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

['Sarah Helen Needs', 'Reading School Of Pharmacy', 'University Of Reading', 'Whiteknights', 'Reading', 'United Kingdom', 'Sirintra Sirivisoot', 'Division Of Dengue Hemorrhagic Fever Research', 'Faculty Of Medicine Siriraj Hospital', 'Mahidol University']

Date: 2022-05

Abstract 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.

Author summary Diagnosis 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.

Citation: Needs SH, Sirivisoot S, Jegouic S, Prommool T, Luangaram P, Srisawat C, et al. (2022) Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples. PLoS Negl Trop Dis 16(4): e0010266. https://doi.org/10.1371/journal.pntd.0010266 Editor: Brett M. Forshey, US Department of Homeland Security, UNITED STATES Received: April 9, 2021; Accepted: February 18, 2022; Published: April 7, 2022 Copyright: © 2022 Needs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was funded by grants from the Newton Fund, British Council Institutional Links IL35237556 (ADE, PA and SS), the Wellcome Trust, Pathfinder Award 204388/Z/16/Z (ADE) and the EPSRC EP/L013983/1 (ADE) and EP/R022410/1 (ADE and SHN) and EP/S010807/1 (ADE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: ADE is one of the inventors of patent application protecting aspects of the novel microfluidic devices tested in this study, and is a director and shareholder in Capillary Film Technology Ltd, a company holding a commercial license to this patent application: WO2016012778 "Capillary assay device with internal hydrophilic coating" AD Edwards, NM Reis.

Introduction Dengue virus (DENV) is a member of Flavivirus genus, which shares some typical RNA genomes with West Nile virus, Zika virus, yellow fever virus, and Japanese encephalitis virus. There are nearly estimated 400 million cases of dengue annually, being endemic in tropical and subtropical countries and efficiently transmitted by mosquitoes, mostly Aedes spp. [1]. DENV genomes contain of three structural (capsid (C), pre-membrane protein (prM), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4b, and NS5) [2]. During viral replication in an infected host, soluble NS1 proteins are secreted in high concentrations into the circulation and to a lesser extent saliva and urine [3,4,5]. NS1 from four serotypes of DENV is used as a common diagnostic marker (up to nine days after onset) in both acute primary and secondary infections [6]. Many commercial DENV NS1 tests are available, based on rapid immunochromatography (lateral flow rapid tests used at point-of-care) and microplate ELISA (in diagnostic laboratory), such as Panbio, Standard Diagnostics, and Bio-Rad. Their overall sensitivity and specificity varied from 45 to 80% and 93 to 100%, respectively [7,8,9,10]. Both methods use only small volumes of sample (blood/serum/plasma) (50–100 μL). When comparing the processing times, the one-step sandwich-format microplate ELISA could provide a result in 120 min, while rapid tests need less than 30 min. Even though the rapid test is faster, lateral flow immunoassays have a much higher limit of detection compared to a microplate ELISA and can therefore only detect higher levels of viral protein. At antigen levels approaching the limit of detection, visual interpretation of lateral flow rapid tests can be ambiguous. However, lateral flow test readers are increasingly available to improve result interpretation [11]. Microplate ELISA avoids visual interpretation as this is routinely measured with a microplate reader but requires laboratory operation. Therefore, a need for higher sensitivity rapid tests that can be performed with minimal laboratory equipment is required. The antibody pairs in DENV NS1 immunoassays that are currently in clinical use detect NS1 from all four serotypes with varying analytical sensitivity. However, by careful selection of antibody clones that bind to conserved epitopes, serotype-selective immunoassays have been developed including microplate ELISA [12,13,14]. A prototype lateral flow immunoassay could differentiate each DENV serotype, and also distinguish DENV from Zika virus without cross reactivity [15], offering the potential for dengue serotyping rapid tests, although NS1 from Zika virus is found at much lower plasma concentrations making lateral flow unlikely to be sensitive enough for Zika diagnosis. This rapid test panel showed sensitivity and specificity to each DENV NS1 serotype ranging from 76–100%, illustrating the potential for serotype-specific antibody pairs to define serotype from plasma NS1. When patients have a secondary infection caused by a different DENV serotype they may be more prone to develop more severe forms of disease—dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)—due to antibody-dependent enhancement (ADE) [16]. If severe dengue patients do not receive appropriate treatment or immediate hospitalization, fatalities are possible. The effect of serotype infection on patient outcome is not fully understood since serotyping is mainly used for surveillance. However, the serotype of secondary infections may indicate disease outcomes [17]. If this could be accurately mapped it is possible that serotype-specific rapid tests could be prognostic for severe disease, however, knowledge of concurrent or secondary infection currently provides better prediction of severe disease [18]. Rapid knowledge of infecting DENV serotype has potential value for early intervention, for disease surveillance and for epidemic control. To identify four serotypes of DENV, conventional virus isolation and characterization, and RT-PCR methods are currently performed [19,20,21]. Within the past decade, microplate DENV serotyping-NS1 ELISAs have also been developed as in-house assays, but prediction of some serotypes was still inaccurate [12,13,14]. Our recent modified serotyping-NS1 microplate ELISA (plus) showed perfect concordance in four serotypes identification in dengue patients’ specimens comparing to RT-PCR [22], indicating that immunoassay methods are capable of accurate serotyping. An ideal DENV diagnostic tool would need to be as sensitive and specific as possible, be able to be interpreted in real-time, should be easy to perform without investment in laboratory instruments, cheap to manufacture, and capable of classifying DENV serotypes. Real-time monitoring of dengue serotype infection will also be helpful for epidemiology, disease surveillance, and public health strategy to control endemic infection and target interventions (e.g., mosquito control) to epidemic outbreaks [23]. Microfluidic technology can fabricate miniature compact bioassay devices offering many benefits to healthcare systems across the world if economical, robust, and reproducible fabrication can be achieved. Microfluidic and lab-on-a-chip devices have been developed to screen, detect, or monitor tumor cells and to measure and detect various protein and nucleic acid biomarkers in human biological samples [24,25,26]. Microcapillary film (MCF) has been shown to be useful for miniaturizing sandwich ELISA to detect biomarkers in clinical samples [27,28,29,30,31]. Produced by melt-extrusion of fluorinated ethylene propylene (FEP), MCF comprises a flat ribbon containing ten parallel microcapillaries. This fluoropolymer plastic film has optical clarity from refractive index matching allowing the bioassay output (color or fluorescence) to be easily observed and simply quantified on a smartphone camera [29,32]. In previous studies, MCF devices have been developed, using an ELISA-based assay format, in both single and multiplex assay strips that were driven by pressure (using a syringe to deliver sample and reagent) and interpreted by a flatbed scanner [27,28] or a smartphone camera [29]. For simpler assays requiring only a sample plus single reagent, MCF test strips used capillary action achieved by coating the inner surface with hydrophilic polymer [33]; this hydrophilic polymer coating is compatible with antibody coating for immunoassays [31,32,33]. Recently, we further reduced the complexity of MCF immunoassay devices by using gravity to flow the sample followed by wash and detection reagents sequentially through microcapillaries [34]. Previous work on protein biomarker detection has identified that immunoassays using MCF devices performed as well as microplate ELISA [30,31], but only with spiked simulated samples. Here, we developed multiplex MCF immunoassays for viral antigen and for the first time evaluated the analytical performance of the tests with human clinical patient samples. We described the transfer of sandwich immunoassays from microplate ELISA to MCF test strips to develop a simple rapid assay method for the detection of serotype specific DENV NS1. We used gravity driven MCF devices to detect and differentiate multiplex serotype-specific DENV NS1 proteins in 205 undiluted patient plasma samples alongside 50 other febrile illness controls. MCF assays were performed using a novel holder, termed Cygnus, that simplified sample and reagent addition to MCF. The Cygnus device can be stacked in microplate compatible spacing to test up to 12 samples (i.e., 120 individual immunoassays) and showed stacked Cygnus devices permit easy-to-use and flexible rapid multiplexed immunoassays in MCF.

Discussion In this study, the Cygnus device was developed to detect DENV infection in plasma through viral NS1 antigen detection as well as DENV serotype identification simultaneously, without the use of complex laboratory equipment and with short time-to-result. The performance of the Cygnus device was evaluated with dengue clinical samples in comparison to an in-house serotyping-NS1 microplate ELISA which provides similar features for DENV NS1 detection and serotyping. Compared to a standard RT-PCR method, both assays showed similar sensitivity for the detection of NS1 regardless of serotype, 83% vs 82% for NS1-microplate ELISA and NS1-Cygnus respectively. Sensitivity to DENV1-4 serotypes compared to RT-PCR detected by NS1-Cygnus ranged from 76 to 80% with DENV3 being the most sensitive. While that of microplate NS1-ELISA showed a range of sensitivity from 76 to 92%, with DENV1 the most sensitive. (Table 6). Overall, specificity of NS1-microplate ELISA was higher than the NS1-Cygnus device, 100% vs 86%. The anti-NS1 mAbs specific to each serotype as well as the pan-serotype mAb are critical reagents for the microcapillary based assay development and were selected based on high reactivity to direct NS1 coated ELISA and low K D from surface plasmon resonance (S1 Table). The low specificity of the Cygnus test was a result of false positive samples assigned to DENV1, which was not found in NS1-microplate ELISA, although the same clones of serotype-specific mAbs were used. This might be explained by the different configuration of antibody pair which were designed for capture and detection between two assays. In the microplate ELISA, a greater flexibility in antibody pairings was possible, at the cost of needing separate plates and sample addition for each serotype assay. The pan-serotype antibody (clone 2E11) could be used as either the capture or detection antibody, but use as the capture antibody gave better performance, presumably because it could bind more effectively to epitopes shared between NS1 of all serotypes [22]. In contrast, the Cygnus platform, serotype-specific mAbs must be used as capture antibodies for multiplexing within each microcapillary test strip, while a pan-serotype mAb (clone 1F11) was needed for detection of all serotypes. This may lead to non-specific binding explaining those false positive cases. Furthermore, after assay optimization to produce the most sensitive assay, a higher concentration of DENV1 antibody (60 μg/ml) than that of other serotypes (40 μg/ml) was used to coat in microcapillary strips which may also increase the potential for background signal resulting in false positives. Noted in the NS1-microplate ELISA, this clone (84B) alone also gave poor sensitivity to DENV1 (31/50; 62%), but the sensitivity was enhanced to 92% by the addition of a further microplate assay using a DENV1/3 subcomplex antibody pair (clone 5F3) (Table 6 and S1 Data). From these lines of evidence, we suggest that the concept of the Cygnus to detect DENV NS1 and serotyping is promising, although DENV1 specific assay requires improvement. Presently there are many commercial test kits to diagnose DENV NS1 by either microplate ELISA or rapid immunochromatographic LFT, for example, SD Bioline Dengue Duo (Alere, USA), Platelia NS1 Ag (Bio-Rad, France), or Panbio Dengue Early ELISA (Alere). Their sensitivity and specificity can vary widely (70–99%) [42,43]. However, some concerns remain about serological cross-reactivity between arboviruses such as newer Flavivirus including Zika that emerge in dengue endemic areas. One study used both SD Bioline Dengue Duo and Platelia Dengue NS1 Ag kit in 65 Zika-infected patients, but none of them showed positive results [44], suggesting high specificity of dengue NS1 assay. Nevertheless, none of the commercially available dengue NS1 test kits can differentiate DENV serotypes. In addition to conventional virus isolation and RT-PCR, in-house NS1 microplate ELISA can be used to differentiate serotypes [12,13,14,22,45]. Only one prototype of NS1 dipstick rapid test was recently developed for DENV serotyping with 76, 89, 100 and 100% sensitivity for DENV1-4 respectively and 89, 98, 100 and 96% specificity for DENV1-4, respectively [15]. Several studies have suggested that sensitivity of the NS1 assays in primary infection is greater than secondary infection cases in which more false negative samples were found [46,47,48,49]. Therefore, in DENV endemic areas where secondary infections are frequent, NS1 assay performance tends to be low. This is potentially due to the presence of NS1 immune complex (IC) which is generated by circulating NS1 and existing anti-NS1 antibodies [50,51,52] which reduces the amount of free NS1 protein. Disassociation of NS1 IC by heat or acid/base methods explored in previous studies [53,54,55] were found to enhance assay sensitivity. However, our NS1-Cygnus device and NS1-microplate ELISA gave above 82% sensitivity despite patient plasma used in this study being mostly from secondary infections (98%). This may suggest high binding ability of the selected anti-NS1 antibody pairs which were used in both assays, or high analytical sensitivity for both immunoassay platforms (microplate and microcapillary). Regarding the development process of the Cygnus device, distinct from the flat plastic capillary film ELISA in previous studies [27,29,31], the Cygnus devices represent an improved and more accessible format for microfluidic immunoassays by coating with hydrophilic polymer (PVOH) allowing fluid flow to be driven entirely by gravity without requiring mechanical operation via the multi-syringe aspirator [31]. As found previously, the excellent optical transparency of fluorinated ethylene propylene (FEP) which has a refractive index equal to water, made it suitable for simple fluorescent substrate detection and straightforward recording of results by smartphone cameras [29]. No significant difference in analytical performance was seen between the different cameras used, confirming previous systematic comparison of cameras for microfluidic immunoassay detection [39]. The imaging processing for quantitation of NS1 levels, is currently performed manually. For greater uptake of the Cygnus test, this analysis should be automated, analogous to a lateral flow reader. Here, we demonstrated that this proof-of-concept test can be imaged using simple and low-cost optics with a smartphone camera, enabling the possibility of smartphone embedded app for automatic digital image analysis alongside scoring the test and reporting results electronically. In summary, we demonstrate that our Cygnus prototype device may represent a promising tool for rapid serotype-specific detection of NS1 for diagnosis and surveillance of dengue infection in clinical samples. Due to its inexpensive mass-manufacture, portability, speed, and simplicity to operate, alongside sensitivity, specificity and accuracy that closely matches microplate ELISA for DENV2-4, we believe these devices could be used for surveillance and routine diagnosis worldwide especially in remote areas with limited access to laboratory infrastructure (e.g., microplate ELISA readers). This device could be helpful as an ancillary test alongside RT-PCR in hospital diagnostic labs or in the field. Moreover, it provides DENV serotyping information and rapid results that are valuable for real-time dengue epidemiology.

Acknowledgments The clinical cohorts were supported by the Office of the Higher Education Commission, Mahidol University under the National Research Universities Initiative and Research Chair Grant, NSTDA, Thailand. We sincerely thank Nattaya Tangthawornchaikul (National Science and Technology Development Agency), Adisak Songjaeng, Arunee Mapralub, Supansa Pakdee, Chotika Kaewpuenk, Dararat Prayongkul and Oranicha Khamprapa (Faculty of Medicine Siriraj Hospital, Mahidol University) for their contribution to dengue hospital cohorts where the collection of dengue clinical specimens and database were used in this study. We likewise offer great appreciation to Watchara Kasinrerk (Chiang Mai University) for his pioneered contribution to generate panels of mouse monoclonal antibodies to dengue proteins.

[END]

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