Introduction
Human fungal infections encompass a broad spectrum of diseases caused by more than 600 species of fungi present in the environment. They can affect any part of the body, producing superficial infections of the skin and nails, mucosal infections, chronic and allergic infections, and, finally, invasive fungal infections (IFIs). IFIs are the most serious clinical entities and are associated with high mortality, as they mainly affect immunosuppressed patients with poor general health.
Diagnosis of these diseases is difficult and frequently delayed due to the lack of suspicion and/or the lack of suitable diagnostic tests. In many parts of the globe, particularly in low income countries, some of these infections are neglected diseases than could be easily solved with skilled personnel and adequate diagnosis (Oladele et al., 2019). In developed countries, the main problem is that these invasive infections occur in highly immunosuppressed patients with a poor outcome in absence of an early diagnosis. The increasing use of immunosuppressive drugs for treating serious medical conditions have favored the rise of opportunistic fungal infections in these regions.
The overall mortality due to fungal infections is around 1,600,000 people per year (Bongomin et al., 2017). The most common species involved in invasive disease are Aspergillus spp., Candida spp., Cryptococcus spp., Pneumocystis jirovecii and fungi responsible of endemic mycoses (Histoplasma capsulatum, Paracoccidioides spp., Coccidioides spp., and Blastomyces dermatitidis). Nevertheless, when a patient has several predisposing factors for a prolonged period of time, almost any fungal species can cause an infection.
The clinical profile of mycosis varies greatly depending on the causal agent, the location of the infection, and the predisposing factors of the patient. Regardless of the patient’s clinic, the diagnosis is always difficult. Classical diagnosis methods have limitations as they lack of a suitable sensitivity and specificity. Recently, new diagnostic tests have been developed but many of them are still used in combination with classical methods because of the lack of validation and consensus among laboratories. Moreover, some of these new techniques are too expensive for resource challenging countries. This review describes the methods routinely employed in the Mycology Laboratories and their usefulness in the diagnosis of the different clinical entities in the field of the Medical Mycology. In addition, it delves into the methods most recently developed and describes their use alone or in combination with classical methods.
Two figures have been included with the purpose of summarizing all these concepts. In Fig. 1 it is shown how, depending on the kind of infection, the number of techniques used is greater and more specialized due to both the difficulty of the diagnosis and the severity of the infection. In Fig. 2, a brief summary of all diagnostic techniques that can be performed has been presented.
Fig1. Main human fungal infections and the corresponding laboratory techniques commonly used for their diagnosis.
Fig2. Advantages and limitations of both classical and new fungal diagnostic methods.
Conventional Diagnostic Methods
Conventional diagnostic methods are those used classically in the Laboratory of Microbiology. Although they are useful, they lack of sensitivity and specificity and should be used in combination with the new tools recently developed.
Culture
Isolating the fungus from a clinical sample using an adequate culture medium is considered the gold standard method for the diagnosis of a fungal infection. Specific media for fungal culture commonly used are Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), malt extract agar (MEA), and, less commonly, brain heart infusion (BHI). Optimally, cultures should be incubated at 301C(711C) during 7 days, but some fungal species require longer incubation periods, up to several weeks (Sutton, 2015). Although this technique is simple and cheap, it is time consuming and its diagnostic performance depends on sample origin, fungal species, underlying disease of the patient, clinical status, etc. Here we cite some examples: (1) Blood cultures, which has been used mainly in Candida spp. invasive infections, are frequently negative in other infections even if they are disseminated (Alexander and Pfaller, 2006), (2) Mucorales species are fast growing but the yield in cultures from clinical samples is low, probably due to the fragility of their hyphae (Ribes et al., 2000), (3) endemic fungi are fastidious and slow-growth organisms that requires 3-4 weeks to growth (Buitrago et al., 2011), etc. Finally, false positive results are common since fungi are usual contaminants of the laboratory and part of the saprophytic human flora (Cuenca-Estrella et al., 2008).
Once the culture is obtained, the identification of the grown fungus is essential, especially in invasive disease, in order to establish an appropriate antifungal therapy. Classical fungal identification requires the visualization and identification of the different fungal structures by microscopy. In the case of yeasts, also biochemical tests of carbon assimilation and/or fermentation are carried out to complement microscopical observations (Willinger et al., 2015). Microscopical identification requires very skilled and specialized personnel that can be common in a reference center but rare in a clinical setting. Recently, however, a fast identification is possible by using alternative techniques such as those based on mass spectrometry (MS) or molecular methods. In the last decade, MALDI-TOF technology has revolutionized microbial identification since it is easy to use and allows reducing costs as well as time response (Posteraro et al., 2013). The sequencing of specific DNA targets, mainly the ITS region of the ribosomal has been very useful for identification of fungi and taxonomical studies being considered the gold standard method for fungal molecular identification (Perlin and Wiederhold, 2017) but this specialized approach is time consuming and requires an appropriate database for sequence comparison (Irinyi et al., 2015).
Direct Visualization and Histopathology
Direct examination of clinical samples by microscopy to detect fungal structures is also a simple, cheap, and rapid method largely used in Microbiology laboratories. Samples are visualized after being treated with KOH 10% or stained with specific dyes. It is a very useful technique for some species as Pneumocystis jirovecii (immunofluorescent staining) (Tasaka and Tokuda, 2013)or Cryptococcus neoformans (Indian ink staining) (Baddley and Dismukes, 2011), but in general, it lacks sensitivity and negative results never rule out an infection by fungi.
Histopathology uses tissue samples to identify fungal structures by microscopy. If fungi are suspected, the specimens are first colored with stains that highlight the fungal wall as Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS). This method, although is an important tool for the microbiologist, has also several limitations. Moreover, obtaining biopsies of the affected tissue is a very invasive procedure and may be contraindicated in certain patients (Guarner and Brandt, 2011; Perfect, 2013). In other cases, the amount of material obtained is very limited making histopathological studies impossible.
Both methods enable the identification of fungal structures and they can provide a presumptive diagnosis while waiting for additional test for identifying the species involved, as the presence of common fungal elements such as hyphae or yeasts in samples or tissues does not provide sufficient information to identify the species (Lease and Alexander, 2011). The sensitivity for microscopic morphological techniques have been reported that varies from 20% to 80% (Guarner and Brandt, 2011). Finally, very skilled personnel is required for both methods as it is difficult to identify fungal structures in tissues and misidentifications are frequent. Diagnosis by these methods should be descriptive and correlated with clinical and epidemiological data.
New Diagnostic Methods
To overcome weaknesses of conventional diagnostic methods, alternative approaches based on the detection of antibodies, antigens, DNA, and other biomarkers have been developed and they are now routinely used as complementary diagnostic tools in clinical settings.
Antibody Detection
Complement fixation (CF), immunodiffusion (ID), and enzyme-immunoassay (EIA) are the most common techniques used to detect antibodies in the serum of patients with suspicion of fungal infection. Nowadays, these tests are mainly used for the diagnosis of endemic mycoses and chronic or allergic forms of aspergillosis. The sensitivity of these methods varies depending on the test (i.e., 59%–88% in chronic pulmonary aspergillosis; Page et al., 2016) and/or the type of disease (i.e., 64%–97% for histoplasmosis; Falci et al., 2017).
The main advantage of serological tests is the requirement of minimally invasive samples. Furthermore, results may be obtained when culture is negative and, if positive, the need of handling potentially infectious fungi is reduced (Kozel and Wickes, 2014). However, sensitivity is very limited in immunosuppressed patients due to the low antibody response and interpretation of serological results could be challenging since seropositivity remains long time after disease (Ramanan et al., 2017; Richardson and Page, 2018).
Antigen Detection
The detection of fungal antigens in human body fluids has revolutionized the early diagnosis of invasive fungal infections. Several tests based on the detection of these antigens have been commercialized and included in the revised EORTC/MSG criteria for diagnosis of IFIs (Donnelly et al., 2019). Table 1 summarizes main characteristics, sensitivity, and limitations of each technique.
Detection of 1,3-b-D-glucan (BDG) for diagnosis of fungal infection
BDG is a component of fungal wall, except in Cryptococcus spp. and Mucorales, which is released during infection. There are several commercial assays developed but only Fungitell (Associates of Cape Code, Inc., East Falmouth, MA, USA), a chromo genic quantitative enzyme immunoassay (EIA), has been approved by FDA (Theel and Doern, 2013). The detection of this antigen in serum has a great sensitivity in some important fungal infections as invasive candidiasis (IC), invasive aspergillosis (IA), and Pneumocystis Pneumonia (PCP) (Ambasta et al., 2015; Cuenca-Estrella et al., 2012; Karageorgopoulos et al., 2011). However, there are many factors affecting the performance of the technique: (1) It cannot discriminate at species level, (2) causes a high rate of false positive results, (3) is presented in a closed commercial system, which commonly implies the shipment of sample to a reference laboratory, and (4) the efficiency of this test is reduced in solid organ transplant (SOT) patients (Cuenca-Estrella et al., 2011; Lu et al., 2011; Perfect, 2013).
Detection of galactomannan (GM) and glycoproteins for diagnosis of IA
The detection of GM, a polysaccharide present in the cell wall of most Aspergillus spp. which is secreted during infection, is commonly performed by using the commercial assay Platelia Aspergillus Ag EIA (Bio-Rad, Marnes-la-Coquette, FR). This technique has a great efficiency in serum of patients with hematological malignancies or hematopoietic stem cell transplant (HSCT) recipients, but sensitivity is drastically reduced in SOT patients, especially lung transplant recipients. In addition to serum, GM can also be efficiently detected in other samples such as bronchoalveolar lavage fluid (BALF), advancing IA diagnosis (Ambasta et al., 2015; Steinbach, 2015). Despite these strengths, several limitations, such as cross-reactivity with other fungi and false positive (i.e., antibiotic treatment) or false negative results (i.e., antifungal treatment), deeply affect sensitivity and specificity of the technique (Lease and Alexander, 2011).
Last years, a new diagnostic technology has been developed based on immuno-chromatographic assays performed in lateral-flow devices (LFD). These “pregnancy tests-like” assays are cheap, sensitive, easy to use, and have low turnaround time (Prattes et al., 2016). The Aspergillus-specific LFD test is based on the detection of an extracellular glycoprotein (different from GM) secreted by Aspergillus spp. constitutively during active growth (Thornton, 2008). This test can be performed in serum and BALF samples with comparable sensitivity to GM detection and higher specificity, although it shows cross-reaction with Penicillium spp. (Heldt and Hoenigl, 2017).
Detection of capsular polysaccharide antigen (CPA) for the diagnosis of cryptococcosis
Latex-agglutination (LA) is the most used method for the detection of the CPA of Cryptococcus spp, particularly a component called glucuronoxylomannan (GXM), which is shed into blood and cerebrospinal fluid (CSF) during infection (Yauch et al., 2005). This method can be performed in both serum and CSF being very useful for the diagnosis of cryptococcosis, especially in AIDS patients with cryptococcal meningitis, however the performance is reduced in patients with only pulmonary involvement (Baddley and Dismukes, 2011). An LFD is also available for the detection of CPA of Cryptococcus spp. with sensitivity even superior to the LA assay (Nalintya et al., 2016).
Detection of mannan (MNN) for diagnosis of invasive candidiasis
The combined detection of MNN, a highly immunogenic component of Candida spp. cell wall that circulates in blood during infection, and anti-MNN Igs can be used for specific detection of Candida spp. in serum samples (Sendid et al., 1999). EIAs quantifying MNN and anti-MNN Igs are commercialized as Platelia Candida Ag Plus and Platelia Candida Ab Plus (Bio-Rad, Marnes-la-Coquette, FR) and the combined detection shows acceptable sensitivity for the diagnosis of IC, although it is reduced in non-albicans Candida infections (Mikulska et al., 2010).
Detection of endemic fungal antigens (EFA) for the diagnosis of endemic fungal infections
The detection of antigens produced by fungal species causing endemic mycoses such as H. capsulatum, P. brasiliensis, C. immitis, and B. dermatitidis represents a breakthrough in the diagnosis of endemic fungal infections.
The H. capsulatum polysaccharide antigen can be detected in both serum and urine samples with similar diagnostic value (Fandino-Devia et al., 2016) and it can be performed by using two commercial assays: (1) Histoplasma Quantitative EIA Test (MiraVista Diagnostics, Indianapolis, IN, USA), and (2) the Histoplasma Antigen EIA kit (IMMY, Norman, OK, USA). MiraVista’stest presents great efficiency in serum and urine samples from patients with disseminated infection, but it is reduced in patients with pulmonary forms of the disease (Hage et al., 2015), in which BALF samples are more suitable for diagnosis (Hage et al., 2011).
This test is only performed in MVista’s facilities then is not accessible out of USA limiting their use. On the other hand, IMMY’stest demonstrates a good agreement with MiraVista’s but it has been only tested in urine samples (Falci et al., 2017). Recently, a promising monoclonal Histoplasma galactomannan enzyme-linked immunosorbent assay has been developed by IMMY showing great performance and reproducibility (Cáceres et al., 2018). MVista Diagnostics has also developed EIA tests for the diagnosis of coccidioidomycosis and blastomycosis. In the case of the detection of Coccidioides spp. antigen, it can be performed, in addition to serum and urine, in CSF samples. This means a great advantage in the diagnosis of coccidioidal meningitis, although sensitivity is lower in single respiratory infections (Kassis et al., 2015). Finally, sensitivity for the detection of B. dermatitidis antigen varies deeply depending on the specimen employed, being more efficient in urine samples, however sample pre-treatment can enhance diagnostic performance (Smith and Gauthier, 2015). The main limitation of many of these assays is the high degree of cross-reaction with other fungi (Malcolm and Chin-Hong, 2013).
DNA-Based Methods/Molecular Methods
Methods based on the detection of DNA have been developed recently for the diagnosis of fungal infections. Their powerful advantages over conventional methods (simplicity, high specificity, and short turnaround time) have made them potential candidates to replace those traditional diagnostic assays (Arvanitis et al., 2014).
PCR-based methods
The most relevant DNA detection methods are those that implement polymerase chain reaction (PCR). This technique is playing an increasingly role in the routine diagnostic of several laboratories of clinical microbiology as they offer a rapid, specificand sensitive detection of pathogens (Buchan and Ledeboer, 2014). Furthermore, quantitative or real-time PCR (qPCR) assays allow for determining the fungal DNA burden in patients by using non-specific DNA-binding dyes or fluorescently labeled probes (Kozel and Wickes, 2014). Despite of the advantages, most reported assays have been developed in house by different laboratories and just a reduced number of tests have been commercialized. Moreover, these techniques have several limitations: (1) The contamination risk of samples or the technique itself by environmental fungi, (2) the moderate amount of DNA in low invasive samples such as blood and serum, (3) the lack of standardization, and (4) the low availability of widely validated commercial systems (Alanio and Bretagne, 2017; Khot and Fredricks, 2009). Due to all these disadvantages, PCR has classically been excluded of the EORTC/MSG criteria for IFI diagnosis, however all efforts made last years have achieved its inclusion in the last update (Donnelly et al., 2019).
Nowadays, there are two approaches for the diagnosis of fungal infections by using PCR:
(1) Specific qPCR assays: The greatest efforts have been made in the detection of species causing important fungal infections such as IC, AI, and PCP (Fig. 3). However, there are considerably fewer qPCR studies for the detection of fungi causing neglected infections as histoplasmosis or emerging fungal infections as those caused by Scedosporium spp. or Fusarium spp. among others. Most developed methods target specific multicopy regions of the ribosomal DNA and are performed by using conventional or real-time PCR. One interesting approach based on specific qPCR assays is multiplex qPCR (MRT-qPCR), since it allows for detecting several pathogens in the same reaction tube. These assays are very useful for the differential diagnosis of fungal species with non-specific symptomatology (Gago et al., 2014) or with different antifungal susceptibility profiles (Alonso et al., 2012; Bernal-Martinez et al., 2013; Foongladda et al., 2014),andalsoallowsforthe detection of mixed infections. Recently, many efforts have been made in Europe, to try to achieve a consensus in the development of PCR diagnostic methods with the purpose of being included in the international guidelines (FPCRI).
(2) Panfungal or broad-range PCR assays (Fig. 3): These assays are very useful when there is not a clear suspicion of the fungus involved in the infection. Universal primers able to amplify any fungal species are used to detect fungal DNA in the clinical sample. Sensitivity values of these assays in paraffin-embedded biopsies range between 86%–94% in culture-proven cases and 64%–89% in cases confirmed by histopathology (Powers-Fletcher and Hanson, 2016). One of the main limitations of these techniques is the delay in response time due to the requirement of sequencing the amplified panfungal PCR product. Furthermore, specimen contamination and commensal fungi colonization of body sites of sampling could deeply affect the specificity of the technique (Khot and Fredricks, 2009). Some recent studies have developed other panfungal methods directed to reduce response time. One of these methods complements the use of panfungal primers with a melting curve analysis and probes able to detect genera or the group of fungi (Valero et al., 2016). Others involve different identification techniques such as DNA microarray (Sakai et al., 2014), electrospray-ionization MS analysis (Massire et al., 2013), or T2 magnetic resonance (Pfaller et al., 2016).
Fig3. Comparative scheme describing main characteristics, advantages, and limitations of both specific and panfungal PCR diagnostic techniques.
Non-PCR based methods
Alternative DNA-based methods for the diagnosis and identification on fungal infections are those based on in situ hybridization of labeled probes. The PNA-FISH (peptide nucleic acid-fluorescence in situ hybridization) method employs fluorescently labeled probes that bind complementary to fungal sequences and has been often used to identify Candida spp. from positive blood culture bottles. The probe color detected gives a preliminary indication of fluconazole susceptibility associated to the species (Klingspor et al., 2018). Similarly, chemiluminescent DNA probes are commercially available for the identification of endemic fungal species from positive cultures (Gomez, 2014)or tissues(Guarner and Brandt, 2011).
Future Directions and Conclusion
The aim of this chapter is to summarize those diagnostic methods commonly used for the diagnosis of fungal infections. As stated before, diagnosis of these infections is difficult and the combination of conventional and new techniques is essential for an early diagnosis. In addition, it is important to know the local epidemiology and prevalence of fungal infections as it can help choose the most appropriate diagnostic method that should be used (Lass-Florl, 2017).
Nevertheless, fungal diagnostics is an area under continuous evolution since novel tests and new applications of already existing techniques are in constant development. In that context, the search for new biomarkers (both from the fungal pathogen and the human host) is a main objective for researchers. The detection of new fungal cell wall polysaccharides and second metabolites such as mycotoxins (Escriva et al., 2017) and volatile organic compounds (VOCs) (Acharige et al., 2018) have been reported in last years. Regarding already established techniques, the application of MALDI-ToF MS technology directly in clinical samples (Rizzato et al., 2015) have also gained great interest. In the case of the study of host factors, the increasing use of proteomic and bioinformatic approaches has promoted the research in this area with the aim of being able to predict susceptibility and risk of acquiring fungal infections as well as offering a personalized diagnosis (Lydon et al., 2018). Although many work should be done in this sense in coming years, several efforts have been already made in concern of some fungal infections such as IA (Oliveira-Coelho et al., 2015).
Alternatively, the development of “point of care” (POC) techniques have increased last years as an attempt to obtain robust and cheap methods to be used in less favored regions of the world. LFDs for the detection of Cryptococcus and Aspergillus are com mercially available as previously mentioned, and one more for the diagnosis of histoplasmosis has just been released by MiraVista Diagnostics (Cáceres et al., 2019).
In summary, fungal infections are currently recognized as a cause of significant morbidity and mortality, especially among immunocompromised patients. Consequently, a proper and early diagnosis is essential to establish a suitable antifungal therapy that could allow for improving patient’s outcome. Although the ideal diagnostic method or strategy is still missing, several efforts have been made in last years, which have led to a new generation of diagnostic tools. Undoubtedly, so much remains to be done in this context, from widely standardizing and validating already developed tests through large multicenter studies to exploring combination testing or adapting antigen and DNA detection methods to POC platforms. All this efforts will end in the construction of a proper panel of fungal diagnostic methods that could be available wherever it is required.
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