The Ohio State University Medical Center, Columbus, Ohio 432103; St. Vincent ... St. Francis Medical Center, Wichita, Kansas 672145; and Centers for Disease ...
Vol. 25, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 165-166
0095-1137/87/010165-02$02.OO/0 Copyright 1987, American Society for Microbiology
Quality Control Limits for Microdilution Susceptibility Tests with Norfloxacin A. L. BARRY,l* T. L. GAVAN,2 L. W. AYERS,3 P. C. FUCHS,4 E. H. GERLACH,' AND C. THORNSBERRY6 The Clinical Microbiology Institute, Tualatin, Oregon 970621; The Cleveland Clinic Foundation, Cleveland, Ohio 441062; The Ohio State University Medical Center, Columbus, Ohio 432103; St. Vincent Medical Center, Portland, Oregon 972254; St. Francis Medical Center, Wichita, Kansas 672145; and Centers for Disease Control, Atlanta, Georgia 303336 Received 4 August 1986/Accepted 15 September 1986
A multilaboratory study was designed to define quality control limits for microdilution susceptibility tests with norfloxacin. The following limits were proposed: for Escherichia coli ATCC 25922, 0.03 to 0.125 ,ug/ml; for Pseudomonas aeruginosa ATCC 27853, 1.0 to 4.0 ,ug/ml; for Staphylococcus aureus ATCC 29213, 0.5 to 2.0 Fg/ml; and for Streptococcus faecalis ATCC 29212, 2.0 to 8.0 ,ug/ml. The latter represents a change in the previously recommended control limits.
To monitor the accuracy and precision of susceptibility testing procedures, laboratories must test standard control strains at regular intervals and document the fact that at least 95% of the test results fall within established control limits. For control of microdilution tests, four standard control strains have been established (Escherichia coli ATCC 25922, Streptococcusfaecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853). For each antimicrobial agent, an acceptable range of MICs must be established for each strain. In the past this has been done by surveying several laboratories and establishing a mode; the control limits were then defined as a range represented by one doubling dilution above or below that estimated mode. In this report we describe a prospective collaborative study that was designed to establish control limits for microdilution tests in a more systematic manner. The study design was similar to that described by Gavan et al. (1) for disk tests. Tests were performed with norfloxacin, a new quinolone antimicrobial agent that is intended for use in treating urinary tract infections. Because of its broad spectrum of activity, all four control strains needed to be evaluated. Norfloxacin powders for in vitro susceptibility testing were provided by the Merck Institute for Therapeutic Research, Rahway, N.J. The testing material and subcultures of the four control strains were distributed to all participants. Each of the five facilities prepared its own microdilution trays containing doubling dilutions of norfloxacin (0.008 to 64 ,ug/ml) in Mueller-Hinton broth supplemented with cations (2). The coordinating facility distributed samples of a sixth lot of trays to all five collaborators. The latter trays provided reagents that were common to all participating laboratories and served as a source of internal control data. The trays were stored at -20°C or colder for the duration of the study (3 to 4 weeks). Microdilution susceptibility tests were performed by carefully following the procedures outlined by the National Committee for Clinical Laboratory Standards (NCCLS; 2). The inoculum was approximately 5 x 105 CFU/ml, and MICs were recorded after 16 to 18 h of incubation at 35°C in ambient air. In each of the five laboratories, 10 separate tests *
were performed with the lot of trays that was common to all participants and 20 separate tests were performed with the test lots of trays that were prepared in-house. The results of these tests are shown in Table 1. With the common lot of trays, the five centers provided remarkably similar results. Within any one laboratory, MICs varied no more than one doubling dilution, and all five centers reported MICs within one doubling dilution above or below the all-laboratory mode. Twice as many MICs were generated with multiple test lots of microdilution trays, but the reproducibility was similar to that obtained with the common lot of trays. With E. coli ATCC 25922, laboratory C reported 5 of 20 MICs that were two doubling dilutions below the mode. All other data were within one dilution interval above or below the all-laboratory mode. When P. aeruginosa ATCC 27853 was tested in the common lot of trays, the overall mode was 4.0 ,ug/ml, but when different test lots were evaluated, the mode was 2.0 ,ug/ml, with a marked skewing toward 1.0 ,ug/ml. The potency of norfloxacin in the two types of trays was found to be essentially identical when the
other control strains were tested, i.e., the modal values were identical. The results of tests with the five test lots and with the common lot of microdilution trays were combined, and summary statistics were calculated (Table 2). In all cases, the mode and geometric mean values were essentially identical. The proposed control limits represent the range of concentrations which include at least 95% of the data that were accumulated in this collaborative study. For tests with norfloxacin, those 95% confidence limits represent a range of one doubling dilution above or below the all-laboratory modes. For monitoring the performance of microdilution trays containing norfloxacin, tests with S. aureus ATCC 29213 and S. faecalis ATCC 29212 are most likely to be useful. Tests with P. aeruginosa ATCC 27853 are also likely to be helpful, but an inordinately large number of test results were at the bottom end of the acceptable range (MIC, 1.0 ,ug/ml). The standard control strain of E. coli (ATCC 25922) is too susceptible to norfloxacin to be of much value for quality control purposes. Most clinical laboratories would not test concentrations that low. The recommendations in Table 2 confirm the utility of the control limits that were tentatively defined in the NCCLS
Corresponding author. 165
166
J. CLIN. MICROBIOL.
NOTES
TABLE 1. Norfloxacin MICs obtained with a single lot of microdilution trays common to all laboratories and with five separate test lots of trays prepared in each of five laboratories Control strain and laboratory(s)
E. coli ATCC 25922 Aa B C D E All
Common lot 0.06
0.125
8 1 10
2 9
9 28
10 1 22
0.5
1.0
S. faecalis ATCC 29212 A B C D E All
2.0
4.0
9 9 9 5 9 41
1 1
1 3
P. aeruginosa ATCC 27853 A B C D E All S. aureus ATCC 29213 A B C D E All a
No. of reports of indicated MIC (,ug/ml) in the following: Test lots
6
4 9 9 9 1 32
9 15
3 6
2 il
7 7 4 9 8 35
8.0
0.015
0.03
0.06
5
7 1 3
5
il
12 15 12 20 17 76
0.125
0.5
1.0
2.0
4.0
1 14 10
19 4 10 20 12 65
4
3 7
1 5
8 33
6
4 17
1 1 1
19 40
3
2 1 9
3 1
5 17
4
8.0
12 10 il 16 15 64
16 2 5 17 1 41
2
2
1 15 3 19
6 9 4
19
Only 19 MICs were reported with the test lot; all others provided 20 datum points.
document (2). However, for tests with S. faecalis ATCC 29212, the NCCLS document recommends control limits that are one doubling dilution lower than those that were defined in this report. In the early developmental work, a modal value for norfloxacin was estimated to be 2.0 ,ug/ml and tentative control limits were defined as a range represented by one doubling dilution on either side of that mode. The more extensive collaborative study reported here defined the mode to be 4.0 ,ug/ml, but there was a marked TABLE 2. Norfloxacin quality control parameters for
microdilution susceptibility tests MIC (~Lg/ml) Control strain
Central tendency
Mode Geometric mean
E. coli ATCC 25922
S. faecalis ATCC 29212 P. aeruginosa ATCC 27853 S. aureus ATCC 29213
Proposed limit
skewing toward 2.0 jxg/ml. Almost 95% of our datum points ,uglml, and no MICs of 1.0 ,ug/ml were
were either 2.0 or 4.0
recorded. We recommend that the NCCLS control limits of 1.0 to 4.0 ,ug/ml be modified to include MICs of 2.0 to 8.0 ,ug/ml. That modification has been reviewed and accepted by the NCCLS subcommittee and should appear in the next document supplement. Such difficulties might have been avoided if the first data were collected with the type of collaborative study that is described in this report. To define the range of acceptable variation in microdilution MICs, replicate tests must be performed in several laboratories, each using different lots of microdilution trays. The common lot of trays provides an internal control of methodologic differences that might skew the data and lead to erroneous conclusions.
% of 150 MICs within limits
0.06 4.0
0.06 3.5
0.03-0.125 2.0-8.0
96.6 100
2.0
2.2
1.0-4.0
98
1.0
0.98
0.5-2.0
100
LITERATURE CITED 1. Gavan, T. L., R. N. Jones, A. L. Barry, P. C. Fuchs, E. H. Gerlach, J. M. Matsen, L. B. Reller, C. Thornsberry, and L. D. Thrupp. 1981. Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion susceptibility tests: a collaborative study. J. Clin. Microbiol. 14:67-72. 2. National Committee for Clinical Laboratory Standards. 1985. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A. National Committee for Clinical Laboratory Standards, Villanova, Pa.
