Neonatal surveillance for congenital Zika infection during the 2016 microcephaly outbreak in Salvador, Brazil: Zika virus detection in asymptomatic newborns

Abstract Objective To identify newborns with congenital Zika infection (CZI) at a maternity hospital in Salvador, Brazil, during the 2016 microcephaly outbreak. Methods A prospective study enrolled microcephalic and normocephalic newborns with suspected CZI between January and December 2016. Serology (immunoglobulins IgM and IgG) and quantitative reverse transcriptase polymerase chain reaction (RT‐qPCR) for the Zika virus were performed. Demographic and clinical characteristics of newborns with and without microcephaly were compared. Results Of the 151 newborns enrolled, 32 (21.2%) were classified as microcephalic. The majority of these cases were born between January and May 2016. IgM and IgG Zika virus antibodies were detected in 5 (23.8%) and 17 (80.9%) microcephalic newborn blood samples, respectively. Six (24%) microcephalic newborns tested positive for Zika virus by RT‐qPCR in urine or placenta samples. Thirteen (11.8%) normocephalic newborns also tested positive for Zika virus by PCR in urine, plasma, or placenta samples, while IgM antibodies against Zika were detected in 4 (4.2%) others. Conclusions Identification of 17 normocephalic CZI cases, confirmed by IgM serology or RT‐qPCR for Zika virus, provides evidence that CZI can present asymptomatically at birth. This finding highlights the need for prenatal and neonatal screening for Zika virus in endemic regions.


| INTRODUCTION
In May 2015, the transmission of Zika virus was confirmed in Brazil. 1 During the Zika outbreak in Salvador, Brazil, 14 835 suspected cases were reported. 2 In total, the Brazilian Ministry of Health estimated that 440 000-1.3 million individuals were infected by Zika virus in 2015. 3 Infection was initially considered benign and self-limiting. 4,5 In late 2015, an unexpected outbreak of newborns with microcephaly occurred in major cities in northeastern Brazil and a state of public health emergency was declared in the country. Between October and December 2015, 2975 cases of microcephalic newborns were notified in Brazil, mostly in the states of Pernambuco (1153 cases), Paraíba (476 cases), and Bahia (271 cases), 6 with 229 cases notified in the city of Salvador-the capital of the state of Bahia. 7 In response to the microcephaly outbreak in northeastern Brazil, the Pan American Health Organization (PAHO) and the World Health Organization (WHO) released an epidemiological alert, 8 and a rapid risk assessment by the European Centre for Disease Prevention and Control (ECDC) highlighted a possible link between increased rates of congenital microcephaly in Brazil and the Zika virus epidemic. 9 The association between microcephaly and congenital Zika infection (CZI) was established by the identification of Zika virus in the amniotic fluid of a pregnant woman in Brazil 10 and in the brain tissue of a fetus whose mother was infected by Zika virus. 11 By the end of 2017, a total of 15 298 notified cases of CZI were reported in Brazil, of which 3071 were confirmed. 12 The aim of the present study was to identify and characterize cases of CZI at a maternity hospital in Salvador, Bahia, during the 2016 microcephaly outbreak.

| MATERIALS AND METHODS
This prospective study was conducted at the Jose Maria Magalhães Netto public maternity hospital located in Salvador, Bahia, Brazil, between January 18 and December 16, 2016. In accordance with the protocol established by the Brazilian Ministry of Health in November 2015, 13 the head circumference of newborns was measured at this maternity hospital by health professionals and cases of microcephaly were identified following specific criteria. Newborns who fulfilled these criteria were reported to local health authorities, and enrollment consent was requested from legal guardians to participate in this study. In addition, enrollment was also requested for newborns without microcephaly whose mothers reported episodes of exanthematous skin rash during pregnancy. All newborns were enrolled within the first 24 hours after birth.
Sociodemographic data and clinical information from mothers were collected through interviews, while clinical data on newborns and birth conditions were obtained by reviewing medical records.
Data entry and data management were performed using REDCap version 6.18.1 (Vanderbilt University, Nashville, TN, USA). Biological samples, including placental tissue, blood, umbilical cord blood, and newborn urine were collected by nurses in the maternity ward and the laboratory team at the hospital. Serological and molecular tests were done at the Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador (IGM-FIOCRUZ) and at the Infectious Diseases Immunology Laboratory of the Federal University of Bahia, Salvador.
During the study period, the Brazilian Ministry of Health changed the protocol for the specific criteria used to identify cases of microcephaly. From January to March 12, 2016, the cutoff for microcephaly notification for term newborns was a head circumference of less than or equal to 32 cm for both sexes; and less than or equal to the third percentile on the Fenton growth chart by gestational age and sex for preterm newborns. Between March 13 and December 2016, new criteria were employed to determine microcephaly cases: head circumference less than or equal to two standard deviations below the average (WHO standard) for term newborns; and less than or equal to two standard deviations below the average (INTERGROWTH-21st) by gestational age and sex for preterm newborns. 14 For data analysis purposes, all enrolled newborns were reclassified to define microcephaly according to the International Fetal and Newborn Growth Consortium for the 21st Century (INTERGROWTH-21st) charts, taking into account the newborn's gender, gestational age, and head circumference at birth. 15 Microcephaly was defined as head circumference measuring less than two standard deviations below the average, while severe microcephaly was considered if head circumference measurements were less than three standard deviations below the average. Newborns were considered normocephalic if head circumference measurements were within two standard deviations.
Newborns were classified as small, appropriate, or large for their gestational age based on gender-specific birth weight for their gestational age using WHO reference curves. 16 Newborns considered small-for-gestational-age (SGA) were defined as having a weight below the 10th percentile of the corresponding standard reference curve.
Transfontanellar ultrasound was performed as a standard procedure by the medical staff at the maternity hospital during admission. Information regarding these procedures was obtained from patient medical records. The legal guardians of all newborns provided written informed consent.

| RESULTS
Between January and December 2016, 172 newborns were identified as eligible by health professionals at the maternity hospital in Salvador, Brazil. Of these, 21 of the infants' guardians declined to participate in the study, resulting in 151 newborns subsequently enrolled: 32 (21.2%) classified as microcephalic and 119 (78.8%) as normocephalic.
The temporal distribution of cases by month of birth is shown in Figure 1. A cluster of cases born between January and May was observed. Maternal sociodemographic data are presented in Table 1. No differences in the mothers' age, race, or educational level were observed between microcephalic and normocephalic newborns (P>0.05). No differences in gestational age at birth were seen between the microcephalic and normocephalic newborns (P=0.30). However, significantly higher proportions of microcephalic newborns had low birth weight (P<0.001) and were SGA (P<0.001) compared with normocephalic newborns (Table 1).  Table 2.

Eight
Detailed individual test results are available as supporting information Table S1.  Table 2.
Detailed individual test results are available as supporting information Table S2.
All mothers and newborns were tested for syphilis and HIV at hos- Twelve (7.9%) newborns were admitted to a neonatal intensive care unit, and 3 (2.0%) died. One of the deaths was attributed to severe microcephaly, and two others to premature birth (both normocephalic newborns).

| DISCUSSION
The present year-long hospital study was conducted in response to an   vided English language revision and manuscript copyediting assistance.

CONFLICTS OF INTEREST
The authors have no conflicts of interest.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.