A strain-specific antigen in Japanese Helicobacter pylori recognized in sera of Japanese children

An enzyme immuno assay (EIA) test based on Japanese strain-derived high-molecular-weight cell-associated proteins (JHM-CAP) was evaluated by comparing with a previously developed EIA test based on a U.S. strain-derived high-molecular-weight cell-associated proteins (HM-CAP). Serum samples of 131 Japanese asymptomatic children (mean age, 5.5 years; range, 0 to 21 years) were tested that include 43 positive and 88 negative children as judged by Helicobacter pylori stool antigen test (HpSA test). Both tests showed comparable and reliable specificities, but the sensitivity of JHM-CAP EIA, at 93.0%, was much higher than that of HM-CAP EIA, at 67.4%. More false-negative results of HM-CAP were obtained in children under 10 years of age. Immunoblot analysis revealed that the JHM-CAP but not the HM-CAP preparation had a 100-kDa antigen recognized by JHM-CAP positive sera. It was concluded that JHM-CAP EIA is highly accurate for the serodiagnosis of H. pylori infection in Japanese young children and that the high sensitivity of JHM-CAP EIA in contrast to HM-CAP EIA is due to the presence of a 100-kDa antigen in Japanese strains that may be recognized by the host immune system at an early stage of infection.     

Evaluation of a Helicobacter pylori Stool Antigen Test for the Diagnosis and Follow-Up of Infections in Children

 The aim of this study was to evaluate the performance of a newly developed enzyme immunoassay kit (HpSA) for detecting Helicobacter pylori antigens in the stool of children. This study was comprised of 58 children referred to various endoscopy units for evaluation of gastrointestinal symptoms and upper gastroduodenal endoscopy and 11 children for post-therapy follow-up. In the first group, 23 children were diagnosed as positive for Helicobacter pylori using bacteriological and/or histological methods. Stool antigens were detected in 20 of these positive patients, for a sensitivity of 86.9% and a negative predictive value of 91.9%. Since only one false-positive reaction was observed with the HpSA kit, the specificity was 97.1% and the positive predictive value 95.2%. Results obtained for post-therapy follow-up were also promising. The HpSA assays were negative for the eight children whose infections were eradicated after therapy, and a positive result was obtained for two of three patients who had a persistent infection.     

Antibiotic resistance of Helicobacter pylori strains in Japanese children

The resistance of Helicobacter pylori to the recently available antibiotic treatment regimens has been a growing problem. We investigated the prevalence of H. pylori resistance to clarithromycin, metronidazole, and amoxicillin among 51 H. pylori isolates from Japanese children. In addition, the mutations of the corresponding gene were studied by PCR and restriction fragment length polymorphism analysis. Primary resistance to clarithromycin, metronidazole, and amoxicillin was detected in 29, 24, and 0% of strains, respectively. The eradication rates in clarithromycin-susceptible and -resistant strains were 89 and 56%, respectively (P < 0.05). The prevalence of strains with acquired resistance to clarithromycin (78%) was higher than that of strains with primary resistance (P < 0.01). Among the clarithromycin-resistant strains studied, 92% showed cross-resistance to azithromycin. No acquired resistance to amoxicillin was demonstrated. The A2144G mutation in the 23S rRNA gene was detected in 11 of 12 (92%) clarithromycin-resistant strains tested, whereas the mutation was not detected in any of the 15 susceptible strains. The deletion of the rdxA gene was not demonstrated in any of the strains. The results indicate that a high prevalence of clarithromycin-resistant strains is associated with eradication failure. Testing of susceptibility to clarithromycin is recommended.     

Posttreatment Follow-Up of Helicobacter pylori Infection Using a Stool Antigen Immunoassay

The Helicobacter pylori stool antigen enzyme immunoassay (HpSA) was evaluated during posttreatment follow-up of patients in a country with a very high prevalence of H. pylori infection. From among 273 dyspeptic individuals (18 to 55 years) initially recruited from a shantytown in Lima, Peru, 238 participants who met the inclusion criteria and were suspected to be H. pylori positive based on ¹⁴C urea breath test (UBT) results underwent endoscopy. Participants with endoscopy-proven infections received standard eradication therapy and were monitored by UBT and HpSA at 1 month following treatment and at 3-month intervals for 9 months posttreatment. A second endoscopy was performed if UBT results showed evidence of treatment failure or H. pylori recurrence. Biopsy results were considered the “gold standard” in all analyses. Among patients who underwent endoscopy, HpSA had a pretreatment sensitivity of 93%. Two-hundred thirty patients completed the treatment regimen, of whom 201 (93%) were considered to have had successful treatment outcomes based on a negative follow-up UBT. Thirty-two patients with UBT-defined treatment failures or H. pylori recurrences at any point during the 9-month follow-up underwent a second endoscopy. In the posttreatment setting, HpSA had an overall sensitivity of 73% and a specificity of 67%. Agreement between UBT and HpSA diminished throughout the follow-up. Among 14 participants in whom HpSA remained positive at 1 month following treatment despite UBT evidence of treatment success, 12 (86%) became HpSA negative within 3 months posttreatment. Although this study confirmed the validity of the HpSA in the initial assessment of dyspeptic patients, the test demonstrated a reduced overall accuracy in the detection of treatment failures and H. pylori recurrences during 9 months of posttreatment follow-up. Furthermore, in some patients it may take up to 3 months after successful eradication for antigen shedding to diminish to levels within the negative HpSA range.     

Experimental gastritis induced by Helicobacter pylori in Japanese monkeys.

We used Japanese monkeys (Macaca fuscata) to establish an experimental model in order to clarify the pathogenicity of Helicobacter pylori in gastric and duodenal disorders. A suspension (5 ml; 10(9) CFU/ml) of H. pylori cells isolated from humans was sprayed around the antrum of the stomach of each of 12 of 17 animals with an endoscope. The remaining five animals were not inoculated; they served as a control group. On days 7, 14, and 28 after inoculation, the gastric mucosa samples were examined grossly and were biopsied for microscopic examination with an endoscope. H. pylori was recovered from 7 of the 12 inoculated animals (58%), and infiltration by neutrophils and monocytes was observed histologically. Macroscopic gastritis with erythema and erosions were noted for five of these animals. On day 28 after inoculation, five animals in the infected group were treated with ampicillin. In two infected but untreated animals, the bacteria persisted for more than 6 months. The result of the gastritis scoring of the antral mucosa and the ammonia concentration in the gastric secretion were significantly higher (P < 0.01 to 0.001) for the infected group than for the control group; however, these values decreased to levels comparable to those for the control group after treatment with ampicillin. Urease activity was positive in gastric biopsy specimens from five of the seven animals in the infected group after 7 days and from four of these animals after 14 days but was negative in all specimens from animals in the control group. The level of antibody (immunoglobulin G) in serum for the infected group was elevated but changed very little for the control group. These results suggest that this M. fuscata model can be used to study H. pylori infection and that H. pylori can induce gastritis.     

Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

Application of a Stool Antigen Test To Evaluate the Incidence of Helicobacter pylori Infection in Children and Adolescents from Tehran, Iran

Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries, where a low-cost, rapid diagnostic technique which is reliable for all age groups may be useful for the management of H. pylori infection. For this purpose, we used an HpSA test (Equipar) to detect H. pylori infection in children and adolescents from Tehran, Iran. Thirty-five children who were positive or negative for H. pylori infection by endoscopy-based tests were used as positive and negative controls for the HpSA test. Stools were collected from 430 randomly selected children and adolescents (4 to 18 years old) from southwest, near the center, and northwest of Tehran. A questionnaire that included presence of recurrent abdominal pain (RAP), family history of infection and/or peptic ulcer disease (PUD), and income of parents was completed. A good agreement was found between the results of endoscopy-based tests and those of the HpSA test; the sensitivity and specificity of the Equipar-HpSA test were 100% and 83.4%, respectively. Among 430 children and adolescents, 47% were positive by the HpSA test, of whom 82% had RAP. No difference in incidence was observed between the two sexes; the various categories of age showed an increasing incidence, ranging from 24% (ages 4 to 6) to 58% (ages 16 to 18). The rate of infection in children and adolescents from the southwest was significantly higher (70%) than the rate in those from the northwest (32%), and a family history of H. pylori infection or PUD was observed in 59% of the HpSA positive subjects. The HpSA test is a useful test to detect H. pylori infection in children and adolescents from developing countries.     

Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii

BACKGROUND. Helicobacter pylori are gram-negative spiral bacteria that are associated with chronic gastritis, a known precursor of gastric carcinoma. Persons at high risk for gastric carcinoma have been shown to have a high prevalence of H. pylori infection. METHODS. We studied the relation of H. pylori infection and gastric carcinoma in a cohort of Japanese American men living in Hawaii. The 5908 men were enrolled and examined from 1967 to 1970. By 1989, 109 cases of pathologically confirmed gastric carcinoma had been identified. The store serum of each patient with gastric carcinoma and of each matched control subject were tested for the presence of serum IgG antibody to H. pylori. RESULTS. Ninety-four percent of the men with gastric carcinoma and 76 percent of the matched control subjects had a positive test for H. pylori antibodies, for an odds ratio of 6.0 (95 percent confidence interval, 2.1 to 17.3). As the level of antibody to H. pylori increased, there was a progressive increase in the risk of gastric carcinoma (P for trend = 0.0009). The association was strong even for men in whom the diagnosis was made 10 or more years after the serum sample was obtained (odds ratio, 10.5; 95 percent confidence interval, 2.5 to 44.8). CONCLUSIONS. Infection with H. pylori is strongly associated with an increased risk of gastric carcinoma. However, most persons infected with H. pylori will never have gastric carcinoma. Therefore, other factors that increase the risk of gastric carcinoma among persons infected with H. pylori need to be identified.     

Lipid A in Helicobacter pylori.

Free lipid A of Helicobacter pylori was characterized with regard to chemical composition, reactivity with anti-lipid A antibodies, and activity in a Limulus lysate assay. The predominant fatty acids of H. pylori lipid A were 3-OH-18:0, 18:0, 3-OH-16:0, 16:0, and 14:0. Hexosamine was present in amounts similar to those in Campylobacter jejuni or Salmonella typhimurium lipid A. The lipopolysaccharide of H. pylori contained 2-keto-3-deoxyoctonic acid, a common constituent of enterobacterial and C. jejuni lipopolysaccharides. In the enzyme-linked immunosorbent assay, the doses of lipid A required to inhibit anti-lipid A by 50% (EI50 values) by absorption of the immune (rabbit) serum were 7.9, 1.2, and 1.4 micrograms of O-deacylated lipid A's from H. pylori, C. jejuni, and S. typhimurium per ml, respectively. The lower reactivity of H. pylori lipid A compared with those of the other two lipid A preparations (as shown by the higher EI50 value) was underscored by the use of a murine monoclonal anti-lipid A antibody in the inhibition assay. An EI50 value was not obtained at the concentrations tested for H. pylori lipid A; the corresponding figures for C. jejuni and S. typhimurium lipid A's were 13 and 14 micrograms/ml, respectively. No inhibition was obtained with H. pylori lipopolysaccharide, which showed a low-molecular-weight profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of H. pylori lipid A in the Limulus assay was approximately 71 and 650 times lower than those of C. jejuni and S. typhimurium lipid A's, respectively. These findings suggest that lipid A is an integral part of the outer cell wall of H. pylori. The lower reactivity of H. pylori lipid A with anti-lipid A antibodies and in the Limulus assay compared with that of C. jejuni or S. typhimurium lipid A may be explained by a different composition of the fatty acids, especially the 3-hydroxy fatty acids, and a possible deviating phosphorylation pattern.     

Effect of Low-Dose Antigen Exposure on Development of Immunity to Helicobacter pylori Infection in Mice

The effect of low-dose antigen exposure on the development of immunity to Helicobacter pylori infection was studied in outbred mice. Animals that were primed with a subinfectious number of H. pylori bacteria exhibited significantly lower bacterial loads after challenge with an infectious dose of pathogen (versus controls, P < 0.05).     

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C₅₀ chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.     

Evaluation of Helicobacter pylori vacA genotype in Japanese patients with gastric cancer.

AIM: To examine the vacA genotypes of Helicobacter pylori strains in Japan and to define whether any specific genotype was associated with gastric cancer. METHODS: The allelic variation of vacA gene was studied using a recently introduced polymerase chain reaction based vacA genotyping system. RESULTS: 80 H pylori strains were isolated from gastric biopsies of 40 patients with gastric cancer and 40 control subjects in a Japanese population. All strains were s1/m1 subtype and 79 of 80 strains were classified as s1a subtype. CONCLUSIONS: The recently proposed vacA genotyping system is applicable to Japanese H pylori strains and most strains have the s1a genotype, associated with increased virulence. While the high frequency of s1a/m1 vacA genotype might play a role in the increased incidence of atrophic gastritis and gastric cancer in Japanese subjects, it precludes its use as a predictor of clinical outcome of H pylori infection in Japan.     

儿童上消化道出血与幽门螺杆菌感染的关系

目的研究儿童上消化道出血与幽门螺杆菌（Helicobacter pylori,Hp）感染之间的关系。方法对98例上消化道出血患儿组和65例非上消化道出血患儿组同时用13C-尿素呼气试验（13C-UBT）和采用ELISA法测定血清Hp抗体来判断Hp感染情况,所有患儿均行电子胃镜检查。结果上消化道出血患儿组和非上消化道出血患儿组的Hp阳性率分别为59.18%、18.46%,两组比较差异有统计学意义（P〈0.001）。结论儿童上消化道出血与幽门螺杆菌感染有密切关系。     

利用小鼠感染模型筛选与鉴定幽门螺杆菌外膜蛋白抗原

目的:利用小鼠感染模型筛选与鉴定幽门螺杆菌SS1株的外膜蛋白抗原。方法:提取SS1株的外膜蛋白进行双向电泳,用幽门螺杆菌感染的小鼠血清作免疫印迹实验,将阳性反应蛋白点进行质谱鉴定分析,将肽质量指纹谱数据输入互联网上的蛋白质数据库进行检索。结果:获得32种抗原相关蛋白。通过与已有报道的幽门螺杆菌感染人抗原比较分析,发现大部分典型的保护性抗原在本实验中都可以检测到。结论:幽门螺杆菌感染的小鼠模型适用于人用保护性幽门螺杆菌抗原的筛选;而且此研究中得到的相关抗原蛋白对于寻找与鉴定幽门螺杆菌未知保护性抗原也有参考价值。     

Production and Application of New Monoclonal Antibodies Specific for a Fecal Helicobacter pylori Antigen

The aim of the present study was to establish monoclonal antibodies that could be used to produce a diagnostic test composed of one kind of monoclonal antibody recognizing a fecal Helicobacter pylori antigen. The need to develop such a test arose from disadvantages of the diagnostic test that uses a polyclonal antibody or plural kinds of monoclonal antibodies, such as the lower specificity for H. pylori antigen and the difficulty of reproduction with consistent quality. Mice were immunized with sonicated cells of the coccoid form of H. pylori, and fecal samples from H. pylori-positive subjects were screened by a direct sandwich enzyme immunoassay (EIA) for antibody production from 32 hybridoma clones. The three stable clones produced antibodies (21G2, 41A5, and 82B9) that reacted with the same soluble antigen. Gel filtration chromatography showed that the molecular masses of the cellular antigen and the fecal antigen were the same, 260 kDa. The antigen was labile in response to sodium dodecyl sulfate and heat treatments. A single-step direct sandwich EIA using a single monoclonal antibody, 21G2, was developed. The EIA could detect the antigen in 41 H. pylori clinical isolates and in fecal samples from seven H. pylori-positive subjects. Several kinds of Helicobacter species (Helicobacter felis, Helicobacter hepaticus, Helicobacter mustelae, and Helicobacter cinaedi) except H. pylori, major bacteria in feces (Campylobacter jejuni, Bacteroides vulgatus, Bifidobacterium breve, Bifidobacterium infantis, and Escherichia coli), and fecal samples from six H. pylori-negative subjects showed negative results. These results indicate that the new monoclonal antibodies and the new specific EIA would be useful as a noninvasive method of diagnosis of H. pylori infection.     

Immune Responses to Differentiated Forms of Helicobacter pylori in Children with Epigastric Pain

Helicobacter pylori infection affects human populations of all ages. This gastric bacterium exists in spiral form and the reported viable but nonculturable coccoid form. The present study aims to examine the probable role of the coccoid form in H. pylori infection by comparing the seroprevalences of the spiral and the coccoid forms in children with epigastric pain. Four hundred eighty-nine children (mean age, 8.5 years) with epigastric pain formed the basis of this study. Five hundred ninety-nine schoolchildren of comparable ages and with no record of dyspepsia served as controls. The seroprevalence of antigens prepared from both morphological forms was examined by enzyme-linked immunosorbent assay. The results showed that 65 (13.3%) and 273 (55.8%) of 489 symptomatic children were seropositive for antigens of the H. pylori spiral and coccoid forms, respectively. In contrast, only 7.0% of the control group had elevated levels of immunoglobulin G antibodies against the spiral form, while 26.5% were positive for antibodies against the coccoid form. There were no significant differences between genders or among ethnic groups. The study showed a rise in seroprevalence corresponding with age: 7.1% for those ≤5 years to 21.4% for those ≥11 years. The seroprevalence of antigens of the H. pylori spiral and coccoid forms in children with epigastric pain was twofold higher than that in the control subjects. Interestingly, there was a fourfold increase in seropositivity for coccoid-form antigen compared to that for the spiral-form antigen among the symptomatic pediatric patients as well as the control group, indicating a possible infective role of the coccoid form of H. pylori in the pediatric patients with epigastric pain.     

根除儿童口腔幽门螺杆菌预防胃内幽门螺杆菌感染的多中心研究

目的:探讨根除儿童口腔幽门螺杆菌（Hp）预防胃内Hp感染的可能性。方法:采用多中心前瞻随机研究,选取口腔Hp阳性但胃内Hp阴性的幼儿园儿童共计427例,随机分为使用“无幽梅”牙膏组与普通牙膏组,分别接受“无幽梅”牙膏和普通牙膏。疗程结束后,再次检测口腔Hp,将口腔Hp阳性及阴性患者各分为一组,1年后行C13呼气试验检查,分析两组患者胃内Hp感染情况。口腔Hp检测方法采用特异度及敏感度双高的套式PCR方法。结果:随访1年,口腔Hp阴性组胃内Hp感染率为0.51%,口腔Hp阳性组胃内Hp感染率为6.51%,两组统计差异具有显著性（P<0.01）。结论:儿童根除口腔Hp可以降低胃内Hp感染的发生。     

Clinical Relevance of the babA2 Genotype of Helicobacter pylori in Japanese Clinical Isolates

Genotypic variation of Helicobacter pylori is speculated to associate with different clinical outcomes. In Western countries, the gene encoding blood group antigen-binding adhesin (BabA), babA2, is of high clinical relevance and is a useful marker to identify patients who are at higher risk for peptic ulceration and gastric adenocarcinoma, as are vacA and cagA. We investigated the presence of babA2 and cagA in 179 Japanese clinical isolates by PCR and Southern blot analysis and looked for correlations with various clinical outcomes (nonulcer dyspepsia, duodenal ulcers, gastric ulcers gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma). The prevalence of the babA2 genotype was 84.9% and that of the cagA genotype was 96.1%. There was no correlation between the babA2 and cagA genotypes, and there was no association between the babA2 or cagA status and clinical outcome. These results indicate that babA2 status is not of high clinical relevance in Japan and that Japanese strains are different from those infecting Western populations.