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Pregnancy outcomes after maternal Zika virus infection in a non-endemic region: prospective cohort study
Clinical Microbiology and Infection 25 (2019) 633.e5e633.e9
Contents lists available at ScienceDirect
Clinical Microbiology and Infection journal homepage: www.clinicalmicrobiologyandinfection.com
Research note
Pregnancy outcomes after maternal Zika virus infection in a non-endemic region: prospective cohort study 1, *, y, A. Suy 1, y, E. Sulleiro 2, y, A. Soriano-Arandes 3, 4, N. Maiz 1, I. García-Ruiz 1, C. Rodo Va zquez Me ndez 5, M. Garrido 6, A. Frick 3, 4, valo 1, A. Rando 2, A. Anton 2, E. S. Are 4 2 1 C. Rodrigo , T. Pumarola , E. Carreras 1) noma de Barcelona, Barcelona, Maternal Fetal Medicine Unit, Department of Obstetrics, Hospital Universitari Vall d’Hebron, Barcelona, Universitat Auto Spain 2) noma de Barcelona, Barcelona, Spain Department of Microbiology, Hospital Universitari Vall d’Hebron, PROSICS Barcelona, Universitat Auto 3) noma de Barcelona, Tropical Medicine and International Health Unit, Hospital Universitari Vall d’Hebron, Barcelona, PROSICS Barcelona, Universitat Auto Barcelona, Spain 4) noma de Barcelona, Barcelona, Pediatric Infectious Diseases and Immunodeficiences Unit, Hospital Universitari Vall d'Hebron, Barcelona, Universitat Auto Spain 5) noma de Barcelona, Barcelona, Spain Department of Paediatric Radiology, Hospital Universitari Vall d’Hebron, Universitat Auto 6) noma de Barcelona, Barcelona, Spain Department of Pathology, Hospital Universitari Vall d’Hebron, Universitat Auto
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2018 Received in revised form 1 February 2019 Accepted 5 February 2019 Available online 14 February 2019
Objectives: The aim was to describe pregnancy outcomes after Zika virus (ZIKV) infection in a nonendemic region. Methods: According to the Spanish protocol issued after the ZIKV outbreak in Brazil in 2015, all pregnant women who had travelled to high-burden countries were screened for ZIKV. Serological and molecular tests were used to identify ZIKV-infected pregnant women. They were classified as confirmed ZIKV infection when reverse transcription (RT) PCR tested positive, or probable ZIKV infection when ZIKV immunoglobulin M and/or immunoglobulin G and ZIKV plaque reduction neutralization tests were positive. Women found positive using molecular or serological tests were prospectively followed-up with ultrasound scans and neurosonograms on a monthly basis until delivery; magnetic resonance imaging and amniotic fluid testing were performed after signed informed consent. Samples of placenta, and fetal and neonatal tissues were obtained. Results: Seventy-two pregnant women tested positive for ZIKV infection: ten were confirmed by RT-PCR, and 62 were probable cases based on serological tests. The prevalence of adverse perinatal outcomes was 33.3% (three out of nine, 95% CI 12.1e64.6%): two cases of congenital ZIKV syndrome (CZS) and one miscarriage, all born to women infected in the first trimester of gestation. All ZIKV-confirmed women had persistent viraemias beyond 2 weeks (median 61.50 days; IQR 35.50e80.75). Amniotic fluid testing was only positive in the two fetuses with anomalies. Conclusion: The prevalence of perinatal adverse outcomes for women with ZIKV-confirmed infection was 33.3%. Amniocentesis for ZIKV RT-PCR is recommended when fetal abnormalities are found. Intensive , prenatal and postnatal follow-up of ZIKV-infected pregnancies is advised in confirmed cases. C. Rodo Clin Microbiol Infect 2019;25:633.e5e633.e9 © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Editor: Emmanuel Bottieau Keywords: Amniotic fluid brain malformation congenital prenatal diagnosis ultrasound Zika virus
Introduction
, Maternal-Fetal Medicine Unit, Department of * Corresponding author. C. Rodo Obstetrics, Hospital Universitari Vall d’Hebron, Ps. Vall d’Hebron 119-129, 08035 Barcelona, Spain. ). E-mail address: [email protected] (C. Rodo y These authors contributed equally to the study.
After the outbreak of Zika virus (ZIKV) in Brazil in 2015, a causal relationship between prenatal ZIKV infection and microcephaly in newborns was reported [1]. A 7% risk of birth defects in symptomatic women with confirmed infection by RT-PCR has been reported in French territories of America [2].
https://doi.org/10.1016/j.cmi.2019.02.008 1198-743X/© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
633.e6
et al. / Clinical Microbiology and Infection 25 (2019) 633.e5e633.e9 C. Rodo
Microcephaly turned out to be one of the signs of the spectrum of congenital infection; therefore, it was suggested that the term congenital Zika virus syndrome (CZS) be used to define the clinical spectrum of ZIKV infection [3]. Molecular techniques such as the reverse transcription (RT) PCR can detect ZIKV RNA in maternal blood during the acute phase of the infection [4]. However, the presence of the virus can be prolonged in pregnant women, as shown by our group and others [5]. Serological diagnosis of ZIKV infection after the acute phase is difficult due the high rates of cross-reactivity with other flaviviruses, even with the greater specificity of plaque reduction neutralization tests (PRNTs) [6]. Considering the high immigration flow from Latin American countries to Spain, both the Spanish and Catalan governments issued guidelines for screening of ZIKV infection in pregnant women. The purpose of this study is to describe the prospective follow-up of ZIKV-infected pregnant women and their fetuses in a tertiary healthcare centre in Barcelona. Methods Cross-sectional survey followed by a prospective cohort study of pregnant women who tested positive for ZIKV in a tertiary healthcare centre in Barcelona (Catalonia, Spain). Protocol description In accordance with the Centers for Disease Control and Prevention (CDC) both the Spanish and the Catalan governments issued guidelines for ZIKV screening during pregnancy [7,8]. Inclusion criteria were pregnant women having travelled to any of the vectoral endemic countries 8 weeks before the last menstrual period or during pregnancy; or pregnant women having had sexual intercourse with men that had travelled to any of the vectoral endemic countries in the past 6 months. As the protocol stated a universal screening policy, no exclusion criteria were considered. Definitions Confirmed case: positive ZIKV RNA RT-PCR in any maternal or fetal sample (serum, urine or amniotic fluid). Probable case: positive immunoglobulin M (IgM) for ZIKV and positive ZIKV PRNT or positive immunoglobulin G (IgG) for ZIKV and positive or indeterminate ZIKV PRNT. The PRNT for Dengue virus (DENV) or other flaviviruses was not available in our country. Gestational age at ZIKV infection: the first day of symptoms for symptomatic women; and the last day before coming to Spain for the non-symptomatic women. Women not reporting symptoms consistent with ZIKV infection were considered asymptomatic for the analysis. Prolonged viraemia: presence of ZIKV RNA in serum beyond 14 days after symptoms onset for symptomatic pregnant women or the presence of ZIKV RNA in serum beyond 21 days after last exposure to ZIKV for asymptomatic pregnant women [9]. Congenital Zika virus infection: newborns with clinical and/or radiological findings consistent with congenital ZIKV syndrome [3]. Adverse perinatal outcome: abortion or miscarriage related to ZIKV or CZS. Pregnancy screening and follow-up Women meeting the inclusion criteria were screened for ZIKV in the first antenatal care visit. They were screened with RT-PCR and serologically within the first 2 weeks after returning from endemic
countries, but only serologically after 2 weeks (please see supplementary material). Women testing positive for ZIKV were transferred to a tertiary healthcare centre and followed-up in a high-risk unit on a monthly basis. They were interviewed regarding the presence/absence of symptoms of ZIKV infection, and the timeline between infection, pregnancy, and first laboratory analysis. Serum and urine RT-PCR for ZIKV RNA were repeated monthly if positive results were obtained. Neutralizing antibody titres against ZIKV were determined using a PRNT with a 90% cut-off value and repeated up to three times in different samples if the result was indeterminate. Detailed fetal anatomic survey was performed for all at-risk women. Ultrasound evaluations and fetal neurosonograms were performed monthly. Fetal magnetic resonance imaging (MRI) was performed in the third trimester. Amniotic fluid testing was offered to all ZIKV-positive women and strongly recommended when fetal abnormalities were found on ultrasound examinations. Women who consented to amniocentesis were tested for ZIKV RT-PCR and other congenital infections (Toxoplasma gondii, Cytomegalovirus, Parvovirus B19, Herpes Virus I and II, Varicella Zoster virus) in amniotic fluid. When fetal anomalies were found, we also performed genetic tests (microarray) in amniotic fluid samples to rule out genetic conditions. Placenta and breast milk were also tested for ZIKV with specific RT-PCR within 24 hr after delivery. The Vall d’Hebron Institut de Recerca (VHIR) Ethics Committee approved the study on 20 May 2016 (PR(AMI)46/2016). Data were collected in a REDCap® platform database (https://projectredcap. org/). A descriptive analysis was carried out calculating medians and interquartile ranges (IQR) for quantitative variables and absolute frequencies and percentages for categorical variables. We tested for a significance at p 0.05 using the ManneWhitney U-test for continuous variables and the chi-squared or Fisher test for categorical variables. Results Between January 2016 and September 2017, 254 pregnant women were tested for ZIKV in our centre. Seventy-two (72/254, 28.3%) were positive for ZIKV. One case with positive ZIKV RT-PCR was unavailable for follow-up after 24 weeks' gestation and was excluded from the analysis. All except five screened women were born in Central or South America and were infected while travelling to their home countries (Table 1). No cases of infections after partner exposure were observed. Nine out 71 women (9/71, 12.6%) tested positive for serum and/ or urine ZIKV RT-PCR (confirmed cases) and 62 (62/71, 87.3%) tested positive for ZIKV IgM and/or ZIKV IgG and positive or indeterminate PRNT (probable cases). Median maternal viraemia for confirmed cases was 61.5 days (IQR 35.50e80.75 days) (range 23e107 days) (Table 1). Twenty-three women (23/71, 32.3%) had symptoms consistent with Zika virus disease and 48 were asymptomatic (48/71, 67.6%). Seven women in the confirmed group reported symptoms consistent with ZIKV infection compared with 16 in the probable group (p 0.001). All women were serologically screened for Toxoplasma gondii, Rubella virus, Hepatitis B and C viruses, syphilis, and HIV, with no active infections detected. There were two cases of congenital disease and one firsttrimester abortion in the confirmed group. Thus, the overall risk of any adverse perinatal outcome in women with confirmed
et al. / Clinical Microbiology and Infection 25 (2019) 633.e5e633.e9 C. Rodo
633.e7
Table 1 Demographic characteristics and laboratory results of the study population Demographic characteristics
Median maternal age at diagnosis (years) Overall adverse outcome (%) Trimester at ZIKV infection First trimester (1e12 weeks) (n, %) Second trimester (13e28 weeks) (n, %) Third trimester (29e41 weeks) (n, %) Median GA at ZIKV symptoms (weeks) Reporting maternal symptoms status Reported (n, %) Not reported (n, %) Kind of symptoms (n ¼ 23) Rash Fever Arthralgia Conjunctival hyperaemia Country of exposure Dominican Republic Honduras Colombia Venezuela Bolivia Ecuador El Salvador Peru Laboratory tests results at diagnosis Positive ZIKV RT-PCR (n, %) Both serum and urine Only serum (urine negative) Only urine (serum negative) Persistent viraemia (n, %) Median persistent ZIKV viraemia (days) Positive ZIKV IgM (n, %) Both RT-PCR and IgM þ Only IgM þ Positive ZIKV IgG (n,%) PRNT (n, %) PRNT positive PRNT indeterminate One time Two times More than two times DENV testing (n, %) DENV IgM þ DENV IgG þ DENV IgG e/IgM e Positive TORCH in maternal serum Amniocentesis (n, %) Positive ZIKV RT-PCR (n, %) GA at technique (weeks) Positive TORCH in amniotic fluid Placental ZIKV RT-PCR þ (n, %) Breast milk ZIKV RT-PCR þ (n, %) Placental pathology positive for ZIKV
Total
Confirmed infection
Probable infection
(n ¼ 71)
(n ¼ 9)
(n ¼ 62)
28.48 (IQR 23.18e32.21) 3 (4.2%)
28.69 (IQR 22.74e36.48) 3 (33.3%)
28.25 (IQR 23.10e32.11) 0
0.563 0.001
16 (22.5%) 41 (57.7%) 14 (19.7%) 19.42 (IQR 15.57e27.14)
3 (33.3%) 6 (66.6%) 0 19.28 (IQR 11.42e28.28)
13 (21%) 35 (56.4%) 14 (22.6%) 19.42 (IQR 13.42e27)
0.241
23 (32.3%) 48 (67.6%)
7 (77.7%) 2 (22.2%)
16 (25.8%) 46 (74.2%)
0.001
21 (84.0%) 15 (65.2%) 12 (60%) 4 (20%)
7 6 3 2
(87.5%) (85.7%) (42.9%) (28.6%)
14 (82.4%) 9 (56.3%) 9 (69.2%) 2 (15.4%)
0.999 0.345 0.356 0.587
26 (36.6%) 15 (21.1%) 8 (11.2%) 7 (9.8%) 5 (7.0%) 6 (8.4%) 3 (4.2%) 1 (1.4%)
4 1 3 0 0 1 0 0
(44.4%) (11.1%) (33.3%)
22 (35.5%) 14 (22.6%) 5 (8.1%) 7 (11.3%) 5 (8.1%) 5 (8.1%) 3 (4.8%) 1 (1.6%)
0.507
9 (12.6%) 3 (4.2%) 6 (8.4%) 0 9 (12.6%) 61.50 (IQR 35.50e80.75) 9 (12.6%) 3 (4.2%) 6 (8.4%) 71 (100%) 71 (100%) 41 (57.7%) 30 (42.2%) 4 (5.6%) 21 (29.2%) 5 (6.9%) 48 (66.6%) 2 (4.2%) 47 (97.9%) 1 (2.1%) 0 18 (25.3%) 2/18 (11.1%) 26.0 (IQR 21.8e30.4) 0 2/44 (4.5%) 0/15 0/48
9 (100%) 3 (33.3%) 6 (66.6%) 0 9 (100%) 61.50 (IQR 35.50e80.75) 4 (40%) 3 (33.3%) 0 9 (100%) 9 (100%) 9 (100%) 0 0 0 0 9 (100%) 1 (11.1%) 9 (100%) 0 0 8 (88.8%) 2/8 (25%) 24.0 (IQR 20.4e29.0) 0 2/7 (28.6%) 0 0
(11.1%)
0 0 0 0 0 0 6 (9.7%) 0 6 62 (100%) 62 (100%) 32 (51.6%) 30 (49.4%) 4 (6.5%) 21 (33.9%) 5 (8.1%) 39 (62.9%) 1 (2.6%) 38 (97.4%) 1 (0.02%) 0 10 (16.1%) 0/10 26.0 (IQR 23.6e32.1) 0 0/37 0 0
p
0.480
<0.0001
<0.0001 <0.0001 0.027
0.040
0.343 0.999
<0.0001 0.183 0.325 0.022
GA, gestational age; IQR, interquartile range; ZIKV, Zika virus; RT-PCR, reverse-transcription polymerase chain reaction; IgM, immunoglobulin M; IgG, immunoglobulin G; PRNT, plaque-reduction neutralization tests; DENV, Dengue virus; TORCH, toxoplasmosis, other (syphilis, varicella-zoster, parvovirus B19), rubella, cytomegalovirus (CMV), and Herpes infections.
infection was 33.3% (3/9; 95% CI, 12.1e64.6%). The three women with adverse perinatal results were infected in the first trimester. None of the confirmed cases was infected in the third trimester of pregnancy. No cases of fetal infection were observed in the probable group. Of the two confirmed cases who did not report symptoms, one delivered a healthy baby but the other decided to terminate the pregnancy because of severe brain malformations. Amniocentesis was performed on 18 women (18/71, 25%), eight in the confirmed group (8/9, 88.8%) and ten in the probable group (10/62, 16.1%) (p <0.0001) (Table 2). Amniotic fluid ZIKV RT-PCR was positive in the two cases of CZS (2/8, 25%) (case 1 and 8,
Table 2). Screening for other congenital infections proved negative in all the analysed samples and genetic tests were normal for all of them. Second-trimester ultrasound was normal in all except three cases: two cases of brain malformations in the confirmed group (2/ 8, 25%), both consistent with CZS (case 1 and 8, Table 2), and one case of congenital heart defect in the probable infection group (p 0.004). Third-trimester ultrasound was normal in all except one case of CZS already diagnosed (case 1, Table 2) and one fetus smallfor-gestational age in the probable group. We performed fetal MRI in 37 women (37/71, 52.1%). MRI confirmed the normality of the ultrasound findings in 35 cases and the abnormal ultrasound
et al. / Clinical Microbiology and Infection 25 (2019) 633.e5e633.e9 C. Rodo
633.e8 Table 2 Confirmed cases of Zika virus infection Case no.
1
2
3
4
5
6
7
8
9
Gestational week at diagnosis Persistent viraemia (days) Clinical signs or symptoms US and MRI anomalies Second trimester fetal US: head circumference (mm) Third trimester US: head circumference (mm) GA at amniocentesis (weeks) Blood/serum Urine Amniotic fluid Placenta Other samples
11.42
26
20.85
28.28
8.14
24.28
15
11.28
17.57
107
89
38
70
23
52
72
28
78
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
No
No
No
Spontaneous abortion
No
Yes
No
No
176
203
178
ZIKV IgM DENV IgM Outcome of pregnancy GA at delivery (weeks) Birth weight (g) Birth weight zscore Birth length (cm) Birth length z-score Head circumference at birth (mm) Head circumference zscore at birth (mm)
153
201.3
251
323
299
310
288
302
19
30
26
34.3
Not performed
26
22
21.1
20.2
Positive Negative Positive Negative Negative: breast milk, membranes, cord Negative Positive Delivery
Positive Negative Negative Negative Negative: breast milk
Positive Positive Negative Negative Not performed
Positive Negative Negative Negative Not performed
Positive Positive Not performed Positive Not performed
Positive Negative Negative Negative Negative: breast milk, cord
Positive Negative Positive Positive Positive: brain, membranes, CSF
Positive Negative Negative Not performed Not performed
Positive Negative Negative Not performed Not performed
Negative Negative Delivery
Positive Negative Delivery
Negative Negative Delivery
Negative Not performed Delivery
Positive Negative Delivery
40
39.2
37.6
37.5
Negative Negative Elective abortion 22.6
Positive Negative Delivery
37
Negative Negative Spontaneous abortion 11.2
36.5
39.6
2540 e2.2
3180 e0.5
2940 e1.15
3060 0.48
d
3070 0.50
325 e2.61
2880 e0.08
3580 0.05
48 e1.09 30
50.5 0.03 35
48 e1.09 34
50 0.97 33.5
d
48.5 0.37 34
25 < e3 16.8
Not reported d Not reported
Not reported d Not reported
e4.0
0.2
e0.125
0.38
d
0.73
e3.57
e0.16
d
GA, gestational age; US, ultrasound; MRI, magnetic resonance imaging; 2T US, second trimester ultrasound; 3T US, third trimester ultrasound; ZIKV, Zika virus; RTPCR, real time polymerase chain reaction; IgM, Immunoglobulin M; IgG, immunoglobulin G.
findings in the two CZS cases (p 0.042) (please see supplementary material). Case 1 delivered a congenitally affected baby at 37 weeks, case 8 opted for termination of pregnancy at 22 weeks and case 6 miscarried at 9 weeks' gestation. Cases 2e5, 7, and 9 delivered healthy babies (Table 2). No cases of CZS were suspected among uneventful pregnancies with available perinatal results (67 cases). Regarding the CZS child (case 1, Table 2), all samples obtained after birth were negative, including RT-PCR in serum, urine, and cerebrospinal fluid (CSF). IgM antibodies were also negative in serum and CSF. However, serum ZIKV IgG antibodies remained positive 24 months after delivery while being negative for other children born to ZIKV-infected mothers. Discussion We have described a cohort of 71 women with confirmed or probable ZIKV infection during pregnancy in a non-endemic country and provide information on the follow-up and perinatal results. The overall risk of an adverse perinatal outcome in pregnant women with confirmed Zika virus infection and no further re-exposure was
33.3%. The distribution of ZIKV-infected pregnant women was similar for all trimesters of pregnancy as previously reported [10]; however, there was a tendency for worse outcomes when the ZIKV infection occurred during the first trimester (p 0.071). All women with confirmed infection had viraemias longer than 14 days [5]. We could not confirm the ZIKV diagnosis in 87% of the cases (probable cases). The lack of adequate laboratory tests to exclude other flavivirus infections in women with suspicion of ZIKV infection and the lack of data on DENV infection for nearly one third of women, despite being stated on the protocol [11], made it very difficult to reach a confirmed diagnosis of Zika infection. Flaviviruses have strong cross-reactivity which generates false positive results in serological tests because of crossreaction with other antibodies [6]. In order to confirm the infection, PRNT was performed. However, PRNT for flaviviruses other than ZIKV is not available in our country; thus, it was not possible to confirm the infection in 30 cases. In order to decrease professionals' and patients' anxiety regarding inconclusive ZIKV testing, intensive pregnancy follow-up in high-risk units should be restricted to women with positive RT-PCR and/ or positive ZIKV IgM.
et al. / Clinical Microbiology and Infection 25 (2019) 633.e5e633.e9 C. Rodo
ZIKV is responsible for other brain abnormalities besides microcephaly, and it has been related to the fetal brain disruption sequence [12]. In case 1 (Table 2), both scalp rugae and prominent occipital bones were observed at delivery. This feature had already been reported in 11 out of 35 infants born with microcephaly in Brazil, suggesting an arrest of cerebral growth but not of the scalp skin. Arthrogryposis, as observed in case 8, has been also reported in four out of 35 infants in Brazil [13]. MRI assessment of the fetal brain did not add new information to the ultrasound findings in the two affected CZS fetuses nor in the non-affected ones. This could be because both fetuses were severely affected. However, MRI may add information to the ultrasound that may change the prognosis and the management of the pregnancy, particularly when ultrasound findings are mild [14]; it should therefore be considered when brain malformations are suspected by ultrasound. The rate of amniocentesis performance was higher in the confirmed group, even in women with no fetal abnormalities. ZIKV RT-PCR in amniotic fluid was positive in the two CZS infected fetuses but at delivery was negative for one of them. In light of our results, amniocentesis should be strongly recommended when fetal malformations are observed. However, more data are required before it can be recommended when the ultrasound examination is normal. It was not possible to demonstrate that ZIKV infection in early pregnancy entails worse outcomes than other trimesters, probably because of the small numbers of the sample. Risk of miscarriage remains a concern because it is difficult to demonstrate that a first-trimester abortion is related to ZIKV, particularly in asymptomatic women, since spontaneous abortion rates in general population are 10e20% [15]. Conclusion We found 33.3% adverse outcome in ZIKV RT-PCR confirmed infection in the first trimester. ZIKV RT-PCR was positive in amniotic fluid in fetuses with congenital malformation. Fetal MRI confirmed findings in affected fetuses but did not provide any additional information to a normal ultrasound examination. We recommend performing amniocentesis if fetal anomalies are observed, and confirming ultrasound findings by MRI when there is a suspicion of pathology. Moreover, we advise for prenatal followup of ZIKV-infected pregnancies and postnatal follow-up of their babies, particularly in confirmed cases. Transparency declaration The authors declare no conflicts of interest. There was no funding source for the study. The investigators had full access to all the data in the study and had final responsibility for the decision to submit for publication.
633.e9
Acknowledgements We thank the National Centre for Microbiology, Instituto de Salud Carlos III (Madrid, Spain) for their help in performing the plaque reduction neutralization tests of the analysed samples.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2019.02.008.
References [1] Rasmussen SA, Jamieson DJ, Honein MA, Petersen LR. Zika virus and birth defects–reviewing the evidence for causality. N Engl J Med 2016;374:1981e7. A, et al. Pregnancy [2] Hoen B, Schaub B, Funk AL, Ardillon V, Boullard M, Cabie outcomes after ZIKV infection in French territories in the Americas. N Engl J Med 2018;378:985e94. [3] de Oliveira Melo AS, Aguiar RS, Amorim MMR, Arruda MB, de Oliveira Melo F, Ribeiro STC, et al. Congenital Zika virus infection: beyond neonatal microcephaly. JAMA Neurol 2016;73:1407e16. [4] Petersen EE, Polen KND, Meaney-Delman D, Ellington SR, Oduyebo T, Cohn A, et al. Update: interim guidance for health care providers caring for women of reproductive age with possible Zika virus exposure–United States, 2016. MMWR Morb Mortal Wkly Rep 2016;65:315e22. C, V [5] Suy A, Sulleiro E, Rodo azquez C, Bocanegra C, Molina I, et al. Prolonged Zika virus viremia during pregnancy. N Engl J Med 2016;375:2611e3. [6] Rabe IB, Staples JE, Villanueva J, Hummel KB, Johnson JA, Rose L, et al. Interim guidance for interpretation of Zika virus antibody test results. MMWR Morb Mortal Wkly Rep 2016;65:543e6. [7] Bocanegra C, Sulleiro E, Soriano-Arandes A, Pou D, Suy A, Llurba E, et al. Zika virus infection in pregnant women in Barcelona, Spain. Clin Microbiol Infect 2016;22:648e50. [8] Bardají-Alonso A, Bern ardez S, Carreras-Moratonas E, Coma-Colom C, Costa davant de casos de febre Sampere D, Falguera-Puig G, et al. Protocol d'actuacio tric i pedi ncia vírica de Zika en l' ambit obste atric de Catalunya. Barcelona: Age de Salut Pública de Catalunya; 2016. https://scientiasalut.gencat.cat/handle/ 11351/1915?locale-attribute¼en. [9] Meaney-Delman D, Oduyebo T, Polen KND, White JL, Bingham AM, Slavinski SA, et al. Prolonged detection of Zika virus RNA in pregnant women. Obstet Gynecol 2016;128:724e30. [10] 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. C, Suy A, Sulleiro E, Soriano-Arandes A, Anto n A, García I, et al. In utero [11] Rodo negativization of Zika virus in a case with serious Central Nervous System abnormalities. Clin Microbiol Infect 2018;24:549.e1e3. [12] More C. Fetal brain disruption sequence. J Pediatr 1990;116:383e6. [13] del Campo M, Feitosa IML, Ribeiro EM, Horovitz DDG, Pessoa ALS, França GVA, et al. The phenotypic spectrum of congenital Zika syndrome. Am J Med Genet A 2017;173:841e57. [14] Rossi AC, Prefumo F. Additional value of fetal magnetic resonance imaging in the prenatal diagnosis of central nervous system anomalies: a systematic review of the literature. Ultrasound Obstet Gynecol 2014;44:388e93. [15] Cohain JS, Buxbaum RE, Mankuta D. Spontaneous first trimester miscarriage rates per woman among parous women with 1 or more pregnancies of 24 weeks or more. BMC Pregnancy Childbirth 2017;17:437 [Internet].
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Clinical Microbiology and Infection journal homepage: www.clinicalmicrobiologyandinfection.com
Research note
Pregnancy outcomes after maternal Zika virus infection in a non-endemic region: prospective cohort study 1, *, y, A. Suy 1, y, E. Sulleiro 2, y, A. Soriano-Arandes 3, 4, N. Maiz 1, I. García-Ruiz 1, C. Rodo Va zquez Me ndez 5, M. Garrido 6, A. Frick 3, 4, valo 1, A. Rando 2, A. Anton 2, E. S. Are 4 2 1 C. Rodrigo , T. Pumarola , E. Carreras 1) noma de Barcelona, Barcelona, Maternal Fetal Medicine Unit, Department of Obstetrics, Hospital Universitari Vall d’Hebron, Barcelona, Universitat Auto Spain 2) noma de Barcelona, Barcelona, Spain Department of Microbiology, Hospital Universitari Vall d’Hebron, PROSICS Barcelona, Universitat Auto 3) noma de Barcelona, Tropical Medicine and International Health Unit, Hospital Universitari Vall d’Hebron, Barcelona, PROSICS Barcelona, Universitat Auto Barcelona, Spain 4) noma de Barcelona, Barcelona, Pediatric Infectious Diseases and Immunodeficiences Unit, Hospital Universitari Vall d'Hebron, Barcelona, Universitat Auto Spain 5) noma de Barcelona, Barcelona, Spain Department of Paediatric Radiology, Hospital Universitari Vall d’Hebron, Universitat Auto 6) noma de Barcelona, Barcelona, Spain Department of Pathology, Hospital Universitari Vall d’Hebron, Universitat Auto
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2018 Received in revised form 1 February 2019 Accepted 5 February 2019 Available online 14 February 2019
Objectives: The aim was to describe pregnancy outcomes after Zika virus (ZIKV) infection in a nonendemic region. Methods: According to the Spanish protocol issued after the ZIKV outbreak in Brazil in 2015, all pregnant women who had travelled to high-burden countries were screened for ZIKV. Serological and molecular tests were used to identify ZIKV-infected pregnant women. They were classified as confirmed ZIKV infection when reverse transcription (RT) PCR tested positive, or probable ZIKV infection when ZIKV immunoglobulin M and/or immunoglobulin G and ZIKV plaque reduction neutralization tests were positive. Women found positive using molecular or serological tests were prospectively followed-up with ultrasound scans and neurosonograms on a monthly basis until delivery; magnetic resonance imaging and amniotic fluid testing were performed after signed informed consent. Samples of placenta, and fetal and neonatal tissues were obtained. Results: Seventy-two pregnant women tested positive for ZIKV infection: ten were confirmed by RT-PCR, and 62 were probable cases based on serological tests. The prevalence of adverse perinatal outcomes was 33.3% (three out of nine, 95% CI 12.1e64.6%): two cases of congenital ZIKV syndrome (CZS) and one miscarriage, all born to women infected in the first trimester of gestation. All ZIKV-confirmed women had persistent viraemias beyond 2 weeks (median 61.50 days; IQR 35.50e80.75). Amniotic fluid testing was only positive in the two fetuses with anomalies. Conclusion: The prevalence of perinatal adverse outcomes for women with ZIKV-confirmed infection was 33.3%. Amniocentesis for ZIKV RT-PCR is recommended when fetal abnormalities are found. Intensive , prenatal and postnatal follow-up of ZIKV-infected pregnancies is advised in confirmed cases. C. Rodo Clin Microbiol Infect 2019;25:633.e5e633.e9 © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Editor: Emmanuel Bottieau Keywords: Amniotic fluid brain malformation congenital prenatal diagnosis ultrasound Zika virus
Introduction
, Maternal-Fetal Medicine Unit, Department of * Corresponding author. C. Rodo Obstetrics, Hospital Universitari Vall d’Hebron, Ps. Vall d’Hebron 119-129, 08035 Barcelona, Spain. ). E-mail address: [email protected] (C. Rodo y These authors contributed equally to the study.
After the outbreak of Zika virus (ZIKV) in Brazil in 2015, a causal relationship between prenatal ZIKV infection and microcephaly in newborns was reported [1]. A 7% risk of birth defects in symptomatic women with confirmed infection by RT-PCR has been reported in French territories of America [2].
https://doi.org/10.1016/j.cmi.2019.02.008 1198-743X/© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
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Microcephaly turned out to be one of the signs of the spectrum of congenital infection; therefore, it was suggested that the term congenital Zika virus syndrome (CZS) be used to define the clinical spectrum of ZIKV infection [3]. Molecular techniques such as the reverse transcription (RT) PCR can detect ZIKV RNA in maternal blood during the acute phase of the infection [4]. However, the presence of the virus can be prolonged in pregnant women, as shown by our group and others [5]. Serological diagnosis of ZIKV infection after the acute phase is difficult due the high rates of cross-reactivity with other flaviviruses, even with the greater specificity of plaque reduction neutralization tests (PRNTs) [6]. Considering the high immigration flow from Latin American countries to Spain, both the Spanish and Catalan governments issued guidelines for screening of ZIKV infection in pregnant women. The purpose of this study is to describe the prospective follow-up of ZIKV-infected pregnant women and their fetuses in a tertiary healthcare centre in Barcelona. Methods Cross-sectional survey followed by a prospective cohort study of pregnant women who tested positive for ZIKV in a tertiary healthcare centre in Barcelona (Catalonia, Spain). Protocol description In accordance with the Centers for Disease Control and Prevention (CDC) both the Spanish and the Catalan governments issued guidelines for ZIKV screening during pregnancy [7,8]. Inclusion criteria were pregnant women having travelled to any of the vectoral endemic countries 8 weeks before the last menstrual period or during pregnancy; or pregnant women having had sexual intercourse with men that had travelled to any of the vectoral endemic countries in the past 6 months. As the protocol stated a universal screening policy, no exclusion criteria were considered. Definitions Confirmed case: positive ZIKV RNA RT-PCR in any maternal or fetal sample (serum, urine or amniotic fluid). Probable case: positive immunoglobulin M (IgM) for ZIKV and positive ZIKV PRNT or positive immunoglobulin G (IgG) for ZIKV and positive or indeterminate ZIKV PRNT. The PRNT for Dengue virus (DENV) or other flaviviruses was not available in our country. Gestational age at ZIKV infection: the first day of symptoms for symptomatic women; and the last day before coming to Spain for the non-symptomatic women. Women not reporting symptoms consistent with ZIKV infection were considered asymptomatic for the analysis. Prolonged viraemia: presence of ZIKV RNA in serum beyond 14 days after symptoms onset for symptomatic pregnant women or the presence of ZIKV RNA in serum beyond 21 days after last exposure to ZIKV for asymptomatic pregnant women [9]. Congenital Zika virus infection: newborns with clinical and/or radiological findings consistent with congenital ZIKV syndrome [3]. Adverse perinatal outcome: abortion or miscarriage related to ZIKV or CZS. Pregnancy screening and follow-up Women meeting the inclusion criteria were screened for ZIKV in the first antenatal care visit. They were screened with RT-PCR and serologically within the first 2 weeks after returning from endemic
countries, but only serologically after 2 weeks (please see supplementary material). Women testing positive for ZIKV were transferred to a tertiary healthcare centre and followed-up in a high-risk unit on a monthly basis. They were interviewed regarding the presence/absence of symptoms of ZIKV infection, and the timeline between infection, pregnancy, and first laboratory analysis. Serum and urine RT-PCR for ZIKV RNA were repeated monthly if positive results were obtained. Neutralizing antibody titres against ZIKV were determined using a PRNT with a 90% cut-off value and repeated up to three times in different samples if the result was indeterminate. Detailed fetal anatomic survey was performed for all at-risk women. Ultrasound evaluations and fetal neurosonograms were performed monthly. Fetal magnetic resonance imaging (MRI) was performed in the third trimester. Amniotic fluid testing was offered to all ZIKV-positive women and strongly recommended when fetal abnormalities were found on ultrasound examinations. Women who consented to amniocentesis were tested for ZIKV RT-PCR and other congenital infections (Toxoplasma gondii, Cytomegalovirus, Parvovirus B19, Herpes Virus I and II, Varicella Zoster virus) in amniotic fluid. When fetal anomalies were found, we also performed genetic tests (microarray) in amniotic fluid samples to rule out genetic conditions. Placenta and breast milk were also tested for ZIKV with specific RT-PCR within 24 hr after delivery. The Vall d’Hebron Institut de Recerca (VHIR) Ethics Committee approved the study on 20 May 2016 (PR(AMI)46/2016). Data were collected in a REDCap® platform database (https://projectredcap. org/). A descriptive analysis was carried out calculating medians and interquartile ranges (IQR) for quantitative variables and absolute frequencies and percentages for categorical variables. We tested for a significance at p 0.05 using the ManneWhitney U-test for continuous variables and the chi-squared or Fisher test for categorical variables. Results Between January 2016 and September 2017, 254 pregnant women were tested for ZIKV in our centre. Seventy-two (72/254, 28.3%) were positive for ZIKV. One case with positive ZIKV RT-PCR was unavailable for follow-up after 24 weeks' gestation and was excluded from the analysis. All except five screened women were born in Central or South America and were infected while travelling to their home countries (Table 1). No cases of infections after partner exposure were observed. Nine out 71 women (9/71, 12.6%) tested positive for serum and/ or urine ZIKV RT-PCR (confirmed cases) and 62 (62/71, 87.3%) tested positive for ZIKV IgM and/or ZIKV IgG and positive or indeterminate PRNT (probable cases). Median maternal viraemia for confirmed cases was 61.5 days (IQR 35.50e80.75 days) (range 23e107 days) (Table 1). Twenty-three women (23/71, 32.3%) had symptoms consistent with Zika virus disease and 48 were asymptomatic (48/71, 67.6%). Seven women in the confirmed group reported symptoms consistent with ZIKV infection compared with 16 in the probable group (p 0.001). All women were serologically screened for Toxoplasma gondii, Rubella virus, Hepatitis B and C viruses, syphilis, and HIV, with no active infections detected. There were two cases of congenital disease and one firsttrimester abortion in the confirmed group. Thus, the overall risk of any adverse perinatal outcome in women with confirmed
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633.e7
Table 1 Demographic characteristics and laboratory results of the study population Demographic characteristics
Median maternal age at diagnosis (years) Overall adverse outcome (%) Trimester at ZIKV infection First trimester (1e12 weeks) (n, %) Second trimester (13e28 weeks) (n, %) Third trimester (29e41 weeks) (n, %) Median GA at ZIKV symptoms (weeks) Reporting maternal symptoms status Reported (n, %) Not reported (n, %) Kind of symptoms (n ¼ 23) Rash Fever Arthralgia Conjunctival hyperaemia Country of exposure Dominican Republic Honduras Colombia Venezuela Bolivia Ecuador El Salvador Peru Laboratory tests results at diagnosis Positive ZIKV RT-PCR (n, %) Both serum and urine Only serum (urine negative) Only urine (serum negative) Persistent viraemia (n, %) Median persistent ZIKV viraemia (days) Positive ZIKV IgM (n, %) Both RT-PCR and IgM þ Only IgM þ Positive ZIKV IgG (n,%) PRNT (n, %) PRNT positive PRNT indeterminate One time Two times More than two times DENV testing (n, %) DENV IgM þ DENV IgG þ DENV IgG e/IgM e Positive TORCH in maternal serum Amniocentesis (n, %) Positive ZIKV RT-PCR (n, %) GA at technique (weeks) Positive TORCH in amniotic fluid Placental ZIKV RT-PCR þ (n, %) Breast milk ZIKV RT-PCR þ (n, %) Placental pathology positive for ZIKV
Total
Confirmed infection
Probable infection
(n ¼ 71)
(n ¼ 9)
(n ¼ 62)
28.48 (IQR 23.18e32.21) 3 (4.2%)
28.69 (IQR 22.74e36.48) 3 (33.3%)
28.25 (IQR 23.10e32.11) 0
0.563 0.001
16 (22.5%) 41 (57.7%) 14 (19.7%) 19.42 (IQR 15.57e27.14)
3 (33.3%) 6 (66.6%) 0 19.28 (IQR 11.42e28.28)
13 (21%) 35 (56.4%) 14 (22.6%) 19.42 (IQR 13.42e27)
0.241
23 (32.3%) 48 (67.6%)
7 (77.7%) 2 (22.2%)
16 (25.8%) 46 (74.2%)
0.001
21 (84.0%) 15 (65.2%) 12 (60%) 4 (20%)
7 6 3 2
(87.5%) (85.7%) (42.9%) (28.6%)
14 (82.4%) 9 (56.3%) 9 (69.2%) 2 (15.4%)
0.999 0.345 0.356 0.587
26 (36.6%) 15 (21.1%) 8 (11.2%) 7 (9.8%) 5 (7.0%) 6 (8.4%) 3 (4.2%) 1 (1.4%)
4 1 3 0 0 1 0 0
(44.4%) (11.1%) (33.3%)
22 (35.5%) 14 (22.6%) 5 (8.1%) 7 (11.3%) 5 (8.1%) 5 (8.1%) 3 (4.8%) 1 (1.6%)
0.507
9 (12.6%) 3 (4.2%) 6 (8.4%) 0 9 (12.6%) 61.50 (IQR 35.50e80.75) 9 (12.6%) 3 (4.2%) 6 (8.4%) 71 (100%) 71 (100%) 41 (57.7%) 30 (42.2%) 4 (5.6%) 21 (29.2%) 5 (6.9%) 48 (66.6%) 2 (4.2%) 47 (97.9%) 1 (2.1%) 0 18 (25.3%) 2/18 (11.1%) 26.0 (IQR 21.8e30.4) 0 2/44 (4.5%) 0/15 0/48
9 (100%) 3 (33.3%) 6 (66.6%) 0 9 (100%) 61.50 (IQR 35.50e80.75) 4 (40%) 3 (33.3%) 0 9 (100%) 9 (100%) 9 (100%) 0 0 0 0 9 (100%) 1 (11.1%) 9 (100%) 0 0 8 (88.8%) 2/8 (25%) 24.0 (IQR 20.4e29.0) 0 2/7 (28.6%) 0 0
(11.1%)
0 0 0 0 0 0 6 (9.7%) 0 6 62 (100%) 62 (100%) 32 (51.6%) 30 (49.4%) 4 (6.5%) 21 (33.9%) 5 (8.1%) 39 (62.9%) 1 (2.6%) 38 (97.4%) 1 (0.02%) 0 10 (16.1%) 0/10 26.0 (IQR 23.6e32.1) 0 0/37 0 0
p
0.480
<0.0001
<0.0001 <0.0001 0.027
0.040
0.343 0.999
<0.0001 0.183 0.325 0.022
GA, gestational age; IQR, interquartile range; ZIKV, Zika virus; RT-PCR, reverse-transcription polymerase chain reaction; IgM, immunoglobulin M; IgG, immunoglobulin G; PRNT, plaque-reduction neutralization tests; DENV, Dengue virus; TORCH, toxoplasmosis, other (syphilis, varicella-zoster, parvovirus B19), rubella, cytomegalovirus (CMV), and Herpes infections.
infection was 33.3% (3/9; 95% CI, 12.1e64.6%). The three women with adverse perinatal results were infected in the first trimester. None of the confirmed cases was infected in the third trimester of pregnancy. No cases of fetal infection were observed in the probable group. Of the two confirmed cases who did not report symptoms, one delivered a healthy baby but the other decided to terminate the pregnancy because of severe brain malformations. Amniocentesis was performed on 18 women (18/71, 25%), eight in the confirmed group (8/9, 88.8%) and ten in the probable group (10/62, 16.1%) (p <0.0001) (Table 2). Amniotic fluid ZIKV RT-PCR was positive in the two cases of CZS (2/8, 25%) (case 1 and 8,
Table 2). Screening for other congenital infections proved negative in all the analysed samples and genetic tests were normal for all of them. Second-trimester ultrasound was normal in all except three cases: two cases of brain malformations in the confirmed group (2/ 8, 25%), both consistent with CZS (case 1 and 8, Table 2), and one case of congenital heart defect in the probable infection group (p 0.004). Third-trimester ultrasound was normal in all except one case of CZS already diagnosed (case 1, Table 2) and one fetus smallfor-gestational age in the probable group. We performed fetal MRI in 37 women (37/71, 52.1%). MRI confirmed the normality of the ultrasound findings in 35 cases and the abnormal ultrasound
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633.e8 Table 2 Confirmed cases of Zika virus infection Case no.
1
2
3
4
5
6
7
8
9
Gestational week at diagnosis Persistent viraemia (days) Clinical signs or symptoms US and MRI anomalies Second trimester fetal US: head circumference (mm) Third trimester US: head circumference (mm) GA at amniocentesis (weeks) Blood/serum Urine Amniotic fluid Placenta Other samples
11.42
26
20.85
28.28
8.14
24.28
15
11.28
17.57
107
89
38
70
23
52
72
28
78
Yes
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
No
No
No
Spontaneous abortion
No
Yes
No
No
176
203
178
ZIKV IgM DENV IgM Outcome of pregnancy GA at delivery (weeks) Birth weight (g) Birth weight zscore Birth length (cm) Birth length z-score Head circumference at birth (mm) Head circumference zscore at birth (mm)
153
201.3
251
323
299
310
288
302
19
30
26
34.3
Not performed
26
22
21.1
20.2
Positive Negative Positive Negative Negative: breast milk, membranes, cord Negative Positive Delivery
Positive Negative Negative Negative Negative: breast milk
Positive Positive Negative Negative Not performed
Positive Negative Negative Negative Not performed
Positive Positive Not performed Positive Not performed
Positive Negative Negative Negative Negative: breast milk, cord
Positive Negative Positive Positive Positive: brain, membranes, CSF
Positive Negative Negative Not performed Not performed
Positive Negative Negative Not performed Not performed
Negative Negative Delivery
Positive Negative Delivery
Negative Negative Delivery
Negative Not performed Delivery
Positive Negative Delivery
40
39.2
37.6
37.5
Negative Negative Elective abortion 22.6
Positive Negative Delivery
37
Negative Negative Spontaneous abortion 11.2
36.5
39.6
2540 e2.2
3180 e0.5
2940 e1.15
3060 0.48
d
3070 0.50
325 e2.61
2880 e0.08
3580 0.05
48 e1.09 30
50.5 0.03 35
48 e1.09 34
50 0.97 33.5
d
48.5 0.37 34
25 < e3 16.8
Not reported d Not reported
Not reported d Not reported
e4.0
0.2
e0.125
0.38
d
0.73
e3.57
e0.16
d
GA, gestational age; US, ultrasound; MRI, magnetic resonance imaging; 2T US, second trimester ultrasound; 3T US, third trimester ultrasound; ZIKV, Zika virus; RTPCR, real time polymerase chain reaction; IgM, Immunoglobulin M; IgG, immunoglobulin G.
findings in the two CZS cases (p 0.042) (please see supplementary material). Case 1 delivered a congenitally affected baby at 37 weeks, case 8 opted for termination of pregnancy at 22 weeks and case 6 miscarried at 9 weeks' gestation. Cases 2e5, 7, and 9 delivered healthy babies (Table 2). No cases of CZS were suspected among uneventful pregnancies with available perinatal results (67 cases). Regarding the CZS child (case 1, Table 2), all samples obtained after birth were negative, including RT-PCR in serum, urine, and cerebrospinal fluid (CSF). IgM antibodies were also negative in serum and CSF. However, serum ZIKV IgG antibodies remained positive 24 months after delivery while being negative for other children born to ZIKV-infected mothers. Discussion We have described a cohort of 71 women with confirmed or probable ZIKV infection during pregnancy in a non-endemic country and provide information on the follow-up and perinatal results. The overall risk of an adverse perinatal outcome in pregnant women with confirmed Zika virus infection and no further re-exposure was
33.3%. The distribution of ZIKV-infected pregnant women was similar for all trimesters of pregnancy as previously reported [10]; however, there was a tendency for worse outcomes when the ZIKV infection occurred during the first trimester (p 0.071). All women with confirmed infection had viraemias longer than 14 days [5]. We could not confirm the ZIKV diagnosis in 87% of the cases (probable cases). The lack of adequate laboratory tests to exclude other flavivirus infections in women with suspicion of ZIKV infection and the lack of data on DENV infection for nearly one third of women, despite being stated on the protocol [11], made it very difficult to reach a confirmed diagnosis of Zika infection. Flaviviruses have strong cross-reactivity which generates false positive results in serological tests because of crossreaction with other antibodies [6]. In order to confirm the infection, PRNT was performed. However, PRNT for flaviviruses other than ZIKV is not available in our country; thus, it was not possible to confirm the infection in 30 cases. In order to decrease professionals' and patients' anxiety regarding inconclusive ZIKV testing, intensive pregnancy follow-up in high-risk units should be restricted to women with positive RT-PCR and/ or positive ZIKV IgM.
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ZIKV is responsible for other brain abnormalities besides microcephaly, and it has been related to the fetal brain disruption sequence [12]. In case 1 (Table 2), both scalp rugae and prominent occipital bones were observed at delivery. This feature had already been reported in 11 out of 35 infants born with microcephaly in Brazil, suggesting an arrest of cerebral growth but not of the scalp skin. Arthrogryposis, as observed in case 8, has been also reported in four out of 35 infants in Brazil [13]. MRI assessment of the fetal brain did not add new information to the ultrasound findings in the two affected CZS fetuses nor in the non-affected ones. This could be because both fetuses were severely affected. However, MRI may add information to the ultrasound that may change the prognosis and the management of the pregnancy, particularly when ultrasound findings are mild [14]; it should therefore be considered when brain malformations are suspected by ultrasound. The rate of amniocentesis performance was higher in the confirmed group, even in women with no fetal abnormalities. ZIKV RT-PCR in amniotic fluid was positive in the two CZS infected fetuses but at delivery was negative for one of them. In light of our results, amniocentesis should be strongly recommended when fetal malformations are observed. However, more data are required before it can be recommended when the ultrasound examination is normal. It was not possible to demonstrate that ZIKV infection in early pregnancy entails worse outcomes than other trimesters, probably because of the small numbers of the sample. Risk of miscarriage remains a concern because it is difficult to demonstrate that a first-trimester abortion is related to ZIKV, particularly in asymptomatic women, since spontaneous abortion rates in general population are 10e20% [15]. Conclusion We found 33.3% adverse outcome in ZIKV RT-PCR confirmed infection in the first trimester. ZIKV RT-PCR was positive in amniotic fluid in fetuses with congenital malformation. Fetal MRI confirmed findings in affected fetuses but did not provide any additional information to a normal ultrasound examination. We recommend performing amniocentesis if fetal anomalies are observed, and confirming ultrasound findings by MRI when there is a suspicion of pathology. Moreover, we advise for prenatal followup of ZIKV-infected pregnancies and postnatal follow-up of their babies, particularly in confirmed cases. Transparency declaration The authors declare no conflicts of interest. There was no funding source for the study. The investigators had full access to all the data in the study and had final responsibility for the decision to submit for publication.
633.e9
Acknowledgements We thank the National Centre for Microbiology, Instituto de Salud Carlos III (Madrid, Spain) for their help in performing the plaque reduction neutralization tests of the analysed samples.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2019.02.008.
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