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.