Vincenzo De Francesco

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

Effects of Helicobacter pylori eradication on early stage gastric mucosa-associated lymphoid tissue lymphoma

BACKGROUND & AIMS Different remission rates of gastric low-grade, B-cell, mucosa-associated lymphoid tissue (MALT) lymphoma have been reported after Helicobacter pylori eradication. We assessed the long-term remission and relapse rates of early stage MALT lymphoma in patients treated only by H pylori eradication and identified factors that might predict outcome. METHODS This systematic review analyzed data from 32 studies, including 1408 patients. RESULTS The MALT lymphoma remission rate was 77.5% (95% confidence interval, 75.3-79.7), and was significantly higher in patients with stage I than stage II(1) lymphoma (78.4% vs 55.6%; P = .0003) and in Asian than in Western groups (84.1% vs 73.8%; P = .0001). Neoplasia confined to the submucosa regressed more frequently than that with deeper invasion (82.2% vs 54.5%; P = .0001); patients with lymphoma localized to the distal stomach experienced regression more frequently than those with lymphoma of the proximal stomach (91.8% vs 75.7%; P = .0037). The remission rate was higher among patients without the API2-MALT1 translocation than in those with this translocation (78% vs 22.2%; P = .0001). In an analysis of data from 994 patients, 7.2% experienced lymphoma relapse during 3253 patient-years of follow-up evaluation, with a yearly recurrence rate of 2.2%. Infection and lymphoma were cured by additional eradication therapy in all patients with H pylori recurrence (16.7%). Five (0.05%) of the patients initially cured of lymphoma developed high-grade lymphoma within 6 to 25 months of therapy. CONCLUSIONS H pylori eradication is effective in treating approximately 75% of patients with early stage gastric lymphoma. Long-term follow-up evaluation of these patients is needed to detect early lymphoma relapse or progression.

Mechanisms of Helicobacter pylori antibiotic resistance: An updated appraisal

Helicobacter pylori (H. pylori) antibiotic resistance is the main factor affecting the efficacy of the current eradicating therapies. The aim of this editorial is to report on the recent information about the mechanisms accounting for the resistance to the different antibiotics currently utilized in H. pylori eradicating treatments. Different mechanisms of resistance to clarithromycin, metronidazole, quinolones, amoxicillin and tetracycline are accurately detailed (point mutations, redox intracellular potential, pump efflux systems, membrane permeability) on the basis of the most recent data available from the literature. The next hope for the future is that by improving the knowledge of resistance mechanisms, the elaboration of rational and efficacious associations for the treatment of the infection will be possible. Another auspicious progress might be the possibility of a cheap, feasible and reliable laboratory test to predict the outcome of a therapeutic scheme.

Standard triple and sequential therapies for Helicobacter pylori eradication: An update

H. pylori infection remains a worldwide spread disease with a definite morbidity and mortality. Unfortunately, no current therapy regimen is able to cure the infection in all treated patients. The efficacy of the widely recommended triple therapies is decreasing, and a novel 10-day sequential therapy has been proposed. Data of 3 previous meta-analyses showed a significantly higher eradication rate following the sequential as compared to the 7-10 days triple therapies. The sequential therapy achieved significantly better results than triple therapies in children, elderly patients, non-ulcer dyspepsia patients, and in those infected with resistant strains towards either clarithromycin or metronidazole. We identified further 10 randomized trials. By pooling data, H. pylori infection was cured in 2,454 (86%; 95% CI: 84.7-87.3) out of 2,853 patients with the sequential therapy and in 2,320 (75.3%; 95% CI: 73.8-76.9) out of 3,079 patients treated with standard triple therapies (p<0.001), corresponding to a number to treat (NNT) of 9. The comparison between the 10-day sequential regimen and 14-day triple therapies deserves further investigations.

Concomitant, sequential, and hybrid therapy for H. pylori eradication: A pilot study

BACKGROUND AND OBJECTIVE Since the efficacy of the standard triple therapies for Helicobacter pylori eradication has decreased, novel antibiotic regimens have been introduced, including concomitant, sequential, and hybrid therapies. We aimed to compare the cure rates achieved by these new therapy regimens. METHODS This was a multicenter, open-label, pilot study enrolling consecutive non-ulcer dyspepsia patients with H. pylori infection never previously treated for the infection. Patients were randomized to receive one of the following treatments: (a) concomitant therapy: omeprazole 20mg, amoxicillin 1g, clarithromycin 500 mg, and tinidazole 500 mg for 5 days; (b) sequential therapy: omeprazole 20mg and amoxicillin 1g for 5 days followed by omeprazole 20mg, clarithromycin 500 mg, and tinidazole 500 mg for 5 days; (c) hybrid therapy: omeprazole 20mg, and amoxicillin 1g for 7 days followed by omeprazole 20mg, amoxicillin 1g, clarithromycin 500 mg, and tinidazole 500 mg, for 7 days. All drugs were administered twice daily. Bacterial eradication was checked 6 weeks after treatment by using a (13)C-urea breath test. A 10-day, second-line therapy with omeprazole 20mg, levofloxacin 250 mg, and amoxicillin 1g, all given twice daily, was offered to the eradication failure patients. RESULTS Overall, 270 patients were enrolled, but 13 patients early interrupted treatment due to side effects. At intention-to-treat (ITT) and per-protocol analysis (PP), the eradication rates were 85.5% and 91.6% with the concomitant regimen, 91.1% and 92.1% with the sequential therapy, and 80% and 85.7% with the hybrid regimen. Differences were not statistically significant. H. pylori infection was cured in 10 (55.6%) patients with the second-line regimen. CONCLUSION In our study, both concomitant and sequential therapy, but not hybrid therapy, reached high eradication rates. The success rate of second-line levofloxacin-based triple therapy is decreasing.

A randomized controlled trial evaluating a new 2-L PEG solution plus ascorbic acid vs 4-L PEG for bowel cleansing prior to colonoscopy

BACKGROUND Bowel preparation is critical for the efficacy and safety of colonoscopy. Poor patient tolerance to bowel preparation has been associated with the high amount of fluid administered. A 2-L polyethylene glycol (PEG) solution containing ascorbic acid has been recently developed. AIMS To compare the efficacy, safety and acceptability of 2-L PEG+ascorbic acid vs 4-L PEG for colonoscopy. METHODS We designed a single blind randomized non-inferiority study in order to compare the two bowel preparations. A blinded assessment of cleansing was made by the endoscopist according to the Aronchick scale. Acceptability was assessed by questionnaire. Intention-to-treat (ITT) and per-protocol (PP) analysis were reported. RESULTS Overall, 169 patients (PP: 166) were selected for the 2-L PEG+ascorbic acid and 170 (PP: 166) for the 4-L PEG. When rating global bowel cleansing at ITT, an excellent-good level was reported in 84.6% (PP: 86.2%) of patients who received 2-L PEG+ascorbic acid and 75.3% (PP: 77%) of patients who received 4-L PEG (p=0.04). Acceptability rate favoured 2-L PEG+ascorbic acid vs 4-L PEG (83% vs 76%; p=0.02). CONCLUSIONS 2-L PEG+ascorbic acid, completed with an additional L of clear fluids, provided bowel cleansing which appeared to be more effective and acceptable than 4-L PEG.

Onset of liver damage after a single administration of infliximab in a patient with refractory ulcerative colitis.

We report the case of a patient with refractory ulcerative colitis who developed cholestatic acute liver damage after a single infusion of infliximab. Unusual aspects of this case were the early onset (after the first administration) of liver damage and the absence of antinuclear antibodies, alcohol intake, hepatotoxic drugs and all known viral and metabolic causes of hepatic injury. Moreover, no serological or morphological findings of primary sclerosing cholangitis were observed. The patient’s liver damage resolved spontaneously within 6 weeks. Although a direct relationship between administration of infliximab and onset of acute liver damage could not be definitely established, our case suggests that infliximab may induce direct liver damage, the course of which is similar to acute cholestatic hepatitis and resolves following withdrawal of the drug.

Cost-effectiveness of endoscopic surveillance for gastric intestinal metaplasia

Background:  Patients with intestinal metaplasia (IM) are at increased risk for gastric cancer. Endoscopic surveillance has been shown to anticipate cancer diagnosis in an earlier stage. Cost‐effectiveness of endoscopic surveillance in IM patients is unknown. To assess the efficacy and cost‐effectiveness of an yearly endoscopic surveillance in patients with IM.

Eradication therapy in helicobacter pylori-negative, gastric low-grade mucosa-associated lymphoid tissue lymphoma patients: A systematic review

Aim: To assess the remission rate of gastric low-grade B-cell mucosa–associated lymphoid tissue lymphoma after an eradication therapy in Helicobacter pylori-negative patients. Methods: We performed a systematic review with pooled analysis of published studies. Data were analyzed according to: (1) number of H. pylori-negative patients treated with only eradication therapy; (2) number of patients in whom the complete lymphoma remission was achieved; and (3) the method used to exclude H. pylori infection. Results: Overall, 11 studies with 110 patients met the inclusion criteria for this pooled analysis. H. pylori infection was excluded in all studies with at least 3 different diagnostic tests. Eradication therapy achieved a complete lymphoma regression in 17 (15.5%; 95% confidence interval, 8.7-22.2) patients. Conclusions: Eradication therapy is successful in a small but distinct subgroup of H. pylori-negative patients with low-grade mucosa–associated lymphoid tissue lymphoma. On the basis of the generally indolent behavior of this neoplasia, before resorting to aggressive, costly, and potentially more toxic oncologic therapies, it would seem reasonable to attempt eradication therapy in all patients, irrespective of their H. pylori status.

Mucosal assessment of tumor necrosis factor alpha levels on paraffined samples: a comparison between immunohistochemistry and real time polymerase chain reaction

Author(s) : Enzo IerardiArticle title : Mucosal assessment of tumor necrosis factor alpha levels on paraffined samples:a comparison between immunohistochemistry and real time polymerase chainreactionArticle no : 483739Dear Author,Please check these proofs carefully. It is the responsibility of the corresponding author to checkagainst the original manuscript and approve or amend these proofs. A second proof is notnormally provided. Informa Healthcare cannot be held responsible for uncorrected errors, even ifintroduced during the composition process. The journal reserves the right to charge for excessiveauthor alterations, or for changes requested after the proofing stage has concluded.The following queries have arisen during the editing of your manuscript and are marked in themargins of the proofs. Unless advised otherwise, submit all corrections using the CATS onlinecorrection form. Once you have added all your corrections, please ensure you press the “SubmitAll Corrections” button.