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.

Figure 1. Maximum likelihood phylogeny of contemporary DENV strains.

Representative DENV strains from currently circulating genotypes considered for primer and probe modifications. Full genome sequences were used. Each taxa is labeled: country of origin, year of isolation, and GenBank accession number. Due to figure space limitations, only a representative subset of strains were used to generate these ML trees. DENV-1 Asian (I) and American-African (III) genotypes , DENV-2 Asian I (IIIa), American-Asian (IIIb), and Cosmopolitan (IV) genotypes , DENV-3 South Pacific (I), Thailand (II), and Indian Subcontinent (III) genotypes , , DENV-4 Southeast (I) and Indonesian (II) genotypes .

Figure 2. Performance comparison.

Performance and sensitivity of the assay was compared to the Johnson et al., 2005 real time RT-PCR assay by detection of (8) ten-fold dilutions of in vitro transcribed RNA derived from target regions, 5 replicas per dilution. Both assays, CDC and Johnson 2005, were performed in singleplex (A) and multiplex (B) formats. Viral RNA was quantified using a standard curve measured in genome copy equivalents per RT-PCR reaction (GCE/rx) log transformed for linear scale plotting. Each data point reflects the average CT of 5 replicates. A linear regression was estimated for CDC (black solid lines) and Johnson 2005 (black dashed lines). Regression coefficient (R²) is shown for every regression and every serotype. Error bars indicate standard deviation.

Figure 3. Limit of detection.

The limit of detection of the Assay was evaluated by detection and quantification of (8) ten-fold dilutions of laboratory-adapted DENV-1–4 strains, 5 replicas per dilution. The Assay was performed in singleplex (A) and multiplex formats (B). Viral RNA concentration was quantified using a standard curve measured in genome copy equivalents per milliliter of serum or plasma (GCE/mL). A linear regression was estimated for dilutions of virus stocks in plasma (dashed lines) or serum (solid line). Regression coefficient (R²) is shown for each serotype. Error bars indicate standard deviation of measurements from 5 replicas in GCE/mL.