Result Interpretation of SARS-CoV-2 RT-PCR Testing

Ching-Yi Tsai, Ph.D. and Chin S. Yang, Ph.D.

Introduction

Since late 2019, a cluster of pneumonia cases caused by a novel coronavirus was discovered in Wuhan, China and has since spread from there throughout the world in a few months, causing a pandemic. The virus was described and called SARS-CoV-2 because of genetic similarity to the virus causing the first SARS disease in 2003-2004. The pandemic has caused tremendous chaos in our daily life. In addition to human testing for the virus, testing for the virus in our indoor environments is often necessary or required because the virus is highly infectious and can stay infectious in the air for hours and on many indoor surfaces for many days to weeks.

Viruses contain either DNA or RNA in their genetic makeup, and cannot duplicate themselves outside of a living host which can be, either a bacterium, a plant, or an animal such as humans. The SARS-CoV-2 is a RNA virus. Its RNA is enveloped in an outer protein shell which binds to the viral RNA and encapsulates it to form the nucleocapsid. Because it must reproduce inside a living host, the only way to detect it is either by detecting their RNA or by infecting a suitable host, such as a cell culture. The cell culture method is complex, time consuming, and impractical in practice. It is important to emphasize that the detection of SARS-CoV-2 RNA does not directly translate into infectious SARS-CoV-2 virus, although it is very often assumed.

There are currently several testing methods available commercially for the detection of the SARS-CoV-2 RNA or its antibodies produced in its hosts, i.e. humans. The antibody testing methods are not suitable for environmental testing. Variations of RNA detection methods have been marketed by different companies since the outbreak of COVID-19. Some rapid test methods, although offering quick turnarounds, may not be the most reliable. The gold standard is the Polymerase Chain Reaction (PCR) method, specifically Reverse Transcriptase PCR (RT-PCR). For more information and details on PCR and RT-PCR, please refer to our technical factsheet titled “PCR and RT-PCR for SARS-CoV-2 Testing.”

Prestige EnviroMicrobiology uses commercially available and CDC approved RT-PCR test kits for the detection of two SARS-CoV-2 genetic markers (N1 and N2) for the analysis of environmental samples. All controls (positive, negative and internal controls) are included in the analysis to assure the process of viral RNA extraction and RT-PCR..

This technical factsheet helps you use and interpret the results derived from the RT-PCR testing.

Results Interpretations:

Prestige EnviroMicrobiology only reports test results that pass all controls and are deemed accurate and reliable.

The first thing in the results is to understand the Ct value or Ct level. Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal in RT-PCR to exceed the threshold (i.e. exceed background level) or to be detected. Ct levels are inversely proportional to the amount of target nucleic acid in the sample. The lower the Ct level the higher the amount of target nucleic acid is in the sample. Generally speaking, Cts <29 are strong positive reactions indicative of abundant target nucleic acid in the sample. Cts of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid. Cts of 38-40 are considered weak reactions indicative of minimal amounts of target nucleic acid which could represent an infectious state or environmental contamination.

The second part of the results is whether one or both of SARS-CoV-2 two genetic markers (N1 and N2) are detected.

1.      A sample is considered negative for SARS-CoV-2 when Ct values for both N1 and N2 markers do not cross the threshold within 40.00 cycles (>40 Ct).

2.      A sample is considered positive for SARS-CoV-2 if Ct values for both N1 and N2 markers cross the threshold within 40.00 cycles (< 40 Ct).

3.      When the Ct values for any one marker (N1 or N2 but not both markers) crosses the threshold within 40.00 cycles (< 40 Ct) the result is inconclusive. The recommendation is to retest the extracted RNA. Prestige routinely re-tests inconclusive samples and reports both sets of results.

How to apply this information to the sampled environment?

• Think of the Ct values as the virus concentrations. The lower the Ct values, the higher the viral concentrations are.

• The environment is considered contaminated, if any indoor sample tests positive and other samples are inclusive or negative.

• If inconclusive results are associated with negative samples but no positive results, consider retesting, or re-clean and retest. An inconclusive result suggests that SARS-CoV-2 particles might have previously been present but are probably damaged and likely noninfectious. If disinfectants are applied in the environment, the disinfectants may have damaged the virus particles. The virus particles may also degrade and lose its infectiousness on the indoor surfaces over time, days to weeks. Because items and surface areas sampled for testing are usually a relatively small portion of the environment, it is possible that intact, infectious virus particles might still be in the environment. 

• If any one sample collected from the environment tests positive, the environment is considered contaminated. It is important to consider this issue prior to your sampling. If several rooms are sampled, each room should be considered as a separate environment (e.g., classrooms, hotel rooms or hospital patient rooms). When two spaces are closely connected (e.g., a classroom and an interior prep room), they are considered one single functional space.

• Another consideration is the design, construction and maintenance of the HVAC system. Several factors, including the length of the supply air-duct, the presence of interior duct liners, whether the return air is ducted or dumped into an un-ducted plenum, and the efficiency of the filters, should be considered.

In the event that an indoor environment tests positive for the virus. The environment must be cleaned, surfaces sanitized or disinfected, and retested for SARS-CoV-2 before occupancy. Please consider the following treatment options.

Surface treatments can be accomplished by using several common disinfectants. The following cleaning/sanitizing agents/disinfectants are known to be very effective against SARS virus, which is genetically about 80% and structurally similar to SARS-CoV-2. The sanitizers/disinfectants are:

1.      Alcohols, including rubbing alcohol or isopropyl alcohol containing at least 60-70% alcohol, can inactivate the virus particles by denaturing the viral proteins However, alcohols are flammable and should only be used with great care. It should not be used in large scale sanitization indoors, or except in properly controlled manner and in well ventilated spaces.

2.      The second disinfectant is hydrogen peroxides. A review of scientific literature suggests that 0.5% hydrogen peroxide can inactivate SARS virus within one minute. Common hydrogen peroxide sanitizer available in drug stores is about 3% hydrogen peroxide. Some mold remediation contractors may have the equipment and capability to apply hydrogen peroxide by fogging.

3.      The third option is sodium hypochlorite solutions or similar chlorine-releasing compounds. It was reported that 0.1% sodium hypochlorite solution can inactivate SARS particles within one minute. Sodium hypochlorite is the active ingredient in household bleach. Commercial bleach solutions usually contain 5-6% sodium hypochlorite. Chlorine-releasing compounds and solutions should always be used with the greatest caution. Chlorine gas is toxic and can be lethal. Never mix bleach solutions with acids, alkali, or other cleaning agents, such as ammonia or Windex. When applying chlorine-releasing compounds indoors, workers must have respiratory protections and increase ventilation in the work area.

4.      Additional disinfectants on the EPA approved list are available at the EPA website.

https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2-covid-19

As always, know your sanitizers and disinfectants. Read instruction labels on the disinfectant containers or MSDSs, if they are available, before using them. When applying sanitizers and disinfectants, make sure paying attentions to recommended dosage and contact time. These disinfectants should only be used and applied by professionals when used on a large scale.

In an indoor environment where contamination is suspected, sanitization with selected sanitizers/disinfectants is recommended. Fogging, surface wiping, or a combination of both is useful. Fogging may reach areas where wiping is difficult to reach. However, wiping on surfaces where frequent human contacts likely occur is also very important.

After sanitization and disinfection treatments, post-treatment is verified by swab sampling to test for the virus or for bacterial and fungal spores as surrogates to the virus. The current testing method for the virus is the same as those used for clinical RT-PCR samples.

Should UV Lights Be Used in SARS-CoV-2 Disinfection?

There have been news and other commercial announcements suggested that UV lights, specifically UVC, can or may inactivate SARS-CoV-2 virus particles in the indoor environment. Currently there are limited peer-reviewed data regarding the wavelength, dose, and duration of UVC radiation required to inactivate the SARS-CoV-2 virus. UVC dosages are inversely proportional to the distance between the UV source and the target. There are other factors that may affect the effectiveness of UVC on killing bacteria or inactivation of SARS-CoV-2 virus. Please visit the FDA webpage (https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/uv-lights-and-lamps-ultraviolet-c-radiation-disinfection-and-coronavirus) for more information when considering the use of UVC indoors.

UV lights can also be a health hazard and are harmful to both the eyes (cornea and conjunctiva injuries) and skin (photokeratitis). The user may not realize the danger until after the exposure has caused damage. Symptoms can occur 4 to 24 hours after exposure. UV lights may emit ozone which can be irritating to the airway or cause asthmatic attack. UV lights can also damage skin and cause skin cancer. For more health and safety of using UV lights, please visit this website https://www.safety.rochester.edu/ih/uvlight.html. UV exposures can cause degradations of certain common indoor materials, such as plastic, polymers, and dyed textile, resulting in fine dusts of unknown chemical nature and of unknown health effects.

What else Can and Should Be Done Before Occupancy?

Another important change that can be implemented before an indoor environment is open for occupancy is to increase ventilation. Use an HVAC (heating, ventilating and air-conditioning) system to bring outdoor air in to purge indoor air. Upgrade filtration efficiency of the HVAC system but make sure that the HVAC operation engineer is consulted. If there is no HVAC system, open windows and doors (particularly those on opposite sides) to facilitate cross-ventilation. However, open windows and doors may only be practical when outdoor weather is suitable with moderate temperature and relative humidity.

Please feel free to email or call us (856-767-8300), if you have additional questions to discuss.

References:

Centers for Disease Control and Prevention. 2020. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. CDC-006-00019, Revision: 03. https://www.fda.gov/media/134922/download