Comparison of Etest,Disk Diffusion,and Broth Macrodilution for In Vitro Susceptibility Testing of Rhodococcus equi

MICs of erythromycin, clarithromycin, azithromycin, rifampin, gentamicin, and doxycycline against 101 isolates of Rhodococcus equi were determined by broth macrodilution, disk diffusion, and Etest. Categorical agreement ranged between 85.1 and 100%. Overall, the agreement between Etest and disk diffusion was better than the agreement between broth macrodilution and the agar-based methods.     

Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds

Invasive mold infections are life-threatening diseases for which appropriate antifungal therapy is crucial. Their epidemiology is evolving, with the emergence of triazole-resistant Aspergillus spp. and multidrug-resistant non-Aspergillus molds. Despite the lack of interpretive criteria, antifungal susceptibility testing of molds may be useful in guiding antifungal therapy. The standard broth microdilution method (BMD) is demanding and requires expertise. We assessed the performance of a commercialized gradient diffusion method (Etest method) as an alternative to BMD. The MICs or minimal effective concentrations (MECs) of amphotericin B, voriconazole, posaconazole, caspofungin, and micafungin were assessed for 290 clinical isolates of the most representative pathogenic molds (154 Aspergillus and 136 non-Aspergillus isolates) with the BMD and Etest methods. Essential agreements (EAs) within ±2 dilutions of ≥90% between the two methods were considered acceptable. EAs for amphotericin B and voriconazole were >90% for most potentially susceptible species. For posaconazole, the correlation was acceptable for Mucoromycotina but Etest MIC values were consistently lower for Aspergillus spp. (EAs of <90%). Excellent EAs were found for echinocandins with highly susceptible (MECs of <0.015 μg/ml) or intrinsically resistant (MECs of >16 μg/ml) strains. However, MEC determinations lacked consistency between methods for strains exhibiting mid-range MECs for echinocandins. We concluded that the Etest method is an appropriate alternative to BMD for antifungal susceptibility testing of molds under specific circumstances, including testing with amphotericin B or triazoles for non-Aspergillus molds (Mucoromycotina and Fusarium spp.). Additional study of molecularly characterized triazole-resistant Aspergillus isolates is required to confirm the ability of the Etest method to detect voriconazole and posaconazole resistance among Aspergillus spp.     

Vancomycin MIC for Methicillin-Resistant Coagulase-Negative Staphylococcus Isolates: Evaluation of the Broth Microdilution and Etest Methods

Vancomycin MIC results were determined by the broth microdilution (BMD) method and by Etest using 130 methicillin-resistant coagulase-negative staphylococcus bloodstream isolates obtained from a tertiary hospital. The majority (98.5%) of MIC results determined by BMD were ≤1 μg/ml, in contrast to MIC results determined by Etest (72.3% were ≥1.5 μg/ml). The MICs obtained by Etest were, in general, 1- to 2-fold higher than the MICs obtained by BMD.Coagulase-negative staphylococci (CoNS) have emerged as important nosocomial pathogens during the last decade, particularly in nosocomial bloodstream infections (4). Resistance to methicillin in CoNS is very common among isolates recovered from hospitalized individuals (4, 19). For this reason, vancomycin is usually the drug of choice for treatment of infections by methicillin-resistant CoNS (MRCoNS) (17).A reduction in the efficacy of vancomycin has been described in studies of methicillin-resistant Staphylococcus aureus (MRSA) infections treated with this antibiotic, and it has been suggested that slight increases in vancomycin MICs of between 1 and 2 μg/ml, which are within the susceptible range, may be related to suboptimal clinical outcomes (12, 16). Therefore, the determination of the MIC of vancomycin has been recommended for these pathogens (3). Some of these studies have used the broth microdilution (BMD) method for determining vancomycin MICs, while others have used the commercial Etest technique (AB Biodisk, Solna, Sweden). It has been reported that the Etest provides higher MICs than those obtained with BMD in S. aureus, mainly MRSA (14, 15). Nevertheless, there is no study comparing both methodologies for determination of the MIC of vancomycin in CoNS.Considering the increasing incidence of MRCoNS, the need for MIC determination of vancomycin, and the absence of studies assessing the performance of the Etest with these organisms, we aimed to compare the Etest and BMD methods for determination of the MICs of vancomycin in MRCoNS isolates.A total of 130 clinical isolates of CoNS recovered from blood samples obtained from patients who were hospitalized from May 2004 to August 2005 at the Hospital de Clínicas de Porto Alegre were analyzed. Only one isolate per patient was included. Blood cultures were performed using BacT/Alert (bioMérieux, Marcy l''Etoile, France). The colony morphology, Gram stain reaction, catalase testing, and absence of the coagulase enzyme were used to identify CoNS. Isolates were identified as Staphylococcus epidermidis by PCR using primers for tuf (10). The isolates that were not identified as S. epidermidis by PCR were identified using the API ID 32 Staph (bioMérieux, Marcy l''Etoile, France) semiautomated system, according to the instructions of the manufacturer. Results yielding a quality of identification of 85% or higher were accepted. The presence of the mec A gene was assessed by PCR using specific primers (13). The MICs of vancomycin were determined in duplicate by reference BMD, as recommended by CLSI, using in-house-prepared panels. The following dilutions of vancomycin were tested: 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/ml. The standard Etest procedure was performed using Mueller-Hinton agar (Becton Dickinson, Sparks, MD), with an inoculum density equivalent to a 0.5 McFarland standard. Vancomycin Etest strips were placed onto the agar with sterile forceps. The cultures were incubated for 24 h at 35°C. S. aureus ATCC 29213 was used for quality control (3).The Wilcoxon test was used to compare the MICs obtained by Etest and BMD. Two sets of comparisons were done, one with the exact MIC values determined by Etest and the other with the MICs determined by Etest rounded up to the next dilution.All 130 isolates of CoNS proved to be mec A positive and were identified as follows: 87 (66.9%) were S. epidermidis isolates, 13 (10.0%) were S. haemolyticus isolates, 12 (9.2%) were S. hominis isolates, and 11 (8.5%) were S. capitis isolates. Seven isolates (5.4%) were not identified to the species level. The MICs of vancomycin ranged from 0.25 to 2 μg/ml by BMD and from 0.38 to 3 μg/ml by Etest. No discrepancies were observed in duplicates performed by BMD. The MIC₅₀s and MIC₉₀s for vancomycin were both 1 μg/ml by BMD and 1.5 and 2 μg/ml, respectively, by Etest (P < 0.001 for both comparisons). Most MICs determined by Etest were ≥1.5 μg/ml (94 isolates, 72.3%), while most MICs determined by BMD were ≤1 μg/ml (128 isolates, 98.5%). A total of 113 (86.9%) and 5 (3.8%) isolates presented MICs determined by Etest that were 1- and 2-fold dilutions higher than those determined by BMD, respectively. Only 10 isolates (7.7%) presented the same MIC using the two methods (Fig. ​(Fig.1).1). When the Etest MICs of 0.38, 0.75, 1.5, and 3 μg/ml were converted to 0.5, 1, 2, and 4 μg/ml, respectively, there was an even higher discrepancy between the two methods. In this case, almost all MRCoNS had MICs of ≥1 μg/ml (124 isolates, 95.4%) (Fig. ​(Fig.2).2). MICs determined by Etest were higher than those determined by BMD for all MRCoNS species, when analyzed separately (Table ​(Table1).1). No isolate had a MIC higher than 4 μg/ml, regardless of the method used.Open in a separate windowFIG. 1.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution and Etest methods.Open in a separate windowFIG. 2.Scattergram of correlation between vancomycin MICs obtained by the broth microdilution method and the Etest method after rounding up.

TABLE 1.

Distribution of vancomycin MICs according to CoNS species

Comparison of Agar Dilution, Disk Diffusion, MicroScan, and Vitek Antimicrobial Susceptibility Testing Methods to Broth Microdilution for Detection of Fluoroquinolone-Resistant Isolates of the Family Enterobacteriaceae

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within ±1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.     

Evaluation of Vancomycin Susceptibility Testing for Methicillin-Resistant Staphylococcus aureus: Comparison of Etest and Three Automated Testing Methods

We evaluated the ability of four commercial MIC testing systems (MicroScan, Vitek 2, Phoenix, and Etest) to detect vancomycin MIC values of ≤1 to ≥2 in 200 methicillin-resistant Staphylococcus aureus (MRSA) strains compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference methods. Compared to the BMD method, absolute agreement (0 ± dilution) was highest for the Phoenix system (66.2%) and the MicroScan turbidity method (61.8%), followed by the Vitek 2 system (54.3%). The Etest produced MIC values 1 to 2 dilutions higher than those produced by the BMD method (36.7% agreement). Of interest, the MicroScan system (prompt method) was more likely to overcall an MIC value of 1 mg/liter (74.1%), whereas the Phoenix (76%) and Vitek 2 (20%) systems had a tendency to undercall an MIC of 2 mg/liter. The ability to correctly identify vancomycin MIC values of 1 and 2 has clinical implications and requires further evaluation.     

Multicenter Study To Determine Disk Diffusion and Broth Microdilution Criteria for Prediction of High- and Low-Level Mupirocin Resistance in Staphylococcus aureus

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of ≥512 μg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-μg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the “gold standard” for HLR, the sensitivity and specificity of a single-well 256 μg/ml BMD test were 97 and 99%, respectively, and those for the 200-μg disk test were 98 and 99%, respectively. Testing with two disks, 200 μg and 5 μg, was evaluated for its ability to distinguish HLR isolates (MICs ≥ 512 μg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 μg/ml), and susceptible isolates (MICs ≤ 4 μg/ml). Using no zone with both disks as an indication of HLR and no zone with the 5-μg disk plus any zone with the 200-μg disk as LLR, only 3 of the 340 isolates were misclassified, with 3 susceptible isolates being classified as LLR. Use of standardized MIC or disk tests could enable the detection of emerging high- and low-level mupirocin resistance in S. aureus.Mupirocin is a topical antibacterial agent that is used both for the treatment of skin infections and for the suppression or elimination of nasal carriage of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) (8). The recommendations of the Healthcare Infection Control Practices Advisory Committee suggest the use of a tiered approach to the prevention and control of infections with multidrug-resistant organisms, including MRSA, in acute-care settings (20). In their recommendations, decolonization is presented as one intervention that may be considered when intensified MRSA control measures are needed; if decolonization is used, susceptibility testing and monitoring for the emergence of resistance to the decolonization agent are recommended in one study (21).There are two levels of resistance to mupirocin: low-level resistance (LLR), for which the MICs are 8 to 256 μg/ml, and high-level resistance (HLR), for which the MICs are ≥512 μg/ml (11). The mupirocin MICs of strains susceptible to mupirocin are MICs ≤4 μg/ml. HLR is associated with the presence of the plasmid-mediated mupA gene, which encodes a mupirocin-resistant isoleucyl-tRNA synthetase, although S. aureus strains with HLR that lack mupA have occurred (this study) and can also be created in the laboratory (23). LLR results from mutation of the native, chromosomal isoleucyl-tRNA synthetase ileS gene (1). Studies suggest that S. aureus strains with HLR to mupirocin cannot be successfully eliminated with mupirocin and that the occurrence of HLR is increasing (22). It has been suggested that S. aureus strains demonstrating LLR could be eliminated by topical application of mupirocin because of the high concentrations achieved locally, but this has not been demonstrated definitively (11, 21).Until recently, methods for testing topical agents have not been included in susceptibility testing documents published by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS), although guidelines for testing by various methods have been suggested by others (9, 10, 12, 13, 16, 17). The British Society for Antimicrobial Chemotherapy has formal recommendations for the testing of mupirocin (www.bsac.org.uk) that include testing of a 5-μg and a 20-μg mupirocin disk. Their recommendations require MIC testing to determine the level of resistance if a 5-μg disk is used alone. An initial investigation at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, showed that a 200-μg mupirocin disk was able to differentiate isolates with LLR from those with HLR (15). We undertook the study described here to determine the MIC and disk diffusion criteria for the detection of S. aureus strains with high- or low-level mupirocin resistance and to validate quality control tests. Using data from this study, a screen test for prediction of high-level mupirocin resistance is now included in CLSI susceptibility testing documents (3, 6, 7).     

Comparative Evaluation of FUNGITEST and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of Candida Species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.The rising prevalence of serious fungal infections and antifungal drug resistance has created an increased demand for reliable methods of in vitro testing of antifungal agents that can assist in their clinical use. The National Committee for Clinical Laboratory Standards (NCCLS) has developed a standardized broth macrodilution method for the testing of Candida spp. and Cryptococcus neoformans (9). This reference method and microdilution adaptations of it have been reported to give almost identical results (1, 3, 4, 11, 18). However, these methods are time-consuming and labor-intensive and have not eliminated the need for simpler and more economical methods of routine testing.The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a commercial procedure for the breakpoint testing of six antifungal drugs (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). Each 16-well microplate contains 2 negative control wells, 2 positive growth control wells, and 12 drug-containing wells. Each drug is provided at two concentrations in dehydrated form and is reconstituted by adding the inoculum suspension in RPMI 1640 medium. The FUNGITEST method is not available in the United States. In this study we compared the FUNGITEST method with a broth microdilution adaptation of the NCCLS reference method using 200 isolates of seven Candida spp. and C. neoformans.(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)     

Comparison of Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methods for Testing Activity of Cefditoren against Streptococcus pneumoniae

This study evaluated the susceptibility of pneumococci to cefditoren by agar dilution and microdilution methods (both in air) and by E-test (AB Biodisk, Solna, Sweden) and disk diffusion methods (both in CO(2)). By the three MIC tests, the MICs at which 50 and 90% of isolates were inhibited (MIC(50)s and MIC(90)s) were, respectively, as follows (in micrograms per milliliter): for the 65 penicillin-susceptible strains tested, 0.016 and 0.03 (by agar dilution), 0.016 and 0.03 (by microdilution), and 0.016 and 0.03 (by E test); for the 68 penicillin-intermediate strains tested, 0.125 and 0.5 (by agar dilution), 0.125 and 0.5 (by microdilution), and 0. 25 and 0.5 (by E test); and for the 67 penicillin-resistant strains tested, 1.0 and 1.0 (by agar dilution), 0.5 and 1.0 (by microdilution), and 1.0 and 1.0 (by E test). With tentative cefditoren breakpoints (in micrograms per milliliter) of /=8.0 (resistant), all strains were susceptible to cefditoren by agar, microdilution, and E-test results; with breakpoints of /=4.0 microg/ml, 97% of strains were cefditoren susceptible by agar dilution results, 98% were susceptible by microdilution results, and 99% were susceptible by E-test results. When microdilution and E-test results were compared to those from the reference agar dilution method, 191 (95.5%) and 183 (91.5%) of strains gave essential agreement (+/-1 log(2) dilution); 8 (2.7%) minor discrepancies were found for both methods with a breakpoint of /=20 (susceptible), 17 to 19 (intermediate), and /=16 mm (susceptible). All three methods for testing the MIC of cefditoren showed excellent correlation.     

Colistin Susceptibility Testing of Gram-Negative Bacilli: Better Performance of Vitek2 System than E-Test Compared to Broth Microdilution Method as the Gold Standard Test

Introduction: Unavailability of optimal susceptibility testing (ST) challenges the clinical use of colistin. Broth microdilution (BMD), which is the reference for colistin ST, is inconvenient for diagnostics. Vitek2 and E-test although technically easier, are no longer recommended. Materials and Methods: For the evaluation of Vitek2 and E-test in reference with BMD, a total of 138 Gram-negative bacilli (GNB) especially carbapenem-resistant isolates from Tata Medical Center, Kolkata, India, were included during 2017–2018. The evaluation was performed only for Enterobacteriaceae (n = 102), but not for non-fermentative GNB (n = 36) due to lack of colistin-resistant (COL^R) isolates. Results and Conclusion: Of 138 isolates, meropenem, colistin and dual resistance were detected in 110 (79.7%), 31 (22.5%) and 21 (15.2%) of isolates, respectively. Using the European Committee on Antimicrobial Susceptibility Testing guidelines (susceptible, ≤2 μg/ml), Vitek2 performed better than E-test (essential agreement, 92.2% vs. 63.7%; categorical agreement, 94.1% vs. 93.1%; very major error [VME], 10% vs. 23.3%). However, Vitek2 overcalled resistance than E-test (major error, 4.2% vs. 0%). Considering Chew et al. proposed breakpoints (susceptible, ≤1 μg/ml), VMEs declined for both test (6.7% vs. 10%), but still remained unacceptable. Of eight colistin-heteroresistant isolates, two VME were categorised by Vitek2, one VME was by E-test, and two were uninterpretable. Both Vitek2 and E-test are unreliable. Further studies correlating minimum inhibitory concentrations with clinical outcome are needed to determine the accurate breakpoints for better patient management.     

Standardized Methods and Quality Control Limits for Agar and Broth Microdilution Susceptibility Testing of Mycoplasma pneumoniae,Mycoplasma hominis,and Ureaplasma urealyticum

An international multilaboratory collaborative study was conducted to develop standard media and consensus methods for the performance and quality control of antimicrobial susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum using broth microdilution and agar dilution techniques. A reference strain from the American Type Culture Collection was designated for each species, which was to be used for quality control purposes. Repeat testing of replicate samples of each reference strain by participating laboratories utilizing both methods and different lots of media enabled a 3- to 4-dilution MIC range to be established for drugs in several different classes, including tetracyclines, macrolides, ketolides, lincosamides, and fluoroquinolones. This represents the first multilaboratory collaboration to standardize susceptibility testing methods and to designate quality control parameters to ensure accurate and reliable assay results for mycoplasmas and ureaplasmas that infect humans.     

Progress in Antifungal Susceptibility Testing of Candida spp. by Use of Clinical and Laboratory Standards Institute Broth Microdilution Methods, 2010 to 2012

Antifungal susceptibility testing of Candida has been standardized and refined and now may play a useful role in managing Candida infections. Important new developments include validation of 24-h reading times for all antifungal agents and the establishment of species-specific epidemiological cutoff values (ECVs) for the systemically active antifungal agents and both common and uncommon species of Candida. The clinical breakpoints (CBPs) for fluconazole, voriconazole, and the echinocandins have been revised to provide species-specific interpretive criteria for the six most common species. The revised CBPs not only are predictive of clinical outcome but also provide a more sensitive means of identifying those strains with acquired or mutational resistance mechanisms. This brief review serves as an update on the new developments in the antifungal susceptibility testing of Candida spp. using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods.     

Fluconazole Susceptibility Testing of Cryptococcus neoformans: Comparison of Two Broth Microdilution Methods and Clinical Correlates among Isolates from Ugandan AIDS Patients

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var. neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.     

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.)     

Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.     

Antifungal Susceptibility of 205 Candida spp. Isolated Primarily during Invasive Candidiasis and Comparison of the Vitek 2 System with the CLSI Broth Microdilution and Etest Methods

Infections due to Candida spp. are frequent, particularly in immunocompromised and intensive care unit patients. Antifungal susceptibility tests are now required to optimize antifungal treatment given the emergence of acquired antifungal resistance in some Candida species. An antifungal susceptibility automated method, the Vitek 2 system (VK2), was evaluated. VK2 was compared to the CLSI broth microdilution reference method and the Etest procedure. For this purpose, 205 clinical isolates of Candida spp., including 11 different species, were tested for fluconazole, voriconazole, and amphotericin B susceptibility. For azoles, essential agreement ranged from 25% to 100%, depending on the method used and the Candida species tested. Categorical agreements for all of the species averaged 92.2% and ranged from 14.3 to 100%, depending on the 24-h or 48-h MIC reading by the Etest and CLSI methods and on the Candida species. Results obtained for Candida albicans showed excellent categorical and essential agreements with the two comparative methods. For Candida glabrata, the essential agreement was high with the CLSI method but low with the Etest method, and several very major errors in interpretation were observed between VK2 and the Etest method for both azoles. Low MICs of fluconazole were obtained for all of the Candida krusei isolates, but the VK2 expert software corrected all of the results obtained to resistant. Amphotericin B results showed MICs of ≤1 mg/liter for 201 (VK2), 190 (CLSI), and 202 (Etest) isolates. The AST-YS01 Vitek 2 card system (bioMérieux) is a reliable and practical standardized automated antifungal susceptibility test. Nevertheless, more assays are needed to better evaluate C. glabrata fluconazole sensitivity.Invasive candidiasis infections are of increasing concern, particularly in immunodeficient or intensive care unit patients. The emergence of antifungal resistance and the development of new echinocandin class and broad-spectrum azole fungal agents have complicated the choice of antifungal treatment for candidiasis.Antifungal choice is first based on Candida species identification, but antifungal susceptibility testing will play an increasingly important role when selecting which antifungal drug to use (3). Standardized methods for antifungal susceptibility testing have been available for many years. The Clinical and Laboratory Standards Institute (CLSI) standardized broth microdilution method remains a reference for antifungal susceptibility testing (4). Indeed, clinically relevant interpretative breakpoints are available and quality control strains are validated (11). Nevertheless, this method is complex and laborious to use as a routine method. Alternative, standardized and reliable methods adapted to hospital laboratories, such as the Etest, are now commonly used (2).The objective of this study was to evaluate and compare a new automated antifungal susceptibility test system (AST-YS01 Vitek 2 cards; bioMérieux) to both the CLSI reference broth microdilution method and Etest procedures. For this purpose, 208 clinical isolates of Candida spp. isolated primarily from patients with invasive candidiasis were tested with fluconazole, voriconazole, and amphotericin B.(Preliminary data were presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 16 to 19 May 2009, abstr. P1306.)