lable at ScienceDirect Clinical Microbiology and Infection 24 (2018) 646e652 Contents lists avai Clinical Microbiology and Infection journal homepage: www.cl in icalmicrobiologyandinfect ion.com Original article Adverse birth outcomes associated with Zika virus exposure during pregnancy in S~ao Jos�e do Rio Preto, Brazil M.L. Nogueira 1, *, N.R.R. Nery Júnior 4, C.F. Estofolete 1, A.C. Bernardes Terzian 1, G.F. Guimar~aes 1, N. Zini 1, R. Alves da Silva 1, G.C. Dutra Silva 1, L.C. Junqueira Franco 1, P. Rahal 2, C. Bittar 2, B. Carneiro 2, P.F.C. Vasconcelos 5, D. Freitas Henriques 5, D.M.U. Barbosa 3, P. Lopes Rombola 3, L. de Grande 3, A.F. Negri Reis 3, S.A. Palomares 3, M. Wakai Catelan 3, L.E.A.A. Cruz 3, S.H. Necchi 3, R.C.V. Mendonça 3, I.N. Penha dos Santos 3, S.B. Alavarse Caron 3, F. Costa 4, 6, 9, F.A. Bozza 7, A. Soares de Souza 1, C.C. Brand~ao de Mattos 1, L.C. de Mattos 1, N. Vasilakis 8, A.H. Oliani 1, D.C.M. Vaz Oliani 1, A.I. Ko 9 1) S~ao Jos�e do Rio Preto School of Medicine, S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil 2) S~ao Paulo State University, S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil 3) Health Secretariat, S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil 4) Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil 5) Evandro Chagas Institute, Ananindeua, Par�a, Brazil 6) Federal University of Bahia, Salvador, Bahia, Brazil 7) Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil 8) University of Texas Medical Branch (UTMB), Galveston, TX, USA 9) Yale School of Public Health, New Haven, CT, USA a r t i c l e i n f o Article history: Received 15 October 2017 Received in revised form 29 October 2017 Accepted 2 November 2017 Available online 10 November 2017 Editor: L. Leibovici Keywords: Adverse outcome Arbovirus Birth Pregnancy Zika virus * Corresponding author. M. L. Nogueira, S~ao Jos�e do (FAMERP), 5416 Brigadeiro Faria Lima Ave, Vila S~ao Pe Paulo, 15090-000, Brazil. E-mail address: mnogueira@famerp.br (M.L. Nogu https://doi.org/10.1016/j.cmi.2017.11.004 1198-743X/© 2017 European Society of Clinical Micro a b s t r a c t Objectives: We aimed to report the first 54 cases of pregnant women infected by Zika virus (ZIKV) and their virologic and clinical outcomes, as well as their newborns' outcomes, in 2016, after the emergence of ZIKV in dengue-endemic areas of S~ao Paulo, Brazil. Methods: This descriptive study was performed from February to October 2016 on 54 quantitative real- time PCR ZIKV-positive pregnant women identified by the public health authority of S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil. The women were followed and had clinical and epidemiologic data collected before and after birth. Adverse outcomes in newborns were analysed and reported. Urine or blood samples from newborns were collected to identify ZIKV infection by reverse transcription PCR (RT-PCR). Results: A total of 216 acute Zika-suspected pregnant women were identified, and 54 had the diagnosis confirmed by RT-PCR. None of the 54 women miscarried. Among the 54 newborns, 15 exhibited adverse outcomes at birth. The highest number of ZIKV infections occurred during the second and third trimesters. No cases of microcephaly were reported, though a broad clinical spectrum of outcomes, including lentic- ulostriate vasculopathy, subependymal cysts, and auditory and ophthalmologic disorders, were identified. ZIKV RNA was detected in 18 of 51 newborns tested and in eight of 15 newborns with adverse outcomes. Conclusions: Although other studies have associated many newborn outcomes to ZIKV infection during pregnancy, these same adverse outcomes were rare or nonexistent in this study. The clinical presentation the newborns we studied was mild compared to other reports, suggesting that there is significant heterogeneity in congenital Zika infection. M.L. Nogueira, Clin Microbiol Infect 2018;24:646 © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. Rio Preto School of Medicine dro, S~ao Jos�e do Rio Preto, S~ao eira). biology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. mailto:mnogueira@famerp.br www.sciencedirect.com/science/journal/1198743X http://www.clinicalmicrobiologyandinfection.com https://doi.org/10.1016/j.cmi.2017.11.004 https://doi.org/10.1016/j.cmi.2017.11.004 https://doi.org/10.1016/j.cmi.2017.11.004 M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652 647 Introduction Zika virus (ZIKV) infection has been associated with severe birth defects, such as newborn microcephaly [1,2], meningoen- cephalitis [3] and Guillain-Barr�e syndrome (http://ecdc.europa.eu/ en/publications/Publications/zika-virus-americas-association-with- microcephaly-rapid-risk-assessment.pdf) [4]. Microcephaly repre- sents a small part of a broad spectrum of teratogenic outcomes of intrauterine ZIKV infection referred to as congenital Zika syn- drome [5]. Intrauterine growth restriction, ocular abnormalities, placental damage, foetal blood anomalies [6] and death are other findings that may be associated with ZIKV infection during preg- nancy [1,2,7]. The city of S~ao Jos�e do Rio Preto in S~ao Paulo State, Brazil, is a region in which several arbovirus circulate [8e10]. In 2016 a ZIKV outbreak was reported in the city [11], and a surveillance system was established to identify illnesses caused by ZIKV. Special atten- tion has been paid to pregnant women in an attempt to ascertain the impact of ZIKV infection on newborns. This study is a report of the first 54 confirmed cases of women infected by ZIKV during pregnancy and their virologic and clinical outcomes, as well as their newborns' outcomes, identified through our surveillance system. Methods Study population From February to October 2016, the city's public health au- thority identified 216 pregnant patients with Zika-like symptoms among 1674 pregnant women receiving elective and emergency services. The Brazilian Ministry of Health defines Zika-suspected cases on the basis of macular or papular rash with two or more of the following signs or symptoms: fever, conjunctival hyperaemia without secretion, pruritus, polyarthralgia or joint oedema [12]. Fifty-seven pregnant women with symptomatic acute Zika- suspected infection between 5 and 38 weeks of pregnancy (gesta- tional age defined as first trimester until week 13, second trimester from weeks 14 to 26, and third trimester after week 27) [13] who attended a health service in S~ao Jos�e do Rio Preto were considered to be Zika-suspected patients and had a blood sample collected during acute infectionwhich was found to be ZIKV RT-PCR positive. These pregnant women were referred to the Children's and Ma- ternity Hospital in S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil, the reference hospital, and were monitored under a protocol approved by the S~ao Jos�e do Rio Preto medical school institutional review board. These blood samples were also tested for toxoplasmosis, other agents (including HIV, when relevant), rubella, cytomegalo- virus, herpes simplex, and syphilis (TORCHS) using molecular and/ or serologic methods. The ZIKV-positive pregnant women were monitored by a multidisciplinary medical team through the use of clinical and radiologic evaluations. After delivery, the newborn's umbilical cord blood and/or urine were collected and tested for the presence of ZIKV bymolecular and serologic methods. The clinical examinations of newborn and anthropometric measurements were performed according to the guidelines of the Brazilian Ministry of Health [12], including defi- nition of microcephaly, as newborns of 37 weeks' gestation or less and cephalic perimeter lower than 2 standard deviations (based on INTERGROWTH-21st Project data) for gestational age and sex [14]; or newborns of 37 weeks' gestation or more and cephalic perimeter of less than or equal to 31.5 cm for girls and 31.9 cm for boys, and equivalent to lower than 2 standard deviations (based on World Health Organization criteria) [15]. The following were considered to be adverse outcomes: lenticulostriate vasculopathy, subependymal cysts, choroidal cyst, bilateral cranial bleed, chorioretinitis, premature birth and abnormal otoacoustic emission (OAE). Ultrasounds were performed with an HDI 5000 convex probe (Philips, Amsterdam, The Netherlands) in order to generate foetal and postnatal images. Magnetic resonance imaging was performed with a Philips Gyroscan Intera 1.5 T scanner, and the images were analysed by specialists in foetal medicine. Special attention was given to the foetus's or newborn's central nervous system. When available, OAE tests and fundus examinations were performed by specialists to identify any auditory or ophthalmologic disorders, respectively. Virus and RNA extraction The virus strain used as positive control was ZIKVBR. It was propagated in C6/36 Aedes albopictus cell cultures [15e17]. Virus RNA was extracted from 140 mL blood and urine samples with the QIAamp Viral RNA Mini kit (Qiagen, Germantown, MD, USA) ac- cording to the manufacturer's instructions. ZIKV quantitative real-time PCR To detect the ZIKV genome in mothers' blood or newborns' umbilical cord blood and/or urine samples, a one-step quantitative real-time, fluorescent probe-based RT-PCR (qPCR) assay was per- formed using primers targeting the envelop (E) gene [18]. All samples with Ct values of 38.5 or less were considered positive for ZIKV. ZIKV ELISA The umbilical cord blood samples found to be positive for ZIKV in qPCR were also tested for the Zika NS1 protein. The Zika Virus NS1 ELISA Kit (BioFront Technologies, Tallahassee, FL, USA) was used to capture anti-ZIKV NS1. All of the assays were performed according to themanufacturer's instructions. Each platewas read at 450 nm using a Spectramax Plus Microplate reader (Molecular Devices, Sunnyvale, CA, USA). Complete genome After RNA extraction, cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). Nineteen fragments were amplified by nested PCR using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific). Fragment sizes ranged from 430 to 1461 bp. Nested PCR products were purified using the DNA Clean & Concentrator Kit (Zymo Research, Irvine, CA, USA). Frag- ments were sequenced using the direct Sangermethodwith BigDye Terminator 3.1 in an ABI 3130XL Genetic Analyzer (Applied Bio- systems). Sequences were assembled and analysed for coverage and quality by SeqMan software from the Lasergene package (DNASTAR, Madison, WI, USA). Phylogenetic reconstruction The evolutionary history was inferred using the maximum likelihood method based on the general time reversible model [19] using a data set compiled of 99 complete open reading frame nucleotide sequences available in GenBank. The tree with the highest log likelihood (�35779.2777) is shown in Supplementary Fig. S1. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial trees for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise http://ecdc.europa.eu/en/publications/Publications/zika-virus-americas-association-with-microcephaly-rapid-risk-assessment.pdf http://ecdc.europa.eu/en/publications/Publications/zika-virus-americas-association-with-microcephaly-rapid-risk-assessment.pdf http://ecdc.europa.eu/en/publications/Publications/zika-virus-americas-association-with-microcephaly-rapid-risk-assessment.pdf M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652648 distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likeli- hood value. A discrete gamma distribution was used to model differences in evolutionary rates among sites (five categories; þG, parameter ¼ 0.2918). The rate variation model allowed for some sites to be evolutionarily invariable ([þI], 0.0010% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codon positions included were Table 1 Characteristics of women in cohort and their pregnancies according to infants' birth out Characteristic Total (n ¼ 54) Adverse b No. of responses No. of positives or median (% or IQR) No. of responses Demographic data Mother's age (years) 54 27.5 (23e34) 15 Ethnicity White 45 31 (69) 14 Mestizo 45 10 (22) 14 Black 45 3 (7) 14 Other 45 1 (2) 14 Educational level completed College education 44 10 (23) 14 High school 44 26 (59) 14 Primary school 44 8 (18) 14 Medical history Paras 37 1 (0e2) 12 Gravidas 37 2 (1.5e3.5) 12 Comorbiditiesb 54 9 (17) 15 STD 45 4 (9) 14 Zika infection during pregnancy Trimester of ZIKV infection First trimester 54 4 (7) 15 Second trimester 54 26 (48) 15 Third trimester 54 24 (44) 15 Rash 53 51 (96) 15 Pruritus 54 34 (63) 15 Headache 54 23 (43) 15 Arthralgias 54 21 (39) 15 Fever 54 18 (33) 15 Myalgias 54 15 (28) 15 Respiratory symptomsc 54 8 (15) 15 Conjunctivitis 54 1 (2) 15 Serum ZIKV RT-PCR positive 53 45 (85) 15 Urine ZIKV RT-PCR positive 52 41 (79) 14 Pregnancy Current alcohol drinker 44 2 (5) 14 Current smoker 44 6 (14) 14 Medicationsd 54 35 (65) 15 Complicationse 44 10 (23) 14 TORCH serology Toxoplasmosis IgM positive 47 2 (4) 13 CMV IgM positive 47 0 (0) 13 Rubella IgM positive 47 4 (9) 13 VDRL positive 49 1 (2) 13 US/MRI exam No. of prenatal US exams 51 3 (3e3) 14 Abnormal prenatal US examf 51 2 (4) 14 Abnormal foetal MRIg 25 6 (24) 8 CMV, cytomegalovirus; IQR, interquartile range; MRI, magnetic resonance imaging; NA, n TORCH, toxoplasmosis, other agents (including HIV, when relevant), rubella, cytomegalo test; ZIKV, Zika virus. a Adverse outcomes: lenticulostriate vasculopathy, subependymal cysts, choroidal c examination. b Comorbidities: with adverse outcomes: hypothyroidism (1), idiopathic thrombocyto comes: hypothyroidism (2), hypertension (3). c Coryza, sore throat or cough. d Medications: with adverse outcomes: levothyroxine (1), prednisone (1), methyldopa levothyroxine (1), methyldopa (2), methyldopa plus metformin (1), levothyroxine plus m e Complications during pregnancy: with adverse outcomes: gestational diabetes (1), he outcomes: gestational diabetes (2), rubella (4); acute toxoplasmosis (1). f US: with adverse outcomes: retro-ovulate haematoma (1), oligohydramnios (1). g MRI (no significant findings): with adverse outcomes: eccentric placental insertion of thyroid lobes (1), increased subtentorial measures plus pericardial effusions (1), right re 1 þ 2 þ 3 þ noncoding. All positions containing gaps and missing data were eliminated. There were a total of 10,208 positions in the final data set. Evolutionary analyseswere conducted inMEGA7 [20]. Statistical analysis All statistical analyses were carried out using the Epi Info soft- ware for Windows (Centers for Disease Control and Prevention, comes irth outcomesa (n ¼ 15) No adverse birth outcomes (n ¼ 39) p No. of positives or median (% or IQR) No. of responses No. of positives or median (% or IQR) 23 (21e38) 39 28 (22e34) 0.68 9 (64) 31 22 (71) 0.83 4 (29) 31 6 (19) d 1 (7) 31 2 (6) d 0 (0) 31 1 (3) d 2 (14) 30 8 (27) 0.51 10 (71) 30 16 (53) d 2 (14) 30 6 (20) d 1 (0.5e2) 25 1 (0e1) 0.60 2 (1.5e3) 25 2 (2.5e3) 0.80 3 (20) 39 6 (15) 0.68 2 (14) 31 2 (6) 0.39 1 (7) 39 3 (8) 0.20 4 (27) 39 22 (56) d 10 (67) 39 14 (36) d 14 (93) 39 37 (95) 0.49 10 (67) 39 24 (62) 0.73 5 (33) 39 18 (46) 0.39 7 (47) 39 14 (36) 0.47 3 (20) 39 15 (38) 0.20 4 (27) 39 11 (28) 0.91 0 (0) 39 8 (20) 0.06 0 (0) 39 1 (3) 0.53 14 (93) 38 31 (82) 0.28 10 (71) 38 31 (82) 0.43 1 (7) 30 1 (3) 0.57 2 (14) 30 4 (13) 0.93 10 (67) 39 25 (64) 0.86 4 (29) 30 6 (20) 0.53 1 (8) 34 1 (3) 0.47 0 (0) 34 0 (0) NA 0 (0) 34 4 (12) 0.20 1 (8) 36 0 (0) 0.09 3 (3e3) 37 3 (3e3) 0.80 2 (14) 37 0 (0) 0.02 1 (13) 17 5 (29) 0.36 ot applicable; RT-PCR, reverse transcription PCR; STD, sexually transmitted disease; virus, herpes simplex; US, ultrasound; VDRL, Venereal Disease Research Laboratory yst, bilateral cranial bleed, chorioretinitis, premature birth, abnormal otoacoustic penic purpura (1), chronic cardiopathy (1), hypertension (1); with no adverse out- (1), sulfadiazine plus pyrimethamine (1), acyclovir (1); with no adverse outcomes: etformin (1), clindamycin (1), spiramycin (1). rpes simplex virus infection (1), syphilis (1), acute toxoplasmosis (1); with no adverse umbilical cord (1); with no adverse outcomes: placental thickening (1), asymmetrical nal cyst in foetus (1), swallowing failure and gastric distention (1). M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652 649 Atlanta, GA, USA).We used chi-square andWilcoxon rank sum tests to compare the characteristics according to birth outcomes for categorical and continuous data, respectively. Results Among 216 symptomatic acute ZIKV-suspected pregnant women in the S~ao Jos�e do Rio Preto public health system between February 2016 and October 2016, this descriptive study included 57 pregnant women (26%) who had ZIKV infection confirmed by RT- PCR in blood. Three pregnant women (5%) were lost to follow-up, resulting in a final sample size of 54 women. ZIKV infection was detected in all trimesters of gestation. Fifteen pregnant women (28%) experienced adverse birth outcomes. The clinical and de- mographic characteristics of the 54 mothers and their respective newborns are shown in Table 1. The distribution of suspected and confirmed cases of ZIKV according to epidemiologic week and gestational week of ZIKV exposure as well as the associations be- tween these data and adverse outcomes are shown in Figs 1 and 2. Fig. 1. Suspected and confirmed cases of Zika virus (ZIKV) infection according to epidemiolo infected pregnant women (panel C) in S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil, in 2016. No pregnant woman in this study miscarried, and only eight (15%) of the foetuses were born at less than 37 weeks' gestation. The Apgar score median of the newborns was 9 of 10 and 10 of 10 at 1 and 5 minutes, respectively; two newborns had Apgar scores lower than 7 at 1 minute and none at 5 minutes, and no abnor- malities were detected in the neurologic examinations. The addi- tional serologic screening performed for infectious diseases during pregnancy is shown in Table 1 and Supplementary Table S4. Find- ings of radiologic examinations are shown in Tables 1 and 2. Almost a quarter of pregnant women (28%, 15/54) who received follow-up care presented adverse foetal/birth outcomes (Supple- mentary Table S1). In three cases (20%) there were histories of comorbidities, and in seven cases the mother reported exposure to alcohol, tobacco or illicit drugs (Table 1 and Supplementary Table S1). One newborn, born prematurely, encountered all of the anthropometric parameters below those expected for gestational age, compatible with intrauterine growth restriction. In this same newborn, unilateral ultrasound, abnormal OAE test results and ZIKV in cord blood (RT-PCR) were all identified, without other gic week (panel A), gestational week of ZIKV exposure (panel B) and birth rate of ZIKV- Fig. 2. Characteristics of maternal cohort, Zika infection and adverse outcomes in S~ao Jos�e do Rio Preto, S~ao Paulo, Brazil, in 2016. M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652650 infectious agents but with exposure to illicit drugs (marijuana) during gestation. The adverse outcomes observed in each case of ZIKV exposure in utero are listed in Supplementary Tables S1eS3. Among the 39 newborns with no adverse birth outcomes, the profile of ZIKV exposure was similar to those with adverse out- comes. Clinical and laboratory data of these newborns are pre- sented in Table 2. The serologic and molecular tests for ZIKV are shown in Supplementary Table S6. Evidence of ZIKV infection was detected in 18 (35%) of 51 newborns who were evaluated by RT-PCR at birth using umbilical cord blood and/or urine samples (Table 3). Among the newborns who did not exhibit adverse outcomes, ZIKV RNA was detected in ten (28%) of 36 (Supplementary Tables S4e7). The complete genome of ZIKV was amplified from one patient and two controls, all adult men, and sequenced. Phylogenetic analyses revealed that the ZIKV identified in our mothers during the 2016 outbreak was clustered together with the same virus circulating in other areas of the country (Supplementary Fig. S1). Discussion On the basis of surveillance alerts, our health centre has been conducting a prospective study on ZIKV in pregnancy and associ- ated birth defects (with a focus on microcephaly) since January 2016. In ten months of surveillance, there were 216 cases of ZIKV- suspected pregnant women in our centre, and here we report 54 cases (26%) of pregnant women who were found to have ZIKV infection confirmed by RT-PCR in blood samples. Fifteen adverse foetal/birth outcomes and 18 cases of congenital ZIKV infection in newborns were reported. Although ZIKV infection in the first trimester of gestation is associatedwithmicrocephaly [1,2], no such cases have been detected thus far among the newborns in our cohort. Most of the adverse neurologic outcomes (14/15, 94%) occurred in the second and third trimesters, and this may have been responsible for these mild outcomes. This is not the first report to associate ZIKV infection after the first trimester with regular head circumference at birth but with adverse clinical outcomes, such as congenital brain injury acquired as a result of ZIKV [21]. The outcomes associated with ZIKV infec- tion during pregnancy may include no effects, miscarriage or foetal infection resulting in congenital Zika syndrome [22]. An important study performed in Brazil in 2016 [1] reported several outcomes in foetuses and newborns exposed to ZIKV during pregnancy, as in- trauterine growth restriction, cerebral calcifications, abnormal arterial flow in the cerebral or umbilical arteries, global cerebral atrophy, microcephaly, macular hypoplasia and scarring, and placental insufficiency. Congenital anomalies, including microcephaly, have a complex and multifactorial aetiology and may be caused by other infections (such as TORCH infections) during pregnancy, as well as chromo- somal disorders, exposure to environmental toxins and metabolic diseases [22,23]. Congenital toxoplasmosis [24], syphilis [25], her- pes simplex virus [26] and rubella [27,28] may affect the central nervous system and cause neurologic deficits. Out of all of the cases in this study inwhich subependymal cysts were observed, only one pregnant woman had a reagent toxoplasmosis IgM result and a newborn who was ZIKV RT-PCR positive in the umbilical cord blood. Among those with vasculopathy, the only infection identi- fied was that of ZIKV. These factors lead us to believe that ZIKV can be the cause of neurologic abnormalities. Knowing the cause of these issues is an important tool for prevention. Because this is a descriptive study, a control group of women with no infection was not defined. A limitation presented by this study was the lack of data in some variables. The data were collected by the attending physician on the basis of a preestablished record, although it was not always completely filled out. The clinical spectrum observed in our newborns differed from those reported in other studies. Lenticulostriate vasculopathy, subependymal cysts, auditory disorder and chorioretinitis were the main out- comes observed, and there were no cases of macular hypoplasia, microcephaly or abnormal neurologic test results after birth. These findings showed that the symptoms of congenital Zika syndrome might be broader than originally thought. The link to ZIKV may not be clearly established nor excluded. In some cases, the only infec- tious agent detected was ZIKV. In cases where other infectious agents were identified by serologic tests, the clinical findings were not usually related to them. In conclusion, our study highlights the importance of ZIKV infection in all trimesters of gestation. Brain abnormalities other than microcephaly, intracerebral calcifications or severe outcomes Table 2 Characteristics of newborn infants according to birth outcome Characteristic Total (n ¼ 54) Adverse birth outcomes (n ¼ 15) No adverse birth outcomes (n ¼ 39) p No. of responses No. of positives or median (% or IQR) No. of responses No. of positives or median (% or IQR) No. of responses No. of positives or median (% or IQR) Birth Gestational age at birth (weeks) 54 38 (37.5e38) 15 38 (37e39) 39 38 (37e38.5) 0.83 Premature (<37 weeks' gestation) 54 8 (15) 15 3 (20) 39 5 (13) 0.51 Male sex 54 30 (56) 15 6 (40) 39 24 (62) 0.15 Caesarean section delivery 35 29 (83) 12 11 (92) 23 18 (78) 0.32 Apgar score (median) At 1 minute 33 9 (9e9) 11 9 (9e9) 22 9 (9e9) 0.30 At 5 minutes 33 10 (9e10) 11 10 (10e10) 22 10 (9.5e10) 0.09 Anthropometric measurements Head circumference Circumference (cm) 53 35 (34e36) 15 35 (34e36) 39 35 (34e36) 0.71 Percentilea 53 89 (77e97) 15 92 (76e98) 39 89 (79e96) 0.78 Microcephalyb 54 0 (0) 15 0 (0) 39 0 0 d Weight Weight (g) 54 3097 (2901e3420) 15 2970 (2894e3486) 39 3098 (2929e3460) 0.62 Percentileb 54 66 (39e82) 15 65 (44e85) 39 66 (39e84) 0.95 Small for gestational ageb 54 0 (0) 15 0 (0) 39 0 (0) d Length Length (cm) 54 48 (46.8e49.5) 15 47 (46e48) 39 48 (47e49) 0.05 Percentile 54 43 (27e71) 15 32 (14e56) 39 48 (34e73) 0.05 Clinical evaluation Abnormal neurologic evaluation 54 0 (0) 14 0 (0) 40 0 (0) NA Abnormal ophthalmologic examc 22 2 (9) 10 2 (20) 12 0 (0) 0.10 Abnormal OAE/AABRd 34 6 (18) 14 6 (43) 20 0 (0) 0.00 Radiologic evaluations Cranial USe 38 7 (18) 14 7 (50) 24 0 (0) 0.00 Cranial MRI 3 0 (0) 1 0 (0) 2 0 (0) NA ZIKV diagnostic testing RT-PCR positive 51 18 (35) 15 8 (53) 36 10 (28) 0.08 Serum 48 14 (29) 15 5 (33) 33 9 (27) 0.67 Serum Ct 14 36.5 (36e37) 5 36.3 (36.2e36.5) 9 36.8 (35.6e37.4) 0.31 Urine 46 4 (9) 15 3 (20) 31 1 (3) 0.06 Urine Ct 4 36.5 (31e36.6) 3 36.4 (31e36.6) 1 37.7 d 0.18 Trimester of infection First trimester 18 2 (11) 8 1 (13) 10 1 (10) 0.20 Second trimester 18 8 (44) 8 2 (25) 10 6 (60) d Third trimester 18 8 (44) 8 5 (63) 10 3 (30) d MAC-ELISA positive ZIKV 16 0 (0) 7 0 (0) 9 0 (0) NA Hospitalization Days 37 2 (2e4) 14 2 (2e4) 23 2 (2.5e4.5) 0.64 NICU admission 54 5 (9) 15 0 (0) 39 5 (13) 0.15 AABR, automated auditory brain stem response; MAC-ELISA, dengue IgM capture enzyme-linked immunosorbent assay; MRI, magnetic resonance imaging; NA, not applicable; OAE, otoacoustic examination; RT-PCR, reverse transcription PCR; US, ultrasound; ZIKV, Zika virus. a Value less than �2 standard deviations head circumference. b Value <10th weight percentile. c Abnormal ophthalmologic exam: unilateral chorioretinitis. d OAE: one case, confirmed by AABR. e US: with adverse outcomes: lenticulostriate vasculopathy (2), subependymal cysts (3), choroidal cyst (1), bilateral cranial bleed (1). Table 3 Outcomes among newborns from mothers exposed to ZIKV during pregnancy Outcome No. of cases Incidence (95% CI) Adverse birth outcome 15/54 28 (17e41) Exposure in first trimester 1/4 25 (0.63e81) Exposure in second trimester 4/26 15 (5e33) Exposure in third trimester 10/24 42 (23e62) ZIKV detected at birth 8/18 44 (23e67) ZIKV not detected at birth 7/15 47 (23e71) ZIKV detection at birth 18/51 35 (22e48) ZIKV exposure in first trimester 2/4 50 (9e91) ZIKV exposure in second trimester 8/26 31 (15e50) ZIKV exposure in third trimester 8/24 33 (14e52) With adverse outcomes 8/15 53 (29e77) No adverse outcomes 10/39 26 (14e41) Cumulative incidence shown as cases per 100 births. CI, confidence interval; ZIKV, Zika virus. M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652 651 detected by imaging examinations during pregnancy may occur, reflecting the marked heterogeneity of exposure to ZIKV during pregnancy. Adverse outcomes were mild or nonexistent in our newborns, but their occurrence may affect neurologic develop- ment, thus having an important negative impact on the patient specifically and on the population more generally. These impacts may only be measured some years after birth. This study provides additional evidence of the association between congenital ZIKV infection and certain foetal outcomes, and it contributes to a better understanding in the pathogenesis of birth defects caused by ZIKV. Transparency declaration Supported by the S~ao Paulo Research Foundation (FAPESP) via grants 2013/21719-3 and 2016/15021-1 toMLN, grant 2015/12295-0 M.L. Nogueira et al. / Clinical Microbiology and Infection 24 (2018) 646e652652 to ACBT and grant 2016/05115-9 to LCM. The opinions, as- sumptions and conclusions or recommendations expressed in this material are the responsibility of the authors and do not necessarily reflect the views of FAPESP. PFCV was supported by the Zika Virus Fast Track program provided by the Coordination for the Improvement of Higher Level Education Personnel and the Brazilian National Council for Scientific and Technological Development (CNPq) by grants 303999/2016-0, 440405/2016-5 and 457664/2013-4. MLM is a CNPq research fellow. All authors report no conflicts of interest relevant to this article. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.cmi.2017.11.004. References [1] Brasil P, Pereira JP, Moreira ME, Ribeiro Nogueira RM, Damasceno L, Wakimoto M, et al. Zika virus infection in pregnant women in Rio de Janeiro. N Engl J Med 2016;375:2321e34. [2] Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M, Mraz J, et al. Zika virus associated with microcephaly. N Engl J Med 2016;374:951e8. [3] Carteaux G, Maquart M, Bedet A, Contou D, Brugieres P, Fourati S, et al. Zika virus associated with meningoencephalitis. N Engl J Med 2016;374:1595e6. [4] Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet 2017;4:2009e109. [5] Moore CA, Staples JE, Dobyns WB, Pessoa A, Ventura CV, Fonseca EB, et al. Characterizing the pattern of anomalies in congenital Zika syndrome for pe- diatric clinicians. JAMA Pediatr 2017;171:288e95. [6] Schaub B, Vouga M, Najioullah F, Gueneret M, Monthieux A, Harte C, et al. Analysis of blood from Zika virus-infected fetuses: a prospective case series. Lancet Infect Dis 2017;17:520e7. [7] Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika virus. N Engl J Med 2016;374:1552e63. [8] Mondini A, Chiaravalloti Neto F, Gallo y Sanches M, Lopes JC. Spatial analysis of dengue transmission in a medium-sized city in Brazil. Rev Saude Publica 2005;39:444e51. [9] Mondini A, Bronzoni RV, Cardeal IL, dos Santos TM, Lazaro E, Nunes SH, et al. Simultaneous infection by DENV-3 and SLEV in Brazil. J Clin Virol 2007;40:84e6. [10] Terzian AC, Mondini A, Bronzoni RV, Drumond BP, Ferro BP, Cabrera EM, et al. Detection of Saint Louis encephalitis virus in dengue-suspected cases during a dengue 3 outbreak. Vector Borne Zoonotic Dis 2011;11:291e300. [11] Fernanda Estofolete C, Terzian AC, Parreira R, Esteves A, Hardman L, Greque GV, et al. Clinical and laboratory profile of Zika virus infection in dengue suspected patients: a case series. J Clin Virol 2016;81:25e30. [12] Nota informativadSVS/MS. Procedimentos a serem adotados para a vigilância da Febre do vírus Zika no Brasil. Brasilia: Ministerio da Saude; 2016. [13] Assistência Pre ́ -natal: manual te ́ cnico. 3rd ed. Brasilia: Secretaria de Poli ́ ticas de Sau ́ de; 2000. p. 66. [14] Villar J, Cheikh Ismail L, Victora CG, Ohuma EO, Bertino E, Altman DG, et al. International standards for newborn weight, length, and head circumference by gestational age and sex: the Newborn Cross-Sectional Study of the INTERGROWTH-21st Project. Lancet 2014;384:857e68. [15] World Health Organization. Assessment of infants with microcephaly in the context of Zika vi ́ rus. Geneva: World Health Organization; 2016. p. 2. [16] Shope RE, Sather GE. Arboviruses. In: Lennet FH, Schimidt NJ, editors. Diagnostic procedures for viral, rickettsial and clamydial infections. 2nd ed. Washington, DC: American Public Health Association; 1979. p. 767e814. [17] Figueiredo LT. The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families. Rev Soc Bras Med Trop 1990;23:13e8. [18] Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008;14:1232e9. [19] Nei M, Kumar S. Molecular evolution and phylogenetics. In: New ed. New York: Oxford University Press; 2000. p. 333 il. [20] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870e4. [21] Soares de Souza A, Moraes Dias C, Braga FD, Terzian AC, Estofolete CF, Oliani AH, et al. Fetal infection by Zika virus in the third trimester: report of 2 cases. Clin Infect Dis 2016;63:1622e5. [22] Silasi M, Cardenas I, Kwon JY, Racicot K, Aldo P, Mor G. Viral infections during pregnancy. Am J Reprod Immunol 2015;73:199e213. [23] Kleber de Oliveira W, Cortez-Escalante J, De Oliveira WT, do Carmo GM, Henriques CM, Coelho GE, et al. Increase in reported prevalence of micro- cephaly in infants born to women living in areas with confirmed Zika virus transmission during the first trimester of pregnancydBrazil, 2015. MMWR Morb Mortal Wkly Rep 2016;65:242e7. [24] Singh S. Congenital toxoplasmosis: clinical features, outcomes, treatment, and prevention. Trop Parasitol 2016;6:113e22. [25] Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 2015;64:138. [26] Enright AM, Prober CG. Neonatal herpes infection: diagnosis, treatment and prevention. Semin Neonatol 2002;7:283e91. [27] Rorke LB. Nervous system lesions in the congenital rubella syndrome. Arch Otolaryngol 1973;98:249e51. [28] Miller E, Cradock-Watson JE, Pollock TM. Consequences of confirmed maternal rubella at successive stages of pregnancy. Lancet 1982;2:781e4. https://doi.org/10.1016/j.cmi.2017.11.004 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref1 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref1 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref1 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref1 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref2 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref2 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref2 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref3 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref3 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref3 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref4 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref4 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref4 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref5 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref5 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref5 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref5 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref6 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref6 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref6 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref6 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref7 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref7 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref7 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref8 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref8 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref8 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref8 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref9 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref9 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref9 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref10 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref10 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref10 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref10 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref11 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref11 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref11 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref11 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref12 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref12 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref12 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref12 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref13 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref13 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref13 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref14 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref14 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref14 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref14 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref14 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref15 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref15 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref16 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref16 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref16 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref16 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref16 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref17 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref17 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref17 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref17 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref18 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref18 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref18 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref18 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref19 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref19 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref20 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref20 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref20 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref21 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref21 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref21 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref21 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref22 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref22 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref22 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref23 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref24 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref24 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref24 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref25 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref25 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref26 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref26 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref26 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref27 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref27 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref27 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref28 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref28 http://refhub.elsevier.com/S1198-743X(17)30634-1/sref28 Adverse birth outcomes associated with Zika virus exposure during pregnancy in São José do Rio Preto, Brazil Introduction Methods Study population Virus and RNA extraction ZIKV quantitative real-time PCR ZIKV ELISA Complete genome Phylogenetic reconstruction Statistical analysis Results Discussion Transparency declaration Appendix A. Supplementary data References