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Dengue Plaque Reduction Neutralization Test (PRNT) in Primary and Secondary Dengue Virus Infections: How Alterations in Assay Conditions Impact Performance

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.

Received December 1, 2008. Accepted for publication July 27, 2009.

Acknowledgments: We thank the volunteers, their parents, and the medical staff of the Queen Sirikit National Institute of Child Health (Bangkok, Thailand) for their valuable participation in this study. We thank the laboratory, project management, and clinical staff of the Department of Virology, Armed Forces Research Institute of Medical Sciences (Bangkok, Thailand) for performing laboratory assays to support the study and coordinating study activities. We also thank Suchitra Nimmannitya for providing expert consultation on the diagnosis and management of dengue cases.

Financial support: This work was funded by the United States Army Military Infectious Diseases Research Program (Fort Detrick, Frederick, MD) and NIH grant P01A134533.

Disclosure: One of the authors is employed by the GlaxoSmithKline Group or Companies. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.

Disclaimer: The opinions or assertions contained herein are the private views of the authors and are not to be construed as reflecting the official views of the United States Army, Royal Thai Army, or the United States Department of Defense.

^* Address correspondence to Stephen J. Thomas, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, APO AP 96546. E-mail: Stephen.thomas{at}afrims.org

Authors’ addresses: Stephen J. Thomas, Richard Jarman, and Robert V. Gibbons, Department of Virology, USAMC-AFRIMS, APO AP 96546, Tel: 662-644-5644, Fax: 662-644-4760, E-mails: Stephen.thomas{at}afrims.org, richard.jarmnan{at}afrims.org, and robert.gibbons{at}afrims.org. Ananda Nisalak, Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, 10400 Thailand, Tel: 662-644-5644, Fax: 662-644-4760, E-mail: anandan{at}afrims.org. Kathryn B. Anderson, Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA 30030, Tel: 404-840-5004, E-mail: kbander{at}learnlink.emory.edu. Daniel H. Libraty, Rm S6-826, UMMS, 55 Lake Ave N., Worcester, MA 01655, Tel: 508-856-4182, Fax: 508-856-4890, E-mail: daniel.libraty{at}umassmed.edu. Siripen Kalayanarooj, Department of Pediatrics, Queen Sirikit National Institute of Child Health, Rajvithi Road, Bangkok 10400 Thailand, E-mail: siripenk{at}gmail.com. David W. Vaughn, GlaxoSmithKline Biologicals, 2301 Renaissance Boulevard, RN0220, PO Box 61540, King of Prussia, PA 19406-2772, Tel: 610-787-3907, Fax: 610-787-7057, E-mail: david.w.vaughn{at}gsk.com. Robert Putnak, Division of Viral Diseases, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, Tel: 301-319-9425, Fax: 301-319-9661, E-mail: robert.putnak{at}us.army.mil. Timothy P. Endy, Infectious Disease Division, Department of Medicine, State University of New York, Upstate Medical University, 725 Irving Avenue, Suite 304, Syracuse, NY 13210, Tel: 315-464-5533, Fax: 315-464-5579, E-mail: endyt{at}upstate.edu.