Laboratory Isolation and Identification of Candida Species
Pei Pei Chong
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, for instance Candida krusei is intrinsically resistant to fluconazole but Candida parapsilosis may be susceptible. 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 and identification starting from the collection of specimen, transporting them to a diagnostic laboratory followed by staining, microscopy, automated blood culture systems, biochemical tests and molecular techniques 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.
May 10, 2011; Accepted: June 23, 2011;
Published: July 22, 2011
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 and Gruby studied Trichophyton schoenleinii
whereas Langenbeck had reported on the causative agent for thrush which was
Candida albicans (Wilson and 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 physicians knowledge
of a drug, drug availability, patients 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.
Fungal infections fall into four broad categories, i.e., the deep-seated systemic
mycoses, cutaneous mycoses, subcutaneous mycoses and superficial mycoses. Systemic
mycoses occur sporadically and are caused by heterogeneous groups of fungi.
Fungal spores that are inhaled may cause histoplasmosis, blastomycosis, sporotrichosis,
coccidioidomycosis, cryptococcosis and paracoccidioidomycosis (Brooks
et al., 2010). Cutanoeus mycoses are infections of the keratinocytes
of the epidermis and their appendages such as hair and nails. Most common are
the dermatophytoses, caused by dermatophytes from the genera Trichophyton,
Microsporon and Epidermophyton (Ichhpujani
and Bhatia, 2007). However, subcutaneous mycoses involve the skin, subcutaneous
tissues and bones resulting from the embedment of saprophytic fungi in these
regions of the body without being disseminated to the internal organs. The principle
mycoses are mycetoma, chromoblastomycosis, sporotrochosis and rhinosporidiosis
(Arora, 2004). Superficial mycoses are only surface infections
of the skin, hair, nail and mucous membranes. They are caused by a variety of
fungi such as dermatophytes, Candida spp., Malassezia, Exophiala,
Trichosporon, Piedraia and many others.
Apart from the above categories of mycoses, opportunistic mycoses are another
major concern among immunocompromised patients. An opportunistic organism causes
disease at any instance when a persons body is in a compromised state.
The overuse of antibacterial antibiotics, immunosuppressive agents, cytotoxins,
irradiation and steroids lead to this new category of systemic mycoses. These
patients have been deprived of immune resistance by the bodys normal flora,
thus develop opportunistic mycoses, for example candidiasis, aspergillosis,
mucormycosis and others. Candida albicans is a common cause of opportunistic
mycosis. Oral candidiasis is common among AIDS patients, poorly nourished patients
and immunosuppressed patients. Incidences of oral thrush among school children
between the age of 6 and 13 years old have been reported in Nigeria (Nneka
and Ebele, 2005).
In Malaysia, 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 C. glabrata, C. lusitaniae, C. famata, C.
krusei and C. parapsilosis. Re-infection from these yeasts varied
among individuals either by identical, similar or different strains or species
of Candida (Chong et al., 2003). In another
report, the most predominantly isolated species from two hospitals in Malaysia
from the year 2004 to 2009 was Candida tropicalis, followed by C.
albicans, Candida parapsilosis, C. krusei, Candida rugosa,
Candida dubliniensis and Candida glabrata (Madhavan
et al., 2010). Other studies have reported that there is an increase
in the number of disseminated candidiasis among acute leukaemia patients following
chemotherapy (Cantu, 2005). Basetti
et al. (2007) reported that the incidence of candidemia within a
one-year period of study was 13% among hospitalized patients in two teaching
hospitals in Italy. 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 leukaemia patients, oral colonisation by Candida
species was found in 90% of the patient population. Treatment with ketoconazole
among these patients showed little effect and complete eradication was only
seen in 9 out of 20 patients treated for 5 days (Rodu et
al., 2006). Related studies have shown that there were increased disseminated
Candida infections among bone marrow transplant patients and neutropenic
patients by Candida krusei since a century ago (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.
SPECIMEN AND TRANSPORT
All specimens should have a laboratory test requisition form each completely
filled by the physician with the patients name, age, sex, specimen source
and patients history (Tortora et al., 2010).
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 manager or technicians should inform
the physician if additional amount of specimen is required. Specimens obtained
from skin, nail clippings and hair should be placed in an envelope, Petri dish
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
and Haleagrahara, 2011). If there should be a delay in transporting the
specimens to the diagnostic laboratory, then the specimens should be incubated
The basic culture media used in isolating clinical Candida species are
blood agar, Potato Dextrose Agar (PDA) or broth (PDB), Sabouraud brain heart
infusion agar, Sabouraud Dextrose Agar (SDA) or broth (SDB), Yeast Nitrogen
Base (YNB) and Yeast Potato Dextrose (YPD) agar or broth. Lees synthetic
medium can be used for mycelial development and yeast formation for Candida
albicans (Jatta et al., 2009; Yang
et al., 2009). Other media that can be used as a selective or differential
media are CHROMagar Candida (Madhavan et al., 2009;
Bernal et al., 1996), CandiSelect4 (Gaschet
et al., 2008) and Pharmamedia (Slifkin, 2000).
Pagano-levin agar can also be used as a differential medium but the presence
of Triphenyltetrazolium Chloride (TTC) was found to inhibit the growth of some
species of Candida (Yamane and Saitoh, 1985;
Samaranayake et al., 1987). Therefore, Candida
Bromocresol Green Agar (BCG Agar) is now used as an alternative as TTC was replaced
with a non-toxic inhibitor, bromocresol blue. Since C. albicans from
C. dubliniensis are closely related species which can be difficult to
differentiate phenotypically, cultivation on Bird-seed agar was found to be
a fast, reliable and sensitive method to discriminate these two species (Pasligh
et al., 2010). 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). Sabourauds 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 blood agar and incubated between 35 to 37°C
for 72 to 96 h. Long term storage for few months can be done using agar slants
(Chakraborty et al., 2005).
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 can be viewed
under reduced-light brightfield microscope or phase-contrast microscope (Aslanzadeh
and Roberts, 1991). Presumptive identification of C. albicans is
done with the germ tube test. In this method, the clinical sample is incubated
in human or animal serum for 2 to 3 h at 30 to 37°C. If C. albicans
is present, short, slender, tubelike structures (germ tube) can be observed
under the microscope (Mackenzie, 1962). Reports of other
species of Candida such as C. tropicalis and C. parapsilosis
were also found to produce similar structures (Campbell
et al., 1998; Freydiere and Guinet, 1997;
Lipperheide et al., 1993; Perry
et al., 1990). However, stains can be used as an aid to view yeasts
in specimens. Pottasium hydroxide solution (10-20% KOH and 10% glycerine) is
the most commonly used stain to detect fungi or yeast cells. KOH digests keratin
and glycerine prevents degradation of yeast. Lactophenol cotton blue can also
be used together with KOH for better observation of yeast under brightfield
microscope. Gram and Giemsa stains are useful in staining yeast cells because
of their small size (3-4 μm). Calcofluor white, a colourless dye can be
used to detect fungal elements even in frozen and paraffin-embedded tissue sections
(Aslanzadeh and Roberts, 1991). The advantage of using
this dye is that stained specimens can be viewed immediately using an improvised
and economical fluorescent microscope with 25 watt halogen lamp rather than
epifluorescent or mercury vapour lamp. Periodic acid Schiff can be also used
to detect fungi in tissues but may take several h to perform the test. However,
methenamine silver is known to be the best method to detect fungi in histology.
Fungal elements would be seen as black against a red or green background. Apart
from these, stains used in cytology and pathology laboratories are hematoxylin-eosin
and papanicolaou which are found to be less effective than methenamine silver
(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. Detection
of Candida cultures may differ from one species to another, example the
mean time for positive detection of Candida albicans is between 35.3
to 85.8 h where as for Candida glabrata is between 80 to 154 h (Fernandez
et al., 2009). Yeasts cultures are usually held for 21 days and blood
cultures for about 5 days.
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 API 20C, API 32C or RapID 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 RapID Yeast Plus system. The resulting
colours at the end of the incubation period are coded and compared with the
RapID Yeast Plus Differential Chart to identify the species. This method is
currently the fastest commercial method for the identification of yeasts which
requires a 4 h incubation period only. However, the identification of Candida
dubliniensis was found better with API 32C than Vitek-2 YST system (Cardenes-Perera
et al., 2004). API 32C was also useful in differentiating C. albicans
from C. dubliniensis as this two species are phenotypically alike. API
32C is based on the assimilation of various carbohydrates and 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 h which is faster than API 20C
and API 32C (Loiez et al., 2006). However, in
another report, there was a necessity for additional tests with CHROMagar and
API 32C to verify the results of Vitek-2 YST system (Pryce
et al., 2006). It was also suggested that conventional diagnostic
methods are not reliable to identify newly emerging Candida pathogens
such as C. haemulonii (Ruan et al., 2009)
and C. kefyr (Gomez-Lopez et al., 2010),
as they were often mis-indentified as other species of Candida. In their
report, it was also stated that although the VITEK 2 system was found better
compared to VITEK 1 and API 32C, it still could not be used to differentiate
C. haemulonii from its sibling species C. pseudohaemulonii.
NUCLEIC ACID-BASED IDENTIFICATION
Nucleic acid sequence of a gene is an important property as it carries the
identity of an organism. Molecular methods are based on the detection of the
nucleic acid sequence of a gene specific to an organism. In probe-based identification,
the organisms single stranded DNA or RNA would bind to a complementary
sequence and form hybrid which is double stranded. Identification of specific
organisms can be done from the clinical specimen, culture, formalin-fixed or
paraffin-embedded tissues (Hong, 2006). The use of rRNAs
is essential for probe-based detection of species as its nucleotide sequence
is well conserved within a species and varies between species. Therefore, it
is used as a target for species identification. Sequencing the amplified regions
of Internal Transcriber Spacer regions (ITS) of the rRNA gene for Candida
gained success in species identification (White et al.,
1990). The early discoveries were in C. dubliniensis (Sullivan
et al., 1995), C. orthopsilosis and C. metapsilosis
(Tavanti et al., 2005) using PCR methods. In
Denmark, differentiation between C. orthopsilosis and C. metapsilosis
was performed using a newly described RFLP method using SADH (secondary dehydrogenase-encoding
gene), 26S rRNA (D1/D2) and ITS1-ITS2 regions (Mirhendi
et al., 2010). In another study, molecular identification using ITS1-ITS4
fungal primers were able to identify C. orthopsilosis which was previously
identified as C. parapsilosis with API 20C test kit (Yong
et al., 2006). Very recently, a rapid real-time PCR method was also
developed to distinguish C. metapsilosis, C. orthopsilosis and
C. parapsilosis. Different melting curve used was able to identify the
three species mentioned above. Hays et al. (2011)
and Gomez-Lopez et al. (2010) reported that a
strain which was identified as C. kefyr with biochemical test method
was discovered to be C. sphaerica by ITS sequencing. In another report,
strains of C. haemulonii was mis-identified as C. sake, Pichia
ohmeri and other Candida spp. by biochemical test kits (Ruan
et al., 2009). However, molecular methods revealed their species
identity. A multiplex PCR method used in another study was able to differentiate
C. nivariensis and C. bracarensis which are phenotypically identical
to C. glabrata (Romeo et al., 2009).
Affirm DNA probe system is a diagnostic test that can detect and differentiates
causative agents of vaginitis, mainly Candida, Gardnerella and
Trichomonas (Hong, 2006). This system uses two
distinct single stranded DNA that is complementary to unique sequences of target
organisms. These probes are known as capture probe and colour development probe.
The Affirm VPIII Microbial Identification Test yields positive results only
for symptomatic vaginitis based on the organisms cell count. This is very
useful in terms of detecting symptomatic vaginitis or vaginosis and providing
appropriate treatment. PNA FISH probe is a new diagnostic technique using fluorescence
in situ hybridization using Peptide-Nucleic Acid (PNA) probes. Peptide nucleic
acids are synthetic compounds containing nucleotide bases attached to a peptide
backbone which targets the rRNA. PNA FISH for Candida albicans has been
approved by FDA and is commercially available. Pulsed-Field Gel Electrophoresis
(PFGE) which was developed in early 1980s is currently used for strain characterization,
to understand the evolution of antimicrobial resistance and epidemiological
analyses. The source of transmission can be identified especially in nosocomial
infections and to curtail disease outbreaks using this technique (Wu
and Della-Latta, 2006). PFGE has been used in genotyping Candida
spp., with or without the use of restriction enzyme digest in its protocol (Espinel-Ingroff
et al., 1999). The identity of the infective strains in a patient
with recurrent Candida infections over a period of time is also possible
with this technique (Bennett et al., 2004). A
combination of molecular typing methods with high and low discriminatory power
is necessary to provide optimal source tracking especially for nosocomial infections
of Candida spp. High discrimination allows detection of strain variants
and low discrimination allows the isogene determination of organisms (Lopez-Ribot
et al., 2000). Therefore, to fulfil both these requirements, Candida
specific DiversiLab Kit and DiversiLab Yeast Kit can be used (Frye
and Healy, 2006).
There are many other techniques that can be used for strain typing or identification
such as Ca3 genetic fingerprinting for Candida albicans (Martinez
et al., 2002; Pujol et al., 2002,
2004), Multilocus Sequence Typing (MLST) (Bougnoux
et al., 2006; Chen et al., 2006; Viviani
et al., 2006) Multilocus Enzyme Electrophoresis (MLEE) (Arnavielhe
et al., 2000; Boldo et al., 2003), Restriction
Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD)
analysis (Chong et al., 2007; Lephart
et al., 2004; Roilides et al., 2003;
Samaranayake et al., 2003), microsatellite-PCR
(Lopes et al., 2007; Correia
et al., 2004; Dalle et al., 2003)
and Amplified Fragment Length Polymorphism (AFLP) (Lopes
et al., 2007). Reverse transcription PCR (RT-PCR) analysis can be
also used for the identification of mutations and gene regulations in Candida
species (Francisca et al., 2011; Yong
et al., 2006; Looi et al., 2005).
However, the use of above stated methods depend on the aim of the identification.
Routine identification in a laboratory using molecular techniques remains challenging
as some of these techniques may be costly, require trained personnel and still
lack sufficient species-specific primers. Therefore, the selection of methods
to identity an organism is not solely based on the effectiveness of the methods
but also the availability and affordability of them.
A well trained mycologist 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.
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