Posaconazole MICs Research Articles

Exploring synergy between azole antifungal drugs and statins for Candida auris.

Global emergence of rapidly developing resistance to multiple antifungal drugs and high mortality pose challenges to the treatment of invasive Candida auris infections. New therapeutic approaches are needed, such as repurposing drugs including combination with antifungals. Statins have been reported to exert antifungal effects against various Candida species. Our study investigated potential synergy between the statins (rosuvastatin and fluvastatin) and azoles (voriconazole, posaconazole and isavuconazole) on clinical isolates of C. auris. Twenty-one clinical isolates of C. auris were obtained. Chequerboard assays based on the CLSI broth microdilution method were used to assess synergy based on FIC index (FICI) calculations of MICs of individual drugs and in combinations. Single drug geometric mean (GM) MICs of fluvastatin and rosuvastatin were ≥128 mg/L in all 21 isolates. GM (range) MICs of posaconazole, voriconazole and isavuconazole were 0.259 (0.016-1 mg/L), 0.469 (0.016-2 mg/L) and 0.085 (0.004-1 mg/L), respectively. Combination of azoles with fluvastatin showed synergy in 70%-90% of C. auris isolates. In particular, voriconazole/fluvastatin resulted in 16-fold reduction in voriconazole MIC and synergy in 14/21 (67%) isolates. Posaconazole/fluvastatin resulted in 8-fold reduction in posaconazole MIC and synergy in 19/21 (90%) isolates.Combining rosuvastatin with the azoles also showed synergy against C. auris in 40%-60% of the isolates and additive effect in 40%-50%. None of the combinations was antagonistic. Our results provide a rationale for pursuing in vivo synergy tests as well as clinical studies to explore tolerability, treatment outcomes, optimal dose and exposure targets.

P093 A novel GAL4-like transcriptional regulator modulate the azoles sensitivity of Exophiala dermatitidis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives Exophiala dermatitidis is an emerging opportunistic pathogen among the ever-increasing numbers of immunocompromised hosts. Understanding its transmission mechanism, pathogenicity, and potential azole-resistant mechanism is important for better understanding of this fatal pathogen and benefits future diagnosis, and treatment.By using a PCR-based fragment fusion approach, we applied phosphotransferase (HPH) resistance gene which amplified from pAN7-1 as a selective gene for generating replacement cassettes. Combined with electroporation transformation and screening on selection medium containing 50 μg/ml HMB, we obtained the gene knockout strain Δ GAL4, a novel GAL4-like transcriptional factor (XP_009154426.1) encoding gene (GeneID:20 306 783) accurately deleted.MethodsGrowth curve determination: Take 200 μL of 2 × 10⁴ CFU/ml bacterial suspension and 200 μL liquid sabourand medium in EP tubes. Shaking at 200 rpm 37°C for 0 h, 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 16 h, 18 h, 24 h, 30 h, 36 h, 48 h. Then add 200 μL cultured bacterial suspension to a 96-well plate and repeat three times. Meanwhile set up blank medium control wells. By using an enzyme marker to measure the absorbance at 504 nm, analyzing with SPSS, and then graphing with GraphPad software.Morphological observations: Activated strains on Sabourand microcultures and incubated at 37 °C for 3d. Observe morphology by using fluorescent microscopy to compare the different strains of wild and knockout strains, and using lactic acid cotton phenol blue/fluorescent dye filming observe the morphology of conidia.E-test drug sensitivity method: Spread fresh bacterial suspension of ΔGAL4 and ATCC34100 containing 1 × 10⁶CFU/ml spores on RPMI 1640 agar plates with sterile cotton swabs, and place drug sensitivity test strips [itraconazole (Itraconazole Liofilchem, ICZ 92148), posaconazole (Posaconazole Liofilchem (PCZ 92152), voriconazole (Mérieux, VRC 532800)] in the center plate, after sealing with a parafilm, incubating in 37°C for 36-48 h, and read the values at the tangent of the drug susceptibility strip and the antibacterial circle.ResultsThe results revealed that the ΔGAL4 showed almost no difference in growth and morphology compared with wild strain, but its sensitivity to azoles was altered. We applied E-test to evaluate the MIC changes of azoles. The MIC of itraconazole, voriconazole, and posaconazole against ΔGAL4 was 0.5 μg/ml, 0.125 μg/ml, and 0.25 μg/ml; against ATCC34100 was 2 μg/ml, 0.19 μg/ml, and 0.38 μg/ml, respectively. △GAL4 MIC suggests that ∆GAL4 showed more sensitivity to azoles compared to its wild-type strain.ConclusionThe present results suggested that a novel GAL4-like transcriptional factor may play an important role in azole sensitivity of E. dermatitidis, which may be through modulating efflux pumps or Cyp51A. Further investigation of this GAL4 related modulating pathway is needed.

Participation of the ABC Transporter CDR1 in Azole Resistance of Candida lusitaniae.

Candida lusitaniae is an opportunistic pathogen in humans that causes infrequent but difficult-to-treat diseases. Antifungal drugs are used in the clinic to treat C. lusitaniae infections, however, this fungus can rapidly acquire antifungal resistance to all known antifungal drugs (multidrug resistance). C. lusitaniae acquires azole resistance by gain-of-function (GOF) mutations in the transcriptional regulator MRR1. MRR1 controls the expression of a major facilitator transporter (MFS7) that is important for fluconazole resistance. Here, we addressed the role of the ATP Binding Cassette (ABC) transporter CDR1 as additional mediator of azole resistance in C. lusitaniae. CDR1 expression in isolates with GOF MRR1 mutations was higher compared to wild types, which suggests that CDR1 is an additional (direct or indirect) target of MRR1. CDR1 deletion in the azole-resistant isolate P3 (V688G GOF) revealed that MICs of long-tailed azoles, itraconazole and posaconazole, were decreased compared to P3, which is consistent with the role of this ABC transporter in the efflux of these azoles. Fluconazole MIC was only decreased when CDR1 was deleted in the background of an mfs7Δ mutant from P3, which underpins the dominant role of MFS7 in the resistance of the short-tailed azole fluconazole. With R6G efflux readout as Cdr1 efflux capacity, our data showed that R6G efflux was increased in P3 compared to an azole-susceptible wild type parent, and diminished to background levels in mutant strains lacking CDR1. Milbemycin oxim A3, a known inhibitor of fungal ABC transporters, mimicked efflux phenotypes of cdr1Δ mutants. We therefore provided evidence that CDR1 is an additional mediator of azole resistance in C. lusitaniae, and that CDR1 regulation is dependent on MRR1 and associated GOF mutations.

Azole-Resistant Aspergillus fumigatus Clinical Isolate Screening in Azole-Containing Agar Plates (EUCAST E.Def 10.1): Low Impact of Plastic Trays Used and Poor Performance in Cryptic Species.

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedure's performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n = 91) and cryptic species (n = 52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multidish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harboring TR34-L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/liter. Isolates harboring G54R and TR46-Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

In Vitro Antifungal Drug Resistance Profiles of Clinically Relevant Members of the Mucorales (Mucoromycota) Especially with the Newer Triazoles.

Mucoromycoses (infections caused by members of the order Mucorales, phylum Mucoromycota [ex-Zygomycota]) are highly destructive, rapidly progressive infections, with dire prognoses especially when they occur in immunocompromised hosts. Current treatment guidelines recommend liposomal formulations of amphotericin B with adjunctive surgery as first line therapy, with the newer triazoles posaconazole or isavuconazole as alternative treatments, or as salvage therapy. Among the many organisms belonging to this order, a limited number of species in the genera Rhizopus, Mucor, Lichtheimia and Rhizomucor are responsible for most cases of human infection. Here, we present the minimum inhibitory concentration data (MICs) for amphotericin B, posaconazole, isavuconazole, itraconazole and voriconazole with a panel of over 300 isolates of the five most common agents of human infection (Lichtheimia corymbifera, Rhizopus arrhizus, R. microsporus, Rhizomucor pusillus and Mucor spp.) determined using the CLSI broth microdilution method. In agreement with previous studies, the most active antifungal drug for all Mucorales was amphotericin B, with MICs within the range that would predict susceptibility with Aspergillus fumigatus. Conversely, MICs for voriconazole against all species tested were high, and above the range associated with clinical efficacy with A. fumigatus. Interestingly, whilst isavuconazole and posaconazole MIC distributions indicated in vitro activity against some members of the Mucorales, activity was species-dependent for both agents. These data underscore the importance of accurate identification of the causative agents of mucoromycosis, coupled with antifungal susceptibility testing of individual isolates, in determining the optimal treatment of infections caused by these aggressive opportunistic human fungal pathogens.

The Role of New Posaconazole Formulations in the Treatment of Candida albicans Infections: Data from an In Vitro Pharmacokinetic-Pharmacodynamic Model.

Posaconazole is more active than fluconazole against Candida albicansin vitro and is approved for the treatment of oropharyngeal candidiasis but not for that of invasive candidiasis (IC). Here, we explored the efficacy of posaconazole against C. albicans in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of IC and determined the probability of pharmacodynamic target attainment for the oral solution and intravenous (i.v.)/tablet formulations. Three clinical C. albicans isolates (posaconazole MICs, 0.008 to 0.25 mg/liter) were studied in the in vitro PK/PD dilution model simulating steady-state posaconazole PK. The in vitro exposure-effect relationship, area under the 24-h free drug concentration curve (fAUC0-24)/MIC, was described and compared with in vivo outcome in animals with IC. PK/PD susceptibility breakpoints and trough levels required for optimal treatment were determined for EUCAST and CLSI 24-h/48-h (CLSI24h/CLSI48h) methods using the fAUC0-24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis for oral solution (400 mg every 12 hours [q12h]) and i.v./tablet formulations (300 mg q24h). The in vitro mean (95% confidence interval [CI]) EI50 was 330 (183 to 597) fAUC0-24/MIC for CLSI24h and 169 (92 to 310) for EUCAST/CLSI48h methods, which are close to the near-stasis in vivo effect. The probability of target attainment for EI50 was estimated; for the wild-type isolates (MIC ≤ 0.06 mg/liter), it was low for the oral solution and higher than 95% for the i.v./tablet formulations for the EUCAST/CLSI48h methods but not for the CLSI 24-h method. Non-wild-type isolates with EUCAST/CLSI48h MICs of 0.125 and 0.25 mg/liter would require trough levels of >1.2 and >2.4 mg/liter, respectively. Posaconazole i.v./tablet formulations may have a role in the therapy of invasive infections by wild-type C. albicans isolates, provided that a steady state is reached quickly. A PK/PD susceptibility breakpoint at the epidemiological cutoff (ECV/ECOFF) of 0.06 mg/liter was determined.

The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR343/L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome sequencing. TR343/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR343, thus supporting the possibility of in-host selection of TR34 variants.

Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation.

Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06-1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil's load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

A Revised Species Concept for Opportunistic Mucor Species Reveals Species-Specific Antifungal Susceptibility Profiles.

Recently, the species concept of opportunistic Mucor circinelloides and its relatives has been revised, resulting in the recognition of its classical formae as independent species and the description of new species. In this study, we used isolates of all clinically relevant Mucor species and performed susceptibility testing using the EUCAST reference method to identify potential species-specific susceptibility patterns. In vitro susceptibility profiles of 101 mucoralean strains belonging to the genus Mucor (72), the closely related species Cokeromyces recurvatus (3), Rhizopus (12), Lichtheimia (10), and Rhizomucor (4) to six antifungals (amphotericin B, natamycin, terbinafine, isavuconazole, itraconazole, and posaconazole) were determined. The most active drug for all Mucorales was amphotericin B. Antifungal susceptibility profiles of pathogenic Mucor species were specific for isavuconazole, itraconazole, and posaconazole. The species formerly united in M. circinelloides showed clear differences in their antifungal susceptibilities. Cokeromyces recurvatus, Mucor ardhlaengiktus, Mucor lusitanicus (M. circinelloides f. lusitanicus), and Mucor ramosissimus exhibited high MICs to all azoles tested. Mucor indicus presented high MICs for isavuconazole and posaconazole, and Mucor amphibiorum and Mucor irregularis showed high MICs for isavuconazole. MIC values of Mucor spp. for posaconazole, isavuconazole, and itraconazole were high compared to those for Rhizopus and the Lichtheimiaceae (Lichtheimia and Rhizomucor). Molecular identification combined with in vitro susceptibility testing is recommended for Mucor species, especially if azoles are applied in treatment.

Clinical characteristics, rapid identification, molecular epidemiology and antifungal susceptibilities of Talaromyces marneffei infections in Shenzhen, China.

Although case series of talaromycosis have been reported in China, their detailed clinical and microbiological characteristics have never been systematically profiled. In this study, we report the clinical characteristics, molecular epidemiology, rapid identification and antifungal susceptibilities of talaromycosis in The University of Hong Kong-Shenzhen Hospital in Shenzhen. Seven cases of talaromycosis were observed since commencement of hospital service in 2012. Three patients were local Shenzhen residents, whereas the other four were immigrants from other parts of China. Two patients were HIV-negative, but with underlying diseases requiring immunosuppressive therapy. Two of the seven patients succumbed. All the seven isolates were successfully identified as T.marneffei by MALDI-TOF MS using Bruker database expanded with in-house generated T.marneffei mass spectra. MLST showed that the seven strains belonged to six different, novel sequences types. Phylogenetic analyses of the concatenated five-locus sequence revealed that the seven strains were scattered amongst other T.marneffei strains. The MICs of itraconazole, isavuconazole, posaconazole and voriconazole against the seven clinical isolates were low but MICs of anidulafungin were high. Underlying diseases other than HIV infection are increasingly important risk factors of talaromycosis. MALDI-TOF MS is useful for rapid identification. Highly diverse T.marneffei sequence types were observed.

In vitro susceptibility of dematiaceous fungi to nine antifungal agents determined by two different methods

The infection rate for dematiaceous fungi has increased rapidly over the most recent decades. However, the treatment for such infections has been lacking in empirical support with oral antifungal agents. To provide a better regimen for dematiaceous infections in future, the Sensititre YeastOne® colorimetric antifungal panels were used and compared with Laboratory Standards Institute (CLSI) M38-A2 reference broth microdilution method. Two methods were used for nine antifungals against 67 dematiaceous fungi. Via two methods, we found that the MICs of itraconazole, voriconazole, posaconazole and amphotericin B were lower than fluconazole. The values obtained with CLSI method for four triazoles, 5-flucytosine and amphotericin B were in high essential agreements with those observed by YeastOne® method. The results of echinocandins across the two methods showed some divergence, which might be attributed to the methodology itself, particularly when sensitivity was determined in the lowest concentration of the drug. In YeastOne® method, the results were defined as MICs, but as MECs in CLSI method. The YeastOne®method appeared to be both easier and more efficient for dematiaceous fungi when compared with the CLSI method, and the agreement between the two methods was good for most common antifungals.

Antifungal susceptibility of cryptococcus neoformans clinical isolates from different cities of Argentina

Background: Long-term fluconazole maintenance therapy in AIDS patients has generated concern that resistance strains might emerge. The clinical breakpoints for Cryptococcus spp. are not yet available. It is well known that the susceptibility vary according to genotyping and geographic regions. Therefore it is mandatory to study the susceptibility of Argentinian isolates to follow therapeutic criteria based on Epidemiological Cutoff Values (ECVs). The aim of the present study was to evaluate the activity of six antifungal drugs against a collection of Cryptococcus neoformans isolated in Argentina. Methods & Materials: C. neoformans clinical isolates (n: 88) were obtained in four cities of the Argentina (Buenos Aires, Rosario, Santa Fe and Paraná). Fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B and flucytosine MICs were determined by the CLSI broth microdilution method document M27-A3/S4. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control. Results: Voriconazole MICs geometric mean was 0.06 μg/ml (range 0.03-0.12), Itraconazole 0.07 μg/ml (0.03-0.25), Posaconazole 0.99 μg/ml (0.03 - 0.25), Amphotericin-B 0.15 μg/ml (0.03-0.5), Flucytosine 3.52 μg/ml (2-8) and Fluconazole 4.13 μg/ml (1-16). Fluconazole and Flucytosine showed the highest number of non-wild type strains. For the azole, 31.8% of the isolates showed MIC values above the ECV while for the pyrimidine analog 10.0% of the strains were included in this category. Cross-resistance was observed in four isolates that were classified as non-Wild Type to Fluconazole and Itraconazole, to Fluconazole and Posaconazole, Flucytosine and Fluconazole and to Amphotericin B and Fluconazole. Only one strain was considered as Non Wild Type for three drugs (Fluconazole, Posaconazole and Flucytosine). Conclusion: The most active azole drug against C .neoformans was Voriconazole followed by Itraconazole, Posaconazole and Fluconazole. Considering that VNI is the most prevalent genotype in Argentina almost one third of the strains should be considered as non-wild type. Therefore it is necessary to investigate the molecular mechanisms involved in azole resistance phenotype in order to have the theoretical basis to design molecular diagnostic tools able to quickly uncover resistant isolates.

In vitro activity of zinc chelators combined with antifungals against Mucormycetes

Background: Mucormycetes are resistant to most antifungals and new treatment approaches are needed. Different combination therapies for mucormycoses were reported, with variable results. Zinc deprivation by hosts during infection represents an important antifungal mechanism. Thus, combination of chelators with antifungals may be a plausible chemotherapeutic alternative. This strategy has been tested against other fungal pathogens, with promising results. In this work we study in vitro interactions between amphotericin B, posaconazole and the zinc chelators Clioquinol (5-chloro-7-iodo-quinolin-8-ol), N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) and 1,10-Phenantroline against six Mucormycetes species. Methods & Materials: Twenty-seven Mucormycetes isolates were studied: 2 Rhizopus oryzae, 18 Rhizopus microsporus, 1 Mucor spp, 1 Lichteimia cornymbifera, 4 Syncephalastrum racemosum and 1 Cunninghamella sp. MIC values (no visible growth) for amphotericin B, posaconazole and the chelators Phenantroline, Clioquinol and TPEN were obtained using the CLSI M38-A2 broth microdilution method. All drugs were purchased at Sigma in the form of standard powders, dissolved in 100% dimethylsulphoxide to make stock solutions and stored at -70 °C until required. The dilution ranges were 0.007 to 4 mg/L for amphotericin, 0.015 to 8 mg/L for posaconazole and 0.03 to 16 mg/L for the chelators. The interactions between the antifungals and the chelators were studied by determining the fractional inhibitory concentration index (FICI) using a checkerboard method. The data was interpreted as synergy (FICI ≤ 0.5), antagonism (FICI > 4) or no interaction (FICI > 0.5–4). Results: Amphotericin and posaconazole MICs ranged between 0.25-4 and 0.5-2 mg/L, respectively. Chelators MICs ranged between 0.25 and > 16 mg/L. The FICI results were strain dependent. TPEN and posaconazole were synergistic against Cunninghamella sp. Clioquinol demonstrated synergy with both antifungals against 41% of R. microsporus isolates. Phenantroline and TPEN with either posaconazole and amphotericin demonstrated no interaction for most isolates (80%). Conclusion: Clioquinol proved to be the most effective zinc chelator against the isolates in our Mucormycetes collection. Combination of posaconazole with this drug showed promising results for further in vivo studies in a model such as Galleria mellonella.

Rezafungin (CD101) demonstrates potent in vitro activity against Aspergillus, including azole-resistant Aspergillus fumigatus isolates and cryptic species.

Rezafungin is an investigational echinocandin under development for the treatment and prevention of invasive fungal infections, with a long half-life in humans (∼130 h) and potent in vitro activity against Aspergillus spp. Our objective was to further evaluate its activity against Aspergillus fumigatus isolates, including azole-resistant isolates and cryptic Aspergillus spp. Clinical isolates of Aspergillus were used, including 15 WT and 31 azole-resistant A. fumigatus, 11 Aspergillus lentulus, 5 each of Aspergillus thermomutatus and Aspergillus udagawae and 11 Aspergillus calidoustus. Minimum effective concentrations (MECs) and MICs of rezafungin, caspofungin, micafungin, posaconazole and voriconazole were determined by CLSI M38-A2 broth microdilution. Differences in geometric mean (GM) MEC/MIC values were assessed for significance by ANOVA. Rezafungin GM MECs for A. fumigatus were 0.024 and 0.043 mg/L for WT and azole-resistant isolates, respectively. Rezafungin was also active against cryptic species, including A. lentulus (0.016 mg/L), A. calidoustus (0.044 mg/L), A. thermomutatus (MEC range ≤0.015-0.25 mg/L) and A. udagawae (≤0.015-0.03 mg/L). This activity was similar to that of caspofungin and micafungin with the exception of A. calidoustus, against which rezafungin was more potent than caspofungin (GM MEC 0.044 versus 0.468 mg/L; P < 0.0001). Rezafungin demonstrated potent in vitro activity against Aspergillus spp., including azole-resistant A. fumigatus isolates and cryptic species with elevated posaconazole and voriconazole MICs. Additional studies are warranted to determine whether the in vitro activity translates into in vivo efficacy against infections caused by resistant Aspergillus isolates.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 μg/ml for the CLSI method and 0.25 μg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 μg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 μg/ml. The tentative ECOFFinder ECV with SYO was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

Antifungal Susceptibility of the Aspergillus viridinutans Complex: Comparison of Two In Vitro Methods.

Cryptic species of Aspergillus fumigatus, including the Aspergillus viridinutans species complex, are increasingly reported to be causes of invasive aspergillosis. Their identification is clinically relevant, as these species frequently have intrinsic resistance to common antifungals. We evaluated the susceptibilities of 90 environmental and clinical isolates from the A. viridinutans species complex, identified by DNA sequencing of the calmodulin gene, to seven antifungals (voriconazole, posaconazole, itraconazole, amphotericin B, anidulafungin, micafungin, and caspofungin) using the reference European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. The majority of species demonstrated elevated MICs of voriconazole (geometric mean [GM] MIC, 4.46 mg/liter) and itraconazole (GM MIC, 9.85 mg/liter) and had variable susceptibility to amphotericin B (GM MIC, 2.5 mg/liter). Overall, the MICs of posaconazole and the minimum effective concentrations of echinocandins were low. The results obtained by the EUCAST method were compared with the results obtained with Sensititre YeastOne (YO) panels. Overall, there was 67% agreement (95% confidence interval [CI], 62 to 72%) between the results obtained by the EUCAST method and those obtained with YO panels when the results were read at 48 h and 82% agreement (95% CI, 78 to 86%) when the results were read at 72 h. There was a significant difference in agreement between antifungals; agreement was high for amphotericin B, voriconazole, and posaconazole (70 to 86% at 48 h and 88 to 93% at 72 h) but was very low for itraconazole (37% at 48 h and 57% at 72 h). The agreement was also variable between species, with the maximum agreement being observed for A. felis isolates (85 and 93% at 48 and 72 h, respectively). Elevated MICs of voriconazole and itraconazole were cross-correlated, but there was no correlation between the other azoles tested.

Comparative evaluation of different gradient diffusion tests for detection of azole resistance in Aspergillus fumigatus

Gradient diffusion assays Etest and MIC Test Strip for itraconazole, posaconazole and voriconazole as well as isavuconazole (MIC Test Strip) were evaluated for Aspergillus fumigatus against EUCAST broth microdilution. Both assays demonstrated generally good performance; however, posaconazole MIC Test Strip showed low agreement with broth microdilution due to MIC overestimation.

Isavuconazole susceptibility of clinical Aspergillus fumigatus isolates and feasibility of isavuconazole dose escalation to treat isolates with elevated MICs.

Isavuconazole is a new triazole approved for the treatment of invasive aspergillosis. We investigated isavuconazole MIC distributions, isavuconazole MIC correlations with those of other azoles and pharmacodynamics of isavuconazole in low-level resistant Aspergillus fumigatus isolates. Isavuconazole, voriconazole, itraconazole and posaconazole susceptibility of 487 clinical A. fumigatus isolates was determined by EUCAST broth microdilution methodology. Using an in vivo estimation of the pharmacodynamic target and a previously published pharmacokinetic model, the probability of target attainment (PTA) was determined for a range of isavuconazole MICs using three dosing regimens (I, 200 mg once daily; II, 300 mg once daily; and III, 400 mg once daily). Two hundred and seventy-nine of 487 isolates were phenotypically WT based on epidemiological cut-offs of voriconazole, itraconazole and posaconazole. Twenty-five of 279 phenotypically WT isolates and 196 of 208 non-WT isolates were classified as isavuconazole resistant based on the EUCAST breakpoint of 1 mg/L. Isavuconazole MICs showed very high correlation with voriconazole MICs, but moderate and low correlation with itraconazole and posaconazole MICs. The PTA for isolates with an isavuconazole MIC of 1 mg/L was 92%-99% for 90% effective concentration (EC90) for the three dosing regimens. For isolates with an MIC of 2 mg/L the PTA decreased to 64%-92% for EC90. Our study indicated that isavuconazole and voriconazole MICs are highly correlated and that high-dose isavuconazole treatment might be an option in patients infected with an A. fumigatus isolate with an isavuconazole MIC of 2 mg/L.

Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans.

The activity of 7 antifungal agents against 3,557 invasive yeasts and molds collected in 29 countries worldwide in 2014 and 2015 was evaluated. Epidemiological cutoff values (ECVs) published in the Clinical and Laboratory Standards Institute (CLSI) M59 document were applied for species with no clinical breakpoints. Echinocandin susceptibility rates were 95.9% to 100.0% for the 5 most common Candida species, except for the rates for Candida parapsilosis to anidulafungin (88.7% susceptible, 100.0% wild type). Rates of fluconazole resistance ranged from 8.0% for Candida glabrata to 0.4% for Candida albicans Seven Candida species displayed 100.0% wild-type amphotericin B MIC results, and Candida dubliniensis and Candida lusitaniae exhibited wild-type echinocandin MIC results. The highest fluconazole, voriconazole, and posaconazole MIC values for Cryptococcus neoformans var. grubii were 8 μg/ml, 0.12 μg/ml, and 0.25 μg/ml, respectively. Aspergillus fumigatus isolates were 100.0% wild type for caspofungin and amphotericin B, but 3 (0.8%) of these isolates were non-wild type to itraconazole (2 isolates) or voriconazole (1 isolate). Mutations in FKS hot spot (HS) regions were detected among 13/20 Candida isolates displaying echinocandin MICs greater than the ECV (16 of these 20 isolates were C. glabrata). Most isolates carrying mutations in FKS HS regions were resistant to 2 or more echinocandins. Five fluconazole-nonsusceptible C. albicans isolates were submitted to whole-genome sequencing analysis. Gain-of-function, Erg11 heterozygous, and Erg3 homozygous mutations were observed in 1 isolate each. One isolate displayed MDR1 promoter allele alterations associated with azole resistance. Elevated levels of expression of MDR1 or CDR2 were observed in 3 isolates and 1 isolate, respectively. Echinocandin and azole resistance is still uncommon among contemporary fungal isolates; however, mechanisms of resistance to antifungals were observed among Candida spp., showing that resistance can emerge and monitoring is warranted.