1 Department of Microbiology, University Hospital, Birmingham; 2 Department of Microbiology, Ninewells Hospital, Dundee; 3 Department of Infection, Guys & St Thomas Hospital, London; 4 Department of Microbiology, Basildon Hospital, Basildon, Essex; 5 Department of Microbiology, Royal Hallamshire Hospital, Glossop Road, Sheffield, UK
Received 3 January 2003; returned 18 February 2003; revised 18 February 2003; accepted 26 February 2003
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Abstract |
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Methods: Laboratories tested a collection of 82 strains selected on the basis of their challenging and characterized resistance mechanisms.
Results: In comparison with the reference MIC method, VITEK 2 gave an essential agreement of 304/315 (enterococci), 1619/1674 (staphylococci) and 2937/3074 (Gram-negative bacilli): overall 96.0% agreement. Corresponding category (SIR) agreements with VITEK 2 were 247/252, 1496/1561 and 2478/2626 (overall 95.1%). Using five routine methodologies, category agreements ranged from 58/63 to 45/45; 222/232 to 174/174, and 333/372 to 250/259 for the three organism groups with an overall agreement of 95.0%. In contrast to VITEK 2 Advanced Expert System (AES), routine microbiology laboratories did not attempt to detect resistance mechanisms for every antibiotic studied. VITEK 2 AES detected all 19 resistance mechanisms in enterococci: where applicable, routine methods detected 14, 10 and 10. Of 30 resistance mechanisms in staphylococci, VITEK 2 AES detected 25 compared with 23, 20, 17 and 18 detected by routine methods. Finally, of 44 resistance mechanisms in Gram-negative bacilli, VITEK 2 AES detected 30 compared with 30, 23, 15 and 10 detected by routine methods.
Conclusions: VITEK 2 performed susceptibility tests accurately and the AES detected and interpreted resistance mechanisms appropriately. Heavy inocula in a liquid medium possibly favour better expression of certain resistance determinants. Although certain routine microbiology methods performed adequately, VITEK 2 AES offers a rapid, standardized method suited to laboratories lacking experience of resistance mechanisms and/or those not testing an appropriate number, or range, of antibiotics to detect resistance phenotypes.
Keywords: VITEK 2; Expert; automated AST
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Introduction |
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Materials and methods |
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VITEK 2 systems were installed and verified in five UK laboratories by bioMérieux Application Specialists. Each laboratory was asked to test the same set of 82 strains (Enterobacteriaceae 36; pseudomonads 8; Staphylococcus aureus 22; coagulase-negative staphylococci 7; enterococci 9) obtained from the Institut Pasteur collection and distributed by bioMérieux; none of these strains had been used for the initial development of VITEK 2 AES. Identification of antibiotic resistance mechanisms was achieved using appropriate biochemical and/or molecular procedures, including determination of the isoelectric focusing substrate profile, determination of the inhibitor profile, recombinant DNA techniques, DNA amplification and sequencing.9 The evaluators were informed of the species, but not of the genotypes or anticipated resistance phenotypes. In total, the 82 strains displayed 95 phenotypes (Table ). MICs were determined, by Professor Courcol (University Hospital of Lille, France), on nine separate occasions using an agar dilution method according to NCCLS guidelines12 and these data were used as gold standard reference values for data comparison.
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Strains were sub-cultured twice, then grown for 1824 h at 35°C on Columbia agar containing 5% horse blood (bioMérieux) in air. Suspensions of these cultures were made in 0.45% saline, adjusted to the turbidity of a 0.6 McFarland Standard, and used to load the test cards for VITEK 2, which was used in accordance with the manufacturers directions. The following test cards were used: Enterobacteriaceae (AST-N020); pseudomonads (AST-N017); staphylococci (AST-P523), and enterococci (AST-P524). Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC 29213 and ATCC 25923 and Enterococcus faecalis ATCC 29212 were used as control strains. Antibiotic resistance phenotypes were determined using VITEK 2 AES.
Susceptibility tests with local methodology
Centres 1 and 2 both used an agar-incorporation, break-point method based on that described by Faiers et al.13 Both centres used Iso-sensitest agar (Oxoid, Basingstoke, UK) with an inoculum of 104 cfu/spot and both read plates using a Mastascan Elite (Mast Group Ltd, Bootle, UK). Centre 1 prepared antibiotic plates using antibiotic powders, of known potency, obtained from the relevant manufacturers and used BSAC interpretive criteria14,15 throughout. Resistance phenotypes were determined using an Expert system running on Microsoft Access [T. Winstanley & K. Humphries (Mast Group Ltd, Bootle, UK), unpublished data] largely popu- lated with published rules.46 Centre 2 prepared plates using Adatabs (Mast Group Ltd) and also used BSAC interpretive criteria14,15 with the exception of ampicillin for enterococci (1 mg/L) and ofloxacin for Enterobacteriaceae (1 mg/L). Lysed horse blood (5%) was added to the medium for testing Gram-positive cocci and the production of ß-lactamase by staphylococci was detected using ß-lactamase test papers (Intralactam; Mast Group Ltd) after induction on methicillin plates (0.5 mg/L). Centre 2 determined antibiotic resistance phenotypes with VITEK 2 only. Centres 3 and 4 followed the BSAC standardized disc diffusion susceptibility method.14,15 Centre 3 read plates manually but used rule-based computerized checking as well as manual interpretation of phenotypic resistances. Centre 4 used an Aura image automated zone reader (Oxoid, Basingstoke, UK)16 to read inhibition zones; disc interaction tests to determine the presence of ß-lactamases17,18 and Etests (Cambridge Diagnostic Services, Cambridge, UK) to confirm glycopeptide resistance in enterococci; resistance phenotypes were interpreted manually. Centre 5 used VITEK (bioMérieux; version VTK-R07.01) according to the manufacturers instructions. GNS-532 test cards were used for Enterobacteriaceae and pseudomonads; GPS-519 cards were used for staphylococci and enterococci; NCCLS criteria were used.12 All Expert rules (XPT-ON) that would routinely be used were enabled. Appropriate control organisms were examined by every method. Retention of key resistances was not confirmed at the time of the study. However, all organisms giving aberrant results in terms of MIC or category of susceptibility, together with a representative sample of those giving concordant results, were re-examined using Etests (Cambridge Diagnostic Services, Cambridge, UK).
Data analysis
The percentage of essential agreement between the test MIC and the MIC determined by the reference agar-dilution method was defined as the percentage of results within ±1 log2 dilution MIC. MIC results were categorized (susceptible, intermediate, resistant) using NCCLS12 or BSAC14,15 interpretive criteria depending on the methodology of the comparator. All test results were analysed before antibiograms being edited on the basis of inferred resistance mechanisms. The relevant susceptibility breakpoints were used to calculate very major, major and minor errors between the agar dilution MIC and the comparator. Very major errors corresponded to organisms for which the MIC indicated resistance and the test method indicated susceptibility (i.e. total number of resistant strains was used as the denominator). Major errors corresponded to organisms for which the MIC indicated susceptibility and the test method indicated resistance; minor errors corresponded to organisms for which the MIC indicated intermediate resistance and the test method indicated susceptibility or resistance and vice versa. Resistance mechanisms inferred by VITEK 2 AES or the individual centres were compared with previous conclusions based on biochemical or genetic analysis (Table ). Results were graded as Agreement when the same mechanism was inferred, as Partial agreement when the correct mechanism amongst others was inferred and as Disagreement when a different mechanism was inferred.
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Results |
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In comparison with the agar-dilution reference method, VITEK 2 gave an overall essential agreement of 96.5%, 96.7% and 95.5% for enterococci, staphylococci and Gram-negative bacilli, respectively (Table ). Inter-laboratory variations were minimal. Results showed that 3.5% of MICs determined for Gram-negative bacilli with VITEK 2 were greater (>1 dilution) but that only 1.0% were lower (<1 dilution) than those obtained with the reference method. This skew in distribution occurred with ofloxacin, gentamicin, tobramycin, amikacin, piperacillin, cefalothin and ceftazidime but was most noticeable with cefotaxime and with resistant rather than with susceptible organisms. Results for these antibiotics accounted for the lowest degrees of agreement. Oxacillin MIC results with staphylococci tended to be one dilution lower than those obtained with the reference method but, overall, the distribution of MICs with staphylococci was towards higher MICs than with the reference method (2.3% versus 0.9%) and essential agreement with individual antibiotics was good. MICs with enterococci were evenly distributed although essential agreement with nitrofurantoin was only 88.9%: five strains (three of these the same strain tested on different occasions) gave MICs higher than the reference MIC.
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The degree of agreement between resistance mechanisms inferred using routine methodology and reference results was 73.7%, 52.6% and 52.6% for enterococci (centres 1, 3 and 4, respectively) compared to 100% with VITEK 2. The degree of agreement for staphylococci was 76.7%, 56.7%, 66.7% and 60.0% (centres 1, 3, 4 and 5, respectively) compared with 83.3% with VITEK 2. In contrast to VITEK 2 AES, all routine methods detected methicillin resistance in all S. aureus strains and in the single strain of S. warneri and centre 4 detected lincosamide nucleotidylation in the strain of S. haemolyticus. No routine method detected teicoplanin resistance in S. aureus. Agreement for Gram-negative bacilli was 68.2%, 34.1%, 52.3% and 22.7% (centres 1, 3, 4 and 5, respectively) with centre 1 performing as well as VITEK 2. Centres 1 and 4 detected one strain with high-level cephalosporinase; centre 1 further detected the E. coli impermeability mutant and carbapenemase production in Enterobacteriaceae resistance mechanisms that were not detected by VITEK 2 AES.
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Discussion |
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A total of 5063 MICs were determined with VITEK 2 in the five test centres. In comparison with the agar-dilution reference method, VITEK 2 gave an overall essential MIC agreement of 96.5%, 96.7% and 95.5% for enterococci, staphylococci and Gram-negative bacilli, respectively. These data conform with those of other published studies3,9 and meet FDA criteria for any new MIC test method.19 Inter-laboratory variations were minimal. In general, for Enterobacteriaceae, MICs determined by VITEK 2 were higher than those determined using an agar-dilution method. Cantón et al.8 reported that VITEK 2 read lower than the reference method (a micro-dilution method) but Leclercq et al.9 also found VITEK 2 to read higher than an agar-dilution method. Several ß-lactam agents (including piperacillin, cefalothin, ceftazidime and cefotaxime) are inoculum dependent in that the heavier the inoculum used, the higher the determined MIC becomes.20 Thus, the use of a heavy inoculum in a broth test should better favour expression of resistance in comparison with an agar-dilution method and will account for relatively lower essential agreement values with these antibiotics in comparison with imipenem, for example. Leclercq et al.9 noticed discrepancies with piperacillin, cefotaxime and ceftazidime with ESBL-producing strains and with cefalothin with penicillinase-producing strains and these corresponded to inoculum effects. For enterococci, the lowest degree of agreement was with nitrofurantoin and this was also found by Leclercq et al.9
Data were analysed by comparison with gold standard agar-dilution MIC data that had been categorized according to the relevant interpretive standards. Results for individual organismantibiotic combinations ranged from 88.9% (gentamicin versus Gram-negative bacilli) to 100%. With VITEK 2, the overall agreements for organism groups were 98.0%, 95.8% and 94.4% for enterococci, staphylococci and Gram-negative bacilli, respectively. The overall mean was 95.1%. Corresponding agreements for organism groups examined by routine methods were 92.1100% (mean 95.3%), 95.7100% (mean 97.1%) and 89.596.5% (mean 93.5%) with an overall mean of 95.0%. With routine methods, there was also a wider range of agreements for individual organismantibiotic combinations (66.7100%) reflecting localized problems. For example, centre 3 had problems with gentamicin and staphylococci subsequently found to be due to a batch of defective antibiotic discs; centre 4 had problems with enterococci relating to vancomycin, and centre 1 had problems with Enterobacteriaceae and co-amoxiclav MICs falling close to the breakpoint. Centre 5 also had problems with enterococci and vancomycin, and with Gram-negative bacilli and both cefotaxime and tobramycin: minor errors were generated as the VITEK uses an intermediate category. Centre 4 used an Oxoid Aura image and this zone reader can interpret zones within 1 mm of the breakpoint as borderline susceptible or borderline resistantresults interpreted using these data may thus improve the performance of the method.
Some laboratories do not attempt to determine resistance mechanisms routinely from raw antibiotic susceptibility data. Other laboratories fail to detect mechanisms, often because insufficient or inappropriate antibiotics are tested as part of the standard antibiotic panel and this is reflected in the data generated during this study. Overall agreement of phenotypic interpretation with reference genotypic data for enterococci, staphylococci and Gram-negative bacilli using VITEK 2 was 100%, 83.3% and 68.2%, respectively. Corresponding values using routine methodologies were 52.673.7%, 56.776.7% and 22.768.2%. Essential agreement between categories of susceptibility determined by VITEK 2 and the routine methods was based on raw unexpertized data. However, most Expert systems are capable of editing antibiograms based on inferred mechanisms and, furthermore, are capable of artificial learning. The strains used in this study are not representative of strains encountered, as a matter of course, in routine microbiology laboratories as they were selected on the basis of their challenging resistance mechanisms. Nonetheless, VITEK 2 performed susceptibility tests accurately and the AES detected and interpreted resistance mechanisms appropriately.
In this study, routine microbiology methods were shown to perform susceptibility tests with comparable accuracy to VITEK 2. Further, a conventional computerized expert system5 or a knowledgeable operator, whilst not completely as effective as the AES, also carried out interpretative reading adequately. Routine methods are also cost-effective, flexible and, for the most part, standardized. However, VITEK 2 offers a standardized method ideally suited to laboratories lacking familiarity with, increasingly, myriad resistance mechanisms4,7 and/or those not testing an appropriate number, or range, of antibiotics to detect resistant phenotypes using interpretative reading.6 The main benefit of the adoption of VITEK 2 is likely to be the provision of rapid results that could bear both clinical and financial rewards.21
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Acknowledgements |
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Footnotes |
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References |
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