title: Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection.
abstract: BACKGROUND
  Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.
  
  
  OBJECTIVE
  In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR).
  
  
  METHODS
  From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.
  
  
  RESULTS
  The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/μL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).
  
  
  CONCLUSIONS
  The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.
authors.affiliation: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Waggoner
authors.affiliation: National Virology Laboratory, Centro Nacional de Diagnóstico y Referencia, Ministry of Health, Managua, Nicaragua.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ballesteros
authors.affiliation: Sustainable Sciences Institute, Managua, Nicaragua.
authors.forename: Lionel
authors.initials: L
authors.lastname: Gresh
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Alisha
authors.initials: A
authors.lastname: Mohamed-Hadley
authors.affiliation: National Virology Laboratory, Centro Nacional de Diagnóstico y Referencia, Ministry of Health, Managua, Nicaragua.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Tellez
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Malaya K
authors.initials: MK
authors.lastname: Sahoo
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Janaki
authors.initials: J
authors.lastname: Abeynayake
authors.affiliation: National Virology Laboratory, Centro Nacional de Diagnóstico y Referencia, Ministry of Health, Managua, Nicaragua.
authors.forename: Angel
authors.initials: A
authors.lastname: Balmaseda
authors.affiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Harris
authors.affiliation: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: bpinsky@stanford.edu.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Pinsky
mesh_terms: D065632:Chikungunya Fever / Q000175:diagnosis*; D002648:Child; D002675:Child, Preschool; D003715:Dengue / Q000175:diagnosis*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009527:Nicaragua; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
keywords: Background; Chikungunya virus; Dengue virus; Molecular diagnostics; Multiplex real-time RT-PCR