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.

On the Home Front: Specialised Reference Testing for Dengue in the Australasian Region

“…The multi-faceted utilization of molecular and serological platforms employed by both PHV and ESR is highly desirable and advantageous for the specific diagnosis of acute-phase samples compared to the use of conventional ELISA alone. As recently described [ 33 ], PHV used a combination of RT-rtPCR/RT-PCR and MIA serological platforms to assess a panel of sera from patients suspected of ZIKV infection. In that study, specific ZIKV infection could be confirmed in 99 of 101 suspected cases and included the differentiation between primary and secondary infections [ 33 ].…”

“…The response rate of clinicians in providing a second patient sample varies, depending on the viral infection suspected or if initiated by a PHU. For ZIKV infections, provision of a convalescent-phase sample was reported in 60.6% of cases [ 33 ]; however, PHV usually only receive convalescent-phase samples in <30% of cases.…”

“…As a reference laboratory, PHV maintains assays that are not in common use and this greatly assists further scrutiny and confirmation of positives that have been screened by alternate commercial assays. This includes multiple RT-rtPCR/RT-PCR assays for DENV and ZIKV and one for CHIKV ( Table 1 ) and flavivirus ELISA/MIA protocols employing whole virus antigens made from 12 different arboviruses [ 33 ] (PHV ELISA Protocols, Supplementary Data ).…”

“…PHV screen sera for anti-flavivirus IgM and IgG antibodies using in-house flavivirus ELISAs (IgM; MAC-ELISA, IgG; FLG-ELISA, Supplementary data ). Samples that were IgM positive were further analysed for specific anti-flavivirus (including DENV 1–4) IgM antibodies using a flavivirus typing ELISA (FLTYP) [ 47 ] (2005–2013) or FLMIA [ 33 ] (2013–2017).…”

Emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development

“…As improvements in technology occur over time, early vaccine assays are often re-designed and bridged to assays with higher throughput, multiplexed detection, reduction of sample volumes, and automation to support testing of large numbers of specimens for late-stage clinical trials. [28][29][30][31][32][33][34][35] Often, a variety of tests are evaluated early in the program and, based on the usefulness of the data, a down-selection occurs so that the most relevant few remain to support large Phase 3 clinical trials and licensure of the vaccine. 2,35,36 Accepted "gold standards" of immunoassays for use at the onset of an EID vaccine program are rare.…”