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

Abstract

a b s t r a c tBackground: 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.Published by Elsevier B.V.

Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor

“…The primary outcomes for each trial was designed to evaluate safety, reactogenicity, and immunogenicity of ZPIV through the primary timepoint of day 57. Participants were assessed on days 1,4,8,15,29,32,36,43, and 157 57 following ZPIV vaccination for ZIKV neutralization. Interim results were published in Modjarrad, K., et al "Preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated Zika virus vaccine candidate: phase 1, randomised, double-blind, placebocontrolled clinical trials", The Lancet 391, 563-571 (2018).…”

“…A subset of mice (n = 5 per group) were deeply anesthetized for blood sampling using cardiocentesis on day 3 post-infection to determine viremia followed by euthanasia to collect the following tissues for virus dissemination: kidney, liver, spleen, brain and axillary lymph node. DENV in sera and homogenized tissues was determined as previously described 43 , with modification (the DENV-2 probe fluorophore was changed to JOE). RNA was extracted using the MagMax-96 Viral RNA isolation kit (Applied Biosystems) on the Kingfisher Flex Purification System (ThermoFisher Scientific).…”

Route of inoculation and mosquito vector exposure modulate dengue virus replication kinetics and immune responses in rhesus macaques

“…DENV in sera was determined as previously described, with modification: the DENV-2 probe fluorophore was changed to JOE. [80] RNA was extracted from sera on days 1-14, 16, 18, and 21 using QIAamp Viral RNA mini on the QIAcube instrument. Eluted RNA was quantified by qRT-PCR using the SuperScript III Platinum One-Step qRT-PCR kit (Thermo Fisher Scientific) on the QuantStudio 7-Flex Real-Time PCR instrument (Thermo Fisher Scientific).…”

Challenges and Opportunities of Laboratory diagnosis of Dengue virus infection: a review

“…We computed sensitivity and specificity of RT-PCR [33,35,[68][69][70][71][72][73][74][75][76], NS1 ELISA [75][76][77][78][79][80], NS1 rapid diagnostic tests [81][82][83][84][85][86], IgM ELISA [87,89,90] and IgM RDT [88,91] 100%, 95% CI against 80.3-99%, 95%CI. These observations concur with the results reported earlier [94].…”