Echinocandin Drugs Research Articles

The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

Candidemia due to uncommon Candida species in children: new threat and impacts on outcomes

Many uncommon Candida spp. (species other than C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei) have been shown to emerge in tertiary care facilities. We aimed to investigate these uncommon candidemia in children. Forty-six cases of candidemia caused by uncommon Candida spp. were identified during 2003–2015 from a medical center in Taiwan. The most common specie was C. guilliermondii (31.2%), followed by C. lusitaniae (18.8%) and C. metapsilosis (18.8%). These cases were analyzed and compared with 148 episodes of C. albicans candidemia. The incidence density of uncommon Candida spp. candidemia and the proportion to all candidemia episodes increased substantively during the study period. Prior exposure to azoles was uncommon in the 30 days prior to infection, but fluconazole resistant strains were significantly more common (n = 19, 41.3%). The increased incidence density of uncommon Candida spp. candidemia was associated with increasing use of antifungal agents. No differences in demographics, underlying comorbidities, risk factors, clinical features, dissemination, and 30-day mortality were found between uncommon Candida spp. and C. albicans candidemia. Patients with uncommon Candida spp. candidemia were more likely to require modifications in antifungal treatment and receive echinocandin drugs (43.5% vs 21.6%, p = 0.007). Candidemia caused by uncommon Candida spp. had poorer response to antifungal treatment, led to longer duration of candidemia (median 4.0 versus 2.5 days, p = 0.008), and had a higher treatment failure rate (56.5% vs 38.5%, p = 0.040).

FKS2 and FKS3 Genes of Opportunistic Human Pathogen Candida albicans Influence Echinocandin Susceptibility.

Candida albicans, a prevailing opportunistic fungal pathogen of humans, has a diploid genome containing three homologous FKS genes that are evolutionarily conserved. One of these, the essential gene FKS1, encodes the catalytic subunit of glucan synthase, which is the target of echinocandin drugs and also serves as a site of drug resistance. The other two glucan synthase-encoding genes, FKS2 and FKS3, are also expressed, but their roles in resistance are considered unimportant. However, we report here that expression of FKS1 is upregulated in strains lacking either FKS2 or FKS3 Furthermore, in contrast to what is observed in heterozygous FKS1 deletion strains, cells lacking FKS2 or FKS3 contain increased amounts of cell wall glucan, are more resistant to echinocandin drugs, and consistently are tolerant to cell wall-damaging agents. Our data indicate that C. albicansFKS2 and FKS3 can act as negative regulators of FKS1, thereby influencing echinocandin susceptibility.

The Gastrointestinal Tract Is a Major Source of Echinocandin Drug Resistance in a Murine Model of Candida glabrata Colonization and Systemic Dissemination.

ABSTRACTCandida species are a part of the human microbiome and can cause systemic infection upon immune suppression. Candida glabrata infections are increasing and have greater rates of antifungal resistance than other species. Here, we present a C. glabrata gastrointestinal (GI) colonization model to explore whether colonized yeast exposed to caspofungin, an echinocandin antifungal, develop characteristic resistance mutations and, upon immunosuppression, breakthrough causing systemic infection. Daily therapeutic dosing (5 mg/kg of body weight) of caspofungin resulted in no reduction in fecal burdens, organ breakthrough rates similar to control groups, and resistance rates (0 to 10%) similar to those reported clinically. Treatment with 20 mg/kg caspofungin initially reduced burdens, but a rebound following 5 to 9 days of treatment was accompanied by high levels of resistance (FKS1/FKS2 mutants). Although breakthrough rates decreased in this group, the same FKS mutants were recovered from organs. In an attempt to negate drug tolerance that is critical for resistance development, we cotreated mice with daily caspofungin and the chitin synthase inhibitor nikkomycin Z. The largest reduction (3 log) in GI burdens was obtained within 3 to 5 days of 20 mg/kg caspofungin plus nikkomycin treatment. Yet, echinocandin resistance, characterized by a novel Fks1-L630R substitution, was identified following 5 to 7 days of treatment. Therapeutic caspofungin plus nikkomycin treatment left GI burdens unchanged but significantly reduced organ breakthrough rates (20%; P < 0.05). Single-dose pharmacokinetics demonstrated low levels of drug penetration into the GI lumen posttreatment with caspofungin. Overall, we show that C. glabrata echinocandin resistance can arise within the GI tract and that resistant mutants can readily disseminate upon immunosuppression.

Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance.

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiae slt2Δ, bck1Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants (sakAΔ, mpkCΔ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

Unraveling Drug Penetration of Echinocandin Antifungals at the Site of Infection in an Intra-abdominal Abscess Model.

ABSTRACTIntra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents; however, their clinical effectiveness is highly variable, with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption ionization mass spectrometry imaging technology, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model. Drug accumulation within lesions was observed with both drugs at their humanized therapeutic doses. CD101, but not micafungin, accumulated in lesions at levels above the mutant prevention concentration of the infecting strain. These findings indicate that current echinocandin drugs are limited by penetration at the site of infection and have implications for clinical outcomes and emergence of resistance in patients with IAC.

Echinocandin Resistance in Candida Species Isolates from Liver Transplant Recipients.

ABSTRACTLiver transplant recipients are at risk of invasive fungal infections, especially candidiasis. Echinocandin is recommended as prophylactic treatment but is increasingly associated with resistance. Our aim was to assess echinocandin drug resistance in Candida spp. isolated from liver transplant recipients treated with this antifungal class. For this, all liver-transplanted patients in a University Hospital (Créteil, France) between January and June of 2013 and 2015 were included. Susceptibilities of Candida isolates to echinocandins were tested by Etest and the EUCAST reference method. Isolates were analyzed by FKS sequencing and genotyped based on microsatellites or multilocus sequence typing (MLST) profiles. Ninety-four patients were included, and 39 patients were colonized or infected and treated with echinocandin. Echinocandin resistance appeared in 3 (8%) of the treated patients within 1 month of treatment. One patient was colonized by resistant Candida glabrata, one by resistant Candida dubliniensis, and one by resistant Candida albicans. Molecular analysis found three mutations in FKS2 HS1 (F659S, S663A, and D666E) for C. glabrata and one mutation in FKS1 HS1 (S645P) for C. dubliniensis and C. albicans. Susceptible and resistant isolates belonged to the same genotype. To our knowledge, this is the first study on echinocandin resistance in Candida spp. in a liver transplant population. Most resistant isolates were found around/in digestive sites, perhaps due to lower diffusion of echinocandin in these sites. This work documents the risk of emergence of resistance to echinocandin, even after short-term treatment.

Unraveling Drug Penetration of Echinocandin Antifungals at the Site of Infection in an Intra-Abdominal Abscess Model

BackgroundIntra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents. Yet, their clinical effectiveness is highly variable with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption/ionization (MALDI) mass spectrometry imaging as well as standard analytical techniques, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model.MethodsFemale 6–8 week old CD1 mice weighing 18–22 g were infected intraperitoneally (IP) with 1 × 107 CFU of C. albicans SC5314 mixed with sterile stool matrix. Single IP doses of CD101 at 5 or 20 mg/kg (equivalent to humanized therapeutic dose) or micafungin at 5 mg/kg (therapeutic dose) were administered to mice at day 3 post-inoculation. Mice were sacrificed at just before antifungal treatment (n= 1), and at 1, 3, 6, 24, and 48 hours post-dose (n = 3 per group per time point). Liver and kidney lesions were collected for MALDI imaging. Laser capture microdissection (LCM) followed by liquid chromatography coupled tandem mass spectrometry (LC/MS-MS) was applied to 6 and 24 hours samples for drug exposure measurement. In a separate experiment, mice were treated with 2 or 3 doses of micafungin (5 mg/kg), or a single dose of CD101 (20 mg/kg). Drug accumulation was analyzed at 48 and 72hours post the first dose.ResultsDrug accumulation within lesions was observed with both drugs at their humanized therapeutic dose. However, micafungin, even at steady-state, failed to approach the mutant prevention concentration (MPC) (16 µg/mL) of the infecting strain. CD101 demonstrated extensive penetration into the lesions after a single dose administration and persisted in lesions at above MPC level of 29.7 µg/mL at 72 hours postdose.ConclusionThese findings indicate that current echinocandin drugs may be limited by penetration at the site of infection, which have implications for clinical outcomes and emergence of resistance in patients with IAC.Disclosures C. J. Clancy, Merck: Received research funding, Research support; Astellas: Received research funding, Research support; Cidara: Received research funding, Research support; Astellas: Scientific Advisor, Advisory board; Merck: Scientific Advisor, Advisory board; Cidara: Scientific Advisor, Advisory board; Medicines Company: Scientific Advisor, Advisory board; D. Perlin, Cidara: Research Contractor and Scientific Advisor, Research grant; Amplyx: Research Contractor and Scientific Advisor, Research grant; Matinas: Scientific Advisor, Research support; Scynexis: Research Contractor and Scientific Advisor, Research grant; Merck: Research Contractor, Research grant; Astellas: Research Contractor, Research grant

CD101: a novel long-acting echinocandin.

SummaryCD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin‐susceptible and ‐resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin‐resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild‐type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long‐lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

Drug resistance mechanisms and their regulation in non-albicans Candida species.

Fungal pathogens use various mechanisms to survive exposure to drugs. Prolonged treatment very often leads to the stepwise acquisition of resistance. The limited number of antifungal therapeutics and their mostly fungistatic rather than fungicidal character facilitates selection of resistant strains. These are able to cope with cytotoxic molecules by acquisition of appropriate mutations, re-wiring gene expression and metabolic adjustments. Recent evidence points to the paramount importance of the permeability barrier and cell wall integrity in the process of adaptation to high drug concentrations. Molecular details of basal and acquired drug resistance are best characterized in the most frequent human fungal pathogen, Candida albicans Effector genes directly related to the acquisition of elevated tolerance of this species to azole and echinocandin drugs are well described. The emergence of high-level drug resistance against intrinsically lower susceptibility to azoles in yeast species other than C. albicans is, however, of particular concern. This is due to their steadily increasing contribution to high mortality rates associated with disseminated infections. Recent findings concerning underlying mechanisms associated with elevated drug resistance suggest a link to cell wall and plasma membrane metabolism in non-albicans Candida species.

Utility of in-house fluconazole disk diffusion susceptibility testing in the treatment of candidemia

Among 302 first candidemia episodes, 210 (69.6%) were initially treated with an echinocandin or polyene (E/P) antifungal drug. In 137 (72.5%) patients with fluconazole-susceptible isolates, treatment was changed to fluconazole based on disk diffusion susceptibility testing. Clinical outcomes were not compromised in patients receiving E/P who were de-escalated to fluconazole for treatment of candidemia based on disk diffusion results.

Echinocandin Resistance in Candida.

Invasive fungal infections are an important infection concern for patients with underlying immunosuppression. Antifungal therapy is a critical component of patient care, but therapeutic choices are limited due to few drug classes. Antifungal resistance, especially among Candida species, aggravates the problem. The echinocandin drugs (micafungin, anidulafungin, and caspofungin) are the preferred choice to treat a range of candidiasis. They target the fungal-specific enzyme glucan synthase, which is responsible for the biosynthesis of a major cell wall polymer. Therapeutic failure involves acquisition of resistance, although it is a rare event among most Candida species. However, in some settings, higher-level resistance has been reported among Candida glabrata, which is also frequently resistant to azole drugs, resulting in difficult-to-treat multidrug-resistant strains. The mechanism of echinocandin resistance involves amino acid changes in "hot spot" regions of FKS-encoded subunits of glucan synthase, which decreases the sensitivity of enzyme to drug, resulting in higher minimum inhibitory concentration values. The cellular processes promoting the formation of resistant FKS strains involve complex stress response pathways that yield a variety of adaptive compensatory genetic responses. Standardized broth microdilution techniques can be used to distinguish FKS mutant strains from wild type, but testing C. glabrata with caspofungin should be approached cautiously. Finally, clinical factors that promote echinocandin resistance include prophylaxis, host reservoirs including biofilms in the gastrointestinal tract, and intra-abdominal infections. An understanding of clinical and molecular factors that promote echinocandin resistance is critical to develop better diagnostic tools and therapeutic strategies to overcome resistance.

Rapid development of Candida krusei echinocandin resistance during caspofungin therapy.

In invasive candidiasis, there has been an epidemiological shift from Candida albicans to non-albicans species infections, including infections with C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Although the prevalence of C. krusei remains low among yeast infections, its intrinsic resistance to fluconazole raises epidemiological and therapeutic concerns. Echinocandins have in vitro activity against most Candida spp. and are the first-line agents in the treatment of candidemia. Although resistance to echinocandin drugs is still rare, individual cases of C. krusei resistance have been reported in recent years, especially with strains that have been under selective pressure. A total of 15 C. krusei strains, isolated from the blood, urine, and soft tissue of an acute lymphocytic leukemia patient, were analyzed. Strains developed echinocandin resistance during 10 days of caspofungin therapy. The molecular epidemiology of the isolates was investigated using two different typing methods: PCR-based amplification of the species-specific repetitive polymorphic CKRS-1 sequence and multilocus sequence typing. All isolates were genetically related, and the mechanism involved in decreased echinocandin susceptibility was characterized. Clinical resistance was associated with an increase in echinocandin MICs in vitro and was related to three different mutations in hot spot 1 of the target enzyme Fks1p. Molecular evidence of the rapid acquisition of resistance by different mutations in FKS1 highlights the need to monitor the development of resistance in C. krusei infections treated with echinocandin drugs.

Mechanisms of echinocandin antifungal drug resistance.

Fungal infections due to Candida and Aspergillus species cause extensive morbidity and mortality, especially among immunosuppressed patients, and antifungal therapy is critical to patient management. Yet only a few drug classes are available to treat invasive fungal diseases, and this problem is compounded by the emergence of antifungal resistance. Echinocandin drugs are the preferred choice to treat candidiasis. They are the first cell wall-active agents and target the fungal-specific enzyme glucan synthase, which catalyzes the biosynthesis of β-1,3-glucan, a key cell wall polymer. Therapeutic failures occur rarely among common Candida species, with the exception of Candida glabrata, which is frequently multidrug resistant. Echinocandin resistance in susceptible species is always acquired during therapy. The mechanism of resistance involves amino acid changes in hot-spot regions of Fks subunits of glucan synthase, which decrease the sensitivity of the enzyme to drug. Cellular stress response pathways lead to drug adaptation, which promotes the formation of resistant fks strains. Clinical factors promoting echinocandin resistance include empiric therapy, prophylaxis, gastrointestinal reservoirs, and intra-abdominal infections. A better understanding of the echinocandin-resistance mechanism, along with cellular and clinical factors promoting resistance, will facilitate more effective strategies to overcome and prevent echinocandin resistance.

Resistance in Candida albicans: Exploring the Cell Wall Barrier by Proteomics

The incidence of candidiasis has dramatically increased and bloodstream infections due to different species of Candida are becoming a prime cause of morbidity and mortality in different types of immunocompromised patients. Azole and echinocandin drug resistance accounts for the dramatic increase in incidence of nosocomial bloodstream candidiasis found in recent years. Cell wall constitutes the barrier between the yeast and the host and resistant strains change the proteome of this compartment. In the last decade different proteomic platforms have been applied to study cell wall and markers of resistance to drugs have been pointed out. Modulation of these proteins seem to suggest that although resistance is based on a specific mutation able to counteract the toxicity of the antifungal drug, a set of other molecular modifications takes place contemporary or subsequently the establishment of the resistance and seems to support the viability of the resistant yeast. Profiled proteins by proteomics may be valuable in design therapy using classical antifungal along with complementary drugs able to abolish pathways that strengthen the resistance and attenuate virulence of the mutated cell.

Surface coatings with covalently attached caspofungin are effective in eliminating fungal pathogens.

In this work we have prepared surface coatings formulated with the antifungal drug caspofungin, an approved pharmaceutical lipopeptide compound of the echinocandin drug class. Our hypothesis was to test whether an antifungal drug with a known cell-wall disrupting effect could be irreversibly tethered to surface coatings and kill (on contact) biofilm-forming fungal human pathogens from Candida spp. The first aim of the study was to use surface analysis to prove that the chemical binding to the surface polymer interlayer was through specific and irreversible bonds (covalent) and not due to non-specific adsorption through weak forces that could be later reversed (physisorption). Secondly, we quantified the antifungal nature of these coatings in a biological assay showing excellent killing against C. albicans and C. tropicalis and moderate killing against C. glabrata and C. parapsilosis. We concluded that caspofungin retains antifungal activity even when it is irreversibly immobilized on a surface, providing a new insight into its mechanism of action. Thus, surface coatings that have echinocandins permanently bound will be useful in preventing the establishment of fungal biofilms on materials.

Echinocandin resistance: an emerging clinical problem?

Echinocandin resistance in Candida is a great concern, as the echinocandin drugs are recommended as first-line therapy for patients with invasive candidiasis. Here, we review recent advances in our understanding of the epidemiology, underlying mechanisms, methods for detection and clinical implications. Echinocandin resistance has emerged over the recent years. It has been found in most clinically relevant Candida spp., but is most common in C. glabrata with rates exceeding 10% at selected institutions. It is most commonly detected after 3-4 weeks of treatment and is associated with a dismal outcome. An extensive list of mutations in hot spot regions of the genes encoding the target has been characterized and associated with species and drug-specific loss of susceptibility. The updated antifungal susceptibility testing reference methods identify echinocandin-resistant isolates reliably, although the performance of commercial tests is somewhat more variable. Alternative technologies are being developed, including molecular detection and matrix-assisted laser desorption ionization-time of flight. Echinocandin resistance is increasingly encountered and its occurrence makes susceptibility testing essential, particularly in patients with prior exposure. The further development of rapid and user-friendly commercially available susceptibility platforms is warranted. Antifungal stewardship is important in order to minimize unnecessary selection pressure.

In vivo Candida glabrata biofilm development on foreign bodies in a rat subcutaneous model.

Biofilm studies have been mostly dedicated to the major human fungal pathogen Candida albicans, whereas much less is known about this virulence factor in Candida glabrata, certainly under in vivo conditions. This study provides a deeper understanding of the biofilm development of C. glabrata, its architecture and susceptibility profile to fluconazole and echinocandins. In vitro and in vivo C. glabrata biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well polystyrene plates or implanted subcutaneously in the back of a rat, respectively. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the biofilm architecture. Quantitative real-time PCR was used to demonstrate the expression profile of EPA1, EPA3, EPA6 and AWP1-AWP7 during in vivo biofilm formation. Mature biofilms were observed within the first 48 h and the amount of biofilm reached its maximum by 6 days. Architecturally, mature C. glabrata biofilms consisted of a thick network of yeast cells embedded in an extracellular matrix. Moreover, in vivo biofilms were susceptible to echinocandin drugs, whereas fluconazole remained ineffective. Gene expression profiling revealed that EPA3, EPA6, AWP2, AWP3 and AWP5 were up-regulated in in vivo biofilms compared with in vitro biofilms. C. glabrata is a unique microorganism, which, despite the lack of transition to the hyphal form, formed thick biofilms inside foreign bodies in vivo. To our knowledge, this is the first study that has described in vivo C. glabrata biofilm development and its architectural changes in detail and provides an insight into the susceptibility profile, as well as the gene expression machinery, of biofilm-associated infections.

Echinocandin resistance, susceptibility testing and prophylaxis: implications for patient management.

This article addresses the emergence of echinocandin resistance among Candida species, mechanisms of resistance, factors that promote resistance and confounding issues surrounding standard susceptibility testing. Fungal infections remain a significant cause of global morbidity and mortality, especially among patients with underlying immunosupression. Antifungal therapy is a critical component of patient management for acute and chronic diseases. Yet, therapeutic choices are limited due to only a few drug classes available to treat systemic disease. Moreover, the problem is exacerbated by the emergence of antifungal resistance, which has resulted in difficult to manage multidrug resistant strains. Echinocandin drugs are now the preferred choice to treat a range of candidiasis. These drugs target and inhibit the fungal-specific enzyme glucan synthase, which is responsible for the biosynthesis of a key cell wall polymer. Therapeutic failures involving acquisition of resistance among susceptible organisms like Candida albicans is largely a rare event. However, in recent years, there is an alarming trend of increased resistance among strains of Candida glabrata, which in many cases are also resistant to azole drugs. Echinocandin resistance is always acquired during therapy and the mechanism of resistance is well established to involve amino acid changes in "hot-spot" regions of the Fks subunits carrying the catalytic portion of glucan synthase. These changes significantly decrease the sensitivity of the enzyme to drug resulting in higher MIC values. A range of drug responses, from complete to partial refractory response, is observed depending on the nature of the amino acid substitution, and clinical responses are recapitulated in pharmacodynamic models of infection. The cellular processes promoting the formation of resistant Fks strains involve complex stress response pathways, which yield a variety of adaptive compensatory genetic responses. Stress-adapted cells become drug tolerant and can form stable drug resistant FKS mutations with continued drug exposure. A major concern for resistance detection is that classical broth microdilution techniques show significant variability among clinical microbiology laboratories for certain echinocandin drugs and Candida species. The consequence is that susceptible strains are misclassified according to established clinical breakpoints, and this has led to confusion in the field. Clinical factors that appear to promote echinocandin resistance include the expanding use of antifungal agents for empiric therapy and prophylaxis. Furthermore, host reservoirs such as biofilms in the gastrointestinal tract or intra-abdominal infections can seed development of resistant organisms during therapy. A fundamental understanding of the primary molecular resistance mechanism, along with cellular and clinical factors that promote resistance emergence, is critical to develop better diagnostic tools and therapeutic strategies to overcome and prevent echinocandin resistance.

Biofilm Formation and its Impact on Antifungal Therapy

Fungi can protect themselves from host defences and antifungal drugs by the production of an extracellular hydrophobic matrix. Candida biofilms exhibit resistance to antifungal agents from all classes including the azoles, echinocandins, amphotericin B complex, and flucytosine. Although demonstrated on polystyrene and bronchial epithelia cells, until today, only indirect evidence for A. fumigatus biofilms in patients is available. The antifungals with the most activity against biofilms are the liposomal formulation of amphotericin B and agents in the echinocandin drug class. Importantly, echinocandins show excellent anti-biofilm activity against C. albicans at therapeutic concentrations. However, other biofilms formed by moulds, including A. fumigatus, are relatively resistant to echinocandins. Multiple mechanisms contribute to the intrinsic and acquired antifungal resistance during the different stages of fungal biofilm development. During the growth phase of the early biofilm various factors account for biofilm resistance. Combinational and sequential antifungal therapy as well as combination with enhancers can improve the effect of a single drug. Further studies are warranted to develop new therapeutic strategies targeting fungal biofilm-specific resistance mechanisms.