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6
S. Banik
Additionally, many antibody-based immunostains are also used for the visualization
of fungal morphology and diagnosis of invasive infections (Rüchel and Schaffrinski
1999; Hamer etal. 2006; Kakoschke etal. 2019; Yao etal. 2019).
Histological examination for the detection of fungal infections is particularly
benecial where culture is not possible. However, the collection of biopsy samples
is an invasive process and may not be possible in critically ill patients or patients
with thrombocytopenia. Moreover, morphological similarities among different fungal pathogens require additional conrmation from more specic diagnostic tests.
Tissue immunohistochemistry and in situ hybridization using antibodies against
fungal species-specic antigens could offer better specicity but lack of commercially available antibodies and high degree of cross-reactivity have kept its application limited (Guarner and Brandt 2011).
1.2.2 Culture
Many commercially available diagnostic systems require prior isolation of Candida
from clinical specimens. Due to its ubiquitous and nonfastidious nature, Candida
species can grow on media commonly used in clinical laboratories. A variety of
clinical specimens such as blood, urine, saliva, sputum, BAL, CSF, and skin swabs
can be used for fungal isolation and identication by culture (Deorukhkar and
Saini 2014).
Sabouraud Dextrose Agar (SDA) is the most widely used culture media for preliminary isolation of Candida species from polymicrobial clinical specimens (Odds
1991). It was developed by Raymond Sabouraud and later named after him in 1892.
On the SDA plate, all species of Candida appear as smooth, creamy, and pasty colonies which can become wrinkled if incubated for longer periods of time (Lynch
1994). SDA is often supplemented with antibiotic chloramphenicol and gentamicin
to suppress bacterial growth (Odds 1991; Coleman et al. 1993; Lynch 1994).
However, SDA cannot distinguish different Candida species present in the same
specimen (Samaranayake et al. 1987). Nonetheless, it is not useful in detecting
infections caused by multiple fungi. A variety of different media such as PaganoLevin agar (SDA supplemented with triphenyltetrazolium chloride), malt peptone
agar, potato dextrose agar, phosphomolybdate agar, and Nickerson’s medium can be
useful for the differentiation of Candida species in polyfungal infections (Nickerson
1953; Pagano etal. 1957; Costa and Brancocde 1964; Bump and Kunz 1968).
There has been a signicant increase in the number of invasive fungal infections
over the last decades due to the prolonged use of antifungals and the increasing
number of immunocompromised patients. Most invasive Candida infections are
caused by C. albicans, followed by C. glabrata, C. tropicalis, C. krusei, and
C. parapsilosis. Increased use of azoles as antifungals has facilitated the emergence
of non-C. albicans species with higher frequency, especially in cancer and HIV
patients. Since different Candida species may differ in their resistance to antifungals, a rapid and presumptive identication of Candida at the species level is crucial
for proper treatment. Several chromogenic media, which are selective and
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
7
differential, have been developed for presumptive identication and speciation of
Candida isolates with good specicity and sensitivity. The use of chromogenic
media in clinical microbiology laboratories has reduced the time for identication
and discrimination of fungal pathogens particularly in polyfungal infections. Since
it was rst reported in 1994, several different chromogenic media have become
available commercially (Table 1.1). The chromogenic substrates present in these
media react with hexosaminidase (C. albicans, C. dubliniensis, and C. tropicalis)
and alkaline phosphatase (C. krusei) enzymes secreted by fungal cells, resulting in
colonies with characteristic pigmentation specic to different Candida species.
Additionally, supplementing with chloramphenicol and/or gentamicin can inhibit
bacterial growth in these media.
CHROMagar Candida (CHROMagar, Paris, France) is the most widely used
chromogenic medium. It contains a chromogenic substrate of hexosaminidase and
can be reliably used for the differentiation of C. albicans, C. dubliniensis, C. tropi-
calis, C. parapsilosis, C. glabrata, and C. krusei on the basis of color and colony
morphology. This medium has the added advantage of detecting polyfungal infections directly from clinical specimens. Recently, another chromogenic medium,
CHROMagar™ Candida Plus, has become available which can differentiate C. auris
from other fungal pathogens. It can be utilized for the preliminary identication of
C. auris in healthcare settings and thus helps to contain the spread of this highly
transmissible pathogen (Borman and Johnson 2013).
Culture remains the gold standard for diagnosis of fungal infection until now.
However, it is time-consuming, requires 24–72 h of incubation and sometimes
weeks for some fungi and less sensitive. In particular, blood culture takes 2–5days
and requires a larger volume due to less number of Candida present in the blood.
Furthermore, the sensitivity depends on the collection, transportation and storage of
clinical samples and can be suppressed by the antifungal agents. Despite all these,
culture offers some advantages. It isolates the infecting fungal pathogen to be used
for further identicationbyphenotypic, biochemical, and molecular assays and for
determining antifungal susceptibility prole (Borman and Johnson 2013). Recently,
Chen etal. developed a rapid enrichment method using magnetic beads coated with
a recombinant human mannan-binding lectin that enabled a direct culture of
Candida sp. from blood with an increasing sensitivity to 90% and shorter time
period to positivity compared to standard blood culture (Chen etal. 2020).
1.2.3 Germ Tube Test
The germ tube test which was rst described more than half a century ago is still the
most widely used microbiological technique for rapid and presumptive identication of C. albicans (Taschdjian etal. 1960). It is also known as the “Reynolds–
Braude phenomenon.” The germ tubes are lamentous overgrowth, that arises from
the side of a blastospore without any constriction at the site of emergence
(Deorukhkar etal. 2012). This test, mostly used for discriminating C. albicans and
C. dubliniensis from other species, is based on their ability to form germ tube-like
8
BIGGY agar
(Nickerson
Agar;
Candida
Ident
agar
CHROMagar
HardyChrom
Candida
Brilliance
Candida
agar
Milipore
Sigma,
USA)
Smooth,
(Milipore
Sigma,
USA)
Light
ChromID
Candida agar
(BioMerieux)
Candida Plus
(Paris,
France)
Green blue Blue to dark
(Hardy
Diagnostics,
CA, USA)
(Thermo
Fisher
Scientic)
Green Smooth
circular
brown-black
colonies; no
color
green
blue
metallic green
to dark
metallic green
diffusion
into
surrounding
medium; no
sheen
Smooth,
discrete
brown-black
colonies
purple
Pink Blue to
Metallic blue
with pink
halo
metallic blue
with a blue
halo
Dark blue Blue to dark
with black
centers;
diffuse
blackening
of medium
after 72h;
sheen
White Light
Pink to
Beige/
brown-
greenish,
purple
yellow/
brown
S. Banik
grey-cream
CHROMAGAR
Candida (Kima)
circular, pale
green
CHROMOGENIC
Candida agar
(Biolife, Italy)
CHROMATIC
Candida
(Liolchem,
Italy)
Green Green Smooth,
CHROMagar
Candida
(Paris,
France)
to turquoise
CandiSelect4
(Bio-Rad,
Hercules, CA,
USA)
Species
Table 1.1 Morphological features of medically important Candida sp. on different agar
C. albicans Pink to purple Leafy green
circular, pale
purple- blue
Blue Blue-violet Smooth,
Dark to
metallic blue
with a purple
mat,
uniformly
C. tropicalis Turquoise,
halo
colored,
convex,
smooth
Smooth,
circular, white
Smooth, circular,
brilliant pink
Pale pink to
white
Smooth,
circular, pale
pink with
Smooth,
circular, pale
green
C.
parapsilosis
grey
periphery
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
Large, at,
wrinkled
silvery
brown-black
colonies
with brown
edge; yellow
halo
Small,
cream,
opaque
9
fuzzy
White Purple,
Pink and
fuzzy
Rough,
spreading
pink
Pink to
brown,
dry,
Flat, rough,
pink with grey
periphery
irregular
white
Mauve White Cream
Smooth, pink
with darker
mauve center
Beige/
yellow/
brown
circular pink to
violet
White
rough
Green Bluish green,
Smooth,
circular, dark
green
Pink, rough, large,
spreading
Pink, pale
edges
Rough, pink,
fuzzy with
whitish
border
blue, rough,
dry, irregular
C. krusei Turquoise-
Dark violet Beige Pink-violet Smooth,
turquoise,
C. glabrata Pale
at, shiny,
convex,
bottle green
Dark green Yellow- green Smooth, circular,
smooth with
dark center
Smooth,
circular, blue
C.
dubliniensis
grey to pink
10
S. Banik
structures when incubated in serum or other proteinaceous medium for 2–4 h
(Sullivan etal. 1996). To perform the germ tube test, a suspension is prepared from
the fresh culture of an isolate, and after a few hours of incubation, it is examined for
germ tube formation under a microscope. At least ve germ tubes should be counted
in the entire wet mount preparation to consider an isolate as germ tube positive
(Deorukhkar etal. 2012).
Although it is simple, economical, and has 98% sensitivity, it requires skills to
differentiate a true germ tube from pseudohyphae (Berardinelli and Opheim 1985;
Hilmioglu etal. 2007; Pincus etal. 2007). More than 90% of the clinical isolates
that have positive germ tube test are C. albicans, but false-positive results can occur
with other non-C. albicans species such as C. tropicalis, C. parapsilosis, and C. afri-
cana whichhave been reported to form germ tube-like structures (Tierno Jr and
Milstoc 1977; Lipperheide etal. 1993; Freydière and Guinet 1997; Campbell etal.
1998). This relatively simple and rapid test is particularly useful as a rst identica-
tion step before conrmatory tests are employed.
1.2.4 Formation ofChlamydospore
Chlamydospore is a morphological form that is developed under stress conditions to
facilitate the survival of certain fungal species including Candida (Sonneborn etal.
1999). The chlamydospore formation test is performed by inoculating Candida spe-
cies on cornmeal agar and observing microscopically for the presence of chlamydospore (Aubertine et al. 2006). Moreover, rice extract agar, sunower seed agar,
casein agar, Staib agar, and tobacco agar can also be used for chlamydospore formation test (Sullivan etal. 1999; Aubertine etal. 2006). An addition of polysorbate 80
or Tween 80 to these agar media further enhances the development of chlamydospores. This test is less subjective than the germ tube test but requires a longer
incubation time of several days. Chlamydospores are produced by C. albicans,
C. dubliniensis, and a few strains of C. tropicalis (Moran etal. 2012). The chla-
mydospore formation in C. albicans differs from that of C. dubliniensis and hence
has been used to differentiate these two closely related species (McCullough
etal. 1999).
The chlamydospore formation test is relatively simple and can be performed in
clinical laboratories for a preliminary identication of Candida spp. in the sample.
However, this test is not always denitive, and additional methods are required for
precise identication.
1.2.5 Carbohydrate Assimilation Test
The yeast carbohydrate assimilation test is one of the most widely used methods in
clinical laboratories for a denitive identication of yeasts including Candida spp.
The ability of different species of Candida to assimilate different carbohydrates as
the sole carbon source for metabolism allows their speciation. The carbohydrate
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
11
assimilation test relies on the use of yeast nitrogen base as the basal medium and the
presence of growth when appropriate carbohydrate is added to the medium.
Since the development of auxanographic technique for carbohydrate assimilation, a variety of methods have been developed for denitive and rapid results. More
than seven decades ago, Wickerham and Burton rst developed a broth method for
carbohydrate assimilation testing of yeasts where the isolate to be identied was
grown in a set of basal media in a tube supplemented with different carbohydrates
(Wickerham 1943, 1946; Wickerham and Burton 1948). Although this method was
precise, it was labor-intensive and time-consuming as it could take up to 4weeks for
yeasts with slow rates of carbon assimilation and often difcult to interpret by turbidity measurements. Moreover, most Wickerham media are not available commercially, thus making it inappropriate for routine use in clinical diagnostic laboratories
(Pincus etal. 2007). Wickerham and Burton’s technique was further modied to
include aeration of the broth, replacement of broth with an agar slant, and addition
of a pH indicator (bromocresol purple) in the agar slant (Ahearn etal. 1960; Martin
and Schneidau Jr 1970; Adams Jr and Cooper 1974). Although these modications
offered quick and decipherable methods, they had several disadvantages. The use of
pH indicators required buffering and monitoring the pH of the medium. Furthermore,
any diffusion of acid in the medium and thereby reversion of the pH indicator
resulted in misinterpretations of test results (Huppert etal. 1975).
Land etal. developed an improved method for comprehensive phenotypic identication of Candida spp. known as dye pour-plate auxanogram (DPPA) which allows
testing of multiple substrates on the same agar plate (Land etal. 1975). In this auxanographic technique, carbohydrate-impregnated discs are placed on an agar medium
which is seeded with the yeast. An enhanced area of yeast growth around a specic
carbohydrate disc is an indicative of the assimilation of that carbohydrate (Moore
etal. 1988). The addition of bromocresol purple in the agar allows for easy interpretation of results. Further modication of this auxanographic technique employed a
higher concentration of yeast nitrogen base medium and elimination of the pH indicator to reduce misinterpretation which was the basis for a commercial method, BBL
Minitek (BBL Microbiology Systems, Cockeysville, MD) (Mickelsen etal. 1977).
The manual auxanographic method was later replaced by many commercial yeast
identication systems such as analytical prole index (API) 20C AUX and ID 32C
(bioMerieux, Hazelwood, MO) which are discussed later.
The carbon assimilation test has long been used as a standard method for the
characterization and taxonomical identication of yeasts and hence provides a valuable tool in clinical laboratories. However, this test may not provide denitive identication for the existence of rare or newly emerging Candida spp. or any mixed
infections. Therefore, supplementary tests are needed for further conrmation.
1.2.6 Nitrogen Assimilation Test
Nitrogen compounds are essential to the growth and metabolism of yeasts. The ability of yeasts to utilize various nitrogen compounds for growth has been used in their
12
classication. A nitrogen assimilation test is preferred due to its high sensitivity and
specicity and can be determined in a broth medium using alpha-naphthylamine
and sulfanilic acid. Yeast carbon base medium is used for the classication of yeasts
based on their nitrogen assimilation (Wickerham 1946).
S. Banik
1.2.7 Carbohydrate Fermentation Test
The carbohydrate fermentation test is used to determine the ability of yeasts to ferment a specic carbohydrate in the medium detected by a pH indicator (phenol red)
and speciate them based on their fermentation pattern. This test is generally performed in a liquid medium in the presence of different carbohydrates and relies on
the production of acid or gas or both (Buesching etal. 1979). Acids are released
during the fermentation of carbohydrates which decreases the pH of the growth
medium and changes the color of the indicator from purple to yellow. A Durham
tube is placed in the liquid medium to collect gas produced during the fermentation
process.
Although the carbohydrate fermentation test is useful in differentiating Candida
spp., this test is less sensitive and reliable than the carbohydrate assimilation test.
Moreover, longer incubation time and laborious processes have limited its use in
routine diagnostic laboratories. Kali etal. developed a cost-effective method for
carbohydrate fermentation in microtiter plates which reduced the turnaround time
without reducing discordant results (Kali etal. 2015).
1.2.8 Urease Test
The urease test is performed to differentiate C. albicans which is urease-negative
from urease-positive C. krusei, C. lipolytica, and C. humicola. This test is based on
the ability of yeasts to produce the enzyme urease and is performed on Christensen’s
urea agar slant for 48h. Urease enzyme, secreted by the yeast, breaks down urea to
ammonia and carbon dioxide which changes the pH of the medium to alkaline. This
change in pH converts the light-orange phenol red indicator to pink or red which is
an indicative of a positive urease test (Christensen 1946; Seeliger 1956).
1.2.9 Tween 80 Opacity Test
Many pathogenic Candida species secrete lipolytic enzymes such as phospholipases and esterases (Wills 1965; Ghannoum 2000). The esterase activities of some
Candida species were rst reported by Pospisil and Kabatova, with the application
of the Tween opacity test using Tween 80 as a substrate (Pospísil and Kabátová
1976). Later, patterns of responses associated with various species of Candida to
Tween opacity tests on various Tween compounds were demonstrated to be useful
in their identication (Rudek 1978).
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
13
Tween 80 agar is used to evaluate the lipolytic activity of Candida species. The
presence of a halo or zone of precipitation around the site of inoculation on the
Tween opacity test agar as observed under transmitted light is evaluated as a positive test, indicating esterase production by the isolate. Upon hydrolysis of the Tween
compounds and cleavage of ester bonds by the esterase enzyme, fatty acids are
released which bind with calcium ions in the medium. These calcium complexes are
insoluble and visible as zones of precipitation around the inoculation site
(Tirunarayanan and Lundbeck 1968). This test differentiates C. albicans which is
esterase positive from esterase-negative C. dubliniensis (Slifkin 2000).
1.3 Nonculture-Based Conventional Methods
Timely and rapid diagnosis of invasive candidiasis is essential for providing
appropriate treatment and obtaining a better outcome (Morrell etal. 2005; Garey
etal. 2006). Blood culture, the gold standard test for diagnosis of candidemia or
invasive candidiasis, has its own limitations. The sensitivity is around 20–70%
depending on the frequency, volume, and timing of the blood drawn (Clancy and
Nguyen 2013). Blood cultures are mostly positive if the samples are collected
during active candidemia when the median concentration of circulating Candida
species is 1CFU/ml (Pfeiffer et al. 2011). Candida spp. multiply slower than
bacteria which is attributed for prolonged time, usually 2–5days, for blood cultures to be positive (Arendrup etal. 2011). However, frequent sampling (once
daily or more frequently if the patient has fever), larger volume for culture, use of
fungal selective media, and collection of blood before starting off antifungal treatment can increase the sensitivity and shorten the turnaround time (Arendrup etal.
2011; Ericson etal. 2012).
Blood cultures are positive in 40% of patients with candidemia associated with
deep-seated candidiasis and negative in deep-seated candidiasis in the absence of
candidemia. The sensitivity of cultures across the spectrum of invasive candidiasis
is only 50% (Clancy and Nguyen 2013). In patients with deep-seated invasive candidiasis who are critical of any invasive diagnostic procedures, a presumptive diagnosis is made using ultrasound, magnetic resonance imaging (MRI), or computed
tomography (CT) (Anttila etal. 1994; Ascioglu etal. 2002). Moreover, the microscopic identication of fungi can also be utilized as it is rapid and sensitive but
requires skill and lacks specicity (Hay etal. 2019; Mendonça etal. 2022). This has
resulted in an increasing use of antifungals as prophylactic treatment resulting in
increased resistance. This has prompted the development of several nonculturebased tests to improve the diagnosis of candidemia and invasive candidiasis.
The diagnosis of fungal pathogens by serological methods is well established
and achieved by the detection of fungal antigens, metabolites and circulating antibodies in serum, urine, and bronchoalveolar uid (Jensen 2021). Commonly used
serological assays for invasive candidiasis are enzyme-linked immunosorbent
assays (ELISA), lateral ow assays (LFA), radio-immunosorbent assays (RIA), and
agglutination assays.
14
S. Banik
1.3.1 Serological Methods
1.3.1.1 Mannan Antigen andAntimannan Antibody
The detection of mannan antigen and antimannan antibody in serum is one of the
earliest nonculture-based diagnostics for invasive candidiasis including pediatric
populations and patients with CNS infection (Oliveri etal. 2008; Biesbroek etal.
2013; Clancy and Nguyen 2013). Mannan is a major component of the Candida cell
wall which comprises 7% of the cell dry weight. It is one of the main Candida antigens that are present in the blood during an infection and hence been used as a
diagnostic biomarker of invasive candidiasis (Klis 1994). A combination of mannan
antigen and antimannan antibody testing provides a sensitivity and specicity of
83% and 86%, respectively, in patients of invasive candidiasis infected with C. albi-
cans, C. tropicalis, and C. glabrata (Mikulska etal. 2010). Both mannan antigen
and antimannan antibody are detected in the serum 6days earlier than blood cultures become positive (Arendrup etal. 2010).
There are several commercially available serological assays for the detection of
mannan antigens and antimannan antibodies. Among them, Platelia™ Candida Ag/
Ab Plus (Bio-Rad, Marnes-la-Coquette, France) and Serion ELISA Antigen
Candida assay (Serio GmbH, Wurzburg, Germany) are widely used in many
European countries. None of these tests are approved by the U.S.Food and Drug
Administration (FDA) as a diagnostic tool in the United States (Clancy and
Nguyen 2018).
1.3.1.2 ß-d-Glucan
Polysaccharides are major structural constituents of fungal cell walls and are composed of glucan, chitin, and mannan. Of these, 1,3-ß--glucan (BDG) is the most
abundant polysaccharide component of the cell wall of most pathogenic fungi
including Candida spp., Aspergillus spp., and Pneumocystis jirovecii (Bowman and
Free 2006). It is used as a surrogate biomarker for the detection of invasive fungal
infections in a chromogenic assay (Lamoth and Alexander 2014). BDG is not
directly detected in these assays, rather they measure the BDG-mediated activation
of a coagulation cascade in a modied limulus amebocyte lysate pathway of the
horseshoe crab. In these assays, BDG in samples activates factor G which in turn
activates a clotting enzyme resulting in the cleavage of a chromogenic substrate,
p-nitroanilide from the synthetic peptide substrate, changing to a yellow color.
Release of p-nitroanilide over time is measured as an end-point absorbance and
used to determine the concentration of BDG in samples (Wright etal. 2011). It has
been observed that some serum samples have an inherent yellow color that may
inuence the end-point absorbance. Therefore, some assays measure the absorbance
of the diazo derivative of p-nitroanilide which is purple in color (Lamoth and
Alexander 2014). As a pretreatment, samples are exposed to an alkaline reagent to
convert the triple-helix form of BDG which is the most abundant form to its singlestrand form. This alkaline pretreatment of the sample also inactivates serine proteases and serine protease inhibitors present in human serum, thereby reducing the
rate of false-positive and false-negative reactions of this assay (Mennink-Kersten
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
15
et al. 2008). BDG assay is not a specic test as it cannot differentiate between
Candida and other fungi.
Four commercial BDG assays are available for the diagnosis of invasive fungal
infections: Fungitell (Associates of Cape Cod, East Falmouth, USA), Fungitec-G
(Seikagaku Biobusiness, Tokyo, Japan), beta-Glucan test (Waco Pure Chemical
Industries, Osaka, Japan), and Maruha (Maruha-Nichiro, Foods Inc., Tokyo, Japan).
These commercial kits use reagents derived from different species of horseshoe
crab, and therefore, cutoff values for positive results range from 11 to 80pg/ml
depending on the assay. Overall sensitivity and specicity of these assays are
58–100% and 57–100%, respectively (Ostrosky-Zeichner etal. 2005; Akamatsu
etal. 2007; De Carolis etal. 2020). Of these, only Fungitell is FDA-approved and
available in the USA and Europe. Fungitell has sensitivity and specicity of
69.9–100% and 73–97.3%, respectively, for invasive candidiasis (Ostrosky-Zeichner
etal. 2005; Mohr etal. 2011; León etal. 2016; De Carolis etal. 2020). The performance of the serum BDG assay is better if positivity is dened by two consecutive
test results, rather than a single test (Hanson etal. 2012).
Although the serum BDG assay is an effective biomarker for the diagnosis of
invasive fungal infections, the major drawback is high false-positive rates among
hospitalized patients which are attributed to bacteremia, hemodialysis with cellulose membranes, human blood products (albumin, plasma or immunoglobulin), certain ß-lactam antibiotics, colonization of Candida or mold without infection, some
drugs and surgical dressings containing glucan and severe mucositis (Racil etal.
2010; Hammarström etal. 2015; León etal. 2016). Moreover, Alcaligenes faecalis,
Streptococcus pneumoniae, and Pseudomonas aeruginosa showed BDG reactivity
with the Fungitell assay and could also contribute to false-positive results (MenninkKersten etal. 2008). The NPV of BDG assays is very high (90–99.8%), reecting a
high sensitivity that can be used to exclude invasive fungal infections in critical care
patients. It provides fast results in a cost-effective and noninvasive way.
1.3.1.3 C. albicans Germ Tube Antibody Assay (CAGTA)
The CAGTA test detects antibody response developed against a hyphal protein
(Hwp1) expressed during biolm formation and tissue invasion by Candida species
(Martínez-Jiménez etal. 2014). In addition to C. albicans, this test was evaluated
for other Candida species, including C. tropicalis, C. parapsilosis, C. krusei,
C. guilliermondii, C. glabrata and C. dubliniensis (Moragues etal. 2004; Martín-
Mazuelos etal. 2015). Invasive candidiasis CAGTA IFA IgG (Vircell Microbiologist
S.L., Granada, Spain) is a commercially available indirect immunouorescence kit
that detects IgG antibodies in human plasma or serum produced against antigens
located on the cell wall surface of the mycelium of Candida species. This test was
shown to be useful for the diagnosis of invasive candidiasis in ICU (intensive care
unit) patients in combination with other biomarkers (Martín-Mazuelos etal. 2015;
León etal. 2016). However, this laborious test was later adapted to an automatic
chemiluminescence assay, the invasive candidiasis VirClia IgG Monotest (Vircell
Microbiologist S.L., Granada, Spain) with ready-to-use reagents for faster results
(Parra-Sánchez etal. 2017). For invasive candidiasis, the sensitivity and specicity