Clinical assessment and brain findings in a cohort of mothers, fetuses and infants infected with ZIKA virus

Accepted Manuscript Clinical Assessment and Brain Findings in a Cohort of Mothers, Fetuses and Infants Infected with Zika Virus Magdalena Sanz Cortes, MD, PhD, Ana Maria Rivera, MD, Mayel Yepez, MD, Carolina V. Guimaraes, MD, Israel Diaz Yunes, MD, Alexander Zarutskie, BS, Ivan Davila, MD, Anil Shetty, PhD, Arun Mahadev, BS, Saray Maria Serrano, MD, Nicolas Castillo, MD, Wesley Lee, MD, Gregory Valentine, MD, Michael Belfort, MD, PhD, Guido Parra, MD, Carrie Mohila, MD, PhD, Kjersti Aagaard, MD, PhD, Miguel Parra, MD, PhD PII:

S0002-9378(18)30013-9

DOI:

10.1016/j.ajog.2018.01.012

Reference:

YMOB 12038

To appear in:

American Journal of Obstetrics and Gynecology

Received Date: 2 November 2017 Revised Date:

4 January 2018

Accepted Date: 8 January 2018

Please cite this article as: Sanz Cortes M, Rivera AM, Yepez M, Guimaraes CV, Diaz Yunes I, Zarutskie A, Davila I, Shetty A, Mahadev A, Serrano SM, Castillo N, Lee W, Valentine G, Belfort M, Parra G, Mohila C, Aagaard K, Parra M, Clinical Assessment and Brain Findings in a Cohort of Mothers, Fetuses and Infants Infected with Zika Virus, American Journal of Obstetrics and Gynecology (2018), doi: 10.1016/j.ajog.2018.01.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT CLINICAL ASSESSMENT AND BRAIN FINDINGS IN A COHORT OF MOTHERS, FETUSES AND INFANTS INFECTED WITH ZIKA VIRUS

Magdalena Sanz Cortes1, MD, PhD; Ana Maria Rivera2, MD; Mayel Yepez1, MD; Carolina V. Guimaraes3, MD; Israel

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Diaz Yunes2, MD; Alexander Zarutskie1, BS; Ivan Davila1, MD; Anil Shetty1, PhD; Arun Mahadev1, BS; Saray Maria Serrano4, MD; Nicolas Castillo2, MD; Wesley Lee1, MD; Gregory Valentine5, MD; Michael Belfort1, MD, PhD; Guido Parra2, MD; Carrie Mohila6, MD, PhD; Kjersti Aagaard1, MD, PhD; Miguel Parra2, MD, PhD

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1. Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine & Texas Children’s Hospital, Houston, TX, United States

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2. Department of Obstetrics and Gynecology. Division of Maternal Fetal Medicine. Cediul-Cedifetal, Barranquilla, Colombia.

3. Department of Radiology, Baylor College of Medicine & Texas Children’s Hospital, Houston, TX, United States 4. Department of Radiology. Cediul-Cedifetal, Barranquilla, Colombia. 5.

Department of Pediatrics, Division of Neonatal Medicine, Baylor College of Medicine &

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Texas Children’s Hospital, Houston, TX, United States

6. Department of Pathology and Laboratory Medicine, Baylor College of Medicine & Texas Children’s Hospital,

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Houston, TX, United States

Financial support: A portion of the effort for this work was funded by the NIH (grant number R01NR014792 to K.M.A) and the March of Dimes Prematurity Research Initiative (K.M.A.). The authors report no conflict of interest.

Paper presentation information: This work was presented as abstracts 73 and 205 at the 37th Annual Pregnancy Meeting, January 23-28, 2017, Las Vegas, Nevada.

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ACCEPTED MANUSCRIPT Corresponding Author: Magda Sanz Cortes, MD, PhD Associate Professor Baylor College of Medicine & Texas Children’s Hospital

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Division of Maternal-Fetal Medicine 6651 Main St Houston, TX, 77030 Phone: 713 798-8467

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Email: [email protected]

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Abstract Word Count: 520

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Word Count: 5581

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ACCEPTED MANUSCRIPT Condensation: Detailed neuroimaging assessment of brain anomalies in fetuses and infants affected by congenital ZIKV infection. Short title: Fetal and infant neuroimaging in congenital ZIKV infection. Implications and Contributions:

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This study was performed to assess brain anomalies detected in congenital ZIKV infection using different fetal and postnatal neuroimaging techniques with advanced MRI processing, to quantify fetal brain volumes and microstructural features.

One of the main findings of this study is that microcephaly is not detected in all cases of congenital ZIKV infection and it

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could be an end-point of the disease resulting from progressive changes related to brain volume loss.

This study adds an objective quantification of brain anomalies detected in fetuses and infants affected with congenital

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ZIKV syndrome that may provide further insight on the potential mechanisms involved in the development of brain

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lesions.

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ACCEPTED MANUSCRIPT ABSTRACT Background: Congenital Zika virus (ZIKV) infection can be detected in both the presence and absence of microcephaly, and manifests as a number of signs and symptoms detected clinically and by neuroimaging. However, to date, qualitative and quantitative measures for the purpose of diagnosis and prognosis are limited.

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Objectives: Main objectives of this study conducted on fetuses and infants with confirmed congenital ZIKV infection and detected brain abnormalities are: 1) To assess the prevalence of microcephaly and the frequency of the anomalies including a detailed description based on ultrasound and magnetic resonance imaging (MRI) in fetuses and ultrasound, MRI and computed tomography imaging postnatally; 2) To provide quantitative measures of fetal and infant brain

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findings by MRI using volumetric analyses and diffusion weighted imaging (DWI); 3) To obtain additional information from placental and fetal histopathological assessments and postnatal clinical evaluations.

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Study design: This is a longitudinal cohort study of ZIKV-infected pregnancies from a single institution in Colombia. Clinical and imaging findings of patients with laboratory confirmed ZIKV infection and fetal brain anomalies were the focus of this study. Patients underwent monthly fetal ultrasound scans, neurosonography and a fetal MRI. Postnatally, infant brain assessment was offered by using ultrasound, MRI and/or computed tomography. Fetal head circumference measurements were compared to different reference ranges using < 2 or 3 standard deviations(SD) below the mean for the

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diagnosis of microcephaly. Fetal and infant MRI images were processed to obtain a quatitative brain volumetric assessment. DWI sequences were processed to assess brain microstruture. Anthropometric, neurological, auditory and

termination.

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visual assessments were performed postnatally. Histopathological assessment was included if patients opted for pregnancy

Results: All subjects (n=214) had been referred for ZIKV symptoms during pregnancy affecting themselves or their

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partners or if fetal anomalies compatible with congenital ZIKV syndrome were detected. A total of 12 pregnant patients with laboratory confirmation of ZIKV infection were diagnosed with fetal brain malformations. Most common findings assessed by prenatal and postnatal imaging were: brain volume loss (92%), calcifications (92%), callosal anomalies (100%), cortical malformations (89%), and ventriculomegaly (92%). Results from fetal brain volumetric assessment by MRI showed how one of the most common findings associated with microencephaly was reduced supratentorial brain parenchyma and increased subarachnoid cerebrospinal fluid. DWI analyses of apparent diffusion coefficient (ADC) values showed microstructural changes. Microcephaly was present in 33.3-58.3% of the cases at referral and present at

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ACCEPTED MANUSCRIPT delivery in 55.6.7-77.8% of cases. At birth, most of the affected neonates (55.6-77.8%) had head circumference measurements more than 3SD below the mean. Postnatal imaging studies confirmed brain malformations detected prenatally. Auditory screening results were normal in 2 cases assessed. Visual screening showed different anomalies 2 of the 3 cases examined. Pathology results obtained from two out of the three cases that opted for termination showed similar

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signs of abnormalities in central nervous system and placental analyses, including brain microcalcifications. Conclusion: Congenital microcephaly is not an optimal screening method for congenital ZIKV syndrome, as it may not accompany other evident and preceding brain findings; microcephaly could be an end-point of the disease resulting from progressive changes related to brain volume loss. Long-term studies are needed to understand the clinical and

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developmental relevance of these findings.

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Key words: Congenital ZIKV infection, neuroimaging, microcephaly, brain malformations, fetal brain MRI, Zika virus,

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neurodevelopment, calcifications, ventriculomegaly, neurotropic virus, congenital infection, pregnancy, flaviviridae.

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ACCEPTED MANUSCRIPT INTRODUCTION

Almost 10% of women with a perinatal infection with Zika virus (ZIKV) result in congenital ZIKV syndrome (CZS)1, inclusive of brain and ocular malformations, microcephaly, congenital deafness, limb contractures and arthrogryposis2–8. It

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has been shown how a rate of 10% ZIKV infection in general population would be expected to produce a prevalence of 13.2% of microcephaly9. The association between maternal ZIKV infection and congenital malformations were first reported in late 2015, as ZIKV spread throughout the Americas and was associated with a 20-fold increase in the reported rate of microcephaly in Northern states in Brazil10. Given these early reports from Brazil, the National Health Institute in

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Colombia began active surveillance for ZIKV, documenting the first cluster of cases in October 201511. Colombia is recognized as heavily endemic, with the city of Barranquilla having a significant prevalence of infection among pregnant

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women11. Similar to observations in Brazil and across the Americas, a four-fold increase in the rate of congenital microcephaly has been observed in Colombia during the current ZIKV pandemic12. Although initial descriptions of the effects of in utero ZIKV infection were centered prominently on the finding of microcephaly13, it is now accepted that signs and symptoms of congenital infection are both present in absence of a reduced head size, and precede microcephaly6. Microcephaly (defined as a head circumference <3 standard deviations

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(SD) below the mean) that is associated with ZIKV infection is likely a time-dependent event. Its antenatal appearance and detection are dependent on both timing of the insult as well as the lag interval from the initial exposure14. There is an absence of consensus over how to reach the diagnosis of microcephaly -as different cutoffs have been proposed- or over

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which nomograms should be used to make this assessment15,16. As a result of these limitations and lack of consensus, screening recommendations for detecting fetuses affected by congenital ZIKV have ranged from screening for

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microcephaly alone to a full neuroanatomical evaluation.15–17 Moreover, limited and largely selective neuroimaging studies have qualitatively described a number of findings in association with congenital ZIKV infection, which may or may not occur with microcephaly. These, have failed to provide associated postnatal imaging or neurodevelopmental clinical exam findings6,8,18–21. Similarly, quantitative assessment using advanced post-processing imaging methods or additional imaging sequences have not yet been reported in the context of congenital ZIKV infection, resulting in largely descriptive and subjective case definitions. Our study includes a cohort of ZIKV-infected pregnant patients with detailed longitudinal neuroimaging assessment of

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ACCEPTED MANUSCRIPT their fetuses and infants prospectively. Advanced MRI processing was performed using fetal and infant’s images to assess quantitatively the effects of CZS in order to improve our knowledge on the underlying mechanisms of perinatal brain injury caused by ZIKV. The aims of this study were: 1) to assess the prevalence of microcephaly in the presence of other brain findings in a cohort

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of infected and affected fetuses and infants, using different standards; 2) to determine the similarity or variation of these findings in a cohort from Colombia compared with other congenital ZIKV series reported to date; 3) to provide quantitative measures of fetal and infant brain findings in congenital ZIKV infection by MRI using volumetric analyses and diffusion weighted imaging. In order to meet these study objectives, we qualitatively and quantitatively characterized

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fetal and infant findings with common and advanced neuroimaging techniques among a prospective cohort of pregnant women with ZIKV-infection in a single institution in Barranquilla, Colombia. Here we report demographic, clinical,

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laboratory, and imaging findings using ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) to

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correlate prenatal screening with postnatal clinical findings.

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ACCEPTED MANUSCRIPT MATERIAL AND METHODS

Study design. A prospective study was performed in Cediul-Cedifetal Clinic, Barranquilla, Colombia from December 2015 through July 2016. Cediul is a referring center for fetal imaging in the Colombian Caribbean coast, performing over

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15,000 fetal ultrasounds per year. Within their staff there are 4 obstetricians and 3 maternal-fetal medicine specialists, each with over 15-year expertise on high-risk fetal imaging. All subjects that were referred for ZIKV symptoms during pregnancy (or 8 weeks prior to last menstrual period) with themselves or their partners were included in this study. Also, pregnant subjects that were diagnosed with fetal anomalies compatible with CZS were also sent to our center. A total of

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214 subjects were evaluated. We used a standard form to collect personal and clinical data. Mothers gave information on illness during pregnancy compatible with ZIKV infection with or without serological confirmation by themselves or their

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sexual partners. The initial case definition for ZIKV was fever and at least one of the following symptoms: non-purulent conjunctivitis, headache, rash, pruritus or arthralgia with no known alternative cause. On December 24th, 2015 the symptom criteria were revised to include fever and rash plus at least one of the following symptoms: non-purulent conjunctivitis, headache, pruritus, arthralgia, myalgia or malaise.

All subjects enrolled in this study gave their written informed consent to participate. This study was approved by the local

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ethics committee of Cediul (IRB 0003-2016). Interpretation of the obtained ultrasound and MRI images and image post processing were performed at Baylor College of Medicine (Houston, USA), under an IRB approved protocol (H-39104). All pregnancies were dated by first trimester ultrasound crown-rump length assessment. Maternal serologies for

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Cytomegalovirus (CMV), Toxoplasma, Venereal Disease Research Laboratory (VDRL), human immunodeficiency virus (HIV), Rubella and Herpes virus serologic testing were abstracted from their prenatal medical records. If maternal

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serologies were positive for ZIKV and/or fetal brain anomalies were detected by ultrasound, an amniocentesis was offered to test amniotic fluid for PCR of ZIKV. In all cases fetal karyotype was also obtained. For those cases with positive maternal serologies for other infections (such as CMV, Toxoplasmosis etc), PCR in amniotic fluid was requested and performed.

Other causes of microcephaly such as exposure to licit and illicit drugs, toxic substances and ionizing radiation were excluded in fetuses/infants categorized as affected by CZS.

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ACCEPTED MANUSCRIPT Fetal ultrasonography. Fetal ultrasound follow up was offered on a monthly basis including fetal growth, Doppler and anatomic assessment (see Suppl. Material). Fetal growth parameters were compared to Hadlock et al. 22and Intergrowth 2123 nomograms. Additionally, head circumference was compared to Kurmanavicious et al. 24, Chernevak et al.25 and local Colombian reference standards. The latter were constructed using a historical cohort (2011-2012) of 772 healthy

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Colombian subjects from the Barranquilla area, that were evaluated at Cediul-Cedifetal prospectively during their pregnancy with normal fetuses. These fetal biometric analyses were used to establish normal reference ranges in a comparable cohort. Fetal head circumference measurements were categorized as less and equal or above 2 and 3 SDs below the mean for gestational age.

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Neurosonography. A detailed scan of the fetal brain was performed at least once during the course of pregnancy, following previously defined guidelines26 by an experienced MFM specialist (M.P.S) as detailed in Suppl. Material.

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Fetal brain MRI. MRI was offered to all cases with abnormal brain ultrasound findings. It was performed on a 1.5 Tesla Phillips Achieva scanner-software version 5.1.7- (Phillips North America, Andover, MA). No maternal or fetal sedation was used. Details on image and diffusion weighted imaging acquisition are described in Suppl. Material. This additional sequence provides indirect information on fetal brain microstructure, based on the diffusivity of the water molecules within the brain.

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Image post-processing. Fetal brain segmentation (Suppl. Fig.1), volumetric assessment and diffusion weighted imaging analysis to obtain apparent diffusion coefficients (ADC) values from different regions of interest, were performed by two

diffusion sequences.

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examiners (A.Z and I.E.D) as described in Suppl. Material. ADC values were assessed on 3 prenatal MRIs with available

In order to compare fetal brain volumetric values and diffusion weighted imaging results from ZIKV cases, a group of 10

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healthy fetuses that underwent fetal brain MRI at a comparable gestational age (+/-1 week) were included as controls (Suppl. Material). Additionally, the obtained ADC values from ZIKV fetuses were compared to previously published nomograms for the same regions at same gestational ages27. Perinatal outcomes. Perinatal results were recorded in all cases. Neonatal head circumferences were compared to two different nomograms (Intergrowth 2128 and Fenton29) and were used to apply the cutoff for microcephaly proposed by the Brazilian government19 (head perimeter ≤ 32 cm for term infants, or < 2 SD below the mean for age and sex on Fenton curves for preterm neonates29). An auditory brainstem response evaluation and visual evoked potentials were performed in

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ACCEPTED MANUSCRIPT only two and 3 cases for hearing and central visual function screening respectively although testing was offered in all cases. Postnatal brain imaging. Parents and legal guardians were invited to continue with the follow up of their infants in our center including different imaging tests (transcranial ultrasound, CT scan and MRI). Imaging acquisition details in Suppl.

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Material. Statistical analysis. Descriptive statistics were used for data analysis, carried out using the Statistical Package for Social Sciences (SPSS), version 21.0 (IBM Corporation, Armonk, NY, USA). Student´s t test for independent samples and Pearson´s chi-squared or Fisher´s exact tests were used to compare quantitative and qualitative data respectively. Results

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were considered to be significant at a p-value <0.05.

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ACCEPTED MANUSCRIPT RESULTS From the 214 subjects that were initially referred, 12 presented with antenatal evidence of significant brain abnormalities and positive laboratory results for ZIKV infection. Details regarding these pregnancies and deliveries are provided in Suppl. Table 2.

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All 12 pregnant patients were able to recall whether they or their partners did or did not have symptoms suggestive of ZIKV infection: 3 of 12 reported no symptoms themselves during pregnancy or in the 2 months preceding last menstrual period, but 2 of these 3 reported symptoms in their partners, and 1 of these 3 denied symptoms in either themselves or their partners. Of the 9 of 12 pregnant patients with symptoms, 8 were symptomatic in the first trimester, 1 in the second

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trimester, and none in the third trimester (Table 1). The most common constellation of symptoms was the appearance of rash and fever. Eight of the 9 symptomatic cases presented with 2 symptoms, and one case developed 6 symptoms. Of the

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3 of 12 asymptomatic pregnant patients (case 3, 4 and 10), case 4 and case 10 reported unprotected sexual intercourse within 1 week from the appearance of symptoms. Additionally, Case 9 was symptomatic at the same time as her partner and both manifest as fever, rash and conjunctivitis (Table 1).

Fetal assessment. All 12 cases were defined by a negative maternal IgM serology for VDRL, toxoplasmosis, herpes virus, rubella and HIV. Only one case (Case 1) was found to have a positive CMV IgM serology but CMV specific PCR

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in amniotic fluid was negative. A karyotype with or without chromosomal microarray (CMA) was obtained in all cases. Case 1 had a normal CMA variant: microduplication 14q32.33. All other cases had normal karyotypes. Laboratory confirmation of ZIKV infection consisted on 7 cases with qRT-PCR positive in maternal serum, 5 cases with positive

blood of the neonate.

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qRT-PCR in amniotic fluid, 2 cases tested qRT-PCR positive in placental tissue, and 1 case was positive in umbilical cord

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Fetal brain abnormalities. All 12 fetuses showed significant brain findings during MRI and ultrasound evaluation. First ultrasound evaluation after enrollment in our study was performed at a mean gestational age of 21.92±5.5 weeks. All images and reporting were reviewed by two experienced MFM specialists with neuroimaging expertise (M.P.S) (M.S.C) and one neuroradiologist with fetal imaging expertise (C.G). Table 2 shows the individual findings based on fetal brain MRI and ultrasound. MRI was performed in 10 cases (two patients declined) at a mean gestational age of 30.01±4.57weeks. All detailed brain anomalies were seen by ultrasound and MRI. However, we relied only on ultrasound

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ACCEPTED MANUSCRIPT imaging to assess the presence and location of fetal brain calcifications and on MRI to detect the presence and type of cortical anomalies. Eleven of 12 cases of congenital ZIKV syndrome in our cohort presented with a similar clinical constellation of brain abnormalities with varying degrees of severity (Figures 1 and 2). Common traits included varying degrees of

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microcephaly, increased cerebrospinal fluid in the subarachnoid space with decreased parenchyma volume in all brain regions. Calcifications were detected in 92% of our cases, being both punctiform and coarse and predominantly present in the subcortical-cortical junction (83%) but also in the periventricular zones (67%) and basal ganglia (42%) (Fig. 1D-F). Malformations of cortical development were present in 89% of the cases most frequently affecting the frontal lobes. Of

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those, abnormal gyral pattern with decreased or absent sulcation (lissencephaly-pachygyria spectrum) (56%), and other focal migrational/postmigrational abnormalities (89%) were among the most frequent findings (Fig. 1G-I).

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Ventriculomegaly was a constant but in a mild-moderate degree in most of the cases, only presenting with > 15mm measurements at the level of the atriums of the posterior horns in two cases. Ventricular dilation was also seen involving the anterior horns of the lateral ventricles, and third ventricle in most of the cases (Fig. 2B and 1J, respectively). Some cases also showed small-sized periventricular cysts, located superior to the anterior horns and also inferior to them. (Fig. 2A-C) Intraventricular synechiae and bands at the level of the frontal, temporal and occipital horns of the ventricular

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system (Fig. 2D-F) were identified in 58% of the cases. Abnormalities in the corpus callosum such as partial agenesis and dysgenesis appeared in 100% of the cases. (Fig. 2G-I) Thinned/hypoplastic brainstem (pons) was also identified in two of the 10 cases that underwent fetal MRI. Other findings such as abnormally redundant posterior scalp skin folding and

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dilated dural sinuses were seen in 60% and 50% of these cases respectively. (Fig. 2J-L) Case 12 was unique in that it did not present any of these common central nervous system (CNS) features suggestive of CZS. However, hypoplasia of the

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cerebellar vermis with an enlarged fourth ventricle, suggestive of a Dandy Walker spectrum was detected in this case (Suppl. Fig. 3A-F). Three cases (Case 1,4 and 7) showed club feet and associated signs of arthrogryposis in the lower extremities. (Suppl. Fig. 4)

Fetal head biometry. When fetal head circumference was assessed at the first and last sonographic evaluation (at enrollment and closest to delivery, respectively) (Table 4), most of the cases (33.3-58.8%) did not show signs of microcephaly at first and most (55.6-77.8%) presented a head circumference that was below the cutoff of -3SD in the last scan.

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ACCEPTED MANUSCRIPT Neurosonography and Doppler ultrasound assessment. Biometrics obtained from different brain structures demonstrated corpus callosum length below the 5th percentile30 in all the cases assessed, anterior horn widths greater than the 95th percentile31 and third ventricular width that were enlarged32 in 75% of the cases. Ventriculomegaly was observed in 83.3% of cases, with 30% defining severe range26 (Suppl. Table 3). Prenatal ultrasound Doppler measurements were obtained in

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three cases, and all were within the normal ranges33–35(Suppl. Table 4). Brain volumetric measurements.

Most pronounced volumetric differences (Table 5) in ZIKV fetuses consisted of a reduced supratentorial brain parenchymal volume (-75.5%) and increased ventricular volume (+48.6%). Although overall subarachnoid cerebrospinal

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fluid volume was decreased in ZIKV fetuses, we observed an increased subarachnoid cerebrospinal fluid volume/ supratentorial parenchymal volume (+150%). This likely quantifies the observed qualitative estimates of brain

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parenchymal loss with a compensatory increase in subarachnoid cerebrospinal fluid volume in CZS. Moreover, the overall Total Brain Volume and Total Intracranial Volume were significantly reduced in ZIKV cases. Fetal brain microstructure assessed by diffusion weighted imaging and ADC measurements. ADC values were overall lower in ZIKV fetuses than controls and also lower than published nomograms for same gestational age27 at scan as

Perinatal course.

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shown in Table 5. A non-significant trend was detected for cerebellar hemispheres and mesencephalon (p=0.06).

As 3 cases opted for termination of pregnancy (TOP), complete perinatal courses were available from 9 of 12 cases.

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(Suppl. Fig. 6 and Suppl. Table 5) All continuing pregnancies delivered after 35.0 weeks, 3 had a vaginal delivery and 6 underwent Cesarean delivery (indication was based on microcephaly or prior Cesarean delivery, electing repeat Cesarean).

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No signs of antenatal nor intrapartum non-reassuring fetal status were encountered. Apgar scores, birth weights, neonatal length and head circumference are detailed in Table 6. When head circumference was analyzed, 55.6% and 77.8% of the cases showed a head circumference ≤ 3 SD below the mean using Intergrowth 2128 and Fenton29 standards, respectively. When the cutoff of head circumference ≤ 2 SD below the mean (using either nomogram) or the Brazilian criteria for microcephaly were applied, 78-89% of the cases were included (Table 6).

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ACCEPTED MANUSCRIPT Mean gestational age at the occurrence of the detection of brain malformations was 28.4±5.3 weeks, and age at the occurrence of microcephaly defined as ≤2 SD or ≤3 SD below the mean (using Intergrowth 21 standards) lagged by over 1 week with means of 29.7±3.8 and 30.2±2.9 weeks, respectively. Perinatal autopsy and histology findings at the time of termination of pregnancy. (Suppl.Table 2) Two of the three

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subjects that opted for TOP authorized an autopsy. Information provided by pathology reports are summarized in Suppl.Table 5. Perinatal pathologic exam of all fetal organs were normal except for CNS. Cases 3 and 4 both showed varying degrees of cortical thinning with apoptosis and scattered microcalcifications. There was only minimal to mild microglial activation. Immunohistochemical stains for ZIKV were negative in both cases. Abnormal pathology results

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were also detected in placental tissue. Interestingly, similar features were detected in both fetuses CNS and placental tissue (Suppl. Fig. 6).

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Postnatal follow up and evaluation.

Only three infants returned for further postnatal imaging and evaluation (Suppl. Table 6) due to geographical restrictions (Suppl. Fig. 7). All three infants had preserved respiratory function with normal oxygen saturations. Case 6 and 10 showed signs of elevated blood pressure. Pupillary reflexes were normal in all three with presence of Babinski reflex in

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Case 12 and 10, but not in Case 6. Signs of moderate hypertonicity were observed in Case 6 and 10. All three cases were adequately breastfed and they did not show signs of dysphagia or any deglutition problems. Regarding the results obtained from hearing and visual screening tests, Case 6 and 10 auditory brainstem response results were within normal limits

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suggesting normal hearing function. All three affected infants had different visual evoked potentials results. Case 6 had absence of evoked potentials in both eyes suggesting gross impairment to the infant’s bilateral optic nerves. Case 10

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demonstrated delayed conduction of the retino-cortical pathway bilaterally. Case 12 had normal findings confirming intact visual pathways.

Postnatal CT neuroimaging. Mean age of the 4 infants that were scanned was 27 days (range 19-36 days). Images showed very similar findings in all of the cases (Table 7). Main findings consisted on marked microcephaly, abnormal cortical sulcation with a smoothed cortical surface (lissencephaly-pachygyria spectrum), thinning of supratentorial parenchyma, ex-vacuo dilatation of the lateral ventricles and multiple parenchymal calcifications.(Fig.3 and 5) Calcifications were located mostly in the subcortical-cortical junction, and fewer were seen within the basal ganglia and periventricular

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ACCEPTED MANUSCRIPT regions. Abnormalities in the posterior fossa were detected in one case, with decreased volume of the cerebellar hemispheres. Most cases had overlapping sutures and a prominent occipital bony bulge. Postnatal MRI neuroimaging. MRI was performed under sedation in two cases at a mean age of 40 days (35-45 days)(Fig. 4, 5 and 6). Significant microcephaly, decreased volume of the supratentorial brain parenchyma and ex-vacuo

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enlargement of lateral and third ventricles were the main common findings detected in both cases. Migration abnormalities were also present in both. Case 6 showed signs of lissencephaly with a markedly thinned corpus callosum.(Table 7) Calcifications in the subcortical-cortical junction and basal ganglia were seen in both cases using T1 sequences. Prominence of the occiput (occipital bulge), flattening of the frontoparietal skull as well as redundant skin

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folds at the neck and occipital region, were common traits in both subjects. No clear abnormalities were seen in brainstem and posterior fossa in the two cases that underwent postnatal MRI. Brain volumetry (Table 8) showed decreased brain

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volumes in all studied areas (except for ventricular volume that was increased) when compared to neonates from published references36. Diffusion weighted imaging sequences were obtained only in one case (Case 8); when ADC values were compared to nomograms37, increased values in cerebellar vermis (by 16%) and mesencephalon (by 23%) were detected in the ZIKV infected case. Anterior fontanelles were closed in Case 6 and 10, limiting the performance of

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transcranial ultrasound to only one case (Case 12). As shown in Suppl Fig. 3G-I, images obtained in this case confirmed

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prenatal imaging findings, showing an isolated cerebellar vermian hypoplasia.

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ACCEPTED MANUSCRIPT DISCUSSION Main findings of the study. In this study we have reported brain findings in a cohort of 12 cases affected by ZIKV from Barranquilla, Colombia. This is the first report in which a detailed exam of pre and postnatal (Figs. 5 and 6) neuroimaging findings are presented in one of the ZIKV epidemic sites outside of Brazil. The main findings of this study include the

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detection of predominantly peripheral cerebral calcifications, ventriculomegaly, cortical malformations and brain volume loss as some of the most frequently detected anomalies in this congenital ZIKV-infected cohort. Microcephaly was not detected in all cases and appeared to temporally follow and lag behind preceding intracranial malformations. Microcephaly is not uniformly present in CZS. After the detection of a 20-fold increase in the rate of congenital

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microcephaly in North East Brazil during the ZIKV epidemic, concerns were raised about a potential association between infection and congenital defects8,38,39. Screening for microcephaly was one of the initial recommendations to identify cases

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potentially affected by ZIKV16, nevertheless there is not an agreement on its definition or on the reference ranges to be used for this purpose40. Brazilian authors have used a head circumference < 2SD below the mean or below the 3rd percentile41, French Polynesia reports have used < 3rd percentile only42,43, whereas it has been defined as < 3SD by different scientific societies15,16. Postnatally, head circumference Z-scores (ZS) of < -2 (moderate) and < -3 (severe)41,44 have been proposed to define this condition in the context of ZIKV; moreover, many Brazilian reports have used a

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standard cutoff for head circumference of 32-33cm or less for term new borns7,13,18,19,45–47. In this study, detection rates of microcephaly, using 2 or 3 SD below the mean as cutoffs for its definition, were similar for the five different nomograms used before and after delivery. Population-based studies may be more appropriate to

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determine the optimal diagnostic criteria for microcephaly as the lack of differences in our study could be explained by our small sized cohort. Based on our results, it seems reasonable that if fetal head circumference falls under -2SD, fetal

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neurosonography should be performed by expert hands as proposed by ISUOG and SMFM guidelines15,16. The implications of being born with a moderately or severely reduced head size in the context of a ZIKV infection still remain unknown. Prospective studies assessing the neurological outcome of these subjects may shed some light on these implications.

Approximately 10% of our cases had a strictly normal head circumference in spite of significant brain abnormalities, which is consistent with 13%19 and 9.7%18 rates reported by others in Brazil. This could be explained by an increased subarachnoid space or ventricular system in association to microencephaly, which can make the head circumference

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ACCEPTED MANUSCRIPT appear bigger than the actual brain volume, therefore masking a small brain size19. With the current level of knowledge about this disease, the association between ZIKV and microcephaly may be on the worse side of disease severity spectrum and head reduction may be an ongoing process that may worsen over time. Supporting this hypothesis, our results show how brain anomalies appear earlier than small fetal head size falling 3SD below the mean. Also, two of the cases that

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were followed up postnatally (Case 6 and 10), showed a decreasing head circumference ZS over the first two months of life with increasing birth weight percentiles (Suppl. Table 6). Case 12 showed a consistent increase in birth weight percentile and head circumference ZS over the study period. Similarly, one case series reported 13 Brazilian infants with CZS who initially had normal head circumference at birth but developed microcephaly in 85% of their cases during the

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first year of life6. Thus, microcephaly alone is not an adequate screening tool as CZS can present in a variety of ways and degrees of severity.

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Spectrum of brain abnormalities detected by ultrasound and MRI in cases of fetal ZIKV infection. The majority of cases had a common pattern of brain anomalies presenting with different degrees of severity that is similar to those reported by others in the type of anomalies encountered and their frequency (Table 9). Calcifications (detected in 92% of the cases) were predominantly located on the subcortical-cortical junction, but also in periventricular and basal ganglia areas. Calcifications in the subcortical-cortical junction are consistently present in

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different congenital ZIKV reports7,18,19,47 and seem to be specific to this infection18,47. Fetal brain calcifications are not typically detected in this location in other neurotropic congenital infections that present otherwise similar features19. A potential explanation may be that the destruction of brain parenchyma in ZIKV can occur by vasculopathy and not by

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ependymitis or bleeding as seen in other infections18.

Ventriculomegaly was detected in 92% of our cases. Similarly, it was reported in 86%19 and 94%18 of the cases of two

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different Brazilian studies. In our cohort, ventricular dilation was moderate and more prominent in the posterior horns, which coincides with previous reports19 and could be related with the marked posterior periventricular volume loss in most cases. We agree with observations from Cavalheiro et al47 that even those cases with severe ventriculomegaly are rarely associated with a ventricular hypertensive pattern. This may reflect the ex vacuo ventriculomegaly nature of this finding rather than an obstructive mechanism. Brain volume loss was found with different degrees in 11 of our 12 cases, which is aligned with the 91-100% rate reported by other studies7,8,18,19,41,42,47 . (Suppl.Fig.8) Parenchymal loss may be one of the initial signs of ZIKV infection,

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ACCEPTED MANUSCRIPT given its known capacity to induce neuronal apoptosis, interfering with one of the initial phases of corticogenesis, such as neural proliferation and migration. This mechanism has been described for other congenital infections that lead to brain atrophy, microcephaly and cortical development anomalies, and may be the common pathway that can explain other brain findings and fetal complications in ZIKV infection7,48–50 .

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Cortical abnormalities and decreased sulcation in particular, were among the most common (89%) diagnoses in our series, in alignment with prior reports7,8,13,18-20,42,51. Most of the cortical abnormalities were detected in the frontal lobes, which is considered a specific trait of CZS19. A potential explanation for the strong association between congenital ZIKV and cortical malformations could rely on its confirmed neurotropism48, suggesting an interference in all phases of

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neurodevelopment: neuronal proliferation, migration, cortical organization and myelination13,39,52–55. Reduced cell proliferation could lead to both microcephaly and a simplified gyral pattern. In other forms of microcephaly, a strong

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correlation between the severity of microcephaly and the simplification of the gyral pattern has been proven56. The global presence of cortical hypogyration, white matter hypomyelination and cerebellar hypoplasia in the majority of the cases, suggest that ZIKV is associated with a disruption in brain development rather than destruction of the brain20.This evidence is supported by results from Tang et al48 who found how ZIKV directly infects human neural progenitor cells with high efficiency, resulting in stunted growth of this cell population, transcriptional dysregulation and apoptosis. Additionally,

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the frequently encountered simplified gyration pattern could be explained by the lack or decreased tension exerted on the developing cortex by axonal connections, that may orchestrate cortical folding in normal conditions57,58. We postulate that decreased neuronal proliferation results in fewer cortical neurons and thus, fewer axons exiting from the cortex; this might

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result in decreased tension arising from axons and consequently fewer and shallower sulci56. Callosal abnormalities were detected in 100% of our cases with different degrees of severity. They have been reported in

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38-100% of the cases by others18,19,47. A decreased number of neuronal cells and callosal axons and/or the interference to neuronal migration potentially caused by ZIKV could explain these findings, similarly to what has been described for HIV and human herpes virus7,49,50,60.

Pseudocysts and intraventricular synechiae were observed in 58% of our cases, mostly in the frontal and occipital areas, similarly to what has been reported18,42,47. These findings could be related to the necrosis or hemorrhage of the germinal matrix. When the destructive process is substantial and cysts coalesce, the ependyma may separate from the adjacent tissue and develop into synechiae similarly to what occurs in congenital CMV infection60.

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ACCEPTED MANUSCRIPT Enlarged confluence of dural venous sinuses was detected in 50% of our cases. Other authors have proposed that this heterogenous material could resemble blood clots by ultrasound and had a hyperattenuated appearance on postnatal CT scan18. Arthrogryposis and club feet were observed in three of our cases. Viral interference with the migration process of motor

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neurons could be responsible for these findings7,13. Dandy Walker spectrum was the only finding in Case 12. Microcephaly did not occur before or after delivery, and no calcifications, ventricular dilation or cortical dysplasias were detected. It is unknown why this clinical variability was encountered. Other cases presented varying forms of cerebellar abnormalities, with a vermian height measurement below

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the 5th percentile for gestational age in 42% of the cases and reduced transcerebellar diameter in half of them. Posterior fossa structures have been reported to be less commonly affected than supratentorial structures18. This finding may be

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explained by the aforementioned theory of a predominant carotid distribution of fetal viremia47. MRI Volumetric analyses were useful not just to confirm the observed signs of microencephaly with decreased supratentorial brain volume and increased subarachnoid space volume (reflecting the compensatory increase of cerebrospinal fluid in the subarachnoid space as part of the typical ex vacuo mechanism), these results provided insight to the areas that were most affected or more “preserved” by the virus. The least affected infratentorial volumes could suggest

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that the viral involvement could spare the vertebrobasilar circulation, affecting mainly the carotid system47. On the other hand, fetal viremia with vasculitis in the carotid brain circulation could be associated with tissue necrosis, which may explain the common neuronal migration abnormalities encountered in CZS19. The use of diffusion weighted imaging

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(ADC values) for the assessment of fetal brain microstructure is now reported for the first time in CZS. A non-significant trend for decreased ADC values was detected, reaching values close to statistical significance for the cerebellar

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hemispheres and mesencephalon. Decreases in ADC values reflect restricted water diffusivity, similar to what has been proposed to explain reduced cerebral ADC values in congenital CMV infection61. Perinatal and postnatal findings. Most of our cases were delivered at term with adequate perinatal results, coinciding with other authors19,41. Our cases were mostly delivered by elective cesarean section, thus it is unknown if similar outcomes would have been encountered if more vaginal deliveries were attempted. Although no ocular structural anomalies were detected in either pre or postnatal imaging studies, two out of the three cases presented with significant bilateral visual defects, which could be associated with unnoticed chorioretinal and optic nerve disease. Up to 34% of

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ACCEPTED MANUSCRIPT congenital ZIKV infected and microcephalic infants from a Brazilian cases series had different forms of eye anomalies, with retinal alterations in up to 69% of the cases with CZS62,63. We did not detect hearing abnormalities in the two cases that were assessed, which again coincides with literature that shows how only 5.8-9% of microcephalic infants had sensorineural hearing loss that varied in severity and laterality in congenital ZIKV64.

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Strengths and limitations to our study. The main strength of this investigation is its prospective nature with pathology results and longitudinal follow up. This is the first time that perinatal ZIKV-related brain anomalies are reported in detail from a cohort of Colombian patients. Moreover, we were able to perform a quantitative analysis of ZIKV brain images, which provides insight on the ongoing processes that may alter brain composition and microstructure. All of our 12 cases

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presented with normal karyotypes which is not commonly reported in other studies19.

We acknowledge that as MRI was performed in only 10 cases, the detection rate of cortical malformations can be

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underreported. One other case that underwent an early MRI scan (at 20 weeks), opted for a TOP. Early MRI scans may not detect cortical abnormalities that could appear later on in pregnancy. Also, information about the moment of clinical symptoms were based on maternal self-report, which could constitute a recall bias. The potential effect of a co-infection may be unrecognized by not testing for Dengue or Chickungunya. Lastly, infants were assessed in a very limited number of cases and very early in infancy, when some subtle neurologic manifestations of disease are difficult to identify.

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Conclusion and clinical take home messages. For the first time in CZS a MRI volumetric assessment shows how there is an association of an increased ventricular system volume, decreased supratentorial brain parenchymal volume and increased cerebrospinal fluid in the subarachnoid space. The combination of these volumes determines fetal head size,

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which can result into microcephaly or not. We observed that congenital microcephaly was not an optimal screening method to detect fetuses affected by CZS as it was only present in 33-58% of our cases at the time of diagnosis, therefore

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we speculate that microcephaly is likely an end-point of this devastating congenital infection, resulting from progressive changes related to brain volume loss. Long-term studies are needed to assess the clinical relevance of brain anomalies that are encountered and the neurodevelopmental sequelae of this devastating condition.

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ACCEPTED MANUSCRIPT Acknowledgements: We would like to express our gratitude for the contributions of Viviana Meza Estrada, Maria del Mar Soriano and Amanda Barrero-Ortega for the coordination and screening of patients. Eduardo De Nubila-Lizcano, Adrian Colmenares and Carlos Bustamante- Zuluaga who provided insight and expertise on the project. Marcela Mercado and Diana Valencia

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from the Instituto Nacional de Salud, contributed with their expertise on the interpretation and release of laboratory results of these patients. We would like to thank Dulfary Serna Aristizabal for her help with the postnatal assessment of the

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patients from this cohort. We are also grateful to Edgar Parra for the assistance with the pathology reports.

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Figure 1- Prenatal cerebral findings shown by ultrasound and MRI. Image A, B and C show a parasagittal, coronal and midsagittal view of the fetal brain respectively on MRI. Significant brain parenchyma volume loss, enlarged subarachnoid space (blue arrow) and ventricular system are

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seen in all three images. Head to face disproportion, suggestive of microcephaly can be observed in A and C; Images D, E and F are obtained from a neurosonography exam, representing

transfrontal coronal, parasagittal and transcerebellar coronal views respectively. Image D and E

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show linear and coarse calcifications located within the frontal (D), parietal, occipital and

temporal lobes (E) at the subcortical-cortical junction. Image F shows the presence of punctate

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calcifications (white arrows); Images G to I: Fetal MRI of a 28 week old fetus demonstrates diffuse malformation of the cortical development with simplified pattern of gyration in the spectrum of lissencephaly as well as focal areas of migration abnormalities (yellow stars) seen on sagittal (G) and coronal views ( H and I). Image H also shows an underopercularized Sylvian fissure (green star). Image J and K are obtained from a neurosonography exam, J is an

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axial transcerebellar view showing a dilated third ventricle (blue arrow) and dilated posterior horn of the ventricular system (yellow star). Image K shows a parasagittal view (three horn view) with a dilation of the frontal, occipital and temporal horns. Image L shows significant ventricular ex

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vacuo dilation and markedly thinned brain parenchyma with increased subarachnoid space shown

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on an axial view of fetal MRI

Figure 2- Prenatal cerebral findings shown on ultrasound and MRI. Image A and B show parasagittal and coronal views of the fetal brain from a neurosonographic exam. Periventricular cyst is seen above the left anterior horn. Image C from a fetal MRI shows the presence of an occipital periventricular cyst (blue arrow) and markedly thinned brain parenchyma seen on sagittal view. Image D and E show an axial and parasagittal view from fetal MRI demonstrating intraventricular synechiae/septations (orange star). Images G, H and I show midsagittal MRI views of different fetuses with different degrees of callosal

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dysgenesis (blue arrows). Image J, K and L show the dilation of the dural sinuses (orange stars) and abnormally redundant nuchal skin folding (blue arrow) that can be seen in this condition.

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Figure 3- Postnatal CT scan images. Axial CT images showing abnormal brain sulcation, marked parenchymal volume loss and multiple supratentorial calcifications. Most of calcifications are

distributed along the peripheral subcortical-cortical junction with lesser involvement of the deep

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structures. Images D and E demonstrates calcifications also located on basal ganglia. Image B

also shows a small cerebellum, which is a less frequent finding in ZIKV affected patients. Image

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F is a 3D reconstruction of the skull.

Figure 4- Postnatal brain MRI. Images A and D represent axial T2 weighted sequence images showing parenchymal volume loss and abnormal brain sulcation (blue arrows) resulting in exvacuo dilatation of lateral ventricles. Images B,C, E and F represent sagittal and coronal T1

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weighted sequences demonstrating marked microcephaly and lissencephaly from two different patients. Images E and F, Additional sagittal T1 sequences of different patients. Image E exemplifies occipital prominence with focal enlargement of the dural sinus (red arrow). Image F

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shows punctate calcifications along the subcortical-cortical junction on a T1-weighted sequence following the common pattern seen in ZIKV affected patients. Images G to I represent the DWI

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sequence in three axial planes. Regions of interest to calculate ADC values have been delineated: Mesencephalon bilaterally in image G, white matter in image H and cerebellar hemispheres in image I. Images J, K and L show 3D volume reconstruction of total intracranial volume (shown in purple), supratentorial brain parenchyma (shown in orange), cerebellum in yellow, brainstem in blue and ventricular system in green.

Figure 5- Pre and postnatal images from Case 8:Images A and B show prenatal ultrasound images at 34.5 weeks. A is an axial transventricular image and B is a coronal transcaudal image. In A,

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ventriculomegaly with thinned occipital brain parenchyma (blue arrow) are pronounced and B shows dilated anterior horns and basal ganglia calcifications (blue arrow). Images C to E are T2weighted images obtained from fetal brain MRI scan performed at 33.4 weeks. All three images

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show the marked brain volume loss, simplified pattern of gyration in the spectrum of lissencephaly and ventriculomegaly. Focal cortical malformation is identified with red arrow. Images F and G show the 3D segmentation volume rendering with i the supratentorial brain

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parenchymal volume in orange, the cerebellum in yellow, and the brainstem in blue. Image G

shows the ventricular system in green. Image H shows a diffusion weighted image in which ADC

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values from mesencephalon are measured. Images I and J represent the appearance of the infant’s head, showing signs of marked microcephaly. Images K to M show postnatal CT scan performed at 45 days of life with coronal (K), sagittal (L) and axial (M) views. Calcifications located on the subcortical-cortical junction (red arrow) and also at the level of basal ganglia. Images N to R are obtained from postnatal brain MRI. Image N shows a T2 weighted axial image at the level of the

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basal ganglia and posterior ventricular horns. A simplified cortical pattern is seen as shown by red arrow. Also signs of severe brain volume loss and increased CSF in the subarachnoid space are seen. Image O shows the infant’s head shape by a 3D reconstruction. Images P and Q show the

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3D segmentation volume rendering that was performed (orange-supratentorial brain parenchyma, yellow- cerebellum, blue-brainstem, green- ventricular system). Image R shows a diffusion

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weighted image obtained from this case, with a delineation of regions of interest at the level of mesencephalon to obtain ADC values.

Figure 6- Pre and postnatal images from Case 6:Images A and B correspond to prenatal ultrasound scan performed at 31.4 weeks. Images A shows transventricular plane with a normal sized posterior horn in the ventricular system. Image B is a transtahlamic plane where head circumference was measured. Images C to G are obtained from fetal brain MRI scan performed at 32.3 weeks. Image C is a midsagittal view showing a microcephalic head with a lissencephalic

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cortex Redundant scalp skin is seen at the level of the occiput (blue arrow) and red arrow indicates a dilated dural sinus. Image D corresponds to an axial view in which we can identify a ventricular synechia (red arrow) in the occipital horn. Image E is a coronal view of the fetal brain

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that shows different areas of abnormal cortical unfolding due to migration anomalies. Image F and G are obtained from a 3D segmentation volume rendering (orange-supratentorial brain

parenchyma, yellow- cerebellum, blue-brainstem, green-ventricular system). Images H and I are

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postnatal images showing the baby’s microcephalic head (H) and image I that was obtained

immediately after birth showing clubbed feet and abnormally positioned lower extremities with

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sign s of arthrogryposis. Images J to M were obtained from the MRI scan performed at 35 postnatal days, Images J to L are T1 weighted sequences in axial, sagittal and coronal planes respectively. Lissencephaly is observed with signs of moderate brain parenchyma volume loss. Image M is a volume reconstruction of the infant’s head, N and O images correspond to 3D segmentation volume rendering (orange-supratentorial brain parenchyma, yellow- cerebellum,

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blue-brainstem, green- ventricular system).

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Table 1. Maternal-Fetal Demographic and Cohort Characteristics Education Level

Medications

Chronic Condition

No

3

Bachelor

Carbamazepine, ferrous sulfate, folic acid.

Epilepsy

ZIKV Symptoms

ZIKV Symptoms in Sexual Partner (GA at symptoms) No

Karyotype

Fetal ultrasound follow up

46, XY

Yes

1

27

1

0

2

30

1

0

31.0

Cienaga

No

1

High school

Ferrous sulfate, folic acid.

No

Yes (10weeks)

No

46,XY

Yes

3

18

1

0

21.0

Santa Marta

No

4

Bachelor

No

No

No

46,XY

Yes

4

28

3

1

29.0

Santa Marta

No

6

Bachelor

Ferrous sulfate, Folic acid. Ferrous sulfate, Folic acid.

No

No

46,XX

Yes

5

20

2

0

27.0

Santa Marta

No

1

Yes

2

1

31.0

Riohacha

No

3

Yes (13.5weeks) Yes (7.5weeks)

46,XY

26

No

46, XX

Yes

7

20

2

0

25.4

Sincelejo

No

1

Technicians

8

20

1

0

24.0

Corozal

No

1

Bachelor

9

25

1

0

18.3

Soledad

No

2

Bachelor

10

37

3

2

29.0

Caucasia Municipio de Antioquia

No

2

Bachelor

11

18

1

0

22.5

Sabana Larga

No

1

Bachelor

12

30

2

1

32.8

Santa Marta

No

3

Bachelor

Ferrous sulfate, Folic acid. Ferrous sulfate, Folic acid, Loratadine Acetaminophen, Ampicillin, Ferrous sulfate, Folic acid. Folic acid, sulfate ferrous. Acetaminophen, Ferrous sulfate, Folic acid. Acetaminophen, Ferrous sulfate, Folic acid , Metronidazole, Nifedipine. Cephalexine, Ferrous sulfate, Folic acid. Ferrous sulfate, Folic acid, Nifedipine, Betamethasone

No

6

Elementary school High school

Yes (1 week after LMP) No

(GA at symptoms) Yes (10weeks)

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Barranquilla

Socioeconomic status*

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City of origin

History of Smoking

Body mass index (kg/m2) 22.0

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Parity

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Gravida

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Age

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Case

No

No

Yes (10weeks)

No

46,XY

Yes

No

Yes (12weeks) Yes (12.1weeks)

No

46,XY

Yes

Yes (10.4weeks)

46,XX

Yes

No

No

No

Yes (2 weeks after LMP)

46,XX

Yes

No

Yes (9.2weeks)

No

46,XY

Yes

No

Yes (14weeks)

No

46,XX

Yes

Abbreviations: GA=Gestational Age. LMP=Last menstrual period. *Socioeconomic status based on the “Official strata divisions” (Bushnell D, Hudson RA. “Social Strata Division”. In Colombia: A Country Study.(Rex A, Hudson, ed.)pp 101-103.Library of Congress Federal Research Division (2010).

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Table 2- Anomalies Detected by Fetal MRI and Ultrasound.

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Location of calcifications in the fetal brain based on fetal Ultrasound

Case

GA at ultrasound detection of abnormalities (weeks)

GA at MRI (weeks)

Enlarged subarachnoid space

Decreased Brain volume

Ventriculo megaly

1

19.3

26.2

Yes

Yes

2

28.2

28.0

Yes

3

25.3

29.0

4

19.3

5

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Findings from fetal MRI/Ultrasound imaging

Corpus callosum

Brain stem Hypoplasia

Cerebellar hypoplasia

Enlarged Cisterna magna

SubcorticalCortical junction

Basal ganglia

Periven tricular

Yes

Yes

Yes

Hypoplasic

No

No

No

Yes

No

No

Yes

Yes

Yes

Yes

Hypoplasic

No

No

No

Yes

No

No

Yes

Yes

Yes

21.0

Yes

No

Yes

33.4

-

Yes

Yes

Yes

6

27.4

32.3

No

Yes

7

27.5

-

Yes

Yes

8

33.4

33.4

Yes

Yes

9

34.2

33.1

Yes

10

33.1

33.2

11

33.2

12

26.5

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MCD

Simplified gyral pattern

Yes

Yes

Hypoplasic

No

No

No

Yes

Yes

Yes

No

No

Hypoplasic

Yes

Yes

Yes

No

No

Yes

-

No

-

No

Yes

Yes

Yes

Yes

Yes

No

No

Yes

No

Yes

Yes

Yes

Yes

-

Yes

Hypoplastic

-

No

No

Yes

No

Yes

Yes

Yes

Yes

Dysgenetic

No

No

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Dysgenetic

No

No

No

Yes

No

No

Yes

Yes

Yes

Yes

Yes

Dysgenetic

No

No

No

Yes

Yes

Yes

36.6

Yes

Yes

Yes

Yes

Yes

Hypoplasic

No

No

Yes

Yes

Yes

27.3

No

No

No

No

No

Normal

No

No Dandy Walker

No

No

No

No

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EP Yes

Dysgenetic Dysgenetic/ hypoplastic

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Abbreviations: GA=gestational age; MCD=Malformations in cortical development. Case 5 and 7 did not undergo fetal MRI and information on fetal brain anomalies was obtained from ultrasound –in bold-(neurosonography only).

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Table 3- Frequency of Fetal Anomalies in Our Cohort Assessed by MRI and Ultrasound.

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1

Ventriculomegaly

(91.7) 11/12

Mild

(41.7) 5/12 (33.3) 4/12

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Moderate

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(91.7) 11/12 (66.7) 8/12 (83.3) 10/12 (41.7) 5/12 (8.3) 1/12 (8.3) 1/12 (100) 12/12 (58.3) 7/12 (41.7) 5/12 (89) 8/9 (56) 5/9 (22) 2/9 (89) 8/9 (44) 4/9

Calcifications Periventricular Cortical–subcortical white matter junction Basal ganglia and/or thalamus Brainstem Cerebellum Corpus Callosum abnormalities Hypoplasic Dysgenetic Cortical abnormalities* Lissencephaly Polymicrogyria or pachygyria Irregular areas of sulci / Migration abnormalities Nodular heterotopia

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Abnormality

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Severe Cerebellum abnormalities Cerebellar hypoplasia Vermian hypoplasia Redundant skin folding** Septations or synechia Occipital Temporal Frontal Increased T2 signal** Parietal Occipital Temporal Heterogeneous material, some of which could be thrombus in the region of the confluence of sinuses** Brainstem hypoplasia and/or atrophy**

(16.7) 2/12 (75) 9/12 (50) 6/12 (41.7) 5/12 (60) 6/10 (58.3) 7/12 (33.3) 4/12 (33.3) 4/12 (25) 3/12 (50) 5/10 (20) 2/10 (50) 5/10 (10) 1/10 (50) 5/10 (20) 2/10

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Frequency of each abnormality is listed as the percentage in parenthesis and proportion. *Cortical abnormalities were only considered in 9 of the 10 cases that underwent MRI as one case was examined at 20 weeks, which is not an appropriate age to assess cortical malformations 1 Measured by ultrasound considering: Mild (10- 12mm), Moderate (12.1-15mm) and Severe (>15mm). (Melchiorre K, Bhide A, Gika AD, Pilu G, Papageorghiou AT. Counseling in isolated mild fetal ventriculomegaly. Ultrasound Obstet Gynecol 2009;34(2):212-224). **Assessed by fetal MRI only.

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Intergrowth23

Kurmanavicius et al24

Chervenak et al 25

4/12 (33.3)

4/12 (33.3)

4/12 (33.3)

4/12 (33.3)

4/12 (33.3)

4/12 (33.3)

7/9 (77.8)

7/9 (77.8)

6/9 (66.7)

5/9 (55.6)

1

4/12 (33.3)

7/12 (58.3)

4/12 (33.3)

4/12 (33.3)

7/9 (77.8)

7/9 (77.8)

7/9 (77.8)

7/9 (77.8)

7/9 (77.8)

7/9 (77.8)

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Last ≤ -2 SD (%) ultrasound head circumference ≤ -3 SD (%) (n=9)1 GA= 31.93± 2.85 weeks

Colombian Cohort**

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ultrasound ≤ -2 SD (%) head circumference (n=12) ≤ -3 SD(%) GA= 21.92± 5.5 weeks

Hadlock et al 22

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First

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Table 4. Ultrasound Head Circumference Comparison of ZIKV Infected Fetuses to Different Nomograms.

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3 cases that opted for Termination of Pregnancy and 1 without follow-up ultrasound were excluded. Abbreviations: GA= Gestational age; US= Ultrasound; HC=Head Circumference. Data is expressed as ratios and percentages in parenthesis. **Established normality range of head circumference values from healthy population of Colombian population.

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N=10 30.72 ± 4.60 8.12 ± 2.98 178.80 ± 80.36

+48.6 -75.5

2.36 ± 0.97 5.87 ± 3.45 60.01 ± 21.40

3.6 ± 1.40 9.05 ± 4.92 102.82 ± 36.41

1.53 ± 0.63

66.17 ± 30.18 126.19 ± 49.80

N=3 1.46 ± 0.05

ADC cerebellar vermis ADC frontal ADC mesencephalon ADC parietal ADC occipital

1.3 ± 0.25 1.61 ± 0.17 1.34 ± 0.12 1.54 ± 0.205 1.56 ± 0.19

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P* 0.73 0.01 <0.01

-34.4 -35.1 -41.6

0.034 0.11 <0.01

0.61 ± 0.1

+150

<0.01

199.59 ± 89.09 302.41 ± 124.86

-67.0 -58.3

<0.01 <0.01

Reference Values27

N=3 1.64 ± 0.11

1.78 ± 0.35

0.06

1.52 ± 0.07 1.63 ± 0.098 1.67 ± 0.19 1.61 ± 0.13 1.51 ± 0.05

1.74 ± 0.38 1.90 ± 0.40 1.62 ± 0.15 2.05 ± 0.35 1.88 ± 0.45

0.23 0.88 0.06 0.65 0.7

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ADC cerebellar hemispheres

% difference**

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Brainstem volume (ml) Cerebellar volume (ml) Subarachnoid cerebrospinal fluid volume (ml) Subarachnoid cerebrospinal fluid volume/supratentorial parenchymal volume Total Brain Volume (ml) Total Intracranial Volume (ml)

Healthy controls

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Gestational age at scan (weeks) Ventricular volume (ml) Supratentorial brain parenchymal volume (ml)

ZIKV infected fetuses N=10 30.01 ± 4.57 12.09 ± 3.35 45.84 ± 27.04

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Table 5. Prenatal MRI Volumes and Diffusion Weighted Imaging Assessment

Abbreviations: Total Brain Volume=Supratentorial brain parenchymal volume + Brainstem + Cerebellar volume ; Total Intracranial Volume =Total Brain Volume +Subarachnoid Cerebrospinal Fluid volume; ADC= apparent diffusion coefficient (µm2/ms).** %difference= (ZIKV volumecontrol volume)/control volume x 100. * t-test comparison from independent samples.

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Table 6. Perinatal Results of ZIKV infected fetuses.

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37.8±1.15 77.8 55.6 0 2615.0 ± 359.37 25.4±14.5 11.1% 21.67 ± 18.74 33.3% 483.33± 26.46 49.01±34.5 11.1% 43.78± 34.94 22.2% 288.39±26.46 7.07±20.6 6.33 ± 19.0 55.6% 77.8% 88.9% 88.9% 77.8%

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Gestational age at delivery (weeks) Cesarean section delivery (%) Male gender (%) Apgar score less 7 at 5 minutes Birth weight (g) Birth weight percentile- IG21* Birth weight <10 percentile (IG21)* Birth weight percentile- Fenton† Birth weight <10 percentile (Fenton)† Neonatal height (mm) Neonatal height percentile- IG21* Neonatal height <10 percentile IG21* Neonatal height percentile- Fenton† Neonatal height < 10 percentile Fenton† Neonatal head circumference (mm) Neonatal head circumference percentile IG21* Neonatal head circumference percentile Fenton† Neonatal head circumference ≤ -3 SD IG21* Neonatal head circumference ≤ -3 SD Fenton† Neonatal head circumference ≤ -2 SD IG21* Neonatal head circumference ≤ -2 SD Fenton† Neonatal head circumference ≤ 32 cm at term or < 2 SD by Fenton if gestational age at birth < 37 weeks.

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N=9

Abbreviations: g: grams; IG-21= Intergrowth 21 standards; mm=millimeter. *Percentiles and z-scores based on Intergrowth-21st standards28. †Percentiles and SD based on the Fenton growth chart for preterm infants29.

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Findings from postnatal MRI and CT scans

Ventriculomegaly

Malformations in cortical development

Simplified gyral pattern

35 (MRI)

No

Yes

Yes

Yes

Yes

7

32 (CT)

Yes

Yes

Yes

Yes

Yes

8

45 (MRI)

Yes

Yes

Yes

Yes

Yes

9

19 (CT) 38 (CT)

Yes

Yes

Yes

Yes

Yes

10

27 (CT)

Yes

Yes

Yes

Yes

Yes

Hypoplastic/ Dysgenetic

Cerebellar hypoplasia

Thinned

Enlarged Cisterna Magna

SubcorticalCortical white matter junction

Basal Ganglia

Brain stem

Periventricular

No

No

Yes

Yes

No

No

NA

No

Yes

Yes

Yes

Yes

No

Yes

Dysgenetic

No

No

No

Yes

Yes

No

No

Dysgenetic

No

No

No

Yes

No

No

No

Dysgenetic

No

No

No

Yes

Yes

No

No

EP

6

Brain Stem Hypoplasia

AC C

Case number

Corpus callosum

SC

Decreased Brain volume

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Enlarged subarachnoid space

Location of calcifications assessed by CT scan imaging

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Age at scan (days)

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Table 7. Postnatal Imaging Findings.

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Table 8. Postnatal MRI Brain Volumes and Diffusion Weighted Imaging Assessment Reference Values*

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ZIKV infected fetuses N=2 Age at MRI (days)

40

Ventricular volume (ml)

18.71± 8.44

Supratentorial brain parenchymal volume (ml)

72.16 ± 10.98

Brainstem volume (ml)

4.09 ± 0.82

Cerebellar volume (ml)

19.76 ± 6.97

26.98 ± 0.37

Cerebrospinal fluid volume in subarachnoid space (ml)

63.68 ± 34.81

-

Total brain volume (ml)

114.72 ± 5.25

425.38 ± 4.81

Total Intracranial Volume (ml)

178.41 ± 40.06

-

2.109 ± 0.15

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SC

370.68 ± 4.18 -

0.65 ± 0.11

-

N=1

Reference Values**

ADC cerebellar hemispheres

1.090

1.17 ± 0.05

ADC cerebellar vermis

1.360

1.17 ± 0.05

ADC frontal lobes

1.453

1.58 ± 0.11

ADC mesencephalon

1.318

1.01 ± 0.07

ADC parietal lobes

1.49

1.62 ± 0.15

ADC occipital lobes

1.637

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Total brain volume/Total intracranial volume

-

1.72 ± 0.10 2

Abbreviations: ADC=Apparent diffusion coefficient (µm /ms)* Reference values by 37 Knickmeyer et al. 200836. ** Reference values by Wolf et al. 2001.

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Table 9- Literature Review of Main Publications Detailing Brain Abnormalities Associated to ZIKV infection.

Soares de Oliveria18 (N=31 feuses;45 neonates; Presumed (P) N=28 / Confirmed N=17 infection) Cavalheiro47 (N=13; Neonates with

CT/MRI

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

100% located on the cortical-subcortical WM junction

91% decreased brain volume

86% Predominant enlargement of posterior portions of lateral ventricles and trigone

95% Frontal predominance (simplified gyral pattern, polymicrogyria, pachyria, hemimegalencephal, periventricular heterotopic GM)

50% had hypoplasia of cerebellum or brain stem

38% hypogenesis and 38% hypoplasia

Yes

Yes

59% calcifications located on BBGG 45% periventricular 36% BS 50% Cerebellum Yes

Postnatal

Prenatal/ Postnatal

CT/US

US/MRI fetuses CT/MRI postnatal

Postnatal

CT/MRI

Location: Periventricular, thalamic and BBGG, Parenchymal

Abnormal Corpus Callosum

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Cerebellar and BS hypoplasia

Others

Hypersignal T2 Parenchymal brain hemorrhage.

88% enlarged cisterna magna 52% Redundant scalp skin

31% Excessive scalp skin 11% Arthrogryposis

33% Neuronal migrational disorders (lissencephaly and pachygyria)

Yes

Yes

GM/WM junction: 88%(100%P) Periventricular: 65%(14%P) Cortical: 24% (14%P) BBGG/Thal 65%(64%P) BS 18%(14%P) Cerebellum 6% (4%P) Yes

100% (100%P)

92.3%

Malformation of cortical development and sulcation

SC

Postnatal

Ventricular enlargement

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US/CT

Cerebral atrophy

TE D

Schuler Faccini13 (N=35; Infants with microcephaly and presumed infection. 27 with neuroimaging studies)

Prenatal/ Postnatal

Calcifications

Yes 94%(96%P)

Yes 94% (100%P)

Mild 24% (18%P)

Lissencephaly 12% (21%P)

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Vasco de Aragao19 (N=23; Infants with microcephaly and presumed infection. 6/23 confirmed infection)

Imaging

AC C

Brasil41 (N=125 cases of confirmed ZIKV infection)

Moment

Yes

Yes

Vermis hypoplasia 59%(68%P)

94% (78%P)

Septations in the occipital horn 29% (11%P)

Cerebellar abnormalities 83% (39%P)

Moderate 41% (32%P)

Poly/pachygyria 65%(50%P)

Severe 29% (46%P)

Irregular areas of sulci and/or gyri 29% (75%P)

BS hypoplasia 70% (21%P)

Yes

Yes

Yes

No

100%

100%.

100% Lissencephaly

Severe 70% (39%P)

Heterogeneous material in the region of confluence of sinuses 53% (28%P)

Skull had frequently collapsed appearance with overlapping sutures and redundant skin folds

Yes Hypoplasia 100%

38.5% intraventriclar septations

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Oliveira Melo8 (N=2; Fetuses with microcephaly and confirmed infection)

Prenatal

Postnatal

Prenatal

US/MRI

CT

US

100% Hypoplasia

Yes 2/3

2 cases with large occipital subependymal psuedocysts

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US/fetal MRI Postnatal MRI/CT

Yes 100%

Yes 90.9%

57.1% BBGG/brain stem/thalami 100% Subcortical 14.2% Periventricular

Increased SA space. Reduced cerebral volume.

Mild, moderate or severe and usually asymmetric.

Yes 100%

Yes 100%

Yes 100%

Yes 100% diffuse polymicrogyria and opercular dysplasia

Yes 2/3 cerebellar hypoplasia 1/3 BS hypoplasia

Yes 100%

Yes 100%

Yes

100% Abnormal hypodensity of WM

Cerebellar hypoplasia 74%

87% that diffusely involved all cerebral lobes.

Yes 100% Frontal lobe (69-78%) Parietal lobe (83-87%). Cortico-medullary junction (53-86%) Punctuate (72-100%) Bandline distribution (56-75%) BBGG (57-65%) Thalamus (39-43%) Yes 100% On WM, Frontal lobe, BBGG and cerebellum.

Yes 100%

27.3% Less severe gyral disorganization 45.4% Pachyria 18.2% Lissencephaly

Severe in 53% of the cases.

Involving only lateral ventricles in 43%

Yes 81.8% hypoplasia of cerebellar vermis

Global hypogyration.

Severe (only Sylvian fissure was visible in 78%)

Yes

Abnormalities of BBGG and thalamus and hypodevelopemnt of BS and cerebellum in most patients

SC

Prenatal/ Postnatal

Arthrogryposis 3/11 Unilateral microphtalmia and bilateral cataracts 1/11 FGR

Yes

TE D

Hazin20 (N=23; Infants with microcephaly. Confirmed in 7/23)

7.7% Periventricular

EP

GuillemetteArtur42 (N=3; Fetuses with brain anomalies and confirmed ZIKV infection)

7.7% with hypertensive pattern

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Oliveira Melo7 (N= 11; Fetuses and Infants with confirmed ZIKV infection and brain anomalies)

Cortical/subcortical junction and BBGG

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microcephaly and confirmed infection and brain anomalies)

Brain stem hypoplasia 2/23

1/23 signs of ischemic stroke of left MCA

Yes

Yes

Yes

Yes

100%

50% Severe unilateral VMG

100% Thinned pons and BS

100% Thinned in one case and absent in other

Cisterna magna in one case. Cataracts in one case.

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US/MRI

Yes

Yes

Yes

Yes

Yes

Yes

80% Enlarged SA space

60%

80% Opercular dysplasia (agyria, polymicrogyria)

60% Vermian dysgenesis

60% Absence or dysgeneis

100% Parenchymal calcification

40% Occiptal subependymal pseudocysts

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Prenatal and postnatal

EP

TE D

M AN U

SC

Abbreviations: BS= Brain stem; WM=White Matter; GM=Gray matter; BBGG=Basal Ganglia; CT=CT scan; US=Ultrasound; SA=Subarachnoid space. * Only the results reported from Group 1a in this reference are in the table, which corresponds to fetuses with cerebral anomalies and microcephaly. This subgroup was selected as it is the only one with laboratory confirmation.

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Besnard43* (N=19; 5 Fetuses with microcephaly. Confirmed in 4 cases)

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SUPPLEMENTARY MATERIAL FETAL ULTRASONOGRAPHY:

Sonographic studies were performed with a Voluson General Electric E8 scanner (GE

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Healthcare, Milwaukee, WI), by an experienced Maternal Fetal Medicine (MFM)

specialist. Images were reviewed by an external MFM specialist for quality assurance. Fetal anatomy was assessed in detail and estimated fetal weight (EFW) was used to

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evaluate growth based on the Hadlock model1 . Doppler assessment included pulsatility index (PI) of the umbilical artery, middle cerebral artery and uterine arteries.

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Cerebroplacental ratio was calculated and mean PI of the uterine arteries were calculated. All the Doppler parameters were compared to reference ranges2.

NEUROSONOGRAPHY:

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Images were obtained by the combination of a transabdominal/transvaginal approach or transabdominal only depending if the fetus was in cephalic or breech presentation respectively. Standard planes were obtained, digitally stored and reviewed by an external MFM specialist (M.S.C). Main fetal brain structures (corpus callosum length3, transcerebellar diameter4, posterior

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fossa anteroposterior width, cerebellar vermis height5, anterior horns width6, atrial width of both posterior ventricular horns7, third ventricle width8 and craniocortical and sinocortical spaces)

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were measured and compared to normograms. Additionally, brain parenchyma was assessed and the presence of calcifications or hyperechogenic areas were reported if present.

FETAL BRAIN MRI AND DWI: MRI was performed on a 1.5 Tesla Phillips Achieva scanner-software version 5.1.7- (Phillips North America, Andover, MA). No maternal or fetal sedation was used. MRIs were acquired using a 6 channel body array coil on maternal abdomen placed as close as possible to the fetus for

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optimal signal. Localizer sequences were obtained using a Balanced Turbo Field Echo (BTFE) sequence in three orthogonal planes (axial, coronal, sagittal). Diagnostic sequences performed: Axial Single Shot Fast Spin Echo (SSFSE) T2-weighted image (slice thickness of 5 mm,

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acquisition time 20seconds, Echo time (TE) was 2ms, and repetition time (TR) was 4ms. SSFSE T2- weighted sagittal images were obtained with 16 seconds for acquisition time, TE: 2ms,

TR:4ms. The last sequence obtained was a coronal Short TI Inversion Recovery (STIR), with 16

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seconds as acquisition time, TE: 73 ms, and TR: 7656ms. An axial he rapid echo-planar Diffusion weighted image (DWI) sequence was also acquired, using a TE 105ms, TR 3410ms, acquisition

IMAGE POST-PROCESSING:

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time 40 secs.

Fetal brain segmentation and volumetric assessment were performed using the acquired T2 weighted images. Due to the inherent motion and intensity corruption it was necessary to perform a super-resolution reconstruction from the three individual T2 weighted image stacks obtained

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during acquisition to get a single 3-D volume, on which volumetric analysis was performed. The process of super-resolution was carried out according to previously established software and protocols9. Steps include first converting the originally acquired raw-data from DICOM to NIfTI

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format. Next, masks were created on axial stacks for each case, in order to rule out extraneous image data. Super-resolved volume was obtained by co-registering the 3 orthogonal T2 weighted

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image (Axial, Coronal, Sagittal) stacks and iteratively performing voxel-based intensity matching using the reconstruction program previously described9. Super-resolved images were then analyzed by performing post-processing manual segmentation (Suppl. Fig 1) using Amira 6.0 software (FEI Visualization Sciences Group, Hillsboro, Oregon). For the purpose of this research, volumetric analysis was performed on supratentorial brain parenchyma, ventricular system, brainstem, cerebellum, and cerebrospinal fluid (CSF) in the subarachnoid space. Total brain volume was calculated as the addition of all volumes except for CSF in subarachnoid space. Total intracranial volume was calculated as Total brain volume in addition to the CSF

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volume in the subarachnoid space. Using the segmentation data, individual volumes were assessed by using the Amira tool, Material Statistics. FETAL BRAIN DWI POSTPROCESSING:

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DW images were visualized through an Osirix MD software version 2.6 (Pixmeo SARL- Geneva, Switzerland). Images were first run through the plugin image filter UMM Diffusion (Heidelberg University- Mannheim, Germany) followed by drawing specific regions of interest for evaluation

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(Suppl. Fig. 2). ROIs that were assessed included: cerebellar hemispheres (bilateral), cerebellar

vermis, frontal/parietal/occipital lobes (bilateral), and mesencephalon (bilateral). Average values

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from right and left areas were calculated. Additionally, the obtained ADC values from ZIKV fetuses were also compared to previously published normality ranges for the same regions at similar gestational ages.10

FETAL BRAIN MRI HEALTHY CONTROLS:

Healthy controls were obtained from a preexisting study from Baylor College of Medicine (IRB

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H-30457) and were considered fetuses with a normal growth, no structural malformations and normal karyotype (Suppl. Table 1).

Fetal brain MRIs in this group of healthy controls were performed at Texas Children’s Hospital,

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Houston, TX, using a 1.5 Tesla Phillips

Ingenia scanner -software version 5.1.7- (Phillips North America, Andover, MA). All MRI

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studies were performed without maternal or fetal sedation. A combined six-channel body array coil was used. Conventional imaging was performed using a non-breath-hold SSFP sequence in three orthogonal planes to provide initial localization. Main images were acquired using a singleshot-T2 (SSH TSE) sequence in three orthogonal planes (axial, coronal sagittal) with the following parameters: 3 mm slice thickness, 1.5 mm slice overlap, 1050ms TR, 140ms TE. Acquisition time was 45 seconds per plane. Rapid echo-planar DWI was acquired in the axial plane using a b-value of 0 and 800 s/mm3 along 3 orthogonal directions. The following parameters were used: TR, 3400 ms; TE, 125 ms; section thickness, 4 mm. Each DWI sequence

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was acquired in 2 minutes. ADC maps were constructed using the assigned b-values. All MRIs shared the following settings: field of view, 280 mm Å~ 280 mm; matrix, 128 Å~ 128; voxel size, 2.5 mm Å~ 2.5 mm Å~ 4.0 mm. Fat suppression was achieved using a frequency selective radio

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frequency pulse. Images were processed in the same way as described above for ZIKV cases. Examiners were blinded to the patient’s clinical information and outcomes. POSTNATAL BRAIN IMAGING:

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• Transcranial ultrasound was performed obtaining the standard planes for this purpose11 using a Voluson General Electric E8 scanner -GE Healthcare, Milwaukee, WI-).

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• CT scans were performed using a Brightspeed GE scanner with 16 coils, using the following settings: slice thickness 1.3mm, kv: 100mA, obtaining a total of 1800 axial slices with coronal and sagittal reconstructions.

• Postnatal MRI scans, were performed on the same device as for prenatal images was used, with a head coil, T1 axial images were obtained with TR 149ms, TE 2ms, acquisition time 20 secs,

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also T2-weighted images were obtained with TR 110ms, TE 4406ms with acquisition time per sequence of 4 min. Postnatal MRI images were processed in the same way as for fetal images, obtaining a volumetric assessment of the same regions, this was compared to normality

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references12. In one case, DWI information was acquired. Same ADC regions as those obtained

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in fetuses, were calculated and compared to normal references values for that age13 (Fig. 2)

REFERENCES

1.

Hadlock FP, Harrist RB, Sharman RS, Deter RL, Park SK. Estimation of fetal weight with

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the use of head, body, and femur measurements-a prospective study. Am J Obstet Gynecol. 1985;151(3):333-337. 2.

Arduini D, Rizzo G. Normal values of Pulsatility Index from fetal vessels: a cross-

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sectional study on 1556 healthy fetuses. J Perinat Med. 1990;18(3):165-172. Achiron R, Achiron A. Development of the human fetal corpus callosum: A highresolution, cross-sectional sonographic study. Ultrasound Obstet. Gynecol.

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2001;18(4):343-347. 4.

Sherer DM, Sokolovski M, Dalloul M, Pezzullo JC, Osho JA, Abulafia O. Nomograms of

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the axial fetal cerebellar hemisphere circumference and area throughout gestation. Ultrasound Obstet Gynecol. 2007;29(1):32-37. 5.

Benn PA, Gainey A, Ingardia CJ, Rodis JF, Egan JFX. The fetal cerebellar vermis: Normal development as shown by transvaginal ultrasound. Prenat Diagn. 2001;21(8):687692.

Brouwer MJ, de Vries LS, Groenendaal F et al. New reference values for the neonatal

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6.

cerebral ventricles. Radiology 2012; 262(1):224-33. 7.

International Society of Ultrasound in Obstetrics and Gynecology Education Committee.

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Sonographic examination of the fetal central nervous system: guidelines for performing the “basic examination” and the “fetal neurosonogram”. Ultrasound Obstet Gynecol

8.

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2007;29(109-16).

Sari A, Ahmetoglu A, Dinc H et al. Fetal biometry: size and configuration of the third

ventricle. Acta Radiol 2005;46(6):631-635.

9.

Kuklisova-Murgasova M, Quaghebeur G, Rutherford MA, Hajnal J V., Schnabel JA. Reconstruction of fetal brain MRI with intensity matching and complete outlier removal. Med. Image Anal. 2012;16(8):1550-1564.

10.

Righini A, Bianchini E, Parazzini C, Gementi P, Ramenghi L, Baldoli C, Nicolini U,

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Mosca F, Triulzi F. Apparent diffusion coefficient determination in normal fetal brain: a prenatal MR imaging study. AJNR. Am. J. Neuroradiol. 2003;24(5):799-804. 11.

AIUM practice guideline for the performance of neurosonography in neonates and infants.

12.

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J Ultrasound Med 2010;29(1):151-156. Knickmeyer RC, Gouttard S, Kang C, Evans D, Wilber K, Smith JK, Hamer RM, Lin W, Gerig G, Gilmore JH. A structural MRI study of human brain development from birth to 2

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years. J. Neurosci. 2008;28(47):12176-12182.

Wolf RL, Zimmerman RA, Clancy R, Haselgrove JH. Quantitative apparent diffusion

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coefficient measurements in term neonates for early detection of hypoxic-ischemic brain

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injury: initial experience. Radiology 2001;218(3):825-33.

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Maternal Age (years) BMI (kg/m2) Avg. Gravida Avg. Parity Primiparity (%) History of Smoking (%) Race* (% Non-white) Gestational age at delivery (weeks) Preterm delivery** (%) Birth weight (grams) Birth weight percentile† Fetal growth restriction Head circumference (mm) Head circumference percentile† Length (mm) Length percentile† Vaginal delivery (%) Termination of Pregnancy (%)

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(N=10) 32.6 ± 4.4 29.66 ± 7.11 2.66 ± 1.87 1.00 ± 1.32 4 (40%) 0 3 (30%) 38.67 ± 2.36 11.1 3517 ± 573 66.95 ± 24.51 0 343 ± 13.1 65.8 ± 22.2 514.5 ± 32.6 74.83 ± 32.6 62.5 0

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Suppl. Table 1- Maternal Demographics and Perinatal Characteristics of 10 healthy aged-matched Controls

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BMI= Body mass index. Avg=Average.* Race defined as White (including Hispanic), Black or African American, American Indian and Alaska Native, Native Hawaiian/Pacific Islander, Other. ** Preterm delivery defined as gestational age at delivery less than 37 weeks. † After using Intergrowth 21 normograms23 .

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Suppl. Table 2. Maternal-Fetal demographic and cohort characteristics

Yes

2

No

3

Yes

Date TOP

27.3

4/11/16

Yes

5

No

6

No

7

No

8

No

9

No

10

No

11

No

12

No

GA delivery (weeks)

Location delivery

Way of delivery

Birth Weight (g)

800

19

204

0

C-section

1800

0.07

280

0

1000

29

235

4

352

4

205

99.26

Vaginal

2410

46.48

240

0

C-section

2810

28.61

295

0.4

Barranquilla 6/12/16

38.5

Barranquilla Santa Marta

29.0 4

Date deliver y

4/30/16 Santa Marta

21.1

5/3/16 Barranquilla 5/14/16

35.2

7/6/16

38.2

6/17/16

38.5

6/24/16

36.6

6/28/16

38.6

8/25/16

38.4

8/7/16

38.0

5/19/16

38.3

Riohacha Barranquilla

Birth weight percentile *

Barranquilla Barranquilla Caucasia

Barranquilla Santa Marta

Head circumference (mm)

Head circumference percentile*

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GA TOP (weeks)

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TOP

C-section

2810

22.9

270

0

Vaginal

2595

31.04

290

0.22

C-section

2920

22.5

290.5

0.01

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Case number

C-section

2660

19

300

1

C-section

3000

46

290

0

C-section

2530

12

340

62

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GA= Gestational age. TOP= Termination of pregnancy. HC=Head circumference. C-section= cesarean section.* Birth weight and Head circumference percentiles using Intergrowth 21 standards23 .

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Suppl. Table 3. Neurosonographic Findings

Gestation al age at scan (weeks)

Head circumference (mm)

Corpus callosum length (mm)

Transcerebellar diameter (mm)

Cerebellar vermian height (mm)

Maximal atrial width in posterior horns of lateral ventricles (mm)

1

26.0

203 ↓

24.0 ↓

11.6 ↓

10.8 ↑

2

28.2

215.4 ↓

29.5 ↓

14.9↓

12.2 ↑

3

27.2

199.4 ↓

28.8 ↓

16

10.4 ↑

4

20.0

60

16.5 ↓

8.7 ↓

11.4 ↑

5

33.6

280.9 ↓

47.4

12.4 ↑

6

31.4

233.6 ↓

35.2 ↓

9.2

7

30.4

231 ↓

29 ↓

17.0 ↑

8

34.5

255 ↓

45.2

9

23.0

197.1

24.4

10

33.2

250 ↓

11

36.6

272.4 ↓

20.0↓

47.8

12

27.2

250

26.2↓

30.3

10.3 ↓

Right anterior horn width (mm) 4.2 ↑

Left anterior horn width (mm) 3.1 ↑

5.5 ↑

8.8 ↑

5.5 ↑

3.8 ↑

7.1 ↑

5.9 ↑

5.1 ↑

6.0 ↑

7.6 ↑

3.1↑

3.3 ↑

5.6 ↑

4.4 ↑

3.5 ↑

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23 ↓

Third ventricle width (mm)

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Case number

15.5 ↑ 11.3 ↑

33.5

15.0 ↑ 10.9 ↑

1.8

7.8 ↑

6.7 ↑

16.5 ↓

6.7

1.2

2.3 ↑

2.4 ↑

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15.7 ↓

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Abbreviations: Arrows indicate abnormal findings: Head circumference less than the 5th percentile; Corpus callosum length less than the 5th percentile; Transcerebellar diameter less than the 5th percentile; Cerebellar vermian height less than the 5th percentile, Atrial width ≥10 mm; Third ventricle width above the 95th percentile; Anterior horns above the 95th percentile.

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Suppl. Table 4- Doppler ultrasound assessment of ZIKV infected and affected fetuses GA 29.2 33.2 23.3

UA PI 1.12 1.2 1.1

MCA PI 2 2.04

CPR 1.78 1.7

mUtA PI 0.76 0.83 0.73

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Case 7 11 12

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Abbreviations: GA= Gestational age; PI= Pulsatility index; UA= umbilical artery; MCA= medial cerebral artery; CPR= cerebroplacental ratio calculated as UA PI/ MCA PI; mUtA: mean uterine artery. All Doppler assessments were considered to be within the normality ranges (UA PI and mUtA PI less than the 95th percentile and MCA PI and CPR above the 5th percentile).

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Suppl. Table 5- Pathology results Case 1

Case 3

Case 4

27.3

29

21.1

Weight at delivery (g)

800

1000

352

Weight at delivery percentile IG21*

19

29

Microcalcifications. Cortical thinning. Increased neuroblast apoptosis and disrupted neuropil.

Microcalcifications. Cortical thinning.

Increased neuroblast apoptosis and disrupted neuropil. Minimal microglial activation.

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Mild microglial activation.

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CNS

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GA at TOP (weeks)

Diffuse congestion and focal microhemorrhage.

Non-homogenous accelerated placental pattern. Excessive perivillous fibrin deposition. Partial obliteration of fetal vessels. Enlarged chorionic villi.

Excessive perivillous fibrin deposition. Partial obliteration of fetal vessels. Hyperplasia of the nodi of syncytial cells. Enlarged chorionic villi.

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Placenta

Diffuse congestion and recent hemorrhage.

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Abbreviations: GA=Gestational age; TOP= Termination of Pregnancy; IG21=Intergrowth 2124. CNS= Central nervous system.* Intergrowth 21 standards23

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Case 12 2 m 22 d 6150 75 560 7 390

0

0

44

present no 180/108 140 95% 1m3w

present present 128/75 96 100% 2m

Present Present 70/45 115 98%

Normal 1m 5d

Normal 0m

Absence of evoked potentials bilaterally in optic nerve

Delayed conduction of retinocortical pathway bilaterally

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Case 10 1 m 15 d 6345 99 540 25 320

0m

Normal

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Age at evaluation Weight (g) Weight percentile* Height (mm) Height percentile* Head circumference (mm) Head circumference percentile* Pupillary reflexes Babinsky reflex Blood Pressure (Hg mm) Heart rate (bpm) O2 Saturation Age at audiology assessment Result audiology report Age at Visual evoked potentials Visual evoked potentials

Case 6 1m5d 4250 44 540 45 320

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Suppl. Table 6- Postnatal follow up assessment.

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Abbreviations: m=month; d= day. * Weight, height and head circumference percentiles were calculated using Intergrowth 21 standards23 .

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