Abstract

On February 1, 2016, Zika virus (ZIKV) was designated as a Public Health Emergency of International Concern by the director of the World Health Organization. Zika virus has spread to numerous countries throughout the Americas, affecting up to an estimated 1.3 million people since the first reports from Brazil in early 2015. Although ZIKV infections are self-limiting, fetal microcephaly and ophthalmic anomalies have been associated with ZIKV infection as a possible result of perinatal transmission. The causal link between maternal ZIKV infection and newborn microcephaly and eye lesions has not been proven beyond doubt and is currently debated. We discuss the possibility of causality by ZIKV using Koch's postulates and the more appropriate Bradford Hill criteria. In this review, we summarize and consolidate the current literature on newborn microcephaly and eye lesions associated with ZIKV infection and discuss current perspectives and controversies.

As of February 1, 2016, Zika virus (ZIKV) is now a Public Health Emergency of International Concern [ 1 ]. The association of ZIKV with microcephaly and ophthalmic anomalies was first reported with the outbreak in Brazil in 2015 [ 2–7 ]. Given the possible association of ZIKV with congenital abnormalities in neonates, the US Centers for Disease Control and Prevention (CDC) recommends that pregnant women should not travel to places with ongoing ZIKV transmission, and male partners of pregnant women should consider refraining from having sex for the duration of the pregnancy if they have traveled or lived in areas where ZIKV has been reported.

Zika virus is an arbovirus first isolated from a rhesus monkey in the Zika Forest near Entebbe, Uganda in 1947 [ 8 ]. In 1952, Uganda and Tanzania reported the first human cases [ 9 ]. From the 1950s to 1980s, ZIKV spread throughout Africa and Southeast (SE) Asia. Until the early 2000s, there were no reported epidemic outbreaks of ZIKV.

The first reported epidemic occurred on the island of Yap in the Federated States of Micronesia in 2007 [ 10 ], and this was soon followed by another epidemic in French Polynesia (FP) in 2013 [ 11 , 12 ]. The FP epidemic was the largest in ZIKV history with more than 28 000 infections in the first 4 months of the epidemic. The first cases of Guillain-Barre Syndrome (GBS) associated with ZIKV were reported during the FP outbreak [ 13 ]. New ZIKV cases were subsequently found in Cook Island, Easter Island, and New Caldonia [ 14–16 ]. The origins of the emerging pandemic began in the northeast regions of Brazil with the first reported cases in May 2015 [ 17 ]. Zika virus has since spread throughout Central and South America and the Caribbean, including the US Virgin Islands and Puerto Rico [ 18–20 ].

The Virus and Entomology

Zika virus is a positive sense, single-stranded ribonucleic acid (RNA) virus of the Flaviviridae family. It is an arbovirus transmitted to humans via the bite of an infected mosquito of the Aedes genus, primarily Aedes aegypti [ 21 ]. Aedes polynesiensis , Aedes albopictus , Aedes africanus , and Aedes hensilli have also been considered as vectors for ZIKV [ 21–23 ]. Aedes aegypti and A albopictus , found throughout much of the Americas, also transmit chikungunya and dengue viruses that have similar clinical presentations. The ZIKV genome contains 10 794 nucleotides encoding 3419 amino acids [ 24 ]. Through genome studies, ZIKV is closest in relation to the Spondweni virus, another flavivirus that is a mosquito-borne pathogen [ 25–27 ].

With the spread of ZIKV initially throughout Africa and SE Asia, genomics of the virus showed separate African and Asian lineages. Genomic testing of ZIKV causing the outbreak on Yap Island in Micronesia found it to be related to the Asian rather than the African lineage [ 28 , 29 ]. The FP strain seems to be related most closely to the SE Asian strains rather than from the Yap Island outbreak, suggesting introduction into FP independent of the Yap island outbreak. The genome of the ZIKV causing the Brazilian epidemic is closest to that of ZIKV from FP, which suggests the continued eastward spread of the Asian lineage [ 17 ].

The potential for ZIKV spread looms large. Mexico, Colombia, and the United States have an estimated 30.5, 23.2, and 22.7 million people, respectively, living in regions that are conducive to the spread of ZIKV by A aegypti and A albopictus [ 30 ]. Given the distribution of A aegypti mosquitos, ZIKV has huge potential to spread throughout these countries in the coming months.

Diagnostic Testing for Zika Virus Infections

Zika virus diagnosis relies mainly upon the detection of ZIKV RNA in body fluid specimens. Reverse-transcriptase polymerase chain reaction (RT-PCR) and viral isolation in blood specimens collected less than 5 days after the onset of symptoms are the reference techniques [ 31 ]. The viremic period in humans can be short, potentially from the 3rd to the 5th day after the first occurrence of symptoms. Several studies have found that ZIKV can be isolated for almost 1 week longer in the urine than compared with the serum [ 32 , 33 ]. Zika virus has also been isolated from the saliva, amniotic fluid, and semen [ 34 , 35 ]. Reverse-transcriptase PCR has also shown the presence of ZIKV in neuronal tissues [ 36 ].

Zika virus serology may also be useful for diagnosis of ZIKV infections, although this may be complicated by cross-reactivity. Zika virus-specific immunoglobulin (Ig)M antibodies can be detected by enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assays after day 5 from the onset of symptoms. The CDC has obtained Emergency Use Authorization from the US Food and Drug Administration (FDA) to use the Zika IgM Antibody Capture ELISA for the detection of IgM antibodies in serum and/or cerebrospinal fluid. It is recommended to test potentially affected individuals as soon as 4 days and up to 12 weeks after potential infection [ 37 ]. Antibodies can cross-react with other genetically related viruses such as other members of the Flaviviridae family (ie, Yellow fever, Dengue, West Nile), making serologic tests difficult to interpret [ 38 , 39 ]. In addition, IgM and IgG levels may be low, making diagnosis difficult [ 24 ]. Therefore, a plaque reduction neutralization test should be used to improve the specificity of the antibodies and eliminate cross-reactivity to other Flaviviridae viruses [ 24 ].

Samples for both the RT-PCR and the serologic studies should be sent via local or regional public health agencies to the CDC. Although commercial tests are currently being tested for use, none are currently recommended by the Pan American Health Organization (PAHO), World Health Organization (WHO), or CDC. However, Quest Diagnostics has developed a commercial PCR test that received emergency use authorization by the FDA as of April 2015. If the healthcare provider is unable to send tests to the CDC, they have the option of sending tests to the WHO Collaborating Center.

Clinical Presentation

An estimated 80% of individuals infected by ZIKV appear to be asymptomatic [ 10 , 24 ]. When symptoms occur, they usually appear approximately 3 to 12 days after the bite of an infected mosquito. Symptoms are described as “dengue-like”, which consists of fever, myalgia, arthralgia, retro-orbital pain, conjunctival hyperemia, maculopapular rash that may include the palms and soles, vertigo, edema of extremities, pruritus, and possibly vomiting. Before the FP epidemic, there were no reports of severe presentations from ZIKV infection. Possible association with GBS was first mentioned in the FP outbreak [ 11 , 12 ]. The potential associations with microcephaly and eye lesions were first raised during the Brazil outbreak with subsequent reports in FP [ 2 , 7 , 40 , 41 ].

Association of Microcephaly With Zika Virus

Microcephaly was first reported during the current outbreak in Brazil. The Brazilian Ministry of Health (MoH) reported an increased birth prevalence of microcephaly in the northeast of Brazil. The previously reported incidence of microcephaly was 0.5 cases per 10 000 live births, estimated by reviewing birth certificates [ 4 ]. The PAHO published an alert regarding an increase in cases of microcephaly in Brazil [ 42 ]. In December 2015, PAHO reported that 2 pregnant women whose fetuses were found to have microcephaly by prenatal ultrasound had positive ZIKV on RT-PCR of the amniotic fluid [ 43 ]. There was another report of positive ZIKV RT-PCR from multiple body tissues in an infant with microcephaly who died soon after birth [ 43 ]. These reports alerted the MoH in Brazil, the European Centre for Disease Prevention and Control, and the CDC of the possible association of microcephaly and ZIKV infection [ 44 ].

The reporting of baseline prevalence of microcephaly in Brazil has several limitations [ 4 ]. First, the expected estimate of 0.5 cases per 10 000 live births is lower than the expected estimate of 1–2 cases per 10 000 live births [ 45 ], which suggests cases of microcephaly are generally underreported on birth certificates in this region. Second, infant head size was not routinely recorded before the MoH alert. Therefore, cases of microcephaly may have previously been missed. In addition, hospitals were on heightened alert after the MoH announcement, and the subsequent media coverage it received could have led to increased surveillance and reporting. Finally, different definitions of microcephaly have been used in Brazil that have affected the reporting of microcephaly prevalence. Before December 8, 2015, Brazil's MoH used the definition of suspected microcephaly to help determine neonates with potential ZIKV infection as either a term neonate with a head circumference (HC) ≤33 cm or a preterm neonate with HC <3rd percentile using Fenton curves. The definition for microcephaly in term neonates was changed on December 8, 2015 by the MoH to HC ≤32 cm, but the MoH did not change the definition for preterm neonates. This small change is believed to have decreased the sensitivity of detecting ZIKV-associated microcephaly cases from 92% to 86% and increased the specificity from 79.3% to 93.8%. The estimated annual number of suspected cases of ZIKV-associated microcephaly cases dropped from approximately 602 thousand to 178 thousand in Brazil [ 46 ].

Nevertheless, the currently reported rate of 20 cases of microcephaly per 10 000 live births is much above the estimated average for microcephaly. The increased incidence of microcephaly that coincided with the ZIKV epidemic suggested an association with ZIKV. Further strengthening this concern, 2 pregnant women from the northeast of Brazil were found to have positive RT-PCR in the amniotic fluid of babies with microcephaly [ 2 ]. One infant had brain atrophy with coarse parenchymal calcifications along with corpus callosum and vermian dysgenesis. The second infant had markedly asymmetric cerebral hemispheres and absence of corpus callosum. This report highlighted the concerns for intrauterine transmission of ZIKV and the association with microcephaly. Further supporting evidence came from the CDC where researchers tested samples from 2 pregnancies that ended in miscarriages as well as infants with microcephaly that died shortly after birth. All individuals were from Brazil and positive for ZIKV. All 4 mothers had reported a febrile illness with rash during their pregnancies. The tissue from the brains of the full-term infants was positive for ZIKV, suggesting intrauterine transmission [ 4 ].

The strong neurotropism of ZIKV was first described in 1952 when intraperitoneally infected mice developed ZIKV infection in the brain, suggesting that ZIKV crosses the blood-brain barrier [ 47 ]. In a separate study, approximately 2 decades later, mice were given intracerebral injections with ZIKV, which led to astrocyte enlargement with destruction of pyriform cells in the area of the brain known as Ammon's horn [ 48 ]. Viral “factories” were found within both neurons and astrocytes and consisted of multiple vesicles within a network of endoplasmic reticulum showing active replication of the virus in the neural tissue [ 48 ]. However, most of the infected neurons did not appear to show evidence of gross destruction. Therefore, it was hypothesized that cell destruction was due to an immunological response to the viral infection. Even though the virus directly infects the brain tissue, it is still unclear whether ZIKV infection and/or replication lead to the development of microcephaly. One author postulated that ZIKV could lead to the development of microcephaly by altered centrosome function or number, which has been shown to induce microcephaly [ 49 , 50 ], even though this hypothesis has not been directly tested for ZIKV.

Further strengthening the argument for ZIKV leading to microcephaly, a microcephalic infant was found to have ZIKV in the brain [ 36 ]. The mother developed a febrile illness with rash during the 13th week of gestation. She had prenatal ultrasounds at 14 and 20 weeks, which were within normal limits, but ultrasound at 29 weeks showed microcephaly with calcifications in the fetal brain and placenta. After elective abortion, ZIKV was found in the fetal brain tissue along with microcephaly in addition to multifocal dystrophic calcifications in the cortex, widened Sylvian fissures, almost complete agyria, and hydrocephalus. The other fetal organs were negative for ZIKV, supporting the theory that ZIKV has strong neurotropism. In addition, cerebral cortex development appeared to have arrested just after 20 weeks gestation. The complete genome sequence of ZIKV isolated from the brain was consistent with the Asian lineage strain currently present in Brazil, supporting the theory that the transmission had occurred in Brazil.

Ophthalmic Anomalies and Implications

In January 2016, the association of ophthalmic abnormalities with ZIKV was reported [ 40 ]. The authors reported 3 children in Brazil, all with microcephaly and intracerebral calcifications, who had unilateral pigment mottling of the macula and loss of foveal reflex. One infant had well defined macular atrophy. Zika virus was diagnosed clinically in both the mothers and their infants, but no serological tests or RT-PCR tests were performed to confirm ZIKV infection. In early February 2016, a report of 29 infants with microcephaly (defined as a cephalic circumference of ≤32 cm) from Bahia, Brazil described 10 (∼34.5%) of the 29 children had eye abnormalities including focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, lens subluxation, and bilateral iris colobomas [ 5 ]. Bilateral eye involvement was reported in 70% of the children with eye abnormalities. Of the 29 mothers, 23 (79.3%) had reported clinical signs and symptoms of ZIKV infection with 18 having signs and symptoms in the 1st trimester and 4 in the 2nd trimester. Neither the mothers nor the infants were tested by serology or RT-PCR to confirm ZIKV infection. Another case series reported 10 infants with microcephaly and intracerebral calcifications who all had ophthalmic abnormalities [ 7 ]. Of the 7 mothers who had clinical symptoms of ZIKV, 6 (85.7%) had symptoms in the 1st trimester and all mothers had normal eye exams. The infants had various abnormalities that included optic nerve hypoplasia, macular pigment mottling, foveal reflex loss, or chorioretinal macular atrophy, but the anterior segments of all eyes were normal. Four of these infants had myopia, a finding that is different than the usual hyperopia seen in normal infants [ 51 ].

Macular and chorioretinal disease in infants can significantly impact visual development in infants. The choroid is the highly vascularized area between the sclera and the retina, and its inflammation may lead to scarring of the retina and visual loss. Chorioretinal scarring or active chorioretinitis is the most characteristic ophthalmic finding in neonates to suggest a congenital infection such as with cytomegalovirus (CMV) and toxoplasmosis infections [ 52–54 ]. Although common congenital infections were ruled out in microcephalic infants with ocular abnormalities in the majority of the studies aforementioned, the lack of ZIKV testing is a significant limitation. Further studies will need to look into the possibility of ZIKV as causative of these ophthalmic abnormalities. However, given the current concern, infants with suspicion for having ZIKV infection should have evaluation and follow up by an ophthalmologist.

Other Possible Associations

Although no studies have reported hearing loss in neonates with ZIKV infection, the CDC recommends that all neonates with suspected ZIKV infection should have routine hearing tested after birth and at 6 months of age. This second recommendation is because infants with CMV infections have shown progressive sensorineural hearing loss that may not be present on the initial hearing screen [ 55 , 56 ].

Possible associations with other congenital anomalies were reported in the first 35 patients enrolled in the Brazilian Society of Medical Genetics-Zika Embryopathy Task Force Registry in Brazil [ 4 ]. Of the 35 patients enrolled, 4 (11%) were diagnosed with arthrogryposis (congenital contractures) and 5 (14%) were diagnosed with talipes (clubfoot), suggesting involvement of the central and peripheral nervous systems.

Developmental delay is also highly likely and may be related to the neuronal injury caused by the ZIKV infection. There are no reports of developmental outcomes of children living in ZIKV-endemic regions or children who have been diagnosed with ZIKV infection. Given that other intrauterine infections can lead to developmental delay [ 57 ], close follow up with a developmental pediatrician is recommended for patients suspected of having ZIKV infection.

The Dispute of Pyriproxyfen

Zika virus has only recently been considered as the etiology behind microcephaly and ophthalmic anomalies seen in neonates. In evaluating for other possible etiologies for these findings in neonates, infectious, genetic, metabolic, or environmental etiologies were considered. The question of whether pyriproxyfen, a mosquito larvicide that was introduced into Brazilian drinking water in the past 18 months, is responsible for microcephaly has been debated, but it is now largely ruled out as a likely etiology. According to the WHO, pyriproxyfen was evaluated in 1999 and 2001. In 1999, a 1-year study in dogs concluded that pyriproxyfen had a low acute toxicity [ 58 ]. In 2001, the safety of pyriproxyfen as a mosquito larvicide in potable water was assessed and found that intake at the target concentration would not create unacceptable risks to humans drinking the water [ 58 ]. In addition, in another document release by the WHO in 2007 entitled, “Pyriproxyfen in Drinking Water” [ 59 ], pyriproxyfen was studied in rats and rabbits for the possibility of adverse neurodevelopmental outcomes. Even at the highest doses of 1000 mg/kg pyriproxyfen, no developmental toxicities were observed. Given the wide distribution of pyriproxyfen throughout the world and the lack of reports of microcephaly elsewhere, the likelihood of pyriproxyfen as the etiology for the microcephaly and ophthalmic abnormalities appears minimal [ 60 ]. However, further research may be required to clarify the role of pyriproxyfen in the causation of microcephaly.

DISCUSSION

The current spatial and temporal associations of microcephaly, ophthalmic abnormalities, and other anomalies with the ZIKV epidemic strongly suggest causality. Koch's [ 61 ] postulates, proposed in 1891, is still used as a benchmark in proving causality of disease by infectious agents. The postulates include isolating the pathogen in pure culture, reinfecting a susceptible person in whom the disease develops, and then reisolating the pathogen [ 61 ]. Koch's [ 61 ] postulates are not satisfied in diseases caused by viruses or prions, where the infectious agent cannot be isolated in pure culture. Moreover, the presence of subclinical or asymptomatic infections with ZIKV makes it difficult to satisfy Koch's [ 61 ] postulates.

Another means of assessing causality is the use of Bradford Hill's [ 62 ] criteria put forth in 1965. Bradford Hill's [ 62 ] criteria are a framework for examining causal relationships and include assessing the association for strength, consistency, specificity, temporality, biological gradient, plausibility, coherence, reversibility (experiment), and analogy [ 63 ]. Causality is inferred by the fulfillment of these criteria, as many as possible. Reports of microcephaly and central nervous system anomalies in Brazil as well as FP satisfy the “consistency” criterion; however, the same association occurs in a variety of settings. The ZIKV epidemic preceded the reports of microcephaly by 5 months, confirming the “temporality” criterion. In addition, reports of afflicted babies born to mothers with infections in the 1st and 2nd trimesters of pregnancy strengthen the temporality criterion. Knowing that other viral intrauterine infections, such as CMV and rubella, lead to similar conditions in neonates, ZIKV intrauterine infection fulfills “coherence” and “analogy” to other similar pathogens. Lastly, confirmation of the presence of ZIKV in amniotic fluid and the fetal brain of affected pregnancies lends credence to “biologic plausibility”.

Although the aforementioned Bradford Hill [ 62 ] criteria seem applicable in the case of ZIKV infections, others are more difficult to appraise. Until more epidemiologic studies on ZIKV infections and disease states are reported, we will not be able to measure the “strength” of the association between exposure to ZIKV and development of congenital anomalies. Because microcephaly and ophthalmic anomalies have a variety of causes, “specificity” cannot be established. “Biologic gradient” may need to be studied in animal models. Finally, if preventative steps to remove the exposure lead to a decrease in the frequency of the effect or disease state, then “reversibility” or “experiment” criterion is fulfilled. Only with time can a decrease in ZIKV transmission in an area demonstrate subsequent decreases in the rates of microcephaly and/or ophthalmic anomalies. Therefore, the experiment criterion has not yet been fulfilled. Although all 9 criteria need not be fulfilled in order to infer causality, strength of causality inference is enhanced by the number of criteria fulfilled.

There is no recommendation as to how many criteria need to be fulfilled to infer causality. However, the more criteria satisfied, the stronger the argument for inferring causality. With regards to ZIKV infection and its causality of neonatal microcephaly and ophthalmic anomalies, the Bradford Hill [ 62 ] criteria strongly suggest causality.

CONCLUSIONS

The causal link between maternal ZIKV infection and newborn microcephaly and eye lesions has not been proven beyond doubt, but many aspects of this epidemic strongly suggest causality. Given the increased incidence of ZIKV infections and the continued spread throughout the Americas, further research into causality of neural and ophthalmic pathology is paramount from a public health perspective.

Acknowledgments

Author contributions. G. V. searched the literature, drafted and revised the manuscript, and approved the final manuscript for submission. L. M. reviewed and revised the drafted manuscript and approved the final manuscript for submission. M. P. developed the concept, assisted in writing the manuscript, and revised and approved the final manuscript for submission.

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

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