Abstract
BackgroundEarly and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.MethodsA single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).ResultsAcute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.ConclusionThe RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.
Highlights
And rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread
Design of DENV-specific reverse transcription-loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay primers In order to develop the single-tube RT-LAMP assay for detection of all four DENV serotypes, a set of nine primers comprising four outer (F3/134, F3/2, B3/123, and B3/4), four inner (FIP/123, FIP/4, BIP/123, and BIP/4), and one loop primers (BLP/1234) were designed
Specificity of RT-LAMP assay No cross-reactivity of the RT-Lamp assay was observed with all other three closely related arboviruses common in the region, including Japanese encephalitis virus (JEV), Chikungunya virus (CHIKV) and Sindbis virus (SINV) (Figure 1)
Summary
And rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic. Studies have shown that heterotypic secondary infection is associated with higher risk of contracting DHF and DSS [6] possibly through antibody-dependent enhancement [7,8], original antigenic sin [9,10], cytokine storm [11], or autoimmune response [12,13]. A sensitive, easy to perform and rapid method for detection of DENV in viremic febrile patients is of paramount importance especially for patient management and immediate vector control measures to prevent large-scale epidemic [15]
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