Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus

Abstract

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Author Summary

Significant expansion of the four DENV serotypes (DENV-1, -2, -3 and -4) has been reported throughout tropical and sub-tropical regions of the world, with estimates of 390 million cases annually. The need has arisen for expanded diagnostic testing for DENV infections in the United States, as dengue infection has been added to the list of national notifiable diseases. Timely and accurate diagnosis of dengue is important for clinical care, disease surveillance, disease prevention, and control activities. However, current testing is performed with laboratory-developed research-based assays available only in a limited number of laboratories that have not been validated or approved for diagnostic testing in the United States. Here we report the development and evaluation of the CDC DENV-1–4 Real Time RT-PCR Assay, the first molecular test approved by the US Food and Drug Administration for the diagnosis and serotyping of DENV in human serum or plasma samples. This test was designed and validated for the detection of contemporary, clinically relevant DENV strains transmitted globally, facilitating the global deployment of the test and increase detection of traveler-associated dengue cases.