The computer program for SRA permits the calculation of the equation constants A and B and the subsequent calculation of zone diameters from MIC values or MIC equivalents from zone diameters.

SRA calculations can for example be used when the disk potency of a new antibiotic is being considered. SRA is also valuable when the regular interpretive zone diameter breakpoints cut through rather homogeneous populations in zone diameter histograms of a single species and antibiotic and therefore give rise to interpretive inconsistencies. SRA will provide a basis for correcting the breakpoints for this combination.

In some rare instances the equation of SRA does not apply, as shown by large differences in the A and B constants for different strains of the same species. If, as shown earlier, there is a correlation of these differences to the MIC values, then a slight variation of the SRA equation can be used. It requires two or more reference strains to be used and the prior calculation of the SRA constants A and B.

There are some important points to remember when using the disk diffusion method for antibiotic susceptibility testing and applying SRA for calibration or analysis.

• Disk diffusion test has to be standardized.
  The disk diffusion test used in the laboratory has to be well standardized in order to provide consistent performance before further calibration is introduced using SRA.
• Reference strain should be representative of the species.
  There is a slight variation among "wild type", susceptible strains of a given species. The reference strain used for calibration should preferably represent a mean performance. Some international control strains do not show a "normal" behaviour in disk testing against some antibiotics.
• MIC-value of reference strain has to be exact!
  The precision of the disk test is greater than the 2-log dilution series commonly used for MIC-testing. Therefore, MIC-values have to be determined for reference strains using also intermediate dilution steps for the necessary accuracy in the SRA calculations. The MIC-values can be calculated as geometric means of repeated tests with intermediate dilutions.
• Disk contents of antibiotic should cover a full linear range.
  The linearity of the disk - zone2 relation has to be checked before using the data for SRA calculations. At least three disk values in the linear range have to be included. Disks giving zone diameters less than 10 mm or greater than 45 mm should be excluded. Such values are definitely outside the linear range.
• SRA has to be performed as any other routine disk test.
  It might seem an unnecessary remark, but so often it has happened that the SRA test is given to the chief technician or the research technician who does not perform the disk test in the very same way as it is done in the routine microbiology laboratory. The data therefore do not reflect the disk test performance which was supposed to be calibrated.

In our experience the need to calibrate the disk diffusion test arises in situations where problem combinations of species and antibiotics have been identified. With a well standardized disk test most interpretations of susceptibility will be correct. We regularly measure the inhibition zones and plot the zone diameters species-wise. Problem combinations of drug/bug are then identified as histogram plots where the interpretive zone diameter breakpoints cut through histogram peaks. This analysis is performed automatically in the ADBact computer program which we use for the total administration of the clinical microbiology laboratory.

For problem combinations of species and antibiotics, species-specific interpretive breakpoints have to be used in the laboratory. SRA calculations can aid in the definition of such breakpoints.

Selected references:

Kronvall, G.: Analysis of a Single Reference Strain for Determination of Gentamicin Regression Line Constants and Inhibition Zone Diameter Breakpoints in Quality Control of Disk Diffusion Antibiotic Susceptibility Testing. Journal of Clinical Microbiology, 16: 784-793, 1982.

Ringertz, S. Kronvall, G. On the theory of the disk diffusion test. Evidence for a nonlinear relationship between critical concentration and MIC, and its practical implications for susceptibility testing of Haemophilus influenzae. APMIS 96: 484-490, 1988.

Kronvall, G. Ringertz, S. Karlsson, I. Göransson, E. Dornbusch, K. Laboratory- and Species-Specific Interpretive Breakpoints for Disk Diffusion Tests of Chloramphenicol Susceptibility of Haemophilus influenzae. Antimicrob. Agents Chemother. 32: 1484-1489, 1988.

Colour graphs © G.Kronvall