Journal of Antimicrobial Chemotherapy (2001) 48, 121-125
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Interpretative criteria and quality control parameters for telithromycin disc diffusion susceptibility tests
The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA
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Abstract |
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In vitro studies were carried out with 4175 Gram-positive clinical isolates, comparing telithromycin broth microdilution MICs with disc diffusion zone diameters. Provisional interpretive criteria for testing staphylococci were
23 mm for susceptible and 
19 mm for resistant. Because pneumococci and other streptococci require increased CO2, the corresponding zone sizes are 3 mm smaller (20 mm and 16 mm). Two collaborative studies were performed in order to propose the following quality control limits: 24–30 mm for Staphylococcus aureus ATCC 25923 and 27–33 mm for Streptococcus pneumoniae ATCC 49619. |
Introduction |
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The ketolide class of antimicrobial agents includes telithromycin, the first of that class to be developed for use as a chemotherapeutic agent.1 In this report we compile data that have been recorded at our institute in order to propose interpretive criteria for the disc diffusion susceptibility test. In addition, we summarize the results of two multilaboratory collaborative studies that were designed to select quality control (QC) limits for telithromycin disc diffusion tests.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Antimicrobial susceptibility tests
Disc diffusion and broth microdilution susceptibility tests were performed as outlined by the National Committee for Clinical Laboratory Standards (NCCLS).2,3 Because structurally related compounds such as the macrolides are tested with a 15 µg disc, initial studies of telithromycin used a disc content of 15 µg. Subsequently, there were no obvious reasons to consider use of other disc potencies. Disc diffusion tests were performed on Mueller–Hinton agar. When testing streptococci, the agar was supplemented with 5% defibrinated sheep blood and the plates were incubated at 35°C with 5–7% CO2. When testing staphylococci, the plates were incubated at 35°C in ambient air. Broth microdilution tests were performed in cation-adjusted Mueller– Hinton broth; 3% lysed horse blood was added when testing streptococci. Microdilution trays were incubated at 35°C without added CO2.
Quality control studies
An initial 10 laboratory study was undertaken in order to propose tentative zone size limits for disc tests of Staphylococcus aureus ATCC 25923 and Streptococcus pneumoniae ATCC 49619. This study followed the protocol described by the NCCLS4 except that we used 10 testing facilities rather than the required seven. This initial study involved replicate tests on six different batches of Mueller–Hinton agar from four manufacturers and two batches of 15 µg telithromycin discs from different manufacturers. This exercise generated 400 zone diameters and tentative zone size limits were calculated using the statistic of Gavan et al.5 A 15 µg erythromycin disc was also tested as an internal control and that generated 200 zone diameters, >95% of which were within established QC ranges. To confirm the validity of the tentative control limits, two batches of telithromycin discs from different manufacturers and one batch of erythromycin discs were distributed to 30 clinical laboratories where both control strains were tested on 20 separate days using media obtained from local suppliers. These zones of inhibition were evaluated to determine how often zones fell within the tentative QC ranges that were defined by the initial study.
Microorganisms
In 1997 and again in 1999, fresh clinical isolates were collected from different medical centres throughout North America and susceptibility tests were performed in a central laboratory. Here we combine the results of broth microdilution and disc diffusion tests accumulated during these two surveys. Data were obtained with 2082 staphylococci (1061 S. aureus and 1021 coagulase-negative species), 1123 Streptococcus pneumoniae and 970 other Streptococcus spp. (413 Streptococcus pyogenes, 297 Streptococcus agalactiae, 165 viridans group, 95 other species). Scattergrams were prepared for each of the three major groups of microorganisms. Because MICs were not evenly distributed over a wide range of concentrations, regression statistics were considered inappropriate. Using an error minimization approach, interpretive criteria were proposed for testing staphylococci in ambient air and for testing streptococci with increased CO2. Zone size breakpoints were those that minimized interpretive discrepancies between the two types of susceptibility tests.
MIC breakpoints
Like the macrolides, telithromycin tends to concentrate in broncho-pulmonary tissues and in white blood cells.6 Consequently, the concentration at the site of infection is likely to greatly exceed the concentration in the patient's blood. Telithromycin is administered orally as a single daily dose. Following a 800 mg dose, peak serum concentrations >2.0 mg/L are anticipated.7 It is possible that microorganisms with MICs of 1.0 or 2.0 mg/L might respond to therapy but those with MICs of 4.0 mg/L are likely to be resistant. We tentatively adapted conservative MIC breakpoints of 0.5 mg/L as susceptible, 1.0 mg/L as intermediate and 2.0 mg/L as resistant. Scattergrams were then prepared in order to select zone size criteria that would best separate categories defined by those MIC breakpoints.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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QC limits for telithromycin disc tests were defined by an initial 10 laboratory study that generated 400 zone diameter measurements. Those limits were further evaluated by a less rigidly structured 30 laboratory study that should have generated 1200 zone diameters. For logistic reasons, not all 1200 zones were obtained. The overall distribution of zone diameters for S. aureus ATCC 25923 and for S. pneumoniae ATCC 49619 are described in the Table
. In both studies, two telithromycin discs from different manufacturers and one erythromycin control disc were placed on each test plate. The Table includes results of all tests with both control strains.
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Under the rigidly controlled conditions of the initial study, zone diameters were clustered within a range including 3 mm on either side of the median zone diameter and >98% of all determinations fell within the proposed 7 mm ranges for each of the control strains. The control erythromycin disc gave 200 zones all within the current QC ranges2 for each control strain. Under less stringently controlled test conditions in a larger variety of clinical laboratories, using media from local suppliers, the overall range of zone diameters was greater. With the erythromycin control disc,
95% of the reported zones were within accepted limits.2 However, telithromycin zones were more broadly distributed and only 90–92% fell within the proposed 7 mm ranges. The proportion of tests that fell within the QC limits would be increased if the control ranges were extended by adding 1 mm to either end (9 mm ranges). The initial 7 mm ranges are those that will be included in the NCCLS document.2
The Figure shows scattergrams for tests with staphylococci, pneumococci and non-pneumococcal streptococci. Zone size interpretive criteria and discrepancy rates are displayed in each scattergram. Because of the distribution of MICs for these strains, the actual MIC breakpoints could be raised by one or two doubling dilutions without greatly influencing the overall percentage susceptibility. Most strains with MICs of 1.0 mg/L tended to give large zones in the susceptible category and, consequently, a breakpoint of 1.0 mg/L might be preferred if supported by clinical data.
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We propose tentative disc test criteria of
19 mm for resistant, 20–22 mm for intermediate and 23 mm for susceptible when testing Staphylococcus spp. Among 2082 staphylococci, 629 strains were resistant (MICs of 2.0 mg/L) and five (0.8%) of those resistant strains were susceptible by the disc test (very major discrepancies, which were not confirmed by retesting). Furthermore, three (0.2%) of 1447 susceptible strains were resistant by the disc test (major discrepancies, which were not retested). There was no obvious explanation for the few discrepancies between broth microdilution and disc diffusion tests.
Because the streptococci were incubated in 5–7% CO2, slightly smaller zone diameters might be anticipated. For testing S. pneumoniae and other streptococci, interpretive zone size criteria can be reduced to 16 mm for resistant, 17–19 mm for intermediate and 20 mm for susceptible. Telithromycin-resistant streptococci were extremely uncommon in our collection of isolates and thus we cannot properly assess the accuracy of the zone size criteria for the resistant category. The overall discrepancy rates for disc tests of pneumococci and other streptococci combined were: 0.1% very major, 0.2% major and 1.3% minor differences. Fourteen of the 15 strains with intermediate MICs of 1.0 mg/L gave large zones of inhibition and appear to be part of the normally distributed susceptible population. Those strains were not subjected to replicate testing in order to determine how often their MICs might move from intermediate to susceptible or resistant categories.
Additional tests with enterococci were not as successful (data not shown). The disc test should not be used for evaluating telithromycin against enterococci because of an unacceptable number of discrepant results. Because enterococcal infections are not likely to be treated with telithromycin, we did not try to set interpretive criteria for that genus. For the other Gram-positive cocci, the disc test appears to be reasonably accurate.
The results presented here are part of the information package that is currently being considered by the US Food and Drug Administration and will soon be presented to the NCCLS subcommittee for antimicrobial susceptibility tests. QC ranges proposed here have been accepted by the NCCLS subcommittee for antimicrobial susceptibility tests. Official judgments concerning interpretive criteria for tests of telithromycin have not yet been established for the NCCLS methods. In the interim, our provisional criteria might be useful to those who wish to test telithromycin by NCCLS methods. Susceptibility tests performed by other procedures may require slightly different interpretive criteria or QC limits.
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Acknowledgments |
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These studies were made possible by grants from Aventis Pharmaceuticals Research and Development, Romainville, France.
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Notes |
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* Corresponding author. Tel: +1-503-682-3232; Fax: +1-503-682-2065; E-mail: cmi{at}hevanet.com
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Bryskier, A., Aqouridas, C. & Chantot, J. F. (1997). Ketolides; a new semi-synthetic 14-member-ring macrolide. In Expanded Indications for the New Macrolides, Azalides and Streptogramins, (Zinner, S. H., Young, L. S., Acar, J. F. & Neu, H. C., Eds), pp. 39–50. Marcel Dekker, New York.
2 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility Tests—Seventh Edition: Approved Standard M2-A7. NCCLS, Wayne, PA.
3 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.
4 . National Committee for Clinical Laboratory Standards. (1998). Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters—Third Edition: Tentative Standard M23-T3. NCCLS, Wayne, PA.
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Gavan, T. L., Jones, R. N., Barry, A. L., Fuchs, P. C., Gerlach, E. H., Matsen, J. M. et al. (1981). Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: a collaborative study. Journal of Clinical Microbiology 14, 67–72.
6 . Serieys, C. M., Cantalloube, C., Soler, P., Gia, H. P. & Brunner, F. (1999). HMR 3647 achieves high and sustained concentrations in broncho-pulmonary tissues. In Twenty-first International Congress of Chemotherapy, Abstract P-78. Journal of Chemotherapy 44, Suppl. A, 57.
7 . Gia, H. P., Roeder, V., Namour, F., Sultan, E. & Lenfant, B. (1999). HMR 3647 achieves high and sustained concentrations in white blood cells in man. In Twenty-first International Congress of Chemotherapy, Abstract P-79. Journal of Chemotherapy 44, Suppl. A, 57.
Received 1 November 2000; returned 8 February 2001; revised 12 March 2001; accepted 27 March 2001
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