The association between microcephaly and congenital Zika virus infection was confirmed. We provide evidence of the absence of an effect of other potential factors, such as exposure to pyriproxyfen or vaccines (tetanus, diphtheria, and acellular pertussis, measles and rubella, or measles, mumps, and rubella) during pregnancy, confirming the findings of an ecological study of pyriproxyfen in Pernambuco and previous studies on the safety of Tdap vaccine administration during pregnancy.

We screened neonates born between Jan 15 and Nov 30, 2016, and prospectively recruited 91 cases and 173 controls. In 32 (35%) cases, congenital Zika virus infection was confirmed by laboratory tests and no controls had confirmed Zika virus infections. 69 (83%) of 83 cases with known birthweight were small for gestational age, compared with eight (5%) of 173 controls. The overall matched odds ratio was 73·1 (95% CI 13·0–∞) for microcephaly and Zika virus infection after adjustments. Neither vaccination during pregnancy or use of the larvicide pyriproxyfen was associated with microcephaly. Results of laboratory tests for Zika virus and brain imaging results were available for 79 (87%) cases; within these cases, ten were positive for Zika virus and had cerebral abnormalities, 13 were positive for Zika infection but had no cerebral abnormalities, and 11 were negative for Zika virus but had cerebral abnormalities.

We did a case-control study in eight public maternity hospitals in Recife, Brazil. Cases were neonates born with microcephaly, defined as a head circumference of 2 SD below the mean. Two controls without microcephaly were matched to each case by expected date of delivery and area of residence. We tested the serum of cases and controls and the CSF of cases for detection of Zika virus genomes with quantitative RT-PCR and for detection of IgM antibodies with capture-IgM ELISA. We also tested maternal serum with plaque reduction neutralisation assays for Zika and dengue viruses. We estimated matched crude and adjusted odds ratios with exact conditional logistic regression to determine the association between microcephaly and Zika virus infection.

A Zika virus epidemic emerged in northeast Brazil in 2015 and was followed by a striking increase in congenital microcephaly cases, triggering a declaration of an international public health emergency. This is the final report of the first case-control study evaluating the potential causes of microcephaly: congenital Zika virus infection, vaccines, and larvicides. The published preliminary report suggested a strong association between microcephaly and congenital Zika virus infection.

Microcephaly was the first postnatal clinical finding to be reported at the beginning of the epidemic.However, rapidly accumulating evidence showed that congenital Zika syndrome could cause more than isolated microcephaly.In the early months of the marked increase in the prevalence of microcephaly, we designed a case-control studyto investigate an association between microcephaly and congenital Zika virus infection and other potential causes. The previously published preliminary reportdocumented a strong association with Zika virus; we now report the final results, with the aim of assessing the association between microcephaly and congenital Zika virus infection, along with a comprehensive investigation of other potential risk factors in an epidemic context in Pernambuco, Brazil.

Birth defects among fetuses and infants of US women with evidence of possible Zika virus infection during pregnancy.

This study supports the magnitude of risk of microcephaly associated with congenital Zika virus infection; provides evidence that neither larvicide or vaccinations during pregnancy caused the epidemic; highlights that neither a negative laboratory result for Zika virus nor an absence of cerebral abnormalities alone are sufficient to discard Zika virus as a cause of individual cases of microcephaly.

This is the final report of a case-control study, with a much larger sample size than a preliminary analysis of a subset of these data. This analysis supports the strength of association with Zika virus and, for the first time, investigates other potential risk factors including use of larvicides and vaccination during pregnancy. We confirm the strong association between Zika virus infection and microcephaly at birth and provide evidence that use of larvicides and vaccines during pregnancy did not increase the risk of microcephaly. We also provide information regarding all cases of microcephaly born during the study period: about half had either laboratory confirmation of Zika virus or typical brain image abnormalities. No controls had laboratory-confirmed Zika virus infection. There was some association between laboratory-confirmed Zika virus infection and cerebral abnormalities; 60% of those with brain abnormalities were negative for Zika virus when tested with specific IgM and PCR, and about half of those who were Zika virus-positive had no cerebral abnormalities. A high proportion of cases of microcephaly were small for gestational age. The high prevalence of serological markers of Zika virus infection in the mothers of controls indicate a high transmission of infection in the study area.

We searched PubMed and LILACS using the search terms “Zika” and “case-control study”. We searched for articles published up to Sept 30, 2017, including publications in English, Portuguese, and Spanish. The causal link between Zika virus infection and microcephaly, as part of the congenital Zika virus syndrome, is now well established; however, we did not identify any case-control studies of Zika virus infection and microcephaly. The final piece of the puzzle, providing epidemiological evidence, was the preliminary finding of a strong association in a case-control study of Zika virus infection and microcephaly in Recife, Pernambuco (Brazil), the hotspot of the microcephaly epidemic. Other risk factors have been suggested but never investigated at individual level, the more crucial being vaccines during pregnancy and use of the larvicide pyroxifen in containers of drinking water for mosquito control.

At the start of this microcephaly epidemic, the main causal hypothesis was Zika virus infection during pregnancy,but other possible causes were proposed; two of these causes were of particular interest because of their potential implications. The first of these possible causes was larvicide use in reservoirs of drinking water to control Aedes aegypti, namely pyriproxyfen, which was introduced in 2014 by the Brazilian Ministry of Health).The other possible cause of interest was vaccine administration during pregnancy.

Pyriproxyfen and the microcephaly epidemic in Brazil—an ecological approach to explore the hypothesis of their association.

In August, 2015, physicians reported a cluster of cases of microcephaly in the state of Pernambuco, northeast Brazil. Microcephaly is an abnormality in birth that was rarely reported before the Zika virus epidemic.Microcephaly is a clinical sign that can reflect abnormal brain development, but it can be also be found in healthy neonates. By definition, microcephaly is any insult that disturbs early brain growth, and it can be caused by genetic variations, teratogenic compounds, or other congenital infections (such as cytomegalovirus, rubella, herpes, or toxoplasmosis).

Practice parameter: diagnostic assessment of the child with microcephaly (an evidence-based review): report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society.

The funders of the study were involved in data interpretation and writing of the report. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication.

We also compared the means of anthropometric variables (head circumference, weight, height, and Z score–weight for gestational age and sex) in four categories of cases. These categories were negative for Zika virus (laboratory tested) and negative for cerebral abnormalities (determined by CT imaging of the neonates' brains); positive for Zika virus and negative for cerebral abnormalities; negative for Zika virus and positive for cerebral abnormalities; and positive for Zika virus and positive for cerebral abnormalities. These anthropometric variables were also recorded in controls, and these groups were compared with analyses of variance and Bonferroni post-hoc test to identify homogeneous subgroups. A χ 2 test was used to compare the characteristics of mothers and neonates, the frequency of abnormalities between neonates who were positive and negative for Zika virus, and smoking between mothers from different socioeconomic classes. Stata (version 14.1) software was used for the statistical analyses.

We estimated the crude mOR and 95% CI for the association between microcephaly and Zika virus infection for all cases, considering the results in any specimen (serum or CSF for livebirths, or macerated tissues for stillbirths). Additionally, crude mORs were independently estimated by sample type (serum or CSF) and microcephaly severity. We also investigated the agreement between qRT-PCR Zika virus-positivity in serum and CSF and between the IgM positivity in serum and CSF.

We applied a median unbiased estimator for binary data in exact conditional logistic regression to control for the fact that all controls tested negative for Zika virus.The model respected matching and included other conditioning variables: “condvars”.The study originally aimed to include 200 cases and 400 controls to have 90% power and 95% precision to detect an association with an odds ratio of 2 or greater, assuming that 67% of cases were exposed.

We investigated the association between microcephaly and each potential risk factor by conditional logistic regression. We included the variables associated with microcephaly with a p value less than or equal to 0·10 in the multivariable analysis by use of a conditional exact logistic regression model. Thus, we calculated matched odds ratio (mOR) for the association between microcephaly (outcome) and Zika virus infection (exposure), adjusted by smoking during pregnancy, skin colour, and having received the tetanus, diphtheria, and acellular pertussis vaccine (Tdap) during pregnancy.

Information that was recorded on demographic and socioeconomic factors included mother's age, number of years of schooling, and skin colour (self-reported). The purchasing power of individuals and families was defined by use of the Brazilian economic classification criteriaof 2015, which defines eight socioeconomic classes from A (highest) to E (lowest). We also collected data on the family history of microcephaly or malformations; vaccination status of the mother; self-reported ingestion of misoprostol (medical abortion pill), epilepsy medication, or folic acid; maternal use of recreational drugs, tobacco, and alcohol during pregnancy; exposure to pyriproxyfen (including in any domestic water reservoir); and the use of insect repellent on skin. Vaccination cards were consulted (when available), and we only considered vaccination during pregnancy.

Laboratory-confirmed Zika virus infection was defined in a neonate as a positive qRT-PCR or an IgM result for Zika virus in any biological specimen (serum, CSF, or macerated tissues). Neonates were considered to be small for gestational age if their birthweight was lower than the teth percentile for gestational age and sex on the Fenton growth chart.

In cases, brain imaging was done by CT scan and was classified as the presence or absence of major cerebral abnormalities, identified by physicians who were specialised in imaging diagnosis. Abnormalities included calcification, ventriculomegaly, malformation of cortical development (such as lissencephaly and polymicrogyria), and presumed vascular abnormalities. Controls were investigated by transfontanellar ultrasonography. Mothers were interviewed with a standardised questionnaire to determine several demographic and socioeconomic factors.

Serum samples of mothers and neonates (cases and controls) and CSF samples (cases) were tested by qRT-PCR for detection of the Zika virus genome,and by capture-IgM ELISA for IgM antibodies.Macerated tissues (from the brain, kidney, or pooled organs) of stillbirth cases were tested by qRT-PCR. The presence of Zika virus and dengue virus (1–4)-specific neutralising antibodies was assessed in the serum samples of mothers and neonates (cases and controls) by the plaque reduction neutralisation test (PRNT),with a 50% cutoff value for positivity. Serum samples were tested for toxoplasmosis, rubella, and cytomegalovirus IgM antibodies, the main infectious causes of congenital microcephaly.

Head circumference was measured in the delivery room with a non-stretch Teflon tape; a second measurement was done 12–24 h after birth to confirm microcephaly by the study neonatologists. At this second measurement, the neonates had a complete clinical examination by the study neonatologist, which included the assessment of reflexes. CSF was collected from cases around 48 hours after birth (but longer in infants who were in an intensive care unit). Umbilical cord blood was collected from cases and controls; when necessary, peripheral blood was collected before the neonate left the hospital. Blood specimens were stored at the Virology and Experimental Therapy Department, Fiocruz Pernambuco (Recife, Brazil).

We estimated gestational age by antenatal fetal ultrasonography. If ultrasounds were not available, we used the date of the last menstrual period recorded on the antenatal care card or reported by the mother. When both ultrasounds and the date of the last menstrual period were not available, we used the Capurro method.

The study was approved by the research ethics committees of the Pan American Health Organization (PAHO-2015-12-0075) and Fiocruz Pernambuco (CAAE: 51849215.9.0000.5190). All mothers provided written informed consent.

The criteria for matching for the expected date of delivery were specific to the gestational age of the cases. For cases born at term and post-term (37 weeks or more), controls were the next eligible neonates born at 37 weeks' gestation or more. For early preterm cases (born at <34 weeks), controls were the next eligible neonates who were born at less than 34 weeks' gestation. For preterm cases born between 34 and 36 weeks' gestation, controls were the next eligible neonates born at 34–36 weeks' gestation.

Controls were selected from the first neonates born after 0800 h on the morning after the birth of a case in one of the study hospitals, where a trained nurse stayed 7 days a week (from 0800 h to 1700 h) and listed the women who were admitted. However, we cannot guarantee that all consecutive neonates were screened.

The study population consisted of neonates born from women residing in Pernambuco, Brazil, and delivered in eight public maternity hospitals in Recife. Cases—neonates with microcephaly (livebirths or stillbirths)—had head circumferences at least 2 SD smaller than the mean for their sex and gestational age on the Fenton growth chart.Microcephaly was considered severe when the head circumference was at least 3 SD smaller than the mean. Exclusion criteria were anencephaly, encephalocele, and confirmation of the phenotype of a well defined congenital syndrome. Controls were live neonates without microcephaly and with no brain abnormalities (determined from transfontanellar ultrasonography) and no major birth defects, determined from physical examination by the study neonatologist. We selected two controls per case, which were matched by health region of residence and expected date of delivery to ensure that cases and controls were conceived at the same stage of the epidemic.

We present the final analysis of our case-control study of neonates who were consecutively recruited at birth. The protocol can be accessed online

Results

13 de Araújo TVB

Rodrigues LC

de Alencar Ximenes RA

et al. Association between Zika virus infection and microcephaly in Brazil, January to May, 2016: preliminary report of a case-control study. Figure Study profile The preliminary analysis included participants recruited from Jan 15 to May 2, 2016;this Article includes participants recruited up to Nov 30, 2016. We did this analysis before reaching 200 cases for two reasons: first, we reached the necessary power for statistical analysis because the proportion of controls who were exposed to Zika virus was lower than expected (as evidenced by the absence of Zika virus infection in all controls); second, the epidemic slowed down in Recife and cases became rarer. We screened 110 eligible cases, which included 92 livebirths and 18 stillbirths ( figure ). Our final analyses included 91 cases (82 livebirths and nine stillbirths) of microcephaly and 173 controls. We initially screened 13 624 neonates (13 531 livebirths and 93 stillbirths) from the study maternity hospitals during the study period; the prevalence of microcephaly at birth was estimated to be 101 (91 cases included in the study and ten excluded livebirths), resulting in an estimated prevalence of 74 cases of microcephaly per 10 000 births (95% CI 60–90).

50 ) than mothers of controls, with a borderline p value (p=0·051). All mothers of cases and controls tested negative for Zika virus by qRT-PCR testing. Table 1 Characteristics of mothers and neonates Cases (n=91) Controls (n=173) p value Mothers Age, years ·· ·· 0·11 13–24 44 (48%) 95 (55%) ·· 25–34 29 (32%) 60 (35%) ·· ≥35 18 (20%) 18 (10%) ·· Number of years in education ·· ·· 0·13 ≤4 17 (19%) 20 (12%) ·· 5–9 36 (40%) 60 (35%) ·· 10–12 33 (36%) 87 (50%) ·· ≥13, higher education 5 (5%) 6 (3%) ·· Reported rash during pregnancy ·· ·· 0·10 No rash 66 (73%) 139 (80%) ·· First trimester 7 (8%) 10 (6%) ·· Second trimester 13 (14%) 10 (6%) ·· Third trimester 5 (5%) 14 (8%) ·· PRNT 50 result ·· ·· 0·051 Zika virus-positive 62 (70%) 99 (57%) ·· Zika virus-negative 27 (30%) 74 (43%) ·· Testing not done 2 0 ·· Neonates Sex ·· ·· <0·0001 Girls 61 (67%) 84 (49%) ·· Boys 29 (32%) 89 (51%) ·· Intersex 1 (1%) 0 ·· Gestational age ·· ·· <0·0001 Term (≥37 weeks) 66 (73%) 153 (88%) ·· Premature (≤36 weeks) 25 (27%) 20 (12%) ·· Birthweight, g ·· ·· <0·0001 ≥2500 21 (23%) 159 (92%) ·· 1500–2499 52 (57%) 14 (8%) ·· <1500 18 (20%) 0 ·· Weight for gestational age ·· ·· < 0·0001 Normal 14 (17%) 165 (95%) ·· Small for gestational age 69 (83%) 8 (5%) ·· Not available * * Not available in eight stillbirths. 8 0 ·· Data are n (%). PRNT 50 =plaque reduction neutralisation test. Cases were more likely to be female, small for gestational age, and premature than controls ( table 1 ). 26 (29%) of 91 cases had severe microcephaly. There were no significant differences in the age or number of years that the mothers of the cases and controls had spent in education. Mothers of cases were slightly more likely to have serological markers of previous Zika virus infection (judged by PRNT) than mothers of controls, with a borderline p value (p=0·051). All mothers of cases and controls tested negative for Zika virus by qRT-PCR testing.

50 -negative mothers of cases, six had a neonate who was seropositive for Zika virus IgM, and five others had a neonate with major cerebral abnormalities on CT; none of these neonates was stillborn. Table 2 Proportion of cases with laboratory confirmation of Zika virus infection qRT-PCR Zika virus-specific IgM Either test CSF 17/70 (24%) 10/70 (14%) 25/71 (35%) Serum 1/78 (1%) 9/79 (11%) 10/79 (13%) Tissue macerate (stillbirth) 7/9 (78%) ·· ·· Any specimen ·· ·· 32/91 (35%) Data are number of positive tests/total number of tests (%), assessed by qRT-PCR or ELISA for Zika-specific IgM. Cerebrospinal fluid, or serum, or both were not collected for nine stillbirths (in which tissue macerate was collected instead), two of the three neonatal deaths, and in 11 cases for other reasons. Approximately a third of cases were positive for Zika virus infection (32 [35%] of 91 cases); confirmation of congenital infection by qRT-PCR or anti-Zika virus IgM ELISA was more frequent in CSF than in serum samples, and more cases were confirmed to have a Zika virus-positive result by qRT-PCR than by capture-IgM ELISA ( table 2 ). There was good agreement between Zika virus IgM positivity in CSF and in serum (OR 0·94, 95% CI 0·82–1·00). Of 27 PRNT-negative mothers of cases, six had a neonate who was seropositive for Zika virus IgM, and five others had a neonate with major cerebral abnormalities on CT; none of these neonates was stillborn.

No neonate tested IgM positive for cytomegalovirus, toxoplasmosis, or rubella (data not shown). Of the nine stillbirths, seven were positive for Zika virus, and five had severe microcephaly. There were three neonatal deaths; all deaths occurred in the intensive care unit and CT scan imaging was not done for these neonates or the stillbirths. However, two of the neonates who died were positive for Zika virus and had severe microcephaly, and the other was negative for Zika virus (data not shown).

Cases with severe microcephaly had a higher usage of intensive or intermediate care units (15 [75%] of 20 livebirths) than did the cases with moderate microcephaly (32 [52%] of 62 livebirths). The proportion of neonates who were small for gestational age was high for cases with severe or moderate microcephaly. 69 (83%) of 83 cases with known birthweight were small for gestational age, compared with eight (5%) of 173 controls. Archaic reflexes did not differ between groups when examined by neonatologists (who assessed suction, Moro, Babkin, and neck tonic reflexes).

2 test comparing the frequency of abnormalities between neonates who were positive and negative for Zika virus). Among the 26 cases with severe microcephaly, 19 (73%) were Zika virus-positive and seven (37%) of these 19 cases had cerebral anomalies. Seven of these 19 cases who were Zika virus-positive did not have CT imaging done (five were stillborn and two died as neonates). Of the seven severe cases who were Zika virus-negative, three had cerebral anomalies and one did not have CT imaging done. In the moderate cases, 13 (20%) of 65 were Zika virus-positive and, of these cases, three (23%) of 13 had cerebral anomalies. Eight (15%) of the 52 moderate cases who were Zika virus-negative had cerebral anomalies. Four of these 52 moderate cases did not have CT imaging done, of which two cases were Zika virus-negative (one was stillborn, one died as a neonate) and two cases were Zika virus-positive and were stillborn. Table 3 Association of cerebral abnormalities with Zika virus infection in cases Positive for Zika virus infection (n=23) Negative for Zika virus infection (n=56) Total p value Abnormalities present 10 * * Five cases with calcification and ventriculomegaly; one case with calcification, ventriculomegaly, and malformation of cortical development; one case with ventriculomegaly; one case with calcification and malformation of cortical development; one case with malformation of cortical development; and one case with vascular abnormality. 11 † † One case with calcification; two cases with ventriculomegaly; one case with ventriculomegaly and vascular abnormality; one case with ventriculomegaly and malformation of cortical development; two cases with malformation of cortical development; and four cases with vascular abnormality. 21 (27%) 0·029 ‡ ‡ χ2=4·74; comparing frequency of abnormalities between cases who were positive and negative for Zika virus. Abnormalities absent 13 (57%) 45 (80%) 58 (73%) ·· Data are n (%). Laboratory tests for Zika virus and brain imaging for cerebral abnormalities were done for 79 cases. 21 (27%) of 79 cases had major cerebral anomalies on CT ( table 3 ). Ten (43%) of 23 cases that were positive for Zika virus had major cerebral abnormalities on CT, compared with 11 (20%) of the 56 cases who tested negative for Zika virus (p=0·029; χtest comparing the frequency of abnormalities between neonates who were positive and negative for Zika virus). Among the 26 cases with severe microcephaly, 19 (73%) were Zika virus-positive and seven (37%) of these 19 cases had cerebral anomalies. Seven of these 19 cases who were Zika virus-positive did not have CT imaging done (five were stillborn and two died as neonates). Of the seven severe cases who were Zika virus-negative, three had cerebral anomalies and one did not have CT imaging done. In the moderate cases, 13 (20%) of 65 were Zika virus-positive and, of these cases, three (23%) of 13 had cerebral anomalies. Eight (15%) of the 52 moderate cases who were Zika virus-negative had cerebral anomalies. Four of these 52 moderate cases did not have CT imaging done, of which two cases were Zika virus-negative (one was stillborn, one died as a neonate) and two cases were Zika virus-positive and were stillborn.

When we compared the anthropometric variables between case categories (positive or negative for Zika virus and for cerebral abnormalities) and against controls, the only difference in anthropometric variables was found between the controls and the four categories of cases (p<0·0001 for all comparisons with an analysis of variance; a Bonferroni post-hoc test identified the controls as different from the case categories). The case categories were not significantly different. Specifically, cases that were Zika-negative and showed no cerebral abnormalities were similar to the other case categories but significantly differed from the control groups.

Table 4 Association between microcephaly and investigated cofactors Case (n=91) * * Data are n (%), unless otherwise indicated. Vaccination status was unknown for measles and rubella and measles, mumps, and rubella in 19 cases and 37 controls, and for tetanus, diphtheria, and acellular pertussis in 12 cases and 21 controls. Control (n=173) * * Data are n (%), unless otherwise indicated. Vaccination status was unknown for measles and rubella and measles, mumps, and rubella in 19 cases and 37 controls, and for tetanus, diphtheria, and acellular pertussis in 12 cases and 21 controls. Odds ratio (95% CI) p value Mother is not white 84 (92%) 141 (82%) 3·5 (1·3–9·5) 0·013 Family per-capita income, US$ ≤56·0 24 (26%) 43 (25%) 1·0 (Ref) 56·0–96·9 22 (24%) 39 (23%) 1·0 (0·5–2·1) 0·97 97·0–168·6 19 (21%) 43 (25%) 0·8 (0·4–1·7) 0·63 >168·6 19 (21%) 44 (25%) 0·8 (0·4–1·6) 0·47 Unknown 7 (8%) 4 (2%) ·· ·· Economic class (ABEP) 18 Associação Brasileira de Empresas de Pesquisa (ABEP)

Critério Brasil. D–E 52 (57%) 83 (48%) 1·0 (Ref) C2 28 (31%) 60 (35%) 0·7 (0·4–1·3) 0·26 B2–C1 11 (12%) 30 (17%) 0·6 (0·3–1·3) 0·17 Siblings with malformation (including microcephaly) No 53 (96%) 89 (98%) 1·0 (Ref) Yes 2 (4%) 2 (2%) 1·7 (0·2–16·4) 0·62 Had no siblings 36 82 ·· ·· Familial history of microcephaly or other malformation 26 (29%) 49 (28%) 1·0 (0·6–1·8) 0·96 Maternal use of folic acid in pregnancy (self-reported) Yes, regularly 56 (63%) 120 (69%) 1·0 (Ref) Yes, occasionally 15 (17%) 18 (10%) 1·7 (0·8–3·4) 0·18 No 18 (20%) 35 (20%) 1·1 (0·6–2·1) 0·79 Unknown 2 0 ·· ·· Maternal use of medication for epilepsy (self-reported) 4 (4%) 9 (5%) 0·8 (0·3–2·5) 0·77 Vaccinated Tetanus, diphtheria, and acellular pertussis 45 (57%) 107 (70%) 0·6 (0·3–1·0) 0·058 Measles and rubella 3 (4%) 6 (4%) 0·9 (0·2–3·3) 0·90 Measles, mumps, and rubella 3 (4%) 5 (4%) 1·1 (0·3–5·0) 0·88 Maternal risk behaviours during pregnancy Smoking 18 (20%) 12 (7%) 3·2 (1·5–7·0) 0·004 Drinking alcohol 16 (18%) 22 (13%) 1·6 (0·8–3·3) 0·21 Recreational drugs 3 (3%) 1 (1%) 5·2 (0·5–50·3) 0·16 Exposure to other substances Larvicides at the water storage site 49 (54%) 92 (53%) 1·0 (0·6–1·8) 0·89 Larvicides elsewhere in the house 14 (15%) 22 (13%) 1·3 (0·6–2·8) 0·45 Daily use of insect repellent on the body 9 (10%) 13 (8%) 0·9 (0·4–2·0) 0·83 Occupational exposure to pesticides 4 (4%) 4 (2%) 1·9 (0·5–7·7) 0·39 Most mothers of cases and controls lived in poverty; around half were classified in the two lowest levels of the socioeconomic scale ( table 4 ). Only two of the 18 investigated factors (other than Zika virus infection) were associated with microcephaly (p<0·05) in the conditional analysis: smoking (OR 3·2, 95% CI 1·5–7·0; p=0·004) and having skin colour that was not white (3·5, 1·3–9·5; p=0·013). The association between microcephaly and Tdap vaccination in pregnancy was at borderline level (0·6 [0·3–1·0]; p=0·06). Only two mothers of controls and no mothers of cases reported having taken misoprostol during pregnancy. There was no increase in the risk of microcephaly with the measles, mumps, and rubella or measles and rubella vaccines.

Table 5 Association between microcephaly and Zika virus infection Cases * * Data are the number of all cases or controls who were positive for Zika virus, assessed by qRT-PCR or Zika virus-specific IgM/total number of patients (%). Controls * * Data are the number of all cases or controls who were positive for Zika virus, assessed by qRT-PCR or Zika virus-specific IgM/total number of patients (%). Matched odds ratio (95% CI) Serum, CSF samples, or macerated tissue Zika-positive, of total cases or controls 32/91 (35%) 0/173 87·0 (15·6–∞) Zika-positive, of total cases or controls, adjusted † † Odds ratio when adjusted by smoking during pregnancy, maternal vaccination against tetanus, diphtheria, and acellular pertussis during pregnancy, and skin colour. ·· ·· 73·1 (13·0–∞) Cases, categorised by severity of microcephaly ‡ ‡ 10 Brasil P

Pereira JP

Moreira ME

et al. Zika virus infection in pregnant women in Rio de Janeiro. , 14 Fenton TR

Kim J A systematic review and meta-analysis to revise the Fenton growth chart for preterm infants. Severe is defined as a head circumference of more than 3 SD smaller than the mean for their sex and gestational age. Severe 19/26 (73%) 0/51 52·4 (9·1–∞) Not severe 13/65 (20%) 0/122 33·7 (5·6–∞) The matched association between microcephaly and Zika virus infection was extremely strong (mOR 87·0, 95% CI 15·6–∞); no controls had laboratory-confirmed Zika virus infection ( table 5 ). The association remained strong (73·1, 13·0–∞) and significant when adjusted by confounders (smoking during pregnancy, skin colour, and receiving Tdap during pregnancy). When controlling for laboratory confirmation of Zika, the association between microcephaly and smoking, having a skin colour that was not white, and having received Tdap vaccine lost significance (p values between 0·07 and 0·10). By subgroups, these associations were mOR 52·4 for severe cases and mOR 33·7 for less severe cases.

We further investigated the association of self-reported smoking and skin colour with economic class. Smoking during pregnancy was more common among the poorest classes in both cases and controls: one (2%) of 41 women in B2–C1, three (3%) of 88 women in C2, and 26 (19%) of 135 women in D–E reported smoking during pregnancy (χ2=17·3; p=0·0002). The proportion of mothers who reported smoking during pregnancy in the D–E category was higher for cases (15 [29%] of 52) than for controls (11 [13%] of 83; p=0·044). Skin colour was not associated with economic class (p=0·51). We also explored the association of small for gestational age and mothers' reported smoking in pregnancy. Among all 30 neonates whose mothers smoked, 17 (57%) were small for gestational age, compared with 63 (27%) of 234 neonates born from mothers who did not smoke. However, among the small for gestational age cases, only 15 (22%) of 69 had a mother who smoked.

In our study, smoking was a potential confounder for the association between congenital Zika virus infection and microcephaly, since smoking was associated with Zika virus congenital infection (p=0·046) and microcephaly (p<0·004). The association between congenital Zika virus infection and microcephaly remained when adjusted for smoking.