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CONCISE COMMUNICATION Validation of commercial dry-form broth microdilution panels for susceptibility testing of AZD2563, a new long-acting oxazolidinone R. N. Jones,1,2, T. R. Anderegg1 and D. J. Biedenbach1 1 2

JMI Laboratories/The Jones Group, 345 Beaver Kreek Center, Suite A, North Liberty, Iowa 52317 and Tufts University School of Medicine, Boston, Massachusetts, USA Tel: 1 319 665 3370

Fax: 319 665 3371

E-mail: [email protected]

The MIC results using a dry-form broth microdilution panel (TREK Diagnostic/Sensititre, Westlake, OH, USA) were validated for AZD2563, a novel oxazolidinone compound. In comparision studies against reference frozen-form panels, the commercial MIC results were the same as the reference calue for 82.7% of organisms and all results were within one log2 dilution. Using 462 organisms, most from three genus groups (enterococci, staphylococci, streptococci), test results indicate that Sensititre MIC values were comparable to the reference test and can be utilized in clinical trials of for routine laboratory use when testing AZD2563 and linezolid, the drug class comparator. Keywords AZD2563, oxazolidinone, validation Accepted 12 June 2002

Clin Microbiol Infect 2003; 9: 543±546

AZD2563 is a potent oxazolidinone described as having activity against all clinically important Gram-positive cocci [1,2]. When directly compared with linezolid (formerly U-100766), AZD2563 was slightly more active, and it has been predicted to have pharmacokinetic features allowing once-daily dosing [3,4]. In preparation for continued clinical development and the use of broth microdilution MIC methods by investigator laboratories, the commercial products containing AZD2563 require structured validation [5]. This investigation utilizes guidelines suggested by the National Committee for Clinical Laboratory Standards (NCCLS) to determine the equivalence and reproducibility of MIC results produced by dry-form panels for the broth microdilution method [5,6]. Minimal criteria for the number of organisms tested [5] were achieved by testing 100 strains of streptococci, staphylococci and enterococci, species against which AZD2563 was active. The actual numbers of strains tested were as follows: b-hemolytic streptococci, 52 strains; viridans group streptococci, 54 strains; Streptococcus pneumoniae, 111 strains; coagulase-negative Staphylococcus spp. (CoNS), 42 strains; Staphylococcus aureus, 63 strains; Enterococcus spp., 100 strains; and 40 strains from

various Gram-negative species used to challenge the method with oxazolidinone-resistant organisms. The Gram-negative bacilli included Enterobacteriaceae (25 strains; 12 species), Pseudomonas aeruginosa (®ve strains), Stenotrophomonas maltophilia (®ve strains), and Acinetobacter spp. (®ve strains). The broth microdilution trays were produced by TREK Diagnostics/Sensititre (Westlake, OH, USA) as dry-form panels containing AZD2563 (dilution range, 0.015±32 mg/L) and linezolid (0.015± 32 mg/L) in a ®nal post-inoculum volume of 0.1 mL. Comparison reference trays were prepared in the same volume of cation-adjusted Mueller±Hinton broth, and frozen at 70 8C or below until used. The same inoculum (5 105 CFU/mL) was used for testing, with the interpretation of endpoints conforming to the criteria of the NCCLS read at organism-speci®c times ranging from 18 to 24 h [6]. Quality control (QC) organisms recommended by the NCCLS [6] were tested concurrently (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Streptococcus pneumoniae ATCC 49619). All recorded QC results for AZD2563 and linezolid (comparison oxazolidi-

ß 2003 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

544 Clinical Microbiology and Infection, Volume 9 Number 6, June 2003 Table 1 Validation experiments comparing results from dry-form AZD2563 trays with reference frozen-form AZD2563 tray MICs Dry-form MIC variations in log2 dilutions

Organisms (no. tested)

2

Streptococci b-hemolytic (52) Viridans group (54) Streptococcus pneumoniae (111) Staphylococci CoNS (42) Staphylococcus aureus (63) Enterococci (100) Other species (40) Totals AZD2563 (462) Linezolid (462)b

1

Same

1

2

0 0 0

0 0 2

50 47 104

2 7 5

0 0 0

0 0

0 9

24 52

18 2

0 0

0 0

1 0

65 40

34 0

0 0

0 0

12a 45a

382a 399a

68a 18a

0 0

a

All AZD2563 results were within 1 log2 dilution, 82.7% having the same MIC value by both tested methods, and for linezolid 86.4% of results were the same. b Linezolid tested as a control drug in the same class.

none) were within published control ranges [7] or those recommended by the manufacturer (AstraZeneca, data on ®le). In the reproducibility phase of this study, 10 strains of Gram-positive cocci were tested three times daily for 3 days. Two QC strains were included (ATCC 29212 and ATCC 29213) were

also tested. Target levels of inter-method accuracy were selected as 95% of results within 1 log2 dilution of the reference test result and 50% having the same MIC value. Acceptable reproducibility was also 95% of results within 1 log2 dilution step of the overall model MIC, analyzed on the same day and between days of testing. Table 1 summarizes the results of testing AZD2563 in commercial dry-form trays and reference NCCLS reagent panels [6]. For each group of Gram-positive organisms (Streptococcus pneumoniae, other streptococci, staphylococci, enterococci, at 100 strains each), identical MIC values were observed with both methods for 65.0% (enterococci) to 93.7% (Streptococcus pneumoniae) of results. Among the enterococcal and CoNS strains, a trend towards slightly higher MIC results was noted for the dry-form method. Overall (462 tests), the dry-form MIC values for AZD2563 were within 1 log2 dilution step of the reference method MIC; 82.7% of comparison MICs were identical. Linezolid comparisons between methods (Table 1) revealed a similar level of acceptable accuracy, with all results within 1 log2 dilution, and 86.4% of MIC comparisons being identical. A trend towards lower linezolid MICs was detected for Streptococcus pneumoniae when using the dry-form panels (data not shown). Results of the reproducibility testing are shown in Table 2 for the AZD2563 MICs performed in

Table 2 AZD2563 dry-form panel reproducibility results when testing 10 Gram-positive organisms (seven species) at three replicates daily for three days, or 90 total results AZD2563 MIC variation in log2 dilutions (linezolid results) Within replicates on same day Organism Streptococcus pneumoniae F377 Streptococcus pneumoniae F477 Viridans streptococcus 11-8649A Streptococcus pyogenes 35-356A CoNSa 15-2091A Staphylococcus aureus 1-253A Staphylococcus aureus ATCC 29213 E. faecium A5138 E. faecalis A2477 E. faecalis ATCC 29212 Total

0 1 0 0 0 0 0 0 2 0 3

Replicates between days

1

Same

1

(0) (1) (0) (0) (0) (0) (0) (0) (1) (0) (2)

9 8 9 9 9 9 9 9 7 9 87

0 0 0 0 0 0 0 0 0 0 0

(9) (8) (9) (9) (9) (9) (9) (9) (8) (9) (88)b

(0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0)

0 1 0 3 0 3 0 0 0 0 7

1

Same

1

(0) (1) (0) (3) (0) (0) (0) (3) (4) (0) (11)

9 8 6 6 9 6 9 9 5 9 76

0 0 3 0 0 0 0 0 4 0 7

(9) (8) (6) (6) (9) (6) (9) (6) (5) (9) (73)c

(0) (0) (3) (0) (0) (3) (0) (0) (0) (0) (6)

a

CoNS, coagulase-negative staphylococcus. bExact replicate-to-replicate reproducibilities were 96.7% and 97.8% for AZD2563 and linezolid, respectively. All MIC results (100.0%) were 1 log2 dilution step for both oxazolidinones. c The exact MIC was achieved between days in 84.4% and 81.1% for AZD2563 and linezolid, respectively. All MIC results (100.0%) were 1 log2 dilution step for both oxazolidinones.

ß 2003 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 9, 543±546

Concise Communication 545

dry-form commercial panels. The same AZD2563 MIC value was obtained for these test variation analyses of tests performed on the `same day' (96.7%) and of those performed `between days' (84.4%). The linezolid reproducibility results were similar (97.8% and 81.1%), and both oxazolidinones showed all reproducibility MIC values within 1 log2 dilution of the established MIC mode for each strain, e.g. acceptable by prede®ned criteria. These studies examined the ability of commercial (TREK Diagnostics) broth microdilution dryform panels to produce accurate MIC results for AZD2563, a new oxazolidinone [1±4]. Validation of this product was achieved by testing suf®cient numbers of genus and species groups as recommended in the guidelines of the NCCLS [5]. Accuracy and reproducibility results showed all MICs (100.0%) to be within acceptable limits, and the same result as obtained with the reference method [6] was produced in 82.4% of inter-method test validation comparisons and 96.7% of reproducibility tests. These results con®rm those reported by the manufacturer (TREK Diagnostics, data on ®le). The clinical use of agents in the oxazolidinone class (linezolid and AZD2563) will require accurate assessments of organism susceptibility to guide therapy and detect episodes of linezolid resistance [8]. To date, very rare occurrences of oxazolidinone-resistant strains have been reported in patients receiving long-term therapy and often having indwelling infected devices [9±11]. Linezolid resistance among initial isolates from patient infections remains extremely rare in the absence of compromised infection control practices [12]. With these validation results for AZD2563, and the linezolid control clinical laboratories using this commercial product, we should be able to accurately and reproducibly detect oxazolidinone-resistant isolates (MIC 8 mg/L) [8], and adequately follow AZD2563 by in vitro susceptibility tests in the clinical trial phases of development. The long-term shelf life of these dry-form panels will also facilitate AZD2563 development by utilization of common lots of reference-quality reagents. ACKNOWLEDGMENTS The following individuals provided excellent technical and editorial support: K. Meyer, P. Turner, L.

Deshpande, P. Rhomberg, M. Beach, and C. Knapp. The study was made possible by a research/educational grant from AstraZeneca. REFERENCES 1. Turner PJ, Wookey A, Greenhalgh JM et al. Investigations into the antibacterial spectrum of the new oxazolidinone, AZD2563, against recent clinical isolates from North America and Europe [abstract F-1024]. In: Program and abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago. Washington, DC: American Society for Microbiology, 2001: 223. 2. Gravestock MB, Betts MJ, Chawner E et al. In vivo studies of novel oxazolidinones with O- and N-linked C-5 heterocyclic side-chains, including AZD2563 [abstract F-1023). In: Program and abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago. Washington, DC: American Society for Microbiology, 2001: 222. 3. Arundel PA. The new oxazolidinone, AZD2563: physiologically-based pharmacokinetic extrapolation to man [abstract F-1039]. In: Program and abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago. Washington, DC: American Society for Microbiology, 2001: 223. 4. Craig WA, Andes DR. Pharmacodynamic characteristics of AZD2563, the new oxazolidinone, in a murine thigh-infection model [abstract F-1037]. In: Program and abstracts of the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago. Washington, DC: American Society for Microbiology, 2001: 226. 5. National Committee for Clinical Laboratory Standards. Development of in vitro susceptibility testing criteria and quality control parameters. Approved guideline M23-A2. Wayne, PA: NCCLS, 2001. 6. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Document M7A5. Wayne, PA: NCCLS, 2000. 7. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. M100-S12. Wayne, PA: NCCLS, 2002. 8. Livermore DM, Mushtaq S, Warner M. Susceptibility testing with linezolid by different methods, in relation to published `general breakpoints'. J Antimicrob Chemother 2001; 48: 452±4. 9. Zurenko GE, Todd WM, Hafkin B et al. Development of linezolid-resistant Enterococcus faecium in two compassionate use program patients treated with linezolid [abstract 848]. In: Program and


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