The CDC requires that samples with presumptive positive, equivocal, or inconclusive IgM test results for the Zika virus must be forwarded for confirmation by PRNT. The PRNT is a serological test which utilizes the ability of a specific antibody to neutralize a virus, in turn, preventing the virus from causing the formation of plaques in a cell monolayer. Typically, the assay involves mixing a constant amount of virus with dilutions of the serum specimen being tested, followed by plating of the mixture onto cells of an appropriate cell line for the individual virus. The concentration of plaque forming units can be determined by the number of plaques formed after a few days. A vital dye (eg, neutral red) is then added for visualization of the plaques and the number of plaques in an individual plate is divided by the original number of virons to calculate the percentage neutralization. Depending on the virus, the plaque forming units are measured by microscopic observations, fluorescent antibodies, or specific dyes that react with the infected cell. Interpretation is typically based on 70% neutralization, which is the last dilution of serum capable of inhibiting 70% of the total plaques (virions).
Currently, the PRNT test is considered the "gold standard" for detecting and measuring antibodies that can neutralize the viruses that cause many diseases. It has a higher sensitivity and is more specific than many other serological tests for the diagnosis of some viruses.
Although the PRNT typically provide the greatest specificity for Zika and other viruses, serological assays tend to be subject to cross-reactivity especially in patients with prior flavivirus infection or immunization history. In addition, the test is relatively cumbersome and time intensive (few days), compared to other EIA tests.
Typically, for laboratories performing PRNT, a four-fold rise in neutralizing antibody titers in the absence of a rise in antibody titer to other flaviviruses is further evidence of a recent Zika virus infection.