Prevalence of Emerging Fungus: Candida Species in Different

Clinical Samples from Patients in a Hospital Setting.

Atencio LDD1, Azucena IT2, Besares SCT3

1
Medical Microbiology, Clinical Laboratory, Department of Pathology, Medical Center Hosptal (MCH), Iloilo City,
Philippines, 5000.
2
Department of Environmental and Natural Resources, Ecosystems Research and Development Bureau-Costal
Resources and Ecotourism Research Development and Extension Research Center (DENR, ERDB-CRERDEC),
Banilad, Mandaue City, Cebu, Philippines, 6014.
3
Faculty of Science Department, Ramon Avancena National High School, Department of Education (RANHS-
DEPED), Iloilo City, Philippines, 5000.

Abstract
The yeast Candida being the main cause of candidiasis is a commonly isolated pathogen
from immunocompromised patients. Successfully identifying the species of Candida is important
in the treatment and management of the disease. The trend in the resistance acquired by some
species of Candida leads to the importance of identification to the species level. This may avoid
prescription of antifungal drugs that may not be available to specific species. There are many
techniques that can be adopted by hospitals or independent diagnostic laboratories to aid in the
identification of this yeast. This study reviews various methods of isolation, identification,
differentiation, characterization and antibiogram. Starting from the collection of specimen,
transporting them to a diagnostic laboratory followed by culture in agar plates, staining,
microscopy, automated blood culture systems, biochemical tests and antimicrobial susceptibility
test that are used for the identification of Candida species. The cost, expertise, laboratory
facilities and the population needs of each geographical region should be considered in adopting
these techniques for the diagnosis of Candida infections.

Key Words
Candida species, Candida albicans, Candida tropicalis, Candida parapsilosis,
candidiasis, candidemia, fungal infection, antifungal agents, antifungal resistance, fungi,
mycology, opportunistic infections.

Introduction
The study of infectious diseases caused by fungi greatly attributes the study of Pasteur
and Koch with pathogenic bacteria. Following their study, in 1839, microbiologists Schonlein
&Gruby studied Trichophyton schoenleinii whereas Langenbeck had reported on the causative
agent for thrush which was Candida albicans (Wilson & Gisvold, 2004). Then, great concern
over bacteriology overshadowed mycology for many years. However, the rise of incidences in
mycoses had received serious attentions to medical mycology. The most suitable first-line
antifungal regimen is still an unknown fact. The choice of drug selection depends on the
physician’s knowledge of a drug, drug availability, patient’s condition, concomitant medications
and cost (Gallagher et al., 2005). Antifungal drugs, mainly those containing azole groups such as
itraconazole, ketoconazole and fluconazole have been used in the treatment of initial and
subsequent Candida infections. However, there have been reports showing the difficulties in
complete eradication of this fungus from patients as they seem susceptible to the antifungal drugs
used. The reason could be due to the genetic differences among the fungal species or simply due
to the overuse of azole drugs. Therefore, it is appropriate for physicians to have information on
the type of Candida species before prescribing antifungal drugs to these patients. This study
reviews laboratory methods that can be employed to identify Candida spp. in fungal infections
(Madhavan, Jamal, & Chong, 2011). In the Philippines, Candida species have been isolated from
women diagnosed with vaginal candidiasis from a teaching hospital and it was found that
Candida albicans was the predominant species among the strains isolated. The other species
were Candida tropicalis and Candida parapsilosis. Re-infection from these yeasts varied among
individuals either by identical, similar or different strains or species of Candida (Madhavan,
Jamal, & Chong, 2011). In another report, the most predominantly isolated species from
hospitals in the Philippines was Candida tropicalis, followed by C. albicans and Candida
parapsilosis (Madhavan, Jamal, & Chong, 2011). Other studies have reported that there is an
increase in the number of disseminated candidiasis among immunocompromised patients (Cantu,
2005). Due to the strain resistance towards amphotericin B, some of these patients were given
fluconazole, caspofungin and voriconazole for re-infections. In other cases of acute
immunocompromised patients, blood, respiratory, urine and sterile fluids colonisation by
Candida species was found in 90% of the patient population. (Rodu, 2006). Related studies have
shown that there were increased disseminated Candida infections among intensive care unit
patients Wingard et al., 1991). The role of a diagnostic laboratory is important in the
management of the fungal infections. The ability to identify the causative agent of the fungal
infection to the genus, species or the strain, to a great extend, can determine the treatment for the
infection and minimize treatment failure or recurrent infections. Candida infections often
associated with high morbidity and mortality have increased remarkably during the couple of
decades (Anaissie, 1992). The incidence of Candida infections is on the rise with the increase in
number of immunocompromised patients due to excessive use of immunosuppressive drugs as
well as the use of medical and surgical interventions (Bernal et., 1998). Although Candida
albicans is the most prevalent species (Blinkhorn et., 1989), an epidemiological shift in Candida
pathogens has been recently noted by the increasing number of infections caused by nonalbicans
Candida species (NAC). The increased species diversity and incidence of infections have
resulted in the need for an accurate and rapid identification of Candida isolates and have become
important for proper patient management as various species respond differently to antifungals
and for the prevention of emergence of drug resistance (Anaissie, 1992).

Materials and Methods

Specimen and Transport

All specimens have a laboratory test requisition form each completely filled by the
physician with the patient’s name, age, sex, specimen source and patient’s history or diagnosis
and with permission under the hospital’s research review ethics committee. Ethical clearance
approved by the ethical committee of the hospital with the defined guidelines for medical
research based on patient information and materials was obtained for this research (Tortora et al.,
2010). Basic demographic data such as age and gender as well as information on the clinical
condition such as underlying disease or predisposing factors were recorded from the patient’s
laboratory requisition form. The age of the patients ranges from 0 – 80 years (Adjapong, 2014).
In order to recover yeast cells in specimen, adequate amount of specimen is necessary to perform
desired tests. For example, 5 mL of cerebrospinal fluid is recommended as an optimum volume
for laboratory diagnosis. In adults with bloodstream infections, 10 mL of blood should be drawn
into each culture bottle. Generally, two aerobic and two anaerobic culture bottles are
recommended (Brooks et al., 2010). The laboratory head, registered medical technologist,
registered microbiologist or laboratory technicians should inform the physician if additional
amount of specimen is required. Specimens obtained like sputum, endotracheal aspirate, wound,
abscess, drainage, urine, and sterile fluids should be placed in a sterile specimen cup. Petri dishes
or any other suitable containers and sealed properly. Specimens from mucous membranes can be
inoculated in a medium or directly smeared on a clean slide using an inoculation loop or a swab.
Slides are covered with cover slips and should be placed in an envelope or a slide box and
sealed. In subcutaneous infections, scrapings, crusts, aspirated pus or tissue biopsies should be
removed aseptically and placed into a sterile container. In systemic infections, specimens can be
drawn from blood, cerebrospinal fluid or other areas directly into appropriate sterile vials
containing blood culture medium (Chakravarthi & Haleagrahara, 2011). If there should be a
delay in transporting the specimens to the diagnostic laboratory, then the specimens should be
incubated at 35°C.

Culture Medium (Isolation)

The basic culture media used in isolating clinical Candida species are Blood Agar Plate
(BAP), Potato Dextrose Agar (PDA), and Sabouraud Dextrose Agar (SDA) (Jatta et al., 2009;
Yang et al., 2009). Agar plates provide a large surface area to perform the streaking method. This
method would enable the identification of any contaminants or transient normal flora. It was also
recommended that the least selective medium should be inoculated first with the most selective
and inhibitory media last. This would prevent carry-over inhibition from one medium to another
(Nye et al., 2006). Sabouraud’s dextrose agar is also available as Mycosel which contains
chloramphenicol and Mycobiotic inhibitory mould agar to inhibit bacterial growth. Sterile body
fluids and tissues samples are recommended to be cultured on Trypticase Soy Broth and
incubated between 35 to 37°C for 72 to 96 hours. Long term storage for few months can be done
using agar slants (Chakraborty et al., 2005).

Automated Blood Culture System

Automated culturing systems detect microbial growth automatically by monitoring the
CO2 production released from the metabolic activity of the microbial cells. The advantages of
this system are it is more sensitive than manual systems and does not require manual inspection
or examination of the culture (Ryan and Murray, 1993; Han, 2006). There are various BACTEC
systems that are used in hospital laboratories to detect many microorganisms. Several systems
can be used for yeast identification in blood such as BacT/Alert standard, BACTEC 9240
standard, BacT/Alert FAN, BACTEC fungal medium and BACTEC Plus Anaerobic/F bottles
(Han, 2006). Blood samples are inoculated directly into 2 bottles with a maximum of 10 mL in
each bottle.
Microscopy (Identification and Differentiation)
The microscope is the best tool for a microbiologist. Distinct features of yeasts can be
identified by observing their morphology. Microscopes can be used for fast identification and
detection of possible yeasts in a clinical sample. Specimens from exudates, sputum, urine and
cerebrospinal fluid and direct from isolates from agar plates using catalase test through Gram’s
staining can be viewed under reduced-light brightfield microscope or phase-contrast microscope
(Aslanzadeh and Roberts, 1991). Presumptive identification and differentiation of Candida
albicans from Candida tropicalis and Candida parapsilosis is done with the germ tube test. In
this method, the clinical sample is incubated in human or animal serum for 2 to 3 hours at 30 to
37°C. If Candida albicans is present, short, slender, tube like structures while Candida tropicalis
and Candida parapsilosis do not have this quality. In germ tube it can be observed under the
microscope (Mackenzie, 1962). Reports of other species of Candida such as Candida tropicalis
and Candida parapsilosis were also found to produce similar structures but not completely with
tube like structures (Campbell et al., 1998; Freydiere and Guinet, 1997; Lipperheide et al., 1993;
Perry et al., 1990).

Biochemical Test and Antimicrobial Susceptibility Test (Characterization and Sensitivity)

Biochemical tests are also routinely done following the initial phenotypic identification
of the cultures on agar media and microscopy. Tests using single enzyme are able to detect the
presence or absence of an enzyme or a biochemical reaction within seconds to minutes. These
tests are economical, rapid and simple to perform (Aslanzadeh, 2006). Various Candida species
can be detected by observing the changes in the indicator colour when the yeast cultures utilize
1% carbohydrates such as glucose, maltose, sucrose, trehalose and raffinose. These tests are now
available as commercial kits such as Yeast Plus systems. Other than carbohydrates, hydrolysis of
1% fatty acid ester, 0.05% aryl-substituted glycosides, 0.3% urea and 0.01% arylamide
substrates can be detected with Vitek-2 Yeast Plus system. The resulting colours at the end of the
incubation period are coded and compared with the Vitek-2 Yeast Plus Differential Chart to
identify the species. This method is currently the fastest commercial method for the identification
of yeasts which requires a 18 hours incubation period and only Vitek-2 YST system is based on
the detection of enzymes in the yeast species. It was reported that Vitek-2 YST system is an
automated new colorimetric card system which could correctly identify Candida species in 18
hours which is faster than API 20C and API 32C (Loiez et al., 2006). In their report, it was also
stated that although the Vitek-2 system was found better compared to VITEK 1 and API 32C in
identifying Candida species. When a patient presents with an illness due to an infectious
organism, knowing the best treatment options requires more than species identification,
especially as many organisms are becoming resistant to antimicrobials. Antimicrobial
Susceptibility Testing (AST) and resistance detection is also important to making the best
patient-care decisions. Designed for Vitek-2 automated instruments, Vitek-2 AST cards offer
rapid, accurate Antimicrobial Susceptibility Test (AST) results. The Vitek-2 Fungal
Susceptibility Card is intended for use with the VITEK 2 systems in clinical laboratories
as an in vitro test to determine the susceptibility of clinically significant yeasts to antifungal
agents when used as instructed. Recognizing the importance of good diagnostic tools for
infectious organisms and the ever-growing needs in the field of microbiology. The antifungal
drugs used for Antimicrobial Susceptibility Test (AST) of Candida species are Amphotericin B,
Caspofungin, Fluconazole, Flucytosine, Micafungin, and Voriconazole. Minimum Inhibitory
Concentration (MIC) are then measured by VITEK 2 systems to know if Candida albicans,
 Candida tropicalis and Candida parapsilosis are resistant, intermediate or susceptible to
antifungal drugs.

Data analyses
All statistical analyses were performed using the statistical package for the social
sciences (SPSS) version 17.0 for windows (SPSS Inc., Chicago, Il, USA). Any relationship
between the observed and expected frequency of the various Candida species was subjected to
Pearson’s chisquare test, and the level of significance was set at P < 0.05. The reproducibility of
AST was assessed by nonparametric correlation coefficient, and AST was considered
reproducible if the correlation coefficient was P < 0.05.

Results
This study used a conventional phenotypic tests and machine biochemical and sensitivity
techniques to isolate, identify, differentiate and characterize Candida species from different
patient groups presenting with various forms of candida infections, determine the various
biological types of Candida species present, assign clades to the Candida albicans, Candida
tropicalis, Candida parapsilosis isolates assayed for this study based on the presence or absence
of general-purpose genotypes, study the phenotypic structure, biochemical test of Candida
species and antifungal susceptibility trend among the Candida species isolates in a hospital
setting..

Isolation and Identification of Candida species

Genus: Candida
Species: albicans
Cultural Characteristics
White creamy colored and foot-like extensions from the margin (Blood Agar Plate),
Smooth creamy colonies after 24-48 hours (Potato Dextrose Agar), and Creamy, pasty colonies,
smooth after 24-48 hours at 25-37°C (Sabouraud Dextrose Agar).
Microscopic Characteristics
Small, oval, measuring 2-4 µm in diameter.
Yeast form, unicellular, reproduce by budding. Single budding of the cells may be seen. Both
yeast and pseudo-hyphae are gram positive. Encapsulated and diploid, also form true hyphae.
Polymorphic fungus (yeast and pseudohyphal form). Can form biofilms. Normal condition:
Yeast. Special condition (pH, Temperature): Pseudohyphae. 80-90% of cell wall is carbohydrate
(Kidd S, Halliday C, Alexiou, H, & Ellis D. 2016).

A B C D
 Figure 1. A. Colonies on BAP (obverse view), B. Colonies on PDA (obverse view), C. Colonies
on SDA (obverse view), D. Yeast form and pseudohyphae (Gram’s stain) (100x).
Genus: Candida
Species: tropicalis
Cultural Characteristics
White creamy colored and foot-like extensions from the margin (Blood Agar Plate),
Smooth creamy colonies after 24-48 hours (Potato Dextrose Agar), and Creamy, pasty colonies,
smooth after 24-48 hours at 25-37°C (Sabouraud Dextrose Agar).
Microscopic Characteristics
Small, oval, measuring 2-4 µm in diameter.Yeast form, unicellular, reproduce by
budding. Single budding of the cells may be seen. Both yeast and pseudo-hyphae are gram
positive. Encapsulated and diploid, also form true hyphae.
Polymorphic fungus (yeast and pseudohyphal form). Can form biofilms. Normal condition:
Yeast. Special condition (pH, Temperature): Pseudohyphae. 80-90% of cell wall is carbohydrate
(Kidd S, Halliday C, Alexiou, H, & Ellis D. 2016).

A B C D
Figure 2. A. Colonies on BAP (obverse view), B. Colonies on PDA (obverse view), C. Colonies
on SDA (obverse view), D. Yeast form and pseudohyphae (Gram’s stain) (100x).
Genus: Candida
Species: parapsilosis
Cultural Characteristics
White creamy colored and foot-like extensions from the margin (Blood Agar Plate),
Smooth creamy colonies after 24-48 hours (Potato Dextrose Agar), and Creamy, pasty colonies,
smooth after 24-48 hours at 25-37°C (Sabouraud Dextrose Agar).
Microscopic Characteristics
Small, oval, measuring 2-4 µm in diameter.
Yeast form, unicellular, reproduce by budding. Single budding of the cells may be seen. Both
yeast and pseudo-hyphae are gram positive. Encapsulated and diploid, also form true hyphae.
Polymorphic fungus (yeast and pseudohyphal form). Can form biofilms. Normal condition:
Yeast. Special condition (pH, Temperature): Pseudohyphae. 80-90% of cell wall is carbohydrate
(Kidd S, Halliday C, Alexiou, H, & Ellis D. 2016).
A B C D
Figure 3. A. Colonies on BAP (obverse view), B. Colonies on PDA (obverse view), C. Colonies
on SDA (obverse view), D. Yeast form and pseudohyphae (Gram’s stain) (100x).
Yeast will grow on bacteriological media like blood agar plate. They may appear as
small, creamy or white colonies that are somewhat more raised than staphylococcal colonies. A
presumptive identification of Candida species can be made by observing pasty, yellow-white
colonies from which feet extend out from the margins into the surrounding agar. Growth of
Candida species on potato dextrose agar are smooth and white colonies. Growth of Candida
species on sabouraud dextrose agar. Colonies with white to cream colored, smooth, glabrous, and
yeast-like in appearance.

Differentiation of Candida species by a Germ-Tube Formation Test

A B C

Figure 4. Germ tube formation test. A Candida albicans, B. Candida tropicalis, C. Candida
parapsilosis (100x).
Candida species can all grow as budding yeast cells (blastospore). These budding yeast
cells are spherical or oval in shape and are approximately 2.5 x 3.7 µm in size (Larone, 2002).
However, some Candida species are innately pleomorphic and can grow in different growth
forms during pathogenesis in humans or in other stress-induced media as a means to compensate
for the changing conditions in the new environment. Morphological switching from yeast to
hyphal or filamentous form is a prerequisite for initiation of infection at different stages of
candidal colonisation/infection (Finkel et al., 2012; Thompson et al., 2011 Saville et al., 2003)
and also connotes virulence in Candida species ((Finkel et al., 2012; Fitzpatrick & Sheppard
2010; Thompson et al., 2011). The test included the formation of a germ-tube in human serum
when incubated at 37o C (Coleman et al., 1997; Odds & Bernaerts, 1994; Pincus et al., 1999).
Characterization – Biochemical Test of Candida species using Vitek 2

C
Figure 5. Biochemical Tests (Vitek 2). A Candida albicans, B. Candida tropicalis, C. Candida
parapsilosis.
The VITEK ID-YST card consists of 64 wells with 47 fluorescent biochemical tests.
They comprise 20 carbohydrate assimilation tests: adonitol (ribitol), d-trehalose, d-cellobiose,
dulcitol, d-galactose, d-glucose, lactose, d-maltose, d-mannitol, d-melibiose, d-melezitose,
palatinose, d-raffinose, l-rhamnose, sucrose, salicine, l-sorbose, d-sorbitol, d-l-lactate, and
succinate. The six organic acid assimilation tests are N-acetyl-glucosamine, methyl-α-d-
glucopyranoside, citrate, d-galacturonate, d-gluconate, and mono-methyl-ester-succinate. The
eight substrates for the detection of the oxidases are coupled with 4-methylumbelliferone (4MU):
α-galactoside–4MU, α-glucoside–4MU, α-mannoside–4MU, β-galactoside–4MU, β-glucoside–
4MU, β-glucuronide–4MU, β-N-acetyl-glucosaminide–4MU, and β-xyloside–4MU. The nine
substrates for the detection of arylamidases are coupled with 7-amino-methylcoumarin (7AMC):
glycine-7AMC, hydroxyproline-7AMC, H-lysine-alanine–7AMC, γ-glutamyl-transferase–
7AMC, H-glycine-glycine–7AMC, histidine-7AMC, isoleucine-7AMC, proline-7AMC, and
valine-7AMC. The four miscellaneous tests are phosphatase, urea, nitrate, and actidione. In cases
of identification with low discrimination (see below), simple additional tests (most of them can
be performed the same day) are suggested by the VITEK 2 system. Additional tests were chosen
to demonstrate blastospores or arthrospores, apiculated cells, polysaccharidic capsule, carotenoid
pigment, convoluted colony, hyphae or pseudohyphae, sporangia, growth at 37°C, and growth
without oil (Graf, Adam, Zill, and Göbel, 2000).
The integrated VITEK 2 instrument automatically filled, sealed and transferred the cards
into an incubator (incubation temperature was 35°C). Every 15 min the cards were automatically
subjected to a fluorescence measurement. Each profile was interpreted according to a specific
algorithm. After an incubation period of 15 h, the profile result was compared to the ID-YST
database, which led to the final identification of the microorganism.

Table 1
Biochemical testing pattern of the Candida spp. isolates by the Vitek-2 system and
Clinical Laboratory Standards Institute broth microdilution method (n =75)

Figure 6. CLSI biochemical testing interpretive in Candida albicans for the month of August,
September, October and November.
Figure 7. CLSI biochemical testing interpretive in Candida parapsilosis for the month of August,
September, October and November.

Figure 8. CLSI biochemical testing interpretive in Candida tropicalis for the month of August,
September, October and November.

With Vitek-2 ID system, 75 Candida species isolates including Candida albicans (n = 16) 21%,
Candida parapsilosis (n = 5) 7%, and Candida tropicalis (n = 54) 72% were correctly identified.

Antibiogram – Antifungal Susceptibility Test (AST)

Table 2
Antifungal susceptibility testing pattern of the Candida spp. isolates by the Vitek-2
system and Clinical Laboratory Standards Institute broth microdilution method (n =75)
Figure 9. CLSI antifungal susceptibility interpretive in Candida species for Amphotericin B,
Caspofungin, Fluconazole, Micafungin and Voriconazole.

Figure 7. CLSI antifungal resistant interpretive in Candida species for Amphotericin B,

Caspofungin, Fluconazole, Micafungin and Voriconazole.

Figure 10. CLSI antifungal intermediate interpretive in Candida species for Amphotericin B,
Caspofungin, Fluconazole, Micafungin and Voriconazole.

In AST for Amphotericin B, Vitek-2 AST system showed that 79.6% isolates of the
Candida species were susceptible, the remaining 18.5% were resistant and 1.9% are
intermediate. While by the CLSI broth microdilution method, Candida species isolates were
susceptible with 68.5%, 31.5% were resistant and 0% intermediate for Caspofungin. While in
Fluconazole, 98.1% are susceptible and 1.9% for resistant and 0% for intermediate. All the
isolates of Candida species were found to be susceptible 100% to Micafungin and Voriconazole
with 0% resistant and and 0% Intermediate. The measurement of percentage agreement between
the Vitek-2 AST system and CLSI broth microdilution method.

Discussion
Candida species is an important cause of systemic mycosis in hospitalized patients, and
morbidity and mortality worldwide, especially in critically ill patients (Sardi et., 2013). Among
Candida species, Candida albicans, Candida tropicalis, Candida parapsilosis were the most
common species encountered in routine clinical laboratory samples. ((Graf, Adam, Zill, and
Göbel, 2000). In our study, we have found Candida tropicalis (72%) as the predominant species
followed by Candida albicans (21%), and parapsilosis (7%).
In this study, we evaluated the new, fully automated VITEK 2 system for rapid
identification of yeasts and yeast-like organisms. The strains were simultaneously tested by the
ID 32C system. Due to its large database and accuracy, the ID 32C has been used by several
authors as a reference method. The ID 32C strip consists of 32 biochemical reactions; the results
were reported after incubation at 30°C for 48 h. In contrast, the ID-YST card of VITEK 2
comprises 47 biochemical tests; the results were obtained after an incubation at 35°C for 15 h.
The possibility to achieve the identification within 15 h due to its more sensitive fluorescence
technology represents a major progress of the new VITEK 2 system. The VITEK 2 system
performed well for the correct identification with or without additional tests (92.1 and 87.6%,
respectively). These data are also in agreement with recent studies of the VITEK 2 system
performed with a manually operated prototype and different panels of strains (Bossy et al., 37th
ICAAC; Lebeau et al., 98th Gen. Meet. Am. Soc. Microbiol.).
In this study, we also evaluated the Vitek-2 AST system with the CLSI broth
microdilution method for Candida species. A majority of Candida isolates were susceptible to
both antifungal drugs tested by AST-YS06 Vitek-2 cards and the CLSI M27-A3 method. All the
isolates of Candida species (100%) were susceptible to Micafungin and Voriconazole with 0%
resistant and and 0% Intermediate. In current clinical management practices, Amphotericin B,
Caspofungin, Fluconazole is not recommended as a treatment option for Candida species
infection [35] or susceptibility testing.[36]
The CCIs values between all the methods were statistically significant (P < 0.05). The
CCIs values for the Vitek-2 AST system and the CLSI broth microdilution method were also
statistically significant. However, the CCIs values for the Vitek-2 AST system were lower than
those observed for the CLSI broth microdilution method; this may be because the ranges of
antifungal agents in the Vitek-2 AST system do not match exactly with the ranges of the CLSI
broth microdilution method.

Conclusion and Recommendations

Differentiating Candida species isolates into their respective species type is paramount
for any mycological analysis for quick therapeutic intervention and effective patient
management. In addition, exact identification might reduce the pressure and gratuitous use of
toxic antifungal agents and thus minimise the formation of resistant strains and long hospital
stays. The present study revealed that phenotypic and machine system screening reduces the
period required for identification and improves the rate of identification of Candida species
isolates. However, conventional identification methods are still considered reference standard
methods, in spite of time-consuming along with ambiguities results, and hence, suited better for
research purposes. Therefore, Vitek-2 system appeared to be an alternative method for
identification and AST for the Candida species to prescribe appropriate antifungal agents for the
better management of opportunistic infection among immunosuppressed or
immunocompromised patients. A well trained mycologist or microbiologist is still an important
criterion to access the results of various diagnostic techniques used in a laboratory. Therefore,
offering a comprehensive diagnostic service not only requires resources but also up-to-date
training for the laboratory staff to encounter the ever evolving diagnostic methods.

Acknowledgement
Atencio, Luke Dranoel D. is supported by the Philippine Academy of Microbiology and
Philippine Society for Microbiology Incorporated. Azucena, Irish T. is supported by the
Department of Environmental and Natural Resources, Ecosystems Research and Development
Bureau-Costal Resources and Ecotourism Research Development and Extension Research
Center. Besales, Shane Catherine T. is supported by the Faculty of Science Department, Ramon
Avancena National High School, Department of Education.

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