Accepted Manuscript

Title: DENGUE INFECTION IN PREGNANCY AND ITS

IMPACT ON THE PLACENTA

Author: <ce:author id="aut0005"

author-id="S120197121730005X-
ea143985da4f1b1a05ab6a77421b7ff7"> Christiane Fernandes
Ribeiro<ce:author id="aut0010"
author-id="S120197121730005X-
ac2dd82e4847d4ac0b9d5c35f908c293"> Vânia Glória Silami
Lopes<ce:author id="aut0015"
author-id="S120197121730005X-
b0b8d14f08271c9ebc271b7d9f137081"> Patrı́cia
Brasil<ce:author id="aut0020"
author-id="S120197121730005X-
bcb7485b5b8ca746ab08981095cb86de"> Andrea Rodrigues
Cordovil Pires<ce:author id="aut0025"
author-id="S120197121730005X-
6ed1fcc800042450fada698f98942dc0"> Roger
Rohloff<ce:author id="aut0030"
author-id="S120197121730005X-
3eec392ce5b43bd5fe0a97cbb671f9eb"> Rita Maria Ribeiro
Nogueira

PII: S1201-9712(17)30005-X
DOI: http://dx.doi.org/doi:10.1016/j.ijid.2017.01.002
Reference: IJID 2827

To appear in: International Journal of Infectious Diseases

Received date: 12-8-2016

Revised date: 24-12-2016
Accepted date: 5-1-2017

Please cite this article as: Ribeiro Christiane Fernandes, Lopes Vânia Glória
Silami, Brasil Patrı́cia, Pires Andrea Rodrigues Cordovil, Rohloff Roger,
Nogueira Rita Maria Ribeiro.DENGUE INFECTION IN PREGNANCY AND
ITS IMPACT ON THE PLACENTA.International Journal of Infectious Diseases
http://dx.doi.org/10.1016/j.ijid.2017.01.002
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 DENGUE INFECTION IN PREGNANCY AND ITS IMPACT ON THE PLACENTA

Christiane Fernandes Ribeiro, Vânia Glória Silami Lopes, Patrícia Brasil, Andrea Rodrigues Cordovil Pires,
Roger Rohloff, Rita Maria Ribeiro Nogueira.

RIBEIRO, C. F. a; SILAMI, V. G. L.b; BRASIL, P c; PIRES, A. R. C.d; ROHLOFF, Re; NOGUEIRA, R. M.

Rf.

Christiane Fernandes Ribeiro, MD PhD, a Departamento Materno-infantil, Universidade Federal

Fluminense,303, Marquês do Paraná Street, 3° floor, Centro, Niterói, Rio de Janeiro 24033-900, Brazil
(corresponding author). Address: Departamento Materno-Infantil, E-mail: chrisribeiro@vm.uff.br, Phone:
+55 (21) 2629-9031.

Vânia Glória Silami Lopes, MD PhD, b Department of Pathology, Universidade Federal Fluminense,303,
Marquês do Paraná Street, Centro, Niterói, Rio de Janeiro,24033-900, Brazil, E-mail: vasilami@oul.com.br.

Patrícia Brasil, MD PhD, c ‘Evandro Chagas’ National Institute of Infectology [Instituto Nacional de
Infectologia Evandro Chagas]/Fiocruz, Rio de Janeiro, Brazil. E-mail: patricia.brasil@ini.fiocruz.br.

Andrea Rodrigues Cordovil Pires, MD, Pathologist, d Department of Pathology, Universidade Federal
Fluminense, 303, Marquês do Paraná Street, Centro, Niterói, Rio de Janeiro,24033-900, Brazil, E-mail:
andreapires@fontemd.com.br.

Roger Rohloff, MD, e Intensivist, Head of maternal-fetal intensive care unit of Perinatal de Laranjeiras and
Barra da Tijuca, Brazil. E-mail: roger.rohloff@gmail.com

Rita Maria Ribeiro Nogueira, MD PhD, f Flavirus Laboratory, Oswaldo Cruz Institute/Fiocruz, Brazil, E-
mail: rita@ioc.fiocruz.br.

No conflicts of interest.

The first draft of the manuscript was written by the corresponding author (Christiane Fernandes Ribeiro;
RIBEIRO, C F).
 2

Highlights
Histopathological and immunohistochemical study in placental tissues and retained products of
conception from 24 patients with confirmed dengue infection during pregnancy.
Immunohistochemistry can be used in placental samples or retained products of conception as a
laboratory confirmation method in dengue endemic areas when embedded material is the only material
available.

ABSTRACT

A histopathological and immunohistochemical study was conducted in placental tissues and retained
products of conception from 24 patients with confirmed dengue infection during pregnancy. The
immunohistochemical assay was positive for dengue virus in 19 placental and three ovular remnants
analyzed. The light microscopic findings were signs of hypoxia, choriodeciduitis, deciduitis and
intervillositis and the viral antigens were found in cytoplasmic of the trophoblast, villous stroma and
decidua.Our results suggest that immunohistochemistry could be used as a laboratory confirmation method
for dengue in pregnant women, especially in endemic areas when embedded material is the only material
available.

Key words or search terms: dengue, pregnancy, vertical transmission, placenta

Introduction
The occurrence of dengue infection in pregnancy has been reported in the literature since 1948. A
recent systematic review (1) reported several cases of antenatal complications associated with dengue,
including miscarriage, stillbirth, and premature deliveries. In addition, maternal death due to dengue
infection in pregnancy has been reported in Brazil (2,3) . In 2013, seven cases of vertical transmission were
reported and confirmed by serology, RT-PCR, and immunohistochemistry (4)
This study describes the fetal outcomes and the histopathological and immunohistochemical findings
in placenta samples and retained products of conception in pregnant women with confirmed dengue
infection.

Materials and Methods

It is a descriptive study of fetal outcome and pathological alterations from 24 pregnant women with
laboratory- confirmed dengue. Confirmation was defined as the presence of IgM by the method of antibody
 3

capture enzyme-linked immunosorbent assay and by the presence of nucleic acid by reverse-transcription
polymerase chain reaction (RT-PCT) in maternal or neonatal serum, in the seasonal epidemics of dengue in
the city of Rio de Janeiro, from January 2002 to May 2010.

Nineteen placentas and five ovular remnants were examined.

Pregnant women with seropositivity to HIV infection, hepatitis B, syphilis, toxoplasmosis, or rubella;
preeclampsia and gestational diabetes were excluded.

Neonates were defined as premature if gestational age was inferior to 37 weeks and low weight if
birth weight was less than 2.500g
 (5). Asphyxia was defined with Apgar score lower than 7 at the fifth
minute of life (6).
Placental weight and disk measurements and some of the characteristics of the fetal membranes,
including color and transparency, were evaluated. The distribution and size of the chorionic vessels were
evaluated in the fetus. The color and appearance of the placental lobes and tissue sections were examined.
The color and thickness of the funicular vessels and Wharton’s jelly were evaluated in the umbilical cord
and the abnormalities recorded. After examination, the placental tissues were excised, and seven sections
were obtained: from the umbilical cord, two cross-sections at opposite ends and a longitudinal section in the
median region; from the membrane, a cross-section of the membrane roll; and from the placental disk, a
sample of the cord insertion site and a sample from the middle and outer disks as well as of any other sites
considered relevant. All of the tissues sections were stained with hematoxylin-eosin (HE).
The fetal membranes, umbilical cord, chorionic plate, villi, intervillous space, and decidua basalis
were examined under a light microscope.
Immunohistochemistry was conducted with the murine IgG2A monoclonal anti-DENV complex
(MAB8705; clone D3–2H2–9-21; Millipore, Billerica, Massachusetts) diluted 1:300 in phosphate-buffered
saline and incubated overnight.

Paraffinized liver tissue from a patient who died from DENV was used as the positive control for the
immunohistochemical analysis. For the negative immunohistochemical control, 2 different procedures were
incorporated: (1) paraffinized liver tissue from the patient who had died of dengue, suppressing the
monoclonal antibody, the primary antibody of the incubation step, using only the diluent solution (bovine
serum albumin) and (2) paraffin-embedded placental tissue of patients without infectious disease on which
the full technique was performed (without the deletion of the MAB).

Results
 4

The average gestational age at delivery was 38 ± 3 weeks, and the average birth weight was 2,881 ± 543
g. Damage to the fetus occurred as miscarriage in 5 cases (20,8%), fetal death in 2 cases (8,3%), prematurity
in 3 cases (12,5%), and 7 symptomatic newborns (29%). Two newborns developed dengue shock syndrome
(DSS) and were discharged without sequelae.
The pathological findings were described on table 1. The light microscopic findings were shown in
figure 1. No changes were observed in the umbilical cord suggestive of maternal blood-borne infections,
even during the vertical transmission of type 2 virus confirmed in cases No. 06,09,13, and 24 in newborns
and case No. 23 in the fetus.

The immunohistochemical analysis in 24 samples. In two cases the immunostaining was negative.
Staining was observed in the following placental cells: decidual cells trophoblasts, and villous stroma cells
(figure 2). All staining was cytoplasmic. In 10 cases (41,6%) with positive immunostaining, a
correspondence was observed between the area with histological changes and the immunostained area.

Discussion
In this study, two possible mechanisms of fetal and neonatal morbidity were proposed: the presence of
hemodynamic changes during pregnancy that could affect the placenta and cause fetal hypoxia, or the direct
effect of infection on the fetus.
Of the 17 newborns, 14 newborns were at term and three were premature corroborating the results of
previous studies on the association between dengue virus infection and increased prematurity (7) .
Histopathological changes were associated with the inflammatory responses, including deciduitis,
choriodeciduitis, intervillositis, focal and multifocal villitis, necrotizing villitis, proliferative villitis, and
multifocal necrotizing villitis, some of this, described in other viral diseases, including rubella,
cytomegalovirus, and varicella zoster (8)(9), but it is the first time it is described in dengue infection.
Pathological changes, related to hypoxia, were observed: edema of the villous stroma, pre-infarction
areas, chorangiosis, and infarcted areas. These findings underline the importance of the hemodynamic
changes experienced by pregnant women during dengue virus infection.
Two of the six patients with sickled erythrocytes in the intervillous space evolved to DSS and died.
Accordingly, sickle cell disease has been reported to be a risk factor for severe dengue (10,11) .

Conclusion
 5

The hemodynamic changes experienced by the mother due to capillary leak syndrome as well as the
vertical transmission of the virus would be responsible to neonatal and fetal higher morbidity. The placenta
proved to be an organ that reflects well the inflammatory response, virus presence and the maternal
hemodynamic alterations.
The positive immunostaining in placental samples in 92 % (22 cases) opens a new perspective for the
diagnosis of dengue in pregnant women, considering that these samples are easily available, especially when
these samples are the only material available.

We acknowledge FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) for

funding the laboratory diagnosis. Janice Coelho, Adriana Gouveia, Licínio Esmeraldo da Silva and Luca
Ugenti Cipiani, for their contribution on data collection.
 6

References

1. POULIOT SH, Xiong X, Harville E, Paz-Soldan V, Tomashek KM, Breart G, et al. Maternal dengue and pregnancy
outcomes: a systematic review. Obstet Gynecol Surv. 2010;65(2):107–18.

2. ALVARENGA C, Silami V, Brasil P, Boechat ME, Coelho J, Nogueira RM. Dengue during Pregnancy: A Study of
Thirteen Cases. Am J Infect Dis. 2009;5(4):295–300.

3. Nunes PCG, Paes MV, de Oliveira CAB, Soares ACG, de Filippis AMB, Lima M da RQ, et al. Detection of dengue NS1
and NS3 proteins in placenta and umbilical cord in fetal and maternal death. J Med Virol. agosto de 2016;88(8):1448–52.

4. RIBEIRO CF, Lopes VGS, Brasil P, Coelho J, Muniz AG, Nogueira RMR. Perinatal Transmission of Dengue: A Report of
7 Cases. The Journal of Pediatrics. novembro de 2013;163(5):1514–6.

5. BATTAGLIA FC, LUBCHENCO LO. A practical classification of newborn infants by weight and gestational age. J
Pediatr. 1967;71(2):159–63.

6. APGAR V. A proposal for a new method of evaluation of the newborn infant. Anesth Analg. 1953;32:260–7.

7. BASURKO C, Carles G, Youssef M, Guindi WEL. Maternal and fetal consequences of dengue fever during pregnancy. Eur
J Obstet Gynecol Reprod Biol. 2009;147(1):29–32.

8. FOX H, SEBIRE N. Pathology of the Placenta, 3e. 3o ed. Saunders; 2007. 576 p.

9. OZONO K, Mushiake S, Takeshima T, Nakayama M. Diagnosis of congenital cytomegalovirus infection by examination of

placenta: application of polymerase chain reaction and in situ hybridization. Pediatr Pathol Lab Med. 1997;17(2):249–58.

10. RIBEIRO CF, Silami VG, Brasil P, Nogueira RMR. Sickle-cell erythrocytes in the placentas of dengue-infected women. Int
J Infect Dis. 2012;16(1):e72.

11. LIMONTA D, González D, Capó V, Torres G, Pérez AB, Rosario D, et al. Fatal severe dengue and cell death in sickle cell
disease during the 2001-2002 Havana dengue epidemic. Int J Infect Dis. 2009;13(2):e77-78.
 7

Figure 1 – Light microscopic findings and immunostaining

A B

C D

E F

A- Loss of trophoblastic epithelium( ) and inflammatory infiltrate in villous stroma( )HE 40X (case 13); B- Immunostaining
in villous stroma cells 20 X (case 13); C-Proliferation and congestion of intravilliary vessels ( )and villous stroma edema (
)HE 40X (case 18); D- Immunostaining in villous stroma cells 40 X (case 18); E- Fibrin deposit in villi( ) , proliferation and
villous vascular congestion ( )HE 20X (case 10); F- Immunostaining in villous stroma cells and trophoblast 20X (case 10).
 8

Figure 2 – Immunostaining in placental tissue

A B C

F
D E
A-Negative control in liver tissue 20 X; B- Negative control in placental tissue 20 X; C- Positive control in liver tissue; D-
Immunostaining in decidual cells 20 X ( case 10); E- Immunostaining in villous stroma cells 40 X (case 17); F- Immunostaining
in trophoblast 20X (case 13).
 9

Table 1. Clinical characteristics and outcomes of pregnant women with dengue, histopathological and immunohistochemical findings in the placenta and outcomes
of the fetus

Patient Age Period Gestational SEC*** Laboratory Signs ofOther

hypoxia****
changes Immunohistochemistry***** Outcome Outcome
(days)* age Mother confirmation on light – staining area of the of the
(weeks) at microscopy mother fetus
birth
Mother newborn
1 17 58 39 Yes IgM+ Yes Choriodeciduitis, Villous stroma Discharge Discharge
multifocal
intervillositis
2 38 1 12 Yes IgM+ No Choriodeciduitis Trophoblast/villous stroma Discharge Miscarriage
3 16 8 34** Yes IgM+ Yes Deciduitis, PPA, Trophoblast Discharge Discharge
sickled
erythrocytes
4 23 4 41 No IgM+ No Focal villitis Decidua Discharge Discharge
5 33 13 34** Yes IgM+ Yes Choriodeciduitis, Trophoblast/villous stroma Discharge Discharge
intervillositis,
multifocal villitis,
villous necrosis
6 34 9 39 Yes IgM+ DENV2 Yes Choriodeciduitis, Trophoblast/villous Discharge Discharge
stroma/decidua
deciduitis,
intervillositis,
villitis, sickled
erythrocytes
7 17 11 40 No IgM+ Yes Choriodeciduitis, Decidua Discharge Discharge
decidua basalis
8 38 16 40 No IgM+ Yes PPA ****** Villous stroma/decidua Discharge Discharge
9 23 0 39 No IgM- IgM+ Yes Deciduitis, villitis Decidua/villous stroma Discharge Discharge
10 38 4 39 No IgM+ Yes Choriodeciduitis, Decidual/trophoblast Discharge Discharge
intervillositis,
villitis, infarction,
intervillous
thrombosis
11 17 0 40 No IgM+ No Deciduitis Decidua/villous stroma Discharge Discharge
12 19 3 38 No IgM+ Yes Villous hydrops Trophoblast Discharge Discharge
13 26 3 39 No IgM+ DENV2 Yes Deciduitis, Trophoblast/villous Discharge Discharge
stroma/decidua
multifocal necro-
proliferative villitis
14 17 5 28 Yes IgM+ Yes Deciduitis, villitis, Trophoblast/decidua Death Death
sickled
erythrocytes
15 19 13 11 Yes IgM+ No Deciduitis Trophoblast Discharge Miscarriage
16 25 7 33** Yes IgM+ Yes Focal intervillositis Villous stroma/decidua Discharge Discharge
17 21 1 40 No IgM+ Yes Irrelevant signs Villous stroma/decidua Discharge Discharge
18 28 6 34 Yes IgM+ Yes Sickled Villous stroma/decidua Death Death
erythrocytes
19 36 3 Yes IgM+ Yes Choriodeciduitis, Negative immunostaining Discharge Miscarriage
decidua basalis,
multifocal villitis,
subchorial
intervillositis
 10

20 35 4 10 Yes IgM+ No Choriodeciduitis, Negative immunostaining Discharge Miscarriage

intervillositis,
diffuse villitis,
decidua basalis
21 17 6 42 Yes IgM+ Yes Choriodeciduitis Trophoblasts/decidua Discharge Discharge
22 24 6 38 No IgM+ Yes Choriodeciduitis, Deciduous/trophoblast Discharge Discharge
PPA
23 32 3 12 No IgM+ No Multifocal Trophoblast/decidua Discharge Miscarriage
intervillositis
24 22 4 39 Yes IgM+ IgM+ Yes PPA Villous stroma Discharge Discharge
*Elapsed period between the onset of symptoms and childbirth
**Premature
***CAPILLARY LEAK SYNDROME IN THE MOTHER: hemoconcentration, hypotension, shock, pleural effusion, or ascites
****HYPOXIA: EDEMA OF THE VILLOUS STROMA, PRE-INFARCTION AREAS (INCREASE IN THE NUMBER OF SYNCYTIAL KNOTS AND
REDUCED INTERVILLOUS SPACE), OR CHORANGIOSIS
***** IHC: Immunohistochemistry
****** PPA: Preterm placental abruption