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Andreas Karameris, MD, PhD Dpt. of Pathology, NIMTS Hospital, M. Petraki 12, 11521 Athens, Greece Athens, January 4, 2013 Dear Editor, Thank you for evaluating our paper entitled ‘Real Time PCR detection and quantitation of Helicobacter pylori clarithromycin-resistance strains in archival material and correlation with Sydney classification’. Please find attached a revised version according to the recommendations of the reviewers and Associate and Section Editor’s comment. General comments: 1) This is a retrospective, molecular mainly analysis in routine archival material. No other data concerning virulence factors (cag, vag, cultures etc), were available. Answer to the reviewers Reviewer Nr 1: Comment 1: In this study, we modified an already known method such as the biprobe ClariResTM RT-PCR assay (which is applicable mainly for detection of H. pylori mutations in stools), to suit at the needs of a modern Surgical Pathology Department. The aforementioned assay is an IVD one (In Vitro Diagnostic). That means that the kit contains all positive and negative controls needed for the secure detection of mutations in the 23S RNA gene of Hp. So, there is no need for extra controls to be included in the assay. Instead of that, in our study we included extra positive and negative controls as mentioned in details in “The real time PCR and genetic analysis” section. The positive controls that used in our lab consisted of microorganisms with known concentration/volume of culture medium. Comment 2: The classical triple therapy is the Standard Triple Therapy based on a PPIi to which two antibiotics (CAM plus amoxicillin - AMPC or MNZ) are associated, for 7 days. The molecular results are clear-cut and not equivocal as you commented, because the kit used in this study is an IVD one. It contains all the necessary positive and negative controls for a step-by-step evaluation of the resistance of H.pylori to clarithromycin, eliminating thus the possibility of leading to wrong results. Of course as already mentioned in your comments, the susceptibility of strains to antibiotics was not fully known and much remained to be clarified. The infection status was determined primarily by histology (mod. Giemsa stain) and furthermore by Q-RT-PCR. Comment 3: We shortened the discussion a great deal, by eliminating ‘excessive’ comments. Comment 4: All pictures were omitted according to yours suggestions. Thank you Reviewer Nr 2: Comment 1: We clarified the aim of the study. The PCR method we used is not a new one as someone should be supposed. In this study, we modified an already known method such as the bi-probe ClariResTM RT-PCR assay (which is applicable mainly for detection of H. pylori mutations in stools), to suit at the needs of a modern Surgical Pathology Department. The aforementioned assay is an IVD one (In Vitro Diagnostic). That means that the kit contains all positive and negative controls needed for the secure detection of mutations in the 23S RNA gene of Hp. Group B of patients is literally a control group, consisted mainly of patients with susceptibility to clarithromycin. Comment 2: It is not our intention in this study to do genotyping of H.pylori in archival material and to perform multivariate analysis between molecular data on one hand, and histology and especially virulence factors on the other, because there is a great deal of publications (more that 25 as revealed in the Medline) on this particularly field, with sometimes controversial results. For example, in an early study of Scholte et al. (Am J Gastrenterol, 2002, 97(7): 1687-1695, they found that the degree of atrophy is associated with vacA S1, m1 and cgaA(+) genotypes, whereas the degree of neutrophil activity was associated with vacA s1 and cagA genotype. vacA S2 and cagA (-) strains appeared more resistant to antibiotic therapy, irrespective of resistance to clarithromycin. In a most recent paper of Agudo et al. (Journal of Clinical Micorbiology, Oct 2010, pp.3703-3707), it seems that s2/m2 strains which are mostly cagA (-) seem to be more resistant to clarithomycin. In our study a detailed histological analysis of gastritis was done according to modified Sydney classification in 2 selectively chosen groups of patients (and not in the general population), and we tried to examine possible correlations between the different histological parameters and molecular data affiliated with the resistance to clarithromycin. To our knowledge so far, only 2 studies in this field were published, focusing mainly on the severity of gastritis (Yang, Katelaris). I would like, at this point, to make a comment related to the density of colonization and molecular/histological changes: In our paper, a significant correlation between the grade of bacterial density estimated by Q-RTPCR and those estimated by histology was noticed. Of course this finding is not new. But the quite un-expending observation is that we also found evidence of Hp presence in (histologically) negative cases, mainly among group B patients. The density of Hp under the microscope in our work seems to be relevant to genetic alterations in 23S rRNA. Is, under these circumstances, possible someone to predict resistance to clarithromycin, looking exclusively to histology? We don’t know yet an it doesn’t seem to be the case so far. What we know is that the eradication rate is not statistically linked with bacterial density nor with gastric activity per se (Tankovic et al. Pathol Biol 2001;49:528-533). Of course there is another paper claimed that Hp density is a predicting factor for eradication, but density was indirectly assessed by urea breath test, stating that urease activity was related to density (Moshkowitz et al. Gut, 1995;36:845-7). However, this relation has not been proved with solid criteria. Taking the above into account, we did add some lines in the discussion section according to Comment 3. Thank you Sincerely, A. Karameris, MD, PhD