Evaluation of three commercial real-time PCR kits for dengue diagnosis

Abstract

Background: Dengue is the most important arthropod-borne viral disease in the world. Since dengue clinical manifestations are similar to those of many other febrile syndromes, the availability of dengue-specific laboratory tests is useful for appropriate management of patients. Dengue is endemoepidemic inMartinique, a French Caribbean island. The island has experienced epidemics caused by DENV-2/DENV-4 in2005, DENV-2 in2007, DENV-1/DENV-4 in2010 and DENV-2/DENV-4 in2013. In our virology unit, RT-PCR (Hemi-nested protocol from Lanciotti et al., J Clin Microbiol 1992) is systematically run for patients consulting at hospital facilities with acute febrile disease and admitted until day 8 of fever onset. This technique is considered as the gold standard but it is time consuming and at high risk of contamination. Moreover there is a need for standardized dengue RT-PCR with internal controls. Methods & Materials: In the present study, we evaluated the performance of three commercialdengue real-time PCR kits (SimplexaTM Dengue PCR assay: Focus Diagnostic, RealStar® Dengue RT-PCR kit 1.0: Altona Diagnostics, Geno-Sen's Dengue 1-4 Real-time PCR kit: Genome Diagnostics Pvt) using 162 samples positive with the hemi-nested RT-PCR (46 DENV-1, 37 DENV-2, 33 DENV-3, and 46 DENV-4). In addition, 70 negative serum specimens were used to evaluate specificity. All three kits included an internal control. RNA extraction was performed using the NucliSens easyMAG® (BioMérieux). Results: Geno-Sen's, RealStar®, and Simplexa™ tests were positive in 138 (85.1%), 135 (83.3%), and 151 (93.2%) samples, respectively. The sensitivity of Simplexa™ was significantly higher than Geno-Sen's (Chi 2, p = 0.009) and RealStar® (p = 0.005). Compared to the in-place reference RT-PCR, the specificity was 100% for Geno-Sen's and Simplexa™ and 98% for Real Star.According to dengue genotypes, the sensitivity was for DENV-1 (91%, 78% and 96%), for DENV-2 (89%, 87%, and 92%) for DENV-3 (91%, 91%, 91%), for DENV-4 (72%, 80% and 94%) by Geno-Sen's, Real Star® and Simplexa™ kits respectively. Simplexa is the only kit providing genotyping of virus. Conclusion: The good performance of the SimplexaTM Dengue PCR assay at the detection and genotyping level in combination with an automated extraction is suitable for diagnosis during dengue outbreaks in tropical areas.