Jean-Louis Fauchère

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.

Implication of the Structure of the Helicobacter pylori cag Pathogenicity Island in Induction of Interleukin-8 Secretion

ABSTRACT Helicobacter pylori virulence is associated with the presence of the cag pathogenicity island (PAI). Thecag PAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa to H. pyloriinfection. The aim of this study was to determine whether the genetic structure of the cag PAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of thecag PAI were performed on 153 strains isolated from adults suffering from ulcers (n = 79) or gastritis (n = 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entire cag PAI, 12 strains (7.8%) had thecag PAI split in two, 49 strains (32%) had nocag PAI, and 9 strains exhibited other structural combinations. The presence of an entire cag PAI was statistically correlated with the presence of IS605(P = 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of the cag PAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of the cag PAI structure suggests that this region is not the only requirement for IL-8 secretion.

Comparative evaluation of 29 commercial Helicobacter pylori serological kits

Serology is a noninvasive diagnostic method for the detection of Helicobacter pylori infection. Many commercial kits are now on the market. It is necessary to assess their performances to help the user to choose the most appropriate.

Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

ABSTRACT We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori—the wild-type sequence and the three mutations conferring clarithromycin resistance—using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.

T2182C mutation is not associated with clarithromycin resistance in Helicobacter pylori.

In a recent article, Khan et al. described a new mutation in the 23S rRNA gene, T2182C, conferring clarithromycin resistance to Helicobacter pylori (1). An A-to-G mutation at either position 2142 or position 2143 and an A-to-C mutation at position 2142 in the 23S rRNA gene have been shown to confer resistance to clarithromycin in H. pylori (5). In 2000, we isolated a clarithromycin-resistant (Clar) strain from a French patient (MIC > 256 μg/ml). Sequencing the 23S rRNA gene revealed a single T2182C mutation. To confirm the role of the T2182C mutation, we used the PCR fragment containing this mutation to transform a susceptible receptor strain. We obtained Clar transformants. These results were accepted for oral presentation at the 11th International Workshop on Campylobacter, Helicobacter and Related Organisms held in 2001 (3), but a final checking of the MIC determined by E-test revealed a mixed set of colonies. We observed small colonies in the susceptible area of the plate and big colonies in the resistant area. Subcultivation of isolated colonies provided two different clones. A Clas clone (MIC, 0.016 μg/ml) harbors the T2182C mutation and a wild-type sequence at positions 2142 and 2143. A Clar clone (MIC, 256 μg/ml) harbors both the T2182C mutation and an A2143G mutation. Randomly amplified polymorphic DNA patterns from the Clas and Clar isolates demonstrated that they are genetically identical. Coming back to the frozen gastric biopsy sample, we isolated 53 single colonies of H. pylori. Fifty-two colonies were from the Clas clone, and only one was from the Clar clone. It is possible that Khan et al. were misled, like we were, by a mixed population of a Clas clone and a Clar clone. It is surprising that they mention in their article the use of “pure culture from a single colony [for] further study” (1). T2182C is not a new mutation, and it has been already described in previous reports (2, 4, 6). In 1998, Wang et al. (6) first reported a Clar strain with A2143G and T2182C mutations. In vitro site-directed mutagenesis experiments suggested that this additional mutation is not associated with clarithromycin resistance (6). In 1999, Matsuoka et al. (4) reported the T2182C mutation in both sensitive and resistant colonies. In 2002, Kim et al. (2) described the same T2182C mutation for four Clar isolates, but they did not indicate that they worked on single-colony isolates. Attention must be paid to experiments used as evidence that a mutation is responsible for resistance in H. pylori. Experiments have to be realized on several isolated colonies, since a mixture of strains can lead to a false determination of sequence. Transformation with PCR fragments could lead to resistant clones growing on antibiotic plates, since the error rate of Taq polymerase spontaneously generates random mutations containing the well-known A2142G or A2143G mutations (5). The existence of a Clas isolate (20-222S) harboring a T2182C mutation in the 23S rRNA genes led to the conclusion that the T2182C mutation is not associated with clarithromycin resistance in H. pylori. The susceptible clone 20-222S is available on request.

Novel Antigens of Helicobacter pylori Correspond to Ulcer-Related Antibody Pattern of Sera from Infected Patients

ABSTRACT Recently, we reported that the patterns of antibodies to Helicobacter pylori protein antigens in serum may be useful for screening patients at high risk for ulcers (P. Aucher et al., J. Clin. Microbiol. 36:931-936, 1998). Here we report the identification, by a combination of electrophoretic, immunochemical, and protein sequencing methods, of five antigens that correspond to this antibody pattern: groEL, catalase A, flagellin A, beta-ketoacyl-acyl carrier protein synthase I (β-ketoacyl-ACP S), and peptidyl prolyl cis-trans isomerase (PPiase). β-Ketoacyl-ACP S and PPiase are reported for the first time as antigens of diagnostic interest in infections by H. pylori. The antigenicity of the five antigens, together with those of CagA and VacA, was tested in an immunoblot assay with water-soluble protein extracts from two H. pylori pathogenic strains (HP 141 and ATCC 43579) and panels of sera from H. pylori-positive patients with gastroduodenal ulcers (GDU), nonulcer dyspepsia (NUD), as well as sera from H. pylori-negative healthy volunteers. For catalase A, groEL, and flagellin A antigens, no overall statistically important values were found making it possible to discriminate between patients with GDU and NUD. For both H. pylori strains, the mean performance indices (MPI) presenting percentages of correctly classified patients with GDU and NUD showed that the most significant antibody patterns were as follows: anti-VacA + anti-β-ketoacyl-ACP S (MPI = 76.1), anti-VacA + anti-PPiase (MPI = 71.8), and anti-CagA + anti-VacA + anti-β-ketoacyl-ACP S (MPI = 70.5). Antibody patterns detected with these antigen profiles may therefore be useful in developing a diagnostic test designed to predict the clinical severity of the H. pylori infection within the adult population of France.

Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer

Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann-Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.

Antibody response of patients with Helicobacter pylori-related gastric adenocarcinoma: significance of anti-cagA antibodies.

ABSTRACT The aim of this study was to search for a specific antibody pattern in sera from patients suffering from Helicobacter pylori-related gastric adenocarcinoma (GAC). The serological response of 22 patients suffering from GAC, 31 patients with gastroduodenal ulcer, and 39 asymptomatic subjects was analyzed using immunoblotting performed with three H. pylori strains: strain ATCC 43579; strain B110, isolated from a patient with ulcers; and strain B225, isolated from a patient with GAC. In addition, the latex agglutination test Pyloriset Dry was used to analyze ambiguous sera. H. pylori seropositivity was 75% in the GAC group, 61.3% in the ulcer group, and 56.4% in the asymptomatic group. Anti-CagA antibodies were found more often in the GAC group (48.8%) and in the ulcer group (47.3%) than in the asymptomatic group (21.2%). These percentages depended on the strain used as an antigen: in the GAC group, the anti-CagA frequencies were 93.3, 40, and 13.3% with strains B225, B110, and ATCC 43579, respectively. Thus the presence of anti-CagA antibodies was increased in patients suffering from H. pylori-related GAC, in particular when the CagA antigen was from a GAC strain. These data suggest the existence of a CagA protein specifically expressed by H. pylori strains isolated from GAC patients.

Modification in the ppk gene of Helicobacter pylori during single and multiple experimental murine infections

ABSTRACT The bacterial pathogen Helicobacterpylori is highly adapted to the human stomach, and a high level of polymorphism is observed among clinical isolates. This polymorphism may be the consequence of adaptive changes during colonization, making a strain better able to survive, to evade the immune system, and to provoke a chronic infection. To investigate the mechanisms involved in the acquisition of diversity in H. pylori, mouse models of single infections, coinfections, and superinfections were developed. These experimental infections were conducted with strain SS1, well known to be mouse adapted, and with two strains freshly isolated from infected patients: Hp141 and Hp145. Genetic modifications occurring in these strains were studied over time by comparing randomly selected colonies of the emerging strains to those of the infecting strains by using randomly amplified polymorphic DNA fingerprinting with six different primers and by using PCR to amplify the vacA and cagA genes. We showed that, regardless of the number of infecting strains, only one emerged from the animals and that the establishment of a first strain thwarted the implantation of a second strain. During both a single infection and a coinfection with SS1, Hp141 was replaced by a genetic variant (Hp141v) that overcame SS1 in coinfection experiments. Hp141v exhibited a deletion of a 102-bp repeated sequence within the ppk gene, which encodes polyphosphate kinase (PPK), an enzyme involved in the physiological adaptation of the microbial cell to nutritional and environmental stresses. The deletion led to higher enzymatic activity of PPK, and the variant exhibited a better capacity to colonize mice. Considering that the modified gene is known to be involved in adaptation to a new environment, our results are consistent with an adaptive change in strain Hp141 and suggest that PPK is an important virulence factor in H. pylori.

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer.

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.