Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infections: How alterations in assay conditions impact performance

Abstract

Dengue virus (DENV) infection is a worsening global health problem. The plaque reduction neutralization test (PRNT) is currently considered to be the "gold standard" to characterize and quantify circulating levels of anti-DENV neutralizing antibody (NAb). Many variations of the PRNT are currently in use and neither the assay nor its performance conditions have been standardized or harmonized between laboratories. We used a well-characterized panel of acute and late convalescent follow-up sera samples from children experiencing primary and secondary DENV infections to evaluate the performance of the dengue PRNT under a variety of testing conditions. Investigators varied cell type, control virus passage, and the use of complement across multiple assay runs of the same sample panel. Our findings indicate wide variation in PRNT titer results in response to varied testing conditions.

Figure 1

Schematic depicting the testing conditions throughout the experiment using a primary DENV-1 infection as an example. DLI = dengue-like illness; D1, 2, 3, 4 = DENV-1, -2, -3, -4; B = BHK-22; L = LLC-MK2; V = Vero cells; −C = without complement; +C = with complement.

Figure 2

A, Interaction of viral strain passage and complement. C+ = with complement; C− = without complement; TC = tissue culture; GMT = geometric mean titer. B, Interaction of cell line and complement. TC = tissue culture; GMT = geometric mean titer. C, Interaction of cell type and viral strain passage. TC = tissue culture; GMT = geometric mean titer. A–C, display geometric mean neutralizing antibody titers (GMT) to DENV 1–4 for all two-way combinations of reference virus, cell type, and the presence or absence of complement. The GMTs for the interactions here are calculated from multivariate mixed-effects models like those presented in Table 3A–D, but with the inclusion of the two-way interaction term of interest (for example, cell line and complement). For convenience, the reference levels for variables other than the conditions being measured were used to estimate the expected antibody titer. As noted in Table 3A–D, referent levels include primary infection, the absence of complement, the use of Vero cells, and prototype virus. P values represent the overall significance of the interaction term in the model for that serotype, controlling for all other variables. Starred P values indicate those that were significant at a α-level of 0.05.