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Testing for Zika virus infection in pregnancy: key concepts to deal with an emerging epidemic

  • Author Footnotes
    1 These authors contributed equally to this article.
    Catherine Eppes
    Footnotes
    1 These authors contributed equally to this article.
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Author Footnotes
    1 These authors contributed equally to this article.
    Martha Rac
    Footnotes
    1 These authors contributed equally to this article.
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • James Dunn
    Affiliations
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX

    Department of Pathology and Immunology, Texas Children’s Hospital, Houston, TX
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  • James Versalovic
    Affiliations
    Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX

    National School for Tropical Medicine, Baylor College of Medicine, Houston, TX

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX

    Department of Pathology and Immunology, Texas Children’s Hospital, Houston, TX

    Department of Pediatrics, Texas Children’s Hospital, Houston, TX
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  • Kristy O. Murray
    Affiliations
    National School for Tropical Medicine, Baylor College of Medicine, Houston, TX

    Department of Pediatrics, Texas Children’s Hospital, Houston, TX
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  • Melissa A. Suter
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Magda Sanz Cortes
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Jimmy Espinoza
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Maxim D. Seferovic
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Wesley Lee
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Peter Hotez
    Affiliations
    National School for Tropical Medicine, Baylor College of Medicine, Houston, TX

    Department of Pediatrics, Texas Children’s Hospital, Houston, TX
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  • Joan Mastrobattista
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Steven L. Clark
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Michael A. Belfort
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    National School for Tropical Medicine, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Kjersti M. Aagaard
    Correspondence
    Corresponding author: Kjersti Aagaard, MD, PhD.
    Affiliations
    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Baylor College of Medicine, Houston, TX

    National School for Tropical Medicine, Baylor College of Medicine, Houston, TX

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX

    Departments of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Texas Children’s Hospital, Houston, TX
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  • Author Footnotes
    1 These authors contributed equally to this article.
Open AccessPublished:January 23, 2017DOI:https://doi.org/10.1016/j.ajog.2017.01.020
      Zika virus is an emerging mosquito-borne (Aedes genus) arbovirus of the Flaviviridae family. Following epidemics in Micronesia and French Polynesia during the past decade, more recent Zika virus infection outbreaks were first reported in South America as early as May 2013 and spread to now 50 countries throughout the Americas. Although no other flavivirus has previously been known to cause major fetal malformations following perinatal infection, reports of a causal link between Zika virus and microcephaly, brain and ocular malformations, and fetal loss emerged from hard-hit regions of Brazil by October 2015. Among the minority of infected women with symptoms, clinical manifestations of Zika virus infection may include fever, headache, arthralgia, myalgia, and maculopapular rash; however, only 1 of every 4–5 people who are infected have any symptoms. Thus, clinical symptom reporting is an ineffective screening tool for the relative risk assessment of Zika virus infection in the majority of patients. As previously occurred with other largely asymptomatic viral infections posing perinatal transmission risk (such as HIV or cytomegalovirus), we must develop and implement rapid, sensitive, and specific screening and diagnostic testing for both viral detection and estimation of timing of exposure. Unfortunately, despite an unprecedented surge in attempts to rapidly advance perinatal clinical testing for a previously obscure arbovirus, there are several ongoing hindrances to molecular- and sonographic-based screening and diagnosis of congenital Zika virus infection. These include the following: (1) difficulty in estimating the timing of exposure for women living in endemic areas and thus limited interpretability of immunoglobulin M serologies; (2) cross-reaction of immunoglobulin serologies with other endemic flaviruses, such as dengue; (3) persistent viremia and viruria in pregnancy weeks to months after primary exposure; and (4) fetal brain malformations and anomalies preceding the sonographic detection of microcephaly. In this commentary, we discuss screening and diagnostic considerations that are grounded not only in the realities of current obstetrical practice in a largely global population but also in basic immunology and virology. We review recent epidemiological data pertaining to the risk of congenital Zika virus malformations based on trimester of exposure and consider side by side with emerging data demonstrating replication of Zika virus in placental and fetal tissue throughout gestation. We discuss limitations to ultrasound based strategies that rely largely or solely on the detection of microcephaly and provide alternative neurosonographic approaches for the detection of malformations that may precede or occur independent of a small head circumference. This expert review provides information that is of value for the following: (1) obstetrician, maternal-fetal medicine specialist, midwife, patient, and family in cases of suspected Zika virus infection; (2) review of the methodology for laboratory testing to explore the presence of the virus and the immune response; (3) ultrasound-based assessment of the fetus suspected to be exposed to Zika virus with particular emphasis on the central nervous system; and (4) identification of areas ready for development.

      Key words

      Click Supplemental Materials under article title in Contents at ajog.org
      Human infection with Zika virus (ZIKV), an emerging mosquito-borne flavivirus (Flaviviridae family, Flavivirus genus), has reached pandemic levels in the Americas, with at least 50 countries or territories, including Puerto Rico, Florida, and Texas, reporting infection over the interval from May 2015 through November 2016 (Figure 1).

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      The recently confirmed causal link between ZIKV and fetal microcephaly now places ZIKV among the relatively small list of infections in pregnant women that lead to congenital anomalies. Coupled with the recent pandemic, this has led to unprecedented numbers of pregnant women at risk for having fetuses with severe abnormalities.
      Figure thumbnail gr1
      Figure 1Schematic map of the United States with reported endemic and nonendemic cases
      Schematic map of the United States and its territories with reported endemic and nonendemic cases (A, current as of December 2016) alongside pictorial images of the Aedes aegypti mosquito (B) and ZIKV particles (C). In image C, the TEM was produced in black and white, with postprocessing digital colorization. Shown in green are cytoplasmic and nuclear elements of host Vero E6 green monkey kidney epithelial cells inoculated with a purified and passaged 1947 Ugandan ZIKV strain. Shown in purple are 40 nm diameter flavivirus particles with a characteristic dense core and outer envelope. The TEM images of fixed cells were obtained after purified ZIKV was used to inoculate Vero E6 cells in vitro. These images were obtained from the CDC public domain image archives. Images are reproduced with the expressed permission of Dr Cynthia Goldsmith and the CDC.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      CDC, Centers for Disease Control and Prevention; TEM, transmission electron microscopy; ZIKV, Zika virus.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.

      Epidemiology and estimates of congenital ZIKV infection

      Previous outbreaks of ZIKV were largely sporadic across Southeast Asia and equatorial African belts but later spread east, resulting in an outbreak in Yap Island in 2007, followed by epidemics in French Polynesia, New Caledonia, the Cook Islands, and Easter Island in 2013 and 2014.
      • Faye O.
      • Freire C.C.
      • Iamarino A.
      • et al.
      Molecular evolution of Zika virus during its emergence in the 20th century.
      • Ioos S.
      • Mallet H.P.
      • Leparc Goffart I.
      • Gauthier V.
      • Cardoso T.
      • Herida M.
      Current Zika virus epidemiology and recent epidemics.
      Until recently ZIKV illness was thought to be self-limiting, resembling a mild version of dengue virus (DENV) or chikungunya virus. ZIKV is transmitted via Aedes spp of mosquitos and is spread via sexual transmission and vertical (mother to child) and blood transfusions (Figure 1).

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      Clinical manifestations of ZIKV infection include fever, headache, arthralgia, myalgia, and maculopapular rash, although only 1 of every 4–5 people who are infected manifest symptoms.
      • Faye O.
      • Freire C.C.
      • Iamarino A.
      • et al.
      Molecular evolution of Zika virus during its emergence in the 20th century.
      • Ioos S.
      • Mallet H.P.
      • Leparc Goffart I.
      • Gauthier V.
      • Cardoso T.
      • Herida M.
      Current Zika virus epidemiology and recent epidemics.
      • Besnard M.
      • Lastere S.
      • Teissier A.
      • Cao-Lormeau V.M.
      • Musso D.
      Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014.
      • Duffy M.R.
      • Chen T.H.
      • Hancock W.T.
      • et al.
      Zika virus outbreak on Yap Island, Federated States of Micronesia.
      Thus, clinical symptoms are not an effective screening tool for the diagnosis or relative risk of ZIKV infection because approximately 80% are likely asymptomatic.
      Based on the initial reports of relatively mild symptoms accompanying infection, ZIKV was not thought to lead to severe consequences. Exceptions included rare cases of Guillian-Barre (73 of 28,000 cases) and even rarer instances of perinatal transmission (2 initial cases, with potentially as great as 1% by later estimates) first reported during the French Polynesia outbreak.
      • Faye O.
      • Freire C.C.
      • Iamarino A.
      • et al.
      Molecular evolution of Zika virus during its emergence in the 20th century.
      • Besnard M.
      • Lastere S.
      • Teissier A.
      • Cao-Lormeau V.M.
      • Musso D.
      Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014.
      However, as ZIKV spread to the Americas in far higher volume (1.3 million autochthonous cases by December 2015), an approximate 20-fold increase in congenital cases of microcephaly with brain and ocular malformations was reported throughout northeast and southeast Brazil.
      European Centre for Disease Prevention and Control
      Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barré syndrome.
      Although no other flavivirus is known to cause disseminated fetal neural malformations in humans, worldwide concern for latent viral disease was raised following several case reports demonstrating ZIKV RNA in the amniotic fluid, placenta, and fetal neural tissue weeks to months after the initial maternal infection (Figure 2).
      • Besnard M.
      • Lastere S.
      • Teissier A.
      • Cao-Lormeau V.M.
      • Musso D.
      Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014.
      European Centre for Disease Prevention and Control
      Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barré syndrome.
      • Ventura C.V.
      • Maia M.
      • Bravo-Fliho V.
      • Gois A.L.
      • Belfort R.
      Zika virus in Brazil and macular atrophy in a child with microcephaly.
      • Mlakar J.
      • Korva M.
      • Tul N.
      • et al.
      Zika virus associated with microcephaly.
      • Oliveira Melo A.S.
      • Malinger G.
      • Ximenes R.
      • Szejnfeld P.O.
      • Alves Sampaio S.
      • Bispo de Filippis A.M.
      Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?.
      • Martines R.B.
      • Bhatnagar J.
      • Keating M.K.
      • et al.
      Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses—Brazil 2015.
      Figure thumbnail gr2
      Figure 2ISH of ZIKV in human fetal brain and placental tissue
      In situ hybridization of ZIKV in human fetal brain and placental tissue. Left panel, Localization of ZIKV RNA by in situ hybridization in brain tissues from infants with microcephaly. A, ISH with use of an antisense probe. ZIKV genomic RNA (red stain) in cerebral cortex of an infant (case patient 66, gestational age 26 weeks). Original magnification, ×10. B, ISH with use of a sense probe. Serial section shows negative-strand replicative RNA intermediates (red stain) in the same areas shown in panel A. Original magnification, ×10. C, ISH with use of an antisense probe. This is a higher magnification of panel A, showing cytoplasmic staining of neural (arrowheads) and glial cells. Original magnification, ×20. D, ISH with use of a sense probe. This is a higher magnification of panel B, showing cytoplasmic staining of neural and glial cells (arrowheads). Original magnification, ×20. E, ISH with use of an antisense probe. This figure shows the localization of negative-strand replicative RNA intermediates in neural cells or neurons (red, arrowheads) of another infant with fatal outcome (case patient 67, gestational age 27 weeks). Original magnification, ×40. F, Immunostaining of neurons (arrowheads) with the use of antibodies against neuronal nuclei in a serial section. Original magnification, ×40. G, Hematoxylin and eosin stain showing cortical neural cells in a serial section. Original magnification, ×40. H, Immunostaining of glial cells (arrowheads) with use of a glial fibrillary acidic protein antibody in the same case. Original magnification, ×40. Right panel, Localization of ZIKV RNA by ISH in placental tissues of women after a spontaneous abortion. A, ISH with use of an antisense probe. ZIKV genomic RNA localization in placental chorionic villi, predominantly within Hofbauer cells (red stain, arrows) of a case patient who had spontaneous abortion at 11 weeks of gestation (case patient 56). Original magnification, ×10). B, ISH with use of a sense probe. Serial section shows negative-strand replicative RNA intermediates (red stain, arrows) in the same cells shown in panel A. Original magnification, ×10. C, Hematoxylin and eosin stain of placental tissue of a case patient who experienced a spontaneous abortion at 8 weeks of gestation (case patient 47). Original magnification, ×20. D, Immunostaining for CD163, highlighting villous Hofbauer cells in a serial section as seen in panel C. Original magnification, ×63. E, ISH with use of an antisense probe. ZIKV genomic RNA as seen in a serial section from the same case patient as in panel C, showing staining within Hofbauer cells (red stain, arrows) of placental chorionic villi, is shown. Original magnification, ×40. F, ISH with use of a sense probe. Serial section shows negative-strand replicative RNA intermediates (red stain, arrows) in the same cells as shown in panel E. Original magnification, ×40. G, Hematoxylin and eosin stain from the same case patient as in panel C, showing inflammatory cell infiltrates in maternal side of placenta. Original magnification, ×63. H, ISH with use of a sense probe. Negative-strand replicative RNA intermediates (red stain, arrows) in inflammatory cells in a serial section is shown. Original magnification, ×63. These images have been reproduced with the expressed permission of the author and publisher (Bhatnagar et al
      • Bhatnagar J.
      • Rabeneck D.B.
      • Martines R.B.
      • et al.
      ZIKV RNA replication and persistence in brain and placental tissue.
      ).
      ISH, in situ hybridization; ZIKV, Zika virus.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      More recently, however, several national and regional cohorts or registries have provided further evidence, suggesting that there are multiple clinical manifestations of ZIKV infection in pregnancy. The first of these studies was published as a preliminary description of a prospective cohort of 88 symptomatic gravidae from Rio de Janeiro, Brazil, that had been followed up throughout gestation,
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      with a further expanded description published in late December 2016 and inclusive of 134 ZIKV positive women.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      In the preliminary report, Brasil et al
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      described that 42 of the 72 symptomatic gravidae who tested positive for ZIKV underwent ultrasound examination, with 29% (12 of 42 ZIKV) demonstrating variable findings on ultrasound, ranging in presumptive severity from central nervous system (CNS) lesions with microcephaly to isolated findings suggestive of placental insufficiency such as fetal growth restriction or abnormal umbilical artery Doppler velocimetry or amniotic fluid volume.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      Fortunately, Brasil et al
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      published an expanded recent follow-up from this prospective cohort with detailed pregnancy and infant outcomes. Inclusion into this expanded study cohort of 345 women was limited to those presenting to a single clinic in Rio de Janeiro with a rash, and all positive ZIKV cases were defined by testing positive within 5 days of rash development for ZIKV viral nucleic acid by polymerase chain reaction (PCR; QuantiTect Probe real-time reverse transcriptase polymerase chain reaction [rRT-PCR]
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      ) on blood specimens, urine specimens, or both. Of the 345 gravidae with a rash enrolled, 182 were PCR positive for ZIKV and 163 were PCR negative. Of the 182 ZIKV-positive, 125 of 134 had delivery and follow-up data; of the 163 ZIKV negative, 61 were followed up through delivery.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      Because only gravidae presenting to the clinic with a rash were enrolled in this cohort study, it is not surprising that ZIKV-negative women were more likely to have positive Chikungunya virus immunoglobulin (Ig) M or PCR results (41.7%, or 25 of 60, vs 2.8%, or 3 of 106 tested, P < .001). Interestingly, more than half of ZIKV-positive women presented with acute infection in the second trimester, and ZIKV-negative women were more likely to have used insect repellent (80% vs 60%, P = .0006).
      They reported adverse pregnancy outcomes in 46.4% (58 of 125 after 9 of the 134 gravidae were lost to follow-up), including a 7% risk of fetal loss (9 of 125; 6 of 9 miscarriages and 3 of 9 stillbirths) and 41.9% (49 of 117 live births) with congenital anomalies noted by the first month of life (49 of 117
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      ). The rate of adverse pregnancy outcomes including fetal loss with laboratory-documented ZIKV infection was similar and did not significantly vary by trimester of exposure (55%, or 11 of 20 of pregnancies in the first trimester, 52%, or 37 of 72 in the second trimester, and in 29%, or 10 of 34 of those in third trimester
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      ).
      In a comparative analysis with the ZIKV-negative pregnancies, a statistically significant difference was observed for the elevated risk of adverse pregnancy outcomes in any trimester with ZIKV-documented infection (46.4% vs 11.5%, P < .001), emergency cesarean delivery (23.5% vs 2.5%, P = .003), and evidence of abnormal neonatal and infant findings (41.9% vs 5.3%, P < .001
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      ).
      Interestingly, Brasil et al
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      described that almost all of the neonatal and infant abnormalities detected in ZIKV-positive gravidae affected the CNS but were not accompanied by microcephaly per se. Positive postnatal findings included microcephaly, cerebral calcifications, cerebral atrophy, ventricular enlargement, hypoplasia of cerebral structures, parenchymal brain hemorrhages, and gross findings on postnatal examination. In fact, among ZIKV-positive gravidae, a total of 31 of 49 (63%) infants available for follow-up displayed 1 or more of the following: hypertonus, spasticity, limb contractures, seizures, persistent cortical thumb sign or clenched fists, redundant scalp skin (even among normocephalic infants), and abnormal funduscopic or audiology examinations.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      Of note, although the data provided are descriptive, the authors found that a number of infants with normal clinical assessments in early infancy had abnormal nonspecific postnatal magnetic resonance imaging (MRI) findings.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      These included diffuse T2 hypersignaling in in the peritrigonal posterior areas and less evident in the frontal parietal white matter, with diffusion sequence hyposignaling. These findings are abnormal in infants and raise concern for cortical tract dysfunction.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      Although the reports of Brasil et al are limited to symptomatic women from a single pregnancy clinic in Rio de Janeiro, a similar spectrum of congenital findings have been described among women exposed in other regions of the Americas.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      Honein et al
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      have provided recent estimates from a US Centers for Disease Control and Prevention (CDC) and health department registry (US Zika Pregnancy Registry) of 442 completed and registered pregnancies.
      These US-based investigators reported that 6% (26 of 442, 95% confidence interval [CI], 4–8%) of maternal ZIKV infections result in congenital birth defects and reach 11% (9 of 85, 95% CI, 6–19%) when exposure was documented exclusively in the first trimester or preconception; no cases of microcephaly or brain malformations were detected unless first-trimester exposure was documented to occur.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      However, gestational age at infections was unknown for 2 of 26 fetuses or infants with birth defects and 27 of the 442 total completed pregnancies; nearly half of all women in the US. Zika registry had exposure during multiple trimesters of pregnancy.
      Of the 26 affected fetuses or infants, 4 had microcephaly but no reported neuroimaging, 14 had microcephaly and brain abnormalities, and 4 had brain abnormalities without microcephaly. Malformations noted included intracranial calcifications, abnormalities of the corpus callosum and cortical formation, cerebral atrophy, ventriculomegaly, hydrocephaly, and cerebellar abnormalities.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      The rate of congenital ZIKV among reported pregnancies did not differ by symptom occurrence or severity (6% in both groups), and asymptomatic registry subjects were just as likely to have an affected infant or fetus as symptomatic subjects (16 of 271 symptomatic, 95% CI, 4–9%, vs 10 of 167, 95% CI, 3–11%
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      ). Interestingly, of the 442 women, 61% (271 of 442) were asymptomatic and 38% (167 of 442) were symptomatic.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      There are several comments pertaining to the US registry report of Honein et al
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      that are worth considering prior to implementing their findings as part of obstetrical clinical counseling or risk assessment. First, registered subjects represented exposure resulting from travel of the subject or their partner to endemic regions in addition to only Brazil and include Barbados, Belize, Colombia, the Dominican Republic, El Salvador, Guatemala, Haiti, Honduras, Mexico, Marshall Islands, and Venezuela.
      Second, registry inclusion necessitated laboratory evidence of possible ZIKV infection and required either physician or public health reporting of positive cases. As with any voluntary registry, there is a strong risk of ascertainment bias. Both performance of testing for exposure to ZIKV and entry into the registry may be biased by either symptoms or ultrasound-based detection of anomalies. Similarly, a positive ZIKV test may have precluded additional genomic testing, including chromosomal microarray and karyotypic analysis.
      In addition, access to and availability of testing likely limited the population denominator in this study, and the human factors related to reporting may have overrepresented the numerator. This possibility is best realized by the reported rate of 61% of registry subjects reporting symptoms, which is 2–3 times the anticipated rate.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      Finally, although no birth defects were reported among the pregnancies with maternal symptoms or exposure only in the second trimester (0 of 76) or third trimester (0 of 31), ongoing follow-up of infants was not reported, and there was insufficient data to adequately estimate the purported affected during the latter 2 trimesters.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      Because there are multiple reports of normocephalic neonates at birth after second- and third-trimester ZIKV exposure who subsequently display postnatal brain malformations,
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • 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.
      • Moura da Silva A.A.
      • Ganz J.S.
      • Sousa P.D.
      • et al.
      Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome.
      • França G.V.
      • Schuler-Faccini L.
      • Oliveira W.K.
      • et al.
      Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation.
      case affect rates arising solely from the current registry ought to be considered preliminary estimates.
      In summary of both the Brazilian symptomatic cohort of Brasil et al
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      and the US-based registry of Honein et al,
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      tremendous gratitude should be extended to these investigative teams for their Herculean efforts aimed at collecting, characterizing, and detailing broad and robust perinatal clinical outcomes in the year since the association between ZIKV and fetal malformations was first largely recognized.
      However, these high-impact reports are limited by hindrances inherent to descriptive cohorts and estimates of relative or absolute risk of congenital ZIKV malformations in any trimester of pregnancy remain preliminary. All counseling should be accordingly framed with an appropriate degrees of uncertainty. This would include acknowledging a persistent but not currently quantifiable attributable risk for congenital ZIKV malformations following infection at any point during gestation and a need for postnatal follow-up. Moreover, until population-based studies are completed with universal testing of both asymptomatic and symptomatic women at risk of exposure, the true attributable risk estimate cannot be determined with any degree of confidence.
      Given the challenges in estimating the likelihood of congenital malformation following perinatal ZIKV exposure, we are left to question to what extent the current ZIKV pandemic will affect the global health burden in the coming decades. In the absence of ZIKV exposure in the population, microcephaly occurs in approximately 7 per 10,000 births.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      Assuming an estimated attack rate of ZIKV as high as 73% (as occurred on the island of Yap
      • Duffy M.R.
      • Chen T.H.
      • Hancock W.T.
      • et al.
      Zika virus outbreak on Yap Island, Federated States of Micronesia.
      ), the burden to both individual families and society in the face of a burgeoning pandemic is potentially staggering, even if the risk of microcephaly or congenital brain malformation is as low as 0.1–11% among symptomatic gravidae. In light of such a potential burden and the need for accurate risk estimates, the importance of accurate and predictive diagnosis is readily evident.

      Who should be tested and how?

      The first step in potentially diagnosing congenital ZIKV infection is the recognition of who should be tested. Because the majority of infected patients will have no or mild symptoms, clinical screening is not a reliable tool. There are 2 key risk factors that make pregnant women eligible for testing: those either with a personal history of exposure risk (either traveling to or residing in an endemic area) or sexual contact with a partner bearing the same exposure risk. Recognition of those at risk for ZIKV is therefore predicated on screening patients for regional travel and residence and sexual history, 2 areas commonly neglected in standard medical visits. Use of prompts within the electronic medical record may aid in screening.
      For those women who have resided in or traveled to a Zika endemic area, the CDC recommends testing inclusive of 8 weeks prior to conception and throughout gestation. This recommendation is based on a doubling of the maximal incubation time in nonpregnant women (3–14 days) plus intervals of viremia (2–10 days

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      ) and viruria (up to 14 days).

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      These rough estimations were empirically demonstrated in a nonpregnant subject case report from our group documenting viral shedding in the vaginal mucosa for 2 months after symptomatic infection.
      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      In addition, women whose male sexual partners have traveled to areas with active ZIKV transmission have a risk of viral acquisition for 6 months.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      However, the exact duration of potential transmission via sexual exposure is currently unknown, although ZIKV RNA has been discovered in semen for up to 188 days after illness onset.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      Important pitfalls to these guidelines include the potential for prolonged viremia and viruria noted in pregnant women.
      • Aagaard-Tillery K.M.
      • Silver R.
      • Dalton J.
      Immunology of normal pregnancy.
      • Driggers R.W.
      • Ho C.Y.
      • Korhonen E.M.
      • et al.
      Zika virus infection with prolonged maternal virmeia and fetal brain abnormalities.
      • Meaney-Delman D.
      • Oduyebo T.
      • Polen K.N.
      • et al.
      U.S. Zika Pregnancy Registry Prolonged Viremia Working Group. Prolonged detection of Zika virus RNA in pregnant women.

      Why should we test and not just screen for microcephaly with ultrasound? Molecular diagnostics for viral pathogens and estimation of risk

      There are 2 primary reasons that screening for microcephaly is insufficient. First, there are multiple malformations that may not entail microcephaly, including intracranial calcifications, abnormalities of the corpus callosum, and cerebellar abnormalities.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      Second, in instances of abnormal cortical formation, cerebral atrophy, and cortical neuronal agenesis, neuronal death will occur over time and microcephaly will be observed with age.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • 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.
      • Moura da Silva A.A.
      • Ganz J.S.
      • Sousa P.D.
      • et al.
      Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome.
      • França G.V.
      • Schuler-Faccini L.
      • Oliveira W.K.
      • et al.
      Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation.
      Fortunately, over the past 2 decades, clinical laboratory medicine has witnessed an unprecedented capacity in the ability to rapidly test for infectious pathogens. It was not long ago when diagnosis relied on weeks of viral cultures, which were often low or nil yield, whereas today’s current clinical laboratory medicine and pathology practice allows for highly sensitive and specific rapid molecular diagnostic testing. Ergo, we are notably handicapped when we cannot reliably identify a potential viral pathogen or establish exposure and immunity with serological testing. In many circumstances diagnostic testing is utterly and fundamentally necessary to both personalized clinical management and public health control measures.
      This is no truer than when a viral pathogen will result in a devastating perinatal transmission, yielding a potentially severely compromised infant faced with lifelong disability. In the short term, diagnoses are important for decisions of pregnancy continuation. However, in the longer term, accurate and early diagnosis becomes the cornerstone of developing targeted and efficacious interventions and estimating the natural history of an infection to give truly informed risk estimates.

      Currently available laboratory-based testing for ZIKV

      There are several limitations to currently available testing for ZIKV. To illustrate these limitations, consider the following hypothetical clinical scenario. Ms Jones is a 35 year old G2P0010 who resides in Texas but works part time in Brazil. Her husband resides and works full time in Brazil. She comes to see you at 16 weeks’ gestation, after she completed a 4 month interval working in Rio de Janeiro with frequent travel to the northern provinces. You have performed an ultrasound, which shows borderline bilateral ventriculomegaly at 11 and 12 mm, with an overall estimated fetal weight at the 24th centile and a head circumference and biparietal diameter at the 15th centile. Per the CDC algorithm,

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      she should be eligible for IgM testing up to 12 weeks from entry back into the United States. However, what if she were actually infected at 4 weeks’ gestation? Her IgM might be negative or indeterminate as a result of natural titer waning. In the absence of an IgG isotype serological test, a negative or indeterminate IgM titer would potentially be interpreted falsely as negative.
      Knowing these limitations, what are the testing options for this patient? Would rRT-PCR testing be helpful? When should each test be performed? How should we interpret our testing results, and what are the current limitations to these interpretations? In the following sections, we discuss testing options; Tables 1 and 2 provide summary guidance.
      Table 1Available ZIKV testing modalities
      Test categorySpecimen sourcesTiming of first positive test
      Based on current information
      Duration of positive test
      Based on current information
      LimitationsInterpretations of positive tests
      Viral serologyIgMSerum





      CSF
      4 d in symptomatic

      7-14 d in asymptomatic



      Unknown
      12 wks





      Unknown
      • Cross-reactivity with other flaviviruses
      • High false-positive rates
      • Risk for false-negative due to delayed seroconversion or titer waning
      • PRNT is needed as follow-up test
      • False positive
      • Recent ZIKV infection
      • Acute other flavivirus infection (cross-reactivity)
      IgGSerum7-14 d
      Extrapolated from West Nile IgG24,25
      >12 wks
      • Currently not available for clinical use
      • Other flavivirus infection >12 wks (cross-reactivity)
      • ZIKV Infection >2 wks, if IgM negative then likely >12 wks
      PRNTSerumWith positive serological testing
      • Available only through the CDC
      • Long turnaround time
      See Table 2
      Viral nucleic acid testingrRT-PCRSerum

      Blood

      Urine

      CSF

      Tissue

      Amniotic fluid
      0-7 d

      0-7 d

      0-14 d

      Unknown

      Unknown

      Unknown
      5-14 d
      Prolonged viremia and viruria noted in pregnant women and neonates.


      5-14 d
      Prolonged viremia and viruria noted in pregnant women and neonates.


      14 d
      Prolonged viremia and viruria noted in pregnant women and neonates.


      Unknown

      Unknown

      Unknown
      • Prolonged viremia possible and poorly understood
      Recent Zika infection
      UltrasoundAmniotic fluid

      Biometry

      Neurological

      Extremities
      VariableOnce present, appears progressive
      • Poor specificity of findings in isolation or in constellation
      When performed in isolation, cannot distinguish anomaly cause. When performed in conjunction with amniocentesis with chromosomal microarray or other infectious pathogen testing, improved specificity; see Table 3.
      Ig, immunoglobulin; PRNT, plaque reduction neutralization test; rRT-PCR, real-time reverse transcriptase polymerase chain reaction; ZIKV, Zika virus.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      a Based on current information
      b Extrapolated from West Nile IgG
      • Brent C.
      • Dunn A.
      • Savage H.
      • et al.
      Preliminary findings from an investigation of Zika virus infection in a patient with no known risk factors—Utah, 2016.
      • Reusken C.
      • Pas S.
      • GeurtsvanKessel C.
      • et al.
      Longitudinal follow-up of Zika virus RNA in semen of a traveler returning from Barbados to The Netherlands with Zika virus disease, March 2016.
      c Prolonged viremia and viruria noted in pregnant women and neonates.
      Table 2Interpretation of serological results
      Modified from Rabe et al.65
      IgM ELISA testingResultInterpretation
      ZIKV IgMDetectedNo evidence of Zika or Dengue infection, false-positive IgM
      ZIKV PRNTNot detected
      DENV PRNTNot detected
      ZIKV IgMPositive or equivocalRecent Zika virus infection
      DENV IgMPositive or equivocal
      ZIKV PRNT≥10
      DENV PRNT<10
      ZIKV IgMPositive or equivocalRecent Dengue virus infection
      DENV IgMPositive or equivocal
      ZIKV PRNT<10
      DENV PRNT≥10
      ZIKV IgMInconclusive in one assay and inconclusive or negative in the otherRecent flavivirus infection; specific virus cannot be determined
      DENV IgM
      ZIKV PRNT≥10
      DENV PRNT≥10
      ZIKV IgMInconclusive in one assay and inconclusive or negative in the otherEvidence of Zika virus; timing cannot be determined
      DENV IgM
      ZIKV PRNT≥10
      DENV PRNT<10
      ZIKV IgMInconclusive in one assay and inconclusive or negative in the otherEvidence of Dengue virus; timing cannot be determined
      DENV IgM
      ZIKV PRNT<10
      DENV PRNT≥10
      ZIKV IgMInconclusive in one assay and inconclusive or negative in the otherEvidence of flavivirus infection; specific virus and timing of infection cannot be determined
      DENV IgM
      ZIKV PRNT≥10
      DENV PRNT≥10
      DENV, Dengue virus; ELISA, enzyme-linked immunosorbent assay; PRNT, plaque reduction neutralization tests, with units of 10-fold higher or lower titer by PRNT used as cutoff values; ZIKV, Zika virus.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      a Modified from Rabe et al.
      • Bhatnagar J.
      • Rabeneck D.B.
      • Martines R.B.
      • et al.
      ZIKV RNA replication and persistence in brain and placental tissue.
      The diagnosis of ZIKV infection currently relies on the detection of viral RNA via rRT-PCR or identifying an IgM serological response.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      Given testing limitations, diagnosis and patient counseling are inexact with poor predictive sensitivity (rRT-PCR) and antibody cross-reactivity (IgM). Because few experience symptoms and travel or residence in endemic areas frequently spans weeks if not months, the exact timing of exposure is often unknown or cannot be reliably estimated.
      This is particularly a concern when the serological testing relies on an initial response antibody, such as the pentavalent IgM isotype antibody. As a consequence of isotype class switching,
      • Aagaard-Tillery K.M.
      • Silver R.
      • Dalton J.
      Immunology of normal pregnancy.
      the first antibody serologically to rise will be the IgM and, given its pentavalency, is often cross-reactive with other viral strains and family members; whether this is true for ZIKV and other related flaviviruses is not yet known. IgM titers may initially be detectable between 4 and 14 days

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      but typically not until 10–14 days. The titers will start to measurably wane by 10–12 weeks. Subsequently, higher avidity IgG isotypes will appear and remain positive for longer periods of time and thus provide ongoing immunity.
      • Aagaard-Tillery K.M.
      • Silver R.
      • Dalton J.
      Immunology of normal pregnancy.
      Similarly, possibly paralleling the variation in clinical symptom severity, even currently symptomatic patients may not test positive in either serum or urine by rRT-PCR.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.

      rRT-PCR testing

      The incubation period for ZIKV is 3–14 days, and in nonpregnant subjects’ serum, viremia lasts for as few as 2 days to as great as 10 days.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      Longer periods of viremia and viruria have been observed during pregnancy, presumably because of fetal-placental infection (Figure 2).
      • Driggers R.W.
      • Ho C.Y.
      • Korhonen E.M.
      • et al.
      Zika virus infection with prolonged maternal virmeia and fetal brain abnormalities.
      • Meaney-Delman D.
      • Oduyebo T.
      • Polen K.N.
      • et al.
      U.S. Zika Pregnancy Registry Prolonged Viremia Working Group. Prolonged detection of Zika virus RNA in pregnant women.
      Virus persists on average 2 weeks longer in urine, and testing for viruria has been shown to improve detection rates.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Interim guidance for Zika virus testing of urine—United States, 2016.
      Because the majority of patients with ZIKV infection (80%) are asymptomatic,

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      identifying patients during the viremic or viruric stage is challenging. Current CDC guidelines recommend testing serum or urine by rRT-PCR when a patient presents within 14 days of their last potential exposure to ZIKV or within 14 days of symptoms.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      Their last potential exposure can be either the date of sexual contact with someone at risk for ZIKV or the last date of travel to a ZIKV endemic region. Strictly adhering to this testing algorithm would by definition miss prolonged viremia, which may be a surrogate of fetal-placenta infection (Figure 2).
      • Driggers R.W.
      • Ho C.Y.
      • Korhonen E.M.
      • et al.
      Zika virus infection with prolonged maternal virmeia and fetal brain abnormalities.
      • Meaney-Delman D.
      • Oduyebo T.
      • Polen K.N.
      • et al.
      U.S. Zika Pregnancy Registry Prolonged Viremia Working Group. Prolonged detection of Zika virus RNA in pregnant women.
      Other body fluids in which ZIKV RNA (but not necessarily infectious virions per se) has been detected include saliva, semen, breast milk, and vaginal and cervical mucous.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      These niches potentially serve as latent portals for continuing infectivity of ZIKV, to either the mother or the fetus. Fortunately, horizontal transmission has been documented only via sexual intercourse (both male to female, female to male, and amongst same-sex partners) and blood transfusions, both in reported infrequent numbers.

      Centers for Disease Control and Prevention. Areas with Zika. Available at: http://www.cdc.gov/zika/geo/index.html. Accessed December. 9, 2016.

      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      • Petersen E.E.
      • Meaney-Delman D.
      • Neblett-Fanfair R.
      • et al.
      Update: interim guidance for preconception counseling and prevention of sexual transmission of Zika virus for persons with possible Zika virus exposure—United States, September 2016.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      • Driggers R.W.
      • Ho C.Y.
      • Korhonen E.M.
      • et al.
      Zika virus infection with prolonged maternal virmeia and fetal brain abnormalities.
      • Meaney-Delman D.
      • Oduyebo T.
      • Polen K.N.
      • et al.
      U.S. Zika Pregnancy Registry Prolonged Viremia Working Group. Prolonged detection of Zika virus RNA in pregnant women.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Interim guidance for Zika virus testing of urine—United States, 2016.
      • Brent C.
      • Dunn A.
      • Savage H.
      • et al.
      Preliminary findings from an investigation of Zika virus infection in a patient with no known risk factors—Utah, 2016.
      • Reusken C.
      • Pas S.
      • GeurtsvanKessel C.
      • et al.
      Longitudinal follow-up of Zika virus RNA in semen of a traveler returning from Barbados to The Netherlands with Zika virus disease, March 2016.
      However, the possibility of transmission from contact with other infected body fluids is certainly a looming concern.
      • Brent C.
      • Dunn A.
      • Savage H.
      • et al.
      Preliminary findings from an investigation of Zika virus infection in a patient with no known risk factors—Utah, 2016.
      Persistence of ZIKV RNA in other body compartments also provides an opportunity to expand testing options.
      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.
      The level of ZIKV is higher in semen than urine, saliva, or serum and is detectable for longer periods of time after acute infection.
      • Reusken C.
      • Pas S.
      • GeurtsvanKessel C.
      • et al.
      Longitudinal follow-up of Zika virus RNA in semen of a traveler returning from Barbados to The Netherlands with Zika virus disease, March 2016.
      This may be of particular interest in family planning. Saliva and urine testing are potential fluids for the detection of ZIKV RNA and may be of interest in certain populations, such as children and neonates (in whom drawing blood is difficult) or in settings of limited resources when the additional cost and availability of phlebotomy services are burdensome.
      • Musso D.
      • Roche C.
      • Nhan T.X.
      • Robin E.
      • Teissier A.
      • Cao-Lormeau V.M.
      Detection of Zika virus in saliva.
      Detection of ZIKV nucleic acid has been observed in breast milk, but no documented cases of transmission have occurred as of yet.
      • Dupont-Rouzeyrol M.
      • Biron A.
      • O’Connor O.
      • Huguon E.
      • Descloux E.
      Infectious Zika viral particles in breastmilk.
      A recent case report from our own institution demonstrated persistence of ZIKV in the red cell compartment for up to 81 days after symptoms, 73 days longer than serum, suggesting that the use of whole blood instead of serum may increase the sensitivity of ZIKV detection, particularly in the asymptomatic patient.
      • Murray K.O.
      • Gorchakov R.
      • Carlson A.R.
      • et al.
      Prolonged detection of zika virus in vaginal secretions and whole blood.

      Serological testing

      Serological testing is recommended 4 or more days after the onset of clinical illness or ≥14 days from the last potential exposure in asymptomatic patients. An IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) is the only FDA-approved serological test and can be performed on serum and cerebrospinal fluid. Evidence from the serological response to other flaviviruses, particularly DENV and West Nile virus, suggests that IgM may be present up to 3 months after the exposure; in some patients with West Nile encephalitis, IgM antibodies were detectable more than 1 year after the infection,
      • Babaliche P.
      • Doshi D.
      Catching dengue early: clinical features and laboratory markers of dengue virus infection.
      • Wahala W.M.P.B.
      • de Silva A.M.
      The human antibody response to dengue virus infection.
      • Prince H.E.
      • Tobler L.H.
      • Yeh C.
      • Gefter N.
      • Custer B.
      • Busch M.P.
      Persistence of West Nile virus–specific antibodies in viremic blood donors.
      • Roehrig J.T.
      • Nash D.
      • Maldin B.
      • et al.
      Persistence of virus-reactive serum immunoglobulin M antibody in confirmed West Nile virus encephalitis cases.
      with one study showing IgM antibodies persisting up to 8 years after the infection.
      • Murray K.O.
      • Garcia M.N.
      • Yan C.
      • Gorchakov R.
      Persistence of detectable immunoglobulin M antibodies up to 8 years after infection with West Nile virus.
      At present, serological testing for ZIKV with IgM is currently recommended up to 12 weeks after the exposure because serological waning and trough intervals are not yet known. IgG antibodies to ZIKV develop shortly after IgM antibodies and are thought to confer life-long immunity with higher avidity. For a brief interval from September through August, at least one commercial laboratory offered ZIKV IgG, but it is presently not available. Undoubtedly availability of IgG serologies with potential avidity testing would be advantageous.
      In addition to waning titers of IgM isotypes, there is often serological cross-reactivity with other flaviviruses in patients who have had a recent or prior flavivirus infection, particularly DENV. This complicates the diagnosis, particularly because ZIKV is emerging in areas in which DENV is endemic. Currently serological differentiation and confirmation rely on a more cumbersome and less available testing method, the plaque reduction neutralization test (PRNT). The PRNT identifies virus-specific neutralizing antibody titers to various related flaviviruses.
      Use of the PRNT necessitates contextual estimations, and a PRNT fold titer of >10 with a competing flavivirus antibody titer (for example, DENV) of a fold titer of <10 would suggest recent infection with ZIKV in the past 3 months (Table 2). However, what often results are PRNT estimates with dual elevations, such as ZIKV PRNT fold titer of >10 and DENV PRNT fold titer of >10, as an acute flavivirus infection, type indeterminate, and may represent either a recent ZIKV or DENV infection or a coinfection (see Tables 1 and 2). To date, PRNT testing is offered only through the CDC, and there is ongoing high demand with prolonged turnaround times.
      In summary, a negative ZIKV IgM test may represent either a patient who is not at risk, is too early in the course of infection and has not yet seroconverted, or who had infection with ZIKV and has already seroconverted to IgG with a physiological waning of their IgM titer (Tables 1 and 2). A positive IgM test indicates one of the following: (1) a false-positive result, (2) acute ZIKV, or (3) another acute flavivirus infection, including coinfection with ZIKV. In patients experiencing symptoms for less than 2 weeks, lack of seroconversion needs to be evaluated by rRT-PCR testing on both a serum and urine specimen as recommended by the CDC. With the development and dissemination of ZIKV-specific IgG serological and avidity testing, the potential for expanded serological testing will be enabled.

      Amniocentesis

      A growing body of literature suggests infants with confirmed fetal infection are at an increased risk of intracranial abnormalities.
      • Soares de Oliveira-Szejnfeld P.
      • Levine D.
      • Melo A.S.
      • et al.
      Congenital brain abnormalities and Zika virus: what the radiologist can expect to see prenatally and postnatally.
      In the setting of a positive ZIKV IgM with positive PRNT, positive rRT-PCR in maternal serum or urine, or with ultrasound abnormalities, amniocentesis to detect ZIKV RNA via rRT-PCR should be considered to evaluate for vertical transmission (Table 1). However, the sensitivity, specificity, and a positive and negative predictive value of rRT-PCR on amniotic fluid is unknown, and these potential benefits and limitations should be discussed openly. In cases in which ultrasound abnormalities are detected, it is our clinical practice to also perform chromosomal microarray
      • Wapner R.J.
      • Martin C.L.
      • Levy B.
      • et al.
      Chromosomal microarray versus karyotyping for prenatal diagnosis.
      and test for other congenital pathogens in the setting of ultrasound abnormalities.

      Ultrasound-based fetal assessment

      The CDC, the American Congress of Obstetricians and Gynecologists,
      • Meaney-Delman D.
      • Rasmussen S.A.
      • Staples E.
      • et al.
      Zika virus and pregnancy: what obstetric health care providers need to know.
      the Society for Maternal-Fetal Medicine (SMFM)
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      , and the International Society of Ultrasound in Obstetrics and Gynecology
      • Papageorghiou A.T.
      • Thilaganathan B.
      • Bilardo C.M.
      • et al.
      ISUOG interim guidance on ultrasound for Zika virus infection in pregnancy: information for healthcare professionals.
      have recommended an ultrasound evaluation for fetal anomalies in pregnant women who have been infected or potentially exposed to ZIKV. Paramount to these recommendations have been the detection of microcephaly using ultrasonography.
      • Oduyebo T.
      • Igbinosa I.
      • Petersen E.E.
      • et al.
      Update: Interim guidance for health care providers caring for pregnant women with possible Zika virus exposure—United States, July 2016.
      • Meaney-Delman D.
      • Rasmussen S.A.
      • Staples E.
      • et al.
      Zika virus and pregnancy: what obstetric health care providers need to know.
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      • Papageorghiou A.T.
      • Thilaganathan B.
      • Bilardo C.M.
      • et al.
      ISUOG interim guidance on ultrasound for Zika virus infection in pregnancy: information for healthcare professionals.
      Microcephaly is defined as a head circumference (HC) >3 SD below the mean for the estimated gestational age, with fewer false-positive results the further the HC falls from the mean.
      A causal criterion between ZIKV and microcephaly was acknowledged by the CDC in April 2016, and multiple other congenital anomalies have been associated with congenital ZIKV.

      CDC Concludes Zika Causes Microcephaly and Other Birth Defects. Available at: https://www.cdc.gov/media/releases/2016/s0413-zika-microcephaly.html. Accessed December 27, 2016.

      • Rasmussen S.A.
      • Jamieson D.J.
      • Honein M.A.
      • Petersen L.R.
      Zika virus and birth defects—reviewing the evidence for causality.
      Thus, it is possible that microcephaly may represent an extreme end point in the spectrum of cerebral cortical hypoplasia and tissue loss (Table 3). Some recent studies have indicated that ZIKV-associated congenital malformations can occur after infection in any trimester or even after birth in infants with a normal HC at delivery
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • 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.
      • Moura da Silva A.A.
      • Ganz J.S.
      • Sousa P.D.
      • et al.
      Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome.
      • França G.V.
      • Schuler-Faccini L.
      • Oliveira W.K.
      • et al.
      Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation.
      a finding that differs from the typical pattern seen in other congenital infections.
      Table 3CNS abnormalities reported with ZIKV during pregnancy
      Ultrasound viewAbnormalitySpecificity for congenital ZIKV infection
      Transventricular (axial view)
      • Ventriculomegaly
      • Septations in occipital horns
      • Poor
      • Poor
      Transthalamic (axial view)
      • Agenesis of the thalami
      • Poor
      Transcerebellar (coronal and axial views)
      • Posterior fossa abnormalities, including Vermian dysgenesis/hypoplasia and an enlarged cisterna magna
      • In isolation, poor
      Midsagittal plane (sagittal view)
      • Brain stem hypoplasia and/or atrophy
      • Agenesis/dysgenesis of the corpus callosum
      • Poor
      • Poor
      Transcaudate (coronal view)
      • Enlargement of anterior horns of ventricular system
      • Enlarged subarachnoid space that can be quantified by measuring sinocortical and craniocortical spaces
      • Poor
      Brain parenchyma and cortex (can be assessed in all views)
      • Calcifications, predominantly located in the gray-white matter junction but also identified in thalamus, basal ganglia, cortex and periventricular regions
      • Brain atrophy with enlarged extraaxial spaces
      • Abnormalities in the cortical development such as delayed sulcation, lissencephaly, polymicrogyria, or pachygyria
      • Enlarged confluence of the dural venous sinuses from intracranial hemorrhage
      • Increased specificity for congenital viral and pathogen malformations but not unique to congenital ZIKV
      • Poor
      • Poor
      • Likely higher specificity for congenital ZIKV infection, but more data are needed
      Head profile
      • Slanted forehead, consistent with microcephaly
      • Increased specificity for congenital viral and pathogen malformations but not unique to congenital ZIKV
      Orbits
      • Ocular defects (asymmetrical microphtalmia, cataracts, and herniation of the orbital fat into the cranial vault)
      • Increased specificity for congenital viral and pathogen malformations but not unique to congenital ZIKV
      Amniotic fluid assessment
      • Oligohydramnios
      • Poor
      Biometry
      • BPD and HC
      • AC
      • FL, HL
      • Microcephaly/diminished head size
      • Asymmetric growth restriction
      • Symmetric growth restriction, or constitutional
      • Poor and may be genomic in origin
      • Poor specificity in isolation
      Extremities
      • Joint contractures (arthrogryposis) and clubbed feet
      • Poor and may be disruptive (ie, Potter’s sequence), genomic, or infectious in etiology
      AC, abdominal circumference; BPD, biparietal diameter; CNS, central nervous system; FL, femur length; HC, head circumference; HL, humerus length; ZIKV, Zika virus.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      It is possible that this is the result of the placenta functioning as a reservoir for ZIKV replication, a pattern that differs from most other congenital infections.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      However, other studies indicate the risk maybe predominantly in the first trimester.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      Thus, microcephaly is likely the end result of cortical agenesis and brain volume loss, rather than a primary consequence of fetal viral infection or calvarium destruction. Ergo, screening for microcephaly as initial evidence of fetal infection is likely too little too late.
      The congenital ZIKV syndrome portends a constellation of findings, inclusive of intracranial abnormalities, which may include microcephaly (Table 3).
      • Costello A.
      • Dua T.
      • Duran P.
      • et al.
      Defining the syndrome associated with congenital Zika virus infection.
      In a review of 438 symptomatic subjects with rash or suspected fetal abnormalities, 17 had a confirmation of fetal infection based on positive amniotic fluid, cord blood, or neonatal brain tissue at autopsy and 28 had presumed infection based on CNS findings. Of these subjects, almost all had intracranial abnormalities with multiple instances of intracranial abnormalities with normal HC measurements.
      • Soares de Oliveira-Szejnfeld P.
      • Levine D.
      • Melo A.S.
      • et al.
      Congenital brain abnormalities and Zika virus: what the radiologist can expect to see prenatally and postnatally.
      Similarly, a second observational study has shown that ventriculomegaly and microcephaly progress as pregnancy advances,

      Sarno M, Aquino M, Pimentel K, et al. Progressive lesions of the central nervous system in microcephalic fetuses with suspected congenital Zika virus syndrome. Ultrasound Obstet Gynecol, in press.

      which would explain the observation from the French Polynesian outbreak that first-trimester infection portends a stronger association with microcephaly than infection later in pregnancy,
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      a finding recently confirmed in the US Zika Registry.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      We now know that ZIKV infection as late as 27 weeks causes continued CNS damage into the first 6 months of life despite a normal HC during pregnancy and at delivery, suggesting that the neurological damage caused by ZIKV is a continuum that may begin in utero but does not necessarily end with delivery.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • 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.
      • Moura da Silva A.A.
      • Ganz J.S.
      • Sousa P.D.
      • et al.
      Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome.
      • França G.V.
      • Schuler-Faccini L.
      • Oliveira W.K.
      • et al.
      Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation.
      • Oliveira D.B.
      • Almeida F.J.
      • Durigon E.L.
      • et al.
      Prolonged shedding of Zika virus associated with congenital infection.
      Limitations to these studies include the small number of cases of microcephaly (n = 8 in French Polynesia) and a small number of women who had delivered by the time of publication (n = 8 deliveries in Rio de Janeiro), with no follow-up data after delivery and theoretic estimates of risk based on mathematical modeling.
      • Besnard M.
      • Lastere S.
      • Teissier A.
      • Cao-Lormeau V.M.
      • Musso D.
      Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014.
      • Ventura C.V.
      • Maia M.
      • Bravo-Fliho V.
      • Gois A.L.
      • Belfort R.
      Zika virus in Brazil and macular atrophy in a child with microcephaly.
      • Martines R.B.
      • Bhatnagar J.
      • Keating M.K.
      • et al.
      Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses—Brazil 2015.
      Given the limitations of findings by ultrasonography alone for the evaluation of congenital ZIKV infection (Table 3), we view sonographic screening as an additional clinical tool (potentially aiding in our diagnosis), which could be complementary to molecular diagnostics. A recent SMFM statement recommended that if the HC by prenatal ultrasound is >2 SD below the mean, a careful evaluation of the fetal intracranial anatomy is indicated. If the intracranial anatomy is normal, SMFM recommends follow-up scans in 3–4 weeks.
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      These recommendations acknowledge that fetal brain malformations and findings of congenital ZIKV disease will occur independent of (and in fact may precede) strictly defined fetal microcephaly at >3 SD below the mean.
      • Mlakar J.
      • Korva M.
      • Tul N.
      • et al.
      Zika virus associated with microcephaly.
      • Oliveira Melo A.S.
      • Malinger G.
      • Ximenes R.
      • Szejnfeld P.O.
      • Alves Sampaio S.
      • Bispo de Filippis A.M.
      Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?.
      • Meaney-Delman D.
      • Rasmussen S.A.
      • Staples E.
      • et al.
      Zika virus and pregnancy: what obstetric health care providers need to know.
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      • Papageorghiou A.T.
      • Thilaganathan B.
      • Bilardo C.M.
      • et al.
      ISUOG interim guidance on ultrasound for Zika virus infection in pregnancy: information for healthcare professionals.
      • Oliveira D.B.
      • Almeida F.J.
      • Durigon E.L.
      • et al.
      Prolonged shedding of Zika virus associated with congenital infection.
      Based on these recommendations by the SMFM and other professional entities, a further discussion regarding fetal neurosonographic imaging is warranted.

      Fetal brain imaging in cases of suspected congenital ZIKV infection

      An evaluation of intracranial anatomy requires the use of conventional sonographic planes including the transventricular, transthalamic, and transcerebellar views and the brain parenchyma to evaluate for structural anomalies are well as calcifications. Given these considerations in total, our current practice at Baylor College of Medicine is to perform neurosonographic (neurosonology) screening in at-risk women at the time of their midtrimester fetal anatomic survey and every 4–6 weeks thereafter.
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      Because fetal brain malformations may be either incident to or causative of microcephaly, we do not predicate indication for neurosonography (neurosonology) on the presence or absence of microcephaly but rather use parallel testing results and at-risk estimations as our indication (Figures 3 and 4 and Table 3).
      Figure thumbnail gr3
      Figure 3Fetal neurosonographic images with anatomic landmarks
      Axial sonographic views recommended by the International Society of Ultrasound in Obstetrics and Gynecology to perform a targeted CNS scan to evaluate the fetal brain.
      • Costello A.
      • Dua T.
      • Duran P.
      • et al.
      Defining the syndrome associated with congenital Zika virus infection.
      Images A, B, and C are axial views; the dashed red lines on images A and C align to the Sylvian fissure, while the dashed red line in image B aligns to the parietoccipital fissure. Image A corresponds to the transthalamic plane, in which the measurement of head circumference and biparietal diameter should be performed; the blue dashed line is placed on the location for optimal measurement of the third ventricle width. Image B is the transventricular plane, in which measurements of the fetal ventricular atria width should be performed (blue dashed line). Image C corresponds to transcerebellar plane, in which measurement of the posterior fossa or cisterna magna and cerebellar width should be performed.

      Sarno M, Aquino M, Pimentel K, et al. Progressive lesions of the central nervous system in microcephalic fetuses with suspected congenital Zika virus syndrome. Ultrasound Obstet Gynecol, in press.

      Images D through G are key views for neurosonographic (neurosonology) imaging aimed at the detection of potential abnormalities detected in the sagittal and coronal planes. Images D and E are obtained from the sagittal views, in which panel D is the midsagittal plane view used to assess and measure the height of the corpus callosum (dashed yellow line) and cerebellar vermian (dashed blue line). Also shown in image D is the cingulate fissure (dashed red line). Image E represents the parasagittal plane views and can assess brain parenchyma and the ventricles. Both F and G images are obtained in coronal planes, in which panel F represents the transcaudal plane, in which the anterior horns of the ventricles (dashed yellow line) and the size of the subarachnoid space (measured by the sinocortical and craniocortical spaces) can be assessed. Panel G arises from the transcerebellar plane and optimally assesses the calcarine fissure (dashed red line) and cerebellum.
      CNS, central nervous system; CSP, cavum septum pellucidum; IHF, interhemispheric fissure.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      Figure thumbnail gr4
      Figure 4Neurosonographic images from second and third-trimester fetuses affect by ZIKV infection
      Representative images from several latter second- and early third-trimester fetuses affected by congenital ZIKV infection, with axial, sagittal, and coronal views in similar planes to with landmarks in white texts; abnormal findings are highlighted in off-white bold text. Images A and B are axial views: image A shows the dilation of the ventricular system at the level of the posterior horns corresponding to the transventricular plane. The parietoccipital fissure is absent, and there is evidence of diffuse parenchymal thinning with linear calcifications located at the WM-GM junction (off-white arrows). The subarachnoid space and posterior horns ventricular system are markedly dilated (off-white asterisks). Image B corresponds to the transcerebellar plane. There is a significant dilation of the entire ventricular system as shown by an enlarged third ventricle and dilated anterior and posterior horns (off-white asterisks). There is some degree of brain parenchymal thinning with coarse calcifications at the level of the basal ganglia (off-white arrow). Images C through E represent the sagittal views: image C shows an abnormally thin and short corpus callosum in the midsagittal plane (off-white line). While the brain stem and cerebellar vermis have a normal appearance, punctiform calcifications are seen in the frontal and temporal lobe (off-white arrow). Images D and E represent parasagittal views of the fetal brain showing linear and coarse calcifications located on the WM-GM junction and the basal ganglia (off-white arrows). There is evidence of an enlarged ventricular system with thinned brain parenchyma and enlarged subarachnoid space (off-white asterisks). Additionally observed are signs compatible with a delayed sulcation based on the abnormally smooth cortical surface for third-trimester fetuses. Images F, G, and H are coronal views: images F and G show the transcaudal plane, with enlarged anterior horns (off-white asterisks), parenchymal calcifications located at the WM-GM junction and basal ganglia (off-white arrows). Periventricular cysts are seen above and below the anterior horns (off-white arrows). Image H shows a transcerebellar plane with enlarged subarachnoid space and significant ventricular dilatation (off-white asterisks) and accompanying parenchymal thinning. Parenchymal calcifications are seen in the WM-GM junction (off-white arrows).
      WM-GM, white matter-gray matter.
      Eppes. Testing for Zika virus infection in pregnancy. Am J Obstet Gynecol 2017.
      Neurosonography (neurosonology
      International Society of Ultrasound in Obstetrics ande Gynecology Education Committee
      Sonographic examination of the fetal central nervous system: guidelines for performing the “basic examination” and the “fetal neurosonogram”.
      ) is best performed via transvaginal sonography if the fetus is in a cephalic presentation, but it can also be performed using the transabdominal approach. Transventricular, transthalamic, and transcerebellar planes should be obtained in the axial view. Midsagittal and parasagittal planes should be obtained in the sagittal view, and transcaudate and transcerebellar planes should be obtained in the coronal views. In each view, brain development and the integrity of its anatomic structures should be assessed.
      • Monteagudo A.
      • Timor Tristch I.E.
      Normal sonographic development of the central nervous system from the second trimester onwards.
      • Monteagudo A.
      • Timor Tristch I.E.
      • Mayberry P.
      Three-dimensional transvaginal neurosonography of the fetal brain: ‘navigating’ in the volume scan.
      • Malinger G.
      • Lev D.
      • Lerman-Sagie T.
      Normal and abnormal development fetal brain development during the third trimester as demonstrated by neurosonography.
      In addition, the head circumference, biparietal diameter, ventricular widths at the level of the anterior and posterior horns, third ventricle, transcerebellar diameter, posterior fossa, vermian height, and callosal length should be measured (Figures 3 and 4).
      International Society of Ultrasound in Obstetrics ande Gynecology Education Committee
      Sonographic examination of the fetal central nervous system: guidelines for performing the “basic examination” and the “fetal neurosonogram”.
      These measurements should be referenced to previously published normality values, considering the gestational age at the time of the examination.
      • Sari A.
      • Ahmetoglu A.
      • Dinc H.
      • et al.
      Fetal biometry: size and configuration of the third ventricle.
      • Sherer D.M.
      • Sokolovski M.
      • Dalloul M.
      • Pezzullo J.C.
      • Osho J.A.
      • Abulafia O.
      Nomograms of the axial fetal cerebellar hemisphere circumference and area throughout gestation.
      • Malinger G.
      • Ginath S.
      • Lerman-Sagie T.
      • Watemberg N.
      • Lev D.
      • Glezerman M.
      The fetal cerebellar vermis: normal development as shown by transvaginal ultrasound.
      • Achiron R.
      • Achiron A.
      Development of the human fetal corpus callosum: a high-resolution, cross-sectional sonographic study.
      For the fetal HC, the SMFM
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.
      advocates for the utilization of Chernevak’s reference values,
      • Chervenak F.A.
      • Rosenberg J.
      • Brightman R.C.
      • Chitkara U.
      • Jeanty P.
      A prospective study of the accuracy of ultrasound in predicting fetal microcephaly.
      whereas the International Society of Ultrasound in Obstetrics and Gynecology advocates for the use of Intergrowth 21 reference values.
      • Papageorghiou A.T.
      • Ohuma E.O.
      • Altman D.G.
      • et al.
      International standards for fetal growth based on serial ultrasound measurements: the Fetal Growth Longitudinal Study of the INTERGROWTH-21st Project.
      Fetal brain sulcation should be assessed in detail, with consideration of the cortical fissures that would normally be observed in subsequent gestational age windows (Figure 4).
      • Toi A.
      • Lister W.S.
      • Fong K.W.
      How early are fetal cerebral sulci visible at the prenatal ultrasound and what is the normal pattern of early fetal sulcal development?.
      • Malinger G.
      • Kidron D.
      • Schreiber L.
      • et al.
      Prenatal diagnosis of malformations of cortical development by dedicated neurosonograhy.
      Fetal MRI can also be performed, either in lieu of or as an adjunct to neurosonology, to better characterize intracranial abnormalities. Fetal MRI may be particularly helpful in the assessment of potential cortical and brain sulcation abnormalities in which the detection and differentiation are limited by using ultrasound imaging alone.
      • Soares de Oliveira-Szejnfeld P.
      • Levine D.
      • Melo A.S.
      • et al.
      Congenital brain abnormalities and Zika virus: what the radiologist can expect to see prenatally and postnatally.
      Referral to a provider or center with experience in neurosonography (neurosonology) and fetal MRI should be considered.

      How do we counsel women with laboratory-based evidence of recent ZIKV infection?

      Unfortunately, at this time the main benefit of ZIKV screening and testing lies in patient counseling, with notable currently present degrees of uncertainty in ascribing absolute or attributable risk estimates. While there is not a current treatment option for maternal or fetal ZIKV infection, this is not dissimilar from antenatal genetic testing, and therefore, the value of diagnosis should not be undervalued.
      Moreover, although we still do not know the true risk of congenital ZIKV with maternal infection, there are several studies from which we can provide both risk and uncertainty estimates to our patients. First, in the United States, asymptomatic women may be at similar risk to symptomatic women.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      There appears to be a wide estimated occurrence and gestational ages of susceptibility reported, ranging from <1% to 29%.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      • 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.
      • Moura da Silva A.A.
      • Ganz J.S.
      • Sousa P.D.
      • et al.
      Early growth and neurologic outcomes of infants with probable congenital Zika virus syndrome.
      • França G.V.
      • Schuler-Faccini L.
      • Oliveira W.K.
      • et al.
      Congenital Zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation.
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      Whether infection in the first trimester has a higher rate of congenital ZIKV syndrome and microcephaly than infection in other trimesters is uncertain, but the risk being exclusive to windows of exposure in the periconeption and first trimester is highly improbable or reported not to be true.
      • Duffy M.R.
      • Chen T.H.
      • Hancock W.T.
      • et al.
      Zika virus outbreak on Yap Island, Federated States of Micronesia.
      • Mlakar J.
      • Korva M.
      • Tul N.
      • et al.
      Zika virus associated with microcephaly.
      • Martines R.B.
      • Bhatnagar J.
      • Keating M.K.
      • et al.
      Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses—Brazil 2015.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      • Meaney-Delman D.
      • Rasmussen S.A.
      • Staples E.
      • et al.
      Zika virus and pregnancy: what obstetric health care providers need to know.
      Society for Maternal-Fetal Medicine (SMFM) Publications Committee
      Ultrasound screening for fetal microcephaly following Zika virus exposure.

      CDC Concludes Zika Causes Microcephaly and Other Birth Defects. Available at: https://www.cdc.gov/media/releases/2016/s0413-zika-microcephaly.html. Accessed December 27, 2016.

      • Rasmussen S.A.
      • Jamieson D.J.
      • Honein M.A.
      • Petersen L.R.
      Zika virus and birth defects—reviewing the evidence for causality.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      • Costello A.
      • Dua T.
      • Duran P.
      • et al.
      Defining the syndrome associated with congenital Zika virus infection.

      Sarno M, Aquino M, Pimentel K, et al. Progressive lesions of the central nervous system in microcephalic fetuses with suspected congenital Zika virus syndrome. Ultrasound Obstet Gynecol, in press.

      • Oliveira D.B.
      • Almeida F.J.
      • Durigon E.L.
      • et al.
      Prolonged shedding of Zika virus associated with congenital infection.
      • Malinger G.
      • Lev D.
      • Lerman-Sagie T.
      Normal and abnormal development fetal brain development during the third trimester as demonstrated by neurosonography.
      • Simonin Y.
      • Loustalot F.
      • Desmetz C.
      • et al.
      Zika virus strains potentially display different infectious profiles in human neural cells.
      • Quicke K.M.
      • Bowen J.R.
      • Johnson E.L.
      • et al.
      Zika virus infects human placental macrophages.
      • Miner J.J.
      • Cao B.
      • Govero J.
      • et al.
      Zika virus infection during pregnancy in mice causes placental damage and fetal demise.
      In other words, at the present time, there is no trimester known to be absent of risk for congenital ZIKV syndrome.
      Regardless of our present inability to accurately or precisely ascribe relative or attributable risk for congenital ZIKV syndrome, there are several grounded statements that can be currently used when counseling women and their partners. First, all evidence to date suggests that just as with other vertical transmissions, only a minority of women with any ZIKV exposure (symptomatic or asymptomatic) will be at risk for congenital anomalies at birth.
      • Duffy M.R.
      • Chen T.H.
      • Hancock W.T.
      • et al.
      Zika virus outbreak on Yap Island, Federated States of Micronesia.
      • Mlakar J.
      • Korva M.
      • Tul N.
      • et al.
      Zika virus associated with microcephaly.
      • Martines R.B.
      • Bhatnagar J.
      • Keating M.K.
      • et al.
      Notes from the field: evidence of Zika virus infection in brain and placental tissues from two congenitally infected newborns and two fetal losses—Brazil 2015.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro—preliminary report.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      • Meaney-Delman D.
      • Rasmussen S.A.
      • Staples E.
      • et al.
      Zika virus and pregnancy: what obstetric health care providers need to know.

      CDC Concludes Zika Causes Microcephaly and Other Birth Defects. Available at: https://www.cdc.gov/media/releases/2016/s0413-zika-microcephaly.html. Accessed December 27, 2016.

      • Rasmussen S.A.
      • Jamieson D.J.
      • Honein M.A.
      • Petersen L.R.
      Zika virus and birth defects—reviewing the evidence for causality.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      • Costello A.
      • Dua T.
      • Duran P.
      • et al.
      Defining the syndrome associated with congenital Zika virus infection.

      Sarno M, Aquino M, Pimentel K, et al. Progressive lesions of the central nervous system in microcephalic fetuses with suspected congenital Zika virus syndrome. Ultrasound Obstet Gynecol, in press.

      • Malinger G.
      • Lev D.
      • Lerman-Sagie T.
      Normal and abnormal development fetal brain development during the third trimester as demonstrated by neurosonography.
      • Simonin Y.
      • Loustalot F.
      • Desmetz C.
      • et al.
      Zika virus strains potentially display different infectious profiles in human neural cells.
      • Quicke K.M.
      • Bowen J.R.
      • Johnson E.L.
      • et al.
      Zika virus infects human placental macrophages.
      • Miner J.J.
      • Cao B.
      • Govero J.
      • et al.
      Zika virus infection during pregnancy in mice causes placental damage and fetal demise.
      Second, we still do not have adequate data to inform counseling regarding the incidence of late congenital infection; therefore, long-term follow-up of neonates is essential.
      • Brasil P.
      • Pereira Jr., J.P.
      • Moreira M.E.
      • et al.
      Zika virus infection in pregnant women in Rio de Janeiro.
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.

      CDC Concludes Zika Causes Microcephaly and Other Birth Defects. Available at: https://www.cdc.gov/media/releases/2016/s0413-zika-microcephaly.html. Accessed December 27, 2016.

      • Rasmussen S.A.
      • Jamieson D.J.
      • Honein M.A.
      • Petersen L.R.
      Zika virus and birth defects—reviewing the evidence for causality.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      • Cauchemez S.
      • Besnard M.
      • Bompard P.
      • et al.
      Association between Zika virus and microcephaly in French Polynesia, 2013–2015: a retrospective study.
      • Costello A.
      • Dua T.
      • Duran P.
      • et al.
      Defining the syndrome associated with congenital Zika virus infection.
      • Oliveira D.B.
      • Almeida F.J.
      • Durigon E.L.
      • et al.
      Prolonged shedding of Zika virus associated with congenital infection.
      Lastly, women with an amniocentesis rRT-PCR positive for ZIKV likely have an appreciably greater risk of congenital ZIKV infection, and the neurocognitive effects are likely to be significant.
      • Dupont-Rouzeyrol M.
      • Biron A.
      • O’Connor O.
      • Huguon E.
      • Descloux E.
      Infectious Zika viral particles in breastmilk.
      However, because of the potential limitations of detection in dilute amniotic fluid volumes, absence of ZIKV by rRT-PCR cannot be considered a reliable means of ruling out congenital ZIKV infection.

      Regulatory challenges

      The mainstay of current diagnostic capabilities include rRT-PCR and IgM serology, with a heavy reliance on PRNT for serological testing interpretation and confirmation (Tables 1 and 2). Missing elements include IgG serologies and avidity testing. Moreover, access to timely diagnosis is limited by regional testing capacity and burden of testing. As more regions in the United States become endemic, this burden of testing and need for improved sensitivity and specificity will only increase.
      Expansion of testing modalities to both hospital-based and commercial laboratories, alongside city, county, and state health departments, will be crucial to capacity expansion. FDA regulations in an emerging pandemic are decidedly important because both test precision and accuracy are of utmost importance. However, these concerns must be balanced with diminished regulatory burden to enable the development of crucially important timely testing in the face of a congenital infectious pandemic.

      Knowing what we do not know and prioritizing what we need to learn

      Despite the advances in understanding the natural history and risk of ZIKV during the past year, including adverse pregnancy outcomes and long-term neurological effects, there are a multitude of questions that remain unanswered, which impede our ability to provide adequate counseling and timely pregnancy management.
      First, we do not know the risk estimates for fetal infection or CNS abnormalities in the setting of either maternal or amniotic fluid infection as measured by rRT-PCR. Second, there are multiple strains of ZIKV and the majority of ZIKV strains isolated in the Americas are of Asian lineage, and all are associated with fetal congenital infection. However, it is unknown whether African strains convey similar risk, which was previously underreported, or whether viral mutations have imparted the capacity for congenital malformations. Similarly, it is unknown whether infection with one ZIKV strain will confer life-long immunity and protection against all ZIKV strains.
      • Simonin Y.
      • Loustalot F.
      • Desmetz C.
      • et al.
      Zika virus strains potentially display different infectious profiles in human neural cells.
      Clarifying these distinct risks and potential immunity may be important in vaccine development, particularly if implemented in pregnant or reproductive-aged populations.
      Third, the exact mechanism of ZIKV entry into the fetal compartment has not been defined. Multiple lines of evidence suggest that placental cells, including placental macrophages, are permissive to replication.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      • Simonin Y.
      • Loustalot F.
      • Desmetz C.
      • et al.
      Zika virus strains potentially display different infectious profiles in human neural cells.
      However, the implications of these findings to fetal infection, particularly as a portal of delayed entry, are currently unknown.
      • Suter M.A.
      • Aagaard K.M.
      Disease watch: Zika virus—placental passage and permissivity for infection.
      • Quicke K.M.
      • Bowen J.R.
      • Johnson E.L.
      • et al.
      Zika virus infects human placental macrophages.
      • Miner J.J.
      • Cao B.
      • Govero J.
      • et al.
      Zika virus infection during pregnancy in mice causes placental damage and fetal demise.
      Fourth, maternal viremia and viruria has been observed to persist much longer during pregnancy than what is observed in the nonpregnant population, and it remains unknown whether this observation is predictive of pregnancies at highest risk of fetal abnormalities or a confounder that results from the pathophysiology and immunological changes during pregnancy.
      Fifth, information regarding consequences of neonatal infection is lacking. Because significant CNS growth and development continues into the third trimester, this may be an age group susceptible to ZIKV infection unique to what has been observed with ZIKV infection later in life. Long-term data regarding late neurological complications from maternal ZIKV infection during pregnancy are crucial for answering these questions. Similar to the long-term cardiovascular comorbidities observed in women who experienced preeclampsia during pregnancy, it remains unknown whether ZIKV portends a similar adverse neurological risk profile.
      Lastly, testing options need to be expanded and interpretation as to the risk of fetal congenital infection needs to be clearly understood. The introduction and validation of IgG testing would further improve determining patients at risk and the timing of infection. Further exploration into the incidence and prognostic value of prolonged maternal viremia is important but likely will not occur if patients are tested only within 2 weeks from exposure. Quantitative measures, similar to HIV viral load, may be able to predict latent infection and the subsequent risk of fetal infection. Similarly, detection and persistence of positive and negative strand ZIKV RNA by single molecule fluorescent in situ hybridization technology in different tissue compartments, akin to active viral replication, may act as a surrogate to viral load quantification that can be used in appropriate resource settings.

      Future prevention and interventions

      Creating therapeutic interventions against maternal-to-child-transmission of ZIKV infection poses a number of challenges. Recent findings that asymptomatic maternal infection can and will pass ZIKV to the unborn fetus at approximately the same rate as symptomatic maternal infection
      • Honein M.A.
      • Dawson A.L.
      • Petersen E.E.
      • 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.
      suggest that any efficacious population-based approaches would likely rely mostly on vaccinating women of reproductive age prior to conception.
      While several prototype ZIKV vaccines are under development and in clinical trials, especially at the National Institute of Allergy and Infectious Diseases, National Institutes of Health,

      Zika Virus Vaccines. Available at: https://www.niaid.nih.gov/diseases-conditions/zika-vaccines. Accessed Dec. 30, 2016.

      we can anticipate that a number of scientific and regulatory hurdles could ultimately slow down an advance toward licensure.

      Vannice KS, Giersing BK, Kaslow DC, et al. Meeting Report: WHO consultation on considerations for regulatory expectations of Zika virus vaccines for use during an emergency. Vaccine. 2016 Dec 1. pii: S0264-410X(16)30969-0. http://dx.doi.org/10.1016/j.vaccine.2016.10.034. [Epub ahead of print].

      Among them are the absence of known correlates of protection and potential safety concerns, including the possibility of vaccine-induced Guillen-Barre syndrome and the general safety and ethical issue surrounding the vaccination of pregnant women or women who plan to become pregnant in the near future. Traditionally such populations have presented a high bar in terms of vaccine regulatory science. Therefore, it is unclear whether we will have a safe and effective vaccine against ZIKV anytime soon.
      Similarly, several recent reports have suggested the use of existing drugs with potential fetal therapeutic potential.
      • Xu M.
      • Lee E.M.
      • Wen Z.
      • et al.
      Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen.
      • Retallack H.
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      Zika virus cell tropism in the developing human brain and inhibition by azithromycin.
      • Pascoalino B.S.
      • Courtemanche G.
      • Cordeiro M.T.
      • Gil L.H.
      • Freitas-Junior L.
      Zika antiviral chemotherapy: identification of drugs and promising starting points for drug discovery from an FDA-approved library.
      These range from drugs with well-characterized safety profiles in pregnancy (ie, erythromycin and niclosamide
      • Xu M.
      • Lee E.M.
      • Wen Z.
      • et al.
      Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen.
      • Retallack H.
      • Di Lullo E.
      • Arias C.
      • et al.
      Zika virus cell tropism in the developing human brain and inhibition by azithromycin.
      ) to the use of so-called orphan FDA-approved drugs with therapeutic potential but unknown fetal and maternal risk (ie, caspace inhibitors and the 5-HT3 antagonist palonosetron
      • Xu M.
      • Lee E.M.
      • Wen Z.
      • et al.
      Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen.
      • Pascoalino B.S.
      • Courtemanche G.
      • Cordeiro M.T.
      • Gil L.H.
      • Freitas-Junior L.
      Zika antiviral chemotherapy: identification of drugs and promising starting points for drug discovery from an FDA-approved library.
      ).
      Given ethical and regulatory hurdles with fetal- and pregnancy-related research in the United States and elsewhere, human clinical intervention trials are likely a long way off. Ergo, the ongoing development of primate ZIKV congenital infection models are of paramount importance because they will provide crucially important preclinical data.

      Conclusion

      In a mere 18 months, ZIKV has rapidly spread to more than 50 countries, including the United States, leaving in its wake devastating fetal and long-term neurological consequences. The push for precise risk estimates and testing of desperately needed preventative vaccine and therapeutic options necessitates highly sensitive and specific molecular antenatal diagnostic testing and parallel sonographic screening. Once these methodologies are readily available, large prospective, population-based observational and therapeutic trials will be both necessary and sufficient to estimate congenital risk with ZIKV infection and develop effective interventions for eradication or prevention. As evidenced by its spread north into Florida and Texas in recent months, the increasing burden to both obstetrical providers and our laboratory medicine colleagues will only increase, and we need to be armed with the diagnostic tools necessary to care for our patients in our local and global community.

        Glossary of Terms

        • Avidity: the overall binding strength between an antibody and an antigen, which is reflective of both the affinity of the antibody for its epitope and the antibody valency. In general, avidity testing is done with IgG isotype antibodies and lower avidity is seen with more recent infection.
        • Arbovirus: viruses transmitted by arthropod (including mosquitoes) vectors.
        • Dengue virus (DENV): DENV is the causative virus of dengue fever. Like ZIKV and DENV, CHIKV is a positive-stranded RNA arbovirus of the Flaviviridae family, genus Flavivirus. Unlike ZIKV, despite well-documented exposures in pregnancy, DENV does not cause congenital malformations.
        • Chikungunya virus (CHIKV): CHIKV is the causative virus of chikungunya. Like ZIKV, DENV is a positive-stranded RNA arbovirus of the Togaviridae family, genus alphavirus. It too is transmitted by the same Aedes spp. of mosquitos and, similar to both ZIKV and DENV infection, causes mild to severe symptoms of fever, rash, arthralgia, and headache. However, unlike ZIKV, it is not known to cause congenital malformations.
        • Flaviviridae family: the family of viruses to which both DENV and ZIKV belong. Humans and other mammals serve as their natural hosts, and they are transmitted primarily through arthropod vectors such as mosquitos and ticks. Other members of the family include yellow fever virus, West Nile virus, and St Louis encephalitis virus (all of the genus Flavivirus) as well as hepatitis C (genus Hepacivirus).
        • Microcephaly: acute or chronic slowing in head growth, resulting in a small head circumference relative to gestational age and body size, strictly defined as measuring greater than 3 SD (or Z scores) below the standardized population mean. There are multiple causes of microcephaly, including congenital infections (such as ZIKV, cytomegalovirus, toxoplasmosis, rubella and herpes virus, syphilis, and HIV), chromosomal abnormalities (including both aneuploidy and structural malformations, such as Down syndrome and microdeletion syndromes), exposure to toxic environmental pollutants and teratogens (such as arsenic and mercury, alcohol, and radiation), and acute trauma with hypoxic-ischemic injury. During the course of the current ZIKV pandemic, different organizations and publications have used a working definition of microcephaly of both –2 and –3 SD to define microcephaly.
        • Nucleic acid test: a generalized term referring to a nucleic acid test (NAT) or nucleic acid amplification test (NAAT), which are molecular-based approaches for detecting and quantifying a particular pathogen (virus or bacterium) in a specimen of blood or other tissue or body fluid.
        • Pandemic: an infectious epidemic that has documented spread across a large region, generally spanning continents.
        • Plaque reduction neutralization test (PRNT): quantification of the titer of neutralizing antibody for a virus. Values are provided as fold higher or lower titers by PRNT.
        • rRT-PCR (real-time reverse transcriptase–polymerase chain reaction): the NAT methodology used currently for ZIKV, rRT-PCR quantitatively monitors the amplification of ZIKV nucleic acid through the creation of complementary transcripts during the PCR (i.e., in real time).
        • Zika virus (ZIKV): the causative viral pathogen of Zika disease and congenital Zika syndrome.

      Acknowledgment

      We are grateful to Drs Ila Singh, Rodion Gorchakov, Diana Racusin, and Bob Garrison for critical review of the manuscript. We are additionally grateful to Drs Bhatnagar and Goldsmith of the CDC for their generous contributions of images in Figures 1 and 2 and Dr Miguel Parra Saavedra (CEDIUL-CEDIFETAL, Barranquilla, Colombia) for his collaborative contributions in Figure 4.

      Supplementary data

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