Immunity to Non-Dengue Flaviviruses Impacts Dengue Virus Immunoglobulin G Enzyme-Linked Immunosorbent Assay Specificity in Cambodia

Background Seroprevalence studies are the standard for disease surveillance, and serology determined eligibility for the first dengue vaccine. Expanding flavivirus co-circulation and vaccination complicate testing. We evaluate the accuracy of a common dengue virus serological assay, examine immunity to non-dengue flaviviruses as a contributor to decreased performance, and assess whether alternative cut points may improve assay performance. Methods Children (n = 770) aged 2–9 years in Kampong Speu, Cambodia were enrolled in a prospective longitudinal study, and PanBio indirect dengue virus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was performed. Plaque reduction neutralization tests (PRNTs) using dengue viruses were performed on a subset to assess the accuracy of the IgG ELISA, and PRNTs with Zika, Japanese encephalitis, and West Nile viruses evaluated immunity to non-dengue flaviviruses. Receiver operating curve analysis identified an alternative cut point to improve IgG ELISA accuracy. Results The dengue IgG ELISA had a lower specificity than previously reported (58% vs 93%–100%). Of those with false-positive IgG results, 46% had detectable neutralizing antibodies against other flaviviruses including 14% against West Nile virus. A higher IgG cut point improved the test accuracy in this population. Conclusions Physicians and public health authorities should be alert for West Nile in Cambodia. Immunity to non-dengue flaviviruses can impact dengue surveillance. Clinical Trials Registration NCT03534245.