Commercial broth microdilution panel validation and reproducibility trials for garenoxacin (BMS-284756), a novel desfluoroquinolone

Results from garenoxacin dry-form broth microdilution MIC panels prepared commercially (Sensititre, TREK Diagnostics) were compared to reference frozen-form MICs to ensure the validity of the longer-shelf-life product. A total of 1078 organisms from seven major organism groups were used in this trial. All commercial MIC results were within +/- one log(2) dilution of reference garenoxacin values, and reproducibility trials produced identical MIC results for 90.5 to 92.1% of garenoxacin MIC comparisons. Control quinolones (ciprofloxacin and gatifloxacin) also performed at a similarly high level of accuracy.     

Comparison of anaerobic susceptibility results obtained by two methods of inoculum preparation

We evaluated the use of inocula prepared directly from blood agar plates in agar dilution susceptibility tests of anaerobic bacteria and compared the results with susceptibility results obtained from the National Committee for Clinical Laboratory Standards proposed thioglycolate broth cultures. The objectives were to evaluate the reproducibility of each of the two methods of inoculum preparation and to compare the MICs obtained by each method. The reproducibility studies were conducted on 14 stock strains. The mode MICs obtained by the direct agar method were identical to those obtained by the reference broth method 74% of the time and within +/- 1 log2 dilution 100% of the time. The degree of reproducibility of each of the two methods was identical (93% +/- 1 log2 dilution). MIC results obtained by the direct agar method agreed with the MICs obtained by the reference broth culture method in 92.9% of 1,125 MIC data pair determinations performed on stock cultures. The reproducibility of the direct agar method within +/- 1 log2 dilution step for 115 fresh clinical isolates was 93%, including 93.4% of the results with the Bacteroides fragilis group. Only two very major discrepancies (false-susceptible by the agar method) were identified among the 708 MIC data pairs on these clinical isolates. Preparation of inocula directly from growth on agar plates provides a rapid and reproducible method for agar dilution susceptibility testing of anaerobes.     

Evaluation of the MICUR system for quantitative antimicrobial susceptibility testing: a multiphasic comparison with reference methods.

Four laboratories participated in a three-phase study to evaluate the MICUR antimicrobial broth microdilution system (Boehringer Mannheim Diagnostics, Inc., Houston, Tex.). The dried-antimicrobial agent MICUR system was compared with a reference broth microdilution method (National Committee for Clinical Laboratory Standards) by using 304 recently isolated clinical strains and two collections of stock or challenge organisms. Of 7,092 minimum inhibitory concentration (MIC) datum pairs derived from the clinical isolates, 96.6% were within an acceptable (+/- 1 log2 dilution) range. MICUR MICs agreed with the reference broth microdilution method MICs in 95.3% of 6,840 MIC pair determinations performed on stock or challenge cultures. The MICUR intralaboratory reproducibility within +/- 1 log2 dilution step for the clinical isolates was 98.4%. The MICUR intralaboratory and interlaboratory reproducibilities for 26 stock cultures were 98.4 and 95.1%, respectively. For 180 challenge cultures (4,199 MIC pairs) which were included in the MICUR testing to provide a wide variety of antimicrobial susceptibility and resistance patterns, the results for 92.5% were in close agreement with the reference broth microdilution results. No specific resistance mechanism went unrecognized by this new commercial system. The MICUR system gives comparable MIC results when evaluated against the reference broth microdilution method, and it would be acceptable for use in clinical microbiology laboratories.     

Evaluation of Etest for susceptibility testing of rapidly growing mycobacteria.

MICs of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, and imipenem were determined by Etest for 100 clinical strains of rapidly growing mycobacteria and compared with MICs determined by a reference agar dilution method. Etest MICs were also determined by an alternative inoculum application (agar overlay) method and compared with MICs determined by the inoculum application method recommended by the manufacturer (swabbing). Agreement between Etest and agar dilution MICs within +/- 1 log2 dilution was 85% (511 of 600), and agreement within +/- 2 log2 dilutions was 97% (580 of 600). The rate of complete category agreement was 88%, and rates of major and minor errors were 2.2 and 11.7%, respectively. No very major errors were detected for Etest MICs. Interlaboratory agreement between MICs determined at two separate laboratories was 81% (121 of 149) within +/- 1 log2 dilution and 92% (137 of 149) within +/- 2 log2 dilutions. Agreement between laboratories by interpretive category was 92%. Exact agreement between agar overlay and swab application MICs was 52.3%, and agreement within +/- 1 log2 dilution was 82.3%. Diffuse ellipse edges and trailing growth were still a problem with the overlay method, and in some cases results were more difficult to interpret than they were with the corresponding swab-prepared plate. In summary, our data suggest that Etest may be an accurate and reproducible method for determining susceptibility of rapidly growing mycobacteria.     

Validation of commercial dry-form broth microdilution panels for susceptibility testing of AZD2563, a new long-acting oxazolidinone

The MIC results using a dry-form broth microdilution panel (TREK Diagnostic/Sensititre, Westlake, OH, USA) were validated for AZD2563, a novel oxazolidinone compound. In comparision studies against reference frozen-form panels, the commercial MIC results were the same as the reference calue for 82.7% of organisms and all results were within ± one log₂ dilution. Using 462 organisms, most from three genus groups (enterococci, staphylococci, streptococci), test results indicate that Sensititre MIC values were comparable to the reference test and can be utilized in clinical trials of for routine laboratory use when testing AZD2563 and linezolid, the drug class comparator.     

Comparison of sensititre broth microdilution and agar dilution susceptibility testing techniques for meropenem to determine accuracy, reproducibility, and predictive values.

The stability, accuracy, reproducibility, and predictive value of Sensititre MIC panels containing meropenem (Merrem) were evaluated by using National Committee for Clinical Laboratory Standards (NCCLS)-recommended American Type Culture Collection (ATCC) strains and 110 selected strains of rapidly growing and fastidious aerobes and anaerobes with various degrees of susceptibility to meropenem. The NCCLS-recommended agar dilution method was used as a standard reference method. Meropenem-containing Sensititre MIC panels were monitored for their stabilities at room temperature and reproducibilities over 24 months by using six ATCC strains. Ninety-nine percent of the MICs of both meropenem and imipenem obtained for NCCLS-recommended ATCC strains were within the established ranges after 2 years. The overall agreement (+/- 1 twofold dilution) between the Sensititre and the agar dilution meropenem MICs was greater than 93%. The predictive value of meropenem MICs for indicating suspeptibility or resistance obtained by the Sensititre method was greater than 90%. No major or very major interpretive errors were observed, and only 5% of meropenem MICs were associated with minor interpretive errors. Problematic organisms were not observed. The Sensititre MIC panels containing meropenem offer a convenient and valid alternative to the NCCLS reference method for the susceptibility testing of potential pathogens likely to be recovered from mixed infections.     

Evaluation of in vitro spectra of activity of azithromycin, clarithromycin, and erythromycin tested against strains of Neisseria gonorrhoeae by reference agar dilution, disk diffusion, and Etest methods.

The macrolide-azilide susceptibility testing (agar dilution, disk diffusion, Etest) criteria for 105 Neisseria gonorrhoeae strains were evaluated. In addition, the potencies of azithromycin, clarithromycin, and erythromycin were studied. The most active macrolide-azilide agent was azithromycin (MIC at which 90% of the isolates are inhibited [MIC90], 0.5 microgram/ml) compared with clarithromycin (MIC90, 1.5 to 2 micrograms/ml) and erythromycin (MIC90, 2 to 4 micrograms/ml). The Etest (AB Biodisk, Solna, Sweden) was observed to produce MIC results very similar to those of the reference agar dilution test (GC agar base), with 100% of the results within 1 log2 dilution step of the reference MICs. The disk diffusion test zone diameters for all three drugs correlated at an acceptable level (r = -0.81 to -0.92) with the reference agar dilution MICs. Interpretive criteria for susceptibility were proposed for azithromycin at a MIC of < or = 2 micrograms/ml and a disk diffusion test zone of > or = 25 mm. No category for resistance was proposed because of the paucity of strains for which MICs were > 2 micrograms/ml. These tentative criteria should be further validated by correlations with clinical trial data for gonococcal strains (as they emerge) that have azithromycin MICs above the proposed susceptible category range.     

Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds

This report presents a semisolid agar antifungal susceptibility (SAAS) method for the rapid susceptibility screening of yeasts and molds. The reproducibility and accuracy of the SAAS method were assessed by comparing the MICs of amphotericin B and fluconazole obtained for 10 candidate quality control (QC) American Type Culture Collection yeast strains in >/=15 replicates with those found by six independent laboratories using the National Committee for Clinical Laboratory Standards (NCCLS) M27-P broth macrodilution method (M. A. Pfaller et al., J. Clin. Microbiol. 33:1104-1107, 1995). Overall, 96% of MICs for both drugs fell within 1 log(2) dilution of the modal MIC for each strain. The MICs for amphotericin B showed 99% agreement with the NCCLS proposed QC ranges within 1 log(2) dilution. Likewise, the MICs for fluconazole at >/=75% growth reduction showed 99% agreement for seven strains. Three strains, Candida albicans ATCC 24333 and ATCC 76615 and Candida tropicalis ATCC 750, showed a less sharp fluconazole endpoint at >/=75% growth reduction, but at >50% growth reduction, the agreement was 98% within 1 log(2) dilution of the proposed range. The MIC agreement within the proposed range for the suggested QC strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 was 100% for fluconazole and 100% within 1 log(2) dilution of the proposed range for amphotericin B. The SAAS method demonstrated the susceptibility or resistance of 25 clinical isolates of filamentous fungi such as Aspergillus fumigatus to amphotericin B, itraconazole, and fluconazole, usually within 48 h. Although the results are preliminary, this SAAS method is promising as a rapid and cost-effective screen and is worthy of concerted investigation.     

Evaluation of anaerobic microdilution MIC results with the Prompt Inoculation System.

The Prompt Inoculation System (3M Co.) was compared with the overnight suspension inoculation procedure used with a broth microdilution anaerobic commercial system (Micro-Media Systems) for differences in MIC. MIC results from both suspension methods using six National Committee for Clinical Laboratory Standards-recommended quality control organisms were identical in 18 instances (75%) and within +/- 1 log2 dilution in 96% of the comparisons. Results with 45 anaerobic clinical isolates also compared satisfactorily; 83% of the results were identical and 97% were within +/- 1 log2 dilution. In addition, the direct (Prompt)-inoculated microdilution trays produced better growth and more valid MIC results; 92% of the clinical isolates produced MIC results versus 79% by the overnight suspension procedure.     

Rapid antimicrobial susceptibility testing of gram-negative bacilli using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A

The MicroScan Rapid Neg MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of Gram-negative bacilli. In a three site clinical study eleven antimicrobial agents were evaluated by comparing results obtained with 741 clinical isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels determined visually. Results for 31%, 40%, 12% and 9% of the isolates were available within 3.5, 4.5, 5.5 and 7.0 hours respectively. Results for 7.3% were not available within that time period. For the seven drugs analyzed using a Minimum Inhibitory Concentration range of dilutions, overall agreement (+/- 1 dilution) was 94%, with 1.5% very major, 0.9% major and 2.5% minor errors. For the four drugs analyzed using a Breakpoint range of dilutions, overall agreement (+/- 1 dilution) was 97%, with two percent very major, and one percent major errors. The MicroScan Rapid Neg MIC system is an accurate and rapid method for same day determination of susceptibility of Gram-negative bacilli.     

Rapid antimicrobial susceptibility testing of gram-positive cocci using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A

The Microscan Rapid Pos MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of staphylococci, streptococci, and Listeria. In a three site clinical study, panels containing 26 antimicrobial agents were evaluated by comparing results obtained with 605 clinically significant isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels. Results for 16%, 40%, 13%, 9%, 8%, 11% and 1% of the isolates were available within 3.5, 4.5, 5.5, 7.0, 8, 11 and 15 h respectively. Results for 2% were not available within that time period. Overall agreement (+/- 1 dilution) for the 14,609 efficacy comparisons was 96%, with 1% each for very major, major and minor errors. Interlaboratory reproducibility testing of 25 isolates in triplicate in each site, showed an overall essential agreement of 97%. The MicroScan Rapid Pos MIC System is an accurate, reproducible and rapid method for same-day determination of susceptibility of Gram-positive cocci.     

Comparison of three commercial MIC systems, E test, fastidious antimicrobial susceptibility panel, and FOX fastidious panel, for confirmation of penicillin and cephalosporin resistance in Streptococcus pneumoniae.

The performances of three commercial broth microdilution MIC assays adapted for use with fastidious organisms--the E test (ET), Fastidious Antimicrobial Susceptibility panel (FAS), and FOX Fastidious panel (FOX)--were compared with a MIC using Mueller-Hinton broth with 5% lysed horse blood (MHLHB) to confirm penicillin and cephalosporin resistance in clinical isolates of Streptococcus pneumoniae. Of the isolates screened for penicillin resistance, 5 (12.8%) were categorized as susceptible, 16 (41.0%) were categorized as intermediate, and 18 (46.2%) were categorized as resistant by MHLHB. Only the isolates exhibiting intermediate-to-resistant MICs were included in the comparison. Agreement within +/- 1 log2 dilution was found in 91, 21, and 76% of the ET, FAS, and FOX MICs, respectively, compared with the MHLHB MIC. No very major or major discrepancies occurred with the ET or FOX; however, two very major interpretive errors occurred with the FAS. Agreement between the ET and MHLHB for cefotaxime, ceftriaxone, and cefuroxime was 88, 85, and 100%, respectively. Less than 50% of cephalosporin MICs categorized as > 0.5 microgram/ml by MHLHB were detected by FAS or FOX. Of the methods compared, the ET was the most reliable alternative for susceptibility testing of pneumococci.     

Detection of penicillin and extended-spectrum cephalosporin resistance among Streptococcus pneumoniae clinical isolates by use of the E test.

Increasing penicillin resistance and the initial recognition of resistance to extended-spectrum cephalosporins among Streptococcus pneumoniae isolates have placed greater emphasis on accurate methods for susceptibility testing of clinical isolates. This study has evaluated the use of the E test (AB Biodisk NA, Piscataway, N.J.) for the detection of penicillin and cefotaxime resistance among 147 pneumococcal clinical isolates in three geographically separate laboratories. These included 42 penicillin-resistant (MIC, > or = 2 micrograms/ml) and 14 cefotaxime-resistant (defined here as an MIC of > or = 2 micrograms/ml) isolates. E test strips were applied to the surface of Mueller-Hinton sheep blood agar plates and incubated at 35 degrees C in 5% CO2 for 20 to 24 h. E test MICs were compared with MICs determined with lysed horse blood-supplemented Mueller-Hinton broth in a microdilution format as recommended by the National Committee for Clinical Laboratory Standards. Penicillin MICs agreed within one log2 dilution for 136 of 147 (92.5%) isolates, and cefotaxime MICs agreed within one log2 dilution for 142 of 147 (96.6%) isolates. No very major or major interpretive errors occurred with either penicillin or cefotaxime E test MIC results. There were 9.5 and 5.4% minor interpretive category errors with penicillin and cefotaxime E test MICs, respectively. These data indicate that the E test represents a convenient and reliable method for the detection of penicillin or cephalosporin resistance in pneumococci.     

Comparison of spiral gradient endpoint and agar dilution methods for susceptibility testing of anaerobic bacteria: a multilaboratory collaborative evaluation.

A multilaboratory collaborative study was carried out to assess the utility of the spiral gradient endpoint (SGE) method for the determination of the antimicrobial susceptibilities of anaerobes and to evaluate the equivalence of the MICs obtained by the SGE method with those obtained by the reference agar dilution method of the National Committee for Clinical Laboratory Standards. The standard deviation of the MIC obtained by the SGE method for the five participating laboratories was +/- 0.26 of a twofold dilution, whereas it was +/- 1 twofold dilution by the reference method. The interlaboratory reproducibility of the results for two control strains tested with imipenem, chloramphenicol, and metronidazole indicated that 96% of the measurements fell within +/- 1 twofold dilution of the mode. The equivalence of the SGE method with the agar dilution method was assessed with a wide variety of anaerobic organisms. The MICs by both methods were within 1 doubling dilution in 93% of the measurements (n = 1,074). Discrepancies generally occurred with those organism-drug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement).     

Comparative assessment of Etest for testing susceptibilities of Neisseria gonorrhoeae to penicillin, tetracycline, ceftriaxone, cefotaxime, and ciprofloxacin: investigation using 510(k) review criteria, recommended by the Food and Drug Administration.

We evaluated the ability of the Etest (AB Biodisk, Solna, Sweden) method to accurately and reproducibly determine the antimicrobial susceptibility of Neisseria gonorrhoeae. One hundred gonococcal isolates were used to evaluate the diagnostic performance of the Etest compared with the reference agar dilution method for penicillin, tetracycline, ciprofloxacin, and ceftriaxone. Between 92 and 99% of Etest MIC results for all drugs were within +/- 1 log2 dilution of the reference MIC. According to recommended interpretive criteria, ceftriaxone, cefotaxime, and ciprofloxacin had 100% categorical agreement, while penicillin (86%) and tetracycline (85%) categorical agreement percentages were lower because of the large number of strains that were within 0.5 to 1 log2 dilution of the susceptible or resistant breakpoints. Reproducibility data also demonstrated that the Etest was precise (99.1%) when subjected to replicate testing. On the basis of these data, the Etest method provides an effective, simple alternative to the reference agar dilution method for the direct quantification of N. gonorrhoeae susceptibility.     

Evaluation of the AutoMicrobic system for susceptibility testing of aminoglycosides and gram-negative bacilli.

The AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) was compared with a reference broth microdilution MIC method to determine the accuracy and reproducibility of aminoglycoside susceptibility testing of gram-negative bacilli. Stock clinical isolates (n = 176) which demonstrated resistance to at least one aminoglycoside, extended-spectrum penicillin, or broad-spectrum cephalosporin (or a combination) were selected for this study. Isolates with moderate susceptibility to the aminoglycosides were also included. Of these isolates, 116 were either resistant or moderately susceptible to one or more of amikacin, gentamicin, netilmicin, and tobramycin. When AMS MIC results for 704 antimicrobial agent-organism combinations were compared with parallel microdilution MIC results, exact agreement (AMS MIC = reference MIC) rates were: amikacin, 71.6%; gentamicin, 71.6%; netilmicin, 83.0%; and tobramycin, 69.3%. Agreement rates within +/- 1 log2 dilution were: amikacin, 96.0%; gentamicin, 93.8%; netilmicin, 97.2%; and tobramycin, 96.0%. When National Committee for Clinical Laboratory Standards criteria were used to qualitatively evaluate performance, the overall agreement rates were: amikacin, 100.0%; gentamicin, 99.4%; netilmicin, 98.9%; and tobramycin, 99.4%. There were only four very major discrepancies, which represented 0.6% of the tests performed, and there were no major discrepancies. The percentages of minor discrepancies were: amikacin, 9.6%; gentamicin, 14.2%; netilmicin, 11.9%; and tobramycin, 10.8%. Of the overall average of 11.6% minor discrepancies, 9.7% occurred even though the AMS MIC was within +/- 1 log2 dilution of the reference MIC. The intralaboratory reproducibility ranged from 93.3 to 100% for the four drugs examined. With this challenge group of gram-negative bacilli, the AMS generated aminoglycoside MIC results that were comparable to those obtained by a reference broth microdilution method.     

Quality control limits for the standard anaerobic reference agar dilution susceptibility test procedure of the National Committee for Clinical Laboratory Standards.

Multilaboratory studies were performed to develop MIC quality control limits for the National Committee for Clinical Laboratory Standards reference agar dilution method for anaerobic susceptibility tests. Acceptable MICs were defined as those which include greater than 95% of all 100 MICs generated by the study. Most MIC control limits included either 2- or 3-dilution intervals rather than the more traditional 3-dilution intervals that are described as the mode +/- 1 doubling dilution.     

Evaluation of mupirocin E-test for determination of isolate susceptibility: comparison with standard agar dilution techniques.

Mupirocin E-test strips have been evaluated for their ease of use and accuracy in determining the susceptibilities of 171 strains of Staphylococcus spp., Streptococcus spp., Haemophilus influenzae, and Moraxella catarrhalis. The susceptibility of each strain was determined on two occasions, using parallel E-test and agar dilution methodologies each time. To ensure similar precisions for statistical analyses, E-test MICs were rounded up to a standard twofold agar dilution scale. Clear, elliptical zones were obtained against Staphylococcus spp. M. catarrhalis also gave clear zones, but the scale intercept was often difficult to interpret because of the irregular shape of the inhibition zone. Poor growth sometimes resulted in less-distinct zones of inhibition against Streptococcus spp. and H. influenzae. Excellent correlation was observed between the the E-test and agar dilution against Staphylococcus spp. and H. influenzae, with > 95% of the E-test values falling within one log2 dilution of the corresponding agar MIC. The correlation was lower for Streptococcus spp. and M. catarrhalis, with 86 and 83%, respectively, of E-test results falling within one log2 dilution of the agar MIC. When E-test MICs did not agree exactly with the corresponding agar MIC against Staphylococcus spp. or Streptococcus spp., there was a tendency for the E-test to give a lower MIC. This bias has little effect upon individual MICs in staphylococci or in the generation of susceptibility interpretation errors ( < 1.5% overall), but it could reduce population geometric mean MICs by factors of 0.78 to 0.83.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of Etest for rapid susceptibility testing of Mycobacterium chelonae and M. fortuitum.

Etest is a new concept for MIC determinations for antimicrobial agents that is based on a predefined antibiotic gradient on a plastic strip calibrated with a continuous logarithmic MIC scale covering 15 twofold dilutions. Etest was compared with a reference agar dilution method for susceptibility testing of clinical isolates of Mycobacterium chelonae and M. fortuitum. Results read after 3 days showed good agreement between MICs obtained with Etest and those obtained with the reference method within +/- 2 dilutions for 90% of all test combinations. All but one of the strains were inhibited by low concentrations of ciprofloxacin or amikacin. Susceptibility to clarithromycin, erythromycin, imipenem, rifampin, doxycycline, and fusidic acid was variable, and all strains were resistant to ceftazidime and trimethoprim. The results suggest that Etest is well suited for studies of drug resistance in rapidly growing mycobacteria.