title: Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene.
abstract: BACKGROUND
  Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy.
  
  
  OBJECTIVE
  This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches.
  
  
  METHODS
  Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens.
  
  
  RESULTS
  The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection.
  
  
  CONCLUSIONS
  The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.
authors.affiliation: Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea.
authors.forename: Je-Hyoung
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Division of Life Science, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Chom-Kyu
authors.initials: CK
authors.lastname: Chong
authors.affiliation: Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur 50586 Malaysia.
authors.forename: Mangalam
authors.initials: M
authors.lastname: Sinniah
authors.affiliation: Hospital Kuala Lumpur, Jalan Pahang, Kuala Lumpur 50586 Malaysia.
authors.forename: Jeyaindran
authors.initials: J
authors.lastname: Sinnadurai
authors.affiliation: Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea. Electronic address: hosong@wku.ac.kr.
authors.forename: Hyun-Ok
authors.initials: HO
authors.lastname: Song
authors.affiliation: Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of Medicine, Iksan, Jeonbuk, Republic of Korea. Electronic address: hyunpk@wku.ac.kr.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
mesh_terms: D003715:Dengue / Q000175:diagnosis*; D042241:Early Diagnosis; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012698:Serologic Tests; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000235:genetics*
chemical_list: C079340:NS1 protein, Dengue virus type 2; C546113:NS1 protein, Dengue virus type 3; C082515:NS1 protein, dengue-1 virus; D017361:Viral Nonstructural Proteins
keywords: Dengue; Early diagnosis; Heterotypic mixed infection; Multiplex RT-PCR; NS1; Quantitation; Serotyping