Zika Virus Infection in Pregnant Women in Rio de Janeiro

BACKGROUND

Zika virus (ZIKV) has been linked to central nervous system malformations in fetuses. To characterize the spectrum of ZIKV disease in pregnant women and infants, we followed patients in Rio de Janeiro to describe clinical manifestations in mothers and repercussions of acute ZIKV infection in infants.

METHODS

We enrolled pregnant women in whom a rash had developed within the previous 5 days and tested blood and urine specimens for ZIKV by reverse-transcriptase–polymerase-chain-reaction assays. We followed women prospectively to obtain data on pregnancy and infant outcomes.

RESULTS

A total of 345 women were enrolled from September 2015 through May 2016; of these, 182 women (53%) tested positive for ZIKV in blood, urine, or both. The timing of acute ZIKV infection ranged from 6 to 39 weeks of gestation. Predominant maternal clinical features included a pruritic descending macular or maculopapular rash, arthralgias, conjunctival injection, and headache; 27% had fever (short-term and low-grade). By July 2016, a total of 134 ZIKV-affected pregnancies and 73 ZIKV-unaffected pregnancies had reached completion, with outcomes known for 125 ZIKV-affected and 61 ZIKV-unaffected pregnancies. Infection with chikungunya virus was identified in 42% of women without ZIKV infection versus 3% of women with ZIKV infection (P<0.001). Rates of fetal death were 7% in both groups; overall adverse outcomes were 46% among offspring of ZIKV-positive women versus 11.5% among offspring of ZIKV-negative women (P<0.001). Among 117 live infants born to 116 ZIKV-positive women, 42% were found to have grossly abnormal clinical or brain imaging findings or both, including 4 infants with microcephaly. Adverse outcomes were noted regardless of the trimester during which the women were infected with ZIKV (55% of pregnancies had adverse outcomes after maternal infection in the first trimester, 52% after infection in the second trimester, and 29% after infection in the third trimester).

CONCLUSIONS

Despite mild clinical symptoms in the mother, ZIKV infection during pregnancy is deleterious to the fetus and is associated with fetal death, fetal growth restriction, and a spectrum of central nervous system abnormalities. (Funded by Ministério da Saúde do Brasil and others.)

STUDY POPULATION

In this cohort study, pregnant women at any week of gestation who presented to the acute febrile illness clinic at the Oswaldo Cruz Foundation with a rash that had developed within the previous 5 days were offered enrollment and were included in the study after they had provided written informed consent. After the women were enrolled, detailed demographic, medical, and prenatal history information, as well as clinical findings, were entered into case-report forms. Laboratory data on rubella, cytomegalovirus, and Venereal Disease Research Laboratory (VDRL) serologic testing were abstracted from prenatal medical records and entered in case-report forms. Serum and urine specimens were obtained at study entry. Weekly follow-up was conducted by telephone, and a second visit was scheduled within 30 days after enrollment for clinical and laboratory follow-up. Women were referred for fetal ultrasonography before 20 weeks of gestation, between 20 and 30 weeks of gestation, and after 30 weeks of gestation. No women had had a diagnosis of fetal malformations in the current pregnancy before enrollment. The study population was generally healthy; women reported no coexisting conditions or medication use. Infants born to ZIKV-positive mothers are being followed prospectively.

STUDY OVERSIGHT

The study protocol was approved by the institutional review boards at Fundação Oswaldo Cruz (Fiocruz) and the University of California, Los Angeles. The authors vouch for the accuracy and completeness of the data and the analyses and for the fidelity of the study to the protocol.

LABORATORY TESTING

Real-time reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays for ZIKV were performed with the QuantiTect Probe RT-PCR kit (Qiagen), as described previously,⁹ with the same primers and cycle times, at the Fiocruz Flavivirus Laboratory; assays were performed on blood specimens, urine specimens, or both that were obtained at the entry visit. The Fiocruz Flavivirus Laboratory is a reference laboratory for flavivirus infections in the region. Serologic testing for IgG antibodies to dengue (Abcam) and IgM antibodies to chikungunya (Euroimmun) were performed on serum specimens obtained at the entry visit. Patient specimens were also tested by RT-PCR for dengue¹⁰ and chikungunya.⁹ Patients were tested for parvovirus B19 and cytomegalovirus by PCR (TaqMan RT PCR, Applied Biosystems) and by serologic testing during regular prenatal care. PCR assays for human immunodeficiency virus (HIV) DNA (Abbott RT HIV Viral Load) were performed on all patient specimens. Screening tests for syphilis were performed during prenatal care with the use of VDRL assays, with treponemal assays (Alere Determine Syphilis) used as confirmatory tests.

FETAL ULTRASONOGRAPHY

All abdominal scanning was performed with a 4-to-8-mHz probe (Voluson 730 Expert/Voluson E6, GE) by perinatologists who were certified by the Brazilian College of Radiology and the Brazilian Federation of Societies of Gynecology and Obstetrics (Febrasgo). The variables that were measured are listed in the Supplementary Appendix, available at NEJM.org. For Doppler studies, the pulsatility index of the umbilical artery and of the middle cerebral artery were used.¹¹ Abnormalities such as cerebral calcifications and microcephaly were noted. Measured fetal ultrasonographic variables were plotted by gestational age according to the nomograms published on www.perinatology.com. Fetal growth restriction was defined as fetal weight estimated according to the Hadlock formula that was below the 10th percentile.¹² Microcephaly in fetal imaging was defined as fetal head measurements (e.g., head circumference) that were two standard deviations below the mean expected at a particular gestational age or below the 3rd percentile.¹³

INFANT CLINICAL ASSESSMENTS

Anthropometric measures at birth (weight, length, and head circumference) were obtained in all live-born infants. History taking and physical examinations were performed for all infants by pediatric specialists. ZIKV-exposed infants were evaluated by a multidisciplinary team that included neonatologists, neurologists, infectious disease specialists, geneticists, ophthalmologists, and physical therapists. All abnormal diagnoses were reviewed by a panel of specialists for confirmation. Microcephaly was defined as a head-circumference z score of less than −2 (moderate) or less than −3 (severe). Small-for-gestational-age infants were defined as infants with body-weight z scores of less than −1.28 at birth.¹⁴

INFANT IMAGING STUDIES

Brain imaging studies were offered for infants born to mothers who had positive PCR results for ZIKV. Transfontanel ultrasonography, computed tomography (CT) of the head with and without contrast, and magnetic resonance imaging (MRI; AERA Siemens, 1.5 tesla) with or without contrast were performed according to the standard of care. If abnormalities were suspected on transfontanel ultrasonography, patients were referred for CT or MRI brain imaging. All infant assessments were conducted with knowledge of ZIKV infection status.

STATISTICAL ANALYSIS

We compared the demographic and clinical variables of pregnant women who were positive for ZIKV on PCR with those who were negative for ZIKV on PCR, using Fisher’s exact test (two-sided); P values of 0.05 or less were considered to indicate statistical significance. We performed similar analyses comparing pregnancy and infant outcomes in ZIKV-infected and ZIKV-uninfected mother–infant pairs. For comparison of medians, an independent-samples median test was used. Comparisons of negative pregnancy outcomes according to maternal trimester of infection between ZIKV-infected and ZIKV-uninfected mother–infant pairs were compared with the use of McNemar’s test.

CHARACTERISTICS OF THE PARTICIPANTS

During the period from September 2015 through May 2016, we enrolled 345 pregnant women and tested blood specimens, urine specimens, or both for ZIKV by qualitative RT-PCR. Of these 345 women, 182 (53%) had positive results for ZIKV on PCR in blood, urine, or both. The current report focuses on 134 ZIKV-positive women and 73 ZIKV-negative women who were expected to deliver by July 31, 2016 (Fig. 1). Among the 134 ZIKV-positive women, quantitative ZIKV PCR results were available for 130 (4 PCR assays were performed at outside facilities). Among women with quantitative PCR results, 85 had positive PCR results in serum specimens, 76 had positive PCR results in urine, and 31 had positive PCR results in both specimens; 45 women had positive results in urine only and 54 had positive results in blood specimens only (median number of PCR cycles for serum specimens, 32.0; inter-quartile range, 30.0 to 34.0; range, 24.2 to 37.0; and median number of PCR cycles for urine specimens, 30.0; interquartile range, 27.0 to 33.0; range, 22.0 to 37.0). Demographic and clinical characteristics are described in Table 1. Among ZIKV-positive women, 38% reported similar illnesses in other family members, and only 14% reported that their partner had been ill. ZIKV infection was present in women of all socioeconomic strata. More than half the women presented with acute infection in the second trimester of pregnancy. ZIKV-negative women were more likely to have used insect repellent than ZIKV-positive women (83% vs. 60%, P = 0.006), but otherwise there were no significant differences in demographic characteristics or medical history between the two groups. ZIKV-positive women resided across multiple neighborhoods and municipalities within the larger metropolitan Rio de Janeiro area (Fig. S1 in the Supplementary Appendix).

CLINICAL PRESENTATION OF THE MOTHERS

All pregnant women had rash as part of their clinical presentation, since rash was an inclusion criterion. A descending macular or maculopapular rash was the most common type of exanthem noted in ZIKV-positive women (Fig. S5 in the Supplementary Appendix). The maculopapular rash was seen far more frequently in ZIKV-positive women than in ZIKV-negative women (P = 0.02). The other prevalent finding was pruritus, which was seen in 90% of ZIKV-positive women in our study. The next most common finding was arthralgia, which was reported in 62% of ZIKV-positive women and in 70% of ZIKV-negative women (P = 0.29). Conjunctival injection was present in 58% of ZIKV-positive women and in a smaller percentage (40%) of ZIKV-negative women (P = 0.03). Fatigue or malaise was described in 52% of ZIKV-positive women versus 75% of ZIKV-negative women (P = 0.002), and myalgia was described in 41% versus 62% (P = 0.005); the higher rates among ZIKV-negative women were probably due to the diagnosis of underlying chikungunya virus in some ZIKV-negative women. Lymphadenopathy (isolated or generalized) was present in both groups (in 38% of ZIKV-positive women and 27% of ZIKV-negative women, P = 0.12). Fever was not a highly prominent finding, occurring in less than a third of the women with acute ZIKV infection but in 58% of ZIKV-negative women (P<0.001). When fever was present, it was generally short-term and low grade (37.5 to 38.0°C). Nausea or vomiting was reported in 31% of ZIKV-positive women and was more common (occurring in 44%) among ZIKV-negative women (P=0.07).

OUTCOMES OF PREGNANCIES

Among 134 women who had positive results for ZIKV on PCR testing, information on confirmed outcomes of pregnancy was available for 125, with 117 live births in 116 pregnancies (one set of twins) between January 1 and July 31, 2016. There were 9 cases of fetal death (Fig. 1 and Table 2, and Table S2 in the Supplementary Appendix): 5 miscarriages in the first trimester of pregnancy, 2 miscarriages in the second trimester, and 2 stillbirths in the third trimester. Among 73 ZIKV-negative women, 61 had known outcomes, including 4 cases of fetal death and 57 live births. In the ZIKV-positive group, 93% of the women remained in the study, whereas retention in the ZIKV-negative group was 84% (P = 0.03).

Three of seven negative pregnancy outcomes in the ZIKV-negative group — two of the four fetal losses and one small-for-gestational-age infant — occurred in women with chikungunya virus. One of the first-trimester miscarriages in the ZIKV-positive cohort occurred in a woman who was coinfected with ZIKV and chikungunya virus. Three ZIKV-infected patients (2.8%) were coinfected with chikungunya virus; however, chikungunya infection was more prevalent in the ZIKV-negative group (42%, P<0.001) (Table 2). No patients in either group were found to have active dengue infection on PCR; however, prior dengue infection was very common, with 88% in the ZIKV-positive group and 86% in the ZIKV-negative group having IgG antibodies to dengue. One woman in each group had a positive treponemal syphilis test; those pregnancies did not have adverse outcomes. No women had evidence of active cytomegalovirus infection by either PCR or IgM detection. Among ZIKV-negative women, 29 other infections were identified, including 23 cases of chikungunya, 4 cases of parvovirus B19, and 1 case of syphilis. None of the women were found to have HIV by RT-PCR testing.

Among 125 pregnancies in ZIKV-positive women, 58 adverse pregnancy outcomes were noted (46.4%); in contrast, 7 of the 61 pregnancies (11.5%) in the ZIKV-negative cohort resulted in adverse outcomes (P<0.001). Despite the high rate of adverse outcomes in our control group of pregnant women with other infectious illnesses, the findings in the ZIKV-positive group were far more striking. Adverse pregnancy outcomes by the timing of maternal ZIKV infection are shown in Figure 2. The timing of maternal ZIKV infection ranged from 6 to 39 weeks of gestation. Adverse outcomes after ZIKV infection occurred regardless of the timing of maternal infection; adverse outcomes occurred in 55% of pregnancies in which the mother was infected in the first trimester (11 of 20 ZIKV-infected pregnancies), in 52% of those in which the mother was infected in the second trimester (37 of 71 ZIKV-infected pregnancies), and in 29% of those in which the mother was infected in the last trimester of pregnancy (10 of 34 ZIKV-infected pregnancies). Among ZIKV-infected pregnancies, there were 5 miscarriages (25% of the 20 pregnancies with first-trimester infection), 2 fetal losses (3% of the 71 pregnancies with second-trimester infection); and 2 stillbirths (6% of the 34 pregnancies with third-trimester infection). Among 117 live births in the ZIKV-positive cohort, 49 infants (42%) were found to have abnormalities on clinical examination, imaging, or both; in contrast, among 57 live births in the ZIKV-negative cohort, 3 infants (5%) had such abnormalities (P<0.001).

Three infants with abnormalities who were born to ZIKV-negative women were small for gestational age; one was born to a woman who was found to have chikungunya virus infection. Because of the large number of chikungunya infections in the control group, adverse pregnancy outcomes were also frequent in this group. There was no significant difference in the rate of fetal loss between ZIKV-positive mothers and ZIKV-negative mothers (7.2% and 6.6%, respectively; P = 1.0). Obstetrical complications were very high in both groups: 39% in ZIKV-positive women and 35% in ZIKV-negative women (P=0.62). ZIKV-positive women, however, were nearly 10 times as likely as ZIKV-negative women to have emergency cesarean sections performed owing to fetal distress (23.5% vs. 2.5%, P = 0.003) (Table 2). Infants born to ZIKV-positive mothers were also nearly 4 times as likely to need critical care assistance immediately after birth (a finding that is reflective of fetal distress) as infants who had not been exposed to ZIKV (21% vs. 6%, P = 0.01).

A total of 153 ultrasound studies were performed in 59 women who were found to be positive for ZIKV during pregnancy; the remaining 75 ZIKV-positive women declined imaging studies either because the obstetrical facility was too far away or because of fear of possible fetal abnormalities related to ZIKV infection. Detailed ultrasonographic findings are described in Table S1 in the Supplementary Appendix. ZIKV-negative women underwent fetal ultrasonography as part of regular prenatal care. All the women in the cohort received prenatal care. Nine women with ZIKV infection (6.7%) were lost to follow-up (i.e., did not seek prenatal care at our obstetrical facility and were unable to be reached for further evaluation despite multiple attempts; pregnancy outcomes are unknown). Included among these 9 women was 1 woman whose fetus had severe abnormalities on fetal ultrasonography. In the ZIKV-negative cohort, 12 women (16%) were lost to follow-up, mainly because of a change in their contact information. ZIKV-negative women did not deliver at our obstetrical facility. In addition, women were less likely to continue follow-up in the study if ZIKV infection was ruled out.

INFANT OUTCOMES

Infants who were small for gestational age, as a potential consequence of fetal growth restriction or poor placental perfusion, constituted 9% of ZIKV-exposed babies and 5.3% of babies in the control group (P = 0.06). Four infants in the ZIKV-positive group (3.4%) were noted to have microcephaly at birth; two were small-for-gestational-age infants with proportionate microcephaly (i.e., the head size is small but is proportional to the weight and length of the infant), and two had disproportionate microcephaly (i.e., the head size is small relative to the weight and length of the infant). None of the infants in the control group had microcephaly. Fetal growth variables for fetuses of ZIKV-positive women who had ultrasonography performed during pregnancy are shown in Figure 3, and anthropometric measurements, including birth weight and head circumference, of live-born infants in both groups are shown in Figure 4 and Table 3.

A total of 49 of the 117 live-born infants (42%) who had been exposed to ZIKV in utero had abnormal findings in the first month of life (Table S2 in the Supplementary Appendix). Almost all the abnormalities affected the central nervous system (CNS). Microcephaly was observed in infants whose mothers were infected in weeks 8, 12, 30, and 38 of gestation. Disproportionate microcephaly was seen only in infants infected in the first trimester of pregnancy; 2 infants infected in later trimesters had proportionate microcephaly and were small for gestational age. One of the infants with disproportionate microcephaly was also small for gestational age. Cerebral calcifications, cerebral atrophy, ventricular enlargement, and hypoplasia of cerebral structures were seen in multiple infants, with cerebral calcifications seen in infants infected as late as 34 weeks of gestation. Parenchymal brain hemorrhages were seen in some infants, including one whose mother was infected shortly before delivery at 39 weeks of gestation. A total of 31 of the 49 infants (63%) had grossly abnormal results on neurologic examinations; hypertonicity, clonus, hyperreflexia, abnormal movements, spasticity, contractures, and seizures were identified. Abnormal funduscopic examinations and abnormal hearing assessments were also noted.

Other features that were identified included persistence of the cortical thumb sign, with maintained clenched fists beyond 3 months of age, which reflects CNS disease; foveas in the knees or elbows due to limb contractures in utero; and redundant scalp skin in infants with normal head circumference. MRIs have not been performed in all infants to date. Conversely, a number of infants with normal clinical assessments in early infancy had abnormal nonspecific MRI findings; a common description was “excessive hypersignaling in T₂ in the white matter, diffuse in the peritrigonal posterior areas and less evident in the frontal parietal white matter with hyposignaling in the diffusion sequence.” These findings are abnormal and may reflect cortical tract dysfunction; nevertheless, close follow-up will be needed to ascertain the degree of CNS involvement.

Two infants were born large for gestational age owing to maternal gestational diabetes; one of these infants, born to a mother who had been infected at 15 weeks of pregnancy, was found to have congenital heart disease, which probably was associated with maternal gestational diabetes, although we cannot exclude the possibility of a potential association with ZIKV infection. Follow-up of ZIKV-exposed infants is ongoing.

Supplement1

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Supplementary Materials