Genotypic determination of resistance and heteroresistance to clarithromycin in Helicobacter pylori isolates from antrum and corpus of Colombian symptomatic patients.

BACKGROUND: The effectiveness of Helicobacter pylori first-line treatment has decreased drastically with the rise of strains resistant to clarithromycin. Therapy failure has also been described in patients with infections by strains with dissimilar antimicrobial susceptibilities. The present study aims to estimate the prevalence of resistance and heteroresistance to clarithromycin in H. pylori isolates from antrum and corpus of Colombian patients. METHODS: The study material included 126 isolates from antrum and corpus biopsies from 63 symptomatic patients over 18 years old who had a gastric endoscopy performed on them between June 2014 to August 2016. PCR amplification and sequencing of the H. pylori 23S rDNA gene was performed to determine the presence of mutations associated with clarithromycin resistance. Random amplified polymorphic DNA analysis was implemented in cases of resistance and heteroresistance. RESULTS: The overall frequency of resistance to clarithromycin was 38.1% (24/63 patients), of which 19 patients had resistant isolates in both stomach segments (14 with A2143G mutation and 5 with A2142G mutation), and 5 patients had a heteroresistant status. The remaining 61.9% (39/63 patients) presented only susceptible isolates. DNA fingerprinting analysis showed different patterns in 4/22 paired isolates. CONCLUSIONS: The high prevalence of H. pylori clarithromycin-resistance obtained (> 15%) constitutes an alert for gastroenterologists and suggests the need for reconsideration of the current eradication regimen for H. pylori in the studied population. The data show that heteroresistance status is an additional factor to be considered in the assessment of resistance. In consequence, it is advisable to examine at least two biopsies from different gastric segments.

Can bacterial virulence factors predict antibiotic resistant Helicobacter pylori infection?

AIM: To evaluate the association between virulence factor status and antibiotic resistance in Helicobacter pylori (H. pylori)-infected patients in Ireland. METHODS: DNA was extracted from antral and corpus biopsies obtained from 165 H. pylori-infected patients. Genotyping for clarithromycin and fluoroquinolone-mediating mutations was performed using the Genotype HelicoDR assay. cagA and vacA genotypes were investigated using PCR. RESULTS: Primary, secondary and overall resistance rates for clarithromycin were 50.5% (n = 53/105), 78.3% (n = 47/60) and 60.6% (n = 100/165), respectively. Primary, secondary and overall resistance rates for fluoroquinolones were 15.2% (n = 16/105) and 28.3% (n = 17/60) and 20% (n = 33/165), respectively. Resistance to both antibiotics was 12.4% (n = 13/105) in treatment-naïve patients, 25% (n = 15/60) in those previously treated and 17% (n = 28/165) overall. A cagA-positive genotype was detected in 22.4% (n = 37/165) of patient samples. The dominant vacA genotype was S1/M2 at 44.8% (n = 74/165), followed by S2/M2 at 26.7% (n = 44/165), S1/M1 at 23.6% (n = 39/165) and S2/M1 at 4.8% (n = 8/165). Primary clarithromycin resistance was significantly lower in cagA-positive strains than in cagA-negative strains [32% (n = 8/25) vs 56.3% (n = 45/80) P = 0.03]. Similarly, in patients infected with more virulent H. pylori strains bearing the vacA s1 genotype, primary clarithromycin resistance was significantly lower than in those infected with less virulent strains bearing the vacA s2 genotype, [41% (n = 32/78) vs 77.8% (n = 21/27) P = 0.0001]. No statistically significant association was found between primary fluoroquinolone resistance and virulence factor status. CONCLUSION: Genotypic H. pylori clarithromycin resistance is high and cagA-negative strains are dominant in our population. Less virulent (cagA-negative and vacA S2-containing) strains of H. pylori are associated with primary clarithromycin resistance.

Different antibiotic susceptibility between antrum and corpus of the stomach, a possible reason for treatment failure of Helicobacter pylori infection.

AIM: To assess whether antibiotic resistance varies between the antrum and corpus of the stomach of patients that are either Helicobacter pylori (H. pylori) therapy-naive or pre-treated. METHODS: H. pylori strains were isolated from antrum and corpus biopsies from 66 patients that received a diagnostic gastroduodenoscopy for variant clinical indications. Antimicrobial susceptibility to amoxicillin, clarithromycin, tetracycline, metronidazole, levofloxacin and rifabutin was tested with the E-test method on Iso-Sensitest agar with 10 vol% defibrinated horse blood. In patients with a different antibiotic susceptibility pattern between the isolates from the antrum and corpus, DNA fingerprinting via random amplified polymorphic DNA analysis was performed to detect differences among DNA patterns of H. pylori isolates. RESULTS: Primary, secondary and tertiary resistance to clarithromycin was 6.9%, 53.8% and 83.3%, retrospectively. Metronidazole and levofloxacin resistance also increased according to the number of previous treatments (17.2%, 69.2%, 83.3%; 13.8%, 23.1%, 33.3%). Tertiary resistance to rifabutin was detected in 12.5% of patients. In none of the 66 patients a resistance against amoxicillin or tetracycline was detectable. Discordant antibiotic susceptibility between antrum and corpus isolates for different antibiotics was seen in 15.2% (10/66) of the patients. Two out of those ten patients were naive to any H. pylori antibiotic treatment. The remaining eight patients previously received at least one eradication therapy. DNA fingerprinting analysis revealed no substantial differences among DNA patterns between antrum and corpus isolates in the majority of patients suggesting an infection with a single H. pylori strain. CONCLUSION: Different antibiotic susceptibility between antrum and corpus biopsies is a common phenomenon and a possible explanation for treatment failure. Resistant H. pylori strains may be missed if just one biopsy from one anatomic site of the stomach is taken for H. pylori susceptibility testing.

Biological activity and microscopic characterization of Lythrum salicaria L.

BACKGROUND: There are several plants have been used worldwide in the folk medicine with high incidence for treatment of human disorders, of which Lythrum salicaria belongs to the Lythraceae family has traditionally reputation for some medicinal usage and recently many biological and pharmacological activity of the plant have been studied. METHODS: In this study, microscopic characterizations of the aerial parts of the plant were determined. Moreover, the plant extract (aqueous methanol 80%) was subjected to an anti-diabetic activity test (in a rat model of streptozocin induced diabetes), anti-Helicobacter pylori (using disc diffusion method) along with antioxidant activity against DPPH (stable free radical) tests. Besides, total flavonoids, phenols, tannins, as well as polysaccharides contents have been assessed using spectroscopic methods. RESULTS: The microscopic properties of the plant fragments revealed anomocytic stomata, conical shape trichomes, and abundant spherical pollen grains as a characteristic pattern for the aerial parts of the plant. The extract of the plant at concentration of 15 g/kg showed mild lowering activity on blood glucose level to 12.6% and 7.3% after 2 and 3 h of administration. Additionally, clinically isolated H. pylori strain was inhibited with the plant extract at concentration of 500 mg/mL (zone of inhibition: 17 ± 0.08 mm). Moreover, IC50 values for DPPH inhibition of the plant extract, vitamin E, BHA were examined as 13.5, 14.2, and 7.8 µg/mL, respectively. Total flavonoids, phenols, tannin, and polysaccharides contents of the extract were successfully evaluated as 5.8 ± 0.4 µg QE/mg EXT, 331 ± 3.7 µg GAE/mg EXT, 340 ± 2.3 µg TAE/mg EXT, 21 ± 0.2 µg GE/mg EXT, respectively. CONCLUSIONS: The results suggested that L. salicaria has low anti-diabetic and anti-Helicobacter pylori effects, but high antioxidant activity, just the same as positive standard (vitamin E), which might be attributed to the high content of phenolic compounds in the extract.

Helicobacter pylori isolates from proximal and distal stomach of patients never treated and already treated show genetic variability and discordant antibiotic resistance.

INTRODUCTION: The treatment failure of Helicobacter pylori (H. pylori) infection may be mainly because of antibiotic resistance and the presence of a mixed infection in the same patient. AIM: To investigate the incidence of mixed infection and discordant antibiotic resistance in patients never treated and already treated. MATERIALS AND METHODS: Susceptibility test to amoxicillin, rifabutin, tinidazole, clarithromycin, levofloxacin, and moxifloxacin was conducted on H. pylori strains culture from 76 patients never treated and 72 patients already treated unsuccessfully. DNA fingerprinting was determined on H. pylori strains harboring in the same patient with a discordant resistance to the same antibiotic. RESULTS: Forty percent of patients never treated and 53% of patients already treated showed a pangastric infection. The overall resistance to amoxicillin, clarithromycin, and tinidazole was significantly higher in patients with pangastric infection in comparison with those with the infection in the antrum (P<0.05). Discordant resistance was present in 33% of patients never treated, and 21% of patients already treated. DNA fingerprinting showed substantial differences among DNA patterns suggesting a mixed infection. CONCLUSION: Culture and susceptibility test should be performed when necessary not only in the antrum but also in the fundus of patients with H. pylori infection.

In vitro and in vivo anti-Helicobacter/Campylobacter activity of the aqueous extract of Enantia chlorantha.

The aqueous extract of Enantia chlorantha Oliver (Annonaceae) stem bark, a plant widely used in Cameroon for the traditional treatment of gastritis and stomach problems, was assessed for in vitro and in vivo anti-Helicobacter/Campylobacter properties using the well diffusion assay, agar dilution assay, and killing rate determination. The in vitro activity was dose-dependent, and the same antimicrobial parameters (MAQ = 0.63 mg; MIC = 0.39 mg/mL; MBC = 1.56 mg/mL; ET(100) = 8 h) were obtained for both H. pylori and C. jejuni/coli. When the plasma active principle concentration equivalence was determined in vitro using plasma from rats exposed to a single dose (3000 mg/kg) of the extract, the peak absorption of E. chlorantha active principle against H. pylori occurred at 2 h. Plasma activity was nil 8 h after extract administration. The in vivo H. pylori eradication potency of the extract was assessed using mice infected with H. pylori. Antral mucus sample cultures from mice treated with E. chlorantha extract (500 and 1000 mg/kg for 3 days) did not yield any growth. The results suggest that in addition to its in vitro activity, E. chlorantha water extract also possesses in vivo antibiotic effects against H. pylori.

Antibodies to Helicobacter pylori and CagA protein are associated with the response to antibacterial therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

The aim of this study was to clarify predictive factors for response to eradication therapy in cases of Helicobacter pylori (H. pylori)-positive API2-MALT1-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Sixty-six patients who were examined for H. pylori infection and the presence of the API2-MALT1 chimeric transcript and who underwent H. pylori eradication therapy as first-line therapy, were enrolled in this study. Immunohistochemical markers (p53, Ki-67, and BCL10), microsatellite instability, loss of heterozygosity, serum levels of antibodies (anti-H. pylori and anti-CagA), and markers for gastritis (gastrin and pepsinogens) were examined, and the results were compared between patients whose tumors regressed completely after eradication therapy (responders) and patients whose tumors did not regress (non-responders). Of the 66 patients with localized gastric MALT lymphoma, 47 (71.2%) showed complete remission after eradication therapy. None of the H. pylori-negative (n = 9) and/or API2-MALT1-positive (n = 7) patients responded to antibacterial treatment. Of 44 patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma, 38 (86.4%) showed complete remission after eradication therapy. Titers of antibodies against H. pylori and CagA protein were significantly higher in the responders than in the non-responders (P = 0.0235 and 0.0089, respectively). No significant difference between the groups was observed for the other factors. In conclusion, measurement of titers of serum antibodies to H. pylori and CagA protein may be useful for predicting the response to eradication therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

Clarithromycin resistance of Helicobacter pylori strains isolated from children' gastric antrum and fundus as assessed by fluorescent in-situ hybridization and culture on four-sector agar plates.

AIM: To assess validity of culture on four-sector agar plates and fluorescent in-situ hybridization (FISH) test, and clarithromycin resistance rate in Helicobacter pylori strains isolated from children in the last 10 years. METHODS: In the last 5 years, gastric biopsy specimens from antrum and fundus were taken from 89 consecutive children (median age 9 years) with H. pylori gastritis and from 21 controls. Culture was performed on 176 gastric biopsies (89 from antrum, 87 from fundus) on four-sector agar plates, and FISH test with DNA ProbeMix. After its validity was evaluated, FISH test was applied on additional 119 biopsies from 68 children (68 from the antrum, 51 from the fundus) stored in the Pathology archive in the previous 5 years. RESULTS: Culture was positive in 157 of 176 biopsies (sensitivity: 89.2%, 95% confidence interval (CI) 85-94). In 33 of 89 children (37%) resistant strains were found in one or both gastric sites. FISH test was positive in 148 of 176 biopsies from infected children (sensitivity 84.1%, 95%CI 79-89) and in none of 42 biopsies from controls (specificity 100%). When applied on archive biopsies, FISH test was positive in 96 of 119 (80.7%, 95%CI 74-88). Total children harboring resistant strains in the last 10 years, as assessed by FISH test, were 66 of 157 (42%). Mixed infection with both sensitive and resistant strains were found in 40 children (25%) and in 12 of them resistant strains were in the fundus only. CONCLUSIONS: Culture on four-sector agar plates and FISH test had a high sensitivity and specificity and showed co-presence of sensitive and resistant strains. In one-third of children with mixed infection, the resistant strains were in the fundus only. Clarithromycin resistance should be assessed in biopsies both from the antrum and the fundus, utilizing antral biopsies only can underestimate its prevalence.

Low-dose rabeprazole, amoxicillin and metronidazole triple therapy for the treatment of Helicobacter pylori infection in Chinese patients.

BACKGROUND: Rabeprazole in combination with amoxicillin and metronidazole (RAM) has been shown to be an effective second-line treatment of Helicobacter pylori infection. The effects were compared of 7-day low-dose and high dose rabeprazole in RAM for the primary treatment of H. pylori infection in Chinese patients. METHODS: Helicobacter pylori-positive dyspeptic patients were randomized to receive either (i) rabeprazole 10 mg, amoxicillin 1000 mg and metronidazole 400 mg (RAM-10) or (ii) high-dose rabeprazole 20 mg, amoxicillin 1000 mg and metronidazole 400 mg (RAM-20), each given twice daily for 7 days. Helicobacter pylori eradication was confirmed by (13)c-urea breath test 5 weeks after stopping medications. side-effects of treatments were documented. RESULTS: A total of 120 patients were eligible for analysis. By intention-to-treat and per-protocol analysis, the eradication rates were 83% and 86% in the RAM-10 group and 75% and 76% in the RAM-20 group, respectively (P = 0.26 and P = 0.17). Both regimens were well-tolerated and compliance was >98% in both groups. CONCLUSIONS: Low-dose rabeprazole in combination with amoxicillin and metronidazole is an effective, economical and well-tolerated therapy for the treatment of H. pylori infection in Chinese population.

Correlation between Helicobacter pylori infection and vitamin C levels in whole blood, plasma, and gastric juice, and the pH of gastric juice in Korean children.

BACKGROUND: It is well known that chronic gastritis induced by Helicobacter pylori may be associated with hypochlorhydria and may also be accompanied by low levels of vitamin C in plasma and gastric juice in adults. This study investigates the relationship between H. pylori infection and vitamin C levels in the blood, plasma and gastric juice and the gastric juice pH of Korean children. METHODS: During a 5-year period, multiple gastric antral biopsies were taken from 452 children who underwent gastroduodenoscopy. The biopsy specimen was inoculated into phenol red buffered urea broth and incubated for 48 hours to detect color changes. The histopathologic findings were evaluated using the Sydney System. Concentrations of vitamin C in whole blood, plasma, and gastric juice aspirate were measured using the 2,4-dinitrophenylhydrazine method. RESULTS: Four hundred fifty-two patients (228 boys, 224 girls) aged 1 to 15 years were enrolled in this study. H. pylori was detected in 112 patients (24.8%) using histology, whereas it was found in 204 patients (45.1%) using the urease test. One hundred seven patients (23.7%) had active gastritis, and 421 patients (93.1%) had chronic gastritis. Vitamin C levels in whole blood, plasma, and gastric juice exhibited significant negative correlation with the age of patients, the histologic density of H. pylori, the degree of active and chronic gastritis, and the severity of H. pylori infection (based on urease positivity and histologic density of H. pylori). Gastric juice pH was correlated with the degree of chronic gastritis and was significantly higher in urease-positive patients. CONCLUSIONS: The data demonstrate that vitamin C levels in whole blood, plasma, and gastric juice and the gastric juice pH in Korean children are closely related to the severity of H. pylori infection and the histologic changes in the stomach. These data suggest that vitamin C may play a role in determining infection and progression, and vitamin C supplementation may be an important axis for the management of H. pylori infection in children.

Gastric transitional zones, areas where Helicobacter treatment fails: results of a treatment trial using the Sydney strain mouse model.

Current combination therapies cure Helicobacter pylori infection in 75 to 85% of cases. However, many treatment failures are not explained by antibiotic resistance. Our goal was to explore treatment failures under in vivo conditions by using the H. pylori Sydney strain (SS1) mouse model. Mice infected with H. pylori (SS1) were treated with monotherapies or combination therapies used in human trials. Bacterial levels and distribution of organisms within the stomach were assessed 24 h after treatment to determine clearance and location of treatment failures and 29 days after treatment to determine cure rates. Except for treatment with metronidazole, mono- and dual therapies did not cure infection but resulted in decreases in bacterial levels and differences in distribution within the stomach. In cases of treatment failure when clarithromycin was used, omeprazole and dual therapy with omeprazole and amoxicillin resulted in organisms being cleared from the antrum, but organisms remained in the antrum-body transitional zone. The triple therapies of OMC and bismuth subcitrate, metronidazole, and tetracycline were successful in eradicating infection. Except for metronidazole monotherapy and triple therapy with OAC, there was good correlation between the Sydney strain mouse model and humans with respect to the success of antimicrobial therapy. The antrum-body transitional zone was identified as a sanctuary site in treatment failure. This could result from antimicrobial agents not functioning effectively at this site or bacteria in this location expressing products that protect them against antimicrobial agents. This is the first demonstration of a possible sanctuary site as a reason for failure of therapy.

Essential role of magnesium ion in water for colonization of Helicobacter pylori in 2-week-old miniature pigs.

This study was designed to determine whether magnesium ion in water would influence the colonization of Helicobacter pylori in 2-week-old miniature pigs. Groups A (2 pigs) and B (1 pig) were both fed a milk diet dissolved in drinking water, Group C (2 pigs) was fed a milk diet dissolved in deionized distilled water (DDW), and Group D (1 pig) was fed a milk diet dissolved in DDW supplemented with MgCl2. Groups B, C, and D were all challenged with H. pylori, and Group A was not. Necropsy was performed on the pigs on postinfection Day 5, and biopsy specimens were taken from 16 sites of the stomach. H. pylori were recovered from 11 of 16 sites in Group B, 1 of 32 sites in Group C, and 13 of 16 sites in Group D. On the other hand, the degree of lymphocyte infiltration increased in the order of Group A < Group B < Group C < Group D. These observations suggest that magnesium ion in drinking water is essential for the colonization of H. pylori in the pig stomach. Possible mechanisms for the lymphocyte infiltration are discussed.

[Zhongxu-qizhi type of chronic gastritis and Campylobacter pyloridis infection].

According to the syndrome differentiation of TCM, one hundred cases of chronic gastritis were recognized as Zhongxu-Qizhi type (Group I, 57 cases) and the other types (Group II, 43 cases), the latter further divided into 36 cases of disharmony of Liver and Stomach type and 7 cases of deficiency of Stomach Yin type. The pathohistological investigation and urease test showed that the campylobacter pyloridis (CP) infected rate in the Group I (92.9%) was very significantly higher than that in Group II (58.1%, P less than 0.01); severe degree and deep location of CP infection occurred more significantly in Zhongxu-Qizhi type (52.8% and 73.6% respectively). Between the two groups, active chronic gastritis and severe invasion of polymorphonuclear cells were significantly different (P less than 0.05). All these findings suggest that there were some relationships between CP infection and syndrome differentiation of TCM.