Clinical evaluation of an in-house-developed real-time RT-PCR assay for serotyping of dengue virus
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BRIEF REPORT
Clinical evaluation of an in‑house‑developed real‑time RT‑PCR assay for serotyping of dengue virus M. B. Kakade1 · N. Shrivastava1 · J. A. Patil1 · D. Parashar1 · P. S. Shah1 · K. Alagarasu1 Received: 29 March 2020 / Accepted: 3 June 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract In the present study, an in-house-developed real-time RT-PCR (rRT-PCR) for serotyping of dengue virus (DENV) was evaluated for its performance, using 612 clinical samples. Compared to the composite reference standard, the in-house-developed rRT-PCR had an overall sensitivity of 97.5% and a specificity of 100%. The assay had a sensitivity of 100%, 95.6%. 96.9% and 100% for detecting DENV-1, DENV-2, DENV-3 and DENV-4, respectively. We recommend periodic evaluation of real-time RT-PCR assays for detecting DENV serotypes with a large number of samples and the use of at least two assays that target different regions of DENV genomes. Dengue, a febrile illness caused by four serotypes of dengue virus, is among the top ten global health threats, affecting more than 100 nations. Choosing the appropriate diagnostic test for dengue depends on the day of illness and the immune status of the patient. Diagnosis at an early post-onset day (POD) of illness is helpful for better clinical management of the case and also contributes to early initiation of vector-control measures in the region from where cases arise. Molecular assays detecting the viral RNA and serological assays detecting the non-structural protein (NS) 1 antigen are the assays of choice during an early POD of illness [1]. However, serological assays detecting NS1 antigen have lower sensitivity during secondary infections [2]. Moreover, with the possible introduction of DENV vaccines in the near future, virological confirmation will be the recommended choice of diagnosis during the vaccine era [3, 4]. Molecular assays detecting individual DENV serotypes are gaining importance, since they provide information on the infecting serotype. Moreover, information on circulating serotypes in a particular geographical region might help in predicting outbreaks if changes in the most prevalent serotype are observed during the early phase of the dengue season. Different real-time RT-PCR assays have been developed Handling Editor: Zhenhai Chen. * K. Alagarasu [email protected] 1
Dengue/Chikungunya Group, ICMR-National Institute of Virology, Pune 411001, Maharashtra, India
for detection of individual DENV serotypes, but a recent in silico analysis has reported that none of the assays have 100% sensitivity, due to the presence of mismatches in the 3’ portion of the primers used in those assays [5]. Increasing diversity of DENV necessitates the use of multiple molecular assays targeting different regions of the genome for detection of the virus, and each assay needs to be evaluated using a large number of samples representing all four serotypes equally. Clinical evaluation of real-time RT-PCR assays allows course corrections to be made in these assays, includ
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