Vaccine 32 (2014) 2570–2573

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Antitoxins for diphtheria and tetanus decline more slowly after

vaccination with DTwP than with DTaP: A study in a Chinese
population
Yuyan Wu a,1 , Yuan Gao a,1 , Bingqing Zhu a , Haijian Zhou a , Zhenhua Shi b ,
Junsheng Wang b , Haipo Wang b , Zhujun Shao a,∗
a
National Institute for Communicable Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Chinese Center
for Disease Control and Prevention, and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, P.O. Box 5, Changping, Beijing
102206, PR China
b
Gaobeidian Center for Disease Control and Prevention, Gaobeidian, Hebei Province 074000, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: DTP vaccines are used for the prevention of pertussis, diphtheria and tetanus. In 2007, in
Received 5 December 2013 Gaobeidian city, China, the DTwP vaccine was replaced with DTaP. This study described the diphtheria
Received in revised form 7 March 2014 and tetanus sero-epidemiology in subjects vaccinated solely with DTwP or DTaP.
Accepted 13 March 2014
Methods: Blood samples were obtained between October 2012 and June 2013 from 587 healthy subjects
Available online 25 March 2014
aged 2–17 years. Serum IgG antibodies against diphtheria and tetanus were determined using ELISA.
Interrupted time series analyses examined the changes in antitoxin levels over time and analyzed the
Keywords:
alterations in diphtheria and tetanus antitoxin levels after the vaccine switch.
Diphtheria and tetanus
Cross-sectional study
Results: Mean concentrations of diphtheria antitoxin and tetanus antitoxin were 0.074 IU/ml (95% CI
Seroprevalence 0.065–0.084) and 0.063 IU/ml (95% CI 0.053–0.076). The protection rates (antitoxins >0.01 IU/ml) for
China diphtheria and tetanus were 88.25% and 82.11%. Mean antitoxin levels for both diphtheria and tetanus
decreased with increasing age, but this decrease was much slower for DTwP than DTaP.
Conclusions: Although the observed protection rates for diphtheria and tetanus were sufficient to prevent
an outbreak at present, the means levels of diphtheria and tetanus antitoxins decreased with increasing
age; therefore, booster vaccinations at 7 and 12 years of age would be strengthened in Gaobeidian city,
China.
© 2014 Published by Elsevier Ltd.

1. Introduction
Diphtheria and tetanus are both vaccine-preventable diseases,
and, in China, are reportable infectious diseases. The main vac-
cines used in the prevention of diphtheria, tetanus and pertussis are
DTwP or DTaP; the main components of these vaccines are diph-
theria toxoid and tetanus toxoid combined with either whole-cell
pertussis (DTwP) or acellular pertussis (DTaP). The basic vaccine
DTP has also been combined with Haemophilus influenzae type b or
inactivated polio virus to create multivalent conjugate vaccines. It
∗ Corresponding author at: 155, Changbai Road, National Institute for Commu-
nicable Disease Control and Prevention, Chinese Center for Disease Control and
Prevention, Changping, Beijing 102206, PR China. Tel.: +86 010 58900752.
E-mail address: shaozhujun@icdc.cn (Z. Shao).
1
These authors contributed equally to this study.
is known that DTaP vaccines have a lower efficacy than DTwP vac-
cines against pertussis [1,2], but little is known about the long-term
efficacy of DTaP against diphtheria and tetanus.
In China, both DTwP and DTaP vaccines were licensed by China
Food and Drug Administration (CFDA). DTaP vaccine in China was
produced with the method of immunoprecipitation [3]. Primary
vaccination of infants and young children consists of three doses
of DTP (DTwP or DTaP) in the 3rd, 4th and 5th months of life fol-
lowed by a fourth dose at 18–24 months, with Diphtheria-Tetanus
vaccine (DT) booster doses being recommended at 7 and 12 years.
DTwP was introduced in 1978 and was replaced by DTaP in early
2007 because of its potentially severe adverse reaction. In China,
primary vaccination with DTP (DTwP or DTaP) is mandatory, while
the DT booster doses are not. According to official estimates, since
2002 the immunization coverage rate achieved with three doses of
the DTP vaccination in childhood has been more than 90% [4]. In
2011 the immunization coverage of four doses was over 99% [5]. In

http://dx.doi.org/10.1016/j.vaccine.2014.03.052
0264-410X/© 2014 Published by Elsevier Ltd.
 Y. Wu et al. / Vaccine 32 (2014) 2570–2573 2571

Gaobeidian city and the first generation of children receiving solely Table 1
Geometric mean of diphtheria and tetanus antitoxin levels plotted against age.
DTaP as their primary vaccination is now aged 6 years.
Antibodies against diphtheria and tetanus toxins decline over Age Number Geometric mean of Geometric mean of
time. In children aged 5–9 years antibodies against diphtheria and of test diphtheria (IU/ml) tetanus (IU/ml)
tetanus are low if the booster dose is not given until they are 10 2 years old 4 0.125 0.361
years old [6–9]. However, an evaluation of the antitoxin against 3 years old 38 0.172 0.449
diphtheria and tetanus has not been carried out in Chinese chil- 4 years old 38 0.147 0.389
5 years old 20 0.086 0.357
dren and adolescents. After the introduction of DTaP, a world-wide
6 years old 63 0.04 0.046
efficacy evaluation of DTaP was conducted, but this focused only on 7 years old 35 0.037 0.055
the sero-conversion rate after vaccination [10,11]. In China, little is 11 years old 95 0.109 0.08
known about the long-term changes in antitoxins against diphthe- 12 years old 92 0.096 0.059
13 years old 45 0.044 0.021
ria and tetanus after the replacement of the DTwP vaccine with
14 years old 41 0.047 0.020
DTaP. 15 years old 39 0.066 0.033
This is a cross-sectional study that measured the levels of IgG 16 years old 48 0.048 0.025
antibodies against diphtheria and tetanus. The aim was to deter- 17 years old 29 0.071 0.039
mine the sero-epidemiology of diphtheria and tetanus among Total 587 0.074 0.063
children aged 2–6 years old and students aged 7–17 years old in
Gaobeidian city, China.
2.3. Statistical analysis

2. Patients and methods
2.1. Subjects
Serum samples were collected between October 2012 and June
2013. The samples used were residual specimens that had been col-
lected for another purpose from all kindergarten (aged 2–6 years),
primary school (7–11 years) and secondary school students (aged
12–17 years) in Gaobeidian city, China during regular physical
examinations, and would otherwise have been discarded. A ran-
dom set of samples were selected for each school. Students aged
8–10 years old were not available for this study.
Subjects were selected by stratified sampling using gender,
school and age. For school sampling, a proportional stratified
sampling method was used according to the size of the school.
All individuals received vaccines according to the immunization
schedule mentioned above and were asymptomatic upon enter-
ing the study. Individuals with any signs of respiratory disease or
immune-compromised conditions were excluded. In total, 587 sub-
jects (301 male, 286 female) aged 2–17 years (mean, 10.46 ± 4.29
years; median, 11 years) from kindergartens, primary and sec-
ondary schools were included in the analysis. Among the 587
children and students, two subjects (one girl and one boy) had
received one booster dose of DTP when they were 7 years old
and none of the subjects had received boosters at 12 years old.
Basic demographic and epidemiological data which were regularly
recorded in regular physical examinations, such as age, gender and
vaccination history, were recorded.
All data were recorded using Microsoft Excel 2007. Statistical
analysis was performed, and graphs created, using SPSS (version
16.0), SAS (version 9.2) and JMP (version 10.0). The prevalence of
diphtheria and tetanus antitoxins, geometric means and 95% con-
fidence intervals (CIs) were calculated. The prevalence rates were
compared using a chi-square test. A value of P < 0.05 was considered
statistically significant. The interrupted time series method was
used to analyze the differences in the changes in serum antitoxin
levels before and after the vaccine replacement; the intervention
point in the time series was the introduction of DTaP in 2007. A
cut-off point of 6 years was chosen, because children younger than
6 years old had received solely DTaP while students older than 11
years old had received solely DTwP. Two students who had received
booster doses of DTP at 7 years old and students aged 7 years old
(receiving both DTwP and DTaP) were excluded from the inter-
rupted time series analysis.
2.4. Ethics statement
Approval for the sero-survey was obtained from the National
Institute for Communicable Disease Control and Prevention.
Informed consents were not obtained as the data were analyzed
anonymously. However, we receive oral approval from the Center
of Disease Control and Prevention, Gaobeidian city.
3. Results
3.1. Antitoxins against diphtheria

2.2. Laboratory methods
The serum concentrations of diphtheria and tetanus IgG
antitoxins were tested quantitatively by commercial ELISA kits
for diphtheria IgG and commercial ELISA kits for tetanus IgG
(Serion ELISA classic, Institut Virion/Serion GmbH, Würzburg,
Germany) coating diphtheria toxoid and tetanus toxoid respec-
tively, according to the manufacturer’s instructions. Three levels
of immunity to diphtheria and tetanus were defined: diphtheria
and tetanus antibody levels of ≤0.01 IU/ml = ‘no immune pro-
tection or sero-negativity or susceptibility’; antibody levels of
0.011–0.099 IU/ml = ‘basic immunity or low sero-positivity or basic
protection’; ≥0.1 IU/ml = ‘full protection or sero-positivity’. A sub-
ject was considered to have ‘protection’ with antibody levels above
0.01 IU/ml, which includes both the basic and full protection cate-
gories above [12–14].
For the 587 subjects tested, the geometric mean of the diph-
theria antitoxin levels was 0.074 IU/ml (95% CI, 0.065–0.084). A
diphtheria antitoxin level giving full protection was found in 248
subjects (42.25%), while 270 (46.00%) had antitoxin levels giving
basic protection, and 69 (11.75%) subjects had low antitoxin levels,
giving no immune protection. The cross-sectional distribution of
the diphtheria geometric mean and protection rate plotted against
age is shown in Table 1 and Fig. 1. Prior to the cut-off point of 6
years old, the rates of full protection declined with increasing age,
and were at their lowest in subjects aged 6 years. In subjects aged
11 years and over, this reduction was not obvious, and the pro-
tection rate at 11 years was significantly higher than in subjects
at 6 years (98.79% vs. 77.78%, P = 0.001). The scatter distribution of
the log transformed diphtheria antitoxin levels plotted against age
is shown in Fig. 2, with regression lines calculated using an inter-
rupted time series method, with a cut-off point of 6 years. Fig. 2
shows that diphtheria antitoxin levels reduced more gradually after
2572 Y. Wu et al. / Vaccine 32 (2014) 2570–2573

Fig. 1. Cross-sectional distribution of diphtheria and tetanus antitoxin levels plotted against age. Susceptibility: diphtheria and tetanus antitoxin levels ≤0.01 IU/ml; basic
protection: diphtheria and tetanus antitoxin levels 0.011–0.099 IU/ml; full protection: diphtheria and tetanus antitoxin levels ≥0.1 IU/ml.

DTwP than after DTaP (0.0067 vs. 0.026), and the geometric mean
level of diphtheria antitoxins was higher at 11 years than at 6 years.
On the other hand, the results presented in Fig. 2 may indicate that
DTwP induced higher antitoxin levels than do DTaP, it declined over
time until age 13 when it leveled off. Because there was a more
rapid decline in antibody titer the first years after vaccination than
observed at later years, like which was found in pertussis toxin
[15,16].
with diphtheria antitoxins, the reduction in tetanus antitoxin levels
was more gradual for DTwP than for DTaP (0.079 vs. 0.0064).
According to the present study, a positive correlation was
observed between diphtheria antitoxin levels and tetanus anti-
toxin levels for DTaP (r = 0.727, P < 0.0001) and DTwP (r = 0.647,
P < 0.0001; data not shown).
4. Discussion

3.2. Antitoxins against tetanus
For the 587 subjects tested, the geometric mean of the tetanus
antitoxin levels was 0.063 IU/ml (95% CI, 0.053–0.076). Tetanus
antitoxin levels giving full protection were found in 248 subjects
(42.25%), while 234 (39.86%) had antitoxin levels giving basic pro-
tection, and 105 (17.89%) subjects had low antitoxin levels giving no
immune protection. The cross-sectional distribution of the tetanus
geometric mean and protection rate plotted against age is shown in
Table 1 and Fig. 1. Among the children under 6 years old, the low-
est full protection rate was observed in subjects aged 6 years old.
In subjects older than 11 years, the full protection rate remained
stable, but the protection rate at 11 years was significantly higher
than at 6 years (93.68% vs. 69.84%, P < 0.001). The scatter distribu-
tion of the log transformed tetanus antitoxin levels plotted against
age is shown in Fig. 2, with regression lines calculated using an
interrupted time series method, with a cut-off point of 6 years. As
The proportion of individuals susceptible to diphtheria with
antitoxin concentrations of ≤0.01 IU/ml varies widely from 0% in
Weifang city, China to 12% in the Netherlands, and 37.6% in the UK
[17–19], though the quality of different ELISA kits used in these
studies may be highly variable. In this study of Chinese subjects,
11.75% had no immune protection, with antitoxin concentrations
≤0.01 IU/ml. The variations between countries can be explained
by the different vaccination programs, and different study designs
and methods. However, the differences between the two Chinese
cities are likely to be caused by differences in the vaccination pro-
grams and the different population awareness levels. In Weifang
city, booster vaccines against diphtheria and tetanus were given
to individuals aged 7 years and also to adolescents entering their
first year at secondary school, and the vaccine coverage was more
than 95% [20]. In Gaobeidian city, however, booster vaccinations
at 7 years old and 12 years old are not mandatory and only two
of the enrolled students had received the booster vaccination. This

Fig. 2. Scatter distribution of log transformed diphtheria and tetanus antitoxin levels plotted against age, and with an interrupted time series regression models for antitoxins
against diphtheria and tetanus for DTwP and DTaP. GMC = geometric mean concentration; X = interrupted time (years) from the last primary vaccination dose (at 1 years).
 Y. Wu et al. / Vaccine 32 (2014) 2570–2573
low-level of uptake for the booster vaccinations might be due to
the low awareness levels in parents. The trend of protection rates
and GMCs for diphtheria antitoxin concentrations confirmed the
low booster coverage in subjects aged 7 years, because there was
no peak observed for these variables at 7 years. Thus, the booster
vaccination is important to reduce the numbers of susceptible indi-
viduals in the population. A threshold protection rate of 75% is
sufficient to prevent an outbreak of diphtheria, which implies that
the present observed protection rates of 84.66% (subjects aged 2–7
years) and 90.96% (subjects aged 11–17 years) is sufficient to pre-
vent an outbreak in Gaobeidian city [21]. However, the protection
rate at 7 years old was only 74.29%, and our data suggest that
the diphtheria antitoxin concentrations declined each year. Thus,
booster vaccinations should be strengthened to children and stu-
dents in Gaobeidian city.
In both subjects younger than 6 years or older than 11 years, we
observed a trend of waning immunity for diphtheria and tetanus
with increasing age, which has been reported widely elsewhere
[12,20–22]. The novel finding in this study was the observation of
a “rise” of the mean antitoxin levels and protective rates against
diphtheria and tetanus in subjects aged 11 years, even though only
two students had received booster vaccinations aged 7 years and no
subjects had received any further booster vaccinations. The results
of the interrupted time series analysis suggested that antitoxins
against diphtheria and tetanus declined more slowly after vaccina-
tion with DTwP than with DTaP. In this study, the protection rate
for diphtheria at 11 years (10 years after DTwP vaccination) was
significantly higher than that at 6 years (5 years after DTaP).
The positive correlation between diphtheria and tetanus for
DTwP and DTaP suggested that the antitoxins against diphtheria
and tetanus come from the same vaccine, and no other related
exposures were responsible for the improvement in antitoxin levels
against diphtheria and tetanus.
We are confident that the antitoxins against diphtheria and
tetanus measured in this study were present as a result of the DTP
vaccination program. In addition to DTwP or DTaP, a vaccine against
diphtheria alone and multivalent conjugate vaccines, which include
antitoxins against diphtheria or tetanus, are used as boosters for
army personnel or for people traveling abroad. None of these vac-
cine types had been used in the subjects included in our study. Also,
during the period of 2003–2010, fewer than 10 diphtheria cases had
been reported [20], and tetanus as a vaccine-preventable disease
was not communicable [12]. This means that the antitoxins against
diphtheria and tetanus in the subjects in this study are likely to be
present due to vaccination with DTwP or DTaP.
Students aged between 8 and 10 years were unavailable in the
present study, and we could not investigate the antitoxin levels of
the last group of children who received DTwP as their primary vac-
cination. Secondly, this was a cross-sectional study, and as such,
does not provide data on long-term comparisons of the effects of
DTwP and DTaP. It is possible that children aged 2–6 years were
exposed to different environmental factors after vaccination than
the students aged 11–17 years, such as better sanitary conditions,
better health resources, or reduced likelihood of exposure to cer-
tain pathogens. This model could be impacted by the influence of
confounding factors, such as individual differences among the age
groups. Thus, it would be useful to conduct a cohort study to mon-
itor the long-term trends for antitoxins levels for diphtheria and
tetanus after DTaP or DTwP vaccinations.
5. Conclusions
In Gaobeidian city, though the protection rate against diph-
theria is sufficient to prevent an outbreak at present, this study
has demonstrated that antitoxin concentrations decline each year;
2573
therefore, booster vaccinations against diphtheria and tetanus
should be strengthened. Although the levels of antitoxins against
diphtheria and tetanus decline with increasing age for both the
DTwP and DTaP vaccinations, this decline was much slower for
DTwP than DTaP.
Authors’ contributions: YY Wu and ZJ Shao planned the study.
YY Wu, Y Gao, ZH Shi, JS Wang, and HP Wang were in charge of
data collection. YY Wu carried out the immunoassays and per-
formed the statistical analysis. YY Wu drafted and edited the
manuscript. BQ Zhu, Yuan Gao and HJ Zhou participated in data
analysis. All authors read and approved the final manuscript. Con-
flict of interests: The authors declare that they have no conflict of
interests.
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