INTRODUCTION
Hepatitis B virus
(HBV) is a double stranded DNA virus belonging to Hepadnaviridae
family. Whose incubation period is between 6 weeks and 6 months 1 .HBV
infection affects over 350 million people worldwide and about 2 million die
annually of HBV- related chronic hepatic disease2,3.
Liver cirrhosis and hepatocellular carcinoma lead to death in these patients2.
The prevalence of HBV
infection according to the geographical area, may be high (≥8%),
intermediate (2-7%), or low (≤2%)3.
Nigeria is classified among the countries that are highly endemic for viral
hepatitis4. The
prevalence of HB s Ag in normal population in Nigeria ranges from 2.7% to 13.3%5,
6
Generally, pregnant
women have depressed immunity, thus infection of HBV is of clinical importance.
Acute Viral hepatitis is one of the medical disorders in pregnancy known to be
associated with to high morbidities and sometimes mortalities because it could
lead to fulminant hepatitis which, though rare (1-2%), is associated with a
dramatically high mortality (63-93%) 7.
Hepatitis B e antigen
(HBeAg) is a soluble non-particulate antigen that is
found only when HBsAg is present1.
Pregnant women who are HB e Ag positive in the third trimester frequently
transmit this infection to the offspring in up to 90% of cases in the absence
of immunoprophylaxis, whereas those who are negative
rarely infect their offspring1, 8. Amongst those infected, 90% will
develop chronic infection and 25% will die due to complication of liver disease
7, 10.
HBV is transmitted
primarily through parenteral and sexual exposure to HB s Ag positive blood or
other body fluid from those who are chronic HBV carriers or who have acute
hepatitis B 3.
HBV is also transmitted
prenatally, with the possibility of vertical transmission8. Since a
large number of these transmitted cases progress to chronicity, infected
infants can initiate new cycle of both horizontal and vertical infection.
Infection acquired during the perinatal period has the highest risk of
chronicity 7, 8 47, 48. Prevention of perinatal transmission is thus
important to prevent chronic carrier state. Recent studies have shown a higher
incidence of low birth weight among infants born to mothers with acute infection
during pregnancy9. However, knowledge about mother to child
transmission of hepatitis B in Jos, which is the researcher’s area of study is
scarce, hence the need for the study.
Statement
of the Problem
Nigeria is classified
among the countries that are highly endemic for viral hepatitis4.
The prevalence of the viral infection amongst pregnant population is as high as
8.3%63 in Zaria and value as high as 13.3%5 has also been
reported. Perinatal transmission is an important route for neonatal infection,
because infection acquired during the perinatal period has the highest risk of
chronicity, thereby perpetuating the vicious cycle47, 48.
Prevention of perinatal transmission is thus important to prevent chronic
carrier state. This would only be possible if the prevalence of mother to child
transmission of hepatitis B virus is identified. There is scarcity of knowledge
in this area of study, so that the problem on ground in relation to the study
is identifying and examining the rate and extent of mother-to-child
transmission of hepatitis B in Jos metropolis.
Study
Hypothesis
This study
hypothesizes that the prevalence of hepatitis B virus infection in pregnancy is
comparable to that in the general population and mother to child transmission
do occur at birth.
Justification
of the Study
The prevalence of HB
s Ag in normal population is high and it is said to be between 2.7% to 13.3% 5,
6.
Hepatitis B infection
in pregnancy is an important health issue because infected mothers can transmit
same to their offspring with such consequences as low birth weight babies,
chronic hepatitis, liver cirrhosis and possible hepatocellular carcinoma7.
Ninety percent of
perinatal infection can be prevented if HB s Ag positive mothers are identified
and their newborn treated promptly with hepatitis B immunoglobulin and HBV
vaccine 10. Transmission of
HBV will add to a large number of carriers in the community and becomes a
public health problem. This study which is aimed at ascertaining the rate of
mother -to- child transmission of hepatitis B markers at delivery,
will help to inform policy formulation and programming in health planning for
women and children in Jos metropolis in particular, and Nigeria (including the
outside world) in general. It is for this reason that this dissertation mother- to- child transmission of Hepatitis
B in Jos metropolis was conducted.
Aim
General: The
aim of the study is to determine if the transmission of hepatitis B virus from
mother to child in Jos metropolis occurs. It is further to identify the extent
to which this occurrence or otherwise is evident, and to proffer solutions as
applicable.
Specific
Objectives Are:
1.
The study is designed to determine the
prevalence of HB s Ag among women at delivery
at Jos
2.
It is to determine the prevalence of HB s Ag on cord blood after delivery at Jos
3.
In addition to the above, it is also to determine the proportion of HB s Ag positive
women that are HB e Ag positive
4.
It is also to determine the proportion of HB
s Ag positive cord blood that are HB e Ag positive
5.
Finally, based on the above, to see if it is
rational to recommend the inclusion of routine
hepatitis B screening test for pregnant women
LITERATURE
REVIEW
Hepatitis
B Virus
HBV is a
small (42-nm) DNA virus that contains partially double-stranded DNA within its
core12. Using its own DNA polymerase for replication, the virus is
able to reproduce within a host's infected hepatocytes, drawing from the cell's
pool of nucleotide precursors12.
Hepatitis
B surface antigen (HBsAg) is the HBV serum marker
that has come to be used most commonly in clinical situations and screening
protocols. Discovered by Blumberg and co-workers in 196513, it
initially was not known to be a virus-associated marker. The antigen, first
isolated in the serum of an Australian aborigine during a study of serum
protein polymorphisms (hence it being labeled the “Australia antigen”) 14,
was found incidentally to cross-react with the serum of patients who have
had multiple blood transfusion. It was later found to be present in the serum
of institutionalized patients and, it was believed to be possibly associated
with Down syndrome.15
Subsequent
work by Blumberg's group and others established a link between the newly
identified antigen and acute hepatitis B, an association confirmed by electron
microscopy identification of particles dense with the antigen in the serum of
patients who were acutely ill with hepatitis.16 Those particles are
now known to represent incomplete portions of the viral envelope, synthesized
in great excess during the process of viral replication12, 16. In
addition, intact viral particles bear the surface antigen on their outer
envelope16. The presence of HBsAg in serum
indicates infectivity16, although such presence alone cannot
distinguish acute from chronic infection, an often confusing exercise that
requires a more complete elaboration of HBV-related serologies.
Although HBsAg is the first antigen detectable in the
course of HBV infections, predating even the appearance of symptoms in those
patients who become clinically ill, it is the predictable appearance and
disappearance of other HBV-associated antigens and antibodies over time that
allows patterns compatible with either acute or chronic infection to be
described.
The
complete hepatitis B viral particle, also known as the Dane particle, after
Dane and co-workers who described it in 197017, consists of the
viral core surrounded by its HBsAg-rich envelope. If
the envelope is removed by the use of detergents in vitro, a viral core
antigen (HBcAg) can be identified. Unlike HBsAg, HBcAg does not circulate
freely in serum and is found in blood only as an integral component of the
internal viral nucleocapsid17. A third antigen, the e antigen (HBeAg), is serologically distinct from both HBsAg and HBcAg. HBeAg is associated primarily with the core antigen in the
virus' internal structure, but unlike HBcAg, it can
be found circulating in serum, frequently in complexes with immunoglobulin.18
All three serologically unique antigens stimulate the production of equally
distinct antibodies (HBsAb, HBcAb,
and HBeAb) in the course of non
chronic host infection.
The presence of HBeAg
has been closely correlated with both the infectivity of a particular patient's
serum and the microscopic detection in serum of the HBV virus itself 19,
20, and an increased risk for chronic liver disease21, 22.
Seropositivity for HBeAg is taken as
a marker of active viral replication and the most infectious phase of the
disease, either in acute or in chronic illness. Practically, however, HBsAg is used in screening protocols because of the high
concentrations of this antigen produced in response to viral presence and
replication. More vigorous HBV serologic testing is performed, along with liver
function evaluation, in HBsAg-positive individuals,
both symptomatic and asymptomatic, to characterize the nature and extent of
their disease. The appearance of HBsAb in the serum
of patients occurs in the setting of resolution of acute infection; it is this
antibody that appears to confer protective immunity. However, both HBcAb and HBeAb have been shown
experimentally to be protective against reinfection 23,24,25,26.
Prevalence
The prevalence of HBV
infection, according to the geographical area, may be high (≥8%),
intermediate (2-7%), or low (≤2%) 3. Nigeria is classified among the
countries highly endemic for viral hepatitis4. The
prevalence of HB s Ag in normal population in Nigeria ranges from 2.7% to 13.3%5,
6
Viral
hepatitis infection remains a public health problem in developing countries. A
12.3% overall prevalence for HBsAg in pregnant women
attending antenatal clinic at General Hospital Minna
has been reported66. This was a descriptive cross-sectional study
using 261 pregnant women to determine social characteristics and seroprevalence of hepatitis B virus. This also corroborates
the World Health Organization (WHO, 1990) report for Nigeria as a highly
endemic area with prevalence greater than 8%. In related studies in different
parts of Nigeria, higher prevalence rates of 11.6% were reported among pregnant
women in including 2.19 % in Benin City 64, 4.3% in Port Harcourt in
2005 and 2.89% in 200665.
There
is a relatively higher hepatitis B virus infection rate in patients co-infected
with HIV. Agbaji et al67 carried a cross
sectional study in Jos amongst 1042 HIV positive patients and reported that
hepatitis B co- infection was found to be 14.2% amongst females and 19.4%
amongst male in the study. Sirisena et al68 quoted a higher co infection rate of 28.7%
in an earlier study
Manifestation of Hepatitis B Infection in the
Host
This can be either an
acute infection (which invariably occurs during pregnancy in a previously
otherwise normal person), or a chronic infection (which is usually preexistent
in either overt or latent form and with pregnancy occurring later)
Acute Hepatitis:
Hepatitis B infection
does not have any special predilection for pregnancy12, 13. In a study of
acute viral hepatitis in pregnancy from North India, HBV infection was observed
in 19% and 18% of the pregnant and nonpregnant females
respectively13. Moreover, acute HBV infection is not more
severe in pregnant women than in non pregnant
individuals12, 14. The presentation, as a rule, does not differ from that in non pregnant women. Persons with overt acute viral hepatitis
initially have non-specific complaints including fatigue, malaise, anorexia,
nausea, headache, myalgia and low grade fever. The nausea and vomiting of
prodromal stage may be confused with the symptoms present in pregnancy without
hepatitis. If the illness resolves before there is sufficient liver cell injury
and consequent secondary dysfunction to cause jaundice, these prodromal
symptoms are passed off as a flu-like viral syndrome or even for the normal
physiological effects of pregnancy itself. Otherwise, jaundice develops within
2 to 10 days of the prodrome. These patients may also
complain of right upper quadrant discomfort and examination may reveal tender
hepatomegaly14. Splenomegaly is noticed in about 10% of cases14,
15. In the later stages of pregnancy, the abdominal examination for hepatosplenomegaly may be difficult. Most often, jaundice and symptoms of liver disease resolve in 6
weeks14. Some of them have a violent course resulting in fulminant
hepatic failure with features of cerebral edema, coagulopathy, multiple organ
system failure and a few others have a persistent course beyond 6 months to
result in chronic hepatitis14, 15.
Acute
hepatitis, particularly late in pregnancy, may induce premature labour, but this seems to have little adverse effect on the
foetus.13 Heiber et
al14 have also
noticed an increase in the incidence of prematurity (31.6%) over that seen in
the general delivery population (10-11%). Apart from this, there are
possibilities of intrapartum or postpartum
hemorrhage, especially if the prothrombin time is
prolonged as in fulminant hepatic failure13. The usual hematologic
examinations are unremarkable. The aminotransferases are high even if the
bilirubin is normal (as in anicteric hepatitis) 13. The viral
markers like HBsAg and IgM
Anti-HBc are helpful in diagnosing acute hepatitis
due to HBV13.
Chronic Hepatitis and Cirrhosis
In most affected
individuals, chronic viral hepatitis is asymptomatic either indefinitely or
until there is sufficient liver damage for the patient to develop
manifestations of end-stage liver disease16. Many cases of chronic
viral hepatitis are therefore diagnosed after serum transaminase levels are incidentally noted to be abnormal16.
This commonly occurs, when an apparently healthy young woman becomes pregnant
and consults an obstetrician. Apart from the abnormal transaminase, the other
investigations are usually normal unless the liver disease is severe16.
Patients with severe chronic hepatitis are usually aware of the liver problem
before deciding about conception. In cirrhosis, the situation is slightly
different; fertility is decreased probably due to amenorrhea and non-ovulating
cycles, and hence pregnancy is less often seen in them.
Physical examination
may be normal or the patient may have subtle physical findings consistent with
early cirrhosis, including palmer erythema, splenomegaly, and a small liver
(sometimes with enlargement of the left lobe). Palmer erythema, if present, may
be confused with that of pregnancy (physiological). Examination of the abdomen
is difficult and may be ambiguous in the later stages of pregnancy, when the
liver and spleen are not palpable. In a minority of patients with end-stage
liver disease, clinical features of liver failure gradually becomes apparent
and may be mistaken for a hepatic complication of pregnancy16.
Chronic HBV carriers
usually have normal pregnancies, unless there is severe chronic hepatitis or
secondary cirrhosis and associated complications.16 Infeld et al had reported pregnancies in at least 28 of
mild chronic hepatitis patients without any harmful maternal effects17.
In cirrhotics, the main maternal risk is related to
the degree of portal hypertension and the likelihood of esophageal variceal haemorrhage18,19.
The onset of labour increases the intraabdominal
pressure thereby increasing the chances of variceal
bleeding. Besides this, the deterioration of hepatic function also occurs.
Liver and renal failures are the other causes of maternal mortality 16
Management
of Acute Viral Hepatitis
The management is
similar to that for non pregnant state. The
management, like in a non- pregnant state consists of supportive measures like
high calorie diet, bed rest and vitamins15. If marked anorexia or
vomiting is present hospitalization may be required for intravenous fluid
administration15. There is no recommended antiviral therapy for
acute viral hepatitis because most adults clear the infection spontaneously15.
Early antiviral treatment may only be required in the 1-2% of patients with
fulminant hepatitis and those who are immunocompromised15.
Management
of Chronic Liver Disease and Cirrhosis
Treatment of chronic
infection may be necessary to reduce the risk of cirrhosis and liver cancer17,18. Supportive management of liver disease is similar
to that in the non pregnant state. There are no
dietary restrictions unless cirrhosis with complications has set in17.
Management of esophageal varices needs special
attention by way of prophylactic banding17. Use of pharmacoprophylaxis for bleeding requires very careful
monitoring of the haemodynamic status18.
Coagulopathy should be managed with administration of fresh frozen plasma.
Antiviral therapy is
considered as applicable to non pregnant state. Of
the three recommended available drugs for chronic hepatitis B, interferon alpha
is not used possibly due to lack of adequate controlled studies even though
reports of its use in HCV infection does
not show significant interferon related fetal malformation 20,21.
The safety of Adefovir, Dipivoxil
in pregnancy has not been clearly established22. Only Lamivudine
which is used in an oral dose of 100mg daily has been found to be safe and has
been advocated for use in pregnancy23.
Vertical
Transmission and Effect on the Fetus
The significance of
HBV infection during pregnancy derives in major part from its potential to be transmitted vertically 24. Ten percent of infants born to women
with acute HBV infection during the first trimester of pregnancy are HBsAg positive at birth25 and 80 to 90% of
neonates become HBsAg positive without prophylactic
therapy if acute maternal infection develops during the third trimester of
pregnancy14,26. This variable rate of
vertical transmission from mothers with acute disease is explained by the fact
that the placenta is a reasonably effective barrier to the spread of HBV
infection27,28. According to Okada et al27, 85% of neonatal HBV infections are
caused by intrapartum exposure to infectious blood
and vaginal secretion, and the remaining 15% by haematogenous
transplacental viral spread. However,
Zhang28 in 2004 showed by measuring concentration of HBsAg & HBcAg in maternal decidual cells, trophoblastic cells, villous mesenchymal cells and villous capillary endothelial cells,
that the main route of HBV transmission from mother to fetus is transplacental, from the mother's side of the placenta, to
the fetal side. He also detected HBV DNA in amniotic fluid samples and vaginal
secretion samples - emphasizing transmission of infection by these during parturition.
Okada et al24,
examined the newborn infants of 139 asymptomatic carriers and did not find HBsAg positivity in the cord blood at any of them. However,
on follow up for more than seven months, eight of the 11 mother-child pairs
tested showed antigenemia, the earliest appearing 5
days after delivery. The sub-type of HBsAg was
identical for each mother-child pair. In the true sense, this could be an
instance of horizontal transmission from the mother during the immediate post
natal period. Subsequently Okada et al27
showed that all babies born to e antigen positive mothers developed surface
antigenemia.
In contrast, all of the babies born to e antibody negative mothers escaped surface antigenemia.
From this it is quite clear that e antigen positivity in the mother is a
reliable marker to predict transmission of infection from mother to child. In
the absence of appropriate prophylaxis, 40% of the neonates of HB e antigen
negative mothers and 90% of the neonates of HB e antigen positive mothers will develop
HBV infection.27, 30 However Sinatra et al29 reported that three infants born to
anti-HB e positive mothers developed acute icteric hepatitis B within 3 months
of birth. Liu et al31 have demonstrated
that HLA-DR3 is positively correlated with chronic HBV carriage and highly
replicative status in pregnant women. HLA-DR13 shows negative correlation with
chronic HBV carriage in pregnant women.
The strong
possibility of vertical transmission lends importance to diagnosing acute or
chronic HBV infection in pregnant women and justifies mandatory ante partum
serum HBsAg screening32. By
doing so, previously unsuspected chronic HBV infection is diagnosed in
otherwise healthy individuals. This has the added benefit of making it possible
to refer them for appropriate antiviral therapy before development of
significant liver damage and associated functional insufficiency.
The
infants of potentially infectious mothers are treated with HBV Human Hyper
globulin (HBIG) at delivery and simultaneously active immunoprophylaxis
is initiated32.
This approach is effective in preventing chronic HBV in approximately 85%
neonates33; it is ineffective in cases of haematogenous transplacental infection (15%)34.
Different measures
have to be adopted to modify (interrupt) the vertical transmission of HBV
Infection.
HBIG – HBIG can be
administered a dose of 200 IU i.m. every week from
28th week of gestation reduces the intrauterine infection to 16.1% against
32.7% (in controls).34
Lamivudine
- Lamivudine has been
used with good safety and efficacy in the last four weeks of pregnancy to
decrease the risks of vertical transmission35. Li et
al34 showed
that the intrauterine infection was reduced to 16.3% against 32.7% (controls)
with its use from 28th week of gestation in a dose of 100mg/day. This is also
substantiated by other studies23. However, there is a report of
failure of vertical transmission of hepatitis B virus despite antenatal
Lamivudine therapy36. Of course, in this case, the authors detected precore mutant in
both the mother and the child and this is to be interpreted appropriately
without undermining the vertical transmission lowering effect of Lamivudine.
Caesarean-section does not show any
extra reduction in the incidence of vertical transmission in comparison to
vaginal delivery37. Apart from vertical transmission, the maternal
HBV infection does not have any effect on the fetal outcome37.
Although there was an increase in incidence of prematurity, it had no effect on
congenital malformations, stillbirths, abortions or intrauterine malnutrition
in comparison to the controls14.
Breastfeeding
Breast feeding is an important outcome of
pregnancy and successful delivery. So, once the HBV infected mother, with all
possible precautions delivers a baby without any evidence of infection at
birth, the next question that comes to mind is: whether breast feeding can be
done safely? Even though, breast milk of infected mother contains HBV DNA, with
appropriate immunoprophylaxis, including hepatitis B
immunoglobulin and hepatitis B vaccine, breast feeding of infants of chronic
HBV carriers (irrespective of replicative status) poses no additional risk for
the transmission of the hepatitis B virus38.
Prevention
Mothers positive for both HBsAg
and HBeAg are at highest risk for transmitting the
virus; 85% to 100% of their offspring become infected, with 70% to 90% becoming
chronic carriers37. Mothers who are HBsAg-positive
but HBeAg-negative, presumably indicating lower
levels of replicating virus, do have a lower risk of transmitting the virus,
but up to 35% of their children still will become carriers in the absence of
neonatal therapy.39,40,41,42. In addition to the long-term risks of
HBV-related sequelae in chronic carriers, such as
cirrhosis and hepatocellular carcinoma, both fulminant fetal neonatal hepatitis 43,
44, 45, and childhood-onset
hepatic carcinoma46 have been described in children born to
HBsAg-positive mothers.
Early attempts at interrupting the perinatal
transmission cycle used HBIG alone, administered in the neonatal period.
Globulin alone had a protective efficacy against the carrier state of 70% to
75%, even though the protection was not permanent, and
many children eventually became infected after the passively acquired antibody
was cleared, undoubtedly through household contact46. With the
advent of the hepatitis B vaccine, trials were established to test its efficacy
when administered in the newborn period, both alone and in conjunction with
HBIG41. A combination of HBIG and vaccine in the newborn period
conferred significantly greater protection against perinatally
transmitted HBV than even the vaccine alone, increasing efficacy from a range
of 75% to 85% up to 90% to 95%,42,47,48,49,50,51,52,53,54,55. A
small but identifiable percentage of babies, who become infected despite even
combined HBV therapy at birth, is believed to
represent intrauterine
infection55, 56.
HBV DNA has been identified in abortus tissue extracted from an HBsAg-positive
mother 57, and other reports show evidence of intrauterine infection
in clinical situations, increasing risks for transplacental
leakage, such as preterm labour associated with
placental abruption55,56. Still,
combination HBV-specific immunotherapy provides the best opportunity to prevent
the chronic carrier state in the offspring of HBsAg-positive
mothers37. In the United States alone, approximately 16,500 births
occur in HBsAg-positive women each year,
approximately 4300 of whom are also HBeAg-positive58. Infants born
to these women should receive HBIG (0.5 ml) intramuscularly (IM), ideally
within 12 hours of birth59. HBV vaccine should be administered
concurrently at a different site (0.5 ml IM) or can be administered up to 7
days after birth if it is not immediately available59. The timing of HBIG appears to be more critical than
that of vaccine in achieving maximal effectiveness of passive–active therapy.
Subsequent vaccination is performed, also 0.5 ml IM, at ages 1 month and 6
months59. Follow-up for these infants is crucial, because one recent
study confirms the concern that in the United States, groups at highest risk
for HBV infection are also least likely to be compliant with follow-up care. It
is the recommendation of the American College of Obstetricians
and Gynecologists that HBsAg screening be performed
as part of routine prenatal testing in all pregnant women32.
MATERIAL
AND METHOD
Study
Area.
This study was
carried out in the maternity unit of the Department of Obstetrics and Gynaecology, Jos University Teaching Hospital (JUTH) Jos.
Jos University Teaching Hospital (JUTH) is a tertiary health institution
situated in Jos. It is one of the two teaching hospitals in the north-central
geopolitical zone of Nigeria, although there are Federal Medical Centres (FMCs) in the other states within the geo-political
zone. Jos is the capital city of Plateau State, which has over 30 different
ethnic groups 11.
The 2006 Nigerian
census puts the population of Plateau State at 2,959,588 with 1,031,662 being
female11.
Plateau State lies
between latitude 70 and 110 North and Longitude 700
and 2500 east11. The capital city is a pear-shaped upland
known as Jos Plateau. This upland stretches for approximately 104km from north
to south, and 80km from east to west, covering an area of about 8,600 sq km11. It has a height of 1,200m above sea
level11. Jos University Teaching Hospital has recently re-located to
its permanent site at Laminga, Jos North Local
Government Area of the State capital, in North central Nigeria. The hospital
has a well established Obstetrics and Gynaecology Department.
Study
Population
The study population were women who present in labour
at the labour ward of Jos University Teaching
Hospital, North Central, Nigeria
Study
Design
The study is a
descriptive cross-sectional study and hospital based. It was conducted between
1st March, 2011 and 31st August ,2011.
The parturients who give an informed consent were
recruited to the study during the last 5 weeks of pregnancy and the
pre–structured questionnaires were administered. As the women came in labour, blood samples were taken from the ante-cubital vein. Cord blood was also collected at delivery of
the baby after cutting the umbilical cord
Inclusion
Criteria
1.
Pregnant women who gave informed consent and
who presented in Labour at the labour
ward of JUTH for delivery and met the criteria.
Exclusion
Criteria
1.
Women who were not pregnant
2.
Pregnant women who have had hepatitis B
immunization
3.
Pregnant women who were HIV positive
4.
Pregnant women who declined to participate in
the study.
Ethical
Consideration
This proposal was presented to the research
and ethical committee of Jos University Teaching Hospital for approval.
Informed consents were obtained from the subjects before enlistment into the
study.
Estimate of
Sample Size
The sample size was calculated using the formula60
N=z2 pq/d2
Where,
N= desired sample size
Z=standard normal deviate 1.96 which
correspond to 95% confidence interval.
P= prevalence expressed as 100% that is,
13.3%5
q=complimentary proportion 1-p
d=degree of accuracy desired=0.05
N=(1.96)2
x 0.133 x 0.867
(0.05)2
=177.2
A minimum of 180 pregnant women were
recruited for the study.
Data
Collection
Collection
Of Blood Sample /Serum Preparation
Blood
samples were collected aseptically by venepuncture
using 5 ml sterile disposable hypodermic syringes and needles from the ante-cubital vein of pregnant women who presented in labour in the labour ward of Jos
University Teaching Hospital. The blood samples collected were dispensed into
pre-labeled specimen bottles. The samples were allowed to clot and centrifuged
at 3,000 rpm for 5mins to separate the serum. The sera were extracted using
micropipette and testing carried out as explained below
Procedure for Detecting HB s Ag
Hepatitis
B surface antigen (HBsAg) detection was done using
the in vitro diagnostic kit manufactured by ACON Laboratories, Inc. ,4108 Sorrento Valley Blvd., San Diego, CA 92121, USA.
The test kit (dipsticks) is a rapid immunochromatographic
assay designed for qualitative determination of HBsAg
in human serum or plasma. Assays were carried out at room temperature. The test
strips were removed from their foil pouches and immersed into serum samples
with arrows pointing towards the samples. The strips were taken out after about
10secs and placed on a clean, dry, non-absorbent surface. This was to allow
time for the reaction to take place. The
specimen was absorbed into the test strips and moved by capillary action upward
towards the control line. Results were read after 10mins post immersion. Positive
samples generated a colour band in the test region of
the strips and another in the control region while negative samples had a colour
band in the control regions only.
Procedure for Detecting HB e Ag
Sample
positive for hepatitis B surface antigen were tested for hepatitis B e antigen to
determine infectivity using CTK ONSITE test kit manufactured by CTK Biotech,
Inc. 6748 Nancy Ridge Drive, San Diego, CA 92121, USA. 2-3 drops of plasma were
dropped in the test kit well using a small plastic dropper. Results were read
after 15 minutes. There are C and T
bands on the test kit. The test is positive if C and T bands show colour development and is negative if only C band shows colour development.
Mothers who are positive for hepatitis B surface antigen shall have the
cord blood of their babies also similarly tested.
Positive
samples were stored at -200C and were later confirmed by using a
commercially prepared recombinant antigen-based Enzyme Linked Immunosolvent Assay (ELISA) test. This was done at the
Immunology Laboratory of the Jos University Teaching Hospital, Jos.
Statistical
Methods for Data Analysis
The results are expressed as means, and
standard deviation. Chi square was used to determine significance of
association. The relationship between HB s Ag
and other variables in the questionnaire were examined using P values.
Significance was determined using P<0.05 at 95% confidence interval. All
analysis were conducted using the SPSS version 15
software.
Limitation
to the Study
1. One of the limitations was poor knowledge
of the disease entity and the myth surrounding Hepatitis B virus infection
which affected consent
2. Repeat testing of the positive infants at
3 months, 6 months and at 12 months
would been necessary to ascertain the actual mother to child transmission. This would be the
basis for future studies
3. Other hepatitis B markers like Anti HBc, Anti HBs and Anti HBe would
have been done to enable proper classification of patients. However, cost of
testing for all the markers is high and therefore, a serious challenge.
The
Benefits of the Study
This study will benefit both patients and
humanity
Patient
The opportunity to have serum hepatitis B
surface antigen test done free –of- charge for these patients by the
researcher.
Arrangement was made for immunization of those
mothers who are negative after weaning for them to be immunized and arrangement
was also made for
co-administration of hepatitis B immunoglobulin and passive hepatitis B
immunization to those babies born to hepatitis B s antigen positive
mothers. Another benefit to the patient
is that those who participated in the study were provided with free haematinics for 6 weeks by the researcher.
Humanity
The result of the study will assist in policy
formulation in assessing the current non -screening of women for hepatitis B
surface antigen during ANC booking.
RESULTS
Tables 1, 2, 3, 4 and 5 show the age
distribution, educational status of mother, occupation of mother, educational
status of father and occupation of father. The analysis did not show any statistically
significant association between the above socio-demographic variables and the seropositivity of HB ' s’Ag. P
values >0.05
Table 6 shows the gravidity of the
participants and serostatus. Also no statistically
significant association was found between gravidity and HB '
s’ Ag. X2=2.266, P
value=0.519
Table 7 shows history of blood transfusion
and HB ' s’ Ag.
Sero status of the 180 enrollee in the study,
8 had history of blood transfusion. One in the eight with history of blood
transfusion was HB ' s’ Ag. positive.
Again this was not statistically significant.
X2= 0.209, P value= 0.648
Table 8 compares the mean gestational age at
delivery between the seropositive and the seronegative
participants. Their mean gestational age was not statistically significantly
different. P value=0.621
Table 9 shows the prevalence of HB ' s’ Ag. in the 180 study
participant of 12.77%.
Table 10 shows the prevalence of HB e Ag in
the 180 study participant of 1.7%.
Table 11 shows the prevalence of HB ' s’ Ag. in the cord blood of
babies of the 180 participant of 2.2%.
Table 12 shows the cross tabulation of the
maternal HB ' s’ Ag. and cord HB '
s’ Ag. Association was statistically significant. X2= 12.346,
P value= 0.000
Table 13 shows the cross tabulation of the
maternal HB ' s’ Ag. and
maternal HB e Ag. In the general study population of 180, 3 (1.67%) tested
positive for HB e Ag. That is 3 in the
23 that tested positive for HB ' s’ Ag. That is
proportion of 23:3 or 13.04%. Association was statistically significant, X2=13.346,
P value=0.000
Table 14 shows the cord HB s Ag and cord HB e
Ag of babies of participants. Of the 4 cord blood that tested positive for HB ' s’ Ag., none was positive for HB e Ag.
DISCUSSION
This study was
undertaken to find out the prevalence of HB ' s’Ag. among women at delivery and to assay the above marker in the
respective cord bloods of their babies. Secondly, it is find the proportion of HB ' s’Ag Positive women that are HB 'e' Ag positive and also,
to document same proportion in the cord bloods of their babies. Hepatitis B, a
viral liver infection remains a public health problem in developing countries
with high endemicity.
A prevalence rate of
12.77% for HB's’Ag was found in this study. This
finding makes Jos an area of high endemicity3 for hepatitis B virus.
This prevalence rate compares to a rate of 12.6% by Jombo
et al (2005) in prevalence study of hepatitis B virus infection in a rural
settlement in Northern Nigeria69. Also in the same north central
Nigeria, Mbaawuaga et al, (2008) find a prevalence of
11% in a study in Markudi, Nigeria involving 300
pregnant women70. Similar
prevalence rates have been reported, 11.6% in Maiduguri, north eastern Nigeria61
and 13.8% in Lagos, south-western Nigeria. But lower prevalence rates have been
reported from studies in the southern part of the Nigeria, 2.19% in Benin City64,
and 4.3% in Port Harcourt65. Agbaji et al (2008) in a cross sectional study in Jos,
Nigeria among 1042 HIV positive patients report a prevalence of HB ' s’Ag of 14.2% among the female and 19.4% among the males in
the study67. Also in a similar study Sirisena
et al (2002) quoted a higher co- infection prevalence rate of 28.7%68.
But these were studies among HIV positive participants and studies have shown
that prevalence of HB ' s’Ag is higher amongst HIV
positive patients67, 68. In this study, the researcher excluded HIV
positive women.
Prevalence rate of
cord HB's’Ag of 2.2% was found in this
study. Of the 23 women that tested positive to HB's’Ag, 4 (17.4%) had their respective cord blood also
positive for HB's’Ag. Though studies assessing
the cord blood seropositivity for HB ' s’Ag at delivery in this part of the world is scarce,
intrauterine infection though said to be rare is known to occur27, 28.
Rates as low as 2.5% for mother-to- child transmission of hepatitis B infection
prenatally, has been70. At delivery, cord blood of babies from HB ' sʼ Ag positive mothers have been found to be positive
in up to 50% of cases7. This is however higher than the 17.4% that
was found in this particular study. Ameer et al,
(2007) found out that of the 300 women that enrolled in their study, 37 were
positive for HB ' sʼ Ag (12.33%). Cord blood was
positive for HB ' s’Ag in 4 out of the 37 HB ' s’ Ag positive women (that is 10.8%). Differences in
the conduct of labour and delivery, and differences
in transplacental transfer of infection, possibility
of delayed sero-conversion could account for the variance. Whatever the reason
for the difference, it is however shown that intrauterine or rather prenatal
transmission making the cord blood to become positive do occur. Zuberi et al, (1989) reported a similar low vertical
transmission72.
The study also
revealed that of the 23 women that were HB ' s’ Ag
positive, 3 were HB 'e’ Ag positive. Thus giving a proportion
of 23:3 or 13.04%, the prevalence rate for HB 'e’ Ag in the study population of
1.7%. HB 'e’Ag is the marker for
infectivity. Being HB 's’Ag positive has a highly statistically significant
association with being HB 'e’Ag positive. X2=
13.346, P value= 0.000. Ameer et al, (2007) found
that HB e Ag was positive in 40.54% of patient that were HB s Ag positive71.
This does not compare to the 13.04% found in this study.
This study showed
that of the 4 babies whose cord bloods were positive for HB S Ag, none tested
positive to HB 'e’Ag. Ameer
et al (2007) find that 75% of cord bloods that were HB s Ag positive were also
HB e Ag positive71. The difference could be due to the natural
history of the disease.
CONCLUSION
The results of the
study showed that the prevalence of HB s Ag among women at delivery in Jos
metropolis is 12.77%. It also revealed that the prevalence of HB s Ag on cord
blood is 2.2% which is an issue of concern. It also revealed that the
Proportion of HB s Ag to HB e Ag in women at delivery is 23:3. This means that 13.04% of those
that were HB s Ag positive were infectious. Association was statistically
significant. There was no HB e Ag positivity in all the 4 cord blood that was
HB s Ag positive.
Recommendations
Following this study,
it is recommended that:
In line with WHO recommendation of routine antenatal screening for HB's’Ag. For pregnant women at booking2, it
recommended based on the finding of this study that routine screening for HB S
Ag on all pregnant women be introduced at the booking clinic. This will help
identify those that are positive. In this particular group further measures or
interventions to protect the unborn babies can be taken
Also government and
non-governmental agencies should intensify efforts to enlighten the public in
general and those within the reproductive age group in particular of the public
health importance of the disease
A larger multicentre study design to follow up babies born to
mothers that are HB S Ag for 6 and 12 months is also recommended so as to
ascertain both horizontal and vertical transmission of hepatitis B virus.
Conflict
of interest: There was no conflict of interest
Funding: There was
no funding
REFERENCES
1.
Guberman C et al .Renal, urinary tract,
gastrointestinal and dermatologic disorders in pregnancy. In: Decherney AL et al (Eds.) Current Diagnosis and treatment Obstetric and Gynaecology 10th
edition. McGraw-Hill Medical Publishing Division.2007; 382-385.
2.
World Health Organization (WHO). Advanced Immunization Management (AIM). Hepatitis B(cited 2005 August.);
available from www.who.int/mediacentre/factsheet/FS204/en/WHO. Assessed
15th August 2010
3.
Maddrey WC. Hepatitis B: an important public health
issue. J med virol. 2000;
61(3): 362-6.
4.
Sirisena ND, Njoku MO, Idoko JA. HBsAg in patients with human immunodeficiency Virus1 (HIV-1) infection
in Jos, Nigeria. Nigerian Medical Practitioner 2002; 41:18-20
5. Kulkarimi S, Alowola FO, Wayo GG. Prevalence of hepatitis B surface antigen in Northern Nigerian
blood donors. Vox Sangunis 1996; 50: 151-153.
6. Muula AS. Tackling HIV/AIDS in Africa-another perspective. African
Health 2000; 23 (1): 5-6
7. Sylvie RR, Francis D. Hepatitis B mother to
child transmission. Expert Rev. Anti-infective ther. 2004; 2(1): 133-145
8. Sriprakash I, Anil
TP. Routine prenatal screening of Indian women for HBsAg:
benefits derived versus cost. Tropical Doctor 1997; 102: 10-15
9. Shepard TH. Catalog of teratogenic agents 9th ed. Baltimore, MD: John Hopkins
University press; 1998.
10. Caruana SR, Keily HA, De silva SL, Chea l, Noun S, Saykav P.
Knowledge about Hepatitis viruses in immigrant and refugees from the Mekong
Region, Aust NZ. J Public Health 2005; 29: 64-68.
11.
Daniel, Jos and Plateau State diff missen 2002. Available at http://www.widernet.org/josproject/josplateauintml.
Assessed 15th August 2010.
12. Khuroo MS, Kamili S. Aetiology, clinical
course and outcome of sporadic acute viral hepatitis in pregnancy. J Viral Hepat 2003; 10: 61-9.
13. Jaiswal SP, Jain
AK, Naik G, et al. Viral Hepatitis during pregnancy. Int J Gynaecol Obstet 2001; 72: 103-8.
14. Hieber JP, Dalton
D, Shorey J, et al. Hepatitis and pregnancy. J Pediatr 1977; 91: 545-9.
15.
Adams RH, Combes
B. Viral hepatitis during pregnancy. JAMA 1965; 192: 195-8.
16.
Schweitzer IL, Peters RL. Pregnancy in Hepatitis B antigen positive cirrhosis. Obstet Gynecol 1976; 48 (suppl I): 53S-56S.
17. Infeld DS, Borkowf HI, Varma RR. Chronic persistent hepatitis and pregnancy. Gastroenterology 1979; 77: 524-7.
18.
Cheng YS. Pregnancy
in liver cirrhosis and/or portal hypertension. Am
J Obstet Gynecol 1977; 128:
812-22.
19.
Russell MA, Craigo
SD. Cirrhosis and portal hypertension in pregnancy. Semin Perinatol
1998; 22: 156-65.
20.
Hiratsuka M, Minakami H, Koshizuka
S, et al. Adminstration of interferon-alpha during
pregnancy: effects on fetus. J Perinat Med 2000; 28:
372-6.
21.
Ozaslan E, Yilmaz R, Simsek H, et al.
Interferon therapy for acute hepatitis C during pregnancy. Ann Pharmacother 2002; 36: 1715-8.
22.
Dando T, Plosker G. Adefovir Dipivoxil: A review of its use in chronic hepatitis B.
Drugs 2003; 63: 2215-34.
23.
Su GG, Pan KH, Zhao NF, et al.
Efficacy and safety of lamivudine treatment for chronic hepatitis B in
pregnancy. World J Gastroenterol 2004; 10: 910-1.
24.
Okada K, Yamada T, Miyakawa
Y, et al. Hepatitis B surface antigen in the serum of infants after deliver
from asymptomatic carrier mothers. J Pediatr
1975; 87: 360-3.
25.
Sweet RL. Hepatitis
B infection in pregnancy. Obstet Gynecol Report 1990; 2: 128.
26.
Reinus J, Leikin E. Viral hepatitis in pregnancy. Clin Liver Dis 1999; 3: 115-30.
27.
Okada K, Kamiyama
I, Inomata M, et al. e antigen and anti-e in the serum of asymptomatic carrier
mothers as indicators of positive and negative transmission of hepatitis B
virus to their infants. N Engl J Med 1976; 294:
746-9.
28.
Zhang SL, Yue
YF, Bai GQ, et al. Mechanism of intrauterine
infection of Hepatitis B virus. World J Gasroenterol
2004; 10: 437-8.
29.
Sinatra FR, Shah P, Weissman JY, et al. Perinatal transmitted acute icteric
Hepatitis B in infants born to Hepatitis B surface antigen-positive
anti-Hepatitis B e positive carrier mothers. Pediatrics 1982; 70: 557-9.
30.
McMahon BJ, Alward
WL, Hall DB, et al. Acute Hepatitis B virus infection: relation of age to the
clinical expression of disease and subsequent development of the carrier state.
J Infect Dis 1985; 151: 599-603.
31.
Liu HY, Kong BH, Luo
X, et al. Study on the association
between maternal-infantile vertical transmission of hepatitis B virus and human
leukocyte antigen DR gene domain. Zhonghua Fu Chan Ke Za Zhi
(Chinese) 2003; 38: 599-603.
32.
ACOG Technical bulletin. Hepatitis in pregnancy. 174 November 1992.
33.
Stevens CE, Taylor PE, Tong MJ, et al.
Yeast-recombinant Hepatitis B vaccine - efficacy with hepatitis B immune
globulin in prevention of perinatal Hepatitis B virus transmission. JAMA 1987; 257: 2612-6.
34. Li XM, Yang YB, Hou
HY, et al. Interruption of HBV intrauterine transmission: a clinical study.
World J Gastroenterol 2003; 9: 1501-3.
35.
Van Zonneveld
M, van Nunen AB, Neisters
HG, et al. Lamivudine treatment during pregnancy to prevent perinatal
transmission of hepatitis B virus infection. J Viral Hepat
2003; 10: 294-7.
36.
Kazim SN, Wakil SM, Khan LA, et al. Vertical transmission of
hepatitis B virus despite maternal lamivudine therapy. Lancet 2002; 359:
1488-9.
37.
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; 115:1510-2.
38.
Hill JB, Sheffield JS, Kim MJ, et al.
Risk of hepatitis B transmission in breast-fed infants of chronic hepatitis B
carriers. Obstet Gynecol
2002; 99: 1049-52
39.
Okada K, Kamiyama I ,Inomata
M et al:The e antigen and Anti-e in the serum of
asymptomatic carrier mothers as indicators of positive and negative
transmission of hepatitis B virus to their infants .N Engl
J Med 1976 ;294:746.
40.
Beasley RP, Trepo C, Steven CE. Et al: The e antigen
and vertical transmission of hepatitis B surface antigen .Am J Epidemiol 1977; 105:94.
41. Steven CE, Toy PT, Tong MJ et al: Perinatal
hepatitis B virus transmission in the United States
:Prevention by passive –active immunization. JAMA 1985;253:1740.
42.
Biswas SC, Gupta I, Gangul NK et
al :Prevalence of Hepatitis B surface antigen in pregnant mothers and its
perinatal transmission .Trans R Soc Trop Med
Hyg.1989;83:698.
43.
Dupuy JM, Frommel D,Alagille D:Severe viral hepatitis B in infancy.Lancet
1975;1:191.
44.
Fawaz KA, Grady GF,Kaplan MM et al:Repetitive
maternal-fetal transmission of fetal hepatitis B.N Engl
J Med 1975;293:1357.
45.
Delaplane D, Yoge VR, Crussi F et al :Fetal hepatitis B in early infancy:The importance of identifying HB s Ag positive
pregnant women and providing immunoprophylaxis for
their newborn .Paediatrics 1983;72:176
46.
Chang MH, Chen DS, Hsu HC et al :Maternal transmission
of hepatitis B virus in childhood hepatocellular carcinoma. Cancer 1989;64:237
47.
Beasley RP, Hwang LY, Lin CC et al: Hepatitis B immune globulin (HBIG) efficacy
in the interruption of perinatal transmission of hepatitis B virus carrier
state: Initial report of a randomized double-blind placebo-controlled trial.
Lancet 1981; 2:388.
48.
Beasley RP, Hwang LY, Lee GC et al: Prevention of perinatally
transmitted hepatitis B virus infections with hepatitis B immune globulin and
hepatitis B vaccine. Lancet 1983;2:1099.
49.
Tada H, Yanagida M, Mishina
J et al: Combined passive and active immunization for preventing perinatal
transmission of hepatitis B virus carrier state. Pediatrics 1982;70:613.
50. Wong VCW, Ip
HMH, Reesink HW et al: Prevention of the HBsAg carrier state in newborn infants of mothers who are
chronic carriers of HBsAg and HBeAg
by administration of hepatitis-B vaccine and hepatitis-B immunoglobulin:
Double-blind randomized placebo-controlled study. Lancet 1984; 2:921.
51. Pongpipat D, Suvatte V, Assateerawatts A:
Efficacy of hepatitis-B immunoglobulin and hepatitis-B vaccine in prevention of
the HBsAg carrier state in newborn infants of mothers
who are chronic carriers of HBsAg and HBeAg. Asian Pac J Allergy Immunol
1986;4:33.
52.
Schaln SW, Mazel JA, deGast
GC et al: Prevention of hepatitis B infection in newborns through mass screening
and delayed vaccination of all infants of mothers with hepatitis B surface
antigen. Pediatrics 1989; 83:1041.
53.
Poovorawan Y, Sanpavat S, Pongpunlert W et al: Protective efficacy of a recombinant
DNA hepatitis B vaccine in neonates of HBe antigen-positive
mothers. JAMA 1989; 269:3278.
54.
Chen DS, Hsu HM, Sung JL et al: Immunization of newborns of HBsAg-positive
mothers: Ag-positive mothers: A successful first step in control of hepatitis B
virus in Taiwan. Hepatology 1989; 9:900.
55.
London WT, O'Connell AP: Transplacental transmission
of hepatitis B virus. Lancet 1:1037, 1986
56.
Tang SX, Yu GL: Intrauterine infection with hepatitis B virus. Lancet 1990;
335:302.
57.
Li L, Shen MH, Li YQ et al: A study of mother-infant
transmission of hepatitis B virus. Shanghai, International Symposium on Liver
Cancer and Hepatitis, 1986
58.
Centers for Disease Control: Recommendations of the Immunization Practices
Advisory Committee. Prevention of perinatal transmission of hepatitis B virus:
Prenatal screening of all pregnant women for hepatitis B surface antigen MMWR Morb Mortal Wkly Rep 37:341, 1988
59.
Jonas MM, Reddy RK, DeMedina M et al: Hepatitis B
infection in a large municipal obstetrical population: Characterization and
prevention of perinatal transmission. Am J Gastroenterol
1990; 85:277.
60.
Araoye MO. Sample size determination.In :Araoye MO (ed) Research
methodology with statistics for Health ans Social Sciences.Nathadex pub.,2003;6:115-121.
61.
Harry TO., Bajani MD., Moses AE. Heaptitis B virus infection among
blood donors and pregnant women in Maiduguri, Nigeria. East Africa
Medical Journal1990;
70: 596-597.
62.
Nasidi A, Harry, TO., Vyazor, SO., Numumbe, GMR., Azzan, BB., Ancinlev, VA. (1983). Prevalence of Hepatitis B infection marker in two different
geographical areas of Nigeria. Proceedings of the
first international conference, 12-15 December 1983, Lagos, Nigeria.
63.
Luka, SA., Ibrahim, MB., Iliya,
SN. Seroprevalence of hepatitis B surface antigen among pregnant women attending Ahmadu Bello University Teaching hospital, Zaria, Nigeria.
Nigerian Journal of Parasitology 2008; 29(1): 38-41
64.
Onakewhor, JUE., Offor, E., Okonofua, FE. Maternal and neonatal seroprevalence of
Hepatitis B surface antigen (HBsAg) in Benin City.
Journal of Obstetrics and Gynaecology 2001; 21(6): 583-586.
65.
Akani, CI., Ojule, A C., Opurum, HC., Ejilemele, AA. Seroprevalence of HBsAg in pregnant women in Port Harcourt. Nigeria. Nigeria
Postgraduate Medical Journal, 2005; 12(4): 266-270
66.
Ndam IS.,Joshua IA., Luka
SA.,Sadiq HO., Epidemiology of hepatitis B infection
amongst pregnant women in Minna,Nigeria.Science World
Journal .2008;3 :3
67.
Agbaji OO,Ladep
GN ,Agaba P, Badung BP, Danung ML, Imade G et al.
Prevalence and characteristics of Hepatitis B Surface Antigenaemia
amongst HIV infected Patient in Jos, Nigeria. Hungarian Medical Journal ,2008;2(1.9):77-82
68.
Sirisena ND, Njoku M, Idoko JA . Hepatitis B Surface antigenaemia in patient with HIV 1 infection in Jos , Nigeria .Nigeria Medical Practitioner 2002;41:18-20.
69.
Jombo GTA, Egah DZ, Banwat EB. Hepatitis B virus infection in
a rural settlement in Northern Nigeria. Niger J med 2005; 4: 524-428.
70.
Mbaawuga EM, Enenebeaku
MNO, Okopi JA, Damen JG. Hepatitis B virus (HBV) infection among pregnant women in Markudi, Nigeria. Afr. J. Biomed.
Res. 2000; 11: 155-159.
71.
Ameer A, Abdul R, Ghulam
QKK. Hepatitis B markers; its transmission in newborn from
mothers. Professional med J 2007; 14(2): 307-311
72.
Zuberi SJ, Lodi P. Pathern
of HB s Ag/ HB e Ag antigenaemia in pregnant women.
JAMA 1989; 39: 160.
