Hepatitis B Virus Mother-to-Child Transmission Among HIV-infected and non-HIV-infected Pregnant women in Jos, Nigeria

 

ABSTRACT

The high prevalence of HIV and Hepatitis B Virus (HBV) co-infection among pregnant women constitute major reservoir in endemic communities for the transmission of Mother-to-Child with HBV. In addition, pregnant women are the interface for their sexual partners and infants in serving as the main mechanism that perpetuates the HBV infection in the population. It is reported that 35% to 50% of chronic HBV carriers in high endemic regions acquired the infection through Mother-to-Child Transmission (MTCT) from HBV infected mothers. Co-infections of HBV and HIV infection is associated with higher rates of HBV-DNA replication thereby increasing the risk of MTCT of HBV infection. There is paucity of data on the burden of HBV MTCT among pregnant women in our environment and the study is aimed at determining the prevalence of HBV MTCT in Jos. A cohort longitudinal study involving pregnant women and their babies with 20 each for HBV infected and HBV/HIV co-infected groups that will be recruited between January 2017 and December, 2017 among those accessing Obstetric services in JUTH, PSSH, and OLA in Jos, Nigeria. Data on socio-demographics, Immunization status, HBsAg, and HIV status as well as HBV DNA level will be recorded. Data will be analyzed using STATA version 14. All statistical tests will be two-sided and Chi-square tests will be conducted among groups to compare their differences. Odds ratio (OR) and 95% Confidence Interval (CI) will be calculated based on logistic regression models. A p-value less than 0.05 will be considered statistically significant. This study will demonstrate the extent to which HBV MTCT contributes to the burden of HBV in Jos, Nigeria.

 

BUDGET:

Description of Items	Institution	Cost (Naira)	Quantity	Total (Naira)
1.0 Personnel Cost/Allowances	Federal GoN	-	4	-
2.0 Equipment/Consumables
HBsAg Rapid Test Kids (Accumen)	STAMINA	7,370 / pack (50 kids)	30 Packs	221,100
HBsAg Combo Kids	STAMINA	16,500 / pack (20 kids)	7 Packs	115,500
HIV Rapid Test Kids (Determine, Unigold & Stat PaK)	CDC Support to Health Facility	-	-	-
Z-10 Bottles	STAMINA	33,000 / set of bottles	1 sets of bottles	33,000
Z- 5 serum Bottles	STAMINA	30,800 / set of bottles	1 sets of bottles	30,800
5 ml Syringes & Lancets	STAMINA		800 pieces of 5 ml syringes 25 packs	45,000
Cotton wool	STAMINA	1,600 / pack	4 packs	6,400
Methylated Spirit	STAMINA	2,000 / Liter	4 Liters	8,000
Disposable Hand Gloves	STAMINA	2500 / pack	5 packs	12,500
Medical Waste Bin	Study Site
ARVs & Hepatitis Vaccines	CDC support & National Immunization Program
Phone Calls & SMSs	STAMINA	2,800 / Month	2 Personnel for 12 Months	63,600
ICT Services/Publications	STAMINA			210,000
Research Assistant	STAMINA	20,000 / Month	1 Assistant for 12 Months	240,000
Local Transportation	STAMINA	8,500	12 Months	102,000
HBV DNA (Viral Load)	SAMINA	24,000/Sample	38 HBsAg Mother-Baby pairs	912,000
Sub-Total				1,999,900

 

BUDGET JUSTIFICATION:

The personnel cost will not be covered by the research grants as the PI and other collaborators are Junior Faculty in the University. A major part of the funds will be used in purchasing HBV screening kids and Test kids for other viral markers.

A Research Assistant will however be contracted to complete the questionnaires and collect blood samples for screening tests. Part of the funds will be used for logistic support by the PI in monitoring data collection in the three sites in Jos.

Antiretroviral drugs for PMTCT of HIV positive mothers will be made available through the standard PEPFAR programs in the study facility and Hepatitis B vaccines will be provided to Infants through the National Immunization Program in Nigeria at designated Child Welfare Clinics in Jos at no cost.

Biohazard equipment for medical waste management will key into the waste management systems of the study heath facility for appropriate disposal of medical waste to be generated from the research activities.  

 

PROJECT NARRATIVE:

The award of the Research Grant will provide the opportunity to determine the burden of chronic HBV infection among HIV infected, non HIV infected pregnant women and their Infants in Jos. There is paucity of data on the burden of this infection resulting from MTCT in Jos, the research will also provide evidence on the (rate of vertical transmission of HBV) and the associated risk factors in Jos. The information from this research will be useful in the development of protocols for the prevention of HBV MTCT in Jos, Nigeria.

 

RESEARCH PLAN:

Specific Aims:

1. To determine and compare the prevalence of HBV infection in HIV-infected and non HIV-infected pregnant women in Jos, Nigeria.
2. To determine the prevalence of Mother-to-Child Transmission of HBV infection among HIV-infected and non HIV-infected pregnant women in Jos, Nigeria.
3. To determine the risk factors associated with Mother-to-Child Transmission of HBV among HIV-infected and non HIV-infected pregnant women in Jos, Nigeria.

 

Background:

The global burden of diseases in a study showed that HIV/AIDS caused 1.47 Million deaths/ year worldwide, tuberculosis caused 1.2 Million deaths/ year and Chronic viral hepatitis caused 1.3 Million deaths/ year out of which 800,000 deaths are attributed to HBV infection. ¹

High rates of co-infection among pregnant women constitute major reservoir in endemic communities for HBV transmission as pregnant women are the interface for their sexual partners and infants in addition to serving as the main mechanism that perpetuates the HBV infection in the population. ^{2, 3, 4} It is reported that 35% to 50% of chronic HBV carriers in high endemic regions acquired the infection through HBV MTCT from HBV infected mothers or in early childhood. ^{5, 6}

Nigeria is classified as endemic nation for both viruses as the country is home to about 3.4 Million PLHIV and 18 Million people are living with chronic HBV infection. ^{7, 8} Globally, 5-10% of HIV infected persons are co-infected with HBV in general population; ⁹ however, studies of HBV/HIV co-infection in pregnancy showed a prevalence of 1.5% and 4.9% in America and Europe respectively, in Africa it has been estimated to be 0.7% to 11.6%, and in Nigeria prevalence is put at 10%. ^{10, 11, 12, 13, 14, 15}

In a study of HBV and HIV Co-infections among pregnant women in Ibadan, Nigeria, 8.9% of 721 HIV positive pregnant women were Hepatitis B surface antigen (HBsAg) positive. ¹⁶ In an earlier study on sero-prevalence of Hepatitis B and HIV infections in pregnant women in Jos, Nigeria; the HBV sero-prevalence for Hepatitis B core antigen (HBcAg) was 63.3% indicating previous or ongoing infection with HBV. ¹⁷ A more recent cross-sectional study of prevalence and immune status of HIV/HBV co-infected pregnant women in two hospitals in Jos in 2008 found the prevalence of HBsAg sero-positivity among HIV positive pregnant women to be 11.8%. ¹³

The presence of Hepatitis B e antigen (HBeAg) in serum is an indicator of viral replication and often used as a marker of ability to spread the virus to other people; with reported rates of transmission from mothers who are positive for HBeAg varying from 7% to 28%. ^{18, 19} Before the introduction of universal hepatitis B vaccinations, the risk of vertical transmission is greatest for infants born to women who are HBsAg positive and HBeAg positive ranging from 70-90% at 6 months of age; about 90% of these infants remain chronically infected. The risk of vertical transmission among infants born to HBsAg positive but HBeAg negative mothers ranging from 10-40% with 40-70% of these infants remaining chronically infected. ^{3, 20}

Infants born to HBV/HIV co-infected women are however, more likely to be infected vertically and are more at greater risk of developing chronic hepatitis than infants born to HBV mono-infected mothers. Although spontaneous clearance of HBV acquired in adulthood occurs in over 90% of immune competent persons, HIV-infected persons are half as likely as HIV-uninfected persons to spontaneously clear HBV. Therefore, chronic HBV infection occurs in 5-10% of HIV-infected individuals who are exposed to HBV, a rate 10 times higher than that for the general population. ²¹

HBV/HIV co-infected persons also have higher levels of HBV replication with higher HBV DNA levels as well as persistence of HBeAg sero-positivity. ²² This is reported in a study in South Africa of HIV/HBV co-infected pregnant women; the HBeAg prevalence was 43% and the rate of HBV MTCT was 28%. ²³ Also in a Malawian study in HIV-infected women and their HIV-exposed uninfected infants showed that 5% of women were positive for HBsAg and among these, 38% were at high risk of transmission (positive for HBeAg). In that study, the rate of transmission of HBV was 10% and all infected infants were born to HBeAg positive women. ²⁴

The most important risk factor for in-utero transmission of HBV infection in pregnancy is the maternal HBV DNA levels (viral load). ²⁵ It has also been shown that failures in MTCT infant post exposure prophylaxis (PEP) usually occur in mothers with HBV DNA levels or thresholds of greater or equal to 10 ⁶ to greater or equal to 10 ⁸ Copies per mL. ²⁶

Justification / Significance:

The risk of MTCT of HBV is greatest for infants born to pregnant women who are both positive for HBsAg and HBeAg and is put at 70% to 90% at 6 months of age; about 90% of these children will remain chronically infected with HBV and 25% will eventually die prematurely from cirrhosis and Liver cancer. ^{3, 20} Co-infections of HBV and HIV in addition to enhancing HBV disease progression, enhances HBV-DNA replication thereby increasing the risk of MTCT of HBV infection. ²⁷

A combination of active and passive immune prophylaxis is the optimum strategy to prevent HBV infection in babies of HBsAg positive and HBeAg positive mothers; and if initiated within 24 hours of birth has been shown to protect 85% to 95% of babies. When HBIG is unavailable, vaccination alone can prevent vertical transmission in 66% to 90% of cases. ^{28, 29} Factors that have been implicated with failure of PEP include maternal HBeAg positivity and high HBV-DNA levels, delay in receipt of the Hepatitis B birth dose, failure to complete the vaccine series, failure to receive HBIG, non response to vaccine and rarely, HBV mutations and for some unknown reasons, MTCT occurs in 5% to 15% of infants despite timely prophylaxis. ^{30, 31}

The administration of ART starting from late pregnancy will reduce maternal HBV viral load and potentially reduce the possible risk of MTCT of HBV infection. ^{32, 33} Tenofovir is a potent antiviral that acts against HBV with a high barrier to resistance. ³⁴ A retrospective study evaluated the efficacy of tenofovir administered in the third trimester of pregnancy at a dose of 300mg once daily to eleven women with high viremia (>10 ⁶   copies/mL). ³⁵ The mean maternal viral load declined from 8.87 log ₁₀ copies/Ml at baseline to 5.25 log ₁₀ copies/mL at the time of delivery. All of the eleven infants received passive/active prophylaxis, and none of them were HBsAg- positive at 28- 36 weeks of age. ³⁵

A second retrospective study enrolled 45 HBeAg- positive pregnant women with high viral loads (HBV-DNA >10 ⁷ copies/mL); 21 of them were treated with tenofovir from week 18- 27 of gestation, and 24 of them were untreated and serve as control. ³⁶ All infants received standard passive/active prophylaxis. At 28 weeks of age, HBsAg was detected in two (8.3%) babies of untreated mothers, but not in any of the babies of treated mothers (p=0.022). The two groups of babies did not differ in terms of adverse events, birth defects, height, or weight. Twenty-eight weeks after delivery, HBV-DNA was undetectable in 62% of the treated mothers, whereas HBD-DNA was detected in all of the controls (p<0.001).

Tenofovir is part of many first line HIV regimens in PEPFAR supported facilities in Nigeria and is therefor available in many centers providing PMTCT of HIV. Unfortunately, in many centers, this drug may not be available to those who are HBV mono-infected. A fixed –dose combination (FDC) ARV regimen consisting of tenofovir (TDF), emtricitabine (FTC) and efaveranz (EFV) is currently recommended for HIV positive pregnant women regardless of their CD4-cell count. Since TDF and ETC are also effective in the treatment of HBV infection, these PMTCT guideline has been been shown to offer simultaneous prevention of HIV and HBV vertical transmission.

Gaps in hospital practices and policies to prevent MTCT of HBV infection exist in resource-poor settings attributed likely to poor perception of the HBV burden. ³⁷ The infrastructure which supports HIV services which includes; rapid testing for HIV, identification of patients who need therapy, follow-up of patients with screening for complications of the disease are well established in Nigeria with over a decade support of PEPFAR and could be leveraged for prevention of HBV MTCT.

Although several studies in Nigeria have shown that the prevalence of HBV and HIV are both high, there is dearth of information on the contribution of HBV MTCT to the burden of HBV in Jos. The study is aim at determining the prevalence of HBV MTCT among HBV/HIV co-infected and HBV infected pregnant women to inform policies for prevention of MTCT of HBV infection in Jos, Nigeria.

Hypothesis:

Null Hypothesis-

There is no difference in the prevalence of HBV MTCT among HIV/HBV-co-infected and HBV-infected pregnant women in Jos.

 

Materials and Methods:

Study Area: The study will be conducted in the Obstetrics and Gynecology departments of the Jos University Teaching Hospital (JUTH), Lamingo-Jos, Plateau State Specialist Hospital (PSSH), and OLA Hospital Jos, Plateau state. The three health facilities are suitable sites for the study due to the availability of large number of study population accessing baseline screening for HBV and HIV at booking as well as availability of other laboratory services. The facilities have a good mixed of personnel including Obstetricians, Physicians and Gastro-enterologist and neonatologist at hand to manage mothers and neonates depending on the HBsAg test results.

Study Design:

The study will be a cohort longitudinal study involving HBsAg positive pregnant women that will be consecutively recruited by simple random sampling technique in the three study sites of JUTH, PSSH, and OLA between January and December, 2017. Pregnant women will be screened for HBsAg and HIV during antenatal clinic attendance at term (38 to 42 weeks) or when admitted for delivery at term in the study facilities; the study will have pregnant women mono-infected with HBsAg in one group and pregnant women co-infected with HBV/HIV in the other group, and their babies screened for HBsAg at birth (mother-baby pair). For each mother-baby pair in the two groups with Positive HBsAg or HBcAg or other HBV makers; maternal and cord blood samples will be obtained at delivery for baseline assay of other HBV markers including; HBeAg, HBsAb, HBcAb and quantification of HBV-DNA (viral load) using a PCR based technique. Intrauterine infection will be defined as HBsAg positivity and/or HBV-DNA in neonatal blood. The HBsAg screening of the baby will be repeated at 6 months of age to identify infants with chronic HBV infection and other HBV markers including anti-HBs to measure HBV vaccination uptake. All babies will be routinely offered HBV vaccination according to the National Program on Immunization Schedule.

Study Population: The study population will consist of pregnant women who are HBsAg positive accessing antenatal care, labor and delivery services and their babies (mother-baby pair) in Jos University Teaching Hospital, PSSH and OLA.

Inclusion and Exclusion Criteria:

Inclusion Criteria:

1. All pregnant women who are HBsAg positive at term (38 to 42 weeks) presenting in antenatal clinic and labor ward with a live baby.
2. All HBsAg positive pregnant women who are HIV positive at term (38 to 42 weeks) presenting in antenatal clinic and labor ward with a lived baby.
3. All eligible pregnant women in the antenatal clinic and labor ward who gave consent for the study.

Exclusion Criteria

1. All pregnant women at term and/or in labor and have chronic medical conditions in pregnancy.

 

Sample Size Determination:

The sample size was calculated for cohort study using OpenEpi, Version 3, open source calculator—SScohort.

Two-sided significance level (1-alpha): 95

Power (1-bete, % chance of detecting): 80

Ratio of sample size, HBV group/HBV/HIV group: 1

Percent of HBV group with outcome: 5

Percent of HBV/HIV group with outcome ^{21, 24}: 50

Odds Ratio: 19

Risk/Prevalence Ratio: 10

Risk/Prevalence difference: 45

Sample Size- HBV/HIV Group: 19

Sample Size- HBV Group: 19

Total Sample Size: 38

The minimum sample size for the study will be 38 mother-baby pairs which will be divided into two groups; one group will be mother-baby pairs with HBV/HIV co-infected mothers and the other group will be mother-baby pairs with HBV mono-infected mothers.

Sampling Procedure:

Study participants will be by simple random sampling method involving consecutive recruitment of all pregnant women at term who are positive for HBsAg and accessing antenatal care, labor and delivery services and their babies as mother-baby pairs at JUTH, PSSH and OLA between January and December 2017. For HBsAg positive mothers, HBV DNA (HBV viral load) will be determined as an important predictor of HBV MTCT. We will leverage on the well established PMTCT services in the study sites with every antenatal visit commencing with health talks and group HIV pre-test counseling. Study participants in the HBV/HIV group will have further evaluation and care based on National PMTCT guidelines and records of HIV viral load, CD4+ cell count and Antiretroviral drug regiment will be recorded for analysis.

Data Collection Technique:

Trained research assistants will collect data through interviewer administered questionnaire in all the research sites. The PI will collate all the questionnaires after going through each to ensure completeness and accuracy. The data will be entered into the computer using excel spread sheet by the data clerk and back up sent to the PI on weekly basis. All the questions will be coded with an identifier to prevent patient identification. Blood samples will also be taken from all pregnant women who consent to participate in the study.

Data Collection instrument:

A self design structured pro-forma will be used to obtain demographic data, clinical information and risk factors associated with HBV infection from eligible participants in the study. The information will be obtained from each participant before collecting specimen for laboratory assessment.

Specimen Collection and Processing:

About 5ml of blood specimen will be collected by a laboratory Scientist by venipuncture from each eligible study participants and umbilical cord blood sample for the new born (mother-baby pair) between January and December 2017, using disposable sterile vacutainer needle and a 5ml vacutainer EDTA (ethylene-diamine tetra-acetic acid which is an anticoagulant) bottle. The plasma will be carefully aspirated into cryovials following centrifugation for 5 minutes and then it will be stored at -4 ^oc. until analysis is carried out. Subsequently, the sera will be stored at -80 ^oc. for future use in research activities.

Detection of HBsAg Using First Response HBV Card:

The specimen will be tested for HBsAg using the first response test card. The test device will be removed from the foil pouch and labeled with the specimen identifier. Twenty-five (25) microliter of plasma will be placed into specimen pot of the cassette. The reading will be taken at 10 minutes (a single red/pink band at the control region indicates a negative result, while two bands each at the control and the patients’ region indicates a positive result, a single band at the patients’ region indicates an invalid test and will need to be repeated).

Analysis of specimen for HIV:

The blood specimen collected will be tested for antibodies to HIV 1 and 2 using the Determine test kits and the HIV positive specimen will be retested using Unigold test kits all in accordance to the manufacturers’ instructions.

Detection of Other Markers of HBV Infection:

The other markers of HBV include hepatitis B surface antibody (anti-HBs), hepatitis B envelope antigen (HBeAg), hepatitis B envelope antibody (anti-HBe), and hepatitis B core antibody (anti-HBc). All specimens that will be reactive to HBV will be further analyzed to detect other markers of HBV infection. The test kit will be removed from the pouch and labeled with the specimen identifier. Four (4) drops of plasma will then be placed in each of the specimen pot. The reading will be taken after 15 minutes {(colored bands appearing in both the control and test region indicates a positive result while only one colored band in the control region and no band in the control region indicates a positive, negative and an invalid result respectively for HBsAg, anti-HBs and HBeAg) (a distinct control colored band appearing in the control region indicates a positive result, two colored bands in the control and test regions indicates a negative result, no band in the control region means an invalid result for HBeAb and HBcAb)}.

HBV-DNA Determination:

HBV DNA quantification will be performed using the COBAS AmpliPre- COBAS Taq-Man HBV test (Roche Diagnostic). Viral loads above the linear range will be determined by dilution, as recommended by the manufacturer. This will be use as determinant of vertical transmission of HBV.

For HBsAg positive mothers, HBV DNA will be extracted from the sera of each mother- and- baby pair using QIAamp DNA mini kit (QIAGEN) and the same could be conducted for the babies in the feature for molecular epidemiology research.

Vaccination of Babies:

Active vaccination will be given to babies according to the Nigerian National Immunization Schedule. The first dose at birth, and the subsequent doses at 2, 4, and 6 months of age to complete the immunization. The level of vaccination will be assessed for each child using the Child Health Records.

Data Analysis:

The data will be analyzed using STATA 14 software. Analysis of frequencies and means of continuous variables will be conducted. A p-value less than 0.05 will be considered statistically significant. All statistical tests will be two-sided and Chi-square tests will be conducted among groups to compare their differences. Odds ratio (OR) and 95% Confidence Interval (CI) will be calculated based on logistic regression models.                                                                                                                     

Benefit of the study to the participants and the community:

What is known from the literature is that the use of certain antiretroviral drugs in PMTCT has benefits in patients with HBV infection as it reduces the viral load thereby reducing vertical transmission. Little is known about the HBV vertical transmission in our setting for both those benefitting from ART and those not benefitting any intervention beside vaccination that is common to both groups. The study will help us document the burden of HBV among HIV infected and non HIV infected pregnant women in Jos and also help assess the prevalence of HBV MTCT in Jos and provide information on the risk factors for HBV MTCT in Jos.

The study will also help in the future how to identify HBV infected pregnant women and their infants who require not only prophylaxis, but who might need ART treatment as well as tracing sexual and household contacts benefit from testing, counseling, vaccination or therapy if indicated.

The information will be useful in planning further grants on strategies to eliminate HBV vertical transmission in resource poor settings as an integral strategy for the Sustainable Development Goals agenda on Health.

The research outcome will pave way for subsequent studies involving HBV molecular epidemiology implicated in mother-to-child transmission in our settings.

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Plan for Protection of Human Subjects:

Written informed consents will be obtained from study participants prior to recruitment. All information obtained will be kept confidential and use for publications. However, women who declined consent to participate in the study will be provided care like any other woman according to available and established standard guidelines for HIV and HBV infection in pregnancy.

Consent for Storage of Serum:

Consent will also be obtained from study participants for storage of serum to be used in further research in future.

Consent for Infant Participants:

A separate permission and consent will be obtained from the Parents of the infant of the infant to participate in the study. Decline of consent from parents will not affect their vaccination services and other child welfare and growth clinic activities.

Project Mentor: Prof. AI Zoakah; Department of Community Medicine, University of Jos.

Pending IRB Approval:

Approval for the study will be obtained from the Health Research Ethical Review Committees of JUTH, PSSH, and OLA. Separate written applications for approval in the three selected study facilities to conduct the study will be followed.