Evaluation of Synergistic Activity of Isavuconazole or Voriconazole plus Anidulafungin and the Occurrence and Genetic Characterization of Candida auris Detected in a Surveillance Program.

A total of 15 Candida auris isolates from the SENTRY antimicrobial surveillance program between 2006 and 2019 were combined with 21 isolates from other collections for the evaluation of antifungal susceptibility and synergy against anidulafungin plus voriconazole or isavuconazole using the checkerboard method. Surveillance isolates were analyzed for genetic relatedness and resistance mechanisms. Applying the tentative statistical epidemiological cutoff values and the Centers for Disease Control tentative breakpoints, 32/36 isolates were resistant to fluconazole, 5/36 were resistant to amphotericin B, 5/36 were non-wild-type (NWT) to anidulafungin, 3/36 were NWT to micafungin, and 1/36 and 10/36 were NWT to isavuconazole and voriconazole, respectively. Of these, 10 isolates were multidrug resistant, which means that these isolates were resistant to 2 antifungal classes. Synergy or partial synergy was noted in 5/36 and 22/36, respectively, of the isolates with the combination of anidulafungin plus voriconazole, and 11/36 and 19/36 isolates, respectively, for the combination of anidulafungin plus isavuconazole. Multilocus sequence type (MLST) analysis of the 15 SENTRY isolates demonstrated that the isolates from the US were genetically related to, but different from, isolates from Latin America (Panama and Colombia) and Germany. Single nucleotide polymorphism (SNP) analysis showed that the 15 SENTRY isolates belonged to the described international clades and had associated Erg11 alterations, including 11 isolates displaying K143R, one displaying F126L, and one displaying Y501H alterations and a fluconazole MIC result of ≥64 mg/liter. Resistance mechanisms were not observed in the two isolates displaying fluconazole MIC values at 4 and 16 mg/liter. Isavuconazole displayed activity and greater synergy when tested with anidulafungin than seen with anidulafungin plus voriconazole against the C. auris clinical isolates that displayed resistance phenotypes.

Activity of a Long-Acting Echinocandin, Rezafungin, and Comparator Antifungal Agents Tested against Contemporary Invasive Fungal Isolates (SENTRY Program, 2016 to 2018).

We evaluated the activity of rezafungin and comparators, using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methods, against a worldwide collection of 2,205 invasive fungal isolates recovered from 2016 to 2018. Candida (n = 1,904 isolates; 6 species), Cryptococcus neoformans (n = 73), Aspergillus fumigatus (n = 183), and Aspergillus flavus (n = 45) isolates were tested for their susceptibility (S) to rezafungin as well as the comparators caspofungin, anidulafungin, micafungin, and azoles. Interpretive criteria were applied following CLSI published clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs). Isolates displaying non-wild-type (non-WT) echinocandin MIC values were sequenced for hot spot (HS) mutations. Rezafungin inhibited 99.8% of Candida albicans isolates (MIC50/90, 0.03/0.06 µg/ml), 95.7% of Candida glabrata isolates (MIC50/90, 0.06/0.12 µg/ml), 97.4% of Candida tropicalis isolates (MIC50/90, 0.03/0.06 µg/ml), 100.0% of Candida krusei isolates (MIC50/90, 0.03/0.06 µg/ml), and 100.0% of Candida dubliniensis isolates (MIC50/90, 0.06/0.12 µg/ml) at ≤0.12 µg/ml. All (329/329 [100.0%]) Candida parapsilosis isolates (MIC50/90,1/2 µg/ml) were inhibited by rezafungin at ≤4 µg/ml. Fluconazole resistance was detected among 8.6% of C. glabrata isolates, 12.5% of C. parapsilosis isolates, 3.2% of C. dubliniensis isolates, and 2.6% of C. tropicalis isolates. The activity of rezafungin against these 6 Candida spp. was similar to the activity of the other echinocandins. Detection of the HS mutation was performed by sequencing echinocandin-resistant or non-WT Candida isolates. Good activity against C. neoformans was observed for fluconazole and the other azoles, whereas the echinocandins, including rezafungin, displayed limited activity. Rezafungin displayed activity similar to that of the other echinocandins against A. fumigatus and A. flavus These in vitro data contribute to accumulating research demonstrating the potential of rezafungin for preventing and treating invasive fungal infections.

Activity of a Long-Acting Echinocandin (CD101) and Seven Comparator Antifungal Agents Tested against a Global Collection of Contemporary Invasive Fungal Isolates in the SENTRY 2014 Antifungal Surveillance Program.

The activity of CD101 and comparator antifungal agents against 606 invasive fungal isolates collected worldwide during 2014 was evaluated using the Clinical and Laboratory Standards Institute (CLSI) method. All Candida albicans (n = 251), Candida tropicalis (n = 51), Candida krusei (n = 16), and Candida dubliniensis (n = 11) isolates were inhibited by ≤0.12 µg/ml of CD101 and were susceptible or showed wild-type susceptibility to the other echinocandins tested. Five C. glabrata isolates (n = 100) displayed CD101 MIC values of 1 to 4 µg/ml, had elevated MICs of caspofungin (2 to >8 µg/ml), anidulafungin (2 to 4 µg/ml), and micafungin (2 to 4 µg/ml), and carried mutations on fks1 and fks2Candida parapsilosis (n = 92) and Candida orthopsilosis (n = 10) displayed higher CD101 MIC values (ranges, 0.5 to 4 µg/ml and 0.12 to 2 µg/ml, respectively), and similar results were observed for the other echinocandins tested. Fluconazole resistance was noted among 11.0% of Candida glabrata isolates, 4.3% of C. parapsilosis isolates, and 2.0% of C. albicans and C. tropicalis isolates. The activity of CD101 against Aspergillus fumigatus (n = 56) was similar to that of micafungin and 2-fold greater than that of caspofungin but less than that of anidulafungin. These isolates had wild-type susceptibility to itraconazole, voriconazole, and posaconazole. The echinocandins had limited activity against Cryptococcus neoformans (n = 19). CD101 was as active as the other echinocandins against common fungal organisms recovered from patients with invasive fungal infections. The long half-life profile is very desirable for the prevention and treatment of serious fungal infections, especially in patients who can then be discharged from the hospital to complete antifungal therapy on an outpatient basis.

Differential Activity of the Oral Glucan Synthase Inhibitor SCY-078 against Wild-Type and Echinocandin-Resistant Strains of Candida Species.

SCY-078 (formerly MK-3118) is a novel orally active inhibitor of fungal ß-(1,3)-glucan synthase (GS). SCY-078 is a derivative of enfumafungin and is structurally distinct from the echinocandin class of antifungal agents. We evaluated the in vitro activity of this compound against wild-type (WT) and echinocandin-resistant isolates containing mutations in the FKS genes of Candida spp. Against 36 Candida spp. FKS mutants tested, 30 (83.3%) were non-WT to 1 or more echinocandins, and only 9 (25.0%) were non-WT (MIC, >WT-upper limit) to SCY-078. Among C. glabrata isolates carrying FKS alterations, 84.0% were non-WT to the echinocandins versus only 24.0% for SCY-078. In contrast to the echinocandin comparators, the activity of SCY-078 was minimally affected by the presence of FKS mutations, suggesting that this agent is useful in the treatment of Candida infections due to echinocandin-resistant strains.

Activity of a long-acting echinocandin, CD101, determined using CLSI and EUCAST reference methods, against Candida and Aspergillus spp., including echinocandin- and azole-resistant isolates.

OBJECTIVES: The objective of this study was to evaluate the in vitro activity of CD101, a novel echinocandin with a long serum elimination half-life, and comparator (anidulafungin and caspofungin) antifungal agents against a collection of Candida and Aspergillus spp. isolates. METHODS: CD101 and comparator agents were tested against 106 Candida spp. and 67 Aspergillus spp. isolates, including 27 isolates of Candida harbouring fks hotspot mutations and 12 itraconazole non-WT Aspergillus, using CLSI and EUCAST reference susceptibility broth microdilution (BMD) methods. RESULTS: Against WT and fks mutant Candida albicans, Candida glabrata and Candida tropicalis, the activity of CD101 [MIC90 = 0.06, 0.12 and 0.03 mg/L, respectively (CLSI method values)] was comparable to that of anidulafungin (MIC90 = 0.03, 0.12 and 0.03 mg/L, respectively) and caspofungin (MIC90 = 0.12, 0.25 and 0.12 mg/L, respectively). WT Candida krusei isolates were very susceptible to CD101 (MIC = 0.06 mg/L). CD101 activity (MIC50/90 = 1/2 mg/L) was comparable to that of anidulafungin (MIC50/90 = 2/2 mg/L) against Candida parapsilosis. CD101 (MIC mode = 0.06 mg/L for C. glabrata) was 2- to 4-fold more active against fks hotspot mutants than caspofungin (MIC mode = 0.5 mg/L). CD101 was active against Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger and Aspergillus flavus (MEC90 range = ≤0.008-0.03 mg/L). The essential agreement between CLSI and EUCAST methods for CD101 was 92.0%-100.0% among Candida spp. and 95.0%-100.0% among Aspergillus spp. CONCLUSIONS: The activity of CD101 is comparable to that of other members of the echinocandin class for the prevention and treatment of serious fungal infections. Similar results for CD101 activity versus Candida and Aspergillus spp. may be obtained with either CLSI or EUCAST BMD methods.

In vitro antifungal susceptibilities of isolates of Candida spp. and Aspergillus spp. from China to nine systemically active antifungal agents: data from the SENTRY antifungal surveillance program, 2010 through 2012.

We report the in vitro activity of nine systemically active antifungal agents against 237 contemporary clinical isolates of yeast and moulds obtained from 13 laboratories in China during 2010 through 2012. Susceptibility testing was performed using CLSI methods. Sequencing of fks hot spots was performed for echinocandin non-wild-type (WT) strains. Isolates included 220 from eight species of Candida, 15 from four species of Aspergillus and one isolate each of Rhodotorula mucilaginosa and Trichosporon asahii. Resistance to amphotericin B (0.0%), flucytosine (0.0-1.7%) and the echinocandins (0.0-3.4%) was distinctly uncommon among C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. pelliculosa. Three C. albicans isolates showed resistance to echinocandins and one harboured a mutation in HS1 of fks1. Resistance to the azoles was much more common with resistance to fluconazole, voriconazole and posaconazole detected among isolates of C. glabrata and C. tropicalis. Both C. parapsilosis and C. pelliculosa exhibited decreased susceptibility to fluconazole. Amphotericin B, the mould-active azoles and the echinocandins were all quite active against isolates of A. fumigatus and A. flavus. Consistent with previous studies from China, resistance to fluconazole is prominent among Candida spp. isolates in this country.

Frequency of fks mutations among Candida glabrata isolates from a 10-year global collection of bloodstream infection isolates.

Among 119 echinocandin non-wild-type (non-WT) Candida glabrata strains from two global surveys, mutations in fks hot spots (HSs) were detected in 28 (from 7 countries and 8 U.S. states): 24 strains (85.7%) had non-WT MICs for micafungin, 22 (78.6%) for anidulafungin, and 25 (89.3%) for caspofungin. The most common FKS substitutions among non-WT strains were at positions F659 (n = 7) and S663 (n = 7). Three isolates displaying WT MIC results had F625Y, L630I, and D632Y substitutions or non-HS mutations. Mutations that have been reported to decrease the echinocandin binding to the 1,3-ß-d-glucan synthase were categorized as resistant by applying the new CLSI breakpoint criteria for all three echinocandins.

Use of micafungin as a surrogate marker to predict susceptibility and resistance to caspofungin among 3,764 clinical isolates of Candida by use of CLSI methods and interpretive criteria.

Due to unacceptably high interlaboratory variation in caspofungin MIC values, we evaluated the use of micafungin as a surrogate marker to predict the susceptibility of Candida spp. to caspofungin using reference methods and species-specific interpretive criteria. The MIC results for 3,764 strains of Candida (eight species), including 73 strains with fks mutations, were used. Caspofungin MIC values and species-specific interpretive criteria were compared with those of micafungin to determine the percent categorical agreement (%CA) and very major error (VME), major error (ME), and minor error rates as well as their ability to detect fks mutant strains of Candida albicans (11 mutants), Candida tropicalis (4 mutants), Candida krusei (3 mutants), and Candida glabrata (55 mutants). Overall, the %CA was 98.8% (0.2% VMEs and MEs, 0.8% minor errors) using micafungin as the surrogate marker. Among the 60 isolates of C. albicans (9 isolates), C. tropicalis (5 isolates), C. krusei (2 isolates), and C. glabrata (44 isolates) that were nonsusceptible (either intermediate or resistant) to both caspofungin and micafungin, 54 (90.0%) contained a mutation in fks1 or fks2. An additional 10 C. glabrata mutants, two C. albicans mutants, and one mutant each of C. tropicalis and C. krusei were classified as susceptible to both antifungal agents. Using the epidemiological cutoff values (ECVs) of 0.12 µg/ml for caspofungin and 0.03 µg/ml for micafungin to differentiate wild-type (WT) from non-WT strains of C. glabrata, 80% of the C. glabrata mutants were non-WT for both agents (96% concordance). Micafungin may serve as an acceptable surrogate marker for the prediction of susceptibility and resistance of Candida to caspofungin.

Isavuconazole and nine comparator antifungal susceptibility profiles for common and uncommon Candida species collected in 2012: application of new CLSI clinical breakpoints and epidemiological cutoff values.

The in vitro activity of isavuconazole and nine antifungal comparator agents was assessed using reference broth microdilution methods against 1,421 common and uncommon species of Candida from a 2012 global survey. Isolates were identified using CHROMagar, biochemical methods and sequencing of ITS and/or 28S regions. Candida spp. were classified as either susceptible or resistant and as wild type (WT) or non-WT using CLSI clinical breakpoints or epidemiological cutoff values, respectively, for the antifungal agents. Isolates included 1,421 organisms from 21 different species of Candida. Among Candida spp., resistance to all 10 tested antifungal agents was low (0.0-7.9 %). The vast majority of each species of Candida, with the exception of Candida glabrata, Candida krusei, and Candida guilliermondii (modal MICs of 0.5 µg/ml), were inhibited by ≤0.12 µg/ml of isavuconazole (99.0 %; range 94.3 % [Candida tropicalis] to 100.0 % [Candida lusitaniae and Candida dubliniensis]). C. glabrata, C. krusei, and C. guilliermondii were largely inhibited by ≤1 µg/ml of isavuconazole (89.7, 96.9 and 92.8 %, respectively). Decreased susceptibility to isavuconazole was most prominent with C. glabrata where the modal MIC for isavuconazole was 0.5 µg/ml for those strains that were SDD to fluconazole or WT to voriconazole, and was 4 µg/ml for those that were either resistant or non-WT to fluconazole or voriconazole, respectively. In conclusion, these data document the activity of isavuconazole and generally the low resistance levels to the available antifungal agents in a large, contemporary (2012), global collection of molecularly characterized species of Candida.

Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations.

BACKGROUND: Fluconazole (FLC) resistance is common in C. glabrata and echinocandins are often used as first-line therapy. Resistance to echinocandin therapy has been associated with FKS1 and FKS2 gene alterations. METHODS: We reviewed records of all patients with C. glabrata bloodstream infection at Duke Hospital over the past decade (2001-2010) and correlated treatment outcome with minimum inhibitory concentration (MIC) results and the presence of FKS gene mutations. For each isolate, MICs to FLC and echinocandins (anidulafungin, caspofungin, and micafungin) and FKS1 and FKS2 gene sequences were determined. RESULTS: Two hundred ninety-three episodes (313 isolates) of C. glabrata bloodstream infection were analyzed. Resistance to echinocandins increased from 4.9% to 12.3% and to FLC from 18% to 30% between 2001 and 2010, respectively. Among the 78 FLC resistant isolates, 14.1% were resistant to 1 or more echinocandin. Twenty-five (7.9%) isolates harbored a FKS mutation. The predictor of a FKS mutant strain was prior echinocandin therapy (stepwise multivariable analysis, odds ratio, 19.647 [95% confidence interval, 7.19-58.1]). Eighty percent (8/10) of patients infected with FKS mutants demonstrating intermediate or resistant MICs to an echinocandin and treated with an echinocandin failed to respond or responded initially but experienced a recurrence. CONCLUSIONS: Echinocandin resistance is increasing, including among FLC-resistant isolates. The new Clinical and Laboratory Standards Institute clinical breakpoints differentiate wild-type from C. glabrata strains bearing clinically significant FKS1/FKS2 mutations. These observations underscore the importance of knowing the local epidemiology and resistance patterns for Candida within institutions and susceptibility testing of echinocandins for C. glabrata to guide therapeutic decision making.

In vitro activity of a new oral glucan synthase inhibitor (MK-3118) tested against Aspergillus spp. by CLSI and EUCAST broth microdilution methods.

MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ≥ 4 µg/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC(90))/MIC(90) values (µg/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fumigatus SC; 0.12, 2, and 0.06 for Aspergillus terreus SC; and 0.06, 1, and 0.03 for Aspergillus niger SC. Essential agreement between the values found by CLSI and EUCAST (± 2 log(2) dilution steps) was 94.3%. MK-3118 was determined to be a potent agent regardless of the in vitro method applied, with excellent activity against contemporary wild-type and itraconazole-resistant strains of Aspergillus spp.

In vitro activities of isavuconazole and comparator antifungal agents tested against a global collection of opportunistic yeasts and molds.

Isavuconazole is a new broad-spectrum triazole with a favorable pharmacokinetic and safety profile. We report the MIC distributions for isavuconazole and 111 isolates of Candida (42 Candida albicans, 25 Candida glabrata, 22 Candida parapsilosis, 14 Candida tropicalis, and 8 Candida krusei isolates), as determined by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution (BMD) methods. Also, the relative activities of isavuconazole, itraconazole, fluconazole, posaconazole, voriconazole, and the three echinocandins were assessed against a recent (2011) global collection of 1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds using CLSI BMD methods. The overall essential agreement (EA) (±2 log2 dilutions) between the CLSI and EUCAST methods was 99.1% (EA at ±1 log2 dilution, 90.1% [range, 80.0 to 100.0%]). The activities of isavuconazole against the larger collection of Candida spp. and Aspergillus spp. were comparable to those of posaconazole and voriconazole; the MIC90 values for isavuconazole, posaconazole, and voriconazole against Candida spp. were 0.5, 1, and 0.25 µg/ml and against Aspergillus spp. were 2, 1, and 1 µg/ml, respectively. Isavuconazole showed good activities against Cryptococcus neoformans (MIC90, 0.12 µg/ml) and other non-Candida yeasts (MIC90, 1 µg/ml) but was less potent against non-Aspergillus molds (MIC90, >8 µg/ml). Isavuconazole MIC values for three mucormycete isolates were 4, 1, and 2 µg/ml, whereas all three were inhibited by 1 µg/ml posaconazole. Isavuconazole demonstrates broad-spectrum activity against this global collection of opportunistic fungi, and the CLSI and EUCAST methods can be used to test this agent against Candida, with highly comparable results.

Echinocandin and triazole antifungal susceptibility profiles for clinical opportunistic yeast and mold isolates collected from 2010 to 2011: application of new CLSI clinical breakpoints and epidemiological cutoff values for characterization of geographic and temporal trends of antifungal resistance.

The SENTRY Antimicrobial Surveillance Program monitors global susceptibility and resistance rates of newer and established antifungal agents. We report the echinocandin and triazole antifungal susceptibility patterns for 3,418 contemporary clinical isolates of yeasts and molds. The isolates were obtained from 98 laboratories in 34 countries during 2010 and 2011. Yeasts not presumptively identified by CHROMagar, the trehalose test, or growth at 42°C and all molds were sequence identified using internal transcribed spacer (ITS) and 28S (yeasts) or ITS, translation elongation factor (TEF), and 28S (molds) genes. Susceptibility testing was performed against 7 antifungals (anidulafungin, caspofungin, micafungin, fluconazole, itraconazole, posaconazole, and voriconazole) using CLSI methods. Rates of resistance to all agents were determined using the new CLSI clinical breakpoints and epidemiological cutoff value criteria, as appropriate. Sequencing of fks hot spots was performed for echinocandin non-wild-type (WT) strains. Isolates included 3,107 from 21 Candida spp., 146 from 9 Aspergillus spp., 84 from Cryptococcus neoformans, 40 from 23 other mold species, and 41 from 9 other yeast species. Among Candida spp., resistance to the echinocandins was low (0.0 to 1.7%). Candida albicans and Candida glabrata that were resistant to anidulafungin, caspofungin, or micafungin were shown to have fks mutations. Resistance to fluconazole was low among the isolates of C. albicans (0.4%), Candida tropicalis (1.3%), and Candida parapsilosis (2.1%); however, 8.8% of C. glabrata isolates were resistant to fluconazole. Among echinocandin-resistant C. glabrata isolates from 2011, 38% were fluconazole resistant. Voriconazole was active against all Candida spp. except C. glabrata (10.5% non-WT), whereas posaconazole showed decreased activity against C. albicans (4.4%) and Candida krusei (15.2% non-WT). All agents except for the echinocandins were active against C. neoformans, and the triazoles were active against other yeasts (MIC90, 2 µg/ml). The echinocandins and triazoles were active against Aspergillus spp. (MIC90/minimum effective concentration [MEC90] range, 0.015 to 2 µg/ml), but the echinocandins were not active against other molds (MEC90 range, 4 to >16 µg/ml). Overall, echinocandin and triazole resistance rates were low; however, the fluconazole and echinocandin coresistance among C. glabrata strains warrants continued close surveillance.

Activity of MK-3118, a new oral glucan synthase inhibitor, tested against Candida spp. by two international methods (CLSI and EUCAST).

OBJECTIVES: To evaluate the activity of the orally bioavailable enfumafungin derivative MK-3118 and comparator antifungal agents tested against a collection of 113 clinical isolates of Candida spp. using CLSI and EUCAST broth microdilution (BMD) methods. METHODS: Candida spp. isolates (n=113) were tested by CLSI and EUCAST methods. The collection contained 29 Candida albicans, 29 Candida glabrata, 21 Candida tropicalis, 15 Candida parapsilosis and 19 Candida krusei, including azole- and echinocandin-resistant isolates. CLSI and EUCAST MIC endpoints of 50% and 100% inhibition were determined using visual reading at 24 and 48 h of incubation and spectrophotometric reading at 24 h of incubation, respectively. RESULTS: MK-3118 CLSI MIC results ranged from 0.06 to 16 mg/L depending on species, duration of incubation and endpoint criteria (EC) used. Comparison of CLSI and EUCAST following 24 h of incubation and either 50% or 100% inhibition revealed an essential agreement (EA; ± 2 doubling dilutions) of 99.1% using the 50% inhibition EC and 93.2% using the 100% inhibition EC. MK-3118 (24 h of incubation and 50% EC) was active against all the species tested and displayed similar potency to caspofungin (using CLSI BMD) against C. albicans (MIC90, 1 and 2 mg/L, respectively), C. tropicalis (1 and 1 mg/L, respectively), C. parapsilosis (0.5 and 0.5 mg/L, respectively) and C. krusei (2 and 1 mg/L, respectively), but was 8-fold more potent than caspofungin against C. glabrata strains (MIC90, 2 and 16 mg/L, respectively). MK-3118 was active against fluconazole-resistant strains as well as caspofungin-resistant strains with documented fks mutations. CONCLUSIONS: MK-3118 was documented to have potent in vitro activity against Candida spp. when tested by both CLSI and EUCAST BMD methods, with the highest overall EA (99.1%) obtained when MK-3118 MIC results were read after 24 h of incubation using a partial inhibition EC (50%).

Significance of molecular identification and antifungal susceptibility of clinically significant yeasts and moulds in a global antifungal surveillance programme.

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45 °C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, ß-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7 %): Aspergillus spp. (64.1 % correct); Candida spp. (60.1 % correct); non-Candida yeasts (58.9 % correct); non-Aspergillus moulds (30.4 % correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0  %), C. lusitaniae (100 %), C. dubliniensis (92.3 %), C. kefyr (100 %), and C. neoformans (90.2 %). Only 50.0 % of isolates of C. albicans and 59.1 % of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.

Candida bloodstream infections: comparison of species distributions and antifungal resistance patterns in community-onset and nosocomial isolates in the SENTRY Antimicrobial Surveillance Program, 2008-2009.

Community-onset (CO) candidemia, defined as a positive blood culture taken at or within 2 days of hospital admission, represents a distinct clinical entity associated with substantial morbidity and mortality. Reference MIC results from the SENTRY Antimicrobial Surveillance Program (2008-2009) were analyzed to compare the antifungal resistance patterns and species distributions from patients with CO and nosocomial bloodstream infections (BSI) in 79 medical centers. Among 1,354 episodes of BSI, 494 (36.5%) were classified as CO and 860 (63.5%) as nosocomial in origin. More than 95% of the isolates from both BSI types were contributed by Candida albicans (48.4%), C. glabrata (18.2%), C. parapsilosis (17.1%), C. tropicalis (10.6%), and C. krusei (2.0%). C. albicans was more common in CO BSI (51.0%) than nosocomial BSI (46.9%), whereas C. parapsilosis and C. krusei were more common in nosocomial BSIs (18.1 and 2.7%, respectively) than in CO BSIs (15.4 and 0.8%, respectively). C. glabrata and C. tropicalis were comparable in both CO (18.4 and 10.5%, respectively) and nosocomial (18.1 and 10.6%, respectively) episodes. Resistance to azoles (fluconazole, posaconazole, and voriconazole) and echinocandins (anidulafungin, caspofungin, and micafungin) was uncommon (<5%) in CO BSI using recently established Clinical and Laboratory Standards Institute breakpoint criteria. Resistance to echinocandins (anidulafungin [3.8%], caspofungin [5.1%], and micafungin [3.2%]) and azoles (fluconazole [7.7%], posaconazole [5.1%], and voriconazole [6.4%]) was most prevalent among nosocomial BSI isolates of C. glabrata. CO candidemia is not uncommon and appears to be increasing worldwide due to changing health care practices. Although resistance to the azoles and echinocandins remains uncommon among CO isolates, we demonstrate the emergence of nosocomial occurrences of C. glabrata expressing resistance to both monitored classes of antifungal agents.

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Aspergillus spp. determined by CLSI and EUCAST broth microdilution methods.

E1210 is a first-in-class broad-spectrum antifungal that suppresses hyphal growth by inhibiting fungal glycophosphatidylinositol (GPI) biosynthesis. In the present study, we extend these findings by examining the activity of E1210 and comparator antifungal agents against Aspergillus spp. by using the methods of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test wild-type (WT) as well as amphotericin B (AMB)-resistant (-R) and azole-R strains (as determined by CLSI methods). Seventy-eight clinical isolates of Aspergillus were tested including 20 isolates of Aspergillus flavus species complex (SC), 22 of A. fumigatus SC, 13 of A. niger SC, and 23 of A. terreus SC. The collection included 15 AMB-R (MIC, ≥ 2 µg/ml) isolates of A. terreus SC and 10 itraconazole-R (MIC, ≥ 4 µg/ml) isolates of A. fumigatus SC (7 isolates), A. niger SC (2 isolates), and A. terreus SC (1 isolate). Comparator antifungal agents included anidulafungin, caspofungin, amphotericin B, itraconazole, posaconzole, and voriconazole. Both CLSI and EUCAST methods were highly concordant for E1210 and all comparators. The essential agreement (EA; ± 2 log(2) dilution steps) was 100% for all comparisons with the exception of posaconazole versus A. terreus SC (EA = 91.3%). The minimum effective concentration (MEC)/MIC(90) values (µg/ml) for E1210, anidulafungin, caspofungin, itraconazole, posaconazole, and voriconazole, respectively, were as follows for each species: for A. flavus SC, 0.03, ≤ 0.008, 0.12, 1, 1, and 1; for A. fumigatus SC, 0.06, 0.015, 0.12, >8, 1, and 4; for A. niger SC, 0.015, 0.03, 0.12, 4, 1, and 2; and for A. terreus SC, 0.06, 0.015, 0.12, 1, 0.5, and 1. E1210 was very active against AMB-R strains of A. terreus SC (MEC range, 0.015 to 0.06 µg/ml) and itraconazole-R strains of A. fumigatus SC (MEC range, 0.03 to 0.12 µg/ml), A. niger SC (MEC, 0.008 µg/ml), and A. terreus SC (MEC, 0.015 µg/ml). In conclusion, E1210 was a very potent and broad-spectrum antifungal agent regardless of in vitro method applied, with excellent activity against AMB-R and itraconazole-R strains of Aspergillus spp.

Geographic variations in species distribution and echinocandin and azole antifungal resistance rates among Candida bloodstream infection isolates: report from the SENTRY Antimicrobial Surveillance Program (2008 to 2009).

Antifungal testing results from the SENTRY Antimicrobial Surveillance Program (2008 to 2009) were analyzed for regional variations of invasive Candida species infections. Among 2,085 cases from the Asian-Pacific (APAC) (51 cases), Latin American (LAM) (348 cases), European (EU) (750 cases), and North American (NAM) (936 cases) regions, Candida albicans predominated (48.4%), followed by C. glabrata (18.0%), C. parapsilosis (17.2%), C. tropicalis (10.5%), and C. krusei (1.9%). Resistance to echinocandins (anidulafungin [2.4%] and micafungin [1.9%]) and azoles (3.5 to 5.6%) was most prevalent among C. glabrata isolates, as determined using recently established CLSI breakpoint criteria. C. glabrata isolates were more common in NAM (23.5%), and C. albicans isolates were more common in APAC (56.9%), with C. parapsilosis (25.6%) and C. tropicalis (17.0%) being more prominent in LAM. Emerging resistance patterns among C. glabrata cases in NAM require focused surveillance.

Triazole and echinocandin MIC distributions with epidemiological cutoff values for differentiation of wild-type strains from non-wild-type strains of six uncommon species of Candida.

When clinical susceptibility breakpoints (CBPs) are absent, establishing wild-type (WT) MIC distributions and epidemiological cutoff values (ECVs) provides a sensitive means for detecting emerging resistance. We determined species-specific ECVs for anidulafungin (ANF), caspofungin (CSF), micafungin (MCF), fluconazole (FLC), posaconazole (PSC), and voriconazole (VRC) for six rarer Candida species (819 strains) using isolates obtained from the ARTEMIS Program and the SENTRY Antimicrobial Surveillance Program, all tested by a reference broth microdilution method. The calculated ECVs, expressed in µg/ml (and the percentages of isolates that had MICs less than or equal to the ECVs), for ANF, CSF, MCF, FLC, PSC, and VRC, respectively, were 0.12 (95.2), 0.12 (97.8), 0.12 (100.0), 0.5 (95.7), 0.12 (98.6), and 0.03 (100.0) for Candida dubliniensis; 4 (100.0), 2 (96.0), 2 (99.1), 8 (95.0), 0.5 (97.5), and 0.25 (98.0) for C. guilliermondii; 0.25 (98.9), 0.03 (98.0), 0.12 (97.5), 1 (99.1), 0.25 (99.1), and 0.015 (100.0) for C. kefyr; 2 (100.0), 1 (99.6), 0.5 (96.6), 2 (96.1), 0.25 (98.6), and 0.03 (96.6) for C. lusitaniae; and 2 (100.0), 0.5 (100.0), 1 (100.0), 2 (98.0), 0.25 (97.1), and 0.06 (98.0) for C. orthopsilosis, but for C. pelliculosa, ECVs could be determined only for CSF (0.12 [94.4]), FLC (4 [98.2]), PSC (2 [98.2]), and VRC (0.25 [98.2]). In the absence of species-specific CBP values, these WT MIC distributions and ECVs will be useful for monitoring the emergence of reduced susceptibility to the triazole and echinocandin antifungals.

Pre-clinical development of antifungal susceptibility test methods for the testing of the novel antifungal agent E1210 versus Candida: comparison of CLSI and European Committee on Antimicrobial Susceptibility Testing methods.

OBJECTIVES: To compare European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI broth microdilution (BMD) methods for testing the novel antifungal E1210 against a recent collection of 102 clinical isolates of Candida spp. METHODS: Candida isolates (102) were tested by CLSI and EUCAST methods; 21 Candida albicans, 20 Candida glabrata, 25 Candida parapsilosis, 24 Candida tropicalis and 12 Candida krusei, including echinocandin- and azole-resistant isolates. CLSI and EUCAST MIC endpoints of 50% and 100% inhibition were determined using visual reading at 24 and 48 h of incubation and spectrophotometric reading at 24 h of incubation, respectively. RESULTS: E1210 CLSI MIC results ranged from ≤0.008 to only 1 mg/L (excluding C. krusei) depending on species, duration of incubation and endpoint criteria (EC). E1210 was not active against C. krusei (MIC(50) >16 mg/L). Overall essential agreement (EA; ±2 doubling dilutions) between the 24 and 48 h CLSI readings was 100% and 97.6% using the 50% and 100% inhibition EC, respectively. Slightly more trailing growth at 48 h was observed with the 100% inhibition EC. Comparison of the 50% and 100% endpoints at 24 h of incubation showed an overall EA of 100%. Comparison of CLSI and EUCAST read at 24 h of incubation and either 50% or 100% inhibition revealed an EA of 97.8% using the 50% inhibition EC and 88.9% using the 100% inhibition EC. CONCLUSIONS: E1210 was found to have potent in vitro activity against Candida spp. when tested by both CLSI and EUCAST BMD methods, with the highest overall EA (97.8%) obtained when E1210 MIC results were read after 24 h of incubation using a partial inhibition EC.