Determination of disk diffusion and MIC quality control parameters for AZD2563, a novel long-acting oxazolidinone

1. Introduction 

Emerging antimicrobial resistance, especially among the Gram-positive pathogens, has led to the development of a new antimicrobial class, the oxazolidinones (Diekema & Jones, 2001). The oxazolidinones have a unique mechanism of action and kill curve results suggest the antimicrobials are predominantly bacteriostatic Diekema and Jones 2001, Jones et al 2002b. The oxazolidinones have been shown to be highly effective against multi-drug resistant pathogens as well as against the rarer or unusual Gram-positive species such as Corynebacterium spp., Listeria spp., Micrococcus spp., and Bacillus spp. AZD2563 is a novel oxazolidinone from a compound series with O- and N-linked C-5 heterocyclic side chains (Gravestock et al., 2001). When comparing AZD2563 to linezolid, the first oxazolidinone released for clinical use, the MIC values show that AZD2563 is slightly more active Anderegg et al 2002a, Anderegg et al 2002b, Jones et al 2002b. The pharmacokinetic features of AZD2563 also suggest less frequency in projected clinical trials, an option not available with linezolid Craig and Andes 2001, Arundel 2001.

The favorable preliminary studies performed using AZD2563 suggest the need for accurate quality control (QC) guidelines for laboratory testing using control strains from the American Type Culture and Collection (ATCC). This study summarizes the results of a National Committee for Clinical Laboratory Standards (NCCLS) M23-A2 qualifying study to establish QC limits for AZD2563 when tested by disk diffusion and MIC methods National Committee for Clinical Laboratory Standards. 2000a, National Committee for Clinical Laboratory Standards. 2000b, National Committee for Clinical Laboratory Standards. 2001.

The QC study design followed the NCCLS (2001) M23-A2 guidelines. A total of eight laboratories (JMI Laboratories, North Liberty, IA; Michigan State University, East Lansing, MI; TREK Diagnostics, Westlake, OH; University of Alberta, Alberta, Canada; Cleveland Clinic Foundation, Cleveland, OH; University of Texas Medical Center, Houston, TX; Strong Memorial Hospital, Rochester, NY; VA Medical Center, Iowa City, IA; and Iowa State University, Ames, IA) provided data for the MIC (M7-A5) and disk diffusion (M2-A7) methods described by the National Committee for Clinical Laboratory Standards. 2000a, National Committee for Clinical Laboratory Standards. 2000b.

QC strains tested for the MIC portion of the study were Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Streptococcus pneumoniae ATCC 49619. The frozen-form, reference broth microdilution panels contained four lots of Mueller-Hinton broth with or without supplemented 5% lysed horse blood. The panels were prepared by TREK Diagnostics (Westlake, OH) and stored at −80°C until used. The antimicrobial agents and their role in the study were as follows: AZD2563 powder obtained from AstraZeneca (Macclesfield, United Kingdom) served as the investigational drug, linezolid powder obtained from Pharmacia and Upjohn (Kalamazoo, MI) served as a class comparator and internal control agent (S. pneumoniae only) and vancomycin powder obtained from Sigma Chemical (St. Louis, MO) served as the internal control agent for all testing. Reference broth microdilution standards were used by all eight laboratories (NCCLS, 2000a). Each laboratory generated 40 MIC results per QC strain per laboratory for a total of 320 MIC results. Colony counts were performed from the broth microdilution panels by quantitative subculturing onto drug-free blood agar plates to determine the inoculum (target of 5.0 × 105 CFU/ml).

QC strains tested for the disk diffusion (NCCLS, 2000b) portion of the study were S. aureus ATCC 25923 and S. pneumoniae ATCC 49619. Depending on the QC strain tested, three different agar lots of Mueller-Hinton agar with or without the supplemented 5% sheep blood were used (prepared by Remel, Lenexa, KS). QC strains were tested using two disk lots of the investigational drug, AZD2563 (BBL; Cockeysville, MD), two disk lots of the class comparator and internal QC drug, linezolid (BBL; Cockeysville, MD and Remel, Lenexa, KS) and one disk lot of the second internal QC drug, vancomycin (Remel, Lenexa, KS). Each laboratory tested the QC strains generating 60 zone diameters per QC strain per laboratory for a total of 480 zone diameters. The internal QC drug, vancomycin produced a total of 210 zone diameters. The colony counts performed from the broth microdilution panels had an average of 2.7 × 105 CFU/ml by all participants with a range of 1.0 × 105 to 4.8 × 105 CFU/ml.

Table 1 shows an example of the data calculated from the MIC values of S. aureus ATCC 29213 tested against AZD2563. Over 70% of the total results were at 2 μg/ml, which was also the modal value for seven of the eight participating laboratories. Each laboratory had a narrow range of reported MICs (one to three log2 dilution step); thus, the proposed range for S. aureus ATCC 29213 was 1–4 μg/ml which would encompass 100% of the results obtained. Similar results were received for E. faecalis ATCC 29212, and the modal value was 2 μg/ml (77.8% of the results) for six of the eight laboratories. The proposed range for E. faecalis ATCC 29212 was also 1-4 μg/ml (includes all reported AZD2563 MICs). The multi-center results for S. pneumoniae ATCC 49619 required a proposed four log2 dilution range (0.25-2 μg/ml) due to the MIC occurrences of 43.8 and 55.0% at 0.5 and 1 μg/ml, respectively.

Table 1. Inter- and intra-laboratory comparisons of AZD2563 MIC results versus S. aureus ATCC 29213 for a eight medical center protocol meeting the study design guidelines found in NCCLS M23-A2 [2001].

	MIC (μg/ml)	Laboratory code (occurrences):								Total (%)
		A	B	C	D	E	F	G	H
	1	21		1		7		11	14	54 (16.9)a
	2	19	40	28	38	33	21	27	23	229 (71.6)a
	4			11	2		19	2	3	37 (11.5)a
	Total	40	40	40	40	40	40	40	40	320
	Mode (μg/ml)	1	2	2	2	2	2	2	2	2
	Range (dilutions)	2	1	3	2	2	2	3	3	3

a Proposed range includes 1–4 μg/ml (100% of reported values were within this range).

Fig. 1 is a representative bar graph that shows the frequency of AZD2563 zone diameter occurrences when testing AZD2563 against S. pneumoniae ATCC 49619. Due to a single laboratory (H) producing aberrant results (consistently smaller zone diameters), all of their results have been omitted from the disk diffusion portion of the QC study. A total of 420 zone diameters were analyzed for S. pneumoniae ATCC 49619 with a mode of 28 mm and a 7 mm range was proposed. This QC range (25–31 mm) for S. pneumoniae ATCC 49619 encompasses 99.5% of the participants’ results. The S. aureus ATCC 29213 results when tested against AZD2563, produce a mode of 26 mm with a 7 mm proposed range (includes 98.6% of results).

View Large Image Download to PowerPoint

Fig. 1. Graph showing frequency of AZD2563 zone diameter occurrences.

In this study, two internal control drugs were used to allow for the re-evaluation of linezolid disk diffusion ranges, specifically for S. aureus ATCC 25923. Thus, vancomycin served as the control drug for linezolid; and vancomycin and linezolid served as the control agent for AZD2563. The linezolid zone diameters for S. aureus ATCC 25923 demonstrated a skewed distribution toward the smaller zones of the existing QC range. A modification in the QC range from 27–31 mm (82.4% of the results; data not shown) appears necessary (publication in press).

The internal control results for vancomycin tested showed that 98.4% of the MIC values and 100.0% of the disk diffusion values were within the published guidelines of NCCLS (2002). One laboratory did produce out of control results for S. pneumoniae ATCC 49619; however, the linezolid (second control drug) results were within the control range (NCCLS, 2002).

Table 2 summarizes all of the proposed QC ranges (four ATCC strains) for the strains to be used by clinical laboratories for susceptibility testing methods National Committee for Clinical Laboratory Standards. 2000a, National Committee for Clinical Laboratory Standards. 2000b. Seven mm ranges for the disk diffusion testing of AZD2563 provided a 98.6–99.5% of all of the participants’ results, and a 3 or 4 log2 dilution range for the broth microdilution methods would encompass all of the participants’ results. This study summarizes results from a standardized NCCLS QC study evaluating AZD2563, a new, long-acting oxazolidinone, against the most commonly used QC strains. The proposed AZD2563 QC guidelines should be utilized during the clinical trials to promote the accuracy of susceptibility testing data.

Table 2. Proposed quality control ranges of AZD2563 disk diffusion and MIC broth microdilution tests.

	Organism	Disk diffusion		Broth microdilution
		Proposed range (mm)	% in range	Proposed range (MIC μg/ml)	% in range
	S. pneumoniae ATCC 49619	25–31	99.5	0.25–2	100.0
	S. aureus ATCC 25923	23–29	98.6	—	—
	S. aureus ATCC 29213	—	—	1–4	100.0
	E. faecalis ATCC 29212	—	—	1–4	100.0