Lurdes Monteiro

Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori

ABSTRACT The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

Diagnosis of Helicobacter pylori Infection: Noninvasive Methods Compared to Invasive Methods and Evaluation of two New Tests

OBJECTIVES:Current guidelines recommending Helicobacter pylori eradication treatment without performing endoscopy in certain patients highlight the importance of noninvasive tests. Our aim was to determine the accuracy of two new tests: the antigen stool test and Helicoblot 2.1 (an immunoblot used on serum) as well as the 13C urea breath test and ELISA serology in comparison to invasive tests for the pretreatment diagnosis of H. pylori infection.METHODS:Helicobacter pylori infection was diagnosed prospectively in 104 untreated patients using eight different tests. Invasive tests included culture, urease test (CLOtest), histology, and PCR; noninvasive tests included the 13C urea breath test, IgG serology (Pyloriset EIA-G), immunoblot (Helicoblot 2.1), and antigen stool detection (Premier Platinum HpSA). A predefined gold standard based on biopsy tests was used to define H. pylori status, as well as an empirical approach.RESULTS:There was no statistically significant difference between the different tests. The sensitivity of the noninvasive tests ranged between 88.9% and 95.6% (stool test: 88.9%, 95% CI: 82.7–95.1, and Helicoblot 2.1: 95.6%, 95% CI: 91.5–99.6) and the specificity ranged between 92.6 and 98.1% (stool test: 94.4%, 95% CI: 84.6–98.8, and Helicoblot 2.1: 92.6%, 95% CI: 91.5–96.2) when a predefined gold standard was used.CONCLUSIONS:Most tests had sensitivities, specificities, and predictive values >90%. The noninvasive tests are accurate for the diagnosis of H. pylori infection. Helicoblot 2.1 performed as well as the best ELISA kit. The HpSA is a promising direct noninvasive test that can be applied easily to evaluate H. pylori status.

Detection of Helicobacterpylori DNA in human feces by PCR: DNA stability and removal of inhibitors

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.

Risk Factors for Death and Severe Neurological Sequelae in Childhood Bacterial Meningitis in Sub-Saharan Africa

We report a morality rate of 33% among 403 children with bacterial meningitis in Angola. A fatal outcome was associated with impaired consciousness, severe dyspnea, and seizures, and severe neurological sequelae (found in 25% of our patients) was associated with delayed presentation to the hospital, impaired consciousness, and seizures. Being underweight was of secondary importance. Treatment with ceftriaxone, rather than with penicillin plus chloramphenicol, did not improve outcome.

Prevalence and incidence of Helicobacter pylori Infection in a healthy pediatric population in the Lisbon area

Background:  Helicobacter pylori is mainly acquired in childhood. Although adult studies reported a high prevalence of H. pylori infection in Portugal, the actual rate in children remains unknown. This study aimed to determine the prevalence and the incidence of H. pylori infection in an asymptomatic pediatric population of the Lisbon area and to correlate prevalence with sociodemographic determinants.

Identification of Markers for Helicobacter pylori Strains Isolated from Children with Peptic Ulcer Disease by Suppressive Subtractive Hybridization

ABSTRACT Peptic ulcer disease (PUD) occurs after a long-term Helicobacter pylori infection. However, the disease can develop earlier, and rare cases have been observed in children, suggesting that these H. pylori strains may be more virulent. We used suppressive subtractive hybridization for comparative genomics between H. pylori strains isolated from a 5-year-old child with duodenal ulcer and from a sex- and age-matched child with gastritis only. The prevalence of the 30 tester-specific subtracted sequences was determined on a collection of H. pylori strains from children (15 ulcers and 30 gastritis) and from adults (46 ulcers and 44 gastritis). Two of these sequences, jhp0562 (80.0% versus 33.3%, P = 0.008) and jhp0870 (80.0% versus 36.7%, P = 0.015), were highly associated with PUD in children and a third sequence, jhp0828, was less associated (40.0% versus 10.0%, P = 0.048). Among adult strains, none of the 30 sequences was associated with PUD. However, both jhp0562 and jhp0870 were less prevalent in adenocarcinoma strains than in PUD strains from children and adults, the difference being statistically significant for jhp0870. In conclusion, two H. pylori genes were identified as being strongly associated with PUD in children, and their putative roles as an outer membrane protein for jhp0870 and in lipopolysaccharide biosynthesis for jhp0562, suggest that they may be novel virulence factors of H. pylori.

Disease association with two Helicobacter pylori duplicate outer membrane protein genes, homB and homA

BackgroundhomB encodes a Helicobacter pylori outer membrane protein. This gene was previously associated with peptic ulcer disease (PUD) and was shown to induce activation of interleukin-8 secretion in vitro, as well as contributing to bacterial adherence. Its 90%-similar gene, homA, was previously correlated with gastritis. The present study aimed to evaluate the gastric disease association with homB and homA, as well as with the H. pylori virulence factors cagA, babA and vacA, in 415 H. pylori strains isolated from patients from East Asian and Western countries. The correlation among these genotypes was also evaluated.ResultsBoth homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains. In Western strains (n = 234, 124 PUD and 110 non-ulcer dyspepsia (NUD), homB, cagA and vacA s1 were all significantly associated with PUD (p = 0.025, p = 0.014, p = 0.039, respectively), and homA was closely correlated with NUD (p = 0.072). In East Asian strains (n = 138, 73 PUD and 65 NUD), homB was found more frequently than homA, and none of these genes was associated with the clinical outcome.Overall, homB was associated with the presence of cagA (p = 0.043) and vacA s1 (p < 0.001), whereas homA was found more frequently in cagA-negative (p = 0.062) and vacA s2 (p < 0.001) strains.Polymorphisms in homB and homA copy number were observed, with a clear geographical specificity, suggesting an involvement of these genes in host adaptation. A correlation between the homB two-copy genotype and PUD was also observed, emphasizing the role of homB in the virulence of the strain.ConclusionThe global results suggest that homB and homA contribute to the determination of clinical outcome.

Antibiotic-resistant Helicobacter pylori strains in Portuguese children.

Background: Data concerning the effectiveness of Helicobacter pylori eradication regimens based in antibiotic susceptibility testing are scanty in children. Aims: To identify the prevalence of antibiotic resistance in H. pylori strains isolated from Portuguese children in 1999–2003; to evaluate eradication rate after antibiotic susceptibility testing-based treatment; and to identify factors associated with resistance and eradication outcome. Methods: Included were 109 children with a gastric biopsy culture positive for H. pylori. First treatment (amoxicillin, omeprazole and clarithromycin or metronidazole) was guided by susceptibility testing (E test), and eradication was assessed by [13C]urea breath test. Results: Strains were susceptible to amoxicillin and tetracycline; 39.4% were resistant to clarithromycin, 16.5% to metronidazole and 4.5% to ciprofloxacin. No significant association was found between resistance and sex, age, clinical status, gastritis scores, H. pylori density scores and genotype. Clarithromycin resistance was significantly associated with European origin [odds ratio (OR), 3.9], previous H. pylori empiric therapy (OR 2.8) and amoxicillin minimal inhibitory concentration, ≥0.016 (OR 6.0). Eradication rate after susceptibility-based treatment was 74.7% (59 of 79; 95% confidence interval, 65.9–82.9), and a significant association was found between eradication failure and presence of resistance to 1 or more antibiotics (P < 0.05). Conclusions: The prevalence of H. pylori antibiotic resistance was high in the studied population. The modest therapeutic success of clarithromycin and metronidazole susceptibility-based regimens suggests that in addition to resistance, other factors may be involved. The need of susceptibility-based treatment studies in children and of antimicrobial resistance surveillance in high prevalence areas for H. pylori are emphasized.

Magnetic Immuno-PCR Assay with Inhibitor Removal for Direct Detection of Helicobacter pylori in Human Feces

ABSTRACT A PCR protocol was developed to detect Helicobacter pylori in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47 H. pylori-positive and 57 H. pylori-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.

Allelic diversity and phylogeny of homB, a novel co-virulence marker of Helicobacter pylori

BackgroundThe homB gene is a Helicobacter pylori disease-marker candidate, strongly associated with peptic ulcer disease, while homA, its paralogue gene with 90% sequence identity, is correlated with non-ulcer dyspepsia. The HomB encoded outer membrane protein was shown to contribute to the proinflammatory properties of H. pylori and also to be involved in bacterial adherence.This study investigated the distribution of homB and homA genes in 455 H. pylori strains from East Asian and Western countries, and carried out sequence comparison and phylogenetic analyses.ResultsBoth homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains.Analysis of homB and homA sequences revealed diversity regarding the number of copies and their genomic localization, with East Asian and Western strains presenting different genotypes. Moreover, homB and homA sequence analysis suggests regulation by phase variation. It also indicates possible recombination events, leading to gene duplication or homB/homA conversion which may as well be implicated in the regulation of these genes. Phylogenetic reconstruction of homB and homA revealed clustering according to the geographic origin of strains. Allelic diversity in the middle region of the genes was observed for both homB and homA, although there was no correlation between any allele and disease. For each gene, a dominant worldwide allele was detected, suggesting that homB/homA allelic variants were independent of the geographical origin of the strain. Moreover, all alleles were demonstrated to be expressed in vivo.ConclusionOverall, these results suggest that homB and homA genes are good candidates to be part of the pool of H. pylori OMPs implicated in host-bacteria interface and also contributing to the generation of antigenic variability, and thus involved in H. pylori persistence.