Clarithromycin-resistant H. pylori primary strains and virulence genotypes in the Northeastern region of Brazil

ABSTRACT The increase of H. pylori resistance to clarithromycin is a concern. This study evaluated the prevalence of H. pylori's primary resistance to clarithromycin and its association with virulence factors in adult dyspeptic patients and asymptomatic children. The gastric mucosa from patients (153 gastritis, 24 gastric cancer, 21 peptic ulcer) and gastric juice obtained by string test from 24 H. pylori and 23S rRNA positive asymptomatic children were included. The clarithromycin resistance was assessed by TaqMan RT-PCR 23S rRNA point mutations, A2142G and/or A2143G, and H. pylori virulence markers by PCR. Overall, the clarithromycin resistance was 14.4% (32/222), 14.2% in adults, and 12% in children, whereas origin, gender, and disease were not distinctive factors. The most prevalent point mutation was A2143G (62.5%). The point mutation was significantly less frequent in cagA-positive (11.4%) than in cagA-negative (23.6%) strains (p=0.03 OR = 0.4 95%CI = 0.19 - 0.91) as well as in cagE-positive (10.2%), cagE-negative (21.2%) (p=0.03 OR: 0.4 I.C:0.20-0.91). No difference was found in iceA or vacA alleles genotypes. Primary resistance to clarithromycin was lower than that reported in Southeast Brazil. The cagA and cagE positive H. pylori samples have few point mutations suggesting that individuals infected with virulent strains may be more susceptible to anti-H. pylori treatment.

Drug resistance and clarithromycin resistance gene mutations in Helicobacter pylori strains circulating in Changchun area / 中华微生物学和免疫学杂志

Objective To investigate the resistance of Helicobacter pylori (H. pylori) strains to common antibiotics and to analyze the sites of genetic mutations carried by clarithromycin-resistant strains in order to provide reference for selecting sensitive antibiotics against H. pylori and for providing individualized treatment for patients in Changchun area. Methods Drug resistance of H. pylori clinical isolates to common antibiotics was detected by disk dilution method. The 23S rRNA genes of clarithromycin-resistant strains were amplified by PCR and then sequenced to analyze the presence of mutations. Results In this study, 69 strains of H. pylori were successfully isolated with a positive rate of 23. 1％ . Results of the drug susceptibility test to seven commonly used antibiotics showed that there were 52. 2％ of the isolates resistant to clarithromy-cin, 47. 8％ to tinidazole, 37. 7％ to levofloxacin, 33. 3％ to tetracycline hydrochloride, 30. 4％ to furazoli-done, 30. 4％ to metronidazole and 5. 8％ to amoxicillin. Amoxicillin could continue to be used as a first-line antimicrobial agent. Seven mutation sites were found in the 23S rRNA genes carried by the clarithromy-cin-resistant strains, which were A1821G, G1826A, T1830C, G1940A, A2143G, T2182C and A2223G. The A2143G site mutation accounted for 54. 2％ and was the predominant mutation resulting in the resistance to clarithromycin of H. pylori strains circulating in this area. Conclusions The H. pylori strains isolated from patients with gastroduodenal diseases in Changchun area had a high resistance rate to clarithromycin, which was mainly caused by the A2143G mutation in 23S rRNA gene.

Usefulness of rapid urease test samples for molecular analysis of clarithromycin resistance in Helicobacter pylori / Utilidad de las muestras de test rápido de ureasa para el análisis molecular de resistencia a claritromicina en Helicobacter pylori

Helicobacter pylori is a gastric pathogen that is widely recognized as a causative agent of gastric disease. Its eradication is variable, mainly due to increased resistance to clarithromycin. Our objective was: to evaluate (i) if the biopsy specimen used for the rapid urease test is a useful sample to detect resistance to clarithromycin by PCR-RFLP and (ii) the distribution of A2142G and A2143G point mutations in the 23S rRNA gene, in relation to virulence factors in our region. Gastric specimens were collected from adult dyspeptic patients (n = 141) and H. pylori was investigated by the rapid urease test, histopathological analysis and PCR for the hsp60 gene. Clarithromycin resistance was detected by PCR-RFLP in 62 H. pylori (+) paired biopsy specimens submitted to molecular analysis and the rapid urease test. H. pylori virulence factors were analyzed by multiplex PCR using specific primers for the cagA, vacA and babA2 genes. Thirteen out of 62 strains (20.9%) were resistant to clarithromycin: 6/13 (46.2%) harbored the A2143G mutation whereas 7/13 (53.8%) carried the A2142G point mutation. vacA m1s1 was the most frequent genotype among the resistant strains. In conclusion, the biopsy specimens used for the rapid urease test were suitable samples for clarithromycin resistance detection in patients infected with H. pylori, which became especially useful in cases where the number or size of the biopsies is limited. In addition, this is the first report of a molecular analysis for clarithromycin resistance performed directly from gastric biopsies in our region.

Helicobacter pylori es un patógeno ampliamente reconocido como causante de enfermedad gástrica. Su erradicación es variable, principalmente debido al incremento de la resistencia a claritromicina. Nuestros objetivos fueron evaluar la utilidad de la biopsia usada para realizar el test rápido de ureasa en la detección de resistencia a claritromicina por PCR-RFLP y conocer la distribución de las mutaciones puntuales A2142G y A2143G en el gen ARNr 23S, en relación con los factores de virulencia en nuestra región. Se recolectaron muestras gástricas (n=141) provenientes de pacientes adultos dispépticos y se investigó la presencia de H. pylori mediante el test rápido de ureasa, análisis histopatológico y PCR para el gen hsp60. La resistencia a claritromicina se analizó por PCR-RFLP en 62 muestras pareadas de biopsias gástricas H. pylori+ destinadas al análisis molecular y al test rápido de ureasa. Los factores de virulencia de H. pylori fueron analizados mediante PCR multiplex usando oligonucleótidos específicos para los genes cagA, vacA y babA2. Trece de 62 cepas (20,9%) fueron resistentes a claritromicina, 6/13 (46,2%) llevaron la mutación A2143G, mientras que 7/13 (53,8%) presentaron la mutación A2142G. El genotipo vacA s1m1 fue el más frecuente entre las cepas resistentes a claritromicina. En conclusión, las biopsias destinadas al test rápido de ureasa fueron muestras apropiadas para la detección de la resistencia a claritromicina en pacientes infectados con H. pylori. Esto es especialmente útil en aquellos casos en los que el número o el tamaño de las muestras son limitados. Además, este es el primer reporte de estudio de resistencia a claritromicina (mediante técnicas moleculares), directamente de biopsias gástricas en nuestra región.

Lack of association between rrl and erm(41) mutations and clarithromycin resistance in Mycobacterium abscessus complex

BACKGROUND Mycobacterium abscessus complex (MABC) includes species with high resistance rates among mycobacterial pathogens. In fact, MABC infections may not respond to clarithromycin treatment, which has historically been very effective against MABC infection. Molecular markers have been proposed to detect both acquired (rrl polymorphisms) and inducible (erm(41) polymorphisms) clarithromycin resistance in MABC isolates. OBJECTIVES This study aimed to evaluate the susceptibility profile and molecular markers of clarithromycin resistance in MABC. METHODS The clarithromycin susceptibility profile was determined by broth microdilution with reads on days 3, 5, 7 and 14. Mutations in the rrl and erm(41) genes were evaluated by polymerase chain reaction (PCR) using specific primers, followed by sequencing. FINDINGS A total of 14 M. abscessus subsp. abscessus isolates and 28 M. abscessus subsp. massiliense isolates were evaluated, and clarithromycin resistance was observed in all isolates for up to three days of incubation. None of the 42 isolates exhibited a point mutation in the rrl gene, while all the isolates had a T28 polymorphism in the erm(41) gene. Moreover, all 28 M. abscessus subsp. massiliense isolates had a deletion in the erm(41) gene. MAIN CONCLUSIONS While all the MABC isolates exhibited acquired clarithromycin resistance, no isolates exhibited a point mutation in the rrl gene in this study. The M. abscessus subsp. massiliense isolates demonstrated clarithromycin resistance, which is an uncommon phenotype. The molecular data for the rrl and erm(41) genes were not consistent with the phenotypic test results of clarithromycin susceptibility, indicating a lack of correlation between molecular clarithromycin resistance markers for both acquired and inducible resistance.

Clinical study on berberine, amoxicillin, lansoprazole, bismuth quadruple therapy for Helicobacter pylori eradication in 283 patients / 中华消化杂志

Objective To assess the efficacy and safety of berberine, amoxicillin, lansoprazole, bismuth quadruple therapy for Helicobacter pylori (H.pylori)eradication.Methods From December 2015 to April 2016,566 patients with initial treatment of H.pylori infection were prospectively enrolled.Patients were divided into observation group (berberine, amoxicillin, lansoprazole, bismuth) and control group (clarithromycin, amoxicillin, lansoprazole, bismuth), 283 cases in each group, and the treatment courses in two groups were both 14 days.Four weeks after completion of the treatment, the eradication rate of H.pylori and adverse effect rate of the two groups were compared.Student t test and Chi square test were performed for comparison between the two groups.Results There was no statistically significant difference in the baseline demographic data including gender,age, body mass index (BMI), symptom score between patients of the two groups (all P>0.05).Four weeks after completion of the treatment, the eradication rates of observation group and control group were 87.5%(244/279) and 87.1%(242/278) according to per-protocol analysis, and which were 86.2%(244/283) and 85.5 %(242/283) according to intention-to-treat analysis.There was no statistically significant difference between the two groups (x2=0.021,0.058;both P>0.05).The adverse effect rates of the two groups were 12.5%(35/279) and 16.5%(46/278), and there was no statistically significant difference (x2=1.795,P=0.180).Conclusions Both the new quadruple regimen containing berberine, amoxicillin and bismuth, and the standard quadruple regimen containing clarithromycin, amoxicillin and bismuth both can effectively eradicate H.pylori infection.The new regimen might be recommended as first-line eradication regimen in Xi′an district or area with high clarithromycin resistance.

Establishment of an assay for identifying mutation of clarithromycin -resistance gene of Helicobacter pylori by real time PCR with a novel fluorescence quencher / 预防医学

Objective To establish a real time PCR assay with a novel fluorescence quencher for identification of mutation of clarithromycin -resistance gene of Helicobacter pylori.Methods Two mutations of 23S rDNA gene in Helicobacter pylori,No.2142 and 2143,were chosen as targets for detection,and then the primers and the probe with a novel fluorescence quencher were designed.The genome DNA of Helicobacter pylori was extracted,and then detected by real time PCR reported here.Meanwhile,the specificity,reproducibility and sensitivity of the assay were evaluated.Finally,the real time PCR described here,the real time PCR based on TaqMan,and a sequencing assay were applied to detect 55 Helicobacter pylori strains isolated from clinical specimens,respectively.The results from three assays were compared with each other in order to further evaluate the applicability of this assay in clinic.Results It indicated that the mutation points related to clarithromycin -resistance,A2142G and A2143G,were identified by real time PCR with a novel fluorescence quencher rapidly and accurately.Moreover the coefficient of variation was less than 5%.The limit of detection was 100 copies/reaction.While this assay was applied directly to detect 55 Helicobacter pylori strains,the results were in accordance with those obtained from a TaqMan real time PCR and a sequencing assay,respectively.Conclusion The real time PCR described here was a simple,reliable and accurate approach and substituted for the TaqMan real time PCR for identification of two mutation points of clarithromycin -resistance,A2142G and A2143G in Helicobacter pylori.Thus,a novel tool for diagnosis of gene mutation was provided and the results might be regarded as a substantial evidence for clinical individual therapy.

Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

We evaluated the antibiotic resistance rates and eradication rates of clarithromycin based triple therapy from 2005 to 2010 retrospectively. In addition, we investigated the mechanism of clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients. Two hundred and twelve strains of H. pylori were isolated from 204 patients. H. pylori ATCC 43504 was used as the standard strain. The eradication rates of H. pylori from 2005 to 2010 were 89.3%, 82.6%, 86.3%, 87.7%, 81.8%, and 84.2%, respectively. Total eradication rate was 84.9%. DNA sequences of the 23S RNA gene in clarithromycin-resistant strains were determined. The resistance rates of H. pylori to amoxicillin, clarithromycin, metronidazole, tetracycline, ciprofloxacin, moxifloxacin, and levofloxacin were 9.0%, 8.5%, 36.3%, 0%, 14.2%, 14.2%, and 14.2%, respectively. The multidrug resistance rate of H. pylori was 16.5%. Sequence analysis of clarithromycin-resistant strains showed an A2144G mutation in 8 of 14 strains (57.1%), a T2183C mutation in 5 of 14 strains (35.7%), and double mutations of both A2144G and T2183C in 1 of 14 strains (7.1%). In the present study, triple therapy may still be an effective eradication therapy for H. pylori infections in Korea. The A2144G and T2183C mutations are mainly present in clarithromycin-resistant isolates.

Multicenter Study for the Frequency of 23S rRNA Point Mutations Associated with Clarithromycin Resistance in Helicobacter pylori in Korea / 대한임상미생물학회지

BACKGROUND: Clarithromycin resistance in Helicobacter pylori is a major cause of eradication therapy failure. The objective of this study was to determine the frequency and type of mutations in the 23S rRNA gene in Korea, which are associated with clarithromycin resistance. METHODS: From January 2008 to March 2008, 353 gastric biopsy specimens were collected from five university hospitals in Seoul and Kyunggido. H. pylori infection was defined as showing a positive result in at least one of the following three tests: a microaerophilic culture, a CLO test, and a Giemsa/silver stain. The frequencies of A2143G, A2142G, and the wild type of 23S rRNA and the presence of H. pylori were determined by Seeplex ClaR-H. pylori PCR (Seegene Inc., Seoul, Korea). Twenty-nine culture isolates were tested for susceptibility to clarithromycin by E-test (AB Biodisk, Solna, Sweden) or the CLSI (Clinical and Laboratory Standards Institute) disk diffusion test. RESULTS: From 176 H. pylori PCR-positive specimens, 23S rRNA gene mutations were detected in 38 isolates (21.6%), including 27 isolates of A2143G and 11 isolates of A2142G. Total mutation rates varied from 15.8% to 31.3% with the frequency of A2143G mutation alone varying from 8.5% to 25.0% among the five hospitals studied. There were 10 clarithromycin-resistant isolates found by susceptibility test and they were all positive for A2143G mutation. But, 3 of the 19 susceptible isolates were also positive for either A2143G or A2142G mutation. CONCLUSION: In Korea, the overall frequency of clarithromycin-resistant H. pylori was 21.6%; however, the type and frequency of the 23S rRNA mutations varied from hospital to hospital.

Detection of 23S rRNA Mutation Associated with Clarithromycin Resistance in Children with Helicobacter pylori Infection / 대한소아소화기영양학회지

PURPOSE: The resistance of H. pylori to clarithromycin is one of the major causes of eradication failure. In H. pylori, clarithromycin resistance is due to point mutation in 23S rRNA. The aims of this study were to investigate the mutation of 23S rRNA and to examine the association of cagA, vacA genotype and clarithromycin resistant genes. METHODS: H. pylori DNA was extracted from antral biopsy specimens from 27 children with H. pylori infection. Specific polymerase chain reaction (PCR) assays were used for cagA and vacA. Mutations associated with clarithromycin resistance were detected by using PCR restriction fragment length polymorphism (RFLP) analysis of 23S rRNA gene. RESULTS: A2143G mutation was detected in one case and A2144G in 4, indicating 18.5% were clarithromycin resistant. Among the total of 27, cagA was present in 25 (93%), vacA s1a/m1 in 6 (22%), s1a/m2 in 3 (11%), s1c/m1 in 16 (59%), and s1c/m2 in 1 (4%). All of the 5 clarithromycin resistant strains were cagA (+), among which 2 were s1a/m1 and 2 were s1c/m1. There was no relation between genotypes and clarithromycin resistant genes. CONCLUSION: Detection of H. pylori resistance to clarithromycin using PCR RFLP from biopsy specimens might be useful for the selection of antibiotics. Clarithromycin resistant genes are not associated with genotypes of cagA and vacA.

Analysis of Clarithromycin Resistance of Helicobacter pylori Isolated in Korea

Forty-four strains of Helicobacter pylori were isolated from Kosin Medical Center were tested of resistance to antimicrobial agents, and the mechanism of resistance to clarithromycin was investigated. We determined the MICs of amoxicillin, amoxicillin/clavulanic acid, clarithromycin, and metronidazole by agar and broth dilution method. To detect the mutations of 23S rRNA which is associated with clarithromycin resistance, a 3'-mismatched polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis with restriction enzymes BbsI and BsaI were performed. The nucleotide sequence of 23S rRNA was determined. All H. pylori strains appeared to be susceptible to amoxicillin/clavulinic acid, but 2.3% of strains (1 strain) are resistant to amoxicillin, 13.6% (6 strains) to clarithromycin, and 15.9% (7 strains) to metronidazole. No PCR products was observed by the 3'-mismatched PCR. A 291 bp of PCR product was not digested by BbsI, but was digested by BsaI, which was a characteristic of the A2143G point mutation in the 23S rRNA gene. The nucleotide sequencing analysis revealed that all resistant strains had A2143G, T2182C, and T2244C mutations in 23S rRNA gene.

Molecular mechanism of Helicobacter pylori resistance to clarithromycin / 中华消化杂志

Objective To investigate the molecular mechanism of Helicobacter pylori (H.pylori) resistance to clarithromycin. Methods The E test was used to determine clarithromycin resistant strains of H.pylori , and PCR Restriction Fragment Length Polymorphism (RFLP) analysis for 23S rRNA domain V gene mutations. Results Of nine clarithromycin resistant stains of H.pylori , including six primary and three acquired resistant strains, eight were found to have an A to G mutation in 23S rRNA domain V at position 2144. Conclusions The results indicated that the majority (88.8%) of clarithromycin resistant isolates of H.pylori in Shanghai have an A2144G mutation in 23S rRNA domain V.