Estimating seroprevalence of vaccine-preventable infections: is it worth standardizing the serological outcomes to adjust for different assays and laboratories?

The aim of the European Sero-Epidemiology Network 2 (ESEN2) project was to estimate age-specific seroprevalence for a number of vaccine-preventable diseases in Europe. To achieve this serosurveys were collected by 22 national laboratories. To adjust for a variety of laboratory methods and assays, all quantitative results were transformed to a reference laboratory's units and were then classified as positive or negative to obtain age-specific seroprevalence. The aim of this study was to assess the value of standardization by comparing the crude and standardized seroprevalence estimates. Seroprevalence was estimated for measles, mumps, rubella, diphtheria, varicella zoster and hepatitis A virus (HAV) and compared before and after serological results had been standardized. The results showed that if no such adjustment had taken place, seroprevalence would have differed by an average of 3·2% (95% bootstrap interval 2·9-3·6) although this percentage varied substantially by antigen. These differences were as high as 16% for some serosurveys (HAV) which means that standardization could have a considerable impact on seroprevalence estimates and should be considered when comparing serosurveys performed in different laboratories using different assay methods.

Comparative hepatitis A seroepidemiology in 10 European countries.

The WHO recommends hepatitis A virus (HAV) immunization according to level of transmission and disease burden. We aimed to identify susceptible age groups by standardized serosurveys to inform HAV vaccination policy in participating countries: Belgium, Czech Republic, England, Finland, Germany, Italy, Lithuania, Malta, Romania, and Slovakia. Each country tested national serum banks (n = 1854-6748), collected during 1996-2004, for anti-HAV antibodies. Local laboratory results were standardized to common units. Forty-one per cent of those aged <30 years and 6% of those aged ≥30 years were susceptible to HAV in Romania; compared to 70-94% and 26-71%, respectively, elsewhere. Romania reported high HAV incidence in children and young adults. Other countries reported HAV disease primarily in older risk groups. The results suggest low level of HAV transmission in most of Europe. Romania, however, appeared as an area with intermediate transmission. Vaccination of risk groups in countries with high susceptibility of young and middle-aged adults needs to be continued.

Are we hitting immunity targets? The 2006 age-specific seroprevalence of measles, mumps, rubella, diphtheria and tetanus in Belgium.

Susceptibility to vaccine-preventable diseases in Belgium in 2006 was estimated from a serum survey. Immunoglobulins against measles, mumps, rubella (MMR) and diphtheria at all available ages (1-65 years), and against tetanus in >40-year-olds, were measured by ELISA. Age-standardized overall seronegativity for MMR was low (3·9%, 8·0%, 10·4%, respectively). However, the World Health Organization's targets for measles elimination were not met in 5- to 24-year-olds and about 1 in 7 women at childbearing age (15-39 years) were seronegative for rubella. In adults >40 years, tetanus immunity (87·2%, >0·16 IU/ml) largely exceeded diphtheria immunity (20-45%, >0·1 IU/ml). Despite free universal vaccination against MMR for more than 20 years and against diphtheria and tetanus for almost 60 years, our study revealed specific age groups remaining at risk for infection with these pathogens.

Comparative immunogenicity of two vaccination schedules of a combined hepatitis A and B vaccine in healthy volunteers.

In 1996, a combined vaccine against both hepatitis A and B was licensed and commercialized and has been recommended for healthcare personnel in Belgium. This study compares the immunogenicity against hepatitis B virus (HBV) and safety of two vaccination schedules (0-1-12 months and 0-1-6 months) with this vaccine. This is a randomized, stratified and controlled study in healthy adult workers, who are not occupationally exposed to HBV. Seroconversion (≥1 IU/L) and seroprotection (≥10 IU/L) rates were compared using Fisher's exact test; geometric mean concentrations (GMCs) of anti-HBs were compared using one-way ANOVA. All statistical analyses were carried out with SPSS 11 on Apple Macintosh. A total of 399 subjects were enrolled in the study, and 356 were analysed according to the protocol. The rate of ≥10 IU/L at 6 months was 70.6% in the group 0-1-12 and 79.9% in the group 0-1-6; this rate decreased to 55.9% at 12 months in the first group. Seroconversion and seroprotective rates against HBV measured at month 13 in group 0-1-12 (98.9% and 95.6%) and measured at month 7 in group 0-1-6 (99.4% and 97.1%) were not statistically significantly different. GMC of anti-HBs after the 0-1-12 schedule was more than two fold higher than after 0-1-6 schedule (P < 0.001). Reported side effects were comparable in both groups with a slight tendency to fewer side effects in the 0-1-12 group after the third dose. The results from our study show that the completed schedule 0-1-12 offers at least equal protective immunogenicity against HBV as the completed 0-1-6 schedule. People not receiving their third dose at 6 months can be given this dose up to 12 months after the first dose. The drawback of this flexibility, however, is the longer time period before the protection becomes effective.

The European Sero-Epidemiology Network 2 (ESEN2): standardization of assay results for hepatitis A virus (HAV) to enable comparisons of seroprevalence data across 15 countries.

The European Sero-Epidemiology Network 2 (ESEN2) aimed to compare serological results of vaccine-preventable diseases across Europe. To ensure direct inter-country comparability of hepatitis A virus antibody (anti-HAV) measurements, a standardization panel of 150 sera was developed by a designated reference laboratory and tested by participating national laboratories using assays of choice; each country's results were subsequently regressed against those of the reference laboratory. Quantitatively, the assays were generally highly correlated (R2>0.90). Nevertheless, qualitative comparisons indicated that results obtained with different assays may differ despite the usage of well-established international and local standards. To a great extent standardization successfully alleviated such differences. The generated standardization equations will be used to convert national serological results into common units to enable direct international comparisons of HAV seroprevalence data. The results of this study are expected to contribute to the evaluation and potential improvement of the currently employed immunization strategies for hepatitis in Europe.

Distribution of hepatitis C virus genotypes among injecting drug users in contact with treatment centers in Belgium, 2004-2005.

The aim of this study was to determine the current prevalence of HCV genotypes in injecting drug users recruited at treatment centers all over Belgium, and to analyze if the distribution of genotypes was correlated with demographic characteristics, at-risk behaviors, and co-infection with other viruses. Therefore 147 anti-HCV-positive serum samples were selected for subsequent HCV RNA detection and genotyping. HCV RNA could be detected in 98 (67%) of the 147 serum samples. Genotype 1 (38%) and 3 (49%) were the most common genotypes followed by genotype 4 (9%) and genotype 2 (2%). One mixed infection (1%) was detected. The subtype could be determined in 80 cases: genotype 3a was the most prevalent (49%), followed by genotype 1a (16%) and genotype 1b (15%). No significant difference was found between the distribution of genotypes and the location of treatment centers, at-risk behaviors and co-infection with other viruses. Nevertheless, a slight variation over time could be identified (P = 0.06): one in two genotype 3 drug users started with their injecting drug use in the last 10 years (33% in the period 1995-1999 and 21% in the period > or =2000) compared to only one in four genotype 1 drug users (20% in the period 1995-1999 and 9% in the period > or =2000).

The comparative sero-epidemiology of varicella zoster virus in 11 countries in the European region.

The European sero-epidemiology network (ESEN2) aims to standardise serological surveillance of varicella zoster virus (VZV) in 11 participant countries. In each country, serum banks were collected between 1996 and 2003 and tested for VZV antibodies. Assay results were standardised so that international comparisons could be made. Age-specific forces of infection were calculated for three age groups (<5, 5-9 and >or=10 years of age) and used to estimate the base reproduction number (R(0)) and the herd immunity threshold (H). Most VZV infection occurred in childhood, but there was a wide variation in transmissibility, with R(0) ranging from 16.9 in the Netherlands to 3.3 in Italy. Herd immunity thresholds varied from 70% in Italy to 94% in the Netherlands. There are substantial differences in VZV sero-epidemiology within the European region, which will need to be taken into account in designing national policies regarding VZV vaccination.

The phosphatidylserine receptor mediates phagocytosis by vascular smooth muscle cells.

Apoptosis participates in every step of atherogenesis, but the process of clearance of apoptotic cells by phagocytosis has been underestimated. Rapid removal of apoptotic cells is critical for tissue homeostasis, in order to avoid accumulation of necrotic material and subsequent inflammation in the pathological vascular wall. We have demonstrated by RT-PCR, western blot and immunocytofluorescence that vascular smooth muscle cells (VSMCs) express the phosphatidylserine receptor (PSR). We then tested the involvement of PSR in the ability of VSMCs to bind and engulf apoptotic cells. We used a model of senescent erythrocytes, which expose PS after 4 days of culture (85% of cells relative to 8% in freshly isolated erythrocytes). The pseudo-peroxidase activity of haemoglobin contained within erythrocytes allowed us to quantify per se both binding and phagocytosis by VSMCs. We have also shown by light and confocal microscopy that VSMCs were able to ingest aged erythrocytes. Addition of a blocking antibody or transfection of VSMCs by a siRNA directed against PSR reduced the binding and engulfment of aged erythrocytes by more than 90%. These results suggest that PSR is involved in phagocytosis of PS-presenting cells. Incubation of aged erythrocytes with VSMCs also significantly increased the expression of PSR, suggesting that the tethering/ingestion of apoptotic cells triggers this process. Immunostaining for PSR in complicated atherosclerotic plaques shows positivity in the media and macrophage-rich areas. The mechanisms underlying phagocytosis and involving PSR in vivo, within the pathological arterial wall, deserve further investigation.

Standardization of measles, mumps and rubella assays to enable comparisons of seroprevalence data across 21 European countries and Australia.

The aim of the European Sero-Epidemiology Network is to establish comparability of the serological surveillance of vaccine-preventable diseases in Europe. The designated reference laboratory (RL) for measles, mumps, rubella (MMR) prepared and tested a panel of 151 sera by the reference enzyme immunoassay (rEIA). Laboratories in 21 countries tested the panel for antibodies against MMR using their usual assay (a total of 16 different EIAs) and the results were plotted against the reference results in order to obtain equations for the standardization of national serum surveys. The RL also tested the panel by the plaque neutralization test (PNT). Large differences in qualitative results were found compared to the RL. Well-fitting standardization equations with R2> or =0.8 were obtained for almost all laboratories through regression of the quantitative results against those of the RL. When compared to PNT, the rEIA had a sensitivity of 95.3%, 92.8% and 100% and a specificity of 100%, 87.1% and 92.8% for measles, mumps and rubella, respectively. The need for standardization was highlighted by substantial inter-country differences. Standardization was successful and the selected standardization equations allowed the conversion of local serological results into common units and enabled direct comparison of seroprevalence data of the participating countries.

Correlation between detection of antibodies against hepatitis C virus in oral fluid and hepatitis C virus RNA in serum.

Reported here are the results of a study designed to determine the correlation between hepatitis C virus (HCV) RNA positivity in serum and the detection of antibodies against HCV in oral fluid by testing paired serum/oral fluid samples. For the 85 serum samples found positive for antibodies against HCV, using a screening assay and a confirmation assay, 70 of the corresponding oral fluid samples tested positive for HCV antibodies using a previously modified screening assay. For 52 of the 59 serum samples found positive for HCV RNA, the corresponding oral fluid samples also tested seropositive for HCV, while 18 of the 26 serum samples that were negative for HCV RNA had corresponding oral fluid samples that tested seropositive for HCV. For the control group of 54 serum samples that were negative for HCV antibodies, all of the corresponding oral fluid samples were also negative for HCV antibodies, while 53 of the serum samples tested negative for HCV RNA. These results suggest that HCV antibody detection in oral fluid has a slightly higher sensitivity when used to test patients whose serum samples are positive for HCV RNA (chi-square test, p=0.035; Mid-P exact, p=0.049).

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines.

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.

Hepatitis B virus (HBV) genotyping in Belgian patients with chronic HBV infection.

Several studies have demonstrated that pathogenic and therapeutic differences exist among hepatitis B virus (HBV) genotypes. Therefore, this study established the prevalence of different HBV genotypes in 128 Belgian patients with chronic HBV infection. The prevalences of genotypes A and D, and mixed genotypes A and D, were 53%, 37% and 8%, respectively, for a group of blood donors, and 54%, 31% and 9%, respectively, for a group of patients from the gastroenterology units. The results indicated that genotypes A and D are the predominant genotypes in Belgian patients with chronic HBV infection.

Hepatitis B vaccination coverage in Belgian health care workers.

BACKGROUND: The hepatitis B virus (HBV) vaccination policy for health care workers (HCW) started in Belgium in 1983. An update of vaccination coverage and rates of seroconversion and seroprotection among HCW can give an insight into the actual status and encourage further development of vaccination programs. PATIENTS AND METHODS: 5,064 HCW were tested for anti-HBs. We considered those who had a positive anti-HBs test as seroconverted (SC) and those who had an anti-HBs titer > 10 IU/l as seroprotected (SP). RESULTS: 4,771 HCW were eligible for vaccination; 84.9% of them were effectively vaccinated. Among high-risk professions (nurses, care and laboratory workers), 94.79% were vaccinated; for other professions the vaccination coverage was 69.26%. Of the 1,015 non-vaccinated persons, 293 were anti-HBs positive. Among these 54.95% declared they had had a previous hepatitis infection that was serologicaLly proven to be HBV (anti-HBc positive). Of the remaining 132 positives, 70.45% had previously undergone surgery and/or transfusion. Among these 1,015 non-vaccinated HCW, 59.03% were anti-HBs positive. Of these, 373 were nurses, care or laboratory workers. This contrasts with the results for HCW in other sectors, where 11.49% were anti-HBs positive. CONCLUSION: In our sample, high vaccination, seroconversion and seroprotection rates were achieved, at least for higher risk HCW. The same conclusion can be drawn if we consider hospital departments which carry a higher risk of blood-borne infections.

Detection of HCV antibodies in oral fluid.

Although conventionally the detection of HCV antibodies is carried out on serum, the collection of oral fluid is non-invasive, safe and cost effective. In this study, the efficacy of the detection of HCV antibodies in oral fluid was assessed. 73 anti-HCV positive and 73 anti-HCV negative paired serum/oral fluid samples, drawn from patients visiting a Belgian academic hospital, were tested using the modified Ortho HCV 3.0 and LIA confirmation assay. Performing the test on oral fluid with the modified protocol, 61/73 anti-HCV positive samples were tested positive, while 73/73 anti-HCV negative samples were tested negative, giving a sensitivity and specificity of 83.6% (95% CI: 72.7-90.9%) and 100.0% (95% CI: 93.8-100.0%), respectively. Comparing S/CO of concordantly positive and negative samples, the cut-off point was lowered by 30% resulting in a sensitivity of 89.0% (95% CI: 79.0-94.8%) while the specificity remained 100.0% (95% CI: 93.8-100.0%). The confirmation assay was carried out as described by the manufacturer, diluting the oral fluid 1:10. Testing paired samples gave a concordance of 85.6% (125/146), yielding no more accurate results. These findings suggested that the modified ELISA method for anti-HCV detection in oral fluid can be used for epidemiological surveys.

The seroepidemiology of herpes simplex virus type 1 and 2 in Europe.

OBJECTIVES: To describe the seroepidemiology of herpes simplex virus (HSV) types 1 and 2 in the general populations of eight European countries to better understand recent reported changes in disease epidemiology. METHODS: Belgium, Bulgaria, Czech Republic, England and Wales, Finland, Germany, Netherlands, and Slovenia conducted national cross sectional serological surveys for HSV-1 and HSV-2 between 1989 and 2000. Survey sizes ranged from 3000 to 7166 sera. External quality control was ensured through reference panel testing. RESULTS: Large intercountry and intracountry differences in HSV-1 and HSV-2 seroprevalence were observed. Age standardised HSV-1 seroprevalence ranged from 52% in Finland, to 57% in the Netherlands, 67% in Belgium, 81% in Czech Republic, and 84% in Bulgaria. Age standardised (>12 years) HSV-2 seroprevalence ranged from 24% in Bulgaria, to 14% in Germany, 13% in Finland, 11% in Belgium, 9% in Netherlands, 6% in Czech Republic, and 4% in England and Wales. In all countries, probability of seropositivity for both infections increased with age. A large proportion of teenagers and young adults remain HSV-1 susceptible particularly in northern Europe. Women were significantly more likely to be HSV-2 seropositive in six of seven (p<0.05) countries and HSV-1 seropositive in four of seven (p<0.05) countries, particularly in northern Europe. No significant evidence of a protective role of HSV-1 for HSV-2 infection was found adjusting for age and sex (p<0.05). CONCLUSIONS: There is large variation in the seroepidemiology of HSV-1 and HSV-2 across Europe. The observation that a significant proportion of adolescents are now HSV-1 susceptible may have implications for transmission and clinical presentation of HSV-1 and HSV-2.

Serotyping and genotyping of hepatitis C virus in Belgium.

BACKGROUND: Given that both pathogenicity and the response to treatment are possibly associated with hepatitis C virus (HCV) serotype, it appeared sensible to establish the prevalence of the different HCV types in Belgium. MATERIALS AND METHODS: The HCV serotypes were determined in 68 HCV-RNA and anti-HCV-positive samples taken from Belgian patients and compared with the results of the genotyping assay. Possible associations with age and sex were investigated. RESULTS: Antibodies were identified in 55 (80.9%) of the 68 samples, with serotype 1 (58.8%) and serotype 3 (19.1%) showing the highest prevalence. 17 samples contained several serotypes with serotype 1 being detected in 82.4% of cases. Nine of the 11 samples undetermined by serotyping could be determined by genotyping. There was no significant difference in the distribution of HCV types with respect to gender. Compared with genotype 3 (p < 0.01) and genotypes 2 and 4 (p = 0.05), genotype 1 was detected among older patients. CONCLUSION: Our data showed a 96.0% correlation between the serotyping and genotyping assays. Genotypes 1 and 3 are the most prevalent types among Belgian patients. The data suggest that genotype 1 spread earlier than genotypes 2, 3 and 4. This corroborates previous European studies.

Non-viral gene transfer of murine spleen cells achieved by in vivo electroporation.

Gene electrotranfer is an attractive physical method to deliver genes to target tissues. The aim of this study was to evaluate in vivo gene electrotransfer into spleen, one of the most important lymphoid organ, in order to create a new tool to modulate the immuno-inflammatory system. C57Bl/6 mice were submitted either to intramuscular electrotransfer (IME) as a reference method or to intrasplenic (ISE) gene electrotransfer. In the naked injected plasmids, the CMV promoter controlled the expression of luciferase, secreted alkaline phosphatase, EGFP, or IFNgamma. The ISE optimal electrotransfer conditions were first determined and ISE was found to be an efficient gene transfer method, which can be used to express secreted or intracellular proteins transiently. Although transfected cells were still present in the spleen 30 days after ISE, transfected spleen cells could recirculate since they were detected in extrasplenic locations. Using a T-lymphocyte-specific promoter controlling the expression of EGFP, splenic T cells could be targeted. Finally, it appeared that ISE procedure does not impair by itself the immune response and does not result in a significant production of antibodies directed to the transgenic proteins in C57Bl/6 mice. This strategy constitutes a new method to manipulate the immune response that can be used in various experimental designs.

Cumulative inactivation of the outward potassium current: a likely mechanism underlying electrical memory in human atrial myocytes.

The influence of the mode of cell stimulation on the outward K+ current (I(o)) was studied in whole-cell patch-clamped human atrial myocytes. Acceleration of the rate of membrane depolarization at 1 Hz or during prolonged 5-s test pulses at 0.1 Hz increased the rate and extent of I(o) inactivation, resulting in enhanced inactivating (4.9+/-0.6 v 6.3+/-0.7 pA/pF) and suppressed maintained (5.9+/-1.2 v 3.2+/-0.3 pA/pF) current components. These alterations were associated with a leftward shift of the voltage-dependency of I(o), and persisted on returning to a control depolarization protocol (750-ms test pulses delivered at 0.1 Hz). The effects of increasing external K+ concentrations (40 m m) on the kinetics of I(o) were more pronounced following both rapid and prolonged depolarization (changes in I(t)/I(o)caused by 40 m m K+: 8.9+/-3.5% v 15.5+/-3.1% before and after prolonged depolarization; and 9.2+/-1.2% v 15.4+/-1.7% before and after rapid depolarization). The phosphatase inhibitor, okadaic acid, enhanced the effect of rapid and prolonged depolarization on I(o)whereas the inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with KN-62 or KN-93, or by intracellular application of the autocamtide-2-related inhibitory peptide, suppressed it. In conclusion, rapid and prolonged membrane depolarization both cause a cumulative increase in the rate and extent of I(o)inactivation. This process involves slow potassium channel inactivation mechanisms, is regulated by CaMK-II, and may contribute to the electrical memory of the atrial myocardium.

Effect of the extract of Ginkgo biloba (EGb 761) on the circulating and cellular profiles of polyunsaturated fatty acids: correlation with the anti-oxidant properties of the extract.

Ginkgo biloba extract (EGb 761) has beneficial effects on cognitive functions in aging patients, and on various pathologies, including cardiovascular diseases. Although the extract is known to have antioxidant properties and improve membrane fluidity, the cellular mechanisms underlying these effects have not been determined. Here, we examined the in vivo effects of EGb 761 on circulating and cellular lipids. EGb 761 treatment induced significant increases in the levels of circulating polyunsaturated fatty acids (PUFAs), and a decrease in the saturation index SI (saturated/polyunsaturated species). Plasma triglycerides and cholesterol were not affected, while phospholipids were slightly increased at the higher dose of EGb 761. EGb 761 treatment also induced a significant increase in the levels of PUFAs in erythrocyte membranes, especially for the eicosapentaenoic acid (EPA omega 3), and a decrease in the saturation index. Moreover, the response of erythrocytes to oxidative stress was improved in EGb 761-treated animals (H(2)O(2)-induced cell lysis decreased by 50%). Considering that PUFAs are known to improve membrane fluidity and response to oxidative damage, and are precursors of signaling molecules such as prostaglandins, the effects of EGb 761 on circulating and cellular PUFAs may explain some of the pharmacological properties of Ginkgo biloba.

PCR detects HCV RNA in a plasma pool contaminated by a single preseroconversion donation of genotype 5a.

BACKGROUND AND OBJECTIVES: To determine the prevalence of HCV-RNA-positive plasma pools in Belgium, to validate our PCR method and to increase the safety of the released blood products. MATERIALS AND METHODS: Plasma pools consisting each of about 5,000 donations from Belgian unpaid volunteer blood donors were analysed by PCR for the presence of HCV RNA. Two different extraction methods were compared and validated. RESULTS: Two out of 367 plasma pools were found to be HCV RNA positive and were discarded. For one of these two pools, the look-back procedure identified an anti-HCV-negative contaminated donation. The HCV genotype of both the contaminated pool and the donation was 5a, a genotype rare in Europe. The viral load of the preseroconverted donation was 2.9 x 10(7) gEq/ml according to the bDNA method. CONCLUSION: In the case of plasma derivatives, various important steps are already included to increase safety. Nucleic acid testing of manufacturing plasma pools ensures that viral load in the starting material is as low as possible.