Koopmans, M.P.G.

Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination (Article)

2013-01-30T00:00:00Z

Background: The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood. Methods: We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination. Results: We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens. Conclusions: Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.

Corrigendum to " Come fly with me: Review of clinically important arboviruses for global travelers" [J. Clin. Virol. 55 (2012) 191-203] (Article)

2013-01-01T00:00:00Z

Come fly with me: Review of clinically important arboviruses for global travelers (Article)

2012-11-01T00:00:00Z

Western tourists are increasingly traveling to exotic locations often located in tropical or subtropical regions of the world. The magnitude of international travel and the constantly changing dynamics of arbovirus diseases across the globe demand up-to-date information about arbovirus threats to travelers and the countries they visit. In this review, the current knowledge on arbovirus threats to global travelers is summarized and prioritized per region. Based on most common clinical syndromes, currently known arboviruses can be grouped to develop diagnostic algorithms to support decision-making in diagnostics. This review systematically combines and structures the current knowledge on medically important travel-related arboviruses and illustrates the necessity of a detailed patient history (travel history, symptoms experienced, vaccination history, engaged activities, tick or mosquito bite and use of repellent and onset of symptoms), to guide the diagnosis.

Sequence-based identification and characterization of nosocomial influenza A(H1N1)pdm09 virus infections (Article)

2012-11-01T00:00:00Z

Background: Highly transmissible viruses such as influenza are a potential source of nosocomial infections and thereby cause increased patient morbidity and mortality. Aim: To assess whether influenza virus sequence data can be used to link nosocomial influenza transmission between individuals. Methods: Dutch A(H1N1)pdm09-positive specimens from one hospital (N = 107) were compared with samples from community cases (N = 685). Gene fragments of haemagglutinin, neuraminidase and PB2 were sequenced and subsequently clustered to detect patients infected with identical influenza viruses. The probability of detecting a second patient was calculated for each hospital cluster against the background diversity observed in hospital and community strains. All clusters were further analysed for possible links between patients. Findings: Seventeen A(H1N1)pdm09 hospital clusters were detected of which eight had a low probability of occurrence compared with background diversity (P < 0.01). Epidemiological analysis confirmed a total of eight nosocomial infections in four of these eight clusters, and a mother-child combination in a fifth cluster. The nine clusters with a high probability of occurrence involved community cases of influenza without a known epidemiological link. Conclusion: If a background sequence dataset is available, the detection of hospital sequence clusters that differ from dominant community strains can be used to select clusters requiring further investigation by hospital hygienists before a nosocomial influenza outbreak is epidemiologically suspected.

Residual viral and bacterial contamination of surfaces after cleaning and disinfection (Article)

2012-11-01T00:00:00Z

Environmental surfaces contaminated with pathogens can be sources of indirect transmission, and cleaning and disinfection are common interventions focused on reducing contamination levels. We determined the efficacy of cleaning and disinfection procedures for reducing contamination by noroviruses, rotavirus, poliovirus, parechovirus, adenovirus, influenza virus, Staphylococcus aureus, and Salmonella enterica from artificially contaminated stainless steel surfaces. After a single wipe with water, liquid soap, or 250-ppm free chlorine solution, the numbers of infective viruses and bacteria were reduced by 1 log10 for poliovirus and close to 4 log10 for influenza virus. There was no significant difference in residual contamination levels after wiping with water, liquid soap, or 250-ppm chlorine solution. When a single wipe with liquid soap was followed by a second wipe using 250- or 1,000-ppm chlorine, an extra 1- to 3-log10 reduction was achieved, and except for rotavirus and norovirus genogroup I, no significant additional effect of 1,000 ppm compared to 250 ppm was found. A reduced correlation between reduction in PCR units (PCRU) and reduction in infectious particles suggests that at least part of the reduction achieved in the second step is due to inactivation instead of removal alone. We used data on infectious doses and transfer efficiencies to estimate a target level to which the residual contamination should be reduced and found that a single wipe with liquid soap followed by a wipe with 250- ppm free chlorine solution was sufficient to reduce the residual contamination to below the target level for most of the pathogens tested.

Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin (Article)

2012-11-01T00:00:00Z

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Differentglycosylation forms of subtype H5 trimeric HA protein (sH53) were produced by expression in insect cells and different mammaliancells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides.The following sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importancewhen developing glycoprotein-based vaccines, such as recombinant HA proteins.

Unrecognized norovirus infections in health care institutions and their clinical impact (Article)

2012-09-01T00:00:00Z

Noroviruses (NoVs) have emerged as the leading cause of acute viral gastroenteritis (GE) in humans. Although diagnostic facilities have greatly improved, significant underdiagnosis of NoV in hospitals may still occur, thereby increasing clinical burden and nosocomial spread. We evaluated the underdiagnosis of sporadic NoV infections in a tertiary care hospital and estimated its clinical impact. From December 2008 until July 2009, fecal samples specifically referred for bacterial but not viral examination were retrospectively tested for NoV by real-time PCR. The clinical and virological data from patients with undiagnosed NoV infection (missed patients) were evaluated and compared with those from patients with recognized NoV. During the study period, 45 patients with undiagnosed NoV were detected, whereas 50 patients were regularly diagnosed. The missed NoV cases were more frequently adults than children (80% versus 46%; P < 0.001). The viral load levels did not differ between the diagnosed and missed patients, but missed patients more frequently presented without diarrhea (20% versus 4%; P < 0.07). The newly admitted missed NoV cases with GE underwent more diagnostic imaging (24% versus 4%; P < 0.01) and tended to be hospitalized longer. When missed-NoV patients were included, the number of nosocomial clusters doubled and missed patients were index cases in 5 of the 6 clusters. These data indicate that NoV infections are frequently missed despite routine laboratory testing and demonstrate that underdiagnosis of NoV patients is associated with costly abdominal imaging and nosocomial clustering. Awareness of NoV infection in adult patients and education about the importance of viral GE should be increased. Copyright

Influenza in the immediate post-pandemic era: A comparison with seasonal and pandemic influenza in hospitalized patients (Article)

2012-06-01T00:00:00Z

Background: Comparative data on severity and treatment of seasonal, pandemic and post-pandemic influenza virus infections are scarce. Objectives: To systematically analyze characteristics of hospitalized patients with influenza in the post-pandemic period compared to seasonal and pandemic influenza. Study design: Clinical and virological data of patients hospitalized in a tertiary referral hospital with post-pandemic influenza (2010-2011) were compared with those during seasonal influenza epidemics (2007-2009) and the influenza A(H1N1)pdm09 pandemic (2009-2010). Results: 82 patients were admitted during the post-pandemic period, compared to 85 during the pandemic and 60 during seasonal influenza epidemics. No differences were observed in the occurrence of complicated illness and the need for intensive care. However, radiographic pneumonia was significantly more often diagnosed in patients with influenza A(H1N1)pdm09 compared to patients with seasonal influenza A (25% versus 71% in pandemic, p= 0.004, and 55% in post-pandemic, p= 0.047). Oseltamivir was more frequently prescribed in post-pandemic and pandemic patients compared to previous influenza seasons (48.9% resp. 76.5% versus 6.5%, p< 0.0001). During the post-pandemic period, patients with influenza B were significantly less often treated with oseltamivir compared to patients with influenza A (27.0% versus 48.9%, p= 0.043), although the course of illness in patients with influenza B was comparable with influenza A. No upsurge of oseltamivir resistance was observed. Conclusions: In our center, severity of illness was comparable for all influenza seasons, although more radiographic pneumonia was diagnosed in patients with influenza A(H1N1)pdm09. Despite the increased use of oseltamivir, no increase in oseltamivir resistance was detected.

Thermal stability of structurally different viruses with proven or potential relevance to food safety (Article)

2012-05-01T00:00:00Z

Aims: To collect comparative data on thermal stability of structurally different viruses with proven or potential relevance to food safety. Methods and Results: Suspensions with poliovirus Sabin1, adenovirus type5, parechovirus1, human norovirus (NoV) GII.4, murine NoV (MNV1) and human influenza A (H1N1) viruses were heated at 56 and 73°C. Infectivity was tested by culture assay for all but human NoV GII.4 that cannot be cultivated in vitro. Time to first log10reduction (TFL-value) was calculated based on best fit using the monophasic, biphasic or Weibull models. The Weibull model provided the best fit at 56°C for all viruses except influenza virus. The TFL at 56°C varied between a high of 27min (parechovirus) to a low of 10s (adenovirus) and ranked parechovirus>influenza>MNV1>poliovirus>adenovirus. The monophasic model best described the behaviour of the viruses at 73°C, in which case the TFL was MNV1(62s)>influenza>adenovirus>parechovirus>poliovirus(14s). Conclusions: Viruses do not follow log-linear thermal inactivation kinetics and the thermostability of parechovirus and influenza virus is similar to that of proven foodborne viruses. Significance and Impact of the Study: Resistant fractions of viruses may remain infectious in thermal inactivation processes and inactivation of newly discovered or enveloped viruses in thermal food preparation processes should not be assumed without further testing. © 2012 The Authors. Journal of Applied Microbiology

Quantifying transmission of norovirus during an outbreak (Article)

2012-03-01T00:00:00Z

Background: Healthcare workers are thought to play a role in nosocomial transmission of norovirus, but the level and direction of norovirus transmission between patients and healthcare workers in sustaining transmission during an outbreak have not been quantified. Methods: We developed a method for finding plausible transmission trees of who acquired their infection from whom. We applied the method to data from an outbreak of norovirus in 4 wards of a psychiatric institution in the Netherlands in 2008. The simulated transmission trees were based on serial intervals for time between symptom onsets, weighted for the number of days that healthcare workers were present. The obtained transmission trees were linked to the Barthel Index, a measure of patient reliance on healthcare in their basic daily activities. Results: The dominant recognized transmission route was from patient to patient (64%), followed by patient to healthcare worker (29%). The overall estimated reproduction number for healthcare workers was low compared with patients (0.25 vs. 1.20; mean difference = 0.95 [95% confidence interval (CI) = 0.60 to 1.30]). The average number of all subsequent cases attributable to the downstream branch of one single infected healthcare worker in the transmission tree was 4.4 compared with 6.5 for cases attributable to one single infected patient (mean difference = 2.1 [95% CI = -4.7 to 8.9]). In the ward with patients requiring the highest level of care from healthcare workers, the attack rate among healthcare workers was highest. Conclusions: This approach provides a framework to quantify the magnitude and direction of transmission between healthcare workers and patients during a norovirus outbreak. The utility of this method in outbreaks of other infections and in different settings should be explored. Copyright

Emergence and epidemic occurrence of enterovirus 68 respiratory infections in The Netherlands in 2010 (Article)

2012-02-05T00:00:00Z

Following an increase in detection of enterovirus 68 (EV68) in community surveillance of respiratory infections in The Netherlands in 2010, epidemiological and virological analyses were performed to investigate the possible public health impact of EV68 infections. We retrospectively tested specimens collected from acute respiratory infections surveillance and through three children cohort studies conducted in The Netherlands from 1994 through 2010. A total of 71 of 13,310 (0.5%) specimens were positive for EV68, of which 67 (94%) were from symptomatic persons. Twenty-four (34%) of the EV68 positive specimens were collected during 2010. EV68-positive patients with respiratory symptoms showed significantly more dyspnea, cough and bronchitis than EV68-negative patients with respiratory symptoms. Phylogenetic analysis showed an increased VP1 gene diversity in 2010, suggesting that the increased number of EV68 detections in 2010 reflects a real epidemic. Clinical laboratories should consider enterovirus diagnostics in the differential diagnosis of patients presenting with respiratory symptoms.

Virucidal efficacy of hydrogen peroxide vapour disinfection (Article)

2012-02-01T00:00:00Z

Background: Viral contamination of surfaces is thought to be important in transmission. Chemical disinfection can be an effective means of intervention, but little is known about the virucidal efficacy of hydrogen peroxide vapour (HPV) against enteric and respiratory viruses. Aim: To measure the virucidal efficacy of HPV against respiratory and enteric viruses on materials representing those found in institutions and homes. Methods: Poliovirus, human norovirus genogroup II.4 (GII.4), murine norovirus 1, rotavirus, adenovirus and influenza A (H1N1) virus dried on to stainless steel, framing panel and gauze carriers were exposed to HPV 127. ppm for 1. h at room temperature in an isolator. Poliovirus was also exposed to HPV at different locations in a room. The virucidal effect was measured by comparing recoverable viral titres against unexposed controls. Polymerase chain reaction was used to evaluate the effect of HPV on viral genome reduction. Findings: HPV disinfection resulted in complete inactivation of all viruses tested, characterized by >4 log10reduction in infectious particles for poliovirus, rotavirus, adenovirus and murine norovirus on stainless steel and framing panel carriers, and >2 log10reduction for influenza A virus on stainless steel and framing panel carriers, and for all viruses on gauze carriers. Complete inactivation of poliovirus was demonstrated at several locations in the room. Reductions in viral genomes were minimal on framing panel and gauze carriers but significant on stainless steel carriers; human norovirus GII.4 genome was most resistant to HPV treatment. Conclusion: HPV could be an effective virucidal against enteric and respiratory viruses contaminating in-house environments.

Costs of gastroenteritis in the Netherlands, with special attention for severe cases (Article)

2011-12-01T00:00:00Z

In 1999, the costs of gastroenteritis in the Netherlands were estimated using data on hospitalizations from national registries, together with data on etiology and self-reported data on health care resource use in a community-based study. Now, more information on hospitalizations is available and these data were used to update the total costs of gastroenteritis in the Netherlands. The costs of severe gastroenteritis in the Netherlands were estimated in more depth using a hospital-based study, with patient questionnaires including a follow-up period of 6 months. The overall costs of gastroenteritis were calculated taking direct medical costs, direct non-medical costs, and indirect non-medical costs into account. The costs for severe gastroenteritis in 2009 were estimated at €2,203 per hospitalized child and €6,834 per hospitalized adult. The overall costs of gastroenteritis in 2009 were estimated at €611-695 million, which is €133-151 per gastroenteritis case or €37-42 per inhabitant. The total health care costs for gastroenteritis were about 50% higher in 2009 compared to 1999, which is mostly due to the rise in health care costs. The costs per gastroenteritis episode in adults are higher compared to children, mainly due to differences in the reasons for hospitalization and course of disease, and productivity losses.

Norovirus disease associated with excess mortality and use of statins: A retrospective cohort study of an outbreak following a pilgrimage to Lourdes (Article)

2011-03-01T00:00:00Z

Although norovirus infection is generally known to be a mild disease, there is some evidence for severe outcome. An outbreak in a Dutch psychiatric institution, originating from pilgrims returning from Lourdes (France), provided an opportunity for performing a retrospective cohort study in order to identify risk factors for norovirus disease and excess mortality. Relative risks (RR) including 95% confidence intervals (CI) showed that attending the pilgrimage (RR 20, 95% CI 14-30) and age >70 (RR 17, 95% CI 12-22) were risk factors for symptomatic infection. In a subset of patients, for whom more detailed information was available, the use of statins was associated with norovirus disease when adjusted for underlying condition (adjusted odds ratio 39, 95% CI 12-130). Mortality was higher in cases infected during the pilgrimage compared to other residents (RR 209, 95% CI 47-938). Norovirus disease can lead to severe outcome. The newly identified risk of statins for contracting norovirus disease may have considerable consequences for the Western world and needs prospective confirmation.

In search of hidden Q-fever outbreaks: Linking syndromic hospital clusters to infected goat farms (Article)

2011-01-01T00:00:00Z

Large Q-fever outbreaks were reported in The Netherlands from May 2007 to 2009, with dairy-goat farms as the putative source. Since Q-fever outbreaks at such farms were first reported in 2005, we explored whether there was evidence of human outbreaks before May 2007. Space-time scan statistics were used to look for clusters of lower-respiratory infections (LRIs), hepatitis, and/or endocarditis in hospitalizations, 2005-2007. We assessed whether these were plausibly caused by Q fever, using patients' age, discharge diagnoses, indications for other causes, and overlap with reported Q fever in goats/humans. For seven detected LRI clusters and one hepatitis cluster, we considered Q fever a plausible cause. One of these clusters reflected the recognized May 2007 outbreak. Real-time syndromic surveillance would have detected four of the other clusters in 2007, one in 2006 and two in 2005, which might have resulted in detection of Q-fever outbreaks up to 2 years earlier.

Syndromic surveillance for local outbreaks of lower-respiratory infections: Would it work? (Article)

2010-09-14T00:00:00Z

Background: Although syndromic surveillance is increasingly used to detect unusual illness, there is a debate whether it is useful for detecting local outbreaks. We evaluated whether syndromic surveillance detects local outbreaks of lower-respiratory infections (LRIs) without swamping true signals by false alarms. Methods and Findings: Using retrospective hospitalization data, we simulated prospective surveillance for LRI-elevations. Between 1999-2006, a total of 290762 LRIs were included by date of hospitalization and patients place of residence (>80% coverage, 16 million population). Two large outbreaks of Legionnaires disease in the Netherlands were used as positive controls to test whether these outbreaks could have been detected as local LRI elevations. We used a space-time permutation scan statistic to detect LRI clusters. We evaluated how many LRI-clusters were detected in 1999-2006 and assessed likely causes for the cluster-signals by looking for significantly higher proportions of specific hospital discharge diagnoses (e.g. Legionnaires disease) and overlap with regional influenza elevations. We also evaluated whether the number of space-time signals can be reduced by restricting the scan statistic in space or time. In 1999-2006 the scan-statistic detected 35 local LRI clusters, representing on average 5 clusters per year. The known Legionnaires' disease outbreaks in 1999 and 2006 were detected as LRI-clusters, since cluster-signals were generated with an increased proportion of Legionnaires disease patients (p:<0.0001). 21 other clusters coincided with local influenza and/or respiratory syncytial virus activity, and 1 cluster appeared to be a data artifact. For 11 clusters no likely cause was defined, some possibly representing as yet undetected LRI-outbreaks. With restrictions on time and spatial windows the scan statistic still detected the Legionnaires' disease outbreaks, without loss of timeliness and with less signals generated in time (up to 42% decline). Conclusions: To our knowledge this is the first study that systematically evaluates the performance of space-time syndromic surveillance with nationwide high coverage data over a longer period. The results show that syndromic surveillance can detect local LRI-outbreaks in a timely manner, independent of laboratory-based outbreak detection. Furthermore, since comparatively few new clusters per year were observed that would prompt investigation, syndromic hospital-surveillance could be a valuable tool for detection of local LRI-outbreaks.

Phylodynamic reconstruction reveals norovirus GII.4 epidemic expansions and their molecular determinants (Article)

2010-08-30T00:00:00Z

Noroviruses are the most common cause of viral gastroenteritis. An increase in the number of globally reported norovirus outbreaks was seen the past decade, especially for outbreaks caused by successive genogroup II genotype 4 (GII.4) variants. Whether this observed increase was due to an upswing in the number of infections, or to a surveillance artifact caused by heightened awareness and concomitant improved reporting, remained unclear. Therefore, we set out to study the population structure and changes thereof of GII.4 strains detected through systematic outbreak surveillance since the early 1990s. We collected 1383 partial polymerase and 194 full capsid GII.4 sequences. A Bayesian MCMC coalescent analysis revealed an increase in the number of GII.4 infections during the last decade. The GII.4 strains included in our analyses evolved at a rate of 4.3-9.0×10-3 mutations per site per year, and share a most recent common ancestor in the early 1980s. Determinants of adaptation in the capsid protein were studied using different maximum likelihood approaches to identify sites subject to diversifying or directional selection and sites that co-evolved. While a number of the computationally determined adaptively evolving sites were on the surface of the capsid and possible subject to immune selection, we also detected sites that were subject to constrained or compensatory evolution due to secondary RNA structures, relevant in virus-replication. We highlight codons that may prove useful in identifying emerging novel variants, and, using these, indicate that the novel 2008 variant is more likely to cause a future epidemic than the 2007 variant. While norovirus infections are generally mild and self-limiting, more severe outcomes of infection frequently occur in elderly and immunocompromized people, and no treatment is available. The observed pattern of continually emerging novel variants of GII.4, causing elevated numbers of infections, is therefore a cause for concern.

Evolutionary trajectory of the VP1 gene of human enterovirus 71 genogroup B and C viruses (Article)

2010-08-01T00:00:00Z

From 1963 to 1986, human enterovirus 71 (HEV71) infections in the Netherlands were successively caused by viruses of subgenogroups B0, B1 and B2. A genogroup shift occurred in 1987, after which viruses of subgenogroups C1 and C2 were detected exclusively. This is in line with HEV71 typing in Australia, Europe and the USA, but is distinct from that in the Asian Pacific region, where HEV71 subgenogroups B3-B5 and C4-C5 have caused large outbreaks since 1997. To understand these observations in HEV71 epidemiology, the VP1-encoding regions of 199 HEV71 strains isolated in the Netherlands between 1963 and 2008 were used to study the detailed evolutionary trajectory and population dynamics of HEV71. Genogroup B viruses showed an epochal evolution, whereas genogroup C viruses evolved independently, which is in line with the co-circulation of C1 and C2 viruses in the Netherlands since 1997. Considering that strains from the Netherlands are interspersed phylogenetically with GenBank reference strains, the evolution of B1-B2, C1-C2 viruses has a global nature. Phylodynamic analysis confirmed that increased reporting of HEV71 infections in 1986 and 2007 reflected true epidemics of B2 and C2 viruses, respectively. Sequence analysis of the complete capsid region of a subset of isolates revealed several (sub)genogroup-specific residues. Subgenogroup B2-specific rabbit antiserum showed cross-neutralization of B0, B1 and B2 viruses, but not of subgenogroup C1 or C2 viruses, probably explaining the global shift to genogroup C in 1987 following a B2 epidemic. Anti-C1 rabbit serum neutralized both genogroup B and C viruses. Global herd immunity against C1 and C2 viruses possibly explains why epidemics with subgenogroups B4 and C4 are restricted to the Asian Pacific region.

Epidemiology and genotype analysis of emerging sapovirus-associated infections across Europe (Article)

2010-06-01T00:00:00Z

Sapoviruses (SaVs) belong to the Caliciviridae family and can cause gastroenteritis in humans and swine. Despite extensive testing, human sapoviruses have been found only in sporadic cases and in one mixed outbreak in children between 1994 and 2007 in the Netherlands. Here we describe a change in sapovirus epidemiology in the Netherlands resulting in sapovirus outbreaks and infections in adults. From November 2007 to January 2009, 478 outbreaks of acute gastroenteritis were reported to the National Institute for Public Health and the Environment in the Netherlands as a part of ongoing surveillance. Sapoviruses were found to be the most likely cause of 19 outbreaks (4%). During the same 2-year period, sapovirus infections were reported in Sweden, Slovenia, and Hungary. In the Netherlands, further characterization of outbreak strains showed that 12 (63%) sapovirus outbreaks were caused by genotype I.2 viruses. Most patients were adults older than 60 years (range, 1 to 100 years). Phylogenetic analysis using all presently available SaV sequences showed high homology between genotype I.2 strains detected in different geographical regions (Sweden, Slovenia, Taiwan, Japan, and Russia) since 2007. These first reported outbreaks of sapovirus infections in adults in the Netherlands were remarkable. Detection of identical genotypes in many samples might suggest that these viruses have the same origin, and since the infection is spreading fast, the prevalence of sapovirus infection may be increasing. The incidence of sapovirus infections in these countries suggests that a substantial part of Europe is affected by this virus. Copyright

Clostridium difficile is not associated with outbreaks of viral gastroenteritis in the elderly in the Netherlands (Article)

2010-06-01T00:00:00Z

The coincidental increase in norovirus outbreaks and Clostridium difficile infection (CDI) raised the question of whether these events could be related, e.g. by enhancing spread by diarrhoeal disease outbreaks. Therefore, we studied the prevalence of C. difficile in outbreaks of viral gastroenteritis in nursing homes for the elderly and characterised enzyme immunoassay (EIA)-positive stool samples. Stool samples from nursing home residents (n=752) in 137 outbreaks of viral aetiology were investigated by EIA for the presence of C. difficile toxins. Positive samples were further tested by a cell neutralisation cytotoxicity test, a second EIA and culture. Cultured isolates were tested for the presence of toxin genes, the production of toxins and characterised by 16S rRNA polymerase chain reaction (PCR) and sequencing. Twenty-four samples (3.2%) tested positive in the EIA. Of these 24 positive samples, only two were positive by cytotoxicity and three by a second EIA. Bacterial culture of 21 available stool samples yielded a toxinogenic C. difficile PCR ribotype 001 in one patient sample only. In conclusion, we found no evidence in this retrospective study for an association between viral gastroenteritis outbreaks and C. difficile. The high rate of false-positive EIA samples emphasises the need for second confirmation tests to diagnose CDI.

High probability of avian influenza virus (H7N7) transmission from poultry to humans active in disease control on infected farms (Article)

2010-05-01T00:00:00Z

An epizootic of avian influenza (H7N7) caused a large number of human infections in The Netherlands in 2003. We used data from this epizootic to estimate infection probabilities for persons involved in disease control on infected farms. Analyses were based on databases containing information on the infected farms, person-visits to these farms, and exposure variables (number of birds present, housing type, poultry type, depopulation method, period during epizootic). Case definition was based on self-reported conjunctivitis and positive response to hemagglutination inhibition assay. A high infection probability was associated with clinical inspection of poultry in the area surrounding infected flocks (7.6%; 95% confidence interval [CI], 1.4%-18.9%) and active culling during depopulation (6.2%; 95% CI, 3.7%-9.6%). Low probabilities were estimated for management of biosecurity (0.0%; 95% CI, 0.0%-1.0%) and cleaning assistance during depopulation (0.0%; 95% CI, 0.0%-9.2%). No significant association was observed between the probability of infection and the exposure variables.

Introduction of virulence markers in PB2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission (Article)

2010-04-01T00:00:00Z

In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans. Copyright

Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling (Article)

2010-03-01T00:00:00Z

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans. Copyright

Corrigendum to "Strengthening the diagnostic capacity to detect Bio Safety Level 3 organisms in unusual respiratory viral outbreaks" [J. Clin. Virol. 45 (2009) 185-190] (DOI:10.1016/j.jcv.2009.05.024) (Article)

2010-02-01T00:00:00Z

Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase (Article)

2010-01-01T00:00:00Z

Background: Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. Objectives: We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. Study design: Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. Results: 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. Conclusions: We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.

Noroviruses in healthcare settings: a challenging problem (Article)

2009-12-01T00:00:00Z

Current knowledge about noroviruses in relation to healthcare-associated infections (HCAIs) merely scratches the surface. Most data come from outbreak-based studies, which represent only a small piece of the puzzle. Nevertheless, the data available show that the clinical impact of noroviruses is particularly severe in outbreaks in healthcare settings, and that it may be increasing. Coupled with the projected increases of the population aged >65 years, especially those needing healthcare, it is timely to consider noroviruses in discussions around HCAIs. In particular, broadening the scope from a field mostly discussing bacteriological problems and a more holistic approach to dealing with HCAIs may be needed to avoid introducing new risks when trying to deal with the antimicrobial resistance problem.

Shedding of vaccine viruses with increased antigenic and genetic divergence after vaccination of newborns with monovalent type 1 oral poliovirus vaccine (Article)

2009-09-22T00:00:00Z

For the final stages in the eradication of poliovirus type 1 (P1), the World Health Organization advocates the selective use of monovalent type 1 oral poliovirus vaccine (mOPV1). To compare the immunogenicity of mOPV1 with that of trivalent OPV (tOPV) in infants, a study was performed in Egypt in 2005. Newborns were vaccinated with mOPV1 or tOPV immediately after birth and were challenged with mOPV1 after 1 month. Vaccination with mOPV1 at birth resulted in significantly higher seroconversion against P1 viruses and lower excretion of P1 viruses than vaccination with tOPV. Intratypic differentiation of the viruses shed by the newborns revealed the presence of remarkably high numbers of antigenically divergent (AD) P1 isolates, especially in the mOPV1 study group. The majority of these AD P1 isolates (71%) were mOPV1 challenge derived and were shed by newborns who did not seroconvert to P1 after the birth dose. Genetic characterization of the viruses revealed that amino acid 60 of the VP3 region was mutated in all AD P1 isolates. Isolates with substitution of residue 99 of the VP1 region had significantly higher numbers of nonsynonymous mutations in the VP1 region than isolates without this substitution and were preferentially shed in the mOPV1 study group. The widespread use of mOPV1 has proven to be a powerful tool for fighting poliovirus circulation in the remaining areas of endemicity. This study provides another justification for the need to achieve high vaccination coverage in order to prevent the circulation of AD strains. Copyright

Dynamics of antiviral-resistant influenza viruses in the Netherlands, 2005-2008 (Article)

2009-09-01T00:00:00Z

In the Netherlands, influenza specific antivirals are used for the therapy of influenza in nursing homes and hospitals, for prophylaxis in high risk groups and neuraminidase inhibitors are stockpiled as part of pandemic preparedness plans. To monitor the antiviral susceptibility profile, human influenza virus isolates derived from the Dutch influenza surveillance in 2005-2006 (n = 87), 2006-2007 (n = 58) and 2007-2008 (n = 128) were analyzed with phenotypic assays and sequencing. For adamantanes, a high proportion (>74%) of A(H3N2) viruses had the S31N mutation in M2 protein, while variation in the HA1region of adamantane-sensitive viruses suggested that adamantane-sensitive variants were reseeded into the Dutch population and re-emerged as drug-sensitive due to M-segment reassortment. For neuraminidase inhibitors oseltamivir and zanamivir, 98% of types A and B influenza viruses prior to 2007-2008 were sensitive for both, whereas 24% of the A(H1N1) viruses obtained in 2007-2008 were oseltamivir-resistant while retaining sensitivity to zanamivir and adamantanes. Furthermore, oseltamivir-resistant A(H1N1) or adamantane-resistant A(H3N2) virus infections were not associated with differences in clinical symptoms compared to infections with sensitive variants. Our data show the dynamic nature of emergence of drug-resistant influenza viruses, stressing the need for surveillance of resistance trends as part of influenza monitoring programs.

Norovirus illness is a global problem: emergence and spread of norovirus gii.4 variants, 2001-2007 (Article)

2009-09-01T00:00:00Z

Background. Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII.4. We studied the global patterns of GII.4 epidemiology in relation to its genetic diversity. Methods. Data from NoV outbreaks with dates of onset from January 2001 through March 2007 were collected from 15 institutions on 5 continents. Partial genome sequences (n = 775) were collected, allowing phylogenetic comparison of data from different countries. Results. The 15 institutions reported 3098 GII.4 outbreaks, 62% of all reported NoV outbreaks. Eight GII.4 variants were identified. Four had a global distribution-the 1996, 2002, 2004, and 2006b variants. The 2003Asia and 2006a variants caused epidemics, but they were geographically limited. Finally, the 2001 Japan and 2001Henry variants were found across the world but at low frequencies. Conclusions. NoV epidemics resulted from the global spread of GII.4 strains that evolved under the influence of population immunity. Lineages show notable (and currently unexplained) differences in geographic prevalence. Establishing a global NoV network by which data on strains with the potential to cause pandemics can be rapidly exchanged may lead to improved prevention and intervention strategies.

Epidemiology of enterovirus 71 in the Netherlands, 1963 to 2008 (Article)

2009-09-01T00:00:00Z

The incidence of enterovirus 71 (EV71) infection has greatly increased in the Asian Pacific region since 1997. Several large outbreaks, caused by different subgenogroups of EV71, occurred with high rates of morbidity and a substantial number of deaths. In 2007, 58 cases of EV71 infection requiring hospitalization were reported in The Netherlands after a period of low endemicity of 21 years. These events triggered a study on the epidemiology of EV71 in The Netherlands. Genetic analysis of the VP1 capsid region of 199 EV71 isolates collected from 1963 to 2008 as part of enterovirus surveillance activities revealed a change in the prevailing subgenogroups over time. From 1963 to 1986 infections were caused by three different and successive lineages belonging to subgenogroup B (the novel lineage designated B0, as well as B1 and B2). In 1987, following a major epidemic the previous year, the B genogroup was replaced by genogroup C strains of lineages C1 and, later, C2. Analyses of the clinical data suggested that there were differences between infection with genogroup B and with genogroup C strains in terms of the age groups affected and the severity of illness. From comparative analysis with genomic data available in the public domain, we concluded that EV71 strain evolution shows a global pattern, which leads to the question of whether the recently emerged C4 lineage strains will also spread outside of Asia. Copyright

Response to imported case of marburg hemorrhagic fever, the Netherlands (Article)

2009-08-01T00:00:00Z

On July 10, 2008, Marburg hemorrhagic fever was con-firmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classi-fication of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact.

Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets (Article)

2009-07-24T00:00:00Z

The swine-origin A(H1N1) influenza virus that has emerged in humans in early 2009 has raised concerns about pandemic developments. In a ferret pathogenesis and transmission model, the 2009 A(H1N1) influenza virus was found to be more pathogenic than a seasonal A(H1N1) virus, with more extensive virus replication occurring in the respiratory tract. Replication of seasonal A(H1N1) virus was confined to the nasal cavity of ferrets, but the 2009 A(H1N1) influenza virus also replicated in the trachea, bronchi, and bronchioles. Virus shedding was more abundant from the upper respiratory tract for 2009 A(H1N1) influenza virus as compared with seasonal virus, and transmission via aerosol or respiratory droplets was equally efficient. These data suggest that the 2009 A(H1N1) influenza virus has the ability to persist in the human population, potentially with more severe clinical consequences.

Strengthening the diagnostic capacity to detect Bio Safety Level 3 organisms in unusual respiratory viral outbreaks (Article)

2009-07-01T00:00:00Z

Background: Experience with a highly pathogenic avian influenza outbreak in the Netherlands (2003) illustrated that the diagnostic demand for respiratory viruses at different biosafety levels (including BSL3), can increase unexpectedly and dramatically. Objectives: We describe the measures taken since, aimed at strengthening national laboratory surge capacity and improving preparedness for dealing with diagnostic demand during outbreaks of (emerging) respiratory virus infections, including pandemic influenza virus. Study design: Academic and peripheral medical-microbiological laboratories collaborated to determine minimal laboratory requirements for the identification of viruses in the early stages of a pandemic or a large outbreak of avian influenza virus. Next, an enhanced collaborative national network of outbreak assistance laboratories (OAL) was set up. An inventory was made of the maximum diagnostic throughput that this network can deliver in a period of intensified demand. For an estimate of the potential magnitude of this surge demand, historical counts were calculated from hospital- and physician-based registries of patients presenting with respiratory symptoms. Results: Number of respiratory physician-visits ranged from 140,000 to 615,000 per month and hospitalizations ranged from 3000 to 11,500 per month. The established OAL-network provides rapid diagnostic response with agreed quality requirements and a maximum throughput capacity of 1275 samples/day (38,000 per month), assuming other routine diagnostic work needs to be maintained. Conclusions: Thus surge demand for diagnostics for hospitalized cases (if not distinguishable from other respiratory illness) could be handled by the OAL network. Assessing etiology of community acquired acute respiratory infection however, may rapidly exceed the capacity of the network. Therefore algorithms are needed for triaging for laboratory diagnostics; currently this is not addressed in pandemic preparedness plans.

Preparing the outbreak assistance laboratory network in the Netherlands for the detection of the influenza virus A(H1N1) variant (Article)

2009-07-01T00:00:00Z

Background: Late April 2009, human infection with variant influenza virus A(H1N1)v emerged in the Northern Americas posing a threat that this virus may become the next pandemic influenza virus. Objectives: To prepare laboratories for surge capacity for molecular diagnosis of patients suspected for A(H1N1)v infection in the Netherlands. Study design: A panel of 10 blinded specimens containing seasonal A(H1N1) or A(H3N2), or A/Netherlands/602/2009(H1N1)v influenza virus, or negative control was distributed to the outbreak assistance laboratories (OAL) together with influenza virus A (M-gene), swine influenza virus A (NP-gene) and influenza virus A(H1N1)v (H1v-gene) specific primers and probes and protocol (CDC Atlanta, USA). Laboratories were asked to implement and test this protocol. Results: All OAL were able to detect A(H1N1)v using the CDC M-gene reagents, the majority with similar sensitivity as the in-house M-gene based assays. RT-PCRs used in routine diagnostic setting in the OAL specifically designed to detect H1, H3, or NS1 from seasonal influenza A viruses, did not or at very low level cross-react with A(H1N1)v. The CDC swine NP-gene and H1v-gene RT-PCRs showed somewhat reduced sensitivity compared to the CDC and in-house M-gene RT-PCRs. In contrast, in-house developed A(H1N1)v specific H1v-gene and N1v-gene RT-PCRs showed equal sensitivity to CDC and in-house M-gene RT-PCRs. Conclusions: The Dutch OAL are prepared for detection and specific identification of A(H1N1)v, although some level of cross-reactivity was observed with seasonal influenza viruses. Additionally, M-gene based generic influenza A virus detection is recommended to be able to detect emerging influenza A viruses in routine settings.

A new generic real-time reverse transcription polymerase chain reaction assay for vesiviruses; vesiviruses were not detected in human samples (Article)

2009-04-01T00:00:00Z

Different viruses belonging to the genus Vesivirus infect a broad range of animals, and cause gastroenteritis, vesicular skin lesions, hemorrhagic disease, respiratory diseases and other conditions. A recent report on Vesivirus viremia, as detected by PCR, in samples from patients with hepatitis of unknown etiology in the USA suggested a zoonotic potential for vesiviruses. These results have not been confirmed by another laboratory. In order to do so, a generic PCR assay on the RNA polymerase region was developed, and validated with RNA from 69 different Vesivirus species. Except SMSV serotype-8, all species tested were detected, including the ones that were suggested to be involved in zoonotic transmission in the USA (SMSV serotype-5). The generic Vesivirus assay was used on RNA extracted from serum samples from patients with hepatitis, stool samples from patients with gastroenteritis, throat-swab specimens of patients with rash illnesses, throat-swab and nose-swabs of patients with acute respiratory diseases, and cell cultures with cytopathologic effect from enterovirus surveillance in which no pathogen was found. None were found positive. In this study a generic Vesivirus assay was developed and it was concluded that vesiviruses are an unlikely cause of common illnesses in humans in the Netherlands.

Norovirus in a Dutch tertiary care hospital (2002-2007): frequent nosocomial transmission and dominance of GIIb strains in young children (Article)

2009-03-01T00:00:00Z

We report a retrospective analysis of norovirus (NoV) infections occurring in patients of a tertiary care hospital during five winter seasons (2002/03 to 2006/07). Data were compared with national surveillance data and with corresponding data for rotavirus. Between July 2002 and June 2007, faecal specimens from 221 (9.0%) of 2458 hospital patients with diarrhoea tested positive for NoV. The incidence in children varied from 2.52 per 1000 admissions in 2004/05 (when testing began to be performed routinely) to 11.9 per 1000 admissions in 2006/07, while the incidence in adults remained stable (mean: 1.49 per 1000 admissions). Two genotypes predominated during the study period: GIIb strains occurred mainly in children below the age of two-and-a-half years [odds ratio (OR): 14.7; P < 0.0001] whereas GII.4 strains affected all age groups. Compared with rotavirus infections, NoV infections in children were more often hospital-acquired (59% vs 39%, OR: 2.29; P < 0.01). Among these cases we identified 22 clusters of NoV infection among inpatients. Twelve of 53 patients from whom follow-up samples were available demonstrated long-term virus shedding. We report a dynamic pattern of sporadic NoV infections in large hospitals, with frequent nosocomial transmission and with the predominance of GIIb-related strains in children. Effective prevention strategies are required to reduce the impact of sporadic NoV infection in vulnerable patients.

High prevalence of prolonged norovirus shedding and illness among hospitalized patients: A model for in vivo molecular evolution (Article)

2008-10-01T00:00:00Z

During a 2-year survey in an academic hospital, 8 (8.4%) of all norovirus (NoV)-positive patients showed prolonged norovirus illness and shedding (duration, 21-182 days). All patients had underlying illnesses, resulting in some level of immunodeficiency in 5. Four patients were admitted to the hospital with gastroenteritis, 2 acquired norovirus while hospitalized, and 2 were outpatients. Genotypes GII.4 and GIIb-GII.3 were found. Reinfection occurred in 3 patients. Full capsid sequences were determined from strains detected in sequentially collected stool specimens to study evolution. The greatest number of amino acid mutations in a given patient was 11; they were detected in NoV isolates recovered over a 119-day period and were mapped to positions at or near putative antigenic sites. In the patient with most severe immune dysfunction, only 5 amino acids mutated over 182 days, suggesting immune-driven selection. The severe impact on patients and hospitals and the potential role of prolonged shedders as a reservoir for viral antigenic variants lead us to stress the importance of confinement of outbreaks of NoV infection that occur in hospitals.

Prevalence of human parechovirus in The Netherlands in 2000 to 2007 (Article)

2008-09-01T00:00:00Z

Infection with human parechovirus 3 (HPeV3) was described for the first time in Japan in 2004 and reportedly is more often associated with severe disease than infection with HPeV1 or HPeV2. In 2004, infections with HPeV3 were observed for the first time in The Netherlands. Genetic analysis showed several different lineages, suggesting endemic circulation. We analyzed 163 cell culture isolates from the same number of patients tested in routine virological laboratories as part of the national enterovirus surveillance program. Isolates were collected between 2000 and 2007 and could not be characterized by routine methods. In total, 155 isolates (95%) were found positive for HPeV by a reverse transcription-PCR assay targeting the 5′ untranslated region, explaining the majority of the diagnostic deficit in enterovirus surveillance for these years. Typing of the isolates by use of partial genome sequencing of the VP1/2A region revealed the presence of 55 HPeV1, 2 HPeV2, 89 HPeV3, 1 HPeV4, and 8 HPeV5 isolates. We compared isolation dates, age groups affected, and clinical pictures, which were reported as part of the routine surveillance. Clear differences in epidemiology were observed, with HPeV3 occurring at intervals of 2 years and in the spring-summer season, whereas HPeV1 was observed in small numbers throughout each year, with a low in the summer months. HPeV3 infection affected younger children than HPeV1 infection and was significantly more often associated with fever, meningitis, and viremia. Copyright

Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006 (Article)

2007-09-01T00:00:00Z

Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains. Copyright

Bolletjes slikken (Inaugural Lecture)

2007-03-08T00:00:00Z

Rede, in verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar in de virologie aan het Erasmus faculteit van de Erasmus Universiteit Rotterdam op 8 maart 2007

Systemic immune response after rotavirus inoculation of neonatal mice depends on source and level of purification of the virus: Implications for the use of heterologous vaccine candidates (Article)

2007-02-01T00:00:00Z

Rotavirus is an important cause of morbidity and mortality worldwide and vaccines are currently under development, with clinical trails conducted in humans worldwide. The immune responses in infant BALB/c mice were examined following oral inoculation with murine rotavirus EDIM (2 × 104focus-forming units) and with three CsCl gradient-purified fractions of heterologous simian rotavirus SA11 (standardized at 2 × 106CCID50) that differed in antigen composition: fraction 1 was enriched for double-layered rotavirus particles, fraction 2 for triple-layered particles and fraction 3 consisted mainly of cell components. Diarrhoea and high IgG responses, but marginal IgA responses, were observed after inoculation with all three SA11 fractions. Virus shedding was observed in all EDIM-inoculated mice, but in none of the SA11 -inoculated mice. Rotavirus-specific IgG1: 2a ratios were similar in mice inoculated with EDIM and SA11 fraction 1, but higher for SA11 fraction 3- and lower for SA11 fraction 2-inoculated mice. A higher IgG1:2a ratio indicates a more Th2-like immune response. This undesirable response is apparently mostly induced by inoculation with heterologous rotavirus in the presence of abundant cell-associated and soluble rotavirus proteins, compared with infection with a more purified preparation or with homologous virus. These data show that, following inoculation with a standardized amount of infectious virus, the composition of the fraction influences the outcome of the immune responses significantly.

General practitioner practices in requesting laboratory tests for patients with gastroenteritis in the Netherlands, 2001–2002 (Article)

2006-10-02T00:00:00Z

Background: The objective of this study was to estimate the (selective) proportion of patients consulting their GP for an episode of gastroenteritis for whom laboratory tests were requested. In addition adherence of GPs to the guidelines for diagnostic test regime was ascertained. Methods: Data were collected from a GP network in the Netherlands. Information was also collected on the reason for requesting the test, test specifications, and test results. Results: For 12% of the GP patients with gastroenteritis, a stool sample was requested and tested for enteric pathogens. In most patients, the duration, followed by severity of complaints or a visit to a specific, high-risk country were reported as reasons to request laboratory diagnostics. Tests were requested most often in summer months and in February. Campylobacter (requested for 87% of the tests), Salmonella (84%), Shigella (78%) and Yersinia (56%) were most frequently included in the stool tests. Campylobacter was detected most often in patients. Conclusion: Test requests did not always comply with existing knowledge of the etiology of gastroenteritis in GP patients and were not always consistent with the Dutch GP guidelines. Therefore, the data of this study can be used to develop educational approaches for GP's as well as for revision of the guidelines.

Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands (Article)

2004-02-21T00:00:00Z

Background: An outbreak of highly pathogenic avian influenza A virus subtype H7N7 started at the end of February, 2003, in commercial poultry farms in the Netherlands. Although the risk of transmission of these viruses to humans was initially thought to be low, an outbreak investigation was launched to assess the extent of transmission of influenza A virus subtype H7N7 from chickens to humans. Methods: All workers in poultry farms, poultry farmers, and their families were asked to report signs of conjunctivitis or influenza-like illness. People with complaints were tested for influenza virus type A subtype H7 (A/H7) infection and completed a health questionnaire about type of symptoms, duration of illness, and possible exposures to infected poultry. Findings: 453 people had health complaints—349 reported conjunctivitis, 90 had influenza-like illness, and 67 had other complaints. We detected A/H7 in conjunctival samples from 78 (26·4%) people with conjunctivitis only, in five (9·4%) with influenza-like illness and conjunctivitis, in two (5·4%) with influenza-like illness only, and in four (6%) who reported other symptoms. Most positive samples had been collected within 5 days of symptom onset. A/H7 infection was confirmed in three contacts (of 83 tested), one of whom developed influenza-like illness. Six people had influenza A/H3N2 infection. After 19 people had been diagnosed with the infection, all workers received mandatory influenza virus vaccination and prophylactic treatment with oseltamivir. More than half (56%) of A/H7 infections reported here arose before the vaccination and treatment programme. Interpretation: We noted an unexpectedly high number of transmissions of avian influenza A virus subtype H7N7 to people directly involved in handling infected poultry, and we noted evidence for person-to-person transmission. Our data emphasise the importance of adequate surveillance, outbreak preparedness, and pandemic planning.

Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. (Article)

2004-02-03T00:00:00Z

Highly pathogenic avian influenza A viruses of subtypes H5 and H7 are the causative agents of fowl plague in poultry. Influenza A viruses of subtype H5N1 also caused severe respiratory disease in humans in Hong Kong in 1997 and 2003, including at least seven fatal cases, posing a serious human pandemic threat. Between the end of February and the end of May 2003, a fowl plague outbreak occurred in The Netherlands. A highly pathogenic avian influenza A virus of subtype H7N7, closely related to low pathogenic virus isolates obtained from wild ducks, was isolated from chickens. The same virus was detected subsequently in 86 humans who handled affected poultry and in three of their family members. Of these 89 patients, 78 presented with conjunctivitis, 5 presented with conjunctivitis and influenza-like illness, 2 presented with influenza-like illness, and 4 did not fit the case definitions. Influenza-like illnesses were generally mild, but a fatal case of pneumonia in combination with acute respiratory distress syndrome occurred also. Most virus isolates obtained from humans, including probable secondary cases, had not accumulated significant mutations. However, the virus isolated from the fatal case displayed 14 amino acid substitutions, some of which may be associated with enhanced disease in this case. Because H7N7 viruses have caused disease in mammals, including horses, seals, and humans, on several occasions in the past, they may be unusual in their zoonotic potential and, thus, form a pandemic threat to humans.

Een fatale infectie door aviair influenza-A(H7N7)-virus en aanpassing van het preventiebeleid (Article)

2004-01-01T00:00:00Z

In February 2003, the highly pathogenic avian influenza-A virus, subtype H7N7, was the causative agent of a large outbreak of fowl plague in the Netherlands. Two days after visiting a poultry farm that was infected by fowl plague, a 57-year-old male veterinarian developed malaise, headache and fever. After 8 days he was admitted to hospital with signs of pneumonia. Five days later, his condition deteriorated alarmingly. Despite extensive pharmacotherapy he died 4 days later of acute pneumonia. Influenza-A virus, subtype H7N7, was identified by means of reverse transcriptase/PCR in broncho-alveolar washings that had been obtained earlier; routine virus culture yielded the isolate A/Nederland/219/03, which differs by 14 amino-acid substitutions from the first isolate in a chicken (A/kip/Nederland/1/03). Partly as a result of this case, the preventive measures were then adjusted; people who came into contact with infected poultry were given increased possibilities for vaccination and the administration of oseltamivir.

Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice (Article)

2003-01-01T00:00:00Z

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.

The Incidence and Genetic Variability of Small Round-Structured Viruses in Outbrreaks of Gastroenteritis in The Netherlands (Article)

1997-01-01T00:00:00Z

The Role of Small Round-Structured Viruses (SRSV) in Outbreaks of Gastroenteritis in The Netherlands (In Proceedings)

1996-01-01T00:00:00Z