Evaluation of the Sceptor microdilution antibiotic susceptibility testing system: a collaborative investigation.

A multiwell, dried antimicrobial agent susceptibility test system, Sceptor (BBL Microbiology Systems, Cockeysville, Md.), was tested. The system was compared directly with a reference microdilution method by using two collections of stock cultures and 305 fresh clinical isolates. Sceptor was found to be in agreement (+/- log2 dilution) with the reference microdilution method in 96.9 to 98.3% of 9,840 minimal inhibitory concentration determinations performed on stock strains and 95.0% of 7,308 minimal inhibitory concentrations obtained from the clinical isolates. The intralaboratory and interlaboratory reproducibility on stock strains was 97.6 and 97.2%, respectively. The intralaboratory reproducibility for the clinical isolates was 96.9%. Sceptor accurately categorized representative challenge strains of methicillin-resistant staphylococci, beta-lactamase-producing bacteria, organisms producing other antimicrobial agent-inactivating enzymes, and permeability mutants as resistant. Only 0.2% very major errors (false-sensitive minimal inhibitory concentrations by Sceptor) were identified among the clinical isolate test results, the majority being clinically insignificant. The product is accurate and reliable, has a long shelf life, and seems applicable for routine use in clinical laboratories.     

Antimicrobial susceptibility testing of veterinary clinical isolates with the Sceptor System.

The Sceptor System (Becton Dickinson) was compared with an agar dilution method for antimicrobial susceptibility testing of veterinary clinical isolates. The results indicate that the Sceptor System may be used to test gram-positive and fastidious gram-negative bacteria.     

Collaborative evaluation of the micro-media systems anaerobe susceptibility panel: comparisons with reference methods and test reproducibility.

The Micro-Media Systems (MMS) anaerobe susceptibility testing panel results from four laboratories were compared for interlaboratory and intralaboratory variations and for the results with the reference agar dilution and a broth microdilution method. The interlaboratory agreement was 98.0% and intralaboratory agreement was 97.3% (+/- 1 log2 dilution). When interpretive criteria for each antimicrobial agent (susceptible, intermediate, and resistant) were assigned, the MMS anaerobic minimum inhibitory concentration data showed an interpretive accuracy of 91.0 and 95.5% for comparisons to the reference agar dilution and the broth methods, respectively. Most significant interpretive errors were considered minor, and nearly half of all errors involved tetracycline, a drug rarely used for serious anaerobic infections. The MMS anaerobe panels appear to be acceptable for selected use in clinical microbiology laboratories.     

Rapid and overnight microdilution antibiotic susceptibility testing with the Sensititre Breakpoint Autoreader system.

The Sensititre Breakpoint Autoreader system (SBAS) is a broth microdilution method with one to three concentrations of each antibiotic and innovative fluorescence technology to define inhibitory endpoints. We tested 248 gram-negative bacilli and 80 gram-positive cocci using both the rapid (5 h) and overnight (18 h) SBAS procedures. Inhibitory endpoints were also determined by visual inspection of the microdilution trays after 18 h of incubation. SBAS results were compared with those obtained by a standardized disk diffusion (SDD) procedure. Agreement between the rapid SBAS and SDD results for all antibiotic-organism combinations was found in 3,730 of 4,571 (81.6%) tests, with 3.9% very major, 6.5% major, and 7.9% minor discrepancies noted. Data analysis by organism group revealed 86.8, 57.3, 71.4, and 62.3% agreement for members of the family Enterobacteriaceae, Pseudomonas spp., staphylococci, and enterococci, respectively. The results of the overnight SBAS and SDD agreed in 4,154 of 4,654 (89.2%) tests, with 2.3% very major, 1.3% major, and 7.1% minor discrepancies recorded. Concordance was noted in 90.4, 78.1, 90.6, and 83.3% of the comparisons for the members of the Enterobacteriaceae, Pseudomonas spp., staphylococci, and enterococci, respectively. The inhibitory endpoints determined with the Autoreader were as reliable as those determined by visual inspection after 18 h of incubation.     

Comparison of the Sceptor Pseudomonas Plus MIC Panel with agar dilution for susceptibility testing of Pseudomonas aeruginosa.

The antimicrobial susceptibilities of 100 clinical isolates of Pseudomonas aeruginosa to gentamicin, amikacin, tobramycin, ticarcillin, piperacillin, and ceftazidime were determined by using the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.), and the results were compared with those obtained using the National Committee for Clinical Laboratory Standards reference agar dilution method. Excellent correlation was observed for the aminoglycosides, with greater than 95% agreement within 1 doubling dilution of the reference agar dilution MIC, while ticarcillin and piperacillin showed lower percent agreement values of 91 and 88%, respectively.     

Evaluation of accuracy and reproducibility of E test for susceptibility testing of Streptococcus pneumoniae to penicillin, cefotaxime, and ceftriaxone.

We evaluated the reproducibility with which technologists perform and interpret the E test (AB Biodisk, North America, Inc., Piscataway, N.J.) for determining the susceptibility of Streptococcus pneumoniae to penicillin, cefotaxime, and ceftriaxone. Four technologists prepared E test assays to test 124 isolates of S. pneumoniae. Each technologist then interpreted the results of the E test blinded to the interpretation of the other technologists. In addition, E test results were compared with the reference method of broth microdilution. Intraobserver and interobserver agreement were assessed by use of the kappa statistic. Interpretation of the E test and broth microdilution results showed substantial to excellent agreement, with kappa values ranging from 0.878 to 0.987. Compared with broth microdilution, no very major errors and only four major errors were made with the E test. Most minor errors with penicillin and ceftriaxone occurred for isolates with intermediate or high-level resistance, whereas for cefotaxime the minor errors were more evenly distributed between susceptible and intermediate resistance and between intermediate and high-level resistance. These results indicate that there is good agreement between technologists for the interpretation of the E test when testing the susceptibility of S. pneumoniae to penicillin, cefotaxime, and ceftriaxone and that the results of the E test agree with those of broth microdilution.     

Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans.

A study was performed in two laboratories to evaluate the effect of growth medium and test methodology on inter- and intralaboratory variations in the MICs of amphotericin B (AMB), flucytosine (5FC), fluconazole (FLU), itraconazole (ITRA), and the triazole Sch 39304 (SCH) against 14 isolates of Candida albicans. Testing was performed by broth microdilution and semisolid agar dilution with the following media, buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS): buffered yeast nitrogen base (BYNB), Eagle's minimal essential medium (EMEM), RPMI 1640 medium (RPMI), and synthetic amino acid medium for fungi (SAAMF). Inocula were standardized spectrophotometrically, and endpoints were defined by the complete absence of growth for AMB and by no more than 25% of the growth in the drug-free control for all other agents. Comparative analyses of median MICs, as determined by each test method, were made for all drug-medium combinations. Both methods yielded similar (+/- 1 twofold dilution) median MICs for AMB in EMEM and RPMI, 5FC in all media, and FLU in EMEM, RPMI, and SAAMF. In contrast, substantial between-method variations in median MICs were seen for AMB in BYNB and SAAMF, FLU In BYNB, and ITRA and SCH in all media. Interlaboratory concordance of median MICs was good for AMB, 5FC, and FLU but poor for ITRA and SCH in all media. Endpoint determinations were analyzed by use of kappa statistical analyses for evaluating the strength of observer agreement. Moderate to almost perfect interlaboratory agreement occurred with AMB and 5FC in all media and with FLU in EMEM, RPMI, and SAAMF, irrespective of the test method. Slight to almost perfect interlaboratory agreement occurred with ITRA and SCH in EMEM, RPMI, and SAAMF when tested by semisolid agar dilution but not broth microdilution. Kappa values assessing intralaboratory agreement between methods were high for 5FC in all media, for AMB in BYNB, ENEM, and RPMI, and for FLU in EMEM, RPMI, and SAAMF. One laboratory, but not the other, reported substantial to almost perfect agreement between methods for ITRA, and SCH in EMEM, RPMI, and SAAMF. Both laboratories reported poor agreement between methods for the azoles in BYNB. Discrepancies noted in azole-BYNB combinations were largely due to the greater inhibitory effect of these agents in BYNB than in other media. These results indicate that the semisolid agar dilution and broth microdilution methods with EMEM or RPMI yield equivalent and reproducible MICs for AMB, 5FC, and FLU but not ITRA and SCH.     

The accuracy and interobserver reproducibility of endometrial dating

Although controversial, diagnosis of luteal phase defect (LPD) includes the morphological assessment of endometrial development. This study was conducted to determine if refresher training in the histological criteria could improve the accuracy and interobserver reproducibility of endometrial dating. Seventy-eight endometrial biopsies were dated by a reference panel of two pathologists and then reviewed twice by a study panel of four pathologists. In the first review, usual practice was applied. Prior to the second review, they studied a standard document of histological criteria. Samples were dated as proliferative, secretory (post-ovulatory day, POD), menstrual, and undatable. Accuracy levels based on the reference dating and agreement levels using kappa values were calculated per review and compared. The kappa for overall dating was 0.683 in the first review and 0.696 in the second. The respective first and second review kappa values were 0.736 and 0.771 for proliferative, and 0.794 and 0.764 for secretory. Amongst those dated as secretory in the first and second reviews, respectively, 31 and 28% were assigned the same POD by any two panellists, 68 and 63% were dated to within 1 day, and 77 and 71% were dated to within 2 days. Accuracy levels per panellist for overall dating were very high in both reviews but were low for individual PODs. Accuracy and interobserver reproducibility were unaffected by refresher training, suggesting the limits of histological dating have been reached.     

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.     

Collaborative evaluation of the UniScept qualitative antimicrobial susceptibility test.

The UniScept system (Analytab Products, Plainview, N.Y.) is a commercially prepared microdilution antimicrobial susceptibility test for the determination of qualitative susceptibility results for gram-negative and gram-positive bacteria. The system showed excellent correlation with the reference agar diffusion approach for organisms from clinical specimens and with stock and reference cultures. Intra- and interlaboratory reproducibility was high.     

Evaluation of the AutoSCAN-3 and Sceptor systems for Enterobacteriaceae identification.

To evaluate the accuracy and cost effectiveness of the AutoSCAN-3 (Micro-Scan Systems of America, Sacramento, Calif.) and Sceptor (BBL Microbiology Systems, Cockeysville, Md.) systems for identification of members of the Enterobacteriaceae, we performed parallel tests on 678 stock cultures of well-characterized clinical isolates of Enterobacteriaceae. Automated results by AutoSCAN-3 correctly identified 95.1% at the genus level and 94.9% at the species level. However, 15 of 42 Shigella isolates were misidentified as members of other genera. In contrast to the automated results, visual interpretation of panels produced 97.9% agreement at the genus level, missing only three Shigella isolates. Sceptor correctly identified 96.8% at the genus level and 93.4% at the species level. Of 42 Shigella isolates, 3 were missed and were designated as Salmonella spp. Although all Salmonella spp. were correctly identified, six other isolates were misidentified as Salmonella spp. Test costs were found to be comparable for each system, with the cost per test increasing markedly with fewer than 10 to 15 tests performed per day.     

Collaborative investigation of the AutoMicrobic System Enterobacteriaceae biochemical card.

The Enterobacteriaceae biochemical card used in six separate laboratories to identify 170 representatives of Enterobacteriaceae. The AutoMicrobic System (Vitek Systems, Inc.) performed with an accuracy of 97.8% as compared with 98.1% by the standard method selected and 97.6% by a commerically prepared manual system approach. During this time, 5,450 clinical isolates belonging to Enterobacteriaceae were analyzed. Compared with the routine methods used in the various laboratories, the AutoMicrobic System identified 96.4% correctly     

Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests.

A collaborative comparison of macro- and microdilution antifungal susceptibility tests was performed in five laboratories. MICs of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined in all five centers against 95 coded isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. A standard protocol with the following National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Testing recommendations was used: an inoculum standardized by spectrophotometer, buffered (RPMI 1640) medium (pH 7.0), incubation at 35 degrees C, and an additive drug dilution procedure. Two inoculum sizes were tested (1 x 10(4) to 5 x 10(3) to 2.5 x 10(3) CFU/ml) and three scoring criteria were evaluated for MIC endpoint determinations, which were scored as 0 (optically clear), < or = 1 (slightly hazy turbidity), and < or = 2 (prominent decrease in turbidity compared with that of the growth control). Overall intra- and interlaboratory reproducibility was optimal with the low-density inoculum, the second-day readings, and MICs scored as either 1 or 2. The microdilution MICs demonstrated interlaboratory agreement with most of the four drugs higher than or similar to that of the macrodilution MICs. In general, there was good interlaboratory agreement with amphotericin B, fluconazole, and flucytosine; ketoconazole gave more variable results.     

Susceptibility testing of multidrug-resistant Staphylococcus aureus with the Sceptor microdilution system.

The antimicrobial susceptibilities of Staphylococcus aureus isolates were concurrently determined by the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.) and by the standard disk diffusion method. For the methicillin-resistant isolates, there was greater than 98% agreement between the two test results with penicillin G, erythromycin, clindamycin, tetracycline, gentamicin, and tobramycin. Major disagreements (susceptible by one method and resistant by the other) were 7% for methicillin, 13.5% for cephalothin, 3.5% for cefamandole, and 27% for amikacin. The major discrepancies for methicillin were eliminated by supplementing the inoculum broth with salt. For methicillin-susceptible isolates, agreement between the two methods was 96 to 100% for all antibiotics except amikacin.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Modification of the Sceptor system for rapid detection of methicillin-resistant staphylococci.

The 24-h Sceptor MIC system (Johnston Laboratories, Inc., Towson, Md.) was modified to allow rapid (6 h) detection of methicillin-resistant staphylococci. For 105 methicillin-resistant staphylococci tested, 90% of the results obtained by the 6-h method agreed with those obtained by disk agar diffusion. In comparison, 88 and 93% of the results obtained by the AutoMicrobic system (Vitek Systems, Inc., Hazelwood, Mo.) and the 24-h conventional Sceptor system, respectively, agreed with disk agar diffusion results. No false-resistant results were observed with 52 methicillin-susceptible staphylococci tested by any of the three methods.     

Collaborative evaluation of the microbial profile system for quantitative antimicrobial susceptibility testing.

This three-center collaborative study was conducted to evaluate samples of the Microbial Profile System (MPS) antimicrobial microdilution panels [previously produced by Minnesota Mining & Manufacturing Co., (3M Co.), St. Paul, Minn. and currently produced by Flow Laboratories, Inc., Rockville, Md.). This was a three-phase study. In phase I, the inter- and intralaboratory agreement was determined by using strains with selected ranges of susceptibility. The MPS and reference microdilution minimum inhibitory concentrations were within acceptable variation, +/- 1 dilution for 97.7% for the MPS and 98.8% for the reference microdilution panels for the intralaboratory comparisons. The percentage of strains with minimum inhibitory concentrations in the acceptable range for the interlaboratory variation was 96.2% for the MPS and 96.0% for the reference microdilution panels. The phase II studies used strains with known resistance mechanisms. The percent agreement with these strains was: Enterobacteriaceae, 94.5%; nonenteric gram-negative rods, 95.4%; staphylococci, 92.3%; and streptococci, 96.6%. The overall agreement within acceptable limits was 94.7% with these strains. When testing 359 clinical isolates, the frequency of strains within the acceptable range of agreement between the two methods was 97.3%. The MPS panels gave results in each of the three study phases equivalent to those obtained with the reference microdilution panels.     

European Gene Mapping Project (EUROGEM): Breakpoint panels for human chromosomes based on the CEPH reference families

Meiotic breakpoint panels for human chromosomes 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 18, 20 and X were constructed from genotypes from the CEPH reference families. Each recombinant chromosome included has a breakpoint well-supported with reference to defined quantitative criteria. The panels were constructed at both a low- resolution, useful for a first-pass localization, and high-resolution, for a more precise placement. The availability of such panels will reduce the number of genotyping experiments necessary to order new polymorphisms with respect to existing genetic markers. This paper shows only a representative sample of the breakpoints detected. The complete data are available on the World Wide Web URL ( http://www.icnet.uk/axp/hgr/eurogem/HTML/data.html ) or by anonymous ftp ( ftp.gene.ucl.ac.uk in/pub/eurogem/maps/breakpoints ).     

Collaborative evaluation of the UniScept quantitative antimicrobial susceptibility test

UniScept (Analytab Products, Plainview, N.Y.) is a commercially prepared microdilution antimicrobial susceptibility system for the determination, in a single tray, of antimicrobial MICs for gram-negative and gram-positive bacteria and those isolated from urinary tract infections. The system showed excellent correlation with the reference microdilution approach for organisms from clinical specimens and with stock and reference cultures. Intra- and interlaboratory reproducibility was high.