Original Research
published: 19 March 2018
doi: 10.3389/fpubh.2018.00079

E

Peter Aaby1,2*, Søren Wengel Mogensen1, Amabelia Rodrigues1 and Christine S. Benn2,31Bandim Health Project, Indepth Network, Bissau, Guinea-Bissau, 2 Research Centre for Vitamins and Vaccines (CVIVA),
Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark, 3 OPEN, Institute of Clinical Research, Universityof Southern Denmark, Odense University Hospital, Odense, Denmark

Edited by:
Rumen Stefanov,
Plovdiv Medical University,
Bulgaria
Reviewed by:
Camille Locht,
INSERM, France
Marisa Theresa Gilles,
Western Australian Center for
Rural Health (WACRH), Australia*Correspondence:Peter Aaby
p.aaby@bandim.org
Specialty section:
This article was submitted to
Public Health Policy,
a section of the journal
Frontiers in Public HealthReceived: 01 August 2017
Accepted: 02 March 2018
Published: 19 March 2018
Citation:
Aaby P, Mogensen SW, Rodrigues A
and Benn CS (2018) Evidence ofIncrease in Mortality After the
Introduction of Diphtheria–Tetanus–
Pertussis Vaccine to Children Aged
6–35 Months in Guinea-Bissau:
A Time for Reflection?
Front. Public Health 6:79.
doi: 10.3389/fpubh.2018.00079

Background: Whole-cell diphtheria–tetanus–pertussis (DTP) and oral polio vaccine(OPV) were introduced to children in Guinea-Bissau in 1981. We previously reported
that DTP in the target age group from 3 to 5 months of age was associated with higher
overall mortality. DTP and OPV were also given to older children and in this study wetested the effect on mortality in children aged 6–35 months.
Methods: In the 1980s, the suburb Bandim in the capital of Guinea-Bissau wasfollowed with demographic surveillance and tri-monthly weighing sessions for children under 3 years of age. From June 1981, routine vaccinations were offered at theweighing sessions. We calculated mortality hazard ratio (HR) for DTP-vaccinated andDTP-unvaccinated children aged 6–35 months using Cox proportional hazard models.
Including this study, the introduction of DTP vaccine and child mortality has been studied
in three studies; we made a meta-estimate of these studies.
results: At the first weighing session after the introduction of vaccines, 6–35-monthold children who received DTP vaccination had better weight-for-age z-scores (WAZ)
than children who did not receive DTP; one unit increase in WAZ was associated withan odds ratio of 1.32 (95% CI = 1.13–1.55) for receiving DTP vaccination. Though
lower mortality compared with not being DTP-vaccinated was, therefore, expected,
DTP vaccination was associated with a non-significant trend in the opposite direction,
the HR being 2.22 (0.82–6.04) adjusted for WAZ. In a sensitivity analysis, including all children weighed at least once before the vaccination program started, DTP(±OPV) as the most recent vaccination compared with live vaccines or no vaccine
was associated with a HR of 1.89 (1.00–3.55). In the three studies of the introductionof DTP in rural and urban Guinea-Bissau, DTP-vaccinated children had an HR of
2.14 (1.42–3.23) compared to DTP-unvaccinated children; this effect was separately
significant for girls [HR = 2.60 (1.57–4.32)], but not for boys [HR = 1.71 (0.99–2.93)](test for interaction p = 0.27).

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Keywords: bias in vaccine studies, diphtheria–tetanus–pertussis vaccine, heterologous effects, measles vaccine,non-specific effects of vaccines, oral polio vaccine

KEY OBSERVATIONS

children. Other reasons for not being vaccinated were that thechildren were temporarily traveling, or that they stayed for
prolonged periods in the rural areas where access to health care
was limited and child mortality was higher. Thus, apart from thespecific disease-protective effect of DTP, inherent biases would
lead one to expect that DTP-vaccinated children had better
survival than DTP-unvaccinated children.

• DTP and oral polio vaccine (OPV) were first introduced tochildren aged 6–35 months in June 1981 in an urban areain Guinea-Bissau. Children who were DTP-vaccinated at
the first weighing session after the introduction of DTP hadsignificantly better weight-for-age z-scores than those notvaccinated.
• Although better survival was expected, the DTP-vaccinatedchildren had twofold higher mortality than DTP-unvaccinatedchildren.
• In a meta-analysis of the three studies of the introductionof DTP in urban and rural Guinea-Bissau, DTP-vaccinatedchildren had twofold higher mortality than DTP-unvaccinatedchildren.

MATERIALS AND METHODS
Background

Bandim Health Project (BHP) has followed an urban community
in the capital of Guinea-Bissau with a demographic surveillance system since December 1978. The national immunization
program in Guinea-Bissau started in 1986 with funding from
UNICEF. From January 1980, BHP conducted tri-monthly
weighing sessions of all children in the community to identify
malnourished children. From June 1981, vaccinations were
offered in connection with these weighing sessions.

INTRODUCTION
Whole-cell diphtheria–tetanus–pertussis (DTP) vaccine is themost commonly used vaccine in low-income countries withpoor health infrastructure, and the coverage for the third doseof DTP-containing vaccines (DTP3) is the main performanceindicator for vaccination programs (1). However, no prospective
study has shown that receiving DTP is associated with betterchild survival (2, 3). On the contrary, in the past 20 years several
studies have suggested that DTP is associated with increased
child mortality, particularly for girls (2–4).
We recently examined what happened when DTP and oral
polio vaccine (OPV) were introduced to infants aged 3–5 months
in Guinea-Bissau in June 1981 in connection with tri-monthly
weighing sessions in an urban community in Bandim (5). In
this age group, the child’s date of birth determined whether achild was vaccinated early or late. Children who were just over
3 months old at the time of the tri-monthly weighing sessions
were vaccinated at that age; those who were just below 3 months
old would only be vaccinated for the first time at almost 6 monthsof age. In this “natural experiment,” DTP-vaccinated children had
fivefold higher mortality between 3 and 6 months of age thanchildren not yet vaccinated with DTP (5).
When we initiated vaccination with DTP and OPV in
Guinea-Bissau in June 1981, we also offered a catch-up program
to children aged 6–35 months attending the weighing sessions.
This situation provides an opportunity to compare the survival ofolder DTP-vaccinated and DTP-unvaccinated children.
In principle, children above 3 months of age attending theweighing sessions were offered vaccination if vaccines andequipment (syringes, sterilization stove) were available. However,nurses and mothers were reluctant to vaccinate sick or weak

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Demographic Surveillance

When the project started in 1978, child mortality was very high.
Malnutrition was assumed to be the main cause and a study was,therefore, initiated to determine why children were malnourished (6–8). The area was mapped and a census was conducted(5). Four female health workers identified pregnant women,
encouraged women to attend the antenatal clinic in the study
area, and followed children with anthropometric measurements
to assess growth patterns and detect malnourished children.
Each health worker followed a population of 1,500–2,000 individuals, the total number of individuals in Bandim being around
6,300 at the beginning of the study. The health worker kept a listof children under 3 years of age in each of the eight sub-districts
in Bandim. An expatriate nurse supervised the health workers.
BHP had no computerized surveillance system when the study
started, but BHP kept an A5 card (“BHP card”) for each child,
where weights and vaccination dates were noted. With a birthrate around 5%, the annual birth cohort was around 300–350
newborns.
The Bandim population was very mobile for many reasons.
First, it was important to maintain contact with the natal village for ceremonial purposes and to secure access to rice, oftenby helping the family during the rice production cycle. Second,
many women tried to obtain cash income by growing fruits orvegetables in the rural areas or by producing cashew wine to be
sold in Bissau. Third, mothers were not supposed to have sexual
relations during breastfeeding as semen is believed to damage
breastmilk causing diarrhea in the child (9). Breastfeeding

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was prolonged in Guinea-Bissau, between 18 and 36 months
in different ethnic groups. Thus, many women preferred tostay in the rural areas with their family while breastfeeding.
These cultural patterns meant that some mothers and children
were away for long periods. Typically, there would be family
members in Bandim, who we could ask about the whereaboutsof the child.

Tri-Monthly Weighing Sessions

We arranged tri-monthly weighing sessions in each sub-district(8). The health worker in charge advised mothers the day before asession. If a child was not present, its vital status was ascertainedby asking the family. The following morning, the child’s weight
was measured on a hanging Salter scale and noted on the child’s
health card and the BHP card.

Vaccinations

There was no community vaccination program in Guinea-Bissau
when BHP started vaccinations. Mother could have taken theirchildren to the clinic of the Mother and Child Health Program intown. This clinic was mainly attended by the urban elite so very
few children from Bandim had received routine vaccinations(5). In June 1981, BHP started to provide vaccinations at the trimonthly weighing sessions. A health center nurse accompanied
the nutrition team and vaccinated eligible children.
Eligible children were between 3 months and 3 years of theage. However, some children in this age group were not vaccinated. Both nurses and mothers thought that sick or otherwise
weak children should not be vaccinated. The BHP card often
indicated that the child was “sick,” “malnourished,” or “orphan”
as an explanation of why an age-eligible child had not been vaccinated. Other reasons for not vaccinating an age-eligible childwere temporary shortages of vaccines or syringes.
The three DTP and OPV doses could be given from 3 monthsof age with an interval of 1 month, but since we only performed
weighing sessions every 3 months, most children had longer
intervals between the three doses. Also, there were several periods where either OPV or DTP was missing [Ref. (5), Figure 1].
The expatriate nurse sometimes organized additional vaccination
sessions in which the children were not weighed, but vaccinations
were noted on the BHP cards.

Figure 1 | Flowchart of study population and children included in theanalyses. DOB, date of birth; [], died during follow-up. Children were only
included in the main analysis if they had taken part in a weighing session
after October 1, 1980.

mortality was very high (10). Children who never attended aweighing session after birth registration were not included in theanalysis, since their mothers had likely left for the rural areas. In
the analyses, we restricted the data set to children taking part in atleast one weighing session after October 1980, 8–9 months before
vaccinations started. This was done to assure that the children had
been seen fairly recently and were, therefore, likely to be aroundwhen the vaccinations started. Since the children were called
every 3 months, the time of death or migration out of the area isfairly accurate.

Vaccination Analyses

Data Control

We conducted three complementary analyses to assess the effectof DTP on child survival.

Weights and vaccinations from the BHP cards were entered into
a computerized system in 1990–1991. For the present analysis,
information on dates of visit, weights, and vaccination dates waschecked against the original cards in 2015.

Analysis 1

We compared DTP-vaccinated children and those who were
not DTP-vaccinated when they came for their first weighing
session after the introduction of vaccinations in June 1981.
Since not all children were included, the analysis had lesspower. We followed children from their first weighing session
and until they received their next vaccination or they migrated,
died, or turned 3 years of age. Thus, children had to be present
at a weighing session to be included in this analysis and we
could adjust for the weight-for-age z-score (WAZ) obtained atthat session.

The Study Cohort and Vaccination
Analyses

We included children born between June 1978 and December
1980 and hence aged 6 and 35 months in June 1981 when DTP
and OPV vaccines became available (Figure 1). Furthermore, it
was an inclusion criterion that children were registered in the area
before the vaccinations started. We excluded orphans, since they
were not breastfed and were likely to have different care; their

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survival bias, we used a landmark approach in all analyses (11);hence, a child’s vaccination status was only updated from the day
the information was collected.

Analysis 2

In this analysis, children were considered DTP-vaccinatedfrom the date they received their first DTP vaccination (withor without OPV) in June 1981, or at one of the subsequent
weighing sessions (Figure 2). Children were considered DTPunvaccinated from the date vaccination was first offered intheir sub-district, irrespective of whether they were present at
the weighing session, and until they were DTP-vaccinated at
a subsequent session, migrated, died, or turned 3 years of age.
(The difference between this analysis and Analysis 1 was thatchildren were considered DTP-unvaccinated if they were ageeligible, irrespective of whether they had attended a weighing
session or not, and vaccination status could change duringfollow-up, so a child could contribute risk time first as DTPunvaccinated and then as DTP-vaccinated.)

Studies of the Introduction of DTP

Including this study, there are only three studies of the introduction of DTP, all from Guinea-Bissau (5, 12). We made a metaestimate for these studies, since they represent an unusual set ofcircumstances in relation to the discussion of potential biases instudies of the non-specific effects of vaccines (13–17). First, inall three studies the nutritional status was worse for children not
vaccinated. Second, we administered nearly all vaccines, so mostdates of vaccination were known precisely. Third, there were no
campaigns with other vaccines or micronutrient supplements
at the time of these studies. Fourth, they represent all the data
sets available on the introduction of DTP in Guinea-Bissau, soreporting bias is not an issue (15).

Analysis 3

In the third analysis, we compared mortality of children according to their most recent vaccination status; DTP-vaccinatedchildren were compared with children who had received novaccination or live vaccine only (MV, OPV, or MV + OPV) as
their most recent vaccination.

RESULTSOf the 890 children aged 6–35 months registered in Bandim in
June 1981, we were not able to locate the BHP card of 107 (12%)
children in 2015; most will not have attended an examination,
but some cards may have been lost. A further 81 had a BHP card,
but had not attended a weighing session since October 1980,
had no precise vaccination dates, or were excluded due to otherconsiderations (see Figure 1). Hence, 702 children were includedin the study cohort; the number of deaths and person-years in
the different vaccine groups was, therefore, limited (Table S1 in
Supplementary Material).
The temporal distribution of weighing sessions in thiscohort is shown in Figure 2. As documented in Table S2 in
Supplementary Material, 82 and 84% received DTP1 and OPV1
before they reached 3 years of age, the median ages of vaccination being 633 and 614 days, respectively. It should be notedthat only 38 and 49% of the children received all three doses of
DTP and OPV, respectively, before 3 years of age. Due to earlier
MV campaigns (6, 7), 82% had received MV at a median age of348 days. There were 42 deaths between 6 and 35 months of age;
14 had fever as the main symptom, 13 had diarrhea or diarrhea
and vomiting, 6 died from measles, 1 had respiratory infection,
1 was malnourished, 1 had anemia, 1 did not eat, and 5 had no
information, most likely because the mother/family had moved.
We compared background factors for DTP-vaccinated children and children who remained DTP-unvaccinated until at least
3 years of age (Table 1). The DTP-vaccinated children were far
more likely to have attended weighing sessions, to have received
measles vaccine (MV) in the campaigns, or to have received DTP
at the Mother and Child Clinic before June 1981 (6, 7). There were
no differences in distribution of the sexes, twins, or ethnic groups.

Statistical Analyses

The survival of different vaccination groups was compared usinga Cox proportional hazard model with age as underlying time.
Thus, age was inherently controlled in this analysis. The WHO
WAZ was used to assess nutritional status. In analysis 1 in which
we compared children who had attended weighing sessions andbeen vaccinated or not vaccinated we adjusted the analysis fornutritional status (WAZ score). Since we provided almost all
vaccines, most vaccinations were known from the date of vaccination, but a few children were vaccinated elsewhere. To avoid

Analysis 1

At the first weighing session after the vaccinations started in June
1981, the WAZ was much higher for the children who received
DTP (WAZ −0.83) than for those children who did not receive
DTP (WAZ −1.17) (Table 1). An increase of one z-score wasassociated with an odds ratio (OR) of 1.32 (95% CI = 1.13–1.55)

Figure 2 | Examinations are plotted on the axes of age and calendar time.
Each dot corresponds to a weighing examination of a child. The gray triangle
illustrates the age groups and periods, where follow-up time was included in
the survival analysis. The approximate tri-monthly regime of examinations isvisible in the distribution of dots on the horizontal axis.

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for being vaccinated at the first weighing session. Compared withnot being DTP-vaccinated, DTP vaccination at the first weighing
session was associated with a non-significant mortality hazard
ratio (aHR) of 2.22 (95% CI = 0.82–6.04) adjusted for WAZ(Table 2), the aHR being 7.03 (0.88–56.04) for girls, and 1.28
(0.38–4.25) for boys (test for interaction p = 0.17).

with children who had received a live vaccine or no vaccine
at all and had a HR of 1.90 (0.92–3.94) if compared only withchildren who had received live vaccine only (Table 4). In asensitivity analysis, including also the 47 children whose mostrecent weighing session had been before October 1980, theHRs for DTP was 1.89 (1.00–3.55) (Table 4), the HR being 2.76
(1.07–7.07) for girls, and 1.34 (0.56–3.22) for boys.
Though the group was small, it is worth noting that children who
received OPV-only had low mortality (Table S1 in Supplementary
Material), the HR for DTP(±OPV)-vaccinated compared with
OPV-only vaccinated children was 3.76 (0.89–15.83).

Analysis 2

Including all children in the cohort, following them to 3 yearsof age and allowing children to change status during follow-up
when new information was collected at a weighing session, having received DTP was associated with a non-significant HR of
1.48 (0.72–3.06) (Table 3). The HR was 2.91 (0.84–10.00) for girls
and 0.88 (0.34–2.62) for boys.

Studies of the Introduction of DTP

In the three studies of introduction of DTP in rural and urban
Guinea-Bissau, DTP vaccination was associated with a HR of
2.14 (1.42–3.23) compared with DTP-unvaccinated children(Figure 3). The negative effect was separately significant for girls[HR = 2.60 (1.57–4.32)], but not for boys [HR = 1.71 (0.99–2.93)](Table S3 in Supplementary Material) (test for interaction
p = 0.27).

Analysis 3Children who received DTP (with or without OPV) as the mostrecent vaccination had an HR of 1.77 (0.93–3.38) compared

Table 1 | Background factors for 6–35 months old children who were
vaccinated or not vaccinated at their first weighing session in June 1981.
Analysis 1

Diphtheria–tetanus–
pertussis (DTP)vaccinated at or
before first session

DTP-unvaccinatedin first
sessionMean weight-for-age z-score(SD) at first examination−0.83 (0.06) [394]¤−1.17 (0.08) [197]¤

Analysis 2

DTP-vaccinatedduringfollow-up

Not DTPvaccinatedduring follow-up

553
51% (282)
3% (15)149
53% (78)2% (3)

51% (282)
15% (84)
34% (187)
71% (391)

48% (71)
17% (25)
36% (53)
58% (86)

6% (32)
6% (33)
2.57 (0.06)#

5% (8)
0.91 (0.09)#

N
Male sexTwin
Ethnic group
Pepel
Balanta
Other ethnic groups
Measles vaccinated before
June 1981
DTP before June 1981
Classified as malnourished
Mean number (SD) of weighing
sessions per year after startof vaccinations#¤

DISCUSSION
Although lower mortality was expected for DTP-vaccinatedchildren compared with the frail unvaccinated children, DTP
vaccination was associated with higher mortality, particularly ingirls.

Strength and Weaknesses

The home-visits preceding each of the tri-monthly weighing sessions ensured that we had follow-up information for all children
and relatively accurate information on the time of movement ordeath. In the initial analyses, we included only children who had
attended the 3-monthly weighing sessions at least once within
the last 8–9 months (5). This meant that children mostly living
outside the area were not included; these children had no accessto community vaccinations and they lived elsewhere where
the mortality risk may well have been much higher. We had to
exclude some children because their card could not be found(Figure 1). The excluded groups (Figure 1) did not have highmortality so the increased mortality of DTP-vaccinated children
is not due to exclusion of unvaccinated children with a particularly high mortality. When we increased the power of the study
by including children only seen before October 1980 (Figure 1),

Comparison p < 0.0001.
Comparison p = 0.001.

Table 2 | Analysis 1: mortality rates (MR) per 100 person-years and hazard ratios (HR) of 6–35 months old children who were either diphtheria–tetanus–pertussis(DTP)-vaccinated or not DTP-vaccinated at their first examination.
Mortality rate (deaths/person-years)
Vaccination status
All children
Girls
Boys

DTP (±OPV)No DTP

9.68 (18/185.9) [394]11.15 (9/80.7) [191]8.58 (9/104.9) [202]4.80 (5/104.1) [197]1.86 (1/53.7) [100]8.07 (4/49.6) [96]HR (95% CI)

HR (95% CI), adjusted for weightfor-agez-scores (WAZ)

2.01 (0.74–5.41)
6.67 (0.84–52.84)
1.04 (0.32–3.40)

2.22 (0.82–6.04)
7.03 (0.88–56.04)
1.28 (0.38–4.25)

Bandim, 1981–1983.
Children who have received DTP before June 2, 1981 were censored from the analysis.

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the HR estimate for DTP-vaccinated versus DTP-unvaccinatedas most recent vaccine changed from 1.77 (0.93–3.38) to 1.89
(1.00–3.55).

The inherent biases in this study are clearly in favor of the
DTP-vaccinated children (2): first, the DTP vaccine protects
against three severe diseases. Second, the DTP-unvaccinatedchildren were usually children deemed too sick or too weak to be
vaccinated, as evidenced by the nurse’s notes on the BHP card andby the fact that these children had worse nutritional status. Third,
DTP-unvaccinated children attended the weighing sessions lessfrequently (Table 1) and were, therefore, more likely to be staying for longer periods in the rural areas where the mortality risk
was higher (12). Noteworthy, we were able to obtain mortality
information from these children because their father and otherrelatives stayed in the study area.
WHO experts have argued that the negative effect of DTP
is exaggerated, because studies have only been conductedin situations with herd immunity against pertussis and where
the benefit of preventing pertussis would not be seen (13).
However, pertussis was endemic in the 1980s before the roll

Table 3 | Analysis 2: mortality rates (MR) per 100 person-years and hazard
ratios (HR) of 6–35 months old DTP-vaccinated and diphtheria–tetanus–pertussis(DTP)-unvaccinated children.
Mortality rate (deaths/person-years)

All
Girls
Boys

DTP(±OPV)No DTP

5.4 (32/590.6) [553]
6.9 (19/273.5) [270]4.1 (13/316.5) [282]4.1 (10/242.5) [327]2.6 (3/116.4) [155]5.6 (7/125.1) [170]HR (95% CI)

1.48 (0.72–3.06)
2.91 (0.84–10.00)
0.88 (0.34–2.62)

Bandim, 1981–1983.
178 children were first DTP-unvaccinated and then received DTP during follow-up.
Three children had no information on sex. If we adjusted for the most recent WAZ
measurement, the HR of 1.48 (0.72–3.06) became 1.52 (0.74–3.15).

Table 4 | Analysis 3: mortality rates (MR) per 100 person-years and hazard ratios (HR) of 6–35 months old children in relation to most recent vaccination.
Mortality rate (deaths/person-years)
Vaccination status

Diphtheria–tetanus–
pertussis (DTP) (±OPV)No DTP

All
Vaccination status

6.2 (28/451.0) [535]DTP(±OPV)All

6.2 (28/451.0) [535]3.7 (14/382.0) [539]
Only live vaccine(MV, OPV, or MV + OPV)
3.3 (10/303.84) [473]

HR (95% CI)
Had weighing session after
October 1, 1980aHR (95% CI)
All childrenb

1.77 (0.93–3.38)

1.89 (1.00–3.55)

1.90 (0.92–3.94)

1.99 (0.96–4.12)

Bandim, 1981–1983.
a
See Figure 1; Adjustment for the most recent weight-for-age z-scores measurement did not change the estimate.
bInclude 47 children whose most recent weighing session prior to June 1981 had been before October 1980.

Figure 3 | Meta-analysis of the three studies of the introduction of diphtheria–tetanus–pertussis. The fixed effects model gave an estimate of 2.14 (1.42–3.23) andthe random effects model gave 2.17 (1.39–3.38).

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out of the vaccination program in Guinea-Bissau, but all threestudies of the introduction of DTP into urban and rural areasof Guinea-Bissau showed excess mortality associated with DTP
vaccination (5, 12).

children should, therefore, have a higher mortality than vaccinated children (2, 3). The WHO review mentioned four potential
biases, which would favor the unvaccinated group (15). First, sickchildren might come more often to a health center for consultation and, therefore, be more likely to receive DTP, since WHO
has recommended vaccination of sick children; this bias wasclearly not relevant in Guinea-Bissau, where neither nurses nor
mothers thought that a sick child should be vaccinated. Second,
starting follow-up from a survey sometime after the actual DTP
vaccinations had been administered, as would often happen in asetting, where vaccination information is collected with intervals,
could potentially mean that frail children in the unvaccinatedgroup had already died, and that the DTP-vaccinated children,
therefore, had an “unnaturally” high mortality (15). The one
study testing this found no evidence for such a bias (20) andmore importantly, several studies, including all three studies of
the introduction of DTP in Guinea-Bissau, started observation at
the date of vaccination for almost all children and found strong
negative effects. Hence, this bias was not relevant in the present
study. Third, censoring follow-up at subsequent MV would
remove some of the best children from the DTP-vaccinatedgroup and, therefore, gives higher mortality in the DTP group(15). Again, the studies that have tested this potential bias havenot found evidence for such a bias (21) but, more importantly,
several studies—like the present one—did not censor for MV
and found equally strong negative effects for DTP (2). Hence,
this bias was not relevant in this study. Fourth, it has been discussed whether a bias in reporting could have played a role (15).
The observation of increased mortality after DTP was reported
more than 15 years ago (22), and has not been contradicted by aproperly conducted prospective study. In contrast, several othergroups have reported that DTP was associated with increased
overall mortality (23–25) or higher female than male mortality(23, 26–28). Hence, reporting bias is a very unlikely explanation.
We have now reported all the possible data sets from when DTP
was introduced in both urban and rural areas of Guinea-Bissau(5, 12); all showed a negative effect of DTP vaccination. Hence,
reporting bias is not relevant in relation to the studies of the introduction of DTP from Guinea-Bissau. Therefore, the three studiesof the introduction of DTP from Guinea-Bissau (5, 12) are notaffected by the theoretical biases used to recommend caution in
the interpretation of observational studies suggesting deleterious
effects from DTP (15–17).
The specific immunological mechanisms explaining why
DTP and OPV have NSEs have not yet been identified. However,there is an increasing evidence that live vaccines (BCG, Vaccinia)
induce innate immune training producing stronger proinflammatory responses which may lead to protection against
unrelated infections (29, 30). In contrast, studies of non-live
vaccine have suggested that they may induce tolerance which
could enhance the susceptibility to unrelated infections (31). The
pattern of worse effects for females than for males have turned
out to be systematic for several non-live vaccines, including DTP(4, 26), inactivated polio vaccine (32), hepatitis B vaccine (33),pentavalent vaccine (34), and RTS,S malaria vaccine (35). Thispattern has not been studied from an immunological perspective
and an explanation has still to be found.

Comparison With Previous Studies
of DTP and OPVThis study was small (Table S1 in Supplementary Material) andmany results were not statistically significant; some variability
was, therefore, also to be expected. Still the results were very
similar to the previous studies of the introduction of DTP and
OPV. All three studies have accurate assessment of vaccination status and prospective follow-up; all three studies havefound that DTP is associated with an increase in all-cause
mortality (3). A previous meta-analysis suggested twofold
higher mortality for DTP-vaccinated children (3). However,this is probably an underestimation of the “true” effect since
the unvaccinated group is usually affected by various negative health selection biases, including frailty bias. In the beststudies, with no selection bias or good control for frailty bias,
DTP has been associated with four to five times higher mortality (2, 5).
As in this study, we have previously found excess female
mortality after DTP vaccination (4). In our previous metaanalysis, we compared DTP-vaccinated females with DTPunvaccinated, but BCG-vaccinated females, and DTP was
associated with an HR of 2.54 (1.68–3.86). In the three studiesof the introduction of DTP very few of the DTP-unvaccinated
had received BCG. Hence, DTP seems to have a marked negative effect for females irrespective of whether one compares
DTP-vaccinated girls with totally unvaccinated or with BCGvaccinated girls.
There have been few studies of OPV administered alone (5).
It is, therefore, worth noting that the small number of chil­dren
who received OPV only had lower mortality than DTP-vaccinatedchildren in this study (Table S1 in Supplementary Material),
although the difference was not statistically significant. We haverecently been able to document marked beneficial effects of OPV
on all-cause mortality in both randomized trials and in naturalexperiments with OPV campaigns (18, 19).

Interpretation

Various WHO committees have previously reviewed the nonspecific effects of vaccines and have dismissed the possibility
that DTP could have negative effects, and have suggested that thenegative effect of DTP is likely to be explained by uncontrolled
confounding or bias (13–17). Recently, the Strategic Advisory
Group of Experts on Immunization sponsored a review of thepotential non-specific effects of BCG, DTP, and MV (15, 16).
Though it was noted that the majority of studies (7/10) showed adeleterious effect of DTP, the evidence was considered inconsistent because two studies showed a beneficial effect. Furthermore,
the review invoked “a high risk of bias” for all the observational
studies (17).
However, it is important to consider the direction of bias. All
documented biases favor the vaccinated group because vaccination
is usually delayed in unhealthy children, and DTP-unvaccinated
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Introduction of DTP to Children Aged 6–35 Months

Implications and Conclusion

TRANSPARENCY

Our data clearly showed that DTP vaccinations were delayedin unhealthy children. Hence, healthier children received DTP
first, and DTP-unvaccinated children should, therefore, have
had a higher mortality rate. Despite this, DTP was associatedwith increased child mortality, particularly for girls. All threestudies of the introduction of DTP vaccine found negative
effects which are different from what should have been expected
due to the disease-preventive effects of the vaccine and theinherent biases favoring vaccinated children (5, 12). The results
are also in stark contrast to the studies of the introduction ofmeasles vaccine, which uniformly show very strong mortality
reductions (6, 7, 15). Hence, the studies of the introduction of
DTP constitute a clear danger signal that DTP may substantially
increase all-cause mortality.
Adding to the danger signal, DTP is associated with increased
female mortality relative to male mortality in all available
studies. Girls did not have higher mortality than boys in the
pre-vaccination era in West Africa (2). Hence, there is a need forfurther research to assess the overall mortality effect of DTP and
how the negative effects of DTP can be removed or modified.
For example, co-administration of BCG and DTP may reduce
the negative effect of DTP (21). Randomized trials have also
shown that MV or BCG administered shortly after DTP may
reduce the negative effect of DTP and lower mortality (2). Suchalternative immunization strategies should be further tested in
randomized trials.
Given the threat from diphtheria, tetanus, and pertussis
and the less-effective acellular pertussis vaccine used in many
countries, it is understandable that there has been reluctance inaccepting that DTP could have negative effects for overall healthin low-income countries. However, the studies from low-income
countries have been consistent in showing deleterious effect ofDTP (3); furthermore, the first studies are now showing that
non-live and live vaccines have differential NSEs on hospital
admissions for infectious diseases in high-income countries(36, 37). Hence, it would seem to be high time to settle whether
DTP has negative effects on overall child health and if it has negative effects to explore whether alternative vaccination schedules
could remove the problem.
In the current global immunization system, the coverage forthe third dose of DTP (DTP3) is used as the main performanceindicator for national immunization programs. This will clearlylead to an emphasis on increasing the coverage for DTP3 (1)more than the coverage for other vaccines. Given that all studies,including the present one, suggest that DTP is associated withincreased female mortality, this is really an illogical position. We
need to use program performance indicators which are positively
associated with better child survival.

The present study and the cleaning of the original data were
supported by a common grant from DANIDA and the Novo
Nordisk Foundation. The work on non-specific effects ofvaccines has been supported by the Danish Council for
Development Research, Ministry of Foreign Affairs, Denmark[grant number 104.Dan.8.f.], Novo Nordisk Foundation,
and European Union FP7 support for OPTIMUNISE (grant:
Health-F3-2011-261375). CSB held a starting grant from the
ERC (ERC-2009-StG-243149). CVIVA is supported by a grantfrom the Danish National Research Foundation (DNRF108).
PA held a research professorship grant from the Novo Nordisk
Foundation.

INDEPENDENCE

SUPPLEMENTARY MATERIAL

The funding agencies had no role in the study design, data collection, data analysis, data interpretation, or the writing of thereport.

The Supplementary Material for this article can be found online at
http://www.frontiersin.org/articles/10.3389/fpubh.2018.00079/
full#supplementary-material.

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The first author affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; thatno important aspects of the study have been omitted; and thatany discrepancies from the study as planned (and, if relevant,registered) have been explained.

DATA SHARING
Through request to the authors.

ETHICS STATEMENT
The study of nutritional status was planned between the SAREC(Swedish Agency for Research Collaboration with Developing
Countries) and the Ministry of Health in Guinea-Bissau. There
were no ethical committees for approval of health research at thetime of the study. The study was explained to the population incommunity meetings organized by the local committee and theresearchers prior to initiation of data collection. Consent was notsought from individual mothers, since the project implemented
intended national policies for nutritional surveillance andimmunization.

AUTHOR CONTRIBUTIONS
CB and PA proposed the study. PA collected the original data.
AR is responsible for the demographic surveillance system.
SM and PA cleaned the data. SM conducted the statisticalanalyses. The first draft was written by PA; all authors contributed to the final version of the paper. PA will act as guarantorof the study.

FUNDING

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Copyright © 2018 Aaby, Mogensen, Rodrigues and Benn. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, provided
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use, distribution or reproduction is permitted which does not comply with these terms.

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

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