Reading School of Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom.
Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
PLoS Negl Trop Dis. 2022 Apr 7;16(4):e0010266. doi: 10.1371/journal.pntd.0010266. eCollection 2022 Apr.
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-μl 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 (κ = 0.68, 95%CI 0.58-0.77) and NS1-LFT (κ = 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.
登革热病毒（DENV）感染的实验室诊断，包括 DENV 血清分型，需要熟练的劳动力和设备齐全的环境。DENV NS1 侧向流动快速检测（LFT）提供了简单性，但缺乏鉴定血清型的能力。仍然需要一种简单、经济、适合现场使用的血清分型设备。我们展示了一种基于重力驱动的、与智能手机兼容的微流控装置，该装置使用微毛细管膜（MCF）进行登革热病毒 NS1 的多重血清型特异性免疫检测。一种名为 Cygnus 的新型设备具有堆叠设计，允许在 40 分钟内同时分析 1 到 12 个样本。开发了一种夹心酶免疫测定法，用于在一个 60μl 血浆样本中特异性检测所有四种 DENV 血清型的 NS1。该测试旨在弥合快速 LFT 和实验室微孔板 ELISA 在灵敏度、可用性、可及性和速度方面的差距。使用 205 例登革热感染患者的回顾性未稀释血浆样本和 50 例发热性疾病阴性对照对 Cygnus NS1 检测进行了评估。与金标准 RT-PCR 相比，Cygnus 的临床灵敏度为 82%（DENV1-4 分别为 78%、78%、80%和 76%），与内部血清分型 NS1 微孔板 ELISA（82% vs 83%）相当，但优于商业 NS1-LFT（82% vs 74%）。Cygnus 装置的特异性为 86%，低于 NS1-微孔板 ELISA 和 NS1-LFT（分别为 100%和 98%）。对于 Cygnus 阳性样本，DENV2-4 血清型的鉴定与 RT-PCR 完全匹配，但 DENV1 毛细管出现假阳性，表明需要改进 DENV1 捕获抗体以提高特异性。Cygnus 的整体性能与 NS1-微孔板 ELISA（κ=0.68，95%CI 0.58-0.77）和 NS1-LFT（κ=0.71，95%CI 0.63-0.80）具有显著一致性。尽管需要进一步改进 DENV-1 NS1 的检测，但该 Cygnus 设备具有多重检测和快速处理时间的优势，可提供即时的 DENV 抗原检测，包括血清分型，用于及时的登革热诊断，以进行流行监测和疫情预测。
