Selection of Zone Size Interpretive Criteria for Disk Diffusion Susceptibility Tests of Three Antibiotics against Streptococcus pneumoniae, Using the New Guidelines of the National Committee for Clinical Laboratory Standards

Disk diffusion and broth microdilution susceptibility testswere performed with cefotaxime, ceftriaxone, telithromycin,and erythromycin (control) against 407 selected isolates ofStreptococcus pneumoniae. Scattergrams were prepared from theresults of these tests, and the current NCCLS guidelines forsetting disk diffusion test interpretive criteria were applied.Erythromycin zone diameter breakpoints were confirmed. Telithromycininterpretive criteria for the disk test could be easily setwith acceptable discrepancy rates. For cefotaxime and ceftriaxone,the minor discrepancy rates for MICs in the intermediate category± 1 dilution were far in excess of the acceptable 40%limit, i.e., 52 and 71%, respectively. We conclude that the30-µg disk of these two drugs cannot be reliably usedto test pneumococci.

INTRODUCTION

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Introduction

Susceptibility test interpretive criteria are determined bya number of considerations. For MIC susceptible and resistantbreakpoints, these considerations include the pharmacokineticand/or pharmacodynamic information of the drug in question,correlation of MICs with clinical outcomes, and the drug's MICdistribution for the species for which the drug is indicated.

The susceptible and resistant breakpoints for the disk diffusiontest are determined largely by correlating the disk diffusioninhibitory zone diameters with MICs. In the past this has oftenbeen accomplished by regression analysis. The ability of regressionanalysis to suggest appropriate zone diameter breakpoints isdependent upon a fairly even distribution of organisms at eachMIC tested, particularly in the range of the intermediate MIC± 2 to 3 twofold dilutions. With many of the newer antibiotics,however, resistant organisms are rare, and the MIC distributionis heavily weighted toward very susceptible MICs, resultingin an unreliable regression line. To overcome this problem theerror rate-bounded method of Metzler and DeHaan (4) as modifiedby Brunden et al. (2) is commonly used to select disk diffusiontest breakpoints, and this method has been accepted by the NationalCommittee for Clinical Laboratory Standards (NCCLS) (5, 6).

In its first approved guidelines (M23-A), the NCCLS indicatedthat very major discrepancies (resistant MIC and susceptiblezone diameter) should be less than 1.5%, and major discrepancies(susceptible MIC and resistant zone diameter) should be lessthan 3%, using the total population as the denominator (5).No limits on minor discrepancies (intermediate by one methodand resistant or susceptible by the other) were provided. Becausethese discrepancy rates are greatly influenced by the MIC distribution,it was also pointed out that it was more meaningful to calculatethe very major and major discrepancy rates with the MIC-resistantand MIC-susceptible populations, respectively, as denominators.However, no limits for discrepancy rates calculated in thismanner were provided.

In the second edition of the NCCLS guidelines (M23-A2), it wasrecognized that because of the ±1 dilution variationof MICs and the ± 3- to 4-mm variation of zone diametersthat are intrinsic to the testing systems, it was appropriateto allow greater discrepancy rates for strains with MICs atthe intermediate MIC ± 1 twofold concentration (I + 1to I - 1) and more limited discrepancy rates for strains withMICs with $>=$ 2 twofold concentrations above ( $>=$ I + 2) or below ( $<=$ I- 2) the intermediate MIC (6). For setting zone diameter criteriawith a challenge set of organisms, acceptable discrepancy ratesfor these three MIC groups have been proposed as follows: forI + 1 to I - 1, <10% major, <10% very major, and <40%minor discrepancies; for $>=$ I + 2, <2% very major and <5%minor discrepancies; and for $<=$ I - 2, <2% major and <5%minor discrepancies. For assessing already established criteriawith routine clinical isolates, <1.5% very major and <3%major discrepancies using the total population as the denominatorwas maintained.

In a recent study we tested the susceptibility of a challengeset of Streptococcus pneumoniae isolates to cefotaxime, ceftriaxone,and the ketolide telithromycin by both broth microdilution anddisk diffusion methods. These three drugs currently have noaccepted disk diffusion interpretive criteria. The purpose ofthis study is to assess the applicability of the latest NCCLSguidelines for setting disk diffusion breakpoints for thesethree drugs when S. pneumoniae is tested.

MATERIALS AND METHODS

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Materials and Methods

Microorganisms. A total of 407 clinical isolates of S. pneumoniae from NorthAmerica and Europe was selected to give a broad range of MICswith a large proportion of isolates with cefotaxime and ceftriaxoneMICs of $>=$ 1.0 µg/ml. Isolates were identified by the laboratoriessubmitting them by their own standard methods and were confirmedin our laboratory by colonial-morphology, optochin susceptibility,and bile solubility tests. Of the 407 isolates, 147 strainsexhibited the MLS phenotype and 109 had the M phenotype.

Antimicrobial agents. The control drug erythromycin, as well as cefotaxime and ceftriaxone,was supplied by PML Microbiologicals, Wilsonville, Oreg. Telithromycinwas provided by Aventis Pharmaceuticals, Paris, France. Fordisk diffusion tests, commercially prepared disks (BBL, Cockeysville,Md.) were used as follows: cefotaxime and ceftriaxone, 30 µg;and erythromycin and telithromycin, 15 µg.

Susceptibility tests. MICs for each drug were determined by the broth microdilutionmethod described by the NCCLS (7). Serial twofold concentrationsof each drug were prepared in cation-adjusted Mueller-Hintonbroth supplemented with 3% lysed horse blood. The concentrationstested are indicated in Fig. 1 to 4. Disk diffusion tests wereperformed as recommended by the NCCLS (8) on Mueller-Hintonagar supplemented with 5% defibrinated sheep blood.

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FIG. 1. Erythromycin MICs versus zone diameters with 407 S. pneumoniae isolates. Horizontal and vertical lines represent established MIC and zone diameter breakpoints, respectively (8).

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FIG. 4. Ceftriaxone MICs versus zone diameters with 407 S. pneumoniae isolates. Horizontal and vertical lines represent established MIC breakpoints (8) and best-fit zone diameter breakpoints, respectively.

Quality control. The quality control strain, S. pneumoniae ATCC 49619 was testedby both methods on each day of testing. All results for erythromycin,cefotaxime, and ceftriaxone as determined by both methods fellwithin the current NCCLS quality control limits (9).

RESULTS AND DISCUSSION

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Results and Discussion

MIC zone diameter scattergrams for each of the four drugs testedare provided in Fig.1 to 4, and discrepancy rates are listedin Table 1.

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TABLE 1. MIC zone diameter discrepancy rates for four antibiotics with 407 S. pneumoniae isolates

Erythromycin. Erythromycin MICs had a striking bimodal distribution (Fig.1),and this correlated with a corresponding bimodal distributionof zone diameters. With the current erythromycin MIC and zonediameter breakpoints (3, 9), there was one (0.3%) very majordiscrepancy, four (1.0%) major discrepancies, and no minor discrepancies.We concluded that the established interpretive criteria forerythromycin are satisfactory. The newer criteria were alsoused to calculate discrepancy rates. The rates for $>=$ I + 2 and $<=$ I - 2 would be acceptable (Table 1), but for the four MICs inI + 1 to I - 1, there was one very major (25%) and two major(50%) discrepancies. There were too few strains in this categoryto calculate meaningful discrepancy rates. Had these two majorand one very major discrepant tests been repeated, as is recommended,it is conceivable that the discrepancies would have vanished.

Telithromycin. MIC interpretive criteria have yet to be established for telithromycin.For purposes of this discussion, we elected to use the breakpointsproposed by the manufacturer: $<=$ 1.0 µg/ml for susceptible,2.0 µg/ml for intermediate, and $>=$ 4.0 µg/ml for resistant.Based on these MIC breakpoints, 98% of our isolates would beconsidered susceptible (Fig. 2).This illustrates the type ofresults commonly seen with new antibiotics today. With the zonediameter breakpoints selected there was one (0.3%) major discrepancyin the $<=$ I - 2 group and five (20.8%) minor discrepancies in theI + 1 to I - 1 group, which would be considered acceptable discrepancyrates.

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FIG. 2. Telithromycin MICs versus zone diameters with 407 S. pneumoniae isolates. Horizontal and vertical lines represent proposed MIC breakpoints and corresponding zone diameter breakpoints, respectively.

Cefotaxime. Although MIC breakpoints for cefotaxime have been establishedfor pneumococci (8), attempts to establish zone diameter breakpointshave not been successful (1, 3). The scattergram in Fig. 3illustrateswhy this is so. The major and very major discrepancy rates fallwithin acceptable limits, but the minor discrepancy rate is51.6% in the I + 1 to I - 1 group and is thus well above the40% limit for this group. Because of the process for selectingthese isolates for this study, nearly 80% of isolates fell inthe I + 1 to I - 1 group. In a previous study, in which thecefotaxime MICs for the pneumococcal isolates were more representativeof what is usually encountered in clinical practice, i.e., only22% of MICs were in the I + 1 to I - 1 group (1). Nevertheless,>40% of those zone diameters showed minor discrepancies.The latter authors also tested a 1-µg cefotaxime disk,and the minor discrepancy rate for the I + 1 to I - 1 groupwas reduced 37%, which would be considered acceptable.

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FIG. 3. Cefotaxime MICs versus zone diameters with 407 S. pneumoniae isolates. Horizontal and vertical lines represent established MIC breakpoints (8) and best-fit zone diameter breakpoints, respectively.

Ceftriaxone. The problem seen with cefotaxime is also apparent with ceftriaxone(3) (Fig. 4).The major and very major discrepancy rates aresimilar to those of cefotaxime, but the minor discrepancy ratein the I + 1 to I - 1 group is even higher (71%).

Conclusions. The newer NCCLS guidelines for calculating discrepancy ratesled us to the following conclusions: (i) the established zonediameter breakpoints for erythromycin are appropriate; (ii)acceptable zone diameter breakpoints for telithromycin couldreadily be set, if we assume that tentative MIC breakpointsare proven to be acceptable; and (iii) the minor discrepancyrates for cefotaxime and ceftriaxone disk tests are too excessiveto consider testing these 30-µg disks against pneumococci.It is conceivable that a smaller disk content (e.g., 1 µg)might produce more reliable results.

We believe the latest NCCLS guidelines for setting zone diameterbreakpoints are a step in the right direction. The acceptablediscrepancy rate limits may need to be fine-tuned in the futureas more experience is gained with this method. Judgment willclearly be called for when certain discrepancy rates exceedthe current limits. For example, if there is one very majordiscrepancy in the $>=$ I + 2 group and there are only 10 isolatesin this group (10% discrepancy rate), it would be reasonableto waive the 2% limit if all else fits well.

ACKNOWLEDGMENTS

This work was supported in part by grants from Aventis Pharmaceuticalsand Roche Laboratories.

FOOTNOTES

* Corresponding author. Mailing address: The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070. Phone: (503) 682-3232. Fax: (503) 682-2065. E-mail: cmi{at}hevanet.com..

REFERENCES

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