Journal of Medical Microbiology Papers in Press. Published November 23, 2014 as doi:10.1099/jmm.0.082297-0 1 1

The future of novel diagnostics in medical mycology

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Running title: Rapid diagnostic tests for medical mycology

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Contents category: review paper

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Fernando Teles1,2, Jorge Seixas2,3

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Group of Mycology / Unit of Medical Microbiology, Institute of Hygiene and Tropical Medicine

(IHMT), Universidade Nova de Lisboa (UNL). Rua da Junqueira, 100, 1349-008 Lisboa, Portugal.

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2

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(IHMT), Universidade Nova de Lisboa (UNL). Rua da Junqueira, 100, 1349-008 Lisboa, Portugal.

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3

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Lisboa (UNL). Rua da Junqueira, 100, 1349-008 Lisboa, Portugal.

Centre for Malaria and Other Tropical Diseases (CMDT), Institute of Hygiene and Tropical Medicine

Tropical Clinic Unit, Institute of Hygiene and Tropical Medicine (IHMT), Universidade Nova de

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Corresponding author: Fernando Teles. Instituto de Higiene e Medicina Tropical. Rua da Junqueira,

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100,

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[email protected]

1349-008

Lisboa,

Portugal.

Telephone:

(351)

213652600

(ext.

318).

E-mail:

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Keywords: fungal diagnosis; lab-on-a-chip; microfluidics; nanotechnology; point-of-care; rapid tests

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Abbreviations: BG, (1 3)-β-D-glucan; CNT, carbon nanotube; FISH, fluorescence in situ hybridization; FDA, Food and Drug Administration; GM, galactomannan; GXM, glucuronoxylomannan; HAART, highly-active antiretroviral therapy; HIV, human immunodeficiency virus; ITS, internal transcribed spacer; IA, invasive aspergillosis; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MB, molecular beacon; NP, nanoparticle; NASBA, nucleic acid sequence-based amplification; PNA, peptide nucleic-acid; POC, point-of-care; QD, quantum dot; SDI, Sexually Transmitted Diseases Diagnostics Initiative; WHO, World Health Organization

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Abstract

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Several fungal diseases have become serious threats to human health and life, especially upon the

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advent of human immunodeficiency virus (HIV)/AIDS epidemics and of other typical

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immunosuppressive conditions of modern life. Accordingly, the burden posed by these diseases and,

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concurrently, by intensive therapeutic regimens against these diseases has increased worldwide. When

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existing and available, rapid tests for point-of-care (POC) diagnosis of important fungal diseases

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enable surpassing the limitations of current laboratory methods for detection and identification of

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medically-important fungi, both in low-income countries and for first-line diagnosis (screening) in

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richer countries. As with conventional diagnostic methods and devices, former immunodiagnostics

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have been challenged by molecular biology-based platforms, as a way to enhance the sensitivity and

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shorten the assay time, thus enabling early and more accurate diagnosis. Most of these tests have been

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developed in-house, without adequate validation and standardization. Another challenge has been the

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DNA extraction step, which is especially critical when dealing with fungi. In this paper, we have

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identified three major research trends in this field: the application of newer biorrecognition

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techniques, often applied in analytical chemistry; the development of new materials with improved

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physicochemical properties; and novel bioanalytical platforms, allowing fully automated testing.

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Keeping up-to-date with the fast technological advances registered in this field, primarily at the proof-

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of-concept level, is essential for wise assessment of those that, more likely, will be more cost-

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effective and, as already observed for bacterial and viral pathogens, may leverage the current tepid

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developmental status of novel and improved diagnostics for medical mycology.

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Introduction

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The development of novel methods and devices for detection, identification and quantification of

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human pathogens is witnessing unprecedented advances, mainly as a result of the combination of

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established molecular biology principles with the recent technological developments on new materials

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and platforms for biosensing. This has resulted, in particular, from the consolidation of the

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microfluidics and lab-on-a-chip concepts as suitable platforms for bioassays and of nanoengineered

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structures with enhanced physicochemical properties for ultrasensitive and specific diagnostics

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(Fortina et al., 2007; Lee et al., 2010). Biorrecognition of bacteria and viruses causative of important

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human diseases has greatly benefited from such advances, but this has not been so for pathologies of

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fungal aetiology. Several reasons for this can be pointed out, either due to the inherent biological

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characteristics of fungi or to the peculiar clinical and epidemiological features of mycotic diseases.

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However, the immunodeficiency virus (HIV)/AIDS epidemics and the increased incidence of other

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immunosuppressive factors, such as chemotherapy, organ transplantation and new immunological

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treatments for autoimmune diseases, have increased the prevalence and severity of opportunistic

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mycoses in the past few decades. This is an obvious burden for management by the health systems of

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low-resource countries. In parallel, developed countries have faced the emergence of antifungal drug

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resistance arising from intensive chemotherapeutic and immunological treatments and regimens

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(World Health Organization, 2014). Under this scenario, there is increased awareness for the

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limitations of current diagnostic procedures and, accordingly, for the need of novel methods, able to

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precociously and accurately detect and identify the major human fungal pathogen species in a fast,

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timely and less invasive manner. This paper briefly overviews the current status and limitations of

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diagnosis in medical mycology, whilst pointing out some expected trends in this field.

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The importance of diagnosis

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Fungal species of Candida, Aspergillus and Cryptococcus are major etiologic agents of invasive

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infections in immunocompromised patients. The global mortality rate by invasive aspergillosis (IA)

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increased more than 350% in the past 25 years. This disease became a leading cause of death in 60-

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90% of immunodepressed individuals (McNeil et al., 2001). Around one million new cases of

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cryptococcal meningitis occur each year, killing more than half a million HIV-infected persons in sub-

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Saharan Africa and in Southeast Asia every year, which corresponds to up to 90% of all yearly deaths

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in the world by cryptococcal meningitis (Park et al., 2009). Concerning Candida, systemic and

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mucosal infections caused by these species have remained high since the 1990s. Importantly, the

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current status of research in medical mycology allows considering all fungi as potentially harmful in

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severely immunocompromised hosts (Alexander & Pfaller, 2006). The burden of these diseases is

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milder in richer countries, with wider access to the highly active antiretroviral therapy (HAART), but

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worrisome in poorer countries (Teles, 2013). In richer countries, the intensive and increased use of

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chemotherapeutic regimens and of new immunological treatments for malignancy, transplantation and

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autoimmune disease management has led to aggressive and often fatal fungal diseases. As such, early

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and accurate disease diagnosis is crucial for prompt beginning of therapy, thus avoiding the long-term

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complications and high death rates currently observed, but also for rapid shifting of therapeutics once

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drug resistant strains are detected, especially in cases of chronic infections (Teles & Martins, 2011).

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Ironically, as the variety and efficacy of antifungal drugs increase, the need for accurate and rapid

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diagnosis increases, in order to preclude inadequate presumptive antifungal therapy. This does not

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only renders less toxic effects, but also better prognosis, due to decreased drug resistance. In parallel,

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the continuous description of new fungal species poses additional challenges to existing detection and

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identification methods, especially when such species exhibit different virulence or antifungal

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susceptibility patterns.

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Limitations of conventional laboratorial diagnosis

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Understanding the current situation of the development of point-of-care (POC) diagnostic tests in

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medical mycology requires prior comprehension about the limitations of conventional diagnosis and

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hence of challenges in the field. Culture and histopathology of infected tissues have been the most

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traditional diagnostic methods for most mycoses, but obtaining biopsies from sterile body sites is a

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highly invasive approach, a serious handicap, especially in severely ill patients. Plus, a positive

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culture from a sterile site may indicate transient colonization and not true infection, especially for

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opportunistic fungi. Culture followed by microscopic examination requires skilled manpower and

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long assay times for cell culturing, when in vitro growing is feasible. New culture media, lysis

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centrifugation and automated blood culture systems have decreased the assay time (Chandrasekar,

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2009), particularly for detection of Candida spp., but without definitely improving the sensitivity of

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the detection (Archibald et al., 2000; Chandrasekar, 2010). Direct examination, whenever possible, is

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simpler, faster and cheaper than culture, but a negative test does not definitely exclude a fungal

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infection. When infected tissue is available, histopathological diagnosis is a common approach, but it

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lacks sensitivity and selectivity, as several filamentous fungi may exhibit undistinguishable

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morphologies. This is particularly troublesome when distinguishing Aspergillus from related fungi

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requiring different therapeutics (Chandrasekar, 2009). Serological techniques may be targeted to

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detect fungal antigens or antibodies in human serum through immunological or biochemical reactions.

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For antigen detection, tests have used to target two cell wall polysaccharides: (1

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and galactomannan (GM). The first has been used to diagnose invasive infections caused by several

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opportunistic fungi (Odabasi et al., 2004). It is especially useful for presumptive diagnosis

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(screening), envisaging early beginning of antifungal therapy, and not for fungal identification

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(Alexander & Pfaller, 2006). Given the ubiquitous presence of glucan in the environment, this test is

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prone to false-positive results (Chandrasekar, 2009). Moreover, it is not useful for detection of

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Cryptococcus and Mucorales spp., because the cell walls of their etiologic fungi have too low

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amounts of this biomarker (Miyazaki et al., 1995). The second test has been applied for the diagnosis

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of IA (Mennink-Kersten et al., 2004). Yet, its sensitivity and accuracy may decrease under antifungal

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drug usage, which causes less GM to be released from fungal cell walls (Maertens et al., 2001). More

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specific serological tests, such as those for cryptococcal meningitis (Vilchez et al., 2002) and for

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disseminated histoplasmosis (Wheat et al., 2002), have also been employed. Antibody detection tests

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usually lack sensitivity in cases of immunocompromised patients due to the low levels of circulating

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antibodies (Teles & Martins, 2011). Plus, diagnosis of an acute infection requires paired sera

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(Alexander & Pfaller, 2006). A general drawback of immunoassays is potential cross-reactivity

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between different circulating antibodies and antigens and hence low selectivity. The reliability of

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antibody assays for diagnosis of invasive candidiasis is particularly limited; the sensitivity is usually

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low for immunocompromised patients (which are at higher risk for infection), the same happening

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with the specificity, as Candida spp. are ubiquitous in the normal human flora (Arvanitis et al., 2014).

→3)-β-D-glucan (BG)

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The advent of molecular biology-based diagnosis

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The growing technical developments in nucleic acid-based molecular diagnostic methods, especially

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PCR-based techniques for genome amplification and genome sequencing, as well as their increasing

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cost-effectiveness, have improved the reliability and performance of these techniques in mycological

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diagnosis, as recently reviewed in the literature (Teles, 2013), with particular descriptions for the

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major human fungal pathogens (Arvanitis et al., 2014; Gómez, 2014). They usually perform better (in

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terms of sensitivity and selectivity) and quicker than more conventional methods, allowing earlier

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diagnosis. PCR has already been reported for the diagnosis of candidemia and of IA (Yeo & Wong,

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2002). Fungal genome amplification by PCR with conserved (panfungal) oligonucleotide primers

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followed by further amplification with species-specific primers (allowing multiplexed PCR, i.e.,

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simultaneous detection and identification of multiple fungal species in a single assay) or by another

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molecular identification technique (e.g., sequencing or restriction endonuclease analysis) has been

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particularly promising for mycological diagnosis (Hsiao et al., 2005; Iwen et al., 2002). Compared to

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more conventional methods, nucleic-acid based bioassays usually provide faster and more sensitive

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results. The exquisite ultrahigh sensitivity of most of these methods coupled to accurate thresholds

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determination enables tailoring quantitative assays, thus differentiating true fungal infections from

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simple colonization, as well as monitoring and predicting clinical outcomes from therapeutic

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interventions (Zhang, 2013). Among them is real-time PCR which, coupled to fluorescent detection,

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allows gains in sensitivity and minimization of contamination, compared to conventional PCR; yet, it

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remains a too complex and expensive technique for rapid and inexpensive testing. Another promising

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PCR-based technique is nucleic-acid sequence-based amplification (NASBA), which allows

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isothermal direct amplification of RNA from organisms with RNA genomes, and has already proven

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usefulness for diagnosing IA (Loeffler et al., 2001). PCR amplification of fungal genetic markers has

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been in the core of DNA barcoding-based fungal identification and classification. However,

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continuous changes in fungal taxonomy and the inexistence of a widely conserved region in fungal

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genomes at the species and genus levels have delayed wider developments on molecular diagnostics

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for fungi. Ribosomal genes have been particularly used as barcodes for fungi, for containing

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sequences common to all fungi, able to be used for fungal infection screening, but also variable and

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highly-variable domains, such as the internal transcribed spacer (ITS) region, which is suitable for

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species identification (Yeo & Wong, 2002). ITS regions provide high taxonomic resolution and are

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widely characterized in popular sequence databases, such as GenBank (Balajee et al., 2009). Recently,

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a study from an international research consortium proposed ITS as the primary fungal barcode marker

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(Schoch et al., 2012). Higher performance in species discrimination can be achieved by using

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additional sets of primers targeting other fungal genome markers. Overall, this constitutes a prior step

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towards future development of standardized molecular methods for fungal diagnosis. In this regard, it

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is also expected that the development of more reliable databases and of sequencing techniques may

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consolidate molecular methods (for which there is currently no formal approval of regulatory entities

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for fungal diagnosis) as valuable alternatives to conventional methods for fungal identification

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(Alexander & Pfaller, 2006). Three alternative options, of development of a consensus protocols by an

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ad-hoc experts working group, of centralization of molecular testing within a certified reference

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laboratory or of commercialization via production of quality-controlled diagnostic kits and regents,

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have been proposed for standardization of molecular methods for fungal diagnosis (Zhang, 2013). A

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fundamental obstacle of molecular diagnostics for fungi is that fungal cells are usually delimited by

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thick walls that constitute challenging physical barriers for isolation of trace amounts of nucleic-acids.

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In the future, full packaging and automation of the nucleic-acid extraction step will likely contribute

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to overcome this problem, as well as to minimize contamination of the biological sample. Such

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automated methods and devices would avoid the need for bulky clean rooms and achieve higher

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performances than manual extraction methods (Kessler et al., 2001).

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Rapid tests for point-of-care diagnosis

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For successful POC and field applications, especially in remote and resource-depleted regions where

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appropriate diagnostic facilities, equipment and trained manpower often lack, diagnostic devices

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should accomplish the ASSURED criteria, as defined by the World Health Organization (WHO)

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Sexually Transmitted Diseases Diagnostics Initiative (SDI): they must be affordable, sensitive,

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specific, user-friendly, rapid, robust, equipment-free and deliverable to end-users (Peeling et al.,

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2006). Rapidity, in particular, means that fewer samples will reach laboratories for analysis, allowing

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improved decision-making. Indeed, the importance of the concept of rapidity and its inherent

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relationship with the other parameters has been responsible for the term of ‘rapid (diagnostic) tests’

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usually assigned to diagnostics meeting these criteria. Commercial diagnostic kits for detection and

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identification of several clinically-important fungi have been available for several years, such as the

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AccuProbe tests (from former Gen-Probe, San Diego/CA, USA). Their development and

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commercialization have greatly benefited from the advances in molecular methods for fungal

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detection and identification, particularly in the case of dimorphic fungi (Alexander & Pfaller, 2006).

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However, they are essentially lab-based techniques rather than targeted for POC; in general, they do

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not fully comply with the ASSURED criteria, because they require labor-intensive sample

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pretreatment and long assay times, as fungal growth in pure cultures is usually required (Teles &

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Martins, 2011). For Candida species identification, for instance, such tests are relatively expensive

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and laborious, requiring incubations longer than two days (Marot-Leblond et al., 2004). Indeed,

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sample pretreatment has been traditionally disregarded in the development of diagnostic tests,

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especially in the case of mycelia-forming fungi. Hence, their usefulness as true POC tests remains

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limited. The advent of lateral-flow tests, usually in the form of strips for qualitative detection of

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antigens of antibodies (immunochromatography), constitutes a successful paradigm in the conception

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of rapid tests. They have been widely developed, commercialized and used for specific diagnosis of

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many human pathogens. The decentralization paradigm of disease diagnosis (related with the concepts

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of POC and in situ testing), allied to high-throughput and multiplexing abilities of novel tests, is

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opening an appetizing commercial market even for the traditionally unattractive branch of medical

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mycology. Yet, the old paradigm of bulky, complex and expensive analytical apparatus is still far

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from perishing. Rapid tests, rather than true competitors, are still, in essence, complementary in their

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application ranges and contexts. They have found particular market niches in POC applications, for

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decentralized and in situ diagnosis (by non-medical healthcare staff or, ultimately, at the bedside

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level). In particular, disease screening has been perhaps the main specific context for their wide

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application worldwide, as first-line diagnostic tools useful to screen for positive results that will be

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further confirmed with conventional techniques in central laboratories. Some successful examples of

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rapid diagnostic tests for the major human fungal pathogens using lateral-flow supports have been

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applied to clinical samples and reported in recent years, as described in Table 1. The test developed to

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differentiate between Candida albicans and C. dubliniensis (Marot-Leblond et al., 2004) employed a

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sandwich system with two immobilized mAbs, one of them binding epitopes specifically expressed by

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the two Candida species, and the other binding only C. albicans. This test precludes the usual need

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for complex molecular biology techniques, although important differences in performance were

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observed depending on the used isolation medium. For Cryptococcus neoformans, two tests were

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reported, able to detect the four main cryptococcal serotypes. The kit commercialized by Immuno-

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Mycologics (IMMY, OK/USA), which employs mAbs against capsular polysaccharide antigens of the

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C. neoformans/C. gattii species complex, allows the use not only of blood, but also of urine, an ideal,

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non-invasive and readily available specimen to use in POC situations, although the limited sensitivity

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observed in this fluid is probably a result of low fungal burden, due to excretion (Lindsley et al.,

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2011). The other, more recent test also employs mAbs, against the cryptococcal capsular

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polysaccharide glucuronoxylomannan (GXM); it is also tailored for using blood and urine, but its

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evaluation was performed with a relatively small sample size, thus requiring further validation in

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larger studies. Moreover, its selectivity was not assessed (Narvis et al., 2011). For detection of

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Aspergillus spp., the first reported lateral-flow device for human serum employed the mAb JF5, which

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specifically targets an extracellular glycoprotein of Aspergillus spp. (Thornton, 2008). The use of this

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mAb avoids the proneness to the false-positive and false-negative results characteristic of common

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immunoassays targeting GM with the mAb EB-A2. Of note, it also enables to discriminate active

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invasive growth from quiescent spore production, a major issue in cases of IA. However, the

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sensitivity of this test decreased almost 30 times when passing from protein-depleted from protein-

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rich serum. Comparison of the performance of this test with that of conventional immunoassays has

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been troublesome since each assay uses different species of immobilized antibodies and targets

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different Aspergillus antigens. The second, more recent test makes use of the IgM mAb476, able to

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detect GM-like antigens from Aspergillus and other molds in urine (Dufresne et al., 2012). Patients

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with IA were successfully tested, but the cohort was too small to assess meaningful performance

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characteristics. The study also reported low amount of these antigens in urine and the inhibitory effect

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of this fluid, thus decreasing the analytical sensitivity of the test. The hypothesis of sample

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concentration and further desalting/dialysis was raised, highlighting the need for processing complex

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biological media prior to pathogen detection. All the above-mentioned tests employed immunological

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reactions as the basis for the biorrecognition events, thus suffering from the general and well-known

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limitations of immunodiagnosis.

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New technologies for diagnostics

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Most current methods available for medical diagnosis of human mycosis still suffer from a high

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degree of invasiveness, relatively long assay times and variable performances (in terms of sensitivity

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and specificity). Understandably, this constitutes a general driving force for the development of

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improved diagnostic techniques. Integration, automation, miniaturization and packaging of the entire

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biorrecognition process are the essential underlying concepts driving the development of advanced

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diagnostic devices. Accordingly, techniques and signal transducers originally applied in conventional

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analytical chemistry have been reported for detection and identification of fungi; some of these, with

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examples from the literature, include spectrophotometry for Paracoccidioides brasiliensis (Martins et

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al., 2012), fluorescence in situ hybridization (FISH) for Aspergillus and Candida (Rickerts et al.,

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2011), matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) for Candida (Lacroix

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et al., 2014), Aspergillus, Fusarium, Mucorales (Carolis et al., 2012) and Cryptococcus spp.

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(McTaggart et al., 2011), microgravimetry for Aspergillus niger and Saccharomyces cerevisiae (as

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nonpathogenic models of, respectively, A. fumigatus and C. albicans) (Nugaeva et al., 2005), and

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electrochemical techniques for C. albicans (Mulero et al., 2009; Villamizar et al., 2009). Among

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them, MALDI-TOF is undoubtedly a step ahead, given its wide use worldwide for diagnosis of fungal

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and other microbial infections. This has been so especially after multicenter clinical trials (Spanu et

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al., 2012) and regulatory approval of the first of these systems by the US Food and Drug

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Administration (FDA) for clinical use (Arvanitis et al., 2014). Another major research trend in this

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regard has been the development of cheaper materials for the construction of analytical platforms,

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thus making disposability (crucial when dealing with infectious agents) economically feasible. In this

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context, many innovations have arisen from three emergent technological fields. The first one is

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nanotechnology, through the generation of nanostructures enabling improved sensitivity and

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specificity, as a result of the unusual or enhanced physicochemical properties of nanosized materials,

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mostly derived from the very high surface-to-volume ratio of these structures. In bioassays,

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nanoparticles (NPs) may act as immobilization supports for biomolecules, labels for signal

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amplification or even as bioprobes for specific recognition of biological targets (Fortina et al., 2007).

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Among NPs, some of the most important and already used in bioassays for bacterial or viral detection

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include Au-NPs, quantum dots (QDs), molecular beacons (MBs), DNA dendrimers, carbon nanotubes

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(CNTs), liposomes, nanowires and peptide nucleic-acids (PNAs) (Algar et al., 2009; Pumera et al.,

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2007); some of these have also been tested and applied for detection of pathogenic fungi, but only

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rarely in clinical samples (Table 2). The second is micro/nanofabrication, commonly inspired in the

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methods used in silicon planar circuitry industry, potentially rendering mass-production of

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inexpensive and very reproducible diagnostic microdevices (biochips), but already through advanced

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techniques of nanofabrication (Fortina et al., 2007). The third field is microfluidics, consisting in

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small sized platforms for fluid propelling through microchannels, in the course of the bioassay;

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ultimately, the goal is the production of lab-on-a-chip analytical platforms, incorporating, in packaged

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and self-powered miniaturized devices, all bioanalytical steps (from sample pretreatment to final

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detection) traditionally performed with bulky equipment in large laboratory settings (Choi et al.,

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2011; Lee et al., 2010). These devices can also be designed for inter- or even intra-specific

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simultaneous analysis of many different pathogens, by taking advantage of the increasing ability to

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miniaturize and build dense arrays of different immobilized probes (multiplexing) onto low-cost and

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portable platforms (such as disposable cartridges). Another remarkable advantage of these devices is

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the possibility of handling and testing sample volumes several orders of magnitude smaller than those

303

used in conventional diagnostic methods. Very few applications of these technologies for fungal

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detection have been reported so far and, even among these, usually for C. albicans only. One of them

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refers to a generic detection of unknown rare blood pathogens (including C. albicans) with a

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microfluidic device, magnetic microbeads coated with an opsonin, able to recognize and capture most

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blood pathogens (Cooper et al., 2014). After this pre-concentration step, fluorescence scanning was

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employed for final detection, with excellent sensitivity (less than 1 yeast cell per ml of human blood).

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The magnetic capture/concentration step did not affect pathogen viability, thus allowing, upon

310

detection and subsequent antifungal susceptibility testing, rapid diagnosis of sepsis. In another work, a

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real-time PCR-based microfluidic platform, from Advanced Liquid Logic, Inc. (Morrisville/NC,

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USA), was employed for detection of C. albicans DNA in human blood (Schell et al., 2012). Unlike

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the first microfluidic devices, with microchannels for fluid flow, this more versatile second-generation

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layout employs the principle of electrowetting, in which fluid flow is directed by computer-controlled

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electrodes (digital microfluidics). This device exhibited slightly less sensitivity than conventional

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microfluidics and excellent specificity (100%) towards non-candidemic blood samples. So far, most

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of these schemes and devices have been employed only at the prototype level, i.e., without industrial

318

production and commercialization. Some of these fungal bioassays have shown not only to be

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potentially cheaper than traditional diagnostic techniques (Dhiman et al., 2011), but also more

320

advantageous towards antifungal therapy costs’ savings (Forrest et al., 2006). Although most of these

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technical and scientific developments have occurred for biorrecognition of bacteria and viruses,

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innovations in fungal biomolecular detection may proceed faster compared to those microorganisms,

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by taking advantage of the valuable examples and accumulated knowledge, from the past and present,

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with such biological agents. Coupled to well-established signal transduction schemes (e.g., of optical

325

or electrochemical nature), new biomaterials with improved properties and suitable platforms for

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cheaper, simpler and faster biorrecognition of infectious agents promise to significantly move

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forward, and even to create a breakthrough in the current paradigm of diagnosis of human infectious

328

agents and of related diseases.

329 9

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Final remarks

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There is nowadays a vastness of available techniques, analytical platforms and biomolecular probes

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and tags already tested, at least at the proof-of-concept level, with a whole range and combination of

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characteristics and performances for detection/identification of many human pathogens and diagnosis

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of related pathologies. Hopefully, in the future, the key-point in decision-making at the level of

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infectious disease diagnosis will not be whether available diagnostic tools suitable for common human

336

infectious diseases will be able to reach required performance thresholds (of sensitivity, selectivity,

337

etc.) or conditions (e.g., type of biological sample, time period after the infection); instead, taking into

338

account that analytical performance usually runs in parallel with complexity and costs, the essential

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issue will probably be what minimum pathogen discriminatory ability and detection limit will be

340

necessary to guarantee quick and effective disease management, especially envisaging the selection of

341

the most appropriate therapeutic regimen for a given pathological condition. Given the relatively low

342

number of fungi able to cause severe diseases in humans (compared to other types of

343

microorganisms), there has been reluctance and difficulty of national health systems for funding and

344

promoting more widely the research, development and implementation of novel and improved

345

diagnostic tests for mycological diagnosis. This is especially true for low-income countries where

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most human pathogenic fungi and related diseases are endemic. In developed and richer countries,

347

important systemic and opportunistic mycoses have posed significant public health burdens,

348

especially due to the emergence of antifungal drug resistance in immunocompromised patients. Even

349

so, and taking into account that many cases of mycotic infections do not develop severe clinical

350

manifestations, the research and development of new diagnostics for fungal diseases has not been

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considered a health priority, especially in times of severe budget haircuts (as nowadays), particularly

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in the private sector. On the other hand, especially for infections caused by filamentous fungi, early

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and accurate diagnosis remains a challenge. Remarkably, profitable market niches may be envisaged

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in the private sector. This will likely constitute a promising driving motor to pursue new

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developments in improved and economically competitive diagnostics for human fungal diseases in the

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future. Fungi have some challenging structural features for successful biomolecular diagnosis, but it is

357

time to learn with the accumulated experiences obtained from other microorganisms, especially

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bacteria and viruses, in order to definitely leverage and improve the current stagnant status of

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diagnosis in medical mycology, in a context of a growing burden posed by very complex disease

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conditions and critically ill patients.

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16

579

Table 1: Immunochromatographic tests reported in the literature for diagnosis of medically-important

580

fungi. Target fungus

Assay time

Reported LOD or sensitivity

(Ref.)

C. albicans; C. dubliniensis

2,5 h

91.4 - 96.6% (C. albicans)

(Marot-Leblond

98.3% (C. dubliniensis)

et al., 2004)

-1

Aspergillus spp.

15 min

37 ng ml

Aspergillus spp.

----

----

10 min

≥ 5 ng ml

C. neoformans

(Thornton, 2008) (Dufresne et al., 2012)

-1

(Narvis

et

al.,

2011) Cryptococcus spp.

5 - 15 min

100% (serum); 70.7 - 92% (urine)

(Lindsley et al., 2011)

581

LOD, limit of detection

582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

17

605

Table 2

606

Tests reported in the literature for diagnosis of medically-important fungi based on nanoengineered

607

structures. Target fungus

P. brasiliensis

Assay

Type

time

nanostructure

----

Au-NP

of

Sample

Reported

LOD

(Ref.)

or sensitivity Fungal DNA

≥ 4 mg μl

-1

(Martins

et

al., 2012) C. albicans

1h

CNT

Fungal

-1

50 CFU ml

solution Candida spp.

30 min

Au-NP

Wastewater

et al., 2009) ----

effluent C. albicans

2,5 h

PNA

Blood culture

(Villamizar

(Naja et al., 2008)

100%

(Rigby et al., 2002)

A. C.

Fumigatus; glabrata;

krusei;

C.

Few

Au-nanowire

Fungal DNA

100 fM

hours

(Yoo et al., 2011)

C.

neoformans Candida spp.

The future of novel diagnostics in medical mycology.

Several fungal diseases have become serious threats to human health and life, especially upon the advent of human immunodeficiency virus/AIDS epidemic...
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