Procedures and Definitions
Maternal demographic, clinical, laboratory, obstetrical, and neonatal data were recorded in standardized forms. Prenatal routine maternal testing was done for toxoplasmosis, hepatitis B and C, human immunodeficiency virus (HIV), and syphilis. Gestational age was determined in completed weeks by obstetrical estimation18. Spontaneous abortions and stillbirths were defined as embryo or fetal losses that occurred before and after 20 completed weeks of pregnancy, respectively.
Newborn data were extracted from medical records. Birth weight and head circumference measured before hospital discharge were classified according to the INTERGROWTH-21stcriterion19 as recommended by the PAHO. Microcephaly was defined as a head circumference (HC)>2z scores below the appropriate mean for a given age, sex, and gestational age, and severe microcephaly was defined as an HC>3z scores below these standards19,20. All infants were initially classified according to the presence of microcephaly, neonatal neurologic dysfunctions, and other birth defects coded as major or minor21.
Combined neonatal abnormalities detected in fetuses/infants were defined as potentially related to congenital ZIKV infection both according to Honein et al.6 and Pomar et al.1criteria as mild/moderate or severe complications compatible with CZS. Typical CZS phenotype was defined as those classified by the former authors as “brain abnormalities with or without microcephaly” and by the latter as those presenting “severe complications compatible with CZS or fetal loss”. We did not to exclude the first-trimester spontaneous abortions from the adverse outcomes but excluded the fetuses/newborns with other known causes for the CNS anomalies from the typical CZS phenotype definition. Both decisions were taken to allow a better understanding of the complete investigation of cases.
Additional assessments were planned for all infants with microcephaly and other major neurologic findings and those who were born or referred within 3 months of life as follows: Hammersmith Neonatal Neurological Examination (HNNE)22; pediatric evaluations at 3-6 weeks and 3+ 1 months focusing on clinical and neurological findings and the developmental milestones; Bayley-III scale of infant and toddler development screening test23 performed by a certified examiner; hearing screening using both Transient Otoacoustic Emissions (TOAE) and automated auditory brainstem evoked responses (AABR-35dBHL); cranial ultrasonography and/or other neuroimaging studies performed by an experienced physician and reviewed by two board-certified radiologists; complete eye examination including indirect ophthalmoscopy by ophthalmologist.
The HNNE optimality compound score is calculated for each of six categories (tone, tone patterns, reflexes, movements, deviant signs, behavior) of a neurological evaluation containing 34 items. Scores between 30.5 and 34 are found in more than 95% of low risk term infants22. For the purpose of this analysis, an optimality score <30.5 at birth were considered suboptimal. For infants with no HNNE complete evaluation, a short neurological exam proforma of this test with 13 categories was applied24and the presence of any abnormal sign was considered suboptimal. Regarding the Bayley screening test, infants were tested according to the application guidelines in five domains: cognitive, communication (receptive and expressive), and motor (fine and gross). Scores were classified as competent or emerging/at risk in each domain23. An infant with at least two of the neurologic/neurodevelopmental evaluations categorized as suboptimal, abnormal, emerging or at risk was classified with a possibleneurologic abnormality.