Mask based screening
Would it be better to run COVID19 assays on surgical masks instead of nasal swabs? There are some compelling reasons why mask based screening may be better.
A test for COVID19 requires the following:
- The patient sample has the virus present when the patient has the virus.
- The assay has access to enough virus (at minimum 1 viral particle) to make a positive call.
Nasal swabs attempt to pull samples from the back of the nasal cavity--a site that is known to have a high density of COVID19. However, nasal swabs present some challenges:
- Only samples one area of the body at one time.
- They are uncomfortable for the patient.
- Swabbing requires trained medical personnel both (1) putting them at risk for exposure and (2) making them unavailable for other tasks.
- If swabbed incorrectly (not deep enough or in the wrong area), can miss the infected area and produce false negative results.
- Dilutes the biological sample into a 2-3 mL sample vial, reducing the concentration of virus.
However, once swabbed and stabilized in the transport medium, the sample is in a stable and familiar liquid form factor for subsequent RT-PCR analysis.
If we view a surgical mask as a collection medium, it has a number of advantages:
- People who fear or suspect COVID19 should already be wearing masks.
- Aerosols from breathing and coughing represent a broad sampling of the patient's airways and a primary mode of infection.
- Over the hours of wearing a mask, it should have accumulated significant virus on the inner lining if the virus was present.
- The mask material itself acts much like a wicking paper filter. Wicking filters have been used to selectively separate virus particles (such asZika virus here ) for PCR based detection.
Some challenges with masks however are:
- Masks themselves can capture virus particles in the environment, so if the patient is surrounded by others with the virus that virus may be detected.
- RT-PCR based assays are based on multistep processes on small liquid volumes and thermocyclers, not in a fabric form factor.
I want to address this last point about laboratory protocol, as I think it could be surmounted
Once a patient sample comes into the lab, one of the first steps for sample preparation is RNA extraction. The intent is to make the RNA more accessible to the reagents to make the amplification steps more efficient. This extraction is done using pre-mixed kits such as QIAGEN EZ1, QIACube, or Roche MagNA Pure.
While these RNA extraction methods work, they have 4 problems:
- Rely on reagents that are increasingly hard to obtain
- Can be costly
- Take time and additional labor
- Require liquid handling
An alternative approach for RNA extraction is to simply use heat. Rosenstierne and Fomsgaard have suggested
that treating a sample by heating it to 95C for 5 minutes and then cooling it to 4C for 2 minutes is nearly equivalent to using a commercial RNA extraction kit.
For mask based assay, RNA extraction could be carried out by placing the rolled mask into a lightly capped tube with some water, placing the tube into a boiling water bath for 5 minutes and then placing the tube on ice.
The mask could further refine RNA extraction by separating the larger cells and debris from the smaller virus particles. If an appropriate mask could be found or made, the inner layer could act as a removable capture layer enriched in virus particles and leaving larger materials on the outer layers.
This use of a paper or fabric filter for virus purification has been demonstrated for Zika in a paper based microfluidic device
. However, rather than creating a horizontal microfluidic channel, the filtration would happen through the depth of the first mask layer, with the end of the "channel" being the middle layer in the mask.
The CDC protocol for COVID19 detection
uses qRT-PCR for detection. This method is well validated is built from standard off the shelf kits such as Quantabio qScript, Promega GoTaq, or Thermofisher TaqPath. In the CDC protocol, 5 microliters of the patient sample are used to run the assay. The assay is run using a quantitative PCR machine that cycles between hot and cold cycles to perform each round of DNA amplification.
While this method clearly works, it suffers from the following problems:
- Uses a very small volume of a diluted patient sample, making detection hard.
- Requires sophisticated and expensive equipment
- Requires significant expertise and calibration to operate correctly
Really all we want in a COVID19 assay is a binary yes or no answer, so the quantification (the "q" in qRT-PCR) is overkill.
An alternative to qRT-PCR is to use a newer, simpler method called isothermal amplification.
These methods don't require thermal cycling, but instead rely on just being in a moderately high temperature (30C to 65C depending on assay) to cause the sample to amplify. Isothermal amplification is not effective for quantification, but is effective for binary tests.
The amplified DNA with an isothermal amplification protocol can be visualized with visible dyes (pH based) or double stranded DNA dyes such as SYBR green.
Furthermore, the isothermal amplification method known as LAMP has already been demonstrated for COVID19 testing by Zhang et al.
For mask based testing, the heat-treated masks could be assayed by applying drops of LAMP reagents (polymerase, primers, and nucleotides) onto a dot on the inner face of the mask. A control dot without primers could be placed next to the first one. The fabric of the mask would act as the reaction vessel. The mask could then be heated in a sealed humid chamber to allow the isothermal amplification to take place.
The mask result could be read visually by color change or using a black light to detect the fluorescent dye.
Because the mask acts as a virus collector (particularly on the inner surface near the mouth and nose), a LAMP assay could potentially be run using a lower concentration of enzyme and primer, thereby saving reagents and allowing a greater assay sensitivity.
- Are there surgical masks on the market that allow sufficient virus capture to detect with a LAMP type assay?
- What instructions for patients would help to capture a better sample? How long to wear the mask? In what environment? In isolation? Away from sunlight?
- Does the middle layer of a three layer mask enrich in virus particles?
- As a diagnostic, how does a mask based LAMP assay compare to the gold standard of a nasal swab + qRT-PCR assay in terms of sensitivity and specificity? Can these results even be compared if they are effectively comparing different tissue compartments?