Sampling for SARS-CoV-2
PCR Versus Culturing
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According to the World Health Organization, as of June 1 there were an estimated 170 million cases of COVID-19 worldwide and over 3.5 million deaths. Governments at all levels, from national and provincial down to municipal and local, have been forced to evaluate their policies to protect public health. Although the same principles for controlling airborne hazards can be used to control infectious aerosols, the transmission route was not well characterized at the beginning of the pandemic and government leaders seemed completely unaware of the occupational hygiene discipline. The most common policy responses in the United States were masking mandates and social distancing. These measures were intended to reduce exposures and transmission by suppressing viral emission at the source and by limiting person-to-person contact. Unfortunately, many lawmakers and citizens misunderstood the purpose of these measures and felt they were too restrictive when disease prevalence was low and ineffective when prevalence became high.
Other control measures such as shelter-in-place orders, strict quarantines, and isolating positive cases can be effective, and all have been used to varying degrees in many locations. Famously, the city of Wuhan, China, implemented a strict quarantine for over a month with extreme limitations on citizens’ movement. At the time, this measure seemed draconian to Western governments and citizens, but the city reopened within two months while much of Europe and the U.S. have fought protracted battles for over a year. Similar but less severe limitations were instituted in Italy, Spain, and New York City following rapid rises in cases at the beginning of the pandemic. New Zealand and Australia were able to keep their case numbers low by severely restricting incoming travel, enforcing two-week quarantines of arriving travelers and requiring negative COVID test results before release. With a lack of scientific evidence specific to SARS-CoV-2 and the need to make swift decisions, governments chose policies they could enact both broadly and quickly. After initial control measures were implemented, permanent engineering controls were explored such as upper-room germicidal ultraviolet (GUV) light, in-room high efficiency particulate air (HEPA) filtration, and increased dilution ventilation along with higher minimum effective reporting value (MERV) filters. However, improving the indoor infrastructure of whole nations in one season was just not feasible. Sampling strategies are a core tool of industrial hygiene, and environmental sampling in particular is used to evaluate hazard controls. Without analytical support capable of measuring SARS-CoV-2, it was difficult to formulate any nuanced approaches based on benchmarks of virus load or known transmission dynamics. There is still no consensus on the infectious dose of this virus other than a vague directive that fifteen minutes of exposure to an infected person is sufficient to cause infection in a susceptible individual. Determining if a control is “good enough” is therefore difficult. Nonetheless, there is still much value in knowing the level of risk reduction provided by certain control measures and policies. TYPES OF TESTING The early months of the pandemic saw the development of rapid and accurate tests for SARS-CoV-2 in human specimens, and tests were also quickly developed for analysis of these viruses in environmental samples such as air and surface samples. By June 2020, AIHA Laboratory Accreditation Programs (AIHA LAP) began offering accreditation for reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) testing of SARS-CoV-2 under its microbiology program, and several accredited microbiology labs have since added this type of molecular testing to their scopes. This development has provided IHs and decision-makers with the analytical support necessary to make informed policy decisions. Detection of the SARS-CoV-2 virus is primarily performed using RT-qPCR. One advantage of RT-qPCR is that it can be made highly specific by using primers that bind to unique DNA segments. As SARS-CoV-2 is an RNA retrovirus and therefore does not have DNA segments, the assay first converts the viral RNA into DNA using reverse transcription. Next, the primers bind to a segment of DNA unique to the virus and the polymerase enzyme begins replicating that portion of viral DNA. A fluorescent molecule is released from each primer when it binds to DNA; therefore, the sample’s fluorescence is related to the number of DNA replications, which is a function of the number of RNA copies at the start of the reaction and the number of replication cycles. The assay can be quantitative, semi-quantitative, or qualitative depending on how the amplification profile is interpreted. The simplest case is the qualitative assay, which indicates the presence or absence of viral DNA. If a sample yields fluorescence above background, then virus was present in the sample. For semi-quantitative analysis, the number of replication cycles needed for the virus to become detectable is compared between samples. Since each replication cycle doubles the number of viral DNA segments, a sample that takes 25 cycles for the virus to become detectable is expected to have 27 times more virus than a sample that takes 32 cycles. For quantitative analysis, the lab must make a calibration curve from a series of known concentrations of viral samples to determine the detection limit of the assay and relate that to the number of replication cycles. Clearly, qPCR is the most stringent and difficult PCR analysis for the lab to conduct. While highly specific to SARS-CoV-2, PCR-based analysis cannot differentiate between viable and non-viable virus. To differentiate between viable and non-viable virus, two different assays are commonly used: an endpoint dilution assay, commonly referred to as median tissue culture infectious dose 50 percent (TCID50); and a plaque assay. A simple explanation of TCID50 is that it estimates the virus concentration from the amount of sample needed to infect a cell culture. In plaque assays, cells in plates become infected with a virus; the virus replicates and spreads to other cells, forming a plaque, or area of cell death. Counting the number of plaques is a common method to determine virus concentration. Sample results are usually reported as plaque forming units per milliliter (PFU/mL), which can be converted to PFU/m3 by using sampled air volume and virus extraction volume. In TCID50, host cells are cultured in a well plate with liquid medium, and then dilutions of viral fluid are added to the wells and incubated. The end measurement is the percentage of infected wells for each dilution. The rule of thumb is that the PFU/mL concentration is 70 percent of the TCID50 concentration, as TCID50 is more sensitive (and more variable). Neither technique is commonly available outside of research arenas due to their highly specialized nature and the need to maintain virus viability throughout the assay. During the pandemic, plaque assays have been hard to source due to supply chain issues; therefore, TCID50 has been the most commonly used assay for cultivability testing of SARS-CoV-2. For agents like SARS-CoV-2, which must be handled in facilities certified as biosafety level 3 (BSL-3), stringent safety precautions, training, facilities, and government approval are required. These factors make both types of live culturing for SARS-CoV-2 prohibitively costly. The alternative to live culturing is PCR analysis of environmental samples. While PCR analysis has quickly become standardized and more widely available, environmental sampling approaches are still emerging as a new tool in the response to SARS-CoV-2. In the remainder of this article, we will briefly explore the variety of air and surface sampling techniques currently available for SARS-CoV-2. SAMPLING TECHNIQUES Just like any other particulate contaminant, viruses can be efficiently collected from a surface with a swab or wipe and captured from the air with simple filtration or impingement. However, industrial hygienists must consider the compatibility of both the sampling technique and the analysis technique, along with the effect of sampling stresses on the target organism. For surface sampling, it is necessary to remember that the analysis procedure is adapted from nasopharyngeal swabs, so using a ghost wipe instead of a swab may not be compatible with the lab’s extraction process. It is also possible that the wipe matrix or chemical additives may interfere with the RT-qPCR reaction. Any sampling matrix should be validated by the laboratory; therefore, it is best to consult the laboratory before collecting samples to ensure you use the same swab or wipe that they have already validated. Swab sampling is usually conducted on a relatively small surface area (approximately 25 cm2) with a moistened nasal swab. This technique applies the same principles as any wipe sampling: thoroughly wipe the whole area vertically and horizontally while rotating the swab to use its entire surface. Mark a standard area or estimate the area of the swabbed object (such as an elevator button, door handle, or finger). After sampling, the swab is usually placed into a sterile container with a transport medium or nucleic acid fixative.
Capturing the virus and detecting by RT-qPCR is relatively simple, but maintaining viability is challenging.
Air sampling for SARS-CoV-2 has been described in several publications, many of which are listed at the end of this article. Polytetrafluoroethylene (PTFE) and gelatin filters are effective when analyzed with both techniques. Although peer-reviewed publications on sampling for SARS-CoV-2 are still sparse, each lab accredited by the AIHA LAP Environmental Microbiology Laboratory Accreditation Program should list the type of air filters it has validated for use with their analysis procedure. In a recent experiment led by K. A. N. Aithinne that was presented at Virtual AIHce EXP 2021, sampling efficiencies were compared within wet methods (impingers and bubblers) and dry methods (filters) at 12.5 L/min for 10 minutes for a high concentration viral aerosol. Aithinne found that wet sampling methods with the SKC BioSampler, AGI-30, and a fritted glass bubbler needed an antifoam agent to prevent foaming and overflowing due to the use of a collection medium that contained serum protein and the serum protein present in the viral stock medium. Wet methods had poor sensitivity and were prone to interference. All samplers had detectable but not quantifiable viral concentration by RT-qPCR before attempts to concentrate the samples. After centrifugal concentration, the antifoam agent interfered with the RT-qPCR analysis. Similarly, concentration by filtration was impractical because the antifoam agent clogged the pores in the PTFE membrane filters. None of the wet techniques yielded viable virus. Aithinne also compared three filters—mixed cellulose ester (MCE), PTFE, and gelatin—and found that MCE was neither RT-qPCR positive nor viable, but both PTFE and gelatin were RT-qPCR positive and viable. They found gelatin was less precise for RT-qPCR analysis but more sensitive for TCID50 analysis. Although other studies found that SARS-CoV-2 was non-viable when sampled with PTFE and gelatin filters, those studies had higher flow rates and much larger sample volumes, which may have overstressed the virus. Hence, it was detectable by RT-qPCR but not viable.
Collecting a larger sample volume increases the ability to detect via RT-qPCR but seems to reduce viability as determined by TCID50. Capturing the virus and detecting by RT-qPCR is relatively simple, but maintaining viability is challenging. Since most industrial hygienists will not have access to TCID50 for SARS-CoV-2, they will need to determine whether viability is necessary for their purposes.
For detection of SARS-CoV-2 by RT-qPCR techniques, the AerosolSense from Thermo Fisher Scientific is a portable, self-contained, impactor-based sampling system. Samples are collected at 200 L/min through a slit impactor onto a self-sealing cartridge. Sample times can be programmed from 5 minutes to 24 hours. Originally designed for capturing anthrax spores, this impactor was recently validated for capture of virus-sized particles. Before using this system, ensure that your analytical lab has validated analysis with the AerosolSense cartridges.
Two recent studies characterized the AerosolSense for SARS-CoV-2 collection in the laboratory and in real-world healthcare environments. A paper published in March 2021 on reported minimum sensitivity of virus aerosol as approximately 30 genome copies per liter (gc/L) in a 75-minute sample (15 m3) and minimum sensitivity of approximately 0.1 gc/L in samples over 8 hours (96 m3). A second paper published by the preprint server medRxiv compared the effectiveness of air sampling with AerosolSense within COVID-19 patient rooms against other forms of environmental sampling. The AerosolSense detected virus in 53.8 percent of air samples compared to 12.1 percent for surface swabs and 14.8 percent from passive plates. Since we now know that aerosol transmission is the primary form of disease transmission, and the AerosolSense has sufficient sensitivity to detect airborne virus, we now have the capability to evaluate certain controls and perform aerosol risk assessments. Another application for low-sensitivity air sampling is tracking viral variants spread through genetic sequencing of captured viruses.
If the sampling strategy requires viable sampling of SARS-CoV-2, Aerosol Devices Inc. has recently developed the BioSpot sampling systems based on condensation growth tube technology. Water vapor is condensed on particles larger than 5 nm and grown to a critical diameter of approximately 3 µm. These large particles are easily captured by impaction or impingement with efficiencies over 95 percent. To our knowledge, at this time the BioSpot is the only commercially available system capable of reliably collecting viable SARS-CoV-2. Aerosol Devices Inc. offers three versions of this system: the BioSpot-VIVAS, which captures into a petri dish at 8 L/min; the BioSpot-GEM, which captures onto a swab at 1.2 L/min; and the BioSpot-BC, which captures into a small vial or onto micro-well plates at 1–1.5 L/min. The capabilities and applications of these products are beyond the scope of typical industrial hygiene sampling campaigns. LIMITATIONS OF SAMPLING Interpreting results is key to obtaining meaning from your samples and understanding the limitations of your selected sampling and analysis method will ensure you obtain useful information. It is paramount to recognize that detection of virus through RT-qPCR does not mean the virus is infectious. RNA viruses are rather delicate; while they may remain present in the environment for weeks, they are not viable for long. Deactivation of virus by a non-destructive process such as alcohol disinfection, desiccation, heat treatment, or ultraviolet C (UVC) irradiation may leave the genome intact for RT-qPCR detection. In addition, infected persons may shed non-viable virus for weeks after recovery; detection of these viruses cannot be differentiated from infectious, viable virus.
Perhaps RT-qPCR detection techniques are best suited to assessing relative risks by comparing the relative viral loads with and without a control such as masking or social distancing. Another useful application of RT-qPCR is comparing the reduction of viral load when increasing fresh air ventilation rates or installing in-room HEPA filters. However, using RT-qPCR detection to assess effectiveness of upper room UVC is ill conceived because UVC deactivates the virus but does not remove it. For nations with both low vaccination and low COVID-19 case rates, another useful application of RT-qPCR would be daily monitoring of viral load in major public spaces such as airports, train stations, and entertainment venues. Daily tracking of virus loads may provide a leading indicator of viral prevalence and could be useful for establishing dynamic, tiered response policies that use near-real-time virus presence instead of case transmission statistics. Furthermore, variant sequencing of these samples could be used to track viral variants and indicate new transmission pathways.
EVAN L. FLOYD, PhD, CIH, is an assistant professor in the Department of Occupational and Environmental Health at the University of Oklahoma Hudson College of Public Health and past chair of the AIHA Sampling and Laboratory Analysis Committee.
K. A. N. AITHINNE, PhD, CIH, GSP, CPH, is an aerosol scientist at Johns Hopkins University Applied Physics Laboratory in the Asymmetric Operations Sector. They are also the current chair of the AIHA Biosafety and Environmental Microbiology Committee.
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