Language delay was associated with a smaller head circumference at birth in asymptomatic infants prenatally exposed to the Zika virus

Our aim was to analyse 12‐month outcomes of children who were prenatally exposed to the Zika virus and asymptomatic at birth.

complete anthropometric data at 12 months of age and missing neurodevelopment assessments.

| Exposure
We related the timing of the exposure to the virus to the gestational age when the clinical symptoms of the Zika virus infection emerged in the pregnant women. These symptoms were fever with a maculopapular or pruritic rash. 10 Exposure to the Zika virus during pregnancy was confirmed by testing the mother's blood and urine, using polymerase chain reaction (RT-PCR) during the symptomatic acute viraemia period.
Anthropometric measurements were performed at birth and at the follow-up visits. Weight was measured with a Filizola digital scale with 5-gram precision (Filizola ® ). Height was measured with an appropriate ruler, with the child in the supine position, and head circumference was measured with an inelastic tape measure. Z-scores were calculated for each measurement using the Intergrowth-21 11 reference curve at birth and the WHO reference curve 12 at one month of age and older.
All the children included in the study were evaluated by the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). 13 The assessment was performed between 12 and 18 months. Ages were adjusted for prematurity when the children were born preterm. The Bayley-III 13 covers three broad domains of neurodevelopment, which evaluate five independent outcomes.
These are cognition, global language, including receptive and expressive language, global motor development and its components and fine and gross motor skills. The assessment was conducted in an enclosed room by trained psychologists. The composite scores for the broad domains range from 40 to 160 points, with a mean of 100 and a standard deviation of 15. This means that 85 would be one standard deviation below the mean and 115 points would be one standard deviation above the mean. A score of below 85 indicates mild delay and below 70 indicates severe delay. Expressive and receptive language scores and fine and gross motor development scores represent the child's performance in a given sub-score and range from 1 to 19, with a mean of 10 and a standard deviation of three. For example, a score of seven represents one standard deviation below the mean and a score of 13 represents one standard deviation above the mean. 13 We evaluated both the continuous scores and the scores that indicated normal or delayed development. Values below one standard deviation of the standardised mean in at least one of the domains were considered to indicate delay. Values below two standard deviations were considered to indicate severe delay.
• Delays were not associated with gestational age at exposure but were associated with smaller head circumferences at birth.

| Variables
The variables that were analysed were gestational age in complete weeks, type of delivery, birth weight, height and head circumference with their respective standard deviations (Z-scores).
The socioeconomic evaluation of the families covered a number of factors, including age, any use of illicit drugs, maternal alcohol use or smoking and household income, which was assessed in relation to the Brazilian minimum wage. Maternal education was stratified into partial high school or less, complete high school to partial higher education and complete higher education and more. High school education normally ends when adolescents are 17 years of age. The following factors were also recorded: breastfeeding, months of exclusive breastfeeding and exclusive milk formula use, the number of people living in the same house and whether the father participated in childcare. All these variables were considered confounding factors with regard to development.

| Sample size
Non-probabilistic convenience sampling was performed and included all children born asymptomatic who were included in the cohort of the main project.

| Ethical considerations
This study was approved by the Institution's Research Ethics Committee (approval number CAAE 526756616000005269) and registered on clinical trials.gov (NCT03255369).
The mothers provided informed consent before being included in the study.

| RE SULTS
We examined 132 newborn infants, born without congenital Zika syndrome, after their mothers had been exposed to the Zika virus ( Figure 1). Of these, 96 met the inclusion and exclusion criteria and were included in the study.  (Table 1). Admission to the neonatal intensive care unit was 19.8%, and the main indication was early respiratory distress.
The median age at anthropometric assessment was 14 months (IQR 3) and ranged from 8 to 22 months, and the mean Z-scores for weight, length and head circumference were within the normal ranges ( Table 2).
Maternal exposure to the virus occurred predominantly in the second trimester (65.3%), with 20.0% exposed in the first trimester and 14.7% in the third.
The median age at the Bayley-III evaluation was 12 months (IQR 1), ranging from 11 to 19 months. Although the mean motor, language and cognitive scores were within the normal ranges for most of the children, the mean language scores were near the lower limit of normality (Table 3). The receptive language scores were lower than the expressive language scores, with 26/96 (27.0%) scoring less than seven for receptive language and 19/96 (19.7%) scoring less than seven for expressive language.
We found that 35.4% of the children had a delay in at least one  birthweight and head circumference at birth. We found a homogeneous distribution of scores that indicated delayed exposure by trimester, with a slight predominance in infections in the first trimester. However, the differences were not significant, according to the chi-square test (p = 0.76) ( Table 4). Despite the high prevalence of language delay, there was no predominant delay in any of the trimesters.

| DISCUSS ION
The offspring of mothers with Zika infections, confirmed by RT-PCR, had median scores in all Bayley-III domains and these fell within the normal range. Mean scores for language, especially receptive language, were within the expected age range. However, they were close to the lower limit, unlike those in the other evaluated domains. We found a high proportion of children with language delay when we evaluated scores below the normal range in this population. The proportion of children with severe delay, defined by a score of less than 70, in the language domain and its sub-scores, was also analysed. This showed that scores below 70 were not common, indicating that impairment in this function was not severe, at least at this early stage of development. Few studies have addressed the development of children exposed to, and infected by, the Zika virus during the foetal period. 4,14-17 One multicentre study used the General Movement Assessment to evaluate 111 infants exposed to the Zika virus in utero and 333 control infants. It reported the absence of fidgety movements in infants with microcephaly and identified adverse outcomes in newborn infants without microcephaly. 5 Other studies of the same population that our sample originated from analysed the developmental outcomes in a cohort of children, with and without microcephaly, whose mothers were diagnosed with the Zika virus during pregnancy. 4,5,8,16 Delays in the motor and cognition domains were found in 16.4% and 9.6% of the assessed children, respectively. 16 Another study conducted in Salvador, Brazil, investigated the development of children vertically exposed to the Zika virus without microcephaly at birth. The results showed that 34% of these infants had delays in at least one of the Bayley-III domains, predominantly language delay (31%). In contrast, motor and cognitive delays were observed in 3% and 14% of the children, respectively. 7 Another important finding of our study was the association between the head circumference Z-score at birth with global language delay and its components, including receptive and expressive language. A study published by our research group analysed the spectrum of clinical findings in a cohort of 219 children with confirmed intrauterine exposure to the Zika virus. We reported associations between smaller head circumferences at birth with below-average cognitive and language scores. 9 Language development is known to be compromised even when brain insults do not result in global or predominant motor delays. [17][18][19] Cognitive impairments have been reported in children with a history of neonatal encephalopathy, but without alterations in their motor skills. 17,18 Although children with neurodevelopmental disorders have a high rate of motor impairment, most of the need for educational support is due to delays in language and reading ability. 20 In our study, language delay was most prevalent, especially receptive language delay. This may be because cognitive changes become more

| Strengths and limitations
Our study was conducted at a federal reference institution for the congenital Zika virus infection, which allowed us to evaluate a relatively large population. This was a prospective study that included children exposed to the Zika virus in utero, who did not have malformations or other clinical symptoms at birth. All children were born from mothers who showed clinical symptoms and had positive RT-PCR results.
A limitation of this study was that we were unable to obtain Bayley-III assessments or anthropometric measurements for approximately 13% of the children. This was because the mothers missed the appointments. These missing data may have caused selection bias and that should be considered when interpreting the results.
Another limiting factor was that we did not have a control group that were not exposed to the virus during foetal life and this restricts the generalisability of the results. The finding of early language delays at 12 months of age was a limitation when it came to interpreting the results, but this may have been an indication of future language impairment.

| CON CLUS ION
This study analysed the 12-month outcomes of children who were prenatally exposed to the Zika virus and asymptomatic at birth. We found that the greatest number of scores that indicated developmental delay were in the language domain, predominantly the receptive language sub-score. This finding may indicate long-term cognitive impairment and suggests that children who are asymptomatic at birth, despite being exposed to the Zika virus during the foetal period, may still be susceptible to adverse health outcomes later in life. This highlights the importance of conducting further studies with long-term follow-up periods, especially with regard to language impairment and learning difficulties. Future studies should also include a control group of children born outside the Zika virus epidemic regions, to assure lack of exposure. This is because it is