Impact of Variations in Test Method Parameters on In Vitro Activity of Surotomycin against Clostridium difficile and Surotomycin Quality Control Limits for Broth Microdilution and Agar Dilution Susceptibility Testing

Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 μg/ml (standard for lipopeptide testing) is recommended. Quality control ranges for Clostridium difficile (0.12 to 1 μg/ml) and Eggerthella lenta (broth, 1 to 4 μg/ml; agar, 1 to 8 μg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.     

Novel Fluorescent Broth Microdilution Method for Fluconazole Susceptibility Testing of Candida albicans

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.     

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.     

Comparison of the E Test and Agar Dilution Method for Antimicrobial Suceptibility Testing of Helicobacter pylori

A multicentre study was carried out in order to validate the E test in comparison with the reference agar dilution method for testing the susceptibility of Helicobacter pylori to amoxicillin, clarithromycin, and metronidazole. Ten clinical isolates and one control collection isolate (Helicobacter pylori ATCC 43504) were tes ted blindly at four centres according to a uniform methodology. The E test showed excellent intra- and inter-laboratory correlations with the agar dilution method for amoxicillin and clarithromycin (>98% agreement within 2 log₂ dilution steps). For metronidazole, however, the E test revealed significantly higher minimum inhibitory concentration values (>2 log₂) against 5 of the 10 Helicobacter pylori strains tested. Overall, neither method was found reliable for testing the susceptibility of Helicobacter pylori to metronidazole, since both tended to lack reproducibility. Electronic Publication     

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.     

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.     

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.     

Effect of Carbon Dioxide on Broth Microdilution Susceptibility Testing of Brucella spp.

Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO₂ incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log₂ dilution higher in CO₂ than in AA; tetracycline-class MICs to be 1 log₂ dilution lower in CO₂.Routine susceptibility testing of Brucella spp. is not recommended since the susceptibility pattern of wild-type Brucella spp. is fairly predictable, the isolates are fastidious, and the organisms are a potential cause of laboratory-acquired infection (3, 12, 17). In addition, Brucella spp. are intracellular pathogens, and like other intracellular pathogens, in vitro susceptibility may not always correlate with clinical outcome (1, 3, 28). Typically, brucellosis is treated with dual-antimicrobial therapy to lower the possibility of relapse. The most common combinations are streptomycin (or gentamicin) and doxycycline and doxycycline combined with rifampin (4, 12, 21, 28). Trimethoprim-sulfamethoxazole is recommended as alternative therapy (28), but use of fluoroquinolone therapy is controversial (12, 18, 25). Although there has been little or no resistance reported to routinely prescribed antimicrobials for brucellosis, relapse is still common (3, 5, 22, 25), and development of laboratory-confirmed rifampin resistance has been reported (11).Brucella suis, Brucella melitensis, and Brucella abortus are considered potential agents of bioterrorism (6, 24). As with other potential bacterial agents of bioterrorism, engineered antimicrobial resistance is a concern. Antimicrobial susceptibility testing of Brucella spp. to identify effective therapeutic and prophylactic agents would be an important response effort in a bioterrorism event. Although no antibiotic regimen has been precisely studied for prophylaxis of brucellosis in humans, combining doxycycline and rifampin or using trimethoprim-sulfamethoxazole alone (for children and pregnant women) has been used successfully in preventing laboratory-acquired disease, although side effects can occur (20, 23, 27).Many different methods of antimicrobial susceptibility testing, using a variety of media and incubation conditions, have been described for testing Brucella spp. (1, 3-5, 12-14, 16, 21, 22, 25, 26). Jevitt et al. (15) developed a standardized method for susceptibility testing of Brucella spp. using brucella broth, a method that was adopted by the Clinical and Laboratory Standards Institute (CLSI) in 2006 (9). This initial CLSI method for Brucella spp. described a broth microdilution (BMD) procedure using incubation at 35 ± 2°C for 48 h in ambient air (AA). The present study describes a multicenter examination to evaluate incubation in ambient air supplemented with 5% CO₂. Incubation in CO₂-supplemented air is particularly important for some strains of Brucella spp. that are especially fastidious, such as B. abortus (19). Since many laboratories may not have a dedicated CO₂ incubator in a biosafety level 3 (BSL3) space, incubation using a CO₂-generating system, such as BBL GasPak CO₂ (BD, Sparks, MD) and the BBL Gaspak CO₂ pouch capnophilic system (BD), was evaluated.Thirty-nine strains of Brucella spp. from the Centers for Disease Control and Prevention (CDC) collection were used for this study: 20 B. melitensis, 11 B. suis, and 8 B. abortus. Antimicrobial susceptibility testing was performed in 4 laboratories at 3 institutions. Two laboratories tested both ambient air and CO₂ incubation conditions, and two laboratories performed testing using only one of the incubation conditions. BMD panels were prepared at the CDC with brucella broth (BBL, Sparks, MD) at pH 7 to 7.2 and were shipped to participating laboratories, where they were stored at −70°C until ready for use; all panels were from the same preparation lot number. Antimicrobial powders were obtained from Sigma (St. Louis, MO). The antimicrobial agents and ranges tested were as follows: doxycycline, 0.015 to 8 μg/ml; gentamicin, 0.015 to 8 μg/ml; rifampin, 0.12 to 8 μg/ml; streptomycin, 0.12 to 64 μg/ml; tetracycline, 0.015 to 8 μg/ml; and trimethoprim-sulfamethoxazole, 0.015/0.285 to 8/152 μg/ml. The MIC incubation temperature was 35°C for both atmospheres. CO₂ incubation was accomplished with a single-use gas-generating system which produces an atmosphere that contains 2.5 to 10% CO₂ (BBL product insert). The BBL GasPak CO₂ system was used in 2 laboratories (sites A and B), and the BBL GasPak CO₂ pouch capnophilic system was used in laboratory test site D. Inocula were prepared by the direct colony suspension method in Mueller-Hinton broth from 24- to 48-h cultures grown on 5% sheep blood agar plates (BBL) and incubated in ambient air or in ambient air supplemented with CO₂ by using a gas-generating system if the isolate was dependent upon CO₂ for growth (8). The final volume of brucella broth in the MIC tray wells was 100 μl per well; 10 μl of diluted inoculum was delivered by a sterile, plastic commercial inoculator system (Dynex, Chantilly, VA). MIC panels were incubated for 48 h, and endpoints were recorded as the lowest concentration of drug demonstrating no macroscopic growth, except for trimethoprim-sulfamethoxazole, where the endpoint was interpreted as the lowest drug concentration inhibiting 80% of the growth when compared to the growth control well.BMD MIC results from AA incubation and CO₂ incubation for the 39 Brucella isolates are shown in Fig. ​Fig.1.1. AA MIC results were not available for 4 of the 39 isolates because these isolates would not grow in AA; CO₂ was required for growth in broth and on agar media. The MIC modes for tetracycline and doxycycline were 1 log₂ dilution lower in CO₂ than in AA, while gentamicin and trimethoprim-sulfamethoxazole modes were 1 log₂ dilution higher in CO₂ than in AA (Table ​(Table1).1). The modes for streptomycin MICs in AA and CO₂ were the same (4 μg/ml), but there were 40 more results for which the MIC was 8 or 16 μg/ml in CO₂ than in AA. Fourteen of these 40 streptomycin results were categorized as nonsusceptible (MIC of 16 μg/ml) in CO₂, whereas no results fell into the nonsusceptible category for AA incubation.Open in a separate windowFIG. 1.Bar graphs of MICs under ambient air and CO₂ conditions for six antimicrobial agents tested against 39 Brucella isolates at three test sites for each atmosphere. For one site, ambient air data were available for only 31 isolates, while the other two sites each had 35 ambient air results (4/39 isolates required CO₂). S, susceptible. Category divisions are based on the original 2006 CLSI breakpoints; there are no published breakpoints for rifampin (9).

TABLE 1.

MIC modes and ranges for six antimicrobial agents tested against 39 Brucella isolates at three test sites for each incubation atmosphere^a

Comparative Evaluation of National Committee for Clinical Laboratory Standards Broth Macrodilution and Agar Dilution Screening Methods for Testing Fluconazole Susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-μg/ml fluconazole plates were predicted to be ≥8 or ≥16 μg/ml, respectively. On medium with 16 μg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 μg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were ≥16 μg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.     

Comparative Evaluation of the Vitek 2 Yeast Susceptibility Test and CLSI Broth Microdilution Reference Method for Testing Antifungal Susceptibility of Invasive Fungal Isolates in Italy: the GISIA3 Study

The newly available AST-YS01 Vitek 2 cards were evaluated, and the results were compared with those obtained by the CLSI M27-A2 microdilution reference method. Clinical fungal isolates, including 614 isolates of Candida spp., 10 Cryptococcus neoformans isolates, 1 Geotrichum capitatum isolate, and 2 quality control strains, were tested for their susceptibilities to amphotericin B, fluconazole, and voriconazole using both methods. The majority of fungal isolates were susceptible to all antifungal agents tested: the MIC₉₀ values determined by the Vitek 2 and CLSI methods were 0.5 and 1 μg/ml, respectively, for amphotericin B; 8 and 16 μg/ml, respectively, for fluconazole; and <0.12 and 0.25 μg/ml, respectively, for voriconazole. Overall there was excellent categorical agreement (CA) between the methods (99.5% for amphotericin B, 92% for fluconazole, 98.2% for voriconazole), but discrepancies were observed within species. The CAs for fluconazole were low for Candida glabrata and Candida krusei when the results of the CLSI method at 48 h were considered. Moreover, the fully automated commercial system did not detect the susceptibility of Cryptococcus neoformans to voriconazole. The Vitek 2 system can be considered a valid support for antifungal susceptibility testing of fungi, but testing of susceptibility to agents not included in the system (e.g., echinocandins and posaconazole) should be performed with other methods.Antifungal susceptibility testing (AFST) has become increasingly common in clinical practice in recent years. This is a result of both the improved performance of antifungal susceptibility testing methods and the introduction of antifungal drugs with various mechanisms of action, such as the echinocandins and triazoles (7, 9, 10). It is generally considered that the outcome of invasive fungal infections, in particular, candidemia, is improved by prompt initiation of appropriate antifungal therapy (13). Treatment of invasive Candida infections is currently based on the updated IDSA guidelines (15), but knowledge of the susceptibilities of local clinical isolates to antifungal agents can further guide physicians'' choice of appropriate and safe antifungal agents, which is especially important for long-term treatment (9).AFST reference methods for fungi have been available since 1997 from the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) and, more recently, from the subcommittee on AFST of the European Committee for Antimicrobial Susceptibility Testing (EUCAST). However, both of these methods are time-consuming and clinical microbiological laboratory personnel may be unfamiliar with the methodologies (3, 6, 11, 12, 14, 20). Commercially available methods demonstrate variable performance compared with the performance of reference methods; two commercial assays have been approved by the U.S. Food and Drug Administration (FDA) for AFST of fungi with several antifungal agents: Etest (bioMérieux SA, Marcy l''Etoile, France) and the Sensititre YeastOne system (Trek Diagnostic Systems Ltd., East Grinstead, England). Recently, bioMérieux expanded its role in this area with a yeast susceptibility test that determines Candida growth spectrophotometrically using the Vitek 2 microbiology systems, performing fully automated testing of susceptibility to flucytosine, amphotericin B, fluconazole, and voriconazole (1, 16-18).To investigate the reliability of the new AST-YS01 Vitek 2 cards, the susceptibilities of clinical fungal isolates to amphotericin B, fluconazole, and voriconazole, as determined by the Vitek 2 system, were compared with those obtained with the reference CLSI (M27-A2) broth microdilution method (14).     

Comparison of Selective Broth Medium Plus Neomycin-Nalidixic Acid Agar and Selective Broth Medium Plus Columbia Colistin-Nalidixic Acid Agar for Detection of Group B Streptococcal Colonization in Women

The combination of neomycin-nalidixic acid (NNA) agar and a selective broth medium (SBM) has recently been shown to improve the sensitivity of screening cultures for group B streptococcal (GBS) carriage in women. Because of the relatively high cost of NNA agar, a study was initiated to determine whether Columbia colistin-nalidixic acid (CNA) agar would be an equally sensitive, more economical alternative. A total of 580 cervical-vaginal and/or rectal specimens submitted for detection of GBS were included in the study. Each was plated onto NNA and CNA agar and then inoculated into SBM. GBS were recovered from 95 of 580 (16.4%) specimens, including 61 isolates from CNA, 74 from NNA, 73 from the CNA-SMB combination, and 86 from the NNA-SMB tandem. Of those, 22 isolates were recovered on NNA but not CNA, 9 were cultured on CNA but not NNA, 52 were isolated on both media, and 12 were recovered from subcultures of SBM only. The overall sensitivity of CNA alone (64. 2%) was statistically significantly less than that of NNA agar (77. 9%), as was the sensitivity of combination of CNA plus SBM (76.8%) compared to that of NNA plus SBM (90.5%). Based on these findings, CNA should not be considered an acceptable alternative to NNA for the detection of GBS colonization in women despite potential cost savings.     

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.     

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.)     

Multisite Reproducibility of Results Obtained by the Broth Microdilution Method for Susceptibility Testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four Mycobacterium fortuitum group, three Mycobacterium abscessus, and three Mycobacterium chelonae isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin (M. chelonae only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode +/- 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of M. fortuitum against clarithromycin; M. abscessus and M. chelonae against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.