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1 Introduction

Opportunistic fungal infections are among the most difficult diseases to manage in patients. These infections frequently endanger the success of cancer treatments, transplant and surgery complications, autoimmune disease therapies, and also intensive care. In spite of several antifungal drugs and prophylaxis available, there is an increase in reported invasive fungal infections (IFIs). Worldwide mortality due to candidiasis, by Candida albicans (C. albicans) and related species, was 46–75 %; aspergillosis, by Aspergillus fumigatus (A. fumigatus) and related species, was 30–95 %; and mortality rate of cryptococcosis mainly caused by Cryptococcus neoformans (C. neoformans) worldwide was 20–70 % (d’Enfert 2009).

Fungal infections are established by fungi that are ubiquitous in nature and routinely inhaled by us in daily life. However, only individuals with compromised or diminished immunity are susceptible to invasive mycoses. Pathogenic fungi mainly C. albicans, A. fumigatus, and C. neoformans are leading pathogens among IFIs in humans. C. albicans is common microflora in human that may cause systemic infections in individuals with immunocompromised immune system leading to high mortality rates approaching up to 40 % (d’Enfert 2009). Other Candida species colonizes specific locations in the hosts, mainly in the gastrointestinal tract, genital tract, and the skin, whereas C. glabrata and C. parapsilosis also cause IFIs. Opportunistic pathogens, such as A. fumigatus and C. neoformans, are ubiquitously present in the soil. Normally host innate immunity, especially alveolar macrophages, manages these fungal spores, yeasts, or mycelial fragments. However, inhaled fungal spores can survive for extended period in macrophages and can establish infection when host immunity is weakened. The common causative agent of invasive aspergillosis is the filamentous mold A. fumigatus, with mortality rates up to 40–90 % (Dagenais and Keller 2009). The other opportunistic fungal pathogen known to cause infections in immunocompromised individuals is C. neoformans that leads to complications in central nervous system (CNS) such as cryptococcal meningitis among acquired immune deficiency syndrome (AIDS) patients (Sloan and Parris 2014). Pneumocystis spp. among other pathogenic fungi causes pneumonia in immunocompromised hosts.

These fungi have developed molecular mechanisms to combat the defense system in immunocompromised hosts. Detailed understanding of the complex interactions between genetic variations and its contributions to the disease phenotype is lacking and essential to study. Insight into host-pathogen interactions with respect to host genetic susceptibility can improve the identification of novel therapeutic targets and the design of better antifungal prophylaxis strategies.

It is a challenge to develop new techniques for early diagnosis of IFIs and effective treatment considering the increased burden of the disease and death in patients with their vulnerable immune status (Panackal et al. 2006). Diagnosis is extremely challenging, due to lack of sensitive or specific diagnostic methods, and results from currently used test are often available too late to be clinically useful. Considering the population at risk for fungal infections and the presence of wide range of fungal pathogens in the environment niche, these opportunistic fungi present significant challenge for diagnostics and therapeutics. In comparison to the bacterial antibiotics, few antifungal drugs have been reported with minimum risk of side effects and the occurrence of resistance.

Resistance to first-line drugs in most of the pathogenic fungi causing invasive infections ranges from 0 to almost 100 %. Though the evaluation of the overall impact of drug resistance in health is difficult, it is quite clear that morbidity and mortality due to resistant pathogens have increased in view less effective treatment [WHO global strategy for containment of antimicrobial resistance, 2001]. Resistance to various antifungal drugs, azoles in particular, has been reported in pathogenic fungi (Warris et al. 2002). Rates of resistance to widely used azole and fluconazole (FLC) have been reported between 10 % and 25 % in invasive candidiasis. Cross-resistance to other azole agents like voriconazole and itraconazole has also been reported among some of these FLC-resistant isolates (Cuenca-Estrella et al. 2006; Arendrup et al. 2013). Resistance rate to triazoles in Aspergillus spp. is less common and assumed to be below 5 % in most of countries (Snelders et al. 2008; Alastruey-Izquierdo et al. 2013). However, resistance in A. fumigatus seems to increase majorly due to evolution of resistant isolates in response to azole fungicides that is being widely used in Europe for crop protection (Snelders et al. 2008). Several factors have been reported to be associated to drug resistance against azoles in pathogenic fungi including upregulation of functional genes controlling drug efflux, alterations in sterol synthesis, mitochondrial dysfunctioning, decreased affinity for the cellular target and chromosomal abnormalities, and high-osmolarity glycerol (HOG) pathway. The major factor for resistance to azoles involves alteration in target enzyme, 14α-demethylase, and specific drug efflux pumps in terms of the quantity or quality (Ghannoum and Rice 1999; Nascimento et al. 2003), mainly two classes of efflux transporters, class ATP-binding cassette (ABC) and major facilitator superfamily (MFS) (Slaven et al. 2002; da Silva Ferreira et al. 2006).

Here, we have focused on incidences of drug resistance and molecular mechanism in three of the major human pathogenic fungi, Candida, Aspergillus, and Cryptococcus, against azole drugs.

2 Pathogenic Fungi and Invasive Fungal Infections

Pathogenic fungal infections are categorized as primary or opportunistic. Primary infections may develop in immunocompetent hosts, while opportunistic fungal infections are frequent in “high-risk” populations of immunocompromised individuals undergoing treatment for cancer, organ transplant cases, and autoimmune disease; in patients who are at risk of infection after prosthetic surgery or under the treatment of broad-spectrum antibiotics leading to changes in the normal flora; and in patients with HIV with immune deficiency (Nanjappa and Klein 2014). Opportunistic fungal infections could be superficial or systemic. IFIs have increased in the last two decades with increased use of steroids. Opportunistic fungal pathogens include Candida, Aspergillus, and Cryptococcus species. Figure 1 shows pathogenic fungi belonging to these genus and percentage of infection caused by these fungi.

Fig. 1
figure 1

Pathogenic fungi causing invasive fungal infections among transplant recipients. Candida spp. predominates among pathogenic fungi followed by Aspergillus spp. and Cryptococcus spp. as reported by Pfaller et al. (2009)

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2.1 Candida spp.

Candida species are Ascomycota group of fungi of the order Saccharomycotina, normally commensal organisms in mucous membranes, mainly gastrointestinal tract. There are a total of 150 Candida spp., and only 15 of them are commonly associated with humans as colonizers or opportunistic pathogens (Kam and Xu 2002). Of these, Candida albicans is the frequently isolated human commensal and pathogen (Krcmery and Barnes 2002). C. albicans, Candida tropicalis, and Candida glabrata are more virulent species, while Candida parapsilosis and Candida krusei are less virulent spp. Infections due to C. parapsilosis are more prevalent among children, and C. glabrata are frequently encountered among older adults (Yapar 2014; Kullberg and Arendrup 2015). These species have the differences in virulence as well as susceptibility to the azoles and the echinocandin drugs.

C. albicans is a dimorphic fungus and has ability to transform into different morphologies such as yeast, hyphae, and pseudohyphae upon perception of environmental signals (van der Meer et al. 2010). Most healthy individuals carry C. albicans, which harmlessly colonizes mucous membranes in different anatomical sites. Candida spp. are predominant constituents of the vaginal microflora (Seed 2014). However, it is an efficient invasive pathogen for establishing infections in individuals with ineffective adaptive cellular immunity and in patients lacking neutrophils. Candida spp. cause invasive candidiasis which is mostly reported among immunocompromised patients. Candidemia, also known as blood-borne systemic candidiasis, generally develops in neutropenic patients or in patients exposed to contaminated indwelling catheters during surgery (Yapar 2014). For this, Candida is majorly the cause of Candidemia associated with healthcare in the USA (Magill et al. 2014). Despite of proper antifungal therapy being given to the patients, mortality rate has still been reported as high as 40 %. Non-albicans Candida species is also gaining attentions as it is emerging resistance to antifungal drugs (Kullberg and Arendrup 2015).

C. albicans infections are usually prevented by our defense system by mucosal tissues and the peripheral circulation. Polymorphic nuclear leukocytes (PMNL) are the first-line defense against blood-borne Candida infections. Neutrophil defects, decrease in neutrophil counts, and dysregulation in Th-cell reactivity are main risk factors which contribute to severe Candida infections (Romani 2004; Netea et al. 2004). C. albicans induces immunosuppression and leads to production of CD4+CD25+ T-regulatory cells. Further, the presence of specific antibodies also protects against fungal infections (Polonelli et al. 2000).

2.2 Aspergillus spp.

The Aspergillus species are Ascomycota group of fungi of the order Eurotiales, which grow on high osmotic concentrations and on carbon-rich sources. Aspergillus has 339 identified species (Samson et al. 2014); however, only few of them, namely, A. fumigatus, A. flavus, A. niger, and A. terreus, have been considered to be pathogenic to humans. A. fumigatus is the common etiological agent of human aspergillosis accounted for ∼90 % of cases; A. flavus, A. niger, and A. terreus are secondary agents. Aspergillus species causes extensive spectrum of diseases with clinical manifestations that ranges from colonization of fungi in the organs leading to asthma, allergic bronchopulmonary aspergillosis (ABPA), and invasive aspergillosis (IA). The major site of Aspergillus infection is the lung (Ellis et al. 2009). A. fumigatus is a ubiquitous, saprophytic mold that releases airborne conidia which are inhaled by humans everyday (Latge 1999). A. fumigatus causes IA which primarily occurs in transplant recipients and hematological malignancy patients. The smaller (∼2 μm) conidial size of A. fumigatus gives the fungus advantage to remain airborne for long periods and enter human alveoli. Conidia are coated with hydrophobic proteins and with the chemoprotectant melanin (Pihet et al. 2009) to withstand harsh environment in air and in vivo. On the onset of their germination, hyphae are recognized by innate immune cells in paranasal sinus or the lung. The airway mucus serves as a physical, chemical, and biological barrier secreting fluids that contain glycoproteins, proteoglycans, lipids, etc. and lead to clearance of fungal conidia (McCormack and Whitsett 2002).

A. fumigatus also produces various metabolites, e.g., gliotoxin, fumagillin, and helvolic acid which damages epithelium and can have inhibitory effects on ciliary movement (Amitani et al. 1995). Host proteins such as pattern recognition receptors (PRRs), lung surfactant proteins A and D (SP-A and SP-D), mannan-binding lectin (MBL), and toll-like receptors (TLRs) (Walsh et al. 2005; Johnson et al. 2014) are significant for the host defense against Aspergillus, and any alterations in these molecules may affect susceptibility to Aspergillus infections in the individuals (Johnson et al. 2014).

2.3 Cryptococcus and Other Pathogenic Fungi

Cryptococcus causes fatal infections in patients with weak immune status such as those associated with T-lymphocyte deficiency, common in AIDS patients. These infections are frequently reported in patients with stem cell recipients and malignancy, though 15–40 % of Cryptococcus cases are reported in HIV-negative patients (Speed and Dunt 1995). Glucuronoxylomannan (GXM) capsule of Cryptococcus is a well-known virulence factor that suppresses the host inflammatory response and prevents phagocytosis of the fungus (Speed and Dunt 1995).

The use of antifungal prophylaxis has become more prevalent for common and also uncommon fungal pathogens thus complicating clinical management. The other fungal pathogens include Fusarium spp., zygomycete, and the opportunistic yeastlike fungi such as Histoplasma capsulatum and Pneumocystis spp.; Fusarium species causes onychomycosis and fungal keratitis. Fusarium infections in the lungs majorly cause allergic bronchopulmonary fusariosis and hypersensitivity pneumonitis. Although radiological indications for fusariosis is similar to invasive aspergillosis, frequent occurrence of disseminated nodular skin lesions and blood culture positivity is the classical marker of Fusarium infection which is to differentiate it from aspergillosis (Nucci et al. 2015). Members of Fusarium genus may affect humans and cause mycotoxicosis by ingestion of toxin-contaminated food (Bennett and Klich 2003). Mucormycosis is another fungal infections usually found in spreading pneumonia due to invasion in blood vessels (Saxena et al. 2015).

3 Current Diagnosis and Clinical Treatment

3.1 Diagnosis

Early diagnosis of the infection along with species differentiation are of great importance for improved treatment of IFIs. Standard criteria for systemic fungal infections are histopathologic examination for the presence of fungus in the tissue or culture and isolation of etiologic agent from clinical sterile specimens (blood, sputum, urine, cerebrospinal fluid, or tissue biopsy). Radiological tests are used for diagnosis of patients where invasive procedures for sample collection are very difficult and risky due to low immune status. Pulmonary lesions or nodules, infiltration, and halo signs are indicative of pulmonary fungal infections such as aspergillosis, fusariosis, scedosporiosis, or zygomycosis (Greene et al. 2007; Godoy et al. 2012). Among non-culture methods, serological tests are considered as standard practice for the diagnosis of fungal infections. Commercially available ELISA kits are available for Candida and Aspergillus antigens which detect mannan and galactomannan, respectively, and demonstrate good specificity but variable sensitivity (Musher et al. 2004). Detection of fungal DNA signatures in body fluids using polymerase chain reaction (PCR) assay in combination with serological test in high-risk patients is also viable diagnostic option. Advances in the qualitative methods, such as panfungal PCR, for fungal DNA in human blood samples, tissues, bronchoalveolar lavage, and other body fluids, are now in clinical practice as reliable test for fungal infections (Orsi et al. 2015).

3.2 Treatment

Standard antifungal drugs in use are polyenes, azoles, and echinocandins. Amphotericin B (AMB) deoxycholate, AMB-D and lipid formulations of AMB, and L-AMB are polyene drugs in use. Fluconazole and voriconazole are mainline azole compounds in use. Caspofungin, micafungin, and anidulafungin are recommended antifungal echinocandins. Fluconazole remains the choice of drug for invasive candidiasis, while for Candida species known to be susceptible to fluconazole, other compounds like echinocandins and AMB or AMB-D are recommended (Pappas et al. 2009a). Voriconazole is recommended for the primary treatment of invasive aspergillosis in most patients including infections resistant to AMB (also with Aspergillus terreus and Aspergillus nidulans) (Walsh et al. 2008). Recommended antifungal therapy for major fungal infections in adults is discussed in Table 1.

Table 1 Summary of recommendations for the treatment of major IFIs in adult
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4 Azoles and Other Antifungal Drugs

The use of potassium iodide (KI) for treating sporotrichosis was the first successful chemotherapy in 1903. After this, nystatin, the polyene compound, was successfully used as antifungal followed by amphotericin B in 1956, which is still the standard antifungal drug to evaluate new systemic antifungals (Al-Mohsen and Hughes 1998). The antifungal drugs include the natural products (polyenes, griseofulvin, and echinocandins) and the synthetic chemicals (azoles, allylamines, flucytosine, and phenylmorpholines). The available antifungal drugs are broadly classified into azoles, polyenes, nucleoside analogues, and echinocandins based on their mechanism of action.

4.1 Azoles

Azole antifungal agents are now considered to be important for therapeutics of systemic fungal infections. Fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), and ketoconazole (KTC) are some of the most widely used antifungal agents (Gallagher et al. 2003). Triazole drugs are more effective against many fungal pathogens with no severe nephrotoxic effects in comparison to AMB. Among azoles, FLC remains an effective and low-cost drug for the treatment of candidiasis and cryptococcosis. It is the first line of drug used for Candida (except Candida krusei and glabrata) and cryptococcal infections including non-meningeal coccidioidal infections. This drug is safe; however, it is not effective against filamentous fungi. VRC has been effective for the treatment of aspergillosis; however, its use is limited by significant drug interactions. It is effective against most of the Candida and Aspergillus spp. ITC is effective in vitro and in vivo against A. fumigatus, Candida spp., C. neoformans, and the dimorphic fungi. Finally, POS is the latest addition to the azole drugs and is effective against the zygomycetes. POS is effective against most Candida and Aspergillus species in vitro. It is also active against C. neoformans and Fusarium spp. (Zonios and Bennett 2008). KTC was the azole that was available for oral usage and was found with constant levels in blood; however, its use is limited due to hepatotoxicity and resistance reported in patients with candidiasis or AIDS and esophageal and oropharyngeal candidiasis.

Azoles inhibit fungal cytochrome P450 demethylase (an enzyme encoded by CYP51 or ERG11 gene) which converts lanosterol to ergosterol. This leads to reduced amount of ergosterol in the cell membrane of the fungus (Andriole and Bodey 1994; Georgopapadakou and Walsh 1996; Andriole 1998, 1999). In order to understand the molecular targets of azole drugs, various high-throughput studies have been carried out. Altered expression of Saccharomyces cerevisiae with different classes of antifungal compounds (KTC, amphotericin B, caspofungin, and 5-fluorocytosine) was carried out to identify altered gene expression specific to each drug. Exposure of KTC led to altered levels of genes involved in ergosterol biosynthesis pathway as well as sterol uptake, while exposure to caspofungin led to altered levels of genes belonging to cell wall integrity, and exposure to 5-FC led to altered levels of genes involved in DNA damage repair, DNA synthesis, protein synthesis, and regulation of cell cycle. On the other hand, exposure to AMB led to altered levels of genes involved in cell stress, membrane reorganization, cell wall integrity, and transport (Agarwal et al. 2003). In order to understand the molecular targets of antifungal azoles, VRC, and ITC, high-throughput techniques have been used. Using microarray hybridization, 2271 genes were found as differentially expressed in wild-type strain which describes decrease in biosynthesis of genes involved in ergosterol biosynthesis and increased mRNA level of gene-encoding transporters (da Silva Ferreira et al. 2006). Proteomic profiling of A. fumigatus on exposure to ITC (ITC) using 2-DE followed by mass spectrometric analysis led to identification of 54 differentially expressed proteins including proteins related to cell stress, carbohydrate metabolism, and amino acid metabolism.

4.2 Other Antifungal Drugs

The polyene antifungal agents are fungicidal with large spectrum of antifungal activity than any other antifungal agents. Amphotericin B, the polyene compound, has been proposed to interact with ergosterol that leads to the production of aqueous pores (Holz 1974). These pores result in dysregulated permeability and leakage of necessary cytoplasmic components leading to the killing of the organism (Kerridge 1980; Kerridge 1985). Intravenous amphotericin B has been recommended for severe and for invasive aspergillosis, Candida infections of CNS, blastomycosis, coccidioidomycosis, and mucormycosis (Andriole and Kravetz 1962; Andriole and Bodey 1994; Andriole 1998, 1999). Liposomal amphotericin B has reduced nephrotoxicity than conventional AMB (Hiemenz and Walsh 1996; Groll et al. 1998).

Flucytosine 5-FC is a fluorinated pyrimidine deregulating RNA and protein synthesis in the fungal cell mainly targeting pyrimidine metabolism (Andriole 1998, 1999; Groll et al. 1998). Flucytosine activity in vitro has been reported against Aspergillus spp., Candida spp., C. glabrata, and C. neoformans. However, if treated with it alone, it is less effective and has increased chances of developing fungal resistance in Candida and cryptococcal organisms. Echinocandins are cyclic lipopeptide agents with fungicidal activity through inhibition of cell wall enzyme, 1,3-β-d-glucan synthase, not expressed in mammalian cells, and hence form a potential antifungal target (Georgopapadakou and Walsh 1996; Groll et al. 1998). The investigational compound, LY 303366, an inhibitor of β-1, 3-beta-d-glucan synthase, is found to be active against Candida and Aspergillus organisms (Andriole 1998, 1999).

5 Drug Resistance to Azoles in Human Fungal Pathogens

Antifungal drug resistance is among the major causes of therapeutic failure in invasive fungal infections other than low bioavailability of the antifungal drug, weakened immune function, or a higher metabolism of the drug. Primary resistance occurs in organisms which are not at all exposed to the specific drug, while secondary or acquired resistance is due to exposure of an organism to the drug. Clinical resistance is reversion of infection due to therapeutic failure in an organism and is not linked to in vitro resistance (Rex et al. 1997).

For effective treatment of IFI, we need to be familiar with the susceptibility of the resistant fungal isolates to the antifungal drugs. The Clinical Laboratory Standard Institute (CLSI) in the USA and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in Europe have recommended protocols for antifungal susceptibility testing. The minimum inhibitory concentration (MIC) of the drug is calculated as the threshold levels of drugs leading to in vitro growth inhibition. The CLSI has recommended antifungal MIC breakpoints to separate susceptible and resistant population for azoles and echinocandins by analyzing the in vitro susceptibility data, in vitro outcome, and pharmacokinetics/pharmacodynamic studies, while EUCAST has defined the breakpoint derived from MIC as the epidemiological cutoff value (ECV) to avoid confusion with clinical breakpoints. ECVs are the MIC values that capture >95 % of the observed population. In this chapter, we discuss antifungal drug susceptibility for three major pathogenic fungal spp. against azole drugs (Fig. 2).

Fig. 2
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Molecular mechanisms of drug resistance to azoles in human pathogenic fungi. Various mechanisms involved in fungal drug resistance are numbered from 1 to 8. Pathogenic fungi are shown with sign closed circle (Candida spp.), closed triangle (Aspergillus spp.), and closed square (Cryptococcus spp.). (1) Mutations in the target enzyme (lanosterol 14α-demethylase) result in the complete inhibition of the binding of the azole drug to its target. (2) The two different drug efflux systems in fungi, i.e., the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS), contribute to azole drug resistance by pumping out the azole drugs out of the fungal cell leading to their less accumulation. (3) Modifications in the composition of plasma membrane affect the membrane asymmetry which alters the uptake of the drug. (4) Increased levels of target enzyme (lanosterol 14α-demethylase) may overwhelm the drug entering the cell, thereby resulting in increased level of resistance. Overproduction of the enzyme also results in cross-resistance between azoles. (5) The cell has a bypass pathway that balances for the loss-of-function inhibition due to the drug activity. (6) The HOG pathway negatively regulates the expression of ergosterol biosynthetic genes. Thus HOG mutants exhibit a decreased sensitivity toward azoles. (7) Chromosomal abnormalities have been linked with azole resistance in Candida and Cryptococcus species. Candida spp. acquire azole resistance by increasing the copy number of ERG11 (disomy) present on chromosome 5, whereas in Cryptococcus azole resistance is associated with chromosomes 1 and 4. (8) Loss of mitochondrial genome and changes in mitochondria membrane structures lead to the potential activation of drug resistance pathway in Candida spp.

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5.1 Candida spp.

In vitro antifungal drug susceptibility of Candida spp. against different azole drugs (FLC, ITC, VRC, KTC) has been carried out each year for an update on the trends on resistant clinical isolates. Table 2 provides the ECV/MIC data compiled from various studies carried out in the last 2 years for five major Candida species. The comparative analysis of resistant isolates for four azole drugs ITC, FLC, VRC, and KTC showed maximum number of resistant isolates to FLC in C. albicans, i.e., 68 isolates (5.7 %) and 83 isolates (89.24 %); C. glabrata, 70 isolates (7.9 %) and 17 isolates (94.4 %); to VRC in C. glabrata, i.e., 96 isolates (18.4 %); C. tropicalis, 36 isolates (17.6 %); to ITC in C. albicans i.e. 8 isolates (8.6 %); C. tropicalis i.e. 4 isolates (4.8 %); to KTC in C. albicans i.e. 23 isolates (24.7 %); C. glabrata i.e. 3 isolates (16.6 %) (Fothergill et al. 2014; Al-mamari et al. 2014; Zhang et al. 2015; Won et al. 2015; 83 isolates (Al-mamari et al. 2014); C. glabrata, 111 isolates (Fothergill et al. 2014); to VRC in C. albicans, i.e., 82 isolates (Fothergill et al. 2014); C. glabrata, 258 isolates (Fothergill et al. 2014); and C. tropicalis, 94 isolates (Fothergill et al. 2014) (refer Table 2). The trend of increasing resistance of FLC leads to therapeutic failure and raises the need of new effective azoles for treatment. Reports of low resistance rates in VRC suggest that VRC may be used with confidence for the treatment of candidiasis (98 % success rates of VRC and 95 % in case of FLC) against FLC-resistant isolates (Ally et al. 2001).

Table 2 Antifungal drug susceptibility against azole drugs in Candida clinical isolates revealing drug resistant isolates
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There are several reports suggesting correlation of resistant isolates against different azoles and mutations in ERG11 gene in Candida spp. ERG11 (CnCYP51) gene encodes lanosterol 14α-demethylase enzyme which appears to be important target for azole antifungal drug. In a detailed study, ERG11 genes from 17 clinical isolates of C. albicans were analyzed for FLC resistance. These strains were observed to have 27 point mutations in ERG11 gene. Out of these, five mutations mainly Y132H, A114S, Y257H, G464S, and F72S substitutions were most prevalent in resistance spp., while two novel substitutions, T285A and S457P, in hotspot regions were relevant (Wang et al. 2015a). In the clinical isolates of C. albicans, the correlation between naturally occurring mutations in a gene for multidrug resistance regulator 2 (MRR2) and FLC resistance was analyzed. In a group of 20 FLC-resistant C. albicans isolates, 12 isolates showed overexpression of Candida drug resistance 1 (CDR1) gene. Of these, only three FLC-resistant isolates showed 11 identical missense mutations in MRR2 gene, 6 of which were among azole-resistant isolates. In addition, the role of MRR2 mutations in CDR1 overexpression and thus to FLC resistance was verified using recombinant strains with mutated MRR2 gene (Wang et al. 2015b). Hence, increasing rates of azole resistance in these species emphasize on the development of antifungal strategies with greater efficacy toward azole class of antifungals.

5.2 Aspergillus spp.

Appearance of azole resistance in strains of A. fumigatus has become a serious public health problem. Antifungal drug susceptibility in Aspergillus against different azoles has been performed by several groups. We looked for the data on antifungal drug susceptibility of Aspergillus spp. against different azole drugs from major studies in the last 10 years in literature. The comparative analysis of resistant isolates for three azole drugs in A. fumigatus, A. flavus, and A. niger showed maximum number of resistant isolates to VRC in A. fumigatus [3.1 % (Espinel-Ingroff et al. 2010), 0.8 % (Pfaller et al. 2009), and 2.2 % (Rodriguez-Tudela et al. 2008)], to ITC in A. flavus (5.6 %), and to POS in A. niger (8.8 %) (Espinel-Ingroff et al. 2010) (see Table 3). The data suggest increased resistance to VRC, first line of therapy for invasive aspergillosis, in A. fumigatus (Mikulska et al. 2012). This is in line with the observation that VRC treatment is failing due to resistant isolates in the patients as mentioned earlier (Verweij et al. 2007).

Table 3 Antifungal drug susceptibility against azole drugs in Aspergillus clinical isolates revealing resistant isolates
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There are several reports suggesting correlation of resistant isolates against different azoles and mutations in CYP51A1 gene in A. fumigatus (Denning et al. 1997). In a study (Bader et al. 2013), 527 clinical isolates of A. fumigatus were analyzed and found that 17 (3.2 %) strains showed increased MIC0 values. Out of these 17 isolates, 14 were found to have resistance to ITC (MIC0 > 32 mg/l) and 1 was highly resistant to POS [MIC0 > 32 mg/l]. All the resistant isolates showed mutations in CYP51A1, and most common mutation was TR/L98H. Recently, ITC resistance also has been found to be increased due to TR/L98H mutations in CYP51A1 (Snelders et al. 2008). In another study, 38 clinical isolates were analyzed, where 3 isolates showed multi-azole resistance to ITC, VRC, and POS. It was found that all three isolates also showed exclusively TR34/L98H mutations in CYP51A1 gene, not present in the remaining 35 azole-susceptible isolates (Wu et al. 2015).

5.3 Cryptococcus spp.

The antifungal drug susceptibility for azoles in C. neoformans and C. gatti is provided in Table 4. Molecular typing for Cryptococcus spp. done using molecular methodologies like AFLP and PCR fingerprinting identified eight molecular types of Cryptococcus. An in vitro antifungal susceptibility test was done for the abovementioned molecular types of Cryptococcus species using the CLSI broth dilution method (Espinel-Ingroff et al. 2012). In C. neoformans strains with high FLC MICs (≥32 μg/ml), it was observed that a point mutation (involving the glycine to serine substitution at 484th position) in the ERG11 (CnCYP51) gene resulted in an alteration in the binding site of the target enzyme (Rodero et al. 2003). Earlier work done by Sanguinetti et al. showed a reduction in concentration of FLC in C. neoformans due to overexpression of the gene C. neoformans Antifungal Resistance 1 (CnAFR1) which codes for an ABC transporter, a membrane efflux pump (Sanguinetti et al. 2006).

Table 4 Antifungal drug susceptibility against azoles for Cryptococcus species revealing resistant isolates
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Cross-resistance among pathogenic fungi against azoles is expected as their target of action is similar, but azole cross-resistance is rarely seen among the Cryptococcal strains. In C. neoformans, cross-resistance between ITC and FLC is not observed due to the dual targets of ITC (the lanosterol 14α-demethylase and the 3-ketosteroid reductase). In C. neoformans isolates, the cross-resistance is also reported between FLC (MICs >64 μg/ml) and VRC (MICs ≥ 2 μg/ml), but no cross-resistance was seen between FLC and ITC. The fluconazole-voriconazole cross-resistance was attributed to a missense mutation [involving tyrosine to phenylalanine (Y145F) substitution] in the ERG11 gene (Espinel-Ingroff et al. 2012). In addition, the level of heteroresistance to FLC was found to be more in C. gattii than in C. neoformans isolates (Kwon-Chung and Rhodes 1986). Heteroresistance is the expression of different resistance profiles in subpopulations of a strain (Nunes et al. 2007).

6 Molecular Mechanism of Drug Resistance to Azoles

Resistance to first line of therapy such as FLC and VRC has increased in recent years. Several high-throughput studies with clinical isolates and in vitro-developed resistant isolates of Candida spp. have been carried out in order to understand the genes/proteins and pathways involved in drug resistance. Table 5 represents high-throughput studies using transcriptomic, proteomic, and lipidomic analysis to understand molecular pathways involved in drug resistance. Here, we discuss the factors associated with drug resistance against azoles in major pathogenic fungi.

Table 5 Various studies carried out to understand the molecular mechanisms of azole resistance in Candida Species
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6.1 Alteration in Ergosterol Biosynthesis Pathway Enzyme

As discussed earlier, azoles generally target lanosterol 14α-demethylase which is a cytochrome P450-dependent enzyme encoded by CYP51 or ERG11 gene. This is an oxidative process which involves the removal of 14α-methyl group from lanosterol. Azole binds to the ferric ion moiety of the heme-binding site and blocks the enzyme’s natural substrate lanosterol, disrupting the biosynthetic pathway (Odds et al. 2003). Amino acid substitutions in the drug target that inhibit drug binding are common azole drug resistance mechanisms in fungi.

Azole resistance in Candida species is attributed to the point mutation in ERG11 gene resulting in the alteration of drug target that inhibits the azole drug from binding to its target (Vandeputte et al. 2012). In a study on Candida, five different mutations in CYP51A1 gene (G129A, Y132H, S405F, G464S, and R467K) were observed. In the clinical isolates of C. albicans, combined mutations in CYP51A1 gene resulted in a greater decrease in binding affinity of the drugs and then single mutations (Sanglard et al. 1998). Studies on C. albicans and C. glabrata also found that overproduction of drug target (lanosterol demethylase) which resulted from genome rearrangement and chromosome duplication was another factor for an increased azole resistance (Marichal et al. 1997; Selmecki et al. 2008). In another study, a zinc transcription factor, CaUpc2, was essential for controlling the regulation of ERG genes in the subsistence of ergosterol biosynthesis inhibitors (Vandeputte et al. 2012). Further, in other study the binding capacity of CaUpc2 to the promoter region of ERG11 in C. albicans was confirmed (MacPherson et al. 2005).

The azole resistance in A. fumigatus is majorly mediated by mutations in gene CYP51A1. Target gene, cyp51 (ERG11), has two copies, i.e., CYP51A1 and CYP51B, each encoding a different protein (Mellado et al. 2001); however, mutations in CYP51A1 gene are reported as major cause of resistance in A. fumigatus (Odds et al. 2003; Diaz-Guerra et al. 2003; Warrilow et al. 2010). In A. fumigatus the first mutation to be identified was the glycine 54 (G54) point mutation detected in clinical isolates resistant to ITC and POS (Mellado et al. 2007). A high-throughput multiplex RT-PCR has been developed for detecting mutations in A. fumigatus CYP51A1 that leads to ITC resistance (Balashov et al. 2005). In another study, clinical isolates of A. fumigatus with reduced susceptibility were identified, and it was seen that the resistance developed among these species may be linked to two factors: firstly due to a point mutation involving substitution of leucine by histidine at 98th position (L98H) in CYP51A1 gene and secondly due to the presence of two copies of a 34-bp tandem repeats (TR) in the CYP51A1 promoter region (Snelders et al. 2010). Methionine 220 (M220) and glycine 138 (G138) are the less common mutations in A. fumigatus azole resistance strains (Slaven et al. 2002). TR/L98H genotype is the most ubiquitous mutation responsible for resistance mechanism observed for azole-resistant strain (Willger et al. 2008). A recently identified mechanism in the CYP51A1 gene that decreases the susceptibility of A. fumigatus against voriconazole consists single polymorphisms mainly substitutions in tandem repeat of 46-bp in the promoter region (Dirr et al. 2010).

But a limited number of mutations in ERG11 has been reported in C. neoformans, including Y145F (Espinel-Ingroff et al. 2012) and G484S (Rodero et al. 2003). Study by Espinel-Ingroff et al. showed by sequencing the ERG11 gene from clinical isolates of MRL862 (C. neoformans strain isolated from a FLC-treated patient) that the strain MRL862 contained five unique mutations compared to reference strain H99. Triazole susceptibility coupled with the molecular changes in the ERG11 gene revealed that a single missense Y145F, tyrosine replaced by phenylalanine, mutation resulted in high FLC resistance of the strain (Espinel-Ingroff et al. 2012). The study by Rodero et al. analyzed five C. neoformans isolates that were sequentially isolated from an AIDS patient with frequent meningitis and found that out of five isolates, four were FLC susceptible while FLC resistance was seen to be developed in the fifth isolate. The analysis further revealed that a point mutation G484S (involving the glycine to serine substitution at 484th position) in the ERG11 gene was the key to the development of FLC resistance in the fifth isolate (Rodero et al. 2003). Studies by various groups have demonstrated that this substitution confers an orientation change in the P450 heme-binding domain that leads to a decrease in the binding affinity of azole drug as well as a decrease in the enzyme catalytic efficiency (Sanglard et al. 1998; Kelly et al. 1999).

In C. albicans, point mutations in ERG3 gene result in the alteration of C5 sterol desaturase enzyme; therefore, 14α-methyl-3,6-diol (toxic sterol) cannot be synthesized. However, in the presence of azoles, ergosterol is replaced by sterol species resulting in functional fungal cell membrane (Sanguinetti et al. 2015).

6.2 Drug Efflux

There are two different drug efflux systems in fungi that contribute to azole drug resistance. They belong to the superfamily ATP-binding cassette (ABC) and the major facilitator superfamily (MFS). ABC proteins are the ATP-dependent transporters, usually arranged in duplicates, comprise of two transmembrane span (TMS) domains and the two cytoplasmic nucleotide-binding domains (NBDs) which facilitate the ATP hydrolysis. In C. albicans drug resistance isolates, it was reported that CDR1 and CDR2 are main contributors of azole resistance which belongs to ABC transporters superfamily (Vandeputte et al. 2012). Studies on regulation of CDR1 and CDR2 showed cis-acting regulatory elements: a basal expression element (BEE), a drug-responsive element (DRE), two steroid responsive elements (SREs), and a negative regulatory element (NRE) in CDR1, while the CDR2 promoter contains only a DRE motif (de Micheli et al. 2002; Karnani et al. 2004; Gaur et al. 2005). Among these DRE was reported to be the exclusively responsible element for overexpression/upregulation of both CDR1 and CDR2. In azole-resistant clinical isolates of C. glabrata, three transporters mainly CgCDR1, CgCDR2, and ABC transporter co-regulated with CgCDR1 and CgCDR2, called SNQ2, were found to be upregulated and involved in azole resistance (Torelli et al. 2008). In A. fumigatus, atrF and AfuMDR4 were found to be upregulated in itraconazole-resistant strains (Vandeputte et al. 2012). In C. albicans, a gene encoding a protein CaNdt80p that was found to regulate CDR1 participating in drug resistance mechanism was reported (Chen et al. 2004). In a study, it was demonstrated that mutations lead to hyperactive alleles in C. albicans and consequent loss of heterozygosity (LOH) at the transcriptional activator of CDR gene (TAC1) and multidrug resistance 1 (MRR1) loci (Coste et al. 2009).

In azole resistance clinical isolates of C. albicans, multidrug resistance 1 (MDR1) transporter was reported as MFS transporter (Ben-Yaacov et al. 1994). Among FLC-resistant isolates of C. albicans, nucleotide region, called MDR1 drug resistance element (MDRE), was found to be responsible for the overexpression of MDR1 (Riggle and Kumamoto 2006; Rognon et al. 2006). A study by Hiller et al. reported three different cis-activating regions (regions 1, 2, and 3) in MDR1, and regions 1 and 3, close to the MDRE region, were reported to be necessary for controlling the expression of MDR1 in an azole-resistant isolate (Vandeputte et al. 2012). Studies of MRR1 gene deletion in azole-resistant strains were shown to diminish the overexpression of MDR1 suggesting MRR1p identification as a main controller of MDR1. The CdMDR1 and CtMDR1 are homologues of MDR1 in C. dubliniensis and C. tropicalis, respectively, which are found to be upregulated in azole-resistant strains (Pinjon et al. 2003, 2005; Vandeputte et al. 2005).

Drug efflux pumps are also responsible for resistance in A. fumigatus as they mediate reduced accumulation of intracellular drugs (Coleman and Mylonakis 2009). A probe derived from CDR1 gene, ABC transporter genes for drug efflux in C. albicans, was used to clone atrF gene from A. fumigatus with characteristics of multidrug resistance motifs. In A. fumigatus isolate, AF72 has approximately fivefold higher levels of expression of atrF compare to the susceptible isolates AF10 and H06-03 with sub-MIC levels of ITC (Slaven et al. 2002). In A. fumigatus, overexpression of multidrug resistance 3 (AfuMDR3) or AfuMDR4 is linked to high-level resistance against itraconazole due to the mutations at the drug target site (Denning et al. 1997; Nascimento et al. 2003). In the biofilms of azole resistance of A. fumigatus efflux pump, AfuMDR4 pump was seen to be upregulated that is responsible for resistance to VRC (Rajendran et al. 2011).

In A. nidulans, high levels and differential expression of AtrA to AtrD genes were reported in the presence of drugs such as camptothecin, imazalil, ITC, hygromycin, and 4-nitroquinoline oxide using real-time RT-PCR. Further, AtrA to AtrD expression levels were verified in the A. nidulans imazalil-resistant mutants (Semighini et al. 2002). One of the four ABC-type transporter genes, abcD, was reported to have two- to sixfold increased mRNA level expression following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide (do Nascimento et al. 2002).

Many ABC proteins are found in C. neoformans in comparison to other pathogenic fungi (Lamping et al. 2010), but only a few are linked to azole drug resistance. The upregulation of the ABC transporter-encoding gene AFR1 in C. neoformans is contributing to the in vitro resistance to FLC (Coleman and Mylonakis 2009) and corelated to less azole accumulation. Sanguinetti et al. generated a set of recombinant strains of C. neoformans BPY22.17 (FLC-resistant mutant strain), BPY444 (afr1 mutant strain), and BPY445 (AFR1-overexpressing mutants), all derived from a FLC-susceptible isolate of C. neoformans strain BPY22. In infectious mice model exposed to these strains showed, strain BPY445 was more virulent than BPY22 and displayed enhanced intracellular survival due to upregulation of AFR1 (Sanguinetti et al. 2006). In spite of several studies on the role of ABC multidrug efflux transporter drug resistance, little is known about the contribution of the Drug:H+ Antiporter (DHA) family in azole resistance. There are nine DHA1 and seven DHA2 transporters present in C. neoformans which play role in antifungal drug resistance, but their role in azole drug resistance has yet not been studied (Costa et al. 2014).

Influx of Drugs into the Cell

Import of azoles occurs via facilitated diffusion mediated by transporters in fungi such as C. albicans and C. neoformans, and mutations in the transporter may greatly influence resistance. A study by Mansfield et al. demonstrated that azole compounds utilized the same mechanism for incorporating drugs inside the cell membrane which was carried out by a transporter. The mutation in that putative transporter resulted in the azole cross-resistance. Among 35 studied clinical isolates of C. albicans, 4 isolates showed overexpression of genes; ERG11, MDR1, CDR1, and/or CDR2. A mutation is reported in ERG11 which significantly alters [3H]-FLC import suggesting that modification of azole import mediated by mutated transporter may be associated with antifungal resistance (Mansfield et al. 2010). Composition alterations in plasma membrane are some other factor that affects fluidity and asymmetry of the membrane that leads to a decreased drug uptake (Parks and Casey 1996).

6.3 Chromosomal Abnormalities: Loss of Heterozygosity and Aneuploidy

Azole drug resistance has been associated with multiple genomic alterations, including loss of heterozygosity (LOH) of specific genomic regions (Coste et al. 2007; Selmecki et al. 2010), increase in the chromosome copy number, as well as segmental or chromosomal aneuploidies.

Comparative genome hybridization (CGH) analysis showed that there were 37 aneuploid chromosomes in C. albicans strains (FLC-resistant and FLC-sensitive strains). This aneuploidy was frequent on chromosome 5, while segmental aneuploidy was present in eight FLC-resistant strains with extra copies of chr5L which was confirmed by Southern blot analysis (Selmecki et al. 2006). In C. albicans, events of LOH were observed antifungal drug-resistant strains (Sanguinetti et al. 2015). It was shown in C. albicans that LOH events were increased during in vitro exposure to heat stress (onefold to 40-fold), oxidative stress (threefold to 72-fold), and treatment with antifungal agents (FLC, 285-fold) (Forche et al. 2011) and led to the development of antifungal drug resistance. Further, it was demonstrated that increasing extremity of stress was associated with increased rates of LOH. Chromosomal rearrangements and duplications also have a role in increased resistance toward azoles.

Numerous analyses of azole-resistant and azole-sensitive strains obtained by comparative genome hybridization (CGH) from clinical and laboratory sources reflects a clear link between “heteroresistance” (an azole-associated acquisition of aneuploidy) and azole resistance (Kwon-Chung and Chang 2012). Among the screened isolates of C. neoformans and C. gatti, it was showed that both the species displayed intrinsic heteroresistance phenotype to FLC (Sionov et al. 2009), which was observed to diminish upon release of drug stress. It was observed that the strain resistance to FLC was always disomic for Chr1, and further elevation in drug level resulted in disomy of Chr4. Infrequent doubling of chromosome 1 has been reported in erg11 or afr1 mutants suggesting their role during times of stress (Sionov et al. 2010). Since both ERG11 and AFR1 were present on Chr1 in C. neoformans, it was seen that the duplication of the chromosome has been an advantage during the development of a resistant strain during FLC stress.

6.4 Mitochondrial Dysfunction

Mitochondrial dysfunction contributes greatly toward the virulence as well as drug tolerance of fungal pathogens. Loss of mitochondrial genome and changes in mitochondria membrane structures lead to the potential activation of drug resistance pathway. In a study, FLC-susceptible and FLC-resistant strains of C. glabrata showed upregulation of the ABC transporter genes, CgCDR1, CgCDR2, and CgSNQ2 (Ferrari et al. 2011). A study by Vazquez et al. showed that deletion of the mitochondrial inner membrane translocase gene, OXA1, essential gene for the assembly of mitochondrial-encoded subunits, activates the drug resistance pathway in C. glabrata. In addition, C. glabrata modification of phosphatidylglycerol synthase gene CgPGS1 responsible for synthesis of the phospholipids in mitochondria has been associated with drug resistance (Shingu-Vazquez and Traven 2011).

6.5 High-Osmolarity Glycerol Pathway

The HOG pathway is often reported to play an important role in controlling various activities like stress response, biosynthesis of ergosterol, production of virulence factor, and differentiation in pathogenic fungi. In C. neoformans, transcriptome analysis of the HOG pathway discovered Hrk1 (gene regulated by Hog1), encoding a putative protein kinase. This gene plays a role in stress, virulence of the fungus in Hog1-dependent and Hog1-independent manner, and thus antifungal drug susceptibility (Kim et al. 2011). The HOG pathway controls expression levels of ergosterol biosynthesis genes and in turn affects azole drug susceptibility. Consequently, the HOG mutants exhibit a decrease in drug sensitivity toward azoles such as FLC and KTC (Ko et al. 2009).

6.6 Other Mechanisms

Stress response can regulate azole resistance in A. fumigatus but very little is known about it. Sterol regulatory element-binding protein, SrbA, a highly conserved transcription factor, was found to be mediating stress responses under hypoxic conditions. It was further reported to be implicated in maintenance of sterol biosynthesis, hyphal morphology, and specifically in azole resistance, such as FLC and VRC (Willger et al. 2008). Another study involving mutants of MAP kinase kinase 2 (Mkk2), which is generally associated with positive regulation of calcium-mediated signaling, cellular response to salt stress, and cell wall integrity pathway, showed increased sensitivity to both POS and VRC (Dirr et al. 2010). The inhabitation of Hsp90 increases the efficiency of VRC in A. fumigatus suggesting that it may have significant role in the azole resistance in A. fumigatus (Cowen 2009). There are several other mutations in protein-coding regions which were found to favor azole resistance in A. fumigatus. HapE gene encodes for CCAAT-binding transcription factor; mutation in this gene that substitutes proline to leucine is one such example (Camps et al. 2012). However, the mechanism of resistance is not clearly understood.

7 Novel Strategies to Combat Drug Resistance

Due to the limited antifungal treatments available, antifungal agents with broad spectrum of fungicidal activity using versatile mechanism of action are often required. Specific treatment of resistant organisms with less toxicity is of paramount importance (Messer et al. 2009). Novel strategies as discussed below may be considered for combating drug resistance.

Understanding the key molecular mechanisms in host-fungal interactions in vitro and in vivo is the key to finding novel therapies. These may also uncover the mechanisms behind the development of drug resistance. The present antifungal agents in use can be altered in a way which can target fungal virulent genes, enzyme inhibitors, and fungal metabolic pathways. Genome-wide analysis studies can be performed to identify the genes responsible in fungal survival and can serve as efficient targets. The catalogue of essential genes for C. albicans and Aspergillus fumigatus has been studied by molecular techniques such as gene replacement and conditional expression (GRACE) or conditional promoter replacement (CPR) (Roemer et al. 2003; Hu et al. 2007). Such analyses provide specific drug targets for designing effective antifungal drugs. Combinatorial antifungal therapy is used as an alternate approach for primary and salvage treatment of fungal infections, e.g., the use of amphotericin B deoxycholate and flucytosine is highly recommended for cryptococcal meningitis treatment (Pappas et al. 2009b). However, the use of combination therapy for in invasive candidiasis and in aspergillosis has mixed review and still awaits studies, which may come in near future.

The use of multifunctional approach using computational modeling and biochemical studies can be employed to screen the large libraries of chemical compounds effective against broad range of fungal pathogen. The use of in vivo model of Caenorhabditis elegans–Candida albicans killing assay presents rapid and economical approach for antifungal discovery (Breger et al. 2007) and presents limited and definite compounds to be tested for in vivo models or in human clinical trials. Compounds from natural sources (plants, sea, microorganisms) and chemical sources should be screened for their antifungal properties and used as another novel approach to overcome current drug resistance. A cysteine-rich antifungal protein (AFP) isolated from Aspergillus giganteus is reported to exert potent antifungal activity against pathogenic fungi without altering the viability of host mammalian cells (Meyer 2008); however, its bioactivity is restricted to filamentous fungi. A chitosan, a polymer isolated from crustacean exoskeletons, is another well-researched compound known to be effective against C. neoformans biofilms formed on indwelling surgical devices (Martinez et al. 2010). A compound named as 25-azalanosterol inhibits the growth of C. albicans in vitro and has potential as a new class of anti-Candida agents with no toxic side effects in the mammalian host (Wang and Wu 2008). Although it will be extremely useful to have new and improved antifungal drugs, the immediate focus should be on improvisation of strategies to use the antifungal treatment we possess at present.

8 Conclusions

There has been an increase in incidences of IFIs and emergence of new human pathogenic fungal species. One of the major factors for increased resistance to antifungal drugs in these pathogenic fungi is due to exposure to the antifungal drugs. In view of this, species identification and antifungal susceptibility testing to identify resistant isolate needs are urgent needs for better treatment management of IFIs. Clinical breakpoints have been defined for Candida species; however, more studies are required for Aspergillus and Cryptococcus species. Various studies to understand the molecular mechanisms of resistance in fungi have been carried out majorly in C. albicans. However, such studies need to be performed in Aspergillus and Cryptococcus species. A combinatorial therapy targeting multiple pathways involved in drug resistance might help to combat drug-resistant isolates. Further, new antifungals targeting survival genes would be helpful to in combating drug resistance and improved treatment management of IFIs. Novel strategies in search for antimycotic compounds that should be cost-effective along with high performance and limited toxicity are required.

9 Opinion

The improved diagnostic method based on molecular and serologic techniques is an urgent need for early diagnosis of IFI. Further, development of diagnostic platform detecting drug-resistant isolates from clinical samples is imperative for better IFI treatment. To improve the detection of antifungal resistance, novel assays identifying fungal isolates, which possess mutations known to be associated with antifungal resistance, should be developed and included in diagnostic platform. A reproducible and clinically relevant antifungal susceptibility testing and its use in routine clinical practice are needed. In view of the increasing resistance to antifungal drugs leading to persistent fungal infections, focused research on understanding molecular mechanisms involved in drug resistance should be promoted. Research outcomes of such studies may be beneficial for designing new antifungal drugs to combat these life-threatening infections and develop tools enabling the rapid detection of resistance in fungal population of diverse origins. Novel antifungal agents with better efficacy are required due to relative shortage of antifungal agents for treating invasive mycoses diseases.