10 research outputs found
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 (R<sup>2</sup>) is shown for each serotype. Error bars indicate standard deviation of measurements from 5 replicas in GCE/mL.</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
Detection of DENV in the presence of potentially interfering substances present in blood.
*<p>AVG CT = average CT value, STD = standard deviation, ND = not detected.</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
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
Comparison of published and oligonucleotides used in CDC DENV-1–4 Real Time RT-PCR Assay.
<p>Note: The sequences of the primers and probes of CDC DENV-1-4 RT-PCR Assay for detection of DENV-1-4 are covered under a pending patent application.</p>*<p>Frequency: number of complete sequence perfect matches over the total number of sequences screened.</p