Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand

Authors

• Chonticha Klungthong, Armed Forces Research Institute of Medical Sciences
• Wudtichai Manasatienkij, Armed Forces Research Institute of Medical Sciences
• Thipwipha Phonpakobsin, Armed Forces Research Institute of Medical Sciences
• Piyawan Chinnawirotpisan, Armed Forces Research Institute of Medical Sciences
• Prinyada Rodpradit, Armed Forces Research Institute of Medical Sciences
• Kittinun Hussem, Armed Forces Research Institute of Medical Sciences
• Butsaya Thaisomboonsuk, Armed Forces Research Institute of Medical Sciences
• Prapapun Ong-ajchaowlerd, Armed Forces Research Institute of Medical Sciences
• Ananda Nisalak, Armed Forces Research Institute of Medical Sciences
• Siripen Kalayanarooj, Queen Sirikit National Institute of Child Health
• Darunee Buddhari, Armed Forces Research Institute of Medical Sciences
• Robert V. Gibbons, US Army Institute of Surgical Research
• Richard G. Jarman, Walter Reed Army Institute of Research
• In-Kyu Yoon, Armed Forces Research Institute of Medical Sciences
• Stefan Fernandez, Armed Forces Research Institute of Medical Sciences

Date of this Version

2015

Citation

Journal of Clinical Virology 63 (2015) 25–31

Comments

U.S. Government Work

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.

Study design: 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.

Results and conclusion: 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.