Comparison of eMAG™ versus NucliSENS® EasyMAG® performance on clinical specimens.

BACKGROUND: eMAG™ (bioMerieux) is a new nucleic acid extraction platform based on magnetic silica technology, like its predecessor, NucliSENS® easyMAG® (bioMerieux). Using the same reagents and disposables, eMAG™ adds further automation, allowing simultaneous extraction of 48 samples directly from primary tubes, and distribution of nucleic acid extracts on PCR strips or in tubes at the end of the extraction process. OBJECTIVE: To compare the performance of eMAG™ and easyMAG® on various clinical specimens. STUDY DESIGN: Respiratory (n=199), whole blood (n=50), plasma (n=25) and urine (n=25) specimens were extracted in parallel on both platforms. Both qualitative (respiratory virus, cell control, CMV, EBV, HHV6 and BKV detection) and quantitative (respiratory virus and cell control cycle thresolds, and CMV, EBV, HHV6 and BKV viral loads) results were compared. RESULTS: Detection of qualitative targets showed good agreement, ranging from 84.6% for whole blood to 95.9% for respiratory specimens. Correlations between quantitative results were good, with R2 ranging from 0.802 to 0.995. Quantitative results showed average overall differences below 0.10 log10 copies/mL between eMAG™ and easyMAG®. CONCLUSIONS: The two platforms showed comparable performance on the types of clinical specimen tested. With higher automation and throughput than easyMAG®, the eMAG™ platform is likely to be advantageous for laboratories performing a large number of molecular analyses.

Clinical Characteristics Are Similar across Type A and B Influenza Virus Infections.

BACKGROUND: Studies that aimed at comparing the clinical presentation of influenza patients across virus types and subtypes/lineages found divergent results, but this was never investigated using data collected over several years in a countrywide, primary care practitioners-based influenza surveillance system. METHODS: The IBVD (Influenza B in Vircases Database) study collected information on signs and symptoms at disease onset from laboratory-confirmed influenza patients of any age who consulted a sentinel practitioner in France. We compared the clinical presentation of influenza patients across age groups (0-4, 5-14, 15-64 and 65+ years), virus types (A, B) and subtypes/lineages (A(H3N2), pandemic A(H1N1), B Victoria, B Yamagata). RESULTS: Overall, 14,423 influenza cases (23.9% of which were influenza B) were included between 2003-2004 and 2012-2013. Influenza A and B accounted for over 50% of total influenza cases during eight and two seasons, respectively. There were minor differences in the distribution of signs and symptoms across influenza virus types and subtypes/lineages. Compared to patients aged 0-4 years, those aged 5-14 years were more likely to have been infected with type B viruses (OR 2.15, 95% CI 1.87-2.47) while those aged 15-64 years were less likely (OR 0.83, 95% CI 0.73-0.96). Males and influenza patients diagnosed during the epidemic period were less likely to be infected with type B viruses. CONCLUSIONS: Despite differences in age distribution, the clinical illness produced by the different influenza virus types and subtypes is indistinguishable among patients that consult a general practitioner for acute respiratory infections.

Human papillomavirus 16 oncoprotein E7 stimulates UBF1-mediated rDNA gene transcription, inhibiting a p53-independent activity of p14ARF.

High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.

Immortalized sebocytes can spontaneously differentiate into a sebaceous-like phenotype when cultured as a 3D epithelium.

Sebocytes originate from the same lineage as keratinocytes, and both cell types may have similarities in terms of growth and differentiation. We were interested in studying the behaviour of human sebocytes when cultured in conditions validated for epidermal reconstruction. For this purpose, we established a HPV16-E6/7-immortalized human sebocyte cell line (SEBO662) growing in keratinocyte defined media. Postconfluent SEBO662 cells in monolayers express the early sebocyte marker, cytokeratin 7 (K7), do not express Epithelia Membrane Antigen (EMA) and do not exhibit strong lipogenic activity. However, when placed at the air-liquid interface, SEBO662 multilayers spontaneously differentiate into a sebaceous-like structure as shown by the strong polarized expression of the late sebaceous marker EMA, the overexpression of some lipogenic markers and lipid production on the upper side of the epithelium. This work highlights the value of simple 3D models for exhibiting spontaneous differentiation and polarization.

Severe ear chondritis due to cowpox virus transmitted by a pet rat.

We describe a case of cowpox virus infection leading to severe acute inflammation and chondritis of the outer ear, complicated by local necrosis and facial cellulitis. Secondary lesions occurred on a finger and the abdomen. Apart from scarring, outcome was favorable after repeated surgical excision of necrotic tissue.

Human papillomavirus genotype distribution in oropharynx and oral cavity cancer in France--The EDiTH VI study.

BACKGROUND: The incidence of oropharyngeal cancers has gradually increased over the last decades. Recent studies suggest an association between human papillomavirus (HPV) infection and several head and neck cancers, especially oropharyngeal and oral cavity invasive carcinomas. OBJECTIVES: The objective was to assess the overall and type specific HPV prevalence in oropharyngeal and oral cavity carcinomas in France. STUDY DESIGN: Paraffin-embedded tumour specimens were retrospectively collected in 12 French centres and centrally tested for HPV detection and genotyping (INNO-LiPA assay). RESULTS: A total of 523 cases (77% males) were collected, among which 60% were oropharyngeal and 40% oral cavity carcinomas. The most frequent anatomical sites were tonsil (58.9%) and base of tongue (13.7%) for the oropharynx and floor of mouth (41.1%) and oral tongue (38.3%) for the oral cavity. Overall HPV prevalence was 46.5% in oropharyngeal carcinomas and 10.5% in oral cavity carcinomas and was higher in female than in male cases (63.5% vs 42.2% in oropharynx and 17.2% vs 8.0% in oral cavity). About 95% of HPV-positive cases were infected by a single HPV type. HPV 16 was the most prevalent type and was found in 89.7% and 95.5% of HPV-positive oropharyngeal and oral cavity carcinoma cases, respectively. All other HPV types had prevalence below 5%. CONCLUSIONS: Our results indicate that HPV is common among oropharyngeal and oral cavity carcinoma cases in France and emphasize the predominance of HPV 16. The potential benefit of HPV vaccination on the occurrence of head and neck carcinomas should be further evaluated.

Human papillomavirus genotype distribution in tonsil cancers.

BACKGROUND: The incidence of tonsil cancers has increased in several countries. French data on HPV prevalence in tonsil cancers are scarce. The objective of this study was thus to assess the overall and type specific HPV prevalence in tonsil histological samples. METHODS: This French retrospective multicenter study involved 12 centres located throughout the country. Were included 185 histological samples collected from year 2000 to 2009 with a validated diagnosis of tonsil invasive carcinomas. HPV prevalence was studied according to gender, age and histological type of cancer. RESULTS: Overall HPV prevalence was 57% in tonsil cancers. Mean age of diagnosis was comparable in HPV positive tonsils cases (60 ± 11.2) and HPV negative tonsil cases (59 ± 9.6). HPV prevalence was significantly higher in female than in male cases (28/35 versus 78/150 in tonsil cases, respectively, P = 0.003). About 53% of tonsil cases were infected by a single HPV type. Only eight (4%) samples were infected by more than one HPV type. Among HPV positive samples, HPV 16 was found in 89% of tonsil cases. All other HPV types had prevalence below 5%. CONCLUSIONS: Our results indicate that HPV is common in tonsil carcinomas and emphasize the predominant role of HPV 16.

Molecular and clinical characterization of rotavirus from diarrheal infants admitted to pediatric emergency units in france.

BACKGROUND: rotaviruses are the major cause of acute gastroenteritis in young children worldwide, and require careful surveillance, especially in the context of vaccination programs. Prospective surveillance is required to monitor and characterize rotavirus infections, including viral and clinical data, and to detect the emergence of potentially epidemic strains. METHODS: between 2006 and 2009, stool samples and clinical records were collected from 2044 children with acute diarrhea admitted to the pediatric emergency units of 13 French university hospitals. Rotaviruses were detected in stools, then genotyped by reverse transcription-polymerase chain reaction with regard to their outer capsid proteins VP4 and VP7. RESULTS: the genotyping of 1947 rotaviruses showed that G1 (61.7%) and G9 (27.4%) strains were predominant and stable, followed by G2 (6.5%), G3 (4.0%), and G4 (2.5%) strains. Most strains were associated with P[8] (92.9%). Overall, 31 uncommon strains and possible zoonotic reassortants were detected including G12 and G8 strains, some being closely related to bovine strains. No difference in clinical presentation and severity was found among genotypes. CONCLUSIONS: the relative stability of rotavirus genotypes currently cocirculating in France may ensure vaccine effectiveness in the short and medium term. However, the likely emergence of G12 and G8 strains should be monitored during ongoing and future vaccination programs, especially as all genotypes can cause severe infections. Special attention should be paid to the emergence of new rotavirus reassortants not included in current rotavirus vaccines.

Infantile hypertrophic pyloric stenosis: are viruses involved?

Infantile hypertrophic pyloric stenosis (IHPS) is characterized by abnormal thickening of the internal circular muscle layer. IHPS is known to be due to a combination of genetic and environmental factors, but its precise causes and pathophysiology are poorly understood. The objective of the study is to determine the prevalence of the principal viruses targeting the respiratory and digestive tracts in children with IHPS. Nasopharyngeal fluids, stools, vomit, and surgical pyloric muscle fragments and swabs were tested by cell culture, viral antigen assay and PCR. IHPS was diagnosed in 23 boys and 8 girls with a mean (± SD) age of 42 ± 15 days (range 20-88 days). There was no seasonal pattern of diagnosis. Twenty-two children (71%) lost weight (mean 246 ± 164 g, range 30-600 g) after the onset of vomiting, and five (16.1%) were dehydrated. Seven (22.6%) infants had been exposed to an infectious contact within 15 days before admission, and one on the day of admission (3.2%). Ear, nose and throat samples and pyloric muscle specimens were negative for all the viruses tested. An adenovirus type 3 was recovered from one stool sample, and RT-PCR was positive for an enterovirus on one vomit sample. This study suggests that the principal viruses targeting the respiratory and digestive tracts are not responsible for IHPS.

Disseminated varicella with multiorgan failure in an immunocompetent adult.

A case of fulminant disseminated varicella is reported in a 28-year-old immunocompetent man. He developed hepatitis, severe pneumonia, rhabdomyolysis and disseminated intravascular coagulation, followed by encephalopathy and multiorgan failure despite acyclovir therapy. He spent a total of 3.5 months in intensive care and rehabilitation units. Real-time PCR yielded a rapid diagnosis of varicella-zoster virus (VZV) infection and was used to monitor plasma viral load for 56 days. Plasma viral load peaked at 7.1 log(10)/ml on day 4 after symptom onset, then gradually declined and became undetectable after between 1 and 2 months; viral load in lung fluid followed a similar pattern. The glycoprotein E variant associated with increased VZV virulence was not detected, and the VZV thymidine kinase gene bore no major mutations associated with acyclovir resistance. This case serves as a reminder that varicella can be life-threatening in adults and that vaccination of individuals at risk remains essential.

Use of replacement blood donors to study the epidemiology of major blood-borne viruses in the general population of Maputo, Mozambique.

The seroprevalence rates of human immunodeficiency virus (HIV), human T-cell leukemia/lymphoma virus (HTLV), hepatitis B virus (HBV), hepatitis D virus (HDV), and hepatitis C virus (HCV) in Mozambique are poorly documented. The epidemiology of these infections was studied in the Maputo region. All donors attending the blood bank during the study period were interviewed and underwent serological and molecular tests for markers of virus exposure. Thus, 1,578 consecutive replacement blood donors were investigated, as they undergo no selection (other than their relation with a patient needing a transfusion), and may thus provide reliable estimates of the prevalence rates in the general population. The age-standardized prevalence rates among 15- to 49-year-old men and women were, respectively, 12.3 and 15.4% for HIV and 0.9 and 1.2% for HTLV. Low educational status (P = 0.014) and tattooing/scarification (P = 0.023) were predictive of HIV infection in multivariate analysis. The age-adjusted prevalence rates of markers of hepatotropic virus among men and women were, respectively, 10.6 and 4.5% for hepatitis B surface antigen (HBsAg), 1.2 and 1.0% for anti-HCV, and 0 and 0% for anti-HDV. Two percent of donors had viral co-infections, involving most frequently the combination of HIV and HBsAg +. A significant association was found between anti-HIV and anti-HBc (P = 0.012). HBsAg was associated with the place of birth (P = 0.011) and a history of transfusion (P = 0.069). Smokers had higher seroprevalence rates than nonsmokers for HIV (P < 0.0001) and HBsAg (P = 0.045). Genotype A was the most frequent HBV genotype (86.3%) followed by E and D. HCV genotypes were 1a, 1b, 3a, and 5a. These results show that HBV vaccination and HIV-preventive measures need to be reinforced in Mozambique.

Lack of correlation between nutritional status and seroprotection against influenza in a long term care facility.

UNLABELLED: We conducted a prospective study in a long-term care facility. Virologic diagnosis was assessed using viral isolation, polymerase chain reaction and serology for all patients with a flu-like syndrome. Albumin, vitamins and trace elements were also measured. RESULTS: The risk of influenza increased 6.5-fold in patients with an antibody titer of 40 during the influenza outbreak (P=0.04). Micronutrients and vitamins deficiencies were important. Patients with antibody titer >1:40 could still be infected by the virus without correlation with the nutritional status. CONCLUSION: Humoral protection with a titer >1:40 might not be protective in the elderly. Nutritional deficiencies were too prevalent to detect any effect on the results.