Novel norovirus recombinants and of GII.4 sub-lineages associated with outbreaks between 2006 and 2010 in Belgium

<p>Abstract</p> <p>Background</p> <p>Noroviruses (NoVs) are an important cause of acute gastroenteritis in humans worldwide. To gain insight into the epidemiologic patterns of NoV outbreaks and to determine the genetic variation of NoVs strains circulating in Belgium, stool samples originating from patients infected with NoVs in foodborne outbreak investigations were analysed between December 2006 and December 2010.</p> <p>Results</p> <p>NoVs were found responsible of 11.8% of all suspected foodborne outbreaks reported in the last 4 years and the number of NoV outbreaks reported increased along the years representing more than 30% of all foodborne outbreaks in 2010. Genogroup II outbreaks largely predominated and represented more than 90% of all outbreaks. Phylogenetic analyses were performed with 63 NoV-positive samples for the partial polymerase (N = 45) and/or capsid gene (N = 35) sequences. For 12 samples, sequences covering the ORF1-ORF2 junction were obtained. A variety of genotypes was found among genogroups I and II; GII.4 was predominant followed in order of importance by GII.2, GII.7, GII.13, GI.4 and GI.7. In the study period, GII.4 NoVs variants 2006a, 2006b, 2007, 2008 and 2010 were identified. Moreover, phylogenetic analyses identified different recombinant NoV strains that were further characterised as intergenotype (GII.e/GII.4 2007, GII.e/GII.3 and GII.g/GII.1) and intersub-genotype (GII.4 2006b/GII.4 2007 and GII.4 2010/GII.4 2010b) recombinants.</p> <p>Conclusions</p> <p>NoVs circulating in the last 4 years in Belgium showed remarkable genetic diversity either by small-scale mutations or genetic recombination. In this period, GII.4 2006b was successfully displaced by the GII.4 2010 subtype, and previously reported epidemic GII.b recombinants seemed to have been superseded by GII.e recombinants in 2009 and GII.g recombinants in 2010. This study showed that the emergence of novel GII.4 variants together with novel GII recombinants could lead to an explosion in NoV outbreaks, likewise to what was observed in 2008 and 2010. Among recombinants detected in this study, two hitherto unreported strains GII.e/GII.3 and GII.g/GII.1 were characterised. Surveillance will remain important to monitor contemporaneously circulating strains in order to adapt preventive and curative strategies.</p

Natalizumab treatment shows low cumulative probabilities of confirmed disability worsening to EDSS milestones in the long-term setting.

Abstract Background Though the Expanded Disability Status Scale (EDSS) is commonly used to assess disability level in relapsing-remitting multiple sclerosis (RRMS), the criteria defining disability progression are used for patients with a wide range of baseline levels of disability in relatively short-term trials. As a result, not all EDSS changes carry the same weight in terms of future disability, and treatment benefits such as decreased risk of reaching particular disability milestones may not be reliably captured. The objectives of this analysis are to assess the probability of confirmed disability worsening to specific EDSS milestones (i.e., EDSS scores ≥3.0, ≥4.0, or ≥6.0) at 288 weeks in the Tysabri Observational Program (TOP) and to examine the impact of relapses occurring during natalizumab therapy in TOP patients who had received natalizumab for ≥24 months. Methods TOP is an ongoing, open-label, observational, prospective study of patients with RRMS in clinical practice. Enrolled patients were naive to natalizumab at treatment initiation or had received ≤3 doses at the time of enrollment. Intravenous natalizumab (300 mg) infusions were given every 4 weeks, and the EDSS was assessed at baseline and every 24 weeks during treatment. Results Of the 4161 patients enrolled in TOP with follow-up of at least 24 months, 3253 patients with available baseline EDSS scores had continued natalizumab treatment and 908 had discontinued (5.4% due to a reported lack of efficacy and 16.4% for other reasons) at the 24-month time point. Those who discontinued due to lack of efficacy had higher baseline EDSS scores (median 4.5 vs. 3.5), higher on-treatment relapse rates (0.82 vs. 0.23), and higher cumulative probabilities of EDSS worsening (16% vs. 9%) at 24 months than those completing therapy. Among 24-month completers, after approximately 5.5 years of natalizumab treatment, the cumulative probabilities of confirmed EDSS worsening by 1.0 and 2.0 points were 18.5% and 7.9%, respectively (24-week confirmation), and 13.5% and 5.3%, respectively (48-week confirmation). The risks of 24- and 48-week confirmed EDSS worsening were significantly higher in patients with on-treatment relapses than in those without relapses. An analysis of time to specific EDSS milestones showed that the probabilities of 48-week confirmed transition from EDSS scores of 0.0–2.0 to ≥3.0, 2.0–3.0 to ≥4.0, and 4.0–5.0 to ≥6.0 at week 288 in TOP were 11.1%, 11.8%, and 9.5%, respectively, with lower probabilities observed among patients without on-treatment relapses (8.1%, 8.4%, and 5.7%, respectively). Conclusions In TOP patients with a median (range) baseline EDSS score of 3.5 (0.0–9.5) who completed 24 months of natalizumab treatment, the rate of 48-week confirmed disability worsening events was below 15%; after approximately 5.5 years of natalizumab treatment, 86.5% and 94.7% of patients did not have EDSS score increases of ≥1.0 or ≥2.0 points, respectively. The presence of relapses was associated with higher rates of overall disability worsening. These results were confirmed by assessing transition to EDSS milestones. Lower rates of overall 48-week confirmed EDSS worsening and of transitioning from EDSS score 4.0–5.0 to ≥6.0 in the absence of relapses suggest that relapses remain a significant driver of disability worsening and that on-treatment relapses in natalizumab-treated patients are of prognostic importance

Molecular detection of HAV by a new one step real time RT-PCR

Introduction and objectives Hepatitis A virus (HAV) is a RNA virus with a single-stranded positive sense genome and the only species of the genus Hepatovirus of the Picornaviridae family. Belgium and European countries in general, are countries with a low prevalence and the majority of adults can be infected. HAV is mainly transmitted by the fecal-oral route and even if foodborne outbreaks account for less than 5 % of the reported cases per year, the source of infection cannot be identified in 50 % of the reported cases. Therefore the contribution of foodborne infection is probably underestimated. Viral loads in food samples are lower than in clinical samples and their detection requires refined molecular detection methods. Methods A one step real-time RT-PCR to detect HAV, with new primers (HAV F2 and HAV R2) and probe (HAV P2) was performed directly on HAV diluted suspensions and on food samples (dates) and was compared with a ready-to-use commercial kit. Before the one step real time RT-PCR, a preliminary step combining concentration of viral particles with polyethyleneglycol and centrifugation was used on food samples. Results Real time RT-PCR one step with HAV F2/R2/P2 is more efficient but less sensitive than the commercial kit. It could be used to confirm a positive sample or to detect HAV in an unknown sample. With cell cultured HAV, the limit of detection (LOD) is 1.25 infectious particles in volume tested by RT-PCR or 102 TCID50/ml. In food samples, LOD is between 25 infectious particles and 250 infectious particles in volume tested by RT-PCR or between 104 and 105 TCID50/ml. Several hypotheses could explain these results: the loss of viral particles during the extraction process, the low efficiency of RNA extraction and interference of food on molecular detection. Conclusion Molecular detection of virus in food samples remains a challenge and the protocol of extraction should be improved and adapted at each food category to increase the sensitivity of detection in food matrices characterized by a low viral contamination.Travifoo

Optimalisation d’une RT-PCR en temps réel pour la détection du virus de l’hépatite A

En Belgique, l’incidence de l’hépatite A (HAV) est de 1,2 cas pour 100000 habitants. La population présente une faible immunité contre le HAV puisque seulement 50 % de la population possèdent des anticorps anti-HAV après l’âge de 30 ans. Ainsi un grand nombre d’individus reste susceptible de contracter une infection par le HAV. Les sources de contamination sont principalement le contact de personne à personne mais aussi la consommation d’aliments (crus ou « ready-to-eat) contaminés. Cette contamination peut provenir de l’eau d’irrigation pour les fruits et légumes, de l’eau de mer pour les mollusques bivalves ou de la manipulation de l’homme lors des différentes étapes entre la récolte et la vente du produit. Dans ce projet, une nouvelle RT-PCR spécifique pour la détection du HAV est développée et évaluée. Pour choisir de nouvelles amorces et sondes, des alignements sont réalisés avec les séquences de 19 souches de HAV (DNASTAR Lasergene) afin de cibler les régions les plus conservées du génome du HAV. Cinq nouveaux couples d’amorces ciblant plusieurs régions hautement conservées du génome de HAV ont été sélectionnés et testés à différentes températures d’hybridation et différentes concentrations en utilisant un agent se liant à l’ADN double brin (SYBR Green). Une sonde fluorescente, de type FAM, spécifique de l’amplicon délimité par le couple d’amorces fournissant les meilleurs résultats a été dessinée et utilisée avec la technologie d’hydrolyse de sonde à deux concentrations différentes. Des dilutions d’un facteur 10 de la suspension de la souche HM175 (HAV) ont été testées par le couple d’amorces et la sonde choisit pour établir ainsi une limite de détection. La spécificité du couple d’amorce choisit a été testé en présence de différents picornavirus et virus entériques et de 3 génotypes de HAV (IA, IB et IIIA). Le plasmide Sybricon019 et ses amorces spécifiques ont été utilisés comme contrôle interne d’amplification (IAC). Un contrôle positif a aussi été créé afin de s’assurer du fonctionnement correct de la PCR en temps réel. Parmi les 5 couples d’amorces sélectionnés, le couple HAV-F2/HAV-R2 permet d’obtenir les valeurs de Ct les plus faibles avec une concentration optimale de 300nM pour les amorces sens et anti-sens. Ce couple d’amorces cible la région VP1/VP3 du génome du HAV. Différentes températures d’hybridation ont été testées et la température la plus élevée (60°C) a été sélectionnée pour limiter le risque d’amplification aspécifique. Pour augmenter la spécificité, une sonde, HAV P2, spécifique de l’amplicon délimité par les amorces HAV-F2 et HAV-R2, est utilisée à la concentration de 250nM. Des dilutions d’un facteur 10 d’une suspension de HAV, 107 à 101 particules infectieuses par ml (déterminées par TCID50), donnent respectivement des valeurs de Ct de 19,2 à 38,4 et les dilutions de 100 à 10-2 particules infectieuses par ml ne donnent aucune amplification. La limite de détection est donc de 10 particules infectieuses par ml. La spécificité des amorces et de la sonde pour la détection du HAV est correcte puisque les trois génotypes de HAV, IA, IB et IIIA, ont été détectés alors que les différents picornavirus et virus entériques n’ont donné aucun signal fluorescent d’amplification. L’optimalisation d’un nouveau couple d’amorce et d’une sonde (HAV-F2, -R2 et -P2) ciblant une région hautement conservée du génome permet de détecter le virus HAV par RT-PCR. La région ciblée (VP1/VP3) diffère de la plupart des méthodes de détection de HAV par PCR en temps réelle décrites à ce jour. La seconde étape consiste à réaliser une série de tests dans différentes matrices alimentaires à risque (fruits de mer, fruits et légumes crus) dans le but de détecter des échantillons naturellement contaminés par le HAV.Travifoo

MOF materials as catalysts for organic transformations and as selective hosts in recognition of organics

status: publishe

MOF materials as catalysts for organic transformations and as selective hosts in recognition of organics

[Cu3(BTC)2] is a highly selective Lewis acid catalyst for isomerizations of terpene derivatives, such as the rearrangement of alpha-pinene oxide to campholenic aldehyde and the cyclization of citronellal to isopulegol. By using the ethylene ketal of 2-bromopropiophenone as a test substrate, the active sites in [Cu3(BTC)2] were proven to be hard Lewis acid sites. Further evidence for the Lewis acid nature of [Cu3(BTC)2] was found in an IR spectroscopic study on adsorbed carbon monoxide and acetonitrile. CO adsorbs on a small number of Cu(I) impurities and particularly on the Cu(II) coordination sites in the framework. With acetonitrile as a probe, a strong interaction with the [Cu3(BTC)2] framework is revealed. As reaction rates vary depending on the solvent, a first exploration of liquid phase adsorption on [Cu3(BTC)2] has been undertaken. Finally, molecular recognition of dichlorinated benzene isomers is demonstrated with [Cu3(BTC)2] as adsorbent.status: publishe