Phylogeny and prevalence of kobuviruses in dogs and cats in the UK.

The kobuviruses represent an emerging genus in the Picornaviridae. Here we have used next generation sequencing and conventional approaches to identify the first canine kobuvirus (CaKoV) from outside the USA. Phylogenetic analysis suggests that a single lineage genotype of CaKoV now exists in Europe and the USA with 94% nucleotide similarity in the coding region. CaKoV was only identified in a single case from a case-control study of canine diarrhoea, suggesting this virus was not a frequent cause of disease in this population. Attempts to grow CaKoV in cell culture failed. Sequence analysis suggested CaKoV was distinct from human Aichi virus (AiV), and unlikely to pose a significant zoonotic risk. Serosurveys by ELISA, immunofluorescence and neutralisation tests, using AiV as antigen, suggested kobuvirus infection is prevalent in dogs. In addition, IgG antibody to AiV was also detected in cat sera, indicating for the first time that cats may also be susceptible to kobuvirus infection.

Novel group A rotavirus G8 P[1] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs.

Rotavirus is a worldwide major cause of diarrhea outbreaks in neonatal ruminants. An outbreak of ovine diarrheic syndrome (ODS) in 50-75 days-old lambs (weaned lambs) is described. Fecal immunochromatography and intestinal immunohistochemistry for rotavirus group A were performed. In addition, semi-nested multiplex RT-PCR for G and P rotavirus genotyping in combination with sequencing were performed, to support the diagnosis and identify the viral strain. A novel ovine rotavirus group A G8 P[1] strain was determined as the main cause of the ODS observed, whereas other pathogens were ruled out.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Enteric calicivirus and rotavirus infections in domestic pigs.

We report the prevalence of rotavirus and calicivirus infections, along with their respective association with diarrhoea in the porcine population of the region of northern Spain. A total of 221 samples were collected at random from different farms in the region and from the main slaughterhouse facility in the city of Zaragoza. Faecal samples were scored as diarrhoeic or normal and grouped into five groups to match general farm management and age criteria: group I (suckling 0-4 weeks), group II (weaning >4-8 weeks), group III (transition >8-16 weeks), group IV (fattening >16-24 weeks) and group V (adults >24 weeks). Group A rotavirus detection and caliciviruses were investigated by reverse transcription-polymerase chain reaction (RT-PCR). Conventional RT-PCR was performed using primers designed to detect rotavirus group A, caliciviruses and/or human noroviruses. A real-time RT-PCR was carried out using TaqMan probes for genogroups GI and GII of noroviruses. Rotaviruses and caliciviruses were detected with an overall prevalence of 16.7% and 12.2%, respectively. Rotavirus detection in faecal samples was associated with both age and faecal consistency, being more frequent in piglets aged <8 weeks with odds ratios (ORs) equal to 4.3 and 4.9, respectively. Calicivirus shedding in faecal samples was homogenously distributed in all ages, showing no significant association with age or faecal consistency (OR 0.87 and 0.99, respectively). A selection of rotavirus-positive stools were genotyped by multiplex nested PCR. G10, P[6], G12 P[8], G9 [p8] and G4 P[23] genotype combinations were found. Three isolates showed a G3 genotype, but their VP4 gene could not be amplified. It should be noted that the G9 genotype was the major G genotype circulating during that period in Spain. None of the porcine samples was positive for norovirus by real-time RT-PCR, despite the ability of this technique to detect at least 18 human norovirus genotypes. Our data indicate that human noroviruses are unlikely to be circulating in the porcine population; however, sapoviruses have been found. Contrary to rotavirus infection, Calicivirus infection is asymptomatic. Specific primers to detect porcine noroviruses are needed.

Epidemiological and clinical features of norovirus gastroenteritis in outbreaks: a population-based study.

Noroviruses are the most frequent cause of acute gastroenteritis in the community. In Catalonia, it is not clear how this type of viral gastroenteritis is evolving, and the objective of this prospective population-based study was to describe the incidence and epidemiological and clinical features of outbreaks of acute gastroenteritis due to norovirus in Catalonia between October 2004 and October 2005. Incidence rates were calculated using the estimated population of Catalonia in 2005. For each outbreak, the mode of transmission, the number of persons affected, demographic variables, clinical presentation, the date and time of onset of symptoms and the duration of symptoms, physician visits and hospitalizations were collected. Sixty viral outbreaks affecting 1791 people were identified, with no distinct seasonality. The mean number of outbreaks per month was 4.6. The global incidence was 24.6 per 100 000 person-years. The incidence was higher in women (25.7 per 100 000 person-years) and in the 5-11 years (52.4 per 100 000 person-years) and > or =65 years (42.4 per 100 000 person-years) age groups. The prevalence of vomiting, abdominal pain and general malaise was higher in children and adolescents, whereas the prevalence of diarrhoea and myalgia was higher in adults. These results suggest that norovirus infection has an important public health impact in Catalonia and that prevention strategies should be designed and implemented.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

[Evaluation of two immunochromatography kits for rapid diagnosis of rotavirus infections]. / Evaluación de dos equipos inmunocromatográficos comerciales para el diagnóstico rápido de la infección por rotavirus.

A prospective study was conducted to evaluate two immunochromatography (ICG) commercial kits for diagnosis of rotavirus infection, VIKIA Rota-Adeno (bioMérieux) and Simple Rota-Adeno (Operon). Reverse transcriptase and polymerase chain reaction (RT-PCR) with specific primers for the VP7 gene of group A rotavirus was used as the reference method. The sensitivity and specificity of the ICG tests compared with those of the reference method were 98.4% and 84.8%, respectively, for Simple Rota-Adeno (Operon), and 100% and 24.2% for VlKIA Rota-Adeno (bioMérieux). It is remarkable the low specificity of the latter method, which yields a high number of false positive results. The predictive value of a positive result by this method was only 71.6%. Most of the detected rotavirus strains corresponded to genotype G9P[8] (65%), followed by G1P[8] (25.4%) and G2P[8] (3.2%).

Evaluación de dos equipos inmunocromatográficos comerciales para el diagnóstico rápido de la infección por rotavirus / Evaluation of two immunochromatography kits for rapid diagnosis of rotavirus infections

Se realizó un estudio prospectivo para evaluar dos equipos comerciales inmunocromatográficos para el diagnóstico rápido de infección por rotavirus a partir de muestras fecales: VIKIA® Rota-Adeno, de bioMérieux, y Simple Rota- Adeno, de Operon. Como método de referencia se utilizó la transcripción reversa y reacción en cadena de la polimerasa (RT-PCR) con cebadores específicos del gen de la proteína VP7 de rotavirus del grupo A. La sensibilidad y la especificidad respecto de la RT-PCR fueron del 98,4% y 84,8% para el Simple Rota-Adeno, y del 100% y 24,2% para el VIKIA® Rota-Adeno. Es de destacar la baja especificidad de este último equipo diagnóstico, que presentó un elevado número de falsos positivos, por lo que el valor predictivo de un resultado positivo es sólo del 71,6%. Asimismo, se identificaron los genotipos de las cepas de rotavirus detectadas; la mayoría de ellas correspondieron al genotipo G9P(8) (65%), seguido de los genotipos G1P(8) (25,4%) y G2P(8) (3,2%).

A prospective study was conducted to evaluate two immunochromatography (ICG) commercial kits for diagnosis of rotavirus infection, VIKIA® Rota-Adeno (bioMérieux) and Simple Rota-Adeno (Operon). Reverse transcriptase and polymerase chain reaction (RT-PCR) with specific primers for the VP7 gene of group A rotavirus was used as the reference method. The sensitivity and specificity of the ICG tests compared with those of the reference method were 98.4% and 84.8%, respectively, for Simple Rota-Adeno (Operon), and 100% and 24.2% for VIKIA® Rota-Adeno (bioMérieux). It is remarkable the low specificity of the latter method, which yields a high number of false positive results. The predictive value of a positive result by this method was only 71.6%. Most of the detected rotavirus strains corresponded to genotype G9P(8) (65%), followed by G1P(8) (25.4%) and G2P(8) (3.2%).

Analysis of integrated virological and epidemiological reports of norovirus outbreaks collected within the Foodborne Viruses in Europe network from 1 July 2001 to 30 June 2006.

The Foodborne Viruses in Europe network has developed integrated epidemiological and virological outbreak reporting with aggregation and sharing of data through a joint database. We analyzed data from reported outbreaks of norovirus (NoV)-caused gastroenteritis from 13 European countries (July 2001 to July 2006) for trends in time and indications of different epidemiology of genotypes and variants. Of the 13 countries participating in this surveillance network, 11 were capable of collecting integrated epidemiological and virological surveillance data and 10 countries reported outbreaks throughout the entire period. Large differences in the numbers and rates of reported outbreaks per country were observed, reflecting the differences in the focus and coverage of national surveillance systems. GII.4 strains predominated throughout the 5-year surveillance period, but the proportion of outbreaks associated with GII.4 rose remarkably during years in which NoV activity was particularly high. Spring and summer peaks indicated the emergence of genetically distinct variants within GII.4 across Europe and were followed by increased NoV activity during the 2002-2003 and 2004-2005 winter seasons. GII.4 viruses predominated in health care settings and in person-to-person transmission. The consecutive emergence of new GII.4 variants is highly indicative of immune-driven selection. Their predominance in health care settings suggests properties that facilitate transmission in settings with a high concentration of people such as higher virus loads in excreta or a higher incidence of vomiting. Understanding the mechanisms driving the changes in epidemiology and clinical impact of these rapidly evolving RNA viruses is essential to design effective intervention and prevention measures.

Data quality of 5 years of central norovirus outbreak reporting in the European Network for food-borne viruses.

BACKGROUND: The food-borne viruses in Europe (FBVE) network database was established in 1999 to monitor trends in outbreaks of gastroenteritis due to noroviruses (NoVs), to identify major transmission routes of NoV infections within and between participating countries and to detect diffuse international food-borne outbreaks. METHODS: We reviewed the total of 9430 NoV outbreak reports from 13 countries with date of onset between 1 January 2002 and 1 January 2007 for representativeness, completeness and timeliness against these objectives. RESULTS: Rates of reporting ranged from a yearly average of 1.8 in 2003 to 11.6 in 2006. Completeness of reporting of an agreed minimum dataset improved over the years, both for epidemiological and virological data. For the 10 countries that provided integrated (epidemiological AND virological) reporting over the 5-year period, the completeness of the minimum dataset rose from 15% in 2003 to 48% in 2006. Two countries have not been able to combine both data types due to the structure of the national surveillance system (England and Wales and Germany). Timeliness of reporting (median days between the onset of an outbreak and the date of reporting to the FBVE database) differed greatly between countries, but gradually improved to 47 days in 2006. CONCLUSION: The outbreaks reported to the FBVE reflect the lack of standardization of surveillance systems across Europe, making direct comparison of data between countries difficult. However, trends in reported outbreaks per country, distribution of NoV genotypes, and detection of diffuse international outbreaks were used as background data in acute questions about NoV illness and the changing genotype distribution during the 5-year period, shown to be of added value. Integrated reporting is essential for these objectives, but could be limited to sentinel countries with surveillance systems that allow this integration. For successful intervention in case of diffuse international outbreaks, completeness and timeliness of reporting would need to be improved and expanded to countries that presently do not participate.

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo.

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.

Phase I/II trial of doxorubicin and fixed dose-rate infusion gemcitabine in advanced soft tissue sarcomas: a GEIS study.

The aim of the study was to determine the dose-limiting toxicity and maximum tolerated dose of a first-line combination of doxorubicin and gemcitabine in adult patients with advanced soft tissue sarcomas and to explore its activity and toxicity, and the presence of possible interactions between these agents. Patients with measurable disease were initially treated with doxorubicin 60 mg m(-2) by i.v. bolus on day 1 followed by gemcitabine at 800 mg m(-2) over 80 min on days 1 and 8, every 21 days. Concentrations of gemcitabine and 2',2'-difluorodeoxyuridine in plasma, and gemcitabine triphosphate levels in peripheral blood mononuclear cells were determined during 8 h after the start of gemcitabine infusion. Myelosuppression and stomatitis were limiting toxicities, and the initial dose level was applied for the Phase II trial, where grade 3-4 granulocytopenia occurred in 70% of patients, grade 3 stomatitis in 46% and febrile neutropenia in 20%. Objective activity in 36 patients was 22% (95% CI: 9-35%), and a 50% remission rate was noted in leiomyosarcomas. Administration of doxorubicin preceding gemcitabine significantly reduced the synthesis of gemcitabine triphosphate. Clinical activity, similar to that of single-agent doxorubicin, and the toxicity encountered do not justify further studies with this schedule of administration.

Simultaneous determination of gemcitabine di- and triphosphate in human blood mononuclear and cancer cells by RP-HPLC and UV detection.

A reverse-phase HPLC method based on ion-pair formation with UV detection was set up for the simultaneous determination of gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) in human cells. The separation was achieved on a Tracer Excel ODSA column (100 mm x 4.6mm i.d., 3 microm particle size) at room temperature. Nine nucleotides were separated by isocratic elution in 26 min. Accuracy, linearity, sensitivity and precision studies for dFdCDP, dFdCTP, adenosine diphosphate (ADP) and triphosphate (ATP) validated this method. This assay was used to provide data from gemcitabine treated patients and in vitro grown human cancer cells.

Phase II Trial of Doxorubicin Plus Escalated High-Dose Ifosfamide in Patients With Advanced Soft Tissue Sarcomas of the Adult: A Study of the Spanish Group for Research on Sarcomas (GEIS).

Background. To explore the tolerance and the activity of high-dose ifosfamide (IFOS) combined with doxorubicin (DXR) at 50 mg/m(2) every 4 weeks in patients with soft tissue sarcomas. Methods. DXR was given IV bolus and IFOS by continuous infusion at 2 g/m(2)/day. Initial IFOS dose (12 g/m(2)) was adjusted to 10, 13, or 14 g/m(2) according to toxicity. Results. Seventy patients received 277 cycles (median 3 cycles, range 1-10), 34% with IFOS dose increased, 30% decreased, and 48% delivered at 12 g/m(2). Toxicity grade 4 occurred on granulocytes (67% of patients) or platelets (19%), 54% had febrile neutropenia, 31% grade 3/4 asthenia, and 26% abandoned the study due to toxicity. Three toxic deaths occurred. In 57 non-GIST patients objective activity was 45.6% (95% CI, 32 to 58%). Conclusion. At least 4 cycles were tolerated by 71% of patients, most receiving DXR 50 mg/m(2) plus IFOS 10-12 g/m(2), with substantial toxicity.

Determination of thiamine and its phosphorylated forms in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer patients.

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.

An outbreak of food poisoning due to a genogroup I norovirus.

Norovirus infection is associated with approximately 90% of epidemic non-bacterial acute gastroenteritis. The objective of this study is to describe an outbreak of norovirus genogroup I gastroenteritis which affected workers in a hospital and was attributed to food prepared by an infected food handler. Forty cases were detected, of whom 80% were interviewed. The index case was the cook employed in the hospital cafeteria. The following symptoms were observed: abdominal pain in 90.6%, vomiting in 71.9%, diarrhoea in 71.9%, general indisposition in 62.5%, headaches in 53.1% and fever in 32.4% of cases. The initial symptoms were abdominal pain in 37% and vomiting in 28%. Of the 14 samples analysed by RT-PCR, 12 (86%) were positive for a genogroup I norovirus. After sequencing the strain was identified as genotype Desert Shield. Many of the foodstuffs consumed were made by hand, favouring transmission from the index case to the cafeteria users.

Phase II Clinical Trial With Pegylated Liposomal Doxorubicin (CAELYX(R)/Doxil(R)) and Quality of Life Evaluation (EORTC QLQ-C30) in Adult Patients With Advanced Soft Tissue Sarcomas: A study of the Spanish Group for Research in Sarcomas (GEIS).

BACKGROUND: Pegylated liposomal doxorubicin (PLD), a formulation with pharmacokinetic differences with respect to doxorubicin (DXR), might benefit patients with advanced soft tissue sarcoma (STS) pretreated with DXR. PATIENTS AND METHODS: Patients with measurable and progressive STS received PLD at 35 mg/(2) every 3 weeks. Quality of life before and during treatment was assessed with EORTC QLQ-C30. RESULTS: Twenty-eight patients, 22 DXR-pretreated, were given 140 cycles (median 3, range 1-18). Activity in 27 patients (5 GIST): one complete and one partial remission (both non-GIST and without prior DXR), 12 stabilizations and 13 progressions (response rate 7.4%, 95% CI: 0-17%). Grade 3 toxicity: palmar-plantar erythrodysesthesia (19% of patients), stomatitis (4%) or cutaneous (4%). Neutropenia grade>/=3 was detected in 16% of patients. Median relative dose intensity was 95%. Progression-free rate at 3 and 6 months was, respectively, 48 and 22%, median progression-free survival 5.8 months and median overall survival 8.7 months. QLQ-C30 at baseline and at weeks 6-11 in 23 and 13 patients, respectively, showed good reliability and validity. Quality of life did not seem to worsen during therapy. CONCLUSIONS: PLD did not induce objective remissions in 22 STS patients pretreated with DXR, but progression-free rate figures support the use of this agent in patients who have not progressed under a DXR-containing regimen. The toxicity observed was comparable to that of other PLD schedules.

Molecular epidemiology of caliciviruses causing outbreaks and sporadic cases of acute gastroenteritis in Spain.

The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a "new variant" designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.