231
Journal of Neonatal-Perinatal Medicine 6 (2013) 231–236 DOI 10.3233/NPM-1366412 IOS Press
Original Research
Perinatal transmission of hepatitis B virus infection in Ile-Ife, South Western, Nigeria O. Atinuke Olaleyea,∗ , O. Kutia,b , N. Olaniyi Makindea,b , A.O. Innocent Ujahc , O. Akinyemi Olaleyed , O. Olusegun Badejokoa,b , A. Akinyemi Akintayoa and R. Auduc a Department
of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria of Obstetrics and Gynecology, Obafemi Awolowo University, Ile-Ife, Nigeria c Department of Human Virology, Nigeria Institute of Medical Research, Lagos, Nigeria d Department of Obstetrics and Gynecology, Premier Specialists’ Medical Centre, Lagos, Nigeria b Department
Received 06 November 2012 Revised 09 April 2013 Accepted 13 June 2013
Abstract. AIM: To determine the perinatal transmission risk of hepatitis B virus (HBV) and the maternal characteristics influencing it. METHOD: During routine antenatal screening, women who tested positive for hepatitis B surface antigen (HBsAg) were identified and followed through pregnancy. Maternal and cord blood samples were obtained at delivery. The sera of each motherbaby pair were analyzed for HBsAg, HBeAg, HBeAb, HBsAb and HBcAb using an immunochromatographic 5-in-1 panel kit. Quantitative HBV-DNA was assessed using polymerase chain reaction technique. Intrauterine infection was defined when neonatal blood test positive for HBsAg positivity and/or HBV-DNA. Confidence level was set at 95% (p < 0.05). RESULTS: Of the 716 pregnant women screened 73 (10.2%) were HBsAg-positive. Fifty of these HBsAg-positive women completed the study. Intrauterine infections were detected in 36 (72%) newborns; of them only twelve (24%) had positive HBsAg whereas all of them (n = 36) neonates had detectable HBV-DNA (>100 copies/ml). High maternal HBV-DNA titre was associated with increased neonatal HBV-DNA titre (p = 0.001). Parity, maternal age, and mode of delivery showed no association with perinatal transmission. CONCLUSION: The risk of perinatal HBV transmission in this study was high. Perinatal transmission was associated with high maternal viremia. Appropriate prophylaxis for HBsAg-positive mothers and their newborns is advocated. Keywords: Hepatitis B, vertical transmission, intra-uterine infection, perinatal, Nigeria
1. Introduction
∗ Corresponding
author: Dr. Olukemi Atinuke Olaleye, Department of Obstetrics and Gynaecology, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Osun state, Nigeria. Tel.: +234 803 351 1909; E-mail: [email protected].
Hepatitis B infection, one of the major infectious diseases of the liver worldwide, is caused by a small enveloped double-stranded DNA virus. Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. It has an incubation period of six weeks to six months
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
232
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[1]. It is estimated that more than two billion people have been infected by HBV worldwide; Nigeria is one of the countries with high prevalence of HBV infection and currently, about 18 million Nigerians are infected [2]. About 10–20% of HBsAg positive pregnant women transmit the virus to their neonates, while women who are seropositive for both HBsAg and HBeAg have vertical transmission of up to 90% [3]. After acute hepatitis resolves 5–10% of adult patients and 90–95% of infected infants develop chronic infection [4]. Symptoms of chronic HBV infection such as palmar rash and weight loss usually manifest with the development of liver cirrhosis or even hepatocellular carcinoma. This happens typically in late adolescence or adulthood, including those with perinatally-acquired infection [5]. Most perinatal transmission is believed to occur at or near the time of birth, because where the prevalence of HBV is high, neonatal vaccination prevents newborn infection in about 85–90% of cases [6]. Risk factors for transplacental transmission of HBV include maternal HBeAg positivity, HBsAg titre and HBV DNA level [6]. Various methods exist for the detection and diagnosis of hepatitis B virus infection. HBsAg assays are commonly used for diagnosis, and also for screening blood donors. The earliest detectable marker of HBV infection is the viral DNA which can be assessed both qualitatively and quantitatively by polymerase chain reaction (PCR) in plasma, blood and tissue [7]. The risk of mother-to-child-transmission (MTCT) in HIV has been quantified and this has led to the amplification of efforts at its prevention. Despite the fact that HBV has a higher infectivity rate than HIV with significant health implications for chronic carriers, similar efforts at prevention are lacking in our environment. Though several studies have been done to determine the prevalence of the carrier state (HBsAg) among pregnant women in various parts of Nigeria [8–10], there is paucity of information on the vertical transmission rate. This has hampered efforts at establishing and implementing appropriate strategies for the prevention of perinatal HBV infection in this environment. Currently, routine screening for HBsAg is seldom done for all pregnant women at booking in our environment. Moreover, guidelines for intervention in HBsAg positive pregnant women are lacking and the prohibitive cost of the hepatitis B immunoglobulin (HBIG) has prevented its routine use for their newborns. In order to quantify the risk of MTCT in maternal HBV
infection and dictate how much effort needs to be redirected towards its prevention, this study was conducted to determine the seroprevalence, risk factors and rate of perinatal transmission of hepatitis B virus (HBV) infection among pregnant women in OAUTHC, IleIfe. It is expected that the results obtained would be informative on the magnitude of maternal and perinatal HBV infection in this setting and the findings may be used in policy formulation and practice.
2. Materials and methods This prospective study was carried out at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, Osun state and was approved by the Nigeria Institute of Medical Research (NIMR), Lagos, Nigeria. Consents were obtained from participants. All pregnant women who were followed for antenatal care at OAUTHC between September and December 2011 underwent routine HBsAg screening. Women who were HBsAg negative, on antiretroviral therapy and those with antepartum hemorrhage were excluded from the study. A pre-tested structured questionnaire was administered to each consenting participant by a trained interviewer and at delivery, 5 mls each of maternal and cord blood was collected into pre-labelled K2 EDTA specimen bottles. The mother-baby pair samples were centrifuged at room temperature to separate the plasma and 0.5 mls of this was used for qualitative analysis. A 5-in-1 HBV panel kit (Lumi Quick Diagnostics Inc., USA) was used to detect HBsAg, HBeAg, HBsAb, HBeAb and HBcAb, following the manufacturer’s instructions. Another 1 ml was stored ◦ at −20 C and transported using ice packs to NIMR for quantification of the HBV DNA levels, using a PCR technique. The COBAS Amplicor HBV monitor test manual (Roche diagnostics GmbH, Mannheim, Germany) Version 2.0, was used. The HBV DNA concentration obtained was expressed in IU/ml and manually converted to copies/ml (oneIU/ml = 5.82 copies/ml), using the WHO International Standard for Hepatitis B Virus DNA for Nucleic Acid Technology (NAT) Assays Testing (NIBSC 97/746) [11]. Intrauterine infection was defined as presence of HBsAg and/or HBV DNA in neonatal blood. Proportions were compared using the Chi-square and Fisher’s exact tests. Multiple and logistic regression models
O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
233
were used to test for associations between maternal viral load, maternal characteristics and neonatal HBV DNA titre. Significance was determined at p < 0.05.
3. Results Of the 716 pregnant women screened over the four month period, 73 (10.2%) tested positive for HBsAg. Fifty of these 73 HBsAg positive women delivered in the study center and were included in the data analysis. However, there were no significant socio-biologic differences between the women who delivered in our facility i.e. respondents and those that did not i.e. nonresponders (Table 1). None of the women had ever received hepatitis B vaccination. Among the respondents, 14 (28%) of them had caesarean delivery, while 36 (72%) had spontaneous vaginal delivery. The birth weights of the infants born ranged from 2.2–4.2 kg, with a mean birth weight of 3.2 kg. There was no stillbirth or congenital abnormality among the newborns. The risk factors for maternal HBV infection identified among the study participants are as shown in Fig. 1, with the commonest risk factors being a history of multiple sexual partners and presence of scarification marks. HBsAg was positive in 24% while HBV DNA was detectable (>100 copies/ml) in 72% of the
Fig. 1. Distribution of risk factors identified in respondents.
Table 1 Socio-biologic characteristics of study participants Variable Age(years) 1)
Neonatal HBV DNA Negative (n = 14)
Frequency
%
Frequency
%
p – value
1 3 6 4 17 3 4 18
2.8 8.3 16.7 11.1 47.2 8.3 11.1 50
2 13 5 1 6 2 3 9
14.3 92.9 35.7 7.1 42.9 14.3 21.4 64.3
0.12a 1.00a 0.14b 1.00a 0.78b 0.61a 0.38a 0.36b
None of the maternal risk factors assessed was significantly associated with a risk of perinatal transmission. a Fisher’s Exact Test; b Chi-square.
The relationship between maternal risk factors for HBV infection and rate of perinatal transmission is as shown in Table 3. History of scarification, sexually transmitted infection (STI), blood transfusion and jaundice were not significantly associated with perinatal transmission (p > 0.05). A multiple regression analysis of the ungrouped maternal and neonatal HBV DNA titres as shown in Table 4 revealed a significant association between these variables (p = 0.001). Though increase in parity and infant birth weight were associated with increase in neonatal HBV DNA titre, this finding was not statistically significant. Similarly, younger maternal age and spontaneous vaginal delivery were associated with increase in neonatal HBV DNA titre, but this was also not statistically significant (p > 0.05).
4. Discussion From the routine antenatal screening of pregnant women at the booking clinics, the maternal HBsAg seroprevalence obtained over the period of study was 10.2%. This figure reflects the high prevalence of hepatitis B in this environment, and corroborates the WHO
report classifying Nigeria as a highly endemic area for viral hepatitis, with prevalence greater than 8% [12]. Other investigators in Nigeria have reported maternal HBV prevalence of 2.19% in Benin City [8], 8.3% in Zaria [9] and 12.3% in Minna [10]. Lower prevalence rates of 3.7% and 2.1% have been reported by researchers in Ethiopia and Turkey respectively [13, 14]. While a previous Nigerian study examined only maternal HBsAg, HBeAg and neonatal HBsAg to determine the risk of neonatal infection [8], this research was taken a step further to also examine HBV DNA in neonatal (cord) serum. The vertical transmission risk of 72% found in our study using HBV DNA is higher than the 42.86% and 51.6% reported with the HBsAg immunoassay by Onakewhor [8] in Benin City and Eke [15] in Nnewi, Nigeria respectively. The presence of a high intra-uterine transmission risk of HBV infection could further increase the prevalence of the chronic infection within the population. Thus, measures to interrupt mother-to-child transmission should be instituted at all levels of health care. The incidence of HBV intra-uterine infection reported by different researchers varies widely, ranging from 2.1 to 50%, due to different sensitivities of the methods used [16]. Zhang and co-workers in
O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
235
Table 4 Multiple regression model depicting effect of maternal characteristics on neonatal HBV DNA level Neonatal HBV DNA titre (Log10 copies /ml) Variable
Regression
t-value
p-value
coefficient Parity Maternal age (years) Infant birth weight (kg) Mode of delivery (Ref. = CS) Maternal HBV DNA titre (Log10 copies/ml)
3.92 −3.68 4.72 −4.62 2.47
0.50 −0.88 1.52 −0.90 4.81
0.622 0.385 0.136 0.376 0.001
95%CI Lower
Upper
−4.41 −4.20 −4.23 −5.13 2.3
4.63 3.79 5.09 4.72 2.7
Maternal HBV DNA titre was significantly associated with neonatal HBV DNA. CI: Confidence interval; CS: Cesarean section.
China in 1998 [16] reported lower incidences of 17.1% when HBsAg was used as a marker, and 41.5% when HBV DNA was used. A lower incidence of 40.1% intra-uterine HBV infection rate using HBV DNA was reported by the same researchers in 2004 [17]. The variation of the findings with those of this study may possibly be due to a difference in sensitivities of the methods used for DNA detection or to a difference in the study populations. In this study, the incidence of neonatal hepatitis B virus positivity was significantly higher with the use of HBV DNA detection method (72%) than with the use of HBsAg screening method (24%) in the cord blood (p < 0.05%). This difference is likely due to a greater sensitivity of the PCR-based method compared to rapid immune-chromatography method. HBV DNA shows directly the condition of replication and infectivity of the virus in vivo, and this can be reflected through HBV DNA quantitation by FQ-PCR test clinically [18]. A possible reason postulated for the increased level of HBV DNA expression in neonatal blood is that the incubative virus replicates in bulk with fetal liver maturation during later gestation [19]. The maternal HBV DNA titre was the only characteristic found to significantly influence the neonatal HBV DNA level, and this effect is particularly seen with high maternal titres (≥107 IU/ml). Parity, maternal age, mode of delivery and infant birth weight had no significant influence on the neonatal HBV DNA level. This shows that use of these characteristics, except the maternal DNA level in predicting risk of intra-uterine infection may not be effective. This finding is in keeping with the reports of previous studies [20]. Though elevated maternal HBV DNA titre was significantly associated with increasing neonatal HBV DNA titre, this occurred with very high mater-
nal DNA levels. This finding therefore may not justify the use of maternal DNA titre alone in determining prophylaxis in view of the prohibitive cost of such tests. Although intra-uterine infection with HBV has been shown from previous studies to be closely related to maternal HBeAg positivity [7, 16], this finding was not demonstrated in our study, as HBV DNA was detected in the newborns of both HBeAg positive and negative mothers. This may be due to the low (6%) incidence of maternal HBeAg positivity seen in this study. Despite the fact that the intra-uterine infection rate found in this study was high, the incidence of chronic HBV infection (i.e. HBsAg or HBV DNA which persists beyond 6months) in these neonates could not be determined due to the time frame available for the study as further follow-up would be required. The relationship of maternal viremia with intrauterine infection found in our study is similar to that reported by Zhang and colleagues [16] where HBV DNA detection was qualitatively assessed with nestedPCR. It was reported that the incidence of HBV intra-uterine infection was higher in newborns of mothers with serum strongly positive for HBV DNA. Though all the mothers and majority of the neonates in our study had detectable levels of HBV DNA, a quantitative DNA assessment showed that a higher level of maternal HBV DNA was significantly associated with a higher neonatal HBV DNA level (p = 0.001). The mode of delivery was not significantly associated with intra-uterine infection in our study. This finding is not unexpected as most researchers hold that the mechanism of HBV intra-uterine infection is transplacental [17] and this route of infection would not be affected by mode of delivery. Moreover, with immunoprophylaxis of the newborn, mode
236
O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
of delivery does not influence the likelihood of HBV transmission [20]. Though maternal characteristics such as parity, multiple sexual partners, history of sexually transmitted infections and tribal marks/tattoos have been reported as risk factors for HBsAg positivity by some researchers [14, 15], they had no influence on intrauterine infection rate in this study. This finding may be due to the relatively small number of our subjects. Larger studies would therefore be required to further ascertain this finding. The findings from this study demonstrate that HBV infection is endemic in Ile-Ife. There is also a high vertical transmission rate which is increased by high maternal viremia. Vertical transmission is not influenced by maternal age, parity, mode of delivery or presence of risk factors for HBV infection. Further research into the effect of immunization on vertical transmission as well as the most appropriate and cost effective interventions for prevention of hepatitis B virus in our low-resource setting is required.
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments The authors are grateful to Roche diagnostics, Germany for providing COBAS Amplicor/Taqman HBV test kits for the HBV DNA analysis. We also express our gratitude to the Nigerian Institute of Medical Research for providing the consumables and Mr Azuka Okwuraiwe for technical support for the HBV DNA analysis.
[12]
[13]
[14]
[15]
Financial disclosure statement The COBAS Amplicor/ Taqman HBV test kits used for the HBV DNA analysis were donated by Roche diagnostics GmbH, Mannheim, Germany.
[16]
[17]
References [1]
[2]
Burroughs AK, Westaby D. Liver, Biliary tract and Pancreatic disease. In: Kumar P, Clark M. (eds) Clinical Medicine 6th edition. Philadelphia, PA, IL: Elsevier Saunders; 2005, 347418. Jombo GTA, Egah DZ, Banwat EB. Hepatitis B Virus infection in a rural Settlement of Northern, Nigeria. Niger J Med 2005;14(4):425-8.
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Petersen J. HBV treatment and pregnancy. J Hepatol 2011;55:1171-3. Finlayson ND, Hayes PC, Simpson K. Diseases of Liver and biliary system. In: Christopher H, John AA, Hunter NA, et al., (eds). Davidson’s Principle and Practice of Medicine. Edinburg, Churchill Livingstone, 18th edition 1999, 683736. Slowik MK, Jhaveri R. Hepatitis B and C viruses in infants and young children. Semin Pediatr Infect Dis 2005;16:296305. Deng M, Zhou X, Gao S, Yang SG, Wang B, Chen HZ, Ruan B. The effects of telbivudine in late pregnancy to prevent intrauterine transmission of the hepatitis B virus: A systematic review and meta-analysis. Virol J 2012;9:185 Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology 2002;36:1408-15. Onakewhor UA, Offor E, Okonofua FE. Maternal and neonatal seroprevalence of hepatitis B surface antigen (HBsAg) in Benin City, Nigeria. J Obstet Gynecol 2001;21:583-6. Luka SA, Ibrahim MB, Iliya SN. Seroprevalence of hepatitis B surface antigen among pregnant women attending Ahmadu Bello University Teaching hospital, Zaria, Nigeria. Nig J Parasitol 2008;29(1):38-41. Ndams IS, Joshua IA, Luka SA, Sadiq HO. Epidemiology of hepatitis B infection among pregnant women in Minna, Nigeria. Sci World J 2008;3(3):5-8. Saldanha J, Gerlich W, Lelie N, Dawson P, Heermann K Heath A, WHO Collaborative Study Group. An international collaborative study to establish a World Health Organisation international standard for hepatitis B virus DNA nucleic acid amplification techniques. Vox Sang 2001;80:63-71. Ugwuja EI. Seroprevalence of hepatitis B surface Antigen and liver function tests among adolescents in Abakaliki, South Eastern Nigeria. J Trop Med 2010;6(2):1-6. Awole M, Gebre-Selassie S. Seroprevalence of hepatitis B surface antigen and its risk factors among pregnant women in Jimma, Southwest Ethiopia. Ethiop J Health Dev 2005;19(1):45-50. Uyar Y, Cabar C, Balci A. Seroprevalence of hepatitis B Virus among Pregnant Women in Northern Turkey. Hepat Mon 2009;9(2):146-9. Eke AC, Eke UA, Okafor CI, Ezebialu IU, Ogbuagu C. Prevalence, correlates and pattern of hepatitis B surface antigen in a low resource setting. Virol J 2011;8:12 Zhang S, Han X, Yue Y. Relationship between HBV viremia level of pregnant women and intra-uterine infection: Nested PCR for detection of HBV DNA. World J Gastroenterol 1998;4(1):61-63. Zhang S, Yue Y, Bai G, Shi L, Jiang H. Mechanism of intrauterine infection of hepatitis B virus. World J Gastroenterol 2004;10(3):437-8. Li XM, Shi MF, Yang YB, et al. Effect of hepatitis B immunoglobulin on interruption of HBV intrauterine infection. World J Gastroenterol 2004;10(21):3215-17. Zhu Y, Mao Y, Wu W, Cai Q, Lin X. Does hepatitis B Virus Prenatal Transmission Result in Postnatal Immunoprophylaxis Failure? Clin Vacc Immunol 2010;17(12):1836-41. Wang J, Zhu Q, Zhang X. Effect of delivery mode on maternal-infant transmission of hepatitis B virus by immunoprophylaxis. Chin Med J (Engl) 2002;115(10):1510-12
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Journal of Neonatal-Perinatal Medicine 6 (2013) 231–236 DOI 10.3233/NPM-1366412 IOS Press
Original Research
Perinatal transmission of hepatitis B virus infection in Ile-Ife, South Western, Nigeria O. Atinuke Olaleyea,∗ , O. Kutia,b , N. Olaniyi Makindea,b , A.O. Innocent Ujahc , O. Akinyemi Olaleyed , O. Olusegun Badejokoa,b , A. Akinyemi Akintayoa and R. Auduc a Department
of Obstetrics and Gynecology, Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria of Obstetrics and Gynecology, Obafemi Awolowo University, Ile-Ife, Nigeria c Department of Human Virology, Nigeria Institute of Medical Research, Lagos, Nigeria d Department of Obstetrics and Gynecology, Premier Specialists’ Medical Centre, Lagos, Nigeria b Department
Received 06 November 2012 Revised 09 April 2013 Accepted 13 June 2013
Abstract. AIM: To determine the perinatal transmission risk of hepatitis B virus (HBV) and the maternal characteristics influencing it. METHOD: During routine antenatal screening, women who tested positive for hepatitis B surface antigen (HBsAg) were identified and followed through pregnancy. Maternal and cord blood samples were obtained at delivery. The sera of each motherbaby pair were analyzed for HBsAg, HBeAg, HBeAb, HBsAb and HBcAb using an immunochromatographic 5-in-1 panel kit. Quantitative HBV-DNA was assessed using polymerase chain reaction technique. Intrauterine infection was defined when neonatal blood test positive for HBsAg positivity and/or HBV-DNA. Confidence level was set at 95% (p < 0.05). RESULTS: Of the 716 pregnant women screened 73 (10.2%) were HBsAg-positive. Fifty of these HBsAg-positive women completed the study. Intrauterine infections were detected in 36 (72%) newborns; of them only twelve (24%) had positive HBsAg whereas all of them (n = 36) neonates had detectable HBV-DNA (>100 copies/ml). High maternal HBV-DNA titre was associated with increased neonatal HBV-DNA titre (p = 0.001). Parity, maternal age, and mode of delivery showed no association with perinatal transmission. CONCLUSION: The risk of perinatal HBV transmission in this study was high. Perinatal transmission was associated with high maternal viremia. Appropriate prophylaxis for HBsAg-positive mothers and their newborns is advocated. Keywords: Hepatitis B, vertical transmission, intra-uterine infection, perinatal, Nigeria
1. Introduction
∗ Corresponding
author: Dr. Olukemi Atinuke Olaleye, Department of Obstetrics and Gynaecology, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Osun state, Nigeria. Tel.: +234 803 351 1909; E-mail: [email protected].
Hepatitis B infection, one of the major infectious diseases of the liver worldwide, is caused by a small enveloped double-stranded DNA virus. Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. It has an incubation period of six weeks to six months
1934-5798/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
232
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[1]. It is estimated that more than two billion people have been infected by HBV worldwide; Nigeria is one of the countries with high prevalence of HBV infection and currently, about 18 million Nigerians are infected [2]. About 10–20% of HBsAg positive pregnant women transmit the virus to their neonates, while women who are seropositive for both HBsAg and HBeAg have vertical transmission of up to 90% [3]. After acute hepatitis resolves 5–10% of adult patients and 90–95% of infected infants develop chronic infection [4]. Symptoms of chronic HBV infection such as palmar rash and weight loss usually manifest with the development of liver cirrhosis or even hepatocellular carcinoma. This happens typically in late adolescence or adulthood, including those with perinatally-acquired infection [5]. Most perinatal transmission is believed to occur at or near the time of birth, because where the prevalence of HBV is high, neonatal vaccination prevents newborn infection in about 85–90% of cases [6]. Risk factors for transplacental transmission of HBV include maternal HBeAg positivity, HBsAg titre and HBV DNA level [6]. Various methods exist for the detection and diagnosis of hepatitis B virus infection. HBsAg assays are commonly used for diagnosis, and also for screening blood donors. The earliest detectable marker of HBV infection is the viral DNA which can be assessed both qualitatively and quantitatively by polymerase chain reaction (PCR) in plasma, blood and tissue [7]. The risk of mother-to-child-transmission (MTCT) in HIV has been quantified and this has led to the amplification of efforts at its prevention. Despite the fact that HBV has a higher infectivity rate than HIV with significant health implications for chronic carriers, similar efforts at prevention are lacking in our environment. Though several studies have been done to determine the prevalence of the carrier state (HBsAg) among pregnant women in various parts of Nigeria [8–10], there is paucity of information on the vertical transmission rate. This has hampered efforts at establishing and implementing appropriate strategies for the prevention of perinatal HBV infection in this environment. Currently, routine screening for HBsAg is seldom done for all pregnant women at booking in our environment. Moreover, guidelines for intervention in HBsAg positive pregnant women are lacking and the prohibitive cost of the hepatitis B immunoglobulin (HBIG) has prevented its routine use for their newborns. In order to quantify the risk of MTCT in maternal HBV
infection and dictate how much effort needs to be redirected towards its prevention, this study was conducted to determine the seroprevalence, risk factors and rate of perinatal transmission of hepatitis B virus (HBV) infection among pregnant women in OAUTHC, IleIfe. It is expected that the results obtained would be informative on the magnitude of maternal and perinatal HBV infection in this setting and the findings may be used in policy formulation and practice.
2. Materials and methods This prospective study was carried out at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, Osun state and was approved by the Nigeria Institute of Medical Research (NIMR), Lagos, Nigeria. Consents were obtained from participants. All pregnant women who were followed for antenatal care at OAUTHC between September and December 2011 underwent routine HBsAg screening. Women who were HBsAg negative, on antiretroviral therapy and those with antepartum hemorrhage were excluded from the study. A pre-tested structured questionnaire was administered to each consenting participant by a trained interviewer and at delivery, 5 mls each of maternal and cord blood was collected into pre-labelled K2 EDTA specimen bottles. The mother-baby pair samples were centrifuged at room temperature to separate the plasma and 0.5 mls of this was used for qualitative analysis. A 5-in-1 HBV panel kit (Lumi Quick Diagnostics Inc., USA) was used to detect HBsAg, HBeAg, HBsAb, HBeAb and HBcAb, following the manufacturer’s instructions. Another 1 ml was stored ◦ at −20 C and transported using ice packs to NIMR for quantification of the HBV DNA levels, using a PCR technique. The COBAS Amplicor HBV monitor test manual (Roche diagnostics GmbH, Mannheim, Germany) Version 2.0, was used. The HBV DNA concentration obtained was expressed in IU/ml and manually converted to copies/ml (oneIU/ml = 5.82 copies/ml), using the WHO International Standard for Hepatitis B Virus DNA for Nucleic Acid Technology (NAT) Assays Testing (NIBSC 97/746) [11]. Intrauterine infection was defined as presence of HBsAg and/or HBV DNA in neonatal blood. Proportions were compared using the Chi-square and Fisher’s exact tests. Multiple and logistic regression models
O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
233
were used to test for associations between maternal viral load, maternal characteristics and neonatal HBV DNA titre. Significance was determined at p < 0.05.
3. Results Of the 716 pregnant women screened over the four month period, 73 (10.2%) tested positive for HBsAg. Fifty of these 73 HBsAg positive women delivered in the study center and were included in the data analysis. However, there were no significant socio-biologic differences between the women who delivered in our facility i.e. respondents and those that did not i.e. nonresponders (Table 1). None of the women had ever received hepatitis B vaccination. Among the respondents, 14 (28%) of them had caesarean delivery, while 36 (72%) had spontaneous vaginal delivery. The birth weights of the infants born ranged from 2.2–4.2 kg, with a mean birth weight of 3.2 kg. There was no stillbirth or congenital abnormality among the newborns. The risk factors for maternal HBV infection identified among the study participants are as shown in Fig. 1, with the commonest risk factors being a history of multiple sexual partners and presence of scarification marks. HBsAg was positive in 24% while HBV DNA was detectable (>100 copies/ml) in 72% of the
Fig. 1. Distribution of risk factors identified in respondents.
Table 1 Socio-biologic characteristics of study participants Variable Age(years) 1)
Neonatal HBV DNA Negative (n = 14)
Frequency
%
Frequency
%
p – value
1 3 6 4 17 3 4 18
2.8 8.3 16.7 11.1 47.2 8.3 11.1 50
2 13 5 1 6 2 3 9
14.3 92.9 35.7 7.1 42.9 14.3 21.4 64.3
0.12a 1.00a 0.14b 1.00a 0.78b 0.61a 0.38a 0.36b
None of the maternal risk factors assessed was significantly associated with a risk of perinatal transmission. a Fisher’s Exact Test; b Chi-square.
The relationship between maternal risk factors for HBV infection and rate of perinatal transmission is as shown in Table 3. History of scarification, sexually transmitted infection (STI), blood transfusion and jaundice were not significantly associated with perinatal transmission (p > 0.05). A multiple regression analysis of the ungrouped maternal and neonatal HBV DNA titres as shown in Table 4 revealed a significant association between these variables (p = 0.001). Though increase in parity and infant birth weight were associated with increase in neonatal HBV DNA titre, this finding was not statistically significant. Similarly, younger maternal age and spontaneous vaginal delivery were associated with increase in neonatal HBV DNA titre, but this was also not statistically significant (p > 0.05).
4. Discussion From the routine antenatal screening of pregnant women at the booking clinics, the maternal HBsAg seroprevalence obtained over the period of study was 10.2%. This figure reflects the high prevalence of hepatitis B in this environment, and corroborates the WHO
report classifying Nigeria as a highly endemic area for viral hepatitis, with prevalence greater than 8% [12]. Other investigators in Nigeria have reported maternal HBV prevalence of 2.19% in Benin City [8], 8.3% in Zaria [9] and 12.3% in Minna [10]. Lower prevalence rates of 3.7% and 2.1% have been reported by researchers in Ethiopia and Turkey respectively [13, 14]. While a previous Nigerian study examined only maternal HBsAg, HBeAg and neonatal HBsAg to determine the risk of neonatal infection [8], this research was taken a step further to also examine HBV DNA in neonatal (cord) serum. The vertical transmission risk of 72% found in our study using HBV DNA is higher than the 42.86% and 51.6% reported with the HBsAg immunoassay by Onakewhor [8] in Benin City and Eke [15] in Nnewi, Nigeria respectively. The presence of a high intra-uterine transmission risk of HBV infection could further increase the prevalence of the chronic infection within the population. Thus, measures to interrupt mother-to-child transmission should be instituted at all levels of health care. The incidence of HBV intra-uterine infection reported by different researchers varies widely, ranging from 2.1 to 50%, due to different sensitivities of the methods used [16]. Zhang and co-workers in
O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
235
Table 4 Multiple regression model depicting effect of maternal characteristics on neonatal HBV DNA level Neonatal HBV DNA titre (Log10 copies /ml) Variable
Regression
t-value
p-value
coefficient Parity Maternal age (years) Infant birth weight (kg) Mode of delivery (Ref. = CS) Maternal HBV DNA titre (Log10 copies/ml)
3.92 −3.68 4.72 −4.62 2.47
0.50 −0.88 1.52 −0.90 4.81
0.622 0.385 0.136 0.376 0.001
95%CI Lower
Upper
−4.41 −4.20 −4.23 −5.13 2.3
4.63 3.79 5.09 4.72 2.7
Maternal HBV DNA titre was significantly associated with neonatal HBV DNA. CI: Confidence interval; CS: Cesarean section.
China in 1998 [16] reported lower incidences of 17.1% when HBsAg was used as a marker, and 41.5% when HBV DNA was used. A lower incidence of 40.1% intra-uterine HBV infection rate using HBV DNA was reported by the same researchers in 2004 [17]. The variation of the findings with those of this study may possibly be due to a difference in sensitivities of the methods used for DNA detection or to a difference in the study populations. In this study, the incidence of neonatal hepatitis B virus positivity was significantly higher with the use of HBV DNA detection method (72%) than with the use of HBsAg screening method (24%) in the cord blood (p < 0.05%). This difference is likely due to a greater sensitivity of the PCR-based method compared to rapid immune-chromatography method. HBV DNA shows directly the condition of replication and infectivity of the virus in vivo, and this can be reflected through HBV DNA quantitation by FQ-PCR test clinically [18]. A possible reason postulated for the increased level of HBV DNA expression in neonatal blood is that the incubative virus replicates in bulk with fetal liver maturation during later gestation [19]. The maternal HBV DNA titre was the only characteristic found to significantly influence the neonatal HBV DNA level, and this effect is particularly seen with high maternal titres (≥107 IU/ml). Parity, maternal age, mode of delivery and infant birth weight had no significant influence on the neonatal HBV DNA level. This shows that use of these characteristics, except the maternal DNA level in predicting risk of intra-uterine infection may not be effective. This finding is in keeping with the reports of previous studies [20]. Though elevated maternal HBV DNA titre was significantly associated with increasing neonatal HBV DNA titre, this occurred with very high mater-
nal DNA levels. This finding therefore may not justify the use of maternal DNA titre alone in determining prophylaxis in view of the prohibitive cost of such tests. Although intra-uterine infection with HBV has been shown from previous studies to be closely related to maternal HBeAg positivity [7, 16], this finding was not demonstrated in our study, as HBV DNA was detected in the newborns of both HBeAg positive and negative mothers. This may be due to the low (6%) incidence of maternal HBeAg positivity seen in this study. Despite the fact that the intra-uterine infection rate found in this study was high, the incidence of chronic HBV infection (i.e. HBsAg or HBV DNA which persists beyond 6months) in these neonates could not be determined due to the time frame available for the study as further follow-up would be required. The relationship of maternal viremia with intrauterine infection found in our study is similar to that reported by Zhang and colleagues [16] where HBV DNA detection was qualitatively assessed with nestedPCR. It was reported that the incidence of HBV intra-uterine infection was higher in newborns of mothers with serum strongly positive for HBV DNA. Though all the mothers and majority of the neonates in our study had detectable levels of HBV DNA, a quantitative DNA assessment showed that a higher level of maternal HBV DNA was significantly associated with a higher neonatal HBV DNA level (p = 0.001). The mode of delivery was not significantly associated with intra-uterine infection in our study. This finding is not unexpected as most researchers hold that the mechanism of HBV intra-uterine infection is transplacental [17] and this route of infection would not be affected by mode of delivery. Moreover, with immunoprophylaxis of the newborn, mode
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O. Atinuke Olaleye et al. / Perinatal HBV transmission in Nigeria
of delivery does not influence the likelihood of HBV transmission [20]. Though maternal characteristics such as parity, multiple sexual partners, history of sexually transmitted infections and tribal marks/tattoos have been reported as risk factors for HBsAg positivity by some researchers [14, 15], they had no influence on intrauterine infection rate in this study. This finding may be due to the relatively small number of our subjects. Larger studies would therefore be required to further ascertain this finding. The findings from this study demonstrate that HBV infection is endemic in Ile-Ife. There is also a high vertical transmission rate which is increased by high maternal viremia. Vertical transmission is not influenced by maternal age, parity, mode of delivery or presence of risk factors for HBV infection. Further research into the effect of immunization on vertical transmission as well as the most appropriate and cost effective interventions for prevention of hepatitis B virus in our low-resource setting is required.
[3] [4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments The authors are grateful to Roche diagnostics, Germany for providing COBAS Amplicor/Taqman HBV test kits for the HBV DNA analysis. We also express our gratitude to the Nigerian Institute of Medical Research for providing the consumables and Mr Azuka Okwuraiwe for technical support for the HBV DNA analysis.
[12]
[13]
[14]
[15]
Financial disclosure statement The COBAS Amplicor/ Taqman HBV test kits used for the HBV DNA analysis were donated by Roche diagnostics GmbH, Mannheim, Germany.
[16]
[17]
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