Emerging Resistance to Azoles and Echinocandins: Clinical Relevance and Laboratory Detection

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance. In vitro resistance is mostly due to genetic mutations (resistance mechanisms), and it is associated with high minimal inhibitory concentrations (MICs), minimal effective concentrations (MECs), and/or clinical failure. Clinical breakpoints (CBPs) and/or epidemiologic cutoff values (ECVs) are useful to detect the in vitro antifungal resistance when isolates are tested by standardized methods. ECVs are available from the Clinical and Laboratory Standards Institute (CLSI) for Candida spp. versus echinocandins (anidulafungin, caspofungin, and micafungin) and triazoles (fluconazole, posaconazole, and voriconazole). Lately, the CLSI has adjusted to species-specific CBPs for Candida spp. versus fluconazole, similar to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and versus echinocandins. However, the available voriconazole EUCAST and CLSI CBPs differ. In the absence of CBPs, EUCAST and CLSI assigned ECVs for various Aspergillus spp. and triazoles. This article reviews emerging resistance, laboratory detection, and clinical relevance as reported in the literature in the past 3 to 4 years.     

Updates in antifungal susceptibility testing of filamentous fungi

The Clinical and Laboratory Standards Institute (CLSI) has standardized broth microdilution and disk diffusion methodology for testing filamentous fungi (molds) that cause invasive disease. Quality control MIC (minimal inhibitory concentration) and MEC (minimal effective concentration; echinocandins only) limits are also available in the recently published CLSI M38-A2 document. Although breakpoints based on correlations of in vitro results and clinical outcome have not been established, MIC or MEC and zone diameter categories for five antifungal agents and various mold species, as well as epidemiologic cutoffs for Aspergillus fumigatus versus the triazoles, have been recently documented. Some insights of the potential clinical value of reference methods also have been reported. During the past few years, the potential utility of various commercial methods has been evaluated by comparing them with reference methodology. This review summarizes and discusses the advantages and disadvantages of these developments.     

Can We Achieve Clinical Breakpoints for the Triazoles in Aspergillosis?

The azoles are first-line agents for the treatment of aspergillosis. A number of recent studies have shown increasing rates of resistance in A. fumigatus. Consequently, reliable in vitro susceptibility testing and breakpoints that appropriately classify resistant isolates are of paramount importance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical Laboratory Standards Institute (CLSI) have developed susceptibility testing standards, but so far no breakpoints have been defined. The following aspects are evaluated during the process of developing EUCAST breakpoints: the most common dosage, the definition of the wild-type population and epidemiologic cutoff values, pharmacokinetic and pharmacodynamic properties, and the correlation between the minimum inhibitory concentration (MIC) and clinical outcome. This article reviews the issues to be considered before breakpoints can be established for azole drugs and Aspergillus and describes how MICs can be interpreted until clinical breakpoints have been defined.     

EUCAST and CLSI: Working Together Towards a Harmonized Method for Antifungal Susceptibility Testing

The U.S. Clinical and Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (AFST-EUCAST) have developed broth microdilution methodologies for testing yeasts and filamentous fungi (molds). The mission of these methodologies is to identify in vitro antifungal resistance, which is accomplished by the use of either clinical breakpoints (CBPs), or to a lesser degree, epidemiologic cutoff values (ECVs). The newly adjusted and species-specific CLSI CBPs for Candida spp. versus fluconazole and voriconazole have ameliorated some of the differences between the two methodologies. In the absence of CBPs for mold testing, CLSI ECVs are available for six Aspergillus species versus the triazoles, caspofungin and amphotericin B. Recently, breakpoints were developed by the EUCAST for certain Aspergillus spp. versus amphotercin B, itraconazole and posaconazole, which to some extent are comparable to ECVs. We summarize these latest accomplishments, which have made possible the harmonization of some susceptibility cutoffs, if not methodologies for some agent/species combinations.     

Nystatin-Intralipid Preparation: Characterization and In Vitro Activity Against Yeasts and Molds

The objective of our studies is the development of a novel formulation of nystatin (NYT) that could be administered systemically and might be used for therapy of invasive mycoses. We developed a formulation of nystatin and intralipid (IL), which is a clinically used food supplement, and this report focuses on the characterization of NYT-IL, assessment of its antifungal activity and in vitro toxicity. We characterized physical properties of the NYT-IL preparation and its stability during storage. Susceptibility of Candida, Aspergillus and Fusarium species was determined using a CLSI technique. In vitro toxicity of NYT-IL was assessed using an assay measuring hemolysis of sheep red blood cells (SRBC) and leakage of potassium. It was found that: (1) the particle size in NYT-IL did not differ from that of IL; (2) over 80% of NYT was in association with IL; and (3) these features did not change during storage. All Candida and Aspergillus strains had lower minimal inhibitory concentration (MIC) values for NYT-IL than that for NYT; the MICs of the Fusarium strains were similar for NYT & NYT-IL. Toxicity assays showed that the NYT-IL formulation is less toxic than NYT. In conclusion, we describe a novel, characterized, stable formulation of nystatin, nystatin-intralipid, with in vitro activity against pathogenic Candida and Aspergillus species.     

Wild-Type MIC Distributions and Epidemiologic Cutoff Values for Fluconazole and <Emphasis Type="Italic">Candida</Emphasis>: Time for New Clinical Breakpoints?

Antifungal susceptibility testing of Candida against fluconazole has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both CLSI and EUCAST have developed clinical breakpoint (CBP) criteria for fluconazole, but these differ in both magnitude and target species. Studies using the EUCAST method have also defined wild-type minimum inhibitory concentration (MIC) distributions and epidemiologic cutoff values (ECVs or ECOFFs) for the common species of Candida. The ECVs serve as a sensitive means of discriminating wild-type strains from those with acquired resistance mechanisms and include MICs of 1 μg/mL for C. albicans, 2 μg/mL for C. tropicalis and C. parapsilosis, 32 μg/mL for C. glabrata, and 128 μg/mL for C. krusei. Because the CLSI CBPs may be too insensitive to detect emerging resistance among strains of C. albicans, C. tropicalis, and C. parapsilosis, and bisect the WT MIC distribution of C. glabrata, we sought to establish the wild-type MIC distribution and ECVs for fluconazole and Candida spp. The establishment of the wild-type MIC distributions and ECVs for fluconazole using CLSI methods will be useful in resistance surveillance and may prove to be an important step in the development of species-specific CBPs for this important antifungal agent.     

Evaluation of Etest Performed in Mueller–Hinton Agar Supplemented with Glucose for Antifungal Susceptibility Testing of Clinical Isolates of Filamentous Fungi

Although reference broth microdilution protocol is currently available for filamentous fungi antifungal susceptibility testing (AFST), simpler alternatives as Etest^® tend to be favoured in clinical routine, making their validation of utmost importance. In this study, Etest^® method using 2 % glucose supplemented Muller–Hinton agar was compared to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol for filamentous fungi AFST. The echinocandins, caspofungin and anidulafungin, the azoles voriconazole and posaconazole, and the polyene amphotericin B were tested against 48 Aspergillus spp., seven Fusarium spp., one Beauveria bassiana and three Paecilomyces lilacinus isolates. The majority of the isolates were susceptible to the antifungals tested, and the overall level of agreement between the CLSI and Etest methods was 71.9 % for one dilution and 99.7 % when using two dilutions. Since interpretative breakpoints for filamentous fungi employing the CLSI or Etest methods are not available yet, the established epidemiological cut-off values for Aspergillus spp. were used to distinguish wild-type isolates from those with acquired resistance mechanisms. Forty-five Aspergillus strains did not evidence resistance mutations.     

The role of second-generation triazole antifungal agents voriconazole and posaconazole in patients with hematologic malignancies

The second-generation triazoles, voriconazole and posaconazole, have found important roles in the management of invasive fungal infections in high-risk patients. Both agents are more active against Candida albicans and the non-albicans Candida species than the first-generation triazoles. They are active against Aspergillus species, including those species less susceptible to polyenes, and against a variety of non-Aspergillus molds. In contrast to posaconazole, voriconazole has no activity against the zygomycetes, and breakthrough infections have been observed. Both are well absorbed, but considerable intra- and interpatient pharmacokinetic variability has raised the question of therapeutic drug monitoring. Both inhibit hepatic cytochrome P450 isoenzymes, which are important in the metabolism of various drugs coadministered in the management of high-risk patients. Clinical trials have demonstrated the safety and efficacy of both agents for antifungal prophylaxis and treatment in invasive candidiasis, invasive aspergillosis, and in invasive fungal infections caused by a variety of non-Aspergillus molds. Posaconazole is the only triazole approved for use in the treatment of invasive zygomycosis. Voriconazole is the accepted standard first-line therapy for invasive aspergillosis.     

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature

Voriconazole (Vfend™) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] <2 μg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to >4 μg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 μg/ml) were lower than amphotericin B (0.25 to 4 μg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against F. oxysporum and solani, most studies showed MICs ranging from 0.25 to 8 μg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC₉₀s < 1.0 μg/ml). The exception was Sporothrix schenckii (MIC₉₀s and geometric mean MICs ≥ 8 μg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to >32 μg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 μg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

Background

Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.

Methods

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.

Results

Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.

Conclusions

The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.     

Advances in molecular detection of Aspergillus: an update

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.     

Antifungal drug resistance in molds: Clinical and microbiological factors

The incidence of fungal infections has increased over the past decade. In addition to classical pathogens, such as Aspergillus spp, new fungal species are increasingly reported. Despite the availability of new antifungals, mortality of invasive fungal infections remains very high. The host immune status is the main factor for survival. However, most of these pathogens have high minimum inhibitory concentrations (MICs) to antifungals, and therefore, the influence of these high MICs in the outcome of the patients have begun to be addressed. Several strains of Aspergillus fumigatus showing resistance to itraconazole have been isolated, and the molecular-resistance mechanisms have been characterized. In addition, attempts to correlate high MICs with patient outcome have been performed. Although correlation is far from perfect, a clear trend between high MICs and poor outcome has been established. Resistance of fungi to antifungals is a health problem requiring support from research agencies.     

Mechanisms of resistance to antifungal agents: yeasts and filamentous fungi

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance; the latter is associated with numerous factors related to the host, the antifungal agent, or the infecting isolate. Recently, a susceptible MIC breakpoint ( < or =2 microg/ml) was designed for Candida spp. to all three available echinocandins, anidulafungin (Pfizer), caspofungin (Merck) and micafungin (Astellas) and treatment failures have been associated with MICs > 2 microg/ml. In some of these cases, clinical failure was associated with the genetic mutations described below. Azole and flucytosine breakpoints, and the echinocandin susceptible breakpoint, are useful when isolates are tested by CLSI standardized methods; breakpoints are also available by the EUCAST method. More recently, in vitro resistant MIC breakpoints have been assigned for filamentous fungi (moulds) vs. five antifungal agents, but these categories are not based on correlations of in vitro with in vivo response to therapy. However, itraconazole (Janssen), amphotericin B (Bristol-Myers) and voriconazole (Pfizer) clinical failures in aspergillosis have been correlated with MICs > 2 microg/ml. This article provides a review of reported resistance molecular mechanisms to antifungal agents since 2005; previous related reviews are also listed.     

Azole Resistance in Aspergillus fumigatus—Current Epidemiology and Future Perspectives

Azole resistance in Aspergillus fumigatus isolates is increasingly reported in different nosologic contexts with variable prevalence in different countries. Mutations in the target of triazoles are widely described in azole-resistant clinical isolates. The recovery of mutated/resistant isolates is described either in patients undergoing long-term azole treatment or after inhalation of environmentally acquired resistant isolates. Acquisition in patients during azole therapy highlights the capacity of this fungus to adapt to its environment, but it has a low impact in terms of public health, as interhuman transmission of A. fumigatus is uncommon. Environmentally acquired resistant isolates may propagate and affect populations at risk. The use of triazoles as first-line therapy or prophylaxis could lead to selection of resistant isolates in patients, because most isolates harbor azole cross-resistance. Although mold-active triazoles have provided major progress in the prophylaxis and treatment of Aspergillus infection, the increase of azole resistance could question their use in humans.     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Antifungal Susceptibility and Pathogenic Potential of Environmental Isolated Filamentous Fungi Compared with Colonizing Agents in Immunocompromised Patients

Infection is a major cause of morbidity and mortality in bone marrow transplant recipients and in patients with hematological malignancies. The source of infection is almost always endogenous flora or the hospital environment. The present study evaluated bone marrow transplant recipients and patients with hematological malignancies colonized and/or infected with filamentous fungi. During 1 year, environmental air samples were also taken from the bone marrow transplant unit by a modification of gravity air-setting plate (GASP) methodology. Fusarium spp. were the most prevalent genus in the fall and Cladosporium spp. in the winter. Clinically isolated strains grew better at 37 °C than environmental strains. According to NCCLS M-38P methods, environmental Aspergillus strains showed higher MICs to miconazol and itraconazol, and clinical Fusarium strains were less susceptible to fluconazole.     

New antifungal agents for the systemic mycoses

The azoles are the prominent broad spectrum oral antifungal agents in use or under clinical investigation for the systemic mycoses. This class of antifungal agents is represented by the marketed drug ketoconazole (Nizoral) and the experimental triazoles furthest along in clinical trials in the United States, itraconazole and fluconazole. Ketoconazole use is limited by its side effect profile and activity spectrum. Itraconazole appears to be better tolerated and less toxic to liver function, does not cause adrenal suppression and is more active against Aspergillus and Sporothrix schenckii. Fluconazole appears to be a highly promising agent due its highly favorable pharmacokinetic profile; it is water soluble, is well tolerated, is not metabolized to inactive constituents, it has a long half-life and, unlike the other azoles, high cerebrospinal fluid levels are readily attained for consideration in meningeal mycoses. It remains to be determined what place these new triazoles have in managing immunosuppressed patients including those with acquired immune deficiency syndrome known as AIDS. Other experimental antifungal agents, including ambruticin, amphotericin B methyl ester and saramycetin are also described. Sales figures are presented of drugs marketed in the United States for the systemic mycoses and reflect the growing problem of fungal diseases in the population.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

Echinocandin-Resistant Candida: Molecular Methods and Phenotypes

We now have a decade of experience with echinocandin drugs. Large-scale epidemiologic antifungal surveillance studies have demonstrated that caspofungin, micafungin, and anidulafungin retain high potency on clinical isolates of Candida, and resistance remains relatively low. Yet reports of breakthrough infections involving strains with a high minimum inhibitory concentration (MIC) are mounting. Mechanism-specific resistance involving amino acid substitutions in the Fks subunit(s) of the drug target glucan synthase results in reduced enzyme sensitivity to drug and high MICs. The mechanism affects all three drugs and is encountered in all Candida species, as well as in Aspergillus. An initial susceptibility testing breakpoint failed to adequately distinguish wild-type susceptible isolates from fks mutant resistant strains. Considering data from epidemiologic, microbiologic, pharmacokinetic/pharmacodynamic, biochemical, and genetic studies that better capture resistant isolates with fks genotypes has resulted in a proposed new breakpoint which provides a more reliable measure of probable therapeutic success.     

Fungal endophthalmitis

Fungal endophthalmitis occurs secondary to trauma, as a complication of intraocular surgery, or as an extension of an adjacent or distant focus of fungal infection. In the United States, Candida species are the most common pathogens isolated, followed by Aspergillus species. Candida infections show a predilection towards vitreous involvement, whereas Aspergillus infections usually manifest as hemorrhagic infarcts of the retinal or choroidal vessels or as infiltration of the subretinal and subretinal pigment epithelial spaces. Endogenous Aspergillus endophthalmitis is associated with a high mortality rate, underscoring the severity of systemic infection. The prognosis of fungal endophthalmitis depends on the virulence of the organism, extent of intraocular involvement, and the timing and mode of interventions. Prompt therapy following early diagnosis helps to reduce significant visual loss. Despite the introduction of new-generation triazoles and echinocandins into clinical practice, successful therapy is limited by the delay in diagnosis and a lack of broad-spectrum antifungals with good intravitreal penetration that lack systemic toxicity.     

Update on Amphotericin B Pharmacology and Dosing for Common Systemic Mycoses

Amphotericin B (AMB) has been used for nearly five decades in the treatment of life-threatening mycoses. While triazoles and echinocandins have largely supplanted the routine use of AMB for common Candida and Aspergillus infections, this prototypical broad-spectrum agent or its lipid formulations are still preferred by many clinicians as the initial empiric antifungal therapy for severely immunosuppressed patients, and remain the preferred treatment in combination with 5-fluorocytosine for cryptococcal meningioencephalitis. This article reviews progress over the last decade in understanding the pharmacology and clinical dosing of AMB formulations for common systemic mycoses, including invasive candidiasis, cryptococcosis, aspergillosis and mucormycosis.