Pylori In Biopsy Specimens Research Articles

Rapid and accurate detection of Helicobacter pylori from biopsy specimens using loop‐mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid-based assay for quick, accurate and cost-effective diagnosis of many infectious agents. The purpose of this study was to assess the diagnostic value of LAMP for rapid and accurate detection of Helicobacter pylori in biopsy specimens. Patients suffering from one or several gastroduodenal disorders were enrolled in the study. Specificity, sensitivity, and the positive and negative predictive values of LAMP were compared with the gold standard result, which was the assembled result of culture, rapid urease test and polymerase chain reaction. Sensitivity, specificity, and the positive and negative predictive values of LAMP in comparison with the gold standard result were 100%, 30.76%, and 87.67% and 100% respectively [%95 CI]. As the diagnostic value of LAMP is favourable, the method is an optimum technique for diagnosis the presence of H.pylori in different clinical and environmental samples.

Helicobacter pylori vacA d1 Genotype Predicts Risk of Gastric Adenocarcinoma and Peptic Ulcers in Northwestern Iran

There is a close relationship between Helicobacter pylori (H pylori)-specific factors and different gastroduodenal diseases. The present study aimed to investigate the prevalence of vacA d1, d2 genotypes in the H pylori isolates from patients with gastric adenocarcinoma, peptic ulcer disease (PUD) and gastritis in East Azerbaijan region, where the incidence of gastric cancer (GC) is high. Strains isolated from this area are likely to be of European ancestry. In this study, genotyping of the vacA d region of 115 isolates obtained from patients with different gastrodoudenal diseases was accomplished by PCR methods. In addition to PCR amplification of H pylori 16S rDNA, rapid urease tests or histological examination were used to confirm the presence of H pylori in biopsy specimens. Data were collected and analyzed using SPSS version 19. Of the total of 83 H pylori isolates, 36 (43.4%) contained the d1 allele and 47 (56.6%) were subtype d2. The results of the multiple linear/logistic regression analysis showed high correlation between allele d1 and gastric adenocarcinoma or PUD. This study suggests that the H pylori vacA d1 genotype helps predict risk for gastric adenocarcinoma and PUD in East Azerbaijan, Iran.

Determination of Helicobacter pylori Virulence Genes in Gastric Biopsies by PCR

Aim. The aim of this study was to identify the presence of H. pylori in biopsy specimens from symptomatic patients by PCR. In addition, the rate of cagA, vacA, iceA1, and iceA2 virulence genes was determined. Materials and Methods. One hundred antral gastric biopsy specimens were collected during endoscopy from patients suffering from gastroduodenal symptoms. The samples were collected by the gastroenterologists in their own clinics in Ramallah, Palestine. DNA was extracted from the biopsies and subsequently used for PCR identification of H. pylori and the virulence genes using specific primers. Results. The rate of positive H. pylori in the collected biopsies was 44%. The rates of the virulence genes in this sample: cagA, vacA, iceA1, and iceA2 were 65.9%, 40.9%, 63.6%, and 84.1%, respectively. Conclusion. The iceA2 gene was the most frequent in this study. Much research is necessary to determine the presence of an association of this gene with gastric pathology. Variation in the rates of the iceA gene in different countries is a strong indication of its geographical distribution. This study would provide important information regarding the prevalence of virulence genes (vacA, cagA, iceA1, and iceA2) in H. pylori strains in the sample tested in this country.

Immunocytochemical Study of Helicobacter Pylori in the Mucosae of the Gastric Antrum and Rectum

We present the results of bacterioscopic and immunocytochemical study of Helicobacter pylori in biopsy specimens from the gastric antrum and smears from the rectum. Predominance of spiral-shaped vegetative form of Helicobacter pylori in the antrum and the presence of cocci with Helicobacter pylori antigens in smears from the rectum were demonstrated in patients infected with Helicobacter pylori. The diagnostic sensitivity of non-invasive immunocytochemical Helicobacter pylori test in rectal smears was 90%, specificity 76%, and efficiency of the test 84%.

Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

Background and AimsHistological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies.Patients and MethodsWe selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens.ResultsAll controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%.ConclusionsReal-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection.

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: A population‐based study

To investigate the relationship between p16 methylation and Helicobacter pylori infection in precancerous gastric lesions, a population-based study was conducted in Linqu County, a high-risk area of gastric cancer in China. Methylation status of p16 was evaluated by methylation-specific polymerase chain reaction in 920 subjects with precancerous gastric lesions. H. pylori status was determined by 13C-urea breath test and the density of H. pylori in biopsy specimens used for detecting methylation status was assessed by the modified Giemsa stain. The frequency of p16 methylation was significantly higher in subjects with H. pylori positive than those with H. pylori negative in each category of gastric lesion (p<0.001, respectively). Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95%CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95%CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95%CI: 1.90-7.10), and dysplasia (OR, 2.48; 95%CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively. Our findings indicate that p16 methylation was significantly associated with H. pylori infection in precancerous gastric lesions, suggesting that H. pylori infection could potently induce methylation of p16 CpG island.

Quantitative Correlation of Helicobacter pylori Stool Antigen (HpSA) Test with the Severity of H. pylori-Related Gastritis

The Helicobacter pylori (H. pylori) load in both stomach and stool and the resulting severity of gastritis are important criteria in validating the status of H. pylori infection. We aimed to assess the reliability of the H. pylori stool antigen (HpSA) test for the primary diagnosis of H. pylori infection by calculating the best cut-off value to obtain the highest sensitivity and specificity in dyspeptic patients. We also investigated the correlation of HpSA test with the severity of gastritis and H. pylori load. The H. pylori statuses of 95 patients were evaluated by the positivity of both rapid urease test and microscopic detection of H. pylori in biopsy specimens, 88 subjects of whom were H. pylori positive. The sensitivity and specificity of the HpSA test were 51.1% (45/88) and 100% (7/7), respectively, according to the manufacturer's recommended cut-off value of 0.16. However, with the best cut-off value of 0.048, calculated by receiver operator characteristics analysis, the sensitivity of the test increased to 92.0% (81/88) with the same specificity. High values of the HpSA test were correlated with high scores of corpus H. pylori load and the severity of antrum and corpus inflammation (p < 0.05). With the best cut-off value of the HpSA test, the primary diagnosis of H. pylori infection can be made with higher sensitivity and specificity. The HpSA test is a helpful tool that evaluates the severity of H. pylori infection and the degree of gastric inflammatory activity and gastric H. pylori load.

Stool antigen tests for the detection of Helicobacter pylori in children

Stool specimens from 102 children (median age = 6.0 years; Helicobacter pylori in biopsy specimens, n = 43) were tested for H. pylori with the monoclonal antibody–based enzyme immunoassay HpStar. In 28 cases, the specimen was obtained with a rectal cotton swab during endoscopy. The sensitivity and specificity of the HpStar were 0.95 and 0.90, respectively, as compared with biopsy-based methods. The rectal cotton swab specimen gave accurate results in 24 of the 28 cases. Storage of specimens for 1, 3, or 7 days ( n = 6) had a minor effect on the results, but one specimen's originally positive value on borderline turned negative when it was kept in room temperature. Forty-eight specimens were tested in parallel with all 3 tests used: HpStar, Premium Platinum HpSA, and the rapid test ImmunoCardSTAT! The accuracy rates of the tests were 98% for the HpSA and HpStar and 96% for the ImmunoCardSTAT! The performance of the stool antigen tests was excellent in young children.

Schneller Nachweis von clarithromycinresistenten Helicobacter pylori in der Magenbiopsie mittels Real-Time-PCR

The main cause for failure of Helicobacter pylori eradication therapy is resistance to clarithromycin which is due to point mutations. The use of real-time PCR allows the detection of these mutations directly on biopsy specimens within a few hours. In our routine laboratory, we compared LightCycler PCR to conventional detection and susceptibility testing of H. pylori by culture. PCR showed a positive result for H. pylori in 74 specimens. PCR was confirmed by culture in 69 specimens. In five specimens which were positive by PCR but negative on culture the (13)C urea breath test confirmed the PCR results. Sensitivity and specificity of our LightCycler assay for the detection of H. pylori in biopsy specimens were both 100 %. In 26 out of 68 specimens conventional susceptibility testing yielded resistance to clarithromycin. Corresponding point mutations were found in 24 of these specimens. Compared to culture, PCR gave a false-resistant, respectively, a false sensitive result in one specimen each. In another specimen, culture yielded a resistant strain whereas PCR detected both a resistant mutant and the wild-type strain. From two other specimens clarithromycin-sensitive strains were cultured but both a wild-type strain and a mutant were detected by PCR. Sensitivity and specificity of LightCycler PCR for resistance to clarithromycin were 96.2 % and 97.6 %, respectively. This assay had an accuracy comparable to culture and could be performed within 3 hours, allowing it to be used before the administration of H. pylori eradication therapy.

Diagnosis of helicobacter pylori infection by polymerase chain reaction: Is it worth it?

The aim of this study was to determine to what degree polymerase chain reaction (PCR) was superior to histology and culture, and whether a noncommercial urease test was of value, in detecting Helicobacter pylori in gastric biopsy specimens. Gastric biopsy specimens from the antrum and corpus of 134 consenting patients were subjected to PCR, targeting the glmM ( ureC) gene, histology, culture, and a rapid urease test. PCR detected H. pylori in the biopsy specimens from 59 patients. All methods showed a high degree of sensitivity and specificity, but histology gave 2 false-negatives, and culture and the urease test gave 1 false-negative compared with PCR. PCR of a glmM gene segment was superior to the other methods for the detection of H. pylori infection and was comparable to histology in terms of cost. Nevertheless, in this study, histology and culture were found to be relatively reliable methods for examining gastric biopsy specimens.

A prospective evaluation of new rapid urease tests before and after eradication treatment of Helicobacter pylori, in comparison with histology, culture and 13C-urea breath test

Background: The rapid urease test is a simple and cost-effective method to detect Helicobacter pylori in biopsy specimens. The aim of this study was to evaluate the accuracy of two new rapid urease tests, Helicocheck and PyloriTek, before and after eradication. Methods: A total of 278 patients, including 115 patients who had not undergone eradication of H pylori and 163 patients after eradication treatment, were enrolled. Eight biopsy specimens were taken from both the antrum and the body of the stomach for histology, culture, and two rapid urease tests. Assessment of H pylori infection was determined by the combination of histology, culture, and 13C-urea breath test. Results: Overall sensitivity, specificity, and positive and negative predictive values of the Helicocheck before eradication were 91.0%, 100%, 100%, and 62.5%; PyloriTek, 92.0%, 100%, 100%, and 65.2%. Those of Helicocheck after eradication were, respectively, 60.5%, 99.2%, 95.8%, and 89.2%; PyloriTek, 60.5%, 99.2%, 95.8%, and 89.2%. For the Helicocheck, determination of the infection status of H pylori by biopsies from the gastric body had a significantly higher sensitivity than antral biopsies. After eradication, the combination of 1 antral biopsy and 1 biopsy from the body was not effective enough to improve the overall sensitivity. Conclusions: Helicocheck and PyloriTek have equally satisfactory overall sensitivity before eradication treatment. However, the sensitivity of these rapid urease tests was lower after eradication than before eradication. (Gastrointest Endosc 2000;51:164-8.)

A novel real-time quantitative PCR assay for detection of Helicobacter pylori in the gastric mucosa

BACKGROUND: Although the assessment of the density of Helicobacter pylori (H. pylori) colonization may be important, precise quantitative evaluation of H. pylori in the gastric mucosa has been difficult. We have developed a novel real-time quantitative PCR technique based on 5' -3' exonuclease assay for detection of H. pylori. Using this system, we evaluated number of H. pylori organisms present in gastric biopsy specimens. METHODS: A primer pair for PCR amplification was made according to the reported sequence of ureA gene of H. pylori, and an oligonucleotide probe labeled with two fluorescent dyes (a reporter dye at the 5' end and a quencher dye at the 3' end) was also designed. While the probe is intact, the quencher dye greatly reduces the fluorescent signal by the reporter dye (Fluorescence resonance energy transfer phenomenon). During the PCR extension phase, the annealed probe is cleaved by 5' -3' exonuclease activity of Taq DNA polymerase, increasing the reporter dye signal. The reporter dye signal during a PCR assay was monitored with a ABI PRISM 7700 Sequence Detector (Perkin-Elmer). Purified PCR products of ureA gene were used as standard samples for making standard curves plotting log starting copy number and threshold cycle (Ct: PCR cycle number at which the reporter fluorescence signal reached above baseline). Biopsy specimens were collected from antral mucosa of consecutive 32 patients underwent gastroscopy (GU 9, DU 9, GC 1, NUD 13). Three biopsy specimens were obtained from each patient to avoid the effect of patchy distribution of H. pylori. DNA was extracted by conventional phenol-chloroform method. Since the size of each biopsy specimen was variable, quantitative PCR assay described above was performed using 0.5 ~tg of DNA extracted from each sample. !~_!LL_.T~: In each amplification reaction, a liner standard curve could be generated in a wide range of starting copy numbers (6xl07 to 6xl0 copies of the target sequences, R 2 > 0.99). Nine of 32 samples (DU 2, NUD 7) showed no increase in the fluorescence signal during PCR process, indicating the absence of H. pylori, which was also confirmed by histological examinations. In H. pylori positive patients, the detected ureA copy numbers (i.e. number of H. pylori organisms) were variable among patients (range 1.4x102 1.2x105 copies/0.5Mg DNA, mean 2.6x104 copies/0.51ag DNA). The average ureA copy numbers of samples from DU and GU patients were not significantly different from that of NUD patients [GU (2.1 _+ l.l)xl04, DU (1.8 _+ 0.6)xl04, NUD (2.6 + 1.1)xl04 copies/0.51ag DNA, respectively]. The sample of one GC patient showed high density of H. pylori colonization (1.2x105 copies/0.5Mg DNA). CONCLUSIONS: Accurate quantitation of H. pylori in biopsy specimens was possible with this PCR system, and it will be useful for 1-1. pylori research.

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.

Diagnosis of Helicobacter pylori in biopsy specimens.

Diagnosis of Helicobacter pylori in biopsy specimens.

Eradicating Helicobacter pylori and symptoms of non-ulcer dyspepsia.

To examine the effect of eradication of Helicobacter pylori on symptoms of non-ulcer dyspepsia. Four week prospective study. One hospital outpatient and endoscopy department. 90 adults with persistent symptoms typical of non-ulcer dyspepsia but no clinical or endoscopic evidence of other peptic, biliary, pancreatic, or malignant disease; all had histological and microbiological evidence of infection with H pylori. 83 patients completed the treatment regimen. Colloidal bismuth subcitrate 120 mg four times a day for four weeks (27 patients); metronidazole 400 mg and amoxycillin 500 mg each three times a day for one week (27); and bismuth subcitrate 120 mg four times a day for four weeks, metronidazole 400 mg three times a day for one week, plus amoxycillin 500 mg three times a day for the first week (29). Change in symptom scores determined with questionnaire; histological evidence of gastritis and microbiological evidence of presence of H pylori in biopsy specimens. Overall, H pylori was eradicated in 41 (49%) patients. Although gastritis scores improved significantly in only patients in whom H pylori had been eradicated (from 1.56 to 0.61, p less than 0.01 v from 1.83 to 1.07, p = 0.52) mean symptom scores after treatment were similar in patients in whom H pylori had or had not been eradicated (3.0 v 2.3, NS). Similarly the mean symptom score improved whether or not gastritis improved (2.8 v 3.1 respectively, p = 0.72). The observations were similar for treatment groups analysed individually. Antral infection with the organism does not seem to have an important aetiological role in non-ulcer dyspepsia short term.