HBV carriage in children born from HIV-seropositive mothers in Senegal: The need of birth-dose HBV vaccination
Abstract
Hepatitis B is a major public health problem in Senegal, a country with high prevalence and a transmission occurring mainly during infancy. Only, one 6–8 weeks vaccination campaign was initiated in 2005 and it was part of the expanded program of immunization. The aim of this study was to determine the prevalence of HBsAg in children born from HIV-seropositive mothers by using dried blood specimens. Specimens were collected between July 2007 and November 2012 from children aged 2–48 weeks in Dakar and decentralized sites working on HIV mother-to-child transmission prevention. HBsAg detection was performed using Architect HBsAg Qualitative II kit (Abbott Diagnostics, Ireland) and for all reactive samples confirmation was done using Architect HBsAg Qualitative II Confirmatory kit (Abbott Diagnostics, Ireland). Nine hundred thirty samples were collected throughout the country with 66% out of Dakar, the capital city. The median age was 20 weeks and 88% of children were less than 1 year of age with a sex ratio of 1.27 in favor of boys. HBsAg was detected in 28 cases giving a global prevalence of 3%. According to age, HBsAg prevalences were 5.1% for children less than 6 weeks, 4.1% and 4.6%, respectively, for those aged 12–18 weeks and 18–24 weeks of age. The HIV prevalence was 2.6% with no HIV/HBV co-infection. This study showed a high rate of HBV infection in children under 24 months, highlighting the need to promote birth-dose HBV vaccination as recommended by WHO. J. Med. Virol. 88:815–819, 2016. © 2015 Wiley Periodicals, Inc.
INTRODUCTION
Hepatitis B virus (HBV) infection represents a worldwide major problem of public health with approximately two billion infected people with 240 millions chronic carriers of hepatitis B surface antigen (HBsAg). More than 780,000 cases of deaths from HBV-related complications especially cirrhosis and hepatocellular carcinoma were reported each year [WHO, 2013a].
The risk of chronicity is 90% in children under 1 year of age and 25–30% in children aged 1–5 years due to familial transmission [Degos, 2005; Marcellin and Lada, 2006]. Sub-Saharan Africa is an area of high endemicity for HBV with more than 8% of HBsAg prevalence [Kramvis and Kew, 2007; Howell et al., 2014]. In this region, HBV is transmitted mostly from mothers to neonates during the perinatal period and in household. The transmission risk is particularly high (70–90%) when the mother has HBV replication markers and decreases to 10–40% in the absence of active replication [Andre, 2000; Lavanchy, 2005]. In Senegal, HBsAg carriage was estimated at 11.6% in pregnant women [Lo et al., 2012] and the prevalence of HBV markers was 59.4% among children aged 0–5 years [Sall Diallo et al., 2004].
Since 2013, WHO has recommended HBV vaccination within 24 hr for all children born from infected mothers. For all other children, vaccination is done through the Expanded Program on Immunization (EPI) conducted in countries [WHO, 2013b]. A large immunization campaigns have shown in many countries significant decrease in the incidence of HBV infection in infants, children, and even adolescents [Vallejo et al., 2008; Mackie et al., 2009; Rey-Cuille et al., 2013]. In Senegal, a massive vaccination campaign was initiated since 2005 as a part of the EPI and Hepatitis B immunization is offered with the hexavalent vaccine (Diphtheria, Tetanus, Pertussis, Polio, Haemophilus b influenzae, and Hepatitis B) at 6–8 weeks after birth [PNLH-Senegal website]. Despite WHO recommendations, HBV vaccination at birth is not systematic, even in a case of pregnant women with HBsAg positive. Recent findings showed a low prevalence of HBsAg carriage in children in the country, due to immunization [Rey-Cuille et al., 2013], whereas a low immune response in vaccinated ones was found in Dakar, the capital city [Rey-Cuille et al., 2012].
To better help policy makers to adopt quickly WHO recommendations, it is essential to estimate the rate of HBV infection in infants and children in the country. HIV early infant diagnosis (EID) offered the opportunity to get dried blood specimens (DBS) from children less than 24 months through the national HIV prevention of mother to child transmission (PMTCT) program. The aim of this study was to determine the prevalence of HBsAg using DBS collected from children born from HIV-seropositive mothers in Senegal.
MATERIALS AND METHODS
Samples Collection and Processing
DBS samples were collected between July 2007 and November 2012 as part of HIV EID from PTMCT sites distributed throughout the country. DBS cards were prepared by collection of blood drops from toe or finger on the predefined circles on Whatman 903™ paper filter (GE HealthCare, Freiburg, Germany). Dried cards were then transferred to the reference Bacteriology–Virology laboratory of the University Teaching Hospital Aristide le Dantec where HIV molecular testing was performed. The remaining DBS cards that is with sufficient spots for further analysis were used to detect HBsAg on the Architect analyzer (Abbott Diagnostics, Ireland).
From each card, three spots of 6 mm were eluted in 600 µl of PBS at 4°C during 24 hr. HBsAg detection was performed using Architect HBsAg Qualitative II kit (Abbott Diagnostics, Ireland) according to manufacturer's instructions. Architect is a microparticle immunoassay technology for the qualitative detection of HBsAg in human serum and plasma by chemiluminescence. All reactive samples need to be confirmed and were retested in duplicate and the double reactive samples were confirmed using Architect HBsAg Qualitative II Confirmatory kit (Abbott Diagnostics, Ireland) according to manufacturer's instructions.
Statistical Analysis
Data were expressed as absolute value and percentage and were analyzed using Epi InfoTM Version 3.5.4. The χ2 test was used to compare the proportions by applying the Yates correction when numbers were low. For field values lower than 5, the Fisher exact test was used to estimate the P-value. To be significant, the P-value should be less than 0.05.
RESULTS
Infant Characteristics
Nine hundred thirty (930) DBS samples were collected from 168 HIV PMTCT sites in Dakar (the capital city) and other regions throughout the country (Thiès, Diourbel, Louga, Saint-Louis, Matam, Fatick, Kaolack, Kaffrine, Tambacounda, Kédougou, Sédhiou, Kolda, Ziguinchor). Sites were 15–600 km far from the reference laboratory. Children were mainly boys with a sex ratio of 1.27 and the median age was 20 weeks [IQR = 8–36]. HIV proviral DNA was positive in 24 cases representing 2.6% of children (Table I).
| Characteristics | Number | Percentage |
|---|---|---|
| HIV status | ||
| Negative | 905 | 97.3 |
| Positive | 24 | 2.6 |
| Undetermined | 1 | 0.1 |
| ELISA architect HBsAg | ||
| Positive | 28 | 3.0 |
| Negative | 902 | 97.0 |
| Total | 930 | 100.0 |
Seroprevalence of HBs Antigen
A total of 28 DBS were positive in Architect HBsAg Qualitative II test with a global prevalence of 3.0% [95%CI: 2.0–4.4%]. According to age, HBsAg rate was higher in children less than 6 weeks of age followed by the group between 12–18 weeks and 18–24 weeks of age with 5.1%, 4.1%, and 4.6%, respectively, without significant difference (P = 0.415). HBsAg rate was 3.7% (15/409) and 2.5% (13/521) among girls and boys, respectively (P = 0.3003) and no case of HIV/HBV co-infection was found (Table II). For mothers, HBeAg and HBV DNA viral load were not documented. HBsAg was the only marker performed and documented for mother in PMTCT sites. It revealed that all of them were positive for HBsAg with the exception of three. Data related to children immunization was not available.
| Characteristics | % HBsAg positive |
|---|---|
| Age groups (n = 912) P = 0.415 | |
| <6 weeks (n = 218) | 11 (5%) |
| 6–12 weeks (n = 163) | 2 (1.2%) |
| 12–18 weeks (n = 74) | 3 (4%) |
| 18–24 weeks (n = 109) | 5 (4.6%) |
| 24–30 weeks (n = 64) | 1 (1.6%) |
| 30–36 weeks (n = 101) | 2 (2%) |
| 36–42 weeks (n = 35) | 0 (0%) |
| 42–48 weeks (n = 53) | 2 (3.8%) |
| >48 weeks (n = 95) | 2 (2.1%) |
| Sex (n = 929) P = 0.302 | |
| Boys (n = 520) | 13 (2.5%) |
| Girls (n = 409) | 15 (3.7%) |
| HIV status (n = 930) P = 0.671 | |
| Negative (n = 905) | 28 (3%) |
| Positive (n = 24) | 0 (0%) |
| Undetermined (n = 1) | 0 (0%) |
Mothers HIV Prophylaxis Regimens
HIV prophylaxis for mothers was recorded in 78.2% (n = 727) cases and was based on tritherapy including lamivudine (3TC) and/or tenofovir (TDF) in 99.6% (Table III). According to this status (3TC/TDF-based prophylaxis or not), HBsAg prevalence was 3.6 times lower among children born from mother under 3TC/TDF-based prophylaxis than in those whose mothers were not (2.2% vs. 7.9%; P = 0.0006).
| Total of mothers (n = 930) | Mothers of HBV infected children (n = 28) |
|---|---|
| 3TC and/or TDF based prophylaxis | n = 28 |
| Yes (n = 724) | 16 (2.2%) |
| No (n = 126) | 10 (7.9%) |
| Not documented (n = 77) | 2 (2.6%) |
| Mothers prophylaxis regimens (n = 727) | n = 16 |
| AZT or TDF/3TC/NVP (n = 409) | 10 (2.5%) |
| AZT or TDF/3TC/EFV (n = 218) | 4 (1.9%) |
| AZT or TDF/3TC/LPVr (n = 92) | 2 (2.5%) |
| TDF/FTC/LPVr (n = 5) | — |
| AZT/NVP or EFV (n = 3) | — |
DISCUSSION
This study aimed to determine HBsAg rate using EID DBS samples collected from children throughout Senegal. DBS were mainly from Dakar (34.4%), probably due to the large size of population and/or to the accessibility of HIV PMTCT sites, and Ziguinchor (14.2%). For Ziguinchor, this could be explained by the high HIV prevalence among pregnant women [CNLS, 2013], inducing a significant enrollment of women through PMTCT.
In this study, DBS were used as blood support for HIV EID due to its usefulness that confer DNA stability on the filter paper allowing samples transportation from far healthcare centers to reference laboratories. For HBV diagnosis, its use is also increasingly described as a new tool for epidemiological research of HBV markers [Gani et al., 2005; Kania et al., 2013; Mohamed et al., 2013]. Studies had first evaluated the use of DBS by comparison to serum using a radioimmunoassay method [Villa et al., 1981]. More recently, DBS samples were successfully performed to investigate the prevalence of HBV infection in risk groups [Brugal et al., 2009; Mahfoud et al., 2010], in endemic areas [Komas et al., 2010] and to evaluate point of care testing [Kania et al., 2013; Njai et al., 2015]. In addition, some studies evaluated automated platform using chemiluminescence technology in HBsAg detection on DBS with good analytical sensitivity and specificity in comparison to the serum [Mohamed et al., 2013; Ross et al., 2013]. The Architect analyzer based on this chemiluminescence technology was used to analyze the 930 DBS samples.
In Senegal, HBsAg was not systematically included in serological surveys, but recent studies estimated a prevalence around 11% of HBsAg carriage in pregnant women [Lo et al., 2012, 2014]. In the other hand, data on HBsAg carriage in children are still limited [Sall Diallo et al., 2004]. In this study, a global prevalence of 3% was found in children that could be linked to the impact of HBV vaccination through the EPI [Rey-Cuille et al., 2013]. Furthermore, no case of HIV/HBV co-infection was found in children, probably related to the low HIV prevalence in the general population and in pregnant women [CNLS, 2013].
To reduce HIV vertical transmission, antiretroviral prophylactic regimens were constantly improved in the country with widely access to antiretroviral therapy (ART) with the scaling up of the B+ option recommended by WHO for all infected pregnant and breastfeeding women [WHO, 2013c; Mangwiro et al., 2014; Mwapasa et al., 2014].
According to prophylaxis, mothers were mainly on tritherapy-based regimen including 3TC/TDF that have also activity against HBV. A lower rate of HBsAg carriage was found in children whose mothers were on 3TC/TDF-based prophylaxis. These results could be related to the reduction of HBV mother-to-child transmission by 3TC/TDF, which inhibits HBV replication in infected mothers. Indeed, several studies have now demonstrated their effectiveness in reducing HBV vertical transmission in infected mothers with high viral loads [Howell et al., 2014].
Currently, to prevent HBV vertical transmission, European guidelines recommend to treat in the third trimester with TDF if maternal HBV DNA levels are over 106 IU/ml [Howell et al., 2014].
In contrary, a high infection rate (7.9%) was found in the group of children without vertical transmission prevention by ART. However, early transmission could also be due to other transmission routes including familial and environmental contact.
To prevent HBV maternal/child and early horizontal transmission of HBV, most sub-Saharan African countries have elected to vaccinate children against HBV through the WHO-sponsored EPI and the current recommendation from WHO-AFRO is birth-dose HBV vaccination [Howell et al., 2014]. This policy allows an important reduction of perinatal transmission as in Taïwan with a decreasing of HBsAg carriage from 10–20% to 1–2% after 30 years of immunization [Chen et al., 2012] and in Gambia with long lasting protection and 83% of vaccination efficacy [van der Sande et al., 2006; Mendy et al., 2013]. In addition, comparing to immunization at 6 weeks (0.4% HBV infection), the effectiveness of vaccination at birth in Côte d'Ivoire showed zero HBV infection [Ekra et al., 2008].
Perinatal transmission still occurs when women have high HBV DNA load [Khamduang et al., 2013; Wiseman et al., 2014], which contributed as major maternal transmission factor even in case of adequate immunoprophylaxis [Singh et al., 2011; Olaleye et al., 2013; Yin et al., 2013]. However, HBV DNA viral load in mothers was not documented from PMTCT sites and the evidence for HBV vertical transmission could not unfortunately be assessed, presenting a limit in this study.
HBsAg prevalence did not vary significantly according to age suggesting limited additional horizontal transmission due to familial or environmental contact as expected [Vardas et al., 1999].
CONCLUSION
Despite the limited size of samples and the lack to provide evidence of HBV mother to child transmission, this study showed a high rate of HBsAg prevalence in children less than 24 months old in Senegal. This high rate, especially in children without prevention based on 3TC/TDF, demonstrated that Senegal remain a high endemic country for HBV. These results highlighted the need to screen all pregnant women for both HIV and HBV, to promote as HIV, HBV prophylaxis based on TDF regimen, and to vaccinate at birth as recommended by WHO. Finally, this study could positively impact the HBV program as data were shared and contributed in the acceleration of vaccination scaling up at birth for children born from HBV-seropositive mothers.
ACKNOWLEDGMENTS
The authors would like to thank Ndèye Diabou Diagne-Gueye, Sada Diallo, and Oumy Diop-Diongue for their technical support, Abdoulaye Aziz Hane and Jean-Pierre N'guessan for editing the manuscript, and Ousmane Diouf for statistical analysis.
