Figure 50-69 The tissue form of Coccidioides immitis (i.e„ the spherule). The external wall of the spherule does not stain with the silver stain, whereas the internal endospores do stain (arrowhead). Also note how the juxtaposed endospores, which have been released from a spherule that has burst, resemble a budding yeast (arrow). GMS stain (400x).

Blastomyces dermatitidis. The diagnosis of blastomycosis may easily be made when a clinical specimen is observed by direct microscopy. Blastomyces dermatitidis appears as large, spherical, thick-walled yeast cells 8 to 15 (xm in diameter, usually with a single bud that is Connected to the parent cell by a broad base (Figure 50-68; also see Figures 50-19 and 50-20). A smaller form (2-8 pm) may be rarely seen.

Coccidioides immitis. In direct microscopic examinations of sputum or other body fluids, C. immitis appears as a nonbudding, thick-walled spherule, 20 to 200 |im in diameter, containing either granular material or numerous small (2 to 5 |xm in diameter), nonbudding endospores (Figure 50-69; also see Figures 50-21 and 50-25). The endospores are freed by rupture of the spherule wall; therefore, empty and collapsed "ghost" spherules may also be present, Small, immature spherules measuring 5 to 20 pm may be confused with H. capsulatum or B. dermatitidis. When two endospores or immature spherules are lying adjacent to one another, it ■may appear that budding yeast is present. When identification of C. immitis is questionable, a wet preparation of the clinical specimen may be made using sterile saline, and the edges of the coverglass may be sealed with petrolatum and incubated overnight. When spherules are present, multiple hyphal strands will be produced from the endospores.

Histoplasma capsulatum. The direct microscopic examination of respiratory tract specimens and other similar specimens often fails to reveal the presence of H. capsulatum. The organism, however, may be detected by an astute laboratorian when examining Wright- or

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Figure 50-70 The small oval yeast cells, which are relatively uniform in size, are characteristic of Histoplasma capsulatum (2000x).

Giemsa-stained specimens of bone marrow and rarely in peripheral blood. Histoplasma capsulatum is found intracellularly within mononuclear cells as small, round to oval yeast cells 2 to 5 pm in diameter (Figure 50-70; also see Figure 50-16).

Paracoccidioides brasiliensis. Specimens submitted for direct microscopic examinations are important for the diagnosis of paracoccidioidomycosis. Large, round or oval, multiply budding yeast cells (8 to 40 nm in diameter) are usually recognized in sputum, mucosal biopsy, and other exudates. Characteristic multiply budding yeast forms resemble a "mariner's wheel" (Figure 50-71). The yeast cells surrounding the periphery of the parent cell range from 8 to 15 («n in

Figure 50-71 Paracoccidioides brasiliensis in a bone marrow aspirate shows a yeast ceil with multiple buds (arrow).

diameter. Some cells may be as small as 2 to 5 urn but still exhibit multiple buds.

Penicillium marneffei. The direct examination of infected tissues and exudates that contain Penicillium marneffei produces small (2 to 6 pm), yeastlike cells that have internal crosswalk; no budding cells are produced (see Rgure 50-4). Like Histoplasma capsulatum, P. marneffei may also be detected in peripheral blood smears in disseminated disease.

Sporothrix schenckii. Exudate aspirated from unopened subcutaneous nodules or from open draining lesions is often submitted for culture and direct microscopic examination. Direct examination of this material is usually of little diagnostic value because it is difficult to demonstrate the rare characteristic yeast forms. If identified, S. schenckii usually appears as small (2 to 5 pm), round to oval to cigar-shaped yeast cells (see Figure 50-22). If stained with the periodic acid-Schiff (FAS) method in histologic section, an amorphous pink material may be seen surrounding the yeast cells (see Figure 50-17).

Antigen-Protein. Immunodiffusion methods (the exoantigen test) may be used to identify isolates of these organisms based on precipitation bands of identity between specific antibodies and fungal antigen extracts. These assays, however, have been largely replaced by the more rapid nucleic acid hybridization reactions described below.

Nucleic Acid Amplification. Nucleic acid amplification assays are not routinely performed but are available in some reference laboratories and in research settings. Real-time or homogeneous, rapid cycle poly merase chain reaction (PCR) assays have been described for H. capsulatum and C. immitis?1,84 These assays have been demonstrated to be suitable for isolate identify cation, but the potential to use them on DNA extracts from direct clinical specimens also exists. This latter application is exciting, given the slow growth of # capsulatum and the severe disease that may be cause by both of these dimorphic pathogens.

Cultivation. Commonly, the dimorphic fungi an regarded as slow-growing organisms that require 7 to i days for visible growth to appear at 25° to 30° C. However, exceptions to this rule occur with some frequency Occasionally cultures of B. dermatitidis and H. capsulatum. are recovered in as short a time as 2 to 5 days when, many organisms are present in the clinical specimen In contrast, when small numbers of colonies of B. dermatitidis and H. capsulatum are present, sometimes 21 to 30 days of incubation are required before they z\ detected. Coccidioides immitis is consistentiy recovered within 3 to 5 days of incubation, but when many organisms are present, colonies may be detected within 48 hours. Cultures of P. brasiliensis are commonly recovered within 5 to 25 days, with a usual incubation period of 10 to 15 days. As one can see, the growth rate, if slow, might lead one to suspect the presence oi i dimorphic fungus; however, considerable variation ii the time for recovery exists. The exceptions to this slow growth are C. immitis and P. marneffei, which may L i- ' recovered within 3 to 5 days.

Textbooks present descriptions for the dimorphic fungi that the reader assumes are typical for each particular organism. As is true in other areas of microbiology variation in the colonial morphologic features also occurs depending on the strain and the type of medium used. One must be aware of this variation and must not rely heavily on colonial morphologic features for the identification of members of this group of fungi

The pigmentation of colonies is sometimes helpful but also varies widely; colonies of B. dermatitidis and . 7. capsulatum are described as being fluffy white, with a-j change in color to tan or buff with age. Some isolates initially appear darkly pigmented, with colors ranging from gray or dark brown to red.62 On media containing? blood enrichment, these organisms may appear heaped^ wrinkled, glabrous, neutral in color, and yeastlike id;! appearance; often tufts of aerial hyphae project from the top of the colony. Some colonies may appear pink to* red, possibly because of the adsorption of hemoglobin from the blood in the medium. Coccidioides immitis isj described as being fluffy white, with scattered areas i hyphae that are adherent to the agar surface so as to« give an overall "cobweb" appearance to the colony. However, numerous morphologic forms, including textureS ranging from woolly to powdery and pigmentation rang-

jug from pink-lavender or yellow to brown or buff, have been reported.

The definitive identification of a dimorphic fungus has traditionally been made by observing both the mold and tissue or parasitic forms of the organism. In general 25° to 30° C is the optimal temperature for the recovery gad identification of the dimorphic fungi from clinical Specimens. Temperature (35* to 37° C), certain nutritional factors, and stimulation of growth in tissue independent of temperature are among the factors necessary to initiate the transformation of the mold form to the tissue form.47,136 Previously, the definitive identification of 23L dermatitidis and H. capsulation was made by the in vitro conversion of a mold fonn to the corresponding yeast form by in vitro conversion on a blood-enriched medium incubated at 35° to 37° C, and for C, immitis conversion to the spherule form by animal Inoculation. The conversion of dimorphic molds to the yeast form (except for C. immitis) can be accomplished with some difficulty, as outlined in Procedure 50-6. Some laboratories use the exoantigen test (Procedure 50-6) to identify the dimorphic pathogens. However, this test requires an extended incubation before cultures may be identified.

Perhaps the most significant advance in clinical mycology in the last few decades was the development of specific nucleic acid probes for the identification of some of the dimorphic fungi (Procedure 50-8). DNA probes are commercially available from (Gen-Probe Inc., San Diego, Calif) that are complementary to spedes-spedfic ribosomal RNA. Fungal cells are heat-killed, disrupted by a lysing agent and sonication, and the nudeic add is exposed to a spedes-specific DNA probe, which has been labeled with a chemiluminescent tag facridinium ester). The labeled DNA probe combines with an ribosomal RNA of the organism to form a stable DNA.-RNA hybrid. All unbound DNA probes are "quenched" and light generated from the DNArRNA hybrids are measured in a luminometer. Total time for testing is less than 1 hour, and young colonies may be tested.151

Nudeic add probe identification is sensitive, specific, and rapid. Colonies that are contaminated with bacteria or other fungi may be tested; however, results from colonies recovered on a blood-enriched media must be interpreted with caution because hemin may cause false-positive chemiluminescence. The major disadvantage of nucleic add probe identification relates to the cost per test. It is recommended that nucleic acid probes be used whenever possible, however, to confirm the identification of an organism suspected of being H. capsulatum, B. dermatitiiis, or c. immitis. A brief consideration of the colonial morphology of the dimorphic systemic pathogens follows.

Blastomyces dermatitidls. Blastomyces dermatitidis commonly requires 5 days to 4 weeks or longer for growth to be detected but may be detected in as short a time as 2 to 3 days. On enriched culture media, the mold form develops initially as a glabrous or waxy appearing colony is off-white to white. With age, the aerial hyphae often turn gray to brown. The more waxy, yeastlike appearance is typified on media enriched with blood. Uifts of hyphae often project upward from the colonies and have been referred to as the "prickly state" of the organism. Some isolates, however; appear fluffy on primary recovery and remain so throughout the incubation period.

Coccidioides immitis. Cultures of C. immitis represent a biohazard to laboratory workers, and strict safety precautions must be followed when examining cultures. Mature colonies may appear within 3 to 5 days of incubation and may be present on most media, induding those used in bacteriology. Procop and colleagues reviewed 1270 fungal isolates recovered from 176,144 Isolator blood cultures and compared them with the recovery fungi on standard bacteriologic media; all the isolates of C. immitis present were recovered on standard bacteriologic media.110 Laboratory workers are cautioned not to open cultures of fluffy white molds unless they are placed inside of a biological safety cabinet. Colonies of C, immitis often appear as delicate, cobweblike growth after 3 to 21 days of incubation. Some portions of the colony will exhibit aerial hyphae, whereas in others the hyphae will be adherent to the agar surface. Most isolates appear fluffy white; however, colonies of varying colors ranging from pink to yellow to purple and black have been reported.43 On blood agar, some colonies exhibit a greenish discoloration, whereas others appear yeastlike, smooth, wrinkled, and tan.

Histopiasma capsulatum. Histoplasma capsulatum is easily cultured from clinical specimens; however, it may be overgrown by bacteria or rapidly growing molds. A procedure useful for the recovery of H. capsulatum, B. dermatitidis, and C. immitis from contaminated specimens (e.g., sputa) utilizes a yeast extract phosphate medium14 and a drop of concentrated ammonium hydroxide (NfyOH) placed on one side of the inoculated plate of medium.121 In the past it has been recommended that specimens not be kept at room temperature before culture, because H. capsulatum would not survive. The organism will survive transit in the mail for as long as 16 days.53 It is, however; recommended that specimens be cultured as soon as possible to ensure the optimal recovery of H. capsulatum and other dimorphic fungi. The above-described method works well with specimens shipped via mail.

Histoplasma capsulatum is usually considered to be a slow-growing mold at 25° to 30° C and commonly requires 2 to 4 weeks or more for colonies to appear. The organism may, however, be recovered in 5 days or less if many yeast cells are present in the clinical specimen. Isolates of H. capsulatum have been reported to be recovered from blood cultures with the Isolator within a mean time of 8 days.J2 Textbooks describe the colonial morphology of H. capsulatum as being a white, fluffy mold that turns brown to buff with age. Some isolates ranging from gray to red have also been reported. The organism also may produces wrinkle, moist, heaped, yeastlike colonies that are soft and cream, tan, or pink. Tufts of hyphae often project upward from the colonies as described with B. dermatitidis. It is not possible to differentiate H. capsulatum and B. dermatitidis from each other using colonial morphologic features.

Paracoccidioides brasiiiensis. Colonies of P. brasi-liensis grow very slowly (21 to 28 days) and are heaped, wrinkled, moist, and yeastlike. With age, colonies may become covered with a short aerial mycelium and turn tan to brown. The surface of colonies is often heaped with crater formations.

Penicilliam marneffei. At 25° C, Penidllium mar-neffei grows rapidly and produces blue-green to yellowish colonies on Sabouraud's agar. A soluble, red to maroon pigment that diffuses into the agar and is often best observed by viewing the reverse of the colony is suggestive of P. marneffei. Although the growth rate and colonial morphologic features may help one to recognize the possibility of the presence of a dimorphic fungus, they should be used in combination with the microscopic morphologic features used to make the identification. Penidllium marneffei cannot be definitively identified by morphologic features alone; thermal conversion studies or nucleic acid-based testing is needed to confirm the identification of this pathogen.

Sporothrix schenckii. Colonies of S. schenckii grow rapidly (3 to 5 days) and are initially usually small, moist, and white to cream-colored. On further incubation, these become membranous, wrinkled, and coarsely matted, with the color becoming irregularly dark brown or black and the colony becoming leathery in consistency. It is not uncommon for the clinical microbiology laboratory to mistake a young culture of S. schenckii for that of a yeast until the microscopic features are observed.

Approach to Identification

Blastomyces dermatitidis. Microscopically, hyphae of the mold form of Blastomyces dermatitidis are

Figure 50-72 The mycelial form of Blastomyces dermatitidis shows oval conidia bome laterally on branching hyphae (lOOOx),

septate and delicate, and measure approximately 2 jan -in diameter. Commonly, ropelike strands of hyphae are seen; however, these are found with most of the dimorphic fungi. The characteristic microscopic morphologic features are single, drcular-to-pyriform conidia produced on short conidiophores that resemble lollipops (Figure 50-72); less commonly, the conidiophores may be elongated. The production of conidia in some isolates is minimal or absent particularly on a medium containing blood enrichment.

When incubated at 37° C, colonies of the yeas! form develop within 7 days and appear waxy and wrinkled, and cream to tan. Microscopically, large, thick-walled yeast cells (8 to 15 jim) with buds attached by a broad base are seen (see Figure 50-68). SoiriP* strains may produce yeast cells as small as 2 to 5 gm, which have been termed microforms. These small fotmn may resemble C. neoformans or H. capsulatum. Although^ these microforms may be present, a thorough seardT should reveal more typical yeast forms. During con^ version, swollen hyphal forms and immature cells with rudimentary buds will likely also be present. Because the conversion of B. dermatitidis is easily accomplished, this is feasible in the clinical laboratory; however, thi? is the most appropriate instance where mold-to-yeast -conversion should be attempted. Blastomyces dermatitidii may also be identified by the presence of a specific (i.e<> A) band in the exoantigen test97 or by nucleic acid probe, testing. In some instances, H. capsulatum, P. boydii, or % rubrum might be confused microscopically with S. dermatitidis. The relatively slow growth rate of B. de matitidis and careful examination of the microscopic morphologic features will usually differentiate it fro® these fungi.

Coccidioides immitis. Microscopically, some cuUi tures show small septate hyphae that often exbibjf gght-angle branches and racquet forms. With age, the hyphae form arthroconidia that are characteristically Hctangular-to-barrel-shaped. The arthroconidia are Vrger than the hyphae from which they were produced and stain darkly with lactophenol cotton or aniline blue. The arthroconidia are separated from one another by iear or lighter staining nonviable cells (disjunctor cells). These types of conidia are referred to as alter-n?te arthroconidia (see Figure 50-64). Arthroco-ni "a have been reported to range from 1.5 to 7.5 pm in width and 1.5 to 30 pm in length, whereas most are 3 to 4.5 pm in width and 3 pm in length. Variation has been reported in the shape of arthroconidia and ranges from rounded to square or rectangular to curved; however, most are barrel-shaped. Even if alternate arthroconidia are observed microscopically, the definitive identification should be made using nucleic acid probe testing. If a culture is suspected of being C. immitis, it should be sealed with tape to prevent chances of laboratory-acquired' infection. gecause C. immitis is the most infectious of all the fungi, extreme caution should be used when handling cultures of this organism.

Safety precautions include the following:

  • lt; If culture dishes are used, they should be handled only in a biological safety cabinet. Cultures should be sealed with tape if the specimen is suspected of containing C. immitis.
  • The use of cotton plug test tubes is discouraged, and screw-capped tubes should be used if culture tubes are preferred. All handling of cultures of C, immitis in screw-capped tubes should be performed inside a biological safety cabinet. }■ All microscopic preparations for examination should be prepared in a biological safety cabinet, i. Cultures should be autoclaved as soon as the final identification of C. immitis is made.

Other, usually nonvirulent fungi that resemble C. immitis microscopically may be found in the environment. Some molds, such as Malbranchea sp., also produce alternate arthroconidia, but these tend to be more rectangular, and it is necessary to consider them when ihaking the identification. Geotrichum candidum and Trichosporon spp. produce hyphae that disassociate into contiguous arthroconidia; these should not be confused with C. immitis (Figure 50-73 and see Figure 50-63). The colonial morphologic features of older cultures of these fungi may resemble C. immitis, but, as noted, the arthroconidia are not alternate. It is also important to remember that if confusion in identification does arise, or when occasional strains of C. immitis that fail to sporulate are encountered, identification by exoantigen or nucleic acid probe testing may be performed.

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