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African Journal of Reproductive Health
Women's Health and Action Research Centre
ISSN: 1118-4841
Vol. 6, Num. 2, 2002, pp. 87-93

African Journal of Reproductive Health, Vol. 6, No. 2, August, 2002 pp. 87-93

Tetanus Immunity among Pregnant Women Attending Antenatal Care in Dar es Salaam, Tanzania

S Aboud1, EF Lyamuya2, EK Kristoffersen3 and R Matre2

1Department of Microbiology and Immunology, Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania, 2Department of Microbiology and Immunology, The Gade Institute, University of Bergen, and 3Blood Bank, Haukeland University Hospital, Bergen, Norway.
Correspondence: Dr S. Aboud, Department of Microbiology and Immunology, Muhimbili University College of Health Sciences, P.O. Box 65007, Dar es Salaam, Tanzania. Fax +255 222 152550 E-mail: saboud@muchs.ac.tz 

Code Number: rh02025

ABSTRACT

This study was conducted to investigate immunity to tetanus among pregnant women with verbal histories or documentation of having been vaccinated under the current five-dose tetanus toxoid (TT) schedule. It examined sera from 176 pregnant women attending antenatal care at Muhimbili Medical Centre in Dar es Salaam, Tanzania. Tetanus antitoxin level of 0.1IU/ml was considered protective. Our findings show that 94.9% of women had tetanus antitoxin > 0.1IU/ml. Multivariate analysis revealed that time after last vaccination, TT doses received and TT vaccination status explained 7.5%, 5.7% and 2.3% of variations in tetanus antitoxin levels respectively. Pregnant women with non-protective levels of tetanus antitoxin (5.1%) pose great risks of neonatal tetanus to their newborns and are also susceptible to maternal tetanus. Proper keeping of TT vaccination records is vitally important to avoid hyper-immunisation. (Afr J Reprod Health 2002; 6[2]: 87–93) 

RÉSUMÉ

Immunité contre le tétanus chez les femmes enceintes qui fréquentent les services de consultation prénatale à Dar-Es-Salam, Tanzanie. Nous avons mené cette enquête pour étudier l'immunité contre le tétanus chez les femmes enceintes dont l'histoire verbale ou les documentations montrent qu'elles ont été vaccinées selon le programme actuel de l'anatoxine tétanique à cinq doses (AT). L'étude a examiné des sérums de 176 femmes enceintes qui fréquentaient les services de consultation prénatale à Muhimbili Medical Centre à Dar-Es-Salam en Tanzanie. Le niveau de l'anatoxine tétanique de 0,1IU/ml, a été considéré comme étant protecteur. Nos résultats ont montré que 94,9% des femmes se sont faites vacciner contre le tétanus > 0,1IU/ml. L'analyse multifactorielle a révélé que pendant la période après la vaccination les doses de AT reçues et l'état de vaccination ont pu expliquer 7,5%, 5,7% et 2,3% des variations dans les niveaux de l'anatoxine tétanique respectivement. Les femmes enceintes qui n'ont pas atteint les niveaux protecteurs de l'anatoxine tétanique (5,1%) constituent des grands risques de tétanos aux nouveaux-nés. Elles sont aussi susceptibles au tétanos maternel. Pour éviter l'hyper-immunisation, il faut absolument que les documents concernant la vaccination de AT soient bien gardés. (Rev Afr Santé Reprod 2002; 6[2]:87–93)

KEY WORDS: Tetanus toxoid, immunisation, vaccination, neonatal tetanus

INTRODUCTION

In industrialised countries, the introduction of tetanus toxoid (TT) immunisation in the 1940s and 1950s led to a significant reduction in tetanus incidence, making it a rare disease.1 In 1996,  estimated deaths from neonatal tetanus (NT) were 438,080 with mortality rates of 4.8–10.7 per 1000 live births in high-risk developing countries.2 In 1997, WHO estimated the global number of NT cases to be four hundred thousand.3  Estimates of pregnant women immunised against tetanus (TT 2+) in developing countries were 48%.2 High coverage with TT has proved to be remarkably successful in preventing NT in many parts of the world.4-8

Immunisation programme has been integrated into maternal and child health (MCH) clinics in Tanzania since 1975. Diphtheria-pertussis-tetanus (DPT) doses are given to infants at 4, 8 and 12 weeks of age. Currently a five-dose TT schedule is administered to previously unimmunised women of childbearing age through MCH clinics, school health programmes or special outreach services as recommended by the Expanded Programme on Immunisation Global Advisory Group9 since 1987. In 1996, the estimated immunisation coverage of three DPT doses and TT 2+ for Dar es Salaam children under five years10 and pregnant women2 were 88% and 31% respectively. However, reported coverage for pregnant women is much lower than the expected despite  nationwide TT immunisation programme since 1975. This observation suggests either under-reporting or improper keeping of vaccination records. To date, NT still occurs in Tanzania. Deaths due to NT were 8% and 5% of all deaths of children under five years, with case fatality rates of 86.2% and 66.7% in 1996 and 1997 respectively.11 A recent study showed that the current DPT immunisation schedule provides adequate tetanus immunity for children under five years though about half of the 6–15-year-old children had no protection against tetanus.12 It was therefore considered of interest to conduct a sero-survey to investigate immunity to tetanus among pregnant women attending Muhimbili Medical Centre (MMC) antenatal clinic, with verbal histories or documentation of having been vaccinated under the current five-TT dose schedule in Dar es Salaam, Tanzania.

METHODS

We examined 176 apparently healthy pregnant women attending antenatal care at MMC in September 1999, with verbal histories or documentation of having been vaccinated under the current five-dose TT schedule in Dar es Salaam, Tanzania. Ethical clearance and informed consents were obtained in advance. After recording age and number of pregnancies conceived, history of vaccination with TT in current/previous pregnancy was obtained either from mothers or extracted from TT documentation held by the mother/available at the clinic. Blood samples were drawn; serum was separated after centrifugation, inactivated at 56oC for 30 min and stored at -20oC. All sera were then transported on dry ice from Dar es Salaam to Bergen, Norway, and were stored at -20oC until the time for assay.

Tetanus Antitoxin Assay  

Tetanus antitoxin level was determined by an antigen competition ELISA assay as described previously.13 The results of the assay correlate well with those of in-vivo neutralisation assay and they are reliably predictive below 0.16 IU/ml. Tetanus antitoxin level of 0.1 IU/ml was taken as protective.14 Briefly, TT at 0.75 LF/ml was bound to standard ELISA plates diluted in carbonate buffer pH 9.6 at 4o overnight. The plates were stored at 4oC for up to four weeks for subsequent use.

The following day, the plate was washed three times with phosphate-buffered saline with 0.05% Tween 20 (PBST) and was blocked with 100 µl of PBST and 2% bovine serum albumin (BSA) in each well. The plate was incubated at 37oC for one hour. After washing three times with PBST, twelve two-fold serial dilutions of each test serum were made in PBST beginning with 1:8. In parallel, there were equivalent dilutions of the test serum diluted in PBST-BSA and 0.1 LF/ml of TT. Similar dilutions of tetanus immunoglobulin 3.3IU/ml (WHO International Laboratory for Biological Standards, Copenhagen, Denmark) were added to the plate. Known positive and negative control sera were also included in every assay. The plate was incubated at 37oC for one hour. After washing three times with PBST, 100 µl of horseradish peroxidase goat anti-human IgG (Dako A/S, Copenhagen, Denmark) diluted 1/1000 in PBST was added to each well and incubated at 37oC for one hour. After three washings, 50µl of orthophenylene diamine (Dako A/S) was added to each well. Adding 50µl of 4N sulphuric acid stopped the reaction. The optical density was determined at 492nm in an ELISA plate reader (Emax, Sunnyvale, USA). A standard curve was constructed by plotting the average optical density of the duplicate for each dilution of the standard against its respective concentration. The lowest detection limit of the assay was 0.002 IU/ml. 

Statistical Analysis

The data obtained were analysed on SPSS version 9.0. Since tetanus antitoxin levels were skewed, log transformation of tetanus antitoxin levels was performed and geometric mean levels were determined. Student t-test was used to evaluate the differences in geometric mean tetanus antitoxin levels with respect to age, number of pregnancies conceived, number of TT doses received, and time after last TT vaccination. We determined correlation coefficient (r) between tetanus antitoxin level and time after last TT vaccination. We then performed multiple regression analysis (linear and stepwise) to study the independent contribution of number of pregnancies, TT vaccination status, TT doses received and time after vaccination to the prediction of tetanus anti-toxin levels. The goodness of fit of the linear model was assessed and expressed as adjusted multiple coefficient of determination (R2). The limits of the confidence in the prediction from a regression equation were determined by standard error (SE) of the estimate. The model was tested for statistical significance by F-ratio test. The effect of each independent variable was adjusted for the possibility of distorting influences from other independent variables and was expressed as standardised regression coefficient (ß). Multi-collinearity of independent variables was assessed by tolerance that when it was close to zero, there was a possibility of multi-collinearity. The alpha level for rejecting the null hypothesis was 0.05.

RESULTS

The median age of pregnant women was 25 years (range 15–41 years). Among participants, 58% had current pregnancies (Table 1). The mean number of pregnancies was 2.7 (range 1–9). Twenty three (13.1%) pregnant women gave verbal histories of TT vaccination and the rest had documentation (Table 2). Pregnant women who had been vaccinated with two TT doses were 38.7%, whereas 57.9% had 3–5 previous TT doses and 3.4% reported > 5 TT doses (range 2–8).

Three (1.7%) pregnant women had undetectable tetanus antitoxin (Table 2) and the levels were below that considered protective ( 0.1 IU/ml) in 6 (3.4%). There was significant difference in the distribution of tetanus antitoxin levels and TT doses between those with verbal histories and those with documented vaccination. Age had no significant effect on tetanus antitoxin levels but the levels measured were highest among 35–44-year-old women (Table 3). Women who had 2–4 pregnancies had significantly higher mean anti-toxin levels than those who had conceived once. Women who had 4–5 TT doses had significantly higher mean tetanus antitoxin levels than those with 2 TT doses.

The mean time after last TT vaccination was 12.7 months (range 1 month–8 years). Mean tetanus antitoxin levels were significantly lower two years after last TT vaccination. Time after last vaccination correlated significantly with tetanus antitoxin levels (r = -0.283, p < 0.01). Time after last vaccination was significantly associated with TT vaccination status (p  0.001) and number of TT doses received (p = 0.003). Long duration after last TT vaccination might be a risk factor for not retaining TT documentation and also affect the number of TT doses to be administered. After controlling for TT vaccination status and TT doses, time after vaccination still correlated significantly with tetanus antitoxin levels (r = -0.208, p = 0.006). In multiple regression analysis, time after last TT vaccination (ß = -0.221, p < 0.001), TT doses received (ß; = 0.262, p <0.001) and TT vaccination status (ß = 0.189, p = 0.020) predicted 7.5%, 5.7% and 2.3% respectively of the variations in tetanus antitoxin levels (F = 12.089, p < 0.001). Multi-collinearity was not observed.

DISCUSSION

Immunisation of women with TT during pregnancy provides protection to newborn babies against NT as well as maternal tetanus. With an increasing proportion of women immunised with TT, women would be immune and more infants would have protective levels of passively acquired tetanus antitoxin. The study has bias in the sampling method because all the pregnant women were users of MCH services and they were more likely to have been vaccinated with TT. In fact, all the pregnant women studied had verbal histories or documentation of having been vaccinated either in current or previous pregnancies under the current five-TT schedule.

Our study findings show that 3 (1.7%) pregnant women had undetectable tetanus antitoxin levels, and the levels were less than 0.1 IU/ml in 6 (3.4%). All these women (previously unvaccinated) except two had been vaccinated in the current pregnancy with only two TT doses at an interval of four weeks and the second TT dose was received one month prior to inclusion. These findings could probably be explained by inadequate immune response to two TT doses received by these women. These findings compare with those reported previously.16-17 It has been documented14 that optimal immune response is achieved when the intervals between the TT doses are long. The first three TT doses are basic, and in women who are not immunised the first dose should be given as early as possible during pregnancy. Unfortunately in Tanzania, like other developing countries, the pregnant women observed in our study reported to antenatal clinic when pregnancy was already advanced, hence the interval between the first and second dose of TT before delivery is too close for the mother to develop an immune response that will protect the newborn.

The interval between doses of TT given to pregnant women influences the level of tetanus antitoxin in cord serum as well. A previous study reported that the cord/maternal ratio of tetanus antibodies increases as the interval between the second dose and delivery is prolonged.18 In order to assure long intervals between TT doses, it is, therefore, necessary to start vaccination as early as possible in pregnancy. The proportion of protected women observed in our study is comparable to those reported elsewhere.17,19-22 The proportion of protection is much higher when compared to findings by other workers.23-27 Early introduction of TT immunisation programme and massive campaigns might have impacts on the observed tetanus immunity among pregnant women.

Our findings also show that pregnant women who had  two pregnancies had higher mean tetanus antitoxin levels than those who had conceived once. This is due to the fact that most women get vaccinated during pregnancy. As the number of pregnancy increases, the number of TT doses received by a woman increases. Targeting pregnant women for at least two TT doses is the most effective strategy. Our findings differ from those reported by other workers21,26 who observed no effect of the number of pregnancies on tetanus immunity level. Maybe the different immunisation schedules adopted could explain these different observations.

TT vaccination status was found to be a significant predictor for the variation in tetanus antitoxin levels. Vaccination status based on verbal history and not written documentation could lead to inaccurate immunisation history. Women who had no TT documentation could not retain the vaccination documents they were given and the records were also not available from antenatal clinic sources. Some studies reported association between lack of written TT documentation and non-protection.28-29 It has been reported that proper documentation of TT immunisation is essential because it is important in guiding the spacing of subsequent doses without hyper-immunising previously vaccinated individuals.14 This underscores the importance of proper keeping of vaccination records.

Number of TT doses received predicted 5.7% of tetanus antitoxin levels. The degree and duration of tetanus immunity increases with increasing number of TT doses, a finding that has been observed previously.19 However, women who reported to have been vaccinated with  more than five TT doses had relatively lower antitoxin levels than those who had 3–5 TT doses. Waning of tetanus antitoxin levels and possibly inappropriate vaccination schedule could explain the findings. Doses given at properly spaced intervals will not only achieve higher levels of antitoxin but also will prolong tetanus immunity.

Time after last TT vaccination explained 7.5% of the variation in tetanus antitoxin levels. This could be explained by waning of tetanus antitoxin that led to non-protective levels in some women. Following vaccination with five TT doses at recommended intervals, immunity to tetanus is expected to last for the whole childbearing period.14

In conclusion, 9 (5.1%) pregnant women with non-protective levels of tetanus antitoxin pose great risks of NT to their newborns and are also susceptible to maternal tetanus. Proper keeping of TT vaccination records and properly spaced intervals between doses are vitally important to identify pregnant women with inadequate vaccination and to avoid hyper-immunisation.

ACKNOWLEDGEMENTS

We extend our sincere thanks to all study participants. We would also like to acknowledge the sister-in-charge, nursing staff and laboratory technicians of MMC antenatal clinic, Dar es Salaam, Tanzania; Britt Edvardsen and Bente Heggø Hansen of Section of Immunology, Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen, Norway, for their invaluable contribution to this work. This study was supported by NUFU research project 44003, grant no. 42.2/91, Medical Microbiology and Immunology.

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