Previous Article | Next Article 
Journal of Clinical Microbiology, April 1998, p. 1157-1159, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Chromogenic Tube Test for Presumptive
Identification or Confirmation of Isolates as Candida
albicans
John
Merlino,1,2,*
Evanthia
Tambosis,1 and
Duncan
Veal2
Department of Microbiology and Infectious
Diseases, Concord Repatriation General Hospital, Concord, New South
Wales 2139,1 and
School of Biological
Sciences, Macquarie University, New South Wales
2109,2 Australia
Received 21 July 1997/Returned for modification 9 September
1997/Accepted 22 December 1997
 |
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.
| |
TEXT |
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.
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.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Chromogenic identification of C. albicans
among 60 random yeast isolates after preliminary growth at 30 and 37°C
|
|
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.
| |
ACKNOWLEDGMENTS |
We thank the Australian Medical Mycology Reference Laboratory at
Royal North Shore Hospital, Sydney, for supplying control strains for
this study. We are grateful for the skilled support of Mohammad
Siddique for medium preparation and the technical assistance of the
Department of Microbiology and Infectious Diseases at Concord
Repatriation General Hospital. Ross Bradbury, Thomas Gottlieb, Glenn
Funnell, Mary Fisher, Steven Siarakas, and Deborah Parkinson critically
reviewed the manuscript. We thank David Ellis for discussions and
suggestions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Infectious Diseases, Concord Repatriation General
Hospital, Hospital Rd., Concord, NSW 2139, Australia. Phone: (612) 9767 6658. Fax: (612) 9767 7868. E-mail:
john{at}micr.crg.cs.nsw.gov.au.
| |
REFERENCES |
| 1.
|
Baumgartner, C.,
A. M. Freydiere, and Y. Gille.
1996.
Direct identification and recognition of yeast species from clinical material by using Albicans ID and CHROMagar Candida plates.
J. Clin. Microbiol.
34:454-456[Abstract].
|
| 2.
|
Beighton, D.,
R. Ludford,
D. T. Clark,
S. R. Brailsford,
C. L. Pankhurst,
G. F. Tinsley,
J. Fiske,
D. Lewis,
B. Daly,
N. Khalifa,
V. Marren, and E. Lynch.
1995.
Use of CHROMagar Candida medium for isolation of yeasts from dental samples.
J. Clin. Microbiol.
33:3025-3027[Abstract].
|
| 3.
|
Bernal, S.,
E. M. Mazuelos,
M. Garcia,
A. I. Aller,
M. A. Martinez, and M. J. Gutierrez.
1996.
Evaluation of CHROMagar Candida medium for the isolation and presumptive identification of species of candida of clinical importance.
Diagn. Microbiol. Infect. Dis.
24:201-204[Medline].
|
| 4.
|
Blazquez, R.,
J. Berenguer,
C. Sanchez-Carillo,
S. Moreno,
T. Pelaez, and E. Bouza.
1995.
Utility of CHROMagar Candida (CAC), a new differential isolation medium in the mycological diagnosis of oropharyngeal candidiasis (OC) in HIV-infected patients (HIV + pts), abstr. D7, p. 67.
In
Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C.
|
| 5.
|
Cooper, B. H., and M. Silva-Hutner.
1985.
Yeasts of medical importance, p. 526-541.
In
E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C.
|
| 6.
|
Freydiere, A. M., and Y. Gille.
1994.
Yeasts: appearance of colonies on CHROMagar Candida, abstr. MS-1/22, p. 323.
In
Abstracts of the 7th International Congress of Mycology Division. International Union of Microbiological Sciences, Prague, Czech Republic.
|
| 7.
|
Marina, N. M.,
P. M. Flynn,
G. K. Rivera, and W. T. Hughes.
1991.
Candida tropicalis and Candida albicans in children with leukemia.
Cancer
68:594-599[Medline].
|
| 8.
|
Moyer, G. J.,
M. Romagnoli, and W. G. Merz.
1995.
CHROMagar for presumptive identification and detection of yeast species in oncology surveillance cultures, abstr. F-117, p. 107.
In
Abstracts of the 95th General Meeting of the American Society for Microbiology 1995. American Society for Microbiology, Washington, D.C.
|
| 9.
|
Odds, F. C., and R. Bernaerts.
1994.
CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species.
J. Clin. Microbiol.
32:1923-1929[Abstract/Free Full Text].
|
| 10.
|
Ogletree, F. A.,
A. T. Abdlel, and D. G. Ahearn.
1978.
Germ tube formation in atypical strains of Candida albicans.
Antonie van Leeuwenhoek
44:15-24[Medline].
|
| 11.
|
Perry, J. L.,
J. S. Matthews, and T. J. Sheets.
1995.
Comparison of rapid kit and selective chromogenic media for the presumptive identification of Candida albicans and other yeasts, abstr. D8, p. 67.
In
Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C.
|
| 12.
|
Pfaller, M. A.,
A. Houston, and S. Coffman.
1996.
Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata.
J. Clin. Microbiol.
34:58-61[Abstract].
|
| 13.
|
Pickett, M. J., and M. M. Pendersen.
1970.
Nonfermentative bacilli associated with man. II. Detection and identification.
Am. J. Pathol.
54:164-177.
|
| 14.
|
Pickett, M. J., and M. M. Pendersen.
1970.
Characterization of saccharolytic nonfermentative bacteria associated with man.
Can. J. Microbiol.
16:351-362[Medline].
|
| 15.
|
Pickett, M. J., and M. M. Pendersen.
1978.
Rapid methods: new methodology for identification of nonfermenters, p. 155-170.
In
G. L. Giraldi (ed.), Glucose nonfermentating gram-negative bacteria in clinical microbiology. CRC Press, Inc., West Palm Beach, Fla.
|
| 16.
|
Quindos, G.,
R. San Millan,
R. Robert,
C. Bernard, and J. Ponton.
1997.
Evaluation of Bichro-latex albicans, a new method for rapid identification of Candida albicans.
J. Clin. Microbiol.
35:1263-1265[Abstract].
|
| 17.
|
Rinaldi, M. G.
1993.
Biology and pathogenicity of Candida species, p. 1-20.
In
G. P. Bodey (ed.), Candidiasis: pathogenisis, diagnosis and treatment, 2nd ed. Raven Press, New York, N.Y.
|
| 18.
|
Salkin, I. F.,
G. A. Land,
N. J. Hurd, and P. R. McGinnis.
1987.
Evaluation of YeastIdent and Uni-Yeast-Tek yeast identification systems.
J. Clin. Microbiol.
25:625-627.
|
| 19.
|
Schär, G.
1995.
Performance of CHROMagarTM Candida in the routine mycology laboratory, abstr. D6, p. 67.
In
Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C.
|
| 20.
|
Shepard, M. G.
1990.
Biology of Candida species, p. 10-20.
In
L. P. Samaranayaka, and T. W. MacFarlane (ed.), Oral candidosis. Wright Press, London, England.
|
| 21.
|
Wade, J. M.
1993.
Epidemiology of Candida infections, p. 85-107.
In
G. P. Bodey (ed.), Candidiasis: pathogenisis, diagnosis and treatment, 2nd ed. Raven Press, New York, N.Y.
|
| 22.
|
Walsh, T. J., and P. A. Pizzo.
1993.
Laboratory diagnosis of candidiasis, p. 109-135.
In
G. P. Bodey (ed.), candidiasis: pathogenisis, diagnosis and treatment, 2nd ed. Raven Press, New York, N.Y.
|
Journal of Clinical Microbiology, April 1998, p. 1157-1159, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Aliyu, S.H., Enoch, D.A., Abubakar, I.I., Ali, R., Carmichael, A.J., Farrington, M., Lever, A.M.L.
(2006). Candidaemia in a large teaching hospital: a clinical audit. QJM
99: 655-663
[Abstract]
[Full Text]
-
Horvath, L. L., Hospenthal, D. R., Murray, C. K., Dooley, D. P.
(2003). Direct Isolation of Candida spp. from Blood Cultures on the Chromogenic Medium CHROMagar Candida. J. Clin. Microbiol.
41: 2629-2632
[Abstract]
[Full Text]
Top
Abstract
Text
