Detection of Specific ZIKV IgM in Travelers Using a Multiplexed Flavivirus Microsphere Immunoassay

Abstract

Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to or vaccinated against other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV.

Keywords: Zika virus; dengue virus; flavivirus; microsphere immunoassay; optimised; serology; validated.

Figure 1

Test algorithm for flavivirus serology requests. When requested, sera are screened for flavivirus IgG and IgM by in-house ELISAs employing a pool of purified flaviviruses. Specimens determined to be reactive or equivocal in the flavivirus MAC-ELISA are then tested by the multiplexed flavivirus IgM typing microsphere immunoassay (flaviMIA). Specific RT-PCR tests may also be conducted on the sample as appropriate.

Figure 2

Multiplexed flavivirus IgM microsphere immunoassay (flaviMIA) result examples highlighting specific and cross-reactive IgM reactivity. (A) The intensity of the anti-ZIKV IgM signal, reported as mean fluorescence intensity (MFI, y-axis) compared to 11 other viruses (x-axis) used in the flaviMIA defined this sample from Patient Reference No. 7 as clearly containing ZIKV-specific IgM. (B) This ZIKV RNA positive representative sample from Patient Reference No. 11 was classified as having cross-reactive IgM towards both ZIKV and DENV. Red shading indicates the MFI signal range from a seronegative result; green shading indicates the region into which seropositive results fall. DENV, Dengue virus; JEV, Japanese encephalitis virus; MVEV, Murray Valley encephalitis virus; KUNV, Kunjin virus; ALFV, Alfuy virus; KOKV, Kokobera virus; STRV, Stratford virus; YFV, Yellow fever virus; ZIKV, Zika virus.