In vitro activity of delafloxacin against clinical levofloxacin-resistant Helicobacter pylori isolates.

BACKGROUND: Increasing antibiotic resistance in Helicobacter pylori necessitates research on new active molecules. In 2017, delafloxacin, a new fluoroquinolone with chemical properties of activity under acidic conditions, was approved for treatment of community-acquired bacterial pneumonia and acute bacterial skin and soft-tissue infections. Mutations in gyrA are responsible for fluoroquinolone resistance, but certain clinical isolates of H. pylori appear to display a dual phenotype: resistance to levofloxacin associated with very low delafloxacin MICs. OBJECTIVES: To estimate epidemiological cut-off (ECOFF) values and to identify mutations in the gyrA gene, specific to FQ resistance, without increasing the MICs of delafloxacin. METHODS: Clinical strains (n = 231) were collected in the bacteriology laboratory of Poitiers University Hospital over a 2 year period to determine the ECOFF of delafloxacin. Retrospectively, 101 clinical strains with an levofloxacin-resistant phenotype (MIC > 1 mg/L) were selected from 2018 to 2022 for delafloxacin MIC determination and QRDR (gyrA) sequencing. RESULTS: The estimated ECOFF of delafloxacin was ≤0.125 mg/L. No H. pylori isolate showed a levofloxacin-sensitive phenotype with a delafloxacin MIC of >0.125 mg/L. Among the levofloxacin-resistant H. pylori isolates, 53.5% had delafloxacin MICs of ≤0.125 mg/L. The N87I mutation was associated with dual levofloxacin/delafloxacin resistance (P < 0.001) in contrast to the N87K and D91N mutations (P > 0.05). Mutations D91G and D91Y were not associated with a delafloxacin resistance phenotype (P > 0.05). CONCLUSIONS: Delafloxacin seems to be a therapeutic alternative for levofloxacin-resistant strains with greater in vitro activity. However, further clinical/biological investigations are required to determine its efficacy in H. pylori eradication.

In vitro efficacy of combinations of antibiotics used in clinical practice on clinical isolates of Helicobacter pylori.

BACKGROUND: The main antibiotics used against Helicobacter pylori have been chosen empirically over time, with few preclinical studies to provide support. The rise in resistance to some of these antibiotics is prompting a reassessment of their use. This work aimed to evaluate the in vitro efficacy of 2 × 2 combinations of the most widely used antibiotics against H. pylori. MATERIALS AND METHODS: J99 reference strains and 19 clinical isolates of H. pylori with various antibiotic resistance phenotypes were used. Minimum inhibitory concentrations were carried out using the microdilution method in 96-well plates. The activity of 15 possible combinations of two antibiotics including amoxicillin, clarithromycin (CLA), levofloxacin, rifampicin, tetracycline, and metronidazole was determined for all strains by the checkerboard method. A mean fractional inhibitory concentration index (FICmean) was calculated for each combination and strain and the type of pharmacodynamic interaction was considered as synergic if FICmean ≤ 0.5, additive if 0.5 < FICmean ≤ 1, indifferent if 1 < FICmean < 4 or antagonistic if FICmean ≥ 4. RESULTS: Most of the 285 pharmacodynamic interactions tested with clinical strains were close to additivity (average FICmean = 0.89 [0.38-1.28]). No interaction was found to be antagonistic. When two antibiotics to which a strain was resistant were combined, the concentrations required to inhibit bacterial growth were higher than their respective breakpoints. CONCLUSION: The present results have shown that in vitro, the different antibiotics used in therapeutics have additive effects. The addition of the effects of two antibiotics to which a strain was resistant was not sufficient to inhibit bacterial growth. In probabilistic treatment, the choice of antibiotics to combine should therefore be based on the local epidemiology of resistance, and on susceptibility testing in the case of CLA therapy, so that at least one antibiotic to which the strain is susceptible is used.

Bacteremia due to Lachnoanaerobaculum umeaense in a patient with acute myeloid leukemia during chemotherapy: A case report, and a review of the literature.

The present case reports a bacteremia due to Lachnoanaerobaculum umeaense (a Gram-positive, filamentous, rod-shaped, anaerobic, spore-forming bacillus present in the human oral microbiota) in a patient treated for acute myeloid leukemia. After failed identification by MALDI-TOF, identification was done by sequencing of 16s rRNA. The patient was successfully treated with Amoxicillin-clavulanic acid and ciprofloxacin for seven days. Comparison of V1-V3 regions of the bacterial 16S rRNA gene gene with published sequences failed to classify the strain as pathogenic or non-pathogenic based on this phylogenetic classification alone. Although Lachnoanaerobaculum gingivalis are known to be associated with bacteremia in patients with acute myeloid leukemia, this clinical case of infection by L. umeaense argues for further studies that will lead to more efficient classification of the infection by these microorganisms.

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study.

BACKGROUND: Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. METHODS: Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March-April 2020). RESULTS: 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs. 5.3%; p < 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs. 6.1%; p <0.05), and after stratification for K. pneumoniae (39.6% vs. 0%; p <0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs. 13.9%; p < 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs. 16.7%; p <0.01) but in COVID- showed more carbapenem resistance (11.1% vs. 0.8%; p <0.05) and greater resistance to at least two aminoglycosides (11.8% vs. 1.4%; p < 0.05) and to quinolones (53.6% vs. 7.0%; p <0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs. 13.8%; p < 0.01). CONCLUSIONS: The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients.

Ulceroglandular Infection and Bacteremia Caused by Francisella salimarina in Immunocompromised Patient, France.

Although Francisella tularensis is a well-known, highly virulent bacterium that causes tularemia in humans, other Francisella species have been associated with sporadic human infections. We describe a human cutaneous infection with bacteremia caused by F. salimarina, a Francisella species recently identified from seawater and fishes, in an immunocompromised patient in France.

Diagnostic Accuracy of a Noninvasive Test for Detection of Helicobacter pylori and Resistance to Clarithromycin in Stool by the Amplidiag H. pylori+ClariR Real-Time PCR Assay.

The noninvasive detection of Helicobacter pylori and its resistance to clarithromycin could revolutionize the management of H. pylori-infected patients by tailoring eradication treatment without any need for endoscopy when histology is not necessary. Several real-time PCR tests performed on stools have been proposed, but their performances were either poor or they were tested on too few patients to be properly evaluated. We conducted a prospective, multicenter study including 1,200 adult patients who were addressed for gastroduodenal endoscopy with gastric biopsies and who were naive for eradication treatment in order to evaluate the performance of the Amplidiag H. pylori+ClariR assay recently developed by Mobidiag (Espoo, Finland). The results of the Amplidiag H. pylori+ClariR assay performed on DNA from stools (automatic extraction with the EasyMag system [bioMérieux]) were compared with those of culture/Etest and quadruplex real-time PCRs performed on two gastric biopsy samples (from the antrum and corpus) to detect the H. pyloriglmM gene and mutations in the 23S rRNA genes conferring clarithromycin resistance. The sensitivity and specificity of the detection of H. pylori were 96.3% (95% confidence interval [CI], 92 to 98%) and 98.7% (95% CI, 97 to 99%), respectively. The positive and negative predictive values were evaluated to be 92.2% (95% CI, 92 to 98%) and 99.3% (95% CI, 98 to 99%), respectively. In this cohort, 160 patients (14.7%) were found to be infected (positive by culture and/or PCR). The sensitivity and specificity for detecting resistance to clarithromycin were 100% (95% CI, 88 to 100%) and 98.4% (95% CI, 94 to 99%), respectively.

First description of bacteremia caused by Oscillibacter valericigenes in a patient hospitalized for leg amputation.

Initially isolated from the alimentary canal of a Japanese corbicula clam, Oscillibacter valericigenes is a Gram-negative rod, of which culture remains very difficult. Herein we present the first case of bacteremia due to Oscillibacter valericigenes, in humans. A 55-year-old man was hospitalized for clinical management of multiple neglected leg wounds (colonized with maggots) that had occurred during a motorcycle accident. Following radiological confirmation of the bone infection, a transfemoral amputation was performed to limit the risk of extended infection. During hospitalization, before the amputation, the patient experienced fever, biological inflammation justifying the sampling of multiple blood cultures. Anaerobic blood culture was positive after 34 hours, without identification by routine procedure (MALDI-TOF), justifying identification by 16S DNA sequencing. In the absence of possible subculture, antibiotic sensitivity testing could not be performed. A pre-emptive treatment by piperacillin-tazobactam was introduced for 14 days. The evolution was good, except for a local disunion. Complete phylogenic analysis of the clinical strain showed that it significantly differed from the reference strain, which is distantly related to the Clostridia cluster IV. Due to the culture conditions and specialized identification method by sequencing, prevalence of O. valericigenes may be underestimated. Optimization of blood culture procedures and utilization of 16S rRNA gene sequencing are tools needed for identification of rare pathogens that could help to optimize clinical management of infected patients.

Probabilistic chemotherapy in knee and hip replacement infection: the place of linezolid.

Prosthetic joint infection (PJI) can occur with a wide range of microorganisms and clinical features. After replacement surgery of prosthetic joint, prescription of probabilistic broad-spectrum antimicrobial therapy is usual, while awaiting microbial culture results. The aim of our study was to describe the antibiotic susceptibility of microorganisms isolated from hip and knee PJI. The data were collected to determine the best alternative to the usual combination of piperacillin-tazobactam (TZP) or cefotaxime (CTX) and vancomycin (VAN). Based on a French prospective, multicenter study, we analyzed microbiological susceptibility to antibiotics of 183 strains isolated from patients with confirmed hip or knee PJI. In vitro susceptibility was evaluated: TZP+VAN, TZP+linezolid (LZD), CTX+VAN, and CTX+LZD. We also analyzed resistance to different antibiotics commonly used as oral alternatives. Among the 183 patients with PJI, 62 (34%) had a total knee prosthesis, and 121 (66%) a hip prosthesis. The main identified bacteria were Staphylococcus aureus (32.2% of isolates), coagulase-negative staphylococci (27.3%), Enterobacteriaceae (14.2%), and Streptococcus (13.7%). Infections were polymicrobial for 28 (15.3%) patients. All combinations were highly effective: CTX+VAN, CTX+LZD, TZP+VAN, and TZP+LZD (93.4%, 94%, 98.4%, and 98.9% of all cases respectively). Use of LZD instead of VAN in combination with a broad-spectrum beta-lactam covers almost all of the bacteria isolated in PJI. This association should be considered in probabilistic chemotherapy, as it is particularly easy to use (oral administration and no vancomycin monitoring).

Molecular diagnosis of Helicobacter pylori infection in gastric biopsies: Evaluation of the Amplidiag® H. pylori + ClariR assay.

BACKGROUND: Adapted treatments for Helicobacter pylori infection, guided by determining antimicrobial resistance, are associated with high eradication rates. We evaluated the performance of the Amplidiag® H. pylori + ClariR PCR assay (Amplidiag® ) for detecting H. pylori and its clarithromycin resistance from gastric biopsies taken during endoscopy in comparison to culture and our "in-house" PCR. MATERIALS AND METHODS: A total of 127 gastric biopsies were analyzed (98 adults; 29 children). Culture, PCR Amplidiag® , and in-house PCR were performed in parallel. The in-house PCR combined amplification and sequencing of a 267-bp fragment of the H. pylori 23S rRNA gene. Discrepancies were controlled by amplification of glmM gene. RESULTS: For detection of H. pylori, Amplidiag® and the in-house PCR were concordant in 118 of 127 of cases: 66 negative and 52 positive. Discrepancies were observed in nine cases, all with low bacterial load: Amplidiag® did not detect seven biopsies positive on in-house PCR but detected two positive biopsies that were negative on in-house PCR. Among the 19 of 52 (36%) H. pylori cases resistant to clarithromycin, only four biopsies with mixed populations exhibited discordant results between the two PCR methods. The A2142T mutation was not detected by Amplidiag® . With the in-house PCR and amplified glmM gene as the reference method, the sensitivity and specificity of Amplidiag® was 88.5% (95% confidence interval 83-94.1) and 100%. CONCLUSION: This study demonstrated the high sensitivity of the PCR-based Amplidiag® H. pylori test, especially with low H. pylori load, and the probability of its clarithromycin resistance analysis. For clinical use, a well-designed trial with a large scale of samples may still be needed.

Performances of the IDEIA HpStAR Stool Antigen Test in Detection of Helicobacter pylori Infection Before and After Eradication Treatment in Algerian Children.

We aimed to evaluate in an Algerian pediatric population the diagnostic performances of the IDEIA HpStAR noninvasive stool antigen test (Oxoid, Cambridge, UK) to detect Helicobacter pylori infection before and after eradication therapy. A prospective study including 158 symptomatic Algerian children was conducted. Patients were initially diagnosed with invasive (culture, histology, and rapid urease test) and noninvasive tests (urea breath test and IDEIA HpStAR test). Infected patients were treated, and 101 were controlled after treatment with two invasive (culture and histology) and two noninvasive tests (urea breath test and IDEIA HpStAR test). In Algerian children, the IDEIA HpStAR test showed good performances for initial detection of H. pylori infection and also for subsequent control of eradication treatment. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of IDEIA HpStAR test before treatment were 93.6%, 100%, 100%, 87.3%, and 96%, respectively, and those after treatment were 100, 92.8, 78.6, 100, and 94.2%, respectively.

Histopathological Diagnosis of Prosthetic Joint Infection: Does a Threshold of 23 Neutrophils Do Better than Classification of the Periprosthetic Membrane in a Prospective Multicenter Study?

No gold standard exists for histopathological diagnosis of a prosthetic joint infection (PJI). The historical criterion considers the presence of neutrophil infiltration upon examination of periprosthetic tissue. Morawietz et al. proposed a classification of periprosthetic membranes (Morawietz et al., Clin Pathol 59:591-597, 2006, https://doi.org/10.1136/jcp.2005.027458) and a more recently described classification with a new cutoff value of 23 neutrophils in 10 high-power fields (Morawietz et al., Histopathology 54:847-853, 2009. https://doi.org/10.1111/j.1365-2559.2009.03313.x). We performed a multicenter prospective study, which compared both methods for the diagnosis of PJI. All suspicions of PJI (n = 264) between December 2010 and March 2012 in seven centers were prospectively included. Five perioperative specimens were collected per patient for cultures, and one was collected for histology. Diagnosis of PJI was made according to the Infectious Diseases Society of America (IDSA) guidelines. Histopathological analysis classified the patients according to the threshold of 23 neutrophils and according to the classification of Morawietz. Performances of both methods were compared by using clinical and/or bacteriological criteria as the gold standard. Among 264 patients with suspected PJI, a diagnosis of infection was confirmed in 215 and unconfirmed in 49 patients. Histopathological analysis was available for 150 confirmed PJI and 40 unconfirmed PJI cases. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.7%, 90.0%, 96.7%, 52.9%, and 81.1%, respectively, for the Morawietz classification, and 82.0%, 90.0%, 96.9%, 57.1%, and 83.7%, respectively, for the 23-neutrophil threshold. The new algorithm using a threshold of 23 neutrophils can be proposed as a new gold standard for the histopathological diagnosis of PJI.

Molecular characterization of Helicobacter pylori resistance to rifamycins.

INTRODUCTION: Antibiotic resistance is a major contributing factor in treatment failure of Helicobacter pylori eradication. Rifabutin (RB) is a rescue treatment and rifampicin (RP) is used to screen RB resistance in vitro. The aim of this study was to evaluate the rate of rifamycins resistance and to determine the mutations in the rpoB gene conferring resistance to discuss the current break point. METHODS: Antimicrobial susceptibility to RP was first determined by E-test for 1015 H. pylori isolates. RP and RB MICs were then determined by agar dilution method for strains with MIC of RP > 1 mg/L, and the rpoB gene was sequenced. RESULTS: Overall, 54 of 1015 strains exhibited a RP MIC > 1 mg/L by agar dilution method. Among these 54 strains, 10 had MICs of RP > 4 mg/L and RB ≥ 1 mg/L. They all carried at least one mutation in the rpoB gene at codons 530, 538, 540, 525 in the RP resistance-determining region (RRDR). Implication of the mutation L547F was confirmed by site-directed mutagenesis experiment. In contrast, among the 44 H. pylori isolates with a MIC of RP comprised between 2 and 4 mg/L, only 4 of 44 (9%) strains exhibited a mutation in rpoB, but outside RRDR (codons 470, 499, 636, or 657). For 31 of 44 tested strains, the RB MICs were ≤0.064 mg/L. CONCLUSION: These results suggest that H. pylori isolates should be classified as resistant to RP for MICs > 4 mg/L. We considered that the optimal cut off for RB was ≥0.125 mg/L. We report a new mutation responsible for rifamycins, resistance, L547F.

Th-17 response and antimicrobial peptide expression are uniformly expressed in gastric mucosa of Helicobacter pylori-infected patients independently of their clinical outcomes.

BACKGROUND: The pathological determinism of H. pylori infection is explained by complex interplay between bacterial virulence and host inflammatory response. In a large prospective multicenter clinical study, Th17 response, expression of antimicrobial peptides (AMPs), cagA and vacA status, and bacterial density were investigated in the gastric mucosa of H. pylori -infected patients. MATERIALS AND METHODS: Gastric inflammatory response was analyzed by RT-qPCR for quantification of Th17 cytokines (IL-17A, IL-22), CXCL-8, and AMPs (BD2 and S100A9) mRNA levels in gastric biopsies. Detection and genotyping of H. pylori strains were achieved by bacterial culture and PCR. RESULTS: Among 787 patients screened for H. pylori, 269 were analyzed (147 H. pylori -infected and 122 uninfected patients). In H. pylori -infected patients, distribution was 83 gastritis, 12 duodenal ulcers, 5 gastric ulcers, and 47 precancerous and cancerous lesions. CXCL-8, IL-17A, BD2, and S100A9 mRNA levels were significantly increased in H. pylori -infected patients but, surprisingly, IL-22 was not, and no difference was shown between H. pylori -related diseases. A positive correlation was identified between S100A9 expression and bacterial density. Although expression of the virulence genes cagA and vacA did not impact inflammatory response, patients infected with a cagA-positive strain were associated with severe H. pylori -related diseases. CONCLUSION: This study showed that CXCL-8, IL-17A, and AMPs are not differently expressed according to the various H. pylori -related diseases. The clinical outcome determinism of H. pylori infection is most likely not driven by gastric inflammation but rather tends to mainly influenced by bacterial virulence factors.

Erythromycin encapsulation in nanoemulsion-based delivery systems for treatment of Helicobacter pylori infection: Protection and synergy.

Poorly water-soluble and unstable compounds are difficult to develop as drug products using conventional formulation techniques. The aim of the present study was to develop and evaluate a nanoformulation prepared by a hot high-pressure homogenization method, which was a scalable and solvent-free process. We successfully prepared stable nanodispersions to protect a labile antibiotic, erythromycin. The mean diameter of the dispersed droplets was approximately 150 nm, and size distribution was unimodal. Dispersion was physically stable at room temperature for over six months. Using erythromycin as a model compound, we studied its antimicrobial activity in vitro on Helicobacter pylori. Results showed that drug encapsulation improves API stability in an acidic environment and is conducive to a synergistic effect between the drug and the formulation.

Epidemiology of Helicobacter pylori infection.

The study of Helicobacter pylori genetic variability brought us interesting data on the history of mankind. Based on multilocus sequence typing and more recently on whole-genome sequencing, paleomicrobiology still attracts the attention of global researchers in relation to its ancestor roots and coexistence with humans. Three studies determining the prevalence of virulence factors illustrates the controversial results obtained since 30 years by studies trying to associate prevalence of different virulence markers and clinical outcomes of H. pylori infection. Three articles analyzed the prevalence and risk of multiple (genetically distinct isolates) and mixed (susceptible and resistant isolates) infections. A number of studies confirm that H. pylori prevalence is falling worldwide especially in the developed world and in children but that the level of infection is higher in certain ethnic minorities and in Migrants. There is little new in identifying the mode of H. pylori transmission though intrafamilial spread appears to be important. There have, however, been some interesting papers on the presence of the organism in food, water, and the oral cavity.

In vitro culture and phenotypic and molecular characterization of gastric stem cells from human stomach.

BACKGROUND: Human gastric mucosa shows continuous self-renewal via differentiation from stem cells that remain poorly characterized. METHODS: We describe an original protocol for culture of gastric stem/progenitor cells from adult human stomach. The molecular characteristics of cells were studied using TaqMan low-density array and qRT-PCR analyses using the well-characterized H1 and H9 embryonic stem cells as reference. Epithelial progenitor cells were challenged with H. pylori to characterize their inflammatory response. RESULTS: Resident gastric stem cells expressed specific molecular markers of embryonic stem cells (SOX2, NANOG, and OCT4), as well as others specific to adult stem cells, particularly LGR5 and CD44. We show that gastric stem cells spontaneously differentiate into epithelial progenitor cells that can be challenged with H. pylori. The epithelial progenitor response to H. pylori showed a cag pathogenicity island-dependent induction of matrix metalloproteinases 1 and 3, chemokine (CXCL1, CXCL5, CXCL8, CCL20) and interleukine 33 expression. CONCLUSION: This study opens new outlooks for investigation of gastric stem cell biology and pathobiology as well as host-H. pylori interactions.

How Many Samples and How Many Culture Media To Diagnose a Prosthetic Joint Infection: a Clinical and Microbiological Prospective Multicenter Study.

Although numerous perioperative samples and culture media are required to diagnose prosthetic joint infection (PJI), their exact number and types have not yet been definitely determined with a high level of proof. We conducted a prospective multicenter study to determine the minimal number of samples and culture media required for accurate diagnosis of PJI. Over a 2-year period, consecutive patients with clinical signs suggesting PJI were included, with five perioperative samples per patient. The bacteriological and PJI diagnosis criteria were assessed using a random selection of two, three, or four samples and compared with those obtained using the recommended five samples (references guidelines). The results obtained with two or three culture media were then compared with those obtained with five culture media for both criteria. The times-to-positivity of the different culture media were calculated. PJI was confirmed in 215/264 suspected cases, with a bacteriological criterion in 192 (89%). The PJI was monomicrobial (85%) or polymicrobial (15%). Percentages of agreement of 98.1% and 99.7%, respectively, for the bacteriological criterion and confirmed PJI diagnosis were obtained when four perioperative samples were considered. The highest percentages of agreement were obtained with the association of three culture media, a blood culture bottle, a chocolate agar plate, and Schaedler broth, incubated for 5, 7, and 14 days, respectively. This new procedure leads to significant cost saving. Our prospective multicenter study showed that four samples seeded on three culture media are sufficient for diagnosing PJI.

First experience of a multicenter external quality assessment of molecular 16S rRNA gene detection in bone and joint infections.

The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI.

Chemokines and antimicrobial peptides have a cag-dependent early response to Helicobacter pylori infection in primary human gastric epithelial cells.

Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.

Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

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.