Validation and Reproducibility Assessment of Tigecycline MIC Determinations by Etest

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2007, p. 2474 2479 Vol. 45, No. 8 0095-1137/07/$08.00 0 doi:10.1128/jcm.00089-07 Copyright 2007, American Society for Microbiology. All Rights Reserved. Validation and Reproducibility Assessment of Tigecycline MIC Determinations by Etest Anne Bolmström, 1 Åsa Karlsson, 1 Anette Engelhardt, 1 Phion Ho, 1 Peter J. Petersen, 2 Patricia A. Bradford, 2 and C. Hal Jones 2 * AB BIODISK, Solna, Sweden, 1 and Infectious Diseases Research, Wyeth Research, Pearl River, New York 2 Received 12 January 2007/Returned for modification 22 March 2007/Accepted 11 May 2007 A multicenter study was conducted to validate Etest tigecycline compared to the Clinical Laboratory Standards Institute reference broth microdilution and agar dilution methodologies. A large collection of gram-negative (n 266) and gram-positive (n 162) aerobic bacteria, a collection of anaerobes (n 385), and selected collections of nonpneumococcal (n 369), Streptococcus pneumoniae (n 372), and Haemophilus influenzae (n 372) were tested. Strains with reduced susceptibility to tigecycline were used with all test methods. The Etest showed excellent inter- and intralaboratory reproducibility for all organism groups tested regardless of the test methodology. The essential agreement values with the reference method ( 1 dilution) were >99% for the collection of gram-negative and gram-positive aerobes; >98% for the S. pneumoniae, H. influenzae, and anaerobe collections; and 100% for the group of nonpneumococcal. These results validate the performance accuracy and utility of Etest tigecycline and verify the reproducibility of this convenient predefined gradient methodology for tigecycline susceptibility determination. Sixty years after the development of the first tetracyclines, this class of antibacterial agents is a benchmark in terms of antibacterial spectrum, ease of use, and safety profile (19). Tigecycline, the novel 9-t-butyl substituted minocycline derivative, is the first-in-class glycylcycline. Tigecycline was recently approved worldwide for use in patients with complicated skin and skin structure infections and complicated intra-abdominal infections. The broad spectrum of activity of tigecycline against gram-negative and gram-positive pathogens matched that seen with classical tetracyclines (1, 4, 9, 13). However, in contrast to the classical tetracyclines, tigecycline was not subject to efflux through the tetracycline specific efflux pumps or affected by the ribosomal protection mechanism of tetracycline resistance (11, 14, 18). In addition, tigecycline showed a good profile against important clinical pathogens: methicillin-resistant Staphylococcus aureus, glycopeptide-intermediate resistant S. aureus (and heterogeneous glycopeptide-intermediate resistant S. aureus), vancomycin-resistant enterococci, penicillin-resistant Streptococcus pneumoniae, and resistant gram-negative aerobic bacilli producing extended-spectrum -lactamases (4, 12 14, 18). The development and validation of reliable methods for antimicrobial susceptibility testing (AST) and MIC determinations of tigecycline are critical to clinical practice, as well as for ongoing surveillance programs, for this novel agent. Clinical microbiology laboratories have a number of AST methodologies available for daily clinical work. In order to validate and assess the reproducibility of Etest tigecycline (2, 5, 6), a multicenter study was conducted to compare MIC determinations with this method to the Clinical Laboratory Standards Institute (CLSI) reference broth microdilution * Corresponding author. Mailing address: Wyeth Reseaarch, 401 N. Middletown Rd., Bldg. 200, Rm. 3219, Pearl River, NY 10965. Phone: (845) 602-4612. Fax: (845) 602-5671. E-mail: jonesh3@wyeth.com. Published ahead of print on 23 May 2007. and agar dilution (AD) assays. Test collections of organisms comprising clinical isolates, as well as strains having a wide range of susceptibilities to tetracyclines, tigecycline, and/or resistance to other antibiotics, were used in the comparative studies. (This study was presented in part previously [A. Bolmström, Å. Karlsson, P. Ho, A. Wanger, and R. Howe, 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, abstr. D-1645, 2005].) MATERIALS AND METHODS Bacteria. An identical set of five bias/precision test organism collections (aerobes, anaerobes, nonpneumococcal, S. pneumoniae, and H. influenzae) were tested once by each of three study sites using Etest. The composition of each group of organisms is detailed in Table 1. Each of the study sites also tested five collections of clinical isolates (aerobes, anaerobes, nonpneumococcal, S. pneumoniae, and H. influenzae); details on each of the five organism groups are presented in Table 2. At least 50% of the strains comprising the clinical collection were defined as fresh clinical isolates, having been on an agar plate for less than 7 days and never frozen. The remainder of the strains could be sourced from existing clinical collections. In addition, study site 1 used five AST challenge collections (aerobes, anaerobes, nonpneumococcal, S. pneumoniae, and H. influenzae) comprising strains from various culture collections with a wide range of susceptibilities to tetracyclines and tigecycline and/or resistance to other antibiotics to specifically evaluate whether Etest tigecycline can reliably detect tigecycline-intermediate and -resistant (nonsusceptible) organisms. The make-up of the AST challenge collections is also detailed in Table 2. Quality control (QC) strains recommended by the CLSI for aerobes, anaerobes, nonpneumococcal, S. pneumoniae, and H. influenzae (Table 3) were used throughout the studies and comprised part of the reproducibility collections. Antimicrobial agents. Tigecycline was provided by Wyeth (Pearl River, NY) and used according to guidelines published by CLSI (7, 8). Etest tigecycline gradient strips (0.016 to 256 g/ml) were provided by AB BIODISK (Solna, Sweden) and used according to the product insert and the manufacturer s instructions. Susceptibility testing. (i) Media. Cation-adjusted Mueller-Hinton II broth (MHB; Becton Dickinson, Sparks, MD) was used for broth microdilution with aerobes and Mueller-Hinton agar (Becton Dickinson) was used for Etest and 2474

VOL. 45, 2007 TIGECYCLINE Etest 2475 TABLE 1. Composition of bias/precision bacterial test groups for Etest validation Test group Organism No. of strains tested Aerobes Acinetobacter baumannii 1 Enterobacter agglomerans 1 Enterobacter cloacae 2 Escherichia coli 2 Citrobacter spp. 1 Klebsiella pneumoniae 2 Klebsiella oxytoca 1 Morganella morganii 1 Pseudomonas aeruginosa 1 Serratia spp. 1 Stenotrophomonas maltophilia 1 Enterococcus faecalis 2 Enterococcus faecium 2 Enterococcus gallinarum 1 Staphylococcus aureus 4 a Staphylococcus epidermidis 1 CNS b 2 c Total 26 Nonpneumococcal Streptococcus pyogenes 9 Streptococcus agalactiae 9 Viridans group 8 Total 26 Anaerobes Bacteroides fragilis 9 Bacteroides ovatus 4 Bacteroides thetaiotaomicron 7 Bacteroides vulgatus 6 Total 26 S. pneumoniae 28 H. influenzae 25 Bias/precision collection total a Two methicillin-susceptible S. aureus and two methicillin-resistant S. aureus strains. b CNS, coagulase-negative Staphylococcus spp. c Both isolates were methicillin resistant. AD with aerobes. A total of 5% lysed horse blood was added to MHB for testing S. pneumoniae and Streptococcus spp. with broth microdilution, and 5% laked sheep blood was added to Mueller-Hinton agar for the Etest. Anaerobes were tested with brucella blood agar base (Becton Dickinson) supplemented with 5% laked sheep blood, hemin, and vitamin K (BBA) according to the CLSI guidelines for AD, and the same agar was used for Etest. Haemophilus test medium (prepared in-house or purchased from Remel, Lenexa, KS) was used for both the broth microdilution and the Etest for H. influenzae. (ii) Dilution procedures. Broth microdilution was carried out according to the CLSI guidelines for tigecycline (7). Specifically, in light of the demonstrated oxygen sensitivity of tigecycline (3, 17), MHB used for MIC determinations was prepared fresh ( 12 h old at the time of use). Microdilution plates were prepared on the day of use, and the freshly prepared tigecycline stock solution was serially diluted in fresh MHB to provide a range of 15 twofold doubling dilutions (0.016 to 256 g/ml) to match the Etest concentration gradient range. The MIC was determined as the lowest concentration of tigecycline that inhibited growth as judged by the unaided eye. The AD method for aerobes was performed according to CLSI guidelines (7, 16). (iii) Etest. Etest tigecycline (0.016 to 256 g/ml; AB BIODISK, Solna, Sweden) was used according to the manufacturer s instructions. Briefly, an inoculum suspension with a turbidity equivalent to 0.5 McFarland standard was prepared by suspending well-isolated colonies in 0.9% saline for aerobes and in MHB for nonpneumococcal, S. pneumoniae and H. influenzae. For anaerobes, bacterial suspension in brucella broth with a turbidity equivalent to 1 McFarland was used. A sterile cotton swab dipped into the suspension was used to evenly streak the agar surface and allowed to dry for approximately 15 min. In the case of anaerobes, exposure to ambient air was minimized ( 15 min). The Etest tigecycline gradient strip was applied to the agar surface, and the plate was 131 TABLE 2. Composition of clinical and challenge bacterial test collections for Etest validation Test Group Organism Site (no. of organisms tested) 1 2 3 No. of TGC nonsusceptible organisms tested a Aerobes Acinetobacter spp. 7 7 7 NA b Citrobacter spp. 10 10 8 E. agglomerans 5 5 3 1 E. cloacae 10 10 10 2 E. coli 18 18 18 K. pneumoniae 20 20 20 3 Serratia spp. 7 7 7 7 E. faecalis 7 7 13 E. faecium 10 10 10 S. aureus 17 17 17 0 CNS c 7 7 7 1 Total 118 118 120 14 Nonpneumococcal S. pyogenes 48 40 39 S. agalactiae 14 15 15 Viridans group 37 30 30 2 S. dysgalactiae 0 15 15 Total 99 100 99 2 Anaerobes B. fragilis 46 42 60 13 B. ovatus 17 13 0 4 B. thetaiotaomicron 23 20 28 6 B. vulgatus 14 10 6 1 Bacteroides spp. 0 15 16 Total 100 100 110 24 S. pneumoniae 97 100 100 NA H. influenzae 97 100 100 NA Total clinical collections Challenge groups 511 518 529 40 Aerobes Acinetobacter spp. 4 NA Citrobacter spp. 4 E. agglomerans 2 E. cloacae 5 E. coli 5 Klebsiella spp. 10 M. morganii 1 P. stuartii 1 1 P. aeruginosa 2 NA S. maltophilia 3 NA Serratia spp. 2 E. faecalis 4 E. faecium 3 E. gallinarum 1 S. aureus 14 1 S. epidermidis 1 CNS 10 1 Total 72 3 Nonpneumococcal S. pyogenes 25 S. agalactiae 25 Viridans group 21 1 Total 71 1 Anaerobes B. fragilis 36 2 B. ovatus 12 2 B. thetaiotaomicron 17 2 B. vulgatus 10 1 Total 75 7 S. pneumoniae 75 NA H. influenzae 75 NA Total challenge 368 11 strains Total all strains tested 1,926 51 a Includes tigecycline-intermediate and -resistant organisms. b NA, not applicable since breakpoints have not been defined for this organism. c CNS, coagulase-negative Staphylococcus spp.

2476 BOLMSTROM ET AL. J. CLIN. MICROBIOL. TABLE 3. Bias and precision of Etest versus broth microdilution Test group No. of EAs per site (%) a Site 1 Site 2 Site 3 incubated in ambient air at 35 C for 18 to 20 h for aerobes; in 5% CO 2 for 20 to 24 h for nonpneumococcal, S. pneumoniae, and H. influenzae; and in an anaerobic chamber for 24 to 48 h for anaerobes. The MIC endpoint was read where the growth inhibition ellipse intersected the MIC on the Etest gradient strip. Whenever different growth inhibition patterns and/or growth trailing were seen, the MIC endpoint of the first point of significant inhibition, as judged by the naked eye, was selected using the reading guidelines and illustrations provided by the manufacturer. QC. The following QC strains, as appropriate, were tested in parallel and on all test occasions for both the reference methods and Etest: S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, S. pneumoniae ATCC 49619, H. influenzae ATCC 49247, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eubacterium lentum ATCC 43055. Inoculum density checks in terms of CFU/ml for all test procedures were performed with colony count assays for each QC strain and method and all bias/precision tests and for 10% of all clinical isolates for the different organism groups at all three study centers. RESULTS Interlaboratory reproducibility (%) b Aerobes 100 96 92 100 Nonpneumococcal 100 100 100 100 Anaerobes 100 100 96 100 S. pneumoniae 100 96 100 100 H. influenzae 96 88 88 100 Total 99.2 96.2 95.4 100 a Etest readings 1 log 2 dilution of the reference method mode. b Etest readings from all three tests sites 1 log 2 dilution of the Etest mode. QC strains. QC data (60 results) for each of the ATCC QC reference strains for all three study centers were reported to be within the acceptable QC ranges as specified by the CLSI (8). In addition, there was complete agreement between the reference dilution methods and Etest; i.e., 100% of Etest QC results were within acceptable tolerance limits (essential agreement [EA]) limits ( 1 log 2 dilution) of the respective reference method. Bias and precision testing. The interlaboratory variability of Etest performance was assessed by comparing the Etest results for each organism reported by each study site for the groups of organisms in the 5-bias/precision collections (aerobes, anaerobes, nonpneumococcal, S. pneumoniae, and H. influenzae). The bias/precision collections were blinded to all three investigator sites. Interlaboratory reproducibility ( 1 log 2 dilution) was 100% for all five groups of organisms (Table 3). Etest MIC results from each study site were also compared to the mode of the reference result (n 3) for each group of organisms (EA). For the 393 readings taken, the EA was between 88 and 100% for the three study sites. Most of the discrepant readings were for the H. influenzae collection, and when this collection was excluded the EA was 92 to 100% for the three sites (Table 3). Clinical and challenge strains. Comparison of Etest results to reference methodologies for the clinical collections from all three sites plus the AST challenge collections from site 1 is presented in Table 4. The EA was greater than 98% for all five of the organism groups from the three study sites. Eight minor TABLE 4. Comparison of Etest and reference method for tigecycline susceptibility testing of the clinical and stock test collections including challenge strains Test group No. organisms tested No. of EAs (%) a Interpretive category discrepancies (%) b Minor Major Very major Aerobes 428 425 (99.3) 8 (1.9)* 0 0 Nonpneumococcal 369 369 (100) 0 0 0 Anaerobes 385 378 (98.2) 12 (3.1)* 0 0 S. pneumoniae 372 368 (98.9) 0 0 0 H. influenzae 372 367 (98.7) 0 0 0 Total 1,926 1907 (99) 20 (1.0)* 0 0 a Etest result 1 log 2 dilution of the reference method result. b For organisms for which the breakpoints have been approved by the U.S. Food and Drug Administration: aerobes 315 isolates. The EA for isolates marked with an asterisk was 100%. errors occurred with the gram-negative and gram-positive aerobes. In the case of the gram-negative aerobes, the seven minor errors were caused by two K. pneumoniae and five Serratia spp. for which the Etest MIC results were 1 dilution lower than the broth microdilution for four of the seven tests (Fig. 1). The one minor error that occurred with the gram-positive aerobes was caused by S. aureus Mu50 (ATCC 700699) for which the broth microdilution result was 1 g/ml and the Etest result 0.38 g/ml. Twelve minor errors occurred with the collection of anaerobes studied (Table 4). The minor errors were caused by three B. fragilis, three B. thetaiotaomicron, five B. ovatus, and one B. uniformis strain. Etest MICs were lower than the AD result for 9 of the 12 tests (Fig. 2). In contrast to the data from the bias/precision collection above, when the clinical collection of 372 H. influenzae isolates was tested the EA was found to be 98.7% with no CA errors (Table 4 and Fig. 3). The AST challenge collections, comprising clinical isolates and culture collections that included tigecycline and/or tetracycline-susceptible, -intermediate, and -resistant isolates and strains resistant to other classes of antibiotics, were used to evaluate the ability of Etest to reliably detect tigecycline nonsusceptible isolates. EA between Etest and reference methods for the aerobes in the collection was 99.3% with 1.9% minor errors (Table 4). The aerobes incorrectly categorized by Etest included three Serratia spp. isolates, two of which tested intermediate (MIC 3 g/ml) by Etest and resistant (MIC 8 g/ml) by the reference method, and a third isolate that tested resistant (MIC 8 g/ml) by Etest and intermediate (MIC 4 g/ml) by use of the reference broth. There were also two minor errors with K. pneumoniae isolates; in both cases the isolates tested susceptible (MIC 2 g/ml) by Etest and intermediate (MIC 4 g/ml) by reference method. The final aerobe that was categorized as susceptible by Etest (MIC 0.38 g/ml) and nonsusceptible (MIC 1 g/ml) by broth microdilution was S. aureus Mu50 isolate described above. In the case of the anaerobes in the collection, EA was 98.2%, and there were 3.1% minor errors (Table 4). Five test isolates (two B. thetaiotaomicron, two B. ovatus, and one B. fragilis) resulted in a susceptible interpretation (MIC 4 g/ml) by Etest and

VOL. 45, 2007 TIGECYCLINE Etest 2477 FIG. 1. Error rate-bounded analysis comparing tigecycline reference broth dilution with tigecycline Etest strips against 221 gram-negative isolates. The datum points that fall along the diagonal line of the scattergram represent EA between the Etest result (x axis) and the broth microdilution result (y axis). Dotted horizontal and vertical lines demarcate the susceptible ( 2 g/ml), intermediate (4 g/ml), and resistant ( 8 g/ml) categories along both axes. The datum points falling within the quadrant lines represent minor errors. Note that an Etest result that falls between twofold dilutions must be rounded up to the next upper twofold value before categorization. The gram-negative collection includes: Citrobacter spp., Enterobacter cloacae, E. coli, Klebsiella oxytoca, K. pneumoniae, Morganella morganii, Providencia stuartii, and Serratia spp. intermediate (MIC 8 g/ml) by the reference method AD. In addition, four isolates (three B. ovatus and one B. thetaiotaomicron) tested as intermediate (MIC 8 g/ml) by Etest and resistant (MIC 16 g/ml) by AD, whereas a single B. fragilis isolate tested resistant (MIC 12 g/ml) by Etest and intermediate (MIC 8 g/ml) by AD. The remaining three test groups in the AST challenge collection nonpneumococcal, S. pneumoniae, and H. influenzae had EA values of 100, 98.9, and 98.7%, respectively, and resulted in no minor errors (Table 4). FIG. 2. Error rate-bounded analysis comparing tigecycline reference broth dilution with tigecycline Etest strips against 386 anaerobes. The datum points that fall along the diagonal line of the scattergram represent EA between the Etest result (x axis) and the AD result (y axis). Dotted horizontal and vertical lines demarcate the susceptible ( 4 g/ml), intermediate (8 g/ml), and resistant ( 16 g/ml) categories. The datum points falling within the quadrant lines represent minor errors. Note that an Etest result that falls between twofold dilutions must be rounded up to the next upper twofold value before categorization. The anaerobe collection includes: Bacteroides caccae, B. distasonis, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis, B. ureolyticus, B. vulgatus, and other Bacteroides spp.

2478 BOLMSTROM ET AL. J. CLIN. MICROBIOL. FIG. 3. Error rate-bounded analysis comparing tigecycline reference broth dilution with tigecycline Etest strips against 374 H. influenzae isolates. The datum points that fall along the diagonal line of the scattergram represent the EA between the Etest result (x axis) and the broth microdilution result (y axis). Horizontal and vertical lines demarcate the hypothetical nonsusceptible MIC (MIC 0.5 g/ml) along both axes. Note that an Etest result that falls between twofold dilutions must be rounded up to the next upper twofold value before categorization. DISCUSSION The importance of a robust, reliable, and simple-to-use AST device for the clinical microbiology laboratory cannot be overstated. This is even more important for the testing of isolates from critical infections and high-risk patients and where dosage considerations are important in targeting therapy. The Etest predefined gradient strip can be set up as easily as a Kirby-Bauer disk diffusion test by most clinical laboratories without the need for specialized equipment. As novel antimicrobial agents become available for clinical use, the commercial availability and performance validations of Etest gradient strips for these agents in comparison to reference methods becomes an essential exercise. The data presented in the present study verify that the tigecycline Etest gradient method was as accurate as the reference methods for all five organism groups tested with excellent EA for the majority of the 1,926 strains tested. In addition, the error rates were very low. These outcomes are well within the target accuracy as suggested by the CLSI (15) and used by the U.S. Food and Drug Administration (10) to assess the substantial equivalence of the performance of alternative methods and products in comparison to reference methods. The ability of Etest tigecycline to reliably detect tigecycline susceptible and resistant isolates was evaluated with a collection of 1926 strains resulting in 1.0% minor errors. It should be noted that EA for the nonsusceptible strains was 100% and the categorical agreement errors are a by-product of clustering at the breakpoints and the inherent tolerance ( 1 dilution) of AST methodologies. U.S. Food and Drug Administration-defined breakpoints were applied in these studies (Wyeth Pharmaceuticals, Tygacil package insert [http://www.fda.gov/cder /foi/label/2005/021821lbl.pdf]): Staphylococcus (susceptible only breakpoint, 0.5 g/ml), Streptococcus (not including S. pneumoniae) and Enterococcus (susceptible only breakpoint, 0.25 g/ml), Enterobacteriaceae (susceptible, 2 g/ml; intermediate, 4 g/ml; resistant, 8 g/ml), and anaerobes (susceptible, 4 g/ml; intermediate, 8 g/ml; resistant, 16 g/ ml). Hypothetical breakpoints were applied for S. pneumoniae and H. influenzae (susceptible only breakpoint, 0.25 g/ml). In the present study, testing was carried out independently at three test sites using collections of recently acquired clinical isolates and an AST challenge collection, including a representation of tigecycline-nonsusceptible isolates. However, in order to evaluate inter- and intralaboratory reproducibility, each site tested a common bias/precision collection of organisms that was blinded. Etest tigecycline demonstrated excellent reproducibility (100%) of the MIC results when the same 131 isolates were tested across all study sites. Only in the case of the H. influenzae group did two of the sites show less than optimal correlation (88%) with the broth microdilution reference method. Interestingly, in all cases, Etest MIC results were 1 log 2 dilution higher than the reference broth dilution method. The less-than-optimal reproducibility observed with H. influenzae could be attributed among other factors to variability in the quality of the Haemophilus test medium agar and ambient incubation of capnophilic organisms in broth that may influence AST results. It should be noted that for the much larger collection of H. influenzae (372 clinical isolates), the EA was 98.7%, with EA rates of 95.9, 99, and 100% for the three respective sites (Table 4 and Fig. 3). In conclusion, Etest tigecycline gradient strips proved to be robust and reliable even when tested with large collections of diverse organism groups in this multicenter analysis and should provide accurate and reproducible MIC results when used in daily clinical practice. REFERENCES 1. Babinchak, T., E. J. Ellis-Grosse, N. Dartois, G. M. Rose, and E. Loh. 2005. The efficacy and safety of tigecycline in the treatment of complicated intra-

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