BACKGROUND
As pathogens such as influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can easily cause pandemics, rapid diagnostic tests are crucial for implementing efficient quarantine measures, providing effective treatments to patients, and preventing or containing a pandemic infection. Here, we developed the immunochromatography-NanoSuit® method, an improved immunochromatography method combined with a conventional scanning electron microscope (SEM), which enables observation of immunocomplexes labeled with a colloidal metal.
OBJECTIVE
A total of 197 clinical samples from patients suspected to be suffering from influenza were provided by a general hospital at the Hamamatsu University School of Medicine for examination using the Flu kit.
METHODS
Immunochromatography kit
The ImunoAce® Flu kit (NP antigen detection), a human influenza commercial diagnosis kit, was purchased from TAUNS Laboratories, Inc. (Shizuoka, Japan). Au/Pt nanoparticles were utilized to visualize the positive lines. A total of 197 clinical samples from patients suspected to be suffering from influenza were provided by a general hospital at the Hamamatsu University School of Medicine for examination using the Flu kit. After macroscopic diagnosis using the Flu kit, the samples were stored in a biosafety box at room temperature (20-25 °C / 68 - 77 °F). The IgM detection immunochromatography kit against SARS-CoV-2 was obtained from Kurabo Industries, Ltd. (Osaka, Japan).
One step rRT-PCR for influenza A
rRT-PCR for influenza A was performed as described previously using Flu A universal primers. A Ct within 38.0 was considered as positive according to the CDC protocol. The primer/probe set targeted the human RNase P gene and served as an internal control for human nucleic acid as described previously.
SEM image acquisition
The immunochromatography kit was covered with a modified NanoSuit® solution based on previously published components (Nisshin EM Co., Ltd., Tokyo, Japan), placed first onto the wide stage of the specimen holder, and then placed in an Lv-SEM (TM4000Plus, Hitachi High-Technologies, Tokyo, Japan). Images were acquired using backscattered electron detectors with 10 or 15 kV at 30 Pa.
Particle counting
In fields containing fewer than 50 particles/field, the particles were counted manually. Otherwise, ImageJ/Fiji software was used for counting. ImageJ/Fiji uses comprehensive particle analysis algorithms that effectively count various particles. Images were then processed and counting was performed according to the protocol.
Diagnosis and statistics
The EM diagnosis and criteria for a positive test were defined as follows: particle numbers from 6 fields from the background area and test-line were statistically analyzed using the t-test. If there were more than 5 particles in one visual field and a significant difference (P < 0.01) was indicated by the t-test, the result was considered positive. Statistical analysis using the t-test was performed in Excel software. Statistical analysis of the assay sensitivity and specificity with a 95% confidence interval (95% CI) was performed using the MedCalc statistical website. The approximate line, correlation coefficient, and null hypothesis were calculated with Excel software.
RESULTS
Our new immunochromatography-NanoSuit® method suppresses cellulose deformity and makes it possible to easily focus and acquire high-resolution images of gold/platinum labeled immunocomplexes of viruses such as influenza A, without the need for conductive treatment as with conventional SEM. Electron microscopy (EM)-based diagnosis of influenza A exhibited 94% clinical sensitivity (29/31) (95% confidence interval [95%CI]: 78.58–99.21%) and 100% clinical specificity (95%CI: 97.80–100%). EM-based diagnosis was significantly more sensitive (71.2%) than macroscopic diagnosis (14.3%), especially in the lower influenza A-RNA copy number group. The detection ability of our method is comparable to that of real-time reverse transcription-polymerase chain reaction.
CONCLUSIONS
This simple and highly sensitive quantitative analysis method involving immunochromatography can be utilized to diagnose various infections in humans and livestock, including highly infectious diseases such as COVID-19.
Statistical analysis of cytogenetic data