The current recommendations for ZIKV diagnostic practices are based on the understanding that ZIKV viremia lasts for less than a week after the onset of infection.15 During the week of symptomatic infection, RNA detection in serum or blood is considered to be the diagnostic method of choice. ZIKV RNA can be detected in urine for some days longer.22,23 ZIKV is also present in semen for an unknown length of time, and scattered reports of sexual transmission of ZIKV have emerged.24-28 ZIKV RNA testing is not recommended for pregnant women after the first week after the onset of clinical disease. The diagnosis is usually based on a ZIKV-specific antibody response with higher IgM and neutralizing-antibody responses to ZIKV than to other flaviviruses.13 However, we have detected ZIKV RNA in the serum of a pregnant woman at 4 weeks and 10 weeks after the clinical onset of ZIKV infection but not after delivery. We suspect that the persistent ZIKV viremia in the patient described here was a consequence of viral replication in the fetus or placenta, which had high viral loads. Therefore, in addition to current ZIKV diagnostics, the use of quantitative RT-PCR methods may be a potential diagnostic approach for ongoing placental or fetal infections in pregnant women. Notably, in this patient, the ZIKV RNA levels were slightly higher in the maternal serum than in the amniotic fluid. The dynamics of ZIKV RNA in the serum of infected pregnant women are not well understood and will need to be assessed in larger studies.

It is estimated that 80% of ZIKV infections are asymptomatic.29 Although the evidence of the association between the presence of ZIKV in pregnant women and fetal brain abnormalities continues to grow, the timing of infection during fetal development and other factors that may have an effect on viral pathogenesis and their effects on the appearance of brain abnormalities on imaging are poorly understood. Oliveira Melo et al.9 described two cases of ZIKV intrauterine infection associated with microcephaly and brain calcifications that were diagnosed by means of ultrasonography during the third trimester. Similar to the fetus in our report, the two fetuses in that study showed abnormal development of the corpus callosum and decreased brain parenchymal volume. In the case described by Mlakar et al.,10 the results of ultrasonography that was performed at 14 weeks and 20 weeks of gestation were normal, but microcephaly, ventriculomegaly, and calcifications were seen on ultrasonography at 29 weeks of gestation.10 In the larger Brazilian cohort, cerebellar atrophy was seen in a fetus at 20 weeks of gestation, but microcephaly was not diagnosed until 27 to 35 weeks in their cohort.11 In our study, a review of three sequential ultrasonographic images between 16 and 20 weeks showed a decrease in the fetal head circumferences from the 47th percentile to the 24th percentile, which suggests a reduction in the rate of brain growth during that period (Figure S3 in the Supplementary Appendix). We suspect these reductions in brain growth would have eventually met the criteria for microcephaly. As this case shows, the latency period between ZIKV infection of the fetal brain and the detection of microcephaly and intracranial calcifications on ultrasonography is likely to be prolonged. Negative ultrasonographic studies during this period would be falsely reassuring and might delay critical time-sensitive decision making. Serial ultrasonographic measurements of head circumference may provide useful predictive information. The superior soft-tissue resolution of fetal brain MRI might be more sensitive to developmental and encephaloclastic changes, thereby expediting the detection of evolving fetal brain anomalies.

This case is an early foray into the histopathological findings associated with ZIKV in the midgestational fetal brain. The overwhelming findings were of loss of intermediately differentiated postmigratory neurons through an apoptotic mechanism. There appeared to be preservation of more differentiated neurons in basal ganglia, limbic region, and dorsal spinal cord. The germinal matrix cells also appeared to be spared. Of note, the germinal matrix consists predominantly of glioblasts at midgestation with the majority of the neuroblasts having already migrated out of the zone. Although we could not evaluate neuronal precursor subtypes other than calretinin-expressing interneuron lineage cells, selective neuronal vulnerability to ZIKV injury requires further investigation.

The successful isolation of infectious ZIKV from human fetal brain fulfills Koch’s second postulate regarding the isolation of pathogens from a diseased organism and strengthens the association between congenital ZIKV infection and fetal brain damage. Although ZIKV RNA was found in several fetal organs and the placenta, the virus could be isolated only from brain tissue. The rapid isolation in a human neuroblastoma cell line suggests a predilection of the ZIKV strain for human neural lineage cells. This hypothesis is in line with the histopathological findings and the results of a recent study showing a high rate of ZIKV infection in cortical neural progenitor cells but not in embryonic or pluripotent stem cells.30 The close genetic relationship between the isolate in our report and Guatemalan ZIKV strains was consistent with the anamnestic knowledge on the likely geographical origin of the infection. We found a relatively high frequency of nonsynonymous mutations between the FB-GWUH-2016 genome and the Guatemalan ZIKV genome (Figure S4 in the Supplementary Appendix), a finding that could indicate viral adaptation to growth in the fetal brain. However, no amino acid changes were identical to previously reported alterations in the ZIKV genome sequenced from fetal brain tissue.10

In conclusion, our study highlights the possible importance of ZIKV RNA testing of serum obtained from pregnant women beyond the first week after symptom onset, as well as a more detailed evaluation of the fetal intracranial anatomy by means of serial fetal ultrasonography or fetal brain MRI. The isolation of ZIKV from fetal brain provides additional evidence for the association between congenital ZIKV infection and fetal brain damage and provides tools for further studies of the pathogenesis of ZIKV-induced microcephaly. Future studies at various gestational ages will offer better insight into the role of ZIKV infection in abnormal brain development and provide markers for its detection.