Vertical Exposure to Zika Virus and Its Consequences for Child Neurodevelopment (ZIKVIRUSIFF)
Vertical Exposure to Zika Virus and Its Consequences for Child Neurodevelopment (ZIKVIRUSIFF)
The recent increase in the number of cases of congenital microcephaly observed in Brazil is a
 reason of great concern. This increase occurred a few months after Zika virus (ZIKV) was
 introduced in the country, which was associated with reports of pregnant women presenting
 fever and rash illness during pregnancy. Thus, the hypothesis of a relationship between ZIKV
 infection and microcephaly became plausible. However, studies on the pathophysiology of
 maternal ZIKV infection, its consequences for the fetus, and the development of severe
 encephalopathy are still needed. Knowledge about the natural history of vertical transmission
 and its association with changes in fetal development in early life is still scarce. Studies
 on factors which determine the severity and clinical evolution, such as inflammatory response
 mechanisms, viral evolution, and development of serological tests to identify ZIKV infection,
 are still needed. The Aedes aegypti is responsible for the transmission of various types of
 viruses of interest to human health. Currently, it is primarily responsible for the
 transmission of the dengue, chikungunya, and ZIKV in epidemic proportions. In addition, it is
 not yet known whether there is an interaction between these viruses and whether the
 interaction can determine the severity of the disease. The aim of this study is to evaluate
 the natural history of ZIKV disease in two cohorts( pregnant women and children) starting
 with pregnant women or newborns or evennursing mothers, identifying risk biomarkers, mapping
 the anti-viral inflammatory response, evaluating the molecular evolution of the virus,which
 areimportant to determine the mechanisms of vertical viral infection and verify children
 neurodevelopment from birth to the end of 3rd year of life.
The mais goal of the study is to assess the natural history of vertical ZIKV transmission
 from pregnancy to the end of third year of life and the existence of risk factors associated
 with the incidence of clinical and neurological disorders, specially microcephaly.

 A longitudinal study will be conducted from gestation to the end of the third year of life,
 during which ZIKV infection will be the main exposure.

 Sample size will be 500 children exposed to zika virus. The sample size was calculated for
 rare events with basis on the usual microcephaly prevalence in Brazil (2:10.000) and expected
 increase in the number of cases among pregnant women exposed to ZIKAV.

 The study will be conducted at the Fernandes Figueira National Institute for Health of Women
 and Children (IFF). The pregnant women will be selected in the prenatal care clinic at IFF
 and in the Family Health Strategy units, at Centro de Saúde Escola Germano Sinval Faria
 (CDEGSF), which are both from FioCruz/RJ. We also will recruit pregnant woman from clinical
 family of SMS Rio de Janeiro.

 Inclusion criteria The study population will be composed of pregnant women who present
 symptoms compatible with ZIKAV infection, with skin rash, arthralgia-associated fever,
 myalgia, non-purulent conjunctivitis, or headache and asymptomatic pregnant women identified
 at the same time of possibility of exposure. We will include patients with RT- PCR positive.

 All pregnant women who are in the prenatal follow up at IFF and CSGSF, irrespective of the
 gestational age, are elegible as a not exposed in the beginning.

 Exclusion criteria Pregnant women with chromosomal abnormalities detected during fetal life
 or birth will be excluded.

 Definition of exposure Given the knowledge that about 80% of ZIKAV infections are
 asymptomatic, the exposure will be confirmed by the positivity in diagnostic tests (PCR or
 serological tests). Considering the future availability of serological tests, some strategies
 will be used to define exposure groups because up to now only the PCR test is available,
 which remains positive only in the acute phase of the disease.

 As described above, the symptomatic women will be referred for infection diagnostic
 confirmation by RT-PCR , and serologic tests for dengue, chikcungunha, CMV, toxoplasmosis,
 and rubella.

 Women with and without symptoms will have their blood sampled for serological tests in the
 three trimesters of pregnancy and at birth. Blood samples will be stored in a biorepository
 for ZIKA serological testing when specific serological tests are available. These sequential
 samples will also be used to validate serological tests that are in the development stage.

 The exposure groups to be compared will be composed with basis on the results of the
 serological tests. The asymptomatic individuals whose blood samples indicate the presence of
 Zika infection in the future, will migrate to the group of exposed ones.

 The pregnant women in the cohort presenting positive results for specific tests (PCR in the
 acute phase and serological tests later) will be considered exposed to infection whereas
 pregnant women with negative results will be considered not exposed.

 This second group can be categorized as asymptomatic and symptomatic. The third group will be
 the normal children born from mothers without zika proved by negative IgG and IgM Each child
 will be followed for three years from birth onwards.

 Outcomes to be analyzed The main outcome will be microcephaly and/or delay in development
 using Bayley Score 3rd edition at 12, 18 and 24-36 months of age.

 The secondary study outcomes will be as follows: pregnancy ultrasounds status, placenta
 injuries, ocular, hearing, cardiac, nutritional status of children and neurodevelopmental
 changes from birth to three years old, death in pregnant women, abortion, fetal death, and
 death during the first three years of life.

 Data collection:

 The inclusion of pregnant women will occur in the first 24 months of the study. To identify
 the outcomes, each child will be followed from birth to completion of three years of
 chronological age. The total study period for data collection will be of 5 years. The study
 will be conducted in the period Jan 2016 - Mar 2022.

 Data will be collected by health professionals who are linked to health services and provide
 assistance to the study population.

 Data on gestation, birth, and the first three years of life will be obtained from an
 interview with each woman using a questionnaire after appointment. In addition, data for
 records will be collected in forms prepared for this purpose.

 The data collection form (CRF/DCF) will be constructed and validated in all its dimensions
 among the services. POPs for the study flow diagram and for each procedure and care level
 will be prepared.

 -  Socioeconomic characteristics (education, per capita family income, housing conditions,
 sanitation, and garbage collection); health conditions (co-morbidities).

 -  History of exposure to ZIKAV: identification of relatives, neighbors, and work
 colleagues with Zika

 Follow up of the study population

 Follow up of the pregnant women:

 The pregnant women included in the study will be followed in the prenatal clinic, at the
 Fernandes Figueira National Brazilian Institute for Health of Women and Children.

 After inclusion of the pregnant women in the study, consultations will occur monthly, and
 exams will be performed according the following protocol:

 -  Fetal ultrasound at each trimester

 -  Serological exams for Zika, dengue, chikungunya, CMV, toxoplasmosis and rubella each
 trimester

 -  PCR for Zika virus if symptomatic pregnant woman

 -  Prenatal examinations as recommended by the Stork Network from Brazilian Minister of
 Health

 -  Prenatal examinations as recommended by the Brazilian Stork Network The amniotic fluid
 can be collected if indicated by the presence of associated congenital anomalies.

 Doppler:

 The umbilical cord will be insonated to obtain a sonogram of the umbilical artery and thus
 assess the pulsatility index (PI), resistance index (RI), and peak systolic velocity (PSV).

 Virus detection in serum and other human secretions For detection of virus in patients, RNA
 is extracted from serum and/or urine (200 ml) using the RTP Pathogen (Stratec Biomedical AG,
 Birkenfeld, Germany) kit according to the manufacturer's instructions. RNA isolated serves as
 a template in in real-time /RT-PCRreactions asdescribed by: Lanciotti et al., 2008 e Faye et
 al., 2013.

 Virus detection in serum and other human secretions For genomic sequencing, specific regions
 of the virus genome will be amplified by the RT-PCR method. The products thusobtained will be
 purified by agarose gel electrophoresis and then extracted from the gel using the QIAquick
 Gel Extraction (Qiagen) kit. After extraction, the material will be quantified in agarose gel
 by comparison between band intensities using the Low DNA Mass Ladder (Life Technologies)
 standard. The amount needed for sequencing will be forwarded to the Fiocruz sequencing
 platform. The sequences will be manually edited using the Bioedit (v. 7.0.5.3) program, and
 the phylogenetic analyses will be performed using the MEGA (v. 5:05) program.

 Analysis of the placenta and immunohistochemistry The fresh placentas will be forwarded to
 the Department of Pathological Anatomy and Cytopathology (DAPC/DPAC) where they will be kept
 in the refrigerator for up to 48 h. They are described, weighed, and photographed according
 to the routine for analysis of placenta. Fragments from the chorionic plate, cord, and
 membranes will be removed and frozen in liquid nitrogen. The placental plate (five areas),
 cord (at least two areas), and chorion (one fragment) will be fixed in buffered formalin
 maximum time: 30 h). They will be then cleaved for histological processing and embedded in
 paraffin.

 Phenotypic characterization of placental cells and maternal and umbilical cord blood and
 child oral swab by flow cytometry Placental fragments will be collected and maintained in
 sterile DMEM (Sigma; St. Louis, MO, [USA]) culture medium (4 °C) and cleaved into several
 fragments (thickness: 1-2 mm). After cleavage, they are washed in DMEM and subjected to
 dissociation cycles in collagenase 1A (Sigma; _) (300 U/mg; in 1% DMEM) enzyme. Dissociation
 will occur in 6-8 cycles (mild agitation; 37 °C, 10 min each). The cell suspension
 thusobtained will be centrifuged (1000 rpm; 4 °C; 10 min) and the pellet will be placed
 /suspended in DMEM with fetal calf serum (FCS; 10%; Sigma; _; 4 °C) to /inactivate /inhibit
 the collagenase enzyme /action /activity?. The dissociated material will [then] be filtered
 /in /through?a membrane ([pore diameter:] 70 mm). Then, the cells will be counted in a
 Neubauer chamber for use in the following procedures. The maternal and umbilical cord blood
 will be collected in /a blood /an appropriate tube containing EDTA as anticoagulant. The
 serum will be used for viral detection, and the leukocyte cells will be marked after lysis of
 red blood cells.\

 Necropsy of Fetuses and Stillbirths Histopathological and immunohistochemical exams and
 imaging morphogenesis study will be performed in brain and other organs from fetuses and
 stillbirths /in consequence of ZIKAV infection. Besides the usual histological processing in
 paraffin, the other organs will be frozen and submitted to extraction of genetic material and
 investigation of viral RNA. The eyeball will also be analyzed to evaluate possible ocular
 changes.

 Follow-up of newborns:

 Newborns will be followed until 3 years of age in the outpatient clinic at IFF, whose routine
 includes at least 6 visits in the first year, 4 visits/year in the second and third years. A
 multidisciplinary outpatient clinic will be established in both Unit of Infectious Diseases
 in Pediatrics and Pediatric Outpatient Clinic to follow the cohort of children with chance of
 vertical exposure to the ZIKA virus. The outpatient clinic will have medical personnel, and
 professionals in the areas of physical therapy, phonoaudiology, psychology, and social
 service. The medical team will be responsible for following the patients, in collaboration
 with specialties as neurology, pediatric cardiology and ophthalmology, and medical genetics
 service. Whenever necessary, admission of these patients to establish [either] the diagnosis
 or initial approach -{of the case} will occur in the DIPe ward.

 The initial evaluation as recommended for patients either known to be exposed to the virus or
 with microcephaly will consist of the following exams and/or evaluations: neurological,
 ocular, hearing, EEG if necessary, ECG, cord blood for RT-PCR and IgM. Anthropometric data
 and physical examination of the newborn infant, with emphasis on precise measurement of head
 circumference using the Intergrowth Curve (2014) as a reference, according to gestational age
 and patient gender. Evaluation by the Genetics to detect dysmorphic characteristics or
 congenital anomalies that can compromise /affect other organs.

 Imaging exams:

 1. transfontanelle sonography (USTF), as first choice

 2. cranial tomography (CT) without contrast, if TF is not technically possible

 3. Abdominal ultrasound

 4. Echocardiogram

 5. Brain magnetic resonance imaging, when indicated. g) Newborn Hearing Screening (TAN):

 1. Evoked optoacoustic emissions (EOAE); 24-48 h of life 2. Auditory Evoked Potential of
 Brain Stem (PEATE/ABR): up to 1 month of life in children with microcephaly. In case of
 failure, retesting must be done within 30 days. In case of retest failure, forward [the NB]
 immediately [to _].

 h) Ocular Neonatal Screening (TON):

 1. Inspection and Testing of retinal Red Reflection.

 2. Eye Fund exam (fundoscopy)

 3. Documentation with RetCam

 In the subsequent consultations, the clinical follow up, PC measurement, development
 evaluation (including early stimulation) by physical therapy, and morbidity and mortality
 evaluation will be maintained. In the first year of life, PEATE will be performed every three
 months. The subsequent clinic visits will occur monthly until 6 months of age, every two
 months in the period 6-12 months, and every three months until three years [of life]. The
 ophthalmologic evaluation (funduscopic examination) will be held every return.

 The neurological follow up will depend on the initial condition of the patient. Patients
 without microcephaly will be evaluated at birth, 30 days, and 2, 4, 6, 9, 12, 18, 24, and 36
 months. Patients with microcephaly will be evaluated at birth, 30 days, and every 2 or 3
 months, or more frequently if necessary.

 Evaluation of motor development will be done by the Dubowitz examination (in the first 72 h),
 Alberta Infant Motor Scale (AIMS; every three months until the child walk or complete 18
 months of life), and Bayley Test-3rd edition at 12,18 and between 12-36 months of age.

 Evaluation of language development will be performed at 12, 24, and 36 months, with regard to
 possible effects on the neurodevelopment, cognitive development, and language skills.
Inclusion Criteria:

 The study population will be composed of pregnant women who present symptoms compatible
 with ZIKAV infection, with skin rash, arthralgia-associated fever, myalgia, non-purulent
 conjunctivitis, or headache and asymptomatic pregnant women identified at the same time of
 possibility of exposure. We will include patients with RT- PCR positive.

 All pregnant women who are in the prenatal follow up at IFF and CSGSF, irrespective of the
 gestational age, are elegible as a not exposed in the beginning.

 Children born during the outbreak by mothers with confirmed infection, suspected infection
 and normal pregnancy

 Exclusion Criteria:

 Pregnant women with chromosomal abnormalities detected during fetal life or birth will be
 excluded.

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