ANNALS OF IBADAN POSTGRADUATE MEDICINE
M.O. Fatokun1, O.O. Enabor1, F.A. Bello1, O.A. Adesina1 and G.O. Arinola2
Background: HIV infection affects millions of women and children, particularly in sub-Saharan Africa. Tetanus also causes significant maternal and neonatal morbidity and mortality in developing countries. Since the main effect of HIV is immunosuppression, there is potential for a negative influence on the host immune response to tetanus in women with HIV.
Objective: This case-control study evaluated the effect of HIV infection on maternal tetanus antibody production and neonatal tetanus antibody levels.
Methods: Thirty registered primigravidae were recruited from the clinic;15 were HIV positive and 15 were HIV negative. Serum samples of maternal and cord blood were obtained from both groups at delivery. Maternal total IgG and cord blood tetanus-specific antibody were estimated by Enzyme Linked Immunosorbent Assay.
Results: There was no significant difference in the total IgG level of HIV positive mothers compared with HIV negative mothers. No significant difference in the tetanus-specific IgG level in the cord blood of babies of HIV positive mothers compared with cord blood of babies of the HIV negative mothers.
Conclusion: HIV infection did not significantly reduce total IgG production in Nigerian primigravidae. Tetanus-specific IgG levels were above protective levels in neonates of HIV positive mothers suggesting adequate protection.
Keywords: Tetanus, Antibody, Pregnancy, Immunisation, HIV
Dr. F.A. Bello
Dept. of Obs. and Gynae,
University College Hospital,
The devastation of Human Immunodeficiency Virus (HIV) infection remains pronounced globally, particularly in sub-Saharan Africa where an estimated 25.8 million people including pregnant women are affected1. In 2010, antenatal client prevalence of HIV in Nigeria was 4.1%2. It was reported that 90% of paediatric HIV cases were due to mother-to-child transmission (MTCT) of HIV3.
Literature reports that the phenomena of inflammation and immunomodulation are involved either in HIV infection4 or during pregnancy5, which can affect vaccine response. Studies have highlighted the involvement of abnormal cellular and humoral immune responses during HIV infection. This includes abnormal pattern of serum protein electrophoresis, polyclonal hypergammaglobulinaemia, hyperproteine-mia and plasma cell dyscrasias6,7. In addition, impaired phagocytosis, reduced number and functions of T-lymphocytes were reported during pregnancy8. Despite maternal hypergammaglobulinaemia in pregnant HIV infected mothers, reduced IgG transplacental transference to the foetus has been reported6,9,10, which in turn, may influence vaccine response and neonatal immunity.
For the protection of mothers and babies, pregnant women in developing countries are usually vaccinated against tetanus. While no vaccine has yet been licenced for the prevention of HIV, maternal and neonatal tetanus prophylaxis has actively been pursued by the World Health Organization (WHO) since 198911,12. Newborn babies are protected from neonatal tetanus by maternal anti-tetanus antibody of the IgG class which is transplacentally transferred from third trimester of gestation following tetanus toxoid vaccination in pregnant women.
Immunization for pregnant women with tetanus toxoid vaccine is the single most effective strategy independent of other interventions in eliminating neonatal tetanus11,12. However, there are conflicting reports of the influence of maternal HIV infection on anti-tetanus antibody production by the mothers as well as its transfer through the placenta to their unborn babies. For instance, lower anti-tetanus antibody levels in HIV infected women were reported from Senegal13 and Brazil14, but not in The Gambia15. Cumberland et al.6 found reduced transplacental transfer of tetanus antibody, and about 50% lower antibody levels in cord serum. In contrast, De Moraes-Pinto et al. from Malawi found that maternal HIV infection had no effect on cord anti-tetanus IgG levels as well as transplacental transfer of anti-tetanus antibody10. In addition, seroprevalence studies have suggested that HIV-infected patients are less likely to have adequate anti-tetanus antibodies. The progressive decline of CD4 levels in HIV-infected individuals could potentially lead to lost immunity to tetanus16,17.
With the potential for HIV infection among women in the reproductive age group, and specifically pregnant women, it is necessary to evaluate the effect of HIV infection on the efficacy of tetanus toxoid in this group of people where tetanus toxoid vaccination is recommended. It is hypothesized that passively transferred immunity to the developing foetus by pregnant mothers will be affected by HIV infection. This study assessed tetanus-specific IgG levels of cord blood from babies born to HIV positive and negative primigravidae who received tetanus immunization.
MATERIALS AND METHODS
This was a case-control study conducted over four months at the labour ward of the University College Hospital (UCH), Ibadan, Nigeria. Ethical approval was obtained from the UI/UCH Ethical Committee (UI/EC/11/0128). The study group consisted of 15 HIV-infected pregnant mothers, while the control group consisted of age-matched HIV-negative pregnant mothers. Maternal HIV status is routinely determined at the antenatal booking visit. Following counselling, HIV infection is diagnosed using rapid screening tests and confirmed by Western Blot at the Virology Laboratory, College of Medicine, University of Ibadan, Ibadan, Nigeria. Only clients who had registered for antenatal care were recruited to ensure that their HIV status was known. All HIV positive clients are routinely placed on antiretroviral therapy (ARV) as part of antenatal care, in line with the protocol for prevention of maternal to child transmission of HIV. Patients were placed on Truvada (emtricitabine and tenofovir disoproxil fumarate) and efavirenz as from first presentation after 14 weeks of gestation.
Compliance was determined by the attending physician enquiring from the patient how many doses had been missed in the past one week. Patients who missed more than one dose were referred to adherence counsellors. The adherence counsellors would attempt to identify any reason for non-adherence. The patient would be assisted to develop a plan to improve adherence and compliance. Challenges and successes with plan would be reviewed periodically.
Consenting HIV-positive clients were recruited as participants, when admitted in active labour. On account of the anticipated small population, a convenience sample of consecutive clients were counselled until the sample size was complete. The next consenting, age-matched, HIV-negative client who presented in active labour (after each study group participant), was recruited as a control. Sample size was calculated based on the difference in antibody transfer between HIV-positive and -negative women obtained from a similar study18. Participants were enrolled based on documentation in the medical records of having received two doses of tetanus toxoid, the second one being at least four weeks before delivery.
A sample of 5 ml of maternal venous blood was obtained in a universal specimen bottle without anticoagulant. After delivery, the baby’s blood was collected after clamping and cutting the umbilical cord. Maternal and baby sera were separated from the whole blood after centrifugation and stored at -200C.
A data collection tool was used to collect information from the participants and their medical records. The following sections were included: bio-demographic data, HIV status, history of index pregnancy, labour, delivery parameters and history of tetanus toxoid administration. The babies’ Apgar scores, birth weights and other anthropometric parameters were also obtained.
Cord blood tetanus specific IgG and maternal total IgG were measured using Enzyme Linked Immunosorbent Assay (ELISA) method with EUROIMMUN (GMbh, Leubeck, Germany). A fixed volume per well of appropriate sample dilution buffer, antigen standard cocktail or an experimental sample was pipetted into microtitre plates. This sample was incubated at room temperature (250 C-270 C). The ELISA immunoplate was washed 3 times with 350µl/well of washing buffer. A concentration of 100µl of diluted Avidin-HRP conjugate was added, after which the plate was incubated for 30 minutes in darkness. The plate was washed four times and 100 µl per well of developing solution was added. The reaction was stopped with 100µl per well of stop solution and the Optical Density (OD) was read at a specific wavelength within 30 minutes of addition of stop solution. The average absorbance of each OD was plotted against corresponding cytokine values to create a standard curve. The average absorbance of each serum sample was used to determine corresponding cytokine values by interpolating from the curve.
The main outcome variable was the antenatal tetanus IgG production and transfer to babies of the participants. All data were collected, cleaned and manually entered into a computer. Analysis was done using the Statistical Package for Social Sciences (SPSS) version 17. Proportions between groups were compared using the ÷-square test with Fisher’s exact correction. Continuous variables were presented as mean±SD and differences between means compared using the students’ T-test. The level of significance was P<0.05.