Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development

Background: Accurate determination of neutralization antibody titers supports epidemiological studies of dengue virus transmission and vaccine trials. Neutralization titers measured using the plaque reduction neutralization test (PRNT) are believed to provide a key measure of immunity to dengue viruses, however, the assay's variability is poorly understood, making it difficult to interpret the significance of any assay reading. In addition there is limited standardization of the neutralization evaluation point or statistical model used to estimate titers across laboratories, with little understanding of the optimum approach. Methodology/Principal Findings: We used repeated assays on the same two pools of serum using five different viruses (2,319 assays) to characterize the variability in the technique under identical experimental conditions. We also assessed the performance of multiple statistical models to interpolate continuous values of neutralization titer from discrete measurements from serial dilutions. We found that the variance in plaque reductions for individual dilutions was 0.016, equivalent to a 95% confidence interval of 0.45–0.95 for an observed plaque reduction of 0.7. We identified PRNT75 as the optimum evaluation point with a variance of 0.025 (log10 scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240–1∶1000 (2.70±0.31 on a log10 scale). The choice of statistical model was not important for the calculation of relative titers, however, cloglog regression out-performed alternatives where absolute titers are of interest. Finally, we estimated that only 0.7% of assays would falsely detect a four-fold difference in titers between acute and convalescent sera where no true difference exists. Conclusions: Estimating and reporting assay uncertainty will aid the interpretation of individual titers. Laboratories should perform a small number of repeat assays to generate their own variability estimates. These could be used to calculate confidence intervals for all reported titers and allow benchmarking of assay performance. Author Summary: Plaque Reduction Neutralization Tests (PRNTs) remain the most popular approach to characterize an individual's ability to neutralize dengue viruses and are widely used in both epidemiological studies and vaccine trials. However, the underlying variability in the assay is poorly understood, hindering the interpretation of PRNT titer estimates. Further, there is little standardization of its use across laboratories limiting our ability to compare results across settings. Here we used many repeated experiments on the same serum under identical laboratory conditions to estimate the variance in titer measurements. We also identified both the optimum PRNT evaluation point and statistical model to calculate titers. By providing an estimate of the variability in the assay, laboratories will be able to provide a confidence bound on individual PRNT readings. In addition by providing recommended statistical approaches that could be used across laboratories, our findings will help the standardization of the assay across settings.