An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay

Highly Sensitive Detection of Dengue Virus Nucleic Acid in Samples from Clinically Ill Patients▿

Dengue virus (DENV) is a major cause of febrile illness and hemorrhagic fever in tropical and subtropical regions. Typically, patients presenting with acute dengue disease are viremic but may not have yet developed detectable titers of antibody. Therefore, early diagnosis depends mostly on detection of viral components, such as the RNA. To define the potential use of transcription-mediated amplification (TMA) DENV RNA as a diagnostic tool, we first compared its analytic sensitivity using a routine real-time reverse transcription (RT)-PCR and found that TMA is approximately 10 to 100 times more sensitive. In addition, we tested acute-phase serum samples (<5 days post-symptom onset) submitted as part of laboratory-based surveillance in Puerto Rico and determined that among patients with serologically confirmed dengue infection, TMA detected DENV RNA in almost 80% of serum specimens that were negative by the RT-PCR test used for diagnosis and in all specimens with positive RT-PCR results. We conclude that TMA is a highly sensitive method which can detect DENV RNA in approximately 89% of clinical, acute-phase serum specimens

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

<div><p>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 <i>in-vitro</i> 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.</p></div

Limit of detection.

<p>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 <b>(A)</b> and multiplex formats <b>(B)</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.</p

Agreement between CDC DENV-1–4 Real Time RT-PCR and sequencing in samples from suspected dengue patients.

*<p>One sample was negative on the CDC-DENV-1–4 RT-PCR assay which was positive for DENV-3 sequencing. All other negative RT-PCR samples were also negative by E gene sequencing.</p

Reproducibility summary for the CDC DENV-1–4 Real-Time RT-PCR Assay including results from three testing sites.

*<p>AVG CT value is based on one or two samples.</p><p>% CV = coefficient of variation.</p

Agreement between CDC DENV-1–4 Real Time RT-PCR and seroconversion in samples from suspected dengue patients.

*<p>One DENV-1 case was positive on the CDC DENV-1–4 Real-Time RT-PCR Assay (multiplex) and confirmed positive by IgM conversion, but not sequenced effectively enough to produce an interpretable result.</p>**<p>Two samples were negative by the CDC-DENV-1–4 Real-Time RT-PCR Assay (multiplex). One of these samples was DENV-3 positive by the CDC-DENV-1–4 Real-Time RT-PCR Assay (singleplex) and was further confirmed DENV-3 positive by sequencing. The other sample was confirmed DENV-3 positive by sequencing.</p>***<p>Four samples were positive RT-PCR results and did not confirmed by seroconversion. Two of these samples obtained positive results for DENV-3 and the other two samples were DENV-4 positive and these results were confirmed by E gene sequencing.</p>†<p>All instances of IgM conversion were demonstrated in paired acute and convalescent samples.</p

Maximum likelihood phylogeny of contemporary DENV strains.

<p>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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002311#pntd.0002311-Zhang1" target="_blank">[55]</a>, DENV-2 Asian I (IIIa), American-Asian (IIIb), and Cosmopolitan (IV) genotypes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002311#pntd.0002311-Twiddy1" target="_blank">[42]</a>, DENV-3 South Pacific (I), Thailand (II), and Indian Subcontinent (III) genotypes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002311#pntd.0002311-Zhang1" target="_blank">[55]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002311#pntd.0002311-Klungthong1" target="_blank">[56]</a>, DENV-4 Southeast (I) and Indonesian (II) genotypes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002311#pntd.0002311-Klungthong1" target="_blank">[56]</a>.</p

CDC DENV-1–4 Real Time RT-PCR Assay specificity against clinically relevant concentrations of other pathogens.

<p>CDC DENV-1–4 Real Time RT-PCR Assay specificity against clinically relevant concentrations of other pathogens.</p