Sampling and detection of airborne influenza virus towards point-of-care applications

Abstract

Airborne transmission of the influenza virus contributes significantly to the spread of this infectious pathogen, particularly over large distances when carried by aerosol droplets with long survival times. Efficient sampling of virus-loaded aerosol in combination with a low limit of detection of the collected virus could enable rapid and early detection of airborne influenza virus at the point-of-care setting. Here, we demonstrate a successful sampling and detection of airborne influenza virus using a system specifically developed for such applications. Our system consists of a custom-made electrostatic precipitation (ESP)-based bioaerosol sampler that is coupled with downstream quantitative polymerase chain reaction (qPCR) analysis. Aerosolized viruses are sampled directly into a miniaturized collector with liquid volume of 150 μL, which constitutes a simple and direct interface with subsequent biological assays. This approach reduces sample dilution by at least one order of magnitude when compared to other liquid-based aerosol bio-samplers. Performance of our ESP-based sampler was evaluated using influenza virus-loaded sub-micron aerosols generated from both cultured and clinical samples. Despite the miniaturized collection volume, we demonstrate a collection efficiency of at least 10% and sensitive detection of a minimum of 3721 RNA copies. Furthermore, we show that an improved extraction protocol can allow viral recovery of down to 303 RNA copies and a maximum sampler collection efficiency of 47%. A device with such a performance would reduce sampling times dramatically, from a few hours with current sampling methods down to a couple of minutes with our ESP-based bioaerosol sampler.

Fig 1. Schematics of the experimental setup (not to scale).

(a) Experiments are conducted in a bio-safety cabinet to keep all aerosolized virus contained. The setup includes a Collison nebulizer to aerosolize using compressed air and viral solutions provided by a syringe pump. Tubing directs the airborne viruses into the sampling unit (b): (1) our ESP sampler or (2) gelatin filters for comparison. A downstream vacuum pump maintains a constant flow and under pressure inside the ESP sampler. (b) Details of the sampling units: (1) Our ESP sampler includes a three-electrode corona discharge electrostatic precipitator to capture the aerosol particle directly into an integrated liquid collector with a miniaturized volume of 150 μL; (2) gelatin filters are used for comparative measurement of the total amount of virus effectively entering the ESP sampler after nebulization. The filters are housed in a specific cassette placed at the aerosol inlet.

Fig 2. Measurements of the total amount of viruses detected with qPCR versus the total amount of viruses entering the ESP sampler.

Linear regressions were performed using the least-square method on data groups obtained using EP1 and EP2. The qPCR LoD reported to the sampling volume is plotted with a horizontal dashed red arrow and enables calculating the theoretical system LoDs, indicated with black arrows. The collection efficiencies for EP1 and EP2 are significantly different with a probability P = 0.0158 < 0.05 and are indicated with the corresponding standard error. *For EP3, only a single measurement was successfully obtained. The corresponding collection efficiency can be estimated using Eq 1.