Non-invasive techniques for the diagnosis of Helicobacter pylori infection

Helicobacter pylori infection can be diagnosed by invasive techniques requiring endoscopy and biopsy (histologic examination, culture, polymerase chain reaction), and non-invasive techniques (serology, urea breath test, urine or blood, detection of H. pylori antigen in stool specimen). However, recent studies have demonstrated that a strategy of 'testing and treating' for H. pylori in uninvestigated, young (<50 years), dyspeptic patients in primary care is safe and reduces the need for endoscopy. Indeed, a number of clinical guidelines recommend non-invasive testing in dyspeptic patients followed by treatment of H. pylori in primary care based on clinical and economic analyses. Several non-invasive tests are currently available on the market. The choice depends on the clinical circumstances, the likelihood ratio of positive and negative tests, the cost-effectiveness of the testing strategy, and, finally, the availability of the tests. Nevertheless, two non-invasive tests are commonly used: the urea breath test, and the stool antigen test.     

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。     

IgG subclass response to Helicobacter pylori and CagA antigens in children

Specific serum IgG subclass antibodies against Helicobacter pylori antigens and recombinant CagA were analysed in 75 symptomatic children with histologically confirmed H. pylori infection. H. pylori stimulated an IgG1 predominant response, and IgG3 titres showed a positive association with peptic ulcer disease, chronicity of antral inflammation and density of H. pylori colonization. Two methods used for assessing serum IgG CagA antibody status, i.e. Western blotting and enzyme-linked immunosorbent assay (ELISA), were concordant. CagA stimulated an IgG1 and IgG3 predominant humoral response. Total CagA IgG titres were higher in children with active and more severe chronic antral inflammation. These findings suggest that in children the systemic humoral immune response to H. pylori infection may reflect gastroduodenal pathology.     

Detection of Helicobacter pylori infection in symptomatic Bulgarian adults

This study assessed the prevalence of Helicobacter pylori in symptomatic Bulgarian adults by means of culture, Gram's stain and an in-house rapid urease test (RUT), and also assessed the H. pylori density by culture. In total, 1441 non-treated and 270 treated patients were evaluated. Most non-treated patients with ulcers (87.7%), gastric malignancy (79.2%) and other gastroduodenal diseases (73.4%) were H. pylori-positive. Among non-treated and treated patients, 75.3% and 54.8%, respectively, of elderly patients, and 78.3% and 56.1%, respectively, of other adults were H. pylori-positive. Two (0.1%) non-treated adults were Helicobacter heilmannii-positive. The accuracy of direct Gram's stain and the in-house RUT were 74.8% and 64.2% in non-treated patients, and 73.7% and 63.0% in treated patients, respectively. Culture was highly accurate (>95%) in both groups. Older age decreased the sensitivity of the RUT in non-treated patients by 10.7% and that of all tests in treated patients by 6.9-8.1%. Incubation for 11 days was required for the growth of 2% and 4% of the strains from treated patients on selective and non-selective medium, respectively. There were no differences in isolation rates between positive fresh (74.2%) and frozen (75.2%) specimens. In non-treated adults, a high H. pylori density (growth in all quadrants of the plates) was more common (43.1%) in ulcer patients than in other patients (25.4%). In conclusion, H. pylori infection was common in Bulgarian patients, and at a high density in >40% of ulcer patients, while H. heilmannii infection was uncommon. Culture provided a highly accurate diagnostic approach. Stomach biopsies from non-treated patients can be frozen for several days. The benefit of reporting H. pylori density, as determined by culture, requires further evaluation.     

Relationship of TT virus and Helicobacter pylori infections in gastric tissues of patients with gastritis

Blood and gastric tissue biopsies of 34 patients with gastritis were tested for the presence of TT virus (TTV), a ubiquitous virus found in the blood of most humans. Thirty-one of these patients were TTV positive, and 27 patients had virus in both tissues. In addition, 13 of the patients who had TTV in gastric tissue were Helicobacter pylori positive. There was an association of higher TTV titers in gastric tissues of patients who were H. pylori positive than in those in whom the bacterium could not be detected. Furthermore, this association was stronger in H. pylori-positive patients with the presence of the cagA protein. Of 10 specimens in which genogroup determination was carried out in the gastric corpus, 5/5 that were H. pylori positive showed the presence of TTV genogroup 3, while for those that were H. pylori negative, 5/5 showed the presence of genogroup 1t. By contrast, genogroup 1 was found in the corpus of only one H. pylori-positive patient, and genogroup 3 in only one H. pylori-negative patient. The histological severity of gastritis did correlate significantly with loads in the gastric tissues. There was no significant difference in TTV titer in blood of patients regardless of H. pylori infection status. These findings pique interest in clarifying the role of TTV, alone or in association with H. pylori infection, in the pathogenesis of gastritis.     

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type

Yasuda A, Uchida T, Nguyen LT, Kawazato H, Tanigawa M, Murakami K, Kishida T, Fujioka T, Moriyama M. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type. APMIS 2009; 117: 893–9. Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA‐positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver‐operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori‐infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up‐regulated by adhesion to epithelial cells.     

Helicobacter pylori antibody titres in serum,plasma and successively thawed specimens: implications for epidemiological and clinical studies.

Agreement between Helicobacter pylori IgG antibodies measured using the Pylori-set EIA-G kit in serum, plasma and successively thawed specimens was studied and the implications for epidemiological and clinical studies assessed. Plasma titres may differ from serum titres by -6% to +8% and therefore may be substituted for serum. The change in titre around the cut off value was -0.31 (se = 5.7, p = 0.96) per thaw. The estimated maximum drop after three thawings, 34.5, would result in only a small decrease in sensitivity (1.3%). For qualitative epidemiological studies, this additional misclassification rate is relatively small. However, positive titres did reduce over successive thawings, with the estimated maximum drop being 11.4% per thaw. Therefore, thawing does need to be considered as a contributing factor when interpreting titre drops in eradication trials. Baseline and follow up specimens from clinical studies should be thawed once only and tested concurrently.     

Assessment of different methods for staining Helicobacter pylori in endoscopic gastric biopsies

The recent implication of Helicobacter pylori in the pathogenesis of gastritis-peptic ulcer syndrome and its relevance for the development of upper gastrointestinal malignancy warrant efficient methods for the detection and demonstration of the organism in biopsy specimens. We have compared 5 staining methods, namely, haematoxylin and eosin (H & E), immunohistochemistry (IHC), the silver staining HpSS, the alcian yellow-toluidine blue (Leung) method (A-Y) and Genta staining, for the demonstration of the organism in gastric biopsies taken from antrum, body and fundus of 118 patients who presented to our hospital with upper gastrointestinal symptoms. We found no significant differences in the efficacy of H & E, IHC, HpSS and A-Y in the demonstration of H. pylori in all 3 gastric sites. The least reproducible stain in our hands was the Genta stain. We conclude that H & E is adequate for the initial assessment of gastric biopsies in symptomatic upper gastrointestinal patients. This is because it is a well-tested, cheap and easy staining method, requiring a relatively short period of time to perform, with highly reproducible results. It has an added advantage of enabling simultaneous assessment of morphological changes accompanying H. pylori infection. When the density of the organism is expected to be low, we recommend addition of HpSS staining because of its high sensitivity and low cost. The disadvantages of the other staining methods (IHC, A-Y and Genta) are discussed.     

High seroprevalence of Helicobacter pylori infection in non-institutionalised children with mental retardation

Eighty-four children with mental retardation (34 boys, 50 girls; age range 2-18 years, median 6 years) and 84 age- and gender-matched outpatient controls were studied. All children were living at home, had never stayed in an institution, and came from the same urban area. Seropositivity for Helicobacter pylori was found in 42 (50%) of 84 mentally retarded children and 16 (19%) of 84 controls (p < 0.01). Socio-economic factors did not differ between the two groups. The findings indicated that a higher prevalence of H. pylori infection occurs in children with mental retardation, regardless of whether they are institutionalised.     

Evaluation of Pyloriset Dry, a new rapid agglutination test for Helicobacter pylori antibody detection.

We evaluated the performance of a new latex agglutination test, Pyloriset Dry (Orion Diagnostica, Espoo, Finland), in the simultaneous detection of immunoglobulin G (IgG), IgA, and IgM antibodies to Helicobacter pylori and compared it with that of the Pyloristat test (BioWhittaker, Fontenay-sous-Bois, France), an enzyme-linked immunosorbent assay detecting IgG to H. pylori, for 96 untreated dyspeptic patients who had undergone gastroduodenal endoscopy. Infection was diagnosed in 56 cases by positive culture and/or positive Giemsa stain and rapid urease test (antral biopsies) and was associated with chronic gastritis in 52 patients. Forty noninfected patients did not have chronic gastritis. The sensitivity of Pyloriset Dry was 91.1%. The sensitivity of Pyloristat was 91.1 or 82.1%, depending on whether equivocal results were considered positive or negative, respectively. Both tests had a specificity of 87.5%. Their performances were not statistically different. Thus, Pyloriset Dry is an alternative to serological tests for adults, particularly when a small number of serum samples has to be tested.     

Relation between IgG and IgA antibody titres against Helicobacter pylori in serum and severity of gastritis in asymptomatic subjects.

AIMS--To investigate whether the absorbance index of IgG and IgA antibodies against Helicobacter pylori is related to a semiquantitative assessment of the density of H pylori colonisation in gastric biopsy specimens and to the severity of gastritis. METHODS--The grade of gastritis was scored separately for antral and fundic mucosa using three different classifications. Serum IgA and IgG antibodies against H pylori were measured by ELISA. The density of gastric H pylori colonisation was graded semiquantitatively from 0 to 3. RESULTS--Among 48 healthy volunteers studied, 17 were found to have gastritis according to Whitehead''s criteria. H pylori was present in the biopsy specimens of 14 of 17 subjects with gastritis. The IgG H pylori antibody absorbance index was significantly (p < 0.05) correlated not only with the density of antral H pylori colonisation, but also with the degree of gastritis of the antrum, as assessed by the Whitehead score, activity, and the Sydney system (p < 0.05). The IgA H pylori antibody absorbance index was significantly correlated with the Whitehead score and Sydney system, but not with the activity score of the antrum or with the density of antral gastric H pylori infection. There were no significant correlations between the IgG H pylori antibody absorbance index and the gastritis scores of the fundus mucosa and the density of H pylori infection of the gastric body. The IgA H pylori antibody absorbance index was only significantly (p < 0.05) correlated with the density of H pylori colonisation and the Sydney system gastritis score of the corpus. CONCLUSIONS--The serological absorbance index of IgG antibodies against H pylori is related to the severity of antral gastritis and the density of antral H pylori colonisation. Thus a high absorbance index of IgG antibodies against H pylori points to severe antral gastritis and dense H pylori colonisation of the antrum.     

Efficacy of two triple eradication regimens in children with Helicobacter pylori infection

Triple therapy with bismuth subsalicylate, amoxicillin, metronidazole (BAM) or with omeprazole, amoxicillin, clarithromycin (OAC) has been commonly used for the eradication of Helicobacter pylori infection. We compared the efficacy of these triple therapies in children with H. pylori infection. We retrospectively analyzed results in 233 children with H. pylori infection and treated with OAC (n=141) or BAM (n=92). Overall eradication rates of triple therapy with OAC and BAM were 74% and 85%, respectively, which showed no statistical difference. Our study showed that the triple therapy with BAM was more effective for the first-line eradication of H. pylori infection in Korean children, but has no statistical difference with OAC regimen.     

Use of PCR with feces for detection of Helicobacter pylori infections in patients.

PCR was performed for the detection of Helicobacter pylori in feces from 24 patients with proven infections. Several precautions were taken to overcome possible inhibition of PCR with feces. In the first 12 patients, feces were examined shortly after endoscopy. In another group of 12 patients, who were treated during 2 weeks with omeprazole (40 mg each day) to increase gastric pH, feces were examined as well. H. pylori target DNA could not be detected in the stools of any of the 24 infected patients. It was concluded that there was no substantial shedding of H. pylori in feces from either group of patients.     

Genotyping of the cagA gene of Helicobacter pylori on immunohistochemistry with East Asian CagA-specific antibody

The cytotoxin-associated antigen A (CagA) of Helicobacter pylori prevalent in East Asian countries, where the mortality rate due to gastric cancer is high, has been reported to be structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. Based on the structural features of the EPIYA motifs located at the carboxyl terminal of the protein, CagA was subdivided into two types: East Asian CagA and Western CagA. A recent study suggested that immunohistochemistry with anti-East Asian-specific antibody (α-EAS Ab), which was specifically immunoreactive with East Asian CagA but not with Western CagA, may be useful for diagnosis of the cagA genotype. To further evaluate the value of this diagnostic method in terms of sensitivity, specificity, and accuracy, 143 gastric biopsy specimens with α-EAS Ab were analyzed on immunohistochemistry and compared with the sequencing of the cagA gene. It was found that diagnosis of the cagA genotype of H. pylori on immunohistochemistry using the α-EAS Ab was highly sensitive (sensitivity 93.2%) and specific (specificity 72.7%), suggesting that immunohistochemical diagnosis of the cagA genotype is useful for diagnosis of the cagA genotype.     

Cytokine expression associated with Helicobacter pylori and Epstein‐Barr virus infection in gastric carcinogenesis

Helicobacter pylori and Epstein‐Barr virus (EBV) infection, and associated cytokines are involved in gastric carcinogenesis. We investigated the expression of these cytokines and their relationship with clinicopathological characteristics. The study included specimens from 207 patients with gastric adenocarcinoma, 56 with chronic gastritis, 32 with metaplasia, and 30 with low‐grade epithelial dysplasia. Tissue microarrays were constructed and immunohistochemical staining for IL‐1β, IL‐6, IL‐10, IL‐17, p16, p21, TNF‐α, and TNFR1 was performed. EBV and H. pylori infection status was determined. IL‐1β, IL‐6, IL‐17, p16, and p21 protein expression was significantly higher in adenocarcinoma cases than in the other cases (p < 0.05). EBV was only noted in adenocarcinoma (13 cases, 6.3%). The H. pylori infection rate in adenocarcinoma was significantly higher than that in the other cases (p < 0.005). IL‐6 expression was associated with improved survival (p < 0.05), whereas IL‐17 expression was associated with decreased survival (p < 0.05). IL‐6 expression was inversely associated with angioinvasion, and disease stage (p < 0.05), whereas IL‐17 expression was associated with disease stage (p < 0.05). IL‐10 expression was correlated with IL‐1β and TNF‐α expression, and p16 expression was correlated with IL‐17 and EBV status. Our results indicate that IL‐6 and IL‐17 are associated with gastric carcinogenesis and may be considered prognostic factors.     

Helicobacter pylori infections in IgA deficiency: lack of role for the secretory immune system

During the last years considerable attention has been focused on the possibility of the development of an oral vaccine against Helicobacter pylori. However, Helicobacter infection is known to be life-long, despite a vigorous immune response, and the hypothesis that an increased local production of secretory IgA in the gastric mucosa, due to vaccination, should protect against the colonization of the bacterium may therefore be questioned. In this study, when comparing the seropositivity and titre against H. pylori in IgA-deficient patients and age-related normal blood donors, it appears that lack of secretory IgA does not seem to have any major influence on the prevalence of the infection, nor is it reflected in titres of specific IgG antibodies. These results may argue against a pivotal role for IgA in the defence against Helicobacter, and raise questions about current strategies for the development of an oral vaccine against H. pylori and may point to a need for alternative therapeutic strategies.     

抗幽门螺杆菌血清IgE和胃粘膜肥大细胞在Hp致病中的作用

目的探讨机体免疫反应在幽门螺杆菌（Ｈｐ）致病中的作用。方法采用间接ＥＬＩＳＡ法检测了１４９例患者血清中抗ＨｐＩｇＥ,并用改良甲苯胺蓝染色法检测其胃粘膜中肥大细胞（ＭＣ）。结果①Ｈｐ阳性者血清抗ＨｐＩｇＥ含量、阳性率和胃粘膜中ＭＣ总数及脱颗粒比均显著高于Ｈｐ阴性者（Ｐ＜０．０１）;②不同胃部疾病之间血清抗ＨｐＩｇＥ含量、阳性率和胃粘膜中ＭＣ总数及脱颗粒比有显著差异,活动性胃炎显著高于非活动性胃炎和消化性溃疡（Ｐ＜０．０１）,中重度胃炎显著高于轻度胃炎（Ｐ＜０．００１）;③血清抗ＨｐＩｇＥ阳性者胃粘膜内ＭＣ总数及脱颗粒比均显著高于抗ＨｐＩｇＥ阴性者（Ｐ＜０．０１）,且血清抗ＨｐＩｇＥ含量与胃粘膜内ＭＣ脱颗粒比呈正相关（ｒ＝０．６０,Ｐ＜０．００１）。结论血清抗ＨｐＩｇＥ参与了Ｈｐ的致病过程,其机制可能为刺激ＭＣ脱颗粒,而致胃粘膜损伤。     

Assessment of the cag pathogenicity island status of Helicobacter pylori infections with serology and PCR

The serological characterisation of Helicobacter pylori strains has been questioned, e.g., when the presence or absence of the cag pathogenicity island (PAI) is determined. This study compared CagA-reactive serum antibodies, assessed with immunoblot, with cag PAI status, as determined by PCR. CagA serology results were available for 101 individuals contributing 202 bacterial samples for cag PAI PCR. There was a high degree of correlation between the two methods (kappa coefficient, 0.82; 95% confidence interval, 0.68-0.97). Combined with suggested biological explanations for the discrepancies, this finding supports the application of well-evaluated serological assays in the assessment of the cag PAI status of H. pylori infections in clinical and epidemiological studies.     

Immunophenotype of peripheral blood natural killer cells and IL‐10 serum levels in relation to Helicobacter pylori status

Recent findings suggest that NK (Natural Killer) cells may directly modulate the antimicrobial immune responses. In this study, we performed immunophenotypic analysis of peripheral blood NK cells with regard to CD56, CD16, Nkp46, and CD25 markers, as well as IL‐10 levels quantification in the sera samples of asymptomatic, H. pylori (Hp)‐infected or uninfected individuals, and combined these results with our previous findings on lymphocyte cytotoxic activity. Twenty healthy volunteers [10 Hp(?);10 Hp(+)] were included in the study. The percentages of classic lymphocytes (CD3⁺) and NK cells (CD3^?CD56⁺, CD3^?Nkp46⁺, CD3^?CD16⁺) with or without CD25 receptor were evaluated by fluorochrome‐conjugated monoclonal antibody staining and flow cytometry analysis. IL‐10 quantification was performed by enzyme‐linked immunosorbent assay‐ELISA. Our study showed elevated levels of IL‐10 and higher NK cell numbers of both CD3^?CD56⁺CD25⁺ and CD3^?Nkp46⁺CD25⁺ phenotypes, as well as CD3⁺CD25⁺ classic lymphocytes in Hp(+) compared with Hp(?) individuals. No differences between Hp(?) and Hp(+) individuals were found either in total number of classic lymphocytes or NK cell subtypes. Our data suggest that in Hp(+) donors, there is a domination of lymphocytes and NK cells co‐expressing CD25 marker, which might be influenced by the regulatory IL‐10. This phenomenon may be a result of H. pylori adaptation to a changing environment in vivo leading to a chronic infection and lack of severe gastric pathologies.     

IgM antibody response to antigens prepared from vegetative and coccoid forms of Helicobacter pylori

This report describes the utility of antigens prepared from different coccoid and spiral forms of Helicobacter pylori in a serological method. The presence of IgM antibody to H. pylori was determined in 22 human sera on antigens prepared from 24 strains of H. pylori. Antigens prepared from spiral form of certain strains of H. pylori detected IgM in all confirmed positive sera. Antigens obtained from the coccoid cells of the same strains could not completely detect IgM in the same sera. Testing sera on boosted antigens of the coccoid cells showed reduction in the number of false negative, indicating that the coccoid cells do not have one or more antigenic fractions essential for accurate detection of antibody. Our data suggest that H. pylori may lose CagA during the coccoid conversion process and regain it in the spiral form. In conclusion, we suggest that the antigen used for the detection of antibodies to H. pylori in serological methods should contain a broad spectrum of antigenic fractions and should be prepared from certain strains and culturable cells of H. pylori.