Culture ofHelicobacter pylori from gastric biopsies transported in biopsy urease test tubes

Gastric biopsy specimens of 57 consecutively observed dyspeptic patients were studied for the presence ofHelicobacter pylori by histological examination, biopsy urease test (BUT) and culture. For culture, biopsy samples were transported in both Stuart media and BUT tubes. All 15 isolates could be cultured from both Stuart and BUT tubes. Thus, if the main reason for culture ofHelicobacter pylori is for antimicrobial susceptibility testing, only positive BUT tubes need to be submitted. This would reduce both the expense and the number of biopsies needed.     

Optimization of a medium for the rapid urease test for detection of Campylobacter pylori in gastric antral biopsies.

We developed a buffered azide-free urea medium which is sensitive, specific, and nontoxic for rapid detection of Campylobacter pylori in gastric biopsies. Detection of urease produced by the organism provides the basis for the test. The substrate is urea in monobasic sodium phosphate buffer, and phenol red provides indication of the pH change that results from urease activity. A rapid change from yellow to red occurs in the presence of C. pylori, even at low concentrations of the organism. A slower color change occurs with higher concentrations of other urease producers, such as Yersinia enterocolitica and Proteus mirabilis. Experience with 51 patients with our medium showed excellent results in detection of C. pylori in gastric mucosal biopsies. In clinical research and practice, a rapid bedside test will be helpful for rapid diagnosis of C. pylori-positive patients.     

幽门螺旋杆菌感染检测：快速尿酶法（RUT）和银染法（W-S）的比较

目的：快速尿酶法(rapid urease test,RUT)是一种快速、简便的幽门螺旋杆菌(Helicobacter pylori, Hp)检测方法,将其与病理诊断中传统的银染法(W-S)进行比较,通过对比二者的阳性率,以传统银染法(W-S)的阳性率为基准,判断快速尿酶法(RUT)是否能在临床诊断中发挥更好的作用,使Hp感染患者能够得到及时的确诊和治疗。方法：选取2012年8月至2013年8月期间,未使用过抗生素、质子泵抑制剂、H2受体阻滞剂等可能影响Hp检测结果药物的患者164例,同步完成快速尿酶法(RUT)和银染法(W-S)检测,对比快速尿酶法(RUT)和银染法(W-S)的阳性率。结果：快速尿酶法(RUT)的阳性率(35.37%)略高于银染法(W-S)的阳性率(32.32%)。结论：快速尿酶法(RUT)操作便捷,但容易受到诸多不稳定因素的影响,更适合Hp感染的初筛。银染法(W-S)操作相对复杂,但病理诊断结果具有更高的准确性,更适合作为Hp感染最终的确诊手段。     

Ultrastructural localization of urease ofHelicobacter pylori

Helicobacter pylori urease was characterized by means of an enzyme histochemical electron microscopic technique. Ultrastructural analysis revealed no urease activity in one strain; in sevenH. pylori strains (43.75%), urease activity was associated with the cell membrane. Eight strains (50.0%) showed reaction product located within the cytoplasm. Urease activity showed no correlation with localization of activity. Our results demonstrate thatH. pylori urease is not uniform in allH. pylori strains, and differences in activity and localization of urease activity may account for different virulence activities.     

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.     

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.     

Comparison of four different primer sets for detection of Helicobacter pylori in gastric biopsies and oral samples by using real-time PCR

AIMS: The presence of Helicobacter pylori. in the oral cavity remains controversial and the most appropriate method for detection of oral H. pylori has yet to be established. The aim of the present study was to compare four different primer sets on the detection of H. pylori in gastric biopsies and oral samples using real-time PCR. MATERIAL AND METHODS: Gastric biopsy and oral samples were collected from eight patients with gastric symptoms. DNA from clinical samples was extracted and analyzed for the presence of H. pylori by real-time PCR (LightCycler 2.0) using four pairs of primers which targeted 16S rRNA (16S rRNA#295; 16S rRNA#120) or glmM (glmM#294; glmM#722) DNA genes. Three H. pylori strains and three clinical isolates served as reference. The specificities of the four primer pairs were examined for seven oral microorganisms and two Helicobacter non-pylori species. RESULTS: Primer pair 16S rRNA#120 showed an acceptable specificity and a high sensitivity. Primer pairs glmM#294 and glmM#722 demonstrated a high specificity but a low sensitivity and primer pair 16S rRNA#295 demonstrated a poor specificity but acceptable sensitivity. Four H. pylori positive gastric biopsies were demonstrated by culture, histology and real-time PCR with primer pairs 16S rRNA#295 or 16S rRNA#120. No H. pylori was detected in oral samples, either by culture or by real-time PCR. CONCLUSION: Of the four different primer pairs examined, 16S rRNA#120 was the most appropriate to detect H. pylori in clinical samples using real-time PCR.     

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.     

Simple spot test for rapid detection of urease activity

A spot test for the detection of urease activity was developed and evaluated with 761 strains of gram-negative bacteria. The test was compared with the conventional Christensen urea agar slants and urease test on the Vitek Enterobacteriaceae card (Vitek Systems, Inc., St. Ana, Mo.). Of the 348 strains of the Proteus-Providencia-Morganella group that were urease positive, 327 (94%) yielded positive results within 1 min, and all strains yielded positive results within 2 min. All these organisms also gave a positive urease reaction on the Vitek Enterobacteriaceae card test within 5 h and on the Christensen urea agar slants in 4 to 48 h. All the bacteria that did not hydrolyze urea by these two tests also remained negative by the spot test.     

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.     

Histology compared with chemical testing for urease for rapid detection of Helicobacter pylori in gastric biopsy specimens.

Gastric biopsy specimens from 283 patients with ulcer and non-ulcer dyspepsia attending five gastroenterology clinics in the northern region of the United Arab Emirates (UAE) were tested by the agar gel test (n = 115) or the ultra-rapid endoscopy room test (n = 168) for the presence of Helicobacter pylori urease. Results were compared with a histological technique using the Romanowsky type (Diff-3) stain for detecting H pylori in both antral and body type gastric mucosa. A sensitivity of 94% and specificity of 100% using the agar gel test compared with 87% sensitivity and 99.3% specificity for the ultra-rapid endoscopy room test. Grading of H pylori in gastric biopsy specimens showed that the higher the histological grade, the more likely that the urease test would be positive. Both forms of urease tests have high specificity for detecting H pylori in gastric biopsy specimens, although the urea agar test has a higher sensitivity than the ultra-rapid test. Low numbers of H pylori in gastric biopsy specimens are the most important determinant of a false negative urease test.     

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.     

Evaluation of a rapid urease test to detect Campylobacter pylori infection.

Seventy-five consecutive patients referred for upper gastrointestinal tract endoscopy were evaluated for Campylobacter pylori infection by pathology, culture, and a biochemical test to detect bacterial urease. Forty-eight patients (64%) had C. pylori present based on pathology or culture or both. Thirty-two urease tests were positive after 1 h, all in patients with C. pylori detected by the two other methods (specificity, 100%; sensitivity, 67%). After 24 h, 47 urease tests were positive, but only 40 had C. pylori present (specificity, 74%; sensitivity, 83%). When read after 1 h, the urease test was highly specific and led to rapid presumptive diagnosis.     

Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies.

Helicobacter pylori is a spiral, gram-negative bacterium which causes chronic gastritis and plays a critical role in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori expresses significant urease activity which is an essential virulence factor. Since a significant fraction of urease activity is located on the surface of the bacterium, the urease molecule is a logical choice as an antigen for a vaccine; currently recombinant urease apoenzyme is being tested as a vaccine in phase II clinical trials. We have recently demonstrated that urease and HspB (a homolog of the GroEL heat shock protein) become associated with the surface of H. pylori in vitro in a novel manner: these cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria, reflecting the unique characteristics of the outer membrane. To determine if similar mechanisms are operative in vivo, we determined the ultrastructural locations of urease and HspB within bacteria present in human gastric biopsies. Our results demonstrate that both urease and HspB are located within the cytoplasm of all bacteria examined in human gastric biopsies. Interestingly, a significant proportion of the bacteria examined also possessed variable amounts of surface-associated urease and HspB antigen (from 5 to 50% of the total antigenic material), indicating that in vivo, H. pylori has surface characteristics which enable it to adsorb cytoplasmic proteins. This is consistent with our altruistic autolysis model in which H. pylori uses genetically programmed bacterial autolysis to release urease and other cytoplasmic proteins which are subsequently adsorbed onto the surface of neighboring viable bacteria. These observations have important implications regarding pathogenesis and development of vaccines for H. pylori.     

Evaluation of a serological test for diagnosis ofHelicobacter pylori infection in children

A serological test for the diagnosis ofHelicobacter pylori infection (Cobas Core Roche, IgG, 2nd Generation; Roche, France) was compared with the examination of biopsy samples (culture and histology) obtained after endoscopy in 115 children to assess its value. In 94 children (42 positive and 52 negative), results were concordant. In 10 children a positive serological test was associated with an absence ofHelicobacter pylori, while in 11 others a negative serological test was associated with a positive culture. Sensitivity of the test was 79.2% and specificity 83.9%. A relationship between IgG titers and age (r=0.31, p<0.05) was found. Serological tests could be useful for the diagnosis ofHelicobacter pylori infection, but a negative test result does not rule out infection, particularly in children under 10 years of age.     

Comparison of ELISA for antibody detection and biopsy urease test against H. pylori in cases of gastoduodenal disorders

Out of 156 cases of various gastro duodenal disorders studied H. pylori was diagnosed in 119 (76.28%) as indicated by Biopsy urease test and IgG ELISA. Biopsy urease test detected higher number of cases 119 when compared to IgG ELISA 107 cases. ELISA being a non invasive technique can be used successfully for the diagnosis of H. pylori infections.     

Diagnostic accuracy of a rapid whole-blood test for detection of Helicobacter pylori.

In this study, we evaluated a rapid whole-blood test, BM-test Helicobacter pylori, for detection of H. pylori infection in 144 and 48 patients with other gastrointestinal symptoms and with gastric cancer, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the test correlated well with the standards used for the calculation, i.e., serology by enzyme-linked immunosorbent assay or culture and histology.