Approach to identification of the Yeasts
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p777-779
2025-12-07
48
The general approach to yeast identification consists of evaluating the carbohydrate and substrate utilization profile with a commercial system and observing the morphology in a cornmeal preparation. This latter aspect is particularly important for discovering any errors in identification that may have been made by the commercial system and prevents the release of an erroneous identification to the clinician. For example, if a commercial system designates an isolate as C. glabrata but pseudohyphae are seen in the cornmeal preparation, additional testing is needed to identify the isolate correctly, because C. glabrata does not produce pseudohyphae. This traditional approach could be modified to use newer methods of confirmation, such as CHROMagar Candida.
Candida spp. C. albicans may be identified by the production of germ tubes or chlamydoconidia (Figure 1). Other Candida spp. are most commonly identified by the utilization of specific substrates and the fermentation or assimilation of particular carbohydrates. For instance, C. glabrata ferments and assimilates only glucose and trehalose, whereas C. tropicalis ferments and assimilates sucrose and maltose. Another method of identifying C. albicans and differentiating it from other Candida spp. is based on the presence of chlamydoconidia (see Figure 1) on cornmeal agar containing 1% Tween 80 and trypan blue incubated at room temperature for 24 to 48 hours. (Many of the finer points of yeast identification are discussed in The Yeasts: A Taxonomic Study, by Kreger-Van Rij.) The morphologic features of yeasts on cornmeal agar containing Tween 80 often allow for tentative identification of selected species and, an important benefit, may detect misidentifications by commercial systems (Table 1).
Fig1. Chlamydoconidia of Candida albicans (arrows).
Table1. Characteristic Microscopic Features of Commonly Encountered Yeasts on Cornmeal Tween 80 Agar
Colonies that appear star-like or possess feet-like projections on agar, as previously described on blood agar, may be identified as C. albicans, according to the Clinical Laboratory Standards Institute document M35-A2. However, this method is not as sensitive as traditional methods when colonies are examined within 18 to 24 hours versus 24- to 48-hour incubation times. In addition, species such as Trichosporon spp. may give false-positive results for possessing the pseudohyphal fringe that appears as starting on blood agar. A Gram stain of the isolate would provide a means of differentiation of the isolate as Trichosporon spp. by the characteristic presence of arthroconidia. The susceptibility profiles of C. albicans and Trichosporon spp. would also be significantly different.
Germ Tube Test
The germ tube test (see Procedure 1 ) is the most generally accepted and economical method used in the clinical laboratory to identify yeasts.
Procedure 1
Approximately 75% of the yeasts recovered from clinical specimens are C. albicans, and the germ tube test usually provides sufficient identification of this organism within 3 hours.
Germ tubes appear as early hyphal-like extensions of yeast cells that are produced without a constriction at the point of origin from the yeast cell. Another Candida species, C. dubliniensis, has been shown to also produce true germ tubes. Although C. dubliniensis is infrequently encountered, supplemental biochemical or morphologic testing may be needed to differentiate it from C. albicans. C. tropicalis produces what has been called “pseudo-germ tubes,” which are constricted at the base or point of germ tube origin from the yeast cell. Unless this is recognized and the laboratorian has developed the skills to distinguish between true germ tubes and pseudo-germ tubes, C. tropicalis isolates will be mis identified as C. albicans.
The search for a more rapid, less subjective method of identifying C. albicans and other Candida spp. continues. C. albicans produces beta-galactose aminidase and L-proline aminopeptidase. Other Candida spp. may produce one enzyme but not both. Assays such as BactiCard Candida (Remel Laboratories, Lenexa, Kansas), were designed to detect these enzymes.
Heelan et al. compared the germ tube test to BactiCard, Murex C. albicans-50, Albicans-sure, and the API 20C AUX yeast identification systems. All rapid enzymatic screening methods were sensitive and specific for rapid identification of C. albicans. Compared with the germ tube test, all required less time (5 to 30 minutes), were more expensive, and required some additional equipment. Overall, all methods provided rapid and objective alternatives to the germ tube test.
CHROMagar Candida is another product that uses enzymatic reactions to differentiate C. albicans and several other yeast species. More recently C. albicans PNA FISH was released for detection and differentiation of C. albicans from non-albicans Candida spp. directly in positive blood cultures that contain yeast. The use of any new or additional testing for identification of yeasts should be submitted to a financial impact and outcomes analysis and should be compared with the traditional identification methods. Thereafter, the medical director, in conjunction with the medical staff, may determine the optimal approach for the laboratory.
Cryptococcus neoformans
Microscopic examination of colonies of C. neoformans may be helpful for providing a tentative identification of C. neoformans, because the cells are spherical and vary considerably in size. A presumptive identification of C. neoformans may be based on rapid urease production and failure to utilize an inorganic nitrate substrate. Final identification of C. neoformans usually is based on typical substrate utilization patterns and, in some laboratories, pigment production on niger seed (thistle or birdseed) agar (Figure 2). Immunocompromised patients suffering from infection die each year, with an estimated 500,000 in Africa alone. Diagnosis is typically made by identifying the encapsulated yeast in the spinal fluid using India ink. The use of serological techniques for the detection of the cryptococcal polysaccharide capsule glucuronoxylamannan (GXM) may be completed using latex agglutination or enzyme-linked immunosorbent assay (ELISA). A recent study demonstrated that plasma and urine can be used to identify cryptococcal antigen, thereby eliminating the need for the invasive spinal tap. The use of alternate specimens has the potential to improve screening of patients infected with HIV and reduced CD4 lymphocyte counts before the exacerbation of symptoms, which may not be apparent early enough for successful treatment of the infection. Additional tests useful for identifying C. neoformans and other species of cryptococci are discussed later in the chapter.
Fig2. Cryptococcus neoformans colonies are brown when grown on niger seed agar.
Rapid Urease Test
The rapid urease test (see Procedure 2 ) is a most useful tool for screening for urease- producing yeasts recovered from respiratory secretions and other clinical specimens. Alternatives to this method include use of a heavy inoculum of the tip of a slant of Christensen’s urea agar and subsequent incubation at 35° to 37°C. In many instances, a positive reaction occurs within several hours; however, 1 to 2 days of incubation may be required. Interestingly, strains of Rhodotorula spp., some Candida spp., and Trichosporon spp. hydrolyze urea with time, so a distinction should be made between a traditional urease test, which takes hours, and the rapid urease test.
Procedure 2
The microscopic morphologic features of the yeast in question are helpful for interpreting the usefulness of the traditional urease test. An alternative to traditional urease testing is the rapid selective urease test (see Procedure 3 ). This method appears to be useful for rapidly detecting C. neoformans. These screening tests are helpful for making a presumptive identification of C. neoformans. When inoculum is limited, the laboratorian must use tests that can be performed and then prepare a subculture so that additional tests can be performed later. Often, inoculating the organism onto the surface of a plate of niger seed agar is just as fast, and results may be obtained during the same day of incubation at 25°C.
Procedure 3
All the tests mentioned provide a tentative identification of C. neoformans; however, they must be supplemented with additional tests (usually the results of a commercial system in conjunction with cornmeal agar morphology) before a final identification can be reported. Additional tests useful in the identification of potential cryptococci are the nitrate reduction test and the detection of phenoloxidase production.
Trichosporon spp.
The presence of contiguous arthroconidia that are rectangular, often with rounded ends, and predominate, along with septate hyaline hyphae, raises the possibility of Trichosporon spp. Blastoconidia are sometimes present but are not seen in all cultures. Urease production is helpful for differentiating Trichosporon spp., which are positive, from Blastoschizomyces and Geotrichum spp., which are negative. Final identification is based on the characteristic substrate utilization.
Malassezia spp.
M. furfur may be recovered from the blood of patients who have fungemia. In most instances the residual lipid (from lipid replacement therapy) is adequate to support primary growth of the organism in the blood culture. However, subculture onto additional media requires overlaying of the inoculum by olive oil or another source of long-chain fatty acids. These findings, in conjunction with the “bowling pin” or “pop bottle” morphology, are sufficient for identification. Other Malassezia spp. do not require long-chain fatty acids and are traditionally identified using substrate utilization analysis in conjunction with cornmeal agar morphology.
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