a Laboratory Specialists, Inc., 1651A Crossings Parkway, Westlake, OH 44145, USA; b St Thomas' Hospital, London SE1 7EH, UK; c SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426, USA
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Abstract |
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Introduction |
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The optimal way of monitoring resistance trends would be to combine data from as many studies as possible. However, it is difficult to compare susceptibility results because of variation in testing methods and antimicrobial concentrations tested. In addition, some studies lack proper quality control and patient demographic information. Finally, standardized reporting is required for the successful combination and analysis of data.
If standardized testing methods were used for surveillance studies, one of the major variables would be controlled. The combination of European data alone presents its own challenges, as multiple methods are used throughout Europe. For example, the British Society for Antimicrobial Chemotherapy (BSAC) method is used in the UK,3 the Deutsches Institut für Normung (DIN) method in Germany,4,5 the Société Française de Microbiologie (SFM) method in France6 and the Swedish Reference Group for Antibiotics (SRGA) method in Sweden (http:/www.ltkronoberg.se/ext/raf).
One primary objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) is to formulate guidelines for a standardized reference susceptibility method among the European Union participants. Most studies in the USA use the standardized methods of the NCCLS. As a first step in harmonizing efforts, a EUCAST representative is an adviser to the NCCLS Subcommittee on Antimicrobial Susceptibility Testing and has requested NCCLS representation at EUCAST meetings.
Similarities and differences between the methods must first be established to determine a baseline for standardization. One of the major variables between methods is the medium used. This study was undertaken as an initial evaluation to determine how Iso-Sensitest broth (ISB), which is recommended by several European National Committees, compares with cation-adjusted Mueller Hinton broth (CAMHB) defined in the NCCLS method. To control all other variables, testing was performed by the NCCLS method with CAMHB and by the NCCLS method with ISB instead of CAMHB.
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Materials and methods |
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One hundred and twenty-four CDC reference strains with established MICs, comprising 20 Pseudomonas aeruginosa, 20 Enterococcus spp., 19 Staphyloccus aureus, 15 coagulase-negative Staphyloccus spp. and 50 Enterobacteriaceae (20 Escherichia coli, eight Klebsiella spp., eight Enterobacter spp., seven Serratia spp. and seven Proteus spp.) were tested. Ten strains from the CDC challenge set were chosen for reproducibility testing.
Testing sites
The two testing laboratories were St Thomas' Hospital in London, UK (laboratory 1) and Laboratory Specialists, Inc., Westlake, OH, USA (laboratory 2).
Susceptibility test methods and materials
The organisms and antimicrobial agents used in this study have been previously reported as having been most affected by variations in the medium.719 MIC custom dehydrated microtitre panels (Dade, Sacramento, CA, USA) containing the antimicrobial agents (amoxycillin clavulanic acid, ampicillin, ciprofloxacin, erythromycin, gentamicin, imipenem, levofloxacin, oxacillin, gemifloxacin, trimethoprimsulphamethoxazole, tetracycline and vancomycin) were used. Microdilution MICs were tested according to current NCCLS guidelines,20 with the only variation being the prepared media that were used for plate rehydration and inoculation: in each tube, 25 mL of CAMHB (PML, Tualatin, OR, USA) or ISB (PML) was used. Each strain was tested with two batches of CAMHB and two batches of ISB by both laboratories. All panels were rehydrated and inoculated with the MicroScan Renok (Dade) using disposable inoculators and incubated under ambient conditions at 35°C for 1624 h. Enterococcus spp. and Staphylococcus spp. were incubated for 24 h. The lowest concentration of antimicrobial agent at which organisms showed no growth was read as the MIC. The 10 reproducibility strains were tested in triplicate on three occasions at laboratory 2 and in triplicate on one occasion at laboratory 1 using the same batch of CAMHB and ISB at both laboratories. Quality control organisms were tested for each microdilution run using both CAMHB and ISB for five quality control organisms: S. aureus ATCC 29213, E. faecalis ATCC 29212, E. coli ATCC 25922 and ATCC 35218 and P. aeruginosa ATCC 27853.
Data collection and analysis
The data for the 124 CDC reference strains were analysed by four different methods. The cumulative MICs for each broth for each antimicrobial agent were plotted against the range of MIC dilutions tested. For statistical evaluation of any differences between broths, a 24 fully specified factorial analysis was performed for each antimicrobial agent.21 For those antimicrobial agents that differed, the MICs from laboratory 1 and batch 1 were further analysed according to organism by determining the log2 difference (doubling dilution difference) between the ISB MIC and the CAMHB MIC. Essential and category agreement rates and number of errors were calculated for each antimicrobial agent.
Because of off-scale trimethoprimsulphamethoxazole results (;0.5 mg/L) with the Gram-positive organisms and the majority of the Gram-negative organisms, only 26 isolates (six Serratia marcescens and 20 P. aeruginosa) were included in the data analysis. Only Gram-positive isolates (a total of 54) were included in the analysis of erythromycin, oxacillin and vancomycin. For all other drugs, all isolates were analysed.
The independent factors included in the statistical analysis were Gram's stain (positive or negative), laboratory (1 or 2), broth (CAMHB or ISB) and batch of medium (1 or 2). The dependent variable was MIC, transformed to a log2 scale.22
The percentage essential agreement is the percentage of results that were within one doubling dilution of each other. As there were no significant differences between laboratories and batches of media, essential agreement rates, category agreement rates and error analyses were performed on CAMHB batch 1 and ISB batch 1 from laboratory 1. Essential agreement rates were determined from on-scale MICs only. Category agreement rates were based on those results that could be interpreted according to current NCCLS breakpoints.23 An error was considered minor when the results obtained with CAMHB and ISB differed by one category (e.g. susceptible versus intermediate). An error was considered major when the result obtained with CAMHB was susceptible and that with ISB was resistant. An error was considered very major when the result with CAMHB was resistant and that with ISB was susceptible. Agreement and error calculations were based on Staphylococcus spp. only for oxacillin and erythromycin, and Gram-positive organisms only for vancomycin. For all other antimicrobial agents, both Gram-negative and Gram-positive results were included.
The reproducibility results, from 10 of the CDC reference isolates, were analysed by combining both CAMHB and ISB results and determining the percentage of results within three doubling dilutions. Quality control results were analysed according to NCCLS expected ranges.23
A qualitative determination of growth was also evaluated by both laboratories. The growth on each batch was compared with that in CAMHB batch 1 and recorded as similar, better or less.
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Results |
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For all on-scale results, all of the results for all antimicrobial agents were within a three-dilution range.
Media comparison studies
For all three quinolones (ciprofloxacin, levofloxacin and gemifloxacin), gentamicin and tetracycline, slightly higher results were obtained with ISB than with CAMHB, according to both statistical and cumulative MIC methods of analysis (Table I). There were no significant differences with amoxycillinclavulanic acid, ampicillin, imipenem, oxacillin, trimethoprimsulphamethoxazole and vancomycin. There were no significant differences between the laboratories and batches of broth.
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Essential agreement for all antimicrobial agents was >90%, with the exception of ampicillin (88.4%). Category agreement for all antimicrobial agents was >93%, with the exception of erythromycin (88.2%). There were very few errors, the majority of which were within one dilution (Table III).
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Quality control
At least one out-of-range quality control result was obtained with all of the antimicrobial agents, except for ampicillin and oxacillin. With the exception of P. aeruginosa tested with tetracycline and S. aureus tested with erythromycin, >95% of CAMHB quality control results were within the expected ranges. The majority of the out-of-range results were obtained with ISB; <95% of ISB quality control results were within the expected ranges for at least one of the quality control organisms for amoxycillinclavulanic acid, ciprofloxacin, gentamicin, imipenem, levofloxacin, gemifloxacin, trimethoprimsulphamethoxazole and tetracycline. All outlying ISB results were higher than the expected range. Quality control results are summarized in Tables IV and V.
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Discussion |
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The antimicrobial agents that differed were those most affected by varying concentrations of cations in the medium, namely quinolones, aminoglycosides and tetracycline.711 The calcium and magnesium ion levels in the CAMHB were equivalent to those recommended in NCCLS guidelines (2025 and 1012.5 mg/L, respectively). The concentrations of calcium and magnesium ions in the ISB were 3.3 and 53.0 mg/L, respectively. In a previous study, addition of 100 mg/L of magnesium and calcium ions to commercial media deficient in these cations increased ciprofloxacin MICs four-fold.24 The concentration of magnesium ions in this study was approximately half that used in the study by Blaser & Luthy,24 therefore the half dilution difference is consistent with earlier observations and is presumably a result of increased concentrations of magnesium ions.
This study indicates that ISB is an acceptable alternative to CAMHB when using the NCCLS MIC method. Although technologists at both laboratories noted better growth with ISB than with CAMHB, the technologists at laboratory 2 perceived ISB to be better more often. This is an important observation as laboratory 2 is more accustomed to using CAMHB than laboratory 1.
Modification of the Mg2+ concentration in the ISB to be consistent with the 10.012.5 mg/L in CAMHB seems appropriate. A further study would be needed to confirm that this cation adjustment would correct the slight shifts in MICs that were observed and bring quality control results within acceptable ranges. In addition, a greater number of on-scale results, together with the testing of additional antimicrobial agents, would be required for complete validation.
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Acknowledgments |
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Notes |
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References |
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Received 17 November 1999; returned 25 January 2000; revised 9 March 2000; accepted 7 April 2000