title: Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays.
abstract: A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/μl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided.
authors: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, USA.
authors: Jesse J
authors: JJ
authors: Waggoner
authors: Janaki
authors: J
authors: Abeynayake
authors: Malaya K
authors: MK
authors: Sahoo
authors: Lionel
authors: L
authors: Gresh
authors: Yolanda
authors: Y
authors: Tellez
authors: Karla
authors: K
authors: Gonzalez
authors: Gabriela
authors: G
authors: Ballesteros
authors: Angel
authors: A
authors: Balmaseda
authors: Kumudu
authors: K
authors: Karunaratne
authors: Eva
authors: E
authors: Harris
authors: Benjamin A
authors: BA
authors: Pinsky
mesh_terms: D015331:Cohort Studies; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D009527:Nicaragua; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013188:Sri Lanka
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