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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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 00:12:15 Printed in Great Britain 315 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 316 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 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 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 http://jmm.sgmjournals.org 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 00:12:15 317 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. 318 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 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. http://jmm.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 00:12:15 319 F. Teles and J. Seixas 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. 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