Evaluation of Immunological Rapid Urease Testing for Detection of Helicobacter pylori

 The aim of this study was to evaluate in clinical specimens the immunological rapid urease test (IRUT), a new diagnostic system for detection of Helicobacter pylori which employs a monoclonal antibody against Helicobacter pylori urease. Helicobacter pylori urease adsorbed on a solid-phase tip coated with a monoclonal antibody against Helicobacter pylori urease after 15 min of incubation with a gastric mucus sample solution was measured by the pH change of the urea solution inside the tip. The detection limit of Helicobacter pylori urease using this system was determined and compared with that of a commercially available rapid urease test. Clinical evaluation of the system was performed in 155 patients. The IRUT could detect 0.25 milli-international units (mIU) of Helicobacter pylori urease per milliliter in less than 20 min. If a patient with at least one positive result in a standard test for Helicobacter pylori was considered to be Helicobacter pylori positive, the sensitivity, specificity, positive and negative predictive values of the system were calculated as 95.2%, 98.9%, 98.4% and 96.8%, respectively. However, 10 of 19 Helicobacter pylori-positive patients in whom the pH change was less than 0.1 had negative results in at least one of the standard tests, whereas the IRUT correctly detected Helicobacter pylori in all but 3 of these 19 patients. The IRUT accurately determined the Helicobacter pylori status of 75 (98.7%) of 76 patients who had completed treatment. This system has high sensitivity for the detection of Helicobacter pylori, especially in patients with low urease activity.     

Microbiological aspects ofHelicobacter pylori (Campylobacter pylori)

The human gastric pathogenCampylobacter pylori has recently been reclassified asHelicobacter pylori, and a related spiral bacterium found in the stomach of ferrets has been designatedHelicobacter mustelae. The general microbiological features ofHelicobacter pylori are delineated here, with details of phenotypic differences betweenHelicobacter pylori andHelicobacter mustelae; comparisons are made withWolinella succinogenes andCampylobacter jejuni. TheHelicobacter organisms possess an external glycocalyx which can be visualised by electron microscopy, and which may be involved in bacterial adherence. The finding of soluble and cell-associated haemagglutinins ofHelicobacter pylori is reported. Detection ofHelicobacter pylori in clinical specimens, susceptibility of the organism to antibacterial agents, and other aspects of practical and clinical significance are briefly reviewed.     

Detection ofHelicobacter pylori in gastric biopsy tissue by polymerase chain reaction

To evaluate the sensitivity of a polymerase chain reaction (PCR) assay using nested primers in detectingHelicobacter 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 ofHelicobacter pylori. Fourteen of the 17 patients were positive forHelicobacter 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 detectingHelicobacter pylori, and that phenol/chloroform extraction is superior to simple boiling in obtaining DNA samples for PCR.     

Transport conditions and number of biopsies necessary for culture ofHelicobacter pylori

Stuart's transport medium with a charcoal impregnated swab was tested for transport of biopsies from the gastric antrum for culture ofHelicobacter pylori. Biopsies were cultured under microaerophilic conditions either within 2 h or after a delay of 24 h at 4 °C. In 65 patients referred for gastroscopy two biopsies were taken.Helicobacter pylori was found in 39 patients. The rate of survival ofHelicobacter pylori was found to be as high in Stuart's transport medium after 24 h at 4 °C as in the paired biopsy which was cultured immediately after transportation in normal saline. In five (13 %) of 39 patients positive forHelicobacter pylori, the organism was cultured from only one of the biopsies. It is therefore recommended that two biopsies be taken for culture.     

Role ofHelicobacter pylori in gastritis and duodenitis in man

AlthoughHelicobacter pylori is now accepted as the major aetiological factor in chronic gastritis in man, many of the factors which determine its pathogenicity are unknown. The organism has adapted to survive in the low-pH environment of the stomach, partly through its ability to buffer hydrogen ion by the hydrolysis of urea and by the presence of lectins on its surface, which bind to gastric mucosa and epithelial cells. After attachment, harmful toxins and enzymes have access to the gastric cells and cellular damage and an immune response ensues. In patients with duodenal ulceration,Helicobacter pylori-related gastritis predominantly affects the gastric antrum and has a high prevalence. Excessive gastrin production has been suggested as a potential aetiological factor linking infection with duodenal ulcer development. Perhaps more important is the association between gastric metaplasia of the duodenal epithelium, which is correlated with acid load and is more extreme inH. pylori positive patients with duodenitis. Organisms may subsequently spread from the gastric antrum into areas of gastric metaplasia in the duodenal bulb, leading to areas of chronic duodenitis and ultimately frank ulceration. It should not be overlooked, however, that other factors such as genetic predisposition, blood group, stress, drugs and smoking all have a role to play in the outcome, given the comparatively small number of patients in the general population infected withH. pylori who develop ulcer disease.     

Development of a rapid PCR method for identification ofHelicobacter pylori in dental plaque and gastric biopsy specimens

A rapid and simple polymerase chain reaction (PCR) method was developed to detectHelicobacter pylori in gastric biopsy specimens and dental plaque samples. The primers were targeted to the 16S rRNA sequence ofHelicobacter pylori strain ATCC 43504. The system was found to have a theoretical detection level of 0.5 to 5Helicobacter pylori cells in a 5 l sample of dental plaque. In the absence of plaque, the detection level was even better: theoretically, 0.05 to 0.5Helicobacter pylori cells were detected in water suspension. However, this appeared to be due to the presence of free bacterial DNA in the culture used for the sensitivity determination. Thus, the actual sensitivity of the system was found to be fewer than fiveHelicobacter pylori cells, irrespective of the type of sample used. The method was then used to analyse 29 dental plaque and gastric biopsy specimens collected from patients with a history of recurrent peptic ulcer disease. Fourteen stomach specimens were positive forHelicobacter pylori when tested with the PCR method, while the respective figures with culture, histological examination and the urease test were 11, 12 and 9. No positive dental plaque samples were observed.     

Effect of specimen collection techniques,transport media,and incubation of cultures on the detection rate ofHelicobacter pylori

Culture and histologic examination are considered gold standard methods for the detection ofHelicobacter pylori, but discrepancies may occur with either method. Failure to detectHelicobacter pylori may be due to sampling error, inappropriate transport or culture media, or insufficient duration of the incubation period. Rates of detection ofHelicobacter pylori by culture and histopathologic examination of gastric mucosal biopsy specimens were determined in 102 consecutive dyspeptic patients. In a separate group of 60 patients, rates of detection ofHelicobacter pylori by culture of antral brushings and the length of incubation required in selective and nonselective culture media were studied. In the first group of 102 patients, the combination of culture and histologic examination detected 54Helicobacter pylori-positive patients, whereas the separate techniques each detected 51Helicobacter pylori-positive patients. In the second group of 60 patients evaluated by culture of antral brushings, the rate of detection ofHelicobacter pylori was 25 of 60 and was similar for culture (25/60) and histologic examination (25/60). In the second group the length of incubation required to detectHelicobacter pylori was different for selective and nonselective media. In nonselective media, incubation of up to ten days was required to detect allHelicobacter pylori infections, whereas in selective media seven days was sufficient. Rates of detection ofHelicobacter pylori by culture, histopathologic examination and culture from brushings were similar, whereas the combination of culture and histopathologic examination achieved a superior rate of detection. The incubation period required for the detection ofHelicobacter pylori by culture was a minimum of seven days and was dependent on the culture medium used.     

Influence of a proton pump inhibitor-based therapy onHelicobacter pylori strain selection

The influence of the proton pump inhibitor lansoprazole on strain diversity inHelicobacter pylori infected patients was investigated. Multiple isolates ofHelicobacter pylori obtained pre- and post-therapy from gastric antral and body biopsies in 22 patients were compared using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) for analysis. Post-therapy strains exhibiting novel RAPD-profiles were found in 5 of 22 patients (4 of 11 patients treated with lansoprazole alone and 1 of 11 patients treated with lansoprazole plus amoxicillin). Proton pump inhibition may affect the microecology of the stomach by influencing the colonisation patterns of specific strains.     

Immune Response to Natural and Recombinant Antigens of Helicobacter pylori in Patients with Dyspeptic Complaints

 Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43–66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxinassociated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.     

Serodiagnosis ofHelicobacter 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 againstHelicobacter 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 immunoblottedHelicobacter pylori proteins showed reproducible immunoreactive bands at molecular weights of 130,93,75 and 67 kDa. The molecular weight protein fractions ofHelicobacter 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 byHelicobacter pylori infection was observed.     

Influence of oropharyngeal flora and specimen pretreatment on the recovery ofHelicobacter pylori

The recovery ofHelicobacter pylori was studied in vitro in the presence of different concentrations of oropharyngeal flora. The effect on the bacterial yield ofHelicobacter pylori and oropharyngeal flora of washing biopsies once, twice or thrice before culture was then studied in biopsies taken from 35 patients. A low rate of recovery of lowHelicobacter pylori concentrations (< 10³ cfu/ml) was related to the presence of relatively high oropharyngeal flora concentrations ( 10⁴ cfu/ml). After three washings a decrease in oropharyngeal flora between 10-fold and 100-fold cfu/biopsy was observed in seven patient samples. In another three specimens a more than 100-fold reduction in cfu/biopsy was observed. In the case ofHelicobacter pylori, a 60-fold decrease in log cfu/biopsy was observed in only one patient. Pretreatment of the specimens by washing of the biopsies improved the recovery of small amounts ofHelicobacter pylori in 10 of 28 patients. For optimal detection ofHelicobacter pylori in gastric mucosal biopsies, washing of the specimens in addition to the use of selective media is recommended.     

Helicobacter pylori Eradication for Prevention of Metachronous Recurrence after Endoscopic Resection of Early Gastric Cancer

Controversies persist regarding the effect of Helicobacter pylori eradication on the development of metachronous gastric cancer after endoscopic resection of early gastric cancer (EGC). The aim of this study was to assess the efficacy of Helicobacter pylori eradication after endoscopic resection of EGC for the prevention of metachronous gastric cancer. A systematic literature review and meta-analysis were conducted using the core databases PubMed, EMBASE, and the Cochrane Library. The rates of development of metachronous gastric cancer between the Helicobacter pylori eradication group vs. the non-eradication group were extracted and analyzed using risk ratios (RRs). A random effect model was applied. The methodological quality of the enrolled studies was assessed by the Risk of Bias table and by the Newcastle-Ottawa Scale. Publication bias was evaluated through the funnel plot with trim and fill method, Egger''s test, and by the rank correlation test. Ten studies (2 randomized and 8 non-randomized/5,914 patients with EGC or dysplasia) were identified and analyzed. Overall, the Helicobacter pylori eradication group showed a RR of 0.467 (95% CI: 0.362-0.602, P < 0.001) for the development of metachronous gastric cancer after endoscopic resection of EGC. Subgroup analyses showed consistent results. Publication bias was not detected. Helicobacter pylori eradication after endoscopic resection of EGC reduces the occurrence of metachronous gastric cancer.     

Epidemiology and role of Helicobacter pylori virulence factors in gastric cancer carcinogenesis

Infection with Helicobacter pylori is associated with the development of gastric cancer. Although the prevalence of gastric cancer has declined throughout years due to improvement in early screening strategy, mortality due to gastric cancer has not changed. Incidence and mortality due to gastric cancer are higher in developing countries as compared to developed countries. Diagnosis and prognosis of gastric cancer are still poor with patients usually diagnosed with cancer at an advanced stage. Eradication of H. pylori is pertinent for the prevention of gastric cancer. However, the rise in antimicrobial resistance among H. pylori isolates has complicated the prevention strategy. H. pylori express multiple virulence factors for survival in the hostile acid gastric environment. The expression of oncogenic protein cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and outer inflammatory protein is essential for H. pylori to exert pathogenesis towards the host. Interestingly, <3% of H. pylori-infected subjects develop gastric cancer, suggesting a unique way of interaction between the host's immune response and H. pylori virulence factors. This article is aimed to review the epidemiology and role of H. pylori in gastric carcinogenesis. A better understanding of the interaction between H. pylori virulence factors and host is required for better gastric cancer prevention.     

Altered Th17 Cytokine Expression in Helicobacter pylori Patients with TLR4 (D299G) Polymorphism

Helicobacter pylori (H. pylori) is associated with gastric ulcer and gastric adenocarcinoma. Polymorphisms in the host genes coding for Toll-like receptors (TLRs) may influence the innate and adaptive immune response to the infection, affecting the susceptibility to H. pylori or the disease outcomes. However, the details and association with different polymorphism and clinical expression of infection remain unclear. A case-control study consisting of 58 patients with H. pylori infection and 44 H. pylori uninfection was conducted. Genomic DNA was extracted and genotypes of TLR4 Asp299Gly polymorphism were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mucosal cytokines expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. The expression of IL-6, IL-17, IL-21, IL-23 and TGF-β1 was signi?cantly higher in patients with D299G polymorphism in TLR4. But the expression of IL-18 between patients with single-nucleotide polymorphisms (SNPs) in TLR4 and patients with the wild-type allele was not signi?cant. In H. pylori-infected patients with gastritis, SNPs in TLR4 may alter cytokine expression toward Th17 immune response in the gastric mucosa and may have increased risk for the development of peptic ulcer.     

Antigastric autoantibodies in Helicobacter pylori gastritis: prevalence, in-situ binding sites and clues for clinical relevance

Colonization of human gastric mucosa with Helicobacter pylori leads to chronic active gastritis and induces the occurrence of an acquired mucosa-associated lymphoid tissue (MALT) in the stomach. This remodelling of the gastric mucosa together with chronic antigen persistence may induce autoimmune reactions. The aim of this study was to investigate humoral autoimmune reactions to human gastric mucosa in H. pylori gastritis and their clinical relevance. Sera from patients with dyspeptic symptoms were tested for presence of IgG immunoglobulins against H. pylori. Gastric infection with H. pylori and alterations of gastric mucosa were demonstrated by histological examination of gastric biopsy specimens. All sera were tested for reactivity against human gastric mucosa by immunohistochemistry. Two different in-situ binding sites of antigastric autoantibodies were observed. Binding to canalicular structures within parietal cells was significantly correlated with antibodies to H. pylori, elevated basal gastrin levels and atrophy of gastric corpus glands. Our data indicate that autoimmune reactions to antigens in the human gastric mucosa occur in H. pylori gastritis and that they may play a role in the pathogenesis of the disease.     

Prevalence of Helicobacter pylori infection, chronic gastritis, and intestinal metaplasia in Mozambican dyspeptic patients

We estimated the prevalence of Helicobacter pylori infection, chronic gastritis, atrophy, and intestinal metaplasia in dyspeptic patients from Maputo Central Hospital, Mozambique and evaluated the relationship between infection and histopathological features of chronic gastritis. Biopsies from 109 consecutive patients observed in 2005–2006 were collected from antrum, incisura angularis, and corpus for histopathological study according to the Modified Sydney system. H. pylori infection was assessed by histology and polymerase chain reaction. H. pylori prevalence was 94.5%. Chronic gastritis was the most frequent diagnosis (90.8%). Degenerative surface epithelial damage was associated with higher H. pylori density. Glandular atrophy (8.3%) and intestinal metaplasia (8.3%) were infrequent. Our results confirm previous observations in African countries with high prevalence of H. pylori infection and low rates of gastric cancer: high frequency of chronic H. pylori-associated gastritis with very low frequency of gastric atrophy and intestinal metaplasia.     

Transport and storage ofHelicobacter pylori from gastric mucosal biopsies and clinical isolates

Various transport and storage conditions for the recovery ofHelicobacter pylori from gastric biopsies were evaluated. Gastric mucosal biopsies from 16Helicobacter pylori-infected patients were stored in cysteine-Albimi medium containing 20 % glycerol in a refrigerator (4°C) for 1 and 2 weeks and in a –20°C laboratory freezer for 4 and 12 weeks. Two clinical isolates were stored in saline, Stuart's transport media, cysteine-Albimi broth with 20 % glycerol, brucella broth with 20 % glycerol and skim milk with 17 % glycerol at room temperature, 4°C, –20°C and –70°C. Storage at 4°C for 1 and 2 weeks resulted inHelicobacter pylori recovery from 81 % and 19 % of biopsies, respectively. Storage at –20°C yieldedHelicobacter pylori recovery in 100 % and 57 % after 4 and 12 weeks, respectively. At room temperature after 6 h, theHelicobacter pylori titer was reduced. The best storage media for frozen isolates were skim milk/glycerol, brucella broth/glycerol and cysteine-Albimi/glycerol (in descending order). Recovery was better at –70°C than –20°C.     

Quantitative polymerase chain reaction for the detection ofHelicobacter pylori in gastric biopsy specimens

A variety of methods, including the polymerase chain reaction (PCR), are available for the detection ofHelicobacter pylori in clinical samples, but none of them can adequately quantify the organism. In the present study, the competitive PCR, a rapid and simple method for quantification ofHelicobacter pylori DNA in gastric biopsies, was used to measure the amount of DNA present inHelicobacter pylori-positive biopsies. This method is based on coamplification of an internal standard and a target DNA sequence with one set of primers. The internal standard was prepared using a nonhomologous fragment of DNA ligated to specific primers used to amplify the target DNA. This competitive DNA fragment of a desired size and containing primer templates is called a PCR MIMIC. To perform a quantitative PCR, PCR amplification reactions were spiked with known quantities of PCR MIMICs containing unknown amounts of DNA fromHelicobacter pylori-positive biopsies. The amount of target DNA was determined by visual comparison of the PCR products after establishment of the correlation between the internal control concentration and the DNA concentration in a competitive amplification reaction. The results were confirmed by a radioactive method. Quantitative PCR can be a reliable method for determining the extent ofHelicobacter pylori infection.     

Evaluation of a monoclonal antibody for detection ofHelicobacter pylori in a direct immunofluorescence test

A monoclonal antibody was developed for detection ofHelicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection ofHelicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.     

Possible evidence of invasiveness ofHelicobacter (Campylobacter)pylori

Gastric/duodenal biopsy material from 52 patients was examined immunohistochemically forHelicobacter (Campylobacter) pylori. Specimens from 34 of the patients harbouredHelicobacter pylori along the mucosal surface and 13 of these featured, in addition, immunopositive material within the lamina propria. The remaining 18 biopsies were non-reactive. This observation suggests thatHelicobacter pylori can penetrate the epithelium and its basement membrane, resulting in the production of specific systemic antibodies.