Initial Quality Control Evaluations for Susceptibility Testing of Dalbavancin (BI397), an Investigational Glycopeptide with Potent Gram-Positive Activity (2003)

As gram-positive pathogens have acquired resistances to traditional therapeutic agents and multidrug-resistant clones have spread globally, vancomycin in the United States and teicoplanin in Europe have been the antimicrobial agents of choice for the treatment of methicillin-resistant Staphylococcus aureus and invasive enterococcal infections (1, 3). During recent years, there has been a profound escalation in resistance among these pathogenic bacteria (1-4). Vancomycin-resistant enterococci, which cause serious infections including endocarditis, transient and invasive bacteremia, and tissue infections, are common and endemic in some institutions in the United States and Europe (1-4). As of 2002, a total of eight vancomycin-intermediate S. aureus from patient infections have been confirmed in the United States (3). Also in 2002, the first vancomycin-resistant S. aureus (VRSA) strain was isolated in a patient with diabetes, peripheral vascular disease, and chronic renal failure (3). In Europe, teicoplanin has been shown to possess a spectrum similar to that of vancomycin and appears to have fewer side effects, which can be advantageous in treating patients with extended treatment regimens (2). However, the emergence of confirmed VRSA in the United States and the previously documented teicoplanin-intermediate or -resistant strains of coagulase-negative staphylococci in Europe underscores the need for antimicrobial agents which can replace contemporary glycopeptides as a “treatment of last resort” for multidrug-resistant gram-positive pathogens (3).

Dalbavancin (formerly BI397) is a semisynthetic derivative of a natural glycopeptide, MDL 62,476 (formerly {type:entrez-nucleotide,attrs:{text:A40926,term_id:2296837,term_text:A40926}}A40926), with a mode of action which disrupts bacterial cell wall biosynthesis (2, 4). The development of dalbavancin showed great promise because of greater potency than either vancomycin or teicoplanin against staphylococci, including some resistant strains. The antistreptococcal activity of dalbavancin, including penicillin-resistant strains, is similar to that of teicoplanin but superior to that of vancomycin (2). The pharmacokinetic properties of dalbavancin are advantageous compared to those of other glycopeptides due to an extended half-life, which allows prolonged dosing intervals, which can be once weekly for some indications (2, 4).

A quality control (QC) study following the guidelines (M23-A2) and broth microdilution test methods (M7-A6) established by the National Committee for Clinical Laboratory Standards (5, 6) was performed by an eight-laboratory study group. The NCCLS reference frozen-form broth microdilution panels containing four Mueller-Hinton broth lots by quality manufacturers (Difco, Detroit, Mich.; Hardy, Santa Maria, Calif.; BBL, Sparks, Md.) were supplemented with or without 5% lysed horse blood. Panels were prepared by TREK Diagnostics (Cleveland, Ohio) and remained frozen at −80°C until used. The dalbavancin standard powder was obtained from Versicor (Fremont, Calif.), and vancomycin powder, which was obtained from Sigma Chemical (St. Louis, Mo.), served as the internal quality control agent. Each laboratory tested three American Type Culture Collection (ATCC) QC strains; Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, and S. aureus ATCC 29213, daily for 10 days (320 MICs) (6).

The procedure used for testing each of the QC strains is as follows: a 0.5 McFarland standard inoculum was prepared and diluted with 1 ml into 29 ml of water containing polysorbate 80. The inoculum suspension was inoculated into the MIC panels using a hand inoculator. The MIC panels were incubated according to NCCLS M7-A6 recommendations (5). Colony counts were performed from the positive control well after inoculation of the panel to ensure that the final inoculum concentration obtained was approximately 5 × 105 CFU/ml. The proposed QC ranges were optimized to contain ≥95% of all results as recommended by the NCCLS M23-A2 guidelines (6). Each of the MIC results was tabulated and compared within and between laboratories. Results from each of the broth media lots were also compared.

As an example of the dalbavancin MIC distribution for S. aureus ATCC 29213 between the eight laboratories, more than 90% of the combined results were at 0.06 μg/ml, which was also the modal value for all of the eight participating laboratories. The MIC range for each laboratory was one to three log2 dilution steps. The proposed range for S. aureus ATCC 29213 is 0.03 to 0.12 μg/ml, which would contain 100% of the reported results. Similar results were obtained for E. faecalis ATCC 29212, with more than 86% of the total results at the modal value (0.06 μg/ml) and a range of 0.03 to 0.12 μg/ml (100% of the reported value). For S. pneumoniae ATCC 49619, more than 78% of the total results were at the modal value (0.015 μg/ml), with a range of 0.008 to 0.03 μg/ml (99.7% of the reported values). When the MIC occurrences by media lots were calculated, the same modal values for S. aureus ATCC 29213, E. faecalis ATCC 29212, and S. pneumoniae ATCC 49619 (0.06, 0.06, 0.015 μg/ml, respectively) were documented (data not shown). The different media lots used did not have any influence on the results.

Table ​Table11 summarizes all of the proposed QC ranges for S. aureus ATCC 29213, E. faecalis ATCC 29212, and S. pneumoniae ATCC 49619 to be used by clinical laboratories for susceptibility testing methods (5). A three-log2 dilution range for the broth microdilution methods would encompass 99.7 to 100.0% of all results produced by the laboratories.



TABLE 1.

Distributions of dalbavancin MICs for all qualifying results from an eight-laboratory study using three NCCLS QC strains 5



Concurrent testing using vancomycin as the internal control agent was performed on all QC organisms, with all results (100.0%) within the NCCLS published guidelines (5); 0.5 to 2 μg/ml for S. aureus ATCC 29213, 1 to 4 μg/ml for E. faecalis ATCC 29212, and 0.12 to 0.5 μg/ml for S. pneumoniae ATCC 49619. Colony counts were performed from the broth microdilution panels by subculturing in a quantitative manner onto drug-free plates, and the counts ranged from 3 × 104 to 1 × 106 CFU/ml with an average for all laboratories at 3.5 × 105 CFU/ml (target inoculum at 5 × 105).

This report summarizes the results from a MIC QC study for dalbavancin, a new long-acting glycopeptide, tested against the most commonly used gram-positive QC strains (5). The proposed dalbavancin QC guidelines should be utilized during the initial clinical trials, especially since there will be a delay in the development of the disk agar diffusion method. These MIC QC ranges, when applied, will promote the accuracy of dalbavancin susceptibility testing data for regulatory purposes.