Seroprevalence of hepatitis B virus serological markers among pregnant Nigerian women.

BACKGROUND: Chronic hepatitis B infection is a global problem; however, Asia and sub-Saharan Africa are most affected by it. Hepatitis B status of pregnant women is essential for the effective management of the disease and prevention of mother to child transmission.
MATERIALS AND METHODS: The study was conducted at the antenatal care unit of four hospitals within Kaduna Metropolis, Nigeria, between August and December 2011. After obtaining ethical clearance, blood samples were collected from 800 consenting pregnant women, the plasma were screened for hepatitis B surface antigen (HBsAg) using first response HBsAg card and the reactive sera were confirmed with enzyme-linked immunosorbent assay. Other serological markers of hepatitis B virus (HBV) were detected using the one-step HBV multi-5 test kit.
RESULTS: Of the 800 pregnant women screened, 31 (3.9%) tested positive for HBsAg. Only one of the 31 HBsAg positive women had developed the hepatitis B surface antibody, 16 (51.6%) had the envelop antibody, 18 (58.1%) had the hepatitis B core antibody (anti-HBc), and two (6.5%) had hepatitis B envelop antigen (HBeAg). The highest prevalence of HBsAg was recorded among women in age group 21-25 years old (P = 0.968). Similarly, married women (P = 0.772), women in their second trimester of pregnancy (P = 0.938), women with tertiary education (P = 0.972), women from the South-East geopolitical zone (P = 0.250) and those whose husbands were in polygamous relationships (P = 0.944) had the highest seroprevalence of HBsAg.
CONCLUSION: HBV was detected with a prevalence of 3.9% among pregnant women in Kaduna Metropolis, Nigeria. About 96.8% (29) of the reactive women had HBeAg negative chronic hepatitis while 6.5% (2) had HBeAg positive chronic hepatitis B infection. About 58.1% of the women had anti-HBc, hence, did not have immunity and probably had chronic infection with reduced risk of vertical transmission. Pregnant women should be screened for HBsAg at the first antenatal clinic visit for appropriate clinical management and effective prevention of vertical transmission.

Introduction

Hepatitis B virus (HBV) infection is an important widespread health problem and the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Chronic HBV infection is a major cause of mortality and 50% of chronic carriers can be expected to die from the disease due to liver cirrhosis or HCC.[1] Epidemiological survey showed that about 5% of the world population is asymptomatic carriers and approximately 350 million people are infected with HBV worldwide.[2] The risk of death from HBV related liver cancer or cirrhosis is approximately 25% for persons who acquire chronic infection at childhood.[12] About 8% to 10% of people in the general population in developing countries become chronically infected.[23] Moreover, 50 million people are chronic carriers of HBV in Africa with the carrier rate ranging from 9% to 20% in sub-Saharan Africa.[34]

The spread of the virus is majorly by percutaneous or mucosal exposure to infected blood and other body fluids with numerous forms of human transmission.[5] The virus has been detected in peripheral mononuclear cells, tissues of pancreas, spleen, kidney and skin, faces, and fluids such as saliva, semen, sweat, breast milk, tears, urine, and vaginal secretion.[5]

Hepatitis B virus can also be transmitted among family members within households from carriers, possibly by contact of nonintact skin or mucous membrane with secretions or saliva containing HBV by the oral route or by sexual or intimate exposure. However, at least 30% of reported hepatitis B among adults cannot be associated with an identifiable risk factor.[5] Factors affecting the prevalence of chronic HBV include age at the time of infection and mode of acquisition, which vary geographically.[23] In the United States and Western Europe, HBV is often acquired in adolescence or adulthood via sexual contact or injection drug use; while in Nigeria and Africa, the major means of HBV acquisition is sexual contact, close personal contact, vertical transmission from mother to child, and transmission in childhood.[26]

Hepatitis B surface antigen (HBsAg), the hallmark of HBV infection, is the first detectable serological marker to appear in acute HBV infection. It is most frequently used to screen for the presence of HBV infection and its persistence for more than 6 months suggests chronic infection.[17] However, early in an infection, this antigen may not be present, and it may be undetectable later in the infection as it is being cleared by the host. The infectious virion contains an inner “core particle” enclosing viral genome also known as hepatitis B core antigen, or HBcAg. During this “window” in which the host remains infected but is successfully clearing the virus, IgM antibodies to the hepatitis B core antigen (anti-HBc IgM) may be the only serological evidence of disease.[58] Shortly after the appearance of the HBsAg, another antigen named as the hepatitis B envelop antigen (HBeAg) will appear. The presence of HBeAg in a host’s serum is associated with much higher rates of viral replication and enhanced infectivity; however, variants of the HBV do not produce the “e” antigen, therefore, this rule does not always hold true. During the natural course of an infection, the HBeAg may be cleared, and antibodies to the hepatitis B envelop antigen (anti-HBe) will arise immediately afterward. This conversion is usually associated with a dramatic decline in viral replication.[58] If the host is able to clear the infection, eventually the HBsAg will become undetectable and will be followed by IgG antibodies to the hepatitis B surface antigen and core antigen (anti-HBs and anti-HBc IgG). A person negative for HBsAg but positive for anti-HBs have either cleared an infection or has been vaccinated previously.[58]

Infection with HBV may adversely affect pregnancy outcome, leading to spontaneous abortion, premature delivery, intrauterine growth restrictions, and low birth weight infants.[5] Without intervention such as immunoprophylaxis, a mother who is positive for HBsAg confers a 20% risk of passing the infection to her offspring at the time of birth. This risk is as high as 90% if the mother is also positive for HBeAg.[9101112] A HBeAg positive test is an indication of the risk of transmission at a rate as high as 70–90% to a neonate, whereas a negative test indicates a transmission rate of <10%.[9] In these mothers with HBeAg, the risk of HBV perinatal transmission is reduced from 70% to 90% to approximately 5–15% when the infant receives postnatal immunoprophylaxis with both hepatitis B immune globulin and hepatitis B vaccine series.[9101112]

Nigeria is classified among the group of countries highly endemic for HBV infection.[1] Studies carried out in Nigeria showed HBV carriage rate in the range of 9% to 39%.[6] Although hepatitis B vaccination is highly effective in preventing infection with HBV and the consequent acute and chronic liver disease,[3] this infection is still a major problem in Nigeria as reported by various workers.[613141516] Previous studies among pregnant women in Nigeria have reported seroprevalence of between 4.3% and 9.3%.[131415171819] In other African studies, seroprevalence rates of 6.2% and 5.6% have been reported among pregnant women in Sierra Leone[20] and Sudan.[21] In spite of this high prevalence of HBsAg among pregnant women in Nigeria, routine antenatal screening for hepatitis B infection is not yet practiced in many Nigerian hospitals. In addition, vertical transmission of HBV infection is thought to be a major route of transmission in low resource areas such as Nigeria.[22] This study was therefore conducted to detect HBV serological markers among the pregnant women attending ante-natal care in Kaduna Metropolis, Nigeria in order to determine the seroprevalence and stage of infection.

Materials and Methods

Study area and population

The study was conducted in Kaduna Metropolis, Kaduna State, Nigeria between August and December 2011. The state covers an area of approximately 45,567 km2, and has an estimated population of more than 6 million people, with three senatorial zones and 23 Local Government Areas. The climatic conditions in Kaduna State are tropical with well-defined wet and dry season. The study population consisted of 800 pregnant women at various trimesters of pregnancy seen at the point of booking to attend ante-natal care in four selected hospitals in Kaduna Metropolis. The hospitals included 44 Nigerian Army Reference Hospital, St. Gerard Hospital, Barau Dikko Specialist Hospital, and Yusuf Dantsoho Memorial Hospital. Informed consent was obtained from each participant and ethical approval was obtained from the Ethical Committee of each of these hospitals. A self-designed semi-structured questionnaire was used to collect demographic data, clinical information, and risk factors associated with HBV infection. A questionnaire was administered to each pregnant woman prior to sample collection.

Sample size determination

The sample size used for the study was determined using the equation n = Z2PQ/d2 described by Naing et al.[23] and a prevalence rate of 18.2% obtained in a previous study.[24] The calculated sample size was 228.76, which was the minimum number of samples to be used for the study. However, to validate the accuracy of the study, a total of 800 samples were collected and used for the study.

Sample collection

About 5 ml of venous blood sample was collected from each of the 800 participants by a laboratory scientist using a disposable sterile vacutainer needle and a 5 ml vacutainer bottle containing an anticoagulant, ethylenediaminetetraacetic acid. The plasma was carefully aspirated into cryovials and then stored at − 20°C until analyzed.

Sample analysis

First response HBsAg card (Premier Medical Corporation Limited, India) was used to screen all the plasma samples for HBV according to the manufacturer’s instructions. This test is an in vitro qualitative one-step immune-chromatographic assay for the detection of HBV. The test selectively detects HBV in serum or plasma using a two site immunometric assay in which a combination of monoclonal and polyclonal antibodies is used. The test was done by placing 25 µl of plasma in the specimen pot of the cassette and the reading was taken at 10 min. A single red/pink band at the control region was taken as a negative result while two bands each at the control and the patients region was taken a positive result. An invalid test was indicated by a single band at the patients region and was repeated.

The HBsAg reactive samples were confirmed with enzyme-linked immunosorbent assay (Beijing Kinghawk Pharmaceutical Co., Ltd., China) according to the manufacturer’s instructions. Samples that were reactive to HBsAg were further screened for other serological markers of HBV using the one-step HBV multi-5 test kit (Beijing easy sweet Biomedicine Science Tech Co., Ltd., China). The multi-5 in 1 Panel rapid test kit is a rapid test based on the principle of immunoassay combined with conjugated colloid gold technology. The The HBV test kit is a diagnostic device to detect the 5 markers associated with hepatitis B infection. The markers include hepatitis B surface antibody (anti-HBs), HBeAg, anti-HBe, and anti-HBc.

Statistical analysis

The data obtained from the questionnaire and the results of the laboratory analysis were analyzed using Statistical package for social sciences (SPSS) Version 17 program (SPSS Inc. 233 South Wacker Drive, 11th Floor Chicago, IL). The Pearson Chi-square test was employed to determine the relationships between the variables and HBsAg infection and P < 0.05 was considered significant at 95% confidence interval.

Results

Of the 800 pregnant women who participated in the study, 31 (3.9%) tested positive for HBsAg [Table 1]. The highest prevalence of 8% (8/100) was recorded among women attending St. Gerald Hospital while the least (2.8%: 7/250) was recorded among those attending Barau Dikko Hospital. The majority of the pregnant women were in their second trimester of pregnancy (55%: 440/800), a trimester, where most of the cases (4.1%: 18/440) were detected. The lowest prevalence of HBsAg was recorded among women in their first trimester (2.6%: 2/76). About 35% (283/800) of the pregnant women were within age group 26-30 years; women within age group 21–25 years had the highest prevalence (4.4%: 10/229), while none of the women above 41 years had been exposed to the virus (P = 0.968) [Table 2].

Table 1

Percentage distribution of HBsAg according to the hospital of sampling

Hospital	Total	Positive (%)	Negative (%)
Barau Dikko	250	7 (2.8)	243 (97.2)
St. Gerard	100	8 (8.0)	92 (92.0)
44 Nigeria Army	200	7 (3.5)	193 (96.5)
Yusuf Dantsoho	250	9 (3.6)	241 (96.4)
Total	800	31 (3.9)	769 (96.1)

HBsAg=Hepatitis B surface antigen

Table 2

Percentage distribution of HBsAg according to the age of mother and gestation

Parameter	Total	Positive (%)	Negative (%)	P
Age group (years)
16-20	102	3 (2.9)	99 (97.1)	0.968
21-25	229	10 (4.4)	219 (95.6)
26-30	283	12 (4.2)	271 (95.8)
31-35	125	4 (3.2)	121 (96.8)
36-40	54	2 (3.7)	52 (96.3)
41-50	7	0 (0.0)	7 (100.0)
Gestational period
Unknown	1	0 (0.0)	1 (96.1)	0.938
1-3	76	2 (2.6)	74 (97.4)
4-6	440	18 (4.1)	422 (95.9)
7-9	283	11 (3.9)	272 (96.1)

HBsAg=Hepatitis B surface antigen

Table 3 show the distribution of HBsAg among the women according to demographic factor. All the HBsAg positive women were married (3.9%: 31/798) and the women with tertiary education had the highest prevalence (4.9%: 9/185). All the risk factors studied were not significantly associated with HBV infection in this study. However, HBsAg was detected with higher frequency among the pregnant women who had been transfused and with the highest frequency among those who had more than two sexual partners in their lifetime [Table 4].

Table 3

Percentage distribution of HBsAg among the women’s according to demographic data (n=800)

Parameter	Total	Positive (%)	Negative (%)	χ2	df	P
Geopolitical zone of origin
North-West	482	13 (2.7)	465 (97.3)	7.843	5	0.250
North-East	53	3 (5.7)	50 (94.3)
North Central	143	9 (6.3)	134 (93.7)
South-East	46	4 (8.7)	42 (91.3)
South-South	29	1 (3.4)	28 (96.6)
South-West	47	1 (2.1)	46 (97.9)
Marital status
Married	798	31 (3.9)	767 (96.1)	0.081	1	0.776
Widowed	2	0 (0.0)	2 (100.0)
Family type
Monogamy	649	25 (3.9)	624 (96.1)	0.005	1	0.944
Polygamy	151	6 (4.0)	145 (96.0)
Educational status
Primary	191	7 (3.7)	184 (96.3)	0.873	4	0.972
Secondary	339	12 (3.5)	327 (95.9)
Tertiary	185	9 (4.9)	176 (95.1)
Quranic	81	3 (3.7)	78 (96.3)
None	4	0 (0.0)	4 (100.0)

HBsAg=Hepatitis B surface antigen

Table 4

Percentage distribution of HBsAg among the pregnant women according to some risk factor

Risk factor	History	Total	Positive (%)	Negative (%)	P
Blood transfusion	Yes	33	2 (6.1)	31 (93.9)	0.506
	No	767	29 (3.8)	738 (96.2)
Scarification	Yes	234	6 (2.6)	228 (97.4)	0.217
	No	566	25 (4.4)	541 (95.1)
STI	Yes	183	7 (3.8)	176 (96.2)	0.968
	No	617	24 (3.9)	593 (96.1)
Unsterilized sharps use	Yes	103	5 (4.9)	98 (95.1)	0.581
	No	697	26 (3.7)	671 (96.3)
Surgery	Yes	70	3 (4.3)	67 (95.7)	0.852
	No	730	28 (3.8)	702 (96.2)
Parity	Primigravida	164	6 (3.7)	158 (96.3)	0.368
	Multigravidia	636	25 (3.9)	611 (96.1)
Previous sexual partner	1	497	18 (3.6)	479 (96.4)	0.263
	2	237	8 (3.4)	229 (96.6)
	>2	66	5 (7.6)	61 (92.4)
	Total	800	31 (3.9)	769 (96.1)

HBsAg=Hepatitis B surface antigen

Of the 31 pregnant women who were reactive to HBsAg, only one had anti-HBs, two had HBeAg, 16 had anti-HBe, and 18 had anti-HBc [Table 5].

Table 5

Distribution of HBV markers in HBsAg reactive pregnant women attending antenatal care in some hospitals in Kaduna Metropolis

HBV marker	HBsAg positive samples; n=31 (%)
	Present	Absent
Anti-HBs	1 (3.2)	30 (96.8)
HBeAg	2 (6.5)	29 (93.5)
Anti-HBe	16 (51.6)	15 (48.4)
Anti-HBc	18 (58.1)	13 (41.9)

Anti-HBs=Hepatitis B surface antibody, HBeAg=Hepatitis B envelop antigen, Anti-HBe=Hepatitis B envelop antibody, Anti-HBc=Hepatitis B core antibody, HBV=Hepatitis B virus, HBsAg=Hepatitis B surface antigen

Discussion

In this study conducted to determine the prevalence of HBsAg and the phase of infection among the pregnant women attending ante-natal care in some selected hospitals within Kaduna Metropolis, Nigeria, a prevalence of 3.9% of HBsAg was obtained. Detection of HBsAg in an individual indicates that the person is infectious.[58] The seroprevalence rate in this study is higher than the 2.1% obtained among women at the point of delivery at the University of Benin Teaching Hospital, Benin City, Nigeria,[25] and the 1.9% obtained among pregnant women in Bali, Indonesia.[26] By contrast, this prevalence rate is slightly lower than the 4.3% and 9.3% prevalence rates reported among pregnant women in Port Harcourt, Rivers State,[14] and Anambra State, South-Eastern Nigeria, respectively.[13] The rate is also lower than the 6.2%, 5.6%, and 4.9%, reported in pregnant women in Sierra Leone,[20] Sudan,[21] and Europe,[27] respectively.

The wide variations in the reported seroprevalence of HBV in the pregnant women may be due to geographical variation, differences in cultural practices, sexual behavior and practices, and differences in the test methods employed to detect HBV infection.[19]

Even though HBV was detected with the highest prevalence among women in their second trimester of pregnancy and with the lowest among those in their first trimester, the neonates born to these positive women are equally at risk. This is because vertical transmission is thought to be a major mode of transmission of HBV in endemic areas such as Nigeria.[82228293031] In addition, once exposed, the risk of developing chronic infection by an individual, defined as the presence of HBsAg for >6 months duration, depends on the age of the individual and is the highest in neonates (90%) as compared to adolescents (50%) and adults (<10%).[82228]

The age of acquiring HBV infection is the major determinant of the incidence and prevalence rates.[2332] In this study, HBsAg infection was not significantly associated with age (P = 0.968) even though HBsAg was detected with the highest prevalence among women aged between 21 and 25 years. This finding contrasts the findings of Olokoba et al.[19] and Ali and Memon[30] who recorded the highest prevalence rates among women in age groups 25–29 years, and 25–35 years, respectively. This difference may be associated with higher sexual activities within these age groups.[19]

In relation to geopolitical zone, although the difference was not statistically significant, women from the South-East zone had the highest seroprevalence while those from the South-West zone had the lowest. This result may be a reflection of the differences in sexual practices and behavior, or due to awareness of HBsAg and testing, as well as, sociocultural practices and accessibility to healthcare as earlier asserted.[1930]

In this study, all the infected women were married which is indicative of infection likely through unprotected heterosexual intercourse or close person-person contact with infected spouse as the virus can be spread through body fluids and close contact.[256]

Although not statistically significant, higher HBsAg seroprevalence was obtained among multigravidae. This may be due to longer marriage period, hence longer sexually active years by the multigravidae as compared to the primigravidae. Again, although not statistically significant, HBsAg was detected with higher frequency among pregnant women who had more than two sexual partners in their lifetime. Based on the cultural setting of the study area, the information obtained from these women on sexual partners may not be reliable; as such information is shrouded with secrecy and may not easily be stated by the study subjects. This could explain the failure to document an association with number of sexual partners in this study. However, data on sexual behaviors have shown risky behaviors to be very common in Nigeria, while condom use remains low due to religious and cultural beliefs.[33]

This study did not find any correlation between formal education, type of marriage, the number of previous pregnancies, and HBsAg seropositivity (P > 0.05). This suggests that these factors were not the main risk factors for acquisition of HBV in this study. The highest seropositivity in this study was, however, recorded among women who had tertiary education, an observation earlier reported by Buseri et al.[31] Nonetheless, a similar study carried out in Jimma, Ethiopia showed high HBsAg seropositivity rate among the illiterate.[34]

In relation to the history of blood transfusion among the seropositive women, 6.1% of the women had been transfused while 3.8% have not. The higher seropositivity among the transfused women may be due to transfusion of improperly screened blood, or seroconversion after blood transfusion. This result, however, contrasts that of Buseri et al.[31] who reported a higher seropositivity among the women that have never undergone a blood transfusion. This finding is, however, similar to that of Adewole et al.[35] who identified the blood transfusion as a major risk factor for co-infection with HBV.

In this study, women with no scars had the highest prevalence of HBsAg, while those with scars had the lowest, implying that scarification was not a risk factor for acquisition of HBV. This finding, however, contrasts that of Adewole et al.[35] who identified the presence of tribal mark and scarification as a major risk factor for infection with HBV.

Only one of the HBsAg reactive pregnant women had developed anti-HBs. This indicates clearance of infection and immunity from HBV infection.[36] Anti-HBs are known to replace HBsAg as acute HBV infection is resolving. Anti-HBs generally persist for a lifetime in over 80% of patients and indicate immunity.[1833] The remaining 30 (96.8%) reactive women did not have anti-HBs, indicating they were in an active infection phase. Two (6.5%) of the reactive women had HBeAg while 93.5% did not have the envelop antigen. The presence of HBeAg indicating that the virus is actively replicating hence a highly infectious phase. there could therefore be, a 70-90% chance of vertical transmission of the virus to the neonates born to these highly infectious women.[9101112]

About 51.6% of the women had developed the envelop antibody (anti-HBe). The presence of anti-HBe correlates to a decreased infectivity as anti-HBe replaces HBeAg in the resolution of the disease.[1826] About 58.1% of the women had anti-HBc. Anti-HBc is the first antibody to appear in hepatitis B infection,[826] it is detected at the onset of symptoms in acute hepatitis B and persists for life.[36] The presence of anti-HBc indicates previous or ongoing infection with HBV in an undefined time frame.[36] Therefore, these women who had no immunity to the virus, were likely having chronic hepatitis B infection but had reduced risk of vertically transmitting the virus to their offspring.

Conclusion

This study has shown a 3.9% prevalence of HBsAg among pregnant women attending ante-natal care within Kaduna Metropolis between August and December 2011. About 96.8% (29) of the reactive women had HBeAg negative chronic hepatitis while 6.5% (2) had HBeAg positive chronic hepatitis B infection. Over half (51.6%) of the women had developed the envelop antibody correlating to decreased infectivity. About 58.1% of the women had anti-HBc hence, did not have immunity and probably have chronic infection with reduced risk of vertical transmission. All the sociodemographic and risk factors studied were not statistically associated with HBsAg in this study. However, the highest prevalence of HBsAg was recorded among the women within age group 21–25 years old, among the women with tertiary education, among the women in their second trimester of pregnancy, and among the women whose husbands were in polygamous relationships.

Recommendations

The findings of this study support the opinion that pregnant women should be screened for HBsAg infection at the first antenatal clinic visit so that adequate clinical management can be planned for them. In addition, at whatever stage a pregnant woman presents herself for antenatal care, her HBsAg status should be confirmed for effective prevention of vertical transmission. The interventions in preventing the consequences of HBV infection and its sequelae can only be applied to a woman whose HBsAg status is known. Therefore, determining the HBsAg status of pregnant women is a key factor to the success of hepatitis B prevention program.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.