Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

Waggoner, Jesse J.; Abeynayake, Janaki; Sahoo, Malaya K.; Gresh, Lionel; Téllez, Yolanda; González, Karla; Ballesteros, Gabriela; Pierro, Anna; Gaibani, Paolo; Guo, Frances P.; Sambri, Vittorio; Balmaseda, Ángel; Karunaratne, Kumudu; Harris, Eva; Pinsky, Benjamin A.

doi:10.1371/journal.pntd.0002116

Cited by 115 publications

(116 citation statements)

References 42 publications

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“…Samples were tested for ZIKV and/or DENV using the ZCD assay, as previously described (46). DENV-positive samples were serotyped using a serotype-specific DENV multiplex assay (46,52). In some laboratories, samples were tested by RT-PCR for ZIKV as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

Grifoni

Pham

Sidney

et al. 2017

J Virol

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154

214

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While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here, we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether preexisting dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with tetravalent dengue attenuated vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors but declines in DENV-preexposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells from DENV-preexposed donors selectively upregulated granzyme B and PD1, unlike DENV-naive donors. Finally, we discovered that ZIKV

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Section: Methodsmentioning

confidence: 99%

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

Grifoni

Pham

Sidney

et al. 2017

J Virol

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154

214

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“…A heminested RT-PCR for the detection and serotyping of DENV, a version of which remains in wide use, was first reported in 1992 (11,12). 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).…”

mentioning

confidence: 99%

“…We also report an independent evaluation of the Altona RealStar dengue RT-PCR kit, which is a commercially produced internally controlled rRT-PCR kit for DENV detection. Furthermore, using 200 clinical samples, we directly compared four molecular tests for DENV: the pan-DENV and Altona assays, a version of the heminested RT-PCR (11), and the serotype-specific DENV multiplex rRT-PCR (16).…”

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confidence: 99%

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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“…Samples with detectable signal only for the 18S target were considered positive for "Plasmodium, nonfalciparum." DENV serotyping was performed using the DENV multiplex assay, as previously described (17).…”

Section: Methodsmentioning

confidence: 99%

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

et al. 2015

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c Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n ‫؍‬ 182) or plasma (n ‫؍‬ 148) from 317 patients was tested; the average sample volume was 70 l (range, 5 to 300 l). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with >75 l of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.T he entirety of Nigeria is considered an area of moderate to high malaria transmission by the WHO (Ն1 case per 1,000 residents), and virtually all cases result from infection with Plasmodium falciparum, the most common cause of severe malaria (1). Despite the high prevalence of malaria in Nigeria, there is concern that it is overdiagnosed in patients presenting with an undifferentiated systemic febrile illness (2). While traditional microscopy remains the primary malaria diagnostic method worldwide, malaria rapid diagnostic tests (mRDTs) are also being deployed for confirmation of suspected malaria cases. However, nucleic acid amplification tests (NAATs) for malaria, which have proven more sensitive than microscopy or mRDTs, are not routinely performed (3, 4). Therefore, it is possible that estimates of overtreatment may be inflated due to the relatively poor sensitivity of microscopy for malaria diagnosis. Although Plasmodium nucleic acids have been detected in serum and plasma, there is little published data describing the performance of malaria NAATs using these specimen types (4-7). Rather, most studies have used whole blood or dried blood spots (DBS) (8-12). While whole blood and DBS provide ample amounts of Plasmodium nucleic acids, they present significant challenges for (i) long-term storage and/or (ii) nucleic acid extrac...

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Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

Cited by 115 publications

References 42 publications

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

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

Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients

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