Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5219_Библиотеки_им_академика_М_И_Перельмана.pdf
16
S. Banik
of the CAGTA IFA IgG assay range from 51% to 69% and 75% to 80%, respectively, whereas VirClia IgG Monotest showed 76% and 75% of sensitivity and specicity, respectively (Parra-Sánchez etal. 2017; Pini etal. 2019). In a prospective
study in 179 nonneutropenic critically ill patients, the overall agreement between
these two assays was 85.3%. However, VirClia IgG Monotest was better than
CAGTA IFA IgG in detecting C. albicans and C. parapsilosis. Overall, the automated VirClia assay was reliable, rapid, and easy to perform and showed better
sensitivity, specicity, and negative predictive values (NPV) than the CAGTA assay
(Parra-Sánchez etal. 2017).
There are few concerns about these serologic tests. They lack sensitivity among
high-risk immunocompromised patients and have shown different sensitivities for
different species of Candida, especially C. krusei and C. parapsilosis. They are
limited by low concentration of antibodies in serum and rapid clearance of mannan
antigen from the bloodstream. These tests have limited specicity and a positive
antibody response may not distinguish candidemia from heavy colonization. Despite
these limitations, serological tests are rapid, inexpensive, and minimally invasive
and performed well in patients with neutrophil and cell-mediated immune deciency.
The nonculture-based diagnostic tests have varying degrees of sensitivity but
offer better sensitivity when used in combination. For instance, positive CAGTA
and BDG tests in a single blood sample or positive BDG tests in two consecutive
blood samples could differentiate invasive candidiasis from colonization in critically ill patients (León etal. 2016). Additionally, combination of BDG and CAGTA
or mannan antigen and CAGTA tests had shown very high NPV and could be used
for discontinuation of empirical antifungal treatment in patients with suspected
invasive candidiasis or candidemia with no negative impact on outcome (MartínezJiménez etal. 2015). However, due to their own limitations, nonculture-based diagnostic tests should be used as an adjunct to cultures.
1.4 Nucleic Acid-Based Detection
1.4.1 Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) and its various derivatives are widely used molecular techniques for the diagnosis of Candida infections. These methods allow for
the rapid, sensitive, and specic detection of Candida DNA directly in clinical samples. Some commercial kits are blood-culture dependent while some are bloodculture independent. Some commercially available blood-culture independent kits
are listed in Table1.2. PCR-based methods have been established as an alternative
and faster diagnostic tool to culture. A variety of PCR-based assays targeting different genes (internal transcribed spacer regions, different subunits of ribosomal DNA,
ribosomal RNA, and mitochondrial DNA) have been developed for fungal detection
in samples including blood, serum, plasma, urine, BAL, and tissues. These assays
target either conserved or species-specic regions of the fungal DNA.Subunits of
ribosomal DNA (rDNA) contain both highly conserved and variable regions,
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
Manufacturer
Molzym
Molecular
Diagnostics,
Bremen,
Germany
Ingenetix,
Vienna,
Gene
PCR type
Total
assay
sterile body
uids, tissues,
target Sample types
and plex
time
swabs
Austria
sterile body
uids, tissues,
parafn-
embedded
Valentin,
tissues, BAL
Whole blood CubeDx, St.
28S
rDNA
microarray
6h PCR;
Austria
Unknown Whole blood Seegene,
Real-time;
6h
Seoul, South
Korea
Ingenetix,
ITS2 Whole blood,
multiplex
including
DNA
extraction
PCR;
2h
Vienna,
aspirates,
multiplex
excluding
Austria
cerebrospinal
uids, tissue,
parafn-
embedded tissue,
DNA
extraction
17
(continued)
BAL
Candida species identied
Assays
Table 1.2 Blood-culture independent commercial PCR assays
Sepsi Test™ UMD Pan-Candida 24h PCR 18S Rrna Whole blood,
MycoReal Fungi Pan-Candida 24h PCR ITS2 Whole blood,
Hybcell Pathogens DNAxB C. albicans, C. parapsilosis, C. tropicalis, C.
glabrata, C. dubliniensis
glabrata, C. krusei
Magicplex™ Sepsis C. albicans, C. parapsilosis, C. tropicalis, C.
MycoReal Candida C. albicans, C. parapsilosis, C. tropicalis, C.
glabrata, C. dubliniensis, C. krusei, C.
lusitaniae
18
Manufacturer
Bruker
Daltonics,
Bremen,
Germany
Bruker
Daltonics,
Bremen,
Germany
Olm
Diagnostics,
Newcastle
upon Tyne,
England
Olm
Diagnostics,
Newcastle
upon Tyne,
England
Daltonics,
Bremen,
Germany
S. Banik
Gene
PCR type
Total
assay
plasma, serum
target Sample types
Unknown Whole blood,
and plex
Real-time;
multiplex
time
excluding
DNA
plasma, serum
Unknown Whole blood,
Real-time;
multiplex
extraction
<2h
excluding
DNA
extraction
plasma, serum,
BAL
Unknown Fungal culture,
qPCR;
multiplex
45min
excluding
DNA
extraction
Fungal culture,
blood
28S
rDNA
qPCR;
uniplex
excluding
DNA
Swabs Bruker
Mating
Real-time;
extraction
locus
alpha
uniplex
excluding
DNA
extraction
Table 1.2 (continued)
Candida species identied
Assays
Fungiplex® Universal Candida species <2h
Fungiplex® Candida C. albicans, C. parapsilosis, C. tropicalis, C.
glabrata, C. dubliniensis, C. krusei
glabrata, C. dubliniensis, C. krusei
CandID® C. albicans, C. parapsilosis, C. tropicalis, C.
AurisID® C. auris 45min
Fungiplex® Candida auris C. auris <2h
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
19
making them the most widely used targets for fungal PCR-based assays. Moreover,
multiple copies of rDNA, ranging from 50 to 100, are present in fungal genomes,
making it a better target to develop a more sensitive assay than single-copy targets
(Lott etal. 1993; Hsu etal. 2003).
In a meta-analysis of 4500 patients, the PCR assay showed a sensitivity and
specicity of 95% and 92%, respectively, for the diagnosis of candidemia (Avni
etal. 2011). However, the sensitivity of PCR is lesser in patients with invasive candidiasis in the absence of candidemia (White etal. 2021). PCR-based molecular
assays are also useful for the identication of Candida strains that are nonculturable
on conventional media (Fontecha etal. 2019). Different PCR techniques have been
utilized to achieve better sensitivity and specicity for the detection of Candida in
clinical samples.
1. Real-Time PCR (qPCR): Real-time PCR allows for the detection and quanti-
cation of Candida DNA in real time during the amplication process. The amplied product can be detected by using uorescent dyes or probes that emit
uorescence upon binding to the amplied DNA and hence are used to monitor
the reaction (Arya et al. 2005). This enables the determination of the initial
amount of Candida DNA in the sample. Assays using SYBR Green or EVA
Green dye have good sensitivity by poor specicity as they can bind to any
double-stranded DNA.The use of sequence-specic TaqMan probes or molecular beacons offers better specicity. The melting temperature (Tm) of the amplied product can be used to differentiate Candida species in the same reaction.
Real-time PCR is more sensitive and rapid than both the conventional and nested
PCR and is less prone to carry-over contamination (Hsu etal. 2003).
2. Nested PCR: Nested PCR involves two rounds of amplication. In the rst
round, a target region is amplied using outer primers. Then, a second round of
PCR is performed using inner primers that target a smaller region within the rst
PCR product. Nested PCR can enhance sensitivity and specicity especially for
the detection of Candida species from blood samples and hence is instrumental
in the diagnosis of candidemia (del Negro etal. 2010; Avni et al. 2011; Taira
etal. 2014). Despite offering better sensitivity than real-time PCR, nested PCR
is time-consuming as it requires two PCR runs and postamplication steps and
prone to false-positive results.
3. Multiplex PCR: Multiplex PCR allows the simultaneous amplication of mul-
tiple targets in a single reaction using different and specic pair of primers for
each target. This approach is useful for detecting different Candida species or
multiple genetic markers within the same sample, providing comprehensive
information in a single assay and saving costs, time, and efforts. A real-time
multiplex PCR can simultaneously detect multiple pathogens using speciesspecic primers and probes tagged with different uorescent dyes for each
pathogen species. There are a large number of commercial multiplex PCR assays
available (Table 1.2) generally targeting ve pathogenic Candida species
(C. albicans, C. parapsilosis, C. glabrata, C. krusei, and C. tropicalis) which are
responsible for most invasive candidiasis (Pfaller etal. 2011). Arastehfar etal.
20
S. Banik
developed a one-step multiplex PCR that can identify nine different Candida
species (Arastehfar etal. 2019). Seminested PCR was used to increase the sensitivity of multiplex PCR in serum with 99% accuracy compared to biochemical
tests (Ahmad etal. 2002).
4. Digital PCR: Digital PCR partitions the PCR reaction into thousands of indi-
vidual reactions, each containing a single target molecule or none and detects the
amplication as real-time PCR or end-point PCR.This allows for the absolute
quantication of target DNA, providing high precision and sensitivity (Vogelstein
etal. 1999). It is highly reproducible and can detect less than ve copies of
Candida DNA in blood samples. The sensitivity of digital PCR is higher
compared to culture and real-time PCR (Chen etal. 2021). Moreover, the digital
PCR is effective in diagnosing invasive candidiasis in neonates (Li etal. 2019).
Other PCR-based methods including PCR-restriction fragment length polymorphism (PCR-RFLP), amplied fragment length polymorphism (AFLP), randomly
amplied polymorphic DNA (RAPD), and nucleic acid sequence-based amplication (NASBA) are useful for the differentiation of clinically relevant Candida species (Borst etal. 2001; Ahmad etal. 2003; Loefer etal. 2003; Ball etal. 2004;
Trost etal. 2004). PCR-based methodologies for Candida diagnosis offer high sensitivity and specicity, enabling the detection of low quantities of fungal DNA in
clinical samples. These methods are valuable for both the identication of Candida
species and the detection of antifungal resistance markers. However, the interpretation of results should be performed in conjunction with clinical information and
other laboratory ndings for accurate diagnosis and patient management. Currently,
there are no FDA-approved PCR assays for the detection of Candida.
1.4.2 Fluorescence InSitu Hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a molecular technique used for the
identication and visualization of specic DNA or RNA sequences within intact
cells or tissue samples. It is well established for the detection of Candida in clinical
specimens (Hayden etal. 2002). In the context of Candida diagnosis, FISH can be
employed to identify and distinguish Candida species directly in clinical specimens
such as blood, tissue, or other bodily uids. The samples are usually xed to preserve cellular structures and permeabilized to allow the FISH probes to penetrate
the cells. Fluorescently labeled DNA or RNA probes are designed to target and
hybridize with unique sequences within the Candida genome, allowing for the visualization of the targeted genetic material. The uorescent signals emitted by the
labeled probes are examined under a uorescence microscope to determine the
presence and location of Candida cells in the specimen.
The FISH technique provides relatively quick results compared to traditional
culture-based methods allowing for timely diagnosis. The FISH probes can be
designed to target specic Candida species or even strains, providing a high level of
specicity. Depending on the design of the assay, it may allow for the quantication
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
21
of Candida cells in a sample. Moreover, FISH can be adapted to identify multiple
Candida species within the same specimen, which is particularly useful in cases of
mixed infections.
While FISH is a powerful tool for Candida diagnosis, it is typically used as a
complementary method alongside other diagnostic techniques. It is important to
note that FISH may not provide information on antifungal susceptibility, and additional tests may be required for a comprehensive assessment of the infection.
Different FISH techniques may be employed based on the specic goals of the
diagnosis. There are some variations of FISH techniques used for Candida diagnosis. Among them, peptide nucleic acid (PNA)-FISH is most commonly used.
1.4.3 Peptide Nucleic Acid FISH (PNA-FISH)
PNA-FISH uses PNA probes, which are synthetic DNA analogs with a peptide
backbone. PNA probes have higher afnity and stability than traditional DNA
probes (Stender 2003). PNA FISH Yeast Trafc Light (OpGen Inc., Gaithersburg,
MD) is a US FDA approved and commercially available kit. It distinguishes most
common Candida species from yeast-positive blood cultures within 90min. This
method utilizes uorescently labeled probes to complement species-specic rRNA
sequences (Fig.1.1). In this method, C. albicans and C. parapsilosis produce green
uorescence, C. glabrata and C. krusei show red uorescence while C. tropicalis
Fig. 1.1 A schematic drawing of the labeling process with the peptide nucleic acid uorescence
in situ hybridization (PNA FISH®) probe (reprinted from (Arafa etal. 2023)) under permission of
Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/
by/4.0/)
22
produces yellow uorescence. As a result, this assay cannot differentiate C. albicans from C. parapsilosis and C. glabrata from C. krusei (Radic et al. 2016).
However, in a study with more than 200 blood culture samples, this assay showed
the correct result in 96% of cases (Klingspor etal. 2018).
The choice of the FISH technique depends on the specic objectives of the diagnostic assay, such as rapid identication, quantication, or the detection of specic Candida
species. Additionally, factors such as probe design, specicity, and the compatibility of
the FISH technique with different clinical specimens are needed to be considered.
S. Banik
1.4.4 PCR-Based Innovative Diagnosis
The FilmArray, Sepsis Flow Chip, and ePlex system are innovative diagnostic platforms that utilize molecular technologies for the detection and identication of
pathogens, including Candida, in sepsis or bloodstream infection scenarios.
1.4.5 FilmArray System
The FilmArray system, developed by BioFire Diagnostics, is a fully integrated and
automated molecular diagnostics platform. It uses multiplex PCR technology to
simultaneously detect multiple pathogens in a single sample. The FilmArray Blood
Culture Identication (BCID) Panel is designed for the rapid identication of pathogens in blood cultures. It includes targets for various Candida species, allowing for
the detection and differentiation of common Candida strains. The FilmArray system
has a simple workow, with minimal hands-on time. Once a positive blood culture is
agged, a sample is prepared, loaded onto the FilmArray pouch, and the system automatically performs the nucleic acid extraction, PCR amplication, and detection.
1.4.6 Sepsis Flow Chip
The Sepsis Flow Chip (Master Diagnostica, Granada, Spain) is a microarray-based
diagnostic assay designed for the simultaneous identication of pathogens causing
bloodstream infections and their key antibiotic resistance markers from positive
blood cultures in 3h. It uses multiplex PCR amplication with biotinylated primers
followed by automated reverse hybridization to a chip membrane containing probes
and immunoenzymatic detection of positive signals. It can identify 40 bloodstream
infection-causing pathogens including Candida. It has sensitivity and specicity of
93% and 100%, respectively, regarding Candida species (Galiana etal. 2017).
1.4.7 ePlex System
The ePlex system, developed by GenMark Diagnostics, Carlsbad, CA, USA, is an
automated multiplex PCR-based system designed for the analysis of positive blood
cultures. It integrates nucleic acid extraction, amplication, and electrochemical
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
23
detection in a closed system. The FDA-approved ePlex BCID system has a choice
of three panels. The fungal pathogen panel targets 11 different Candida species
including C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. guilliermondii,
C. tropicalis, C. parapsilosis, C. auris, C. lusitaniae, C. kefyrand C. famata as well
as few other fungal pathogens. It provides results within 90min and has shown
specicity and sensitivity of almost 100% for fungal pathogens (Zhang etal. 2020).
The combination of blood culture and ePlex has a rapid turnaround time, making it
suitable for timely and accurate diagnosis and providing better clinical management
of patients with bloodstream infections (Huang etal. 2019).
1.4.8 The T2 Candida Assay
The T2 Candida (T2 Biosystems, Inc., Wilmington, MA, USA) is a nonculturebased qualitative assay for the diagnosis of candidemia and is the only test approved
by the FDA.The assay relies on both the magnetic resonance and PCR amplication to detect the presence of Candida in blood samples. It can identify the ve most
prevalent Candida species (C. albicans, C. tropicalis, C. parapsilosis, C. glabrata,
and C. krusei) which account for >95% of total candidemia cases directly from
whole blood within 4h. Unlike traditional blood culture methods that require days
for pathogen growth, the T2MR assay enables the direct detection of Candida DNA
in whole blood without the need for amplication or culture steps (Neely etal.
2013; Pfaller etal. 2016). The new T2 C. auris panel is 100-fold more sensitive
when compared to available diagnostic tests for C. auris with a detection limit of
5CFU/ml (Kordalewska etal. 2017; Sexton etal. 2018). Moreover, the T2 C. auris
panel has shown signicant advantage compared to culture in detecting C. auris
which has been recognized by the CDC as a serious global health threat due to its
resistance to major classes of antifungals.
The T2 Candida assay uses superparamagnetic nanoparticles and magnetic resonance signal amplication to achieve high sensitivity. Magnetic nanoparticles are
coated with target-specic probes that bind to Candida DNA.When the target DNA
is present in the sample, the nanoparticles become bound to it. The T2MR technology allows for the amplication of the magnetic resonance signal generated by the
bound nanoparticles. This amplication enhances the detection sensitivity of the
assay (Neely etal. 2013). The EDTA tubes containing blood sample (2–4mL) are
inserted directly into the automated T2Dx instrument (T2 Biosystems, Inc.,
Wilmington, MA, USA) which lyses the Candida cells and amplies the DNA.The
magnetic nanoparticles with target-specic probes bind to the target DNA, and this
nanoparticle clustering changes the T2 relaxation time which is then analyzed by a
magnetic resonance instrument (T2MR). The magnetic resonance signal is measured, and the presence of Candida DNA is detected based on the signal generated
by the bound nanoparticles.
The T2 Candidaoffers a rapid turnaround time with a signicantly faster diagnosis compared to traditional blood culture methods, which may take days to provide
results. It is a highly sensitive assay as it can detect as low as 1CFU/ml of Candida
sp. in blood, enabling the detection of low fungal burdens (Pfaller etal. 2016). The
assay does not require blood culture or amplication steps, providing a direct and
24
rapid detection of Candida pathogens in whole blood. The rapid results facilitate an
early initiation of targeted antifungal therapy, which can be crucial in reducing the
complications associated with invasive candidiasis and therefore in improving
patient outcomes (Monday etal. 2021).
S. Banik
1.5 Rapid Identification Systems
The conventional methods used in the identication of fungal pathogens are timeconsuming. The global increased incidence of fungal infections has prompted the
development of rapid and commercial identication systems. These systems should
offer the following advantages: (Turner and Butler 2014) rapid and accurate identication from various types of clinical specimens, (Sardi etal. 2013) user-friendly
and fast processing of samples, and (Deorukhkar and Saini 2014) ability to identify
unique and rare isolates. Several biochemical identication systems are commercially available for the identication of fungal isolates. These systems rely on the
ability of fungi to assimilate different carbohydrates and organic acids, antifungals,
and substrates for specic enzymes. All these different biochemical tests are organized in different microwells in a card, read by the system and compared with a
reference database. ID 32C system (bioMérieux, Marcy l’Etoile, France) is a commercial kit commonly used in European countries. On the other hand, API 20C
yeast identication system (bioMérieux Vitek, Inc., Hazelwood, MO) and the
VITEK automated system ((bioMérieux Vitek, Inc., Hazelwood, MO) are widely
used in the USA.Some manual and automated commercial identication systems
are listed (Tables 1.3 and 1.4) and some are discussed in the following section.
1.5.1 Manual Rapid Identification System
1.5.1.1 The API System
The API system or Analytical Prole Index system (bioMerieux Vitek, Inc.,
Hazelwood, MO, USA) is a commercial system designed for the identication of
microorganisms, including fungi. It is a standardized system that relies on the observation of various biochemical reactions to identify and differentiate microorganisms. The API system consists of a series of microtubes or wells, each containing
specic substrates or chemicals that support the growth of microorganisms and
induce characteristic metabolic reactions. As Candida grows in the medium, it
metabolizes these substrates, leading to observable changes in the appearance of the
medium. Changes in color, turbidity, gas production, or other visible indicators are
recorded and compared to a database or interpretation chart provided by the manufacturer (Fenn etal. 1994). The pattern of reactions is used to identify the Candida
species. Each species has a unique biochemical prole, and the API system provides
a code or prole number that corresponds to the identity of the microorganism. In
some cases, additional tests may be required to conrm the identication obtained
through the API system. Molecular methods such as PCR or other specialized tests
1 Diagnostics of Candida and Candidiasis: Current Methods and Future…
Table 1.3 Manual commercial identication kits
Accuracy
Incubation
Number and type
Kit
API 20C
AUX
API
Candida
Auxacolor 13 carbohydrate
Fungichrom 7 carbohydrate
Fungifast 6 carbohydrate
RapID
Yeast Plus
Uni-YeastTek
Adopted and modied from Pincus etal. (2007)
of tests
19 carbon
assimilation
5 carbohydrate
assimilation, 6
glycosidase, 1
urease
assimilation, 1
phenoloxidase, 1
cycloheximide
resistance
assimilation, 4
aminopeptidase, 2
glycosidase, 1
phenoloxidase, 1
urease, 1
cycloheximide
resistance
assimilation, 1
aminopeptidase, 1
glycosidase, 1
phenoloxidase, 1
urease
5 carbohydrate
assimilation, 3
aminopeptidase, 6
glycosidase, 1
urease, 3
miscellaneous
enzymes
7 carbohydrate
assimilation, 1
urease, 1 KNO3, 1
cornmeal agar
temperature
(°C)
30 48–72 93–100 bioMerieux
37 24–48 68–97.4 bioMerieux
30 24–48 63.8–95.2 Bio-Rad,
30 24–48 65–96 International
37 24–48 65–98 International
30 4–6 84–99 Thermo
22–30 2-7 87–99.8 Thermo
Incubation
time (h)
compared to
conventional
method (%)
25
Manufacturer
Hercules, CA
Microbio,
Signes,
France
Microbio,
Signes,
France
Fisher
Scientic,
Waltham, MA
Fisher
Scientic,
Waltham, MA
may be employed for further conrmation, especially when dealing with closely
related Candida species (Pincus etal. 2007).
The API system is advantageous for its simplicity, standardization, and ability to
rapidly identify a wide range of microorganisms. However, it is important to note
that while the API system can accurately identify many common Candida species,
molecular methods may be required for precise identication, especially in cases
involving nonalbicans Candida species or atypical strains. Healthcare professionals