Zone Breakpoints Research Articles

Antimicrobial susceptibility testing of Haemophilus influenzae Improvement of accuracy of the disc diffusion test

A national quality control study was performed in 1986 to investigate the standard of performance of the disc diffusion antimicrobial susceptibility testing of Haemophilus influenzae in Sweden. The accuracy of susceptibility interpretations was unacceptably low. A new standardized method for susceptibility testing of H. influenzae was then worked out, and a new method of setting interpretive zone breakpoints was introduced. The susceptibility category of the main population of clinical isolates was determined according to the MIC50 of the strains. The zone histograms of clinical isolates from five reference laboratories were used for the calculation of interpretive breakpoints. For instance, for susceptible strains the mean of the combined zone values from these laboratories +/- 2 S.D. covered the zone range of the susceptible group, and one more S.D. below covered the intermediate/indeterminate group. The new zone breakpoints would place the main population of clinical isolates in the correct susceptibility group, and even make it possible to detect strains with different degrees of reduced susceptibility in the routine test. In a follow-up quality control study in 1988 the interpretive errors for clinical isolates were eliminated for all antibiotics except doxycycline in some laboratories. Laboratory-related zone breakpoints for doxycycline calculated by the single strain regression analysis method led to correct susceptibility interpretations also in these cases.

Types of interpretive errors in susceptibility testing. Zone breakpoints for norfloxacin disk diffusion testing.

A total of 548 strains of the eleven most common urinary tract pathogens were investigated for possible errors in norfloxacin susceptibility tests comparing MIC determinations with disk diffusion assays. Most strains were found to be sensitive with MIC-90 values below 1.0 for the Enterobacteriaceae while the classical nalidixic acid resistant species, the gram-positive bacteria and Pseudomonas aeruginosa, were less susceptible to norfloxacin with MIC-90 above 1.0 mg/l. MIC-values close to interpretive MIC-limits were recorded for S. faecalis and S. agalactiae using the recommendations of the national Committee for Clinical Laboratory Standards (NCCLS) (susceptible, S less than or equal to 4.0) and for P. aeruginosa and S. aureus using the Swedish Reference Group for Antibiotics (SRGA) standards (S less than or equal to 1.0). Susceptibility interpretations for these species showed a lack of accuracy consistent with methodological problems of reproducibility, an error called type I. The changes in the MIC-limits required for these strains to correct the error would be S less than or equal to 4 for P. aeruginosa and S. aureus, S less than or equal to 8 for S. agalactiae and S less than or equal to 0.5 for S. faecalis. A type II error, occurring when a bacterial species shows a regression line different from the regular line, was also identified for S. saprophyticus. The use of breakpoints derived from single strains regression analysis corrected this error and also reduced the frequency of similar misinterpretations in other species. The term "species-specific MIC-limits" should be introduced along with the established concept of "species-specific interpretive zone breakpoints" to allow for the correction of type I interpretive errors. Type II errors can be corrected by using species-specific interpretive breakpoints, either issued by reference laboratories or derived by calculations from single-strain regression analysis in the individual laboratory.

Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria.

Twelve Capnocytophaga and seven DF-2 strains were tested for their susceptibility to 14 antimicrobial agents using an agar dilution and an agar diffusion method. Twenty-three other antibiotics were evaluated using the diffusion test only. All strains were fully susceptible to penicillin, ampicillin, cefuroxime, cefotaxime, erythromycin, clindamycin, chloramphenicol, doxycycline, rifamycin and ofloxacin using both methods. Clindamycin, rifamycin and cefotaxime were most active. Using agar dilution some strains were susceptible to gentamicin, but agar diffusion showed total resistance. One Capnocytophaga strain was susceptible and another moderately susceptible to metronidazole, other strains were resistant. The agar diffusion test showed that both Capnocytophaga and DF-2 were resistant to most other aminoglycosides, to fosfomycin, polymyxin and trimethoprim. All strains of both taxa were fully susceptible to piperacillin, cefoxitin, imipenem and fusidic acid and showed different susceptibilities to the other agents. Susceptibility testing by means of agar diffusion using an enriched chocolate agar and 5% CO2 atmosphere could be used to test Capnocytophaga and DF-2 strains and gives sufficient accuracy for routine use, when revised inhibition zone breakpoints are employed.

Criteria for interpretation of agar diffusion susceptibility tests of norfloxacin performed by the ICS method.

Using regression analysis to correlate MIC values and inhibition zone diameters, an attempt was made to establish criteria for interpreting the results of the agar diffusion test with norfloxacin discs (10 mcg). The test was performed on DST agar in accordance with ICS recommendations. The correlation was moderate (r = -0.8547); the regression equation was x = -0.30y + 15.80, where x was log2 MIC + 9 and y the inhibition zone diameter. The position of the MIC breakpoints of norfloxacin and the distribution of the MIC values of the bacterial population did not permit precise inhibition zone breakpoints to be established. To allow a safety margin it is recommended that for the meantime norfloxacin inhibition zones be interpreted as sensitive from 15 mm onwards.