CLSI Reference Method Research Articles

Fixed-Ratio Combination Testing of an Echinocandin, Anidulafungin, and an Azole, Voriconazole, against 1,467 Candida Species Isolates

Anidulafungin is a newer echinocandin antifungal agent, approved by the United States Food and Drug Administration (U.S. FDA) in 2006 (18), with well-documented potency against Candida spp. and a MIC90 value of 0.06 μg/ml reported for a sample of 2,869 Candida albicans isolates across a 6-year collection (16). In common with other echinocandins, anidulafungin MIC90 values for C. parapsilosis and C. guilliermondii can be elevated (1 to 4 μg/ml) (8, 10, 16, 17). Voriconazole is a triazole antifungal that is structurally related to fluconazole (1). Voriconazole has broad-spectrum activity against Candida spp. and has been approved by the U.S. FDA for the treatment of esophageal candidiasis and invasive candidiasis in nonneutropenic patients (11, 19). Compared to fluconazole, voriconazole demonstrates enhanced activity against Candida spp. (5, 15). Voriconazole is active against the commonly isolated species of Candida, including C. albicans (MIC90, 0.015 to 0.06 μg/ml), C. tropicalis (MIC90, 0.12 to 0.25 μg/ml), and C. parapsilosis (MIC90, 0.06 to 0.12 μg/ml) (14, 15). Notably, voriconazole is also active against C. krusei (MIC90, 0.5 μg/ml), a species considered to be intrinsically resistant to fluconazole (MIC90, 64 μg/ml) (14, 15). Although activity has also been demonstrated against C. glabrata, MICs are higher (MIC90, 1 to 2 μg/ml) and resistance is more common than with other species (4, 14, 15). This protocol was designed to (i) quantify the potency and spectrum of anidulafungin and voriconazole using CLSI reference methods (2, 3) and (ii) assess the activity of anidulafungin combined with voriconazole (2:1 ratio) against a large collection of Candida spp. (8). The results were analyzed by directly comparing the MIC values of each agent tested alone or as a component of the 2:1 ratio test. The ratio was selected to mimic the concurrent exposures (area under the curve [AUC] and maximum drug concentration in serum [Cmax]) to each antifungal agent as listed in the U.S. FDA product package inserts (18-20). A total of 1,467 Candida sp. fungemia isolates were submitted by participating medical centers in North America, Europe, Latin America, and the Asia-Pacific region to the central monitoring laboratory (JMI Laboratories, North Liberty, IA) for testing (8). All strains were tested by the reference broth microdilution methods for yeasts as recommended by the CLSI M27-A3 approved standard (2, 3). Interpretive criteria used for yeasts when testing voriconazole and anidulafungin were those of the CLSI (M27-S3, 2008) (2, 3, 17). The quality control (QC) strains utilized included C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 (2, 3, 7); all QC results were within published MIC limits (3, 7). The activities of the two agents tested against the over 1,400 Candida spp. (MIC50/90 and percent susceptible) were as follows: anidulafungin, 0.03/1 μg/ml and 99.0%; voriconazole, ≤0.06/0.25 μg/ml and 98.3%. The combination of anidulafungin and voriconazole demonstrated enhanced activity when tested at a 2:1 ratio against these Candida spp. (Table ​(Table1).1). Anidulafungin tested alone had a MIC90 of 1 μg/ml, whereas that value was reduced to 0.06 μg/ml when it was combined with voriconazole. Similarly, the voriconazole MIC90 of 0.25 μg/ml was decreased to ≤0.03 μg/ml when it was tested with anidulafungin. The combination was 8- to 16-fold more potent than each agent was alone (MIC90 comparison), and all anidulafungin/voriconazole (2:1 ratio) MIC test values were ≤1/0.5 μg/ml, i.e., below the suggested susceptibility breakpoint for one or both agents (3, 17). TABLE 1. Comparisons of anidulafungin and voriconazole tested alone or in a 2:1 ratio combination against 1,467 Candida spp. clinical isolatesd The combination of anidulafungin and voriconazole resulted in improved activity against the putatively resistant species C. glabrata (voriconazole) and C. parapsilosis (anidulafungin) (Table ​(Table1).1). The MIC90 for C. glabrata decreased from 1 μg/ml (90.2%) for voriconazole alone to 0.06 μg/ml (97.0%) when it was combined with anidulafungin. Likewise, the MIC90 for C. parapsilosis decreased from 2 μg/ml (93.9%) for anidulafungin alone to 0.12 μg/ml (90.0%) when it was combined with voriconazole. Recently, Garcia-Effron et al. (6) suggested that the susceptibility breakpoint for the echinocandins be lowered from ≤2 μg/ml to ≤0.25 μg/ml to improve the detection of fks mutant strains of Candida. As shown in Table ​Table1,1, the combination of anidulafungin and voriconazole would capture 99.0% of the isolates tested at an MIC of ≤0.25 μg/ml versus only 82.3% when anidulafungin was tested alone. These in vitro results markedly expand upon what has been reported elsewhere (12) concerning the activity of anidulafungin and voriconazole in combination versus Candida and suggest a favorable interaction of these agents when they are used together against Candida spp. As resistances emerge among these fungal pathogens (6, 9, 13), combination treatments could assume a valuable therapeutic role (11).

Trends in antifungal susceptibility testing using CLSI reference and commercial methods

Antifungal susceptibility testing should identify in vitro resistance among a normally susceptible population or should detect the development of resistance during therapy. The Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards [NCCLS]) has developed reproducible methods for testing the activity of antifungal agents against yeasts (the M27-A3, M27-S3, M44-A and M44-S2 documents) and filamentous fungi (molds; the M38-A2 document). The yeast documents describe both quality control (QC) and interpretative breakpoint parameters for several antifungal agents versus Candida spp. In vitro breakpoints, as well as a QC and reference strain, has been recently described for mold testing. Standard guidelines for testing echinocandins against molds, guidelines for testing dermatophytes, as well as a disk-diffusion method for molds, have been developed recently. However, standard parameters for minimum fungicidal concentration determinations and selection of QC strains and guidelines for testing Malassezia spp. are under development. Breakpoints for Cryptococcus neoformans, dermatophytes and Malassezia spp. versus any antifungal agent and for posaconazole and amphotericin B versus yeasts are not available.

Comparative evaluation ofTrichosporon asahiisusceptibility using ASTY colorimetric microdilution and CLSI M27‐A2 broth microdilution reference methods

The in vitro activity ofAMPH-B, 5-FC, FLCZ, MCZ, ITCZ, and VRCZ against 50 isolates of T. asahii was determined using CLSI M27-A2 microdilution and ASTY colorimetric methods. Observed agreement ranged from 96 to 100% according to the drug. Overall, the agreement between two methods was 97.7%. The ASTY colorimetric method was thus determined to be comparable to the CLSI reference method when testing the susceptibility of T. asahii to a variety of antifungal agents.

Comparison of disc diffusion assay with the CLSI reference method (M27-A2) for testing in vitro posaconazole activity against common and uncommon yeasts

To evaluate the suitability of disc diffusion (DD) assay for testing posaconazole activity and to corroborate its activity against recently isolated yeasts by the CLSI reference microdilution M27-A2 method. A total of 224 yeast isolates (7 species with 52 to 11 isolates each, and 15 species with 1 to 6 isolates) were evaluated, 125 were recent bloodstream isolates, 30 isolates from other sources and six ATCC isolates that included amphotericin B-resistant Candida albicans ATCC 200955, Candida lusitaniae (ATCC 200950, 200951, 200952 and 200953) and amphotericin B- and itraconazole-resistant Candida tropicalis ATCC 200956. MICs were determined at 24 and 48 h by following the CLSI guidelines, document M27-A2. DD testing was performed by following CLSI M44-A document with 5 microg posaconazole discs. Inhibition zone diameters were measured at the transition point at which growth decreased at both 24 and 48 h. DD showed very good reproducibility, with coefficient of variability median value 4.56. Posaconazole demonstrated good in vitro activity against all clinical isolates, including the emerging species and amphotericin B-resistant ATCC isolates except for C. tropicalis ATCC 200956 (posaconazole MIC >or= 16 mg/L). Only 1.5% and 4.1% of isolates were inhibited by >2 mg/L posaconazole at 24 and 48 h. Good correlation was obtained between methods (R = 0.763 at 24 h and 0.602 at 48 h). DD detected posaconazole-resistant isolates (MIC > 2 mg/L). DD could be an alternative to the microdilution reference method, as no major discrepancies were detected.

A colorimetric and spectrophotometric method forin vitrosusceptibility testing ofAspergillusspecies against caspofungin

The in vitro susceptibility of 45 Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus isolates against caspofungin (CAS) was assessed by the CLSI reference method with spectrophotometric reading and by a colorimetric method that employed the dye MTT. Perfect agreement was found between the minimal effective concentrations (MECs) and the MIC-2 values (50% growth reduction) obtained by the MTT method. The agreement found between the MICs obtained by the CLSI and the MTT method was dependent on the MIC endpoint used, the antifungal agent tested, and the species investigated.

Rapid antifungal susceptibility testing of fluconazole and amphotericin B by flow cytometry using FUN-1 ®: a preliminary study

Objective The increasing incidence of severe fungal infections and the difficulties encountered with the existing antifungal susceptibility testing methods led us to develop a new technique based on flow cytometry, using the fluorochrome FUN -1 ®. Materials and methods We tested the susceptibility of 14 Candida spp. strains (5 reference and 9 clinical strains) to amphotericin B and fluconazole by flow cytometry versus M27-A2 CLSI reference method. Fluorescence was measured with a FACScalibur ® flow cytometer (Becton Dickinson). Amphotericin B susceptibility was studied on the 14 strains whereas fluconazole one was only performed on the five reference strains. Results Results were obtained after 30 min incubation for amphotericin B and 6 h for fluconazole. Reproducibility and repeatability tests have been performed on C. albicans ATCC 90028 and were > 95% and > 98%, respectively. The flow cytometry method resulted in clear-cut endpoints, which abolishes the source of variability of the M27-A2 reading. The results obtained showed that the patterns of susceptibility to amphotericin B were the same with the 2 methods used for all the strains. Our preliminary study with fluconazole gave the same results with the 5 control strains. Conclusion FUN-1 ® staining seems to be a rapid alternative to conventional methods to assess susceptibility of Candida spp. clinical isolates.

Antimicrobial and toxicological profile of the new biocide Akacid plus(R)

Akacid plus is a new member of the polymeric guanidine family of disinfectants. It was especially developed to enhance the antimicrobial activity of this class with significantly less toxicity. The in vitro activity of Akacid plus compared with chlorhexidine digluconate and mupirocin was tested against a total of 369 recent clinical isolates. The organisms tested by CLSI reference methods included the following: Staphylococcus aureus (98), Staphylococcus epidermidis (9), Bacillus spp. (2), Enterococcus faecalis (32), Klebsiella spp. (45), Enterobacter spp. (20), Escherichia coli (65), Salmonella spp. (6), Shigella spp. (2), Yersinia enterocolitica (1), Acinetobacter spp. (4), Proteus spp. (7), Pseudomonas aeruginosa (59), Stenotrophomonas maltophilia (4), Candida spp. (10) and Aspergillus spp. (7). In vitro selection of resistance to Akacid plus was carried out on 24 strains. Toxicological analyses were also performed. All tested agents were more effective against Staphylococcus spp. and Bacillus spp. than against E. faecalis and Gram-negative bacteria. The MIC90s of chlorhexidine and mupirocin showed a 4-fold and 32-fold increase for methicillin-resistant S. aureus in comparison with methicillin-susceptible strains, while MIC values of Akacid plus were similar for antibiotic-susceptible and multiresistant strains. Bactericidal action of Akacid plus was observed at 1-2x MIC. The in vitro selection of resistance test showed no increase in MIC values of Akacid plus for any isolate after 30 passages. In addition, Akacid plus showed low oral and dermal toxicity. These preliminary results demonstrate the broad antimicrobial properties of Akacid plus, which makes it a promising tool for topical application in the prophylaxis and treatment of bacterial and fungal infections.

Activities of Micafungin against 315 Invasive Clinical Isolates of Fluconazole-Resistant Candida spp

Micafungin is a new echinocandin exhibiting broad-spectrum activity against Candida spp. The activity of the echinocandins against Candida species known to express intrinsic or acquired resistance to fluconazole is of interest. We determined the MICs of micafungin and caspofungin against 315 invasive clinical (bloodstream and other sterile-site) isolates of fluconazole-resistant Candida species obtained from geographically diverse medical centers between 2001 and 2004. MICs were determined using broth microdilution according to the CLSI reference method M27-A2. RPMI 1640 was used as the test medium, and we used the MIC endpoint of prominent growth reduction at 24 h. Among the 315 fluconazole-resistant Candida isolates, 146 (46%) were C. krusei, 110 (35%) were C. glabrata, 41 (13%) were C. albicans, and 18 (6%) were less frequently isolated species. Micafungin had good in vitro activity against all fluconazole-resistant Candida spp. tested; the MICs at which 50% (MIC(50)) and 90% (MIC(90)) of isolates were inhibited were 0.03 microg/ml and 0.06 microg/ml, respectively. All the fluconazole-resistant Candida spp. were inhibited at a micafungin MIC that was </=1 microg/ml. Among the most common fluconazole-resistant Candida spp. tested in the collection, C. glabrata exhibited the lowest micafungin MICs (MIC(90), </=0.015 microg/ml), followed by C. albicans (MIC(90), 0.03 microg/ml) and C. krusei (MIC(90), 0.06 microg/ml). The new echinocandin micafungin has excellent in vitro activity against 315 invasive clinical isolates of fluconazole-resistant Candida, which represents the largest collection to date of fluconazole-resistant Candida isolates tested against micafungin. Micafungin may prove useful in the treatment of infections due to azole-resistant Candida.

Outcomes evaluation of patients with ESBL- and non–ESBL-producing Escherichia coli and Klebsiella species as defined by CLSI reference methods: report from the SENTRY Antimicrobial Surveillance Program

As extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae continue to emerge worldwide, selection of empiric treatment modalities is an increasing challenge. Data describing the clinical outcomes associated with different treatment regimens have been limited. Using data from centers with confirmed ESBL- and non–ESBL-producing Escherichia coli and Klebsiella species isolates in North America, Latin America, and Europe, potential risk factors for the occurrence of invasive ESBL- and non–ESBL-producing Enterobacteriaceae infections and factors associated with clinical outcome were evaluated. Of the 175 cases considered evaluable, 77% were ESBL-producing organisms. Underlying comorbidities and potential risk factors were generally similar between ESBL and non-ESBL cases with a statistically greater proportion of ESBL cases requiring gastrostomy or jejunostomy tubes, ventilatory assistance, or care in the intensive care unit before culture ( P ≤ 0.008). Among ESBL cases, carbapenem monotherapy and combination therapy were often selected for treatment (32.6% and 13.3%, respectively). Among non-ESBL cases, fluoroquinolones and β-lactam/β-lactamase inhibitor combination agents accounted for the highest proportion of treatment regimens (25.0% and 22.5%, respectively), whereas cephalosporin monotherapy and combination therapy were each used as treatment for 10% of cases. Clinical success was similar between patients with ESBL and non–ESBL-producing isolates (83% and 80%, respectively). Although infections arising from E. coli and Klebsiella species are associated with significant mortality, ESBL production alone did not appear to be an independent risk factor for treatment failure.