L. Boyken

Caspofungin Activity against Clinical Isolates of Fluconazole-Resistant Candida

ABSTRACT A total of 7,837 clinical isolates of Candida were tested against fluconazole, and 351 resistant (fluconazole MIC ≥ 64 μg/ml) isolates were identified (4% of the total tested). All fluconazole-resistant isolates were inhibited by caspofungin at concentrations that can be exceeded by standard doses (MIC at which 90% of the isolates were inhibited, 1 μg/ml; 99% of the MICs were ≤2 μg/ml).

Use of Epidemiological Cutoff Values To Examine 9-Year Trends in Susceptibility of Aspergillus Species to the Triazoles

ABSTRACT In the absence of clinical breakpoints, epidemiological cutoff values (ECVs) have been established to distinguish wild-type (WT) isolates of Aspergillus spp. from those that may harbor resistance mutations. Recently, the CLSI has developed ECVs for triazoles (itraconazole, posaconazole, and voriconazole) and common Aspergillus species. We applied the triazole ECVs to 1,789 Aspergillus isolates collected from 63 centers worldwide from 2001 to 2009 to determine the frequency of non-WT strains of each species. Temporal trends were evaluated for Aspergillus fumigatus and Aspergillus flavus over the 9-year period for each drug. The collection included 1,312 isolates of A. fumigatus, 235 of A. flavus, 162 of Aspergillus niger, 64 of Aspergillus terreus, and 15 of Aspergillus versicolor. Using the ECVs, the percentages of non-WT isolates for itraconazole, posaconazole, and voriconazole, respectively, were as follows: A. fumigatus (2.0%, 3.5%, and 1.4%), A. flavus (0.8%, 5.1%, and 1.7%), A. niger (17.3%, 3.7%, and 0.6%), A. terreus (0.0%, 1.6%, and 3.2%), and A. versicolor (6.3%, 0.0%, and 0.0%). Among 49 Aspergillus isolates for which itraconazole MICs were >2 μg/ml, the posaconazole and voriconazole MICs were greater than the ECVs for 14 and 12 isolates, respectively. The percentages of isolates for which MICs were greater than the ECVs ranged from 1.1 to 5.7% for posaconazole, 0.0 to 1.6% for voriconazole, and 0.7 to 4.0% for itraconazole. There was no consistent trend toward decreased susceptibility for any triazole and A. fumigatus or A. flavus over time. Decreased susceptibility among Aspergillus spp. was observed for each of the extended-spectrum triazoles and varied by species over the 9-year study period.

Use of Epidemiological Cutoff Values To Examine 9-Year Trends in Susceptibility of Candida Species to Anidulafungin, Caspofungin, and Micafungin

ABSTRACT The CLSI clinical breakpoint (CBP) for echinocandin susceptibility (S; MICs of ≤2 μg/ml) may classify isolates with acquired resistance (R) mutations as susceptible. Epidemiological cutoff values (ECVs) have been established to distinguish wild-type (WT) Candida strains from those that may exhibit R mutations. The CLSI-developed ECVs for anidulafungin, caspofungin, and micafungin were applied to 15,269 isolates of Candida spp. collected from over 100 centers worldwide between 2001 and 2009 to determine the frequency of non-WT strains of each species. The collection included 8,378 isolates of Candida albicans, 2,352 isolates of C. glabrata, 2,195 isolates of C. parapsilosis, 1,841 isolates of C. tropicalis, and 503 isolates of C. krusei. The mean percentages of non-WT isolates per year for anidulafungin, caspofungin, and micafungin, respectively, were as follows: for C. albicans, 0.3, 0.1, and 2.1; for C. glabrata, 0.8, 1.3, and 1.6; for C. parapsilosis, 0.0, 1.5, and 0.5; for C. tropicalis, 0.9, 0.7, and 0.9; and for C. krusei, 0.5, 6.4, and 3.5. We noted increases in the percentage of non-WT isolates, from 0.5% (2001) to 3.1% (2009) for caspofungin and C. parapsilosis, from 0.4% (2004) to 1.8% (2009) for anidulafungin and C. glabrata, from 2.4% (2004) to 5.7% (2009) for micafungin and C. krusei, and from 0.0% (2004) to 3.1% (2009) for micafungin and C. parapsilosis. No trends were noted for any species and drug when we used the CBP. Echinocandin CBPs are insensitive for detecting emerging R. Although uncommon, decreased S among Candida isolates was observed for each of the echinocandins and varied by species. Using ECVs is important in determining R trends among echinocandins and Candida.

Wild-Type MIC Distributions and Epidemiological Cutoff Values for Posaconazole and Voriconazole and Candida spp. as Determined by 24-Hour CLSI Broth Microdilution

ABSTRACT We tested 16,191 strains of Candida against posaconazole and voriconazole, using the CLSI M27-A3 broth microdilution (BMD) method (24-h incubation), in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2001 to 2009, 8,619 isolates of Candida albicans, 2,415 isolates of C. glabrata, 2,278 isolates of C. parapsilosis, 1,895 isolates of C. tropicalis, 508 isolates of C. krusei, 205 isolates of C. lusitaniae, 177 isolates of C. guilliermondii, and 93 isolates of C. kefyr were obtained from over 100 centers worldwide. The modal MICs (μg/ml) for posaconazole and voriconazole, respectively, were as follows: for C. albicans, 0.016 and 0.007; for C. glabrata, 0.5 and 0.06; for C. parapsilosis, 0.06 and 0.007; for C. tropicalis, 0.03 and 0.015; for C. krusei, 0.25 and 0.12; for C. lusitaniae, 0.03 and 0.007; for C. guilliermondii, 0.12 and 0.03; and for C. kefyr, 0.06 and 0.007. The ECVs (μg/ml [% of isolates that had MICs equal to or less than the ECV]) for posaconazole and voriconazole, respectively, were as follows: 0.06 (98.5) and 0.03 (98.9) for C. albicans, 2 (96.2) and 0.5 (90.4%) for C. glabrata, 0.25 (99.3) and 0.12 (97.9) for C. parapsilosis, 0.12 (97.6) and 0.06 (97.2) for C. tropicalis, 0.5 (99.8) and 0.5 (99.4) for C. krusei, 0.12 (95.6) and 0.03 (96.6) for C. lusitaniae, 0.5 (98.9) and 0.25 (98.3) for C. guilliermondii, and 0.25 (100.0) and 0.015 (100.0) for C. kefyr. In the absence of clinical breakpoints (CBPs) for posaconazole, these WT distributions and ECVs will be useful in surveillance for emergence of reduced susceptibility to posaconazole among Candida spp. Whereas a CBP for susceptibility of ≤1 μg/ml has been established for voriconazole and all species of Candida, it is notable that ECVs for this agent range from 10- to >100-fold lower than the CBP, depending on the species of Candida. The CBP is inadequate in detecting the emergence of voriconazole resistance among most Candida species encountered clinically. The CBPs for voriconazole should be reassessed, with consideration for development of species-specific CBPs.

Epidemiology of Staphylococcus Aureus Infections in Patients on Hemodialysis

OBJECTIVE To determine the epidemiology of Staphylococcus aureus infections in hemodialysis patients. METHOD S aureus isolates from surveillance cultures and from sites of infection were evaluated by both bacteriophage typing and restriction endonuclease digestion of plasmid DNA. SETTING A hemodialysis unit in Brugge, Belgium. ORGANISMS: S aureus isolates from 11 chronic hemodialysis patients who had participated in the placebo arm of a previously reported placebo-mupirocin comparative study. RESULTS Of 75 S aureus isolates evaluated, 63 were from cultures of nares and 12 from infections (three arteriovenous fistula infections, four wound infections, and five bacteremias). All isolates were typed by bacteriophages and 56 (75%) had plasmids. Three patients developed 12 infections. Eleven infections were caused by isolates previously identified in surveillance cultures. Only one infection was caused by a strain not identified previously in surveillance cultures. CONCLUSION These results support the hypothesis that S aureus isolates causing infections in hemodialysis patients are of endogenous origin.

Strain-Relatedness of Methicillin- Resistant Staphylococcus aureus Isolates Recovered from Patients with Repeated Infection

Invasive disease following methicillin-resistant Staphylococcus aureus (MRSA) detection is common, regardless of whether initial detection involves colonization or infection. We assessed the genetic relatedness of isolates obtained > or =2 weeks apart representing either repeated infections or colonization-infection sets to determine if infections are likely to be caused by previously harbored strains. We found that MRSA infection following initial colonization or infection is caused by the same strain in most cases, suggesting that a single successful attempt at decolonization may prevent the majority of later infection.

Molecular Epidemiology of Coagulase-Negative Staphylococci Isolated from Immunocompromised Patients

OBJECTIVE To define the source of invasive coagulase-negative staphylococci (CNS) and the epidemiology of strain variation in immunocompromised patients. DESIGN Weekly microbial surveillance cultures were obtained from the nares, throat, skin, rectum, and urine. Plasmid pattern analysis was performed on all coagulase-negative staphylococci isolated from blood cultures and on selected strains from the surveillance sites. SETTING A 902-bed, university-owned, tertiary-care referral hospital. PARTICIPANTS Forty-four patients on the bone marrow transplant or hematologic malignancy services. RESULTS Plasmid pattern analysis was performed on 340 surveillance isolates (median = 7 per patient) and 201 bloodstream isolates (median = 3 per patient). Patients were colonized with numerous unique strains (median = 5 per patient) of coagulase-negative staphylococci. The 44 patients had 108 episodes of positive blood cultures, 20 of which were preceded by colonization with the same strain. Isolation of the matching strain from surveillance cultures preceded the positive blood culture by 1 to 8 days in 9 episodes and 18 to 389 days in 11 episodes. The matching strain was isolated from the skin in only 6 (30%) of those episodes and from mucosal sites in 70%. Of the 108 episodes of positive blood cultures, 21 were identified as nosocomial bloodstream infections. Four of the 21 nosocomial bloodstream infections were preceded by colonization with the same strain. In all 4 episodes, the infecting strain was cultured from the nares before the blood cultures were obtained. CONCLUSIONS Our results suggest that mucous membranes might be sources for strains of CNS causing bacteremia.

Comparison of the Broth Microdilution (BMD) Method of the European Committee on Antimicrobial Susceptibility Testing with the 24-Hour CLSI BMD Method for Testing Susceptibility of Candida Species to Fluconazole, Posaconazole, and Voriconazole by Use of Epidemiological Cutoff Values

ABSTRACT The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with CLSI BMD method M27-A3 for fluconazole, posaconazole, and voriconazole susceptibility testing of 1,056 isolates of Candida. The isolates were obtained in 2009 from more than 60 centers worldwide and included 560 isolates of C. albicans, 175 of C. glabrata, 162 of C. parapsilosis, 124 of C. tropicalis, and 35 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 96.9% (voriconazole) to 98.6% (fluconazole). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined epidemiological cutoff values (ECVs). The ECVs (expressed as μg/ml) for fluconazole, posaconazole, and voriconazole, respectively, were as follows: 0.12, 0.06, and 0.03 for C. albicans; 32, 2, and 0.5 for C. glabrata; 2, 0.25, and 0.12 for C. parapsilosis; 2, 0.12, and 0.06 for C. tropicalis; 64, 0.5, and 0.5 for C. krusei. Excellent CA was observed for all comparisons between the EUCAST and CLSI results for fluconazole, posaconazole, and voriconazole, respectively, for each species: 98.9%, 93.6%, and 98.6% for C. albicans; 96.0%, 98.9%, and 93.7% for C. glabrata; 90.8%, 98.1%, and 98.1% for C. parapsilosis; 99.2%, 99.2%, and 96.8% for C. tropicalis; 97.1%, 97.1%, and 97.1% for C. krusei. We demonstrate high levels of EA and CA between the CLSI and EUCAST BMD methods for testing of triazoles against Candida when the MICs were determined after 24 h and ECVs were used to differentiate wild-type (WT) from non-WT strains. These results provide additional data in favor of the harmonization of these two methods.

In Vitro Susceptibility Testing of Filamentous Fungi: Comparison of Etest and Reference M38-A Microdilution Methods for Determining Posaconazole MICs

The performance of the Etest for posaconazole susceptibility testing of 72 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution method (M38-A). The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48h at 35 degrees C. The isolates included Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, A. versicolor, A. oryzae, A. terreus, Cladosporium spp., Curvularia sp., Exophiala sp., Fusarium spp., Paecilomyces spp., Pithomyces sp., Penicillium spp. and Scedosporium apiospermum. Overall agreement between Etest and microdilution MICs was 84% for Aspergillus spp. and 100% for the less common opportunistic molds, with the exception of Penicillium spp. (67%). Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values. The Etest method using RPMI agar appears to be a useful method for determining posaconazole susceptibilities of filamentous fungi.

In vitro selection of resistance to vancomycin in bloodstream isolates of Staphylococcus haemolyticus and Staphylococcus epidermidis.

The purpose of the study was to determine whether vancomycin-resistant strains ofStaphylococcus haemolyticus could be selected regardless of the initial MIC of vancomycin. Twenty-one bloodstream isolates ofStaphylococcus haemolyticus were studied by broth and agar selection methods. The broth method selected strains for which MICs of vancomycin ranged from 4 to 32 µg/ml and MBCs from 16 to>128 µg/ml. The agar method selected strains for which MICs ranged from 8 to 32 µg/ml and MBCs from 8 to>128 µg/ml. For comparison, seven strains ofStaphylococcus epidermidis were evaluated by the agar selection method. Final MICs of vancomycin ranged from 8 to 16 µg/ml; MBCs ranged from 16 to 64 µg/ml. Clearly, in vitro exposure to vancomycin can select strains ofStaphylococcus haemolyticus andStaphylococcus epidermidis for which MIC values are beyond the susceptible breakpoint.