Development of Doxycycline MIC and Disk Diffusion Interpretive Breakpoints and Revision of Tetracycline Breakpoints for Streptococcus pneumoniae

A study was performed to derive susceptibility testing interpretive breakpoints for doxycycline with Streptococcus pneumoniae and to reassess breakpoints for tetracycline using the requirements defined in Clinical and Laboratory Standards Institute (CLSI) document M23-A3. Tetracycline and doxycycline MICs and disk diffusion zone sizes were determined on 189 isolates selected from the 2009-2010 CDC Active Bacterial Core surveillance strain collection according to the testing methods described in CLSI documents M07-A8 and M02-A10. Tetracycline and doxycycline MICs and zones were compared to each other directly, and the reproducibility of MICs and zone diameters for both drugs was determined. Scattergrams of tetracycline MICs versus corresponding zone diameters and doxycycline MICs versus zones were prepared, and analysis indicated that the present CLSI tetracycline MIC and disk breakpoints did not fit the susceptibility data for doxycycline. Doxycycline was 1 to 3 dilutions more potent than tetracycline, especially in strains harboring the tetM resistance determinant. tetM was detected in ≥90% of isolates having tetracycline MICs of ≥4 μg/ml and in ≥90% with doxycycline MICs of ≥1. Limited pharmacokinetic/pharmacodynamic (PK/PD) data coupled with application of the error-rate bounded method of analysis suggested doxycycline-susceptible breakpoints of either ≤0.25 μg/ml or ≤0.5 μg/ml, with intermediate and resistant breakpoints 1 and 2 dilutions higher, respectively. The disk diffusion zone diameter correlates were susceptible at ≥28 mm, intermediate at 25 to 27 mm, and resistant at ≤24 mm. Revised lower tetracycline MIC breakpoints were suggested as susceptible at ≤1 μg/ml, intermediate at 2 μg/ml, and resistant at ≥4 μg/ml. Suggested tetracycline disk diffusion zones were identical to those of doxycycline.     

In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance

The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive Candida sp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of Candida spp. collected from over 90 medical centers worldwide from 1 January 2001 to 31 December 2006. We performed susceptibility testing according to the CLSI M27-A2 method and used RPMI 1640 broth, 24-h incubation, and a prominent inhibition endpoint for determination of the MICs. Of 5,346 invasive Candida sp. isolates, species distribution was 54% C. albicans, 14% C. parapsilosis, 14% C. glabrata, 12% C. tropicalis, 3% C. krusei, 1% C. guilliermondii, and 2% other Candida spp. Overall, all three echinocandins were very active against Candida: anidulafungin (MIC₅₀, 0.06 μg/ml; MIC₉₀, 2 μg/ml), caspofungin (MIC₅₀, 0.03 μg/ml; MIC₉₀, 0.25 μg/ml), micafungin (MIC₅₀, 0.015 μg/ml; MIC₉₀, 1 μg/ml). More than 99% of isolates were inhibited by ≤2 μg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by ≤2 μg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for C. albicans, 99.6%, 100%, and 100%; for C. parapsilosis, 92.5%, 99.9%, and 100%; for C. glabrata, 99.9%, 99.9%, and 100%; for C. tropicalis, 100%, 99.8%, and 100%; for C. krusei, 100%, 100%, and 100%; and for C. guilliermondii, 90.2%, 95.1%, and 100%. There was no significant change in the activities of the three echinocandins over the 6-year study period and no difference in activity by geographic region. All three echinocandins have excellent in vitro activities against invasive strains of Candida isolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candida spp.     

In Vitro Activity of Amikacin against Isolates of Mycobacterium avium Complex with Proposed MIC Breakpoints and Finding of a 16S rRNA Gene Mutation in Treated Isolates

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.     

Breakthrough Invasive Candidiasis in Patients on Micafungin

For Candida species, a bimodal wild-type MIC distribution for echinocandins exists, but resistance to echinocandins is rare. We characterized isolates from patients with invasive candidiasis (IC) breaking through ≥3 doses of micafungin therapy during the first 28 months of its use at our center: MICs were determined and hot-spot regions within FKS genes were sequenced. Eleven of 12 breakthrough IC cases identified were in transplant recipients. The median duration of micafungin exposure prior to breakthrough was 33 days (range, 5 to 165). Seventeen breakthrough isolates were recovered: FKS hot-spot mutations were found in 5 C. glabrata and 2 C. tropicalis isolates; of these, 5 (including all C. glabrata isolates) had micafungin MICs of >2 μg/ml, but all demonstrated caspofungin MICs of >2 μg/ml. Five C. parapsilosis isolates had wild-type FKS sequences and caspofungin MICs of 0.5 to 1 μg/ml, but 4/5 had micafungin MICs of >2 μg/ml. The remaining isolates retained echinocandin MICs of ≤2 μg/ml and wild-type FKS gene sequences. Breakthrough IC on micafungin treatment occurred predominantly in severely immunosuppressed patients with heavy prior micafungin exposure. The majority of cases were due to C. glabrata with an FKS mutation or wild-type C. parapsilosis with elevated micafungin MICs. MIC testing with caspofungin identified all mutant strains. Whether the naturally occurring polymorphism within the C. parapsilosis FKS1 gene responsible for the bimodal wild-type MIC distribution is also responsible for micafungin MICs of >2 μg/ml and clinical breakthrough or an alternative mechanism contributes to the nonsusceptible echinocandin MICs in C. parapsilosis requires further study.Invasive candidiasis (IC) is an important, life-threatening infection in hospitalized patients. The echinocandins (micafungin, caspofungin, and anidulafungin) are the newest class of medications approved for the prophylaxis and treatment of IC. They act via noncompetitive inhibition of β-1,3-glucan synthase, the enzyme responsible for producing β-1,3-d-glucan in the fungal cell wall (41). These drugs have low toxicity and few drug-drug interactions and possess a broad spectrum of antifungal activity against Candida species, including those resistant to fluconazole. In clinical trials, the echinocandins have demonstrated noninferiority for the treatment of IC versus amphotericin B deoxycholate, liposomal amphotericin B, and fluconazole (25, 32, 44). The echinocandins are considered interchangeable for clinical use, and a recent study comparing micafungin to caspofungin for IC supports this notion (38). Based on the accumulated experience, echinocandins are now considered a first-line therapeutic choice for IC (37).The echinocandins exhibit a bimodal MIC distribution among Candida species. MICs of C. parapsilosis, C. guilliermondii, and C. famata MICs (MIC₉₀, 0.25 to 2 μg/ml) are up to 133 times higher than those of C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. kefyr (MIC₉₀, 0.015 to 0.25 μg/ml) (42). However, this difference has not translated into consistent clinical failure (25, 38, 44), and the MIC breakpoint for echinocandin susceptibility was set at ≤2 μg/ml, which is inclusive of 99% of the wild-type distribution of all Candida species (9). Organisms with MICs of >2 μg/ml are considered “nonsusceptible,” but the breakpoint for resistance has yet to be determined owing to the paucity of clinical isolates available from patients failing echinocandin therapy and with MICs of >2 μg/ml.As echinocandin use has escalated, cases of echinocandin breakthrough IC have been described (6, 7, 13, 25, 39, 50), and nonsusceptible isolates (MIC > 2 μg/ml) have been recovered from patients who demonstrated treatment failure (9). Moreover, several of these nonsusceptible isolates possess nonsynonymous point mutations in genes encoding the β-1,3-glucan synthase enzyme complex (Fksp) (4, 13, 39, 47). These specific FKS “hot-spot” mutations reduce the susceptibility of the β-1,3-glucan synthase enzyme complex to echinocandin drugs, supporting a biological mechanism of resistance (14).In February 2006, micafungin became the formulary echinocandin at our hospital, a tertiary care center with multiple intensive care units, two dedicated hematopoietic stem cell transplant (HSCT) units, and an active solid organ transplant (SOT) service. Multiple patients with breakthrough IC while receiving micafungin therapy were noted. These cases were reviewed, and the Candida isolates recovered from these patients were screened for FKS gene mutations; results were correlated with MIC values.(This work was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 to 14 September 2009 [slide presentation M-1243]).     

Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Echinocandins and Candida spp.

We tested a global collection of Candida sp. strains against anidulafungin, caspofungin, and micafungin, using CLSI M27-A3 broth microdilution (BMD) methods, in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2003 to 2007, 8,271 isolates of Candida spp. (4,283 C. albicans, 1,236 C. glabrata, 1,238 C. parapsilosis, 996 C. tropicalis, 270 C. krusei, 99 C. lusitaniae, 88 C. guilliermondii, and 61 C. kefyr isolates) were obtained from over 100 centers worldwide. The modal MICs (in μg/ml) for anidulafungin, caspofungin, and micafungin, respectively, for each species were as follows: C. albicans, 0.03, 0.03, 0.015; C. glabrata, 0.06, 0.03, 0.015; C. tropicalis, 0.03, 0.03, 0.015; C. kefyr, 0.06, 0.015, 0.06; C. krusei, 0.03, 0.06, 0.06; C. lusitaniae, 0.05, 0.25, 0.12; C. parapsilosis, 2, 0.25, 1; and C. guilliermondii, 2, 0.5. 05. The ECVs, expressed in μg/ml (percentage of isolates that had MICs that were less than or equal to the ECV is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, were as follows: 0.12 (99.7%), 0.12 (99.8%), and 0.03 (97.7%) for C. albicans; 0.25 (99.4%), 0.12 (98.5%), and 0.03 (98.2%) for C. glabrata; 0.12 (98.9%), 0.12 (99.4%), and 0.12 (99.1%) for C. tropicalis; 0.25(100%), 0.03 (100%), and 0.12 (100%) for C. kefyr; 0.12 (99.3%), 0.25 (96.3%), and 0.12 (97.8%) for C. krusei; 2 (100%), 0.5 (98.0%), and 0.5 (99.0%) for C. lusitaniae; 4 (100%), 1 (98.6%), and 4 (100%) for C. parapsilosis; 16 (100%), 4 (95.5%), and 4 (98.9%) for C. guilliermondii. These WT MIC distributions and ECVs will be useful in surveillance for emerging reduced echinocandin susceptibility among Candida spp. and for determining the importance of various FKS1 or other mutations.The members of the echinocandin class of antifungal agents (anidulafungin, caspofungin, and micafungin) are now well recognized as the preferred, systemically active antifungal agents for the treatment of invasive candidiasis (IC), including candidemia (19). The in vitro activity of these agents against Candida spp. is also well-known (17, 24), and the Clinical and Laboratory Standards Institute (CLSI) Antifungal Subcommittee has established a clinical breakpoint (CBP) for susceptibility of ≤2 μg/ml for all three agents and all species of Candida (3, 4, 25). Recently, however, it has become evident that Candida infections involving strains with mutations in FKS1 (encodes the echinocandin target) do not necessarily have MICs above this CBP (2, 5-8, 14, 28). Likewise, kinetic studies of the glucan synthesis enzyme complex suggest that a lower MIC cutoff of 0.5 μg/ml may be more sensitive in detecting those strains with FKS1 mutations (7, 8). Given these considerations, we have conducted global surveillance of Candida spp. by using CLSI broth microdilution (BMD) methods to ascertain the wild-type (WT) MIC distribution for the three echinocandins and the eight most common species of Candida causing bloodstream infections (BSI). This information allows us to establish epidemiological cutoff values (ECVs) that may be used to assess the emergence of strains with FKS1 mutations and the decreased susceptibility to these agents (10, 27, 30).     

Utility of Sequencing the erm(41) Gene in Isolates of Mycobacterium abscessus subsp. abscessus with Low and Intermediate Clarithromycin MICs

The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 μg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 μg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 μg/ml, including ATCC 19977^T, revealed 10 sequevars. The sequence in ATCC 19977^T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 μg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) μg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 μg/ml and that isolates with an MIC of 8 μg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.     

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.In January 2006, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for vancomycin and Staphylococcus aureus. The breakpoints were lowered from ≤4 μg/ml to ≤2 μg/ml for susceptible, 8 to 16 μg/ml to 4 to 8 μg/ml for intermediate, and ≥32 μg/ml to ≥16 μg/ml for resistant (2). The vancomycin breakpoints for coagulase-negative staphylococci were not changed. The rationale for lowering the S. aureus intermediate breakpoint to 4 μg/ml was (i) that intermediate S. aureus isolates, although they are rare, likely represented a population of organisms that demonstrate heteroresistance, and (ii) limited outcome data suggested that infections with these isolates are likely to fail vancomycin therapy (9). The results of broth microdilution performed by use of the CLSI reference method were the primary S. aureus susceptibility data evaluated before the CLSI breakpoint change was made. We undertook the study described here to determine the accuracy of commercial systems and reference methods for the detection of decreased vancomycin susceptibility among isolates of S. aureus.(This work was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

Pharmacodynamics of Doxycycline and Tetracycline against Staphylococcus pseudintermedius: Proposal of Canine-Specific Breakpoints for Doxycycline

Doxycycline is a tetracycline that has been licensed for veterinary use in some countries, but no clinical breakpoints are available for veterinary pathogens. The objectives of this study were (i) to establish breakpoints for doxycycline and (ii) to evaluate the use of tetracycline as a surrogate to predict the doxycycline susceptibility of Staphylococcus pseudintermedius isolates. MICs and inhibition zone diameters were determined for 168 canine S. pseudintermedius isolates according to Clinical and Laboratory Standards Institute (CLSI) standards. Tetracycline resistance genes were detected by PCR, and time-kill curves were determined for representative strains. In vitro pharmacodynamic and target animal pharmacokinetic data were analyzed by Monte Carlo simulation (MCS) for the development of MIC interpretive criteria. Optimal zone diameter breakpoints were defined using the standard error rate-bounded method. The two drugs displayed bacteriostatic activity and bimodal MIC distributions. Doxycycline was more active than tetracycline in non-wild-type strains. MCS and target attainment analysis indicated a certainty of ≥90% for attaining an area under the curve (AUC)/MIC ratio of >25 with a standard dosage of doxycycline (5 mg/kg of body weight every 12 h) for strains with MICs of ≤0.125 μg/ml. Tetracycline predicted doxycycline susceptibility, but current tetracycline breakpoints were inappropriate for the interpretation of doxycycline susceptibility results. Accordingly, canine-specific doxycycline MIC breakpoints (susceptible, ≤0.125 μg/ml; intermediate, 0.25 μg/ml; resistant, ≥0.5 μg/ml) and zone diameter breakpoints (susceptible, ≥25 mm; intermediate, 21 to 24 mm; resistant, ≤20 mm) and surrogate tetracycline MIC breakpoints (susceptible, ≤0.25 μg/ml; intermediate, 0.5 μg/ml; resistant, ≥1 μg/ml) and zone diameter breakpoints (susceptible, ≥23 mm; intermediate, 18 to 22 mm; resistant, ≤17 mm) were proposed based on the data generated in this study.     

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.     

Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to 2005

Candida krusei is well known as a fungal pathogen for patients with hematologic malignancies and for transplant recipients. Using the ARTEMIS Antifungal Surveillance Program database, we describe geographic and temporal trends in the isolation of C. krusei from clinical specimens and the in vitro susceptibilities of 3,448 isolates to voriconazole as determined by CLSI (formerly NCCLS) disk diffusion testing. In addition, we report the in vitro susceptibilities of bloodstream infection isolates of C. krusei to amphotericin B (304 isolates), flucytosine (254 isolates), anidulafungin (121 isolates), caspofungin (300 isolates), and micafungin (102 isolates) as determined by CLSI broth microdilution methods. Geographic differences in isolation were apparent; the highest frequency of isolation was seen for the Czech Republic (7.6%) and the lowest for Indonesia, South Korea, and Thailand (0 to 0.3%). Overall, 83% of isolates were susceptible to voriconazole, ranging from 74.8% in Latin America to 92.3% in North America. C. krusei was most commonly isolated from hematology-oncology services, where only 76.7% of isolates were susceptible to voriconazole. There was no evidence of increasing resistance of C. krusei to voriconazole from 2001 to 2005. Decreased susceptibilities to amphotericin B (MIC at which 90% of isolates were inhibited [MIC₉₀], 4 μg/ml) and flucytosine (MIC₉₀, 16 μg/ml) were noted, whereas 100% of isolates were inhibited by ≤2 μg/ml of anidulafungin (MIC₉₀, 0.06 μg/ml), micafungin (MIC₉₀, 0.12 μg/ml) or caspofungin (MIC₉₀, 0.25 μg/ml). C. krusei is an uncommon but multidrug-resistant fungal pathogen. Among the systemically active antifungal agents, the echinocandins appear to be the most active against this important pathogen.     

Effects of Treated versus Untreated Polystyrene on Caspofungin In Vitro Activity against Candida Species

Significant interlaboratory variability is observed in testing the caspofungin susceptibility of Candida species by both the CLSI and EUCAST broth microdilution methodologies. We evaluated the influence of treated versus untreated polystyrene microtiter trays on caspofungin MICs using 209 isolates of four Candida species, including 16 C. albicans and 11 C. glabrata isolates with defined FKS mutations. Caspofungin MICs were also determined using the commercially available YeastOne and Etest assays and 102 isolates. All C. glabrata isolates had caspofungin MICs of ≥0.5 μg/ml, the clinical breakpoint for caspofungin resistance in this species, measured using trays made of treated polystyrene, regardless of the FKS status. In contrast, susceptible isolates could readily be distinguished from resistant/non-wild-type isolates when caspofungin MICs were measured using untreated polystyrene trays and both the YeastOne and Etest assays. Similar results were also observed for C. krusei isolates, as all isolates had caspofungin MICs above the threshold for resistance measured using treated polystyrene trays. In contrast, C. albicans isolates could be correctly identified as susceptible or resistant when caspofungin MICs were measured with treated or untreated trays and with the YeastOne and Etest assays. MICs falsely elevated above the resistance breakpoint were also not observed for C. tropicalis isolates. These results demonstrated that the use of treated polystyrene may be one factor that leads to falsely elevated caspofungin in vitro susceptibility results and that this may also be a greater issue for some Candida species than for others.     

Trends in species distribution and susceptibility of bloodstream isolates of Candida collected in Monterrey, Mexico, to seven antifungal agents: results of a 3-year (2004 to 2007) surveillance study

During a 3-year surveillance program (2004 to 2007) in Monterrey, Mexico, 398 isolates of Candida spp. were collected from five hospitals. We established the species distribution and in vitro susceptibilities of these isolates. The species included 127 Candida albicans strains, 151 C. parapsilosis strains, 59 C. tropicalis strains, 32 C. glabrata strains, 11 C. krusei strains, 5 C. guilliermondii strains, 4 C. famata strains, 2 C. utilis strains, 2 C. zeylanoides strains, 2 C. rugosa strains, 2 C. lusitaniae strains, and 1 C. boidinii strain. The species distribution differed with the age of the patients. The proportion of candidemias caused by C. parapsilosis was higher among infants ≤1 year old, and the proportion of candidemias caused by C. glabrata increased with patient age (>45 years old). MICs were calculated following the criteria of the Clinical Laboratory Standards Institute reference broth macrodilution method. Overall, C. albicans, C. parapsilosis, and C. tropicalis isolates were susceptible to fluconazole and amphotericin B. However, 31.3% of C. glabrata isolates were resistant to fluconazole (MIC ≥ 64 μg/ml), 43.3% were resistant to itraconazole (MIC ≥ 1 μg/ml), and 12.5% displayed resistance to amphotericin B (MIC ≥ 2 μg/ml). Newer triazoles, namely, voriconazole, posaconazole, and ravuconazole, had a notable in vitro activity against all Candida species tested. Also, caspofungin was active against Candida sp. isolates (MIC₉₀ ≤ 0.5 μg/ml) except C. parapsilosis (MIC₉₀ = 2 μg/ml). It is imperative to promote a national-level surveillance program to monitor this important microorganism.     

Effects of Aggregate and Individual Antibiotic Exposure on Vancomycin MICs for Staphylococcus aureus Isolates Recovered from Pediatric Patients

We evaluated the evolution of vancomycin MICs for Staphylococcus aureus and their relationship with vancomycin use among hospitalized children. S. aureus isolates recovered from sterile sites were prospectively tested for vancomycin susceptibility using the Etest between 1 April 2000 and 31 March 2008. Vancomycin MICs were grouped into three categories: ≤1, 1.5, and 2 μg/ml. The association between vancomycin MICs and aggregate vancomycin use and individual patient vancomycin exposure 6 months prior to the documented infection was assessed. The geometric mean values for vancomycin MICs for S. aureus fluctuated over time without a significant trend (P = 0.146). Of the 436 patients included in the study, 363 (83%) had methicillin-susceptible S. aureus (MSSA) and 73 (17%) had methicillin-resistant S. aureus (MRSA) infections. The rate of isolates with a vancomycin MIC of 2 μg/ml increased from 4% (2 of 46) in 2000 to 2001 to 24% (11 of 46) in 2007 to 2008, despite a decrease in vancomycin use (r = −0.11; P = 0.825). The percentage of isolates with a vancomycin MIC of 2 μg/ml was higher for MRSA (15%; 11 of 73) than for MSSA strains (5.2%; 19 of 363) (χ² = 9.2; P = 0.01). Individual patient vancomycin exposure was not associated with a higher vancomycin MIC. In the unadjusted model, in which we compared patients with S. aureus infections with MICs of ≤1 μg/ml, the odds ratios of exposure rates for patients with isolates with MICs of 1.5 μg/ml and 2 μg/ml were 1.02 (P = 0.929) and 1.13 (P = 0.767), respectively. In our experience, the geometric means of vancomycin MICs from S. aureus isolates recovered from hospitalized children oscillated over time and were not associated with previous individual patient vancomycin exposure or aggregate vancomycin use.     

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.     

Fluconazole Disk Diffusion Susceptibility Testing of Candida Species

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-μg fluconazole disks. With 25-μg disks, zones of inhibition of ≥20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were ≤8 μg/ml, and with 50-μg disks, zones of ≥27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 μg/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.     

Characterization of Staphylococci with Reduced Susceptibilities to Vancomycin and Other Glycopeptides

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were ≥4 μg/ml or for which the teicoplanin MICs were ≥8 μg/ml and 24 control strains for which the vancomycin MICs were ≤2 μg/ml or for which the teicoplanin MICs were ≤4 μg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 μg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 μg/ml were noted for these isolates. Vitek results were 4 μg/ml for all strains for which the vancomycin MICs were ≥4 μg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either ≤2 or ≥16 μg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 μg of vancomycin per ml consistently differentiated those strains inhibited by 8 μg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.     

Characterization of a New Clinical Yeast Species,Candida tunisiensis sp. nov., Isolated from a Strain Collection from Tunisian Hospitals

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.     

Determining the Optimal Ceftriaxone MIC for Triggering Extended-Spectrum β-Lactamase Confirmatory Testing

As routine testing of clinical isolates for extended-spectrum β-lactamase (ESBL) production (screen plus phenotypic confirmatory testing) is no longer required by the Clinical and Laboratory Standards Institute (CLSI), a number of clinical microbiology laboratories use ceftriaxone MICs as a proxy means of identifying bacteria as potential ESBL producers. Data from 1,386 clinical isolates suggest that a ceftriaxone MIC cutoff of 8 μg/ml is an excellent predictor of ESBL production, with a positive predictive value and negative predictive value approaching 100% and 99.5%, respectively.     

Rapid Screening for Penicillin Susceptibility of Systemic Pneumococcal Isolates by Restriction Enzyme Profiling of the pbp2B Gene

Restriction digest profiling of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. The pbp2b amplicon of all pneumococcal isolates for which the MICs of penicillin were ≤0.03 μg/ml had one of two different susceptible restriction profiles, and all 33 isolates for which MICs were 0.5 μg/ml or greater had one of seven distinct resistant profiles. Low-concentration penicillin resistance (MICs = 0.06 μg/ml to 0.25 μg/ml) was associated with sensitive HaeIII profiles in some isolates; however, RsaI profiling and pbp2b sequence analysis of such isolates revealed that some isolates contained low-level resistant pbp2b alleles, while others had susceptible pbp2b alleles. This data indicates that low-level penicillin resistance is sometimes conferred by determinants other than pbp2b.     

Comparison between the standardized clinical and laboratory standards institute M38-A2 method and a 2,3-Bis(2-Methoxy-4-Nitro-5-[(Sulphenylamino)Carbonyl]-2H-tetrazolium hydroxide- based method for testing antifungal susceptibility of dermatophytes

In this study, we determined the utility of a 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based assay for determining antifungal susceptibilities of dermatophytes to terbinafine, ciclopirox, and voriconazole in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. Forty-eight dermatophyte isolates, including Trichophyton rubrum (n = 15), Trichophyton mentagrophytes (n = 7), Trichophyton tonsurans (n = 11), and Epidermophyton floccosum (n = 13), and two quality control strains, were tested. In the XTT-based method, MICs were determined spectrophotometrically at 490 nm after addition of XTT and menadione. For the CLSI method, the MICs were determined visually. With T. rubrum, the XTT assay revealed MIC ranges of 0.004 to >64 μg/ml, 0.125 to 0.25 μg/ml, and 0.008 to 0.025 μg/ml for terbinafine, ciclopirox, and voriconazole, respectively. Similar MIC ranges were obtained against T. rubrum by using the CLSI method. Additionally, when tested with T. mentagrophytes, T. tonsurans, and E. floccosum isolates, the XTT and CLSI methods resulted in comparable MIC ranges. Both methods revealed similar lowest drug concentrations that inhibited 90% of the isolates for the majority of tested drug-dermatophyte combinations. The levels of agreement within 1 dilution between both methods were as follows: 100% with terbinafine, 97.8% with ciclopirox, and 89.1% with voriconazole. However, the agreement within 2 dilutions between these two methods was 100% for all tested drugs. Our results revealed that the XTT assay can be a useful tool for antifungal susceptibility testing of dermatophytes.