Comparative evaluation of 29 commercial Helicobacter pylori serological kits.

BACKGROUND: Serology is a noninvasive diagnostic method for the detection of Helicobacter pylori infection. Many commercial kits are now on the market. It is necessary to assess their performances to help the user to choose the most appropriate. MATERIAL AND METHODS: The performances of 29 commercial serological tests detecting antibodies to Helicobacter pylori (17 enzyme-linked immunosorbent assay and 12 near-patient tests) were evaluated using sera from 108 patients prospectively selected from gastroenterology departments of five French hospital centers. These patients were infected (45) or uninfected (47) by H. pylori, or had doubtful results (16), according to the gold standard (culture or histology plus rapid urease test or urea breath test). The tests were evaluated by determining the usual parameters of performance: sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. Two analyzes were performed including or not the 16 patients with doubtful infection as uninfected or not analyzed. RESULTS: Depending on the type of analysis, four or two of the 17 enzyme-linked immunosorbent assay tests presented excellent results with the five performance parameters >90%. Calculation of the Youden index allowed to show significantly better performances for one of the 4. Performances of the 12 near-patient tests were lower with accuracies <90% for all except one test. CONCLUSION: These data should help the users to choose the kit the most appropriate to their goals.

Detection of Campylobacter jejuni by culture and real-time PCR in a French cohort of patients with Guillain-Barre syndrome.

Bacteriological culture and real-time PCR (RT-PCR) were used to detect Campylobacter jejuni in fecal samples from a French cohort of 237 patients with Guillain-Barré syndrome (GBS). We provide evidence that diverse serotypes and genotypes of C. jejuni are a major trigger of GBS in France.

Proteomic Helicobacter pylori biomarkers discriminating between duodenal ulcer and gastric cancer.

Protein patterns of 129 Helicobacter pylori strains isolated from Korean and Colombian patients suffering from duodenal ulcer or gastric cancer were analyzed by the high-throughput methodology SELDI-TOF-MS. Eighteen statistically significant candidate biomarkers discriminating between the two clinical outcomes were selected by using the Mann-Whitney test. Three biomarker proteins were purified and identified as a neutrophil-activating protein NapA (HU HPAG1_0821), a RNA-binding protein (HPAG1_0813), and a DNA-binding histone-like protein HU, respectively (jhp0228). These novel biomarkers can be used for development of diagnostic assays predicting the evolution to gastric cancer in H. pylori-infected patients.

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer.

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori-infected patients, the extremely small group at risk for developing low-grade gastric MALT lymphoma (LG-MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori-associated diseases, duodenal ulcer (DU, n=29) and LG-MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high-throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC-MS/MS. Univariate analysis (Mann-Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG-MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG-MALT strains and seven - in DU strains. Two biomarker proteins, one overexpressed in LG-MALT strains (13.2 kDa) and another one - overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC-MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori-associated clinical outcomes.

Quadruplex real-time PCR assay using allele-specific scorpion primers for detection of mutations conferring clarithromycin resistance to Helicobacter pylori.

We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori--the wild-type sequence and the three mutations conferring clarithromycin resistance--using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.

Antimicrobial resistance data on 16,756 Streptococcus pneumoniae isolates in 1999: A Pan-Regional Multicenter Surveillance Study in France.

The rising prevalence of antibiotic-resistant Streptococcus pneumoniae is a phenomenon observed to different degrees around the world. The present national surveillance study report analyzes a total of 16,756 strains of S. pneumoniae collected across France in 1999. The overall prevalence of S. pneumoniae with decreased susceptibility to penicillin was 44%, to amoxicillin 26%, and to cefotaxime 17%. The proportion of high-level resistant strains to penicillin (MIC > 1 mg/L), amoxicillin and cefotaxime (MIC > 2 mg/L) remained low: 12.3%, 1.8%, and 0.4% respectively. Prevalence of resistance to other antibiotics was high: 53% to erythromycin, 41.7% to cotrimoxazole, 31.8% to tetracycline, and 24.6% to chloramphenicol. Prevalence of penicillin-resistant S. pneumoniae varied according to subject age and specimen source. It was higher in children (52.7%) than in adults (39.8%) and higher in strains isolated from middle ear fluid (63.6%) than from blood cultures (41.8%) in children. S. pneumoniae resistant to other antibiotics were more common in children than in adults, although figures showed geographical variations. Comparison with a previous study realized in 1997 in the same regions confirms a rising trend in the prevalence of resistant bacteria. Therefore, we conclude that prevalence of antibiotic-resistant S. pneumoniae in 1999 continued to rise in France, although strains with high-level resistance to penicillin remained stable.

Polyphosphate kinase: a new colonization factor of Helicobacter pylori.

In order to elucidate the role of polyphosphate kinase (PPK) during the course of an infection by Helicobacter pylori, PPK deficient mutants were constructed using two genetic backgrounds: Hp141v and X47-2AL. The efficiencies of the parental strains and the derivative mutants at colonizing the gastric mucosa of mice were compared. When animals received the Hp141v and the X47-2AL parental strains, 100% of the mice remained colonized for the duration of the 45 days experiment. In contrast, none of the mice that were given the PPK deficient X47-2AL derivative strain had a detectable bacterial load in their gastric mucosa, while the deficient Hp141v derivative strain was detected in 100%, 20% and 40% of the mice at days 3, 15 and 45 post-inoculation (p.i.), respectively. The absence of PPK expression did not impair the in vitro growth of the ppk mutants. However, the reduced ability of the ppk defective mutants to colonize mice was associated with a significant decrease in both motility and in an accumulation of polyP in the bacterial cells. These results are consistent with an essential role of PPK during the initial steps of colonisation of the mouse gastric mucosa and confirm that PPK may act on the virulence of H. pylori partly through an energy dependent mechanism.

Colonization of lower respiratory tract with anaerobic bacteria in mechanically ventilated patients.

OBJECTIVE: To study lower respiratory tract colonization by anaerobic bacteria in ICU patients on prolonged mechanical ventilation using two types of protected tracheal sampling methods. DESIGN AND SETTING: Prospective clinical investigation in the intensive care unit of a university hospital. PATIENTS: Twenty-six consecutive patients mechanically ventilated within 24 h after their admission in ICU and with expected duration of mechanical ventilation longer than 7 days. MEASUREMENTS AND RESULTS: Two types of protected tracheal sampling methods were obtained without the use of bronchoscopic guidance on the day following intubation and twice a week until extubation: protected tracheal aspiration and protected tracheal specimen brush. Specific methods for anaerobic isolation were used. Early colonization was defined if colonization occurred within the first 5 days after intubation. Of the 26 patients studied 22 were colonized by at least one bacterial strain. Twenty-one patients were colonized by aerobic and 15 by anaerobic bacteria. Twenty-eight anaerobic strains were identified, with bacterial counts higher than 10(3) cfu/ml in 11 cases. Of the 15 patients colonized by anaerobes 14 were also colonized by aerobic bacteria. The use of protected specimens ruled out oropharyngeal contamination. Early onset colonization occurred in 16 of 22 patients colonized by aerobes and in 8 of 15 patients colonized by anaerobes. Five patients developed ventilatory-acquired pneumonia following colonization (by anaerobic bacteria in two cases). In eight patients colonization by anaerobic bacteria occurred despite antimicrobial therapy. CONCLUSIONS: These results show that anaerobic bacteria frequently colonize the lower respiratory tract of mechanically ventilated patients and underline the potential importance of the anaerobic bacteria in ventilatory acquired pneumonia.

Modification in the ppk gene of Helicobacter pylori during single and multiple experimental murine infections.

The bacterial pathogen Helicobacter pylori is highly adapted to the human stomach, and a high level of polymorphism is observed among clinical isolates. This polymorphism may be the consequence of adaptive changes during colonization, making a strain better able to survive, to evade the immune system, and to provoke a chronic infection. To investigate the mechanisms involved in the acquisition of diversity in H. pylori, mouse models of single infections, coinfections, and superinfections were developed. These experimental infections were conducted with strain SS1, well known to be mouse adapted, and with two strains freshly isolated from infected patients: Hp141 and Hp145. Genetic modifications occurring in these strains were studied over time by comparing randomly selected colonies of the emerging strains to those of the infecting strains by using randomly amplified polymorphic DNA fingerprinting with six different primers and by using PCR to amplify the vacA and cagA genes. We showed that, regardless of the number of infecting strains, only one emerged from the animals and that the establishment of a first strain thwarted the implantation of a second strain. During both a single infection and a coinfection with SS1, Hp141 was replaced by a genetic variant (Hp141v) that overcame SS1 in coinfection experiments. Hp141v exhibited a deletion of a 102-bp repeated sequence within the ppk gene, which encodes polyphosphate kinase (PPK), an enzyme involved in the physiological adaptation of the microbial cell to nutritional and environmental stresses. The deletion led to higher enzymatic activity of PPK, and the variant exhibited a better capacity to colonize mice. Considering that the modified gene is known to be involved in adaptation to a new environment, our results are consistent with an adaptive change in strain Hp141 and suggest that PPK is an important virulence factor in H. pylori.

Experimental colonization of mice by fresh clinical isolates of Helicobacter pylori is not influenced by the cagA status and the vacA genotype.

Developing murine models of infection by Helicobacter pylori is quite useful but not all the strains are able to colonize the mouse. In order to study the influence of the two main virulence factors, CagA and VacA, on the establishment of H. pylori in mice, we have inoculated C57BL/6 mice with 15 strains randomly chosen among clinical strains freshly isolated from biopsy specimens of infected patients and five reference strains. Only six of the clinical strains and two of the reference strains could infect the animals regardless of the cagA status and the vacA genotype. We concluded that 40% of the H. pylori strains are able to infect mice and that the capacity of colonization is not influenced by the cagA status and the vacA genotype. These factors cannot be used to predict the success of an experimental infection.

Novel antigens of Helicobacter pylori correspond to ulcer-related antibody pattern of sera from infected patients.

Recently, we reported that the patterns of antibodies to Helicobacter pylori protein antigens in serum may be useful for screening patients at high risk for ulcers (P. Aucher et al., J. Clin. Microbiol. 36:931-936, 1998). Here we report the identification, by a combination of electrophoretic, immunochemical, and protein sequencing methods, of five antigens that correspond to this antibody pattern: groEL, catalase A, flagellin A, beta-ketoacyl-acyl carrier protein synthase I (beta-ketoacyl-ACP S), and peptidyl prolyl cis-trans isomerase (PPiase). Beta-Ketoacyl-ACP S and PPiase are reported for the first time as antigens of diagnostic interest in infections by H. pylori. The antigenicity of the five antigens, together with those of CagA and VacA, was tested in an immunoblot assay with water-soluble protein extracts from two H. pylori pathogenic strains (HP 141 and ATCC 43579) and panels of sera from H. pylori-positive patients with gastroduodenal ulcers (GDU), nonulcer dyspepsia (NUD), as well as sera from H. pylori-negative healthy volunteers. For catalase A, groEL, and flagellin A antigens, no overall statistically important values were found making it possible to discriminate between patients with GDU and NUD. For both H. pylori strains, the mean performance indices (MPI) presenting percentages of correctly classified patients with GDU and NUD showed that the most significant antibody patterns were as follows: anti-VacA + anti-beta-ketoacyl-ACP S (MPI = 76.1), anti-VacA + anti-PPiase (MPI = 71.8), and anti-CagA + anti-VacA + anti-beta-ketoacyl-ACP S (MPI = 70.5). Antibody patterns detected with these antigen profiles may therefore be useful in developing a diagnostic test designed to predict the clinical severity of the H. pylori infection within the adult population of France.