Guidance and Recommended Practices for Working with Inactivated SARS-COV-2 Materials

BACKGROUND & PURPOSE

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) is a newly identified viral pathogen that causes the disease COVID-19 (novel Coronavirus Disease-2019). SARS CoV‐2 is a positive strand, or sense strand, single-stranded RNA virus. SARS‐CoV-2 is highly infectious and has affected millions of people worldwide resulting in a pandemic.  Boston University has established a COVID-19 surveillance program to test students, faculty and staff for COVID-19.  Nasal swabs are regularly collected and tested by RT-PCR for the presence of SARS CoV-2 nucleic acids; specifically, the nucleocapsid (N protein) gene.

The sensitivity of the RT-PCR assay poses obstacles for researchers working with recombinant, plasmid, and PCR amplicons as they comply with testing practices. Trace amounts of nucleic acid can potentially spillover into the test sample and produce a positive result that is not indicative of viable virus or an ongoing infection.

SCOPE & APPLICATION

This guidance document describes recommended practices when working with DNA and amplicons derived from SARS-CoV-2, inactivated SARS-CoV-2 materials and other non-infectious viral components in BSL-2 laboratories at Boston University.

This guidance does not apply to:

  • Work with clinical samples from COVID-19 patients. For such samples, follow BSL-2 plus procedures approved by IBC.
  • Work with the SARS-CoV-2 virus at Boston University which is performed in BSL-3 and BSL-4 containment laboratories.

Herein, we describe the potential risks of inactivated material that could result in a positive surveillance test in the absence of disease and identify mitigation practices that may aid in preventing this outcome.

DEFINITIONS

  • 10% Bleach Solution: a solution of freshly diluted bleach that contains one part bleach and nine parts water.
  • BSC: Biosafety Cabinet
  • BSL-2 plus: enhanced procedures or PPE used to minimize the production and/or contain aerosols when working in BSL-2
  • BSO: Biosafety Officer
  • BPHC: Boston Public Health Commission
  • cDNA: Complementary DNA generated from RNA as a template.
  • Clean Bench: is a laminar flow work cabinet or similar enclosure that provides filtered air across the work surface to protect against contamination. The clean bench was originally created to supplement clean room technology.
  • COVID-19 clinical samples: samples obtained from infected patients through an approved IRB and handled with BSL-2 plus procedures approved by IBC.
  • IBCInstitutional Biosafety Committee The IBC is an instituti...: Institutional Biosafety Committee
  • Inactivation: A procedure used to render an agent or nucleic acids as non-viable while retaining characteristic(s) of interest for future use.
  • Risk: as the term is used in this document, refers to the risk of obtaining a positive test outcome in the absence of disease; it is not intended to mean risk of infection or disease.
  • SARS CoV-2 material: material originated from risk group 3 SARS CoV-2 or SARS CoV-2 nucleic acids that have been approved by IBC and BPHC to be used at BSL-2 laboratories after undergoing a verified and/or validated procedure for inactivation.   This guidance document also applies to plasmid preparations containing SARS CoV-2 genomic materials, cDNA genomic material generated by PCR, or amplicons containing DNA sequences for the SARS CoV-2 nucleocapsid gene.
  • Viable agents: replicating virus capable of generating infection or disease.

RECOMMENDED PRACTICES & PROCEDURES

Formalin fixed materials

Formalin and glutaraldehyde chemically cross-link proteins and nucleic acids, fixing them in place such they are not free to aerosolize in any significant amount. Unless such material is purposely manipulated to extract nucleic acids and reconstitute them, it poses very low risk of resulting in a positive test. Standard practices in place for working with such inactivated material, using standard BSL-2 PPE (lab coat and disposable gloves) are therefore sufficient. If the material is treated to allow extraction of nucleic acids, it would be categorized as below.

Treatments that extract and procedures that produce virus related nucleic acids.

The inactivation of viruses by chaotropic agents is robust and enables isolation of nucleic acid material. Furthermore, production of plasmid DNA and synthetic RNA or cDNA that are copies of virus sequences allow necessary research on the virus. An example of a chaotrope that inactivates viruses is TRIzol, which has been shown to render coronavirus RNA non-infectious (Darnell et al., 2004 121:85-91 J. Virol. Meth. and Kumar et al., 2015 223:13-18, J. Virol. Meth.).  These sources for RNA/cDNA or plasmid DNA preparations increase the risk of exposure to nucleic acids that may result in a positive PCR test in the absence of viable agent or disease. This risk increases as the amount of nucleic acid increases. It is important to consider that while as few as 10 molecules of RNA or cDNA may give rise to a positive test, natural RNAses and DNAses on the skin and mucosal surfaces rapidly degrade nucleic acids to subcomponents (reviewed in Gupta et al. 2012; Innate Immunity 19(1) 86–97). Studies using RNAses from mice and human skin showed that microgram quantities of RNA were rapidly degraded within 15-60 minutes (Probst et al., Genet Vaccines Ther. 2006; 4:4). The amount of virus RNA isolated from 1 mL of culture supernatant or 106 cells is large, in the microgram range. However, degradation occurs rapidly in contact with skin RNases and DNases. Even after accidental contamination of a worker, this amount of RNA would be expected to be degraded overnight. Larger volumes of material or DNA increase risk.

RNA work. Based on this information, for microgram amounts of virus RNA, working at the bench with appropriate PPE and cleaning after work would be expected to mitigate risk. Disposable gloves, lab coat, eye protection (as required for all recombinant DNA work at BU) with a surgical face mask (as required by BU COVID-19 policy) accompanied by regular cleaning of work surfaces with 10% bleach followed by 70% alcohol or with RNA and DNA removing detergents such as Minerva labs PCR clean is recommended.

PCR and Plasmid work. Since plasmid DNA is more resistant to degradation than RNA above, microgram quantities will be more persistent. A PCR isolation workstation or a Biosafety Cabinet (BSC) would help prevent the spread of aerosol when handling such material. PCR workstation units are designed to absorb nucleic acids on a specialized filter and are simple to operate. BSCs have been shown to absorb DNA (Held et al. Journal of ABSA International, 2018, Vol. 23(2) 91-95) down to very low amounts.  The BSO must be consulted about their use for containment of nucleic acids. As above, personnel should wear appropriate PPE to include gloves, lab coat, eye protection, face mask but also a face-shield. Clean-up will include wiping down with 10% bleach solution or RNA/DNA cleaning detergent.  Once the material is transferred to smaller amounts, it can be used with the practices above for that amount of material.  After PCR amplification, the copy number of amplicons exceeds 109.  Manipulation of amplicons for restriction digestion, PCR purification, gel identification, etc. requires BSL-2 PPE with eye protection and face mask, and special attention paid to decontamination of surfaces and tubes during transfer in and out of equipment and after completion of the work.  This type of work should be performed at a designated lab bench that can be easily decontaminated after the procedure is completed with cleaning supplies stored locally. Changing gloves when moving between different areas in the lab can help reduce the potential spread of contaminants.

Inactivated viral supernatants: supernatants contain high (107 non-viable virus) quantities of viral/RNA material that can potentially trigger a positive test as indicated above.  While manipulating this type of material, the same approach as above must be followed and clean up with 10% bleach followed with 70% alcohol after use must be employed.  BSL-2 PPE include both eye protection and a face mask.

Dedicated space

A section or sections of the lab bench must be dedicated for work with the materials indicated above (RNA and DNA et.) so that PPE and clean up materials can be on hand for ease of clean up.  The space designated for this work must be free of unnecessary items including boxes, packages, and other items to avoid cluttering.  The clutter-free space will make it easier to clean and prevent spread of contamination.  Additionally, the work surfaces may be covered with disposable liner (i.e. scienceware-labmat-bench-liner).  This will help protect surfaces from potential contamination and minimizes the amount of clean up.  Workflow process should be implemented so that personnel that worked with the materials do not return to other areas.

Dedicated equipment

Equipment that can become contaminated with virus nucleic acids must be cleaned regularly. Since such equipment may be sensitive to corrosion by bleach, ensure that tubes, before being placed in the equipment, are wiped clean with a recommended cleaning solution and cleaning materials are disposed in biohazard waste. Such equipment includes but is not limited to PCR machines and centrifuges. The investigator is responsible to ensure that the equipment is left in a cleaned state. Common practices to work with DNA and RNA such as segregated pipettes with aerosol barrier tips, gel boxes, etc. for work with inactivated SARS-CoV-2 material must be followed and used. Make sure surfaces are wiped clean with the cleaning solutions and cleaning materials recommended above.

Engineering controls

Centrifugation of RNA or DNA materials must be performed using sealed centrifuge rotors or sample cups to contain aerosols. Open these using the guidelines described above defined by the type and amount of material handled.  Use a BSC or a PCR workstation when handling and manipulating the materials.  Aerosols that may be produced are captured by the equipment filters.  Use positive-displacement pipettes and/or aerosol-resistant filtered pipette tips.

NOTE: DO NOT use a clean bench to perform the work described.  A clean bench maintains a continuous flow of positive air inside the cabinet.  The air is blown out of the clean bench and directed towards the user.  DNA or RNA materials could get on and contaminate the user. Additionally, ensure that the PCR hood used does not function like a clean bench.

Recommended cleaning solution

Clean equipment and surfaces routinely with a fresh solution of 10% bleach (or equivalent decontamination agents that removes nucleic acids) followed by 70% ethanol after each use. Make sure that cleaning materials are compatible with the equipment used. DNA decontamination reagent (e.g. LookOut DNA Erase or DNAZap TM) may be used for removal of any DNA.  Consult your BSO if you have questions on what to use.

Disinfect all areas in which research has been present, including all touched surfaces in clean areas, and any lab surfaces with which they had direct contact (lab benches, chairs, chemical cabinets, doorknobs, keyboards, laptops, fume hoods, etc.). Use professional judgement concerning disinfecting sensitive equipment.  More information on cleaning is available in the “Cleaning/Disinfection SOP for Research Laboratories for DNA contamination”.

Personal Protective Equipment (PPE)

Disposable gloves, lab coat, eye protection (as required for all recombinant DNA work at BU), face-shield, and a surgical face mask (as required by BU COVID-19 policy) must be worn while manipulating inactivated SARS CoV-2 material that have undergone amplification (i.e. PCR amplicons, plasmids after replication, or inactivated viral supernatants).

Work Practices

  • Open tubes in the BSC or PCR workstation
  • Do not touch the underside of the tube cap and the lip
  • Wipe down and disinfect the exterior of the tube after recapping
  • Change the gloves frequently and when they become contaminated
  • Do not touch your face
  • Reusable items must be decontaminated before removing outside of the BSC
  • Soak test tube racks in bleach solution for 30 minutes rather than just spraying down. If possible, keep the dedicated racks in the PCR isolation workstation or BSC and transfer the tubes to a fresh rack before removing from the PCR workstation or BSC.
  • Follow pipetting techniques that reduce splashing or spraying to reduce producing aerosols.
  • Remove and replace PPE when contaminated
  • Clean up spills immediately

Spills

Cover spill with paper towels and carefully pour freshly prepared 10% bleach solution or a reagent specific for decontamination of DNA on and around the location of the spill, taking care to not create aerosols while pouring. Bleach solution should be made fresh daily to ensure maximum effectiveness. Remove all contaminated materials into a biohazard bag.

In case of large spill, alert lab members, leave the area and notify control center:

Boston University Medical Campus:             617-358-4144

Charles River Campus:                                   617-353-2105

For spills Inside BSC while working with inactivated virus supernatants:  Immediately stop all work. Leave BSC blower fan on during clean- up. Cover spill with paper towels and carefully pour 10% bleach solution on top of the paper towel covering the spill. The operator must be wearing PPE during this procedure. Bleach solution should be made fresh daily to ensure maximum effectiveness. Wipe down BSC walls, work surfaces, and equipment with disinfectant wipes. Wipe up all excess disinfectant. Discard contaminated materials in a biohazard bag.

Waste Disposal

Accumulated trash should be bagged, and surface decontaminated with 10% freshly made bleach before its removal from the PCR hood or BSC for disposal.  Discard solid waste in a biohazard box with a lid lined with double red biohazard bags.

Liquids treated with a final concentration of 10% bleach for 30 minutes may be disposed down the drain.

Housekeeping

Laboratory staff is responsible for housekeeping. All regular trash will be collected and placed in the dedicated bins.

Record Keeping

Inactivation records for SARS-CoV-2 material should be kept until the material has been used up or destroyed.  Cleaning and decontamination of high-touch areas and lab equipment must be logged and posted in the laboratory.

Separation of handling nucleic acids and other inactivated SARS CoV-2 materials and personnel testing.

Researchers working with purified virus RNA, cDNA, PCR amplicons, plasmids or inactivated viral supernatants need to plan for at least overnight between an experiment and diagnostic testing. RNA and DNA are rapidly degraded on mucosal surfaces but does require time. Having the test first thing in the morning before work is started, and after showering or bathing and wearing a fresh face mask, is highly encouraged to prevent the unintended detection of inactivated SARS CoV-2 material.

Use of Personal Devices and Items

Leave personal items such as cell phones, headphones, earbuds, and other similar personal devices outside of the laboratory work areas where the above materials are handled and used.

Personnel Training

Research personnel that perform and handle these materials must be trained on this guidance by the Principal Investigator (PI).  All new lab personnel must complete their training prior to conducting this work.  Your BSO is available for consultation on best practices.

DEVIATIONS FROM GUIDANCE

Some experimental procedures may require deviations from this guidance.  Deviation must be approved by the BU IBC.  Beforehand, it is strongly recommended to consult with the BSO to evaluate any alternative procedure that may require a departure from those outlined in this guidance to be sure that those procedures minimize risk.

    ENVIRONMENTAL SAMPLING PLAN

    Contamination of the laboratory environment and personnel from DNA, RNA, cDNA, PCR amplicons, or plasmids from the above materials pose a risk of interfering with the personnel COVID testing and monitoring. Although these materials are not infectious, personnel contamination could result in a positive COVID-19 test. COVID-19 testing is part of BU’s overall plan to monitor the current pandemic internally. Should a laboratory member test positive, environmental sampling is considered. When appropriate, EHS will coordinate and work with the PI or designee to perform sampling. Samples are submitted to the CILSE COVID-19 testing lab for analyses.

     

    UPDATED JAN 29, 2021

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