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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 viru...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072537/ https://www.ncbi.nlm.nih.gov/pubmed/24967885 http://dx.doi.org/10.1371/journal.pntd.0002952 |
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author | Salje, Henrik Rodríguez-Barraquer, Isabel Rainwater-Lovett, Kaitlin Nisalak, Ananda Thaisomboonsuk, Butsaya Thomas, Stephen J. Fernandez, Stefan Jarman, Richard G. Yoon, In-Kyu Cummings, Derek A. T. |
author_facet | Salje, Henrik Rodríguez-Barraquer, Isabel Rainwater-Lovett, Kaitlin Nisalak, Ananda Thaisomboonsuk, Butsaya Thomas, Stephen J. Fernandez, Stefan Jarman, Richard G. Yoon, In-Kyu Cummings, Derek A. T. |
author_sort | Salje, Henrik |
collection | PubMed |
description | 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 PRNT(75) as the optimum evaluation point with a variance of 0.025 (log(10) scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240–1∶1000 (2.70±0.31 on a log(10) 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. |
format | Online Article Text |
id | pubmed-4072537 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-40725372014-07-02 Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development Salje, Henrik Rodríguez-Barraquer, Isabel Rainwater-Lovett, Kaitlin Nisalak, Ananda Thaisomboonsuk, Butsaya Thomas, Stephen J. Fernandez, Stefan Jarman, Richard G. Yoon, In-Kyu Cummings, Derek A. T. PLoS Negl Trop Dis Research Article 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 PRNT(75) as the optimum evaluation point with a variance of 0.025 (log(10) scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240–1∶1000 (2.70±0.31 on a log(10) 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. Public Library of Science 2014-06-26 /pmc/articles/PMC4072537/ /pubmed/24967885 http://dx.doi.org/10.1371/journal.pntd.0002952 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Salje, Henrik Rodríguez-Barraquer, Isabel Rainwater-Lovett, Kaitlin Nisalak, Ananda Thaisomboonsuk, Butsaya Thomas, Stephen J. Fernandez, Stefan Jarman, Richard G. Yoon, In-Kyu Cummings, Derek A. T. Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title | Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title_full | Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title_fullStr | Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title_full_unstemmed | Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title_short | Variability in Dengue Titer Estimates from Plaque Reduction Neutralization Tests Poses a Challenge to Epidemiological Studies and Vaccine Development |
title_sort | variability in dengue titer estimates from plaque reduction neutralization tests poses a challenge to epidemiological studies and vaccine development |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072537/ https://www.ncbi.nlm.nih.gov/pubmed/24967885 http://dx.doi.org/10.1371/journal.pntd.0002952 |
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