Hepatitis B virus (HBV) infection causes an estimated 620,000 deaths annually throughout the world.9 Overall, approximately 45% of the world’s population lives in areas of high prevalence of chronic HBV.5 In sub-Saharan Africa, exposure to HBV, as measured by the prevalence of anti-HBc antibodies, varies from 65% to 85%.12 The prevalence of HBV chronic infection as measured by the presence of the surface antigen HBs is particularly high in these countries, ranging from 8% to 20% positivity among the adult general population.2 In Senegal, previous studies have shown that HBV chronic infection prevalence reaches 17% among blood donors,29 11.8% among children 13 years old.3 The prevalence of HBV exposure reported varies between 53% and 60% among children between ages 0 and 5 years.3,23 In Cameroon, the prevalence of hepatitis B surface antigen (HBsAg) positivity varies from 12% among Pygmies7 to 20% among primary school age children6 and 25% among a population ≥4 years.8 In the Central African Republic (CAR), reports show a 14% of HBsAg positivity among young adults presenting at national centers for sexually transmitted diseases18 and 15% among hospitalized patients.16 A more recent study conducted in CAR revealed a prevalence of 8% of HBsAg positivity among children 0–15 years old.11
Following World Health Organization (WHO) recommendations, HBV vaccination policy is increasingly active in countries with high incidence of hepatitis. Senegal and Cameroon integrated the HBV vaccine in their Expanded Program on Immunization (EPI) in 2005, and CAR followed suit in 2008. The HBV vaccination schedule in the EPI includes 3 injections: the first dose 6 weeks after birth and at intervals of 30 days minimum between the following 2 injections.
Although horizontal transmission of the virus during infancy and early childhood persists in sub-Saharan Africa, only a few epidemiological studies have evaluated the HBV transmission rates among children in these countries.1,4,14,19,20,22 We conducted an observational cross-sectional study among hospitalized children between 3 months and 6 years old in Cameroon, CAR and Senegal to assess the prevalence of HBV exposure and infection, as well as factors associated with HBV exposure.
Five pediatric hospitals participated in the study, 2 in Yaoundé (Cameroon), 1 in Bangui (CAR) and 2 in Dakar (Senegal). Between April 2009 and May 2010, all consecutive children—from 3 months to 6 years—hospitalized for any reason—with a blood sample prescribed during hospitalization—health conditions to withstand an extended blood sample of 2 mL minimum—parent or legal guardian’s written consent were consecutively enrolled in this cross-sectional study.
This study was approved by the Senegalese Health Research National Council, the National Ethics Committee of Cameroon and the Scientific and Ethical Committee responsible for validation protocols and study results in CAR. All children anti-HBs negative and anti-HBc negative were offered HBV vaccination free of charge [3 doses of HBV vaccine Euvax B (Sanofi Pasteur, Lyon, France) administered at 0-, 1- and 6-month intervals].
The following data were collected: (1) general characteristics (age, sex and weight), (2) clinical features (reasons for hospitalization, vaccination records on the immunization card when available) and (3) family’s socioeconomic characteristics: mother’s education, personal transportation, electricity, running water and toilet types.
Growth status was expressed separately for men and women by the weight-for-age Z-score.19 Moderate or severe malnutrition was defined from the weight-for-age Z-score value ≤−2.
All samples were tested for anti-HBc by a competitive enzyme immunoassay (EIA) test (ETI-AB-COREK plus; DiaSorin, Saluggia, Italy) and quantified for anti-HBs by a sandwich EIA (ETI-AB-AUK-3; DiaSorin). The level of anti-HBs <10 mIU/mL was considered to be negative. All children anti-HBs negative and anti-HBc positive were further tested for HBsAg by EIA onto AxSYM automat (Abbott, Chicago, IL). HBsAg-positive samples were tested for the presence of HBeAg by EIA (ETI-EBK plus; DiaSorin).
HBV PCR Amplification Assay
The presence of DNA in the plasma was detected and quantified using the Roche Cobas AmpliPrep and Cobas TaqMan (Roche Diagnostics Molecular Systems, Alameda, CA) for extraction and amplification according to the manufacturer’s recommendations. The sensitivity threshold of detection was established at 20 IU/mL.
HBV exposure (past infection) was estimated by the number of children older than 12 months found to be anti-HBc positive, whether anti-HBs positive or negative. HBV infection, acute or chronic, was measured by the presence of HBsAg among the children anti-HBs negative and anti-HBc positive. Quantitative data were expressed as median (interquartile range); qualitative data were expressed as percentages. Univariate analyses were based on χ2 test or Fisher exact test for discrete variables. Continuous variables were compared by analysis of variance or Student t test if the distribution was normal, and otherwise with the Kruskal-Wallis or Mann-Whitney test.
To eliminate maternal antibody transmission, only children older than 12 months were included in the analysis performed to identify factors associated with anti-HBc onset. All variables associated with anti-HBc positivity in the univariate analysis (P < 0.25) were included in a backward stepwise logistic regression model. The age was dichotomized according to the median. Interactions were sought, with all the variables found to correlate with the onset of the anti-HBc antibodies (P < 0.05). In the case of high correlation between variables, only the most correlated variable in the univariate analysis was included in the multivariate model. A P value of ≤0.05 was considered to denote statistical significance. Data were analyzed with STATA software version 12.0 (Stata Corporation, College Station, TX).
General Characteristics of the Children
Between April 2009 and May 2010, 763 children were recruited in Cameroon, 535 children in CAR and 485 in Senegal. An immunization card was available for 428 children and among these, 49% (20/41), 99% (176/177) and 100% (210/210) received an HBV vaccination in CAR, Cameroon and Senegal, respectively.
General characteristics of the children are summarized in Table 1. Forty-four percent of children were female, and the median age was 21 months (12–36 months). The main differences between the 3 countries were age (children in CAR were significantly older than Senegalese and Cameroonian children; P < 0.001), socioeconomic level (Cameroonian children were significantly wealthier than CAR or Senegalese children, with inter alia, 43% living in households with flushing toilets compared with 3% in CAR and 18% in Senegal; P < 0.001) and reasons for hospitalization [the main causes of the children’s hospitalization were malaria in CAR (54%), infectious syndrome and gastrointestinal infection in Cameroon (56%) and respiratory infection or others (63%) in Senegal; P < 0.001]. In Cameroon and CAR, many children lived in the capital, (95%) and (82%), respectively, whereas in Senegal, 74% lived in the suburbs (P < 0.001).
Prevalence of Anti-HBc Antibodies
Among the 1783 children recruited, 346 (19.4%) were anti-HBc positive [213 (16.6%) were children over 12 months]. Of the 346 anti-HBc-positive children, 180 (52%) were anti-HBs positive and 166 (48%) were anti-HBs negative. CAR and Senegal showed significantly higher percentages of anti-HBc-positive children compared with Cameroon: 26% (137/535), 21% (104/485) and 14% (105/763), respectively (P < 0.001).
Anti-HBc positivity distribution was given according to the age groups of the children and country in Table 2 and Figure 1. The prevalence of anti-HBc-positive children was high in children younger than 6 months in the 3 countries: 49.0% (25/51), 44.4% (40/90) and 25.0% (3/12), in Senegal, Cameroon and CAR, respectively. CAR showed a lower rate, although not significant, compared with the 2 other countries, but there were only 12 children recruited at this age. In all 3 countries, a steady decline in anti-HBc positivity was observed until the age of 12 months.
An increase in anti-HBc prevalence occurred in CAR from 12 months old, peaking at 33.8% between 36 and 48 months old. This increase is observed at later ages in Senegal and Cameroon, 18 and 36 months, respectively, reaching a stable level between 10% and 15%.
The distribution of anti-HBc according to age was also performed among the 406 children with complete vaccination (3 injections reported in the immunization card; Figure 2). Although we observed a similar strong decrease in anti-HBc antibodies after the age of 12 months compared with the overall population, we did not observe an increase in the antibodies among children over 24 months old in this subgroup.
Risk Factors Associated with HBV Exposure
The prevalence of anti-HBc among the children older than 12 months, reflecting HBV exposure, was 16.6%, with a significant difference among the 3 countries [26.9% (128/476) in CAR, 13% (42/323) in Senegal and 8.9% (43/485) in Cameroon; P < 0.001)].
Multivariate analysis results showed that being older than 28 months, living in CAR and in a household without flushing toilets were factors independently associated with an increased risk of HBV exposure, defined as anti-HBc positivity among children older than 12 months (Table 3).
Seroprevalence of HBsAg Positivity
Among the 166 anti-HBc-positive and anti-HBs-negative children, 33 were found positive for HBsAg: 27 in CAR, 5 in Cameroon and 1 in Senegal. Thirty-two children had no immunization card, and an immunization card from 1 boy aged 15 months originated from CAR indicated that he was vaccinated, but not with the HBV vaccine. Only 1 child was between 7 and 12 months of age, 9 (27%) were between 13 and 24 months of age and the remaining 23 children (70%) were older than 24 months. Twenty-three children were born before HBV vaccine introduction in the respective EPIs (2008 in CAR and 2005 in Cameroon) and 8 were born during the year of introduction and thus probably did not receive the vaccine.
The percentage of HBsAg-positive children was significantly higher in CAR compared with Cameroon and Senegal (respectively, 5.1%, 0.7% and 0.2%; P < 0.001). Among the HBsAg-positive children, 22/33 were HBeAg positive [20/27 (74%) in CAR, 1/3 in Cameroon and only 1 child HBsAg positive in Senegal]. Viral load median was 8 log IU/mL with values ranging from 5.2 to 8.2 log IU/mL. Figure 3 shows after a rapid decrease in anti-HBc until the age of 12 months, a parallel evolution between the prevalence of HBsAg and anti-HBc after the age of 18 months and an increase in the 2 markers between 18 and 36 months old, with stabilization after 36 months. In Senegal, while the prevalence of exposed children (anti-HBc-positive) was relatively high (21.4%), only 1 child was HBsAg positive.
Our results show a 19.4% anti-HBc-positive prevalence in children aged 3 months to 6 years old in 5 hospitals in 3 African countries and 16.6% among children older than 12 months. The overall percentage of children anti-HBc positive observed among the children younger than 6 months was particularly high (44.4%), decreasing rapidly and consistently reaching 18.8% between 6 and 12 months old. It is generally accepted that maternal antibodies are detectable until 18 months of age. Here, we show that maternal anti-HBc disappeared earlier, before the age of 12 months, in our population of children born to exposed mothers and that almost 50% of the mothers have been exposed to the virus HBV. This rapid decline of anti-HBc antibodies suggests that many antibodies detected at these ages were of maternal origin, with a time-declining affinity and avidity of passively transmitted antibodies. In the assay procedure, children’s serum samples and recombinant HBcAg are incubated in antibody-coated microwells. If anti-HBc antibodies are present in a specimen, they compete with the antibody coated in the microwells for the recombinant HBcAg. The quantity of tracer that binds to the solid phase via recombinant HBcAg and the consequent enzyme activity are inversely related to the anti-HBc concentration present in the specimen. So, the use of a competitive EIA test to detect passively transmitted antibodies such as anti-HBc in this study is particularly well adapted to differentiate between the passively transmitted low affinity antibodies versus newly synthesized higher affinity antibodies against the core of the HBV. This explains the rapid decline in anti-HBc reactivity observed with the competitive EIA, as previously observed with other antibodies passively transmitted to children in HIV-infected mothers.26
Our results confirm those obtained by Barin et al3 before the administration of HBV vaccine in Senegal in 1981, who reported a similar rapid decline of the anti-HBc antibodies between the ages of 6 and 12 months. After declining during the first 12 months of life, the anti-HBc positivity increased in different ways in the 3 countries. Although the prevalence of anti-HBc increased immediately after 12 months in CAR and continued rising steadily until 72 months, the anti-HBc prevalence increased later in Cameroon (36 months) and Senegal (18 months). The profile observed in CAR, which shows that HBV exposure occurs as early as 12 months, is in accordance with the late introduction of HBV vaccine in the EPI (2008) and is consistent with data reported in South Africa before the integration of the HBV vaccine.28
In Cameroon, HBV exposure occurs among children older than 36 months, a result consistent with integration of the HBV vaccine to the EPI in 2005, when these children were not vaccinated against HBV. The 10% HBV exposure observed after the age of 12 months is consistent with the generally observed 10% nonresponse observed among children vaccinated with HBV vaccine.21,25,27
In contrast to Cameroon, in Senegal, there was a peak of anti-HBc among children between 18 and 36 months old. This increase in anti-HBc among children born after integration of HBV vaccine can be explained by the fact that these children were not protected against HBV infection. This hypothesis is supported by our previously published results from this cohort of patients,21 which showed that a high proportion of Senegalese children vaccinated during the years 2006 and 2007 did not respond to HBV vaccine (43%). This low level of protected children contrasted with 86% HBV vaccine coverage reported by the WHO in Senegal for the same years.30 This lack of response to HBV vaccination could be due to problems with storage conditions in Senegal, or to a quality problem with the vaccine preparation, or it may be related to children’s nutritional status, particularly frequent in this population of Senegalese children. Moreover, there may have been a delay between the time the vaccine was officially introduced in the EPI and when it was actually available in all vaccination centers. Despite the peak of anti-HBc observed between 18 and 36 months, the antibody level was low in Senegal compared with that reported by Barin in 1981 before the introduction of the HBV vaccination.3
When repeating the same analysis globally in all 3 countries among the 406 children vaccinated against HBV with 3 injections according to immunization cards, we confirmed the same decrease in anti-HBc at 12 months old. We did not observe an increase in the anti-HBc after the age of 12 months. The low number of children with an immunization card did not permit a separate analysis by country.
We identified 3 factors that positively correlated with exposure: (1) age, with the older children being the most infected, reflecting the year that HBV vaccine was integrated into the EPI; (2) living in CAR, revealing the positive impact of anti-HBV vaccination in Senegal and Cameroon and (3) non-flushing toilet. Having a flushing toilet in Africa may reflect more than a means of human waste disposal. The hypothesis for HBV contamination in childhood is based on prolonged contact with infected people, by inoculation of HBV into cutaneous scratches, abrasions or other lesions.25 A higher risk among children with low socioeconomic levels may explain a higher risk of HBV transmission. It has been suggested that HBV exposure occurring during the first 4 years of life resulted from mother-children transmission, whereas infection occurring after this age was due to horizontal transmission at home or school.3,14 However, the mode of HBV horizontal transmission is not fully understood.10
HBsAg was found in 33 of 1783 children (1.9%), with different percentages depending on the country. The prevalence of HBsAg-positive children reached 5.1% in CAR, whereas it was only 0.7% in Cameroon and 0.2% in Senegal.
Our results show that all HBsAg are detected only after 6 months of age among the population of children anti-HBc positive and anti-HBs negative, suggesting that the children were infected by horizontal transmission and that immunization at 6 weeks should prevent HBV. However, because of the small number12 of children younger than 6 months in CAR, these results should be confirmed with further studies that involve more children.
The viral load among HBsAg-positive children was high, a result consistent with an immunotolerant stage of HBV. This stage, characterized by a high viral load and HBeAg positivity, displays neither inflammatory activity nor the onset of fibrosis. It occurs among those infected at birth or during early infancy.13
Only children anti-HBc positive and anti-HBs negative were tested for the HBsAg. This lack of systematic HBsAg research may have resulted in an underestimation of HBV infection. Nevertheless, the strong correlation between the presence of anti-HBc and HBsAg during HBV infection confirms the relevance of using anti-HBc detection to highlight any exposure to HBV among these children.
Our results obtained in Cameroon and Senegal between 2009 and 2010, 4 years after the introduction of the HBV vaccine into the EPI, show a decrease in anti-HBc antibodies among children born after the 2005 integration of the HBV vaccine into the EPI in Senegal and Cameroon, suggesting a positive impact on HBV exposure. In addition, the prevalence of HBsAg is lower in Cameroon and Senegal among our population of children compared with the historical data,3,6 suggesting a positive impact on HBV infection. These findings are certainly debatable, because the seromarkers were not measured in a comparable population of hospitalized children before the beginning of vaccination. Besides, because our children were recruited in emergency rooms of pediatric hospitals, the prevalence of anti-HBc and HBsAg positivity that we observed may have been overestimated, even if most reasons for hospitalization were not directly related to hepatitis. Finally, the introduction of the HBV vaccine into the EPI in our 3 countries is certainly too recent to assess its impact on the risks of HBV exposure in noninfected subjects.
This cross-sectional study, conducted without following up on included children, limits our ability to estimate true anti-HBc prevalence but suggest a positive impact of the integration of the HBV vaccine on HBV infection. Long-term follow-up studies have shown the effectiveness of HBV immunization programs on HBV exposure and infection by measuring the anti-HBc and HBsAg.15,17,24 However, because cohort studies are time-consuming and costly, and because the public health significance of anti-HBc measurement is debatable, serological surveys of HBsAg and anti-HBc prevalence should be conducted to evaluate the effectiveness of the HBV vaccine after its integration into the EPI, as recommended by the WHO.31
As soon as children exceed the age of 12 months, it is possible to differentiate between passive transfer of anti-HBc maternal antibodies and antibody responses to HBV exposure. Decrease in anti-HBc antibodies among children born after the 2005 integration of the HBV vaccine into the EPI in Senegal and Cameroon, as well as the low prevalence of HBsAg, suggests a positive impact of HBV vaccination. Immunization coverage and the prevalence of HBV infection observed in our studies are encouraging for health authorities to continue their important work in strengthening vaccination programmes in Africa.
The authors thank all the children and their families for the time and effort they devoted to the study. They are also grateful for the cooperation and support of the field staff, health sector personal and laboratory personnel. They wish to thank Dr. Tamara Giles-Vernick for rereading the manuscript.
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HBV markers; HBV exposure; HBV vaccine; childhood
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