Acute-phase serum or plasma samples were collected during the initial visit for study enrollment and transported to the IICS-UNA laboratory. Samples were tested for DENV NS1 antigen using the Standard Q Dengue Duo rapid immunochromatographic test (SD Biosensor, Suwon, South Korea) according to manufacturer recommendations. Qualitative antibody data acquired using this method was not evaluated in this study, see antibody section below. Primary samples were then aliquoted and stored at −80°C until later use or shipment on dry ice to Emory University for additional testing. For molecular testing, total nucleic acids were extracted from 200μL of sample on an EMAG instrument and eluted into 50μL of buffer. Samples were tested for Zika virus, chikungunya virus and DENV by real-time RT-PCR (rRT-PCR) using a validated and published multiplex assay (the ZCD assay) [60 (link)], and DENV serotype and viral load were determined with a published DENV multiplex assay [61 (link), 62 (link)]. Both rRT-PCRs were performed as previously described [60 (link)–62 (link)].
Serologic testing was performed on acute-phase samples using two different methods. First, anti-DENV IgG and IgM were analyzed using commercial ELISA kits [Dengue ELISA IgG (G1018) and Dengue ELISA IgM Capture (M1018), Vircell Microbiologists, Granada, Spain] according to manufacturer recommendations (interpretation: IgM or IgG index >11 positive, 9–11 indeterminate, <9 negative). Second, a 5μL aliquot of serum from 139 participants with sufficient sample was tested in the pGOLD assay (Nirmidas Biotech, Inc, Palo Alto, CA), which is a multiplex serological assay for IgM and IgG against DENV (DENV-2 whole virus antigen) and ZIKV (NS1 antigen). The pGOLD assay was performed as previously described [59 (link), 63 (link)]. In each well of the pGOLD slide, antigens are spotted in triplicate, and average signals are used during analysis. For IgG, the negative control signal was subtracted from the sample signal, and the difference was divided by the average signal of four IgG control spots included in each well. For IgM, a similar calculation was performed using the signal from a known anti-DENV IgM positive control sample included on each run. A positive threshold ratio of 0.1 was established for each isotype, which was ≥ 3 standard deviations above the mean of the negative control.
Chymase and LBP levels were determined using commercial ELISA kits (G-Biosciences, St. Louis, MO, USA), following the manufacturer’s instructions. Complete blood counts and chemistries were performed at the clinical site at the discretion of the care team, and results were included if the sample was obtained within ±1 day of enrollment.
Serologic testing was performed on acute-phase samples using two different methods. First, anti-DENV IgG and IgM were analyzed using commercial ELISA kits [Dengue ELISA IgG (G1018) and Dengue ELISA IgM Capture (M1018), Vircell Microbiologists, Granada, Spain] according to manufacturer recommendations (interpretation: IgM or IgG index >11 positive, 9–11 indeterminate, <9 negative). Second, a 5μL aliquot of serum from 139 participants with sufficient sample was tested in the pGOLD assay (Nirmidas Biotech, Inc, Palo Alto, CA), which is a multiplex serological assay for IgM and IgG against DENV (DENV-2 whole virus antigen) and ZIKV (NS1 antigen). The pGOLD assay was performed as previously described [59 (link), 63 (link)]. In each well of the pGOLD slide, antigens are spotted in triplicate, and average signals are used during analysis. For IgG, the negative control signal was subtracted from the sample signal, and the difference was divided by the average signal of four IgG control spots included in each well. For IgM, a similar calculation was performed using the signal from a known anti-DENV IgM positive control sample included on each run. A positive threshold ratio of 0.1 was established for each isotype, which was ≥ 3 standard deviations above the mean of the negative control.
Chymase and LBP levels were determined using commercial ELISA kits (G-Biosciences, St. Louis, MO, USA), following the manufacturer’s instructions. Complete blood counts and chemistries were performed at the clinical site at the discretion of the care team, and results were included if the sample was obtained within ±1 day of enrollment.
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