Disposable Autonomous Device for Swab-to-Result Diagnosis of Influenza

Abstract

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

Figure 1

Molecular stacks for the detection of NP in a sandwich immunoassay format. At the test lines, capture antibodies bind NP (either A or B, depending on the specificity of the antibody) from lysed virus. The surface-bound NP is subsequently labeled by a mouse-derived antibody pre-conjugated to multiple HRP molecules, and visualized by DAB color development. At the control line, a goat anti-mouse IgG is immobilized; it will bind to the same indicator Ab/HRP conjugate in the absence of NP, and detection Ab complex is then visualized by DAB color development.

Figure 2

Imaging system used to hold the disposable device, record signal development in the device, and to monitor user activities during device operation at Seattle Children's Hospital.

Figure 3

Device operation at Seattle Children's Hospital. Clinical staff collected patient samples and stored the swab in a swab 4 storage tube. The actual device test occurred in a room nearby within the hospital. The cumulative time required for test completion was ∼35 min. The image system continuously recorded the images of the lateral flow test strips for ∼55 min.

Figure 4

Internal device operation. a) Cartoon of the NP detection assay chemistry at the analyte capture lines, which is based on NP binding to HRP-conjugated antibodies. Next, the NP conjugate was captured by the membrane bound capture antibody, followed by HRP turnover of the chromogenic substrate, DAB in the presence of H₂O₂. b) 3D model of the internal components of device. Sample introduction by swab into a swab port containing lysis buffer is followed by activation of the device, which causes puncture of aqueous reagent container and release of aqueous reagents into legs of the 2DPN. The 2DPN automates rehydration of dry reagents, splitting the sample into two channels (one for influenza A detection, the other for influenza B detection), and sequential delivery of subsequent assay reagents.

Figure 5

Examples of the extracted signal profiles for strong, weak, and negative results. The row-averaged pixel intensities in the blue channel of test strip images were used to generate plot profiles for each test strip (blue lines). Sixth-order polynomials (black lines) were fit to the background regions of each plot profile. Test lines were detected as thresholded (dotted black lines) excursions (red asterisks) of the background-subtracted plot profiles (red lines) beyond three standard deviations of the mean background-subtracted intensity (dotted black lines).

Figure 6

Summary of 25 device results vs. gold-standard qRT-PCR test results. The qRT-PCR was used to determine whether the sample was influenza positive or negative. Yellow boxes show disagreement between qRT-PCR test results and the device results.