It has been estimated that about 2 billion individuals worldwide have been infected with the hepatitis B virus (HBV) and about 350 million individuals live with chronic HBV infection. Approximately 600 000 deaths arise from HBV-related cirrhosis and hepatocellular carcinoma every year. Therefore, chronic hepatitis B ranks as the 10th leading cause of death worldwide 1–3. HBV-related cirrhosis represents the main indication for liver transplantation in Romania, where the standardized rate of mortality still ranks first within the European Union 4.
Although the prevalence of HBV infection has already been investigated in various groups (healthy donors, healthcare workers, pregnant women, institutionalized children), its prevalence and risk factors in the general population in Romania remain largely unknown. Previous studies have shown a 6% prevalence rate of hepatitis B surface antigen (HBsAg) carriers in North-Western Transylvania 5, confirming the intermediate endemicity of HBV infection in our country. In a prospective multicenter study assessing the etiological profile of chronic hepatitis and liver cirrhosis, carried out on 2022 patients covering all geographical regions of Romania, HBV infection has been identified as an important cause of chronic hepatitis (35.9%) and liver cirrhosis (44.2%) 6. A recent evaluation of seroepidemiology of hepatitis B in 10 European countries showed a 5.6% prevalence of HBsAg and a 20.5% prevalence of antibodies to the hepatitis B core antigens (anti-HBcAb) in 1259 residual sera samples collected between 1996 and 2003 in Romania 7.
The recognized risk factors for HBV infection include transfusions of blood and blood products, recycling of contaminated needles and equipment during medical procedures, acupuncture, piercing or tattooing, needle sharing by intravenous drug users (IVDU), sexual transmission, perinatal transmission, men who had sex with men, promiscuous heterosexuals, immunosuppressed patients, patients on a hemodialysis program, tissue and organ transplantation, and healthcare transmission. The improvement in socioeconomic status and hygienic conditions, the introduction of general vaccination for newborns and children from 1995 and for risk groups in the beginning of the 1990s, as well as public health policies emphasizing the use of disposable needles and medical equipment have led to a sharp decline in acute hepatitis B in Romania over the last two decades (the overall incidence decreased from 43/100 000 in 1989 to 8.5/100 000 in 2004) 8. However, the burden of HBV infection in Romania still represents an issue of great concern for at least three main reasons: (a) there are no national routine surveillance and registration systems for chronic hepatitis B and for viral-related liver cancer; (b) data on the prevalence of HBV are obtained from limited studies focusing on selected population groups (blood donors, pregnant women, individuals living in small geographical regions); and (c) knowledge of the actual prevalence of HBV infection in the general population is crucial to understand the extent and importance of this public health problem and to foresee its future impact on the health system and ensure an adequate allocation of financial resources.
The aim of this project was to evaluate the prevalence and risk factors of HBV infection in an adult general population in Romania.
Materials and methods
Study population and sample design
The seroprevalence and associated risk factors of HBV infection were investigated by a nationwide cross-sectional survey conducted between 2006 and 2008. This survey was designed as part of a collaborative project between the Romanian Society of Gastroenterology and Hepatology and Roche Romania aimed to assess the impact of viral hepatitis B and C infections in our country (ML 21277/2006).
The participants were chosen using a stratified, multistage sampling design to obtain a representative sample of the adult general population of Romania.
The survey covered a random sex-stratified and age-stratified sample with 17 600 individuals, aged 18–69 years, selected from 88 family physician (FP) praxis lists. We chose randomly from the database provided by the national MEDINET network of family physicians two FP praxes for each of the 41 counties of Romania and one FP praxis from each of the six urban districts of Bucharest.
One FP praxis was from a rural area and another was from an urban area in each county, given that ∼45% of the population of Romania lives in rural areas.
A letter with detailed information about the study was sent to the selected participants to invite them to participate. Individuals who were not found or refused participation were not replaced. A written informed consent was obtained from each participant before their enrollment.
The survey was approved by the National Ethics Committee (No. 4154/26.10.2006) and conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
Serological testing and questionnaire
Blood specimens collected by FP were packed in temperature-controlled medical transport bags and were delivered to the clinical laboratory on the same day by express delivery companies specialized in the transport of biological specimens. Serum samples from all participants were tested both for antibody to the hepatitis B core antigen (anti-HBcAb) and the HBsAg using commercially available enzyme-linked immunosorbent assays (Abbott laboratories, Abbott Park, Illinois, USA).
The following definition was used for past HBV infection – presence of anti-HBcAb and absence of HBsAg. Chronic HBV infection was defined as the presence of anti-HBcAb and HBsAg.
A questionnaire was used to collect information on the sociodemographic characteristics of the participants and the potential risk factors for HBV transmission. The face-to-face interviews were conducted by the FP at the same time as the blood sample collection. Sociodemographic information included age, sex, urban or rural and geographical area of residence, marital status, ethnicity, educational level, and employment. The questions on the risk factors for HBV infection included the following: the presence of infected individuals with HBV or hepatitis C virus (HCV) or individuals with chronic hepatitis B or C in the household; personal history of chronic hepatitis B, antihepatitis B vaccination, and personal history of medical invasive procedures [dental treatment, elective and emergency surgery, frequent injections at a medical facility or at home (‘frequent injection’ was defined as someone who had had more than six ‘injections’/year before the introduction of disposable needles and syringes), blood and blood product transfusions, hemodialysis, solid organ transplantation, etc]; ear and body piercing, tattooing, acupuncture, and attending beauty salons (contaminated cutting instruments); sharing a toothbrush or razor blades with other family or community members; accidents or injuries with serious trauma; occupational hazard; a history of IVDU; age of first sexual intercourse; sexual risk behavior (sex with prostitutes or unknown individuals, men who have sex with men, more than 10 lifetime sexual partners); time spent in prison; and alcohol abuse.
Because of logistic and financial constraints, fieldwork was conducted over a 2-year period (October 2006–October 2008) sequentially in Muntenia and Dobrogea (October 2006–January 2007), Moldavia (November 2007–January 2008), and Transylvania (June–October 2008).
The crude prevalence of past HBV infection or chronic HBV infection by sex, age groups (18–29 years, 30–39 years, 40–49 years, 50–59 years, and 60–69 years), geographical regions of residence, area of residence (rural, urban), ethnic groups, educational levels, marital, social status, and for all investigated risk factors was calculated. Univariate comparisons between categorical variables were carried out using the χ 2-test and the Wilcoxon rank-sum test, as appropriate. The trend across ordered groups such as age groups and education level was calculated using a Wilcoxon-type test developed by Cuzick. We evaluated the association between the risk of having had a past HBV infection or chronic HBV infection and the characteristics of the participants or risk factors using an unconditional multiple logistic regression model. The dependent variable of our model was one of the outcomes (presence or absence of markers of a past HBV infection and of chronic HBV infection, respectively). The independent variables were the characteristics or risk factors to be investigated together with sex, age group, and area of residence (rural, urban). We tested separately the association for each investigated characteristic or risk factor. Odds ratios (ORs) adjusted for sex, age groups, and area of residence (urban, rural) were calculated with the corresponding 95% confidence intervals (CIs). All statistical tests were two-sided and a value of P less than 0.05 was used to indicate statistical significance. We did not use Bonferroni correction adjustment for multiple testing. All statistical analyses were carried out using STATA/SE 11 software (StataCorp, College Station, Texas, USA).
Out of the 17 600 individuals from 88 centers chosen randomly as the study population, 13 460 individuals provided informed consent. The patients with compromized blood samples, incomplete data collection, or inconclusive serological results were excluded from the final analysis. Finally, the prevalence of chronic HBV infection (HBsAg-positive and anti-HBcAb-positive samples) was assessed in 13 127 individuals (74.6%), whereas past HBV infection (anti-HBcAb-positive but HBsAg-negative samples) was assessed in 12 470 individuals (70.5%).
Prevalence of hepatitis B virus chronic infection
We identified 576 HBsAg-positive individuals from the 13 127 samples assessed. The overall prevalence of HBV chronic infection assessed by HBsAg seropositivity in our sample was 4.4% (CI: 4.0–4.8%). The prevalence of HBsAg was significantly lower among the participants from Transylvania and Banat (3.1%, CI: 2.6–3.6%; P=0.002) as compared with those from Moldavia (4.5%, CI: 3.7–5.5%). The highest prevalence of HBsAg seropositivity was observed in the individuals living in Muntenia and Dobrogea (5.4%, CI: 4.8–6.0%; with P=0.00001 when compared with those living in Transylvania and Banat) (Table 1).
The prevalence of HBsAg was higher among men than in women (5.5 vs. 3.6%; P=0.00001) and those from rural areas compared with those from urban areas (4.78 vs. 4.01%; P=0.031).
HBsAg positivity showed a statistically significant increasing trend (P trend =0.001) with age groups (18–29 years, 30–39 years, 40–49 years, and 50–69 years). The lowest chronic HBV infection of 1.9% was found among Hungarian individuals who, in large majority, live in Transylvania and Banat.
The prevalence of HBsAg positivity showed a statistically significant decreasing trend (P trend =0.001) with the education level expressed as the number of years of education. Unemployed individuals had a significantly higher HBV prevalence of 7.8%, with P=0.006, compared with employers/self-employed individuals, with 3.4%. No association was found between marital status and HBsAg seropositivity.
As shown in Table 2, an increased prevalence of infection is associated with familial exposure both to HCV or HBV infection (OR=1.87, CI: 1.47–2.34; P=0.0001) and to chronic B or C liver disease (OR=1.65, CI: 1.23–2.23; P=0.001). No association was found with HBV vaccination.
A personal history of blood or blood product transfusion (OR=1.37, CI: 1.37–1.80; P=0.022), of surgical interventions (OR=1.21, CI: 1.02–1.44; P=0.028), tattooing (OR=1.41, CI: 1.03–1.94; P=0.033), of sexually transmitted diseases (OR=2.17, CI: 1.33–3.52; P=0.002), and alcohol consumption over 60 g/day (OR=1.44, CI: 1.16–1.78; P=0.001) were risk factors associated with HBsAg seropositivity.
At the same time, we found a higher prevalence of HBsAg for individuals who were imprisoned as compared with those who were not (9.6 vs. 4.4%; P=0.016), for those who had first sexual intercourse at younger than 18 years of age vs. those who had first sexual intercourse when they were older than 18 years of age (5.0 vs. 4.2%; P=0.026), and for individuals with multiple sexual partners vs. those with no such sexual habits (5.5 vs. 4.2%; P=0.024).
No association was found between a hepatitis C diagnostic and occupational exposure to blood products, frequent hospitalizations, organ transplantation, hemodialysis, IVDU, unsafe parenteral injections, invasive dental procedures, serious traffic, work and domestic accidents, acupuncture, body piercing, attending beauty salons, sharing tooth brushes and blades, or chronic HBV or HCV infection in their sexual partner.
Prevalence of past hepatitis B virus infection
Anti-HBcAb-positive samples with negative results for HBsAg were detected in 3370 individuals from the 12 470 individuals with negative results for HBsAg. The overall prevalence of past HBV infection in the Romanian sample aged 18–69 years, tested by anti-HBcAb seropositivity, was 27.0% (CI: 26.2–27.8%). The lowest anti-HBcAb prevalence of 24.8% (CI: 23.6–26.0%) was found among the individuals living in Transylvania and Banat. The participants from Muntenia and Dobrogea showed a significantly higher (P=0.0002) prevalence of 28.0% (CI: 26.8–29.2%), whereas those from Moldavia had the highest prevalence ratio of 29.4% (CI: 27.5–31.5%; P=0.0001) compared with the individuals from Transylvania and Banat (Table 3).
The proportion of individuals with past HBV infection (anti-HBcAb positive) showed a statistically significant increasing trend with age (P trend =0.001) as shown in Fig. 1. We found no significant differences in anti-HBcAb seroprevalence among men and women or among those living in rural areas in comparison with urban areas. The lowest proportion of anti-HBc-positive individuals (20.0%) was found in Hungarian ethnics (P=0.00001), and the highest proportion (38.7%) in Romany individuals (P=0.0002), in comparison with Romanian ethnics (27.4%).
The anti-HBcAb prevalence showed a statistically significant decreasing trend (P trend =0.001) with the education level expressed as the number of years of education. Unmarried participants had the lowest anti-HBcAb prevalence of 17.6% (P=0.00001), whereas widowed individuals had the highest anti-HBcAb prevalence of 40.0% (P=0.00001) compared with married individuals (27.6%).
HBV vaccination resulted in a statistically decreased prevalence of anti-HBc positivity, with OR=0.73 (CI: 0.58–0.91; P=0.007). Familial exposure to chronic B or C liver disease is associated with an increased prevalence of anti-HBcAb (OR=1.23, CI: 1.05–1.44; P=0.011). Individuals diagnosed with hepatitis C diagnostic are at a higher risk for HBV infection/exposure with OR=2.18 (CI: 1.47–3.23; P=0.0001) to be anti-HBcAb positive.
As shown in Table 4, a personal history of blood or blood product transfusion (OR=1.21, CI: 1.06–1.37; P=0.005), of surgical interventions (OR=1.15, CI: 1.06–1.24; P=0.0001), tattooing (OR=1.37, CI: 1.15–1.62; P=0.0001), age at first sexual intercourse before the age of 18 years (OR=1.14, CI: 1.04–1.24; P=0.004), and alcohol consumption over 60 g/day (OR=1.13, CI: 1.13–1.27; P=0.034) were risk factors associated with anti-HBcAb seropositivity.
At the same time, we found a higher prevalence of anti-HBcAb among individuals who underwent emergency surgery as compared with those who did not undergo this type of intervention (31.2 vs. 28.6%; P=0.041), and for those who had required frequent hospitalizations vs. individuals who had not (32.9 vs. 26.5%; P=0.00001) (Table 4).
No association was found between occupational exposure to blood products, imprisonment, organ transplantation, hemodialysis, unsafe parenteral injections, IVDU, invasive dental procedures, serious traffic, work and domestic accidents, acupuncture, body piercing, attending beauty salons, sharing tooth brushes and blades, sexually transmitted diseases, multiple sexual partners, or chronic HBV or HCV infection in their sexual partner.
This is the first population-based study to report the seroprevalence and risk factors associated with past HBV infection, or with chronic HBV infection in Romania, assessed for the first time using representative national data. The sample population in this survey represents the population of Romania aged between 18 and 69 years, residing in different geographic areas throughout the country. The stratified, multistage sampling method used in our study was preferred because it ensured a higher participation rate, lower costs, easier logistics, a smaller and well-trained operative team, and because it could be finalized in a shorter period of time (1–3 years) 9. One of the flaws of this sampling method is that individuals without health insurance were not included in the study, but this is an unusual occurrence in Romania where national health insurances cover ∼83–87% of the population. To minimize the selection bias of our sampling method, we increased the sample size to 17 600 individuals to ensure an accurate assessment of the prevalence of HBV. The participation rate of 74.6% in this study is in agreement with those reported by previous studies (55–85.5%) 10–14. There might also be an underestimation of exposure related to IVDU, imprisonment, alcohol abuse, and sexual behavior, because of socially desirable responding.
The lack of accurate data on the prevalence of hepatitis inhibits more effective prevention and control programs at both international and national levels. This study yielded several important findings. First, this nationwide cross-sectional survey confirmed the intermediate prevalence of HBV chronic infection (in the range of 2–8%) in the adult general population in Romania, but lower than that reported previously (4.4% compared with 5.6–6%) 5,7. It is noteworthy that there are some previous published studies 5,7 showing an intermediate endemicity of HBV infection in Romania, but they are cross-sectional in design and have been carried out in selected populations. There is a wide variation in reported HBsAg carriage in Europe, increasing west to east and north to south. Our data highlight the same pattern of HBsAg prevalence. This geographical variation is further reflected in the estimated mortality from cirrhosis and hepatocellular carcinoma because of HBV chronic infection, with about 200 attributed deaths annually in northwestern Europe compared with nearly 19 000 in Central and Eastern Europe 15.
Second, this population survey shows that the main source of moderate endemicity of HBV infection in Romania is primarily unsafe medical procedures in the past associated with contaminated needles and reuse of medical instruments. Significant differences in the prevalence of HBV infection were found between participants from different geographical regions, with the highest rate of HBsAg in individuals from Muntenia and Dobrogea and the lowest rate in those from Transylvania and Banat. Geographical differences in prevalence were consistent with variations in the safety of healthcare-related procedures, the quality of disinfection techniques, use of qualified medical services vs. empirical treatment methods, the proportion of populations with low social and economic status, and low educational level.
Furthermore, in the present study, HBsAg seropositivity has shown a statistically significant increasing trend with age groups, similar to other studies 16–18, but in contrast to the one reported by Nardone et al. 7. This is because of the childhood vaccination program implemented in Europe since 1991 and in Romania since 1995. This trend indicates a cumulative risk of HBV infection over time, suggesting at the same time a cohort phenomenon with reduced transmission in recent years because of vaccination and continuous improvement in healthcare conditions. Since the mid-1990s, disposable syringes, materials, and instruments were broadly introduced, blood was systematically tested for HBV infection, access to medical services has improved, and, as a consequence, the rate of nosocomial and parenteral transmission of HBV infection has decreased.
Statistically significant sex differences (males>females) in HBsAg carriage were reported in Romania in agreement with other studies 7,18,19. No sex differences were observed in any of the countries for past exposure to HBV 7,20 and the same was true for our study.
Educational and socioeconomic status also plays an important, although indirect, role in the transmission of HBV infection. In this study, educational level was inversely related to the prevalence of HBsAg and anti-HBcAb. This might be because individuals with a lower educational level are more likely to have a low income, an unhealthy lifestyle, limited access to healthcare, and be less well informed about the prevention of diseases. Consequently, their rates of morbidity are higher than average, exposing them to infection because of reutilized and virus-contaminated instruments.
Between other risk factors for HBsAg seropositivity, we found young age (<18 years old) at the time of first sexual intercourse and multiple sexual partners. Sexual transmission is important, but does not represent the most common route of transmission as reported in the Netherlands 21. Occupational transmission of HBV was not significant in our study in contrast to others 22,23, suggesting an effective immunization against HBV in healthcare workers in Romania.
Transmission of HBV through transfusion of blood products has been largely eliminated in most parts of the world by screening blood donors and implementing techniques that ensure viral inactivation of products prepared from blood 24. However, in Romania, there was a positive correlation between previous morbidity (blood transfusion, surgery, past liver diseases) and the presence of serum markers of HBV infection, similar to Bulgaria 25.
Previous HBV infection is common among HCV carriers and may exert a negative impact on the natural history of HCV infection (liver enzymes flare, advanced hepatic fibrosis, and cirrhosis), as well as on response to antiviral therapy. Our results indicate that individuals with a diagnosis of chronic hepatitis C are at a higher risk for HBV exposure (OR=2.18; P=0.0001), similar to certain other studies 25,26, but in contrast to an Asian one 20.
In terms of exposure to HBV infection (i.e. anti-HBcAb seropositivity), it was found to be 27.0%, higher than those reported by Nardone et al. 7 among the 10 European countries analyzed. Similar to HBsAg positivity, the same increasing tendency with age was reported for anti-HBc positivity, from 4.0% in those younger than 30 years to 22.5% older than 50 years of age in an Asian hyperendemic country for HBV 20. The highest rate of past exposure to HBV infection in our sample was found in individuals older than 60 years of age (39.4%). In both Germany and Italy, the highest prevalence of past exposure to HBV infection was observed in those aged older than 40 years (18.8%), but much lower than that in our country (26.7% for individuals 40–49 years of age) 7. Age was independently associated with the likelihood of anti-HBc positivity in a Greek study (the prevalence was 21.4% in individuals ≥42 years) 27. HBV vaccination is also responsible for the decreased prevalence of anti-HBc-positive antibodies in young individuals 28,29, as shown by our study.
The results of this first population-based survey conducted in Romania significantly contribute toward the understanding of the actual epidemiology of HBV infection in Europe, classifying Romania as an intermediate endemic zone. These data are critical for a better understanding of the burden of an infection that is still a public health problem in Romania. The advantage of this study is that it used high-quality laboratory tests and covered the adult general population (from 18 to 69 years of age) and in the entire country of Romania.
Some national programs to control the spread and consequences of HBV infection, such as vaccination of newborns and high-risk individuals or promotion of safe sex campaigns, are working, as indicated by the fact that older individuals show a much higher prevalence than younger generations; nevertheless, our study suggests the need for urgent measures for the prevention and control of HBV infection: (a) to increase the awareness of HBV infection and the knowledge of its routes of transmission through public campaigns funded by the government; (b) to lower the cost of HBV tests; (c) to use more sensitive HBV tests; (d) to make the screening of anti-HBcAb in blood donors mandatory; (e) to screen HCV-infected patients; (f) to widen and strengthen vaccination policies and programs; and (g) to offer the HBV vaccine to HCV-positive individuals.
The Medinet Epidemiological Study Team: D. Toma, S. Mariutan, S. Georgescu, M. Vasile, C. Popescu, E.D. Casariu, C. Isar, M. Bataiosu, R.G. Lupu, S. Moia, A. Cara, L. Catrinoiu, V. Fronie, D.V. Sandu, I. Dogaru, G. Comisel, E. Gogonea, F. Meirosu, M. Pripoae, E.D. Neacsu, M. Paraschiv, G. Ciuche, C. Tintaru, L. Hritcu, P. Serban, I.L. Brinza, V. Naumov, M.A. Silion, F. Baicu, R.F. Miftode, I. Picioreanu, I.D. Timofte, C. Neagu, R.M. Papuc, M.L. Oniceanu, M. Ilisei, A.N. Grom, M.A. Gheorghiu, M.D. Andrei, S. Dinisoara, C. Atanasiu, M.G. Moisescu, A. Gidoiu, S.P. Maciuca, N. Nasalciuc, C.M. Tronea, D.A. Petcu, I.V. Radulescu, I.M. Cojocaru, C. Baraganescu, C.M. Cristea, M.N. Bezman, A. Badica, C. Nistor, M. Rapeanu, O Stancu, M. Petrea, G. Haber, C. Kadar, I. Ionescu, M. Capatana, E. Pop, Z. Farcas Pall, D. Suteu, L. Szep, A. Parpalea, S. Nicolae, M. Stanescu, L. Neagu, V. Tatu, V. Moldovan, E. Sipos, A. Badulescu, A. Rusnac, T. Melinda, C. Grindean, D. Bena, G. Lascu, M. Prisacaru, D. Juncu, G.A. Moldovan, E. Pop, T. Horincar, D. Holobutiu, C. Ruba, I. Jipa, F. Hoadrea, D. Petrovici, C. Ciresan.
Conflicts of interest
There are no conflicts of interest.
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