Zika virus (ZKV) is an arbovirus that has rapidly spread in recent years with significant impact in the Americas. ZKV has gained considerable public health relevance being the first infective agent linked to human congenital defects discovered in the last half-century.1 A growing body of evidence has described vertical transmission resulting in neurologic, musculoskeletal, ophthalmic and auditory consequences in the unborn child,2–4 producing the so-called congenital Zika syndrome.5,6 Despite thorough reports on the consequences of this congenital infection,7,8 the full spectrum of clinical manifestations is still an ongoing field of study.
With approximately 100,000 cases reported, Colombia was the second most-affected South American country during the epidemic peak of 2015–2017, surpassed only by Brazil.9 Over 19,000 of the cases meeting the clinical criteria for ZKV occurred in pregnant women. The department of Valle del Cauca in southwestern Colombia reported 3166 pregnant women with symptoms suggesting gestational ZKV, the most in any Colombian department.9,10
Major evaluations of the clinical outcomes of perinatal exposure were made using government records with limited description of signs that are only evident upon a detailed physical examination. Furthermore, many of these reports are based on newborn infants affected by congenital Zika syndrome, who are highly symptomatic,11,12 and subtler manifestations have not been described in detail. A detailed description of signs and symptoms in infants exposed to ZKV during pregnancy will allow us to expand our current knowledge for diagnostic, therapeutic and prognostic purposes. The aim of our study was to describe the adverse clinical outcomes for fetuses, neonates and infants exposed to confirmed ZKV infection in different trimesters of gestational age during the recent epidemic peak in Valle del Cauca, Colombia.
Study Design and Participants
We carried out a prospective observational study of fetuses, neonates and infants of mothers who developed symptomatic ZKV infection during pregnancy in Valle del Cauca during the epidemic peak between November, 2015 and January, 2017. The department of Valle del Cauca has almost 4,500,000 inhabitants with nearly 2,300,000 inhabitants in the capital, Cali, an additional 565,000 in adjacent municipalities and 1,635,000 in distant municipalities.13
The protocol was approved by the Independent Ethics Committee of Corporación Científica Pediátrica, and conducted in accordance with Good Clinical Practices and Declaration of Helsinki guidelines. Each participant’s parent(s) or legal guardian(s) provided written informed consent.
During the epidemic peak, all pregnant women with symptoms suggestive of ZKV infection consulting any primary care center, hospital or clinic in the state had blood drawn for ZKV real-time polymerase chain reaction (RT-PCR), which was a singleplex PCR performed at the Public Health Department according to a previously described protocol.14 Demographic data of all pregnant women with positive RT-PCR were recorded in an official database for public health use. From July, 2016, we contacted all pregnant women with positive RT-PCR results to invite them to participate in the study.
The study sample was made up of pregnancy losses (for which cases a detailed telephone survey was conducted), and those exposed neonatal or infant patients who attended a first postnatal assessment. Information on pregnancy, delivery and the postnatal development of the patient were obtained. Complete physical examinations and evaluations by a pediatric neurologist using the Hammersmith test (HST) were performed, as well as ophthalmologic and auditory evaluations. To obtain reliable measurements, a single investigator obtained head circumference (HC) measurements according to World Health Organization standardized protocols.15 No samples were obtained from patients.
Patients were monitored prospectively until 19 months of age, when they received a neuropsychologic evaluation to determine a Bayley score in an ongoing analysis. Here, we present their clinical characteristics upon the first postnatal assessment.
The HST is a scorable, standardized and well-studied neurologic examination in healthy or high-risk infants.16 It consists of 2 evaluations; one neonatal intended for patients under 2 months of age assesses 6 items: posture and tone; tone patterns; reflexes; type and quality of movements; auditory and visual orientation and the presence of abnormal neurologic signs. The second, used for patients over 2 months, was the infants scale that evaluates 7 items: functionality of cranial nerves; posture of various body parts; quality and quantity of movements; global tone; reflexes; and development and behavior milestones. According to the score obtained, each item can be classified as adequate or inadequate for the age of a patient.17 Neurocognitive dysfunction was considered mild when a patient scored 1–3 abnormal items on the HST, moderate with 4 or 5 abnormal items and severe with 6 or 7 abnormal items.
Ophthalmologic and Auditory Evaluation
Each patient received a complete ophthalmologic examination to assess visual acuity for age, ocular motility and alignment and evaluations of the anterior and posterior segments of each eyeball. Hearing evaluations were performed with brainstem auditory evoked potentials (GSI Audera; Grason-Stadler Eden Prairie, MN).
Primary outcomes were measured in children or fetuses whose mothers had ZKV during different trimesters of pregnancy. Outcomes were assessed during the first postnatal assessment or telephone interaction in the cases of pregnancy losses or neonatal deaths. Outcomes were classified as adverse outcomes possibly related to ZKV according to a recently described surveillance case definition,8 and included microcephaly, specific ocular abnormalities, severe neurocognitive dysfunction (6 or 7 abnormal items in the HST) without microcephaly and consequences of central nervous system (CNS) dysfunction such as severe neurosensory hearing loss, congenital bilateral hip dysplasia or clubfoot.7 Voluntary pregnancy interruption because of known ZKV-related fetal malformations or pregnancy losses when the chronology of events suggested a ZKV-related outcome was also considered possibly related.
Adverse events of uncertain relation to ZKV included intrauterine growth retardation (IUGR), craniofacial malformations, mild or moderate neurocognitive dysfunction (1–5 abnormal items in the HST), mild neurosensory hearing loss, ocular lesions not included in the above category, voluntary pregnancy interruption because of fetal malformations unlikely to be related to ZKV and early neonatal deaths without detectable CNS malformations.
Microcephaly was defined as HC more than 2 standard deviations (SDs) below the mean for the age, gender and gestational age of an individual.18 Neurodevelopmental risk was defined in patients with an HC between −2 SD and −1 SD, or with HC greater than +2 SD.18 IUGR was defined as an estimated fetal weight less than the 10th percentile.19
Quantitative variables were presented as means with SDs if the distribution was normal, or as medians and interquartile ranges when the distribution did not meet normality criteria. Frequencies of the various outcomes (adverse outcomes possibly related to ZKV, adverse events of uncertain relation to ZKV and no adverse outcomes) were compared using the χ2 test according to the trimester of pregnancy when the pregnant woman was exposed to the virus. We estimated the risk of adverse outcomes according to the trimester of pregnancy, and compared risks of adverse outcomes among mothers exposed during the first trimester versus the second or third trimesters through risk ratios and 95% CIs. Risk ratios were also assessed excluding pregnancy losses when the fetal characteristics were not known. Stata 13 software (StataCorp, College Station, TX) was used to perform the analysis.
We were able to contact 268 (42.9%) of the 625 pregnant women with positive ZKV RT-PCR in the public health database during the epidemic peak between November 2015 and January 2017. Of those contacted, we obtained information from 170 pregnant women, including one with a twin pregnancy. Figure 1 shows the flow of infected pregnant women who were contacted and enrolled in the study and their pregnancy outcomes. Of these 171 pregnancy outcomes, there were 17 pregnancy losses and 154 patients who attended the first postnatal assessment. Table 1 shows their demographic and perinatal data. Ninety of the 171 products of gestation (52.6%) had an adverse outcome. There were 58 (33.9%) adverse events of uncertain relation to ZKV, and 32 (18.7%) adverse outcomes possibly related to ZKV, including 16 pregnancy losses and 16 patients (Fig. 2).
Gestation at the Time of Zika Infection
Median gestational age at the time of infection was 23 weeks, most occurring during the second trimester (48.7%, n = 75). The predominant symptom was rash, and fewer than half the patients had fever. Prenatal ultrasounds performed in 139 (90.3%) of the 154 pregnant women found abnormalities in 20 (14.4%); IUGR (n = 9, 6.5%) and microcephaly (n = 5, 3.6%) were the predominant abnormalities (Table 2).
Of the 17 pregnancy losses, 16 were possibly related to ZKV infection; 10 miscarriages, 4 fetal deaths and 2 voluntary terminations. One mother who underwent a voluntary termination presented ZKV at week 18, with severe fetal spinal malformations detected at week 20. This outcome was classified as an adverse event of uncertain relation to ZKV.
Of the 10 miscarriages, 8 were infected with ZKV before week 12, and the others being infected between weeks 13 and 14. Pregnancy losses occurred at a median time of 2.6 weeks (interquartile range 1–6.3) after ZKV infection. Of the 4 pregnancies ending in fetal death, 3 developed ZKV in weeks 3, 18 and 29, with fetal deaths being diagnosed in weeks 21, 22 and 33, respectively. Fetal characteristics are not known for any of these cases. One mother with a twin pregnancy was infected at week 8; fetal death was diagnosed for 1 fetus in week 24. Expectant management was performed, without morbidity on the surviving fetus who was born via C-section in week 38. The dead fetus had a cystic hygroma, omphalocele and anencephaly.
Two voluntary pregnancy interruptions possibly related to ZKV occurred: one had ZKV infection at week 11 of pregnancy, and severe microcephaly and cerebral calcifications were detected by ultrasound at week 28. The second was infected at week 9, with unspecified severe malformations, including CNS abnormalities, detected in week 19.
Data from First Evaluations
Mean age at the first evaluation of the 154 patients was 7.6 months (SD = 4.3). Seventy-three (47.4%) presented adverse outcomes, including 16 (10.4%) with adverse outcomes possibly related to ZKV and 57 (37%) with adverse events of uncertain relation to ZKV.
Of the 16 exposed patients with adverse outcomes possibly related to ZKV, 1 patient had all the abnormal items tested for by HST, without microcephaly, 2 had skeletal malformations (one of them with bilateral hip dysplasia and another with bilateral clubfoot, both without neurologic abnormalities), 4 had ophthalmologic conditions related to ZKV without any neurologic deficit, 2 had severe isolated hypoacusia in auditory evoked potentials without other neurologic deficits, and 7 (4.5%) had microcephaly, 6 with severe neurocognitive dysfunction and 1 with mild dysfunction. Five of the 7 children with microcephaly developed epilepsy.
Of the 57 patients with adverse outcomes of uncertain relation, 49 presented mild neurologic abnormalities (1–3 abnormal items in the HST), 4 presented isolated IUGR at birth without neurologic deficits, 2 with mild hypoacusia in evoked potentials without other abnormalities, 1 with isolated ophthalmologic lesions of uncertain relation and 1 with a complete unilateral fissure of the lip and the hard and soft palate, persistent ductus arteriosus and patent foramen ovale, without neurologic abnormalities.
Twelve patients were evaluated using the HST neonatal scale. Seven presented at least 1 abnormal HST item, including 6 with tone abnormalities. The remaining 142 patients were evaluated using the infant scale. Of these, 50 (35%) presented abnormalities, mainly a delay in the acquisition of motor milestones (n = 27, 19%), followed by cranial nerve abnormalities (n = 19, 13.3%) and reflex abnormalities (n = 18, 12.6%).
Nineteen infants had neurodevelopmental risk, 4 with an HC greater than +2 SD (macrocrania). Of these 19 infants, one presented abnormalities with respect to all the HST items, 9 presented 1–3 abnormal HST items and 9 presented no abnormalities upon receiving the neurologic examination.
Visual and Auditory Monitoring
Auditory evaluations performed on 68 patients (44.1%) found abnormalities in 6 (8.8%) of them. All these patients had normal neurologic examination and HST results.
Ophthalmologic evaluation conducted in 118 patients (76.6%) revealed abnormalities in 14 (11.8%). Of these, 7 had abnormalities possibly related to ZKV, including 4 without neurologic deficits (Table 3).
Adverse Outcomes According to Gestational Age
A higher percentage of women infected during the first trimester experienced adverse outcomes possibly related to ZKV compared with mothers infected during the second or third trimesters (Fig. 2 and Table 4). The risks of developing adverse outcomes possibly related to ZKV compared with no adverse events or adverse events of uncertain relation to ZKV were 3.1 (95% CI: 2.4–4.1) and 3.3 (95% CI: 2.5–4.2) times higher in those mothers exposed during the first trimester, compared with the second or third trimesters, respectively. After excluding pregnancy losses when the fetal characteristics were not known, risk ratios were 2.44 (95% CI: 2.0–3.0) and 2.2 (95% CI: 1.78–2.71).
We report adverse outcomes in 52.6% of a cohort of fetuses, neonates and infants exposed to gestational ZKV. Although some of these adverse outcomes were mild, of uncertain relation, or not conclusively associated with ZKV infection, the frequency of microcephaly in the first postnatal assessment was 4.5%, a 200-fold increase over previously known microcephaly rates in the region, and similar to those reported in studies in the United States, Brazil and The French territories in the Americas.7,20,21 Pregnancy loss was another frequently detected severe outcome in this cohort. When infection occurred in the first 2 trimesters, especially the first, 10% of the pregnancies were lost, representing a 53% increase over baseline rates of pregnancy losses in Colombia before the ZKV epidemic.22 Although it has yet to be demonstrated that ZKV infection increases the risk of pregnancy loss, there is evidence that symptomatic dengue infections during pregnancy increases the risk of fetal death by 1.8–1.9 times.23 Consequently, it is probable that symptomatic gestational ZKV may be associated with many of the losses observed in this cohort.
In total, 18.7% of pregnancies had an adverse outcome possibly related to ZKV, a higher rate than reported in the French territories, but lower than in Brazil.7,20 These differences may be related to the defined outcomes in each study, although differences in the impact of ZKV infection during pregnancy among different regions or countries have been described previously and the reasons for these discrepancies are not clear.24
Different studies have reported contradictory findings on the association between gestational age at the moment of infection and pregnancy outcomes,20,21 although most recent data suggest a greater risk of adverse outcomes with early infections.7,25 In this study, we found a strong association between infection during the first trimester and adverse outcomes possibly related with ZKV, compared with infection in the second or third trimesters. The high frequency of severe outcomes in early infections seems to correspond to the virus’ tropism in relation to the neuronal precursors and the early phases of cerebral differentiation,26,27 similar to other congenital infections such as cytomegalovirus.28 However, ZKV can also replicate in mature neurons,29 which would explain the high frequency of adverse outcomes described for infections occurring in the third trimester.
Although microcephaly has been the highest profile and most-studied finding, the impact of ZKV infection is wide ranging. Detection of patients with ophthalmic and auditory abnormalities possibly related with ZKV with normal neurologic exams or mild neurocognitive dysfunction confirms that ZKV has a broad spectrum of presentation. Infants exposed during pregnancy should receive close neurologic, ophthalmologic and audiologic monitoring, even in the absence of microcephaly. In addition, some manifestations may be less severe, but might still have great impact on the infant´s development. Our study used the HST, a brief neurologic examination designed to assess neurologic development at 12 months but may be used to examine neonates and infants,16 and we found abnormalities in 57 (37%) of the patients evaluated, including 52 with only 1–3 abnormal items (mild neurocognitive dysfunction). Finally, the prevalence of neurodevelopmental risk was 12.3% (n = 19), 95% of these patients displaying no or only mild abnormalities upon neurologic examination. The implications of these subtle abnormalities in the HST or HC are not clear, and only long-term monitoring will determine if these effects will persist or progress and what impact they will have on the development of the child
Of note, there were 7 patients with microcephaly identified at their first postnatal assessment, including 2 who were born with normal HC. This finding is in line with the possibility of active replication of the virus in more mature neural tissue and even in the postnatal stage,30,31 and emphasizes the need for close monitoring of children of mothers exposed to ZKV during pregnancy.
One of the main strengths of this study is the detailed description of the outcomes evidenced in children of mothers infected with ZKV during the different trimesters of pregnancy. During future epidemics, many pregnant women will develop symptomatic ZKV infections, and the description of different adverse events related with this infection will contribute to the growing amount of information available to guide informed decisions by health professionals.
The findings of our study, however, should be interpreted in the context of the following limitations. First, the absence of a control group prevents us from knowing with any certainty how high the risk of congenital abnormalities is in women infected during pregnancy. Second, the lack of confirmatory studies of infection in newborns, or other studies evaluating other congenital infections or genetic abnormalities, prevents us from establishing the exact cause of the adverse outcomes described in this cohort. In addition, only 170 pregnant women with ZKV participated in the study. Although this might have introduced selection bias if the participants were not representative of the 625 pregnant women who were registered in the public health database, there was no information in the nonparticipants to be able to minimize this limitation. Finally, our study was based on symptomatic pregnant women, so it is not possible to apply this information to populations during disease outbreaks as most women infected by ZKV during pregnancy will be asymptomatic.21
Although these limitations prevent us from confirming that the outcomes described are caused by gestational ZKV, the temporal relationship, the characteristics of the described adverse outcomes24 and the association between gestational age when the infection occurs with the frequency of adverse outcomes are biologically plausible and have been described previously.32,33 This suggests a possible association, and that ZKV infection is responsible for the majority of the adverse outcomes reported.
In conclusion, we found a high frequency of gestational and neonatal complications in mothers infected with ZKV any time during gestation, but especially in the first trimester. These complications present a broad spectrum of manifestations ranging from mild abnormalities and isolated ophthalmologic and auditory conditions to severe conditions that can be life threatening and affect multiple organs. The long-term consequences of this congenital infection are not currently known, especially in infants with a normal or mildly abnormal neurologic examination; prospective anthropometric and neurologic evaluation of this cohort is currently underway. Although ZKV is not in active circulation, large regions of the world are at risk with the possibility of outbreaks when a new cohort of susceptible subjects accumulates. In areas where the virus circulates, it is important to strengthen preventative strategies, such as avoiding mosquito bites in pregnant women, vector control and the development of safe and effective vaccines.
The authors would like to acknowledge Heidy Medina for her hard work during data collection and the families who participated in this study.
1. Petersen LR, Jamieson DJ, Powers AM, et al. Zika
virus. N Engl J Med. 2016;374:1552–1563.
2. de Souza AS, de Oliveira-Szjenfeld PS, de Oliveira Melo AS, et al. Imaging findings in congenital Zika
virus infection syndrome: an update. Childs Nerv Syst 2018;3485–93
3. Yepez JB, Murati FA, Pettito M, et al; Johns Hopkins Zika
Center. Ophthalmic manifestations of congenital zika
syndrome in Colombia and Venezuela. JAMA Ophthalmol. 2017;135:440–445.
4. Mittal R, Fifer RC, Liu XZ. A possible association between hearing loss and Zika
virus infections. JAMA Otolaryngol Neck Surg. 2017;33136:2013–2014.
5. Platt DJ, Miner JJ. Consequences of congenital Zika
virus infection. Curr Opin Virol. 2017;27:1–7.
7. Hoen B, Schaub B, Funk AL, et al. Pregnancy
outcomes after ZIKV infection in french territories in the Americas. N Engl J Med. 2018;378:985–994.
8. Rice ME, Galang RR, Roth NM, et al. Vital signs: Zika
-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital Zika
virus infection—U.S. territories and freely associated States, 2018. MMWR Morb Mortal Wkly Rep. 2018;67:858–867.
10. Rodriguez-Morales AJ, Galindo-Marquez ML, García-Loaiza CJ, et al. Mapping Zika
virus disease incidence in Valle del Cauca. Infection. 2017;45:93–102.
11. Del Campo M, Feitosa IM, Ribeiro EM, et al; Zika
Embryopathy Task Force-Brazilian Society of Medical Genetics ZETF-SBGM. The phenotypic spectrum of congenital Zika
syndrome. Am J Med Genet A. 2017;173:841–857.
12. França GV, Schuler-Faccini L, Oliveira WK, et al. Congenital Zika
virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation. Lancet. 2016;388:891–897.
14. Lanciotti RS, Kosoy OL, Laven JJ, et al. Genetic and serologic properties of Zika
Virus associated with an epidemic, Yap State, Micronesia 2007. Emerg Infect Dis. 2008;14:1232–1239.
15. de Onis M, Onyango AW, Van den Broeck J, et al. Measurement and standardization protocols for anthropometry used in the construction of a new international growth reference. Food Nutr Bull. 2004;25(Suppl 1):S27–S36.
16. Maitre NL, Chorna O, Romeo DM, et al. Implementation of the hammersmith infant neurological examination in a high-risk infant follow-up program. Pediatr Neurol. 2016;65:31–38.
17. Haataja L, Mercuri E, Regev R, et al. Optimality score for the neurologic
examination of the infant at 12 and 18 months of age. J Pediatr. 1999;135(2 pt 1):153–161.
19. Suhag A, Berghella V. Intrauterine Growth Restriction (IUGR): etiology and diagnosis. Curr Obstet Gynecol Rep. 2013;2:102–111.
20. Brasil P, Pereira JP Jr, Moreira ME, et al. Zika
virus infection in pregnant women in Rio de Janeiro. N Engl J Med. 2016;375:2321–2334.
21. Honein MA, Dawson AL, Petersen EE, et al; US Zika Pregnancy
Registry Collaboration. Birth defects among fetuses and infants of US women with evidence of possible Zika
virus infection during pregnancy
. JAMA. 2017;317:59–68.
23. Paixão ES, Costa MDCN, Teixeira MG, et al. Symptomatic dengue infection during pregnancy
and the risk of stillbirth in Brazil, 2006-12: a matched case-control study. Lancet Infect Dis. 2017;17:957–964.
24. de Oliveira WK, de França GVA, Carmo EH, et al. Infection-related microcephaly
after the 2015 and 2016 Zika
virus outbreaks in Brazil: a surveillance-based analysis. Lancet. 2017;390:861–870.
25. Shapiro-Mendoza CK, Rice ME, Galang RR, et al; Zika Pregnancy
and Infant Registries Working Group. Pregnancy
outcomes after maternal Zika
virus infection during pregnancy
—U.S. territories, January 1, 2016-April 25, 2017. MMWR Morb Mortal Wkly Rep. 2017;66:615–621.
26. Faizan MI, Abdullah M, Ali S, et al. Zika
and its possible molecular mechanism. Intervirology. 2016;59:152–158.
27. Tang H, Hammack C, Ogden SC, et al. Zika
virus infect human cortical neural precursors and attenuates their growth. Cell Stem Cell. 2016;18:587–590.
28. Boppana SB, Ross SA, Fowler KB. Congenital
cytomegalovirus infection: clinical outcome. Clin Infect Dis. 2013;57(Suppl 4):S178–S181.
29. Lanko K, Eggermont K, Patel A, et al. Replication of the Zika
virus in different iPSC-derived neuronal cells and implications to assess efficacy of antivirals. Antiviral Res. 2017;145:82–86.
30. van der Linden V, Pessoa A, Dobyns W, et al. Description of 13 infants born during October 2015-January 2016 with congenital Zika
virus infection without microcephaly
at birth—Brazil. MMWR Morb Mortal Wkly Rep. 2016;65:1343–1348.
31. Brito CAA, Henriques-Souza A, Soares CRP, et al. Persistent detection of Zika
virus RNA from an infant with severe microcephaly
—a case report. BMC Infect Dis. 2018;18:388.
32. Cauchemez S, Besnard M, Bompard P, et al. Association between Zika
virus and microcephaly
in French Polynesia, 2013-15: a retrospective study. Lancet. 2016;387:2125–2132.
33. Jaenisch T, Rosenberger KD, Brito C, et al. Risk of microcephaly
virus infection in Brazil, 2015 to 2016. Bull World Health Organ. 2017;95:191–198.