Multilaboratory comparison of proficiencies in susceptibility testing of Helicobacter pylori and correlation between agar dilution and E test methods

Abstract

Susceptibility testing was performed at seven Canadian microbiology laboratories and the Helicobacter Reference Laboratory, Halifax, Nova Scotia, Canada, to assess susceptibility testing proficiency and the reproducibility of the results for clarithromycin and metronidazole and to compare the Epsilometer test (E test) method to the agar dilution reference method. Control strain Helicobacter pylori ATCC 43504 (American Type Culture Collection) and 13 clinical isolates (plus duplicates of four of these strains including ATCC 43504) were tested blindly. The National Committee for Clinical Laboratory Standards (NCCLS) guidelines for agar dilution testing were followed, and the same suspension of organisms was used for agar dilution and E test. Antimicrobials and E test strips were provided to the investigators. Methods were provided on a website (www.Helicobactercanada.org). Each center reported MICs within the stated range for strain ATCC 43504. Compared to the average MICs, interlaboratory agreements within 2 log(2) dilutions were 90% (range, 69 to 100%) for clarithromycin by agar dilution, with seven very major errors [VMEs], and 85% (range, 65 to 100%) by E test, with three VMEs. Interlaboratory agreements within 2 log(2) dilutions were 83% (range, 50 to 100%) for metronidazole by agar dilution, with six VMEs and eight major errors (MEs), and 75% (range, 50 to 94%) by E test, with four VMEs and four MEs. At lower and higher concentrations of antibiotic, E test MICs were slightly different from agar dilution MICs, but these differences did not result in errors. When a standardized protocol based on NCCLS guidelines was used, most participants in this study correctly identified clarithromycin- and metronidazole-susceptible and -resistant strains of H. pylori 93% of the time by either the agar dilution or E test method, and the numbers of errors were relatively equivalent by both methods.

FIG. 1.

Errors for clarithromycin (CLA) and metronidazole (MET) MICs by agar dilution (AD) and E test (ET) relative to the average MIC for each H. pylori strain by method (A) and site (B). VMEs are resistance classified as sensitivity by the test, and MEs are sensitivity classified as resistance by the test. There were 32 errors in total.

FIG. 2.

Percent distribution of clarithromycin (CLA) and metronidazole (MET) log₂ dilution MICs by agar dilution (AD) and E test (ET) relative to the average agar dilution MICs for all strains of H. pylori from all sites. A dilution at 0 is equivalent to the average MIC for that strain of H. pylori. Results at −3 and 3 include dilutions equal to and less than or greater than 3 log₂ dilutions different from the average, respectively. Compared to the reference agar dilution method, E test MICs tended to be lower than the average, but the numbers of unacceptable results at −3 or 3 were relatively equivalent by all methods.

FIG. 3.

Comparison of the average of all paired agar dilution (AD) and E test (ET) MICs of clarithromycin (A) and metronidazole (B) for each of the 13 H. pylori strains. R^a, the NCCLS cutoff for clarithromycin resistance is an MIC equal to or greater than 1 μg/ml; R^b, the generally accepted cutoff for metronidazole resistance is an MIC greater than 8 μg/ml. For clarithromycin the overall correlation between the agar dilution method and E test was slightly higher than that for metronidazole, but by both methods with clarithromycin and metronidazole, there was a clear distinction between sensitive and resistant strains.

FIG. 4.

Correlation of E test (ET) and agar dilution (AD) MIC results for all pairs of tests with clarithromycin (A) and metronidazole (B). Values greater than 6 log₂ dilutions different from the average MIC by both methods were excluded from the analysis.