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Original research
Antitetanus toxoid antibodies in mothers and neonates: a single-centre study from Uganda
  1. Nicholas Mugagga1,
  2. Bernard Ssentalo Bagaya2,3,
  3. Mary Nantongo2,
  4. Fahad Muwanda2,
  5. David Mukunya4,5,
  6. Milton W Musaba6,
  7. Annette Olivia Nakimuli1,
  8. Moses Musooko1,
  9. Musa Sekikubo7
  1. 1 Obstetrics and Gynaecology, Makerere University College of Health Sciences, Kampala, Uganda
  2. 2 Immunology and Molecular Biology, Makerere University College of Health Sciences, Kampala, Uganda
  3. 3 Department of Research and Innovation, BMK Medical Laboratory Services, Mityana, Uganda
  4. 4 Community and Public Health, Busitema University, Mbale, Uganda
  5. 5 Department of Research, Nikao Medical Center, Kampala, Uganda
  6. 6 Department of Obstetrics and Gynaecology, Busitema University, Mbale, Uganda
  7. 7 Obstetrics and Gynaecology, College of Health Sciences, Makerere University, Kampala, Uganda
  1. Correspondence to Dr Bernard Ssentalo Bagaya; bernard.bagaya{at}


Background Neonatal mortality due to tetanus persists in Uganda despite the mandatory vaccination of pregnant mothers. Maternal antibodies wane within a year. Uganda’s maternal vaccination guidelines do not specify the timing or frequency of tetanus shots, contributing to suboptimal transfer of tetanus antibodies to neonates. We aimed to determine the prevalence and factors associated with protective tetanus antibodies among newborns at Kawempe National Referral Hospital.

Methods We conducted a cross-sectional study among 293 mother-newborn pairs. At delivery, neonatal cord and maternal venous blood were collected and titred for antitetanus antibodies using a quantitative ELISA kit. The primary outcome of the study was the proportion of newborn babies with tetanus antibodies ≥0.1 IU/mL. Associated factors were determined using generalised linear models for the Poisson family with a log link and robust variance estimation.

Results A total of 258/293 (88.1%) newborns had protective antibody titres. Factors associated with adequate protective antibodies in the newborn included: high (≥0.1 IU/mL) maternal antibody titres, first antenatal visit ≥12 weeks of gestation and receiving a tetanus toxoid (TT) shot ≥28 weeks of gestation. However, number of doses received before current pregnancy was not associated with adequate protective antibody titres.

Conclusion There is a high prevalence of adequate protective levels of antibodies among TT-vaccinated mothers. Maternal titres and a third trimester TT dose correlate with adequate levels of protective anti-TT antibodies among newborns. A third trimester TT dose is recommended.

  • neonatology
  • epidemiology
  • health services research
  • microbiology

Data availability statement

Data are available on reasonable request. Interested researchers may submit queries related to data access to corresponding author.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See:

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What is already known on this topic

  • In tetanus-endemic regions including sub-Saharan Africa, mortality due to neonatal tetanus is 80%–100%.

  • Uganda gives up to five doses of TT shots for women aged 15–49 years, with an assumption of full mother/neonate protection at that milestone.

What this study adds

  • This study shows an association between third trimester TT dose and adequate antibody levels in the newborn.

How this study might affect research, practice or policy

  • The findings of this study should serve as foundational research for empirical studies on appropriate timing of TT vaccination in pregnancy.

  • Researchers, practioners, and policy makers should undertake cost-effectiveness studies that compare one last trimester TT vaccination to the current regimen of multiple vaccinations throughout pregnancy.


Neonatal tetanus has an 80%–100% case fatality1 but has been eliminated in most of the high-income and middle-income countries through maternal vaccination.2 3 Every year 34 000 neonates, mostly from low-income and middle-income countries, die from neonatal tetanus.4 5 In 2018, 45 of 59 priority countries, Uganda inclusive, were validated by WHO as having achieved maternal and neonatal tetanus (MNT) elimination. Relentless implementation of MNT elimination strategies like hygienic childbirth, cord care practices, skilled birth attendance and maternal immunisation programmes need to be maintained and strengthened.6 7

Immunisation during pregnancy elicits antitetanus antibodies that protect the mother and the neonates through placental transfer of IgG.8 9 Antitoxin antibody titres of 0.1–0.15 IU/mL are considered protective10 11 but neonates born to mothers with suboptimal levels are at risk of death due to neonatal tetanus.12 Placental transfer of antibodies is a dynamic process beginning around week 17 of gestation,13 14 with efficiency remaining poor until 32–34 weeks of gestation. Efficiency of placental transfer is dependent on maternal antibody levels,15 placental function, maternal co-infections, IgG subclass16 and timing of vaccination. The Advisory Committee on Immunization Practices (ACIP) recommends that women receive a dose of tetanus toxoid (TT) during the third trimester, between weeks 27 and 36 of gestation in order to provide adequate protection to neonates.17 Vaccination during the third trimester provides the highest level of transferable antibodies to the neonates. Although WHO does not recommend routine adult booster vaccination for tetanus after completion of childhood vaccination series, many countries including Uganda continue to provide up to five routine booster vaccinations to females of reproductive age (15–49 years).18 However, this means that mothers receive vaccination before they are pregnant, and in their early pregnancy, often at the first antenatal care (ANC) contact, periods at which the placenta may not be able to transfer protective antibodies to the fetus adequately.15 Equally, prevalent maternal poor nutrition and infectious diseases burden may synergistically affect vaccine efficacy. We aimed to determine the prevalence of and factors associated with protective tetanus antibodies among newborns at Kawempe National Referral Hospital (KNRH).

Materials and methods

Study design and setting

We conducted a cross-sectional study at KNRH from 1 February to 31 March 2020. KNRH is a teaching hospital for Makerere University School of Medicine. Specimen processing and experiments were performed at the Immunology Laboratory of the Department of Immunology and Molecular Biology, Makerere University College of Health Sciences.

Study setting

Uganda is one of the countries that administers booster TT/tetanus diphtheria (TD) doses for girls and women of reproductive age (15–49 years)18 and TT/TD remains a mandatory vaccine given to all pregnant women attending ANC visits. KNRH is a level 5 hospital which has delivery rooms and operating theatres. Mothers in labour are triaged at admission and only mothers in active labour are admitted in the delivery rooms. Those that needed emergency caesarean sections are transferred to theatre for surgery. The hospital is about 6.4 km from the central business district. The hospital is open 24 hours, 7 days a week. An average of 70–100 mothers are delivered daily and 30 000 babies annually with a caesarean section rate of 22%.19 The mothers who deliver within this labour suite are often referred from lower-level facilities due to complicated/high-risk pregnancies or difficult deliveries.

Sample size and sampling

We calculated a sample size of 293 mother-baby pairs using the formula for sample size of a single proportion at a 5% level of significance. We assumed that 74.4% of babies had protective tetanus antibodies basing on a study from Nigeria.20 We approached every fifth mother recorded in the vertical maternity register for enrolment until sample size was reached. The first participant was chosen randomly from the first five mothers registered that day.

Eligibility criteria

The study enrolled mothers and their newborns delivered at KNRH who consented to study activities during the period of the study. We excluded all babies born before arrival to KNRH, as they already had their umbilical cords ligated and placentae detached. We also excluded mothers admitted in second stage of labour, mothers with intrauterine fetal deaths, mothers too sick to consent and mothers with significant mental disabilities.

Blood sample processing

Two millilitres of venous or cord blood was collected from the mothers and newborns at delivery. To collect the cord blood, the umbilical cord was clamped immediately after delivery of the baby and a needle was used by the obstetrician to collect the blood from the umbilicus. Blood was centrifuged at 1500× g for 15 min and two 0.5 mL aliquots made and stored frozen at −20°C until analysed.

Questionnaire administration

An interviewer-administered questionnaire was used to collect factors associated with maternal transfer of protective antitetanus antibodies, including sociodemographic characteristics of the mother, obstetric, neonatal factors, history of TD vaccination.

Antibody titration

Antitetanus antibody titres were determined using a commercial ELISA kit (MyBioSource, USA catalogue # MBS494626) following manufacturer’s instructions. Serum samples were assayed in duplicate, 100 µL/well and at 1:100 dilution. Plates were washed on an ELx50 plate washer (BioTek Instruments) while the plates were read on a SpectraMax Microplate Reader (Molecular Devices). All reagents and plates were supplied with the commercial kit. Mean optical densities (ODs) of the five standard samples were used to generate standard curves for each plate and mean ODs minus background of each test serum was used to determine the antibody titres of mothers and babies. The results were expressed as IU/mL.

Study variables

The primary outcome was the presence of a protetive level of anti-TT antibodies in umbilical blood, categorised as yes (≥ 0.1 IU/mL) and no (<0.1 IU/mL). The explanatory variables included factors that were associated with transfer of protective antibodies. Sociodemographic factors were age, level of education, marital status, history of previous vaccination. Medical factors were chronic illnesses such as HIV, hypertension (blood pressure >140/90 mm Hg), febrile illness (body temperature >37.5°C), diabetes, malaria in pregnancy. Obstetric factors included parity, gestational age, gestation age at vaccination, gestation age at delivery, birth weight, number of antenatal clinics attended. Biomedical factors were maternal tetanus antibody levels.

Data analysis

Data were summarised using means and SD for normally distributed data and median and IQR for skewed data. The prevalence of protective tetanus antibodies was determined as the proportion of newborns with protective anti-TT antibodies (>0.1 IU/mL). Data were entered into Epidata V.3.1 and exported to Stata V.17.0 for analysis. Factors associated with protective tetanus antibodies among newborns were analysed by generalised linear regression model for the Poisson family, with a log link and robust variance estimation. The strength of the association of the factors with levels of protective levels of anti-TT antibodies was determined from the crude prevalence ratio (cPR) from the regression analyses. The cPR was adjusted to adjusted prevalence ratio (aPR) by controlling for other independent variables in the regression re-analyses. We included all factors that are known to affect the anti-TT antibody levels in the newborn in the final model, based on biological plausibility.

Patient and public involvement

Patients and the public were not involved in the design and conduct of the study. Participants were individually informed of their TT antibody titres. A policy brief was prepared and shared with the Uganda National Expanded Programme on Immunisation, the body responsible for planning management of immunisations in the country.


Characteristic of study participants

We examined serum samples from 293 mother-baby pairs. Majority (185/293, 62.8%) of the mothers were aged 21–30 years with a median age of 25 years (IQR 21–30). More than 60% of mothers were at two or more parity and 22.9% (67/293) were at three or more parity. A half of the mothers had a secondary level of education (156/293, 53.2%). Almost all maternal participants (283/293, 96.6%) reported ever receiving a TT vaccine shot in their lifetime. Less than a half (123/293, 42%) had four or more ANC contacts during the current pregnancy despite WHO recommending eight contacts. HIV and diabetes occurred at a prevalence of 10.3% (30/293) and 2.44 (7/293), respectively among the mothers. Majority of the mothers (58.2%) did not have a source of income but close to 90% declared that they were married. The median birth weight was 3.2 kg (IQR 2.85–3.5) and 77% (225/293) of the neonates were born at term. Further characteristics are shown in table 1 and table 2.

Table 1

Sociodemographic characteristics of study participants

Table 2

Tetanus diphtheria (TD) vaccination characteristics among mother-infant pairs in Uganda

Protective antitetanus antibodies among newborns

The prevalence of protective levels of antitetanus antibody titres among neonates was 258/293 (88.1%: (95% CI 83.8 to 91.3)). The mean (SD) antibody titres were 3.37±2.32 IU/mL.

Factors associated with adequate levels of protective antibodies among newborns

Mothers with antitetanus antibodies ≥0.1 IU/mL were at 3 times as likely to have newborns with protective antibody titres (aPR 3.1, 95% CI 1.5 to 6.4) compared with mothers with antibodies <0.1 IU/mL. Newborns born to mothers who received their last TT/TD dose at 28 or more weeks of gestation were 1.1 times as likely to have protective antibody titres as newborns born to mothers who received their last TT/TD dose before 28 weeks of gestation (aPR 1.1, 95% CI 1.0 to 1.3). Newborns whose mothers received two or more TT/TD doses during current pregnancy were 1.1 times as likely to have protective antibody titres as newborns whose mothers received one or no TT/TD (table 3) (aPR 1.1, 95% CI 1.0 to 1.2). However, our results show no association of previous history of TD doses (before current pregnancy) with protective antibody levels among newborns irrespective of whether ≥5 (aPR 0.94, 95% CI 0.85 to 1.0) doses had been received before the current pregnancy. We observed comparable antitetanus antibody levels between neonates born to mothers aged 20 years and below and those of above 30 years, although neonates of mothers aged 21–30 yearsshowed a slightly elevated levels than both groups. The results of the findings are shown in table 3.

Table 3

Factors associated with adequate levels of protective antitetanus antibodies among newborns


We found a high (88%) prevalence of protective antibody titres among neonates born at KNRH. Uganda is one of the countries that administers booster TT/TD doses for girls and women of reproductive age (15–49 years)18 and TT/TD remains a mandatory vaccine given to all pregnant women attending ANC visits. This could explain the high prevalence of protective antibody titres among newborns in our study. This is supported by the fact that higher maternal antitetanus antibody titres were associated with presence of protective antibody titres among newborns. This finding has also been reported by Sangpetchsong et al, who show that mothers who had protective levels of tetanus antitoxin transferred protection to almost all of the newborns (97%–100%) and levels transferred correlated with maternal titres.21 22

Poor, inconsistent and/or late ANC attendance, sometimes past 28 weeks of gestation, is prevalent in Uganda,23 especially in mothers from low-income settings who deliver in public health facilities like KNRH where this study was conducted. Although the ACIP recommends a TD shot to pregnant mothers at 27–36 weeks of gestation irrespective of their past vaccination status,1 in practice expectant mothers in Uganda are given a TD shot at their earliest ANC visit, sometimes earlier than 12 weeks for fear that they may never return for additional ANC contacts. In the event that they return, a booster TD shot is administered again, sometimes completing the ideal two doses earlier than the recommended 27–36 weeks when the placenta is not well developed to ensure adequate transfer of antibodies to the fetus.14 Our study showed association between a TD dose at ≥28 weeks and protective levels of anti-TT antibodies among neonates, although the effect seen here was small. Nonetheless the result supports the ACIP recommendation of timing of TD shots in expectant mothers together with recommendations of several other similar studies.24 This finding is supported by data from another similar study in Texas, USA, which demonstrated that neonates of mothers immunised preconception or in early pregnancy had insufficient antibodies to protect against tetanus infection.25 Unfortunately, the Ministry of Health in Uganda has not yet adopted the ACIP guidelines as of 2021.

Our finding of comparable TD vaccine-specific antibody responses between HIV-infected and HIV-uninfected counterparts agrees with other studies which have also found protection rates of 80%–100% among HIV-infected TD vaccine recipients.26 Of surprise, however was our disagreement with another Ugandan study conducted among young males seeking safe male circumcision that found HIV-infected TD recipients had elicited poorer tetanus antibody responses.27 The difference between the genders enrolled in both studies perhaps might account for the observed differences. The fact that in Uganda TT/TD booster doses are administered to females as early as 15 years of age, but not to their male counterparts might also explain the discrepancy.

Our study demonstrated that vaccination received before the current pregnancy was not protective, regardless of the number of historical doses received. This is a very important finding in Uganda’s setting because it is widely presumed that completion of the immunisation schedule of 5 TT/TD doses between 15 and 49 years offers lifelong protection, which may not be the case in the context of our results. Our study findings agree with the most recent (2020) published ACIP general recommendation that pregnant women should receive one dose of TD during each pregnancy, irrespective of their history of previously receiving the vaccine.1 Nonetheless, in our context of Uganda, pregestation TD doses for the young girls and mothers may play a role in increasing the odds of boosting vaccine responses and the transplacental transfer of tetanus antibodies to the newborn in those who receive TT/TD vaccination during pregnancy.

Low birth weight was found to have an effect on levels of antitetanus antibodies supporting another West African study that also concluded that babies with low birth weight or those born prematurely28 tended to have lower antibody levels compared with their normal weight counterparts. This observation in part could be as a result of impaired maternal-to-neonate antibody transfer due to the fact that placental transfer of IgG occurs in an exponential fashion as pregnancy progresses, with minimal transfer in the first trimester and the highest transfer in the third trimester.8 29 30 Okoko et al demonstrated lower transplacental transport of IgG antibodies in the preterm neonates compared with term neonates.28 The nutritional status of the mothers could play a role in their immunological responses to vaccines, including TT/TD and subsequent transfer of antibodies to neonates,31 although in this study we never established the nutritional status of the mothers.

It was reassuring to find that regardless of maternal age, neonates had comparable antibodies against tetanus. It is known that performance of one’s immune system diminishes with age, including responses to adulthood vaccination, a situation that would impact umbilical maternal-to-neonate transfer of antibodies. Kugelman et al for instance reported an inverse relationship between maternal age with transfer of BNT162b2 messenger RNA COVID-19 vaccine-specific antibodies to neonates, with an unprecendented 2.7% reduction per 1 year increment in maternal age.32 This age-specific effect on maternal antibody levels, their transfer to and subsequently neonatal tetanus antibodies seems to be an exception as evidenced by our data. Our findings are supported by Oguti et al, who equally found no maternal-specific differences in anti-TD/TT antibodies or in the rate of anti-TT/TD antibody level half-life in mothers or their infants.33


Gestational age was difficult to assess reliably in our setting, especially since majority of the mothers did not have a first trimester ultrasound scan. It was hard to determine whether mothers truly had chronic illness like hypertension, diabetes, HIV, kidney disease since most mothers do not routinely go for check-up before pregnancy.

The CI was large due to a low sample size.


We found a high (93.9%) prevalence of protective levels of tetanus antibodies among mothers, with an 88.1% prevalence of transfer of protective tetanus antibodies to their neonates born at KNRH, Kampala, Uganda. Prevalence of protective levels of antibodies in newborns was associated and correlated with high levels of maternal antibodies, receiving TT/TD vaccine dose within the third trimester, the number of ANC contacts and birth weight of newborn. We also found that previous vaccinations received before the current pregnancy may not be protective to the newborn. Our finding add on the evidence that elicitation of strong antibody titres through booster TT/TD shots during pregnancy will remain a necessary requirement for control and elimination of neonatal tetanus infections as well as mortality.

We therefore recommend more studies with a larger sample size to ascertain the ideal timing for TD immunisation during pregnancy. Also, the notion that accumulate of five or more TD doses accords sufficient subsequent protection against tetanus infection should be discarded, and mothers given TD vaccination at every pregnancy.

Data availability statement

Data are available on reasonable request. Interested researchers may submit queries related to data access to corresponding author.

Ethics statements

Patient consent for publication

Ethics approval

The protocol was reviewed and approved by the School of Medicine Research Ethics Committee (SOMREC) (No. REC REF 2020-025) of Makerere University College of Health Sciences. All participants provided written informed consent.



  • Contributors Conceptualisation: NM, MM, MS, BSB. Methodology: NM, MM, MS, BSB, MN, FM. Data collection: NM, BSB, MN, FM. Software and analysis: NM, MN, MD, BSB. Writing—original draft: NM, MD, MM, MS, AON. Writing—review and editing: NM, MM, MD, MS, AON, BSB. BSB is the guarantor.

  • Competing interests No, there are no competing interests.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.