Christophe Burucoa

High level of antimicrobial resistance in French Helicobacter pylori isolates.

Background: Helicobacter pylori is a human pathogen responsible for serious diseases including peptic ulcer disease and gastric cancer. The recommended triple therapy included clarithromycin but increasing resistance has undermined its effectiveness. It is therefore important to be aware of the local prevalence of antimicrobial resistance to adjust treatment strategy.

Meningitis Due to Capnocytophaga canimorsus after Receipt of a Dog Bite: Case Report and Review of the Literature

We describe a case of meningitis due to Capnocytophaga canimorsus and review 18 cases with attention to risk factors, clinical features, diagnosis, treatment, and outcome. In most of the reported cases, contact with dogs and predisposing factors were found. Clinical manifestations and the findings of examinations of cerebrospinal fluid specimens were similar to those of classic bacterial meningitis; however, the mortality rate for C. canimorsus meningitis very low when compared with the rate for C. canimorsus septicemia (5% vs. 30%).

Implication of the Structure of the Helicobacter pylori cag Pathogenicity Island in Induction of Interleukin-8 Secretion

ABSTRACT Helicobacter pylori virulence is associated with the presence of the cag pathogenicity island (PAI). Thecag PAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa to H. pyloriinfection. The aim of this study was to determine whether the genetic structure of the cag PAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of thecag PAI were performed on 153 strains isolated from adults suffering from ulcers (n = 79) or gastritis (n = 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entire cag PAI, 12 strains (7.8%) had thecag PAI split in two, 49 strains (32%) had nocag PAI, and 9 strains exhibited other structural combinations. The presence of an entire cag PAI was statistically correlated with the presence of IS605(P = 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of the cag PAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of the cag PAI structure suggests that this region is not the only requirement for IL-8 secretion.

Clinical and laboratory characteristics of infective endocarditis when associated with spondylodiscitis.

Abstract.Spondylodiscitis is rarely observed in association with infective endocarditis (IE). In the study presented here, 92 cases of definite IE were examined. Spondylodiscitis was present in 14 (15%) cases. The mean age of patients with spondylodiscitis was 69.1±13.6 years (range, 33–87 years). The male-to-female ratio was 8:6. Predisposing heart disease was found in nine (64.3%) cases. Back pain was reported in all cases. Spondylodiscitis was diagnosed before endocarditis in all cases. The infection affected the lumbar spine in 10 (71%) cases. A bacterium was isolated in all cases: group D Streptococcus (n=5; 35.7%), coagulase-negative Staphylococcus (n=4; 28.6%), and others (n=5). Endocarditis affected predominantly the aortic valve (43%). The outcome was favourable in 12 cases. No differences in clinical features, evolution of disease, or laboratory values were found between IE patients with and IE patients without spondylodiscitis. Spondylodiscitis does not appear to worsen prognosis of IE, although the need for cardiac valve replacement seems to be more frequent in IE patients with spondylodiscitis. IE should be included in the differential diagnosis in patients with infectious spondylodiscitis and risk factors for endocarditis. In such patients, echocardiography should be performed routinely.

Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

ABSTRACT There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.

Comparative evaluation of 29 commercial Helicobacter pylori serological kits

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.

Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

ABSTRACT 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.

From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma

Backgroundelicobacter pylori infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.ResultsA set of 254 H. pylori genes was used to perform array-based comparative genomic hybridization among 120 French H. pylori strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with cag PAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released H. pylori genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest H. pylori genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the vacA s2m2 allele and lacks the genes encoding the major virulence factors (absence of cag PAI, bab B, bab C, sab B, and hom B). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 H. pylori sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.ConclusionThese isolates are deprived of the main H. pylori virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.

Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients

BackgroundDistinct virulence factors of H. pylori have been described: the vaculating cytotoxin (vacA), the cytotoxin associated gene (cagA), the induced by contact with epithelium factor Antigen (iceA gene) and the outer membrane protein oipA. In Tunisia, there are no data regarding the pattern of H. pylori genotypes; therefore, this prospective and multicentre study was the first to be done in Tunisia and aimed to investigate the prevalence of the vacA, cagA, iceA and oipA genotypes of H. pylori isolates from Tunisian patients with peptic ulceration, gastric cancer, MALT lymphoma and gastritis.MethodsH. pylori was cultured from endoscopic biopsies obtained from 281 Tunisian patients. The vacA alleles, cagA, iceA and oipA genotypes were determined by PCR.ResultsThe vacA s1m1, s1m2 and s2m2 were respectively found in 10.7%, 12.5% and 45.6% of strains. The s2m1 genotype was not detected in our study. The cagA was found in 61.6% of isolates. The iceA1 and the iceA2 genotypes were respectively isolated in 60.2% and in 16% of strains. The oipA genotype was detected in 90.8% of strains. Considering the vacA and iceA genotypes, the presence of multiple H. pylori strains in a single biopsy specimen was found respectively in 31.4% and 23.8%. The comparison between strains isolated from antrum and fundus showed that Tunisian patients were infected with two or more strains of different cagA, vacA, iceA and oipA genotypes and the discordance was respectively in 9.6%, 4.6%, 8.9% and 8.5% of strains.ConclusionOur results showed that in 46% (131 strains among 281), the H. pylori strains were highly virulent in relation of the three or four virulent factors they could carry. These finding were described before in the literature. Tunisian patients were colonized by one or multiple strains of H. pylori in the same time in relation of presence of vacA m1/m2 and iceA1/iceA2 in the same biopsy. The discordance between strains isolated from antrum and fundus was high, and it is in favour of multicolonization.

The rtxA toxin gene of Kingella kingae: a pertinent target for molecular diagnosis of osteoarticular infections

ABSTRACT Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection.