桂西地区幽门螺杆菌对克拉霉素的耐药性分析

目的 分析桂西地区幽门螺杆菌(Helicobacter pylori,HP)菌株对克拉霉素的耐药情况,并探讨Hp对克拉霉素耐药与23S rRNA基因点突变的关系.方法 分离培养Hp,纸片扩散法进行药敏实验,PCR方法扩增23S rRNA基因,用聚合酶链反应.限制性片段长度多态性(PCR-RFLP)检测克拉霉素耐药菌株的点突变,同时进行基因测序确定突变位点.结果 桂西地区Hp菌株对克拉霉素耐药率为22.2%(28/126);PCR-RFLP检测10株对克拉霉素耐药的Hp,均存在23S rRNA基因的A2143G、A2144G点突变,10株敏感菌株均无23S rRNA的点突变,基因测序显示耐药菌株有A2143G、A2144G突变,其他位置未发现突变.结论 桂西地区Hp菌株对克拉霉素耐药率(22.2%)略高于北京、上海等地区;Hp的23S rRNA基因A2143G、A2144G点突变与克拉霉素的耐药相关.     

Primary antibiotic resistance of Helicobacter pylori strains among adults and children in a tertiary referral centre in Lithuania

The study evaluated primary antibiotic resistance of Helicobacter pylori within the period 2013–2015 and trends of antibiotic consumption over the last decade in Lithuania; 242 adults and 55 children were included in the study. E‐tests were performed for amoxicillin, metronidazole, clarithromycin, ciprofloxacin, rifampicin and tetracycline. The presence of H. pylori and clarithromycin resistance was additionally tested by PCR. Helicobacter pylori culture was positive in 67 of 242 (28%) adult and in 12 of 55 (21.8%) children samples. Resistance rates among adults by E‐tests were as follows: metronidazole – 32.8% (95% confidence interval (CI): 22.7–44.7%), ciprofloxacin – 7.5% (95% CI: 3.2–16.3%), rifampicin – 7.5% (95% CI: 3.2–16.3%), amoxicillin – 0%, whereas resistance rates in children were as follows: metronidazole – 25% (95% CI: 8.9–53.2%), rifampicin – 8.3% (CI: 1.5–35.4%), amoxicillin and ciprofloxacin – 0%. Accumulated clarithromycin resistance rates by E‐tests and PCR were 8.2% (95% CI: 4.1–16.0%) in adults and 17.7% (95% CI: 6.2–41.0%) in children. Total use of macrolides and lincosamides in Lithuania increased from 1.26 to 1.86 defined daily dose (DDD)/1000 inhabitants/day among adults, while it has doubled from 1.10 to 2.22 DDD/1000/children/day in children within 2003–2015. There are no significant changes in the susceptibility of H. pylori to the most widely used antibiotics in adults over the last years in Lithuania; however, clarithromycin resistance among children exceeds 15% and mandates further larger‐scale studies in paediatric population.     

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type

Yasuda A, Uchida T, Nguyen LT, Kawazato H, Tanigawa M, Murakami K, Kishida T, Fujioka T, Moriyama M. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type. APMIS 2009; 117: 893–9. Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA‐positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver‐operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori‐infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up‐regulated by adhesion to epithelial cells.     

PCR using 3'-mismatched primers to detect A2142C mutation in 23S rRNA conferring resistance to clarithromycin in Helicobacter pylori clinical isolates

Twenty-five clarithromycin-resistant Helicobacter pylori strains (selected by agar dilution) were studied to detect A2142G and A2143G mutations in the 23S rRNA gene by a PCR-restriction fragment length polymorphism method and an A2142C mutation by PCR using a 3'-mismatched specific primer. A 700-bp amplified fragment was obtained by the mismatched PCR only in strains without an A2142G or A2143G mutation, indicating that those strains had the A2142C mutation.     

Cytokine expression associated with Helicobacter pylori and Epstein‐Barr virus infection in gastric carcinogenesis

Helicobacter pylori and Epstein‐Barr virus (EBV) infection, and associated cytokines are involved in gastric carcinogenesis. We investigated the expression of these cytokines and their relationship with clinicopathological characteristics. The study included specimens from 207 patients with gastric adenocarcinoma, 56 with chronic gastritis, 32 with metaplasia, and 30 with low‐grade epithelial dysplasia. Tissue microarrays were constructed and immunohistochemical staining for IL‐1β, IL‐6, IL‐10, IL‐17, p16, p21, TNF‐α, and TNFR1 was performed. EBV and H. pylori infection status was determined. IL‐1β, IL‐6, IL‐17, p16, and p21 protein expression was significantly higher in adenocarcinoma cases than in the other cases (p < 0.05). EBV was only noted in adenocarcinoma (13 cases, 6.3%). The H. pylori infection rate in adenocarcinoma was significantly higher than that in the other cases (p < 0.005). IL‐6 expression was associated with improved survival (p < 0.05), whereas IL‐17 expression was associated with decreased survival (p < 0.05). IL‐6 expression was inversely associated with angioinvasion, and disease stage (p < 0.05), whereas IL‐17 expression was associated with disease stage (p < 0.05). IL‐10 expression was correlated with IL‐1β and TNF‐α expression, and p16 expression was correlated with IL‐17 and EBV status. Our results indicate that IL‐6 and IL‐17 are associated with gastric carcinogenesis and may be considered prognostic factors.     

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.     

Evolution of Helicobacter pylori susceptibility to antibiotics during a 10‐year period in Lithuania

The study evaluated the changes in the prevalence of Helicobacter pylori strains with primary resistance to antibiotics during the last 10 years in Lithuania. H. pylori susceptibilities to antibiotics were tested in 89 patients in 1998, in 81 patients in 2001 and in 90 patients in 2007/2008. Susceptibility to metronidazole, clarithromycin, amoxicillin and tetracycline was tested using E‐test or agar dilution method. Susceptibility to ciprofloxacin was only tested in 2007/2008. Data about utilization of all authorized and available on market macrolides and clindamycin in Lithuania during 2003–2007 were evaluated using WHO ATC/DDD methodology. A total of 260 H. pylori strains cultured from untreated adult patients were investigated. Primary resistance rates (1998, 2001 and 2007/2008) for metronidazole were 24.7%, 33.3%, and 35.6%, for clarithromycin 1.1%, 3.7%, and 3.3% and for tetracycline 0%, 2.5% and 0% respectively. No cases of amoxicillin resistance have been detected. The resistance rate for ciprofloxacin was 5.6% in 2007/2008. Data of total macrolides and clarithromycin utilization in Lithuania revealed that despite an increase of consumption of these drugs in Lithuania during 2003–2007 in 1.5 times, the total macrolide consumption remains one of the lowest in Europe. We have not observed any significant changes in the susceptibility of H. pylori to the most widely used antibiotics during the recent 10‐year period. The low resistance rate to clarithromycin might be related to the policy to avoid use of macrolides as first‐line treatment for pulmonary and other infections.     

Saccharomyces boulardii expresses neuraminidase activity selective for α2,3‐linked sialic acid that decreases Helicobacter pylori adhesion to host cells

Helicobacter pylori is a major causative agent of gastritis and peptic ulcer disease and is an established risk factor for gastric malignancy. Antibiotic combination therapy can eradicate H. pylori. As these same regimens can evoke adverse effects and resistance, new alternative therapies or adjunctive treatments are needed. A probiotic approach may provide a novel strategy for H. pylori treatment. In the current study, two probiotic bacteria, Lactobacillus acidophilus and Lactobacillus reuteri, and a probiotic yeast, Saccharomyces boulardii, were evaluated for their ability to influence H. pylori viability, adherence to gastric and duodenal cells, as well as the effect of S. boulardii on cell surface expression of sialic acid. Our results indicate that S. boulardii contains neuraminidase activity selective for α(2‐3)‐linked sialic acid. This neuraminidase activity removes surface α(2‐3)‐linked sialic acid, the ligand for the sialic acid‐binding H. pylori adhesin, which in turn, inhibits H. pylori adherence to duodenal epithelial cells.     

寡核苷酸微阵列技术用于检测幽门螺杆菌克拉霉素耐药相关23S rRNA基因多态性

目的 幽门螺杆菌（Hp）克拉霉素耐药与23S rRNA基因A2142G、A2143G和C2182T点突变相关。本研究旨在探索建立一种快速、简便的检测方法，获取Hp克拉霉素耐药相关23S rRNA基因多态性的流行病学资料，为临床合理用药提供依据。方法收集胃黏膜标本，用于病理检查、提取DNA。根据核苷酸位点设计相应探针，探针3′端以氨基修饰，氨基与探针序列之间以间隔臂相连，将探针用芯片点样仪点至玻片上。下游引物5′末端用Cy3荧光标记，进行不对称PCR扩增。其产物与芯片杂交，杂交后洗涤、扫描芯片，观察信号强度。非荧光标记引物扩增PCR产物克隆至T载体，测序验证芯片结果。结果（1）寡核苷酸微阵列技术与测序检测Hb 23S rRNA基因多态性结果完全一致。（2）经病理和PCR证实为场阳性的54例标本，杂交结果显示A2142位点均为野生型（54/54）；A2143G突变率为11．11％（6/54），尚未发现A2143C和A2143T的突变；C2182T突变率为12．96％（7／54），尚未发现C2182A和C2182G的突变，其余均为野生型。结论（1）利用寡核苷酸微阵列技术检测坳克拉霉素耐药的23S rRNA基因多态性，快速、简便而准确，可以高通量并直接检测胃黏膜而不需进行细菌培养，为根除Hp选择用药提供科学依据，推动个体化治疗方案的实施。（2）本研究没有发现场23SrRNA基因2142点突变，A2143G和C2182T突变率分别为11．11％和12．96％。     

Analysis of the correlation between P27 expression and Helicobacter pylori infection in gastric cancer

We aimed to explore the correlation between P27 expression and Helicobacter pylori (H. pylori) infection in gastric cancer, so as to provide evidence for understanding the pathogenesis of gastric cancer caused by H. pylori infection. A total of 82 samples of gastric cancer tissues and 56 samples of tumor-adjacent normal tissues collected from the gastrectomy were enrolled in this study. Then, 14C-urease breathing test was carried out to evaluate the infection of H. pylori in gastric cancer tissues, the expression of P27 in the tissue samples was detected by the immunohistochemistry staining, and the correlation between the H. pylori infection and P27 expression in gastric cancer was analyzed. Of 82 gastric cancer patients, there were 53 patients with H. pylori infection (64.63%). Among the patients with highly or moderately differentiated gastric cancer, the expression of P27 was much higher than that of patients with poorly differentiated gastric cancer (p < 0.01). Besides, comparison of the P27 expression between males and females, among different age groups, tumor sizes, TNM stages, tumor infiltration degrees, or lymph node metastasis, showed no significant differences (p > 0.05). Analysis of the correlation revealed that P27 expression was negatively correlated with the infection of H. pylori (p < 0.01). Multifactorial logistics regression analysis indicated that tumor differentiation was a risk factor of P27-positive expression in gastric cancer tissues (p < 0.01). In addition, P27 expression in the gastric cancer tissues was lower than that in the tumor-adjacent normal tissues (p < 0.01). In gastric cancer patients, expression of P27 is correlated with H. pylori infection which, via downregulating P27, can cause the cancerization of gastric mucosa, and P27, for its role in the development and progression of gastric cancer, is a potential auxiliary indicator for clinical diagnosis whether gastric cancer is complicated with H. pylori infection. So, P27 is a key indicator for diagnosis, treatment, and prognostic evaluation of disease in the advanced stage.     

CD4+ Foxp3+ regulatory T‐cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4⁺Foxp3⁺ Treg cells in stomachs and spleens of H. pylori‐infected mice. The expression levels of Foxp3 as well as anti‐ and pro‐inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4⁺Foxp3⁺ Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL‐10 and TGF‐β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN‐γ level was observed in H. pylori‐infected mice. In addition, gastric tissues of the infection group contained more Foxp3⁺ cells. These results indicate that the percentage of CD4⁺Foxp3⁺ Treg cells are increased in H. pylori‐infected mice, suggesting a role of Treg cells in H. pylori‐induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.     

Effects of Helicobacter pylori,geohelminth infection and selected commensal bacteria on the risk of allergic disease and sensitization in 3‐year‐old Ethiopian children

Background Epidemiological studies have suggested that gastro‐intestinal infections including Helicobacter pylori, intestinal microflora (commensal bacteria) and geohelminths may influence the risk of asthma and allergy but data from early life are lacking. Objective We aimed to determine the independent effects of these infections on allergic disease symptoms and sensitization in an Ethiopian birth cohort. Methods In 2008/09, 878 children (87% of the 1006 original singletons in a population‐based birth cohort) were followed up at age 3 and interview data obtained on allergic symptoms and potential confounders. Allergen skin tests to Dermatophagoides pteronyssinus and cockroach were performed, levels of Der p 1 and Bla g 1 in the child's bedding measured and stool samples analysed for geohelminths and, in a random subsample, enterococci, lactobacilli, bifidobacteria and H. pylori antigen. The independent effects of each exposure on wheeze, eczema, hayfever and sensitization were determined using multiple logistic regression. Results Children were commonly infected with H. pylori (41%; 253/616), enterococci (38.1%; 207/544), lactobacilli (31.1%; 169/544) and bifidobacteria (18.9%; 103/544) whereas geohelminths were only found in 8.5% (75/866). H. pylori infection was associated with a borderline significant reduced risk of eczema (adjusted OR 0.49, 95% CI 0.24–1.01, P=0.05) and D. pteronyssinus sensitization (adjusted OR 0.42, 95% CI 0.17–1.08, P=0.07). Geohelminths and intestinal microflora were not significantly associated with any of the outcomes measured. Conclusion and Clinical Relevance Among young children in a developing country, we found evidence to support the hypothesis of a protective effect of H. pylori infection on the risk of allergic disease. Further investigation of the mechanism of this effect is therefore of potential therapeutic and preventive value. Cite this as: A. Amberbir, G. Medhin, W. Erku, A. Alem, R. Simms, K. Robinson, A. Fogarty, J. Britton, A. Venn, and G. Davey, Clinical & Experimental Allergy, 2011 (41) 1422–1430.     

Resistance rates of metronidazole and other antibacterials in Helicobacter pylori from previously untreated patients in Norway

The aim of the study was to describe the antimicrobial resistance rate of Helicobacter pylori isolated from previously untreated patients in Norway, including the application of two different methods for the determination of metronidazole susceptibility. Altogether 102 isolates obtained in 2008 and 2009 from previously untreated patients suspected of H. pylori related disease, were examined applying a standardized European study protocol. The activity of amoxicillin, tetracycline, clarithromycin, metronidazole, rifabutin and levofloxacin was recorded after an incubation period of 72–96 h in a microaerobic atmosphere. Strains resistant to metronidazole were re‐examined for metronidazole resistance applying anaerobic conditions for the first 24 h. None of the isolates were resistant to amoxicillin or tetracycline, whereas 5, 9% were resistant to clarithromycin and 22, 5% resistant to metronidazole tested conventionally. Applying local standards the metronidazole resistance rate fell to 7, 8%, highlighting the importance of the methodology applied for metronidazole susceptibility testing.     

Autoantibodies to a specific peptide epitope of human Hsp60 (ATVLA) with homology to Helicobacter pylori HspB in H. pylori‐infected patients

Helicobacter pylori (Hp) may initiate autoimmunity as a result of molecular mimicry. The aim of this study was to compare the level of IgG antibodies to a specific epitope (P1 peptide) of human heat shock protein (Hsp)60 homologous to Hp Hsp60 (HspB) in the sera of healthy donors (HD), patients with Hp‐related gastritis or coronary heart disease (CHD), uninfected or with Hp infection confirmed by rapid urease test, histological examination (dyspeptic patients) the ¹³C urea breath test (¹³C UBT), and anti‐Hp antibodies (healthy donors, CHD patients). The Anti‐P1 IgG induction by Hp was verified by adsorption of sera with these bacteria and by experimental immunization of Caviae porcellus with Hp. Cytokine secretion by THP‐1Blue? monocytes in response to P1 was also assessed. Anti‐P1 antibodies were detected in patients with gastritis or CHD infected with Hp and they were not found in uninfected individuals or asymptomatic carriers. No antibodies were raised against P2 in any group. Reduced cross‐reactivity to P1 was exhibited by sera adsorbed with Hp. Caviae porcellus infected with Hp produced anti‐P1 autoantibodies. THP‐1XBlue? monocytes responded to P1 by production of proinflammatory cytokines. Autoantibodies against P1 in Hp‐positive patients with gastritis or CHD and upregulation of proinflammatory cytokines by P1 may contribute to the pathogenesis of Hp infection.     

Immunophenotype of peripheral blood natural killer cells and IL‐10 serum levels in relation to Helicobacter pylori status

Recent findings suggest that NK (Natural Killer) cells may directly modulate the antimicrobial immune responses. In this study, we performed immunophenotypic analysis of peripheral blood NK cells with regard to CD56, CD16, Nkp46, and CD25 markers, as well as IL‐10 levels quantification in the sera samples of asymptomatic, H. pylori (Hp)‐infected or uninfected individuals, and combined these results with our previous findings on lymphocyte cytotoxic activity. Twenty healthy volunteers [10 Hp(?);10 Hp(+)] were included in the study. The percentages of classic lymphocytes (CD3⁺) and NK cells (CD3^?CD56⁺, CD3^?Nkp46⁺, CD3^?CD16⁺) with or without CD25 receptor were evaluated by fluorochrome‐conjugated monoclonal antibody staining and flow cytometry analysis. IL‐10 quantification was performed by enzyme‐linked immunosorbent assay‐ELISA. Our study showed elevated levels of IL‐10 and higher NK cell numbers of both CD3^?CD56⁺CD25⁺ and CD3^?Nkp46⁺CD25⁺ phenotypes, as well as CD3⁺CD25⁺ classic lymphocytes in Hp(+) compared with Hp(?) individuals. No differences between Hp(?) and Hp(+) individuals were found either in total number of classic lymphocytes or NK cell subtypes. Our data suggest that in Hp(+) donors, there is a domination of lymphocytes and NK cells co‐expressing CD25 marker, which might be influenced by the regulatory IL‐10. This phenomenon may be a result of H. pylori adaptation to a changing environment in vivo leading to a chronic infection and lack of severe gastric pathologies.     

Decreased susceptibility to chlorhexidine and prevalence of disinfectant resistance genes among clinical isolates of Staphylococcus epidermidis

Staphylococcus epidermidis is a versatile agent, being both a commensal and a nosocomial pathogen usually with an opportunistic role in association with implanted foreign body materials. Pre‐operative antiseptic preparation is an important strategy for reducing the risk of complications such as surgical site infection (SSI). Currently, the most widely used antiseptics are alcohols, quaternary ammonium compounds (QACs), and the bisbiguanide chlorhexidine. Occurrence of resistance to the latter agent has drawn increasing attention. The aim of this study was to investigate if decreased susceptibility to chlorhexidine among S. epidermidis was present in our setting, a Swedish university hospital. Staphylococcus epidermidis (n = 143), retrospectively collected, were obtained from prosthetic joint infections (PJI) (n = 61), post‐operative infections after cardiac surgery (n = 31), and the skin of the chest after routine disinfection prior to cardiac surgery (n = 27). In addition, 24 commensal isolates were included. Minimum inhibitory concentration (MIC) of chlorhexidine was determined on Mueller Hinton agar plates supplemented with serial dilutions of chlorhexidine. Five QAC resistance genes, qacA/B, smr, qacH, qacJ, and qacG, were detected using PCR. Decreased susceptibility to chlorhexidine was found in 54% of PJI isolates, 68% of cardiac isolates, 21% of commensal isolates, and 7% of skin isolates from cardiac patients, respectively. The qacA/B gene was present in 62/143 isolates (43%), smr in 8/143 (6%), and qacH in one isolate (0.7%). The qacA/B gene was found in 52% of PJI isolates, 61% of cardiac isolates, 25% of commensal isolates, and 19% of the skin isolates. In conclusion, decreased susceptibility to chlorhexidine, as well as QAC resistance genes, were prevalent among S. epidermidis isolates associated with deep SSIs.     

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin‐fixed, paraffin‐embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty‐seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin‐resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates.