Dengue Literature Review

RT-PCR

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RT-PCR

Overview

Reverse transcription polymerase chain reaction (RT-PCR) is the reference standard molecular method for dengue diagnosis during the acute viraemic phase. It detects dengue virus (DENV) RNA in serum, plasma, or whole blood, can identify the infecting serotype, and is highly sensitive during the first 5 days of illness. Quantitative real-time RT-PCR (qRT-PCR) additionally allows viraemia quantification and is used in research settings to define asymptomatic dengue by virological confirmation.

Key Points from Literature

Diagnostic performance and timing

  • Detection window: DENV RNA is detectable from approximately 24–48 hours before fever onset through days 5–6 of illness (see Guzman2016 - Dengue Infection)
  • Most sensitive during the first 5 days of illness; sensitivity declines sharply as viraemia clears
  • After day 5–6, IgM-IgG Serology ELISA becomes the diagnostic method of choice; serological and molecular testing are complementary
  • RT-PCR should not be used as the sole test when illness onset is >5 days ago; a negative result in that window cannot exclude dengue

Serotyping

  • RT-PCR can identify the infecting DENV serotype (DENV-1, -2, -3, or -4) — critical for epidemiological surveillance and for interpreting secondary infection risk
  • Serotype identification by conventional or multiplex RT-PCR is standard practice in reference laboratories
  • Sequencing of RT-PCR amplicons allows genotype identification (e.g., distinguishing Asian from American DENV-2 genotype)

Quantitative RT-PCR (qRT-PCR)

  • Allows measurement of viraemia titre; useful in research to assess correlates of disease severity
  • Used as the definitional criterion for asymptomatic dengue infection in the Sungnak2025 Thailand cohort — positive DENV-specific qRT-PCR in individuals with no clinical symptoms (see Asymptomatic Dengue Infection)
  • Viraemia levels correlate with disease severity particularly in secondary infection

Placement in WHO diagnostic tiered structure

  • Reference laboratory level: RT-PCR and virus isolation (plaque assay, C6/36 cell culture)
  • Intermediate level: NS1 ELISA, IgM ELISA (less resource-intensive than RT-PCR)
  • Field level: rapid diagnostic tests (NS1 RDTs, combined NS1/IgM/IgG RDTs)
  • This tiered structure reflects the resource requirements of RT-PCR: thermocycler, RNA extraction, molecular reagents, and trained staff

Comparison with other acute-phase tests

MethodStart dayEnd dayAdvantagesDisadvantages
RT-PCR−2 (pre-fever)~5–6Highest sensitivity early; serotypingResource-intensive; reference lab required
NS1 antigen~1~5–9Point-of-care formats availableVariable sensitivity by serotype/timing
Virus isolation~1~5Gold standard virological confirmationSlow (7–10 days); BSL-2 required
IgM ELISA~5–6WeeksWidely available; point-of-care formatsMisses first 5 days; Zika cross-reactivity

Contradictions & Debates

  • Sensitivity of RT-PCR in primary vs secondary infection: secondary infection may have shorter or lower viraemia, potentially reducing sensitivity in some cases
  • Zika virus RNA is co-detected by some dengue RT-PCR assays designed before 2015 (related flavivirus); dual testing now recommended where Zika circulates

Sources

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