Evaluation of 5 Commercially Available Zika Virus Immunoassays

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Z ika virus is a mosquito-borne member of the fam-
ily Flaviviridae, genus Flavivirus, that was originally discovered in 1947 in Uganda (1). For several decades, Zika virus seemed to be geographically restricted to equatorial Africa with a few documented incursions into Asia (2,3). Although several studies demonstrated serologic evidence of human exposures to Zika virus across Africa, it was believed that this virus was not a major public health threat. However, in 2007, the epidemic potential of Zika virus became apparent when it was identified as the causative agent of an outbreak in Yap State, Federated States of Micronesia, which consisted of 49 confirmed cases, 59 probable infections, and dozens more suspected cases (4,5). Since 2007, several epidemics have occurred across the Pacific Ocean Region, including an outbreak in 2013-14 with thousands of confirmed cases in French Polynesia (6).
In 2015, the first cases of Zika virus infection were confirmed in Brazil, which indicated the beginning of the largest outbreak recorded with autochthonous vectorborne transmission documented in >65 countries across the Americas (2,3,7). Although it is still widely believed that most Zika virus infections in humans are asymptomatic or mild with self-limiting clinical manifestations, it is now documented that Zika virus infections can lead to major complications and long-term sequelae, including congenital birth defects, neurologic disorders, and prolonged risk for sexual transmission of this virus (3,8).
Before 2007, only 14 laboratory-confirmed cases of Zika virus infection had been documented worldwide. Thus, it is not surprising that diagnostics for Zika virus were conducted only in specialized arbovirus reference laboratories (2). During the outbreak on Yap Island, samples were sent to the Arbovirus Diagnostic Laboratory, Centers for Disease Control and Prevention (Fort Collins, CO, USA), where molecular and serologic assays were quickly developed for confirmatory testing (5). Many of these in-house methods developed in 2007, including real-time molecular assays, an IgM-capture ELISA (MAC-ELISA), and a plaque reduction neutralization test (PRNT), have been used during the current outbreak. However, the magnitude of the outbreak, combined with the in-house production of key reagents involved in diagnostics of Zika virus infection, has taxed the few reference laboratories capable of producing, standardizing, and distributing such materials. Therefore, application and evaluation of sensitive and specific diagnostic assays, particularly those that can be used in frontline laboratories, has become a top public health priority.
Several laboratories and commercial vendors have developed and evaluated molecular assays for rapid identification of Zika virus RNA, and, in some instances, other clinically relevant arboviruses, such as dengue virus (DENV) and chikungunya virus, in acute-phase clinical specimens (9). However, high-throughput commercially produced immunoassays have proven to be more challenging because of strong serologic cross-reactivity of closely related flaviviruses, such as DENV. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocols of Zika virus MAC-ELISA and PRNT.

The Study
Samples were submitted to the National Microbiology Laboratory of the Public Health Agency of Canada (Winnipeg, Manitoba, Canada) for arbovirus diagnostic testing. All samples were obtained from Canadian travelers who visited areas with known Zika virus transmission and consulted their physicians after symptoms consistent with Zika virus infection developed upon return.
We obtained deidentified samples from 75 patients. Thirty samples were from patients with serologically confirmed Zika virus infections; 10 from patients with

Evaluation of 5 Commercially Available
Zika Virus Immunoassays Most tests evaluated provided algorithms that resulted in positive, negative, or equivocal results. However, the In-Bios kits account for antigenics in associated with secondary flavivirus infections and reports results as Zika virus positive, possible Zika virus positive, or presumptive other flavivirus positive or negative on the basis of calculations of optical density ratios obtained from a sample with the 3 The assays generally showed reproducible results during independent evaluations, although specificity and sensitivity of each varied ( Table 1). The Euroimmun IgM and IgG ELISAs and the Abcam IgM ELISA showed a specificity of 100% for negative specimens with similar results (>90%) for confirmed DENV-positive samples ( Table 2, https://wwwnc. cdc.gov/EID/article/23/9/16-2043-T2.htm). The NovaTec ELISA showed a specificity of 66% for negative specimens and 70% for DENV-positive specimens. Although the In-Bios Zika Detect ELISA showed similar specificity results for flavivirus-seronegative specimens, it showed decreased specificity for DENV-positive samples. This assay incorrectly identified 40% of these samples as Zika virus IgM positive and 40% as possible Zika virus positive.
Although specificity is a key factor, for a front-line diagnostic test, sensitivity is a major factor in determining its usefulness. With appropriate diagnostic testing in place, including use of Zika virus conformational cross-PRNTs, false-positive results caused by specificity issues can usually be overcome. However, poor sensitivity will lead to false-negative results that might not be followed up by testing of additional sample collections. When compared with the in-house diagnostics (MAC-ELISA with PRNT confirmation), the IgM assays of Euroimmun, Abcam, and Novatec demonstrated sensitivities of 37%, 57%, and 65%, respectively. When we combined results of the Euroimmun IgM and IgG ELISAs, sensitivity increased to 82%. The InBios Zika Virus Detect IgM assay correctly identified all confirmed Zika IgM-positive samples identified by the recommended diagnostic assays, resulting in a sensitivity of 100%. The InBios ELISA also detected IgM in 50% of samples that were positive for Zika virus by RT-PCR, whereas the other assays did not detect IgM in most of these samples (Table 3).

Conclusions
The low sensitivity of most immunoassays evaluated could be improved by testing a repeat sample collected a few weeks after the initial specimen, although this sampling is not always practical, particularly if resources are limited. When performed in combination, the Euroimmun Zika Virus IgM and IgG ELISAs provide improved sensitivity. However, interpretation of recent versus past infections could be problematic, particularly when IgM results are negative and IgG results are positive. On the basis of our findings, the InBios Zika Virus Detect MAC-ELISA provides diagnostic results comparable to the CDC-based in-house MAC-ELISA for specimens collected from patients with primary flavivirus exposures (i.e., no detectable background immunity to DENV). A needed follow-up to our study will be further evaluation of IgM detection by commercial ELISAs involving cases of secondary flavivirus exposures or previous immunization to related viruses, such as yellow fever virus.