UJMR, Volume 5 Number 1, June, 2020, pp 36 - 42 ISSN: 2616 – 0668

https://doi.org/10.47430/ujmr.2051.006

[[[[ Received: 11th May, 2020 Accepted: 17th June, 2020

Antibiogram of Candida Species using Different Susceptibility Testing Techniques:
A Systematic Review

Sule, H., Kumurya, A. S. and Ibrahim, A.

Department of Medical laboratory Science, Faculty of Allied Health Sciences, College of Health
Sciences, Bayero University, Kano
Corresponding Author: [email protected]; +2348036062021
Abstract
Susceptibility pattern determination is a valuable key towards successful treatment of
infectious diseases. Susceptibility of Candida species to different antifungal agents are also a
common practice, however, different methods are employed to achieve the same goal. Some of
the different techniques include: Disc diffusion method, Epsilometre test (E-test), Candfast,
Microdilution technique among others. Fluconazole, Voriconazole, Itraconazole and
Amphotericin B are some of the antifungal agents commonly used for treatment of infections
due to Candida species. Most of these antifungals are also covered in this review. The
commonly encountered Candida species in clinical settings include: C. parapsilosis, C.
tropicalis, C. albicans, C. krusei, and C. glabrata. This review also gave some insight into their
variable susceptibility pattern, as it affects different methods of susceptibility testing. There
were reports of resistance from researchers from different regions across the globe; this
therefore, signifies the importance of availability of data with regards to susceptibility of
these species.
Key words: Susceptibility, Candida species, Antifungal agents, testing techniques

INTRODUCTION from 1998 to 2000, it was discovered in the first

Knowledge of drug susceptibility pattern is an surveillance period that, C. albicans accounted
important factor of help, in the progress of for 52% of the isolates while C. glabrata
treatment and other aspects of patient accounted for only 12%. In the second round of
management. In case of mycotic diseases, the surveillance period, C. albicans accounted
various antifungal agents are employed in for 45% but C. glabrata had a shift up, to 24%
treatment and their efficacy is continually (Punithavathy and Nalina, 2012)
being tested using various methods like disk- Another study also showed that, among the non
diffusion method, which involved the use filter albicans species, Candida glabrata, recorded
disc impregnated with known concentration of higher incidence, which account for the
antifungal agent, Epsilometer test (E- test) decrease in dominance of Candida albicans as
which a strip containing exponential gradient of the cause of invasive fungal disease due to
the agent to be tested, Candida species (Trick et al., 2002).
Broth microdilution method that involved There is a document developed by Clinical and
dilution of different concentration of the agent Laboratory Standards Institute (CLSI) on
and the Candifast microbroth kit consists of a Candida species-specific clinical breakpoints for
tray with two rows of eight wells each to test some antifungal agents like echinocandins,
against the organism, among other methods fluconazole and voriconazole and is believed
(Aher, 2014). that use of such breakpoints can change the
It has been observed over the years that, previously known Candida species sensitivity
Candida species have become prominent impact patterns and consequently, the
nosocomial pathogens (Abi-Said et al., 1997). management of the patients (Pfaller and
Over the years, available data suggested a shift Diekema, 2012).
from Candida albicans as the major cause of Antifungal treatment selection is a factor of the
invasive fungal infections toward non-C. causative agent identification and in vitro
albicans species (Pfaller et al., 2000 Kao et al., susceptibility testing which provide valuable
2001). information for patient management. In
The Centers for Disease Control and Prevention resource-limited settings, echinocandins,
(CDC) conducted population-based surveillance voriconazole and liposomal formulations of
for Candida bloodstream infections over two amphotericin B are either unvailable or if
different time periods: in 1992 and 1993 and available, not affordable.
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 UJMR, Volume 5 Number 1, June, 2020, pp 36 - 42 ISSN: 2616 – 0668

Presently, fluconazole and amphotericin B between the NCCLS M27-A and EUCAST
deoxycholate are the drugs most commonly microdilution procedures, the study
used in some nations. Several studies indicated demonstrated an overall agreement rate of 92%
that Candida species distribution and in vitro (Cuenca-Estrella et al., 2002).
drug susceptibility vary among nations and even Because they involved rather complex methods
in different regions of the same country for susceptibility testing, standard reference
(Nishikaku et al., 2010; Cleveland et al., 2012), procedures are mostly not practicable routinely
even though most of these researches were in clinical laboratories, for this reason most
done in the United States and/or in Europe, microbiologists engage other systems that have
they revealed a consistently decreased levels of numerous advantages, like rapid results, ease
susceptibility to fluconazole by both Candida of performance, and reduced financial
albicans and non-albicans candida species and implication. For these reasons therefore,
such is expected even in other parts of the several techniques like the use of a
world unless proved otherwise (Arendrup et al., colorimetric oxidation–reduction indicator and
2013). those based on agar diffusion are used.
It has been observed that limited information is Accordingly, some of these techniques are
available from many parts of the world, which commercially made available which are rapid
means no large studies that evaluated species and simple alternatives to those procedures
distribution and antifungal susceptibility developed by either the NCCLS or the EUCAST
pattern of yeast isolates in some infections. (Arikan et al., 1997).
The only limited information at hand mostly The significance of use of reference procedures
suggests that non-C. albicans species are in is to provide a standard measure from which
most cases the aetiologic agents of blood other methods can be developed and
stream infections (BSIs) in some countries than compared, as a results, many studies have
expected (Becerra et al., 2010). analysed the correlation between the NCCLS
The information may have serious implications procedure and various commercially available
for infection treatment against the Candida systems (Espinel-Ingroff et al., 1999), this
species, because Candida glabrata is also includes some suitable for susceptibility testing
considered a fluconazole acquired resistant of Candida species.
species, Candida parapsilosis may display high The expanding use of some new antifungal
MIC values for echinocandins while C. krusei is agents, and the emergence of antifungal
also considered to be a resistant specie to resistance as one of the known important
fluconazole irrespective of its MIC (Pfaller et clinical problems, calls for the need of
al., 2012a). reproducible, and clinically relevant antifungal
In a swift effort, the Antifungal Susceptibility susceptibility testing, due also to continued
Testing Subcommittee of the European challenge by the increasing number of invasive
Committee on Antibiotic Susceptibility Testing, fungal infections (Pfaller, 2012).
the (AFST-EUCAST) has developed a standard It is of value that, the collaborative efforts of
broth microdilution protocol for the numerous researchers and the Clinical and
determination of antifungal MICs for Laboratory Standards Institute (CLSI), known
fermentative yeast species (Rodriguez-Tudela before as, National Committee for Clinical
et al., 2003). The standard developed was Laboratory Standards (NCCLS), Subcommittee
based on the guidelines of National Committee on Antifungal Susceptibility Testing have
for Clinical Laboratory Standard (NCCLS), the generated an agreed consensus documents
procedure described in document M27-A2 describing standardized methods for broth- and
(NCCLS, 2002), but the procedure includes agar-based antifungal susceptibility testing
some modifications that allows for automation (CLSI 2009; CLSI, 2010 CLSI, 2017). Based on
of the method and also permit the incubation these, in vitro antifungal susceptibility testing
period to be reduced from 48 to 24 h. remain a model, which: (i) Guides therapeutic
Reproducibility of 94% has been observed in the decision making (ii) Serves as an antifungal
EUCAST procedure for antifungal susceptibility resistance tracker in epidemiologic studies and
testing according to a multicentre evaluation (iii) Aid in drug development studies (Kanafani
study (Cuenca-Estrella et al., 2003). and Perfect 2008). The aim of the review was
Additionally, a panel of 109 bloodstream to survey some susceptibility testing techniques
isolates of Candida species were evaluated used some regions in the world.
against some antifungal agents: fluconazole, The different susceptibility pattern studies in
itraconazole, amphotericin B, and flucytosine some regions of the world
by a two-laboratory study which correlated
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 UJMR, Volume 5 Number 1, June, 2020, pp 36 - 42 ISSN: 2616 – 0668
India (Asia): A study conducted by Sumana et exhibited resistance while 92% were sensitive
al. (2017), 50 isolates of different candida it. In the same vein, 4% of C. parapsilosis, 2% of
species were exposed to variety of antifungal C. albicans, 2% of C. glabrata, 2% of C.
agents and pattern of susceptibility using agar tropicalis, and 2% of C. haemulonii were
disc diffusion method to these antifungals were resistant to Amphotericin B, but 88% proved
noted: Resistance to Fluconazole been observed sensitive to it. Susceptibility to Ketoconazole
were in the following rates; 4% for C. was observed to be as follows: 2% of C. albicans
parapsilosis, 4% for C. tropicalis , 4% for C. and 2% of C. haemulonii were found resistance
albicans, 2% for C. krusei, and 2% for C. while 96% were sensitive. The next antifungal
glabrata, while the greater percentage of the agent, Itraconazole proved the most effective
species 84% were sensitive to it. For in vitro with all isolates (100%) sensitive to it. A
Voriconazole, 2% each, of C. albicans, C. sample of disc diffusion technique used is given
tropicalis, C. glabrata, and C. haemulonii in Fig. 1

Fig 1: Disc Diffusion Method, Source: Rosco diagnostica, 2011

Das et al. (2016) in India identified variable beds school hospital in Parana,´Brazil.
efficacy of some antifungal agents when tested According to the results, non Candida albicans-
against different Candida isolates using disc candida species (NCAC) were found to overtake
diffusion method, they found 9.3% (n=6) of C. candida albicans in the study, with up to 61% of
albicans isolates to be resistant to both the yeasts isolates. From the results, among
voriconazole and fluconazole where as the NCAC species, C. glabrata and C. tropicalis
Fungitest method revealed no fluconazole were identified to be more prevalent than the
resistance among these isolates, except one other NCAC species. Using Epslometre test (E-
intermediate result. However, none of the non- test), amphotericin B proved the only
albicans Candida isolates showed resistance to antifungal drug which had MIC values under the
fluconazole and voriconazole using disk- reference limit (01 mg/ml) for all isolates
diffusion method, but some of these isolates except one C. albicans and one C. glabrata
were found to have intermediate result to with (MIC532mg/ml) and (MIC56ug/ml)
azoles by Fungitest method. In the same vein, respectively. Incidentally, all tested strains,
(100%) of the isolates were found to be when assessed using E-Test (ET) method,
susceptible to amphotericin B and revealed higher MICs compared with
fluorocytosine. It was also discovered that none Microdilution (MD) method for: fluconazole,
of the Candida species isolates from HIV itraconazole, and amphotericin B. When the
positive subjects with previous exposure to isolates were compared, on the basis of
antifungal agents show resistance to any colonization (COL) and infection (INFC), it was
antifungal agent tested in the study. found that COL yeasts were more susceptible to
Brazil (Latin America): A study in Brazil that the 3 antifungal agents than the INFC yeasts in
involved 70 Candida species, in which, 67% of the ET method. It was also observed that, NCAC
them were from infection (either candiduria or species exhibited smaller inhibitory zone
candidemia) and 33% were neither of the two, diameters when disc diffusion method was used
in the University Hospital (UH), Maringa, a 116 in relation to C. albicans. Among the three
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 UJMR, Volume 5 Number 1, June, 2020, pp 36 - 42 ISSN: 2616 – 0668
methods studied, itraconazole proved to be species.
having the highest number of resistant NCAC
The overall concordance (based on the MIC Test and DD methods had high categorical
value obtained within two dilutions) between agreement (Negri et al., 2009). A sample of The
the ET and MD methods was 83% for Epslometre test (E-test) is given below in
amphotericin B, 63% for itraconazole, and 64% figures 2 and 3:
for fluconazole. Considering the breakpoint, E-

Fig 2:Fluconazole (FL) Etest reading patterns for C. albicans . (A) Growth of microcolonies inside
the entire inhibition zone (ellipse); MIC, 0.38 ug/ml. (B) Clear ellipse on Casitone agar; MIC, 0.5
ug/ml. The numbers on the scale correspond to the fluconazole concentrations on the strip (in
micrograms per milliliter). Pfaller et al., Evaluation of the Etest Method for Determining
Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media M.
A. Pfaller,1* S. A. Messer,1 Å. Karlsson,2 and A. Bolmstro ¨M21998. Fig.3: The Epslometre test (E-
test) pointing to MIC point on the test strips, Source: (George et al., 2014).

U.S.A. (America): A study on different methods method at either time points had a 96%
of susceptibility based on MICs and zone likelihood of testing susceptible or susceptible
diameters and for 24 and 48 h incubation dose dependent by the MD method. On the
period was reported, by Matar et al. (2003), other hand the MICs for isolates resistant by the
based on the result, it was generally observed disc diffusion and ET method tend to be almost
that, ET method revealed slightly higher always in the susceptible-dose dependent or
fluconazole MICs than the MD method at both resistant category by the MD method, whereas
incubation times, whereas the voriconazole isolates testing resistant by the MD method
MICs by the MD and ET methods were similar at produced results by the agar-based method
both time points. It was also observed that, the ranging from susceptible to resistant. After 48
overall levels of agreement between the MICs h, trailing growth, for fluconazole tend to
obtained according to both ET and MD methods generate higher MICs. Use of Mueller-Hinton
at 24 and 48 h were satisfactory for both agar flooded with glucose-methylene blue
fluconazole and voriconazole. For both drugs, (GMB) enhanced growth and simplified reading
the agreement between the ET, MIC and the relative to the MD method.
reference (MD) MIC, at 48 h were 93%. This suggest that, the results for isolates that
However, a better agreement percentage was appear to be resistant by any method should be
noticed for the readings obtained at 24 h, when carefully reviewed and that, such isolates may
the rate of agreement was 98% for the two merit repeat testing and/or testing by an
drugs. For some isolates, there was some alternative method. Although more work needs
difference in which the ET MICs were found to to be done with less susceptible isolates, the
be lower while the MD method was lower at 24 aggregate data suggest that agar-based
h but the MICs tend to be elevated at 48 h. methods appear to produce a more consistent
Comparisons of the results as indicated by the in vitro-in vivo correlation than the reference
different methods showed that isolates Microdilusion method, example of the
susceptible by the disc diffusion method or ET microdilusion test can be seen in Figure 4.

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Figure 4: Microdilusion Technique, Source: (Punithavathy et al., 2012)

Egypt: El-Mashad et al.(2011), in a study on the presented as the MIC ranges: The MIC50 and
in vitro antifungal susceptibility of ampotricin B MIC90 values, the numbers of susceptible
(AMB) and Fluconazole (FCZ) on 30 Candida isolates, and the numbers of resistant isolates.
species by 3 methods (Standard broth dilution, Overall, fluconazole exhibited promising
E-test and candifast) found that, for AMB, the activity against most species isolated. In
agreement of Etest with standard method was particular, C. albicans, C. parapsilosis, C.
100% in tested species except C. glabrata tropicalis, and C. lusitaniae were quite
(85.7%) and C. parapsilosis (66.6%). The susceptible to fluconazole. In contrast, C.
number of susceptible isolates in those 2 glabrata was less susceptible to fluconazole
species was lower when tested by E-test (MIC90, 16g/ml). The agents,echinocandins,
method than standard method. The percentage caspofungin and micafungin, exhibited
of agreement of candifast was 100% in all excellent activity against all species of
species except C. glabrata (85.7%). The overall Candida, with the overall rate of susceptibility
modal MICs obtained were 1.0 μg/ml for both of 99.8%. The rate of resistance was low, at
standard method and E test. For FCZ, the 0.2%. The echinocandins were quite potent
percentage of agreement of E-test with the against all species except C. parapsilosis, for
standard method was 100% for each of C. which the MIC90 was 1g/ml. When the
glabrata and C. tropicalis and 92.8% and 66.6% epidemiological cutoff values (ECVs) were
for C. albicans and C. parapsilosis respectively. applied to the echinocandins, the overall rates
The number of susceptible isolates in those 2 of resistance increased slightly. However, most
species was lower when tested by Etest than isolates were slightly more resistant to
standard method. The percentage of agreement caspofungin than to micafungin. Overall,
of candifast method was 100% in all species 133(2.3%) isolates were resistant to fluconazole
except C. albicans, C. glabrata which was 92.8 (MIC 64 g/ml). The numbers of isolates of
and 85.7% respectively. The overall modal MICs specific species resistant to fluconazole were as
obtained for fluconazole were 0.25μg/ml for follows: C. albicans, 30(1.2%) isolates; C.
the standard methods and 0.5μg/ml for E-test. glabrata, 87(5.9%) isolates; C. parapsilosis,
The overall percentage of agreement of E-test 3(0.3%) isolates; C. tropicalis, 2(0.4%) isolates;
with standard method was 90% for each of AMB and C. lusitaniae, 0(0%) isolates.
and FCZ. The overall percentage of agreement
of candifast with standard broth method was CONCLUSION
96.6% for AMB and 93.3% for FCZ. Among the reviewed techniques disc diffusion
According to Lyon et al. (2011), a susceptibility method remain the best for routine work, being
testing based on 24-h incubation for the 6 the easiest and less expensive than E-test and
commonly encountered Candida species (C. microdillution techniques and it gives good
albicans, C. glabrata, C. parapsilosis, C. results. Among the antifungal agents reviewed,
tropicalis, C. krusei, and C. lusitaniae), using fluconazole and itraconazole were shown to be
microdilution method, revealed different MIC promising against the Candida species among
status of the involved isolates. The results were other agents.
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 UJMR, Volume 5 Number 1, June, 2020, pp 36 - 42 ISSN: 2616 – 0668
According to available data on susceptibility conventional antifungal agents throughout the
pattern of Candida species from different world, with many reports of resistance. There
regions of the world, it is evident that, these is therefore the need for global concerted
species display variable reactions (with effort to adequately handle diseases caused by
different susceptibility techniques) to the these organisms.

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