Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Characterization of clarithromycin resistance in Malaysian isolates of Helicobacter pylori

AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test(RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nestedASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23 SrR NA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates thanASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87(87.88%) and 67(67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Polymerase chain reaction-based tests for detecting Helicobacter pylori clarithromycin resistance in stool samples: A meta-analysis

BACKGROUND Helicobacter pylori(H.pylori)infection is closely associated with the etiology of a variety of gastric diseases.The effective eradication of H.pylori infection has been shown to reduce the incidence of gastric carcinoma.However,the rate of H.pylori eradication has significantly declined due to its increasing resistance to antibiotics,especially to clarithromycin.Therefore,the detection of clarithromycin resistance is necessary prior to the treatment of H.pylori.Although many studies have been conducted on the use of polymerase chain reaction(PCR)-based tests to detect clarithromycin resistance in stool samples,no accurate data on the feasibility of these tests are available.Here,we performed a meta-analysis to assess the feasibility of these noninvasive tests.AIM To evaluate the reliability of PCR-based tests for detecting H.pylori clarithromycin resistance in stool samples.METHODS We searched PubMed,Medline,Embase,and other databases for articles that evaluated the value of the PCR analysis of stool samples for detecting the resistance of H.pylori to clarithromycin.We collected cross-sectional studies that met the inclusion criteria.Diagnostic accuracy measures were pooled using a random-effects model.The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool.Subgroup analysis was also conducted according to PCR type,purification technique,reference standard,mutation site,sample weight,number of patients,and age group,and the clinical utility of diagnostic tests was evaluated using the Likelihood Ratio Scatter Graph.RESULTS Out of the 1818 identified studies,only 11 met the eligibility criteria,with a total of 592 patients assessed.A meta-analysis of the random-effect model showed that PCR-based analysis of stool samples had high diagnostic accuracy for detecting clarithromycin resistance in patients infected with H.pylori.The combined sensitivity was 0.91[95%confidence interval(CI):0.83-0.95],Q=30.34,and I2=67.04,and the combined specificity was 0.97(95%CI:0.62-1.00),Q=279.54,and I2=96.42.The likelihood ratio for a positive test was 33.25(95%CI:1.69-652.77),and that for a negative test was 0.10(95%CI:0.05-0.18),with an area under the curve of 0.94.The diagnostic odds ratio was 347.68(95%CI:17.29-6991.26).There was significant statistical heterogeneity,and the sub-analyses showed significant differences in the number of patients,sample weight,purification methods,PCR types,mutation points,and reference standards.The included studies showed no risk of publication bias.CONCLUSION PCR-based tests on stool samples have high diagnostic accuracy for detecting H.pylori clarithromycin resistance.

Detection of genotypic clarithromycin-resistant Helicobacter pylori by string tests

AIM: To evaluate the utility of the string test to detect genotypic clarithromycin-resistant Helicobacter pylori (H. pylori) by polymerase chain reaction (PCR)-restriction fragment length polymorphism.

Antibiotic resistance and cagA gene correlation:A looming crisis of Helicobacter pylori

AIM:To determine antibiotic resistance of Helicobacter pylori(H.pylori) in Pakistan and its correlation with host and pathogen associated factors.METHODS:A total of 178 strains of H.pylori were isolated from gastric biopsies of dyspeptic patients.Susceptibility patterns against first and second-line antibiotics were determined and trends of resistance were analyzed in relation to the sampling period,gastric conditions and cagA gene carriage.The effect of cagA gene on the acquisition of resistance was investigated by mutant selection assay.RESULTS:The observations showed that monoresistant strains were prevalent with rates of 89% for metronidazole,36% for clarithromycin,37% for amoxicillin,18.5% for ofloxacin and 12% for tetracycline.Furthermore,clarithromycin resistance was on the rise from 2005 to 2008(32% vs 38%,P = 0.004) and it is significantly observed in non ulcerative dyspeptic patients compared to gastritis,gastric ulcer and duodenal ulcer cases(53% vs 20%,18% and 19%,P = 0.000).On the contrary,metronidazole and ofloxacin resistance were more common in gastritis and gastric ulcer cases.Distribution analysis and frequencies of resistant mutants in vitro correlated with the absence of cagA gene with metronidazole and ofloxacin resistance.CONCLUSION:The study confirms the alarming levels of antibiotic resistance associated with the degree of gastric inflammation and cagA gene carriage in H.pylori strains.

Susceptibility patterns and virulence genotypes of Helicobacter pylori affecting eradication therapy outcomes among Egyptian patients with gastroduodenal diseases

BACKGROUND Helicobacter pylori(H.pylori)is a significant human pathogen that is responsible for a variety of illnesses,including mucosa-associated lymphoid tissue lymphoma,gastric cancer,peptic ulcers,and gastritis.AIM To investigate the frequency of H.pylori infection and its resistance patterns among Egyptian patients and to determine the influence of H.pylori virulence genetic determinants on the eradication success of 14-d triple therapy regimen.METHODS H.pylori infections were investigated in 72 patients with gastroduodenal complications suggestive of H.pylori infection.The cagA and vacA genotypes of cultured strains were studied using polymerase chain reaction.The patients underwent 14 d of triple-therapy treatment.The treatment response was examined using histology and a rapid urease test 6 wk after therapy discontinuation.RESULTS The intention-to-treat eradication rate was 59.2%(95%CI:48.2%-70.3%).Rates of H.pylori resistance to clarithromycin,amoxicillin,and metronidazole were 52.8%,81.9%,and 100%,respectively.Successful eradication of H.pylori was more significantly associated with vacA s1-positive strains[adjusted odds ratio(aOR)=0.507,95%CI:0.175-0.822].A significant association was found between failed eradication rate and H.pylori strains resistant to clarithromycin(aOR=0.204,95%CI:-0.005 to 0.412)and amoxicillin(aOR=0.223,95%CI:0.026-0.537).CONCLUSION This study’s low H.pylori eradication rate following 14-d triple therapy is concerning and worrying.H.pylori pan-resistance to metronidazole followed by the high resistance to ciprofloxacin,amoxicillin,and clarithromycin in this research is challenging and of great concern.

Comparative effectiveness of first-line therapies for eradication of antibiotic-resistant Helicobacter pylori strains:A network metaanalysis

BACKGROUND As a first-line treatment regimen for Helicobacter pylori(H.pylori)infection,antibiotic therapy is widely used worldwide.However,the question of increasing antibiotic resistance must be considered.Given this issue,we need to find ways to reduce drug resistance.This study examined all currently available first-line regimens and compared them with standard triple treatment through a network meta-analysis of randomized controlled trials(RCTs).AIM To compare first-line treatment regimens for eradication of antibiotic-resistant H.pylori strains.METHODS To compare the effectiveness of the first-line regimens for treating H.pylori infection,a Bayesian network meta-analysis was applied to process data extracted from RCTs.The plausible ranking for each regimen was assessed by the surface under the cumulative ranking curve(SUCRA).In addition,we conducted a relevant search by reference citation analysis.RESULTS Twenty-five RCTs involving 12029 participants[including 1602 infected with clarithromycin(CAM)-resistant strains and 1716 infected with metronidazole(MNZ)-resistant strains]were included,in which a total of seven regimens were used for H.pylori eradication.The results showed that dual therapy containing a high-dose proton pump inhibitor(HDDT)[odds ratio(OR):4.20,95%confidence interval(CI):2.29-8.13]was superior to other therapies for all patients,including those with CAM/MNZ-resistant H.pylori infection.In the comparative effectiveness ranking,for CAM-resistant H.pylori,HDDT(OR:96.80,95%CI:22.46-521.9)had the best results,whereas standard triple therapy ranked last(SUCRA:98.7%vs 0.3%).In the subgroup of high cure rates(≥90%),HDDT was also generally better than other therapies.CONCLUSION For the eradication of CAM-and MNZ-resistant H.pylori strains,HDDT exhibited considerable advantages.The studies of CAM-resistant H.pylori were based on small samples due to a lack of antibiotic sensitivity tests in many RCTs,but the results showed that all patients,including those with CAM-resistant H.pylori infection,had a concordant trend.Overall,HDDT may be a reference for RCTs and other studies of H.pylori eradication.