Egypt. J. Med. Lab. Sci. August 2019, Vol.

28 (2) ISSN 1110-5593

Evaluation of Different Phenotypic

Methods for Identification of Candida
Original Article
Species Isolated from Clinical Samples

Mai M. El-Ashmony1, Hala M. Hafez2, Dalia H.

Abdelhamid2, Ola A. Abd El-Rahman1, Fatma Rasslan1*
1Microbiology and Immunology Department, Faculty of Pharmacy (Girls), Al-Azhar University,
Cairo, Egypt.
2Clinical Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt.

Corresponding author:
Fatma Rasslan, Microbiology and Immunology Department, Faculty of Pharmacy (Girls), Al-Azhar
University
Email: fatma_rasslan@yahoo.com

ABSTRACT
Background and Aim: The incidence of
candidiasis has increased greatly in recent years.
Candida albicans (C. albicans) is the most
common pathogenic species responsible for
various types of candidiasis. Recently, several
studies have reported a drastic increase in the
incidence of non albicans Candida (NAC) spp.
Rapid and correct identification of Candida
isolates at the species level is very beneficial for
initiation of early and adequate antifungal
treatment. The aim of our study is to compare
between different identification methods of
Candida spp. to choose the most accurate, fast and
cost-effective method.
Materials & Methods: Candida spp. isolated
from 134 different clinical specimens were
identified by conventional methods as germ tube
test and cornmeal agar, by CHROMagar Candida
and Vitek-2 system.
Results: CHROMagar Candida accurately
identified the three widespread Candida spp., C.
tropicalis, C. albicans and C. krusei, with
sensitivity and specificity as 100%. It was found
that poly yeast specimen can be detected
simultaneously by using CHROMagar Candida.
Conclusion: Therefore, CHROMagar Candida can
be used as a primary isolation medium for prompt
identification and speciation of Candida spp. in a
short time.
Keywords: Candida spp. - Germ tube - Cornmeal
Agar - CHROMAgar Candida - Vitek-2 system.
INTRODUCTION
Candida species are human commensals which
colonize skin, mucus membrane and
gastrointestinal tract without causing any notable
harm or damage in healthy individuals. However,
in certain circumstances, they can cause a wide
range of disease (Ami, 2018 and Sardi et al.,
2013). In recent years, the incidence of fungal
infections caused by Candida spp. (candidiasis) has
increased dramatically, due to the rise in number of
immunocompromised individuals, such as AIDS
patients, patients undergoing organ transplantation,
cytotoxic chemotherapy or immunosuppression
therapies and excessive usage of broad-spectrum
antibiotics (Dadar et al., 2018; Mnge et al., 2017;
Arendrup, 2010 and Pfaller and Diekema, 2010).
Candidiasis has a wide clinical spectrum ranging

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Egypt. J. Med. Lab. Sci. August 2019, Vol.28 (2)
from superficial mucocutaneous infections to life
threatening invasive disease associated with
candidemia (Pappas et al., 2018). Currently,
candidiasis represents the fourth leading cause of
nosocomial infections, and mortality due to
systemic candidiasis remains high, ranging from
15 to 35% depending on the infecting Candida
spp. (Mnge et al., 2017; Szweda et al., 2015 and
Pfaller, and Diekema, 2007). Candida albicans
(C. albicans) is the most common pathogenic
species responsible for various types of
candidiasis. However, in recent years, there is a
drastic increase in incidence of non albicans
Candida (NAC) spp. (Al-Dorzi et al., 2018; Ami,
2018; Arendrup, 2010 and Pfaller, and Diekema,
2007).
Many of the NAC spp. such as C. krusei and C.
glabrata exhibit resistance to traditional triazole
antifungals such as, fluconazole and may also
demonstrate cross-resistance to newer triazoles
(Magill et al., 2006). Accordingly, the
identification of Candida isolates at the species
level is necessary in order to select the appropriate
antifungal agent for the treatment of serious
infections caused by Candida spp. (Souza et al.,
2015; Kumari et al., 2014 and Eraso et al., 2006).
Sabouraud Dextrose Agar (SDA) medium is the
most widely used for primary isolation of Candida
spp. from clinical specimens. However, it is not a
differential medium; as it can't differentiate
between Candida spp. (Mahmoudabadi, 2000).
Several tests with different techniques from
conventional to molecular methods are available
for identification of Candida spp. (Bharathi,
2018). Conventional method as germ tube test,
culture on cornmeal agar and sugar assimilation
test are time consuming and labor intensive (Jain
et al., 2012). Newer methods such as
CHROMagar, API systems, Vitek-2 ID system
and molecular methods have been developed for
identification of yeast spp. Chromogenic medium
contains substrates that react with enzymes
secreted by the target microorganisms to yield
different colored colonies (Mehta and
Wyawahare, 2016 and Jain et al., 2012). In view
of not all laboratories have automated or semi-
automated systems, and molecular biology-
dedicated departments, it is worth to use a manual
method that is fast, reliable and has high
sensitivity and specificity (Souza et al., 2015).
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Therefore, the aim of our study was to compare
between different identification methods of
Candida spp. in term of sensitivity, specificity and
the time required for proper identification in order
to choose the most accurate, fast and cost-effective
method.
MATERIALS AND METHODS
Specimen Collection
A total of 134 different clinical specimens were
obtained from different wards including the
intensive care unit in Ain Shams University
Hospitals from August 2018 to January 2019 for a
period of six months. Specimens included urine
(112), sputum (10), pus (5), wound swab (3),
central line (3) and one vaginal swab. Specimens
were submitted to the Microbiology laboratory of
Ain Shams University Hospitals.
Methods of Identification
Specimens were inoculated on Sabouraud dextrose
agar (SDA) (Oxoid, England) and incubated
aerobically at 37°C for 24-48 hours. Colonies from
SDA were subjected to Gram staining and then
identified on the species level depending on
standard mycological protocol including germ tube
test and chlamydospores production on cornmeal
agar (Raju and Rajappa, 2011). The colonies were
also subjected to biochemical identification using
Vitek-2 system for spp. confirmation which
considered as gold standard method (Mishra et al.,
2017).
CHROMagar Candida
CHROMagar Candida medium (HiMedia, India)
was prepared and sterilized according to the
manufacturer's instructions. Specimens were
inoculated on CHROMagar Candida plates and
incubated aerobically at 37°C for 48 hours. The
morphology and color of colonies produced were
recorded as described by the manufacturer and
compared with the results of standard mycological
protocol. The colored colonies from CHROMagar
Candida were biochemically identified using
automated Vitek-2 system.
Germ Tube Test Method
Very small inoculum from an isolated colony of
Candida spp. was picked up with a sterile
inoculating loop and was suspended in a test tube
Egypt. J. Med. Lab. Sci. August 2019, Vol.28 (2)
containing normal human serum (0.3-0.5 ml) by
rubbing the inoculated loop against the wall of the
test tube. This helps in diluting the pasty colonies
by giving turbid appearance in the serum. The
mixture was incubated at 42°C for 2-3 hours. A
drop of mixture was placed in a clean glass slide
and covered with a clean cover slip and examined
under high-power objective of the microscope
(Gupta et al., 2019 and Saigal et al., 2011).
Culture on Cornmeal Agar
Isolates were inoculated in a cross hatch pattern
on cornmeal agar and overlaid with a sterile
cover slip and then was incubated for 48 hours
at 37°C. The presence of chlamydospore
was examined microscopically (Raju and
Rajappa, 2011).
Vitek-2 System
The yeast inoculum was prepared by adjusting
turbidity to 2 McFarland standard with a Vitek-2
DensiChek instrument according to the
manufacturer's recommendations. This
standardized suspension was aspirated into the
identification cards (YST-21343), and then the
cards were sealed and incubated by the Vitek-2
instrument for 18 hours.
Statistical Analysis
Sensitivity, specificity and predictive values of
conventional methods (germ tube and cornmeal
agar) and CHROMagar Candida were calculated
and compared to the Vitek-2 system (gold
standard).
Sensitivity = [(true positive X 100)/ (true positive
+ false negative)] Specificity = [(true negative X
100)/ (true negative + false positive)
Positive predicted value (PPV) = [(true positive X
100)/ (true positive + false positive)]
Negative predicted value (NPV) = [(true negative
X 100)/ (true negative + false negative)
(Trevethan, 2017).
RESULTS:
A total of 153 Candida spp. were isolated during
the study period from 134 different clinical
specimens. More than one Candida spp. were
isolated from the same specimen. Among these,
40.5% of the isolates were from males and 59.5%
were from females. The mean age of male and
female patients was 32.93 ± 20.14 years and
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40.36 ± 21.34 years, respectively. The most
common isolated species was C. albicans
(43.79%) followed by C. tropicalis (41.17%), C.
krusei (7.8%), C. glabrata (5.22%) and C.
parasilosis (1.96%). The percent of non albicans
Candida (NAC) (56.2%) was higher than that of C.
albicans (43.79%) (Table 1).
Isolation of Candida spp. from different clinical
specimens showed the majority in urine samples
(81.7%) followed by sputum (8.5%), pus (4.6%),
wound swab (2%), central lines (2.6%) and vaginal
swab (0.6%). Table (2) shows the distribution
frequency of Candida spp. among different clinical
specimens.
Non-albicans C. tropicalis was the most
predominant spp. among ICU patients (47%),
while C. albicans was the predominant (54. 54%)
among other hospital units. The prevalence of
Candida spp. among ICU and other different units
in the hospital is shown in table 3.
Both SDA and CHROMagar Candida were used
for the primary isolation of Candida spp. from
various specimens. There were no discrepancies
between SDA and CHROMagar Candida in
promoting the growth of Candida isolates. Both
media supported growth of all 153 Candida
isolates.
All specimens develop cream, smooth, pasty
convex colonies on SDA with no difference
between Candida spp. On the other hand, germ
tube test and cornmeal agar differentiated Candida
spp. into C. albicans and NAC, where C.
albicans form germ tube in human serum and
form chlamydospore on cornmeal agar after 48
hours’ incubation (Figure 1).
Moreover, it was found that CHROMagar Candida
can differentiate Candida spp. according to the
color of the colony where C. albicans gave light
green colonies, C. tropicalis gave metallic blue
colonies, C. krusei gave purple fuzzy colonies, C.
glabrata gave creamy to white smooth colonies
and C. parasilosis gave creamy colored colonies
with tinge mauve (Figure 2)
(www.chromagar.com). The definitive color of the
colonies was obtained for all species only after
incubation for 48 hours as the color of the colonies
Egypt. J. Med. Lab. Sci. August 2019, Vol.28 (2)
of all Candida spp. were deepened by increasing
time.
The frequencies of identification of various
Candida spp. by the different methods is
described in table 4.
We calculated the performance of the germ tube
test for C. albicans, and the results were 94.9%
sensitivity, 100% specificity, 100% positive
predictive value (PPV) and 96.15% negative
predictive value (NPV) (Table 5). Also, the
performance of cornmeal agar and CHROMagar
for identification of Candida spp. is shown in
table 5 and 6 respectively.
DISCUSSION:
Candidiasis is the most prevalent fungal infection
affecting populations in the world (de Oliveira
Santos et al., 2018) and it represents the fourth
leading cause of nosocomial infections (Szweda
et al., 2015). C. albicans was considered as the
most common cause of candidiasis, and recently
it was observed that there is a significant increase
in the prevalence of non albicans Candida spp.
(NAC) (Kaur et al., 2016 and Deorukhkar et al.,
2014). Such emergence of NAC has higlighted
the importance of identification of Candida spp.
as they differ in anti-fungal susceptibility and
the expression of putative virulence factors (Lee
et al., 2018; Sathiya et al., 2015 and Souza et al.,
2015).
Rapid and accurate identification of Candida
isolates down to the species level is a key for the
selection of proper therapeutic and prophylactic
anti-fungal agents and for patient management
(Decat et al., 2013). In the present study, five
different Candida spp. were identified, and the
majority of species were isolated from urine
(81.7%) and sputum (8.5%), suggesting the
greater occurrence and distribution of Candida
spp. causing urinary tract and respiratory tract
infections. This result is in accordance with
Khadka et al (Khadka et al., 2017). C. albicans
was the most frequently isolated spp. (54.5%)
among hospitalized patient which is in
compliance with Kadry et al (Kadry et al., 2018)
and El-Mashad et al (El-Mashad et al., 2018).
Among NAC spp., C. tropicalis represented the
most predominant spp. (31%) followed by C.
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glabrata (10.9%) which in agreement with Amer
et al (Amer et al., 2015), Camacho-Cardoso et al
(Camacho-Cardoso et al., 2017) and Sharma et al
(Sharma et al., 2017). On the other hand, the
incidence of candidiasis in patients admitted to
ICU caused by NAC spp. (62.3%) was higher than
C. albicans (37.7%). C. tropicalis represented the
most common spp. (47%) followed by C. krusei
(11.22%), C. parapsilosis and C. glabrata
(2.04%). These results are compatible with many
studies carried out recently and reported shift
predominance of C. albicans to NAC spp. (Amr et
al., 2019; Wani et al., 2019 and Kaur et al.,
2016).
Our study reported several mixed yeast infections
especially in ICU patients which may be attributed
to long stay in ICU, extensive use of intravascular
catheters, use of broad-spectrum antibiotics and
miss identification of mixed specimen and hence
inappropriate therapy.
Several methods for detecting and differentiating
Candida spp. from specimens have been suggested
(Sadrossadati et al., 2018). Conventional and
biochemical methods are time consuming and
require skilled laboratory personnel (Wani et al.,
2019 and Sadrossadati et al., 2018), while
chromogenic agar is a rapid, cost effective and
technically simple method compared to
conventional methods (Dadhich et al., 2016;
Mehta and Wyawahare, 2016 and Nazir and
Kanth, 2016).
In the present study, the CHROMagar Candida
medium was used as primary isolation and
identification medium parallel to conventional
method. By using CHROMagar Candida medium,
several species of Candida as C. albicans, C.
tropicalis and C. krusei, C. parasilosis and C.
glabrata can be identified easily and directly from
clinical specimens due to difference in the color of
the colonies. C. albicans produce β-
hexosaminidase which breaks down substrate in
the medium producing green color, while C. krusei
produces phosphatase or β-glucosidase enzyme
which break down another chromogenic substrate
giving pink color. C. tropicalis produces both
enzymes giving metallic blue colonies. In other
Candida spp. neither of these enzymes are
produced and grow as white colonies (Perry,
2017). CHROMagar Candida differentiated three
Egypt. J. Med. Lab. Sci. August 2019, Vol.28 (2)
prevalent species of Candida accurately, C.
albicans, C. tropicalis and C. krusei, with
sensitivity and specificity as 100% when
compared to Vitek-2 system as the gold standard
method. The values obtained in this study are
similar to studies conducted by Bharathi
(Bharathi, 2018) and Nazir and Kanth (Nazir and
Kanth, 2016) who reported 100% sensitivity and
96% specificity for C. albicans. Regarding C.
tropicalis, Bharathi (Bharathi, 2018) reported
100% sensitivity and 96.77% specificity, while
Nazir and Kanth (Nazir and Kanth, 2016)
reported lower sensitivity of 92.3% and 100%
specificity. Concerning C. krusei, Bharathi
(Bharathi, 2018), reported 100% sensitivity and
100% specificity while Nazir and Kanth (Nazir
and Kanth, 2016) reported lower sensitivity of
92.3% and 100% specificity. CHROMagar
Candida showed higher positive predictive value
(PPV) (100%) in identifying C. albicans, C.
tropicalis and C. krusei, indicating that the
probability of achieving success is 100% when
the test result is positive. It was observed that
negative predictive value (NPV) of CHROMagar
Candida for these species identification is also
100%, i.e., the probability of the negative result in
the absence of disease is 100%. A study
conducted by Souza et al (Souza et al., 2015)
reported lower sensitivity, specificity, PPV and
NPV of CHROMagar Candida in identifying C.
albicans (78.6%, 88.5%, 75.9% and 90.0%,
respectively) and C. tropicalis (46.2%, 95.2%,
80% and 81.1%, respectively).
Sensitivity, specificity, PPV and NPV of
cornmeal agar for identification of C. albicans
and C. tropicalis were almost the same as that
obtained from CHROMagar Candida. However,
the total number and frequency of C. albicans and
C. tropicalis isolates obtained from each method
differ greatly as CHROMagar Candida can
identify several species at the same time
according to colony color. For C. krusei,
cornmeal agar also showed a very good
performance of 100% sensitivity, 99.2%
specificity, 90% PPV and 100% NPV.
Concerning C. glabrata, CHROMagar Candida
was more sensitive than cornmeal agar (72.72%
and 57.1% respectively), however, the
specificities were similar (100% and 98.4%,
respectively). CHROMagar Candida also
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ISSN 1110-5593
displayed higher PPV than cornmeal agar (100%
and 66.66% respectively) and similar NPV of
97.97% and 97.7%. Nazir and Kanth (Nazir and
Kanth, 2016) reported 66. 66% sensitivity and
100% specificity which is similar to our study. On
the other hand, Bharathi (Bharathi, 2018) reported
different results of 100% sensitivity and 84.21%
specificity of chromogenic media (Hichrome)
against cornmeal agar. Charles et al (Charles et
al., 2015) reported 100% sensitivity, 100%
specificity, 100% PPV and 100% NPV for
Hichrom medium in identifying C. glabrata.
For C. parasilosis, both CHROMagar Candida and
cornmeal agar exhibited similar sensitivity,
specificity, PPV and NPV which may be due to a
small number of C. parasilosis isolates.
Germ tube test identified only C. albicans and not
able to identify NAC on the species level. The
results for C. albicans showed a high performance
of 94.9% sensitivity, 100% specificity, 100% PPV
and 96.15% NPV that is too much the same as
Souza et al (Souza et al., 2015).
So, CHROMagar Candida can identify five
commonly found Candida spp. efficiently. A
study by Sariguzel et al (Sariguzel et al., 2015)
was carried out to compare conventional methods,
CHROMagar Candida, VITEK2 YST card and
VITEKRMS system for the identification of
Candida strains. They found that CHROMagar
Candida correctly identified 96.2% while VITEK2
YST automated card system correctly identified
90.7% of all strains. VITEKRMS system correctly
identified 100% of the strains. Also, Patel et al
(Patel et al., 2016) found that CHROMagar was
able to identify 93.75% isolates of Candida to
species level while VITEK 2 system was able to
identify 75% isolates.
Sadrossadati et al found that the results of
Candida spp. identification by molecular method
were consistent with those of the phenotypic
methods in 97.5% of the cases and CHROMagar
Candida showed a better function in species
identification than other phenotypic methods used
in this study (Sadrossadati et al., 2018). Also,
Fatima et al concluded that the results of
conventional phenotypic identification methods
including Hichrom agar were 100% accurate when
compared to molecular method and the time taken
Egypt. J. Med. Lab. Sci. August 2019, Vol.28 (2)
by both methods was the same. In addition,
conventional methods were found to be cost-
effective and still cheaper than molecular method
(Fatima et al., 2017).
Also, in the current study, CHROMagar Candida
detected poly Candida spp. specimens, an
advantage that is not provided by any other
conventional methods (SDA and hence, cornmeal
agar and Vitek-2 system), as stated by Mathavi et
al (Mathavi et al., 2016) Sadrossadati et al
(Sadrossadati et al., 2018). Detection of mixed
cultures by CHROMagar Candida causes
variation in the number of Candida spp. isolates
and hence the total number of identified isolates.
So, CHROMagar Candida may be recommended
to replace conventional methods as germ tube
test, cornmeal agar and even automated system
Vitek-2 for identification of these major species,
as it reduces the time and cost required for
identification of these spp.
CONCLUSION
The current study revealed that the incidence of
NAC is constantly increased. CHROMagar
Candida could be considered as the best method
for the identification of Candida spp. in a short
time compared to conventional method, cornmeal
agar and automated Vitek-2 system that require
previous culture of specimen on SDA. Also,
CHROMagar Candida can detect poly yeast
specimen which can't be identified by traditional
SDA, that can help the clinicians to initiate early
appropriate anti-fungal therapy.
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Tables and Figures

Table 1: Distribution of Candida albicans and non albicans Candida isolates

Candida spp. NO %
C. albicans 67 43.79%
C. tropicalis 63 41.17%
C. krusei 12 7.8%
C. glabrata 8 5.22%
C. parasilosis 3 1.96%
Total 153 100%

Table 2: Candida spp. distribution frequency obtained from different clinical specimens

Specimen (No.) C. albicans C. tropicalis C. krusei C. glabrata C. Total (%)

parasilosis
Urine (112) 53 53 8 8 3 125 (81.7)
Sputum (10) 6 5 2 - - 13 (8.5)
Pus (5) 3 3 1 - - 7 (4.6)
Wound swab (3) 1 1 1 - - 3 (2)
Central line (3) 3 1 - - - 4 (2.6)
Vaginal swab (1) 1 - - - - 1 (0.6)
Total (134) 67 63 12 8 3 153 (100)

Table 3: The prevalence of Candida spp. among ICU and other units in hospital
Candida spp. ICU (80 specimens) Other units (54 specimens)
C. albicans 37 (37.7%) 30 (54.5%)
C. tropicalis 46 (47%) 17 (31%)
C. krusei 11 (11. 22%) 1 (1.8%)
C. parasilosis 2 (2.04%) 1 (1.8%)
C. glabrata 2 (2.04%) 6 (10.9%)
Total isolates (153) 98 (64%) 55 (36%)

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Table 4: Identification of Candida spp. by various methods

Vitek-2 Vitek-2
Cornmeal
Species Germ tube Without CHROMagar After
agar CHROMagar CHROMagar
C. albicans 56 59 59 67 67
C. tropicalis - 57 58 63 63
C. krusei - 10 9 12 12
C. glabrata - 6 7 5 8
C. parasilosis - 2 1 6 3
Total 56 134 134 153 153

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Table 5: Diagnostic efficiency of conventional methods (sensitivity, specificity and predictive value) and
agreement between the methods with regard to Vitek-2
Conventional Vitek-2 Vitek-2 Sensitivity Specificity PPV NPV
Species Total
method (+) (-) (%) (%) (%) (%)
Gem tube (+) 56 - 56
94.9 100 100 96.15
Gem tube (-) 3 75 78
C. Total 59 75 134
albicans Cornmeal agar (+) 59 - 59
100 100 100 100
Cornmeal agar (-) - 75 75
Total 59 75 134
Cornmeal agar (+) 57 - 57
C. 98.3 100 100 98.7
Cornmeal agar (-) 1 76 76
tropicalis
Total 58 76 134
Cornmeal agar (+) 9 1 10
100 99.2 90 100
C. kruzi Cornmeal agar (-) - 124 124
Total 9 125 134
Cornmeal agar (+) 4 2 6
C. 57.1 98.4 66.67 97.7
Cornmeal agar (-) 3 125 128
glabrata
Total 7 127 134
Cornmeal agar (+) 1 1 2
C. 100 99.2 50 100
Cornmeal agar (-) - 132 132
parasilosis
Total 1 133 134

Table 6: Diagnostic efficiency of CHROMagar Candida (sensitivity, specificity and predictive value)
against Vitek-2
Vitek-2 Vitek-2 Sensitivity Specificity PPV NPV
Species CHROMagar Total
(+) (-) (%) (%) (%) (%)
CHROMagar (+) 67 - 67
C. 100 100 100 100
CHROMagar (-) - 86 86
albicans
Total 67 86 153
CHROMagar (+) 63 - 63
C. 100 100 100 100
CHROMagar (-) - 90 90
tropicalis
Total 63 90 153
CHROMagar (+) 12 - 12
100 100 100 100
C. kruzi CHROMagar (-) - 141 141
Total 12 141 153
CHROMagar (+) 5 - 5
C. 62.5 100 100 97.97
CHROMagar (-) 3 145 148
glabrata
Total 8 145 153
CHROMagar (+) 3 3 6
C. 100 98 50 100
CHROMagar (-) - 147 147
parasilosis
Total 3 150 153

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