(2019) 12:103
Auerswald et al. Parasites Vectors
https://doi.org/10.1186/s13071-019-3357-3
Parasites & Vectors
Open Access
RESEARCH
First dengue virus seroprevalence study
on Madeira Island after the 2012 outbreak
indicates unreported dengue circulation
Heidi Auerswald1†, Ana de Jesus2,3†, Gonçalo Seixas2,3, Teresa Nazareth2,3, Saraden In1, Sokthearom Mao1,
Veasna Duong1, Ana Clara Silva4,5, Richard Paul6,7, Philippe Dussart1*‡ and Carla Alexandra Sousa2,3‡
Abstract
Background: In 2012, the first dengue virus outbreak was reported on the Portuguese island of Madeira with 1080
confirmed cases. Dengue virus of serotype 1 (DENV-1), probably imported from Venezuela, caused this outbreak with
autochthonous transmission by invasive Aedes aegypti mosquitoes.
Results: We investigated the seroprevalence among the population on Madeira Island four years after the outbreak.
Study participants (n = 358), representative of the island population regarding their age and gender, were enrolled in
2012 in a cross-sectional study. Dengue antibodies were detected with an in-house enzyme-linked immunosorbent
assay (ELISA) using the dimer of domain III (ED3) of the DENV-1 envelope protein as well as commercial Panbio indirect and capture IgG ELISAs. Positive ELISA results were validated with a neutralization test. The overall seroprevalence
was found to be 7.8% (28/358) with the in-house ELISA, whereas the commercial DENV indirect ELISA detected IgG
antibodies in 8.9% of the individuals (32/358). The results of the foci reduction neutralization test confirmed DENV-1
imported from South America as the causative agent of the 2012 epidemic. Additionally, we found a higher seroprevalence in study participants with an age above 60 years old and probable secondary DENV infected individuals
indicating unreported dengue circulation before or after 2012 on Madeira Island.
Conclusions: This study revealed that the number of infections might have been much higher than estimated from
only confirmed cases in 2012/2013. These mainly DENV-1 immune individuals are not protected from a secondary
DENV infection and the majority of the population of Madeira Island is still naïve for DENV. Surveillance of mosquitoes
and arboviruses should be continued on Madeira Island as well as in other European areas where invasive vector
mosquitoes are present.
Keywords: Dengue virus, Seroprevalence, Madeira Island, Serotype
Background
Dengue fever is the most widespread mosquito-borne
viral disease causing an annual estimated 390 million
infections [1] and 25,000 deaths [2]. Infection by any one
of four antigenically distinct serotypes of dengue virus
*Correspondence: pdussart@pasteur-kh.org
†
Heidi Auerswald and Ana de Jesus: Equal contributors
‡
Philippe Dussart and Carla Alexandra Sousa contributed equally to this
work
1
Virology Unit, Institut Pasteur du Cambodge, Institut Pasteur
International Network, PO Box 983, Phnom Penh, Cambodia
Full list of author information is available at the end of the article
(DENV) can lead to symptoms including high fever or
more severe disease with haemorrhage and plasma leakage. However, the majority (~80%) of infections results in
a mild or subclinical outcome [3].
The virus is mainly transmitted by the mosquito species Aedes aegypti, inhabiting the tropics and subtropics, but can also be transmitted by Ae. albopictus, which
is an invasive species present in several European countries [4, 5]. Due to the increasing number of imported
DENV infections [6, 7], there is a growing likelihood of
autochthonous infections where competent vectors are
present. Such autochthonous transmission of DENV
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Auerswald et al. Parasites Vectors
(2019) 12:103
in regions with a naïve population has occurred intermittently in France [8–10], Croatia [11, 12] and the
USA [13, 14] over the past decade. Furthermore, Japan
recently experienced an unprecedented epidemic in
Tokyo [15, 16]. However, Europe has not witnessed a
large dengue outbreak since the epidemic in Greece in
1927/28 [17].
In September 2012, a unique European dengue outbreak occurred on Madeira Island, the larger of the two
inhabited islands of the Portuguese autonomous region
in the Atlantic Ocean. Madeira has been classified as
having a Mediterranean climate, meaning it has mild
year-round temperatures; the average annual temperature at the weather station of the capital city of Funchal
was 19.6 °C for the period 1980–2010. On the highest
windward slopes of Madeira, rainfall exceeds 1250 mm
per year, mostly falling between October and April
(according to the Meteorology Institute of Portugal,
IPMA). The dengue epidemic resulted in 2168 probable cases and 1080 confirmed infections from September 2012 to March 2013, affecting mainly residents
from Funchal and the neighbouring provinces, Câmara
de Lobos, Sao Martinho and Caniço [18]. The majority
of the ≈270,000 inhabitants of this main island live on
the south coast around the capital city of Funchal. In
contrast to the small autochthonous dengue outbreaks
on the European mainland, the outbreak in Madeira
was spread by Ae. aegypti mosquitoes, which were first
reported to be present on the island in 2004 [19]. As
this was the first dengue epidemic that hit the Portuguese island, the population of Madeira was naïve for
DENV infections. The DENV-1 serotype imported
from Venezuela in South America and belonging to
genotype V was believed to have been responsible for
the outbreak [20, 21].
This study was conducted to investigate the seroprevalence among the population on Madeira Island and thus
the actual extent of the dengue epidemic. We aimed to
verify DENV-1 as the causative agent of the epidemic by
performing foci reduction neutralization tests (FRNT)
against all four DENV serotypes, and additionally against
a DENV-1 genotype V isolate originally isolated in 2009
in French Guiana [22]. A secondary objective was the
evaluation of an in-house enzyme-linked immuno-sorbent assay (ELISA) against a commercial indirect ELISA
and the neutralization test, the gold standard for serological flavivirus diagnostics (Additional file 1: STROBE
Statement).
Methods
Study design and sampling
Serum samples from inhabitants of Madeira Island were
collected in 2016. The minimum number of participants
Page 2 of 11
was estimated to be representative of the Madeira population in 2012 (258,686 inhabitants) regarding all ages and
both genders. The total number of participants for this
seroprevalence study for the 235,233 people above the
age of 10 years-old (study age range: 11–92 years), stratified into gender and decennial age group, was calculated
as previously described, applying the formula from Luiz
& Magnanini [23]. Given that there were 1080 DENV
confirmed cases during the 2012/2013 outbreak [18] and
based on the assumption that only 20% infections led to
symptomatic dengue fever cases, an estimated 5400 people might actually have had dengue during the epidemic
and the probable prevalence rate of viral infection would
be 2.1%. Therefore, the minimum sample size was calculated to be 237 participants (197 + 20% reserves), following the age/gender distribution shown in Additional
file 2: Table S1.
The participants were recruited by convenience sampling at the Henriques de Gouveia laboratory and the
Madeira Medical Centre among patients coming for routine blood analyses in November in 2015. After presenting the purpose of our study, each volunteer who agreed
to participate filled out a questionnaire with personal
data and relevant health information: age, gender, weight,
county of residence, history of travel to dengue endemic
countries and history of yellow fever and Japanese
encephalitis vaccination. Blood samples were collected
from each participant using BD Vacutainer tubes, and
one extra-tube using a BD Vacutainer SST II Advance
8.5 ml was collected for the purpose of the study. The
total blood volume was collected according to body
weight of the participant as previously described [24]. On
the day of collection, the blood samples dedicated to the
study were centrifuged and an aliquot of 300 µl of serum
of each sample was taken. All the samples were frozen
and stored at -20 °C until laboratory analyses.
Enzyme‑linked immunosorbent assays
Both of the commercial Panbio ELISAs (Alere Inc.,
Waltham, MA, USA), Dengue IgG indirect ELISA and
Dengue IgG capture ELISA, were performed according
to the manufacturer’s instructions [25, 26]. The Panbio
Dengue IgG indirect ELISA is used for the qualitative
detection of IgG antibodies to DENV antigens of all
four serotypes, whereas the Panbio Dengue IgG capture
ELISA is used specifically for the qualitative detection of
IgG antibodies in secondary DENV infections [25]. The
latter is achieved by a higher cut-off value for positive
results [27]. This testing strategy of using both commercial Panbio ELISAs allowed not only the detection of previous DENV infection within the population of Madeira
Island, but also the exploration of possible unreported
secondary DENV infections. The in-house ELISA test is
Auerswald et al. Parasites Vectors
(2019) 12:103
an indirect assay developed at Institut Pasteur and uses
the stabilized dimer of domain III (ED3) of the DENV-1
envelope protein for the detection of IgG antibodies
against DENV in human serum [28].
The ELISA was performed in 96-well plates (Greiner
Bio One, Kremsmünster, Austria) with a 100 µl/well processing volume. The plates were coated overnight at 4 °C
with 100 µl of recombinant antigen diluted to 0.3 µg/ml in
PBS (Sigma-Aldrich, Steinheim, Germany). The next day
the coated plates were washed with PBS + 0.1% Tween 20
(PBS-T) and then incubated for 1 h at 37 °C with 100 µl/
well of blocking buffer (PBS-T + 5% low-fat dried milk).
Subsequently, the plates were washed with PBS-T and
100 µl serum samples diluted 1:100 in blocking buffer
were added to wells in duplicate. The plates were incubated for 1 h at 37 °C. Next, serum dilutions were discarded, and plates were washed four times with PBS-T.
Then, 100 µl of goat horseradish peroxidase-conjugated
anti-human IgG antibody (Sigma-Aldrich) diluted 1:1000
in blocking buffer was added to each well and incubated
for 1 h at 37 °C. Afterwards, plates were washed four
times with PBS-T and 100 µl TMB substrate (SigmaAldrich) was added to each well and left for 10–15 min.
The colour reaction was stopped by adding 100 µl/well of
0.5 N sulfuric acid and the optical density was measured
at 450 nm with an Infinite 200 PRO NanoQuant spectrophotometer (Tecan, Männedorf, Switzerland).
Cell lines for viral growth and neutralization studies
The monkey cell line LLC-MK2 was used for the detection of neutralizing antibodies via a foci reduction neutralization test [29]. Cells were cultivated in Dulbecco’s
modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco,
Gaithersburg, MD, USA) and 100 U/ml penicillin-streptomycin (Gibco) at 37 °C and in a 5% CO2 atmosphere.
All viruses were grown in Ae. albopictus C6/36 cells and
harvested from the supernatant. These mosquito cells
were cultured in Leibovitz 15 medium (Sigma-Aldrich)
supplemented with 10% FBS, 1% L glutamine (Gibco),
10% tryptose-phosphate (Gibco) and 100 U/ml penicillin-streptomycin at 28 °C.
Viruses
The reference viruses used for the neutralization test were
the following: DENV-1 Hawaii (GenBank: AF425619),
DENV-2 New Guinea C (GenBank: AF038403), DENV-3
strain H87 (GenBank: M93130), DENV-4 H241 (GenBank: AY947539) and JEV strain Nakayama (GenBank:
EF571853). As no DENV-1 isolate from the Madeira epidemic was available, we used a virus that was isolated
from a patient in French Guiana in 2009 (FGU 2009;
GenBank: MH279620) that belongs to genotype V [22]
Page 3 of 11
(Additional file 3: Figure S1). The recombinant DENV-1
ED3 protein was produced based on the DENV-1 strain
FGA/89 (accession no. AF226687) [28].
Foci reduction neutralization test
The FRNT micro-neutralization assay, used as the gold
standard, determined the level of neutralizing antibodies
against different viruses. A subset of sera was tested by
FRNT including (i) all sera formerly tested positive with
at least one of the above mentioned ELISAs (n = 32); (ii)
sera with an undetermined ELISA result (n = 3); and (iii)
sera with negative results in all ELISAs randomly selected
and representing 10% of the total study population, with
similar numbers of sera for each age group (n = 36). The
serum samples were analysed by FRNT as previously
described [30], but modified by using LLC-MK2 cells and
virus-specific polyclonal mouse hyperimmune ascites
fluids (Institut Pasteur in Cambodia). Neutralization was
defined as the serum dilution that induced a 90% reduction in the number of virus-induced foci (foci reduction
neutralization test 90%; FRNT90) compared to controls
(virus alone and flavivirus-negative control serum alone)
and was calculated via log probit regression analysis
(SPSS for Windows v.16.0; SPSS Inc., Chicago, USA). The
first serum dilution used for the assay was 1:10 which
resulted in a final in-test dilution of 1:20 after adding an
equivalent volume of virus. Subsequently, the lower limit
of quantification for the FRNT90 titer was defined as 20,
which follows the WHO recommendations for flavivirus FRNTs [31]. The DENV that induced at least a 4-fold
higher titer compared to the other DENVs determined
the DENV serotype responsible for the dengue infection.
Statistical analysis
Risk factor analysis of the association of age (continuous), gender, yellow fever vaccination (yes/no), travel history outside of Madeira (yes/no) and geographical site
(11 districts) on dengue seropositivity using the results
from the indirect Panbio IgG was performed by fitting
a generalized linear model with binomial error structure (i.e. logistic regression) using GenStat 15th Edition,
(VSN International Ltd., Hemel Hempstead, UK). Wald
test in the context of this logistic regression was used to
determine whether a certain predictor variable was significant or not. Wald statistics, which approximate to a
Chi-square distribution, are given. A dispersion parameter was estimated to account for over-dispersion in the
data. There were only two individuals with JEV vaccination and therefore this variable was not included in the
analysis. Mean FRNT90 titers were compared using the
2-tailed Mann-Whitney test to determine if differences
were significant. The agreement of the FRNT results
with the results of the diverse ELISAs was performed
Auerswald et al. Parasites Vectors
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via Cohen’s kappa test. All statistical analyses were performed with a significance level of α = 0.05.
An estimated number of total infections was calculated
by multiplying the number of people of a determined age
group (Additional file 2: Table S1) by the proportion of
seropositive results of that age group in the test population. The process was repeated for all the pre-determined
age groups and summed to give the estimated number of infections. For example, if we had 15,000 people
aged from 20–29 on Madeira Island at the time, and we
had collected 100 blood samples from people of that age
group of which 15 had a positive result, then we estimate
that 2250 people of that age group have been infected by
dengue. This calculation was carried out for every studied age group and then summed to generate an overall
estimated number of infections.
Results
Study population
The study enrolled 358 individuals aged from 11 to 92
(mean age: 49) and a male:female ratio of 0.61 (Table 1).
The study included residents from all 11 districts of
Madeira although the majority of the study participants
resided in Funchal (229/358, 64.0%). In total, 8.7% of the
study participants (31/358) self-reported a former vaccination against the yellow fever virus (YFV), and 31.8%
(114/358) had travelled to DENV endemic countries.
Only two participants were previously vaccinated against
Japanese encephalitis virus (JEV).
Seroprevalence
The dengue seroprevalence of the study participants
was tested by in-house DENV IgG indirect ELISA using
dimer of Domain III (ED3) of the DENV-1 envelope protein and the DENV IgG indirect ELISA from Panbio. In
total, the in-house ELISA detected 7.8% (28/358) of the
study participants as positive, whereas the commercial
DENV indirect ELISA detected IgG antibodies in 8.9%
of the individuals (32/358; Table 2). For three samples
the result of the indirect Panbio ELISA was undetermined. A subset of 71 sera was tested by FRNT including
all sera with a positive (n = 36) or undetermined (n = 3)
result in any of the two indirect ELISAs (Additional file 4:
Table S2) and 32 sera with negative results in all ELISAs.
Individuals were considered positive for DENV antibodies if they showed a FRNT90 titer ≥ 20 for one or more
DENV reference strains. Based on these criteria, 28 study
participants were found positive for DENV neutralizing
antibodies (Table 2).
Among the DENV seropositive samples, the DENV
capture IgG ELISA from Panbio designed to identify
secondary dengue infection detected five positive individuals (5/28, 17.9%). Additionally, four people (4/28,
Page 4 of 11
Table 1 Demographic characteristics of study participants
Participants
DENV seropositivea DENV seronegative Total
n (%)
n (%)
n (%)
Total number
28 (7.8)
330 (92.2)
358 (100)
Male
11 (8.1)
125 (91.9)
136 (38.0)
Female
17 (7.7)
205 (92.3)
222 (62.0)
49
49
Median age (years) 57.5
10–19
0 (0)
19 (100)
19 (5.3)
20–29
3 (7.9)
35 (92.1)
38 (10.6)
30–39
3 (4.8)
59 (95.2)
62 (17.3)
40–49
6 (9.8)
55 (90.2)
61 (17.0)
50–59
3 (4.2)
68 (95.8)
71 (19.9)
60+
13 (12.1)
94 (87.9)
107 (29.9)
Travel historyb
9 (7.9)
105 (92.1)
114 (31.8)
No travel historyb
19 (7.8)
225 (92.2)
244 (68.2)
YFV vaccination
3 (9.7)
28 (90.3)
31 (8.7)
No YFV vaccination 25 (7.6)
302 (92.4)
327 (91.3)
Funchal
20 (8.7)
209 (91.3)
229 (64.0)
Câmara de Lobos
1 (4.5)
21 (95.5)
22 (6.1)
Santa Cruz
3 (6.1)
46 (93.9)
49 (13.7)
Sao Vicente
1 (16.7)
5 (83.3)
6 (1.7)
Porto Moniz
1 (50)
1 (50)
2 (0.6)
Machico
0 (0)
8 (100)
8 (2.2)
Ribeira Brava
0 (0)
8 (100)
8 (2.2)
Calheta
0 (0)
6 (100)
6 (1.7)
Santana
0 (0)
4 (100)
4 (1.1)
Ponta do Sol
0 (0)
4 (100)
4 (1.1)
Porto Santo
0 (0)
2 (100)
2 (0.6)
Missing data
2 (11.1)
16 (88.8)
18 (5.0)
a
Dengue seropositive patient identified by at least one ELISA assay and
confirmed with seroneutralization assay (FRNT)
b
To dengue endemic countries
Abbreviations: DENV, dengue virus; YFV, yellow fever virus
14.3%) showed inconclusive results and therefore the
dengue immune status could not be determined with
this assay. Based on these results, most of the seropositive study participants (19/28, 67.8%) were considered
to have had a primary DENV infection.
Seropositive individuals were found in only five of
the eleven districts of Madeira, largely reflecting sampling effort. Most of the seropositive individuals (20/28,
71.4%) were residents of Funchal, with ever decreasing numbers in the other Southern districts (Ponta do
Sol, Ribeira Brava, Câmara de Lobos and Santa Cruz)
(Fig. 1). We detected only two cases from the north, one
from Porto Moniz and one from Sao Vicente (Table 1).
Risk factor analysis of the indirect Panbio ELISA seropositivity revealed no significant association with gender (Wald test: χ2 = 0.28, df = 1, P = 0.595), yellow fever
vaccination (Wald test: χ2 = 0.72, df = 1, P = 0.397),
Auerswald et al. Parasites Vectors
(2019) 12:103
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Table 2 Comparison of serology results obtained with the different enzyme-linked immuno-sorbent assays used (n = 358) and the
foci reduction neutralization test (n = 71)
Serological assays used
Positive samples
n (%)
Negative samples
n (%)
Undetermined
n (%)
n = 358
DENV-1 ED3 dimer ELISA (in-house)
28 (7.8)
330 (92.2)
–
DENV IgG indirect ELISA (Panbio)
32 (8.9)
323 (90.3)
3 (0.8)
DENV IgG capture ELISA (Panbio)
5 (1.4)
349 (97.5)
4 (1.1)
n = 71a
DENV-1 ED3 dimer ELISA (in-house)
28 (39.4)
43 (60.6)
–
DENV IgG indirect ELISA (Panbio)
32 (45.1)
36 (50.7)
3 (4.2)
DENV IgG capture ELISA (Panbio)
5 (7.1)
62 (87.3)
4 (5.6)
FRNTb
28 (39.4)
43 (60.6)
–
DENV-1c
24 (33.8)
47 (66.2)
–
DENV-2c
5 (7.0)
66 (93.0)
–
DENV-3c
5 (7.0)
66 (93.0)
–
DENV-4c
7 (9.9)
64 (90.1)
–
DENV-1 genotype V
27 (38.0)
44 (62.0)
–
a
Subset of additional samples tested by FRNT
b
FRNT90 titer ≥ 20
c
Dengue reference strain
Abbreviations: DENV, dengue virus; ELISA, enzyme-linked immuno-sorbent assay; FRNT, foci reduction neutralization test
Fig. 1 Geographical distribution of the seroprevalence across Madeira Island. Districts with dengue seropositive study participants are shown in
blue and the respective residences are marked with red dots. The map was created using QGIS 2.14.3 and the base layer data were obtained from
DIVA GIS (http://www.diva-gis.org/gdata)
travel history to DENV endemic countries (Wald test:
χ2 = 0.01, df = 1, P = 0.94) or place of residence (Wald
test: χ2 = 6.69, df = 10, P = 0.753). However, there was
a significantly increased risk with increasing age (Wald
test: χ2 = 12.57, df = 1, P < 0.001; odds ratio per year of age
1.03; 95% CI: 1.01–1.05).
The seroprevalence results were extrapolated to obtain
the estimated number of infections during the dengue
outbreak. Based on the population in 2012 (total number of residents: 258,686) and the FRNT confirmed seroprevalence of 7.8%, the total number of infections was
estimated to be 16,606 (95% binomial CI: 9221–41,303).
Auerswald et al. Parasites Vectors
(2019) 12:103
The 60+ years old group contributed an estimated > 6000
cases due to the high seroprevalence observed (12.1%; 13
seropositive out of 107 study participants with an age of
60+).
FRNT with DENV reference strains and DENV‑1 genotype V
strain
A subset of 71 sera was tested by FRNT using all four
dengue serotypes to measure the levels of neutralizing
antibodies (FRNT90 titer; Table 2) as well as the serotype. The DENV serotype was identified as the virus
which induced the highest FRNT90 titer. Overall, the
FRNT using DENV reference strains detected 28 individuals with neutralizing antibodies; all these individuals formerly tested positive with at least one of the three
describes ELISAs. None of the ELISA negative samples
was detected positive with the FRNT. Among the 28
FRNT positive samples, 24 (85.7%) were detected positive for neutralizing antibodies against DENV-1 reference
strain. Among these, we identified 21 individuals with
DENV-1 serotype (indicated by the highest FRNT90 titer
against DENV-1 reference strain compared to the other
DENVs). Moreover, three study participants were identified with neutralizing antibodies against DENV-2 (n = 1),
DENV-3 (n = 1) or DENV-4 (n = 1).
After using the reference strains for all four DENVs
(DENV 1-4) we investigated further the immune response
to DENV-1 genotype V strain, as this was the proposed
cause of the 2012 outbreak in Madeira Island. Among
the 71 sera tested by FRNT with a DENV-1 genotype
V strain, we identified one study participant with a low
FRNT90 titer of 30 against this DENV-1 genotype V strain
that was formerly negative against all four DENV reference strains (Additional file 4: Table S2). This increased
the total number of FRNT positive study participants
to 29 (Table 3, Fig. 2). Two other individuals were negative for antibodies against the DENV-1 reference strain
but showed detectable levels of neutralizing antibodies
against the DENV-1 genotype V strain (FRNT90 titer of
73 and 71, respectively). One of these also had neutralizing antibodies against DENV-3 (FRNT90 = 58); the
other individual was also positive for both DENV-3
(FRNT90 = 24) and DENV-4 (FRNT90 = 81).
Four study participants had similar FRNT90 titers
against two serotypes (one with DENV-1 + DENV-2, one
with DENV-3 + DENV-4, two with DENV-1 + DENV4). The FRNT90 titers for the DENV-1 reference strain
(mean FRNT90 = 201) of the seropositive sera (n = 29)
were significantly higher than against DENV-2 (mean
FRNT90 = 13; Mann-Whitney U = 114.5, n1 = n2 = 29,
P < 0.0001, two-tailed), DENV-3 (mean FRNT90 = 12;
Mann-Whitney U = 110, n1 = n2 = 29, P < 0.0001,
Page 6 of 11
Table 3 Mean neutralization titers for seropositive samples by
foci reduction neutralization test (n = 29)
Virus used
Positive samples with FRNT
Mean FRNT90
95% CI
DENV-1 reference strain
201
127–275
DENV-1 genotype V strain
444
DENV-2 reference strain
13
262–626
5.5–21
DENV-3 reference strain
12
6–18
DENV-4 reference strain
18
6–31
Abbreviations: 95% CI, 95% confidence interval; DENV, dengue virus; FRNT, foci
reduction neutralization test
two-tailed) and DENV-4 (mean FRNT90 = 18; MannWhitney U = 117, n1 = n2 = 29, P < 0.0001, two-tailed;
Table 3, Fig. 2). The FRNT90 titers against the genotype V (mean FRNT90 = 444) were twice as high as
the titers against the DENV-1 reference strain (mean
FRNT90 = 201) belonging to genotype I in the positive
individuals (n = 29). As observed before with the DENV-1
reference strain, the mean FRNT90 titer against the
DENV-1 genotype V strain was significantly higher than
the mean neutralization titers against the other DENV
serotypes (DENV-2: Mann-Whitney U = 42, n1 = n2 = 29,
P < 0.0001, two-tailed; DENV-3: Mann-Whitney U = 39,
n1 = n2 = 29, P < 0.0001, two-tailed; DENV-4: MannWhitney U = 53.5, n1 = n2 = 29, P < 0.0001, two-tailed;
Table 3, Fig. 2).
None of the 71 tested sera showed any detectable neutralizing antibodies against JEV. The results of the 32
serum samples formerly tested negative in both indirect
ELISAs (in-house and Panbio) were confirmed negative by FRNT with all DENV reference strains and the
DENV-1 genotype V isolate.
ELISAs in comparison to FRNT
The second objective of this study was the evaluation
of the DENV-1 ED3 dimer indirect ELISA in comparison to the commercial IgG indirect ELISA from Panbio
and the FRNT. This comparative analysis was done with
a subset of 71 sera tested in all above mentioned assays.
Within these, the in-house DENV-1 ED3 dimer indirect
ELISA detected 28 (39.4%) positive individuals, whereas
the commercial DENV indirect ELISA detected IgG antibodies in 32 of the individuals (45.1%; Table 2). For three
samples, the results with the Panbio indirect ELISA were
undetermined. The FRNT confirmed 78.6% (22/28) of the
positive results from the in-house indirect ELISA results
and 90.6% (29/32) of the Panbio indirect ELISA positive
results (Additional file 4: Table S2). Taking the FRNT as
the gold standard assay, the in-house ELISA produced six
Auerswald et al. Parasites Vectors
(2019) 12:103
Fig. 2 Individual FRNT90 titers of all FRNT positive study participants
(n = 29). Mean FRNT90 titers with standard error for FRNT positive
samples. Overall, 28 samples were positive in the FRNT using the
reference strains of DENV-1 (red), DENV-2 (blue), DENV-3 (green) and
DENV-4 (yellow). An additional FRNT with DENV-1 genotype V (pink)
added one more seropositive study participant. The dashed line
indicates threshold of 20. Asterisks indicate the statistically significant
different mean FRNT90 titers between the DENV-1 reference strain
and the DENV-1 genotype V strain with the other DENV serotypes
(Mann-Whitney test, P < 0.0001)
false positive and seven false negative results. The Panbio indirect ELISA produced three false positive results.
Interestingly, one individual who tested positive with
the indirect Panbio ELISA was negative when tested by
the in-house ELISA and the FRNT using DENV reference strains, but had detectable neutralizing antibodies
against DENV-1 genotype V (FRNT90 = 30). Including
all 71 samples tested with FRNT, the DENV-1 in-house
ELISA led to 58 congruent results (81.7%) with the
FRNT (κ = 0.619; 95% CI: 0.433–0.806). The kappa test
revealed 91.5% agreement (65/71 results) between the
Panbio indirect ELISA and the FRNT (κ = 0.836, 95% CI:
0.716–0.956).
The five individuals identified as having had a secondary DENV infection with the IgG capture ELISA from
Panbio showed no significantly higher titers of neutralizing antibodies in the FRNT. However, these individuals with suspected secondary DENV infections had more
often a broad neutralization pattern, where the FRNT
analysis did not identify a single serotype but detected
multiple serotypes. Three of these secondary infected
individuals belong to the age group of 60+.
Discussion
The results of our dengue seroprevalence study on the
population of Madeira Island are in agreement with
investigations made during or directly after the outbreak.
The extent of the outbreak and the fact that Madeira is a
favourite tourist destination led to 81 exported cases to
mainland Europe after returning from Madeira: 11 cases
to mainland Portugal and 70 in other European countries [18] including Finland [32], Belgium [33], Romania
[34], Germany and the UK [35]. Partial sequencing and
Page 7 of 11
phylogenetic investigations of autochthonous cases from
Madeira and imported cases to other European countries
identified DENV serotype 1 (DENV-1) genotype V as
the causative agent of the outbreak [32, 36], highly likely
imported from Venezuela [20, 34].
Our study using FRNT confirmed this suspicion,
revealing that the concentration of neutralizing antibodies was higher against genotype V than against the
reference isolate of genotype I. DENV-1 genotype V is
epidemiologically very important due to its broad distribution in the Americas, Asia, Oceania and Africa over
the last 75 years [37, 38], and because it can cause infections with a severe clinical outcome [39].
The distribution of the seropositive individuals found in
this study confirmed the higher attack rate in the southern districts, especially in Funchal, as reported after the
ECDC outbreak investigation [18]. This is not surprising as the densities of both the human and Ae. aegypti
populations are highest on the southern coastline. The
high seroprevalence in individuals aged 60+ could be due
to unreported previous exposure to DENV. This is supported by our findings that three out of the five identified
secondary infections occurred in this age group.
We found neutralizing antibodies, mainly directed
against DENV-1, in 7.8% of our study population,
whereas the infection rate based on the 1080 confirmed
cases of the outbreak was only 0.4% (and ~1% for probable cases). A similarly high seroprevalence rate (5%) was
observed in individuals without recollection of symptoms in the vicinity of the Tokyo Yoyogi Park outbreak
that led to 162 confirmed cases [16]. A 7:1 ratio of subclinical to symptomatic infection outcome is within the
range previously observed [3, 40]. Due to the high number of subclinical or inapparent infections, especially in
naïve populations, seroprevalence studies are important
for risk assessment if DENV-1 re-emerges or if another
DENV serotype is imported [3]. From our seroprevalence
studies, global estimates of infection in the Madeira population suggest a very high number of individuals have
been exposed to the virus during this relatively short epidemic. This is significant as secondary infections lead to
an increase in severity of disease [41].
Overall, the cross-reactivity of DENV is not fully understood and there are contrary findings. The specificity of
antibodies is not directly correlated with their neutralization capacity. Serotype-specific antibodies represent only
a small fraction of the neutralizing antibodies [42] and
cross-reacting antibodies contribute significantly to the
neutralization capacity [43, 44]. Additionally, neutralizing
antibodies are an important correlate for protection [45]
but are not the only determining factor. Moreover, recent
findings highlight the importance of the cellular immune
response for protective immunity [46]. It is assumed that
Auerswald et al. Parasites Vectors
(2019) 12:103
a primary DENV infection induces a short-term crossreacting immune response against all serotypes [47, 48],
but no long-term protection against heterotypic secondary DENV infection [49]. However, there is long-term
homotypic humoral immunity (e.g. DENV-1 antibodies were found even 60 years after the infection [50])
although there are recent reports of homotypic DENV
re-infections with a clinical outcome [51, 52]. Even crossprotection across genotypes within one serotype varies,
as is known for DENV-2 [53, 54] and DENV-3 [55]. On
the other hand vaccination studies in macaques showed
cross-protection between the two DENV-1 genotypes
IV and V [56]. Cross-neutralization among the DENV-1
genotypes was also observed in patients infected with
genotype I or IV [57]. However, studies with mouse
monoclonal antibodies against DENV-1 genotype II
showed a reduced neutralization capacity against heterologous genotypes [58]. Additionally, Shrestha et al. [58]
investigated DENV-1 monoclonal antibodies from mice
infected with DENV-1 genotype II and found only two
out of 76 monoclonal antibodies that showed a strong
neutralization against all five DENV-1 genotypes. Such
partial lack of intra-serotype cross-neutralization can be
explained by the high diversity of the DENV-1 serotype
[59] and may explain our observed differences in neutralization to genotypes I and V of DENV-1.
The second goal of this investigation was the evaluation
of the previously developed ED3 dimer indirect ELISA
[28]. This assay showed a good agreement of 81.7% with
the gold standard for flavivirus serological diagnostics,
the neutralization test, performed in this study as FRNT
with DENV reference strains. The commercial indirect
ELISA of Panbio showed 91.5% congruent results with
the FRNT using samples from a non-flavivirus endemic
area. This study demonstrates the feasibility of seroprevalence analysis with in-house ED3 dimer indirect
ELISA. The lower sensitivity compared to the Panbio
indirect ELISA and the FRNT could be explained by the
use of the DENV-1 ED3 antigen alone and not in combination with the respective antigens of the other serotypes. A combined application of recombinant antigens
for all four serotypes could lead to the detection of the
individuals with more cross-reactive antibodies, as we
observed study participants with FRNT serotypes other
than DENV-1 and four individuals with similar titers of
neutralizing antibodies against two serotypes. The strategy of combining recombinant antigens of all four DENV
serotypes has been already used successfully in diagnostic assays [60–62]. Additionally, as described by Zidane
et al. [28], the DENV-1 sequence used for the production
of the DENV-1 ED3 antigen is closest to the PCP-consensus sequence derived from 600 DENV strains including
all four serotypes [63].
Page 8 of 11
Further surveillance of invasive mosquitoes and vectorborne diseases on Madeira Island is important not only
regarding the still existing risk of (re-)emerging DENV,
but also due to the high danger of importation of other
arboviruses such as chikungunya or Zika. For the latter,
a recent investigation by Jupille et al. [64] showed that
Ae. aegypti from Madeira Island are efficient vectors.
The mosquito surveillance in Funchal observed increasing populations from 2006 and 2008 [65] and the highest
mosquito density around Funchal during the beginning
of the DENV outbreak [66], likely due to mild climatic
conditions.
However, our study has some limitations. For further characterization of the immune response we used
a DENV-1 genotype V isolated from a patient in French
Guiana in 2009. This isolate belongs to the same genotype as the virus that was partially sequenced during
the Madeira outbreak. Nevertheless, to our knowledge
the Madeira virus was never isolated, which hindered
the direct analysis of the immune response against this
virus. Another limitation is that not all districts were
represented equally. Most of the study participants were
residents of Funchal, which might lead to an underrepresentation of cases in the other districts.
Conclusions
We performed the first seroprevalence study after the
DENV-1 outbreak on Madeira Island. We observed a predominant immune response against DENV-1, especially
against genotype V, in seropositive study participants.
Our study also revealed that the number of infections
might have been much higher than estimated from only
confirmed cases in 2012/2013, as we observed a seroprevalence of 7.8%. These mainly DENV-1 immune individuals are not protected from a secondary DENV infection
and the majority of the population of Madeira Island is
still naïve for DENV and other arboviruses. Therefore,
the surveillance of mosquitoes and arboviruses should be
continued on Madeira Island as well as in other European
areas where invasive vector mosquitoes are present.
Additional files
Additional file 1. STROBE Statement. Checklist for reports of observational studies.
Additional file 2: Table S1. Expected population sample distribution, by
gender and age group.
Additional file 3: Figure S1. Phylogenetic tree of complete sequence
of the E gene of DENV-1 from Madeira. The complete sequence of the E
gene was analysed and assembled by using the CLC Main Workbench 5.5
package (CLC bio A/S, Aarhus, Denmark). MAFFT alignment software was
Auerswald et al. Parasites Vectors
(2019) 12:103
Page 9 of 11
used to perform multiple sequences alignment of Madeira DENV-1 strains
with other reference strains from genotypes I, IV and V available in GenBank. Phylogenetic analyses were performed in MEGA7 software using the
maximum-likelihood method with a general time reversible model. Bootstrap values, indicated at the nodes, were obtained from 1000 bootstrap
replicates and are reported as percentages. DENV-1 French Guiana 2009
strain (genotype V) and DENV-1 Hawaii reference strain (genotype I) used
or for the foci reduction neutralization test are indicated by red colour. The
DENV-1 strains from Madeira outbreak (genotype V) in 2012 are indicated
by blue colour. The ID of each sequence is structured as follows: GenBank
accession no_country_name_year of isolation. The scale-bar indicates
nucleotide substitutions per site.
Author details
1
Virology Unit, Institut Pasteur du Cambodge, Institut Pasteur International
Network, PO Box 983, Phnom Penh, Cambodia. 2 GHTM-Global Health
and Tropical Medicine, 1349-008 Lisbon, Portugal. 3 UEI Medical Parasitology,
Institute of Hygiene and Tropical Medicine of Lisbon, Universidade Nova de
Lisboa, Lisbon, Portugal. 4 Departamento de Saúde, Planeamento e Administração Geral, Instituto de Administração da Saúde e Assuntos Sociais, IP-RAM,
Funchal, Madeira, Portugal. 5 Madeira Regional Government, Institute of Health
and Social Affairs, Av. Zarco, Funchal, Madeira, Portugal. 6 Functional Genetics
of Infectious Diseases Unit, Department of Genomes and Genetics, Institut
Pasteur, 75015 Paris, France. 7 Génomique évolutive, modélisation et santé
UMR 2000, Centre National de la Recherche Scientifique (CNRS), 75724 Paris
Cedex 15, France.
Additional file 4: Table S2. Serological results of ELISA-positive individuals and comparison with foci reduction neutralization test (FRNT) (n = 39).
Received: 28 November 2018 Accepted: 26 February 2019
Abbreviations
CI: confidence interval; DENV: dengue virus; YFV: yellow fever virus; ELISA:
enzyme-linked immunosorbent assay; FRNT: foci reduction neutralization test;
IgG: immunoglobulin G.
Acknowledgements
The authors thank all study participants on Madeira Island as well as the
medical personnel at the LANA Laboratory, Dr Henriques de Gouveia, and
the Madeira Medical Centre/SynLAB Clinical Centre, Dr Castro Fernandes, for
collecting the serum samples and questionnaires. We also thank Anna-Bella
Failloux from the Department of Virology, Arboviruses and Insect Vectors,
Institut Pasteur who kindly provided the DENV-1 genotype V strain.
Funding
This work was partially funded The DENFREE Consortium (European Union FP7
grant no. 282378), Agence National de la Recherche PANIC (Grant no. ANR 14
CE 02 0015 03), Fundação para a Ciência e Tecnologia (FCT) through GHTMUID/Multi/04413/2013 and by the Virology Unit of Institut Pasteur du Cambodge. GS was supported by a FCT doctoral grant (SFRH/BD/98873/2013).
The postdoctoral fellowship of HA was supported by the Calmette and Yersin
Programme of the Institut Pasteur Department of International Affairs.
Availability of data and materials
Data supporting the conclusions of this article are included within the article
and its additional files. The raw datasets used and/or analysed during this
study are available from the corresponding author upon reasonable request.
Authors’ contributions
RP, PD, AJ, ACS and CAS conceived and designed the study and the laboratory
investigations. AJ, HA, ACS, TN, CAS, RP and PD designed the sample collection
and performed data analysis. AJ, HA, SI, SM, VD and GS carried out laboratory
investigations. HA, AJ, RP and PD wrote the paper. HA and AJ as well as PD and
CAS contributed equally to this work. All authors read and approved the final
manuscript.
Ethics approval and consent to participate
For all participants (or respective guardians for underage participants) a consent form was signed in addition to the questionnaire. All data were handled
confidentially and anonymously. This study was reviewed and approved by
the Ethics Committee of the Institute of Hygiene and Tropical Medicine of
Lisbon, Portugal (no. 1-2016).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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