Mycopathologia81, 63-64 (1983). 0301 486x/83/81/0063-2/$00.20/0.

9 Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

Preliminary evaluation of a new test for the rapid differentiation of Candida albicans and
Candida stellatoidea

M. C a s a l & M . J. Linares
Department ofMicrobiology, School ofMedicin~ C6rdoba University, C6rdoba, Spain

Abstract

A preliminary investigation test was carried out concerning the use of esculin hydrolysis to differentiate
Candida albicans from Candida stellatoidea is described. This test provides a rapid and reliable means of
distinguishing C. albicans from C. stellatoidea.

A m o n g hospital acquired nosocomial infections,
opportunistic microorganisms play a very impor-
tant part. Due to a great number of favouring risk
factors, the yeasts, among all these microorga-
nisms, are becoming increasingly more important
because of serious diseases that they can cause in
patients. Of all these - yeasts Candida albicans is
without doubt the most important species due to its
frequency and capacity to produce disease.
The problem which arises is that the differential
diagnosis is difficult with another species, Candida
stellatoidea, - which is also pathogenic. Identifica-
tion is usually based on chlamydospore production,
germ tube formation, whose results are not always
100% accurate. Therefore carbohydrate assimila-
tion tests with sucrose have to be used for definitive
identification.
For this reason it was considered important to
find a rapid differential diagnostic test that could
unequivically differentiate these species. With this
in mind, a new medium with~cycleheximide had
been proposed recently to differentiate these species
(7). However the results of the works of other au-
thors do not seem to agree (6).
In this paper we hypothosize that esculin decom-
position could be used as a possible differential
character between C. albicans and C. stellatoidea.
We used a total of 206 yeasts isolates of which 186
were C. albicans and 20 C. stellatoidea. They all
belonged to the culture collection of our depart-
ment and had been obtained from the yeast collec-
tion of the Wadley Institutes for Molecular Medi-
cine, Dallas, Texas or had been isolated in our own
laboratory and in Dr. Calbo Torrecillas's in Mfila-
ga, from clinical material.
The conservation media used for the cultures
were Sabouraud's dextrose agar or Sabouraud's
dextrose agar with chloramphenicol or both. The
cultures were incubated at 25-28 ~C until the mo-
ment for use.
The medium used for the study esculin hydrolysis
was composed of peptone water (100 ml) esculin
(0.1 g) and ferric citrate (0.05 g). The test was car-
ried out by making an emulsion of a young culture
of the yeast to be studied in 3 ml of the medium (5).
The incubation was at 37 ~ and the reading was
made on the 2nd and 7th day. The other methods of
identification such as chlamydospore production,
germ tube formation, fermentation and assimila-
tion of carbohydrates were made according to me-
thods previously described (1-4).
With these tests the preliminary identification of
cultures as C. albicans or C. stellatoidea was made.
The results are summarized in Table 1. All the C.
albicans isolates tested were positive for esculin
hydrolysis within 48 hours. However this was not so
for C. stellatoidea as none of the isolates hydro-
lyzed esculin in 48 hours nor after many days of
incubation (Table I).
64
Table 1. Comparative study of the different tests used.
Species N ~ of Chlamydospores
isolates
C. albicans 186 94
C. stellatoidea 20 85
On the basis of our results it seems that esculin
hydrolysis can be used as a test to differentiate C.
albicans and C. stellatoidea with 100% effective-
ness, which makes it even more effective than the
sucrose assimilation test. The esculin has advan-
tages compared with other standard morphological
and physiological methods of identification: 100%
accuracy and rapidity. It does not require difficult
or expensive media to prepare or interpret.
It seems that this test should be incorporated in
the routine system of all hospital laboratories which
work with these yeasts as an extra test to differen-
tiate between C. albicans and C. stellatoideal For
this reason we can recommend as a system for their
identification two tests to he performed at the same
time, the chlamydospore production test, and the
esculin hydrolysis test so that in 48 hours it can be
possible to read and interpret the tests. If the first
one is positive and the second is negative or both
prove to be negative, it would indicate presence of
C. stellatoidea.
This test saves time and money so therefore the
test with sugars could be reserved for use only in
cases where a Candida species is not albicans or
stellatoidea.
As this is a preliminary investigation, it would be
interesting to repeat and amplify this study with
more isolates to confirm the effectiveness of the
esculin.
% of positivity
Germ tube Sucrose esculin
98 97 100
98 0 0
Acknowledgements
We thank W. H. Fleming, Wadley Institutes of
Molecular Medicine, Dallas, Texas, U.S.A.; and F.
Calbo Torrecillas Department of Microbiology,
School of Medicine, Malaga University, Spain, for
providing some of the cultures used in this study.
We are grateful to Dr. L. Ajello, Director My-
cology Division, CDC, Atlanta, GA, U.S.A., for
his helpful advice on the preparation of this paper.
References
1. Casal, M. & Linares, M. J., 1980. Contribucion al diagnosti-
co de los hongos levaduriformes de importancia clinica hos-
pitalaria. Med. Clin. 75: 384-386.
2. Casal, M. & Linares, M. J., 1980. Investigaci6n comparativa
de diferentes medios de estudio de la fermentaci6n de carbo-
hidratos por los hongos levaduriformes pat6genos para el
hombre. Laboratorio 69: 321-332.
3. Casal, M. & Linares, M . J . , 1981. The comparison of six
media for chlamydospore production by Candida albicans.
Mycopathologia 76: 125-128.
4. Casal, M. & Linares, M. J., 1981. lnteres de la utilizaci6n de
la leche en el diagn6stico r/tpido de Candida albicans. Labo-
ratorio 71:305-311.
5. Mac Faddin, J. F., 1980. Biochemical Test for Identification
of Medical Bacteria, 2nd. ed. The Williams and Wilkins Co.
Baltimore.
6. Saez, H., 1979. Interet de la resistence a l'actidione et de la
temperature maximale dans la diagnose des Candida. Rev.
Inst. Pasteur 12: 361-370.
7. Salkin, 1. F., 1979. New medium for differentiation of Can-
dida albicans from Candida stellatoidea. J. Clin. Microbiol.
9:551-553.