Specimen collection, transport, and processing of The Yeasts
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p774-777
2025-12-07
72
Stains
Candida spp. Direct microscopic examination of clinical specimens containing Candida organisms reveals budding yeast cells (blastoconidia) 2 to 4 μm in diameter and/or pseudohyphae (Figure 1) showing regular points of constriction, resembling links of sausage. True septate hyphae (filamentation) may also be produced by C. albicans and C. dubliniensis. The blastoconidia, hyphae, and pseudohyphae are strongly gram positive. The approximate number of such forms should be reported, because the presence of large numbers in a fresh clinical specimen may have diagnostic significance. Microscopically C. glabrata blastoconidia are notably smaller (at 1 to 4 µm) than those of other medically significant Candida spp.
Fig1. Periodic acid-Schiff (PAS) staining of urine demonstrates blastoconidia and pseudohyphae of Candida albicans.
Cryptococcus spp. Traditionally, the India ink preparation has been the most widely used method for the rapid detection of C. neoformans in clinical specimens. This method is still used as a rapid and inexpensive assessment tool in many institutions and has considerable diagnostic value in resource-poor settings. This method delineates the large capsule of C. neoformans, because the ink particles cannot penetrate the capsular polysaccharide material. Although this test is useful, many laboratories have replaced it with the more sensitive cryptococcal latex agglutination test that detects cryptococcal antigen. (The cryptococcal antigen detection [CAD)]test is described later in the chapter.) The India ink preparation is commonly positive in specimens from patients with AIDS and has been shown to have a sensitivity of 50% in patients who do not have HIV or AIDS. Laboratories that examine many specimens from these patients may want to continue using this procedure in combination with the CAD test and culturing.
Microscopic examination of other clinical specimens, including respiratory secretions, can be valuable for making a diagnosis of cryptococcosis. C. neoformans appears as a spherical, single or multiple budding, thick-walled yeast 2 to 15 μm in diameter. It usually is surrounded by a wide, refractile polysaccharide capsule (Figure 2). Perhaps the most important characteristic of C. neoformans is the extreme variation in the size of the yeast cells; this is unrelated to the amount of polysaccharide capsule present. It is important to remember that not all isolates of C. neoformans exhibit a discernible capsule.
Fig2. Potassium hydroxide preparation of pleural fluid shows the encapsulated, variably sized, spherical yeast cells (arrow) of Cryptococcus neoformans (phase-contrast microscopy).
Trichosporon spp. Microscopic examination of clinical specimens that contain Trichosporon spp. reveals hyaline hyphae, numerous round to rectangular arthroconidia, and occasionally a few blastoconidia. Usually hyphae and arthroconidia predominate. In white piedra, white nodules are removed and observed using the potassium hydroxide (KOH) preparation after light pressure is applied to the coverslip to crush the nodule. Hyaline hyphae 2 to 4 μm wide and arthroconidia are found in the preparation of the cementlike material that binds the hyphae together. The organism may be identified in culture by the presence of true hyphae, blastoconidia, and arthroconidia in conjunction with a positive urease. Although Trichosporon asahii may be distinguished from other Trichosporon species by its bio physical profile (carbohydrate and substrate utilization), these organisms are likely best distinguished at the species level with molecular tools, such as DNA sequencing.
Malassezia spp. M. furfur most often is detected through direct microscopic examination of skin scrapings. The organism is easily recognized as oval- or bottle-shaped cells that exhibit monopolar budding in the presence of a cell wall with a septum at the site of the bud scar. Small hyphal fragments also are observed (Figure3); the morphology is commonly described as “spaghetti and meatballs.” In cases of fungemia, the morphologic form seen in direct examination of blood cultures is small yeasts without the presence of pseudohyphae.
Fig3. Potassium hydroxide preparation of a skin scraping from a patient with tinea versicolor demonstrates spherical yeast cells (A) and short hyphal fragments (B) of Malassezia furfur. (Phase contrast microscopy; ×500.)
Antigen Detection
The CAD test for C. neoformans may be performed on CSF or serum. In many laboratories this assay has replaced the use of India ink to screen for C. neoformans. It should be noted that Trichosporon spp. produce an antigen similar to that of C. neoformans. Sera from patients who have trichosporonosis often yield false-positive CAD tests when latex agglutination methods are used.
Molecular Assays
Nucleic acid amplification tests (NAATs) have been developed for a variety of yeast species. However, these are usually performed in research settings. Most are labeled as “laboratory-developed tests” or “home-brewed tests,” and few have been approved by the U.S. Food and Drug Administration (FDA). Real-time polymerase chain reaction (PCR) methods are now commercially available in the TaqMan system (Applied Biosystems, USA) and LightCycler (Roche Molecular Systems, Indianapolis, Indiana). These methods are very expensive but have proven to be much more specific than conventional yeast identification methods. Ligouri et al. recently demonstrated the Multiplex PCR method for identifying Candida spp. that has high concordance with commercially available phenotypic identification systems, such as the API 20C AUX (bioMérieux, Durham, North Carolina) and Vitek 2 YST card (bioMérieux, Durham, North Carolina). The Multiplex PCR method is much faster and more sensitive than the current phenotypic tests. A newer molecular test, the PNA FISH kit, uses in situ hybridization to detect Candida organisms in blood culture bottles by targeting specific rRNA sequences. Sub culturing should follow this method, because whether more than one species is present cannot be determined. Colonies may also display protrusions from the colony, resembling a star or foot-like projections on blood agar.
Cultivation
Candida spp. The colonial and microscopic morphologic features of Candida spp. have little value for making a definitive identification. Most Candida spp. produce smooth, creamy white colonies, but some produce dry, wrinkled, dull colonies. In 50% of autopsy-proven cases of invasive candidiasis, organisms could not be isolated from blood culture bottles. Newer blood culture systems, such as the automated BACTEC (Becton Dickinson, Franklin Lakes, NJ) and BacT/ALERT (Biomerieux, Durham, N.C.), have increased the recovery of Candida spp.
Cryptococcus spp. C. neoformans is easily cultured on routine fungal culture media without cycloheximide. The organism is inhibited by the presence of cycloheximide at 25° to 30°C. For optimal recovery of C. neoformans from cerebrospinal fluid, a 0.45-mm membrane filter should be used with a sterile syringe. The filter is placed on the surface of the culture medium and is removed at daily intervals so that growth under the filter can be visualized. An alternative to the membrane filter technique is culture after centrifugation.
Colonies of C. neoformans usually appear on culture media within 1 to 5 days. The growth begins as a smooth, white to tan colony that may be mucoid to creamy (Figure 4). It is important to recognize the colonial morphology on different culture media, because variation does occur; for example, on inhibitory mold agar, C. neoformans appears as a golden yellow, nonmucoid colony. Textbooks typically characterize the colonial morphology as being Klebsiella-like because of the large amount of polysaccharide capsule material present. In reality, most isolates of C. neoformans do not have large capsules and may not have the typical mucoid appearance.
Fig4. Colonies of Cryptococcus neoformans appear shiny and mucoid because of the presence of a polysaccharide capsule.
Trichosporon spp. Colonies of Trichosporon spp. vary in their morphology; however, most are cream colored, heaped, dry to moist, and wrinkled. Some may appear white, dry, and powdery.
Malassezia spp. M. furfur is infrequently cultured in the clinical laboratory. Recovery of the organism is not required to establish a diagnosis (in skin infections), and it is seldom attempted for this purpose. Cultivation, such as from a positive blood culture, requires an agar medium overlaid with a long-chain fatty acid (olive oil). The colonies are small compared with the colonies of C. albicans and are creamy and white to off-white.
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