Detection of Zika virus infection among asymptomatic pregnant women in the North of Peru
Weilg?et?al. BMC Res Notes
Detection of?Zika virus infection among?asymptomatic pregnant women in?the?North of?Peru
Claudia Weilg 0 1 2
Lucinda Troyes 3
Zoila Villegas 3
Wilmer Silva?Caso 0 1 2
Fernando Mazulis 0 1 2
Ammy Febres 3
Mario Troyes 3
Miguel Angel Aguilar?Luis 0 1 2 4
Juana del Valle?Mendoza 0 1 2
0 School of Medicine, Research and Innovation Centre of the Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas , Av. San Marcos Cuadra 2, Chorrillos, Lima , Peru
1 School of Medicine, Research and Innovation Centre of the Faculty of Health Sciences, Universidad Peruana de Ciencias Aplicadas , Av. San Marcos Cuadra
2 Laboratorio de Biologi?a Molecular, Instituto de Investigacio?n Nutricional , Lima , Peru
3 Direccio?n Subregional de Salud de Jae?n, Ministerio de Salud , Cajamarca , Peru
4 Instituto de Investigacio?n de Enfermedades Infecciosas , Lima , Peru
Objective: To report an outbreak of ZIKV infection among asymptomatic pregnant women during 2016 in the city of Jaen, Cajamarca. Results: Zika virus RNA was detected in 3.2% (n = 36) of cases by RT? PCR. The mean age of patients positive for ZIKV infection was 29.6 years. 7 patients (19.4%) infected with ZIKV were in their first? trimester of gestation, 13 (36.1%) were in their second? trimester, and 16 (44%) were in their third? trimester. All of the infected pregnant women were asymptomatic. ZIKV infection remains a major public health issue that calls for constant epidemiological surveillance. It can cause the congenital Zika virus syndrome in the newborns of infected mothers. The lack of molecular diagnostic methods in isolated localities and the similarity of symptoms to other arboviral infections, lead to an under? diagnosis of this disease in endemic areas.
Zika virus; Pregnant women; Outbreak of Zika; Peru
Zika virus (ZIKV) is an emergent arthropod-borne
flavivirus associated with neurologic complications [
first reports of human disease were described in Uganda
and Tanzania during an outbreak in 1952 [
February 2016, the World Health Organization (WHO)
declared Zika virus infection a Public Health Emergency
of International Concern (PHEIC) due to the increasing
number of cases and its associated complications.
Currently, ZIKV is responsible for the ongoing outbreaks in
the Caribbean and South America following the initial
outbreak in Brazil [
The primary route of human ZIKV transmission is
through the bite of mosquitoes Aedes aegypti and Aedes
albopictus in tropical and temperate regions [
is also transmitted via transfusion of blood products,
organ transplantation, laboratory exposure, sexual and,
vertical maternal?fetal transmission [
]. After an
incubation period of 2?14?days, patients develop an acute
febrile syndrome associated with unspecific symptoms
such as headaches, arthralgia, pruritic rash, nonpurulent
conjunctivitis, myalgia, anorexia, asthenia, dysesthesia
and retro-orbital pain [
]. Despite the low frequency
of fatalities and hospitalization due to severe disease,
ZIKV infection is associated with a high frequency of
neurologic complications such as congenital
microcephaly  and Guillain?Barre syndrome [
infection during pregnancy can cause a set of fetal
developmental complications, known as the congenital Zika
virus syndrome [
]. Along with this syndrome, ZIKV is
also associated with a higher rate of pregnancy loss and
stillbirths among infected women [
In Peru, the first ZIKV infection reported was a
Venezuelan immigrant in January of 2016 [
On March of 2016, a middle-aged woman from Lima,
infected with ZIKV via sexual transmission, became
the first autochthonous case in Peru reported by the
Ministry of Health . In the latest epidemiological
update submitted by the PAHO in August of 2017, they
describe a decreasing trend in the number of cases
confirmed in South America since March of 2016 [
In contrast, some Peruvian regions such as Cajamarca,
Loreto, Ica, Tumbes, La Libertad, and Lima, 8080
cases have reported with an increasing trend of
confirmed cases since 2016 [
]. To date, no pregnancy
complications or congenital microcephaly have been
attributed to a ZIKV infection in Peru, according to
the Peruvian Ministry of Health . This study aims
to describe an outbreak of ZIKV infection in a newly
endemic region of Peru among asymptomatic pregnant
women and evaluate newborn congenital
complications in the city of Cajamarca in 2016.
This study was carried out between May and July of
2016 in the province of Jaen. This town has a
population of 135,021, is located in the North of Peru in the
city of Cajamarca and is endemic to other arboviral
diseases such as dengue. Cajamarca is one the poorest
regions in Peru, with a 56% of its population living in
poverty compared to the national average of 36% [
Cajamarca is also one of the regions with a higher
incidence of maternal deaths with 31 cases reported a year
prior this study in 2015 [
Venous and urine samples of 1116 asymptomatic
pregnant patients, were collected in the Regional
Laboratory of the Jaen Medical Health Network as part of
the pregnancy screening program. The inclusion
criteria were pregnant patients with a current or recent
history of residence in Jaen. Pregnant women with acute
undifferentiated febrile illness with symptoms such as
headaches, muscle pain, ocular and/or joint pain, rash,
nausea, asthenia, anorexia, or any other symptom that
could suggest an infection.
Patients positive to ZIKV were included in a
microcephaly protocol with ultrasound evaluation upon
diagnosis and every three thereafter. The cephalic
perimeter of the newborn was measured at birth and
at 24? h. Microcephaly was defined as an
occipitofrontal diameter greater than two standard deviations
below the mean or less than the 3rd percentile based
on standard growth charts for sex, age, and gestational
age at birth [
]. Newborns were followed up 6?months
later by a pediatric neurologist.
Samples were collected following a written informed
consent signed by all participants before enrollment
or by the parents or respective guardians in the case of
underage patients below 18?years of age. The study was
approved by the Research Ethics Board of the Hospital
Docente Regional de Cajamarca in Peru.
A venous blood sample was collected per patient by
using Vacuette? EDTA blood collection tubes (Greiner
Bio-One GmbH, Frickenhausen, Germany) and these
were stored at 4??C until processing.
The RNA was extracted from 200? ?l of blood samples
using High Pure Kit Preparation template (Roche
Diagnostics GmbH, Mannheim, Germany).
Real?time RT?PCR assay for?Detection ZIKV with?taqman probe
A one-step RT-PCR was performed as described by
Alva et?al. [
]. The primers and the probe used are the
ones described by Faye et?al. [
1116 samples of pregnant women were studied from
May to July 2016. None of the recruited samples were
excluded from the data analysis due to exclusion
criteria. Of all the samples, 3.2% (n = 36) of cases were
positive for ZIKV by RT-PCR.
Of the positive cases for infection by ZIKV confirmed
by RT?PCR, the mean age of patients was 29.6? years
and 8.3% (n = 3) were below 18? years of age. In
relation to the gestational age of the infected mothers, 7
patients (19.4%) were in the first trimester, 13 (36.1%)
were in the second trimester and 16 (44%) were in their
third trimester of pregnancy (Table?1).
All the ultrasound evaluations (n = 33) were negative
for microcephaly, and the head circumference
measured at birth and during the postnatal controls were
within normal limits. According to the evaluation of
normal growth and development of the newborns of
the infected mothers, there was no anomaly found.
Three infected pregnant women with a diagnosis
during their third trimester were lost before the follow-up.
Zika virus infection has emerged as a major public
health issue in the Americas. The broad geographical
range of the vector and the emerging complications of
the ZIKV infection have made this flavivirus a growing
global concern that calls for constant epidemiological
surveillance and a strategic response plan [
]. One of
the target populations for the public health strategies
are pregnant women because of the associated
complications that ZIKV infection could have on them and
their newborns [
]. Our findings show a prevalence
of 3.2% (n = 36) of ZIKV infection among the
asymptomatic pregnant women population of Jaen in the
region of Cajamarca, which is higher than the 31
confirmed cases of ZIKV in asymptomatic women during
2016 in Peru [
]. The mentioned findings may
indicate that there is an underestimation of ZIKV
infection that could be explained by the lack of sensitive
and specific molecular diagnostic methods in isolated
localities. Furthermore, the prevalence in this study
highlights the importance of epidemiological
surveillance of ZIKV infection in pregnant women that live in
newly ZIKV endemic regions such as Jaen. These
findings cannot be extrapolated to the ZIKV infection rates
among pregnant women in other endemic regions of
Peru because of the lack of similar molecular diagnostic
studies in other Peruvian localities. In South America,
the incidence of ZIKV peaked in February 2016 and
subsequently showed a declining tendency with the
exception of Ecuador [
] Peruvian data showed an
opposite trend that may be due to an underestimation
of the prevalence during 2016 caused by the lack of a
consolidated surveillance program.
Congenital ZIKV syndrome may include
microcephaly, ventriculomegaly, intracranial calcifications,
extra-axial fluid, decreased brain parenchymal volume,
lissencephaly, cerebellar hypoplasia, delayed myelination
and hypoplasia of the corpus callosum [
Furthermore, newborns of infected mothers can develop
cardiac anomalies with septum defects [
], hearing loss
], seizures due to the underlying brain malformations,
neuromotor abnormalities such as spasticity and feeding
], ocular abnormalities  and can be
born small for the gestational age [
The most plausible ZIKV pathogenesis hypothesis of
neurological complications describe that placental
colonization and injury following maternal infection leads to
the transmission of the virus to the fetal brain [
where ZIKV preferentially infects neural progenitor cells
]. As a result, this arbovirus disrupts neuronal growth,
migration, proliferation, and differentiation, thus
causing thinning of the cortex, macroscopic signs of
microcephaly and impairment of normal brain development in
the fetus [
]. This hypothesis also suggests the
possibility that the placental inflammatory process contributes
to the development of mentioned neurological
The maternal age is also strongly associated with
different pregnancy outcomes [
]. The risk of having a
child with a congenital anomaly increases with advanced
maternal age [
], and it is not always related to the
recognized increase in aneuploidy rate . The US
National Birth Defects Prevention Study found that
newborns of women older than 40?years old had an increased
risk of cardiac defects, craniosynostosis, esophageal
atresia and hypospadias [
]. Also, studies consistently report
that women older than 35? years old have a significantly
increased risk of spontaneous abortion [
stillbirth compared to younger women [
]. ZIKV infection
of pregnant women can also cause pregnancy losses and
congenital malformations such as cardiac defects [
and craniosynostosis [
], which may cause bias when
estimating the risk of congenital ZIKV syndrome among
women older than 35? years old. In our study, the mean
age of infected pregnant women was 29.6?years and only
5.6% (n = 2) were older than 35?years.
The risk of vertical transmission and congenital ZIKV
syndrome exists for both symptomatic and asymptomatic
]. It is important to mention that only
20% of the patients positive for ZIKV infection are
], which is why international guidelines
recommend health care providers to screen all pregnant
women for possible exposure to ZIKV at each prenatal
visit, and to test those with ZIKV exposure risk three
times during pregnancy even if they are asymptomatic
The frequency of birth defects resulting from vertical
transmission of ZIKV is not well established [
However, the risk of developing the congenital ZIKV
syndrome is higher during the first and second trimester,
even though severe fetal or newborn sequelae can
also occur within the third-trimester infection [
]. A prospective study in Brazil found that adverse
outcomes were reported in 55% of pregnancies after
first-trimester maternal infection, 52% after
second-trimester maternal infection, and 29% after
third-trimester maternal infection . Our study found that most
of the infected pregnant women (44%, n = 16) got ZIKV
infection during the third trimester and that none of
the newborns developed microcephaly. These findings
are similar to those published by the Peruvian National
Epidemiological Center for Control and Prevention of
] and can explain why there are no reported
cases of congenital ZIKV syndrome in Peru yet.
Nonetheless, 19.4% (n = 7) and 36.1% (n = 13) of pregnant
women were infected during their first and second
trimester, respectively. This fact highlights the importance
of prenatal ultrasonography to evaluate fetal
abnormalities consistent with congenital ZIKV syndrome [
], and the need of careful clinical evaluation of these
infants in ruling out microcephaly, ocular
abnormalities, hearing loss, neurologic and positional
abnormalities, craniofacial disproportion or cardiac defects .
In conclusion, the disease caused by ZIKV is an
emerging infection that remains a major public health
issue in South America due to their endemicity in
isolated localities. This study reports the first
molecularly confirmed outbreak in the region of Cajamarca
in Peru among asymptomatic pregnant women and
describes a higher frequency of infection in comparison
to the national surveillance report. The unavailability
of molecular diagnostic methods in isolated localities
and the similarity of symptoms to other arboviruses
leads to an under-diagnosis of this infection in
arboviral endemic regions therefore, it is molecular
diagnosis of ZIKV infection in pregnancy screening programs
to evaluate and measure the impact of fetal anomalies
associated with this virus, especially amongst
asymptomatic patients in endemic regions.
This study has two main limitations. First, the
followup evaluation of the infant carried out by a pediatric
neurologist, only occurred at 6? months of age. Second,
there wasn?t a follow up protocol looking for other
ZIKV associated malformations for the newborns of
ZIKV: Zika virus; RT?PCR: reverse transcription?polymerase chain reaction; RNA:
ribonucleic acid; bp: base pairs.
JdVM LT and ZV designed the study protocol. CW, WS, FM, MAAL performed
the RT?PCR and data analysis. JdVM and MAAL are responsible for obtaining
funding and laboratory work supervision. ZV, LT, MT and AF was responsible
for the clinical assessment, samples collection and database completion.
JdVM, WS, FM and CW drafted the manuscript. All authors critically revised the
manuscript for intellectual content. All authors read and approved the final
We thank to the staff of the health network from Jaen, Cajamarca, Peru.
We thank Brandy Russell at the Centers for Disease Control and Prevention
(CDC, Fort Collins, CO, USA) provided positive controls for ZIKV.
The authors declare that they have no competing interests.
Availability of data and materials
Abstraction format used in the study and dataset are available and accessible
from corresponding author upon request in the link: https://figshare.com/artic
Consent to publish
Ethics approval and consent to participate
This study has been approved by two independent Ethics Committees from
Hospital Regional Docente de Cajamarca. All samples were analyzed after a
written informed consent signed by all participants before enrollment or by
the parents or children?s caregivers in the case of underage patients below
18 years of age.
This work was supported by Programa Nacional de Innovaci?n para la Com?
petitividad y Productividad (Inn?vate Per?), under the Contract 116?PNICP ?
PIAP?2015 and Cienciactiva of CONCYTEC under the Contract 164?2016?FON?
DECYT. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Springer Nature remains neutral with regard to jurisdictional claims in pub?
lished maps and institutional affiliations.
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