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Application of Bead-ELISA method to detect Helicobacter pylori VacA.

Helicobacter pylori is an etiological agent of gastritis, gastric ulcer and gastric cancer. In order to clarify the significance of vacuolating cytotoxin (VacA) for the pathogenesis of Helicobacter pylori infection, we established and applied the sandwich bead enzyme-linked immunosorbent assay (Bead-ELISA) for quantitative determination of VacA in the culture mediums of H. pylori and other species of Helicobacter. The minimum concentration of VacA in culture medium detected by Bead-ELISA was 25 pg VacA/ml and its sensitivity was found to be quite high compared to vacuolation assay and Western blot analysis, e.g. the minimum concentrations of VacA in culture medium required for detection by vacuolation assay and Western blotting were 11 ng/ml and 38 ng/ml, respectively. All the H. pylori strains used were found to produce VacA in the culture medium by Bead ELISA, even though some strains were negative by Western blot and vacuolation assay. The results obtained by Bead-ELISA was consistent with those by PCR amplification of a 785 bp vacA fragments. A toxin immunologically similar to VacA produced by other strains of Helicobacter such as H. muridarum (ATCC49282), H. mustelae (F10) and H. felis (ATCC49179) could not be detected by Bead-ELISA as well as Western Blot.

Animals↗

Different interdigestive antroduodenal motility patterns in chronic antral gastritis with and without Helicobacter pylori infection.

Fasting antroduodenal motor activity was studied in 15 dyspeptic patients with chronic superficial antral gastritis and Helicobacter pylori infection (group A), 10 dyspeptic patients with chronic superficial antral gastritis without Helicobacter pylori infection (group B), and eight healthy control subjects (group C) by manometric recording of phases of the interdigestive migrating motor complex (MMC) prolonged over 240 min. A significantly lower incidence of activity fronts (phase III of MMC) starting from the antrum was observed in patients with gastritis and Helicobacter pylori infection vs patients without bacterial colonization (P = 0.013) and in these latter vs control subjects (P = 0.013). Likewise, the overall number of activity fronts was smaller in patients with gastritis than in healthy subjects (P = 0.034). Symptomatic evaluation was performed in the two groups of dyspeptic patients, without detecting any differences in frequency and severity of complaints. Our results show a significant reduction in the occurrence of interdigestive antral phase III of MMC in chronic gastritis associated with Helicobacter pylori infection, suggesting a possible relationship between fasting motility and bacterial colonization.

Adolescent↗

Serodiagnosis of Helicobacter pylori infections by detection of immunoglobulin G antibodies using an immunoblot technique and enzyme immunoassay.

A transferable solid phase enzyme immunoassay (TSP-EIA) and an immunoblot technique were evaluated for the detection of IgG antibodies against Helicobacter pylori. Using the biopsy urease test as reference method, the sensitivity and specificity of the EIA were 96% and 100%, respectively. Immunoblot analysis was carried out by testing sera from patients with a positive urease test who suffered from type B gastritis, gastric and duodenal ulcers, and a negative control group. The immunoblotted Helicobacter pylori proteins showed reproducible immunoreactive bands at molecular weights of 130, 93, 75 and 67 kDa. The molecular weight protein fractions of Helicobacter pylori of 180 kDa and higher were found to be of minor immunological significance. Proteins of less than 60 kDa exhibited wide serum-specific variations in reactivity after immunostaining. No correlation between specific immunoblot patterns and clinical signs induced by Helicobacter pylori infection was observed.

Adult↗

Detection of Helicobacter pylori in gastric biopsy tissue by polymerase chain reaction.

To evaluate the sensitivity of a polymerase chain reaction (PCR) assay using nested primers in detecting Helicobacter pylori, gastric tissue biopsy specimens were collected on endoscopy from 17 patients with a duodenal ulcer. DNA was extracted by phenol/chloroform treatment or boiling in water, and then subjected to a nested PCR using two primer pairs from the urease gene of Helicobacter pylori. Fourteen of the 17 patients were positive for Helicobacter pylori using DNA samples extracted by either method. The PCR results correlated well with the results of an enzyme immunoassay to detect IgG antibody. However, there were two culture negative patients. The three PCR negative patients were both culture negative and serologically negative. DNA from 9 of the 14 patients was randomly selected and subjected to semiquantification by serial dilutions, and then PCR. The results showed that phenol/chloroform extraction yielded 10-1000 times more DNA than the boiling method. It is concluded that the PCR assay is a rapid and sensitive method for detecting Helicobacter pylori, and that phenol/chloroform extraction is superior to simple boiling in obtaining DNA samples for PCR.

Antibodies, Bacterial↗

Elevated risk of Helicobacter pylori infection in submarine crews.

In a prospective study designed to elucidate the route of transmission of Helicobacter pylori, the seroprevalence and incidence of Helicobacter pylori infection was determined in the following branches of the armed forces presumed to be at increased risk of acquiring transmissible diseases by the fecal-oral or oral-oral route: German submarine crews (n = 64, mean age 26.2 years) and regular officers of the French infantry (n = 51, mean age 26.5 years) who had served for a minimum of three years. The submarine crews were compared with air force staff (n = 74, mean age 23.7 years), and the French officers with French infantry recruits (n = 135, mean age 20.5 years) who started their service at the beginning of the study. The frequency of IgG and IgA antibody responses to the 120, 88, 86 and 82 kDa proteins was determined by the immunoblot method. The frequency of a positive antibody response was strongly dependent on age (p less than 0.001). When results were controlled for age by the logistic regression analysis, the submarine crews revealed significantly increased frequencies of the IgG and IgA responses compared to air force staff. The antibody responses of French officers and recruits were not significantly different. It is concluded that submarine crews serving during their missions in an overcrowded space with extremely limited sanitary facilities must be considered a high-risk group for Helicobacter pylori infection. These results strongly suggest person-to-person transmission of Helicobacter pylori, by either the oral-oral or the fecal-oral route.

Adult↗

Mechanism of Helicobacter pylori-associated gastric mucosal injury.

Helicobacter pylori infection is associated with gastric mucosal damage and the infiltration of neutrophils. Myeloperoxidase from neutrophils produces hypochlorous acid, which yields monochloramine in the presence of ammonia produced by urease enzyme of Helicobacter pylori. The target cells of gastric mucosal damage are gastric mucosal cells and endothelial cells. We therefore tested the hypothesis that ammonium, hypochlorous acid, and monochloramine damage the target cells. We studied the in vitro cytotoxic effects of ammonium chloride, sodium hypochlorite, monochloramine, and activated neutrophils on the target cells. Cytotoxicity was measured by a 51Cr-release assay. Ammonium chloride, sodium hypochlorite, and monochloramine were toxic to labeled cells in a concentration dependent manner. The toxicity of these agents was in the order monochloramine > sodium hypochlorite >> ammonium chloride. Incubation of labeled cells with activated neutrophils, Helicobacter pylori, and urea resulted in cytolysis. These cytotoxicities were significantly inhibited by the scavenger of hypochlorous acid, taurine. Monochloramine is more toxic to the target cells than ammonium chloride. Although ammonium chloride at neutral pH by itself has little direct damaging effect on the gastric mucosa, it is damaging to the gastric mucosa through a reaction with hypochlorous acid, suggesting that it plays a role in Helicobacter pylori-associated gastric damage.

Animals↗

High seroprevalence of Helicobacter pylori in diabetes mellitus patients.

To assess the prevalence of Helicobacter pylori in diabetes mellitus, a serological test used to detect antibodies to H. Pylori in patients with diabetes mellitus. Within six months, 45 insulin-dependent, 98 non-insulin-dependent, and a control group of 159 outpatients were enrolled in this study. The age adjusted seroprevalence rate of Helicobacter pylori were determined using a commercial anti-Helicobacter pylori IgC and IgA ELISA (Bio-Rad). The prevalence rates increased with age in all age groups until 60-70 years. In diabetic patients, the frequency of Helicobacter pylori infection was higher than control subjects in nearly all age groups, reaching significance in three age categories of NIDDM patients and in one age category in IDDM patients. This higher seroprevalence could not be explained by differences in socioeconomic status or use of antibiotics.

Adult↗

[Helicobacter '98--epidemiology and significance in carcinogenesis].

Gastritis caused by infection with Helicobacter pylori is one of the most common infectious diseases worldwide. There are data on the epidemiology, pathophysiology and histology of this disease that show that Helicobacter pylori gastritis plays an important role in gastric carcinogenesis. However, we must remember that only a very few among those infected with Helicobacter pylori will develop gastric cancer. Hence, one of the main targets of future research will be to identify individuals who carry a greater risk for developing gastric cancer and may therefore benefit from eradication of Helicobacter pylori in terms of gastric cancer prevention.

Cross-Sectional Studies↗

PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice.

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.

Animals↗

Evaluation of a single-step serological assay for laboratory diagnosis of Helicobacter pylori infection.

A rapid, single-step, in-laboratory qualitative test for the detection of IgG antibodies to Helicobacter pylori in serum (TestPack Plus; Abbott Laboratories, Germany) was evaluated. This test may be used as an alternative to enzyme immunoassays (EIAs). Of 153 adult patients, 110 were defined as Helicobacter pylori positive and 43 as Helicobacter pylori negative by the gold standard, a combination of three tests. The performance characteristics of the TestPack Plus, i.e. sensitivity, specificity, and positive and negative predictive values, were not significantly different from the corresponding values obtained with an EIA used for comparative purposes, the Pyloriset EIA-G test (Orion Diagnostica, Finland). The high positive predictive value (93%) of the TestPack Plus single-step serological test makes it a valuable tool for rapid in-laboratory screening purposes, especially in countries with a high prevalence of Helicobacter pylori infection.

Adolescent↗

Comparison of the E test and agar dilution method for antimicrobial suceptibility testing of Helicobacter pylori.

A multicentre study was carried out in order to validate the E test in comparison with the reference agar dilution method for testing the susceptibility of Helicobacter pylori to amoxicillin, clarithromycin, and metronidazole. Ten clinical isolates and one control collection isolate ( Helicobacter pylori ATCC 43504) were tested blindly at four centres according to a uniform methodology. The E test showed excellent intra- and inter-laboratory correlations with the agar dilution method for amoxicillin and clarithromycin (>98% agreement within 2 log(2) dilution steps). For metronidazole, however, the E test revealed significantly higher minimum inhibitory concentration values (>2 log(2)) against 5 of the 10 Helicobacter pylori strains tested. Overall, neither method was found reliable for testing the susceptibility of Helicobacter pylori to metronidazole, since both tended to lack reproducibility.

Amoxicillin↗

Serological methods for diagnosis of Helicobacter pylori infection and monitoring of eradication therapy.

Several methods can be used to diagnose Helicobacter pylori infection. Invasive methods include detection of the bacterium in gastric biopsy specimens by culture, immunohistochemistry, rapid urease tests, or the polymerase chain reaction. Noninvasive or less invasive detection methods include the urea breath test and serological methods. The urea breath test is based on the detection of 13CO2 or 14CO2 in breath, produced by bacterial urease in the stomach after labelled urea is swallowed. Serological methods are based on the detection of Helicobacter pylori-specific antibodies in serum, saliva, or urine. In this review, the performance and diagnostic value of several serological methods, such as enzyme immunoassay, rapid office-based assays, and Western blot, will be discussed in relation to biopsy-based methods and the urea breath test. In addition, the value of serological assays for monitoring eradication of Helicobacter pylori infection following treatment will be discussed. The diagnostic performance of properly evaluated serological assays is comparable to that of biopsy-based methods and the urea breath test. To monitor eradication of Helicobacter pylori infection following therapy, quantitative enzyme immunoassays can be used, especially in patients with high pretreatment antibody titres.

Antibodies, Bacterial↗

The effect of intragastric ammonia production on titratable gastric acid output in Helicobacter pylori-infected patients with chronic gastritis.

The purpose of this study was to assess whether intragastric neutralization of HCl by ammonia in Helicobacter pylori-infected patients could meaningfully affect the titratable acid output as a measure of gastric acid secretion in a relation to the severity of infection. In 79 patients with different degrees of Helicobacter pylori infection and chronic gastritis, the basal acid output (BAO) and maximal acid output (MAO) after pentagastrin (6 microg/kg s.c.) was estimated. Cl- and NH4+ contents in these fractions were also assayed. H+/Cl- ratio in the MAO fraction was diminished in markedly infected patients (68.1 +/- 3.9%, vs 84.1 +/- 3.3% in noninfected patients; P < 0.005). Ammonium content was maximal in patients with marked infection (0.912 +/- 0.086 vs 0.149 +/- 0.034 mmol/hr in MAO [P < 0.001] and 0.475 +/- 0.063 vs 0.105 +/- 0.016 mmol/hr in BAO of noninfected patients [P < 0.001]), with intermediate values in mild and moderate infection. The NH4 +/(H+ + NH4+) ratio reached 27.01 +/- 7.34% in the BAO of moderately infected patients, vs 10.22 +/- 3.81% in noninfected patients (P = 0.05), and 7.25 +/- 1.06% in the MAO of markedly infected patients, vs 1.14 +/- 0.33% in noninfected patients (P < 0.001). The intragastric ammonia production affects the titratable acid output in Helicobacter pylori-infected patients dependent on the severity of infection. Therefore this factor should be taken into consideration in the evaluation of gastric secretory function in Helicobacter pylori-infected patients.

Adult↗

The gastric cardia in Helicobacter pylori infection.

Helicobacter pylori gastritis is believed to be both more prevalent and more severe in the antrum than in the corpus. The mucosa of the cardia is architecturally similar to that of the antrum. This study was designed to test the hypothesis that intensity of H pylori infection and the associated inflammation are similar in the cardia and the antrum and are greater in these locations than in the corpus. A total of 445 gastric biopsy specimens were obtained from predetermined sites from 50 subjects (42 with demonstrated H pylori infection). Slides were stained with a combined stain to simultaneously visualize H pylori, mucosal morphology, and inflammatory infiltrate. Numbers of H pylori, inflammatory responses, and intestinal metaplasia were graded on a scale from 0 to 5. Helicobacter pylori was detected in the cardia of 40 of the 42 infected subjects (95%). Helicobacter pylori density was similar in the three gastric regions. The intensity of chronic active gastritis was similar in the antrum and the cardia, and was higher in these locations than in the corpus (P < .005). Lymphoid follicles were significantly less prevalent in the cardia and in the corpus than in the antrum (P < .05). Helicobacter pylori infection was as prevalent in the cardia as in the rest of the stomach and its density was similar in all sites of antrum, corpus, and cardia. The inflammatory responses characteristic of chronic active gastritis, except for lymphoid follicles, were similar in the antrum and the cardia and in both these locations were more intense than in the corpus. The significance of these findings is discussed with respect to the relationship between H pylori infection and the genesis of gastric adenocarcinoma and lymphoma.

Adult↗

Genetic evidence for host specificity in the adhesin-encoding genes hxaA of Helicobacter acinonyx, hnaA of H. nemestrinae and hpaA of H. pylori.

Gastric and non-gastric species of Helicobacter were examined for the presence of the adhesin-encoding gene, hpaA, from the human-associated gastric Helicobacter H. pylori (Hp), and for adhesin subunit protein HpaA. Amplification of a 375-bp internal DNA fragment of hpaA by PCR demonstrated the presence of the gene in Hp and in two closely related gastric Helicobacters, H. nemestrinae (Hn) and H. acinonyx (Hx), but not in the more distantly related H. felis (Hf) and H. mustelae (Hm). The non-gastric Helicobacters, H. canis (Hc), H. muridarum (Hr), H. fennelliae (He) and H. cinaedi (Hi), were all negative for hpaA. An immunoblot assay of water extracts with adhesin-specific antibody confirmed these results. The deduced amino acid (aa) sequences of Hp HpaA and Hn adhesin A (hereafter termed HnaA) are very similar, having identical receptor-binding motifs (rbm); also, the hemagglutination (HA) properties of Hn and Hp cells were indistinguishable. In contrast, the rbm of Hx adhesin A (hereafter termed HxaA), compared to that of Hp, contained a non-conservative aa substitution (Ile to Thr); also, there was variance in five consecutive aa from 10 to 14 residues upstream from the rbm. We conclude that these aa substitutions in HxaA are probably responsible for the difference in receptor recognition of this adhesin, as evidenced by the resistance of Hx HA to inhibition with N-acetylneuraminyl-alpha(2,3)-lactose. These results are consistent with the biological similarity between the natural host(s) of Hp and Hn; i.e., human and non-human primates, and the dissimilarity between these hosts and the feline host, the cheetah.

Acinonyx↗

The prevalence of Helicobacter pylori positivity in a symptom-free population, aged 1 to 40 years.

Epidemiologic data about the prevalence of Helicobacter pylori positivity as determined by serology are presented in a large series of asymptomatic subjects (n = 1046). Serology is nowadays generally accepted as a valid non-invasive screening method for the detection of a Helicobacter pylori infection. Blood samples were taken from apparently healthy children (n = 480; aged 1-17 years) and pregnant women (n = 566; age 18-40 years) who were admitted for routine pre-surgery or pre-natal blood analysis. 162 (15.5%) had a positive titer for Helicobacter pylori. There was a significant increase in the number of positive patients with increasing age (ranging from 6.2% in the age group of the 1-5 years old to 31.0% in the age group of the 36-40 years old (p < 0.01). The meaning of this relatively high prevalence of "asymptomatic carriers" remains to be evaluated. A long-term follow-up of these patients should be considered to evaluate if they will develop clinical manifestations suggestive for Helicobacter pylori infection.

Adolescent↗

Detection of Helicobacter pylori in bile of cats.

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.

Animals↗

The high prevalence of Helicobacter sp. in porcine pyloric mucosa and its histopathological and molecular characteristics.

This study examined the prevalence of Helicobacter infection in the pyloric mucosa of pigs and its histopathological and molecular characteristics. Forty porcine pyloric samples were examined for Helicobacter infection by silver staining and PCR assay. The PCR product (376 bp) was digested with NdeII to differentiate between Helicobacter heilmannii and Helicobacter pylori. Another PCR assay run to produce an 1157 bp fragment was performed using a primer set designed from the 16S rRNA gene of Candidatus H. suis, and its product was cloned and sequenced. Infection rates were 62.5% (25/40) and 95.0% (38/40) as determined by silver staining and the PCR assay, respectively. On histopathological examination, lymphoid follicle aggregation in the pyloric mucosa and granulocytic migration into the lumen of pyloric glands were observed in 24 (60.0%) and 33 (82.5%) gastric samples, respectively. All PCR products, except that of H. pylori, were cut into two fragments of 147 and 229 bp by enzymatic digestion with NdeII. Sequencing of the 16S rRNA gene showed that the bacterium had 99.57% (1152 bp/1157 bp) homology to the 16S rRNA gene of Candidatus H. suis.

Animals↗