Different latent class models were used and evaluated for assessing the accuracy of campylobacter diagnostic tests: overcoming imperfect reference standards?

In the absence of perfect reference standard, classical techniques result in biased diagnostic accuracy and prevalence estimates. By statistically defining the true disease status, latent class models (LCM) constitute a promising alternative. However, LCM is a complex method which relies on parametric assumptions, including usually a conditional independence between tests and might suffer from data sparseness. We carefully applied LCMs to assess new campylobacter infection detection tests for which bacteriological culture is an imperfect reference standard. Five diagnostic tests (culture, polymerase chain reaction and three immunoenzymatic tests) of campylobacter infection were collected in 623 patients from Bordeaux and Lyon Hospitals, France. Their diagnostic accuracy were estimated with standard and extended LCMs with a thorough examination of models goodness-of-fit. The model including a residual dependence specific to the immunoenzymatic tests best complied with LCM assumptions. Asymptotic results of goodness-of-fit statistics were substantially impaired by data sparseness and empirical distributions were preferred. Results confirmed moderate sensitivity of the culture and high performances of immunoenzymatic tests. LCMs can be used to estimate diagnostic tests accuracy in the absence of perfect reference standard. However, their implementation and assessment require specific attention due to data sparseness and limitations of existing software.

Pandemic influenza A(H1N1) 2009: molecular characterisation and duration of viral shedding in intensive care patients in Bordeaux, south-west France, May 2009 to January 2010.

From May 2009 to January 2010, the Virology Laboratory at the University Hospital of Bordeaux received more than 4,000 nasopharyngeal samples from the Aquitaine region (south-west France) for the diagnosis of pandemic influenza A(H1N1)2009. Eighty-three infected patients deteriorated and were admitted to intensive care units. Our study focused on 24 of these patients. Positivity for influenza A(H1N1)2009 was monitored by realtime PCR and duration of viral shedding was determined. The first available sample of each patient was analysed for bacterial, fungal and viral co-infection. We observed six bacterial (or bacterial/fungal) co-infections and one viral co-infection with respiratory syncytial virus. The samples were analysed for the presence of the neuraminidase H275Y (N1 numbering) mutation, which confers resistance to oseltamivir, by realtime PCR of the neuraminidase gene. No H275Y mutation was observed in any of the viral strains screened in this study. In parallel, a fragment of the haemagglutinin gene encoding amino acid residues 173 to 362 was sequenced to detect mutations that had been reported to increase the severity of the disease. Two patients were infected by strains bearing the D222G (H3 numbering) mutation. The viral shedding of A(H1N1)2009 in this study ranged from four to 28 days with a median of 11 days.

Evaluation of the combination of the NucliSENS easyMAG and the EasyQ applications for the detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory tract specimens.

Mycoplasma pneumoniae and Chlamydia pneumoniae are respiratory tract pathogens frequently involved in community-acquired pneumonia, but are fastidious microorganisms. Their direct detection mainly requires molecular amplification techniques. A nucleic acid extraction system, NucliSENS easyMAG, and a real-time nucleic acid sequence-based amplification (NASBA) technique, NucliSENS EasyQ, were recently developed by bioMérieux to detect both bacteria. The aim of our study was to compare the easyMAG/EasyQ combination with our in-house combination, MagNA Pure extraction (Roche) and real-time polymerase chain reaction (PCR), to detect both bacteria in respiratory tract specimens. The analytical specificities of both combinations were similar. A higher analytical sensitivity was found for C. pneumoniae using the easyMAG/EasyQ combination, since the easyMAG/EasyQ system detected nucleic acid extracts 10(6) times more diluted than the in-house combination. Both combinations were equivalent when detecting M. pneumoniae in positive respiratory tract samples. Finally, the easyMAG/EasyQ combination is a potential useful tool for the detection of both bacteria regarding sensitivity, specificity, monitoring, and standardization of the procedure.

Epidemiologic cutoff values to separate wild-type from non-wild-type Campylobacter fetus to ciprofloxacin.

The aim of the present study was to propose epidemiologic cutoffs that could be used in routine practice to separate wild-type from non-wild-type Campylobacter fetus to ciprofloxacin. A total of 123 C. fetus isolates obtained from human samples were used for this purpose. Based on the determination of inhibition zone diameter, minimum inhibitory concentration, and sequencing of the quinolone resistance determining region in the gyraseA gene, for all tested isolates, the following cutoffs were proposed: ciprofloxacin-wild type if the inhibition zone diameter was ≥22 mm or the minimum inhibitory concentration was ≤0.5 mg/L.

Helicobacter pylori resistance to antibiotics in 2014 in France detected by phenotypic and genotypic methods.

A large survey of antimicrobial resistance of Helicobacter pylori was performed in France in 2014: 984 patients were enrolled by 75 gastroenterologists all over the country. Among the 783 patients who had never received eradication treatment before, 266 (33.9%) were H. pylori positive. The strains showed a high rate of clarithromycin resistance (22.2%), moderate rate of resistance to levofloxacin (15.4%) and high rate of resistance to metronidazole (45.9%). In all, 187 patients had received previous treatment, of which 115 were H. pylori positive with very high resistance to clarithromycin (73.9%) and metronidazole (78.3%). None of the patients receiving PYLERA (Bismuth salt-Tetracycline HCl-Metronidazole) proton-pump inhibitor developed resistance to tetracycline. A real-time PCR applied to gastric biopsy specimens detected all the cases that were positive by culture as well as 30 additional cases. A good correlation was found between the clarithromycin resistance detected by phenotypic methods and the associated mutations for clarithromycin resistance, which has continued to increase in the last decade but at a lower rate than previously observed.

Proper use of carbapenems: Role of the infectious disease specialist.

OBJECTIVE: One of the objectives of the French national plan on antibiotics is to preserve antibiotic effectiveness. A group of infectious disease specialists of the University hospital of Bordeaux aimed to monitor the prescriptions of broad-spectrum antibiotics. Particular attention was paid to carbapenem (CBP) prescriptions given the increase in betalactamase- and carbapenemase-producing bacteria. PATIENTS AND METHODS: We carried out a three-step Professional Practice Evaluation (PPE): evaluation of CBP prescriptions made at the hospital between January and June 2013; CBP prescription training for prescribers; and another evaluation of CBP prescriptions between January and June 2014. RESULTS: Although the number of admissions remained stable between the two evaluation periods, CBP prescriptions decreased by 16%. The mean treatment duration was stable (9.6 days). Physicians asked for the infectious disease specialist's advice for 82% of CBP prescriptions in 2013 and for 83% in 2014. The number of case patients discussed at the multidisciplinary staff meetings for approval of CBP prescriptions increased from 16% in 2013 to 39% in 2014. Antibiotic de-escalation increased by 61% between the two periods. CONCLUSION: Professional Practice Evaluation, supervised by an infectious disease specialist, is a useful addition to weekly multidisciplinary staff meetings to improve CBP prescription.

Helicobacter pylori infection and gastric carcinoma.

Helicobacter pylori infection is considered to be the main cause of gastric cancer and the most frequent infection-induced cancer. H. pylori is a heterogeneous species which can harbour pathogenic factors such as a cytotoxin, a pathogenicity island (cag) encoding a type 4 secretion system, and the first bacterial oncoprotein, CagA. This oncoprotein appears to be involved in the carcinogenic process in addition to the inflammation generated. This process may concern either local progenitors via an epithelial-mesenchymal transition, or recruited bone marrow-derived mesenchymal cells. There are also environmental factors such as iron deficiency or high-salt diets which interact with the bacterial factors to increase the risk of gastric cancer as well as genetic polymorphism of certain cytokines, e.g. IL-Iß. Recent data suggest that a break in coevolution of a particular phylogeographic lineage of H. pylori and its usual host may also be a risk factor. Studies are currently being performed to assess the feasibility of organized H. pylori eradication programmes to prevent gastric cancer.

Helicobacter pylori infection and stem cells at the origin of gastric cancer.

Helicobacter pylori infection is now recognized as the main and specific infectious cause of cancer in the world. It is responsible for gastric adenocarcinomas of both intestinal and diffuse types, which are the long-term consequences of the chronic infection of the gastric mucosa. Case-control studies have shown an association between the two, recognized as early as 1994 and further substantiated by interventional studies in which H. pylori eradication has led to the prevention of at least part of the gastric cancers. Experimental studies have highlighted the role of bone marrow-derived cells (BMDCs) and particularly mesenchymal stem cells, in the neoplastic process in about a quarter of the cases and possibly an epithelial-mesenchymal transition (EMT) in the other cases. Different studies have confirmed that chronic infection with H. pylori induces a chronic inflammation and subsequent damage of the gastric epithelial mucosa, leading to BMDC recruitment. Once recruited, these cells home and differentiate by cell-cell fusion with local gastric epithelial cells, bearing local stem cell failure and participating in tissue regeneration. The context of chronic infection and inflammation leads to an EMT and altered tissue regeneration and differentiation from both local epithelial stem cells and BMDC. EMT induces the emergence of CD44+ cells possessing mesenchymal and stem cell properties, resulting in metaplastic and dysplastic lesions to give rise, after additional epigenetic and mutational events, to the emergence of cancer stem cells (CSCs) and adenocarcinoma.

Usefulness of pneumococcal antigen urinary testing in the intensive care unit?

OBJECTIVES: The use of pneumococcal antigen urinary tests is substantially increasing and is associated with a significant cost. The relevant use of this test in the intensive care unit (ICU) should be better defined. Our aim was to define the role of this test in relation to other microbiological tests. We described a series of patients admitted to the ICU for an invasive pneumococcal disease (IPD). PATIENTS AND METHODS: We conducted a retrospective and descriptive study of the microbiological tests used to diagnose IPD in patients admitted to the ICU of the University Hospital in Bordeaux. Our aim was to measure the sensitivity of these bacteriological tests and of the BinaxNOWS. pneumoniae test. RESULTS: Between 2009 and 2013, 148 patients were admitted for an IPD. A lower respiratory tract infection was diagnosed in 96.6% of them (143 patients). The overall ICU case fatality rate was 17.6%. The sensitivity of the pneumococcal antigen urinary test, sputum bacteriological examination, and blood cultures was respectively 83%, 37.6%, and 29.7%. S. pneumoniae was isolated from at least one bacteriological sample in 48.6% of patients, but in 51.4%, the diagnosis was only based on the results of the pneumococcal antigen urinary test. CONCLUSION: We suggest performing a pneumococcal antigen urinary test when an IPD is suspected, only if the bacteriological tests are still negative after 48hours. This strategy would result in a substantial cost saving. Patients would not face any additional risks as the result of the pneumococcal antigen urinary test does not have any impact on the initially prescribed antibiotic therapy.

Helicobacter pylori generates cells with cancer stem cell properties via epithelial-mesenchymal transition-like changes.

Helicobacter pylori infection is the major risk factor for gastric adenocarcinoma. The link with gastric adenocarcinoma is partly due to the H. pylori CagA oncoprotein. CagA is responsible for a particular cell phenotype in vitro, the 'hummingbird' phenotype, that corresponds to an elongation of the cells, mimicking an epithelial-mesenchymal transition (EMT). EMT participates in the carcinogenesis process, and is involved in the generation of cancer stem cells (CSCs). However, its involvement in gastric carcinogenesis has yet not been studied. Therefore, the aim of this study was to determine the role of H. pylori in EMT and in the emergence of gastric CSCs. For this purpose, gastric epithelial cells were cocultured with a cagA-positive H. pylori strain or its isogenic-deleted mutants or were transfected with CagA expression vectors. Study of the expression of epithelial and mesenchymal markers showed that H. pylori, via CagA, is responsible for an EMT phenotype associated with an increase in mesenchymal markers as well as CD44 expression, a known gastric CSC marker. Moreover, infection led to an increased ability to migrate, to invade and to form tumorspheres. Cell sorting experiments showed that only the CD44(high) cells induced by H. pylori infection displayed the mesenchymal phenotype and CSC properties in vitro, and had higher tumorigenic properties than CD44(low) cells in xenografted mice. Immunohistochemistry analyses on human and mouse gastric mucosa tissue samples confirmed a high expression of CD44 and mesenchymal markers in H. pylori-infected cases, and in gastric dysplasia and carcinoma. All of these data suggest that H. pylori, via CagA, unveils CSC-like properties by induction of EMT-like changes in gastric epithelial cells.

Identification of Campylobacter species and related organisms by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

The identification of Campylobacter species and related organisms at the species level has always been difficult using phenotypic methods because of their low metabolic activity, whereas molecular methods are more reliable but time-consuming. In this study, 1007 different strains were identified using three different methods: conventional methods, molecular biology (real-time PCR and sequencing) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Molecular methods were considered the gold standard. The accuracy of MALDI-TOF mass spectrometry reached 100% compared with the gold standard for all of the Campylobacter species, except Campylobacter jejuni (99.4%). The accuracy of conventional methods compared with the gold standard ranged from 0% to 100% depending on the species. However, MALDI-TOF mass spectrometry was not able to identify a mixture of two different species present in the same sample in four instances. Finally, MALDI-TOF mass spectrometry is highly recommended to identify Campylobacter spp. as only 0.4% discrepancy was found, whereas conventional methods led to 4.5% discrepancy.

Matrix-assisted laser-desorption/ionization biotyper: experience in the routine of a University hospital.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) is positioned at the forefront of bacterial identification in the future. Its performance needed to be evaluated in a routine Bacteriology laboratory to determine its true benefits. A prospective study was carried out in the Bacteriology laboratory of the Pellegrin University Hospital in Bordeaux, France, from April to May 2009. Bacterial isolates from clinical samples were identified by conventional phenotypic bacteriological methods [Phoenix (Becton-Dickinson) or API strips (bioMérieux)] and in parallel with a mass spectrometer (Ultraflex III TOF/TOF and the biotyper database from Bruker Daltonics). In case of a discrepancy between these results at the genus level, a 16S rRNA and/or rpoB gene sequencing was performed. Of the 1013 bacteria tested, 837 (82.6%) were correctly identified at the species level by MALDI-TOF mass spectrometry (MS) without extraction and 189 after extraction, i.e. 986 (97.3%) were correctly identified at the species level by MALDI-TOF MS, vs. 945 (93.2%) by phenotypic methods. Indeed, the extraction step was necessary for only 15% of the isolates. These results were even better when considering the genus, reaching almost 99% with MALDI-TOF MS and 98% with phenotypic methods. The performance of MALDI-TOF MS is very attractive considering its efficiency and rapidity, and the technique constitutes a precious tool for bacteriological identification in a routine laboratory.