Sampling Virus in Air Webinar

Dr. Martin Harper – Director of Scientific Research
Dr. Martin Harper served as the Chief of the Exposure Assessment Branch in the Health Effects Laboratory Division of NIOSH for 14 years before joining Cole-Parmer / Zefon. He has degrees and diplomas in Geology (from Oxford University), Pollution Control, Environmental Science, and Analytical Chemistry, and obtained his PhD in occupational health research from the London School of Hygiene and Tropical Medicine. He is a Certified Industrial Hygienist and Fellow of the American Industrial Hygiene Association and a Chartered Chemist and Fellow of the Royal Society of Chemistry. He has over 150 peer-reviewed publications and has received several awards each from CDC, NIOSH, ASTM and AIHA. He is considered a Distinguished Lecturer by the AIHA and he is a Courtesy Professor in the Department of Environmental Engineering Sciences at the University of Florida.

Webinar Q&A

What is the preferred method for analyzing media in a RT-qPCR? Is it possible to swab the media, or should the media be soaked in a solution?

First, the virus must be eluted from the filter. We examined two procedures and we provided details of the one with the best recovery in the presentation. Then, the RNA must be extracted from the virus. A typical procedure for PCR uses phenol-chloroform, but the actual procedure will be determined by the laboratory.

What is the average cost of an air sample analysis?

That will be determined by the laboratories offering the service.

How are you aerosolizing the virus? Is there specific equipment you would recommend?

Liquid suspension with the inactivated virus was aerosolized using a three-jet Collison nebulizer with a polycarbonate jar and operated at a flow rate, QA = 5 LPM (pressure of 19 psia). The use of a polycarbonate jar and low aerosolization pressure minimizes damage to biological particles.

While the differences were not statistically significant, do you have an idea as to why the 60 minutes (600L) showed lower levels?

The 3 LPM experiments show a similar trend. It is possible that there was a slow degradation of RNA during extended sampling. However, the results are not significantly different, so this is just speculation.

Has this system been used for other organisms in addition to viruses (e.g. fungal)?

Other microorganisms can be collected on PTFE filters. There is much more literature published with reference to other microorganisms than for virus sampling.

What are your thoughts on using bacteriophage such as Phi6 and MS2 as surrogates of SARS-CoV-2?

The most valid experiments would be using the SARS-CoV-2 virus itself. Any surrogate is a step away from that. As the surrogate becomes more dissimilar, confidence that the results are generalizable becomes lower. We selected the OC43 virus as being the closest in nature to SARS-CoV-2 without compromising safety. However, as OC43 is infectious, it was still necessary to inactivate it before use.

Detection of RNA doesn't translate to infectivity. Isn’t determining whether infective bioaerosol virions are present as bioaerosols more important than detection of virion RNA/DNA? Was the sampling system validated for infective (i.e. viable)?

Of course, viability is important, and systems are under development for maximizing the preservation of viability in air samples. Filter collection does not preserve viability as well as these other sampling systems. However, determining viability in a sample is a much more exacting task than detecting RNA.

A personal test using the Vira-Pore can take 10 to 40 minutes, correct? Is there a possibility to administer a test faster in the future since swab tests only take a few minutes?

The smaller the sampling volume, the less likelihood enough sample will be collected for detection. We selected 10 minutes at 10 LPM as a typical short-term sampling system, based on the typical maximum flow rate of pumps currently available that can be worn on a person (personal).

Using the Vira-Pore, what is the recommended sampling time and flow rate?

We have shown recovery relative to unit sample volume to be similar over the ranges 10 to 60 minutes at 10 LPM and 60 to 960 minutes at 3 LPM. It is likely that similar sample volumes achieved by different flow rates would give similar results.

What is PCR?

Polymerase Chain Reaction is a means of replicating very small quantities of DNA until the DNA is detectable.

When requesting the analysis, what is the analysis to be requested? Is it SARS-COV-2? Is it Corona Virus? Is it COVID-19?

We have validated the Vira-Pore for sampling OC43 virus. Any conclusion with respect to extrapolation to any other virus is the responsibility of the user.

Is there a way to differentiate between viable vs. nonviable, i.e. ability to infect people?

Yes, but not by this sampling and analysis procedure.

If used for room air sampling, I assume one would need a pre-filter? Do you have data on how quickly the filter loads up?

An environment needs to be very dusty to load up a 37 mm open-face filter so that it no longer functions properly as a filter. Dust will contain RNA and DNA from different sources. The primers selected for the PCR analysis are selected to minimize interference from other sources.

What are the detection limits? What is the LOD for this procedure?

The detection limit of this procedure using PCR analysis depends on the PCR system. EN 13908 mentions mainly the polycarbonate filter. Is the use of this filter an issue? I was unable to locate this Standard. However, polycarbonate filters have holes through which smaller particles can pass. The aerosol we generated in our tests had a distribution around an electrical mobility diameter of 50 nm.

Is the binder in the filter material an interfering component for the analysis?

The filter is pure PTFE (no binders and no other polymer).

Is there a procedure for disinfecting the pumps to be reused without damaging the pumps? And has any work been done on seeing whether sampling equipment is contaminated with coronavirus after sampling?

Some pump manufacturers have recommended cleaning with a cloth dipped in 99% isopropanol, but the manufacturer of a specific pump should be contacted for their recommended procedure.

Would it be possible to develop a badge for personal sampling as opposed to using the Vira-Pore cassettes and a personal sampling pump?

Probably. For example, see Han, Thomas and Mainelis (2017) Aerosol Sci. Technol., 51:8, 903-915.

Semi-quantitative analysis (expanded uncertainty 50% or even higher) should be fit for purpose, I would think? Is expanded uncertainty of 30% or less probably overkill? Do you have any comments on this?

Method uncertainty is applicable to quantitative methods. This method can only be validated as fully quantitative when an orthogonal quantitative procedure for determining virus concentration exists, which is not currently the case. If quantitative RT-PCR is used for analysis then relative values could be obtained, but they would not be quantitative measures of concentration of virus in the air.

Do you know any center to test air-filters with OC43 in Canada or US?

The work we performed was at Rutgers, the State University of New Jersey. You could inquire of Professor Mainelis if he is interested in pursuing further work.

Can filter cassettes be regenerated for reuse?

The cassettes are not intended for reuse.

Do I understand correctly that Vira-Pore only detects the presence of a virus and does not speciate to identify a particular virus (e.g. COVID-19)?

We ran tests using a total RNA analysis because we were sure the only RNA in our system was from the virus we aerosolized. In a field situation, there may be multiple sources of interference and that is why PCR with primers specific to the target organism must be used for field samples. Keep in mind our validation was for OC43 virus. It is the responsibility of the user to decide whether validation for this virus can be extrapolated to others.

How did you deactivate this virus?

The virus was inactivated by heating its suspension in a water bath at 56°C for 30 minutes. The absence of plaques in a plaque assay with Vero E6 cells confirmed inactivation.

What is the size of the Zefon filter, e.g. 37 mm?

While the Vira-Pore cassette filter is 37 mm diameter, other sizes could be made available.

Would the reduction in count on samples not kept refrigerated be due to type viable / nonviable RNA?

Since there is no statistically significant reduction in RNA during storage in our experiments so far, we cannot really answer that. Until further work is performed, refrigeration for transportation and storage is a recommendation based on prudence.

Does HVAC equipment or filtration devices (personal space cleaners) when sampling affect results or are is there anything that may result in false positive or negative results?

If air cleaning systems are effective at removing virus particles, then none will be collected on the filters. Ozone generated by certain air cleaning systems may degrade viral RNA, but we did not test this.

How much degradation might occur if we sample for 24 hours straight before storing in refrigerator?

Any degradation of virus that may occur is likely to be a result of the passage of air through virus already on the filter. Thus, virus that is collected at the beginning of sampling would be most susceptible to degradation. To account for this, we loaded samplers (at 3 LPM) with one hour of virus collection and followed up with 5 and 15 hours of clean air. There were no statistically significant losses compared to filters analyzed immediately. We did not go all the way to 24 hours.

Was a cyclone attached to the filter cassette shown for personal sampling in slide 18?

No, that was just a stock photograph to show a small personal sampling pump being worn. We did not have a similar picture with the Vira-Pore cassette at the time.

Do you sample with the cassette open faced or closed face?

We used open-face cassettes because it spreads the deposit more evenly across the filter.

The sampling pump first shown is a Zefon mold air sampling pump which is set at 15 LPM. Is using this pump at 15 LPM harmful to the virus?

That pump was illustrated as a general example of air sample collection. It is designed for use with an impactor; it cannot be used with the Vira-Pore cassette.

Where would someone want to be taking air samples? In what settings do you anticipate using this sampling? Do you have examples of situations in which the testing of air is pertinent? Will there be testing performed at public places like at a college or other school settings? How many samples should be taken per cubic feet to show there is a virus in the sample area or zone? Is there a strategy for collecting control samples vs suspect area sampling? How do you know the duration and L/flow rate to operate at?

The answers to all these important questions depend on the objective of the study, which involves the judgment of the study designer.

What is needed besides the cassette to take a sample?

An air sampling pump, either mains (electric) or battery, appropriate to the required flow rate, and flexible incompressible (e.g. Tygon®) tubing to connect the two. A suitable calibrator will also be necessary to set and verify the flow rate.

Is Vira-Pore validated by any international or local body?

No. We hope that further validation will proceed at some level to result in a government method or national/international standard.

How long does the aerosolized virus remain in the air during validation?

The aerosol was continuously generated for up to 60 minutes in our experiments.

What special precautions should be taken in sending international samples to the US for analysis?

We are not qualified to answer questions related to international transportation. You should consult your carrier for this information.

In collecting a sample say for COVID-19, at what point in the sampling procedure would the virus starts to die?

Viruses are strictly not living and therefore they cannot “die”. However, they can be inactivated so that they are no longer viable. This may occur the moment the virus is collected on the filter, or it may be hours later. That is why viable viruses are not determined by filter sampling. Keep in mind that our validation was for OC43 virus. It is the responsibility of the user to decide whether validation for this virus can be extrapolated to others.

Which PTFE properties make PTFE superior for virus capture? Are other materials comparable?

PTFE filters are inert and highly efficient at collecting even small particles. Other filters may be able to be used if demonstrated through appropriate validation.

How do you grow a virus that has been deactivated? I mean, the cold virus that is used as a surrogate, you said it was inactivated. Does that mean that it is dead, so to speak? And if it is dead, how are you able to grow it or replicate it for the PCR to measure it?

We grew (cultured) it and then deactivated it. PCR operates on the RNA in the virus, which is not all destroyed by the inactivation process.

Is 10 LPM the limit of this sampler? Can you use higher flow so more air can be pulled through during the same time period? SARS-CoV-2 concentration has been shown very low even in some hotspots.

The maximum flow rate at which air can be pulled through a filter is typically determined by the strength of the pump and mains-operated vacuum pumps would be required to exceed 10 LPM. Higher flow rates also result in higher velocities of air through the filter, which may be more damaging to the RNA. We have not tested this.

Which air pump would you recommend for the sampling?

Most high-flow personal air sampling pumps can achieve 3 LPM, while some can achieve 10 LPM with this system, which is why those flow rate values were chosen. Vacuum pumps can also be used.

What does it mean by excellent collection efficiency and at what aerodynamic equivalent diameter?

Air sampling filters are tested using 300 nm aerodynamic equivalent diameter (AED) particles as this is the most penetrating size. Above this size collection becomes more efficient by impaction, and below this size more efficient by diffusion. All PTFE filters with nominal pore-sizes below 3 microns have better than 99% collection efficiency for 300 nm AED particles.

In your expertise, what is more important: the sampling volume per minute or the surface velocity at the filter?

The critical parameter is sample volume. This is achieved by a combination of time and flow rate. The surface velocity increases with the flow rate, but this does not materially change particle collection efficiency over the range used for air sampling. The pressure difference across the filter does increase with velocity, so it is necessary to match the pump capability.

If the virus has been deactivated, how then do you store it and expect to replicate it when it is not viable?

In our experiments virus was grown, deactivated, and used in small batches so there was no appreciable storage time. Our technique only looks at the presence of RNA, which is present whether the virus is active or not.

What is the level of method specific selectivity?

In our experiments, because our virus RNA was the only RNA present, we could use a total RNA detection method, which is faster and cheaper than PCR. In field use PCR is necessary for selectivity and the degree of selectivity will be controlled by the primers selected.

What eluent is used, and how is it processed before PCR? Can the same PCR buffer as used for swabs, be used?

The eluent we used was Primestore® MTM. I believe the same eluent has been used for swabs. After elution, the RNA needs to be released. We used the Qiagen QIAamp mini kit, which has a 30% to 40% recovery compared to the standard phenol-chloroform extraction typically used for PCR.

Have you compared the Vira-Pore system with classical PTFE or quartz filters? I was wondering if the collection efficiency was tested on reference 300 nm particles and what the percentage of efficiency was?

We have not done such a comparison, as it would double or triple the number of experiments and the costs of the project. Collection efficiency of PTFE filters has been determined for a range of pores sizes and this information has been published, e.g. Soo et al., 2016, Aerosol Sci. Technol. 50:76-87.

Is it better to use total suspended particle sampling or aerodynamic size segregated aerosols in terms of yield? For example, would a NIOSH BC 251 be efficient compared to your system?

The NIOSH sampler separates the sample into three different size fractions. I believe that when it was used to collect samples, virus could be found in multiple fractions. As a research tool this may be useful information, but for practical purposes it dilutes the sample leading to lower sensitivity and triples the analysis cost.

Could the system be used to collect in very humid environments?

We performed tests around 40% to 50% relative humidity. We do not see a reason why higher humidity would make a difference, but it has not been tested.

What are the downsides of this method when trying to isolate live virus?

Virus does not maintain its viability on surfaces over the long term, and the stable period may be considerably reduced by dry air passing through the filter. Other methods to preserve viability are preferred over filter collection.

Does the method test for presence and absence of the virus or does it give the actual viral concentration in the air?

Quantitative PCR in theory could be used to measure viral concentration, but there is a great deal of complexity involved in this. As a first consideration one would need to know the true concentration of virus in the air when conducting experiments, and this is difficult to achieve.

Can the PTFE filter be subjected to gravimetrical analysis after conditioning?

PTFE filters can be weighed before and after sampling, but there are difficulties involved, not the least of which is handling a filter that may have viable virus on it. PTFE filters are not easy to weigh properly. See Lawless and Rodes, 1999, J. Air & Waste Manage. Assoc., 49:9, 1039-1049.

What is the best method to sample bacteria in air? Is there is any US standard to follow? What equipment is recommended to use? Flow rate? Media?

This is a complex question, which requires a lengthy answer. A good reference to consult is “Bioaerosols: Assessment and Control” published by ACGIH®.

Do you think it will be possible to evaluate the recovery of OC43 virus when dispersed in artificial lung fluid/nasal fluid? And are these additional proteins and biological materials likely to alter recovery?

Sorry, this question is outside of my field of expertise. PTFE filters have a hydrophobic side. Is this an issue for isolating SARS-CoV-2?

The ZePore filter is hydrophobic on both sides and was used in these experiments. What about the experimental proof to find virus in exhaled aerosol under 1-uM diameter?

Filters are tested at 300 nm, the most penetrating size. If the efficiency of collection is better than 99% at this size, it is even higher at other sizes. The aerosolized virus used in our experiments had an electrical mobility size distribution consistent with sub-micrometer virus particles.

How in the field can you ensure sterility of the equipment prior to sampling? (Whilst setting up the equipment - background: we set up in a 'green zone' to ensure minimum time spent in the 'red zone')?

Sampling equipment can be assembled outside of a contaminated zone and then taken into the contaminated zone. It is appropriate to decontaminate all equipment before removal from the contaminated zone. Pumps, tubing, samplers, etc., should be cleaned off in accordance with protocols for removing other materials from the contaminated zone.

And what is the minimum experimental dimension of aerosol that you have found in a virus?

The electrical mobility diameter of the virus aerosol we created had a size distribution with a peak number concentration at around 50 nm, consistent with projected physical diameters that have been measured for viruses.

Does the volume of air pulled through the sampler overwhelm any contributing contaminating genetic material?

It could. It depends on the specificity of the primer used in PCR.

Have you compared normal hydrophobic PTFE and modified hydrophilic PTFE?

We have not.

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