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All of the following are examples of appropriate specimens for the recovery of fungi except:
Swab
Specimens for fungal culture must be kept in a moist, sterile environment. Swabs that are dried out or submitted with insufficient material on them should be rejected. Generally, swabs are inadequate for the recovery of fungi because they are easily contaminated by surrounding skin flora.
For which clinical specimens is the KOH direct mount technique for examination of fungal elements used?
Skin
A solution of 10% KOH is used for contaminated specimens such as skin, nail scrapings, hair, and sputum to clear away background debris that may resemble fungal elements. Normally sterile specimens (CSF, blood, and bone marrow) do not require KOH for clearing.
The India ink stain is used as a presumptive test for the presence of which organism?
Cryptococcus neoformans in CSF
Meningitis caused by C. neoformans is diagnosed through culture, biochemical reactions, and rapid agglutination tests for cryptococcal antigen. The India ink test is not diagnostic for cryptococcal meningitis because positive staining results are demonstrated in less than 50% of confirmed cases. A positive India ink test shows yeast cells in CSF with a surrounding clear area (the capsule) because the capsule of C. neoformans is not penetrated by ink particles.
Cutaneous disease involving skin, hair, and nails usually indicates an infection with a:
Dermatophyte
Superficial dermatophytes rarely invade the
deeper tissues and are the cause of most cutaneous fungal infections. Fungal infections of the skin are most often caused by Microsporum spp., Trichophyton spp., and Epidermophyton spp., although Candida spp. are sometimes implicated as the cause of nail infections.
What is the first step to be performed in the identification of an unknown yeast isolate?
Germ tube test
The true germ tube (filamentous extension from a yeast cell) is approximately one-half the width and three to four times the length of the cell with no true hyphae constriction at the point of origin. C. albicans produce germ tubes (95%), and a positive test is considered a presumptive identification.
An isolate produced a constriction that was interpreted as a positive germ tube, but Candida albicans was ruled out when confirmatory tests were performed. Which of the following fungi is the most likely identification?
Candida tropicalis
C. tropicalis forms pseudohyphae that resemble true germ tubes by producing a constriction at the point of origin of the yeast cell. Germ tubes represent true hyphae without constriction, and therefore the test should have been repeated along with carbohydrate tests before making a presumptive identification. The other three species of yeast listed do not form hyphae.
Cornmeal agar with Tween 80 is used to identify which characteristic of an unknown yeast isolate?
Hyphae (true and pseudo), blastoconidia and arthroconidia, chlamydospores
Cornmeal agar with Tween 80 (polysorbate) reduces the surface tension and allows for enhanced formation of hyphae, blastospores, and chlamydospores.
Blastoconidia are the beginning of which structures?
Pseudohyphae
Pseudohyphae are the result of a pinching-off process, blastoconidiation, with the growth of filaments with constrictions. Germ tubes are
the beginning of true hyphae (no constrictions). Arthrospores are the result of a breaking-off process of true septate hyphae resulting in square conidia.
An isolate from CSF growing on cornmeal agar produces the following structures:
Blastoconidia = +
Pseudohyphae = Neg
Chlamydospores = Neg
Arthroconidia= Neg
Which tests should be performed next?
Birdseed agar and urease
A yeast isolated from the CSF producing blastospores is most likely to be C. neoformans, which is positive for urease and produces brown colonies on birdseed agar.
Which of the following yeast enzymes is detected using birdseed (niger seed) agar?
Phenol oxidase
Most isolates of C. neoformans produce phenol oxidase when grown on Guizotia abyssinica medium (birdseed medium), producing brown to black pigmented colonies. C. neoformans is the only Cryptococcus species that oxidizes o-diphenol to melanin, which is responsible for the color.
Which of the following yeasts is characteristically positive for germ tube production?
Candida albicans
C. albicans and Candida dubliniensis, a variant of
C. albicans, are the only yeasts that produce germ tubes within 1-3 hours of incubation at 37Ā°C. C. tropicalis produces pseudohyphae after incubation for 3 hours, which may be mistaken for germ tubes. A careful evaluation of the tube origin for constriction is required to avoid a false-positive interpretation.
Arthroconidia production is used to differentiate which two yeast isolates?
Trichosporon cutaneum and Cryptococcus
neoformans
T. cutaneum and C. neoformans are both urease positive, but T. pullulans produces arthroconidia and C. neoformans does not. In addition to Trichosporon spp., arthroconidia are produced by Geotrichum spp.
The urease test, niger seed agar test, and the germ tube test are all used for the presumptive identification of:
Cryptococcus neoformans
Germ tube-negative isolates producing dark brown to black colonies on niger seed agar and a positive urease test are presumptive of C. neoformans. A positive germ tube test is a presumptive identification for C. albicans as well as for C. dubliniensis. See the chart above.
Which of the following yeasts produces only blastoconidia on cornmeal Tween 80 agar?
Cryptococcus spp.
Cryptococcus spp. do not form either pseudohyphae or arthroconidia. Candida spp. produce blastoconidia or pseudohyphae. Trichosporon spp. produce pseudohyphae, blastoconidia, and arthroconidia. See the chart below.
Ascospores are formed by which yeast isolate?
Saccharomyces cerevisiae
Sexual spore production is a characteristic of the Ascomycotina, which produce an ascus (saclike structure) after the union of two nuclei. The resulting spore is termed an ascospore. S. cerevisiae produces ascospores when grown on ascospore agar for 10 days at 25Ā°C.
A germ tube-negative, pink yeast isolate was recovered from the respiratory secretions and urine of a patient with AIDS. Given the following results, what is the most likely identification?
CORNMEAL TWEEN 80 AGAR
Blastoconidia = +
Arthroconidia= Neg
Pseudohyphae = Neg
Urease = +
Rhodotorula spp.
Rhodotorula spp. produce pink- to coral-colored colonies on Sabouraud's agar and cornmeal agar. It is usually considered a contaminant but is an opportunistic pathogen, and must be identified when found in specimens from immunosuppressed patients.
Chlamydospore production is demonstrated by which Candida species?
C. albicans
Cornmeal Tween 80 agar supports the growth of
C. albicans and the formation of its distinctive thick-walled, usually single, terminal chlamydospores. C. dubliniensis also produces chlamydospores (in pairs, triplets, and clusters).
Carbohydrate assimilation tests are used for the identification of yeast isolates by inoculating media:
Free of carbohydrates
The yeast isolate is inoculated directly into the molten agar base free of carbohydrates or is poured as a suspension onto a yeast nitrogen agar base plate. Carbohydrate disks are then added to the surface of the agar, and the plates are incubated for 24-48 hours at 30Ā°C. Growth around the disk indicates the ability of the yeast to utilize the carbohydrate(s) as a sole source of carbon.
Yeast recovered from the urine of a catheterized patient receiving chemotherapy for cancer gave the following results:
CORNMEAL TWEEN 80 AGAR
Germ tube = +
Blastoconidia = +
Pseudohyphae = +
Arthroconidia= Neg
Chlamydospores = +
What further testing is necessary?
No further testing is needed for identification
This isolate is C. albicans, which also produces some true hyphae along with pseudohyphae. A positive germ tube is a presumptive identification along with the production of blastoconidia, terminal chlamydospores, and pseudohyphae.
A blood agar plate inoculated with sputum from a patient with diabetes mellitus grew very few bacterial flora and a predominance of yeast. Given the following results, what is the most likely identification of the yeast isolate?
CORNMEAL TWEEN 80 AGAR
Germ tube = Neg
Arthroconidia = Neg
Chlamydospores = Neg
Pseudohyphae = +
Blastoconidia = + (arranged along pseudohyphae)
Candida tropicalis
C. tropicalis and C. kefyr (pseudotropicalis) differ in their arrangement of blastoconidia along the pseudohyphae. C. kefyr (pseudotropicalis) forms elongated blastoconidia arranged in parallel clusters that simulate logs in a stream. Trichosporon spp. and Geotrichum spp. form arthroconidia.
Dimorphic molds are found in infected tissue in which form?
Yeast phase
Dimorphic molds are in the yeast form in infected tissues because they are in the yeast form at 37Ā°C. Specimens are cultured and incubated at both room temperature and 35Ā°C-37Ā°C. To prove that a mold growing at room temperature
(or 30Ā°C) is a dimorphic fungus, conversion to the yeast form must be demonstrated via subculture and incubation at 37Ā°C.
The mycelial form of which dimorphic mold produces thick-walled, rectangular, or barrel-shaped alternate arthroconidia?
Coccidioides immitis
The mold form of C. immitis shows barrel-shaped arthroconidia separated by empty cells (ghost cells) that cause an uneven staining effect when they are examined under a microscope. S. schenckii, H. capsulatum, and B. dermatitidis produce conidia that are round or oval in shape in the mold phase.
The yeast form of which dimorphic fungus appears as oval or elongated cigar shapes?
Histoplasma capsulatum
S. schenckii is usually acquired by humans through thorns or splinters because it is commonly found on living or dead vegetation. It is called "rose gardener's disease" because gardeners, florists, and farmers are most often infected. S. schenckii is often recovered from exudates of unopened subcutaneous nodules or open draining lesions.
The mycelial form of Histoplasma capsulatum seen on agar resembles:
Sepedonium spp.
Sepedonium spp. are saprophytic molds that do not have a yeast phase and produce large spherical tuberculate macroconidia like H. capsulatum. Histoplasmosis is a chronic granulomatous infection primarily found in the lungs that invades the reticuloendothelial system. Infection occurs via spores released from decaying bird or chicken droppings that are inhaled when disturbed.
The yeast form of which dimorphic mold shows a large parent yeast cell surrounded by smaller budding yeast cells?
Paracoccidioides brasiliensis
P. brasiliensis yeast forms are sometimes seen as a "mariner's wheel" because multiple budding cells completely surround the periphery of the parent cell.
Which group of molds can be ruled out when septate hyphae are observed in a culture?
Zygomycetes
Zygomycetes commonly recovered from clinical specimens are Rhizopus spp. and Mucor spp. Both display aseptate hyphae, while the other groups above display septate hyphae. Zygomycetes usually not encountered in clinical specimens are also aseptate and include Absidia spp., Rhizomucor spp., Cunninghamella spp., and Syncephalastrum spp.
Tinea versicolor is a skin infection caused by:
Malassezia furfur
M. furfur has a worldwide distribution and causes a superficial, brownish, dry, scaly patch on the skin of light-skinned persons and lighter patches on persons with dark skin. M. furfur is not cultured because diagnosis can be made from microscopic examination of the skin scales. Skin scrapings prepared in KOH show oval or bottle-shaped cells that exhibit monopolar budding in the presence of a cell wall and also produce small hyphae.
Which of the following structures is invaded by the genus Trichophyton?
Hair, nails, and skin
Trichophyton spp., Microsporum spp., and Epidermophyton spp. are the organisms causing human dermatomycoses or cutaneous infections. Trichophyton spp. infect hair and nails as well as skin. Infections with members of the genus Microsporum are confined to the hair and skin, while infections caused by the genus Epidermophyton are seen only on the skin and nails.
An organism cultured from the skin produces colonies displaying a cherry-red color on Sabouraud dextrose agar after 3-4 weeks and teardrop-shaped microconidia along the sides of the hyphae. The most likely identification is:
Trichophyton rubrum
Members of the genus Microsporum produce club-shaped microconidia and are usually pigmented white, buff, yellow, or brown. Epidermophyton does not display microconidia and produces yellow-green or yellow-tan colonies. T. rubrum can be differentiated from the other members of the genus by its distinctive cherry-red color. Trichophyton mentagrophytes may also produce a red pigment, but it is usually rose colored or orange, or deep red. T. tonsurans produces white-tan to yellow suedelike colonies. T. schoenleinii produces white to cream-colored colonies, and T. violaceum produces port wine to deep violet colonies.
Which Microsporum species causes an epidemic form of tinea capitis in children?
Microsporum audouinii
M. audouinii and T. tonsurans may both cause epidemic tinea capitis in children. M. audouinii causes a chronic infection transmitted directly via infected hairs on caps, hats, combs, upholstery, and hair clippers. Infected hair shafts fluoresce yellow-green under a Wood's lamp. M. audouinii does not usually sporulate in culture and forms atypical vegetative forms such as antler and racquet hyphae and terminal chlamydospores. In contrast, M. canis produces spindle-shaped, thick-walled multicelled macroconidia, and M. gypseum produces ellipsoidal, multicellular macroconidia.
Microscopic examination of a fungus cultured from a patient with athlete's foot showed large, smooth-walled, club-shaped macroconidia appearing singly or in clusters of two to three from the tips of short conidiophores. The colonies did not produce microconidia. What is the most likely identification?
Epidermophyton spp.
Epidermophyton spp. do not produce microconidia; this differentiates them from Trichophyton spp. and Microsporum spp. Alternaria is not a dermatophyte. Epidermophyton floccosum is the most frequently isolated member of the genus and infects the skin but not the hair.
Which Trichophyton species causes the favus type of tinea capitis seen in the Scandinavian countries and in the Appalachian region of the United States?
T. schoenleinii
T. schoenleinii is identified microscopically by its characteristic antler-shaped hyphae and chlamydospores in the absence of conidia.
The Hair Baiting Test is used to differentiate which two species of Trichophyton that produce red colonies on Sabouraud agar plates?
T. mentagrophytes and T. rubrum
T. mentagrophytes may produce a deep red pigment seen through the reverse side of the agar plate that resembles the cherry-red pigment produced by T. rubrum. However, T. mentagrophytes can be differentiated by its ability to invade the hair shaft. T. rubrum grows on the surface of the hair but does not penetrate the shaft.
A mold that produces colonies with a dark brown, green-black, or black appearance of both the surface and reverse side is classified as a:
Dematiaceous mold
The dematiaceous molds are easily recognized and confirmed by observing dark yellow or brown septate hyphae upon microscopic examination.
A rapidly growing hyaline mold began as a white colony but soon developed a black "pepper" effect on the agar surface. The older colony produced a black matte, making it resemble a dematiaceous mold. What is the most likely identification?
Aspergillus niger
A. niger is the only species listed producing black conidia, which causes a "pepper" effect as the colony grows. The reverse side of the agar plate remains buff or cream colored, which differentiates it from the dematiaceous (dark) molds
Which dematiaceous mold forms flask-shaped phialides, each with a flask-shaped collarette?
Phialophora spp.
Phialophora, Exophiala, and Wangiella all produce phialides, but the last two genera form elongated, tubelike phialides without a collarette, as opposed to the flask-shaped phialides of Phialophora, which contain clusters of conidia at the tips.
Which Aspergillus species, recovered from sputum or bronchial mucus, is the most common cause of pulmonary aspergillosis?
A. fumigatus
A. fumigatus is most often associated with compost piles and is found in the soil of potted plants. A. niger is the cause of cavitary fungus ball lesions of the lungs and nasal passages.
A hyaline mold recovered from a patient with AIDS produced rose-colored colonies with lavender centers on Sabouraud dextrose agar. Microscopic examination showed multiseptate macroconidia appearing as sickles or canoes. What is the most likely identification?
Fusarium spp.
Fusarium spp. are usually a contaminant but are sometimes seen as a cause of mycotic eye, nail, or skin infection in debilitated patients. Fusarium spp. is a hyaline (light) mold and grows on Sabouraud agar plates at 30Ā°C within 4 days. The other three organisms are members of the Dematiaceae family (dark molds).
Material from a fungus-ball infection produced colonies with a green surface on Sabouraud agar in 5 days at 30Ā°C. Microscopic examination showed club-shaped vesicles with sporulation only from the top half of the vesicle. This hyaline mold is most probably which Aspergillus spp.?
A. fumigatus
A. fumigatus is the most common cause of aspergillosis. It is characterized by sporulation only from the upper half or two-thirds of the vesicle. Colonies of A. niger are white with black pepper growth and produce phialides over the entire vesicle, forming the classic "radiate" head. A. flavus colonies are yellow to yellow-green and produce phialides that cover the entire vesicle and point out in all directions. A. terreus produces brown colonies and phialides that also cover the entire vesicle.
A rapidly growing nonseptate mold produced colonies with a gray surface resembling cotton candy that covered the entire plate. Microscopic examination revealed sporangiophores arising between, not opposite, the rhizoids and producing pear-shaped sporangia. What is the most likely identification?
Absidia spp.
Absidia spp. are similar to Rhizopus spp. except for the location of rhizoids (rootlike hyphae). The rhizoids of Rhizopus spp. are located at the point where the stolons and sporangiophores meet, whereas those of Absidia spp. arise at a point on the stolon between the rhizoids. Penicillium spp. and Aspergillus spp. do not form rhizoids.
An India ink test was performed on CSF from an HIV-infected male patient. Many encapsulated yeast cells were seen in the centrifuged sample. Further testing revealed a positive urease test and growth of brown colonies on niger-seed agar. The diagnosis of meningitis was caused by which yeast?
Cryptococcus neoformans
Immunocompromised patients are at risk for invasion of Cryptococcus neoformans. The polysaccharide capsule of C. neoformans is not recognized by phagocytes, which allow patients with impaired cell-mediated immunity to become readily infected with C. neoformans.
A bone marrow sample obtained from an immunocompromised patient revealed small intracellular cells using a Wright's stain preparation. Growth on Sabouraud-dextrose agar plates of a mold phase at 25Ā°C and a yeast phase at 37Ā°C designates the organism as dimorphic. The mold phase produced thick, spherical tuberculated macroconidia. What is the most likely identification?
Histoplasma capsulatum
Thermally dimorphic Histoplasma capsulatum produce microconidia and hyphal fragments at 37Ā°C (yeast phase), whereas at 25Ā°C (mold phase) the organism displays large, thick-walled, round macroconidia with knobby or knoblike projections. The yeast form is able to survive within circulating monocytes or tissue macrophages that can be demonstrated with Giemsa's or Wright's stain.
A lung biopsy obtained from an immunocompromised patient showed many "cup-shaped" cysts (gray to black) in a foamy exudate (green background) using Gomori methenamine silver (GMS) stain. The organism cannot be cultured because it does not grow on routine culture media for molds. The patient was diagnosed with pneumonia that resisted antibiotic treatment. The most likely identification is?
Pneumocystis jirovecci (carinii)
Pneumocystis jirovecii (carinii), most recently classified as a fungus but formerly as a parasite, is best recovered by bronchoalveolar lavage or induced sputum in immunocompromised patients. Open lung biopsy sample was the specimen of choice before the AIDS epidemic. Gomori methenamine silver stain is used to identify the organism; it stains the cyst form but not the trophozoites.
Upon direct examination of a sputum specimen, several spherules were noted that contained endospores. Growth on Sabouraud-dextrose agar showed aerial mycelial elements. The septate hyphae produced barrel-shaped arthroconidia. What is the most likely identification?
Coccidioides immitis
Coccidioides immitis endospores are often confused with yeast cells but they do not bud. C. immitis is endemic in the southwestern United States. Since the arthroconidia are highly infectious, an open plate should not be used, and a slide culture test should not be performed. Rather, tubed media is used for testing, and all work should be performed in a biological safety cabinet.
A bone marrow specimen was obtained from an immunocompromised patient who tested positive for HIV. The organism grew rapidly at 3 days showing a mold form (at 25Ā°C), displaying conidiophores with four to five terminal metulae with each having four to six phialides. The conidia at the end of the phialides were oval and in short chains. They appear as a fan or broom when viewing under 10Ć and 40Ć. At 37Ā°C, the yeast form grew more slowly, showing conidia that formed hyphal elements breaking at the septa to produce oval arthroconidia. This thermo- dimorphic mold is most likely:
Penicillium marneffei
Other Penicillium spp. are differentiated from
P. marneffei (thermally dimorphic) through conversion from the mold to yeast phase. P. marneffei are seen as yeast at 35Ā°C-37Ā°C on 5% sheep blood agar or in BHI broth. Other Penicillium spp. do not display a yeast phase. P. marneffei are recovered from blood, skin, lymph nodes, bone marrow, and internal organs of immunocompromised patients.
What is the specimen of choice for the initial diagnosis of Pneumocystis jirovecii (carinii) in an immunocompromised patient, such as someone with AIDS?
Induced sputum
Open lung biopsy is not recommended for persons with AIDS because of the increased risk of death, infection, and complications associated with this surgical procedure. Persons with AIDS and Pneumocystis jirovecii (carinii) pneumonia (PCP) can be diagnosed by chest x-ray, special stains, and PCR. Non-AIDS patients have a much lower yield, and therefore, bronchoalveolar lavage, tracheal aspiration, endoscopic lung biopsy, and open lung biopsy are more often indicated.
A transplant patient is suspected of having invasive aspergillosis on the basis of clinical and radiological findings. Which specimen is best for the initial identification of aspergillosis by soluble antigen testing?
Serum or urine
To determine an early diagnosis, the detection of soluble antigens of Aspergillus spp. by methods such as ELISA, latex agglutination, and immunoblotting using serum or other body fluids is used frequently. Blood and sputum cultures are frequently negative, and tissue biopsy is associated with a significantly higher risk of complications.
What is the most common cause of mucormycosis infection in humans?
Rhizopus spp.
Vascular invasion followed by thrombosis, tissue infarction, and necrosis are the most common clinical manifestations of cutaneous infections caused by Rhizopus spp. Infections occur mainly in patients who are already being treated for other conditions including lymphoma, leukemia, neutropenia, renal failure, and diabetic ketoacidosis.
A thermally dimorphic fungus shows a filamentous mold form with tuberculate macroconidia at room temperature, and a yeast form above 35Ā°C. Which organism best fits this description?
Histoplasma capsulatum
The yeast phase of H. capsulatum develops as small oval budding cells seen in macrophages. The yeast phase of Paracoccidioides brasiliensis develops large cells that bud creating a structure resembling a mariner's wheel, and Coccidioides immitis produces very large spherules. At 25Ā°C, P. brasiliensis produces small single conidia, and C. immitis produces arthroconidia. Candida spp. are not dimorphic.
H. capsulatum is found in soil containing excrement of birds and bats. It is endemic in the Ohio and Mississippi river valleys, and the most common systemic mycosis in North America.
After a vacation to the Southwestern United States, a midwesterner complained of flulike symptoms with fever, chills, nonproductive cough, and chest pain. Microscopic exam of sputum, cleared with KOH, revealed large, thick-walled spherules containing endospores. Upon culture, the mold phase showed septate hyphae and alternating barrel-shaped arthroconidia. Which organism is most likely the cause of this pneumonia?
Coccidioides immitis
Coccidioides immitis is a soil fungus and is endemic to the Southwestern United States. Infection results from inhaling the arthroconidia, which form endospore-forming spherules and leads to infection within 1-3 weeks. Nonimmunocompromised people usually do not require treatment, and are immune to reinfection. However, immunodeficient persons may develop symptomatic pulmonary and multi-organ infections.
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