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Journal of Clinical Microbiology, August 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 Infectious Diseases Research, Wyeth Research, Pearl River, New York²

Received 12 January 2007/ Returned for modification 22 March 2007/ Accepted 11 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 streptococci (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 streptococci. 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.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 ß-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 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].)

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Bacteria. An identical set of five bias/precision test organism collections (aerobes, anaerobes, nonpneumococcal streptococci, 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 streptococci, 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 streptococci, 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 streptococci, S. pneumoniae, and H. influenzae (Table 3) were used throughout the studies and comprised part of the reproducibility collections.

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TABLE 1. Composition of bias/precision bacterial test groups for Etest validation

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TABLE 2. Composition of clinical and challenge bacterial test collections for Etest validation

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TABLE 3. Bias and precision of Etest versus broth microdilution

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 µg/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 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 µg/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 µg/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 streptococci, 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 incubated in ambient air at 35°C for 18 to 20 h for aerobes; in 5% CO₂ for 20 to 24 h for nonpneumococcal streptococci, 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.

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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 log₂ 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 streptococci, S. pneumoniae, and H. influenzae). The bias/precision collections were blinded to all three investigator sites. Interlaboratory reproducibility (±1 log₂ 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 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 µg/ml and the Etest result 0.38 µg/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).

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TABLE 4. Comparison of Etest and reference method for tigecycline susceptibility testing of the clinical and stock test collections including challenge strains

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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 µg/ml), intermediate (4 µg/ml), and resistant (8 µg/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.

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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 µg/ml), intermediate (8 µg/ml), and resistant (16 µg/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.

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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 µg/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.

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 µg/ml) by Etest and resistant (MIC = 8 µg/ml) by the reference method, and a third isolate that tested resistant (MIC = 8 µg/ml) by Etest and intermediate (MIC = 4 µg/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 µg/ml) by Etest and intermediate (MIC = 4 µg/ml) by reference method. The final aerobe that was categorized as susceptible by Etest (MIC = 0.38 µg/ml) and nonsusceptible (MIC = 1 µg/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 µg/ml) by Etest and intermediate (MIC = 8 µg/ml) by the reference method AD. In addition, four isolates (three B. ovatus and one B. thetaiotaomicron) tested as intermediate (MIC = 8 µg/ml) by Etest and resistant (MIC = 16 µg/ml) by AD, whereas a single B. fragilis isolate tested resistant (MIC = 12 µg/ml) by Etest and intermediate (MIC = 8 µg/ml) by AD. The remaining three test groups in the AST challenge collection—nonpneumococcal streptococci, S. pneumoniae, and H. influenzae—had EA values of 100, 98.9, and 98.7%, respectively, and resulted in no minor errors (Table 4).

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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 µg/ml), Streptococcus (not including S. pneumoniae) and Enterococcus (susceptible only breakpoint, 0.25 µg/ml), Enterobacteriaceae (susceptible, 2 µg/ml; intermediate, 4 µg/ml; resistant, 8 µg/ml), and anaerobes (susceptible, 4 µg/ml; intermediate, 8 µg/ml; resistant, 16 µg/ml). Hypothetical breakpoints were applied for S. pneumoniae and H. influenzae (susceptible only breakpoint, 0.25 µg/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 log₂ 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.

 
* 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{at}wyeth.com

Published ahead of print on 23 May 2007.

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Journal of Clinical Microbiology, August 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.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bolmström, A. Articles by Jones, C. H. Search for Related Content PubMed PubMed Citation Articles by Bolmström, A. Articles by Jones, C. H.