A Direct from Blood/Plasma Reverse Transcription-Polymerase Chain Reaction for Dengue Virus Detection in Point-of-Care Settings.

Authors:
Ninad Mehta
Ninad Mehta
Li Ka Shing Institute of Virology
Kimberly Martin
Kimberly Martin
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University
United States
Tom C Hobman
Tom C Hobman
University of Alberta
Canada

Am J Trop Med Hyg 2019 Apr 15. Epub 2019 Apr 15.

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada.

Infection with dengue virus (DENV) is widespread across tropical regions and can result in severe disease. Early diagnosis is important both for patient management and to differentiate infections that present with similar symptoms, such as malaria, chikungunya, and Zika. Rapid diagnostic tests that are used presently for point-of-care detection of DENV antigens lack the sensitivity of molecular diagnostics that detect viral RNA. However, no molecular diagnostic test for DENV is available for use in field settings. In this study, we developed and validated a reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV adapted for use in field settings. Reverse transcription-polymerase chain reaction was performed directly from plasma samples without RNA extraction. The assay detected all four serotypes of DENV spiked into blood or plasma. Our RT-PCR does not cross-react with pathogens that cause symptoms that overlap with dengue infection. The test performed equally well in a conventional laboratory qPCR instrument and a small, low-cost portable instrument that can be used in a field setting. The lower limit of detection for the assay was 1 × 10 genome copy equivalents/mL in blood. Finally, we validated our test using 126 archived patient samples. The sensitivity of our RT-PCR was 76.7% (95% CI: 65.8-87.9%) on the conventional instrument, and 78.3% (95% CI: 65.8-87.9%) on the field instrument, when compared with the RealStar Dengue RT-PCR Kit 2.0. The molecular test described here is user friendly, low cost, and can be used in regions with limited laboratory capabilities.

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Source
http://dx.doi.org/10.4269/ajtmh.19-0138DOI Listing
April 2019
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