Differential Shedding and Antibody Kinetics of Zika and Chikungunya Viruses, Brazil

In seroconversion panels obtained from patients from Brazil, diagnostic testing for Zika virus infection was improved by combining multiple antibody isotypes, techniques, and antigens, but sensitivity remained suboptimal. In contrast, chikungunya virus diagnostic testing was unambiguous. Recurrent recent arbovirus infections suggested by serologic data and unspecific symptoms highlight the need for exhaustive virologic testing.

by commercial and in-house serologic tests. In a widely used nonstructural (NS) protein 1 antigen-based ELISA, Zika virus IgM seroconversion was low (33% [5/15]), whereas CHIKV IgM seroconversion was 100% using an envelope-based ELISA (p<0.0001 by Fisher exact test) (Figure 2, panel A; Appendix Tables 1,2). Use of an in-house envelope-based ELISA increased the Zika virus IgM detection rate to 50% (7/14), and use of a commercially available μ-capture ELISA increased it to 43% (6/14) (Figure 2, panel A). Despite differential sensitivity, concordant results from different assays suggest comparable specificity of IgM detection (Appendix Table 1). The use of NS1-based IgA as a marker of acute infection increased the detection rate to 53% (8/15) over that of the NS1-based IgM ELISA. All IgM-positive patients also showed IgA, which increased during acute and subacute phases of infection and decreased during convalescence ( Figure 2, panel B; Appendix Figure 3). This finding supports the usability of IgA-based serologic methods as an alternative or additional marker to IgM-based methods to detect acute Zika virus infection.
The detection rate increased 2-fold when we used NS1based IgA from when we used NS1-based IgM 5-9 dpo, suggesting that IgA could be used at later stages of infection (Appendix Figures 1,5). Our findings indicate that serologic detection of acute Zika virus infection can be improved ≈2-fold by use of different antibody classes and antigens but remains poorly sensitive in flavivirusendemic areas.
All Zika virus-infected patients showed IgG responses across the 4 time points in >1 assay ( Figure 2, panels C, D). Plaque reduction neutralization tests (PRNTs) were negative for 2 of 14 rRT-PCR-confirmed Zika virus cases detected by NS1-based IgG ELISA. Without rRT-PCR confirmation, these cases would have been classified false positive (Appendix Table 1). This observation might be explained by differential sensitivity of PRNT and ELISA (10) or false-positive results of the Zika virus NS1-based ELISA in secondary flavivirus infections (6). Similarly, the antibody kinetics of Zika virus NS1-based IgG, envelopebased IgG, and PRNT suggested either relatively early IgG seroconversion or cross-reactivity during acute stages of infection resulting from unspecific immune responses against other flaviviruses (11) (Figure 2, panel D). In contrast, CHIKV IgG seroconversion occurred at later stages ( Figure 2, panel D; Appendix Figure 5), possibly associated with strong and long-lasting CHIKV-specific IgM responses (Appendix Figure 4).

Conclusions
We provide pivotal data on Zika virus and CHIKV diagnostic challenges in a Latin American setting. Limitations of our study include the relatively small number of patients, sampling at heterogeneous dpo and heterogeneous numbers of samples per dpo, and lack of acutely DENVinfected patients to assess test specificity. The strengths of our study include rRT-PCR-confirmed infections, waiving the need to define serologic assays prone to crossreactivity as standards, sampling during Zika virus and CHIKV outbreaks (1,2), sequential sampling of patients up to 90 dpo, use of multiple antigens and immunoglobulin classes, and the combination of molecular and serologic testing methods.
Our data suggest reliable diagnostic testing for acute CHIKV infections by IgM detection from 5 dpo onward. This finding might enable waiving labor-intense and costly molecular protocols in many patients, minimizing costs for public health systems and cohort studies investigating arbovirus pathogenesis. However, reliability of CHIKV serologic diagnostic tests must be reevaluated for co-circulating genotypes (12) and for the antigenically related Mayaro virus (13) if it emerges in Latin America.
The difficulties of adequately diagnosing Zika virus infections in areas to which it is endemic have major implications for public health. Reliable testing for flaviviruses in such areas will be key for epidemiologic studies on Zika virus and assessments of the safety of flavivirus vaccination programs, as illustrated by more severe dengue infections in DENV-seronegative individuals who received a live attenuated dengue vaccine (14). For pregnant women and couples intending pregnancy, accurate diagnosis of acute or past Zika virus infection is crucial. The steep increase in requests for abortion in Latin America illustrates the effect of the Zika virus outbreak on reproductive medicine (15).
Our results highlight that definite exclusion of acute Zika virus infections is challenging in a considerable proportion of patients. However, although limited by a small number of samples, our data highlight the attainability of more accurate Zika virus diagnostic testing by combining molecular and serologic tests using different antibody classes, antigens, and methods and by monitoring an increase of IgG titers in follow-up serum samples. Our data will help clinicians and health authorities build reliable diagnostic algorithms for Zika virus and CHIKV and highlight that exhaustive testing of arboviral infections is required for attributing frequencies of infection-specific symptoms. This work was supported by the European Union's Horizon 2020 Research and Innovation Programme through the ZIKAlliance project (grant agreement no. 734548), the German Centre for Infection Research through the ZIKApath project, by the Conselho Nacional de Desenvolvimento e Pesquisa (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).     Figure 1.