Download The future of novel diagnostics in medical mycology

Journal of Medical Microbiology (2015), 64, 315–322
Review
DOI 10.1099/jmm.0.082297-0
The future of novel diagnostics in medical mycology
Fernando Teles1,2 and Jorge Seixas2,3
Correspondence
Fernando Teles
[email protected]
1
Mycology Group/Unit of Medical Microbiology, Institute of Hygiene and Tropical Medicine,
Universidade Nova de Lisboa, 100 Rua da Junqueira, 1349-008 Lisbon, Portugal
2
Centre for Malaria and Other Tropical Diseases, Institute of Hygiene and Tropical Medicine,
Universidade Nova de Lisboa, 100 Rua da Junqueira, 1349-008 Lisbon, Portugal
3
Tropical Clinic Unit, Institute of Hygiene and Tropical Medicine (IHMT),
Universidade Nova de Lisboa, 100 Rua da Junqueira, 1349-008 Lisbon, Portugal
Received 8 August 2014
Accepted 17 November 2014
Several fungal diseases have become serious threats to human health and life, especially upon the
advent of human immunodeficiency virus/AIDS epidemics and of other typical immunosuppressive
conditions of modern life. Accordingly, the burden posed by these diseases and, concurrently, by
intensive therapeutic regimens against these diseases has increased worldwide. Existing and
available rapid tests for point-of-care diagnosis of important fungal diseases could enable the
limitations of current laboratory methods for detection and identification of medically important
fungi to be surpassed, both in low-income countries and for first-line diagnosis (screening) in
richer countries. As with conventional diagnostic methods and devices, former
immunodiagnostics have been challenged by molecular biology-based platforms, as a way to
enhance the sensitivity and shorten the assay time, thus enabling early and more accurate
diagnosis. Most of these tests have been developed in-house, without adequate validation and
standardization. Another challenge has been the DNA extraction step, which is especially critical
when dealing with fungi. In this paper, we have identified three major research trends in this field:
(1) the application of newer biorecognition techniques, often applied in analytical chemistry;
(2) the development of new materials with improved physico-chemical properties; and (3) novel
bioanalytical platforms, allowing fully automated testing. Keeping up to date with the fast
technological advances registered in this field, primarily at the proof-of-concept level, is essential
for wise assessment of those that are likely to be more cost effective and, as already observed for
bacterial and viral pathogens, may provide leverage to the current tepid developmental status of
novel and improved diagnostics for medical mycology.
Introduction
The development of novel methods and devices for detection,
identification and quantification of human pathogens is
witnessing unprecedented advances, mainly as a result of the
combination of established molecular biology principles
with the recent technological developments in new materials and platforms for biosensing. This has resulted, in
particular, from the consolidation of the microfluidics and
lab-on-a-chip concepts as suitable platforms for bioassays
and of nanoengineered structures with enhanced physicochemical properties for ultrasensitive and specific diagnostics (Fortina et al., 2007; Lee et al., 2010). Biorecognition of
bacteria and viruses causative of important human diseases
has greatly benefited from such advances, but this has not
been so for pathologies of fungal aetiology. Several reasons
for this can be pointed out, concerning either the inherent
Abbreviations: HIV, human immunodeficiency virus; IA, invasive
aspergillosis; POC, point of care.
082297 G 2015 The Authors
biological characteristics of fungi or the peculiar clinical and
epidemiological features of mycotic diseases. However, the
human immunodeficiency virus (HIV)/AIDS epidemic and
the increased incidence of other immunosuppressive factors,
such as chemotherapy, organ transplantation and new
immunological treatments for autoimmune diseases, have
increased the prevalence and severity of opportunistic
mycoses in the past few decades. This is an obvious burden
for management by the health systems of low-resource
countries. In parallel, developed countries have faced the
emergence of antifungal drug resistance arising from
intensive chemotherapeutic and immunological treatments
and regimens (WHO, 2014). Under this scenario, there is
increased awareness of the limitations of current diagnostic
procedures and, accordingly, of the need for novel methods,
able to precociously and accurately detect and identify
the major human fungal pathogen species in a fast, timely
and less-invasive manner. This paper briefly overviews
the current status and limitations of diagnosis in medical
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F. Teles and J. Seixas
mycology, whilst pointing out some expected trends in this
field.
The importance of diagnosis
Fungal species of Candida, Aspergillus and Cryptococcus are
major aetiological agents of invasive infections in immunocompromised patients. The global mortality rate due to
invasive aspergillosis (IA) has increased by more than
350 % in the past 25 years. This disease has become a
leading cause of death in 60–90 % of immunodepressed
individuals (McNeil et al., 2001). Around 1 million new
cases of cryptococcal meningitis occur each year, killing
more than half a million HIV-infected persons in subSaharan Africa and in Southeast Asia every year, which
corresponds to up to 90 % of all yearly deaths in the
world due to cryptococcal meningitis (Park et al., 2009).
Concerning Candida, systemic and mucosal infections
caused by these species have remained high since the 1990s.
Importantly, the current status of research in medical mycology allows all fungi to be considered potentially harmful
in severely immunocompromised hosts (Alexander &
Pfaller, 2006). The burden of these diseases is milder in
richer countries, with wider access to the highly active
antiretroviral therapy, but worrisome in poorer countries
(Teles, 2013). In richer countries, the intensive and increased
use of chemotherapeutic regimens and of new immunological treatments for malignancy, transplantation and
autoimmune disease management has led to aggressive
and often fatal fungal diseases. As such, early and accurate
disease diagnosis is crucial for prompt beginning of therapy,
thus avoiding the long-term complications and high death
rates currently observed, but also for rapid shifting of
therapeutics once drug-resistant strains are detected,
especially in cases of chronic infections (Teles & Martins,
2011). Ironically, as the variety and efficacy of antifungal
drugs increase, the need for accurate and rapid diagnosis
increases, in order to preclude inadequate presumptive
antifungal therapy. This renders not only less toxic effects,
but also better prognosis, due to decreased drug resistance.
In parallel, the continuous description of new fungal species
poses additional challenges to existing detection and
identification methods, especially when such species exhibit
different virulence or antifungal susceptibility patterns.
Limitations of conventional laboratory diagnosis
Understanding the current situation of the development of
point-of-care (POC) diagnostic tests in medical mycology
requires prior comprehension of the limitations of conventional diagnosis and hence of challenges in the field.
Culture and histopathology of infected tissues have been
the most traditional diagnostic methods for most mycoses,
but obtaining biopsies from sterile body sites is a highly
invasive approach, a serious handicap, especially in severely
ill patients. Plus, a positive culture from a sterile site may
indicate transient colonization and not true infection,
especially for opportunistic fungi. Culture followed by
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microscopic examination requires skilled manpower and
long assay times for cell culturing, when in vitro growth is
feasible. New culture media, lysis centrifugation and
automated blood culture systems have decreased the assay
time (Chandrasekar, 2010), particularly for detection
of Candida spp., but without definitely improving the
sensitivity of the detection (Archibald et al., 2000;
Chandrasekar, 2010). Direct examination, whenever possible, is simpler, faster and cheaper than culture, but a
negative test does not definitely exclude a fungal infection.
When infected tissue is available, histopathological diagnosis is a common approach, but it lacks sensitivity
and selectivity, as several filamentous fungi may exhibit
undistinguishable morphologies. This is particularly troublesome when distinguishing Aspergillus from related fungi
requiring different therapeutics (Chandrasekar, 2010).
Serological techniques may be targeted to detect fungal
antigens or antibodies in human serum through immunological or biochemical reactions. For antigen detection, tests
have been used to target two cell-wall polysaccharides:
(1A3)-b-D-glucan and galactomannan. The first has been
used to diagnose invasive infections caused by several
opportunistic fungi (Odabasi et al., 2004). It is especially
useful for presumptive diagnosis (screening), envisaging
early beginning of antifungal therapy, but not for
fungal identification (Alexander & Pfaller, 2006). Given the
ubiquitous presence of glucan in the environment, this test is
prone to false-positive results (Chandrasekar, 2010).
Moreover, it is not useful for detection of Cryptococcus and
Mucorales spp., because the cell walls of these aetiological
fungi have too low amounts of this biomarker (Miyazaki
et al., 1995). The second test has been applied to the diagnosis
of IA (Mennink-Kersten et al., 2004). Yet, its sensitivity and
accuracy may decrease under antifungal drug usage, which
causes less galactomannan to be released from fungal cell
walls (Maertens et al., 2001). More specific serological tests,
such as those for cryptococcal meningitis (Vilchez et al.,
2002) and for disseminated histoplasmosis (Wheat et al.,
2002), have also been employed. Antibody detection tests
usually lack sensitivity in cases of immunocompromised
patients owing to the low levels of circulating antibodies
(Teles & Martins, 2011). Plus, diagnosis of an acute infection
requires paired sera (Alexander & Pfaller, 2006). A general
drawback of immunoassays is potential cross-reactivity
between different circulating antibodies and antigens and
hence low selectivity. The reliability of antibody assays for
diagnosis of invasive candidiasis is particularly limited; the
sensitivity is usually low for immunocompromised patients
(which are at higher risk for infection), and the same is true
for the specificity, as Candida spp. are ubiquitous in the
normal human flora (Arvanitis et al., 2014).
The advent of molecular biology-based diagnosis
The growing technical developments in nucleic acid-based
molecular diagnostic methods, especially PCR-based techniques for genome amplification and genome sequencing,
as well as their increasing cost-effectiveness, have improved
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Journal of Medical Microbiology 64
Rapid diagnostic tests for medical mycology
the reliability and performance of these techniques in
mycological diagnosis, as recently reviewed by Teles (2013),
with particular descriptions for the major human fungal
pathogens (Arvanitis et al., 2014; Gómez, 2014). Such
techniques usually perform better (in terms of sensitivity
and selectivity) and are quicker than more conventional
methods, allowing earlier diagnosis. PCR has already been
reported for the diagnosis of candidaemia and of IA (Yeo &
Wong, 2002). Fungal genome amplification by PCR with
conserved (panfungal) oligonucleotide primers followed by
further amplification with species-specific primers (allowing multiplexed PCR, i.e. simultaneous detection and
identification of multiple fungal species in a single assay) or
by another molecular identification technique (e.g. sequencing or restriction endonuclease analysis) is particularly
promising for mycological diagnosis (Hsiao et al., 2005;
Iwen et al., 2002). Compared with more conventional
methods, nucleic acid-based bioassays usually provide
faster and more sensitive results. The exquisite ultrahigh
sensitivity of most of these methods coupled to accurate
threshold determination enables tailoring of quantitative
assays, thus differentiating true fungal infections from
simple colonization, as well as monitoring and predicting
clinical outcomes from therapeutic interventions (Zhang,
2013). Among them is real-time PCR which, coupled to
fluorescent detection, allows gains in sensitivity and
minimization of contamination, compared with conventional PCR; yet, it remains a too complex and expensive
technique for rapid and inexpensive testing. Another
promising PCR-based technique is nucleic acid sequencebased amplification, which allows isothermal direct
amplification of RNA from organisms with RNA genomes,
and has already proven useful for diagnosing IA (Loeffler
et al., 2001). PCR amplification of fungal genetic markers
has been at the core of DNA barcoding-based fungal
identification and classification. However, continuous
changes in fungal taxonomy and the lack of existence of
a widely conserved region in fungal genomes at the species
and genus levels have delayed wider developments on
molecular diagnostics for fungi. Ribosomal genes particularly have been used as barcodes for fungi, as they contain
sequences common to all fungi, able to be used for fungal
infection screening, but also variable and highly variable
domains, such as the internal transcribed spacer (ITS)
region, which is suitable for species identification (Yeo
& Wong, 2002). ITS regions provide high taxonomic
resolution and are widely characterized in popular
sequence databases, such as GenBank (Balajee et al.,
2009). Recently, a study from an international research
consortium proposed ITS as the primary fungal barcode
marker (Schoch et al., 2012). Higher performance in
species discrimination can be achieved by using additional
sets of primers targeting other fungal genome markers.
Overall, this constitutes a first step towards future
development of standardized molecular methods for fungal
diagnosis. In this regard, it is also expected that the
development of more reliable databases and of sequencing
techniques may consolidate molecular methods (for which
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there is currently no formal approval by regulatory entities
for fungal diagnosis) as valuable alternatives to conventional methods for fungal identification (Alexander &
Pfaller, 2006). Three alternative options, development of
a consensus protocols by an ad hoc expert working
group, centralization of molecular testing within a certified
reference laboratory or commercialization via production
of quality-controlled diagnostic kits and regents, have been
proposed for standardization of molecular methods for
fungal diagnosis (Zhang, 2013). A fundamental obstacle to
molecular diagnostics for fungi is that fungal cells are
usually delimited by thick walls that constitute challenging
physical barriers for isolation of trace amounts of nucleic
acids. In the future, full packaging and automation of
the nucleic acid extraction step will likely contribute
to overcoming this problem, as well as to minimizing
contamination of the biological sample. Such automated
methods and devices would avoid the need for bulky clean
rooms and achieve higher performances than manual
extraction methods (Kessler et al., 2001).
Rapid tests for POC diagnosis
For successful POC and field applications, especially in
remote and resource-depleted regions where appropriate
diagnostic facilities, equipment and trained manpower
are often lacking, diagnostic devices should accomplish
the ASSURED criteria, as defined by the World Health
Organization Sexually Transmitted Diseases Diagnostics
Initiative: they must be affordable, sensitive, specific, userfriendly, rapid, robust, equipment-free and deliverable
to end-users (Peeling et al., 2006). Rapidity, in particular,
means that fewer samples will need to be sent to laboratories for analysis, allowing improved decision-making.
Indeed, the importance of the concept of rapidity and its
inherent relationship with the other parameters is responsible for the term ‘rapid (diagnostic) tests’ usually assigned
to diagnostics meeting these criteria. Commercial diagnostic kits for detection and identification of several
clinically important fungi have been available for several
years, such as the AccuProbe tests (from former GenProbe). Their development and commercialization have
greatly benefited from the advances in molecular methods
for fungal detection and identification, particularly in
the case of dimorphic fungi (Alexander & Pfaller, 2006).
However, they are essentially laboratory-based techniques
rather than targeted at POC; in general, they do not fully
comply with the ASSURED criteria, because they require
labour-intensive sample pre-treatment and long assay
times, as fungal growth in pure cultures is usually required
(Teles & Martins, 2011). For Candida species identification, for instance, such tests are relatively expensive
and laborious, requiring incubations longer than 2 days
(Marot-Leblond et al., 2004). Indeed, sample pre-treatment
has traditionally been disregarded in the development of
diagnostic tests, especially in the case of mycelia-forming
fungi. Hence, their usefulness as true POC tests remains
limited. The advent of lateral-flow tests, usually in the form
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F. Teles and J. Seixas
of strips for qualitative detection or antigens or antibodies
(immunochromatography), constitutes a successful paradigm in the conception of rapid tests. They have been
widely developed, commercialized and used for specific
diagnosis of many human pathogens. The decentralization
paradigm of disease diagnosis (related with the concepts of
POC and in situ testing), allied to high-throughput and
multiplexing abilities of novel tests, is opening an appetizing
commercial market even for the traditionally unattractive
area of medical mycology. Yet, the old paradigm of bulky,
complex and expensive analytical apparatus is still far from
perishing. Rapid tests, rather than true competitors, are still,
in essence, complementary in their application ranges and
contexts. They have found particular market niches in POC
applications, for decentralized and in situ diagnosis (by nonmedical healthcare staff or, ultimately, at the bedside level).
In particular, disease screening has been perhaps the main
specific context for their extensive application worldwide, as
first-line diagnostic tools useful for screening for positive
results to be further confirmed with conventional techniques
in central laboratories. Some successful examples of rapid
diagnostic tests for the major human fungal pathogens using
lateral-flow supports applied to clinical samples have been
reported in recent years, as described in Table 1. The test
developed to differentiate between Candida albicans and
Candida dubliniensis (Marot-Leblond et al., 2004) employed
a sandwich system with two immobilized mAbs, one of them
binding epitopes specifically expressed by the two Candida
species, and the other binding only Candida albicans. This
test precludes the usual need for complex molecular biology
techniques, although important differences in performance
were observed depending on the isolation medium used.
For Cryptococcus neoformans, two tests were reported, able
to detect the four main cryptococcal serotypes. The kit
commercialized by Immuno-Mycologics (IMMY), which
employs mAbs against capsular polysaccharide antigens of
the Candida neoformans/Candida gattii species complex,
allows the use not only of blood, but also of urine, an ideal,
non-invasive and readily available specimen to use in POC
situations, although the limited sensitivity observed in this
fluid is probably a result of low fungal burden due to
excretion (Lindsley et al., 2011). The other, more recent test
also employs mAbs, against the cryptococcal capsular
polysaccharide glucuronoxylomannan; it is also tailored
for using both blood and urine, but its evaluation was
performed with a relatively small sample size, thus requiring
further validation in larger studies. Moreover, its selectivity
was not assessed (Jarvis et al., 2011). For detection of
Aspergillus spp., the first reported lateral-flow device for
human serum employed the mAb JF5, which specifically
targets an extracellular glycoprotein of Aspergillus spp.
(Thornton, 2008). The use of this mAb avoids the proneness
to the false-positive and false-negative results characteristic
of common immunoassays targeting galactomannan with
the mAb EB-A2. Of note, it also enables discrimination of
active invasive growth from quiescent spore production, a
major issue in cases of IA. However, the sensitivity of
this test decreased almost 30 times when passing from
protein-depleted to protein-rich serum. Comparison of
the performance of this test with that of conventional
immunoassays has been troublesome since each assay uses
different species of immobilized antibodies and targets
different Aspergillus antigens. The second, more recent, test
makes use of the IgM mAb476, able to detect galactomannan-like antigens from Aspergillus and other moulds in urine
(Dufresne et al., 2012). Patients with IA were successfully
tested, but the cohort was too small to assess meaningful
performance characteristics. The study also reported low
amounts of these antigens in urine and the inhibitory effect
of this fluid, thus decreasing the analytical sensitivity of the
test. The suggestion of sample concentration and further
desalting/dialysis was raised, highlighting the need for
processing complex biological media prior to pathogen
detection. All the above-mentioned tests employed immunological reactions as the basis for the biorecognition events,
thus suffering from the general and well-known limitations
of immunodiagnosis.
New technologies for diagnostics
Most current methods available for medical diagnosis of
human mycosis still suffer from a high degree of invasiveness, relatively long assay times and variable performance
(in terms of sensitivity and specificity). Understandably, this
constitutes a general driving force for the development of
improved diagnostic techniques. Integration, automation,
miniaturization and packaging of the entire biorecognition
process are the essential underlying concepts driving the
development of advanced diagnostic devices. Accordingly,
techniques and signal transducers originally applied in
Table 1. Immunochromatographic tests reported in the literature for diagnosis of medically important fungi
Target fungus
Candida albicans, Candida dubliniensis
Aspergillus spp.
Aspergillus spp.
Cryptococcus neoformans
Cryptococcus spp.
Assay time
Reported LOD or sensitivity
Reference
2.5 h
15 min
–
10 min
5–15 min
91.4–96.6 % (C. albicans), 98.3 % (C. dubliniensis)
37 ng ml21
–
¢5 ng ml21
100 % (serum), 70.7–92 % (urine)
Marot-Leblond et al. (2004)
Thornton (2008)
Dufresne et al. (2012)
Jarvis et al. (2011)
Lindsley et al. (2011)
LOD, limit of detection.
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conventional analytical chemistry have been reported for
detection and identification of fungi; some of these, with
examples from the literature, include spectrophotometry for
Paracoccidioides brasiliensis (Martins et al., 2012), fluorescence in situ hybridization for Aspergillus and Candida
(Rickerts et al., 2011), matrix-assisted laser desorptionionization time of flight (MALDI-TOF) for Candida
(Lacroix et al., 2014), Aspergillus, Fusarium, Mucorales (De
Carolis et al., 2012) and Cryptococcus spp. (McTaggart
et al., 2011), microgravimetry for Aspergillus niger and
Saccharomyces cerevisiae (as non-pathogenic models of,
respectively, A. fumigatus and C. albicans) (Nugaeva et al.,
2005), and electrochemical techniques for C. albicans
(Mulero et al., 2009; Villamizar et al., 2009). Among them,
MALDI-TOF is undoubtedly a step ahead, given its wide use
worldwide for diagnosis of fungal and other microbial
infections, especially after multicentre clinical trials (Spanu
et al., 2012) and regulatory approval of the first of these
systems by the US Food and Drug Administration for
clinical use (Arvanitis et al., 2014). Another major research
trend in this regard has been the development of cheaper
materials for the construction of analytical platforms, thus
making disposability (crucial when dealing with infectious
agents) economically feasible. In this context, many
innovations have arisen from three emerging technological
fields. The first field is nanotechnology, through the
generation of nanostructures enabling improved sensitivity
and specificity, as a result of the unusual or enhanced
physico-chemical properties of nanosize materials, mostly
derived from the very high surface-to-volume ratio of these
structures. In bioassays, nanoparticles (NPs) may act as
immobilization supports for biomolecules, as labels for
signal amplification or even as bioprobes for specific
recognition of biological targets (Fortina et al., 2007).
Among NPs, some of the most important and already used
in bioassays for bacterial or viral detection include Au-NPs,
quantum dots, molecular beacons, DNA dendrimers, carbon
nanotubes, liposomes, nanowires and peptide nucleic acids
(Algar et al., 2009; Pumera et al., 2007); some of these have
also been tested and applied for detection of pathogenic
fungi, but only rarely in clinical samples (Table 2). The
second field is micro/nanofabrication, commonly inspired
by the methods used in the silicon planar circuitry industry,
potentially rendering mass production of inexpensive and
very reproducible diagnostic microdevices (biochips), but
already available through advanced techniques of nanofabrication (Fortina et al., 2007). The third field is microfluidics, consisting in small-sized platforms for fluid
propulsion through microchannels in the course of the
bioassay; ultimately, the goal is the production of lab-on-achip analytical platforms, incorporating, in packaged and
self-powered miniaturized devices, all bioanalytical steps
(from sample pre-treatment to final detection) traditionally
performed with bulky equipment in large laboratory settings
(Choi et al., 2011; Lee et al., 2010). These devices can also be
designed for inter- or even intra-specific simultaneous
analysis of many different pathogens, by taking advantage of
the increasing ability to miniaturize and build dense arrays
of different immobilized probes (multiplexing) onto lowcost and portable platforms (such as disposable cartridges).
Another remarkable advantage of these devices is the
possibility of handling and testing sample volumes several
orders of magnitude smaller than those used in conventional
diagnostic methods. Very few applications of these technologies for fungal detection have been reported so far and
even among these few, most are for C. albicans only. One of
them refers to generic detection of unknown rare blood
pathogens (including C. albicans) with a microfluidic device,
magnetic microbeads coated with an opsonin, able to
recognize and capture most blood pathogens (Cooper et al.,
2014). After this pre-concentration step, fluorescence
scanning was employed for final detection, with excellent
sensitivity (less than one yeast cell per millilitre of human
blood). The magnetic capture/concentration step did not
affect pathogen viability, thus allowing, upon detection
and subsequent antifungal susceptibility testing, rapid
diagnosis of sepsis. In another work, a real-time PCR-based
microfluidic platform, from Advanced Liquid Logic, was
Table 2. Tests reported in the literature for diagnosis of medically important fungi based on nanoengineered structures
Target fungus
Assay time
Paracoccidioides brasiliensis
Candida albicans
Candida spp.
Candida albicans
A. fumigatus, Candida
glabrata, C. krusei,
Cryptococcus neoformans
Candida spp.
Aspergillus spp.
–
1h
30 min
2.5 h
A few hours
Type of
nanostructure
Au-NP
CNT
Au-NP
PNA
Au-nanowire
,3 h
NPs
A few minutes Colloidal Au and Ag
Sample
Reported LOD or
sensitivity
Reference
Fungal DNA
Fungal solution
Wastewater effluent
Blood culture
Fungal DNA
¢4 mg ml21
50 c.f.u. ml21
–
100 %
100 fM
Martins et al. (2012)
Villamizar et al. (2009)
Naja et al. (2008)
Rigby et al. (2002)
Yoo et al. (2011)
Whole blood
Exhaled air
1 c.f.u. ml21
100 %
Neely et al. (2013)
de Heer et al. (2013)
CNT, carbon nanotube; LOD, limit of detection; NP, nanoparticle; PNA, peptide nucleic acid.
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employed for detection of C. albicans DNA in human blood
(Schell et al., 2012). Unlike the first microfluidic devices,
with microchannels for fluid flow, this more versatile
second-generation layout employs the principle of electrowetting, in which fluid flow is directed by computercontrolled electrodes (digital microfluidics). This device
exhibited slightly less sensitivity than conventional microfluidics but excellent specificity (100 %) towards noncandidaemic blood samples. So far, most of these schemes
and devices have been employed only at the prototype level,
i.e. without industrial production and commercialization.
Some of these fungal bioassays have been shown to be not
only potentially cheaper than traditional diagnostic techniques (Dhiman et al., 2011), but also more advantageous
towards antifungal therapy cost savings (Forrest et al., 2006).
Although most of these technical and scientific developments have been for biorecognition of bacteria and viruses,
innovations in fungal biomolecular detection may proceed
faster than for those micro-organisms, by taking advantage
of the valuable examples and accumulated knowledge, from
the past and present, of such biological agents. Coupled to
well-established signal transduction schemes (e.g. of an
optical or electrochemical nature), new biomaterials with
improved properties and suitable platforms for cheaper,
simpler and faster biorecognition of infectious agents
promise to significantly move forward, and even to create
a breakthrough in, the current paradigm of diagnosis of
human infectious agents and of related diseases.
and related diseases are endemic. In developed and richer
countries, important systemic and opportunistic mycoses
have posed significant public health burdens, especially
due to the emergence of antifungal drug resistance in
immunocompromised patients. Even so, and taking into
account that many cases of mycotic infections do not
develop severe clinical manifestations, the research and
development of new diagnostics for fungal diseases has not
been considered a health priority, especially in times of
severe budget cuts (as nowadays), particularly in the
private sector. On the other hand, early and accurate
diagnosis remains a challenge, especially for infections
caused by filamentous fungi. Remarkably, profitable
market niches may be envisaged in the private sector.
This will likely constitute a promising driving motor to
pursue new developments in improved and economically
competitive diagnostics for human fungal diseases in the
future. Fungi have some challenging structural features for
successful biomolecular diagnosis, but it is time to learn
from the accumulated experience obtained with other
micro-organisms, especially bacteria and viruses, in order
to definitely leverage and improve on the current stagnant
status of diagnosis in medical mycology, in the context of a
growing burden posed by very complex disease conditions
and critically ill patients.
References
Alexander, B. D. & Pfaller, M. A. (2006). Contemporary tools for the
diagnosis and management of invasive mycosis. Clin Infect Dis 43
(Suppl. 1), S15–S27.
Final remarks
There is nowadays a vast array of available techniques,
analytical platforms and biomolecular probes and tags that
have already been tested, at least at the proof-of-concept
level, with a whole range and combination of characteristics and performance for detection/identification of many
human pathogens and diagnosis of related pathologies.
Hopefully, in the future, the key-point in decision-making
at the level of infectious disease diagnosis will not be
whether available diagnostic tools suitable for common
human infectious diseases will be able to reach required
performance thresholds (of sensitivity, selectivity, etc.) or
conditions (e.g. type of biological sample, time since
infection); instead, taking into account that analytical
performance usually runs in parallel with complexity and
costs, the essential issue will probably be what minimum
pathogen discriminatory ability and detection limit will be
necessary to guarantee quick and effective disease management, especially envisaging the selection of the most
appropriate therapeutic regimen for a given pathological
condition. Given the relatively low number of fungi able to
cause severe diseases in humans (compared with other
types of micro-organisms), there has been reluctance and
difficulty on the part of national health systems in funding
and promoting more widely the research, development and
implementation of novel and improved diagnostic tests for
mycological diagnosis. This is especially true for lowincome countries, where most human pathogenic fungi
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Algar, W. R., Massey, M. & Krull, U. J. (2009). The application of
quantum dots, gold nanoparticles and molecular switches to optical
nucleic-acid diagnostics. Trends Analyt Chem 28, 292–306.
Archibald, L. K., McDonald, L. C., Addison, R. M., McKnight, C.,
Byrne, T., Dobbie, H., Nwanyanwu, O., Kazembe, P., Reller, L. B. &
Jarvis, W. R. (2000). Comparison of BACTEC MYCO/F LYTIC and
WAMPOLE ISOLATOR 10 (lysis-centrifugation) systems for detection of bacteremia, mycobacteremia, and fungemia in a developing
country. J Clin Microbiol 38, 2994–2997.
Arvanitis, M., Anagnostou, T., Fuchs, B. B., Caliendo, A. M. &
Mylonakis, E. (2014). Molecular and nonmolecular diagnostic
methods for invasive fungal infections. Clin Microbiol Rev 27, 490–
526.
Balajee, S. A., Borman, A. M., Brandt, M. E., Cano, J., CuencaEstrella, M., Dannaoui, E., Guarro, J., Haase, G., Kibbler, C. C. &
other authors (2009). Sequence-based identification of Aspergillus,
Fusarium, and Mucorales species in the clinical mycology laboratory:
where are we and where should we go from here? J Clin Microbiol 47,
877–884.
Chandrasekar, P. (2010). Diagnostic challenges and recent advances
in the early management of invasive fungal infections. Eur J Haematol
84, 281–290.
Choi, S., Goryll, M., Sin, L. Y. M., Wong, P. K. & Chae, J. (2011).
Microfluidic-based biosensors toward point-of-care detection of
nucleic acids and proteins. Microfluidics Nanofluidics 10, 231–247.
Cooper, R. M., Leslie, D. C., Domansky, K., Jain, A., Yung, C., Cho, M.,
Workman, S., Super, M. & Ingber, D. E. (2014). A microdevice for
rapid optical detection of magnetically captured rare blood pathogens. Lab Chip 14, 182–188.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 00:12:15
Journal of Medical Microbiology 64
Rapid diagnostic tests for medical mycology
De Carolis, E., Posteraro, B., Lass-Flörl, C., Vella, A., Florio, A. R.,
Torelli, R., Girmenia, C., Colozza, C., Tortorano, A. M. & other authors
(2012). Species identification of Aspergillus, Fusarium and Mucorales
with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infect 18, 475–
484.
de Heer, K., van der Schee, M. P., Zwinderman, K., van den Berk, I.
A. H., Visser, C. E., van Oers, R. & Sterk, P. J. (2013). Electronic nose
technology for detection of invasive pulmonary aspergillosis in
prolonged chemotherapy-induced neutropenia: a proof-of-principle
study. J Clin Microbiol 51, 1490–1495.
Dhiman, N., Hall, L., Wohlfiel, S. L., Buckwalter, S. P. & Wengenack,
N. L. (2011). Performance and cost analysis of matrix-assisted laser
desorption ionization-time of flight mass spectrometry for routine
identification of yeast. J Clin Microbiol 49, 1614–1616.
Dufresne, S. F., Datta, K., Li, X., Dadachova, E., Staab, J. F.,
Patterson, T. F., Feldmesser, M. & Marr, K. A. (2012). Detection of
urinary excreted fungal galactomannan-like antigens for diagnosis of
invasive aspergillosis. PLoS ONE 7, e42736.
Forrest, G. N., Mankes, K., Jabra-Rizk, M. A., Weekes, E., Johnson,
J. K., Lincalis, D. P. & Venezia, R. A. (2006). Peptide nucleic acid
fluorescence in situ hybridization-based identification of Candida
albicans and its impact on mortality and antifungal therapy costs.
J Clin Microbiol 44, 3381–3383.
Fortina, P., Wang, J., Surrey, S., Park, J. Y. & Kricka, L. J. (2007).
Beyond microtechnology – nanotechnology in molecular diagnosis.
In Integrated Biochips for DNA Analysis, pp. 187–197. Edited by
R. H. Liu & A. P. Lee. New York, NY: Landes Bioscience and Springer
Science+Business Media.
Maertens, J., Verhaegen, J., Lagrou, K., Van Eldere, J. & Boogaerts,
M. (2001). Screening for circulating galactomannan as a noninvasive
diagnostic tool for invasive aspergillosis in prolonged neutropenic
patients and stem cell transplantation recipients: a prospective validation. Blood 97, 1604–1610.
Marot-Leblond, A., Grimaud, L., David, S., Sullivan, D. J., Coleman,
D. C., Ponton, J. & Robert, R. (2004). Evaluation of a rapid
immunochromatographic assay for identification of Candida albicans
and Candida dubliniensis. J Clin Microbiol 42, 4956–4960.
Martins, J. F. S., Castilho, M. L., Cardoso, M. A. G., Carreiro, A. P.,
Martin, A. A. & Raniero, L. (2012). Identification of Paracoccidioides
brasiliensis by gold nanoprobes. Proc SPIE 8219, 82190Z.
McNeil, M. M., Nash, S. L., Hajjeh, R. A., Phelan, M. A., Conn, L. A.,
Plikaytis, B. D. & Warnock, D. W. (2001). Trends in mortality due to
invasive mycotic diseases in the United States, 1980–1997. Clin Infect
Dis 33, 641–647.
McTaggart, L. R., Lei, E., Richardson, S. E., Hoang, L., Fothergill, A. &
Zhang, S. X. (2011). Rapid identification of Cryptococcus neoformans
and Cryptococcus gattii by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. J Clin Microbiol 49, 3050–3053.
Mennink-Kersten, M. A. S. H., Donnelly, J. P. & Verweij, P. E. (2004).
Detection of circulating galactomannan for the diagnosis and
management of invasive aspergillosis. Lancet Infect Dis 4, 349–357.
Miyazaki, T., Kohno, S., Mitsutake, K., Maesaki, S., Tanaka, K.,
Ishikawa, N. & Hara, K. (1995). Plasma (1A3)-b-D-glucan and fungal
antigenemia in patients with candidemia, aspergillosis, and cryptococcosis. J Clin Microbiol 33, 3115–3118.
Mulero, R., Lee, D. H., Kutzler, M. A., Jacobson, J. M. & Kim, M. J.
(2009). Ultra-fast low concentration detection of Candida pathogens
Gómez, B. L. (2014). Molecular diagnosis of endemic and invasive
mycoses: advances and challenges. Rev Iberoam Micol 31, 35–41.
utilizing high resolution micropore chips. Sensors (Basel) 9, 1590–
1598.
Hsiao, C. R., Huang, L., Bouchara, J. P., Barton, R., Li, H. C. & Chang,
T. C. (2005). Identification of medically important molds by an
Naja, G., Hrapovic, S., Male, K., Bouvrette, P. & Luong, J. H. (2008).
oligonucleotide array. J Clin Microbiol 43, 3760–3768.
Iwen, P. C., Hinrichs, S. H. & Rupp, M. E. (2002). Utilization of the
internal transcribed spacer regions as molecular targets to detect and
identify human fungal pathogens. Med Mycol 40, 87–109.
Jarvis, J. N., Percival, A., Bauman, S., Pelfrey, J., Meintjes, G.,
Williams, G. N., Longley, N., Harrison, T. S. & Kozel, T. R. (2011).
Evaluation of a novel point-of-care cryptococcal antigen test on
serum, plasma, and urine from patients with HIV-associated
cryptococcal meningitis. Clin Infect Dis 53, 1019–1023.
Kessler, H. H., Mühlbauer, G., Stelzl, E., Daghofer, E., Santner, B. I. &
Marth, E. (2001). Fully automated nucleic acid extraction: MagNA
Rapid detection of microorganisms with nanoparticles and electron
microscopy. Microsc Res Tech 71, 742–748.
Neely, L. A., Audeh, M., Phung, N. A., Min, M., Suchocki, A., Plourde,
D., Blanco, M., Demas, V., Skewis, L. R. & other authors (2013). T2
magnetic resonance enables nanoparticle-mediated rapid detection of
candidemia in whole blood. Sci Transl Med 5, 182ra54.
Nugaeva, N., Gfeller, K. Y., Backmann, N., Lang, H. P., Düggelin, M. &
Hegner, M. (2005). Micromechanical cantilever array sensors for
selective fungal immobilization and fast growth detection. Biosens
Bioelectron 21, 849–856.
Pure LC. Clin Chem 47, 1124–1126.
Odabasi, Z., Mattiuzzi, G., Estey, E., Kantarjian, H., Saeki, F., Ridge,
R. J., Ketchum, P. A., Finkelman, M. A., Rex, J. H. & OstroskyZeichner, L. (2004). Beta-D-glucan as a diagnostic adjunct for invasive
Lacroix, C., Gicquel, A., Sendid, B., Meyer, J., Accoceberry, I.,
François, N., Morio, F., Desoubeaux, G., Chandenier, J. & other
authors (2014). Evaluation of two matrix-assisted laser desorption
fungal infections: validation, cutoff development, and performance
in patients with acute myelogenous leukemia and myelodysplastic
syndrome. Clin Infect Dis 39, 199–205.
ionization-time of flight mass spectrometry (MALDI-TOF MS)
systems for the identification of Candida species. Clin Microbiol
Infect 20, 153–158.
Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N.,
Pappas, P. G. & Chiller, T. M. (2009). Estimation of the current global
Lee, W. G., Kim, Y.-G., Chung, B. G., Demirci, U. & Khademhosseini,
A. (2010). Nano/microfluidics for diagnosis of infectious diseases in
developing countries. Adv Drug Deliv Rev 62, 449–457.
burden of cryptococcal meningitis among persons living with HIV/
AIDS. AIDS 23, 525–530.
Peeling, R. W., Holmes, K. K., Mabey, D. & Ronald, A. (2006). Rapid
tests for sexually transmitted infections (STIs): the way forward. Sex
Transm Infect 82 (Suppl. 5), v1–v6.
Lindsley, M. D., Mekha, N., Baggett, H. C., Surinthong, Y.,
Autthateinchai, R., Sawatwong, P., Harris, J. R., Park, B. J., Chiller,
T. & other authors (2011). Evaluation of a newly developed lateral
Pumera, M., Sánchez, S., Ichinose, I. & Tang, J. (2007). Electro-
flow immunoassay for the diagnosis of cryptococcosis. Clin Infect Dis
53, 321–325.
Rickerts, V., Khot, P. D., Myerson, D., Ko, D. L., Lambrecht, E. &
Fredricks, D. N. (2011). Comparison of quantitative real time PCR
Loeffler, J., Hebart, H., Cox, P., Flues, N., Schumacher, U. & Einsele,
H. (2001). Nucleic acid sequence-based amplification of Aspergillus
with sequencing and ribosomal RNA-FISH for the identification of
fungi in formalin fixed, paraffin-embedded tissue specimens. BMC
Infect Dis 11, 202.
RNA in blood samples. J Clin Microbiol 39, 1626–1629.
http://jmm.sgmjournals.org
chemical nanobiosensors. Sensors Actuators B 123, 1195–1205.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 00:12:15
321
F. Teles and J. Seixas
Rigby, S., Procop, G. W., Haase, G., Wilson, D., Hall, G., Kurtzman, C.,
Oliveira, K., Von Oy, S., Hyldig-Nielsen, J. J. & other authors (2002).
Fluorescence in situ hybridization with peptide nucleic acid probes for
rapid identification of Candida albicans directly from blood culture
bottles. J Clin Microbiol 40, 2182–2186.
Schell, W. A., Benton, J. L., Smith, P. B., Poore, M., Rouse, J. L., Boles, D. J.,
Johnson, M. D., Alexander, B. D., Pamula, V. K. & other authors (2012).
Evaluation of a digital microfluidic real-time PCR platform to detect DNA
of Candida albicans in blood. Eur J Clin Microbiol Infect Dis 31, 2237–2245.
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L.,
Levesque, C. A., Chen, W., Bolchacova, E., Voigt, K. & other authors
(2012). Nuclear ribosomal internal transcribed spacer (ITS) region as
a universal DNA barcode marker for fungi. Proc Natl Acad Sci U S A
109, 6241–6246.
Spanu, T., Posteraro, B., Fiori, B., D’Inzeo, T., Campoli, S., Ruggeri,
A., Tumbarello, M., Canu, G., Trecarichi, E. M. & other authors
(2012). Direct MALDI-TOF mass spectrometry assay of blood culture
Thornton, C. R. (2008). Development of an immunochromatographic
lateral-flow device for rapid serodiagnosis of invasive aspergillosis.
Clin Vaccine Immunol 15, 1095–1105.
Vilchez, R. A., Fung, J. & Kusne, S. (2002). Cryptococcosis in
organ transplant recipients: an overview. Am J Transplant 2, 575–
580.
Villamizar, R. A., Maroto, A. & Rius, F. X. (2009). Improved detection
of Candida albicans with carbon nanotube field-effect transistors.
Sensors Actuators B 136, 451–457.
Wheat, L. J., Garringer, T., Brizendine, E. & Connolly, P. (2002).
Diagnosis of histoplasmosis by antigen detection based upon experience
at the histoplasmosis reference laboratory. Diagn Microbiol Infect Dis 43,
29–37.
WHO (2014). Antimicrobial resistance: global report on surveillance
2014. Geneva, Switzerland: World Health Organization. http://www.
who.int/drugresistance/documents/surveillancereport/en/
broths for rapid identification of Candida species causing bloodstream infections: an observational study in two large microbiology
laboratories. J Clin Microbiol 50, 176–179.
Yeo, S. F. & Wong, B. (2002). Current status of nonculture methods for
diagnosis of invasive fungal infections. Clin Microbiol Rev 15, 465–
484.
Teles, F. (2013). Biosensors for medical mycology. In Biosensors and
Yoo, S. M., Kang, T., Kang, H., Lee, H., Kang, M., Lee, S. Y. & Kim, B.
(2011). Combining a nanowire SERRS sensor and a target recycling
their Application in Healthcare, pp. 88–111. Edited by D. OzkanAriksoysal. London: Future Science.
Teles, F. R. R. & Martins, M. L. (2011). Laboratorial diagnosis of
paracoccidioidomycosis and new insights for the future of fungal
diagnosis. Talanta 85, 2254–2264.
322
reaction for ultrasensitive and multiplex identification of pathogenic
fungi. Small 7, 3371–3376.
Zhang, S. X. (2013). Enhancing molecular approaches for diagnosis of
fungal infections. Future Microbiol 8, 1599–1611.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 00:12:15
Journal of Medical Microbiology 64