopic Exam of Fungi

The mycology laboratory can provide this service in many cases by direct examination (particularly with the Gram stain) of the clinical specimen submitted for culture​ The potassium hydroxide preparation has been the recommended method for direct microscopic examination of specimens. ​ The calcofluor white stain now is believed to be superior. Slides prepared by this method may be observed using fluorescent or bright-field microscopy.

Dematiaceous fungi - Approach to ID

Superficial infection​ H. werneckii - produces yeast-like cells that are 1 or 2 celled​ Conidia are produced by annellophores that bear successive rings (annelids) that are difficult to see microscopically​ Piedra hortae - doesn't sporulate on routine mycology media but has highly septate dematiaceous hyphae and swollen intercalary cells​ White Grain Mycetoma: hyaline molds that produce septate hyphae​ Pseudoallescheria boydii - both sexual and asexual reproduction (pleoanamorphism)​ Sexual form - produces cleistothecia, called Psuedoallescheria boydii​ Asexual form - called Scedosporium apiospermum, conidia are golden brown, elliptical to pyriform, borne singly from tips of annelophores​ Graphium form - less common, clusters of conidiophores with conidia at the ends​ Teleomorphic form - brown to black cleistothecia, which are pseudoparenchymatous, saclike structures containing asci and ascospores when fully developed are 50-200 µm that release asci and ascospores when they rupture​ Can be induced to form cleistothecia by culturing on plain water agar​ Resistant to Amphotericin B​. White Grain Mycetoma: hyaline molds that produce septate hyphae​ Scedosporium prolificans​ Differs from Scedosporium apiospermum by producing inflated, flask-shaped annellpophores​ Resistant to most or all commonly used antifungal agents​ Acremonium species​ Hyaline hyphae with simple, unbranched, erect conidiophores​ Single-celled conidia produced loosely or in gelatinous masses at tip of conidiaphore​ Intercalary and terminal chlamydoconidia may be produced​. Black grain mycetoma​ Exophiala jeanselmei, Curvularia species, and Madurella species​ Madurella species produce sterile hyphae on enriched fungal media, nutritionally poor media used for sporulation​ Long tapering phialides with collarettes and sclerotia may be seen​ Temperature tolerance, biochemical hydrolysis, and assimilation studies are used to differentiate Madurella mycetomatis from Tinea grisea​ Chromoblastomycosis​ Cladosporium, Phialophora, and Fonsecaea spp.​ Cladophialophora - long chains of budding, fusiform elliptical conidia that have a dark septal scar (see Figure 60-5)​ Phialophora - short, flask-shaped to tubular phialides each with a well-developed collarette, clusters of conidia produced through an apical pore and stay nearby in a gelatinous mass​ Colonies are wooly and olive-brown to brownish gray, some strains have concentric zones of color​ On slides, hyphae are dematiaceous with common sporulation​ Phialophora verrucose - cup- or flask-shaped phialides (Figure 60-6)​ Pleurostomophora richardsiae - phialides with flattened or saucerlike collarettes​ Fonsecaea - conidial heads with sympodial arrangement of conidia with secondary conidia (Figure 60-7), sometimes Phialophora-type or Rhinocladiella-type sporulation​.

Dermatophytes: Trichophyton mentagrophytes

T. mentagrophytes produces two distinct colonial forms: the downy variety recovered from patients with tinea pedis and the granular variety recovered from lesions acquired by contact with animals Colonies may appear as white to cream-colored or yellow, cottony or downy, and coarsely granular to powdery The granular colonies may show evidence of red pigmentation. The reverse side of the colony is usually rose-brown, occasionally orange to deep red Granular colonies sporulate freely, with numerous small, spherical microconidia in grapelike clusters and thin-walled, smooth-walled, cigarshaped macroconidia measuring 6 X 20 µm to 8 X 50 µm, with two to five septa. T. mentagrophytes produces urease within 2 to 3 days after inoculation onto Christensen's urea agar. T. mentagrophytes perforates hair a feature that may be used to distinguish between T. mentagrophytes and T. rubrum when differentiation is difficult.

Dermatophytes: Trichophyton Rubrum

T. rubrum is a slow-growing organism that produces a flat or heaped-up colony, generally white to reddish, with a cottony or velvety surface. The characteristic cherry-red color is best observed on the reverse side of the colony; however, this is produced only after 3 to 4 weeks of incubation Two types of colonies may be produced: fluffy and granular. Microconidia are uncommon in most of the fluffy strains and more common in the granular strains; they occur as small, teardrop-shaped conidia often borne laterally along the sides of the hyphae It does not perforate hair in vitro or produce urease.

Dermatophytes: Trichophyton schoenleinii

T. schoenleinii causes a severe type of infection called favus. It is characterized by the formation of yellowish cup-shaped crusts, or scutulae, on the scalp; considerable scarring of the scalp; and sometimes permanent alopecia. T. schoenleinii is a slowly growing organism (30 days or longer) that produces a white to light gray colony with a waxy surface. Colonies have an irregular border consisting mostly of submerged hyphae, which tend to crack the agar. The surface of the colony is usually nonpigmented or tan, furrowed, and irregularly folded. The reverse side of the colony is usually tan or nonpigmented. The hyphae tend to become knobby and club-shaped at the terminal ends, with the production of many short lateral and terminal branches . All strains of T. schoenleinii may be grown in a vitamin-free medium and grow equally well at room temperature or at 35°C to 37°C.

Dermatophytes: Trichophyton tonsurans

T. tonsurans is responsible for an epidemic form of tinea capitis that commonly occurs in children and occasionally in adults The fungus causes a low-grade superficial lesion of varying severity and produces circular, scaly patches of alopecia (loss of hair) . The stubs of hair remain in the epidermis of the scalp which may give the typical "black dot" ringworm appearance Cultures of T. tonsurans develop slowly and are typically buff to brown, wrinkled and suedelike in appearance The colony surface shows radial folds and often develops a craterlike depression in the center with deep fissures. The reverse side of the colony is yellowish to reddish brown.

Dermatophytes: Trichophyton verucosum

T. verrucosum causes a variety of lesions in cattle and in humans; it is most often seen in farmers, who acquire the infection from cattle. The lesions are found chiefly on the beard, neck, wrist, and back of the hands; they are deep, pustular, and inflammatory. With pressure, short stubs of hair may be recovered from the purulent lesion. Direct examination of the hair shaft reveals sheaths of isolated chains of large spores (5-10 µm in diameter) surrounding the hair shaft (ectothrix) and hyphae within the hair (endothrix). T. verrucosum grows slowly (14-30 days); growth is enhanced at 35°C to 37°C and on media enriched with thiamine and inositol. Microscopically, chlamydoconidia in chains and antler hyphae may be the only structures observed microscopically in cultures of T. verrucosum. Chlamydoconidia may be abundant at 35°C to 37° Microconidia may be produced by some cultures if the medium is enriched with yeast extract or a vitamin

Dermatiaceious Fungi: Superficial Infections

Tinea nigra - caused by Hortaea werneckii​ Halophilic organism​ Blackish brown macular patches on palms and soles of feet​ Black Piedra - hair, scalp, axillary and pubic hair​ Piedraia hortae​ Hyperkeratosis - Neoscytalidium dimidiatum​ Infections of the skin and nails leading to thickening of epidermis usually in Africa, Asia, and Latin America​ Other superficial infections - mild skin/nail infections​ Cyphellophora species, Phialophora europea, and Knufia epidermidis​

Opportunistic Mycoses

Tissue-invasive opportunistic mycoses are a group of fungal infections that occur almost exclusively in immunocompromised patients- lymphoma, leukemia, diabetes mellitus, or another defect of the immune system Because most of the organisms known to cause infection in this group of patients are commonly encountered in the clinical laboratory as saprobes (saprophytic fungi), it may be impossible for the laboratorian to determine the clinical significance of these isolates recovered from clinical specimens Many of the organisms associated with opportunistic infections are acquired during construction, demolition, or remodeling of buildings or are hospital-acquired.

Chromoblastomycosis

Traumatic inoculation, thorn or splintere puncture wounds development of papule that grows to form warty, tumorlike lesions resembling cauliflower, can spread to lymphatics​ Susceptible to secondary infections​ Usually feet and legs​ Histologic studies show sclerotic bodies - "copper-colored, septate cells that appear to be dividing by binary fission and resemble copper pennies"​ Hyperplasia of epidermis to differentiate from squamous cell carcinoma​ Cerebral chromoblastomycosis - fungal grain abscess caused by dematiaceious fungi​ Tropical and subtropical areas, occasionally in the US​ Cladophialophora carrionii, Fonsecaea pedrosi, and Phialophora verrucosa​.

Trichophyton violaceum

Trichophyton violaceum causes an infection of the scalp and body and is seen primarily in people living in the Mediterranean region, the Middle and Far East, and Africa The typical "black dot" type of tinea capitis is observed clinically. Direct microscopic examination of a calcofluor white or potassium hydroxide preparation of the nonfluorescing hairs shows dark, thick hairs filled with masses of arthroconidia arranged in chains, similar to those seen in T. tonsurans infections Colonies of T. violaceum are very slow growing, beginning as cone-shaped, cream-colored, glabrous colonies. Later these become heaped up, verrucous (warty), violet to purple, and waxy The reverse side of the colony is purple or nonpigmented. Microconidia and macroconidia generally are not present; only sterile, distorted hyphae and chlamydoconidia are found

Trichosporon spp.

Trichosporonosis - 37 species, almost exclusively found in immunocompromised patients​ Disseminated infections, skin lesions, endocarditis, endophthalmitis, brain abscesses​ Transient fungal colonization can be found in respiratory tract, skin, oropharynx, or stool​ White Piedra ​ soft, yellow or pale brown aggregations around hair shafts in axillary, facial, genital, and scalp regions, invades the cortex of the hair causing damage​ White nodules removed and observed with KOH after light crushing with coverslip​ Microscopic examination shows hyaline hyphae, many round to rectangular arthroconidia, and few blastoconidia​ Positive for urease production​ Colonies are cream colored, heaped, dry to moist, and wrinkled, some appear white, dry, and powdery. ​

Pneumocystis jiroveci - Pathogenesis and spectrum of disease

Trophic form is inhaled and adheres to type I pneumocytes and replicates extracellularly, filling alveolar spaces with foamy eosinophilic material Methenamine silver can identify cyst form in tissue Causes impaired oxygen-diffusing capacity, hypoxemia, and interstitial mononuclear inflammatory response known as interstitial plasma cell pneumonia Nonproductive cough, low-grade fever, dyspnea, chest tightness, night sweats During treatment, CD4+ cells increase with symptom improvement followed by exaggerated immune response called immune reconstitution inflammatory syndrome Extrapulmonary infections are possible - lymph nodes, spleen, bone marrow, liver, other sites. These are associated with rapid disease progression with a fatal outcome

Laboratory Diagnosis: Blood

several automated blood culture systems, including the BACTEC (Becton Dickinson, Sparks, MD), BacT/ ALERT (bioMérieux, Durham, NC), and VersaTREK (Thermo Scientific, TREK Diagnostics, Cleveland, OH), are adequate systems for the recovery of yeasts, except Malassezia spp.​ Laboratories that frequently recover dimorphic fungi from blood are encouraged to use the lysis-centrifugation system, the Isolator (Alere Inc, Waltham, MA), or special fungal media. The Isolator has been proven optimal for the recovery of H. capsulatum and other filamentous fungi. ​ With this system, red blood cells and white blood cells, which may contain the microorganisms, are lysed, and centrifugation concentrates the organisms before culturing. ​ The concentrate is inoculated onto the surface of appropriate culture media, and most fungi are detected within the first 4 days of incubation​. The optimal temperature for fungal blood cultures is 30°C, and the suggested incubation time is 21 days.​

Yeast General Characteristics

Unicellular, 2-60 µm, microscopy not useful for differentiation of yeasts unless using growth from cornmeal agar in conjunction with biochemical characteristics​ Useful morphologic characteristics: presence or absence of a capsule, broad-based or narrow-necked budding​. Reproduce asexually by blastoconidia formation (budding) (Figure 62-1) and sexually by production of ascospores or basidiospores​ Different morphologies in different environments​ Germ tube - outpouching of cell wall that becomes tubular and does not have a constriction at its base, shows initial stage of hyphae formation (Figure 62-2) ​ Pseudohyphae - buds elongate, fail to dissociate, and form subsequent buds (Figure 62-3), can resemble links of sausage​ ​ Colonies appear moist, creamy, may produce a capsule showing as a shiny or mucoid colony​ can be pigmented, hyaline, or melanized ​ Phenotypic characteristics do not always help with speciation, MALDI and nucleic acid testing helps​ Commercial ID systems are available​ Rapid screening tests available for Cryptococcus neoformans and Candida albicans​ CHROMagar Candida​ Candida albicans peptide nucleic acid fluorescent in situ hybridization (PNA FISH)​ Some yeasts are resistant to common antifungal medications​

Laboratory diagnosis: Urine

Urine samples collected for fungal culture should be processed as soon after collection as possible.​ The 24-hour urine sample is unacceptable for culture. ​ All urine samples should be centrifuged and the sediment cultured using a loop to provide adequate isolation of colonies. ​ Media containing antibacterial agents must be used to ensure the recovery of fungi.​ If processing is completed within 2 hours, samples may remain at room temperature. However, if processing is delayed, specimens should be refrigerated at 4°C. If a urine transport system is used, samples may be stored at room temperature for up to 72 hours.​

Saccharomyces cerevisae

Used in baking, prototypical experimental organism for basic science research​ Isolated from thrush, vulvovaginitis, and BSI's.

Laboratory Diagnosis: Vaginal

Vaginal samples should be transported to the laboratory within 24 hours of collection using culture transport swabs. ​ Swabs should be kept moist in sterile tubes. This method of collection provides a specimen suitable for a wet preparation.​ Both selective and inhibitory agars should be plated. Vaginal cultures should be screened for yeasts and incubated at 30°C for 7 days.​ If processing is completed within 2 hours, samples may remain at room temperature. However, if processing is delayed, specimens should be refrigerated at 4°C. If a urine transport system is used, samples may be stored at room temperature for up to 72 hours.​

Histoplasma capsulatum

capsulatum-associated with activities that disperse aerosolized conidia or small hyphal fragments. Infection is acquired through inhalation of these infective structures from the environment. reported in people who clean out an old chicken coop or barn that has been undisturbed for long periods and in individuals who work in or clean areas that have served as roosting places for starlings Spelunkers (i.e., cave explorers) are commonly exposed to the organism when it is aerosolized from bat guano in caves. Pathogenesis: H. capsulatum most commonly produces a chronic, granulomatous infection (histoplasmosis) that is primary and begins in the lung and eventually invades the reticuloendothelial system. Approximately 95% of cases are asymptomatic and self-limited The primary sites of dissemination are the lymph nodes, liver, spleen, and bone marrow. Infections of the kidneys and meninges are also possible Ulcerative lesions of the upper respiratory tract may occur in both immunocompetent and immunocompromised hosts.

Ascomycota

include many fungi that reproduce asexually by the formation of conidia (asexual spores) and sexually by the production of ascospores. All produce true septate hyphae. They may exhibit a sexual form (teleomorph) but also exist in an asexual form (anamorph)​. For example, Pseudallescheria boydii complex , in addition to having the Scedosporium apiospermum anamorph , may exhibit a Graphium anamorph​ Numerous yeast species also belong to the Ascomycota; these include Saccharomyces spp. and some species of Candida. In addition, the phylum contains the teleomorphs of the anamorphic genera Aspergillus and Penicillium.​

Basidiomycota

includes fungi that reproduce sexually through the formation of basidiospores on a specialized structure called the basidia.​ Rarely cause disease in human​. This group includes smuts, rusts, mushrooms, and the Cryptococcus neoformans complex​

Zygomycota

includes organisms that produce sparsely septate hyphae and exhibit asexual reproduction by sporangiospores and sexual reproduction by the production of zygospores. Some of the clinically important genera in this phylum are Rhizopus, Mucor, Rhizomucor, Lichtheimia (Absidia) and Cunninghamella​

Talaromyces marneffei

is an emerging dimorphic pathogenic fungus endemic to Southeast Asia, particularly the Guangxi Zhuang Autonomous Region of the People's Republic of China T. marneffei has been associated with the bamboo rat (Rhizomys pruinosus) and the Vietnamese bamboo rat (Rhizomys sinensis). Pathogenesis: T. marneffei is an emerging pathogen that commonly infects immunosuppressed individuals. The organism causes either a focal cutaneous or mucocutaneous infection, or it may produce a progressive disseminated and commonly fatal infection. Granulomatous, suppurative, and necrotizing inflammatory responses have been demonstrated. The mode of transmission- bamboo rat.

Emmonsia spp.

rare causes of human infection. primarily reported in North, Central, and South America (Brazil) as well as several European and Asian countries. Pathogenesis: E. crescens, produces 25 to 400 µm adiaspores (spores that increase in size inside an animal host) in vitro on brain-heart infusion (BHI) agar incubated at 37°C In the natural environment the conidia are approximately 2 to 4 µm in diameter but may grow to 500 µm when inhaled into the human lung. The condition associated with inhaled conidia from Emmonsia spp. is referred to as adiaspiromycosis. Symptoms include fever, cough, dyspnea, hemoptysis, weight loss, fatigue, and possible respiratory failure

Laboratory Diagnosis: Respiratory Tract Secretions

sputum, induced sputum, bronchial washings, bronchoalveolar lavage, and tracheal aspirations​. To ensure optimal recovery of fungi, a media without antibiotic and a media containing antibacterial antibiotics should be used to prevent overgrowth by contaminants.​ Antifungal agent cycloheximide prevents overgrowth by rapidly growing molds and should be included in at least one of the culture media​. Specimens may be stored at room temperature if processing is completed within 2 hours; if processing is delayed, specimens should be refrigerated at 4°C.​

Laboratory Diagnosis: Hair, skin, and nail scrapings

usually submitted for dermatophyte culture and are contaminated with bacteria or rapidly growing fungi or both. ​ Only the leading edge of skin lesions should be sampled, because the centers often contain nonviable organisms​ These specimens should be placed in a sterile container; they should not be refrigerated. ​ Mycosel agar, which contains chloramphenicol and cycloheximide, is satisfactory for the recovery of dermatophytes. ​ Cultures should be incubated for a minimum of 21 days at 30°C before being reported as negative. ​

Blastoschizomyces

white to cream colored moist colonies that may have radiating margins​ Produce hyphae, pseudohyphae, and annelloconidia​ Mostly found in immunocompromised patients, emerging pathogen typically found in the blood​.