title: Variability in dengue titer estimates from plaque reduction neutralization tests poses a challenge to epidemiological studies and vaccine development.
abstract: 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.
  
  
  RESULTS
  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.
authors: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors: Henrik
authors: H
authors: Salje
authors: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors: Isabel
authors: I
authors: Rodríguez-Barraquer
authors: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors: Kaitlin
authors: K
authors: Rainwater-Lovett
authors: Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors: Ananda
authors: A
authors: Nisalak
authors: Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors: Butsaya
authors: B
authors: Thaisomboonsuk
authors: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.
authors: Stephen J
authors: SJ
authors: Thomas
authors: Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors: Stefan
authors: S
authors: Fernandez
authors: Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America.
authors: Richard G
authors: RG
authors: Jarman
authors: Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
authors: In-Kyu
authors: IK
authors: Yoon
authors: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
authors: Derek A T
authors: DA
authors: Cummings
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood*; D002460:Cell Line; D003716:Dengue Virus / Q000254:growth & development*; D006801:Humans; D008253:Macaca mulatta; D015233:Models, Statistical; D009500:Neutralization Tests / Q000592:standards; D010948:Viral Plaque Assay / Q000592:standards
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral