Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses

Leparc-Goffart, Isabelle; Baragatti, Meïli; Temmam, Sarah; Tuiskunen, Anne; Moureau, Grégory; Charrel, Rémi N.; Lamballerie, Xavier de

doi:10.1016/j.jcv.2009.02.010

Cited by 124 publications

(103 citation statements)

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“…In cases where such patients are tested, the sensitivity of DENV detection has generally been poor, ranging from 0 to 69% (20,22,(43)(44)(45). The improved sensitivities of the pan-DENV and multiplex rRT-PCR assays may result from the use of a highly conserved target region in the 5= UTR and capsid gene, compared to previous assays that utilized targets in other regions of the genome, including the CprM, E, NS3, NS5, and 3= UTR (11,13,15,20,24).…”

Section: Discussionmentioning

confidence: 99%

“…Though numerous DENV species-specific and serotype-specific assays have since been developed, direct comparisons between these assays are notably rare (11,(13)(14)(15)(16)(17). Rather, studies evaluating new DENV molecular diagnostic tests typically use samples collected within the first 5 days of fever from patients who had positive testing by viral isolation, seroconversion, or both (10,(18)(19)(20)(21)(22)(23)(24)(25). This practice ensures that only the highest viral loads are evaluated and it likely does not reflect clinical reality, where patients may present at any time during their illness, including five or more days after fever onset.…”

mentioning

confidence: 99%

“…The majority of DENV NAATs have been designed with the dual goals of sensitive DENV detection and the ability to provide a serotype-specific diagnosis (11,13,14,23,24). While serotyping capability is useful for epidemiologic surveillance, the relative contribution of serotype-specific information to the care of individual patients remains unclear (1,10,26).…”

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confidence: 99%

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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

et al. 2013

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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...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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

et al. 2013

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“…Dengue NS1 antigen and dengue IgG were positive, whereas dengue IgM was negative using a lateral-flow diagnostic assay (SD BIOLINE Dengue Duo NS1 Ag þ Ab Combo, ALERE, France). DEN-4 virus RNA was detected (Cycle threshold [Ct] at 32) using four serotype-specific inhouse real-time reverse transcription polymerase chain reaction (rt-RT-PCR) while DEN-1, DEN-2 and DEN-3 tests were negative, and rt-RT-PCR for chikungunya was also negative [11]. The serum sample was inoculated onto Vero cells and despite cytopathic effect was not observed after 5 days, virus replication was demonstrated based on positive rt-RT-PCR for DEN-4 (Ct < 20).…”

Section: Case Reportmentioning

confidence: 99%

A secondary dengue 4 infection in a traveler returning from Haiti confirmed by virus isolation, complete genome sequencing and neutralisation assay: A brief report

Ménard

Ninove

Zandotti

et al. 2015

Travel Medicine and Infectious Disease

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“…These studies have provided details of the genotypes or clades circulating in Thailand [10][11][12]. Although the nested RT-PCR method is more time-consuming and has a higher risk of contamination than realtime RT-PCR (rRT-PCR) [13][14][15][16][17][18][19][20], the cost of the assay is significantly lower making it the preferred method for DENV detection in many laboratories.…”

Section: Introductionmentioning

confidence: 99%

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand

Klungthong¹,

Manasatienkij²,

Phonpakobsin³

et al. 2015

Journal of Clinical Virology

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a b s t r a c tBackground: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. Objective: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. Study design: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. Results and conclusion: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.Published by Elsevier B.V.

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Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses

Cited by 124 publications

References 9 publications

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

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

A secondary dengue 4 infection in a traveler returning from Haiti confirmed by virus isolation, complete genome sequencing and neutralisation assay: A brief report

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand

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