Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.
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.
['Klungthong|Chonticha|C|', 'Manasatienkij|Wudtichai|W|', 'Phonpakobsin|Thipwipha|T|', 'Chinnawirotpisan|Piyawan|P|', 'Rodpradit|Prinyada|P|', 'Hussem|Kittinun|K|', 'Thaisomboonsuk|Butsaya|B|', 'Ong-ajchaowlerd|Prapapun|P|', 'Nisalak|Ananda|A|', 'Kalayanarooj|Siripen|S|', 'Buddhari|Darunee|D|', 'Gibbons|Robert V|RV|', 'Jarman|Richard G|RG|', 'Yoon|In-Kyu|IK|', 'Fernandez|Stefan|S|']
D017931:DNA Primers D003715:Dengue D003716:Dengue Virus D062665:Epidemiological Monitoring D014644:Genetic Variation D006801:Humans D025202:Molecular Diagnostic Techniques D016133:Polymerase Chain Reaction D012189:Retrospective Studies D020133:Reverse Transcriptase Polymerase Chain Reaction D012680:Sensitivity and Specificity D013785:Thailand
RT-PCR Dengue surveillance Dengue virus