Correlation between viral load, multiplicity of infection, and persistence of HPV16 and HPV18 infection in a Dutch cohort of young women.

BACKGROUND: Persistent high-risk human papillomavirus infection precedes the development of cervical cancer. Here we evaluated the contribution of HPV16/18 viral load and the presence of infections with multiple HPV types to persistence and clearance of HPV16/18 infections. METHODS: Vaginal self-swabs were obtained from young women (16-29 y) with one year interval. HPV genotyping was performed using the highly sensitive SPF10-DEIA-LiPA25 system. HPV16/18 DNA loads were quantified via an adapted, highly sensitive qPCR protocol targeting the L1 gene. RESULTS: We identified 227 HPV16 and 111 HPV18 infections with follow-up. For HPV16 132/227 (58%) were persistent and 95/227 cleared. For HPV18 49/111 (44%) infections were persistent and 62/111 cleared. Baseline viral load was significantly higher in persistent infections than in clearing infections for both HPV16 (p=0.022) and HPV18 (p=0.013). At baseline, only HPV16 viral load was significantly higher in multiple HPV infections compared with single infections (p=0.003). In logistic regression analysis HPV16 and HPV18 viral load were found to contribute to persistency with OR=1.279 (95%CI=1.074-1.524) and OR=1.256 (95%CI=1.028-1.533) per log-unit increase HPV16 and HPV18 viral load respectively. The presence of multiple HPV type infections was not associated with higher persistency. CONCLUSION: HPV16/18 viral load might be used as a marker for persisting infections and is affected by the presence of multiple HPV infections. Evaluation of these parameters at the population level may be of value to assess the presence of persistent or clearing HPV16/18 infections as an early marker, and may provide useful quantitative information in (epidemiological) vaccine monitoring studies.

Genetic Diversity in the Major Capsid L1 Protein of HPV-16 and HPV-18 in the Netherlands.

OBJECTIVES: Intratypic molecular variants of human papillomavirus (HPV) type-16 and -18 exist. In the Netherlands, a bivalent vaccine, composed of recombinant L1 proteins from HPV-16 and -18, is used to prevent cervical cancer since 2009. Long-term vaccination could lead to changes in HPV-16 and -18 virus population, thereby hampering vaccination strategies. We determined the genetic diversity of the L1 gene in HPV-16 and -18 viral strains circulating in the Netherlands at the start of vaccination in order to understand the baseline genetic diversity in the Dutch population. METHODS: DNA sequences of the L1 gene were determined in HPV-16 (n = 241) and HPV-18 (n = 108) positive anogenital samples collected in 2009 and 2011 among Dutch 16- to 24-year old female and male attendees of the sexually transmitted infection (STI) clinics. Phylogenetic analysis was performed and sequences were compared to reference sequences HPV-16 (AF536179) and HPV-18 (X05015) using BioNumerics 7.1. RESULTS: For HPV-16, ninety-five single nucleotide polymorphism (SNPs) were identified, twenty-seven (28%) were non-synonymous variations. For HPV-18, seventy-one SNPs were identified, twenty-nine (41%) were non-synonymous. The majority of the non-silent variations were located in sequences encoding alpha helix, beta sheet or surface loops, in particular in the immunodominant FG loop, and may influence the protein secondary structure and immune recognition. CONCLUSIONS: This study provides unique pre-vaccination/baseline data on the genetic L1 diversity of HPV-16 and -18 viruses circulating in the Netherlands among adolescents and young adults.

Correction: Genetic Diversity in the Major Capsid L1 Protein of HPV-16 and HPV-18 in the Netherlands.

[This corrects the article DOI: 10.1371/journal.pone.0152782.].

Six-month incidence and persistence of oral HPV infection in HIV-negative and HIV-infected men who have sex with men.

OBJECTIVES: Our aim was to assess incidence and persistence of oral HPV infection in HIV-negative and HIV-infected men who have sex with men (MSM). METHODS: MSM aged ≥18 years were included in Amsterdam (the Netherlands) in 2010-2011, and followed up 6 months later. Participants completed risk factor questionnaires. HPV DNA was analyzed in oral-rinse and gargle specimens using the SPF10-PCR DEIA/LiPA25 system (version 1). A subset of oral samples was subjected to SPF10 sequencing to identify additional HPV types. Multivariable logistic regression analyses using generalized estimating equations (GEE) were performed to assess determinants for oral high-risk HPV incidence and persistence. RESULTS: 689/795 participant MSM provided both baseline and 6-month data. Baseline prevalence of high-risk HPV was 9.4% in HIV-negative and 23.9% in HIV-infected MSM (P<0.001). 56/689 MSM acquired ≥1 high-risk HPV infection (6-month incidence 8.1%; 95%CI 6.2-10.4%); incidence was 4.1% in HIV-negative and 14.1% in HIV-infected MSM (P<0.001). HIV infection and recent use of cannabis were both independently associated with high-risk HPV incidence. Persistent high-risk HPV was observed in 48/130 (36.9%) infections. CONCLUSION: Incidence of oral high-risk HPV infection in MSM is substantial, and is associated with HIV infection. Over a third of HPV infections persisted over a 6-month period.

Varicella zoster virus infection occurs at a relatively young age in The Netherlands.

INTRODUCTION: To date, there is no universal varicella vaccination in the Netherlands. We studied the seroprevalence of varicella zoster virus (VZV) specific antibodies and determinants for seropositivity among participants of a serosurveillance study, conducted in 2006/2007 among Dutch inhabitants 0-79 years of age. MATERIALS AND METHODS: Serological testing of 6386 blood samples for VZV was performed with a fluorescent bead-based multiplex immunoassay. Seroprevalence and geometric mean concentration (GMC) were weighted for age, sex, ethnicity, and urbanization rate to the total Dutch population. Determinants for VZV seropositivity were identified among children younger than 6 years of age using a logistic regression model. RESULTS: The overall seroprevalence of VZV specific antibodies in the Dutch population was 94.6% (95% CI: 93.2-96.0%). This seroprevalence increased rapidly with age: at 6 years of age, more than 95% were seropositive. Determinants associated with lower VZV seropositivity were: young age, first-generation non-Dutch ethnicity, and low frequency of attendance at a day care center or nursery school. The GMC increased with age and was lower for women than for men from the age of 20 years onwards. CONCLUSIONS: This study confirmed that VZV infection occurs at a younger age in the Netherlands compared to other countries, which might explain the low disease burden due to varicella. Introduction of universal varicella vaccination is not a foregone conclusion in the Netherlands. Changes in migration and day care usage will influence the age-specific risk on varicella and should therefore be monitored. Further research might elucidate the sex differences in VZV specific GMC.

Selective hepatitis B virus vaccination has reduced hepatitis B virus transmission in the Netherlands.

BACKGROUND & AIMS: In the Netherlands, a selective hepatitis B virus (HBV) vaccination programme started in 2002 for men having sex with men, drug users, commercial sex workers and heterosexuals with frequent partner changes. We assessed the programme's effectiveness to guide policy on HBV prevention. METHODS: We analysed reports of acute HBV infection in the Netherlands between 2004 and 2010 requesting serum from patients for HBV-genome S- and C-region sequencing. We used coalescence analyses to assess genetic diversity of nonimported genotype-A cases over time. RESULTS: 1687 patients with acute HBV infection were reported between 2004 and 2010. The incidence of reported acute HBV infection decreased from 1.8 to 1.2 per 100,000 inhabitants, mostly due to a reduction in the number of cases in men who have sex with men. Men were overrepresented among cases with an unknown route of transmission, especially among genotype A2 cases mainly associated with transmission through male homosexual contact. The genetic diversity of nonimported genotype-A strains obtained from men who have sex with men decreased from 2006 onwards, suggesting HBV incidence in this group decreased. CONCLUSIONS: The selective HBV-vaccination programme for behavioural high-risk groups very likely reduced the incidence of HBV infection in the Netherlands mainly by preventing HBV infections in men who have sex with men. A considerable proportion of cases in men who did not report risk behaviour was probably acquired through homosexual contact. Our findings support continuation of the programme, and adopting similar approaches in other countries where HBV transmission is focused in high-risk adults.

Patterns of human papillomavirus DNA and antibody positivity in young males and females, suggesting a site-specific natural course of infection.

BACKGROUND: To monitor the impact of human papillomavirus types 16 and 18 vaccine on HPV infection dynamics in the Netherlands, we started an ongoing study in sexually transmitted infection (STI) clinics in 2009. Here, we analyze baseline type-specific HPV DNA and HPV-specific antibody positivity rates. METHODS: We enrolled 3569 men and women, 16-24 years of age, from 14 STI clinics, and estimated genital and anal HPV DNA and antibody positivity rates of 7 main carcinogenic HPV types. Generalized estimating equations regression analyses were applied to determine risk factors for, and associations between, type-specific HPV DNA and antibody positivity. RESULTS: Genital HPV DNA positivity rates were higher in women than in men; anal HPV DNA was especially high in men who have sex with men (MSM). HPV antibody seropositivity rates were also highest in women and MSM. High-risk sexual behavior was predictive of both HPV DNA and antibody positivity. Despite a strong correlation in serological profiles for multiple HPV types, seropositivity was independently associated with homologous HPV DNA detection. CONCLUSIONS: HPV DNA and antibody positivity rates are higher in women and MSM than in heterosexual men, but their association is similar across gender. This suggests a site-specific natural course of infection.

Prevalence, incidence and persistence of genital HPV infections in a large cohort of sexually active young women in the Netherlands.

BACKGROUND: We assessed age- and type-specific HPV prevalence, incidence and persistence and their associated risk factors in young women prior to vaccination, to enable monitoring of the impact of introduction of HPV vaccination in the years before participation in the cervical screening program. METHODS: The HPV status was assessed in 3282 women aged 16-29 who participated in a Chlamydia trachomatis screening implementation program, of which 2014 women (61%) participated in two rounds (one year apart). Self-collected vaginal swab were analyzed by SPF(10) LiPA on the presence of HPV DNA. Risk factors for prevalent, incident and persistent HPV infections were calculated using generalized estimating equation. RESULTS: The prevalence of any HPV in the first round amounted to 54%, while 34% of the women who participated in the second round had a persistent infection and 45% an incident infection. The five most common HPV types found in this study were HPV16, -51, -52, -31 and -53. HPV16 and/or HPV18 prevalence, incidence and persistence in the second round were 15%, 8% and 9%, respectively and for HPV6 and/or HPV11 6%, 4% and 2%, respectively. Relatively to other HPV genotypes, hrHPV types were found more often as a persistent infection than as an incident infection. Furthermore, there is an age-dependent increase within this age range for persistent infections but not for incident infections. CONCLUSION: The HPV prevalence (54%), incidence (45%) and persistence (34%) is high among sexually active young women in the Netherlands. The different HPV type distribution and risk factors for prevalent, incident and persistent infections, as well as the observed age-trends should be taken into account in interpreting data obtained after vaccine introduction. Repeating measurements post-immunization are particularly relevant until the age when screening starts (i.e. 30 years in the Netherlands).

Oral human papillomavirus infection in HIV-negative and HIV-infected MSM.

OBJECTIVE: Oral infection with human papillomavirus (HPV) is associated with a subset of head and neck cancers. We compared prevalence of, and risk factors for, oral HPV infection among HIV-negative and HIV-infected MSM. DESIGN: Analysis of baseline data from a prospective cohort study. METHODS: MSM aged 18 years or older were recruited from three study sites in Amsterdam, the Netherlands. Participants completed a self-administered risk-factor questionnaire. Oral-rinse and gargle specimens were analyzed for HPV DNA and genotyped using a highly sensitive PCR and reverse line blot assay [short PCR fragment (SPF)10-PCR-DNA Enzyme Immuno Assay (DEIA)/LiPA25 system]. RESULTS: In 2010-2011, 794 MSM were included, of whom 767 participants had sufficient data for analysis. Median age was 40.1 years [interquartile range (IQR) 34.8-47.5] and 314 men were HIV-infected (40.9%). Any of 25 typable HPV types was present in 24.4% of all oral samples. Oncogenic HPV types were detected in 24.8 and 8.8% of oral samples from HIV-infected and HIV-negative MSM, respectively (P < 0.001). Of these high-risk types, HPV-16 was the most common (overall 3.4%). Oral infection with high-risk HPV was associated with HIV infection in multivariable analysis (P < 0.001). Increasing age was significantly associated with oral HPV infection in HIV-negative, but not in HIV-infected MSM. CONCLUSION: Oral HPV infection is very common among MSM. HIV infection was independently associated with high-risk oral HPV infection, suggesting an important role of HIV in oral HPV infection.

Changes in antibody seroprevalence of seven high-risk HPV types between nationwide surveillance studies from 1995-96 and 2006-07 in The Netherlands.

OBJECTIVE: This study evaluates trends in antibody seroprevalences of seven high-risk human papillomavirus (hr-HPV) serotypes (HPV16, 18, 31, 33, 45, 52, and 58) between the 1995-96 and 2006-07 sero-surveys among the Dutch general population in the pre-vaccination era. METHODS: Serum samples of men and women (0-79 years of age) from two cross-sectional population-based serosurveillance studies performed in 1995-96 (n = 3303) and 2006-07 (n = 6384) were tested for HPV-specific antibodies in a VLP-based multiplex immunoassay. RESULTS: HPV16-specific antibody seroprevalence increased during adolescence and shifted to younger ages in the 2006-07 survey compared to the 1995-96 survey. This step-up in HPV16 seroprevalence was most pronounced in women, while a more gradual increase was observed in men. Also in cohorts older than 49 years, HPV16 seroprevalence was higher in 2006-07 as compared to 1995-96 survey. A higher overall seroprevalence in individuals older than 15 years of age was found for HPV16, 18, 31 and 45 in 2006-07 as compared to 1995-96. For HPV33, 52 and 58 seroprevalences were comparable over this 11-year time period. Seropositivity for one or more HPV types was significantly higher in 2006-07 (23.1%) than in 1995-96 (20.0%) (p = 0.013). Multi-seropositivity increased from 7.1% in 1995-96 up to 10.2% in 2006-07 (p<0.0001). Differences in HPV seropositivity for at least one of the seven HPV types between both surveys could be explained in addition to demographic characteristics (age, sex, urbanization degree and ethnicity), also by changes in sexual behaviour (marital status, age of sexual debut and ever reported an STI). CONCLUSION: The observed increase in particular HPV16 seroprevalence could be due to changes in sexual behaviour over the years, and especially in age of sexual debut. Seroprevalence studies provide insight into the distribution of HPV types and infection dynamics in the general population over time, which is important to assess the impact of HPV-vaccination.

Seroprevalence of seven high-risk HPV types in The Netherlands.

BACKGROUND: To obtain insight into the age-specific seroprevalence for seven high-risk human papillomavirus (hr-HPV) serotypes (HPV16, 18, 31, 33, 45, 52, and 58) among the general population in the pre-vaccination era in The Netherlands. METHODS: From a cross-sectional population-based study (ISRCTN 20164309) performed in 2006/2007 6384 sera of men, women and children were tested for seven hr-HPV specific antibodies using a fluorescent bead-based multiplex immunoassay with virus-like particles of the seven HPV serotypes. RESULTS: An increase in seroprevalence was observed in adolescents, especially for the most prevalent HPV type 16 (up to 11.3%). The increase was most pronounced in women, but was less clear for the other six HPV serotypes. Relatively stable seroprevalences were found in the middle aged cohorts and a slight decrease in the elderly. For the age cohorts >14 years, the seroprevalence among women (25.2%) was higher compared with men (20.3%) (p=0.0002). We found that 10.1% of the population was seropositive for multiple HPV serotypes. CONCLUSIONS: The HPV vaccination program is targeted at preadolescents as is justified by the results in this study in which a step-up in HPV seroprevalence is observed at ages of sexual debut. Although direct interpretation of seroprevalence data are hampered by cross-reactivity and seroconversion rate, these data are useful as baseline to evaluate long-term population effects of the HPV16/18 vaccination program.

Global co-existence of two evolutionary lineages of parvovirus B19 1a, different in genome-wide synonymous positions.

Parvovirus B19 (B19V) can cause infection in humans. To date, three genotypes of B19V, with subtypes, are known, of which genotype 1a is the most prevalent genotype in the Western world. We sequenced the genome of B19V strains of 65 asymptomatic, recently infected Dutch blood donors, to investigate the spatio-temporal distribution of B19V strains, in the years 2003-2009. The sequences were compared to B19V sequences from Dutch patients with fifth disease, and to global B19V sequences as available from GenBank. All Dutch B19V strains belonged to genotype 1a. Phylogenetic analysis of the strains from Dutch blood donors showed that two groups of genotype 1a co-exist. A clear-cut division into the two groups was also found among the B19V strains from Dutch patients, and among the B19V sequences in GenBank. The two groups of genotype 1a co-exist around the world and do not appear to differ in their ability to cause disease. Strikingly, the two groups of B19V predominantly differ in synonymous mutations, distributed throughout the entire genome of B19V. We propose to call the two groups of B19V genotype 1a respectively subtype 1a1 and 1a2.

Immunogenicity of a hexavalent vaccine co-administered with 7-valent pneumococcal conjugate vaccine. Findings from the National Immunisation Programme in The Netherlands.

The hexavalent vaccine Infanrix hexa was introduced into the national childhood vaccination schedule in the Netherlands in 2006. It is offered, concomitantly with pneumococcal vaccine (Prevenar), to children at increased risk of hepatitis B, administered in a 4-dose schedule at 2, 3, 4 and 11 months of age. We assessed the immunogenicity of the HBV component of Infanrix hexa co-administered with Prevenar, and compared pertussis and Hib components in Infanrix hexa with the standard Infanrix-IPV+Hib vaccine. Target thresholds for immune responses were achieved for all antigens studied. Over 99% (163/164) of children vaccinated with Infanrix hexa achieved an adequate immune response (≥ 10 mIU/ml) to the HBV component and peak anti-HBs geometric mean concentration (GMC) was 2264 mIU/ml (95%CI:1850-2771 mIU/ml). The GMC of a pertussis component, filamentous hemagglutinin (FHA), of Infanrix-hexa was significantly lower in children vaccinated with Infanrix hexa and Prevenar than in children vaccinated with Infanrix-IPV+Hib. Universal infant HBV vaccination using Infanrix hexa was introduced in The Netherlands in 2011. Despite very high rates of seroconversion for the HBV component of Infanrix hexa, its long term immunogenicity and effectiveness should be monitored after concomitant vaccination.

Mumps vaccine effectiveness in primary schools and households, the Netherlands, 2008.

To estimate the mumps vaccine effectiveness (VE) during a large genotype D mumps outbreak, we conducted a cross-sectional study in eight primary schools and associated households in the Netherlands. Questionnaires were used to collect information on the occurrence of mumps. Multivariate analyses were used to estimate VE. Among schoolchildren we estimated the VE against mumps. Among household contacts where the schoolchild was the index case we estimated the VE against mumps and against mumps infectiousness. In total 1175 children and 2281 household contacts participated in the study. The mumps attack rate among schoolchildren was 17%. The mumps VE in schoolchildren was 92% [95% confidence interval (CI) 83-96%] and 93% [85-97%] for one and two doses of the measles, mumps, rubella (MMR) vaccine, respectively. The adjusted mumps VE among household contacts was 67% [65-95%] and 11% [-4 to 88%] against mumps and mumps infectiousness, respectively. Our study indicates that the mumps component of the MMR vaccine offered adequate protection against mumps among schoolchildren. The relatively low VE among household contacts is of concern.

Hepatitis C virus seroprevalence in the Netherlands.

A population-based anti-hepatitis C virus (HCV) prevalence is important for surveillance purposes and it provides an insight into the burden of disease. In The Netherlands, a recent HCV seroprevalence estimate is not available. This national population-based cross-sectional serosurvey (PIENTER-2) resulted in a weighted national HCV seroprevalence of 0.30% (95% confidence interval 0.05-0.55%). About 70% of the HCV positive individuals found were born in an HCV-endemic country.

Prevention of perinatal hepatitis B virus transmission in the Netherlands, 2003-2007: children of Chinese mothers are at increased risk of breakthrough infection.

BACKGROUND: In the Netherlands, different hepatitis B vaccination schedules have been used for children born to HBV-infected mothers. All schedules included a birth dose of hepatitis B immunoglobuline (HBIg). We assessed determinants of perinatal HBV transmission and determinants of anti-HBs titers in infants born to HBsAg positive mothers. METHODS: We included infants born to HBV infected mothers between 1.1.2003 and 30.6.2007, using national databases and a separate database for Amsterdam. Risk factors for perinatal transmission and determinants of the anti-HBs titer were studied using logistic and linear regression, respectively. RESULTS: Of 2657 infants registered in the national database, 91% were registered to have received HBIg and at least three hepatitis B vaccinations. In Amsterdam, this coverage among 413 children at risk was higher (96%, p<0.01). Serological test results for 2121 infants (80%) indicated that 13 (0.6%) were HBsAg positive. A mother of Chinese descent was the only risk factor for perinatal HBV infection identified (RR 9.1, 95% CI 3.1-26.8). Receiving a birth dose of hepatitis B vaccine later than in the first week of life was not associated with an increased risk of perinatal HBV infection. A shorter period between last vaccination and testing, and having received more doses of hepatitis B vaccine were independently associated with a higher anti-HBs titer. CONCLUSIONS: Infants born to Chinese mothers were at increased risk of perinatal HBV infection. All HBsAg positive pregnant women of Chinese origin should be assessed to determine whether there is an indication for anti-viral treatment during pregnancy. Among infants who received HBIg at birth, we did not detect an increased risk of perinatal HBV infection when the first dose of hepatitis B vaccine was administered after the first week of life.

Type-specific human papillomavirus infections among young heterosexual male and female STI clinic attendees.

BACKGROUND: Baseline genotype-specific human papillomavirus (HPV) prevalence rates and associated risk factors per gender enable future assessment of the impact of vaccination on HPV dynamics. METHODS: Before the start of national HPV vaccination for girls, data were collected cross-sectionally in nationwide Dutch sexually transmitted infections (STI) clinics among heterosexual males (n = 430) and females (n = 1136) aged 16 to 24 years. Self-collected vaginal or penile swabs were analyzed by a sensitive polymerase chain reaction (SPF10) and genotyped with line probe assay. Logistic regression was applied to estimate determinants of HPV prevalent infections. RESULTS: HPV prevalence was 54% among males and 72% among females. High-risk (HR) HPV was present in males and females, 40% and 58%, respectively. Independent risk factors for HR-HPV infection were female gender, number of lifetime sex partners and a history of chlamydia or gonorrhea. In addition, not having a casual partner and consistent condom use were protective factors in women, but not in men. For low-risk (LR) HPV, the odds were smaller. Multiple HR-HPV and sexual risk behavior showed a stronger association compared with a single HR-HPV infection. CONCLUSIONS: Prevalence of HR-HPV is high in both genders. Infection with multiple HR-HPV types was more associated with high-risk sexual behavior than infection with LR-HPV types or a single HR-HPV type.

Assessment of serological evidence for mumps virus infection in vaccinated children.

It is estimated that at least one-third of mumps virus infections in non-vaccinated individuals are asymptomatic. Little information is available whether this proportion is the same among those vaccinated. We validated a commercial oral fluid mumps IgG-specific Enzyme Immunoassays (EIA) with vaccinated control groups to identify symptomatic and asymptomatic mumps virus infections in vaccinated individuals during a mumps outbreak in The Netherlands. A vaccinated control group was required to define a new cutoff value for the assay, because of the presence of low but significant levels of IgG antibodies in oral fluid as a result of mumps vaccination in the past. With a new cutoff, calculated using receiver operator characteristic analysis, we identified an attack rate of 7-10% compared to 2.7% based on clinical symptoms among vaccinated children. This finding has important implications when studying transmission patterns, strain virulence, as well as mumps vaccine effectiveness to protect from infection rather than disease.

Transmission of mumps virus from mumps-vaccinated individuals to close contacts.

During a recent mumps epidemic in the Netherlands caused by a genotype D mumps virus strain, we investigated the potential of vaccinated people to spread mumps disease to close contacts. We compared mumps viral titers of oral fluid specimens obtained by quantitative PCR from vaccinated (n=60) and unvaccinated (n=111) mumps patients. We also investigated the occurrence of mumps infection among the household contacts of vaccinated mumps patients. We found that viral titers are higher for unvaccinated patients than for vaccinated patients during the 1st 3 days after onset of disease. While no symptomatic cases were reported among the household contacts (n=164) of vaccinated mumps patients (n=36), there were cases with serological evidence of asymptomatic infection among vaccinated household contacts (9 of 66 vaccinated siblings). For two of these siblings, the vaccinated index patient was the most probable source of infection. We conclude that, in this particular outbreak, the risk of a close contact becoming infected by vaccinated patients was small, but present.