Development of an Internally Controlled Real-Time Reverse Transcriptase PCR Assay for Pan-Dengue Virus Detection and Comparison of Four Molecular Dengue Virus Detection Assays

Abstract

A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log 10 cDNA equivalents/l and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/l, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P < 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided. Dengue virus (DENV) is the most common vector-borne human viral pathogen worldwide (1). Infection with one or more of the four closely related virus serotypes (designated DENV-1 to -4) results in a range of clinical manifestations spanning asymptomatic infection, dengue fever (DF), and severe dengue, a category that includes entities previously classified as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (1, 2). Infection with one serotype (primary infection) results in immunity to that serotype, but infection can still occur with any of the remaining serotypes (secondary infection). Secondary DENV infection has been shown to be a significant risk factor for the development of DHF or DSS (3, 4). DENV transmission largely occurs in the tropical and subtropical regions of the world, though the number of countries where DENV is endemic has been increasing (1, 5). Recent reports estimate that 230 million DENV infections occur annually worldwide, including 2 million cases of severe disease and 21,000 deaths (6). Over 3.6 billion people live in regions where this pathogen is endemic and are at risk for infection (6). DF is also one of the most common causes of a systemic febrile illness in travelers returning from countries where the virus is endemic and remains a major concern for military personnel stationed in these areas (7-9).A wide array o...

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

“…These are assays either not quantitative or use a two-step method. Other real-time dengue virus RT-PCR assays use either a three-or fourstep cycling method, which increases assay time, or use a SYBR green-based method, which is prone to nonspecific reaction products leading to increased background and false positives (20,(32)(33)(34). In 2010, the international external quality control assessment for the molecular diagnosis of dengue infections published their findings for 37 laboratories performing 46 tests and showed that 80% of these tests lacked sensitivity, specificity, or both (35).…”

“…In early-stage infection, while the patient is viremic, nucleic acid amplification by PCR is the most rapid diagnostic tool. The use of molecular methods has increased, and several PCR-based assays have been developed for the diagnosis of DENV and CHIKV with human samples (20)(21)(22)(23)(24)(25). The objective of this study was to develop a multiplex assay for the rapid, differential, quantitative diagnosis of DENV and CHIKV in a single-reaction format.…”

Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

“…The DENV and RNase P primer and probe sequences were previously published (24). The UFI assay incorporated the following modifications: the DENV probe concentration in the final reaction mixture was decreased to 100 nM, the RNase P primer concentration was decreased to 50 nM, and the RNase P probe concentration was increased to 100 nM.…”

“…For Leptospira 16S rRNA, Pfr364, and Plasmodium 18S rRNA, quantitated plasmid DNA was utilized. Amplicons generated from monoplex reactions for Leptospira, P. falciparum (Pfr364 target), and P. vivax (18S rRNA gene target) were cloned and sequenced using M13 primers as described previously (24,32). Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130 (ATCC BAA-1198D-5; ATCC, Manassas, VA), template genomic DNA was kindly provided by Alexandria Boehm (Stanford University).…”

“…While the reported sensitivities of individual assays have varied, NAATs have proved to be the most sensitive diagnostic tests for these pathogens in the setting of acute infection (8,17,(20)(21)(22)(23). Recently, our group reported the development of an internally controlled, real-time RT-PCR (rRT-PCR) assay for pan-DENV detection (24). This pan-DENV assay detects all four DENV serotypes using hydrolysis probes labeled with a single fluor and targets RNase P as the internal control (IC).…”