Déborah Talmud

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

ABSTRACT 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.

Rapid Detection of Respiratory Tract Viral Infections and Coinfections in Patients with Influenza-Like Illnesses by Use of Reverse Transcription-PCR DNA Microarray Systems

ABSTRACT 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.

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 Crohns diseases). Among the patients with IBD studied, patients with evidence of CMV infection were older (P = &!thinsp;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. J. Med. Virol. 82:1694–1700, 2010.

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, O2 supply, O2 saturation percentage, O2 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. J. Med. Virol. 84:979–985, 2012.

Rapid Virological Diagnosis of Central Nervous System Infections by Use of a Multiplex Reverse Transcription-PCR DNA Microarray

ABSTRACT 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.

Quantitative Genomic and Antigenomic Enterovirus RNA Detection in Explanted Heart Tissue Samples from Patients with End-Stage Idiopathic Dilated Cardiomyopathy

ABSTRACT 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.

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.

Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

ABSTRACT 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 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 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.

Evaluation of a new rapid test for the detection of influenza A and B viruses and pandemic (H1N1) 2009 virus subtyping in respiratory samples.

The emergence in 2009 of a novel influenza A (H1N1) virus was responsible for a pandemic outbreak of febrile respiratory infections worldwide (NSOIAVIT, 2009). During the first pandemic wave, specific real-time RT-PCR (rRT-PCR) assays were the reference methods for the detection of the pandemic (H1N1) 2009 influenza virus, leading to the overload of laboratory facilities due to the large number of clinical samples analysed (Duchamp et al., 2010). The re-emergence during winter 2011 of the pandemic (H1N1) 2009 virus highlighted the need for commercially available and readily performed influenza diagnostic tests capable of detecting and subtyping influenza A viral strains in respiratory samples. The previously reported sensitivity of commercially available rapid influenza antigen tests (RIATs) for pandemic (H1N1) 2009 virus was 11 to 80 %, and generally evaluated at around 60 % for clinical specimens (Drexler et al., 2009; Lee et al., 2010; Lévêque et al., 2010; Louie et al., 2010). Moreover, currently available RIATs are only able to type influenza A and B viruses, and do not specifically subtype the pandemic (H1N1) 2009 virus. The aim of this study was thus to assess the diagnostic performances, comparatively to the rRTPCR assay, of a new RIAT, the ONE STEP influenza virus A/B/A(H1N1) pandemic rapid test (Standard Diagnostics; Bioline), which can detect the influenza viruses A and B, and subtype the pandemic (H1N1) 2009 in respiratory samples.