title: Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.
abstract: BACKGROUND
  AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA.
  
  
  OBJECTIVE
  To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance.
  
  
  METHODS
  A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013.
  
  
  CONCLUSIONS
  The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand. Electronic address: Chontichak@afrims.org.
authors.forename: Chonticha
authors.initials: C
authors.lastname: Klungthong
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Wudtichai
authors.initials: W
authors.lastname: Manasatienkij
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Thipwipha
authors.initials: T
authors.lastname: Phonpakobsin
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Piyawan
authors.initials: P
authors.lastname: Chinnawirotpisan
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Prinyada
authors.initials: P
authors.lastname: Rodpradit
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Kittinun
authors.initials: K
authors.lastname: Hussem
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Butsaya
authors.initials: B
authors.lastname: Thaisomboonsuk
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Prapapun
authors.initials: P
authors.lastname: Ong-ajchaowlerd
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Ananda
authors.initials: A
authors.lastname: Nisalak
authors.affiliation: Queen Sirikit National Institute of Child Health, Bangkok, Thailand.
authors.forename: Siripen
authors.initials: S
authors.lastname: Kalayanarooj
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Darunee
authors.initials: D
authors.lastname: Buddhari
authors.affiliation: US Army Institute of Surgical Research, 3698 Chambers Pass Suite B, JBSA Fort Sam Houston, TX, USA.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Gibbons
authors.affiliation: Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Jarman
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: In-Kyu
authors.initials: IK
authors.lastname: Yoon
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, Thailand.
authors.forename: Stefan
authors.initials: S
authors.lastname: Fernandez
mesh_terms: D017931:DNA Primers / Q000235:genetics; D003715:Dengue / Q000175:diagnosis* / Q000453:epidemiology*; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D062665:Epidemiological Monitoring; D014644:Genetic Variation; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013785:Thailand / Q000453:epidemiology
chemical_list: D017931:DNA Primers
keywords: Dengue surveillance; Dengue virus; RT-PCR