A practical single sample dry latex agglutination test for Helicobacter pylori antibody detection.

Assessment of a single serum sample for Helicobacter pylori antibodies is frequently requested in routine diagnostic laboratories. Current enzyme linked immunosorbent assay (ELISA) kits are not ideal for testing small numbers of serum samples and some have low sensitivities, specificities or large grey zones. A panel of 90 serum samples from patients who had presented for routine upper endoscopy was used to compare three kits for the detection of H pylori antibodies: (1) Pyloriset Dry, total antibody latex agglutination, Orion Diagnostica, Espoo, Finland; (2) Pyloriset enzyme immunoassay (EIA), IgG ELISA, Orion; and (3) Hel-p, IgG ELISA, Amrad, Kew, Victoria, Australia. Diagnosis of H pylori positivity was made if culture results and either rapid urease test or histopathology were positive. The sensitivity, specificity, positive, and negative predictive value for each test was as follows: Orion: latex 93.3%, 95.6%, 95.5%, 93.3%, respectively; Orion: EIA-G 84.4%, 97.8%, 97.4%, 84.4%, respectively; and Amrad: EIA-G 100%, 88.9%, 90%, 100%, respectively. The latex test performed better than the EIAs with respect to sensitivity and specificity.

Validation of a modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of Helicobacter pylori.

Triple antimicrobial therapy that includes metronidazole has been recommended as a first-line therapy for Helicobacter pylori because it has the highest eradication rates. However, resistance in H. pylori to metronidazole has been reported worldwide and its presence may reduce the efficacy of triple therapy. Various methods for testing H. pylori against metronidazole have been used including agar dilution, disk diffusion and the Etest but there has been little standardization of methods. One hundred isolates of H. pylori from different patients were tested for susceptibility to metronidazole by agar dilution, Etest and disk diffusion (5 micrograms disk). The agar dilution results confirmed the MIC susceptibility breakpoint to be < or = 8 micrograms/ml. Using this breakpoint there was close agreement (98%) between Etest and agar dilution results. For susceptible strains, MICs by E-test were generally one twofold dilution lower. Using the error-rate bounded method, agreement between disk diffusion zone diameter and MIC was 98% for agar dilution with breakpoints of > or = 12 mm and < or = 8 micrograms/ml and 100% for Etest with breakpoints of > or = 12 mm and < or = 8 micrograms/ml. The Etest discriminated better than agar dilution between susceptible and resistant strains and was simple to perform. The disk diffusion test is a reliable and cheaper alternative to the Etest with susceptibility being a zone diameter > or = 12 mm with a 5 micrograms disk. The prevalence of metronidazole resistance in this study was 40% by Etest.

Novel bismuth compounds have in vitro activity against Helicobacter pylori.

The increasing antimicrobial resistance in Helicobacter pylori has led to the search for new therapeutic agents. In this in vitro study, novel compounds combining bismuth with sialic acid inhibitors were investigated for bactericidal activity using time-kill methodology. The activity of these compounds was compared to bismuth subcitrate against a type strain and a clinical isolate of H. pylori. The compounds tested showed cidal activity which was related to the bismuth component of each drug. These compounds may offer a potential advantage over current bismuth preparations with the sialic acid inhibitor moiety interfering with adhesion of H. pylori to gastric epithelium.

Detection of extended spectrum beta-lactamases in the routine clinical microbiology laboratory.

AIMS: To compare three methods of confirming the presence of an extended spectrum beta-lactamase (ESBL) enzyme with the initial detection (i.e., screening) by the Vitek AMS. METHODS: Gram-negative bacteria which flagged as ESBL-positive in the Vitek GNS card, or were suspected of harbouring an enzyme, were further tested by each of the following methods: (a) combination disc test using cefpodoxime, ceftazidime and cefotaxime with and without clavulanate; (b) cefotaxime ESBL Etest; and (c) Jarlier keyhole method with cefpodoxime (10 microg), cefotaxime (5 microg) and aztreonam (30 microg) placed 15mm away from an augmentin (30 microg) disc. RESULTS: A total of 52 isolates were investigated, representing an 18-month time period. Fifty of these were positive by Vitek. Twenty-eight (56%) were confirmed by other methods (true positives). Of the 44% Vitek-positive/confirmatory test-negative (false positives), eight were Escherichia coli which was 53% of all E. coli tested. The majority of other false-positive isolates were Klebsiella oxytoca (24% overall) which were all Vitek- and Etest-positive but negative by the combination disc test. CONCLUSIONS: All ESBL-positive strains by Vitek should be confirmed by the combination disc test using all three antibiotics. This will enable differentiation of 'true' ESBLs from false-positive organisms, including K1 hyperbetalactamase-producing Klebsiella oxytoca and AmpC-producing organisms. The cefpodoxime combination discs gave the best differentiation in this study with only one ESBL organism being missed.

Antimicrobial resistance testing of Helicobacter pylori: a comparison of Etest and disk diffusion methods.

Routine antimicrobial resistance testing of Helicobacter pylori is more commonly performed since the correlation between metronidazole resistance and failure to eradicate using this drug, has been made. While resistance testing of H. pylori by Etest is simple to perform, it is expensive compared to disk diffusion methods. In this study the Etest was compared with a modified Kirby-Bauer disk diffusion (NCCLS) method for routine resistance screening of H. pylori. Fifty one pre-treatment isolates were tested against amoxycillin, metronidazole, tetracycline and erythromycin by both Etest and disk diffusion using NCCLS guideline strength disks. Clarithromycin was tested by Etest only. Nitroimidazole and macrolide resistance were detected using the modified Kirby-Bauer disk diffusion method which correlated with Etest minimum inhibitory concentration (MIC). Resistance rates were 49% for metronidazole and 8% for clarithromycin. Cross resistance occurs with macrolides against H. pylori and allows testing of erythromycin to predict resistance to clarithromycin. The very low MICs obtained with H. pylori against amoxycillin and tetracycline require the use of Etest or lower strength disk methods to be used.

Bactericidal activity and synergy studies of proton pump inhibitors and antibiotics against Helicobacter pylori in vitro.

The bactericidal activity of the proton pump inhibitors omeprazole and lansoprazole alone and in combination with a beta-lactam or macrolide antibiotic were investigated in vitro. Time-kill curves against Helicobacter pylori NCTC 11637 and a recent clinical isolate revealed significant concentration-dependent killing with all drugs other than amoxycillin. Combinations of proton pump inhibitor and erythromycin showed synergic activity. In contrast, proton pump inhibitor plus amoxycillin showed additive activity against the clinical isolate only. Bactericidal investigations of anti-helicobacter drugs in vitro may suggest optimum treatment strategies for this common infectious disease.

Metronidazole resistance: a predictor of failure of Helicobacter pylori eradication by triple therapy.

Triple therapy (bismuth and two antibiotics) will eradicate Helicobacter pylori infection in 70-90% of subjects. Treatment failure has been attributed to patient compliance and antimicrobial drug resistance. The aim of this study was to examine factors influencing the eradication of H. pylori following triple therapy. Thirty seven subjects with H. pylori cultured from antral biopsies were treated with colloidal bismuth subcitrate (120 mg qid for 2 weeks), metronidazole (400 mg tid for 1 week) and amoxycillin (500 mg tid for 1 week). Pretreatment isolates of H. pylori were tested for metronidazole susceptibility by agar dilution according to the National Committee for Clinical Laboratory Standards guidelines. Factors including age, sex, clinical diagnosis and metronidazole resistance were evaluated in relation to H. pylori. The overall metronidazole resistance was 32%. Metronidazole resistant strains were more frequent in females, with a resistance rate of 54%. Helicobacter pylori eradication occurred in 68% of patients with a metronidazole susceptible stain and only 17% of patients with a metronidazole resistant strain (P < 0.03). Helicobacter pylori eradication is dependent upon susceptibility to metronidazole. This data would support the role for routine metronidazole susceptibility testing using appropriate standardized methods when triple therapy is to be considered.

Antimicrobial resistance in Helicobacter pylori isolates from children.

OBJECTIVE: To determine the resistance rate to four antimicrobial agents commonly used in the treatment of Helicobacter pylori infection in children. METHODS: Between July 1997 and January 2000, all H. pylori isolates from children undergoing gastroscopy were prospectively collected and subcultured to yield the susceptibility to four antimicrobial agents by E-test. In all, 23 isolates were tested. Demographic data, presenting symptoms, treatment regimen and clinical improvement after treatment were collected retrospectively. RESULTS: The resistance rate of H. pylori to metronidazole and clarithromycin were 43.5% and 8.7%, respectively. No H. pylori strains were resistant to amoxycillin or tetracycline. There were no statistically significant differences in age, sex, ethnicity, presenting symptoms or clinical improvement after treatment between antimicrobial-susceptible and antimicrobial-resistant groups. CONCLUSIONS: The frequent resistance of H. pylori to metronidazole and moderate resistance to clarithromycin in children are comparable with local adult data. The incidence of resistance tended to be higher in patients of non-European ethnicity, but this was not statistically significant. Given that the primary goal of therapy is eradication, and that local resistance rates are high, recommendations for H. pylori management may need to be modified to include sensitivity testing and/or determination of eradication in all patients.

Oxygen concentration influences proton pump inhibitor activity against Helicobacter pylori in vitro.

Omeprazole and lansoprazole are proton pump inhibitors that have shown activity against Helicobacter pylori and other Helicobacter species when tested by agar dilution. Lansoprazole was more active against H. pylori than was omeprazole, and the activity was independent of urease production. Disk susceptibility tests and agar dilution MIC determinations were performed to investigate the effects of incubation under different sets of atmospheric conditions on H. pylori inhibition. Oxygen concentration was found to influence proton pump inhibitor activity in vitro, with higher concentrations leading to greater susceptibility. The method of testing is important in determining the anti-Helicobacter activity of proton pump inhibitors.

In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria.

In this study the effects of both pH and organic acids on Helicobacter pylori NCTC 11637 were tested. Lactobacillus acidophilus, Lact. casei, Lact. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were assayed for their lactic acid production, pH and inhibition of H. pylori growth. A standard antimicrobial plate well diffusion assay was employed to examine inhibitory effects. Lactic, acetic and hydrochloric acids demonstrated inhibition of H. pylori growth in a concentration-dependent manner with the lactic acid demonstrating the greatest inhibition. This inhibition was due both to the pH of the solution and its concentration. Six strains of Lact. acidophilus and one strain of Lact. casei subsp. rhamnosus inhibited H. pylori growth where as Bifidobacterium bifidus, Ped. pentosaceus and Lact. bulgaricus did not. Concentrations of lactic acid produced by these strains ranged from 50 to 156 mmol l-1 and correlated with H. pylori inhibition. The role of probiotic organisms and their metabolic by-products in the eradication of H. pylori in vivo remains to be determined.

Use of a dry latex agglutination test to measure eradication of Helicobacter pylori infection.

BACKGROUND AND METHODS: A total antibody latex serology test was compared with enzyme immunoassay serology after treatment for Helicobacter pylori infection in 22 patients. RESULTS: Nineteen patients were cured of infection, but only nine (47%) were negative by the latex test after 6 months. However a significant decline in immunoglobulin (Ig)G was seen in 90% of the cured patients. CONCLUSIONS: Although the latex test is suitable for initial diagnosis of H. pylori infection, it is not suitable for monitoring treatment success. A decline in IgG of more than 40% correlates well with successful eradication of H. pylori.