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A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections

We have systematically evaluated a dry-format, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay developed by Tetracore Inc. for the Cepheid SmartCycler platform to facilitate rapid diagnosis of dengue virus infections. A panel of related flaviviruses was used to evaluate the clinical specificity of the assay, and it was found to be specific to dengue. Eighty-one clinical samples previously confirmed dengue positive by virus isolation, along with 25 dengue negative control specimens were used to validate this new diagnostic assay. Using these clinical samples, the assay exhibited 98.77% sensitivity and 100% specificity. Over 85% of the clinical specimen exhibited viral loads ranging from 10³ to 10⁷ plaque-forming units per milliliter (PFU/mL). In addition, this dry-format assay is stable at ambient temperatures and requires minimal technical expertise to perform in a small thermocycler platform. These characteristics make it a promising candidate for diagnosis of dengue in mobile laboratories in the field.

Received November 20, 2007. Accepted for publication June 18, 2008.

Acknowledgments: We thank Ms. Ravithat Putvatana for excellent technical help and Dr. Charmagne Beckett for providing us with reagents. This work was done when Dr. Subhamoy Pal was supported by a National Research Council Fellowship.

Financial support: This research was funded by U.S. Naval Medical Research Center work unit number 6000.RAD1.L.A0311 and the U.S. Army Medical Research and Materiel Command.

Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. The study protocol was approved by the Naval Medical Research Center Institutional Review Board (NMRC.2005.0007 EXEMPT) in compliance with all applicable federal regulations governing the protection of human subjects.

Disclosure: Subhamoy Pal, Shuenn-Jue Wu, Kanakatte Raviprakash, Tadeusz Kochel, Kevin Porter, and Curtis Hayes are military service members (or employees of the U.S. Government). This work was prepared as part of their official duties. Some of the authors are employed by Tetracore, Inc. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interest.

^* Address correspondence to Subhamoy Pal, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500. E-mail: pal.subhamoy{at}med.navy.mil

Authors’ addresses: Shuenn-Jue Wu, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: 301-319-7442, Fax: 301-319-7451, E-mail: Shuenn-Jue.Wu{at}med.navy.mil. Subhamoy Pal, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: 301-319-3068, Fax: 301-319-7451, E-mail: pal.subhamoy{at}med.navy.mil. Sajeewane Ekanayake and David Greenwald, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: 301-319-9597, Fax: 301-319-7451, E-mails: sajeewane{at}gmail.com and dave.greenwald{at}gmail.com. Silvia Lara, Tetracore Inc., 9901 Bel-ward Campus Drive, Suite 300, Rockville, MD 20850, Tel: 240-268-5400, Fax: 240-268-1107, E-mail: slara{at}tetracore.com. Kanakatte Raviprakash, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: 301-319-7454, Fax: 301-319-7451, E-mail: K.Raviprakash{at}med.navy.mil. Ta-deusz Kochel, U.S. Naval Medical Research Center Detachment, Lima, Peru, Tel: 011-511-562-3848, ext. 157, E-mail: kochel{at}nmrcd.med.navy.mil. Kevin Porter, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Tel: 301-319-7450, Fax: 301-319-7451, E-mail: Kevin.Porter @med.navy.mil. Curtis Hayes, Viral and Rickettsial Diseases Department (Code 41), Infectious Diseases Directorate, Naval Medical Research Center, 503 Robert Grant Avenue Silver Spring, MD 20910-7500, Tel: 301-319-7455, Fax: 301-319-7451, E-mail: Curtis.Hayes @med.navy.mil. William Nelson, Tetracore Inc., 9901 Belward Campus Drive, Suite 300, Rockville, MD 20850, Tel: 240-268-5400, Fax: 240-268-1107, E-mail: wnelson{at}tetracore.com. Johnny Callahan, Tet-racore Inc., 9901 Belward Campus Drive, Suite 300, Rockville, MD 20850, Tel: 240-268-5400, ext. 5411, Fax: 240-268-1107, E-mail: jcallahan{at}tetracore.com.

Note: A Supplemental Table (Dengue Clinical Sample Assay Results) appears online at www.ajtmh.org.