E-test for Metronidazole Susceptibility Q1 in H pylori: Use of the Wrong Standard May Have Led to the Wrong Conclusion: Reply

Dear Sir, We are very grateful for the interest Dr. Osato and Graham (1) showed in our work (2).We would like to point out a few points. (1) We start from an inoculum size of approximately 108 CFU/ml as recommended by the NCCLSS-approved agar dilution method. With fast-growing bacteria, such a concentration corresponds to the opacity standard McFarland 0.5 With H. pylori, in order to obtain 108 vital organisms per ml, you have to suspend the biomass up to McFarland opacity standard # 4. (2) You cited as example the result published by Megraud et al. (3), and we agree with you, but, unfortunately, they used an opacity standard of McFarland # 3, prepared in brucella broth from 48-h agar plate cultures and read after 48 h, instead of McFarland # 2 prepared in a saline solution from a 72-h agar plate and read after 72 h, described in NCCLS procedures; is that work wrong? (3) Anyway, preliminary tests assessed in about 20 strains showed no discrepancies in the pattern of susceptibility between inocula corresponding to McFarland # 2 and # 4. (4) We showed, together with other authors (4, 5), that the reliability of E-test is linked to the presence of mixed infection, and the different geographic prevalences of bacterial subpopulation as well as the increase of mixed infection in the treated patients are well known. So the different results published in the literature may be due to these two factors. In fact, we assessed the presence of mixed infection in 13.9% of the studies, while this percentage ranged from zero to 80% in other studies. (5) Moreover, we specified that the strains tested for the studywere isolated from patientswho had never been treated. Finally, wan der Wouden (4), Piccolomini (6), Glupczynski (7), and Hirschl (8) found a good correlation between agar dilution and E-test, so we think that when a lot of “small” studies show similar results they have to be considered