Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with BsaI and BbsI restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).     

Application of real-time PCR stool assay for Helicobacter pylori detection and clarithromycin susceptibility testing in Brazilian children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real‐time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real‐time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin‐resistant strains is suspected.     

PCR detection of Helicobacter pylori in string-absorbed gastric juice

Molecular methods for detection of Helicobacter pylori infection have been shown to be highly sensitive in gastric biopsies and cultures. The objective of this work was to compare PCR detection of H. pylori DNA in string-absorbed gastric juice and in gastric biopsies. The study was performed in 47 dyspeptic adult patients undergoing endoscopy, and infection was detected by amplification of a segment of H. pylori ureA gene. Of the 29 patients positive in biopsy analysis, 23 (79%) were also positive in the gastric string. PCR analysis of gastric strings is a sensitive and safe procedure to detect H. pylori when endoscopy is not indicated, and may be of great clinical and epidemiological usefulness in determining effectiveness of eradication therapies, typing virulence genes and detecting antibiotic resistance mutations.     

Whole genome sequencing reveals novel genetic mutations of Helicobacter pylori associating with resistance to clarithromycin and levofloxacin

Background

Detection of mutations in one or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of Helicobacter pylori to clarithromycin (CLA) and levofloxacin (LVX). We aimed to perform whole genome sequencing to explore novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests.

Methods

Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based antibacterial susceptibility test, H. pylori strains were divided into three groups, with nine strains in each group: CLA single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all antibacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and amino acid mutations were screened, and potential candidate genes related to CLA and LVX resistance were identified.

Results

The median age of study subjects was 35 years (IQR: 31–40), and 17 (63.0%) were male. All nine CLA-resistant strains had A2143G mutations in 23S rRNA, while none of nine sensitive strains had the mutation. Six of nine strains in group L and six of nine strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the three groups, we identified five mutated positions belonging to four genes related to CLA resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CLA resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated.

Conclusion

Whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CLA and LVX including fliJ and cheA. Further studies to correlate these findings with treatment outcome should be performed.     

口腔中幽门螺杆菌PCR检测方法的建立

全世界大约有50%的人口感染幽门螺杆菌(Helicobacter pylori, Hp),Hp感染容易造成慢性胃炎、消化性溃疡、低度恶性胃MALT淋巴瘤及胃癌,对人类造成严重的危害。为寻求一种简便、准确、非创伤性的方法用于口腔中Hp的检测,本研究建立了口腔中幽门螺杆菌的PCR检测方法,并对建立的PCR方法的敏感性、特异性进行分析,并应用于检测唾液样品和牙菌斑共50份。结果显示,50份样品中,阳性率为82%。结果表明,本研究建立的口腔中Hp的PCR检测方法特异性强、敏感性高,可以用于临床上Hp诊断的重要参考。     

Methodology and transport medium for collection of Helicobacter pylori on a string test in remote locations

BACKGROUND: Helicobacter pylori can be isolated from patients using the string test but contaminating oral and nasopharyngeal microflora need to be suppressed by rapid plating out onto selective culture media. Recently, use of this diagnostic method was enhanced by using a novel transport medium to collect specimens from subjects in a remote Australian clinic over 1300 km from the laboratory. METHODS: Retrieved string tests were transported to the laboratory in chilled polystyrene boxes in 5 ml screw-cap bottles with 3 ml of a brain heart infusion broth plus antibiotics. These were 20 g/ml vancomycin, 10 g/ml trimethoprim, 10 g/ml cefsulodin, and 10 g/ml amphotericin B. A comparison was made between subjects who gargled with a chlorhexidine mouthwash before swallowing the string test and those who did not. RESULTS: Forty-five urea breath test-positive subjects were tested and H. pylori was isolated from 34 of them. Successful culture was achieved from string tests that were in transit for up to 29 hours and where the maximum temperature in the transport box was 14 degrees C. The additional use of a mouthwash had a marked effect on the isolation rate. H. pylori was cultured from 75% of subjects who gargled but only from 39% who did not. CONCLUSIONS: This methodology and transport medium can broaden the use of the string test to more remote geographic areas where endoscopy is not feasible so that H. pylori isolates may still be obtained for diagnostic and epidemiologic studies. The value of this promising methodology of collection and transport should be assessed in a controlled study.     

Detection of Helicobacter pylori DNA by a simple stool PCR method in adult dyspeptic patients

INTRODUCTION: Helicobacter pylori is the major agent causing peptic ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) gastric lymphoma. A simple stool polymerase chain reaction (PCR) method was performed and compared with the gold standards for the diagnosis of H. pylori infection. MATERIAL AND METHODS: A total of 54 adult patients (mean age, 46.41 +/- 13.12 years) with dyspeptic symptoms from Gastroenterology at Dokuz Eylül University Hospital between May and November 2003 were included. Two antrum and corpus biopsies were taken from each patient. Infection by H. pylori was defined as positivity and negativity of the gold standards. DNA extraction of stool specimens was done using QIAamp DNA Stool Mini Kit (QIAGEN) and PCR conditions included amplification and reamplification steps using the H. pylori ureA gene specific primers (HPU1, HPU2) and were visualized on 1% agarose gel stained with ethidium bromide. RESULTS: Forty-six of 54 patients (85.2%) were diagnosed positive and eight (14.8%) were negative for H. pylori infection by the gold standard methods. Thirty-two patients were positive (59.3%) and 22 of them (40.7%) were detected negative by stool PCR method. The stool PCR method and gold standard methods showed a statistical difference for the detection of H. pylori infection (p < .0001). Sensitivity, specificity, likelihood ratio, and positive and negative predictive values were 65.22%, 75%, 2.61%, 93.75%, and 27.7%, respectively. DISCUSSION: The PCR on the stool specimens resulted as being a very specific test. We suggest that a simple stool PCR method that we developed can be used to detect H. pylori, virulence genes, and in drug resistance studies either first line diagnostic methods in the laboratory or in the clinical management of dyspeptic patients.     

Identification of Helicobacter pylori and the cagA genotype in gastric biopsies using highly sensitive real-time PCR as a new diagnostic tool

The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection.     

The effects of probiotics on PPI-triple therapy for Helicobacter pylori eradication

Background: This study was performed to evaluate whether the addition of probiotics to proton pump inhibitor (PPI)‐based triple therapy increases the likelihood of successful Helicobacter pylori eradication. Materials and Methods: Three hundred and forty‐seven H. pylori‐infected patients were randomized into a triple‐plus‐yogurt group (yogurt group, n = 168) or a triple‐only group (control group, n = 179). Triple therapy consisted of PPI b.i.d., clarithromycin 500 mg b.i.d., and amoxicillin 1 g b.i.d. for 7 days. Yogurt group received triple therapy for 1 week and one bottle of Will yogurt per day for at 3 weeks, starting on the first day of triple therapy. Will yogurt (a Korean brand) contains Lactobacillus acidophilus HY2177, Lactobacillus casei HY2743, Bifidobacterium longum HY8001, and Streptococcus thermophilus B‐1. ¹³C‐urea breath test was performed at least 4 weeks after completion of triple therapy. Eradication rates, compliances, and adverse events were compared. Results: By intention‐to treat analysis the H. pylori eradication rates in the yogurt group 79.2% (133 of 168) was similar to that in the control group 72.1% (129 of 179) (p = .124). However, by per‐protocol (PP) analysis, the eradication rate in the yogurt group, 87.5% (133 of 152) was higher than that in the control group, 78.7% (129 of 164) (p = .037). Common adverse events were metallic taste (11.8%) and diarrhea (8.6%). The frequency of adverse effects in the yogurt group 41.1% (69/168) were higher than in the control group, 26.3% (47 of 179) (p = .003). However, most adverse events were mild to moderate in intensity, and the severities of adverse effects were similar in both groups (p = .401). Conclusions: The addition of Will yogurt to triple therapy did not reduce the side‐effects of triple therapy. But it increased the H. pylori eradication rate by PP analysis, encouraging more research in this field.     

Quantitative detection of Helicobacter pylori in water samples by real-time PCR amplification of the cag pathogenicity island gene, cagE

Aims:  A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila , was developed and validated for the detection of H. pylori in environmental samples.
Methods and Results:  The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori . The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive.
Conclusions:  This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples.
Significance and Impact of the Study:  The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.     

Amoxicillin resistance in Helicobacter pylori: studies from Tokyo, Japan from 1985 to 2003

BACKGROUND: Previous reports revealed no resistant strains of amoxicillin (AMPC), which is usually used in eradication therapy for H. pylori infection. However, the frequency and evolution of natural AMPC-resistant strains in the Japanese population remains unknown. AIM: To assess the prevalence of H. pylori resistance against AMPC in the Tokyo area, a collection of 648 H. pylori strains isolated from patients with GI diseases from 1985 to 2003 was tested for their sensitivity to AMPC. METHODS: The susceptibility of the strains was assessed by determination of the minimal inhibitory concentration (MIC) using the E-test and/or the Dry-plate method. The susceptibility breakpoints of AMPC for H. pylori were: sensitive (AMPC-S); MIC < 0.04 microg/ml, intermittent resistance (AMPC-I); 0.04-1, resistant (AMPC-R); > 1. RESULTS: No AMPC-R strains were detected in the strains isolated between 1985 and 1996, while the rate of resistance was determined to be 1.1%, 2.1%, 5.4%, 5.6%, 0%, 8.8%, and 1.5% every year, respectively, from 1997 to 2003. The percentage of AMPC-I strains increased from 2000 to 2003. The total eradication rate of H. pylori in the patients who received triple therapy containing AMPC was 81.4% (214/263). Classified as above, the rates of AMPC-S, AMPC-I, and AMPC-R were 84.6%, 77.8%, 25%, respectively. CONCLUSION: H. pylori resistance to AMPC is still rare in Japan, although the percentage of AMPC-I strains has increased over the last 4 years. The frequency of isolation of strains showing true resistance to AMPC may increase in the future, along with an increase in the frequency of isolation of AMPC-I strains.     

Steroids mediate resistance to the bactericidal effect of phosphatidylcholines against Helicobacter pylori

Helicobacter pylori assimilates various steroids as membrane lipid components, but it can also survive in the absence of steroids. It thus remains to be clarified as to why the organism relies on steroid physiologically. In this study, we have found that phosphatidylcholine carrying a linoleic acid molecule or arachidonic acid molecule has the potential to kill steroid-free H. pylori . The bactericidal action of phosphatidylcholines against H. pylori was due to the lytic activity of the phosphatidylcholines themselves and not due to the lytic activity of the unsaturated fatty acids or lyso-phosphatidylcholine resulting from the hydrolysis of the phosphatidylcholines. In contrast to the steroid-free H. pylori , the organism that absorbed and glucosylated free cholesterol was unaffected by the bactericidal action of the phosphatidylcholines. Similarly, H. pylori that absorbed estrone without glucosylating it also resisted the bactericidal action of the phosphatidylcholines. The steroids absorbed by H. pylori existed in both the outer and inner membranes, while the glucosyl-steroids produced via the steroid absorption were localized in the outer membrane rather than in the inner membrane. These results indicate that H. pylori absorbs the steroids to reinforce the membrane lipid barrier and thereby expresses resistance to the bacteriolytic action of hydrophobic compounds such as phosphatidylcholine.     

酪酸梭菌辅助根除幽门螺杆菌感染的Meta分析

目的 系统评价酪酸梭菌辅助根除幽门螺杆菌（Helicobacter pylori,H. pylori）感染的疗效和安全性。方法 计算机检索PubMed、CENTRAL（2016年7期）、CBM、CNKI、万方数据库、维普数据库,同时追溯纳入文献和相关综述的参考文献,收集关于酪酸梭菌辅助根除H. pylori感染的随机对照试验（RCT）,检索时间均从建库至2016年7月30日。由两名评价员根据纳入排除标准筛选文献、提取资料、评价纳入研究的偏倚风险,采取RevMan 5.3进行Meta分析。结果 最终纳入13个RCT,共1 372例患者（其中试验组742例,对照组630例）。Meta分析结果显示：与标准根除方案相比,酪酸梭菌辅助治疗组有利于提高H. pylori的根除率（RR=1.26,95%CI：1.18~1.34,P＜0.00001）,并且降低总不良反应（RR=0.41,95%CI：0.32~0.52,P＜0.00001）、腹泻（RR=0.31,95%CI：0.16~0.58,P=0.0003）、味觉紊乱的发生率（RR=0.35,95%CI：0.17~0.73,P=0.005）;然而,在腹胀、恶心的发生率上,酪酸梭菌辅助治疗组虽低于标准方案组,但差异无统计学意义。结论 当前证据显示酪酸梭菌联合标准方案不仅可提高H. pylori的根除率,还能降低总不良反应、腹泻及味觉紊乱的发生。受纳入研究的质量和数量的限制,上述结论有待更多高质量的研究予以验证。     

Quantitative analysis of representative proteome components and clustering of Helicobacter pylori clinical strains

BACKGROUND: Several Helicobacter pylori proteins have been reported to be associated with severe symptoms of gastric disease. However, expression levels of most of these disease-associated proteins require further evaluation in order to clarify their relationships with gastric disease patterns. Representative proteome components of 71 clinical isolates of H. pylori were analyzed quantitatively to determine whether the protein expression levels were associated with gastric diseases and to cluster clinical isolates. METHODS: After two-dimensional electrophoresis (2-DE) of H. pylori isolates, spot intensities were analyzed using pdquest 2-D Gel Analysis Software. The intensities of 10 representative protein spots, identified by peptide fingerprinting using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using quadrupole TOF MS, were subjected to the nonparametric Mann-Whitney test and hierarchical agglomerative cluster analysis. The relationship between clusters and gastric diseases was analyzed by the chi-squared test. RESULTS: Although the spot intensities of the 10 representative proteins were highly variable within each gastric disease group, the expression levels of CagA, UreB, GroEL, EF-Tu, EF-P, TagD, and FldA showed some significant differences among the gastric disease patterns. On the basis of the 10 target protein intensities, hierarchical agglomerative cluster analysis generated a dendrogram with clusters indicative of chronic gastritis/gastric cancers and gastric/duodenal ulcers. CONCLUSION: These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.     

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia

Background

Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H. pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance.

Methods

Strains were isolated from the gastric mucosa of patients infected with H. pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby–Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed.

Results

We isolated 1955 H. pylori strains. The resistance rates of H. pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement.

Conclusion

H. pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H. pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H. pylori infection in Ningxia.