Table 1.

Salient features of serologic assays for diagnosing ZIKV infection and considerations for assay selection.

Serologic Basis	Assay	Mechanism and Output	Advantages	Limitations	Utility in Zika Intervention Trials
IgM	MAC-ELISA	Captures human IgM from serum or other fluid (CSF), tests for binding to virus or VLP. Result expressed as P/N ratio.	Substantial experience with assay type Clear guidance on assigning positive and negative results	Flavivirus cross-reactivity reduces specificity IgM may persist beyond acute phase infection Requires local lab optimization	Most useful in detecting acute symptomatic Zika Paired testing of acute and convalescent samples that demonstrates seroconversion or increasing signal is most consistent with acute ZIKV infection A single positive result is typically only supportive of a Zika diagnosis A single positive result from a fetal or neonatal specimen is presumably very useful and specific for ZIKV infection
	In-house	Platform may vary. Can arrange as IgM capture, Ag capture, or direct Ag coating. Output could be P/N or continuous output of background-subtracted OD	May be less expensive than commercial options Can accommodate Ag substitution in the same platform Can give a magnitude of positivity, not simply a binary result	Unlikely to be robustly validated for clinical trial use Similar limitations as MAC-ELISA Methods may not be widely reproduced or adopted by other similar studies	Similar implementation considerations as MAC-ELISA Likely most useful in smaller trials assessing symptomatic Zika infection OR when IgM results are part of secondary outcomes measures rather than the primary outcome
	Kits	Prefab buffers and plates used. Ag may be prM/E or NS1. Readouts typically categorical if not binary based on simple colorimetric reading.	Easy to use Higher throughput vs MAC-ELISA Standardized interpretation Deployable to most lab and field settings	Each kit has to be considered individually Only a few assays with EUA level FDA approval May be costly May not be extensively evaluated for populations where multiple flaviviruses are endemic	Good option for high throughput IgM testing with consistent quality of acute symptomatic subjects (not for testing paired acute-convalescent specimens) Good option for on-site testing in resource-limiting settings Must give attention to how quickly maximum sensitivity is achieved after symptom onset Ultimate case designation should still meet other criteria (as true for other IgM-based tests)
IgG	In-house	Platform may vary. Can arrange as IgG capture, Ag capture, or direct Ag coating. Output could be P/N, typically continuous output of background-subtracted OD	May be less expensive than commercial options Can accommodate Ag substitution in the same platform Can give a magnitude of positivity, not simply a binary result Also can determine total Ab titer to assay Ag	Unlikely to be robustly validated for clinical trial use Flavivirus cross-reactivity reduces specificity	Particularly useful in large scale assessment of incident ZIKV infections (seroconversions) in areas with low ZIKV and DENV seroprevalence IgG seroconversion may be an efficient method for identifying inapparent ZIKV infections Paired testing of acute and convalescent samples that demonstrates seroconversion or increasing signal is consistent with acute ZIKV infection Data on magnitude or Ab titer can be useful in investigating correlates of immunity
	Kits	Prefab buffers and plates used. Ag may be prM/E or NS1. Readouts typically categorical if not binary based on simple colorimetric reading.	Easy to use High throughput similar to In-house Standardized interpretation Deployable to most lab and field settings	Flavivirus cross-reactivity reduces specificity Only a few assays with EUA level FDA approval May be costly May not be extensively evaluated for populations where multiple flaviviruses are endemic	In contrast to IgM kits, IgG kits may be good option for testing paired acute-convalescent specimens to support assessment of symptomatic Zika, though would not be adequate for “lab confirmation” Good option for on-site testing in resource-limiting settings, but these assays are generally not designed or validated for seroprevalence determination in late convalescence, limiting their utility
	Novel IgG	Variable “Novel” here refers to assays that leverage technologic advances	May increase specificity, throughput, automation, reduce sample volume May use multiplexing to simultaneously assess several outcomes	Not widely validated May still be costly or require sophisticated lab capacity/analytic equipment (though there are serious efforts to engineer low costs tests deployable to resource-limited settings)	No novel tests with sufficient development and translational research to favor use in clinical trials currently These assays may constitute key methodologic advances that warrant implementation in near future, perhaps sooner in smaller trials and pilots
IgM or IgG	PRNT FRNT	Measures the functional activity of serum to inhibit live virus infection of target cells. An Ab titer is calculated (i.e., FRNT50)	Most accepted method for serologic diagnosis of flaviviruses Greater specificity compared to IgM and IgG assays Gives information about the flavivirus exposure history, may differentiate primary and secondary flavivirus infections	Requires BLS-2 and culturing infectious virus Labor intensive Several days turn-around time A number of assay components and conditions lead to large lab-to-lab variation Specificity may be reduced in secondary flavivirus infection, particularly in early convalescence	Likely most appropriate to start with neutralization testing to assess convalescent serostatus in areas with high DENV or ZIKV seroprevalence Paired testing of interval samples can detect inapparent ZIKV infection with good specificity for distinguishing from DENV Data on neutralization titer can be very useful in investigating correlates of protection (neutralizing Ab are often hypothesized to be the mediators of lasting protective immunity against ZIKV infection)
	NS1 BOB	Measures the ability of Ab in serum to block the binding of a ZIKV NS1-specific mAb. Readout is percent blockade (0–100%), but initial report includes an ROC curve analysis for binary outcome	High sensitivity and specificity for ZIKV Deployable to most lab settings	Insensitive in 1st week of symptoms Unknown long-term sensitivity (i.e., could there be seroreversion after 1 or more years?)	Could be very useful in assessing recent ZIKV infection in seroprevalence/sero-incidence studies Can be used for testing paired acute-convalescent specimens to serologically diagnose symptomatic Zika May not have sufficient validation for primary outcome measure, but could be efficient and broadly deployable assay for assessing secondary outcomes Particularly useful in monitoring for ZIKV infection in vaccine trials in which subject are immunized with vaccines that lack NS1
	Novel neut	Variable “Novel neut” here refers to neutralization assays that leverage technologic advances	May allow neutralization testing without requiring live virus May improve throughput and automation May be less challenging to standardize than PRNT/FRNT	May require specialized lab capacity or analytic equipment Limited validation to date May have similar reduced specificity in secondary and early convalescent flavivirus infection	Novel neutralization tests could enter trial design strategies more quickly than IgG assays. Validation that the methods give results that are consistently relatable to PRNT/FRNT titers may be sufficient to favor implementation without extensive testing in large populations

ZIKV, Zika virus; ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; IgG, immunoglobulin G; CSF, cerebrospinal fluid; P/N, a ratio of the optical density of a test sample reacted with ZIKV antigen divided by that of the negative control; prM/E, precursor membrane protein/envelope protein; NS1, nonstructural protein 1; EUA, emergency use authorization; FDA, Food and Drug Administration; DENV, dengue virus; Ab, antibody; Ag, antigen;BSL-2, biosafety level-2; PRNT, plaque reduction neutralization test; FRNT, focus reduction neutralization test; FRNT50, calculated serum dilution at which 50% of maximal neutralization activity occurs; NS1 BOB, NS1 blockade of binding; ROC, receiver operating characteristic.