Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Author summaryDiagnosis of the important mosquito-transmitted dengue virus (DENV) requires laboratory assays to detect viral genome (RT-PCR), viral NS1 protein (immunoassay) or DENV specific antibodies. Current point-of-care NS1 tests cannot distinguish serotype, so laboratory tests are still essential to determine which of 4 DENV serotypes is present. Here we present a rapid serotype-specific NS1 test in a portable microfluidic format. Ten parallel 0.2 mm tubes inside a flat plastic ribbon perform multiplex NS1 immunoassays. A simple cassette delivers sample and reagents sequentially through the microcapillaries by gravity. By stacking cassettes, 12 tests could be performed in under 40 minutes, with results recorded by smartphone. When evaluated with 205 patients plus 50 control samples, and results compared to conventional RT-PCR, the sensitivity for DENV1 to 4 was 78%, 78%, 80%, and 76%, respectively, with specificity of 100% for DENV2-4. DENV1 showed some false positives due to cross-reactivity of the capture antibody. Serotyping performance with MCF-Cygnus devices showed substantial agreement to the serotyping-NS1 microplate ELISA. Therefore, these simple and portable microcapillary immunoassay devices could support dengue NS1 serotyping with potential benefits for near-patient diagnosis, real-time epidemic surveillance and outbreak mapping. Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device-termed Cygnus-with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-mu l plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (kappa = 0.68, 95%CI 0.58-0.77) and NS1-LFT (kappa = 0.71, 95%CI 0.63-0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.