Recovery of Helicobacter pylori from gastric biopsy specimens is not dependent on the transport medium used.

The survival of six clinical isolates of Helicobacter pylori at room temperature was investigated after suspension in five different media: brucella broth with 5% lysed horse blood (BBLH), phosphate-buffered saline (PBS), 20% glucose, Stuart medium, and PBS with 10% Fildes enrichment (PBS-F). Only in BBLH and PBS-F no decrease in mean bacterial numbers was observed during the 24-h study period. No H. pylori isolates could be cultured from Stuart medium after 7 h of incubation. In contrast, the recovery rates in PBS-F or Stuart medium of H. pylori isolates from gastric tissue specimens collected from 19 H. pylori-positive patients were not significantly different even after a delay of culture of up to 24 h. Our data show that the medium composition is not critical for the survival of H. pylori within gastric tissue specimens.     

Detection of Helicobacter pylori in gastric biopsy and resection specimens.

AIMS: To compare the sensitivity of detecting Helicobacter pylori in gastric biopsy and resection specimens using tinctorial and silver impregnation stains, immunohistochemistry and the polymerase chain reaction (PCR). METHODS: Formalin fixed, paraffin wax embedded tissue from 33 gastric biopsy specimens (26 showing chronic gastritis and seven showing low grade mucosa associated lymphoid tissue (MALT) lymphoma) together with blocks of uninvolved mucosa from gastrectomy specimens for MALT lymphoma (five cases) were studied. Consecutive sections were stained using haematoxylin and eosin, Giemsa, the Warthin-Starry silver stain, and a polyclonal antibody directed against H pylori using an immunoperoxidase technique following heat induced antigen retrieval. PCR analysis of DNA extracted from a further section was carried out using primers which amplified a 411 base pair fragment of the urease A gene. RESULTS: H pylori was detected in 14 (37%) sections stained with haematoxylin and eosin, 21 (55%) with Giemsa, 23 (61%) with Warthin-Starry, and 25 (66%) stained with the antibody. Seventeen (45%) cases were positive on PCR. Immunohistochemistry was positive in all cases in which H pylori was detected by other methods. CONCLUSION: Immunohistochemistry using an immunoperoxidase technique following heat induced antigen retrieval for detecting H pylori in gastric biopsy and resection specimens is highly sensitive and easy to use.     

Optimal combination of media for primary isolation of Helicobacter pylori from gastric biopsy specimens.

The aim of the present study was to compare eight media, four nonselective and four selective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 5 months, mucosal antral biopsy specimens were obtained from 222 consecutive dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all eight media. Egg yolk emulsion agar (EYE), Skirrow's medium, Dent's medium, and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase soy agar (TSA), brucella agar, and brain heart infusion agar were used as nonselective media. Overall, by using these eight media, H. pylori was recovered from biopsy specimens from 114 of 222 patients, yielding an isolation rate of 51%. Comparison of all possible combinations of the eight media showed that the highest rate of isolation of H. pylori was 100% (114 of 114) with EYE-MCHOC, followed by 96.5% (110 of 114) when EYE-TSA was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 95%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs.     

Characterization of chromosomal DNA profiles from Helicobacter pylori strains isolated from sequential gastric biopsy specimens.

The restriction endonuclease profiles of DNAs from Helicobacter pylori strains isolated from 20 patients in two or more consecutive biopsy specimens over a period of up to 2 years were analyzed by pulsed-field gel electrophoresis with NotI and NruI. H. pylori strains possess a high degree of genomic diversity which was not observed to occur in vivo, and attempts to observe it in vitro were not successful.     

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.     

Isolation of Helicobacter pylori after transport from a regional laboratory of gastric biopsy specimens in saline, Portagerm pylori or cultured on chocolate agar

Multiple gastric biopsies were taken from 288 patients in Port Lincoln, South Australia. One biopsy was used for a CLOtest and the other three were transported to a central laboratory in Adelaide in physiological saline, Portagerm pylori transport medium or after culture on a chocolate agar plate which was placed in a Biobag. Helicobacter pylori was isolated from 18.3% of patients. There was a 95.7% concordance between culture results and the CLOtest result. Recovery rates after transport on chocolate agar, Portagerm pylori and in saline were 90.2, 90.2 and 84.3%, respectively.     

Genotyping of Helicobacter pylori strains in formalin-fixed or Formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens.

Different genotypes of Helicobacter pylori can play a role in the development of atrophic gastritis, peptic ulcer disease, and gastric carcinomas. In this study the authors developed polymerase chain reaction assays for the detection and identification of vacA (s- and m-regions), cagA, and iceA genotypes of H. pylori in formalin-fixed or formaldehyde-sublimate-fixed paraffin-embedded gastric biopsy specimens. Polymerase chain reaction products were analyzed by reverse hybridization on a line-probe assay. Complete genotyping was achieved in 26 of 28 samples (93%), and multiple genotypes, indicating the presence of multiple strains, were detected in nine samples (32%). This genotyping method offers the possibility for long-term retrospective studies on H. pylori genotypes and gastric pathology in the same archival gastric tissue specimens.     

Helicobacter pylori in gastric biopsy specimens. Comparison of culture, modified giemsa stain, and immunohistochemistry. A retrospective study

Antral biopsy specimens of 302 different endoscopic investigations of 200 patients with non-ulcer dyspepsia were studied for the presence of Helicobacter pylori in order to determine the most sensitive detection method. Part of the biopsy was cultured, and part stained using a modification of the Giemsa stain, and with an immunoperoxidase technique using a polyclonal rabbit anti-H. pylori antiserum. Cross-reactivity of this antiserum with other Campylobacter species was minimal. Material from 244 investigations was studied using all three detection methods. Culture was positive in 44 per cent, Giemsa in 78 per cent, and immunoperoxidase in 89 per cent of these biopsy specimens. Only five positive Giemsa stains with negative immunoperoxidase stain were found, whereas in 32 cases a negative Giemsa stain with a positive immunoperoxidase stain was seen. In the latter cases, the bacterial load was very low. The specimens revealed bacteria only sporadically, always confined to the deep layers of the gastric pits. Culture results correlated significantly with the bacterial load observed in the Giemsa stain. It is concluded that culture of H. pylori is the least sensitive detection method, whereas immunoperoxidase staining is the most sensitive. For daily practice the modified Giemsa stain, however, appears to be sufficient to diagnose the presence of the micro-organism.     

Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens.

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.     

Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens

The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H. pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains. Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR. The levels of anti-H. pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island. For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE. H. pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H. pylori, and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H. pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H. pylori infections.     

Comparison of genotyping Helicobacter pylori directly from biopsy specimens and genotyping from bacterial cultures

PCR for vacA and cagA genotypes of Helicobacter pylori using DNA isolated from infected gastric biopsy specimens was approximately equal to genotyping using bacterial DNA from cultures. Inconsistent results were associated with low H. pylori density in biopsies. A higher proportion of mixed infection was found when biopsies were used.     

Helicobacter pylori stains and association between H. pylori and inflammation in gastric specimens

Gastric cancer has been strongly associated with presence of the bacterium Helicobacter pylori. To improve techniques in identifying H. pylori so that gastric cancer may be predicted early, this project was formulated to determine whether one particular stain is more effective in displaying H. pylori microscopically. In addition, this study attempted to determine whether the degree of inflammatory elements present in tissue could be used to predict the likelihood of H. pylori presence. Protocols for the staining techniques, Steiner and alcian yellow/toluidine blue (AY/TB), were employed on specimens to semi-quantitate H. pylori presence. Serial sections from the same specimens were stained with hematoxylin and eosin to determine the amount of inflammation. Spearman rho correlation was used to evaluate the association between amount of H. pylori and inflammation in each case. It was determined that AY/TB was more easily performed, more effective in demonstrating H. pylori, and more cost effective than the Steiner stain. Additionally, it was determined that a moderate positive association was indicated between high levels of inflammation and marked presence of H. pylori.     

Ribotyping of Helicobacter pylori from clinical specimens.

Ribotyping is a method used to type strains of bacteria by analyzing the restriction enzyme digestion patterns of the rRNA genes. This method was applied to 126 strains of Helicobacter pylori from 100 unrelated symptomatic patients who had endoscopies done and to 15 strains from 15 infected subjects from seven families. Analysis of the rRNA gene patterns revealed 77 distinct ribotypes from the 100 patients. From 15 of these subjects, isolates were recovered from antral mucosal biopsies at follow-up endoscopy. All follow-up isolates from the same patient, with one exception, yielded identical digest patterns. This patient had strains with two distinct digest patterns obtained from a set of three isolates cultured from biopsy specimens taken at different times. Five patients who had isolates recovered from different sites in the stomach (antrum, gastric body, duodenum, and pyloric channel) showed ribotyping patterns which were identical for each patient yet distinct between patients. In seven family groups studied, identical digest patterns were detected in members of two families, with variability in strains detected among members of the remaining families. This study demonstrates that ribotyping provides a useful, reliable, reproducible, and highly discriminatory typing scheme for the study of H. 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.     

Comparative evaluation of three selective media and a nonselective medium for the culture of Helicobacter pylori from gastric biopsies.

Plating on solid media is the standard technique used in most laboratories for the isolation of Helicobacter pylori from gastric biopsies. Recently, various selective media were developed for this purpose. We compared and evaluated three selective media, Skirrow's, Dent's CP, and modified Glupczynski's Brussels campylobacter charcoal media, and chocolate agar medium for the isolation of H. pylori. Gastric biopsies taken from a total of 203 patients were plated in parallel on all four media. An isolation rate of 51% (104 of 203) was obtained with a combination of all four media. Of the 104, 92 (88%) were positive with Dent's medium and with modified Glupczynski's medium. Skirrow's medium gave the highest isolation rate, 96% (100 of 104). However, growth of H. pylori was scant (only one to five colonies) when growth occurred on Skirrow's medium alone. Overall, modified Glupczynski's medium provided significantly heavier growth. Chocolate agar medium yielded a 76% (79 of 104) positivity rate. We recommend the use of a combination of two selective media for the maximum recovery of H. pylori from antral biopsies.