Hervé Lamouliatte

Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori

ABSTRACT The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

Diagnosis of Helicobacter pylori Infection: Noninvasive Methods Compared to Invasive Methods and Evaluation of two New Tests

OBJECTIVES:Current guidelines recommending Helicobacter pylori eradication treatment without performing endoscopy in certain patients highlight the importance of noninvasive tests. Our aim was to determine the accuracy of two new tests: the antigen stool test and Helicoblot 2.1 (an immunoblot used on serum) as well as the 13C urea breath test and ELISA serology in comparison to invasive tests for the pretreatment diagnosis of H. pylori infection.METHODS:Helicobacter pylori infection was diagnosed prospectively in 104 untreated patients using eight different tests. Invasive tests included culture, urease test (CLOtest), histology, and PCR; noninvasive tests included the 13C urea breath test, IgG serology (Pyloriset EIA-G), immunoblot (Helicoblot 2.1), and antigen stool detection (Premier Platinum HpSA). A predefined gold standard based on biopsy tests was used to define H. pylori status, as well as an empirical approach.RESULTS:There was no statistically significant difference between the different tests. The sensitivity of the noninvasive tests ranged between 88.9% and 95.6% (stool test: 88.9%, 95% CI: 82.7–95.1, and Helicoblot 2.1: 95.6%, 95% CI: 91.5–99.6) and the specificity ranged between 92.6 and 98.1% (stool test: 94.4%, 95% CI: 84.6–98.8, and Helicoblot 2.1: 92.6%, 95% CI: 91.5–96.2) when a predefined gold standard was used.CONCLUSIONS:Most tests had sensitivities, specificities, and predictive values >90%. The noninvasive tests are accurate for the diagnosis of H. pylori infection. Helicoblot 2.1 performed as well as the best ELISA kit. The HpSA is a promising direct noninvasive test that can be applied easily to evaluate H. pylori status.

Repeated Pneumatic Dilations as Long-Term Maintenance Therapy for Esophageal Achalasia

INTRODUCTION:In esophageal achalasia, pneumatic dilations (PD) provide short-term and long-term remission rates of 60–90% and 40–50%, respectively. The aim of this study was to evaluate the long-term efficacy of repeated PD as long-term maintenance therapy.PATIENTS AND METHODS:From 1992 to 2004, 150 patients with esophageal achalasia treated by PD were included in this retrospective study (78 males, mean age 57 ± 20 yr). PD were performed until remission was achieved (symptom score ≤3, each item < 2) and subsequently when symptomatic recurrence occurred. A standardized symptoms questionnaire was sent to patients lost to follow-up. Results are expressed as mean ± SD.RESULTS:Initial remission was achieved in 137 of 150 (91.3%) patients with 2.67 ± 1.59 dilations [range 1–12]. Failure and perforation rates were 7.3% (n = 11) and 1.3% (n = 2), respectively. After initial remission, 48 of 137 (35%) patients had recurrent symptoms; the probability to be in remission at 5 and 10 yr was 67% and 50%, respectively. At the end of follow-up (45 ± 38 months, ext. 2–144) 108 of 137 (78.8%) patients were in remission. Among 112 patients whose symptoms could be treated by repeated PD (per protocol analysis), 108 (96.4%) were in remission (3.5 ± 2.1 PD, ext. 2–12). In this group, the probability of being in remission after repeated PD at 5 and 10 yr was 96.8% and 93.4%, respectively. No pretherapeutic factor influenced long-term remission rate. The overall prevalence of gastroesophageal reflux was 34.7%.CONCLUSION:One-third of the patients with esophageal achalasia treated by PD will experience symptomatic recurrence during a 4-yr period. Long-term remission can be achieved in virtually all the patients treated by repeated PD according to an “on-demand” strategy based on symptom recurrence.

Second-line treatment for failure to eradicate Helicobacter pylori: a randomized trial comparing four treatment strategies

Aim : To compare the efficacy of different regimens in patients in whom previous Helicobacter pylori eradication therapy has failed.

Pathogen Evolution In Vivo: Genome Dynamics of Two Isolates Obtained 9 Years Apart from a Duodenal Ulcer Patient Infected with a Single Helicobacter pylori Strain

ABSTRACT The survival and microevolution of Helicobacter pylori strains in the niches of the stomach after eradication therapy have largely been unexplored. We analyzed genomic signatures for two successive isolates obtained 9 years apart from a duodenal ulcer patient who underwent eradication therapy for H. pylori. These isolates were genotyped based on 50 different parameters involving three different fingerprinting approaches and several evolutionarily significant and virulence-associated landmarks in the genome, including nine informative gene loci, the cag pathogenicity island and its right junction, members of the plasticity region cluster, and vacA and iceA alleles. Our observations reveal that the two isolates were derived from the same strain that colonized the patient for almost a decade and were almost identical. Microevolution, however, was observed in the cagA gene and its right junction, the vacA m1 allele, and a member of the plasticity region cluster (JHP926). These results suggest that H. pylori has a great ability to survive and reemerge as a microevolved strain posteradication, thereby hinting at the requirement for follow-up of patients after therapy.

Long-term effects of Helicobacter pylori eradication on gastric antral mucosa in duodenal ulcer patients.

Objectives The aim of this study was to assess the consequences of prolonged Helicobacter pylori eradication on gastric antral mucosa in duodenal ulcer patients. Patients and methods Forty‐three duodenal ulcer patients with confirmed H. pylori eradication after one year of follow‐up were included in this retrospective study. Before H. pylori eradication and during the follow‐up, four antral prepyloric biopsy samples were taken for histopathological examination and culture. Histopathological lesions were graded semi‐quantitatively according to the updated Sydney System for activity, chronic inflammation, glandular atrophy and intestinal metaplasia (IM), as well as presence of lymphoid follicles. Results After a mean follow‐up of 43 ± 23 months, H. pylori eradication statistically improved all gastritis scores, including the atrophy score and the lymphoid follicle score but excluding the IM score. H. pylori eradication resulted in normalization of gastric mucosa in 51.2% of patients and a significantly lower proportion of patients with non‐atrophic gastritis and atrophic gastritis without IM. Atrophy totally disappeared in 16/29 patients (55.2%) in whom IM was absent. No predictive factor for regression of atrophy or normalization of gastric mucosa was identified. Conclusion In duodenal ulcer patients, prolonged absence (more than one year) of H. pylori can lead to normalization of the antral mucosa and the disappearance of mucosaassociated lymphoid tissue, as well as the regression of antral atrophy. Long‐term studies involving selected patients with atrophy and IM which persist after H. pylori eradication are needed to determine the potential benefits of treating H. pylori gastritis with regard to gastric cancer prevention. Eur J Gastroenterol Hepatol 12:719‐725

Agnogenic Myeloid Metaplasia, Portal Hypertension, and Sinusoidal Abnormalities

A patient with agnogenic myeloid metaplasia suffered from gastrointestinal bleeding due to ruptured esophageal varices. The portal vein and its intrahepatic branches were patent. Except for the presence of myeloid cells, mainly megakaryocytes, in the sinusoids, liver histology was more or less normal. However, on Sirius red staining there was marked perisinusoidal fibrosis. In addition to numerous collagen bundles in the Disse space, electron microscopy also revealed the presence of hemopoietic cells, the transformation of perisinusoidal cells into fibroblasticlike or myofibroblasticlike cells, or both, and fragmentary deposits of basement membrane-like material. In the pathogenesis of sinusoidal hypertension as it occurs in agnogenic myeloid metaplasia, all the factors mentioned above should probably be taken into consideration.

Predictors of response to infliximab in luminal Crohn's disease.

AIMS To identify predictive factors of response to infliximab in luminal Crohns disease (CD). PATIENTS AND METHODS All consecutive patients with luminal CD treated with infliximab between October 1999 and March 2003 in Bordeauxs referral centers were included. All had at least 3 months follow-up post infliximab infusion and no prior treatment with infliximab. Response rates were determined 2 and 8 weeks after infusion according to Crohns Disease Activity Index (CDAI) (remission=CDAI<150 and response=CDAI decrease more than 100). RESULTS Among 44 patients (33 female; mean age 35 +/- 14 yr.), 39 (88%) had a clinical response 2 weeks after infusion (79% in remission). At week 8, the rate of response was 61.4% and exclusive colonic involvement predicted sustained response to treatment (P=0.03). The probability of remission at 56 weeks was 21.4%. Multivariate analysis demonstrated that the only factor associated with response duration was initiating immunosuppressive (IS) therapy in women (RR=3.61 95%CI[1.25-10.41], P=0.017). CONCLUSION Exclusive colonic involvement is the only predictive factor of sustained response to infliximab in luminal CD. At the time of infliximab infusion, initiation or modification of IS therapy may favor sustained response, at least in women.

Long-term follow-up of patients undergoing capsule and double-balloon enteroscopy for identification and treatment of small-bowel vascular lesions: a prospective, multicenter study

BACKGROUND AND STUDY AIMS Few data are available concerning the long-term outcome of patients treated endoscopically for bleeding small-bowel vascular lesions (SBVL). The aim of this study was to evaluate the risk of rebleeding after endoscopic therapy for SBVLs detected by video capsule enteroscopy (VCE). The secondary aim was to assess risk factors for rebleeding. PATIENTS AND METHODS A prospective, multicenter study (15 centers) was conducted, involving patients with obscure gastrointestinal bleeding and SBVL on VCE who were treated during double-balloon enteroscopy (DBE). The likelihood of bleeding was defined according to VCE findings, as high or low. RESULTS A total of 183 patients underwent endotherapy during DBE, and 64 (35 %) had rebleeding during the 1 year follow-up period. Multivariate analysis indicated that cardiac disease (hazard ratio [HR] 2.04, 95 % confidence interval [CI] 1.20 - 3.48; P < 0.01) and the presence of overt bleeding (HR 1.78, 95 %CI 1.07 - 2.97; P = 0.03) at presentation were associated with the risk of rebleeding. The association between chronic renal failure and the risk of rebleeding was close to statistical significance (HR 1.77, 95 %CI 0.94 - 3.33; P = 0.08). Kaplan-Meier analysis suggested that patients treated during DBE for a lesion with low likelihood of bleeding on VCE had higher rebleeding rates than those with a high likelihood of bleeding (HR 1.87, 95 %CI 0.94 - 3.37; P = 0.07). CONCLUSION Despite long-term remission in most patients, about one-third had rebleeding at 1 year. Independent risk factors for rebleeding were cardiac disease and overt bleeding at original presentation. The lesion characteristics on VCE may be useful to evaluate the bleeding potential of the lesion and may be used for better selection of patients for DBE.

Prospective, randomized comparison of two small-bowel capsule endoscopy systems in patients with obscure GI bleeding

BACKGROUND Video capsule endoscopy is the first-intention examination in patients with obscure GI bleeding. The new MiroCam capsule, when using electric-field propagation for transmission, has been poorly evaluated in a clinical setting, in contrast with the PillCam SB2 capsule. OBJECTIVE To evaluate the diagnostic concordance (κ value) between PillCam SB2 and MiroCam capsule examinations performed in the same patients. DESIGN AND SETTING Prospective, randomized study in 7 endoscopy units. PATIENTS AND INTERVENTION Eighty-three consecutive patients, ingesting the 2 capsules at a 1-hour interval. RESULTS Seventy-three patients were analyzed (10 technical issues). There were 31 concordant negative cases (42.4%) and 30 concordant positive cases (41.1%). The study showed satisfactory diagnostic concordance between the 2 systems (κ = 0.66). In 12 patients (16.4%), the final diagnosis was different: 9 patients had positive findings on MiroCam examination but no image detected with PillCam SB2, 2 had positive findings on PillCam examination only, and 1 patient had 2 different diagnoses. A positive diagnosis was obtained in 46.6% and 56.2% of patients with PillCam SB2 and MiroCam capsule, respectively, so that the procedures identified 78.6% and 95.2% of positive cases, respectively (P = .02). Small-bowel transit time and capsule reading time were significantly longer in MiroCam procedures. LIMITATIONS Technical failures possibly related to capsule interference. CONCLUSION This study shows at least comparable efficiency of the MiroCam compared with the PillCam SB2 capsule system for the diagnosis of obscure GI bleeding.