Hepatitis C virus prevalence in The Netherlands: migrants account for most infections.

A population-based anti-hepatitis C virus (HCV) prevalence is important for surveillance purposes and it provides insight into the burden of disease. The outcomes of recent studies in the general Dutch population as well as recent HCV data from specific risk groups including migrants, men who have sex with men (MSM) and injecting drug users (IDUs), were implemented in a modified version of the Workbook Method (a spreadsheet originally designed for HIV estimations), to estimate Dutch HCV seroprevalence. The estimated national seroprevalence of HCV was 0·22% (min 0·07%, max 0·37%), corresponding to 28 100 (min n = 9600, max n = 48 000) HCV-infected individuals in The Netherlands. Of these, first-generation migrants from HCV-endemic countries (HCV prevalence ≥2%) accounted for the largest HCV-infected group, followed by IDUs and HIV-positive MSM.

Prevalence of genital HPV infections and HPV serology in adolescent girls, prior to vaccination.

INTRODUCTION: Monitoring the prevalence of type-specific HPV-DNA infections before and shortly after introduction of routine HPV vaccination offers the opportunity to evaluate early effects of the vaccination program. With this aim a cohort study was set up of 14- to 16-year-old girls eligible for HPV vaccination in the Netherlands. Annually, HPV-DNA and antibody status in vaginal self-samples and in serum respectively, will be studied among vaccinated (58%) and unvaccinated girls (42%). Here we present baseline data on vaginal HPV-DNA status in relation to serum antibodies. METHODS: The 1800 enrolled girls filled out an internet-based questionnaire and provided a vaginal self-sample for genotype specific HPV-DNA detection using SPF(10) PCR amplification and reverse line probe hybridization. Furthermore, 64% of the girls provided a blood sample for HPV antibody analysis. IgG antibodies against virus-like particles were determined for 7 HPV genotypes. RESULTS: At baseline, type-specific HPV-DNA was detected in 4.4% (n = 79) of the 1800 girls: 2.7% (n = 49) concerned a high risk HPV type (hrHPV-DNA). The three most common types were HPV type 16, 18 and 51 (40%). Out of the hrHPV-DNA positive girls, 32% was seropositive vs. 12% in HPV-DNA negative girls (p<0.001). Risk factors independently associated with hrHPV-DNA infection among the sexually active girls were age >15 years vs. 14-15 years (OR = 2.6 (1.2-5.9)), age of sexual debut <14 vs. above 14 years (OR = 3.0 (1.1-8.2)), total number of lifetime partners above two vs. less than two partners (OR = 3.2 (1.3-8.0)) and age of partner >17 vs. under 17 years (OR = 4.2 (1.5-13.0)). CONCLUSION: A low hrHPV-DNA prevalence was found in the adolescent girls. The observed vs. expected age-related increase in HPV-DNA prevalence in this cohort in the coming years (with increased sexual activity) will provide understanding of the effect of HPV vaccination. Furthermore, this cohort study will offer the opportunity to improve knowledge of antibody responses following natural infection and vaccination.

Comparison of the hepatitis B virus core, surface and polymerase gene substitution rates in chronically infected patients.

For phylogenetic comparison of hepatitis B virus (HBV) isolates, often a region of the HBV surface gene is analysed. Because the surface gene completely overlaps the polymerase gene, its evolution is constrained, and it may not be the best choice for genetic comparison of HBV isolates. Analysing serial sample pairs of 33 chronically HBV-infected, untreated patients, with a cumulative follow-up of 184 years, the synonymous and nonsynonymous substitution rates of a part of the overlapping HBV surface and polymerase genes were compared to those of a nonoverlapping part of the HBV core gene. The substitution rate of the HBV core gene was higher (8.15 × 10(-4) vs 4.57 × 10(-4) substitutions/site/year) than that of the surface gene. The difference was mainly due to a significantly lower synonymous substitution rate in the surface gene, with dN/dS ratios of 0.412 in the core gene and 0.986 in the surface gene. Contrary to the core gene, the number of substitutions in the surface gene was higher in low viraemic hosts, who control HBV infection by suppressing replication. The number of substitutions in the core gene correlated more strongly with the duration of follow-up. The overlapping HBV surface and polymerase genes experience strong negative selection, which limits the number of substitutions. Because the HBV core gene reflects the duration of infection more accurately, it is more suitable for the analysis of short-term viral evolution and of hepatitis B transmission chains.

Prevalence of hepatitis B virus infection in The Netherlands in 1996 and 2007.

We aimed to assess differences in the prevalence of hepatitis B virus (HBV) infection in The Netherlands between 1996 and 2007, and to identify risk factors for HBV infection in 2007. Representative samples of the Dutch population in 1996 and 2007 were tested for antibodies to hepatitis B core antigen (anti-HBc), hepatitis B surface antigen (HBsAg) and HBV-DNA. In 2007, the weighted anti-HBc prevalence was 3·5% (95% CI 2·2-5·5) and the HBsAg prevalence was 0·2% (95% CI 0·1-0·4). In indigenous Dutch participants, the anti-HBc prevalence was lower in 2007 than in 1996 (P=0·06). First-generation migrants (FGMs) had a 13-fold greater risk of being HBsAg- and/or HBV-DNA-positive than indigenous Dutch participants. In indigenous Dutch participants, risk factors for anti-HBc positivity were older age and having received a blood product before 1990. In FGMs, being of Asian origin was a risk factor. In second-generation migrants, having a foreign-born partner and injecting drug use were risk factors. FGMs are the main target group for secondary HBV prevention in The Netherlands.

Mumps epidemic in orthodox religious low-vaccination communities in the Netherlands and Canada, 2007 to 2009.

We assessed the epidemiological characteristics of a mumps virus epidemic (genotype D) that occurred in the Netherlands between August 2007 and May 2009 and its association with a subsequent mumps outbreak in Canada. In the Netherlands, five data sources were used: notifications (only mandatory since the end of 2008) (56 cases), laboratory confirmation data (177 cases), a sentinel general practitioner (GP) database (275 cases), hospitalisation data (29 cases) and weekly virological reports (96 cases). The median age of cases in the notification, laboratory and GP databases ranged from 13 to 15 years. The proportion of cases that were unvaccinated ranged from 65% to 85% in the notification, laboratory and GP databases. Having orthodox Protestant beliefs was the main reason for not being vaccinated. In Canada, a mumps virus strain indistinguishable from the Dutch epidemic strain was detected between February and October 2008 in an orthodox Protestant community with historical and family links to the affected community in the Netherlands, suggesting that spread to Canada had occurred. Prevention and control of vaccine-preventable diseases among population subgroups with low vaccination coverage remains a priority.

Changing risk profile of hepatitis A in The Netherlands: a comparison of seroprevalence in 1995-1996 and 2006-2007.

The prevalence of antibodies to hepatitis A virus (HAV) was assessed in a nationwide sample (n=6229) in The Netherlands in 2006-2007, and compared to the seroprevalence in a similar study in 1995-1996 (n=7376). The overall seroprevalence increased from 34% in 1995-1996 to 39% in 2006-2007, mainly due to vaccination of travellers and an increased immigrant population. Risk factors remain travelling to, and originating from, endemic regions, and vaccination is targeted currently at these risk groups. Our results show a trend of increasing age of the susceptible population. These people would also benefit from HAV vaccination because they are likely to develop clinically serious symptoms after infection, and are increasingly at risk of exposure through imported viruses through foods or travellers. The cost-effectiveness of adding elderly people born after the Second World War as a target group for prophylactic vaccination to reduce morbidity and mortality after HAV infection should be assessed.

HBsAg-negative mono-infection with hepatitis B virus genotype G.

Infection with a genotype G strain of hepatitis B virus (HBV-G) often occurs as a co-infection with HBV genotype A. In mono-infection with HBV-G, the production of hepatitis B surface antigen (HBsAg), HBe antigen and anti-HBe seems diminished, hampering the serological diagnosis of HBV-G mono-infection. To corroborate this notion, we studied in detail a series of samples of a blood donor with transient HBV-G infection. In this donor, during the temporary presence of HBV DNA and the seroconversion to HBcore antibodies (anti-HBc), no HBsAg or hepatitis B e antigen was detected. During follow-up, no anti-HBe appeared. Multiple resistance mutations to lamivudine were present, demonstrating primary infection with a resistant HBV strain. Cloning and sequencing indicated that no other HBV genotype but genotype G was present. Like other HBV-G isolates, the DNA sequence of the HBsAg a-determinant showed no mutations that could explain the failure to detect HBsAg. Our findings demonstrate that HBV genotype G mono-infection occurs and that routine serology is unsuitable for its detection.

Persistent and transient hepatitis B virus (HBV) infections in children born to HBV-infected mothers despite active and passive vaccination.

Combined passive and active immunization for newborns very effectively prevents perinatal hepatitis B virus (HBV) infections. In the Netherlands, babies born to hepatitis B surface antigen (HBsAg)-positive women receive passive immunization with hepatitis B and at least three active HBsAg vaccinations. Serological testing for the presence of HBV markers was offered for all infants born to HBsAg-positive mothers between January 2003 and July 2007, after completion of their vaccination schedule. About 75% of the infants (n = 1743) completed their HB-vaccination schedule and participated in the serologic evaluation. Twelve of them (0.7%) were found to be HBV infected. Furthermore, we identified three older children with high levels of anti-HBc, anti-HBs and anti-HBe, while they were HBsAg and HBV DNA negative. This serologic profile is evidence for a resolved HBV infection. In the group of older children (1.5-5 years of age, n = 728), about half of the HBV-infected children (3 of 7) had already cleared their infection at the time of sampling. For a proper evaluation of the efficacy of a new intervention programme to prevent vertical HBV transmission, it is also important to analyse the HBV markers in serum collected when the children are older than 1.5 years. In a programmatic setting, all children born to HBV-infected mothers should be tested not only for the level of anti-HBs but also for the absence of HBsAg, because 2 of the 12 HBV-infected children (17%) had a high level of anti-HBs.

Hepatitis B vaccination targeted at behavioural risk groups in the Netherlands: does it work?

In November 2002, the Netherlands adopted a vaccination program targeted at behavioural risk groups. Between January 2003 and December 2007, 1386 patients acutely infected with HBV were reported. Reported cases declined from 326 in 2003 to 220 in 2007. Sexual intercourse was the most frequently reported mode of transmission (65%), especially among men having sex with men. Genotypes A and D remained predominant. In total, 40,600 participants were fully vaccinated, the overall compliance was 62%, and the estimated overall program coverage was 12% of the at-risk population. With more effort, more susceptibles may be reached, but the program will not be sufficient to substantially reduce HBV in the Netherlands. Therefore, universal vaccination should be considered.

A comparison of hepatitis B seroepidemiology in ten European countries.

To inform current and future vaccination strategies, we describe the seroepidemiology of hepatitis B virus (HBV) infection in ten representative European countries using standardized serology that allowed international comparisons. Between 1996 and 2003, national serum banks were compiled by collecting residual sera or by community sampling; sera were then tested by each country using its preferred enzyme immunoassays and testing algorithm, and assay results were standardized. Information on current and past HBV vaccination programmes in each country was also collected. Of the ten countries, six reported low levels (<3%) of antibodies against HBV core antigen (anti-HBc). Of the eight countries testing for HBV surface antigen (HBsAg), the highest prevalence was reported in Romania (5.6%) and in the remaining seven countries prevalence was <1%. Universal HBV vaccination programmes had been established in seven countries as recommended by the World Health Organization, but the seroprevalence of antibodies against HBsAg (anti-HBs) was lower than the reported vaccine coverage in three countries. Regular serological surveys to ascertain HBV status within a population, such as reported here, provide important data to assess the need for and to evaluate universal HBV vaccination programmes.

Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia (<10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An unusually large number of amino acid mutations was present in the immunodominant a-determinant of HBsAg (respectively 3, 6, 7, 10, and 10 mutations). Comparison of the HBV genomes in two donors to a consensus HBV genotype D sequence showed a most prominent hotspot of genetic variation in HBV nucleotides 480-570, encoding the HBsAg a-determinant. The phylogenetic comparison of separate donor HBV genes to the HBV genes of 11 reference strains (genotypes A-H) showed the donor HBV surface genes to form an outgroup, while the HBV polymerase, core and X genes closely cluster with the HBV genotype D reference strain. Maybe the HBV strains in this study represent a natural end-stage of seemingly cleared HBV infection, in which HBV maintains a low level of possibly non-infectious replication, after sacrificing its immunologically offending surface antigen, thus avoiding final clearance by the immune system.

Substitution rate of the hepatitis B virus surface gene.

Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10(3) to 10(9) cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0-8 per patient), resulting in an average substitution rate of 5.1 x 10(-4) nucleotide changes/site/year (range: 0-1.3 x 10(-2)). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA > or =10(8) cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 x 10(-4)), while in the 10 patients with viremia below 10(4) cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 x 10(-4)). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.

[Hepatitis B virus transmission patterns in the Netherlands, 2004]. / Transmissiepatronen van hepatitis B-virus in Nederland, 2004.

OBJECTIVE: To gain insight into hepatitis B virus (HBV) transmission in the Netherlands. DESIGN: Descriptive. METHOD: During 2004, epidemiological data and blood samples (if available) were collected for all reported cases of acute HBV infections in the Netherlands. Following DNA isolation and amplification a 648 base pairs fragment of the HBV S gene was sequenced and subjected to phylogenetic analysis. The sequencing details were also linked to epidemiological information. RESULTS: In 2004, 291 cases ofacute HBV infections were reported. Blood samples were received from 171 patients (59%), and the genotype could be determined for 158 patients (54%). 6 genotypes were identified: A (64%), B (3%), C (3%), D (21%), E (5%) and F (4%). Of all patients with genotype A, 52% had been infected via homosexual or bisexual contact and 16% via heterosexual contact. Of all patients with genotype D, 42% had been infected via heterosexual contact and 15% via homosexual or bisexual contact. The genotype A cluster was extremely homogeneous with many identical sequences, while genotype B-E clusters were more heterogeneous. 4 identical sequences were found within genotype F, but the patients could not be epidemiologically linked. CONCLUSION: Sexual transmission, particularly via homosexual or bisexual contact in men, formed the most important risk factor for acquiring an acute HBV infection. Genotype A was predominant in the Netherlands, especially among homosexual or bisexual men. Most infections within genotype D occurred as a result of heterosexual contact. The results show that there was ongoing transmission of HBV in homosexual or bisexual men, while in heterosexuals more cases of new introduction were seen, possibly via chronic carriers from areas where HBV is endemic.

[Hepatitis B surveillance in the Netherlands, 2002-2005: acute infection is mainly via sexual contact while chronic infection is via vertical transmission through mothers from endemic regions]. / Surveillance van hepatitis B in Nederland, 2002-2005: acute infectie vooral via seksueel contact, chronische via verticale transmissie door moeders uit endemische gebieden.

OBJECTIVE: To study the trends in the prevalence of hepatitis B infections in the Netherlands on the basis of reported cases. DESIGN: Retrospective, descriptive. METHOD: Analysis of data collected from the obligatory notification of hepatitis B to the Dutch Public Health Services in the Netherlands in the period 2002-2005. RESULTS: In the period from January 2002 to December 2005, 7352 hepatitis B virus (HBV) infections were reported, of which 1168 (16%) were acute and 5849 (80%) were chronic infections. Of the acute HBV infections, 34% were transmitted by homo- or bisexual contact and 25% by heterosexual contact. The number of reports of acute HBV infection due to heterosexual transmission increased significantly and originated relatively more often in Dutch patients. The number of reports of chronic HBV infection in men increased significantly; in women there was a decrease over time. Of the chronic HBV infections, 40% were transmitted from mother to child; this was reported especially often by patients from HBV endemic areas. CONCLUSION: Sexual contact was the most important risk factor for the transmission of acute HBV infections, whereas vertical transmission was the greatest risk factor by far for chronic HBV infection. Transmission via heterosexual contact had become increasingly important in the transmission of acute HBV; transmission by homo- or bisexual contact remained constant. Immigration continued to play an important role in the epidemiology of HBV in the Netherlands; the majority of the chronic carriers had been born and infected in an HBV endemic area.

Molecular epidemiology of acute hepatitis B in the Netherlands in 2004: nationwide survey.

To gain insight into hepatitis B virus (HBV) transmission in the Netherlands, epidemiological data and sera were collected from reported cases of acute HBV infections in the Netherlands in 2004. Cases were classified according to mode of transmission. A fragment of the S-gene of HBV (648 bp) was amplified, sequenced, and subjected to phylogenetic analysis. Of the 291 acute HBV cases reported in 2004, 158 (54%) were available for genotyping. Phylogenetic analysis identified 6 genotypes: A (64%), B (3%), C (3%), D (21%), E (5%) and F (5%). Of HBV infected men having sex with men, 86% were infected with genotype A, accounting for 43% of all patients infected with this genotype. There were only three reported cases of injecting drug use of which one was available for sequencing (genotype A). Unlike the genotype A cluster, sequences within the genotype B-E clusters were heterogenic. Within genotype F, several isolates had identical sequences, but patients could not be epidemiologically linked. Sexual transmission, particularly by men having sex with men was the most important transmission route for HBV. Injecting drug use plays a minor role. Genotype A is predominant in the Netherlands, especially among men having sex with men. In addition to imported strains, there seems to be a pool of related but non-identical strains circulating among chronic carriers in the migrant population, from which occasionally new patients are infected, primarily by heterosexual transmission.

The European Sero-Epidemiology Network 2: standardization of assay results for hepatitis B virus.

The aim of the European Sero-Epidemiology Network 2 was to coordinate and standardize the serological surveillance of vaccine-preventable diseases in Europe. In this study, the standardization of hepatitis B virus (HBV) results is described. The 15 participating national laboratories tested a unique panel of 172 sera established by the Greek reference centre for HBV surface antigen (HBsAg), antibodies to HBsAg (anti-HBs) and/or to the HBV core antigen (anti-HBc) by assay methods of their choice. Country-specific quantitative measurements for anti-HBs and anti-HBc were transformed into common units using standardization equations derived by regressing each country's panel results against the reference centre's results, thus adjusting for interassay and interlaboratory variability. For HBsAg, a qualitative analysis (positive/negative) showed at least 99% agreement with the reference laboratory for all countries. By combining these standardized and qualitative results for the markers mentioned earlier, it was possible to achieve comparable estimates of the proportion of the population susceptible to HBV, vaccinated against HBV, with a past HBV infection, and with a current infection or chronic carrier state. Standardization is a very important tool that allows for international serological comparisons to assess the current vaccination policies and the progress of HBV control in Europe.

[Vaccination against pneumococci and hepatitis B in the Dutch National Immunisation Programme]. / Pneumokokken- en hepatitis B-vaccinatie in het Rijksvaccinatieprogramma.

All infants in the Netherlands, which are born after March 2006, receive additional vaccinations at the age of 2, 3, 4 and 11 months to protect them against pneumococcal infections. During the same visit to a consultation bureau, the children also receive a combination vaccine against diphtheria, pertussis, tetanus, poliomyelitis and Haemophilus influenzae (DTPa-IPV-Hib). Children of which at least one parent was born in a country where hepatitis B occurs relatively often are also vaccinated in the Netherlands against hepatitis B. This currently pertains to about 15% of all newborns. These children now receive a new combination vaccine in which a hepatitis B component has been added to the DTPa-IPV-Hib components. They will receive this combination vaccine 4 times. This combination vaccine is given during the same visit as the pneumococcal vaccination. Although pneumococcal vaccination may have a somewhat negative effect on the immune response to hepatitis B, it is expected that the new 4-fold vaccination schedule will induce good and long-lasting protection against hepatitis B in the vast majority of the children. About 700 children are born out of mothers infected with hepatitis B each year in the Netherlands. In the new vaccination schedule, they now receive 5 active vaccinations against hepatitis B and are examined serologically on an individual basis in order to detect breakthrough infections. This will also generate greater insight into the efficacy of the different vaccination schemes and intervention programmes to prevent vertical transmission of the virus.

[Objective registration and interpretation of adverse events caused by vaccines: an essential element of vaccination]. / Objectieve registratie en interpretatie van vaccinbijwerkingen: een essentieel element van vaccinatie.

In this era of accelerated communication, information on adverse events following vaccination is spreading more rapidly and becoming accessible to more lay people than ever before. The pace of development and the introduction of new vaccines has also increased considerably in the last decade. Therefore, it is increasingly important to objectively register and interpret possible adverse events following vaccination. In the Netherlands, information on adverse events is collected through passive reporting systems. Two recent reports on adverse events have provided reassuring results, and no new types of adverse reactions were identified. Since the transition from whole-cell pertussis vaccines to acellular vaccines, children have experienced fewer adverse events. A univocal, familiar, and easily accessible system of passive reporting is required to maintain both confidence in the vaccination programme and high vaccination coverage. The current situation in which two separate organisations (the National Institute for Public Health and Environment (RIVM) and the Netherlands Pharmacovigilance Centre Lareb) are responsible for collecting and interpreting reported adverse events following vaccination is suboptimal.

The enhanced virulence of very virulent infectious bursal disease virus is partly determined by its B-segment.

There is a remarkable difference in virulence of infectious bursal disease virus (IBDV) strains ranging from sub-clinical infections for serotype 2 and cell culture adapted serotype 1 strains, to 100% mortality for very virulent serotype 1 strains in young SPF chickens. It is known that cell culture adaptation related attenuation is determined by distinct mutations in the hypervariable region of the VP2 outer capsid protein, encoded on the A-segment. Amino acid mutations in the hypervariable VP2 region however, offer no explanation for the difference in virulence of classical and very virulent serotype 1 strains. Here we show by in vitro and in vivo analysis of rescued segment re-asserted IBDVs that virulence factors are not only located on the A-segment, but on the RNA Dependent RNA Polymerase (VP1) encoding B-segment as well. Insight into the virulence factors of very virulent IBDV will contribute to the improvement of live IBDV vaccines.

VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.