Introduction

Since October 2015, Brazil has had an alarming number of suspected cases of newborns with microcephaly. During that period, the concomitance with the zika virus infection epidemic in the country aroused the suspicion of an association between these conditions. Therefore, the World Health Organization (WHO) declared a public health emergency in February 2016 [1, 2].

Microcephaly is defined as a clinical sign of congenital malformation in children presenting occipitofrontal circumference two standard deviations lower than the mean compared to children of the same age, sex, and ethnicity [3]. It is a rare condition, with an incidence of 5.9 cases per 10,000 live births in the United States of America [4], and its diagnosis can be confirmed as delayed brain development identified by imaging tests, such as intrauterine ultrasonography (US) or computer tomography (CT) after childbirth.

This congenital malformation has many causes, such as genetic factors and congenital infections, and it is also associated with social factors. Among the congenital infections, cytomegalovirus [5], rubella [6, 7, 8], toxoplasmosis [9], and syphilis [10] stand out as the leading agents. Low education, alcohol abuse, and inadequate prenatal care are also associated with microcephaly [4]. However, a study revealed that 41% of microcephaly cases do not have a definitive cause and are categorized as idiopathic cases [11].

Current studies have confirmed the biological plausibility of a link between zika virus and microcephaly: case reports have confirmed stillbirths infected by zika virus [12]; other studies have shown brain tissue tropism caused by zika virus [13], the vertical transmission of zika through the placenta [12, 14], an ecological association [15], and a higher incidence of malformations in pregnancies with confirmed Zika infections [16].

According to many authors [17, 18], studies must be developed to obtain more information about this malformation outbreak, including the temporal patterns of the complications after the epidemic waves [19] and its repercussion in the present and future generations.

This present case-control study was performed in the state of Ceará, Northeastern Brazil, to contribute to the understanding of the association between zika virus infection and microcephaly and to draft new political-administrative measures for its management.

Methods

Study design

This exploratory case-control study intended to identify protective factors and risk factors for microcephaly in the state of Ceará, Northeastern Brazil – the Brazilian region with the highest incidence of microcephaly.

The cases were defined as children with a clinical diagnosis of microcephaly with an imaging diagnosis of microcephaly performed by computed tomography (CT) or intrauterine ultrasonography (US). The children were born after October 2015, correlating the first trimester of pregnancy with the period of the zika virus epidemic. These newborns were identified not only in public and private maternity records but also in reference services for tracking and infant development until June 2017.

The controls were children without microcephaly, showing a cephalic perimeter larger than the 95% percentile for age according to WHO curves, as well as no signs or symptoms of congenital malformations. They were identified near the residence of each child in the group of cases, with the same age or within a two-month range, ensuring the homogeneity of temporal and environmental exposure in both groups.

Study setting and population

The study population included children of both sexes born from October 2015 until June 2017 who live in the State of Ceará.

Ceará is one of the poorest States in Brazil, ranking 17th in the Human Development Index (0.682) out of the 27 States in the country [20]. Ceará is in the northeastern region of Brazil, and approximately 93% of its land area of 148,000 km² presents a semiarid climate [21]. With a population of 8 million inhabitants [18], and an annual average of 128,000 live births [21], the state is the fourth in total confirmed microcephaly cases (137 cases) and the first in total of fetal and neonatal deaths resulting from microcephaly (24 cases, 16.9%) [22]. The territory of Ceará comprises 184 municipalities, and there have been confirmed cases of microcephaly in 53 (28.8%) of them [22].

Study sample

This study of prevalent cases aimed to enroll a sample of children with a confirmed diagnosis of microcephaly born in Ceará through June 2017. The control group of children was recruited using a ratio of two controls for each case to increase the sample power [23]. The data were collected from 58 randomly selected cases, and from this sample size, it is possible to detect an odds ratio (OR) of 2.3, with a 5% significance level and 80% sample power for exposure with 28% prevalence in the control group.

Data collection

Children diagnosed with microcephaly were identified by the infant care network, collecting mothers’ names and their respective addresses. We obtained the data from these units: the Assis Chateaubriand Maternity School – MEAC, the main public maternity of Ceará; the Albert Sabin Children’s Hospital – HIAS, reference for infant diseases of greater complexity in the Northeastern region of Brazil; and the Core of Treatment and Precocious Stimulation – NUTEP, reference service for children’s precocious stimulation in Ceará.

The data collection was conducted in the children’s households through a questionnaire containing data from the mothers, the children, and their families. The field research team composition was one supervisor and six interviewers, all of them with nursing or physiotherapy degrees and with proper training for the interview and anthropometric measurements. The team made five attempts to find the mother and her child at home to collect the data.

The anthropometric measurements of the children (cases and controls) were carried out just after the mother’s interview. The occipitofrontal perimeter was measured using a retractable inelastic measuring tape with a security lock (1.5 m in size and 1 mm precision). A portable digital scale measured the weight with a capacity of 180 kg and an accuracy of 100 g, and the weight was obtained with the child on the mother’s lap followed by subtracting the mother’s weight. The measurement of the children’s length was performed with the child lying down on an infant anthropometer, with a size of 1.5 m and precision of 1 mm.

Quality control

To reduce memory bias, we checked the data from the pregnancy and the prenatal care in the Pregnancy Booklet (official medical record of pregnant women in Brazil), which is provided by the maternity ward presenting the gestation registers and the prenatal consultations. Children’s data, such as cephalic perimeter and weight at birth, were confirmed by the Child’s Health Booklet (official medical record for children in Brazil), which is provided by the Ministry of Health to all children at birth.

The scales were calibrated according to the producer’s indication. A random sample of 10% of the questionnaires was repeated by the field supervisor to identify possible errors and fill the gaps to validate the quality of the data collection.

The data was double entered in the software program to solve typing errors or data loss.

Variables

The dependent variable, the outcome, refers to the presence or absence of microcephaly, with a confirmed diagnosis by clinical or imaging criteria (CT scan or intrauterine US) in the hospital or maternity ward, following the government protocol [24].

The independent variables were grouped and categorized into the blocks described below.

Diseases during pregnancy included the following – self-reported and confirmed by the pregnancy booklet: zika virus infection diagnosed by a medical doctor, joint pain, clinical syndromes with a skin rash, eye pain, cytomegalovirus, syphilis, rubella, toxoplasmosis, AIDS, flu or other viruses.

The exposures during pregnancy included the following – self-reported: if one had contact with solvents, inks, varnishes, poisons, hair dye or smoothing agents with ammonia; if the mother underwent tests with radiation during pregnancy (X-ray or CT), magnetic resonance imaging, tobacco use, and alcohol consumption.

Data analysis

Bivariate analysis was performed, calculating the proportions of categorical variables and measures of central tendency and dispersion for numeric variables. The differences found between the cases and controls were evaluated for statistical significance using the chi-square test for categorical variables, students’ t-test was used for numeric variables with normal distribution, and the Mann-Whitney test was used for numeric variables with nonnormal distribution. The Kolmogorov-Smirnoff test evaluated the normality of variables. Additionally, if one cell registered fewer than five cases, we used Fisher’s exact test.

For the control of confounders, the variables with a p-value lower than 0.1 were included in a logistic regression model with adjusted odds ratio calculations.

Analyses were performed using SPSS software, version 17, SPSS Inc., considering a p-value ≤0.05.

Ethical aspects

The study was submitted to the Ethics Committee through the Brazil Platform, following all the norms of the 466/2012 Resolution of the National Council of Health of the Ministry of Health [25], and it was approved under protocol number 1.449.427. All the mothers who agreed to participate in the research signed informed consent forms.

Results

Children’s baseline characteristics

The median age and sex of cases and controls were not different (nine months and 57% male). However, there was a difference between cephalic perimeters (35 and 31 centimeters), birth weights (2.67 and 3.22 kg), and gestational ages, as shown in Table 1.

Table 1

Baseline and birth characteristics of the sample.

Condition p value

Case Control Total

n or median % or interquartile n or median % or interquartile n or median % or interquartile

Sex Male 29 50.9% 69 60.0% 98 57.0% 0.194
Female 28 49.1% 46 40.0% 74 43.0%
Age in months 6.50 4.0–21.0 11.00 4.0–20.0 9 4.0–20.0 0.474
Birth weight 2.67 2.3–3.0 3.22 2.8–3.7 3.010 2.6–3.4 <0.001
Cephalic perimeter 31.00 29.5–32.0 35.00 34.0–35.5 33.0 31.0–35.0 <0.001
Timing of birth Normal 45 77.6% 94 81.7% 139 80.3% 0.025
Preterm 12 20.7% 9 7.8% 21 12.1%
Post term 1 1.7% 9 7.8% 10 5.8%
Not specified 0 0.0% 3 2.6% 3 1.7%

Diseases during pregnancy

The mothers of the patients had two-times more fevers than the mothers of the controls (p-value = 0.022), and these cases of illness were earlier in the first trimester in cases (p = 0.032). Among the women who received a diagnosis during pregnancy, there was a predominance of zika virus syndrome in the pregnant women from the cases, while the majority of the mothers of the controls had urinary tract infections or conditions other than zika (p = 0.040). The signs and symptoms were muscular pain, joint pain, weakness, and skin changes, also showing a difference between the groups (0.028, 0.002, 0.041, <0.001, respectively). Mothers of the cases showed almost twice as many reports of insect bites during pregnancy. The odds ratio of having zika virus of the cases compared to the controls was 10.35 (p-value < 0.001) (Table 2).

Table 2

Ratios of diseases presented by the mother during pregnancy in cases and controls.

Condition p value

Case Control

n or median % or interquartile n or median % or interquartile

Fever Yes, once 16 28.1% 16 13.9% 0.022
Yes, more than once 9 15.8% 11 9.6%
No fever 32 56.1% 88 76.5%
Gestation month of fever 3 2.0–6.0 5 4.0–7.0 0.032
Days of fever 2 1.0–3.0 3 3.0–5.0 0.057
Fever intensity Low 11 50.0% 6 26.1% 0.151
Moderate 8 36.4% 9 39.1%
High 3 13.6% 8 34.8%
Any appointment with a physician Yes 18 58.1% 26 66.7% 0.449
No 12 38.7% 13 33.3%
Do not remember 1 3.2% 0 0.0%
Diseases during pregnancy Other 8 47.0% 12 46.1% 0.040
Urinary infection 1 5.2% 12 46.1%
Zika 8 47.0% 2 7.6%
Headache intensity Low 8 15.1% 16 17.4% 0.865
Moderate 10 18.9% 20 21.7%
High 14 26.4% 19 20.7%
None 21 39.6% 37 40,2%
Stiff neck Yes 5 9.6% 14 14.4% 0.599
No 47 90.4% 81 83.5%
Do not remember 0 0.0% 1 1.0%
Eye pain Yes 12 22.2% 14 15.2% 0.227
No 41 75.9% 78 84.8%
Do not remember 1 1.9% 0 0.0%
Photophobia Yes 10 18.5% 13 13.5% 0.417
No 44 81.5% 83 86.5%
Seizures Generalized 2 3.7% 1 1.0% 0.527
Focal 2 3.7% 4 4.1%
No 50 92.6% 92 94.8%
Disorientations Yes 0 0.0% 5 5.2% 0.172
No 54 100.0% 90 93.8%
Do not remember 0 0.0% 1 1.0%
Amnesia Yes 2 3.8% 5 5.4% 0.678
No 51 96.2% 87 93.5%
Do not remember 0 0.0% 1 1.1%
Behavioral changes Yes 13 24.1% 29 30.2% 0.422
No 41 75.9% 67 69.8%
Dyspnea Yes 16 30.2% 27 28.7% 0.851
No 37 69.8% 67 71.3%
Precordialgia Yes 4 7.4% 14 14.4% 0.202
No 50 92.6% 83 85.6%
Dysarthria Yes 0 0.0% 6 6.3% 0.062
No 53 100.0% 89 93.7%
Epigastralgia Yes 10 18.5% 17 17.5% 0.879
No 44 81.5% 80 82.5%
Diarrhea Yes 5 9.4% 10 10.3% 0.864
No 48 90.6% 87 89.7%
Nausea Yes 33 61.1% 58 60.4% 0.933
No 21 38.9% 38 39.6%
Sore throat Yes 6 11.1% 21 21.6% 0.105
No 48 88.9% 76 78.4%
Cough Yes 13 24.1% 23 24.0% 0.987
No 41 75.9% 73 76.0%
Back pain Yes 27 50.0% 58 59.8% 0.245
No 27 50.0% 39 40.2%
Muscle pain Yes 14 26.9% 12 12.5% 0.028
No 38 73.1% 84 87.5%
Joint pain Yes 23 42.6% 19 19.6% 0.002
No 31 57.4% 78 80.4%
Difficulty moving Yes 12 22.6% 14 14.9% 0.237
No 41 77.4% 80 85.1%
Joint edema Yes 17 31.5% 25 26.0% 0.600
No 37 68.5% 70 72.9%
Do not remember 0 0.0% 1 1.0%
Paralysis Ascending 1 1.9% 3 3.2% 0.629
No 53 98.1% 91 96.8%
Weakness Generalized 13 24.1% 9 9.6% 0.041
Focal 4 7.4% 13 13.8%
No 37 68.5% 72 76.6%
Conjunctivitis Yes 0 0.0% 2 2.1% 0.290
No 52 100.0% 92 97.9%
Bleeding Yes 10 19.6% 16 17.2% 0.720
No 41 80.4% 77 82.8%
Skin changes Yes 25 48.1% 14 14.7% <0.001
No 27 51.9% 81 85.3%
Thick skin Yes 11 42.3% 5 27.8% 0.325
No 15 57.7% 13 72.2%
Spot with a border Yes 3 12.0% 0 0.0% 0.128
No 22 88.0% 18 100.0%
Erythema Yes 17 68.0% 7 38.9% 0.058
No 8 32.0% 11 61.1%
Vesicles Yes 1 4.0% 0 0.0% 0.391
No 24 96.0% 18 100.0%
Itchy spots Yes 11 44.0% 6 33.3% 0.480
No 14 56.0% 12 66.7%
Petechiae Yes 16 64.0% 7 38.9% 0.103
No 9 36.0% 11 61.1%
Bruises Yes 3 12.0% 0 0.0% 0.128
No 22 88.0% 18 100.0%
Stings during pregnancy Yes 17 30.9% 18 16.4% 0.031
No 38 69.1% 92 83.6%
Zika infection Yes 27 46.6% 9 7.8% <0.001
No 31 53.4% 107 92.2%
Diagnosis of zika by a health professional Yes 18 81.8% 6 66.7% 0.360
No 4 18.2% 3 33.3%
Gestation month of zika diagnosis 3 2.0–4.0 5 3.0–6.0 0.246
What was the severity of zika? Low 14 63.6% 7 87.5% 0.360
Moderate 4 18.2% 1 12.5%
High 4 18.2% 0 0.0%
Internment due to zika Yes 0 0.0% 1 11.1% 0.120
No 21 100.0% 8 88.9%
Had dengue fever Yes 1 1.8% 1 0.9% 0.606
No 56 98.2% 115 99.1%
Had chikungunya Yes 1 1.8% 0 0.0% 0.153
No 56 98.2% 116 100.0%
Had rubella No 57 100.0% 116 100.0%
Had toxoplasmosis infection Yes 2 3.5% 3 2.6% 0.734
No 55 96.5% 113 97.4%
Had cytomegalovirus infection Yes 2 3.5% 0 0.0% 0.042
No 55 96.5% 116 100.0%
Had herpes Yes 2 3.5% 1 0,9% 0.210
No 55 96.5% 115 99.1%
Had syphilis Yes 1 1.8% 1 0.9% 0.606
No 56 98.2% 115 99.1%
Had AIDS No 56 100.0% 116 100.0%
Had hypertension Yes 3 5.5% 18 15.8% 0.056
No 52 94.5% 96 84.2%
Had eclampsia Yes 1 3.3% 2 3.3% 1.000
No 29 96.7% 58 96.7%
Had preeclampsia Yes 0 0.0% 6 10.0% 0.078
No 29 100.0% 54 90.0%
Had diabetes Yes 1 1.8% 6 5.3% 0.292
No 54 98.2% 108 94.7%
Had kidney disease Yes 1 3.3% 5 8.3% 0.370
No 29 96.7% 55 91.7%
Had anemia Yes 12 21.1% 23 20.2% 0.534
No 45 78.9% 91 79.8%
Had the flu Yes 18 31.6% 44 38.3% 0.390
No 39 68.4% 71 61.7%
Had diarrhea Yes 8 14.0% 7 6.1% 0.082
No 49 86.0% 108 93.9%
Had an allergy Yes 5 8.9% 5 4.3% 0.225
No 51 91.1% 111 95.7%
Had asthma Yes 1 3.3% 1 1.7% 0.613
No 29 96.7% 59 98.3%

Exposures during pregnancy

From all the evaluated exposures, only the exposure to radiation before and during pregnancy had different odds between the groups (0.010 and 0.036 for radiography and tomography, respectively). Contact with chemical agents, repellents, and other substances did not present significant p values (Table 3).

Table 3

Ratios of exposures of the mother during pregnancy in cases and controls.

Condition p value

Case Control

n or median % or interquartile n or median % or interquartile

Worked during pregnancy Yes, outside home 20 35.1% 31 27.2% 0.235
Yes, at home 8 14.0% 10 8.8%
No 29 50.9% 73 64.0%
Contact with ink Yes, during pregnancy 16 27.6% 35 30.7% 0.822
Yes, before 1 1.7% 1 0.9%
No 41 70.7% 78 68.4%
Contact with varnishes Yes, during pregnancy 8 13.8% 9 7.9% 0.168
Yes, before 1 1.7% 0 0.0%
No 49 84.5% 105 92.1%
Contact with solvents Yes, during pregnancy 8 13.8% 12 10.4% 0.291
Yes, before 1 1.7% 0 0.0%
No 49 84.5% 103 89.6%
Contact with tails Yes, during pregnancy 9 15.8% 14 12.2% 0.637
Yes, before 0 0.0% 1 0.9%
No 48 84.2% 100 87.0%
Contact with repellents Yes, during pregnancy 20 34.5% 55 47.8% 0.095
No 38 65.5% 60 52.2%
Contact with pesticides Yes, during pregnancy 4 7.0% 7 6.1% 0.814
No 53 93.0% 108 93.9%
Contact with poisons Yes, during pregnancy 7 12.3% 19 16.8% 0.438
No 50 87.7% 94 83.2%
Contact with pesticides Yes, during pregnancy 0 0.0% 2 1.8% 0.314
No 57 100.0% 112 98.2%
Contact with hair dye Yes, during pregnancy 8 13.8% 21 18.4% 0.443
No 50 86.2% 93 81.6%
Contact with enamels Yes, during pregnancy 33 56.9% 67 59.3% 0.953
Yes, before 1 1.7% 2 1.8%
No 24 41.4% 44 38.9%
Contact with capillary smoothing with ammonia Yes, during pregnancy 2 3.4% 6 5.3% 0.667
Yes, before 0 0.0% 1 0.9%
No 56 96.6% 107 93.9%
Contact with capillary straighteners without ammonia Yes, during pregnancy 2 3.4% 6 5.2% 0.672
Yes, before 0 0.0% 1 0.9%
No 56 96.6% 108 93.9%
Performed radiographs Yes, during pregnancy 6 10.5% 2 1.8% 0.010
No 51 89.5% 112 98.2%
Performed CT scans Yes, during pregnancy 0 0.0% 1 0.9% 0.036
Yes, before 3 5.4% 0 0.0%
No 53 94.6% 113 99.1%
Performed Magnetic Yes, during pregnancy 2 4.8% 0 0.0% 0.074
Resonance Yes, before 1 2.4% 1 1.0%
Imaging No 39 92.9% 98 99.0%
Smoked during pregnancy Yes, all the days 1 17% 5 4.3% 0.272
Yes, some days 2 3.4% 1 0.9%
No, stopped smoking 3 5.2% 2 1.7%
No, never smoked 52 89.7% 108 93.1%
Number of cigarettes per day 15 10.0–20.0 7 4.0–10.0 0.333
Alcohol consumption during pregnancy Yes, a little 5 8.8% 11 9.5% 0.999
Yes, moderate 1 1.8% 2 1.7%
Yes, a lot 1 1.8% 2 1.7%
No 50 87.7% 101 87.1%

Multivariate analysis

After the multivariate analysis, the variable infection by zika virus remained independently associated, with a p-value of 0.018 and odds ratio of 14.68 (1.59–134.83) (Table 4).

Table 4

Multivariate analysis of the determinants of microcephaly.

B S.E. Adjusted OR Adjusted OR CI 95% p-value

Inferior Superior

Zika virus 2.686 1.131 14.680 1.598 134.833 0.018
Muscle pain 0.480 0.711 1.617 0.401 6.517 0.499
Joint pain 0.731 0.602 2.078 0.638 6.768 0.225
Skin changes –0.321 0.905 0.725 0.123 4.277 0.723
Sting during pregnancy 0.115 0.796 1.122 0.236 5.345 0.885
Radiography 0.261 0.908 1.298 0.219 7.692 0.774
CT Scan –0.434 0.899 0.648 0.111 3.777 0.630

Block 1: Zika virus, muscle pain, joint pain, skin changes, stings during pregnancy.

Block 2: Radiography, CT scan.

Discussion

The results of this case-control study indicate that infection by zika virus diagnosed by a physician in the first trimester was, independently, the risk factor most strongly associated with microcephaly, with an adjusted OR of 14.68 (95% CI 1.59–134.83) and high statistical significance (p < 0.001) despite the small sample size. The prevalence of zika virus infection in mothers of children born with microcephaly, confirmed by diagnostic methods suggested by the WHO, was 46.6%, against only 7.8% of mothers who had healthy children during the same period, as suggested by case series [16] and ecological [15] studies and also from another case-control study, in a state near Ceará [26].

Symptoms of the infectious arboviruses syndrome, mainly occurring during the first trimester of pregnancy, associated with skin rash or joint pain, have shown an association with the development of microcephaly in the bivariate analysis. Additionally, there was a higher risk of mosquito bites during pregnancy reported by the mothers of children with microcephaly.

A preliminary laboratory case-control study conducted in another state in the northeastern region of Brazil identified a high prevalence of zika virus infection in mothers of infants with microcephaly (80%) and the mothers of controls (64%) using RT-PCR and new serological methods. However, zika virus infection did not occur in any children in the control group [27].

This study contributed to the investigation of epidemiological factors, not only the maternal infections and exposures already studied, but also identified factors such as radiation exposure. Additionally, it found an association of microcephaly with maternal exposure to radiological examinations during pregnancy, although this association did not remain after controlling for confounders. Other authors [28, 29] have reported this association with radiation and congenital disabilities; however, this is not fully established. We have not yet found mention in the literature of studies that have specifically evaluated this association.

This study was validated by the difference in the means of cephalic perimeter (CP) among the case and control groups. The average CP of the children with microcephaly was below that found by Rocha et al. (2016), who performed an evaluation study to check the normality parameters of CP in children born at term in the Brazilian Northeast, before the context of the epidemic of microcephaly [30]. Other biological criteria evaluated ensured uniformity between the groups in addition to CP, including gestational age and sex.

Despite alcohol abuse and smoking during pregnancy being causes of congenital malformations [4, 31], no association with microcephaly was found in this study.

As Von der Hagen et al. (2014) discussed, 41% of microcephaly cases are idiopathic [11]. The absence of a significant correlation in this study between the classic factors of teratogenesis, such as alcohol or tobacco [32], led to an investigation of other causes, such as the epidemic of the zika virus.

Microcephaly may not be the only outcome in children infected with zika virus, and other neurological disorders can develop after birth [33, 34].

This study was developed with a community design that is well-suited for other risk factors beyond zika infections but with their environmental and socioeconomically correlated factors. Additionally, the emergency situation requires an exploratory study of a broad spectrum. In this sense, we evaluated more than 200 variables, including various epidemiological factors.

Limitations

The memory bias was considered during the data collection using health records whenever possible. Confirmation bias is possible, but there was great diffusion in Brazil of the possible association between zika virus and microcephaly, which has generated a widespread awareness further than the mothers of infants with microcephaly.