Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

Quantitative genomic and antigenomic enterovirus RNA detection in explanted heart tissue samples from patients with end-stage idiopathic dilated cardiomyopathy.

Standardized one-step real-time RT-PCR assay detected enterovirus RNA in cardiac biopsy samples from 4 of 20 patients suffering from idiopathic dilated cardiomyopathy (IDCM). The median viral load was 287 copies per microgram of total extracted nucleic acids, with positive- to negative-strand RNA ratios ranging from 2 to 20. These results demonstrate enterovirus persistence in the heart of IDCM patients, characterized by low viral loads and low positive- to negative-RNA ratios.

Broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex RT-PCR DNA microarray system.

Newly available molecular tools allow a sensitive detection of a broad panel of viruses in respiratory tract specimens. In the present study, the application of a multiplex RT-PCR DNA microarray in diagnosis and epidemiological survey of viral infections in infants hospitalized for bronchiolitis was assessed. One hundred and thirty-eight nasopharyngeal aspirates collected from October 2007 to September 2008 were tested by direct immunofluorescence and viral culture, a combination of referenced RT-PCRs and the DNA microarray. One or more viruses were detected in 96, 126 and 126 of the specimens by direct immunofluorescence and viral culture, RT-PCRs and DNA microarray, respectively (70 vs. 91 vs. 91%, P < 10(-3)). The RT-PCRs and the DNA microarray yielded concordant results for 99% of specimens and identified mixed viral infections in 85 (62%). The most common associations were: human bocavirus and respiratory syncytial virus (32%), adenovirus and respiratory syncytial virus (30%), and parainfluenza virus type 3 and respiratory syncytial virus (23%). None of the bronchiolitis severity parameters including intensive care unit admission, O(2) supply, O(2) saturation percentage, O(2) length and length of stay at the hospital appeared to be significantly increased in multiple viral infections compared to single viral infections (P > 0.1). In conclusion, the use of this DNA microarray in clinical virology practice allows rapid and accurate identification of common and uncommon viral respiratory pathogens in infants hospitalized for bronchiolitis. It should improve the clinical management, the epidemiological survey, and the prevention of the nosocomial transmission of respiratory viruses in pediatric wards.

Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay.

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per µg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.

Rapid virological diagnosis of central nervous system infections by use of a multiplex reverse transcription-PCR DNA microarray.

Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS.

Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection.

The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors.

[Two adult cases of influenza A/H1N1v virus pneumonia hospitalized in the Intensive Care Unit of the Reims University Medical Centre]. / À propos de deux cas de pneumonie à influenza virus A/H1N1v pris en charge en réanimation au CHU de Reims durant l'hiver 2009.

During the 2009-2010 winter season, the new pandemic influenza A/H1N1 virus (S-OIV) was responsible for 1,334 hospitalized severe infection cases including 312 (23.4%) deaths in metropolitan France. In the Champagne-Ardenne area (north eastern) this new epidemic strain was detected in the respiratory samples of 14 severe S-OIV infection cases resulting in 5 deaths. Here we report two of these 14 cases who were suffering from a bilateral pneumonia related to S-OIV infection and who were hospitalized in the Intensive Care Unit (ICU) of the Reims University Medical Centre during December 2009. These two patients were male with at least one known risk factor for severe S-OIV infection (chronic obstructive pulmonary disease (COPD) and morbid obesity, respectively); the COPD patient developed an acute respiratory distress syndrome. The etiological diagnosis of S-OIV infection was performed by use of a real time RT-PCR (rRT-PCR) assay allowing the detection of all the known human influenza A viruses (rRT-PCR targeting the influenza gene M) and of the new influenza A/H1N1 pandemic strain. This rRT-PCR assay was positive in bronchoalveolar lavage samples taken from the two patients, whereas the nasal swab (using Virocult® collection system) appeared to be positive for only one of them. For both patients, a presumptive treatment combining oseltamivir and broad-spectrum antibiotics was started at the time of hospital admission, 24 hours at least before obtaining the results of the virological and bacteriological analyses. These two patients did not develop any secondary bacterial pneumonia and their clinical outcome was good after one and six weeks of hospitalization in ICU, respectively.

Low frequency of cytomegalovirus infection during exacerbations of inflammatory bowel diseases.

Although numerous reports have described inflammatory bowel diseases (IBDs) complicated with cytomegalovirus (CMV) infection, the virus participation as an exacerbating factor remains unclear. The aim of this study was thus to clarify the clinical significance of CMV infection complicating exacerbation and to correlate CMV detection with various characteristics in IBD patients. Sixty-seven colonic biopsies obtained from 53 patients admitted for IBD exacerbation were retrospectively analyzed by real-time PCR assay. The CMV genome was detected in seven (10.4%) colonic biopsies related to seven patients (three ulcerative colitis and four Crohn's diseases). Among the patients with IBD studied, patients with evidence of CMV infection were older (P = 0.047), were more likely male gender (relative risk [RR] 4.48; 95% confidence interval [CI] 0.94-21.36), received corticosteroids (RR 3.2; CI 0.79-13.02) or azathioprine (RR 3.17; CI 0.80-12.57) treatments, presented more extended lesions (RR for rectum-sigmoid-left colon 3.75 (0.0-69.37) and for pancolitis 2.45 (0.36-16.23)), and had a more severe disease (RR 3.3; CI 0.87-12.48) than those without CMV infection. Viral loads measured in the colonic mucosa of infected patient ranged from 5 to 236961 genome copies by microgram of total extracted DNA. No relationship was observed between the severity of the disease and the viral load level. Furthermore, CMV disappeared in five infected IBD patients in remission without antiviral agents. In conclusion, these results showed infrequent CMV detection in colonic biopsies of IBD patients during exacerbation leaving open the question of the relationship between CMV reactivation and the onset or the severity of IBD exacerbation.

Rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-PCR DNA microarray systems.

We prospectively tested 95 nasal swabs or nasopharyngeal aspirates taken from 56 adults and 39 children visiting the Reims University Medical Centre (northern France) for influenza-like illnesses (ILI) during the early stage of the French influenza A/H1N1v pandemic (October 2009). Respiratory samples were tested using a combination of two commercially available reverse transcription-PCR (RT-PCR) DNA microarray systems allowing rapid detection of influenza A virus strains, including the new A/H1N1v strain as well as 20 other common or newly discovered respiratory viruses. Concomitantly, a generic and classical real-time RT-PCR assay was performed to detect all circulating influenza A virus strains in the same samples. Of the 95 respiratory samples tested, 30 (31%) were positive for the detection of influenza A/H1N1v virus infection by both RT-PCR DNA microarray and classical real-time RT-PCR detection assays. Among the infections, 25 (83%) were monoinfections, whereas 5 (17%) were multiple infections associating influenza A/H1N1v virus with coronavirus (CoV), human bocavirus (HBoV), respiratory syncytial virus (RSV), or human rhinoviruses (HRVs). Of the 95 respiratory samples tested, 35 (37%) were positive for respiratory viruses other than influenza A/H1N1v virus. Among these infections, we observed 30 monoinfections (HRVs [63%], parainfluenza viruses [PIVs] [20%]), influenza A/H3N2 virus [6%], coronavirus [4%], and HBoV [4%]) and 5 multiple infections, in which HRVs and PIVs were the most frequently detected viruses. No specific single or mixed viral infections appeared to be associated significantly with secondary hospitalization in infectious disease or intensive care departments during the study period (P > 0.5). The use of RT-PCR DNA microarray systems in clinical virology practice allows the rapid and accurate detection of conventional and newly discovered viral respiratory pathogens in patients suffering from ILI and therefore could be of major interest for development of new epidemiological survey systems for respiratory viral infections.

Respiratory echovirus 30 and coxsackievirus B5 can induce production of RANTES, MCP-1 and IL-8 by human bronchial epithelial cells.

Human Enteroviruses (HEV) (picornaviridae) are considered as one the major viral causes of childhood acute respiratory wheezing illnesses including bronchiolitis and asthma exacerbation. To identify the mechanisms that can regulate the development of airway mucosa inflammation during HEV respiratory lower tract infection, we investigated the profile and the levels of mRNA and protein secretion for CC and CXC human chemokines by HEV-infected primary human bronchial epithelial cells (SAE cells) using RT-PCR array and Bio-Plex assay. Cultures of SAE cells were infected by reference and wild-type HEV respiratory strains, demonstrating a replicative and productive viral infection. We observed that the replicative infection of the SAE cells by reference and wild-type HEV strains induced specific dose and time-dependent increases in mRNA and protein secretion only for RANTES, MCP-1 and IL-8 and not for all other CC and CXC human chemokines tested. The protein secretion of these chemokines appeared to be significantly increased at 48 or 72h post-infection in cultures treated by low-doses of IFN-gamma comparatively to mock-infected cells (P<0.001), and was correlated to the viral replication activity. In conclusion, our findings demonstrated a selective production of RANTES, IL-8 and MCP-1 released by HEV-infected epithelial cells of the small bronchioles along with mechanisms of amplification mediated by IFN-gamma.

Prevalence of rotavirus, adenovirus, norovirus, and astrovirus infections and coinfections among hospitalized children in northern France.

From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (P=0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments.