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Defining cases of Zika virus (ZIKV) infection is a critical challenge for epidemiological research. Due to ZIKV’s overlapping clinical features and potential immunologic cross-reactivity with other flaviviruses and the current lack of an optimal ZIKV-specific diagnostic assay, varying approaches for identifying ZIKV infections have been employed to date. This paper presents the laboratory results and diagnostic criteria developed by the Microcephaly Epidemic Research Group for defining cases of maternal ZIKV infection in a cohort of pregnant women with rash (N = 694) recruited during the declining 2015–2017 epidemic in northeast Brazil. For this investigation, we tested maternal sera for ZIKV by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Immunoglobulin (Ig) M and IgG3 enzyme-linked immunosorbent assays (ELISAs), and Plaque Reduction Neutralization Test (PRNT50). Overall, 23.8% of participants tested positive by qRT-PCR during pregnancy (range of detection: 0–72 days after rash onset). However, the inter-assay concordance was lower than expected. Among women with qRT-PCR-confirmed ZIKV and further testing, only 10.1% had positive IgM tests within 90 days of rash, and only 48.5% had ZIKV-specific PRNT50 titers ≥20 within 1 year of rash. Given the complexity of these data, we convened a panel of experts to propose an algorithm for identifying ZIKV infections in pregnancy based on all available lines of evidence. When the diagnostic algorithm was applied to the cohort, 26.9% of participants were classified as having robust evidence of a ZIKV infection during pregnancy, 4.0% as having moderate evidence, 13.3% as having limited evidence of a ZIKV infection but with uncertain timing, and 19.5% as having evidence of an unspecified flavivirus infection before or during pregnancy. Our findings suggest that integrating longitudinal data from nucleic acid and serologic testing may enhance diagnostic sensitivity and underscore the need for an on-going dialogue regarding the optimization of strategies for defining cases of ZIKV in research., Author summary On February 1, 2016, the World Health Organization declared a Public Health Emergency of International Concern following a “cluster of microcephaly cases and other neurological disorders in Brazil” and highlighted the “urgent need” for coordinated international efforts to investigate the relationship between maternal Zika virus (ZIKV) infections and microcephaly. Due to the lack of a standard algorithm, resultant epidemiological investigations have utilized different strategies for defining cases of ZIKV infections in pregnancy. Here, we report the experience of the Microcephaly Epidemic Research Group in Pernambuco, Brazil, in evaluating 694 pregnant women presenting with rash (i.e., a common sign of ZIKV infection) during the 2015–2017 Latin American outbreak. Integrating time-sensitive data from both nucleic acid amplification testing and serologic assays, a panel of experts developed an evidence-graded set of criteria for identifying cases of maternal infection. When applied to the cohort, nearly one-third of the participants were categorized as having robust or moderate evidence of being infected with ZIKV in pregnancy. The classifications described in this investigation will enable scientists to investigate maternal ZIKV infection and estimate the absolute and relative risks of adverse pregnancy outcomes. The results also underscore the importance of on-going efforts to develop robust diagnostic assays for ZIKV.

Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections.