Pylori 16S rRNA Research Articles

Role of MIC levels and 23S rRNA mutation sites to clarithromycin in 14-day clarithromycin bismuth quadruple therapy for Helicobacter pylori eradication: A prospective trial in Beijing

BackgroundRising clarithromycin resistance undermines Helicobacter pylori (H. pylori) treatment efficacy. We aimed to determine clarithromycin's minimum inhibitory concentration (MIC) levels and identify specific mutation sites in the 23S ribosomal subunit (23S rRNA) that predict treatment outcomes in a 14-day regimen of clarithromycin bismuth quadruple therapy (amoxicillin 1g, clarithromycin 500 mg, rabeprazole 10 mg, and colloidal bismuth pectin 200 mg). Materials and methodsWe included adult H. pylori patients who hadn't previously undergone clarithromycin-based treatment, either as initial or rescue therapy. Exclusions were made for penicillin allergy, recent use of related medications, severe illnesses, or inability to cooperate. Patients underwent a 14-day clarithromycin bismuth quadruple therapy. Gastric mucosa specimens were obtained during endoscopy before eradication. MIC against amoxicillin and clarithromycin was determined using the E-test method. The receiver operating characteristic (ROC) curve helped to find the optimal clarithromycin resistance MIC breakpoint. Genetic sequences of H. pylori 23S rRNA were identified through Sanger Sequencing. (ChiCTR2200061476) ResultsOut of 196 patients recruited, 92 met the inclusion criteria for the per-protocol (PP) population. The overall intention-to-treat (ITT) eradication rate was 80.00 % (84/105), while the modified intention-to-treat (MITT) and PP eradication rates were 90.32 % (84/93) and 91.30 % (84/92) respectively. No amoxicillin resistance was observed, but clarithromycin resistance rates were 36.19 % (38/105), 35.48 % (33/93), and 34.78 % (33/92) in the ITT, MITT, and PP populations respectively. Compared with the traditional clarithromycin resistance breakpoint of 0.25 μg/mL, a MIC threshold of 12 μg/mL predicted better eradication. Among 173 mutations on 152 sites in the 23S rRNA gene, only the 2143A > G mutation could predict eradication outcomes (p < 0.000). ConclusionsInterpretation of elevated MIC values is crucial in susceptibility testing, rather than a binary "susceptible" or "resistant" classification. The 2143A > G mutation has limited specificity in predicting eradication outcomes, necessitating further investigation into additional mutation sites associated with clarithromycin resistance.

Intracellular presence and genetic relationship of Helicobacter pylori within neonates' fecal yeasts and their mothers' vaginal yeasts.

Helicobacter pylori are transmissible from person to person and among family members. Mother-to-child transmission is the main intrafamilial route of H. pylori transmission. However, how it transmits from mother to child is still being determined. Vaginal yeast often transmits to neonates during delivery. Therefore, H. pylori hosted in yeast might follow the same transmission route. This study aimed to detect intracellular H. pylori in vaginal and fecal yeasts isolates and explore the role of yeast in H. pylori transmission. Yeast was isolated from the mothers' vaginal discharge and neonates' feces and identified by internal transcribed spacer (ITS) sequencing. H. pylori 16S rRNA and antigen were detected in yeast isolates by polymerase chain reaction and direct immunofluorescence assay. Genetic relationships of Candida strains isolated from seven mothers and their corresponding neonates were determined by random amplified polymorphic DNA (RAPD) fingerprinting and ITS alignment. The Candida isolates from four mother-neonate pairs had identical RAPD patterns and highly homologous ITS sequences. The current study showed H. pylori could be sheltered within yeast colonizing the vagina, and fecal yeast from neonates is genetically related to the vaginal yeast from their mothers. Thus, vaginal yeast presents a potential reservoir of H. pylori and plays a vital role in the transmission from mother to neonate.

Estimating the Incidence Frequency of Helicobacter pylori Co-infection with Hepatitis C Virus among Thalassemia Individual in Wasit Governorate of Iraq

Objective(s): Helicobacter pylori (H. pylori) is a gram-negative bacterium that plays a significant role in developing gastrointestinal diseases such as peptic ulcers. H. pylori infection affects more than half of the world’s population. This pathogen incidence becomes more effective among thalassemia patients, a common genetic disorder characterized by a point mutation that leads to impaired production of the β-globin chain. Chronic hepatitis C is a chronic disease caused by the hepatitis C virus (HCV) that has significantly increased morbidity and mortality rates in these patients due to liver failure or hepatocellular carcinoma. Methods: Current study included 90 β-thalassemia patients admitted to the thalassemia hematology center in Al-Kut women and children Hospital, Wasit province, Iraq from September 2020 to November 2021. The patients are divided into two groups (β-thalassemia with HCV group and β-thalassemia without HCV). First and second groups contain 32 and 58 specimens of blood and stool samples, respectively. The patient diagnosed with thalassemia syndrome regularly attended the thalassemia hematology center for transfusion and chelation and follow up on Hb level and iron status. This study aimed at serological and molecular diagnosis of co-infection H. pylori with Hepatitis C virus (HCV) among thalassemia patients and diagnosis infection rate. Diagnosis was used Serological enzyme-linked immunoassay (ELISA) techniques for HP-IgG, Level of ferritin, liver function test will be determined and Nested PCR method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool samples. Results: 90 β-thalassemia patients sample were diagnosed by Serological ELISA techniques for HP-IgG and the result showed 30 samples were positive (18 from the β-thalassemia with HCV group and 12 from the β-thalassemia without HCV)and 60 samples negative for HP-IgG and Nested polymerase chain reaction (PCR) method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool for 30 stool samples revealed 13 samples positive (9 from the β-thalassemia with HCV and 4 from β-thalassemia without HCV) while 17 samples were negative. Conclusions: The predominance of H. pylori among patients in Wasit was 43.3%. Levels of ferritin have significant correlation with H. pylori in HCV patients. There was a positive correlation between hepatitis C status and high H. pylori rate when compared with β-thalassemia without HCV. Splenoctomy was a positive correlation with H. pylori in β-thalassemia with HCV. Moreover, these results recommend study about the virulence factor and genes. Other molecular studies are needed for detecting more virulence genes in H. pylori bacteria such as real-time PCR and sequencing of H. pylori in patients with thalassemia, especially those with high serum ferritin.

Helicobacter pylori 23S rRNA gene A2142G, A2143G, T2182C, and C2195T mutations associated with clarithromycin resistance detected in Sudanese patients

BackgroundClarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms.Materials and methodsTwo hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primer targeting 16S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations.ResultsOut of 288 samples, H. pylori was detected in 88 (~ 30.6%) samples by 16 s RNA. Allele-specific PCR detected the variant A2142G in 9/53 (~ 17%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 36% (9/25) of samples; the A2142G was present in one sample, A2143G in 5 samples and T2182C in 4 samples. Additionally, another point mutation (C2195T) was detected in 3 samples. There was no association of 23S rRNA gene point mutations with gender, age group, and patients’ geographical distribution.ConclusionThis study revealed a high frequency (36%) of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene’s V domain. This information should be taken into consideration to avoid eradication therapy failing.

Effects of probiotics or broccoli supplementation on Helicobacter pylori eradication with standard clarithromycin-based triple therapy.

Background/AimsThe eradication failure rate of standard triple therapy (proton pump inhibitor, clarithromycin, and amoxicillin) for Helicobacter pylori infection has increased owing to antibiotic resistance in Korea. We assessed whether Saccharomyces boulardii probiotic or broccoli sprout extract sulforaphane supplementation could increase the H. pylori eradication rate and/or reduce antibiotic-associated adverse events.MethodsA total of 217 patients with H. pylori-positive chronic gastritis or peptic ulcer disease were recruited. Clarithromycin resistance was assessed in all patients by testing for A2142G and A2143G point mutations in H. pylori 23S rRNA using a dual-priming polymerase chain reaction (PCR) oligonucleotide. Thirty-four patients (17.3%) were clarithromycin-resistant and were excluded from the study. Finally, 183 patients with infections not resistant to clarithromycin were randomly assigned to triple therapy only (group A, n = 61), triple therapy plus probiotics (group B, n = 61), or triple therapy plus sulforaphane (group C, n = 61) groups. CYP2C19 polymorphisms were examined at position G681A of exon 5 and G636A of exon 4 by PCR with restriction fragment length polymorphism (PCR-RFLP) analysis. H. pylori eradication was assessed by 13C-urea breath test 4 weeks after treatment completion.ResultsThe eradication rates were similar among the groups both in the intention-to-treat (A = 85.2%, B = 89.6%, and C = 81.6%) and per-protocol (A = 89.2%, B = 86.8%, and C = 96.3%) analyses. The frequencies of overall adverse events in the groups also did not differ (A vs. B: p = 0.574; A vs. C: p = 1.000).ConclusionsProbiotic or sulforaphane with triple therapy for H. pylori infection neither increased the eradication rate nor reduced the occurrence of adverse events.

A variety of Helicobacter pylori strains colonize the stomach of non-bleeding Egyptian patients with upper gastrointestinal disorders

BackgroundChronic infection with Helicobacter pylori is associated with protean manifestations, which vary from no symptoms to multiple gastric diseases. Other H. pylori infections could provide protection against reflux esophagitis, and lower esophageal cancer. The current study aims to scan H. pylori strains that colonize the stomach of Egyptian patients with upper gastrointestinal disorders and its association with the endoscopic outcomes. Identification of H. pylori strains was done by PCR amplification of the 16s rRNA gene from gastric biopsies, proved to be positive for H. pylori by both Giemsa stain and histopathology. PCR products were purified, sequenced, and aligned to GenBank.ResultsBLAST results of H. pylori 16s rRNA gene sequences showed identity between Egyptian H. pylori isolates and four H. pylori strain subpopulations: hspSAfrica, hspEAsia, hpEurope, hspWAfrica. The frequency of H. pylori isolates that showed identity to hspEAsia subpopulation was significantly higher in Ulcerative lesions. H. pylori isolates from ulcerative and neoplasm specimens illustrate base substitutions in 16s rRNA gene variable 9 region compared to the consensus sequence of H. pylori 43504 16s rRNA.ConclusionDifferent H. pylori strains may be associated with differences in the clinical manifestations and could be used as a prognostic marker to predict the outcome of the H. pylori-associated diseases.

Phylogenetic Analysis of Helicobacter pylori 16S rRNA Gene in Gastric Biopsy from Patients with Dyspepsia

Phylogenetic Analysis of <i>Helicobacter pylori</i> 16S rRNA Gene in Gastric Biopsy from Patients with Dyspepsia

Clinical relevance of point mutations in the 23S rRNA gene in Helicobacter pylori eradication: A prospective, observational study.

Clarithromycin-based triple therapy is prescribed worldwide for Helicobacter pylori eradication. However, increases in the clarithromycin resistance of H pylori are thought to be responsible for eradication failure. Here, we studied whether point mutations in domain V of the 23S rRNA gene can affect H pylori eradication failure in a prospective, open-label, observational study. Of the 755 enrolled patients, 299 patients (39.6%) had positive Campylobacter-like organism (CLO) tests. DNA sequencing analysis of H pylori 23S rRNA in 295 patients revealed that 2143G was the most frequent point mutation (24.7% of patients), followed by the 2182T mutation (11.5%). The overall eradication failure rate was 20.9% (42/201) in clarithromycin-based triple therapy. Patients with the 2143G had an approximately 60% eradication failure rate, which suggested that 2143G was a high-risk genotype for eradication failure. Patients with the 2182C genotype without 2143G had an 8.7% failure rate, and patients without 2143G or 2182C had only a 4.3% failure rate. The presence of 2143G, which was associated with previous eradication history and female sex, was an independent risk factor for eradication failure. In conclusion, the 2143G point mutation in the 23S rRNA of H pylori was an independent risk factor for eradication failure in clarithromycin-based triple therapy. Personalized tailored therapy based on the genotypes of 23S rRNA can increase eradication success rates in H pylori infections.

Clarithromycin resistance and female gender affect Helicobacter pylori eradication failure in chronic gastritis.

Background/AimsThe eradication rate of the first-line triple therapy (a proton pump inhibitor, clarithromycin, and amoxicillin) for Helicobacter pylori infection has gradually decreased in Korea. We evaluated whether clinical parameters, clarithromycin resistance, and CYP2C19 genotype can affect the eradication failure.MethodsA total of 203 patients with H. pylori-positive chronic gastritis were consecutively enrolled. They received clarithromycin-based triple therapy for 7 days. A clarithromycin resistance test was performed by detection of A2142G and A2143G point mutations in H. pylori 23S rRNA. The CYP2C19 genotype was examined for polymorphism G681A of exon 5 and G636A of exon 4 by polymerase chain reaction with restriction fragment length polymorphism. Eradication was assessed by a 13C-urea breath test 4 weeks after treatment.ResultsOf 203 patients, 190 completed the study. The eradication rate was 64.0% according to intention-to-treat analysis and 68.4% by per-protocol analysis. CYP2C19 genotypes were identified as follows: 75 poor metabolizers, 75 intermediate metabolizers, and 40 rapid metabolizers. Nonetheless, this polymorphism was not significantly associated with eradication failure (p = 0.682). Clarithromycin resistance was detected in 33/190 patients (17.4%), and their eradication rate was zero. Clarithromycin resistance (odds ratio [OR], 19.13; 95% confidence interval [CI], 9.35 to 35.09) and female gender (OR, 1.73; 95% CI, 1.15 to 4.25) were significantly associated with eradication failure. The other clinical parameters such as age, cigarette smoking, alcohol intake, the body mass index, hypertension, and diabetes were not significantly associated with eradication.ConclusionsClarithromycin resistance and female gender are factors affecting H. pylori eradication failure in patients with chronic gastritis.

Mutations affecting domain V of the 23S rRNA gene in Helicobacter pylori from Cairo, Egypt

Background: Clarithromycin is a main component of the recommended first-line triple therapy for Helicobacter pylori in Egypt. We aimed in our study to investigate the prevalence of clarithromycin-resistant H. pylori strains due to the point mutations at domain V of the H. pylori 23S rRNA among the Egyptian population using the polymerase chain reaction/restricted fragment length polymorphism (PCR/RFLP) assay.Methods: Gastric biopsies obtained from 100 dyspeptic patients who consecutively attended at Cairo University Hospital during the period from January to November 2013 were subjected to PCR/RFLP in order to detect the point mutations at domain V of the H. pylori 23S rRNA associated with clarithromycin resistance. The PCR amplicon of the 23S H. pylori rRNA is restricted with MboII for detection of A2142G mutation and with BsaI for A2143G mutation.Results: The prevalence of H. pylori infection among 100 patients was 70%; clarithromycin resistance was detected in 39/70 (57.7%) of positive H. pylori isolates. Occurrence of 23S rRNA A2142G mutations resulted in two DNA fragments (418 and 350 bp) by PCR-RFLP; on the other hand, no A2143G mutations were detected.Conclusions: The high prevalence of clarithromycin resistance (57.7%) caused by A2142G mutations at domain V of the H. pylori 23S rRNA may mandate changing of the standard clarithromycin-containing triple therapy. The PCR/RFLP assay was a rapid and accurate method for molecular detection of H. pylori infection in addition to determination of different nucleotide mutations causing clarithromycin resistance.

Helicobacter pylori Clarithromycin Resistance and Treatment Failure Are Common in the USA.

Helicobacter pylori antibiotic resistance leads to frequent treatment failure. However, the current US prevalence of H. pylori clarithromycin resistance and treatment failure is unknown. To determine the prevalence of clarithromycin-resistant H. pylori and its impact on treatment failure in the USA. A multicenter, retrospective, cohort study for clarithromycin-resistant H. pylori was conducted over four academic medical centers in different geographic regions of the USA. Gastric biopsy material, residual from standard clinical pathologic examination, was examined for clarithromycin resistance by DNA sequencing of H. pylori 23S rRNA. One hundred and twenty-four cases of H. pylori gastritis were examined from medical centers in four different geographic regions of the USA. The overall prevalence of clarithromycin resistance was 32.3% (range 23.1-45.8%). There was no significant difference in the prevalence of clarithromycin resistance by study site, gender, age, or race/ethnicity. In a subset of 67 patients that had clinical follow-up data, the overall prevalence of clarithromycin resistance was 31.3%. There was a 2.9-fold increase (p=0.002) in treatment failure for cases with clarithromycin resistance (57.1%) compared to wildtype H. pylori (19.6%). H. pylori clarithromycin resistance in the USA exceeds the estimated 20% prevalence compatible with successful empiric antibiotic therapy. This resistance resulted in a significant rate of treatment failure in all sites surveyed. Empiric therapy in the USA should be used with caution until there is better regional or local determination of H. pylori antibiotic resistance.

GENETIC MUTATIONS AFFECTING THE FIRST LINE ERADICATION THERAPY OF Helicobacter pylori-INFECTED EGYPTIAN PATIENTS.

SUMMARYIntroduction: Several genetic mutations affect the first-line triple therapy for Helicobacter pylori. We aimed to study the most common genetic mutations affecting the metronidazole and clarithromycin therapy for H. pylori-infected Egyptian patients.Patients and Methods: In our study, we included 100 successive dyspeptic patients scheduled for diagnosis through upper gastroscopy at Cairo's University Hospital, Egypt. Gastric biopsies were tested for the presence of H. pylori by detection of the 16S rRNA gene. Positive biopsies were further studied for the presence of the rdxA gene deletion by Polymerase Chain Reaction (PCR), while clarithromycin resistance was investigated by the presence of nucleotide substitutions within H. pylori 23S rRNA V domain using MboII and BsaI to carry out a Restricted Fragment Length Polymorphism (RFLP) assay.Results: Among 70 H. pylori positive biopsies, the rdxA gene deletion was detected in 44/70 (62.9%) samples, while predominance of the A2142G mutations within the H. pylori 23S rRNA V domain was evidenced in 39/70 (55.7%) of the positive H. pylori cases. No statistically significant difference was found between the presence of gene mutations and different factors such as patients 'age, gender, geographic distribution, symptoms and endoscopic findings.Conclusion: Infection with mutated H. pylori strains is considerably high, a finding that imposes care in the use of the triple therapy to treat H. pylori in Egypt, since the guidelines recommend to abandon the standard triple therapy when the primary clarithromycin resistance rate is over 20%1.

Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA

Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies.

The Evolution of Helicobacter Pylori Antibiotics Resistance Over 10 Years in Greece

Background: Increasingly, over time, antibiotic resistance is considered a problem for the efficacy of H. pylori eradication treatment. The aim of our study was to evaluate the changes in clarithromycin and levofloxacin resistance of H. pylori strains in Greek patients in two different time periods (in 2000 and in 2010). Methods: Gastric biopsies of consecutive H. pylori-positive patients were investigated retrospectively. Mutations in H. pylori 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively, were determined by allelic specific polymerase chain reaction. Results: In the first time period (2000), H. pylori resistance patterns were evaluated in 50 and in the second period (2010) in 57 patients. During the first time period 30 and 0% of patients were infected with clarithromycin- or quinolone-resistant strains, respectively. In the second time period (2010), the percentage of patients infected with clarythromycin or quinolone resistance strains increased to 42 and 5.3%, respectively. Conclusions: Our study showed an increase in the prevalence of both clarithromycin and quinolones resistance of H. pylori. Although the resistance rate to quinolones increased over the years, it is relatively low justifying its use for the eradication of H. pylori infections. © 2015 S. Karger AG, Basel

Effect of Helicobacter pylori and its virulence factors on portal hypertensive gastropathy and interleukin (IL)-8, IL-10, and tumor necrosis factor-alpha levels

Background/Aim:We aimed to assess the influence of Helicobacter pylori and its virulent factors, cytotoxin associated gene (cag) A and E, on portal hypertensive gastropathy (PHG) and the levels of interleukin (IL)-8, IL-10, and tumor necrosis factor-alpha (TNF-α).Patients and Methods:The patients with cirrhosis underwent screening endoscopy and the lesions related to PHG were graded. Biopsies were obtained for histology, and polymerase chain reaction (PCR) of H. pylori 16S rRNA, cagA, cagE, and tissue cytokine levels was carried out. Absent or mild PHG was compared with moderate to severe PHG.Results:One hundred and forty patients with cirrhosis were studied; males numbered 92 and the mean age of the patients was 50.3 ± 12.0 years, H. pylori positivity in 87 (62.1%) patients was associated with male gender (P = 0.032), younger age (P = 0.029), hepatitis D etiology (P = 0.005), higher serum albumin (0.000), lower Child Pugh score (P = 0.001), and lower portal vein diameter (P = 0.001). There was no significant difference in the levels of TNF-α and IL-8. However, a decrease in the anti-inflammatory cytokine IL-10 was noted with moderate to severe gastropathy. Four H. pylori strains were positive for both cagA and cagE, while four were positive for cagA only. All the four patients with both virulent factors had mild gastropathy only.Conclusion:The presence of H. pylori infection neither affected the severity of PHG nor augmented the IL-8 and TNF-α levels. There was a decline of virulent H. pylori strains and IL-10 levels in patients with advanced PHG.

Esophageal intraepithelial invasion of Helicobacter pylori correlates with atypical hyperplasia

Helicobacter pylori (H. pylori), a common pathogen residing in the gastrointestinal tract, has been well characterized in stomach cancer,while its correlation with esophageal cancer remains poorly understood. In this study, we aim to assess the relationship between esophageal intraepithelial H. pylori invasion and inflammation as well as atypical hyperplasia in esophageal squamous epithelial tissues. Esophageal squamous cell carcinoma (ESCC) tissue samples from 196 individuals from both southern and northern esophageal carcinoma high-risk areas in China were examined (125 from northern high-risk areas, 71 from southern high-risk area), while additional 30 samples were collected adjacent to the esophageal squamous cell carcinoma (A-ESCC). H. pylori infection was identified by Giemsa staining, immuno-histochemical staining, and H. pylori 16S rRNA-based PCR. A significant increase of H. pylori infection was found in tumor tissues (including ESCC and A-ESCC samples) compared to that of non-tumor tissues (p < 0.05). The positive rate of H. pylori 16S rRNA in ESCC, A-ESCC, and normal groups were 62.5, 74.1, and 26.7%, respectively. The PCR results showed that the positive incidence of the H. pylori virulence factor CagA gene in tumor (ESCC and A-ESCC) and normal groups was 54.9 and 20%, respectively (p < 0.05). To explore the possible causes of CagA+ H. pylori infection leading to carcinogenesis, we found that CagA+ H. pylori filtrate induced DNA strand breaks in esophageal epithelial NE3 cells, suggesting that H. pylori infection may be an original cause leading to atypical hyperplasia of esophageal squamous epithelial tissues and contributed to pathological carcinogenesis of ESCC.

The Fate of Helicobacter pylori Phagocytized by Acanthamoeba polyphaga Demonstrated by Fluorescent In Situ Hybridization and Quantitative Polymerization Chain Reaction Tests

Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pylori and BacLight™-stained cells. Following phagocytosis, the fate of cells was assessed by fluorescent in situ hybridization with an oligonucleotide targeting H. pylori 16S rRNA and by quantitative-polymerase chain reaction (qPCR) tests with primers to 16S rDNA. Fluorescent in situ hybridization tests were inconclusive with only a small percentage of amoebae apparently containing active intracellular H. pylori. Furthermore, no increase in bacterial cells was detected by qPCR. Additional research is required to elucidate the mechanisms by which amoebae phagocytize this important bacterial pathogen.

Non‐invasive Genotyping of Helicobacter pylori cagA, vacA, and hopQ from Asymptomatic Children

Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non-invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy. H. pylori status of 86 asymptomatic children was assessed by (13) C-urea breath test (UBT) and PCR. H. pylori 16S rRNA, cagA, hopQ, and vacA genes were amplified from stool DNA samples and sequenced. UBT was positive in 69 (80.2%) of 86 children; in stool DNA analysis, 78.3% were positive by 16S rRNA PCR. cagA, vacA, and hopQ were detected in 66.1%, 84.6%, and 72.3% of stool DNA samples from 16S rRNA-positive children. Of the children's DNA samples, which revealed vacA and hopQ alleles, 91.7% showed vacA s1 and 73.7% showed type I hopQ. Type I hopQ alleles were associated with cagA positivity and vacA s1 genotypes (p < 0.0001). Using stool DNA samples, virulence markers of H. pylori were successfully genotyped in a high percentage of the asymptomatic infected children, revealing a high prevalence of genotypes associated with virulence. Type I hopQ alleles were associated with the presence of cagA and the vacA s1 genotype.

Resistance of Helicobacter pylori to quinolones and clarithromycin assessed by genetic testing in Japan

As bacterial resistance to clarithromycin limits the efficacy of clarithromycin-based regimens for Helicobacter pylori infection, attention has turned to quinolone-based rescue therapies. Resistance of H. pylori to both clarithromycin and quinolone can be predicted by genetic testing. Here, we used this approach to evaluate the prevalence of clarithromycin- and quinolone-resistant strains of H. pylori in Japan. DNA was extracted from gastric tissue samples obtained from 153 patients infected with H. pylori (103 naive for eradication therapy and 50 with previous eradication failure following triple proton pump inhibitor/amoxicillin/clarithromycin therapy). Mutations in H. pylori 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively, were determined. Of 153 patients, 85 (55.6%) were infected with clarithromycin-resistant strains. The prevalence of clarithromycin-resistant strains in patients with previous eradication failure (90.0%, 45/50) was significantly higher than that (38.8%, 40/103) of those naive for eradication therapy (P < 0.001). Fifty-nine patients (38.6%) were infected with strains resistant to quinolones. The incidence of quinolone-resistant strains also appeared higher in patients with eradication failure (48.0%, 24/50) than in those who had not undergone therapy (34.0%, 35/103); however, the difference was not statistically significant (P = 0.112). The incidence of quinolone-resistance in clarithromycin-resistant strains (44/85, 51.8%) was significantly higher than that in clarithromycin-sensitive strains (15/68, 22.1%) (P < 0.001). A high incidence of quinolone-resistance was found in clarithromycin-resistant strains of H. pylori, particularly in patients with previous eradication failure. Our results suggest that testing for susceptibility of H. pylori to quinolones is useful for determining the optimal rescue eradication regimen.

Change in Antibiotic Resistance of Helicobacter pylori Strains and the Effect of A2143G Point Mutation of 23S rRNA on the Eradication of H. pylori in a Single Center of Korea

The current prevalence of primary antibiotic resistance of H. pylori is not known in Korea. This study was done to evaluate the prevalence of primary antibiotic resistance of H. pylori, and to evaluate the effect of point mutations of 23S rRNA on the rate of eradication of H. pylori. H. pylori were isolated from gastric mucosal biopsy specimens obtained from 222 Koreans. The susceptibilities of the H. pylori isolates to amoxicillin, clarithromycin, metronidazole, tetracycline, ciprofloxacin, and levofloxacin were examined using the agar dilution method. DNA sequencing was carried out to detect H. pylori 23S rRNA mutations. The resistance to clarithromycin, tetracycline, ciprofloxacin, and levofloxacin increased during the period of 2007 to 2009 compared with 2003 to 2005 (P<0.05). However, amoxicillin and metronidazole resistance slightly decreased. The rates of eradication were 95.5% for the clarithromycin-sensitive strains, which was higher than the 67.9% for the clarithromycin-resistant strains (P=0.001). By contrast, the eradication rate was 100% in patients with amoxicillin-resistant H. pylori. Among 26 clarithromyin-resistant strains, 6 (23%) had A2143G mutations, and all of the cases in which these mutations were present were not eradicated by proton pump inhibitor-based triple therapy (P=0.0004). By contrast, none of the 26 clarithromyin-sensitive strains had A2143G mutations. The T2183C and A2223G mutations were frequently found in the sensitive strains and in the resistant strains. Clarithromycin resistance of H. pylori, which determined the efficacy of H. pylori eradication of proton pump inhibitor triple regimen, was found to be increased in a single center study. A2143G was an important 23S rRNA mutation associated with clarithromycin resistance and affected the H. pylori eradication efficacy.