O. Burattini

Clarithromycin-Resistant Genotypes and Eradication of Helicobacter pylori

Context Point mutations in the peptidyltransferase region of the 23S ribosomal RNA gene may be responsible for Helicobacter pylori clarithromycin resistance. Contribution This study related mutations to eradication rates in 156 adults treated with clarithromycin regimens for H. pylori infection. Eradication was successful in 14 of 15 patients with either A2142G or A2142C point mutations but in only 11 of 23 patients with the A2143G point mutation. Cautions This was a post hoc subgroup study of selected participants in a multicenter randomized trial. Implications The A2143G point mutation may be associated with a low eradication rate of H. pylori infection. The Editors Helicobacter pylori infection plays a major role in peptic ulcer disease, low-grade mucosa-associated lymphoid tissue lymphoma, and gastric cancer (1), and its eradication dramatically affects the natural history of both peptic ulcer and gastric lymphoma (2). European and U.S. guidelines advised the use of triple therapies (proton-pump inhibitor, clarithromycin plus amoxicillin, or metronidazole) for 7 to 14 days to cure this infection (3, 4). However, H. pylori resistance against clarithromycin is increasing worldwide, reducing the success rate of standard triple therapies to mean values as low as 18% to 44% (5-7). Novel culture-free polymerase chain reaction (PCR)based assays have allowed the detection of the genetic mutations that are involved in the mechanisms of clarithromycin resistance (8, 9). In detail, A2143G and A2142G transitions are the most prevalent point mutations in Europe and the United States (10, 11), while the A2144G mutation is more frequent in Asia (12, 13). Although such genetic mutations have been associated with different degrees of bacterial resistance in vitro, data are still conflicting (7, 14). Moreover, no study has assessed the role of these different mutations on H. pylori treatment outcome. In a recent multicenter study, a novel sequential regimen, consisting of a simple dual therapy given for the first 5 days followed by a triple therapy for the remaining 5 days, achieved a very high cure rate as compared with standard triple therapy (92% vs. 74%) (15). Whether such a high cure rate may depend on increased efficacy of the sequential regimen against the clarithromycin-resistant strains is unknown. We wanted to evaluate the role of different point mutations in the success of eradication therapy and to compare the efficacy of standard triple therapy and the sequential regimen for these mutations. Methods Study Design To assess the role of primary clarithromycin resistance in therapeutic outcome, we designed a post hoc subgroup analysis of a previous study involving 8 Italian centers (15). In detail, we selected patients from those who were previously enrolled by our 2 centers to participate in a multicenter study between January and December 2001 (Figure). Demographic and clinical characteristics of patients enrolled in our substudy were similar to those of patients enrolled in the original multicenter study. Briefly, in the original study, Zullo and colleagues (15) allocated patients who were never treated for H. pylori infection, according to a computer-generated randomization list drawn in each center, to receive standard 7-day treatment (20 mg of rabeprazole, 500 mg of clarithromycin, and 1 g of amoxicillin twice daily) or 10-day sequential therapy (20 mg of rabeprazole plus 1 g of amoxicillin twice daily for the first 5 days followed by 20 mg of rabeprazole, 500 mg of clarithromycin, and 500 mg of tinidazole twice daily for the remaining 5 days). To assess H. pylori status, the investigators performed upper endoscopy with several gastric biopsies for histology (Giemsa staining), a rapid urease test, and a standard 13C-urea breath test at entry and at 4 to 6 weeks after therapy. Investigators considered infections to be eradicated when all 3 test results were negative and considered treatment to have failed when at least 1 test result was positive. The local ethics committee approved the protocol, and all participants gave written informed consent. Figure. Flow chart showing data of patients recruited in this substudy from the previous multicenter study. For our current study, we selected 75 of 192 patients who were treated with standard triple therapy and 81 of 185 patients who were treated with the sequential regimen in the 2 participating centers. We recruited patients consecutively from the randomization lists of the previous study, independent of the eradication status. The final study sample included 58 of 140 patients whose infections were eradicated and 17 of 52 patients whose infections were not eradicated after standard triple therapy and 76 of 177 patients whose infections were eradicated and 5 of 8 whose infections were not eradicated after the sequential regimen. Clarithromycin Resistance Assessment We assessed the 3 point mutations (A2142C, A2142G, and A2143G) of clarithromycin resistance by using a validated real-time PCR, as reported elsewhere (16). Briefly, we extracted the DNA by using NucleoSpin Tissue (Macherey-Nagel GmbH & Co., Dren, Germany), according to the manufacturers instructions, applied on paraffin-embedded sections. We applied the same procedure to homogeneous bacterial cultures of H. pylori (positive and negative controls), for which clarithromycin resistance had been previously assessed with Etest (AB BIODISK, Solna, Sweden). We estimated final DNA concentrations by ultraviolet absorbance at 260 nm. Preparation of the Probes and Primers We designed TaqMan minor groove binder (MGB) probes and primers to hybridize with wild-type and mutant DNA by using the Primer Express program and Custom TaqMan SNP Genotyping Assay service (Applied Biosystems, Foster City, California) that synthesized the primers and probes for each mutation. Genotyping Assay The assay reagents for the genotyping single nucleotide mutation from the Assays-by-Design service (Applied Biosystems) consisted of a 40X mix of unlabeled PCR primers and TaqMan MGB probes (FAM and VIC fluorochrome dyelabeled). These assays were designed for the genotyping of specific mutations. Each assay enables scoring of both genotypes in a single well. Since a recent study showed that the conjugation of MGB to oligonucleotides stabilizes nucleic acid duplexes, causing a dramatic increase in oligonucleotide melting temperature (17, 18), we used an attachment of the MGB, which enables the use of shorter fluorogenic probes, thus resulting in improved mismatch discrimination. Our probes were distinguished by being labeled with different fluorescent reporter dyes (FAM dye and VIC dye). A substantial increase in FAM or VIC dye fluorescence indicated homozygosity for the FAM- or VIC-specific allele, while an increase in both signals indicated heterozygosity (19). Real-Time PCR Assay and Allelic Discrimination We performed the real-time PCR procedure according to the method of Wada and colleagues (20). We enclosed positive and negative controls in each assay. We analyzed fluorescence of hybridized probes by multicomponent graphics, where we examined dye-labeled (FAM and VIC), background, and passive control (ROX fluorochrome dye-labeled) fluorescence and expressed them as normalized reporter signal (Rn). We clustered all samples by using the maximum likelihood algorithm based on the ratio of normalized reporter dye signal. The result of the analysis yields 3 major clusters corresponding to the 3 genotypic constituents: wild-type homozygous, mutated-type homozygous, and heterozygous. Characterization of Positive and Negative Controls by Amplification and Sequencing of the Hp23S Fragment We obtained the Hp23S fragment by PCR amplification of H. pylori extracted DNA from homogeneous bacterial cultures (strains with and without clarithromycin resistance, previously assessed by Etest) by using primer Hp23-F (5-CCACAGCGAT GTG GTCTCAG-3) and Hp23-R (5-CTCCATAAGAGCCAAAGCCC-3) according to conventional PCR assay (21). Before sequencing, we purified the PCR products by using the Wizard PCR preps (Promega, Madison, Wisconsin). We performed the sequencing reaction with the same primers for PCR, as described by Sanger and colleagues (22), by using the Dye Terminator 3.1 Ready Reaction Kit (Applied Biosystems) as indicated by the manufacturer. We performed sequencing on the 2 strands of each PCR product with the automated ABI Prism 377 DNA Sequencer (Applied Biosystems) and aligned the resulting nucleotide sequence by using the Sequence Navigator software package (Applied Biosystems). Statistical Analysis We determined sample size before the start of the study on the basis of the available data in the literature. In detail, an eradication rate ranging from 18% to 44% was reported after standard triple therapy in patients with primary clarithromycin-resistant strains (5-7), whereas the sequential regimen eradicated the infection in 79% of such patients (15). Assuming a high eradication rate for the triple therapy (45%) and a relatively poor success rate for the sequential regimen (70%) in patients with primary clarithromycin-resistant strains, we calculated that at least 68 patients per group were needed to detect a statistically significant difference with 0.8 power and an level of 0.05 (2-sided). After the study was completed, we realized that our sample size estimate provided the necessary number of clarithromycin-resistant patients and should have been inflated, on the basis of a presumed overall rate of clarithromycin resistance, to provide an estimate of total sample size. We compared eradication rates by H. pylori clarithromycin-resistant strain mutation (A2142C, A2142G, and A2143G) by using the Fisher exact test or chi-square test, as appropriate. We determined point mutation groupings after reviewing eradication rates by individual mutation. We compared clinical characteristics among the different groups by using the Student t-test for unpaired da

Sequential treatment for Helicobacter pylori does not share the risk factors of triple therapy failure.

Background : Predicting factors for the outcome of conventional Helicobacter pylori triple therapy have been identified. Of these, the presence of the CagA gene is a strong predictor of successful treatment. Our preliminary data show that this factor becomes irrelevant when sequential therapy is used.

Epithelial cell proliferation of the colonic mucosa in diverticular disease: a case–control study

Background : A higher risk of both advanced adenoma and carcinoma occurs in the sigmoid colon of patients with diverticular disease, for which bacterial carcinogens have been claimed to play a role.

Epithelial Proliferation and ras p21 Oncoprotein Expression in Rectal Mucosa of Patients with Ulcerative Colitis

In ulcerative colitis (UC), epithelial proliferation plays a role in crypt repair and neoplastic evolution. Proliferative status is predominantly connoted in active disease, but not defined in remission. Histologically, remission is characterized by normalization of the picture or development of atrophy. Mutation of the ras oncogene is involved in intestinal carcinogenesis. Aim of this work was to assess the proliferative pattern of rectal epithelium in UC during disease activity and in remission and correlate it with ras oncoprotein p21. The study was performed retrospectively in rectal biopsies from four groups each of 10 patients: active ulcerative colitis (AUC), remission with a normal histology (RUC), remission with rectal atrophy (ARUC), and irritable bowel syndrome (C, control group). In all, immunohistostain was employed to evaluate the proliferation cell nuclear antigen labeling index (PCNA LI) and ras p21. Statistical analysis was performed by ANOVA and Student-Neumann-Keuls tests. %PCNA LI was significantly higher in AUC and ARUC than in RUC and C. Positive cells were predominant in the lower zone of crypts in RUC and C, while a significant expression of PCNA was also observed in the upper areas in AUC and ARUC. Oncoprotein p21 was expressed on the apical surface of the epithelium in 3/10 AUC patients, in all 10 ARUC patients and in none of RUC and C. %The persistently increased epithelial proliferation associated with ras p21 expression in ARUC may be due to the action of an abnormal, mutated ras gene that could play a role in UC-related tumorigenesis.

A new semiquantitative method of quantifying Helicobacter pylori in antigen stools.

Stool antigen test for Helicobacter pylori, a noninvasive assay, is emerging as a strong competitor to urea breath test (UBT). Nevertheless, although the UBT delta value is a semiquantitative indicator of H. pylori intragastric load, until now the H. pylori stool antigen test has been used only as a qualitative investigation. We report here the results of a study performed with the aim of obtaining a semiquantitative measurement of bacterial amount in stools. We studied 15 patients with dyspepsia using H. pylori positivity at histology, the rapid urease test, UBT, and the H. pylori stool antigen test. The result of this last test was expressed by a numerical value we obtained by applying the principle of “standard points” to the absorbance units at spectrophotometric reading. This measurement was previously validated by testing probe sampling of H. pylori stool antigen with known pure and stool-mixed bacterial amounts. A numerical result for H. pylori stool antigen was correlated to UBT delta for each patient using Pearsons r test. Finally, a Student t test was performed to investigate possible differences in UBT and H. pylori stool antigen test values between anti-CagA–positive and -negative patients. We obtained a curve of saturation with both known amount of pure and stool-mixed bacteria. Pearsons r test showed a significant correlation between UBT delta value and H. pylori stool antigen measurement (r = 0.77;p < 0.001). Urea breath test delta and H. pylori stool antigen test values were significantly higher in anti-CagA–positive patients. Our data suggest that a numerical estimation of H. pylori stool antigen may be feasible. This evaluation, similarly to UBT delta, may represent a semiquantitative determination of bacterial intragastric load.

Gastric epithelial cell proliferation and ras oncogene p21 expression in first-degree relatives of gastric cancer patients : a case-control study

Objectives Individuals with a family history of gastric cancer have an increased risk of developing such neoplasia. This study aimed to assess epithelial cell proliferation and ras oncogene mutation in such individuals. Methods Twenty dyspeptic, first-degree relatives of patients with gastric cancer and 20 matched controls were enrolled. Endoscopy with biopsies was performed in all cases. Gastric specimens were used to look for Helicobacter pylori infection and to assess both epithelial cell proliferation and ras oncogene expression by immunohistochemistry. Results Cell proliferation values were not significantly different between the patient and control groups (18.1±7.1 versus 18.9±7.4; P=0.7). Overall, ras mutation was detected in five out of 40 cases, and its distribution was similar between patients and controls (20 versus 10%; P=0.9), as well as between H. pylori-positive and negative patients (22 versus 9%; P=0.2). Cell proliferation values tended to be higher in cases with ras mutation than in those without (25.2±9.4 versus 16.8±5.8; P=0.08). Cell proliferation values were significantly higher in H. pylori-positive cases compared with uninfected cases, in both patient (24.7±4.7 versus 12.5±2.4; P=0.0003) and control (25.9±4.8 versus 13.3±2.8; P=0.0003) groups. Conclusions Both gastric cell proliferation values and ras mutation prevalence did not differ between first-degree relatives of gastric cancer patients and controls. H. pylori infection similarly increased the proliferation index of gastric mucosa in both groups.

Immunohistostaining of hepatitis C virus non-structural protein 4 in ependymocytes of uninfected mice: an antigenic mimicry?

IntroductionIn 2003, a review by Forton et al. suggested thepossibility of finding cerebral dysfuctions in patientswith hepatitis C virus (HCV) infection [1]. Aprevious study, moreover, demonstrated that pa-tients with HCV infection showed slight but sig-nificant neurocognitive impairment, possiblyindicating a further extrahepatic manifestation ofchronic C hepatitis [2]. In addition, Main et al.showed by proton magnetic resonance spectroscopythat choline/creatine ratio is increased in basalganglia and white matter of patients with HCVinfection compared to healthy subjects [3]. There-fore a direct viral effect on the central nervoussystem was hypothesized and this assumption wassupported by the demonstration of HCV nucleotidesequences in cerebrospinal fluid [4] and in braintissue obtained at autopsy in patients with recurrenthepatitis C after liver transplantation [5]. However,the possibility of mimicry between viral antigens andpeptidic molecules has been invoked to explaindemyelinating neuropathies [6] and a link betweenHCV and autoimmune hepatitis has been empha-sized [7].On these bases, we investigated, by immunohis-tochemistry, the possibility of an antigenic mimicrybetween HCV and brain tissue in an animal modelshowing two main peculiarities: unreceptiveness tothe infection [8]; brain size allowing observation ofthe whole organ in a single histological section.Material and methodsFive BALB/c mice with normal serum transaminaselevels and negative antibodies to HCV were sacri-ficed. The whole brain was divided into two symme-trical portions, fixed in 10% buffered formalin andembedded in paraffin. Histological sections of 4 mmwere incubated with mouse monoclonal antibody toHCV-NS4 (Argene Biosoft, Varilhes, France)diluted 1:30 in phosphate buffered saline (PBS).The dilution was decided on the basis of theimmunohistochemical performance obtained in po-sitive controls using different dilutions ranging from1:10 to 1:100 as well as on the basis of our previousexperience [9]. The reaction was then revealed usinga specific mouse-on-mouse (M.O.M.) immunode-tection kit (M.O.M. peroxidase; Vector Labora-tories, Burlingame, Calif., USA). This method hasbeen developed specifically to localize mouse pri-mary monoclonal antibodies on mouse tissues,precluding the inability of anti-mouse secondaryantibody to distinguish between the mouse primary