When compared with antibacterial research, little progress has been made in the development of new antifungal agents, which has been justified by the low occurrence of fungal infections. However, the current increase in incidence of fungal infections has led to aggressive research on new antifungal agents as evidenced by the rise in the number of publications since the 1960s (Maertens, 2004; Ngo et al., 2016). Another reason for the slow development of antifungal agents is the fact that fungi are eukaryotic, with a close evolutionary relationship with human hosts, which complicates the search for antifungal targets. Nonetheless, detailed knowledge regarding the structure, composition and biochemistry of fungal cells, in addition to various facets of fungal infections, has contributed to our understanding about the mechanism of action of many antifungal agents (Borgers, 1980; Kanafani and Perfect, 2008). Typically a long period of 8 to 10 years is required for an antifungal to be approved for clinical use. Reducing toxicity, enhancing bioavailability, improving the antifungal spectrum and combating resistance are efforts that are expected to increase the efficacy of the available antifungals. Indeed, elucidation of the mode of action of a potential antifungal compound can shorten the time from lead to candidate drug. Small antifungal molecules from natural products could represent structural templates for structure-activity relationship studies, thus providing more information to optimize potential new antifungal agents (Sheng and Zhang, 2011). Overall, new strategies regarding antifungal therapy, target identification and rational drug design technologies can significantly accelerate the process of new antifungal development, reducing the time to cure or providing better quality of life to patients.

Although the commercially available antifungal agents to date have targets that are restricted to the plasma membrane and the cell wall (Odds et al., 2003; Sundriyal et al., 2006; Ngo et al., 2016), a certain diversity of targets has been discovered. To develop new therapies, recent studies have focused on the inhibition of fungal virulence factors. Some mechanisms of action are described below, and an overview is presented in Figure 1.

The widespread use of antifungal agents and the limited arsenal associated with the increased number of opportunistic infections have resulted in the progression of resistance to available drugs. The antifungal resistance mechanism may occur through different conditions such as a decrease in the effective drug concentration, changes or overexpression of the drug targets, and metabolic bypasses (Sanglard, 2016). Figure 2 depicts an overview of several antifungal resistance mechanisms described for Candida spp.

The ability of many fungi to form biofilms is one of the reasons for antifungal drug resistance. Many medically important fungi are described as biofilm-forming organisms, such as Candida spp. (Chandra et al., 2005; Hinrichsen et al., 2008; Finkel and Mitchell, 2011; Pires et al., 2011), Pneumocystis spp. (Cushion et al., 2009), Coccidioides spp. (Davis et al., 2002), Aspergillus spp. (Mowat et al., 2009; Kaur and Singh, 2014), Zygomycetes (Singh et al., 2011), Malassezia spp. (Cannizzo et al., 2007; Figueredo et al., 2012), Trichosporon spp. (Di Bonaventura et al., 2006; Colombo et al., 2011), Cryptococcus (Walsh et al., 1986; Martinez and Casadevall, 2007), Histoplasma capsulatum (Pitangui et al., 2012), Trichophyton spp. (Costa-Orlandi et al., 2014) and Paracoccidioides spp. (Sardi et al., 2015).

Biofilms are highly structured and complex microbial communities embedded in a self-produced extracellular matrix (ECM) that attach to a wide range of surfaces and (Fanning and Mitchell, 2012). Several factors contribute to initial surface attachment, such as pH, temperature, osmolarity, flow of the surrounding biologic medium, host immune factors and even the presence of antimicrobial agents (Baillie and Douglas, 2000; Chandra et al., 2001; Ramage et al., 2008).

Fungal biofilm formation occurs through a sequential process including planktonic cell adhesion to an appropriate substratum, colonization, ECM production, biofilm maturation, and dispersion (Fanning and Mitchell, 2012). Despite these specific characteristics, all types of fungal biofilms have distinct properties from planktonic yeast cells and increase antifungal drug resistance up to 1000-fold (Ramage et al., 2001; Uppuluri et al., 2010). Indeed, several studies have shown the inefficacy of antifungal therapy against different fungal biofilms.

Multiple other biofilm-specific factors contribute simultaneously to the resistance of yeasts to antifungal drugs, including cell density, quorum sensing, efflux pump activity, persister cells, ECM presence, stress responses and overexpression of drug targets.

The cell density is an important factor that contributes to the antifungal resistance of biofilms. However, some studies show that this is not a biofilm-specific resistance mechanism because a similar trend was observed for planktonic cells. Perumal et al. (2007) studied the efficacy of different azoles, AmB and caspofungin on planktonic cells at densities similar to those found in biofilms. The susceptibility of dissociated biofilm cells was similar to that of planktonic cells at the same cell density, and this susceptibility decreased as the density of the cells increased (Perumal et al., 2007). This phenomenon was also demonstrated by Seneviratne et al. (2008), who noted the density-dependent susceptibility of planktonic or biofilm for ketoconazole and 5-FC. Lass-Florl et al. (2003) showed similar drug resistance results by increasing the inoculum sizes of Aspergillus species, supporting the idea that the physical density of the cells influences antifungal agent activity.

By providing microorganisms with the ability to communicate and coordinate population growth/morphology via the secretion of signaling molecules, cell density can be considered a key aspect of the quorum-sensing process (Ramage et al., 2009, 2012). QS for fungi was first described in C. albicans by Hornby et al. (2001), who identified farnesol as a molecule that inhibits the hyphal transitional stage of C. albicans and increases adhesion (Saville et al., 2003; Ramage et al., 2009). Exposing C. albicans to farnesol also resulted in alterations in gene expression involving hyphal developmental genes (TUP1 and CRK1), cell surface hydrophobicity genes and those involved in drug resistance (FCR1 and PDR16) (Cao et al., 2005; Enjalbert and Whiteway, 2005). In addition, farnesol induces apoptosis in both A. nidulans and Fusarium graminearum (Semighini et al., 2006, 2008). Tyrosol, the second QS molecule identified in C. albicans, promotes germ tube formation. According to Alem et al. (2006), tyrosol enhances the early phase of biofilm formation and may also inhibit farnesol activity, thereby controlling cell population morphology.

Khot et al. (2006) for the first time studied the mRNA levels of genes involved in ergosterol biosynthesis (ERG genes) and in β-1,6-glucan biosynthesis (SKN1 and KRE genes) in comparison between planktonic and biofilm-associated cells. The authors described the appearance of a unique transcript profile in a subpopulation of AmB-resistant blastospores with significant upregulation of ERG25, SKN1, and KRE1 and downregulation of ERG1. Mukherjee et al. (2003) reported that ergosterol levels were significantly decreased in intermediate and mature phases when compared to early-phase biofilms, suggesting that the biofilm resistance to azole might be explained by ergosterol alterations in biofilm membranes. Subsequent studies (Borecká-Melkusová et al., 2009; Nett et al., 2009; Nailis et al., 2010) compared the exposure of young and mature biofilms to fluconazole, concluding that both induce downregulation of genes encoding enzymes involved in ergosterol biosynthesis (CaERG1, CaERG3, CaERG11, and CaERG25). In addition, treatment of both young and mature biofilms with AmB predominantly resulted in overexpression of CaSKN1, with only modest upregulation of CaKRE1 (Nailis et al., 2010).

Induction of ergosterol pathway genes has been described in different biofilms of Candida species. For example, incubation with fluconazole caused upregulation of CdERG3 and CdERG25 in C. dubliniensis (Borecká-Melkusová et al., 2009) and of genes involved in ergosterol biosynthesis in C. parapsilosis, resulting in antifungal resistance (Rossignol et al., 2009).

Moreover, drug efflux pumps also participate in drug resistance of biofilms. Ramage et al. (2002) investigated the role of efflux pumps, ABC and MFS transporters, of C. albicans biofilm. Expression of CDR genes predominated at the beginning of biofilm formation (24 h), whereas MDR1 was solely overexpressed after 24 h. Expression of CDR1, CDR2, and MDR1 showed that C. albicans was susceptible to azoles when grown planktonically but exhibiting resistance when grown in a biofilm. Several subsequent studies confirmed these results, suggesting that the expression of these genes is necessary for biofilm resistance (Mukherjee et al., 2003; Perumal et al., 2007). Gao et al. (2014) showed that fluconazole increased the expression of CDR1, CDR2 and MDR1 and that the combination with doxycycline downregulated the gene overexpression induced by FLZ.

Later, Nett et al. (2009) performed an in vivo study of C. albicans biofilm formation on implanted catheters and showed upregulation of genes during biofilm development: CDR2 at 12 h and MDR1 at both 12 and 24 h. Similar results were reported for other Candida species, including C. glabrata, in which the expression of CDR1 and CDR2 was found at early (6 h) and intermediate (15 h) biofilm stages, even though neither gene was upregulated at the mature phase (48 h) (Song et al., 2009). C. tropicalis showed increased expression of MDR1 after 24 h of biofilm formation (Bizerra et al., 2008). In A. fumigatus, the initial phase of biofilm formation was associated with increased activity of the efflux pump MDR and gene upregulation at 8 h when treated with voriconazole (Rajendran et al., 2011). Thus, all reports support the idea that efflux pump expression is a mechanism of biofilm resistance, especially in the early phase of biofilm growth until ECM production.

The ECM is considered one of the essential mechanisms of biofilms resistance, conferring enhanced antimicrobial resistance and protection from host immune responses (Sutherland, 2001; Davies, 2003). The ECM may act as an adsorbent, reducing the amount of antimicrobial available to interact with the biofilm, and the structure physically reduces the penetration of antimicrobial agents by walling off access to regions of the biofilm (Taraszkiewicz et al., 2013). The matrix is composed of a variety of proteins, nucleic acids, phospholipids, lipids, amyloid fibers, humid substances, and in some cases, surprising amounts of extracellular DNA (e-DNA) (Estrela et al., 2009; Dogsa et al., 2013). The ECM confers important characteristics to biofilm, such as providing for mechanical stability, an external digestive system, and intense cell interactions, including cell–cell communication and synergistic microconsortia, and serving as a nutrient, energy, and recycling source (Flemming and Wingender, 2010).

During biofilm formation, β-1,3 glucan is one of the principle carbohydrate components of the ECM (Ramage et al., 2012). It is responsible for sequestering azoles, echinocandins, pyrimidines, and polyenes, acting as a “drug sponge,” and confers resistance to C. albicans biofilms (Nett et al., 2010a,b). In addition, the ECM of non-albicans Candida strains also contains β-1,3 glucan, which contributes to azole resistance via specific binding (Mitchell et al., 2013). FKS1 was the first gene described as encoding β-1,3 glucan synthase of C. albicans (Nett et al., 2010a). Other genes essential for the C. albicans ECM are SMI1 and RLM1, which are involved in the protein kinase C cell-wall integrity pathway, controlling the cell wall glucan content in response to stress. Recently, Taff et al. (2012) showed that glucan transferases and exoglucanase are crucial for the accumulation and delivery of β-1,3 glucan to the matrix.

Biofilm presents persister cells, which are directly correlated with accumulating high concentrations of antimicrobial agents. In essence, a persister is a dormant cell that with little or no cell wall synthesis; drugs bind to their target molecules but are unable to promote cell death (Lewis, 2007). The simplest route to form a dormant persister cell might be through the overproduction of proteins that are toxic to the cell and inhibit growth (Lewis, 2010). Different from bacteria, these cells have only been detected in yeast biofilms and not in planktonic populations (LaFleur et al., 2006). Due to the ECM, the persisters present in the biofilm can withstand both antifungal treatment and the immune system (Lewis, 2007). Persisters may be mainly responsible for re-infection once when the concentration of antimicrobial decreases, and they can repopulate the biofilm (Lewis, 2001). Recently, a dose-dependent study involving AmB and chlorhexidine against C. albicans in planktonic and biofilm forms reported complete elimination of planktonic cells in both exponential and stationary stages. However, biphasic killing occurred in the mature biofilm, suggesting the presence of persisters. After AmB treatment, surviving C. albicans in the biofilm capable of producing a new biofilm with a new subpopulation of persisters, suggesting that yeast persisters are not mutants but phenotypic variants of the wild-type population (LaFleur et al., 2006). In addition, Sun et al. (2016) showed that C. albicans AMB-tolerant persisters were produced mainly during the adhesion phase and that the maintenance of these cells was dependent of surface adhesion. Further studies showed the presence of persister cells after the treatment of C. krusei and C. parapsilosis biofilms with AmB. However, the biofilm of C. albicans SC5314 under the same treatment condition did not reveal surviving cells, indicating a lack of persister cells (Al-Dhaheri and Douglas, 2008).

Based on the above, it is possible to affirm that biofilm confers antifungal resistance and that this occurs through various mechanisms. Accordingly, the discovery of new strategies to overcome these microbial communities has been deeply researched.

The development of new drug formulations is one of the main options of studies, including echinocandins and AmB lipid forms that are inhibit fungal biofilm both in vitro (Kuhn et al., 2002; Ramage et al., 2013) and in vivo (Mukherjee et al., 2009; Kucharikova et al., 2013).

The association of drugs is another example of a strategy against fungal biofilms. Some studies have reported the efficacy of a combination of AmB and aspirin (Zhou et al., 2012), FLZ and doxycycline (Gao et al., 2013), and caspofungin and diclofenac (Bink et al., 2012). The sensitization of C. albicans biofilms to different antifungals by the immunosuppressant drug cyclosporine A also resulted in enhanced biofilm inhibition (Shinde et al., 2012).

The presence of e-DNA, which is an important component of the ECM, has prompted some strategies using DNase to decrease biofilm biomass and to enhance the activity of antifungal agents (Martins et al., 2010). Lactonases and α-amylases are also used to control fungal biofilms (Taraszkiewicz et al., 2013). Moreover, an interesting therapeutic option is to block persister survival. Bink et al. (2011) discovered that superoxide dismutase (SOD) may be an inhibitor of N,N’-diethyldithiocarbamate (DDC) in C. albicans biofilms, reducing the miconazole-resistant persister fraction by 18-fold (Bink et al., 2011).

The application of antimicrobial photodynamic therapy has been investigated for anti-fungal biofilm properties with regard to the efficiency of inhibiting several microorganisms, with minimal damage to the host cell (Biel, 2010). This therapy involves the combination of photosensitizer (PS), light and molecular oxygen (de Melo et al., 2013). In addition, strategies involving nanoparticles (Allaker, 2010; Ramasamy et al., 2016), plant extracts (Wojnicz et al., 2012), and chitosan (Carlson et al., 2008) have been applied against microbial biofilm, with significant results. Further studies are necessary for identifying means to overcome fungal biofilm resistance.

Based on the problems discussed above with regard to antifungal treatment, one of the options is the combination of drugs. Using more than one drug can increase efficacy due to the possibility of action on more than one target; in addition, toxicity is reduced because less of the drug is used (Chen et al., 2014). Drug combinations can lead to improved activity, such as synergist activity, or decrease antagonist action. Evaluation of the effect of a drug combination in vitro can be realized by the checkerboard method (Johnson et al., 2004).

Most studies on the in vitro and in vivo combination of azoles with AmB do not show synergistic activity, though previous treatment with azole can influence the action of polyenes. This can be explained by the fact that both drugs have the same target: ergosterol. However, because azoles inhibit ergosterol biosynthesis, less ergosterol is available for polyene to bind (Louie et al., 2001; Sanglard, 2002). Nonetheless, invasive mucormycosis has been successfully treat with an antifungal combination of AmB and posaconazole (Pagano et al., 2013).

There are numerous in vitro studies of antifungal combinations using triazole with echinocandin or triazole with AmB (Elefanti et al., 2013; Katragkou et al., 2014). Regardless, the clinical results of combinatory treatment remain unclear and are generally described for infections caused by fungi, which present difficulty in treatment, such as in aspergillosis and mucormycosis (Belanger et al., 2015). The combination of caspofungin and voriconazole showed in vitro synergistic activity against Aspergillus spp. (Walsh et al., 2008). Moreover, this combination showed efficacy in a mammalian model (Kirkpatrick et al., 2002). Treatment of patients with invasive aspergillosis with a combination of voriconazole and anidulafungin improved survival in comparison with treatment with voriconazole monotherapy (Marr et al., 2015). However, in another study, triazole and echinocandin combination showed the same effect as triazole alone (Raad et al., 2015).

The antifungal 5-FC in combination with AmB or with azole did not show synergistic activity for Candida sp. (Scheid et al., 2012). However, this was efficient for the treatment of cryptococcal meningitis. It should be noted that the lack of availability of 5-FC results in the use of less effective combinations in many countries (Perfect et al., 2010). Moreover, this compound is associated with rapid development of resistance (Vandeputte et al., 2011).

Interaction with non-antifungal agents has also been described as potentiating antifungal activity. Triclosan, a compound exhibiting antimicrobial activity, is widely used in soap, toothpaste, and other personal care products. This molecule showed in vitro synergistic activity with FLZ against C. albicans (Yu et al., 2011). Triclosan is also active against C. neoformans by activating the apoptosis pathway and also shows synergic activity with AmB and FLZ (Movahed et al., 2016). As ion homeostasis is a fundamental factor in the development of fungal disease, ion chelators have been used in the treatment of fungal infections. However, synergistic activity with antifungal drugs did not result in favorable responses in vitro (Lai et al., 2016). Treatment of cardio-vascular disorders has been realized with calcium channel blockers, which also demonstrate antifungal activity (Yu et al., 2014). Recent work shows that the calcium channel blockers amlodipine, nifedipine, benidipine, and flunarizine present synergistic activity with FLZ in C. albicans isolates resistant to FLZ via a mechanism not related to efflux pump inactivation (Liu S. et al., 2016).

Natural source molecules with antifungal activity are also described as having in vitro synergistic activity with traditional antifungal drugs, reducing the concentration of both substances (Soares et al., 2014; Sardi et al., 2016; Wang et al., 2016). Use of the natural substance beauvericin with traditional antifungals was able to inhibit efflux pumps and morphogenesis in FLZ-resistant C. albicans and potentiate the action of this azole (Shekhar-Guturja et al., 2016).

Two different strategies have been developed to increase the therapeutic index of antifungal agents: chemical modifications and/or elaboration of new formulations of antifungal agents to obtain less toxic derivatives (Sheng and Zhang, 2011).

Theoretical and experimental studies on the mechanism of action of AmB and its derivatives were performed by Borowski (2000). Two generations of derivatives were developed. First-generation compounds are modified at the carboxyl group, which improves selective toxicity based on disturbance of the hydrogen bond network in complex with sterols. Second-generation compounds have introduction of a bulky substituent, resulting in an appropriate steric hindrance effect that disturbs interaction with cholesterol but not with ergosterol, leading to improved selective toxicity (Borowski, 2000). Among second-generation derivatives, N-methyl-N-D-fructosyl AMB methyl ester (MFAME) is considered the most interesting compound because it is able to form water-soluble salts, has a broad antifungal spectrum, and lower toxicity than AMB toward animal cells in in vitro and in vivo experiments (Szlinder-Richert et al., 2001).

Despite the huge effort made to decrease fungal resistance and the toxicity of AmB, the development of rational chemical modification of known antifungal agents was insufficient to solve these problems. Thus, new delivery systems have been evaluated to reach this goal (Borowski, 2000; Sheng and Zhang, 2011).

Since 1990, nanostructured systems have been studied as carriers of antifungal agents. Clinically, the intravenous dosage form of AmB-deoxycholate has adverse effects, mainly nephrotoxicity. The synthesis of AmB analogs such as AmB esters or a preparation including an AmB lipid complex, AmB colloidal dispersion, liposomal AmB and intralipid AmB have been generated to improve the therapeutic index and lower toxicity (Gupta and Tomas, 2003; Vyas and Gupta, 2006; Voltan et al., 2016).

In addition, other delivery systems, such as carriers based on solid and nanostructure lipids, synthetic and natural polymers, inorganic and metal nanostructure lipids, dendrimers, silica, and carbon materials (magnetic nanoparticles), have been pursued. These delivery systems are able to improve bioavailability and reduce toxicity and present specificity for target tissues; however, there is an associated high cost of production (Vyas and Gupta, 2006; Sato et al., 2015; Voltan et al., 2016).

Recent work shows that the composition of nanoparticles used as delivery vehicles is fundamental for increasing antifungal activity. Ahmad et al. (2016) produced a conjugate system with AmB and metal nanoparticles that displayed synergistic antifungal activity due to the antimicrobial property of silver against C. albicans and C. tropicalis. Niemirowicz et al. (2016) reported synergistic activity for the combination of polyenes and magnetic nanoparticles. This bio-active nano-sized formulation exhibited enhanced efficiency against two clinical isolates of Candida species in planktonic and biofilm states.

For many years, the only available antifungal for invasive fungal infections was AmB, which has been incorporated into three lipid formulations. However, the imidazole class offers new treatment options because it is proven to be less toxic and in some cases more effective than AmB (Shalini et al., 2011). Although the imidazole class has been available for a decade, improvements in safety were necessary. Accordingly, this class was also subjected to rational chemical modifications. The triazole class was generated by the addition of a nitrogen atom to a cyclic ring. This modification provided a broad spectrum of activity as well as improved safety and pharmacokinetic profile (Allen et al., 2015). In this sense, the introduction of triazoles accelerated the pace of drug development.

New azoles have been developed to combat resistant pathogens to improve the tolerability and administration. Voriconazole is structurally similar to FLZ, with the exception of a fluoropyrimidine group in place of a triazole moiety, which leads to better bioavailability. Alternatively, posaconazole has a spectrum of antifungal activity comparable to that of voriconazole, but the molecular structure with a hydroxylated analog is similar to that of itraconazole. Currently, isavuconazole, ravuconazole, albaconazole, and efinaconazole are the four best studied agents in research aiming to identify an ideal antifungal with a wide spectrum and reliable pharmacokinetics, parenteral and oral dosage forms, and a favorable adverse effect profile (Shalini et al., 2011; Allen et al., 2015).

In addition, Moazeni et al. (2016) reported that the combination of solid lipid nanoparticles with FLZ was able to avoid recognition by efflux pump proteins, preventing extrusion when tested against FLZ-resistant Candida isolates. In another study, an aqueous nano-suspension of itraconazole for the treatment of bronchopulmonary aspergillosis solved pharmacokinetic problem of adequate concentration (Rundfeldt et al., 2013).

In addition to all azole agents, echinocandins are among the newest class of antifungal agents and act by inhibiting glucan synthesis in the fungal cell wall. Echinocandins are composed of a complex hexapeptide core with an N-terminus acylated by a long hydrophobic chain. Although echinocandins are fungicidal with good selectivity, they cannot be orally administered because of their complex lipopeptide structure. To address the limitation, several small molecule glucan synthesis inhibitors have been discovered. However, none is under clinical evaluation thus far (Sheng and Zhang, 2011; Liu N. et al., 2016).

Given their increasing frequency and unacceptably high morbidity and mortality rates, prevention of invasive fungal infections has become of vital importance (Spellberg, 2011; Medici and Del Poeta, 2015). Vaccination of high-risk groups is a particularly promising strategy to prevent invasive fungal infections because easily identifiable risk factors are clearly defined for many such infections (Perlroth et al., 2007; Spellberg, 2011).

Advances in our understanding of the host defense and pathogenic mechanisms underlying fungal infections has supported the development of effective vaccines to combat these diseases. Accordingly, researchers have dedicated studies to developing robust, durable and safe fungal vaccines, especially those that may be useful for endemic infections or in chronic or superimposed infections in intensive care patients (Santos and Levitz, 2014; Shahid, 2016).

Although very few clinical trials have been performed in humans, a growing number of antifungal vaccine candidates are being evaluated in pre-clinical studies. This may be due to the renewed interest in the potential use of vaccines, replacing or associated with chemotherapy, to reduce antifungal drug use and consequently limit drug resistance and toxicity.

For most active vaccines studied against invasive fungal infections, the key to protection has been the induction of cell-mediated, pro-inflammatory, Th1 or Th17 responses, which improve phagocytic killing of the fungus. It is also clear that antigens targeted for vaccination need not be restricted to virulence factors, markedly increasing the antigen repertoire available for testing. Additionally, the concept of niche vaccination of acutely at-risk patients or patients in restricted geographical areas is a new idea that opens doors in vaccinology (Spellberg, 2011).

The greatest advances in fungal vaccines are with regard to studies of invasive candidiasis, and two promising vaccines are presently in the clinical trial phase. The first, containing the rAls3p-N antigen, is in Phase IIa and prevents fungal adhesion and invasion in immunized hosts. Protection with this vaccine has been found to largely be mediated by T cells via neutrophil recruitment and a specific antibody in vaccinated hosts (Edwards, 2012; Shahid, 2016).

The second candidal vaccine in clinical trial phase is a virosome-based vaccine containing a Sap2 antigen/truncated recombinant Sap2 antigen. The truncated form is stable, immunogenic and harmless. The Sap2 vaccine administered intra-muscularly or intra-vaginally induces systemic and 100% mucosal protective immunity (Vecchiarelli et al., 2012; Shahid, 2016).

Another important molecule that has been studied in candidiasis vaccination is heat shock protein 90 (Hsp90-CA). Raska et al. (2005) tested an Hsp90-CA DNA vaccine and found that vaccinated mice exhibited survival times prolonged by 64% compared with the untreated control. In addition, vaccination with the recombinant protein r-hsp90-CA significantly increased survival compared to the control groups.

A monoclonal antibody that binds to the immunodominant epitope of C. albicans Hsp90p was tested pre-clinically. A synergistic action with AMB against FLZ-sensitive and -resistant C. albicans strains was demonstrated in in vivo and in vitro experiments. The same antibody was used in a multicenter double-blind placebo-controlled trial of patients with invasive candidiasis, and complete mycological resolution reached 84% in the combination therapy group compared to 48% in the AMB monotherapy group. Moreover, clearance of infections in the combination therapy group was twice that of AMB therapy alone, resulting in 4 and 18% mortality, respectively (Matthews et al., 2003; Bugli et al., 2013; Wang et al., 2015).

In a different approach, S. cerevisiae cells were genetically engineered to display Enolase 1 (Eno1p) antigens of C. albicans on their surface. Oral administration of these cells could elicit an immune response and aid the survival of mice challenged with C. albicans (Vecchiarelli et al., 2012; Shibasaki et al., 2013; Shahid, 2016). Interestingly, by sharing some elements with Aspergillus species, Candida enolase also exerts a protective function against aspergillosis.

Studies on vaccines protective against other fungi are still in the early pre-clinical phase (Fernandes et al., 2011; Hsieh et al., 2011; Edwards, 2012; Hole and Wormley, 2012; Shahid, 2016).

For protection against aspergillosis, studies have shown that the type 1, cell-mediated immune response, which is efficient in protecting against this disease, can be induced by recombinant protein antigens from Aspergillus, as observed by Bozza et al. (2002). Administration of recombinant allergen Asp 16 f in conjunction with CpG oligonucleotides improved the survival of mice infected by inhaled A. fumigatus. Vaccination with crude antigen preparations from A. fumigatus was also tested, and it was observed that such vaccination improved the survival of mice infected by inhaled and intravenously administered fungi, even in those that were subsequently immunocompromised (Cenci et al., 2000; Ito and Lyons, 2002).

In the case of Cryptococcus, Devi et al. (1991) proposed the use of an anti-phagocytic antigen from the capsule of C. neoformans, glucuronoxylomannan (GMX), as a vaccine to elicit antibody-mediated protection. In a different approach, Wozniak et al. (2011) administered T-cell depleted mice, mimicking a T-cell-deficient host, with an engineered strain of C. neoformans that could express IFN-g. After the immunization, a secondary pulmonary infection using a pathogenic strain was produced, and the mice were protected against the infection. The results demonstrate that it is possible to generate a protective immune response to Cryptococcus even after becoming immunocompromised, such as in cases of HIV.

Recently, Rella et al. (2015) proposed the use of a live attenuated mutant strain lacking sterol glucosidase enzyme (Δsgl1) as a vaccine. In this study, immunization of mice with Δsgl1 cells led to strong protection against challenge with C. neoformans and C. gattii and could also elicit protective immunity in mice deficient for T CD4+ cells, a frequent condition in cryptococcosis patients.

Another possible therapeutic agent in the prophylaxis of Cryptococcus is glycosphingolipid glucosylceramide (GlcCer), a C. neoformans virulence factor. In a recently study, Mor et al. (2016) demonstrated that administration of GlcCer prior to infection with C. neoformans in a murine model prevented dissemination of the fungi from the lungs to the brain and led to 60% mouse survival while preventing side effects such as hepatic injury, a common and great problem in the use of the antifungal therapy currently available.

A peptide derived from the gp43 adhesin of P. brasiliensis, named p10, protects mice from paracoccidioidomycosis (PCM), and combination of immunization with this peptide and different antifungal drugs showed an additive protective effect. The finding suggests that this is an important molecule to be tested in clinical trials against PCM (Taborda et al., 1998; Marques et al., 2006; Marques et al., 2008; Rittner et al., 2012).

To date, few vaccines have reached the clinical stage. However, the importance of mycosis to public health around the world indicates that efforts should be given to the discovery of new molecules and compounds that can be used in the prophylaxis and treatment of these diseases. The challenges are numerous, but the efforts offer hope for controlling these diseases that tend to increase in incidence over time.

Classically, mammalian models are considered the gold standard for drug discovery, virulence and immune response studies. However, in 1959, the concept of the “alternative animal model” was described by Burch and Russell with “the three R’s: Refinement, Reduction, and Replacement” (Russell and Burch, 1959). In Table 1 are described the three R concept and the actions required for its application. In recent decades, the use of alternative animals, also called “mini hosts” or “non-conventional animal models” have been encouraging for in vivo testing. Amoeba, insects, nematodes, fish and chicken embryos are used for the following reasons: (1) because the neural systems of these animals are poorly developed, they are almost painless; (2) a large number of animals can be used for each experiment; (3) maintenance is cheaper than for traditional animals; (4) there is a good correlation between alternative animals and mammalian animals (Trevijano-Contador and Zaragoza, 2014).

Chicken embryos have been used since 1971 for fungal virulence studies (Partridge et al., 1971), and many studies have been published using this model to evaluate virulence of different fungi such as Candida spp., Rhizopus, and A. fumigatus (Jacobsen et al., 2010, 2011; Kaerger et al., 2015). Moreover, with this model, it is possible to evaluate the role of genes using mutants and to study the immune response of the host during infection (Jacobsen et al., 2010). Despite the importance of this model to date, experiments of antifungal efficacy have not been validated. Although chicken embryos are not consider worldwide to be living organisms and the ethical issues for this type of experimentation are less complicated, it is important to highlight that the euthanasia performed after experiments should be administered properly using anesthesia (Aleksandrowicz and Herr, 2015).

Another non-conventional model widely used for in vivo testing is zebrafish embryos, larvae, and adults. The main advantage is that it is possible to analyze different biological processes due to the presence of organs and systems, providing complete raw data analysis for different fields of science (MacRae and Peterson, 2015). This model was useful for studying of the C. albicans virulence (Chao et al., 2010; Brothers et al., 2011; Chen et al., 2015) and C. neoformans pathogenesis (Tenor et al., 2015). Similar to chicken embryos, there is no description thus far of the evaluation of antifungal efficacy using zebrafish. However, toxicity assays and teratogenic testing have provided important information. It is essential to highlight that after 120 h post-fertilization (hpf), ethical regulations are required for zebrafish experiments (Strähle et al., 2012).

Insects can also be used as an alternative animal model. The fruit fly Drosophila melanogaster is described as a model for investigating the virulence of human pathogens, due to similarities between its immune system and that of mammals, and for verifying the efficacy of novel antifungal compounds. Infection can be achieved by injection, rolling contact or ingestion (Lionakis and Kontoyiannis, 2012). Different Candida species have been evaluated using this model; the virulence of C. parapsilosis was found to be lower than that of C. albicans, similar to observations in humans; FLZ also increased the survival of susceptible strains (Chamilos et al., 2006). This model is also suitable for virulence evaluation of C. neoformans mutants (Apidianakis et al., 2004). The virulence of clinical and environmental isolates of A. flavus have been evaluated, as well as different mating types (Ramírez-Camejo et al., 2014). In Drosophila melanogaster, antifungal treatment is realized by ingestion, mixed with food. This model showed a correlation with mammalian models with regard to synergism of tacrolimus with posaconazole for the treatment of Rhizopus oryzae (Lewis et al., 2013). Fusarium moniliforme and Scedosporium apiospermum are able to infect D. melanogaster, and ingestion of food containing voriconazole increased the survival of infected flies (Lamaris et al., 2007).

Galleria mellonella has been described in different fields of science for numerous purposes, and the number of studies is increasing every year. Concerning virulence fungal assays, studies have been reported using G. mellonella as an infection model for different Candida species (Scorzoni et al., 2013; Frenkel et al., 2016; Moralez et al., 2016), Cryptococcus spp. (Benaducci et al., 2016), Sporothrix schenckii (Clavijo-Giraldo et al., 2016), Paracoccidioides spp. (Thomaz et al., 2013; Scorzoni et al., 2015), Histoplasma capsulatum (Thomaz et al., 2013), and Fusarium sp. (Coleman et al., 2011) and also for biofilm formation and behavior studies (Fuchs et al., 2010a; Benaducci et al., 2016).

The advances of this model are due to the ease of manipulation, low cost, and large range of larval incubation temperatures (25–37°C) with the possibility of mimicking the human body temperature. However, one disadvantage is that the genome is not fully sequenced, resulting in a lack of mutant strains to study host responses. Most of the work related to infection and treatment of G. mellonella has been via injection through one of the prolegs (Fuchs et al., 2010b). The benefit of this is that the inoculum and the treatment are controlled. G. mellonella is useful for in vivo treatment with conventional antifungal drugs (Coleman et al., 2011; de Lacorte Singulani et al., 2016), for assessing antifungal synergistic activity (Gu et al., 2016; Sangalli-Leite et al., 2016) and for evaluating the in vivo efficacy of new drug candidates (Browne et al., 2014).

The nematode Caenorhabditis elegans is been used for decades in different fields of biology. This organism has a short life cycle, produces a large number of progeny, and is transparent; moreover, the genome is fully sequenced with the possibility of studying host pathways through RNA interference (RNAi) techniques or constructing transgenic strains. C. elegans is maintained on agar medium and fed non-pathogenic E. coli (Muhammed et al., 2016). C. elegans is susceptible to infection by human pathogenic yeasts and filamentous fungi (Johnson et al., 2009; Pukkila-Worley et al., 2009; Desalermos et al., 2015). For this reason, this organism can be used for high-throughput screening of antifungal drugs candidates. The C. elegans model of infection is well described in antifungal drug discovery. A study with 1,266 compounds identified 15 anti-candidal substances that prolonged the survival of infected larvae and inhibited in vivo filamentation of the yeast (Breger et al., 2007). In another screen with 3,228 candidate substances, nine compounds with potential antifungal activity were identified (Okoli et al., 2009). Synergistic antifungal activity of tyrocidines and caspofungin was successfully evaluated in vitro and in vivo using C. elegans (Troskie et al., 2014). Moreover, C. elegans is a suitable model for studying the immune response because it is able to produce different antimicrobial peptides that are regulated upon infection; these peptides can also serve as new antimicrobial candidates (Ewbank and Zugasti, 2011).

Although different types of animal models are discussed here, it is important to evaluate which is the best system for each study. Regardless, although alternative models have benefits, mammalian models are still considered the gold standard, and many additional studies are necessary before the complete substitution of mammalian models for alternative animals. At present, alternative animals are suitable for screening prior to further studies in mammals.

Despite the increasing number of reports regarding advances in antifungal therapy, the number of cases of infection and antifungal resistance are still alarmingly high, and control of antifungal disease is far from being achieved. Important new advances have been made in the discovery of antimicrobial fungal targets; however, many years are necessary from discovery to clinical use. Because of this, improving existing molecules and developing new formulations and alternative therapy for prevention and treatment are important for treating fungal infections and increasing treatment options and quality of life.

All the authors contributed selecting the topics, reviewing the literature, selecting important and atual informations. Moreover the authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.