Chromogenic Tube Test for Presumptive Identification or Confirmation of Isolates as Candida albicans

Abstract

This report describes a new, modified, simple, and cost-effective method for the use of CHROMagar Candida (CHROMagar Company, Paris, France) for the presumptive identification of isolates as Candida albicans after preliminary growth. Sixty randomly selected clinical isolates were evaluated, including 38 of C. albicans. With incubation at 37°C for 24 h, the sensitivity and specificity appeared to be excellent and the test performed better than the traditional germ tube test. However, at earlier times, C. tropicalis isolates gave false-positive results.

Candida albicans is one of the most frequently isolated yeasts in clinical laboratories. Studies have shown that this organism can account for up to 75% of the yeasts recovered from sites of infection (17, 21). The isolation of C. albicans has been associated with infections, as well as colonization, in both immunocompromised and immunocompetent patients. Predisposing factors for isolation include leukemia, organ transplantation, and human immunodeficiency virus infection, along with extensive surgical procedures or prolonged antibacterial administration (7, 17, 20–22).

Traditionally, in the clinical laboratory, the most helpful, rapid, and cost-effective identification system for the presumptive identification of isolates as C. albicans has been the germ tube test (GTT). As described by Rinaldi, this germ tube-like formation in C. albicans requires (i) that yeasts be in the correct nutrient state, (ii) the presence of an inducer (e.g., serum or other chemical formulations that mimic serum biochemically), (iii) an elevated temperature (>33°C), and (iv) a nearly neutral pH (17). Although this is a rapid test, problems with misinterpretation of elongated blastoconidia for positive germ tubes or false positives due to increased incubation time and false negatives due to a heavy inoculum may prove to be a problem. This, coupled with the time needed for detailed microscopic examination, may make this test liable to error in busy laboratories. It has also been reported that up to 5% of C. albicans isolates are germ tube negative (16, 18).

Recent reports (1–4, 6, 8, 9, 11, 12, 19) have described the use of CHROMagar Candida, a new chromogenic medium, as a primary isolation plate medium for commonly encountered yeast species from clinical specimens. This medium is effective in presumptively identifying C. albicans and other common yeast species. However, its expense as a single plate for routine direct isolation of yeasts may discourage many laboratories from its use. This study describes a new, modified, simple, and cost-effective method for the use of CHROMagar Candida based on a tube test for presumptive identification or as a confirmatory supplementary test in conjunction with the traditional GTT for C. albicans from primary plates.

Yeast cultures and identification.

Sixty randomly selected yeast isolates were examined in this study. They were isolated from clinical material received by the Department of Microbiology and Infectious Diseases at Concord Repatriation General Hospital. Isolates were grown on Sabouraud dextrose agar (Oxoid, Basingstoke, United Kingdom) at 37°C for 24 h. Each isolate was first presumptively identified by the CHROMagar tube test as described in this report. On completion, isolates were presumptively identified by the GTT method (5, 10, 22) and later this identification was confirmed by examination of conventional morphological characteristics on cornmeal agar, carbohydrate assimilation testing, and (only if the findings were discrepant) the use of ID 32 C (bioMerieux Vitek-Australia) strips (5, 22). Initially, each organism was coded and tested in a blind fashion and results were compiled at the end of the study. Reference controls C. albicans AMMRL 36.30, C. tropicalis AMMRL 36.16, C. (Torulopsis) glabrata ATCC 90030, and C. parapsilosis ATCC 90018 were used for quality control testing.

CHROMagar Candida was obtained from the CHROMagar Company, Paris, France, through Dutec Diagnostics (Sydney, Australia). The medium was prepared as instructed by the manufacturer. Since the medium does not require sterilization by autoclaving, after reconstitution, it was kept in liquid form in a water bath. With a glass pipette, 1 ml was dispensed into 2-ml clear, nonsterile plastic tubes, allowed to set at room temperature, capped, and stored at 4°C for up to 1 month. Sabouraud dextrose agar was prepared as instructed by the manufacturer. Germ tube broth was prepared as described by Ogletree et al. (10). The CHROMagar Candida tubes were inoculated with a nonsterile applicator stick. A well-isolated test colony was removed with the end of the applicator stick, which was then stabbed (1 to 2 mm) and spread onto the surface of the medium within the tube. Inoculated tubes were placed into an incubator in air at 30 and 37°C in the dark and viewed at intervals of 2.5, 4, 8, and 24 h with a final confirmatory reading at 48 h. Reading at various times was performed independently by three different scientists, and any variation was recorded. Green color development, as originally described by Odds and Bernaerts (9) and others (1–4, 6, 8, 11, 12, 19), at any stage indicated a positive test for C. albicans. Any other color was interpreted as being negative for C. albicans. Germ tube broths were inoculated, incubated, and interpreted as described by Walsh and Pizzo (22).

All four of the reference control strains employed in this study grew in the CHROMagar Candida tubes at 30 and 37°C. The C. albicans control strain was clearly distinguished from all of the other strains by its distinctive green color after 2.5 h of incubation. The color was more pronounced at 37°C than at 30°C. From 2.5 to 8 h at 30°C, the C. tropicalis control strain could have been easily mistaken for a C. albicans strain, but it was easily visually differentiated by its blue color after 8 and 24 h of incubation, especially at 37°C.

Comparative identification analysis.

As shown in Table 1, 38 (63%) of the 60 yeast isolates tested were identified as C. albicans. These results were later confirmed by the GTT, examination of morphological characteristics, and carbohydrate assimilation testing. There were no discrepancies. Two strains of C. tropicalis initially gave false-positive GTT results. The tests were repeated with both serum and nonserum formulations, and the results were confirmed by further assimilation testing. Both strains were clearly distinct from the C. albicans isolates by the CHROMagar tube test after 24 h (blue) at 30 and 37°C. One isolate could be clearly visually distinguished within 8 h at both temperatures. No dehydration of the medium due to prolonged incubation at 30°C was observed; however, some dehydration of the medium was observed at 48 h of incubation at 37°C.

TABLE 1.

Identification of C. albicans by the chromogenic tube test

Species	No. of isolatesa
	Tested	Positive by chromogenic tube test after 24 h of incubation	Positive by GTT	Positive by conventional assimilation and morphoogical ID
Candida albicans	38	38b	38	38
Candida (Torulopsis) glabrata	13	0	0	13
Candida tropicalis	3	0c	2d	3
Candida parapsilosis	2	0	0	2
Candida krusei	2	0	0	2
Candida kefyr	1	0	0	1
Candida lusitaniae	1	0	0	1

^aA total of 60 isolates were tested. 

^bFalse-negative results can occur below this incubation time. 

^cFalse-positive results can occur below this incubation time. 

^dTwo false positives were detected. Both were isolates identified later as C. tropicalis. 

The findings in this study show that when a small volume (1 ml) of CHROMagar Candida medium was inoculated directly with isolated colonies from a primary plate such as a Sabouraud dextrose agar plate, the inocula provided the chromogenic medium with a higher concentration of species-specific enzymes. This results in faster utilization of enzyme substrates visualized by early release of the chromophore at 30 or 37°C. This decreases the long incubation period reported previously (1–4, 6, 8, 9, 11, 12, 19).

C. tropicalis strains are also recognized clinical pathogens (7). Results obtained with the limited number of strains tested in this study show that C. tropicalis strains can interfere with CHROMagar tube test color development at incubation times of 2.5 to 8 h and that incubation for less than 8 h can easily cause C. tropicalis to be mistaken for C. albicans and vice versa. Therefore, it is recommended that a 24-h incubation time be used before results are interpreted. After 24 h of incubation at 30 and 37°C, the CHROMagar tube test for C. albicans and other candida strains revealed no false-positive or -negative results, as shown in Table 2.

TABLE 2.

Chromogenic identification of C. albicans among 60 random yeast isolates after preliminary growth at 30 and 37°C

Incubation temp (°C)	No. of CHROMagar Candida tube test-positive results at:
	2.5 h	4 h	8 h	24 h	48 h
30	38a	40b	39c	38	38
37	40d	40e	39f	38	38

^aTwo false negatives and two false positives were detected. Two strains of C. albicans gave no color at this incubation time, and two strains of C. tropicalis gave a green color. 

^bTwo false positives were detected, both strains of C. tropicalis. 

^cOne false positive was detected. One strain of C. tropicalis remained green at this incubation temperature. 

^dTwo false positives, both C. tropicalis, and no false negatives were detected. 

^eTwo false positives, both C. tropicalis, were detected. 

^fOne false-positive strain of C. tropicalis was detected. 

The GTT offers a simple, rapid, easy-to-perform, and well-known test for identification of C. albicans infections for microbiology laboratories without adequately trained mycology personnel. However, results from this study and others (5, 10, 17, 18, 22) show that false positives could easily occur. If a CHROMagar tube test is performed as a supplementary test in conjunction with the GTT, it offers an added confirmatory interpretation while still being cost-effective.

The advantages of this technique are that (i) it is cost-effective compared to other methods, e.g., 20 tests can be performed for the cost of an equivalent volume (20 ml) of a single CHROMagar Candida plate; (ii) it is easy to perform as a single test or in conjunction with the GTT as a supplementary test; (iii) the incubation time is shorter than those of other assimilation tests for C. albicans identification; and (iv) it is less labor intensive during interpretation, and the test can be left overnight without the possibility of false positives compared to the GTT. The major disadvantage of this technique is that unlike a plate medium, the tube test does not allow mixed cultures to be detected. Hence, as with the GTT, pure cultures are essential for testing with an inoculum taken from a single colony.

This is a rapid phenotypic presumptive test for germ tube-positive yeasts. Further tests, such as sucrose assimilation, cycloheximide susceptibility, or genotypic tests, may be needed to distinguish C. albicans isolates from less common, closely related species such as C. stellatoidea or C. dubliniensis.

In summary, very similar to the Pickett scheme for identification of gram-negative, nonfermenting bacteria (13–15), the CHROMagar tube technique, incorporating the use of a concentrated inoculum from preliminary growth, offers another alternative cost-effective use for this chromogenic medium for the phenotypic distinction of C. albicans from other commonly encountered yeast species after 8 to 24 h of incubation. Less common yeast isolates need further evaluation with this procedure. The traditional methodology as described by Odds and Bernaerts (9) is still recommended for culturing direct clinical specimens.