The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach

Abstract

Background: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum.

Aims: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test.

Methodology: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples.

Key results: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers.

Conclusions: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Figure 1. Representative sensorgrams for binding of monoclonal antibody Ab3 and DENV-1 NS1.

Increasing concentrations of dengue-1 NS1 were sequentially injected onto 70 RU of Ab3 captured by anti-mouse IgG. The reference-subtracted data (black curve) were fitted using 1∶1 binding algorithm (red curve).

Figure 2. Analytical sensitivity of Dengue Early Rapid test for recombinant dengue NS1.

The test line signal intensity was measured against a serial two-fold dilution series of DENV-1 NS1 (A), DENV-2 NS1 (B), DENV-3 NS1 (C) and DENV-4 NS1 (D) from 512 ng/mL to 0.5 ng/mL. The analytical limit of detection was determined as the lowest concentration required to produce a positive result of 8.5 mABS (absorbance) units using interpolation from a non-linear regression. An absorbance value of 8.5 mABS was determined to be the cut-off value for visualisation of the test line.

Figure 3. Accuracy of dengue rapid tests at the Malaysian study site.

Diagnosis was confirmed using a combination of HI, virus isolation, RT-PCR and IgM ELISA (n = 293). Sensitivity (%) between groups was compared using Fisher's exact test.

Figure 4. Clinical sensitivity of rapid tests for acute dengue over the course of illness.

Data was obtained from laboratory-confirmed cases of dengue at the Malaysian study site (n = 263).