Cynthia C. Knapp

Correlation of MIC with Outcome for Candida Species Tested against Caspofungin, Anidulafungin, and Micafungin: Analysis and Proposal for Interpretive MIC Breakpoints

ABSTRACT The CLSI Antifungal Subcommittee followed the M23-A2 “blueprint” to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against Candida species. MICs of ≤2 μg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of Candida spp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to Candida spp. for which MICs are as high as 2 μg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 μg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were >2 μg/ml (two C. parapsilosis isolates at 4 μg/ml and one C. rugosa isolate at 8 μg/ml). Based on these data, the CLSI subcommittee has decided to recommend a “susceptible only” breakpoint MIC of ≤2 μg/ml due to the lack of echinocandin resistance in the population of Candida isolates thus far. Isolates for which MICs exceed 2 μg/ml should be designated “nonsusceptible” (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.

Wild-Type MIC Distribution and Epidemiological Cutoff Values for Aspergillus fumigatus and Three Triazoles as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Methods

ABSTRACT Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were ≤2 μg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of ≤1 μg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of ≤0.25 μg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

Clinical breakpoints for voriconazole and Candida spp. revisited: review of microbiologic, molecular, pharmacodynamic, and clinical data as they pertain to the development of species-specific interpretive criteria

We reassessed the Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) for voriconazole. We examined i) the essential (EA: ±2 dilutions) and categorical agreement between 24-h CLSI and EUCAST methods for voriconazole testing of Candida, ii) wild-type (WT) MICs and epidemiologic cutoff values (ECVs) for voriconazole by both CLSI and EUCAST methods, and iii) correlation of MICs with outcomes from previously published data using CLSI methods. We applied these findings to propose new 24-h species-specific CLSI CBPs. Adjusted 24-h CBPs for voriconazole and C. albicans, C. tropicalis, and C. parapsilosis (susceptible, ≤ 0.125 μg/mL; intermediate, 0.25-0.5 μg/mL; resistant, ≥ 1 μg/mL) should be more sensitive for detecting emerging resistance among common Candida species and provide consistency with EUCAST CBPs. In the absence of CBPs for voriconazole and C. glabrata (and less common species), we recommend that their respective ECVs be used to detect the emergence of non-WT strains.

Multicenter Comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A2 Reference Method for Testing New Antifungal Agents against Clinical Isolates of Candida spp.

ABSTRACT A multicenter (three centers) study compared MICs obtained by the Sensititre YeastOne Colorimetric Antifungal plate to reference microdilution broth (NCCLS M27-A2 document) MICs of three new triazoles (posaconazole, ravuconazole, and voriconazole) and the echinocandin caspofungin acetate for 100 isolates of Candida spp. In addition, amphotericin B and fluconazole were tested as control drugs. Colorimetric MICs of caspofungin and amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first slightly purple or blue well. Two comparisons of MIC pairs by the two methods were evaluated: 24-h colorimetric MICs were compared to NCCLS MICs at 24 and at 48 h. The interlaboratory reproducibility of YeastOne and reference MICs was also examined. The best performance of the YeastOne plate was with 24-h MICs (overall, 95 to 99% agreement) for all the species and antifungal agents. These results suggest the potential value of the YeastOne plate for use in the clinical laboratory for the four new antifungal agents evaluated.

Comparison of the Sensititre YeastOne colorimetric antifungal panel with CLSI microdilution for antifungal susceptibility testing of the echinocandins against Candida spp., using new clinical breakpoints and epidemiological cutoff values.

A commercially prepared dried colorimetric microdilution panel (Sensititre Yeast One, TREK Diagnostic Systems, Cleveland, OH, USA) was compared in 3 different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method by testing 2 quality control strains, 25 reproducibility strains, and 404 isolates of Candida spp. against anidulafungin, caspofungin, and micafungin. Reference CLSI BMD MIC end points and YeastOne colorimetric end points were read after 24 h of incubation. Excellent (100%) essential agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Categorical agreement (CA) between the 2 methods was assessed using the new species-specific clinical breakpoints (CBPs): susceptible (S), ≤0.25 μg/mL; intermediate (I), 0.5 μg/mL; and resistant (R), ≥1 μg/mL, for C. albicans, C. tropicalis, and C. krusei, and ≤2 μg/mL (S), 4 μg/mL (I), and ≥8 μg/mL (R) for C. parapsilosis and all 3 echinocandins. The new CBPs for anidulafungin and caspofungin and C. glabrata are ≤0.12 μg/mL (S), 0.25 μg/mL (I), and ≥0.5 μg/mL (R), whereas those for micafungin are ≤0.06 μg/mL (S), 0.12 μg/mL (I), and ≥0.25 μg/mL (R). Due to the lack of CBPs for any of the echinocandins and C. lusitaniae, the epidemiological cutoff values (ECVs) were used for this species to categorize the isolates as wild-type (WT; MIC ≤ECV) and non-WT (MIC >ECV), respectively, for anidulafungin (≤2 μg/mL/>2 μg/mL), caspofungin (≤1 μg/mL/>1 μg/mL), and micafungin (≤0.5 μg/mL/>0.5 μg/mL). CA ranged from 93.6% (caspofungin) to 99.6% (micafungin) with less than 1% very major or major errors. The YeastOne colorimetric method remains comparable to the CLSI BMD reference method for testing the susceptibility of Candida spp. to the echinocandins when using the new (lower) CBPs and ECVs. Further study using defined fks mutant strains of Candida is warranted.

Clinical Evaluation of the Sensititre YeastOne Colorimetric Antifungal Panel for Antifungal Susceptibility Testing of the Echinocandins Anidulafungin, Caspofungin, and Micafungin

ABSTRACT A commercially prepared, dried colorimetric microdilution panel (Sensititre YeastOne Trek Diagnostic Systems, Cleveland, OH) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method by testing 2 quality control strains, 25 reproducibility strains, and 404 isolates of Candida spp. against anidulafungin, caspofungin, and micafungin. Reference MIC endpoints and YeastOne colorimetric endpoints were read after 24 h of incubation. YeastOne endpoints were determined to be the lowest concentration at which the color in the well changed from red (positive, indicating growth) to blue (negative, indicating no growth). Excellent essential agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 100% for all three agents. Categorical agreement ranged from 99.3% (anidulafungin) to 100% (caspofungin, micafungin) and interlaboratory reproducibility was 99%. The YeastOne colorimetric method appears to be comparable to the CLSI reference method for testing the susceptibility of Candida spp. to the echinocandins anidulafungin, caspofungin, and micafungin.

The Emergence of Neisseria gonorrhoeae with Decreased Susceptibility to Ciprofloxacin in Cleveland, Ohio: Epidemiology and Risk Factors

Antimicrobial-resistant Neisseria gonorrhoeae is an increasing and costly public heath problem. In 1987, the Centers for Disease Control and Prevention (CDC) recommended that tetracyclines and penicillins no longer be used for the primary treatment of gonorrhea because of emerging resistance to these antibiotic agents [1]. The CDC currently advocates third-generation cephalosporins or selected fluoroquinolones, including ciprofloxacin and ofloxacin, as first-line therapies for uncomplicated gonorrhea [2]. Until recently, almost all isolates of N. gonorrhoeae that had been tested in the United States were susceptible to fluoroquinolones [3], including ciprofloxacin (at a minimal inhibitory concentration [MIC] 0.06 g/mL [4]. The first reports of fluoroquinolone-resistant N. gonorrhoeae in North America were published in 1990 and involved four Canadian men who had acquired their infections in Southeast Asia [5]. Before 1992, the Gonococcal Isolate Surveillance Project (GISP) reported only sporadic isolates of gonococci with decreased susceptibility to fluoroquinolones from patients in Ohio, Hawaii, Texas, Alaska, California, New Mexico, and Massachusetts [6]. This CDC-sponsored sentinel surveillance project, which involves 26 sexually transmitted disease clinics nationwide, has monitored the susceptibility of N. gonorrhoeae to antimicrobial agents since 1986. We recently reported the emergence in Ohio of gonorrhea caused by isolates with decreased susceptibility to fluoroquinolones [3, 7, 8]. Here, we describe the findings of an epidemiologic investigation that assessed risk factors associated with the presence of N. gonorrhoeae that had decreased susceptibility to ciprofloxacin in men with gonorrhea at one sexually transmitted disease clinic in Cleveland, Ohio. Methods This study was done at a public clinic for sexually transmitted diseases in Cleveland, Ohio (clinic X), that has participated in GISP since 1991. Clinic X serves a county with a population of 500 000 persons on a walk-in basis 5 days per week, and it receives approximately 5000 visits annually. One laboratory technician immediately read Gram stains of all urethral discharges to assess the presence of polymorphonuclear leukocytes and gram-negative diplococci. From 1986 through 1993, primary treatment for uncomplicated gonorrhea at clinic X was ceftriaxone, 250 mg administered intramuscularly. In 1994, the dose of ceftriaxone was reduced to 125 mg. Spectinomycin, 2.0 g administered intramuscularly, was used as primary therapy for gonorrhea in patients who had a history of allergy to penicillin. All patients suspected of having gonorrhea also received a 7-day course of doxycycline, 100 mg twice daily. Fluoroquinolones have never been used to treat gonorrhea in clinic X. Urethral isolates from the first 20 men with gonococcal infections who attended clinic X were submitted each month to the regional GISP laboratory for susceptibility testing. Isolates were collected from the first 25 men; isolates 21 through 25 were used to replace any isolates missing from the sequence of isolates 1 through 20. The Section of Microbiology at the Cleveland Clinic Foundation (Cleveland, Ohio) has served as the regional laboratory for clinic X since September 1991. Definition of Case-Patients and Ascertainment and Selection of Controls A case-patient was defined as any male patient who attended clinic X between January 1992 and March 1994 (the study period) and had a urethral N. gonorrhoeae isolate that was submitted to GISP and showed decreased susceptibility to ciprofloxacin (mean inhibitory concentration 0.12 g/mL and 0.25 g/mL). Controls were selected from among male patients who had attended clinic X and who had a urethral N. gonorrhoeae isolate that was submitted to GISP and showed susceptibility to ciprofloxacin (MIC 0.06 g/mL). The records at clinic X for 51 case-patients and 106 controls were abstracted by two investigators. Abstracted information included demographic data, the reason for visiting the clinic, history of sexually transmitted diseases, sexual history, results of clinical examination and assessment, and results of serum rapid plasma reagent assays. Results of serologic tests for human immunodeficiency virus (HIV) were kept in separate medical records that were not reviewed. Laboratory Methods The urethral specimens were plated on selective Martin-Lewis agar and processed; this processing included the identification of gonococci, testing for -lactamase, and the recording and assignment of a GISP number. All isolates were identified as N. gonorrhoeae by established methods [9]. Gonococcal isolates were subcultured from the selective primary medium to a chocolate agar with 1% IsoVitaleX (Becton Dickinson, Sparks, Maryland) supplement. Organisms from the subculture were suspended in tryptic soy broth that contained 20% glycerol. Isolates were then frozen at 20C and shipped on dry ice to the microbiology laboratory at the Cleveland Clinic Foundation, where they were kept frozen at 70C. The laboratory determined MICs by using the agar dilution methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS) with GC II agar base (Becton Dickinson) and 1% IsoVitaleX (Becton Dickinson) supplement [10]. The isolates were tested against penicillin, tetracycline, spectinomycin, ceftriaxone, cefixime, ciprofloxacin, erythromycin, and azithromycin using standard sterile powders supplied by the manufacturers. Susceptibilities were interpreted according to the recommendations of the NCCLS, which define gonococcal isolates as susceptible to ciprofloxacin if the MIC of ciprofloxacin is 0.06 g/mL or less. In our study, isolates were defined as having decreased susceptibility to ciprofloxacin if the MIC was 0.125 g/mL or more. The CDC [11] recently proposed interpretative criteria for defining an isolate as resistant to ciprofloxacin if the MIC is 1.0 g/mL or more. Culture and DNA Preparation for Pulsed-Field Gel Electrophoresis The methods used were modifications of those previously published [12]. Isolates were stored at 70C until needed and were then plated twice onto chocolate agar (Becton Dickinson) at 36.5 C in 5% CO2. Bacterial growth 18 to 22 hours after the second chocolate agar transfer was inoculated into sterile saline to a density approximately equal to McFarland number 4, sonicated, and centrifuged. The pellet was resuspended in 1 mL of cold cell-suspension buffer (Bio-Rad, Hercules, California). The pellets were molded in gel plugs as recommended by the manufacturer of the Gene-Path reagent kit (Bio-Rad). Solidified blocks were treated with lysozyme and incubated for 1 hour to release nucleic acid. Plugs were washed with buffer for 30 minutes and then soaked in buffer that contained 4 mg of phenylmethyl-sulfonyl fluoride per mL (Sigma, St. Louis, Missouri) at room temperature for 1 hour. This was followed by three washes with buffer at room temperature. Pulsed-Field Gel Electrophoresis Assay Gel plugs were digested with 8.3 L of Spe I (a restriction enzyme with a rare recognition site) overnight in 300 L of restriction buffer. The digested DNA plugs were placed in wells that contained 1% agarose gel with 1% low-molecular-weight protein agarose. Electrophoresis was done on the digested DNA plugs in a contour-clamped homogenous electric field apparatus (Gene-Path, Bio-Rad). Gels were stained with ethidium bromide and photographed under ultraviolet light. Bands of 15 to 500 kb in 12 to 15 fragments were produced per organism. Chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis were interpreted on the basis of criteria proposed by Tenover and colleagues [13]. If the pulsed-field gel restriction patterns were identical, the isolates were considered to be indistinguishable; if the patterns differed by 1 to 3 bands, the isolates were considered to be closely related; and if the patterns differed by more than 3 bands, the isolates were considered to be unrelated. Expanded Surveillance for Fluoroquinolone-Resistant Neisseria gonorrhoeae in Cleveland A point prevalence survey was done in the summer of 1994 to determine the prevalence of ciprofloxacin-resistant N. gonorrhoeae urethral isolates obtained from patients who attended two public clinics for sexually transmitted diseases (other than clinic X) in Cleveland (25 isolates) and from patients treated at the Cleveland Clinic Foundation (29 isolates). Statistical Analysis All data were entered into a computer database for analysis (Epi Info, version 5; CDC, Atlanta, Georgia). The Student t-test and the Fisher two-tailed exact test were used for univariate analyses of the significance of associations. Multivariable logistic regression was done using PC SAS/STAT (SAS Institute, Cary, North Carolina). Differences were considered statistically significant at a P value of 0.05 or less. Results From September 1991 through September 1995, 1378 isolates of N. gonorrhoeae from clinic X were tested; 140 (10.4%) had decreased susceptibility to ciprofloxacin. The incidence of gonococcal infections with these strains increased significantly during this period, from 2% in 1991 to 16% in 1994 (Figure 1). During the study period (1 January 1992 to 31 March 1994), 746 isolates of N. gonorrhoeae from patients in the GISP sample from clinic X were tested; 55 isolates (7.4%) had decreased susceptibility to ciprofloxacin. Figure 1. Incidence rates of urethral isolates of Neisseria gonorrhoeae with decreased susceptibility to ciprofloxacin among men treated at one sexually transmitted disease clinic in Cleveland, Ohio, between January 1991 and September 1995. P Description of Case-Patients The clinical records of 51 of the 55 patients whose N. gonorrhoeae isolates had decreased susceptibility to ciprofloxacin were available for review and were included in the epidemiologic study. All patients were black and heterosexual men, and most were unmarried (94%). The mean age was 28.7 ye

Multisite reproducibility of MIC results by the Sensititre® YeaStone Colorimetric Antifungal Susceptibility Panel ☆

Reproducibility of MIC results between laboratories, a major performance criterion used for evaluation of any susceptibility test method, was determined at three test sites using the Sensititre YeastOne Antifungal Panel, which incorporates Alamar Blue as a colorimetric indicator. MICs of five antifungals were determined using a set of 10 isolates of Candida species. Each isolate was tested a total of nine times against each antifungal agent in each of the three laboratories. A total of 1350 MICs were evaluated. MICs were read visually after incubation at 35 degrees C for 24 and 48 h. Overall, 99 to 100% of MIC values were encompassed by a range defined by the modal MIC +/- 1 dilution for each antifungal agent tested at both 24 h and 48 h. Replicate testing of the quality control isolates recommended by the National Committee for Clinical Laboratory Standards demonstrated excellent agreement between results obtained with the Sensititre YeastOne panel and the MIC reference range for each antifungal agent. These studies demonstrated that the Sensititre YeastOne Antifungal Panel may be used to generate MIC values for at least five different antifungal agents with a high degree of intra- and interlaboratory reproducibility.

Timed killing kinetic studies of the interaction between ciprofloxacin and β-lactams against gram-negative bacilli

Timed killing kinetic studies were performed with ciprofloxacin in combination with aztreonam, ceftazidime, piperacillin/tazobactam, and ticarcillin/clavulanic acid against three isolates each of Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa. Each antimicrobial agent in the combination was tested at its MIC and at one-half and one-quarter of its MIC. Colony counts were determined at 0, 3, 5, and 7 hours. Synergy occurred most frequently at 7 hours and, when present, was most likely to occur when ciprofloxacin and the beta-lactam were tested at concentrations equal to their respective MICs. Synergy after 3 hours of incubation was not predictive of a synergestic interaction at 5 or 7 hours. Antagonism was noted in several instances, particularly when ciprofloxacin and the beta-lactam were combined at one-quarter of their respective MICs.

Detection of Inducible Clindamycin Resistance in Staphylococci by Broth Microdilution Using Erythromycin-Clindamycin Combination Wells

ABSTRACT A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 μg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 μg/ml ERY and 0.5 μg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 μg/ml correlated well with the D-zone test.