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.

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.

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.

A simplified method for the preparation of rat thyroxine-binding prealbumin. Factors influencing its circulating level.

Rat thyroxine-binding prealbumin (TBPA) was isolated in three simple steps by means of a serum precipitation by a 5% phenol solution and two consecutive semi-preparative polyacrylamide gel electrophoreses. The overall yield was 15% and the TBPA preparation contained less than 1% impurities. In addition a monospecific antiserum was raised in the rabbit. In polyacrylamide gel, rat TBPA, as with its human counterpart, migrated anodally to albumin while in agarose gel, its electrophoretic mobility was similar to that of albumin. Serum TBPA measured in adult male Wistar rats did not exhibit a circadian rhythm. However, a significant 13% decrease was observed between 9 and 15 h, followed by the restoration of the initial value by 21 h. TBPA concentration was measured in 1-, 15- and 28-day-old male and female pups as well as in adult rats. The level of this protein increased from 1 to 28 days of age and did not display any sexual difference. Yet, while TBPA concentrations in adult males were similar to those recorded in the 28-day-old pups, for adult females, they returned to the levels measured in the 1-day-old pups.

Conformational changes in rodent and human alpha-fetoprotein: influence of fatty acids.

Binding, spectral and immunological studies were performed to demonstrate the conformational changes in rodent and human alpha-fetoprotein (AFP) induced by a free fatty acid environment. Scatchard analysis of estradiol (E2) binding to purified rat AFP indicated that unsaturated fatty acids changed the number of binding E2 sites and the apparent E2 equilibrium dissociation constant which varied non-linearly with docosahexaenoic acid concentration. UV spectral analysis of rodent and human AFPs showed that the absorbance minimum of AFP incubated with unsaturated fatty acid (L-AFP) was red-shifted, broadened and less pronounced than that of purified native AFP (N-AFP). Immunochemical studies with specific polyclonal antibodies to purified rodent and human AFPs (N-AFP antibodies) showed that these proteins lost immunoreactivity after incubation with unsaturated fatty acid. N-AFP antibodies recognized fewer epitopes on L-AFP than on N-AFP, whatever the species. Specific anti-rat L-AFP antibodies were used to demonstrate specific epitopes on rat L-AFP. Rat L-AFP antibodies did not recognize rat N-AFP. Saturated fatty acids were without effect on the binding, spectral and immunological properties of rodent and human AFPs. RIA or ELISA values for human AFP from fetal serum, hepatoma serum, and cord serum, were reduced 80, 50 and 5%, respectively, by unsaturated fatty acids. This decrease correlated with the relative percentage of polyunsaturated fatty acid in each biological fluid. Such results indicate that an unsaturated fatty acid environment induces conformational changes in AFP which may modulate the endocrine and immune functions of this protein.

Thyroxine-binding globulin and thyroxine-binding prealbumin in hypothyroid and hyperthyroid developing rats.

We present evidence based on equilibrium and non-equilibrium binding studies, as well as on immunological techniques, that of the two rat specific thyroid-hormone-binding proteins, i.e., thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA), TBG but not TBPA is regulated by the thyroid hormones (TH). Hypothyroidism, induced from the day of birth by daily treatment with propylthiouracil (PTU-rats), leads to dramatic and sustained increases of the TH-binding abilities of the sera measured at equilibrium, whereas hyperthyroidism, induced by treatment with thyroxine (T4-rats), leads to the decrease of these abilities. Polyacrylamide gel electrophoresis and isoelectrofocalisation of radioiodinated T4-labelled sera, together with immunoassay of TBPA, demonstrate that both effects are due to TBG, the levels of which rise in PTU-rats and decline in T4-rats, while TBPA levels do not respond to either depletion or excess of the thyroid hormones. TBG rather than TBPA appears as the key thyroid-hormone-binding protein of the rat, inasmuch as it alone expresses a regulatory function of the thyroid hormones at protein synthesis level.

A senescence up-regulated protein: the rat thyroxine-binding globulin (TBG).

Thyroxine-binding globulin (TBG), the major carrier of thyroid hormones in human serum, was thought to be absent in most species, including rodents. We demonstrated recently that in fact the rat possesses a TBG gene, virtually non-expressed in young adults, but actively transcribed during post-natal development. We now find that the TBG gene is also increasingly re-expressed during senescence. Evidence is presented suggesting that physiologically decreased thyroid hormone levels, characteristic of neonates and of ageing rats, might constitute a common factor inducing up-regulation of TBG in both developmental and ageing processes. Rat TBG is to our knowledge the first biochemical 'positive' (i.e. increasing) marker of non-pathological senescence, expressed at both biosynthetic and bloodstream levels.

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.

alpha-Fetoprotein and transcortin behave as acute phase reactants in the maternal and fetal compartments of the inflammatory pregnant mouse.

Immunological and binding methods have been used to demonstrate that acute inflammation induced in the pregnant mouse by a single sc turpentine injection elicits plasma protein responses in the fetal as well as in the maternal compartment. The maternal response involves, along with the classical pattern of positive and negative acute phase reactants seen in the inflammatory nonpregnant animal, a highly specific approximately 2-fold increase of alpha-fetoprotein (AFP) concentrations. In addition, the high pregnancy-associated corticosteroid binding globulin (CBG) levels drop dramatically (2-3 times) in response to inflammation. The fetal response is characterized by small (10-25%) but statistically significant declines of AFP, CBG, and albumin concentrations, without any increase in levels of the positive classical acute phase reactants. The divergent responses of the estrophilic mouse AFP on the two sides of the placental barrier result in a 3- to 4-fold enrichment of the maternal serum vs. an approximately 20% impoverishment of the fetal serum in high affinity estrogen binding sites. The similar decrease in levels of CBG in mother and fetus leads to marked losses of high affinity corticosteroid sites for both. Neither the affinity constants for the estrogen-AFP interactions nor those for the corticosterone-CBG interactions are affected by inflammation. This is the first report of AFP as a positive marker of acute inflammation, of AFP as a pregnancy-specific inflammatory reactant in the mouse, and of a plasma protein response of the fetus in utero to an inflammatory stress undergone by the mother.

The microheterogeneity of rat TBG.

Isoelectric focusing (IEF) of native sera from immature or adult rats and of purified or partially purified rat serum thyroid hormone-binding proteins, demonstrates that rat TBG is a microheterogeneous protein. Autoradiography and radioactivity scans of the IEF plates show that it consists of at least four main isoforms, with bands at pH 4.35, 4.45, 4.5 and 4.55. This pattern is remarkably similar to that reported for human TBG. This is the first demonstration of the polymorphism of this recently discovered major binding protein of the rat.

Inhibition of lipoprotein lipase activity by a monoclonal immunoglobulin in autoimmune hyperlipidemia.

Autoimmune hyperlipidemia (AIH) may be induced a variety of antibodies which inhibit different stages of the lipolytic process by which the lipid load is removed from the circulating lipoproteins. In a patient having a monoclonal gammopathy and a nephrotic syndrome with a glomerulonephritis and a marked hypertriglyceridemia, it was found previously that the monoclonal IgG gamma Lac. reacted with human VLDL as well as with human serum albumin. Here it is demonstrated that the purified IgG gamma inhibits the lipolysis of triglyceride substrates by reacting with a substance (Lac. S) necessary for lipoprotein lipase activity. The interaction of IgG lambda Lac. with serum or HDL-activated triglyceride substrates inhibits the lipolytic activity of human and rat plasma post heparin and also adipose tissue lipases. It slightly inhibits the activity of swine pancreatic lipases. The Lac S. which reacts with IgG Lac. is associated to whole and delipidated VLDL and HDL and not to LDL or purified APo-A. It may be an Apo-C or a non-peptidic co-factor of the lipases which remains bound to the apoprotein core after delipidation. Its lack of species specificity and its presence as traces in HSA preparations favors the latter hypothesis. The Lac. substances is different from the Pg and As substances which were found to react with IgA anti-Pg and IgG anti-As antibodies in previously reported antilipoprotein AIH.

Protective efficacy of a recombinant deoxyribonucleic acid hepatitis B vaccine in institutionalized mentally handicapped clients.

Mentally handicapped clients in institutions are at high risk for hepatitis B virus (HBV) infection. In 1985, 770 mentally handicapped residents from four institutions in the Antwerp area were screened for HBV markers. The prevalence of hepatitis B surface antigen was 10.3 percent (range, 6.1 to 15.2 percent); 42.3 percent (range, 11.5 to 60.1 percent) had antibodies to hepatitis B surface antigen and the hepatitis B core antigen. In 1986, 275 seronegative mentally handicapped residents were vaccinated intramuscularly in the deltoid region with 20 micrograms (1.0 ml) of a recombinant deoxyribonucleic acid yeast-derived hepatitis B vaccine (Engerix-B, SmithKline Biologicals, Rixensart, Belgium) on a zero-, one-, six-month schedule. Serum samples were collected at Months 1, 2, 7, 12, and 24 and were tested for HBV markers by radioimmunoassay. The seroconversion rates for hepatitis B surface antigen antibodies were 39 percent at Month 1 (geometric mean concentration, 6.4 IU/liter), 82 percent at Month 2 (geometric mean concentration, 23.4 IU/liter), 97 percent at Month 7 (geometric mean concentration, 1,034 IU/liter), and 96 percent at Month 12 (geometric mean concentration, 269 IU/liter). Among the 214 residents evaluated at Month 12, 69 percent had hepatitis B surface antigen antibody levels greater than 100 IU/liter (geometric mean concentration, 603 IU/liter). No significant adverse reactions were observed. Within the first seven months of observation, HBV infection was detected in eight of 271 subjects (estimated annual incidence of 5 percent). During this period, none of the clients developed clinical hepatitis or showed biochemical evidence of liver damage. Between eight and 24 months, no additional HBV infections were detected. These data can be compared with an annual incidence of HBV infection of 8.7 percent in a historical cohort of mentally handicapped residents in one of the four institutions.

A seroepidemiological survey of HTLV-I/HTLV-II in selected Belgian populations.

A preliminary survey of the prevalence of HTLV-I antibody during 1987 and 1988 in Belgium revealed no positive results among selected blood donors who sided for some length of time in areas where HTLV-I/HTLV-II is endemic (n = 270), in development-aid workers (n = 145), or in missionaries (n = 35) serving in developing countries. Among foreign groups, African students (n = 212), and political refugees (n = 311), HTLV-I/HTLV-II antibodies were detected and confirmed in 3 cases. Limited data from 1987 and 1988 suggest that HTLV-I/HTLV-II poses no major public health problem in Belgium.

Regulation of rat thyroxine-binding globulin and transthyretin: studies in thyroidectomized and hypophysectomized rats given tri-iodothyronine or/and growth hormone.

We have investigated the role of the thyroid compared with the hypophysis in the regulation of the two saturable thyroid hormone carriers of rat serum, thyroxine-binding globulin (TBG) and transthyretin (TTR). We examined, at serum and hepatic mRNA level, the responses of TBG and TTR to thyroidectomy (Tx), hypophysectomy (Hx) and replacement treatments with tri-iodothyronine (T3) or/and GH, both hormones which are depleted when the thyroid or hypophysis are removed. The studies were performed on male rats at the age of 8 weeks, when the developmentally regulated TBG becomes undetectable after its transient postnatal rise, while the nondevelopmentally regulated TTR presents its normal, age-independent level of expression. Tx-induced TBG re-expression was completely reversed by T3 replacement and unresponsive to GH replacement. TTR in the serum, on the other hand, was not affected by Tx or T3 replacement, moderately reduced by Tx in terms of the amount of mRNA, and markedly reduced by GH replacement. GH treatment, moreover, inhibited the expression of TTR in euthyroid controls. Hx, like Tx, induced TBG re-expression, an effect efficiently antagonized by T3 replacement. However, TBG synthesis was higher in Hx than in Tx rats and less effectively antagonized by T3 replacement. Most unexpectedly, GH induced a dramatic further increase in TBG synthesis, and the TBG synthesized in the GH-replaced Hx rats was entirely resistant to down-regulation by T3 replacement. TTR was markedly decreased at both serum and hepatic levels by Hx, unaffected by T3 and further decreased by GH replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of rat alpha-1-acid glycoprotein by phenobarbital is independent of a general acute-phase response.

Phenobarbital (PB) induces transcription of the alpha 1-acid glycoprotein (AGP) gene, one of the major positive acute-phase proteins, the expression of which is controlled by a specific combination of glucocorticoids and cytokines. This raises questions as to the involvement of glucocorticoids and cytokine pathways in the PB-mediated effect on AGP gene expression. We found that the pattern of whole-serum proteins in PB-treated rats differed markedly from that observed during a typical acute inflammatory response (in turpentine-treated rats): levels of some positive acute-phase proteins (APP) increased slightly (alpha 1-acid glycoprotein, haptoglobin, hemopexin and T-kininogen), while levels of alpha 2 macroglobulin, the most sensitive marker of the acute-phase reaction, decreased. Among the negative APP, neither albumin nor prealbumin decreased while CBG increased. The cytokines involved in AGP gene regulation (mainly IL1, IL6 and TNF alpha) do not therefore seem to mediate the effect of PB on acute-phase protein expression. Glucocorticoid involvement is also ruled out by the observed enhancement of the effect of PB on AGP expression in adrenalectomized animals. Our results suggest that phenobarbital acts on AGP expression by a mechanism independent of the inflammatory pathway.

Prevalence of hepatitis C antibodies in a large sample of Belgian healthcare workers.

OBJECTIVE: To assess the risk of hepatitis C virus (HCV) infection among Belgian (Flemish) healthcare workers. DESIGN: A seroprevalence survey of HCV IgG antibodies. SETTING AND PARTICIPANTS: A systematic sample of 5,064 employees from 22 general hospitals in Flanders and Brussels, Belgium, was tested at the annual occupational medical examination. Together with demographic and occupational data, information was collected on the frequency of blood contact, needlestick injuries, and medical and surgical history. The blood samples were tested using the third-generation Abbott Screen Kit test, with confirmation by Matrix, LIA, and an in-house polymerase chain reaction and the Quantiplex-HCV b-DNA test. RESULTS: 21 persons were found to be positive for HCV markers. The overall prevalence was 0.41% (95% confidence interval [CI95], 0.24-0.59). A statistically significant association was found with a history of blood transfusion (odds ratio [OR], 4.14; CI95, 1.67-10.31) and with history of a clinically apparent hepatitis (OR, 3.98; CI95, 1.60-9.90). Although the ORs for the frequency of blood contact were slightly elevated (between 1.17 and 2.73), this association was not significant. Moreover, a history of needlestick injuries showed a nonsignificant OR of 1.28 (CI95, 0.53-3.09), and no statistically significant difference was found with a variety of duties and tasks. The ORs for potential occupational risk factors were adjusted according to age, gender, antecedents, and other confounders using a logistic regression analysis. Based on this procedure, the ORs decreased slightly. CONCLUSIONS: Flemish healthcare workers showed a lower HCV seropositivity than is seen in the general population; a history of blood transfusion and of clinically apparent hepatitis was most strongly associated with the presence of HCV markers. We concluded that employees in Flemish regional general hospitals are not at an overall increased risk for HCV infection, although occasional transmission through percutaneous injuries is possible, and prevention therefore remains imperative.

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.

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.

Immunochemical characterization and quantitation of human sex steroid binding plasma protein.

The interest in the measurement of human sex steroid binding plasma protein (h-SBP) is now increasing since it allows the estimation of the free fraction of circulating hormones in plasma. Up to this date, this protein could only be determined by measuring the total binding capacity of serum for dihydrotestosterone (DHT). The purpose of the present work was to purify the protein, to prepare a rabbit monospecific antiserum and to develop an immunoelectrophoretic assay of h-SBP. The immunological assay is specific, accurate and sensitive. A good correlation with the radioligand assay was found. The h-SBP levels obtained by immunoelectrophoretic assay of different serum samples were 5.3 +/- 1.4 (SEM) mg/L in normal men and 13.4 +/- 2.6 (SEM) mg/L in normal women.