Covid-19 Diagnostic

COVID-19 DIAGNOSTIC

Standard Operating Procedure and Risk Assessment

Prepared by: Maria Isabel Veiga Alexandra Fraga Maria Belém Marques Eduarda Correia Ana Sofia Lima

Approved by: João Carlos Sousa

V1.4 | 06.04.2020 CONTENTS CONTENTS ...... 1 ABREVIATIONS ...... 2 CODING SYSTEM ...... 3 SECTION A – SCOPE ...... 4 SECTION B – GRAPHICAL PRESENTATION OF THE WORKFLOW ...... 5 SECTION C – PERSONNEL ENTRANCE REGISTRATION AND BELONGINGS ...... 5 SECTION D - DATABASES AND LABELLING OF SAMPLES ...... 6 SECTION E – DETAILED STANDARD OPERATING PROCEDURE ...... 6 E3. 1 – VIRUS INACTIVATION AND RNA EXTRACTION USING GeneJET Viral DNA and RNA Purification Kit (ThermoScientific) ...... 9 E3.2 – VIRUS INACTIVATION AND RNA EXTRACTION USING NZYTech Total RNA Isolation Kit ... 19 E3. 3 – VIRUS INACTIVATION AND RNA EXTRACTION USING THE TRIZOL (NZYTechh) METHOD27 E4. RT-qPCR RUN ...... 35 E4. 1 RT-qPCR USING SensiFAST Probe One-Step kit, no ROX (BioLine) (This section of the document is still being updated) ...... 35 E4. 2 RT-qPCR TEAM USING – One-Step NZYSpeedy RT-qPCR Probe kit, no ROX (NZYTech) ..... 42 E4. RESULTS COMMUNICATION ...... 51 SECTION F - RISK ASSESSMENT AND CONTINGENCY MEASURES ...... 53 ATTACHED DOCUMENTS ...... 58

ABREVIATIONS

SOP – Standard Operating Procedures

ICVS – Instituto de Investigação em Ciências da Vida e Saúde

BSC – Biosafety Cabinet

PPE – Personnel Protective Equipment

PCR – Polymerase Chain Reaction

BSL3 – Biosafety Level 3

BSL2 – Biosafety Level 2

RT-qPCR – Real-Time quantitative Polymerase Chain Reaction PC – Positive Control

NTC – Non-Template Control

EtOH – Ethanol

WHO – World Health Organization

EP – Emergency Procedures

CDC – Centers for Disease Control and Prevention

CODING SYSTEM Date dd mm.yyyy

Entry Time hh:mm

Swab Boxes – Original Swab Box #XXX

ICVS ID – ICVS ID XXXX

Samples’ RNA Boxes – Covid19 Action Sample RNA box #XXX

RT-qPCR Run Files – SARS-CoV-2 Diagnostic_ICVSID_dd-mm.yy

SECTION A – SCOPE

This document presents the Standard Operating Procedure (SOP) and the Risk Assessment Protocol prepared by the Life and Health Sciences Research Institute (ICVS) from the School of Medicine, University of Minho in order to assist on the diagnostic for COVID-19. The samples consist of specimens from suspected and/or confirmed cases for COVID-19.

All ICVS staff participating in this procedure must follow the Standard Operations Procedures and Risk Control Measures listed in this document.

This document will be made available in the ICVS webpage. Under no circumstances is ICVS liable for any decisions regarding the procedures implemented by third parties.

!!!NOTICE!!!

This is an evolving document subject to frequent updates. For the more recent version please go to: http://www.icvs.uminho.pt/services-resources/covid19-diagnostic.

SECTION B – GRAPHICAL PRESENTATION OF THE WORKFLOW

SECTION C – PERSONNEL ENTRANCE REGISTRATION AND BELONGINGS

Upon arrival to the ICVS, according to the team schedule, the staff must go to the meeting room (I1.14) and register in the login sheet. Personal belongings should be left at the meeting room. Cell phones are exceptionally allowed if an urgent call is expected. In such case the phone must be placed in a ziplock bag and left by the computer present in each room.

SECTION D - DATABASES AND LABELLING OF SAMPLES

D1 – VOLUNTEERS’ DATABASE

All volunteers' personal data (name, phone and e-mail), availability and details about lab experience (the type of tasks and years of experience) are included in a spreadsheet to allow distribution of volunteers in teams according to the number of samples to be processed. All personal data will be destroyed after the end of the COVID19 Diagnostic action.

D2 – SAMPLES’ DATABASE

Upon arrival, samples' information will be registered in the Sample ID form. Sample ID form spreadsheet is deposited in a shared folder accessible only to the ICVS overseers registered for the molecular detection of SARS-CoV-2 protocol described in this SOP, using their own ICVS login credentials.

The Sample ID form spreadsheet is protected against unintended editing and overseers may only include information regarding their own station.

Original Swab Boxes will be stored at –80°C WHERE?

Samples RNA will be stored at –80°C WHERE?

What to do in the end?

SECTION E – DETAILED STANDARD OPERATING PROCEDURE

E1 – SAMPLES’ TRANSPORTATION TO ICVS Transport of biological samples should follow the rules for packaging of infectious substances recommended by the WHO - category B (UN 3373). A triple packaging system should be used with the following characteristics:

a. Primary container is the one that contains the sample; must be properly identified and must be liquid and solid-tight; must be packed in enough absorbent material to absorb the entire contents in case of breakage or spill; b. Secondary container is the one that holds the primary containers; should be resistant, waterproof and leakproof to liquids and solids; may contain several sample tubes as long as they are individually and separately packed, in order to avoid contact, and protected with absorbent material and shock absorber; c. Outer container is the transport packaging with adequate padding and ice cold blocks, where the secondary containers are placed.

Samples will enter through the main door of the school of Medicine. The meeting point is next to security guard. Samples will be delivered ONLY to authorized ICVS researchers. Samples will be delivered to the PCR Team, that will do the samples’ register in the ICVS database. Then, the PCR team overseer transports samples to the third floor of the ICVS. (Overseer should wear a labcoat and gloves (surgical mask is optional)). The PCR Team overseer delivers the secondary container to the overseer in the Virus Inactivation Room.

E2. SAMPLE LABELING

1. Samples arrive with specific documentation. Samples will be delivered to the BSL3 for Virus Inactivation, while specific information of the samples is registered in the Sample ID form, by the PCR Team: a. Date and entry time, b. Origin of the samples (Health Care/Care Taker Entity), c. Person delivering the samples to the ICVS, d. Health Care/Care Taker Entity ID number. 2. In the Virus Inactivation Room, overseer reads out loud the Health Care/Care Taker Entity ID of each sample and assigns the sample an internal ICVS ID. Operator labels each swab falcon tube with its respective ICVS ID. 3. In the RNA Extraction Room, the overseer will be able to: a. add the name of the operators, overseers and notes if needed, b. print final RNA Eppendorf tube labels containing the ICVS ID as well as the original Health Care/Care Taker Entity ID 4. In the RT-qPCR Room, the overseer will be able to add information on PCR ID, PCR operators, PCR overseer, Test conclusion, sample storage and Notes.

E3. VIRUS INACTIVATION AND RNA EXTRACTION

IMPORTANT CONSIDERATIONS

Concerning the actual situation of pandemic, the reagents’ supply is often delayed or out-of-stock. To overcome the issue of possible stock rupture, we developed and validated alternative protocols to continuously provide the diagnostic of SARS-CoV-2 at ICVS. Protocols to be used for Virus Inactivation and Extraction are, in order of preference, E3.1 – GeneJet Viral DNA and RNA Purification Kit (ThermoScientific), E3.2 – NZYTech Kit and E3.3 - Trizol RNA Extraction Method and for RT-qPCR are, in order of preference, E4.1 SensiFAST One-Step Probe kit (BioLine) and E4.2 One-Step NZYSpeedy RT-qPCR Probe kit (NZYTech).

E3. 1 – VIRUS INACTIVATION AND RNA EXTRACTION USING GeneJET Viral DNA and RNA Purification Kit (ThermoScientific)

E3. 1 A – VIRUS INACTIVATION ROOM

Room: Virus Inactivation Room – BSL3- (I3γ04), ICVS 3rd floor Virus Inactivation Station: Team:

BSC 1: Virus Inactivation station 1 - One operator and one overseer for BSC 1

- One operator and one overseer for BSC 2

BSC 2: Virus Inactivation station 2 - One overseer

-An extra person may come to the antechamber temporarily to drop or pick-up samples, but never enter the virus room.

PPE:

a. coveralls

b. FFP2 or N95 face mask

c. hair cover Maximum capacity with 2 BSC running: 4 people

d. two pairs of gloves Minimum capacity 1 BSC working: 2 people

e. clogs

f. two pairs of shoe covers

g. sleeve covers/disposable gowns

h. safety glasses/face visor

IMPORTANT INFORMATION

ALL FOLLOWING RECOMMENDATIONS FOR THE SARS-COV-2 INACTIVATION PROTOCOL ADD ON TO THE BSL3 GUIDELINES ALREADY IMPLEMENTED AT THE ICVS - IN CASE OF EMERGENCY (SPILL, POWER FAILURE…), PLEASE FOLLOW EMERGENCY PLANS DESCRIBED IN THE ICVS BSL3 MANUAL. SEE ALSO GUIDELINES IN THE RISK ASSESSMENT SECTION BELOW. ONLY AUTHORIZED BSL3 USERS ARE ALLOWED TO ENTER THE BSL3 AND PERFORM THE FOLLOWING VIRUS INACTIVATION PROTOCOL.

E3. 1 A1 VIRUS INACTIVATION TEAM: RESPONSIBILITIES

OPERATORS

Operators will proceed with the inactivation of the virus with the lysis protocol in the BSC. Operators are also responsible for setting up the BSC. All essential reagents, materials and equipment are provided in a checklist:

a. Benchcoat b. Vortex c. Solid waste bag d. Liquid waste boat with 4% deconnex e. 1000 µL and 200 µL pipette tip Box f. 1000 µL and 200 µL pipette g. Disposable/sterile pasteur pipette h. Disinfectant wipes i. Disinfectant squirt bottle j. EtOH squirt bottle k. Rack 1 and Rack 2 for eppendorfs l. Rack A and Rack B for swab falcon tubes m. RNA-free eppendorfs n. Parafilm strips o. Lysis Solution supplemented with Carrier RNA (to be prepared by the rna extraction overseer) p. Proteinase K q. Heated thermoblock at 56ºC r. Rack 3 and Rack C on adjacent table.

OVERSEER The overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and must be ready to help in any emergency. In addition, the overseer must:

a. pick up samples from the antechamber of the Virus Inactivation Room (BSL3); b. fill in the Sample ID form with internal ICVS ID; c. place Rack 3 with inactivated samples in the clean area of the antechamber and call the RNA Extraction Room for sample pick up.

E3. 1 A2 DETAILED PROCEDURES FOR VIRUS INACTIVATION – GeneJET Viral DNA and RNA Purification Kit

MATERIAL PREPARATION

1. Inside the BSC, prepare and label eppendorf tubes with ICVS ID and place them on Rack 1 (only prepare the necessary number to match the number of samples to inactivate - this information is given by the overseer). 2. Close flask of eppendorf tubes and move it to the side to be immediately removed from BSC by overseer. 3. Close eppendorf tubes in Rack 1 and move Rack 1 to the back of the benchcoat area.

SAMPLE CHECK-IN

1. Samples arrive at the antechamber in a screwtop, leakproof container, containing the swab falcon tubes in viral transport medium. 2. Overseer collects samples from antechamber and moves sample container into the working area (onto the benchcoat). 3. Operator opens the container and remove swab falcon tubes. 4. Operator wipes the swab falcon tubes with desinfectant wipes, check that the lid is tightly screwed on, and place tubes onto Rack A. 5. Discard any plastic bag(s) in solid waste bag. 6. Overseer collects Rack A and brings samples to check-in area. 7. Overseer registers check-in team details: a. the person who deposits the sample container in the antechamber room; b. BSL3 operators’ and overseers’ name. 8. In parallel for all samples (one-by-one): a. Operator reads the Health Care/Care Taker Entity ID on the swab falcon tube - each tube should be removed from the Rack to be annotated, to avoid errors. b. Overseer registers a corresponding ICVS ID to the Health Care/Care Taker Entity ID already filled in the Sample ID form. c. Operator labels the swab falcon tube with the internal ICVS ID.

SAMPLE TRANSFER (SWAB TUBE TO EPPENDORF) AND LYSIS

9. Overseer brings Rack A to the BSC. 10. Operator vortexes each swab tube for 10 seconds and return to Rack A. 11. For all samples one-by-one: a. Pick-up plastic Pasteur pipette. Unwrap and discard wrap in solid waste, b. Open the eppendorf tube with ICVS ID and leave it on Rack 1, c. Open the swab falcon tube with the same ICVS ID, holding the swab falcon tube at an elevated position so that the operator is protected by the BSC sash, d. Transfer 200 µl of the sample from swab tube to eppendorf tube, e. Return the swab falcon tube to Rack A, f. Discard plastic Pasteur pipette in liquid waste container, g. Close the swab falcon tube, 12. Close the eppendorf tube. Move Rack A with the swab falcon tubes to the back of the BSC but leave it in the working area. 13. For all samples on Rack 1, one-by-one: a. Open the Buffer tube, Proteinase K tube and Tips Box, b. Put a tip onto the P1000 pipette, c. Open the eppendorf tube facing rear of hood, d. Pipette 200 µL of Lysis solution supplemented with Carrier RNA and add to the eppendorf tube, e. Discard the tip into liquid waste, f. Put a new tip onto the P200 pipette, g. Pipette 50 µl of Proteinase K and add to the eppendorf tube, h. Close the eppendorf tube and put it back in the same position in Rack 2, i. Discard the tip into liquid waste, j. Repeat for each eppendorf tube. 14. Close the liquid waste bottle, close tip box, close buffer tubes. 15. Vortex each eppendorf tube for 10 seconds and return to the same position. 16. Wipe the eppendorf tubes, one-by-one, and incubate for 15 minutes at 56ºC in a thermoblock within the BSC. 17. Wipe the swab falcon tubes, one-by-one, with wipes and move to Rack B. 18. Put parafilm on the swab falcon tubes. 19. When all the swab tubes are disinfected and parafilmed in Rack B, remove outer gloves and place in the liquid waste boat with 4% deconnex. 20. Put on new gloves.

EXIT OF INACTIVATED MATERIAL

21. Move the swab falcon tubes from Rack B to Rack C, being held by the overseer outside of the BSC. 22. Move eppendorfs tubes from Rack 2 to Rack 3, being held by the overseer outside of the BSC. 23. Overseer places Rack 3 in the antechamber in the clean zone. 24. Overseer calls RNA Extraction Room to announce samples are ready for pick up.

CLEANING PROCEDURES: END OF SHIFT OR END OF DAY

If closing for the day OR for the next team, proceed with full decontamination procedure: 25. Wipe all items OUTSIDE the working area with bleach wipes (squirt bottles, box of bleach wipes, solid waste container, liquid waste container, samples container, parafilm box, Rack 2 and Rack B). 26. Wipe all items INSIDE the working area with bleach wipes (Rack A, Tip Box, Pipette, Buffer, Buffer Rack). 27. Carefully close the benchcoat of the working area and throw it in the solid waste bag. 28. Wipe the entire surface of the hood with disinfectant. 29. Wipe the surface of Hood with 70% ethanol to remove traces of disinfectant. 30. Discard all wipes in the solid waste bag. 31. Close the solid waste bag with tape inside the hood (do not close too tightly), disinfect surface area and place trash to autoclave. 32. Overseer closes the sash, turns off light and air flow, and turns ON the UVs. 33. After each exit of the Virus Inactivation Room, all PPE should be discarded or properly disinfected. 34. The swab falcon tubes on Rack C are placed in a designated box stored at -80ºC, labelled “Original Swab Box #X”.

E3. 1 B RNA EXTRACTION ROOM

Room: ICVS 3rd floor, room I3α05 (BSL2)

RNA Extraction Station Team:

BSC A: RNA extraction station A - One operator for BSC A

BSC B: RNA extraction station B - One operator for BSC B

- One overseer for BSC A and BSC B

PPE: Maximum capacity with 2 Hoods running: 3 people

a. labcoat Minimum capacity 1 Hood working: 2 people

b. gloves

c. surgical mask

ALL FOLLOWING RECOMMENDATIONS FOR THE SARS-COV-2 INACTIVATION PROTOCOL ADD ON TO THE BSL2 GUIDELINES ALREADY IMPLEMENTED AT THE ICVS (adicionar o link).SEE ALSO GUIDELINES IN THE RISK ASSESSMENT SECTION BELOW.

ONLY AUTHORIZED BSL2 USERS ARE ALLOWED TO ENTER THE BSL3 AND PERFORM THE FOLLOWING VIRUS INACTIVATION PROTOCOL.

E3. 1 B1 RNA EXTRACTION TEAM – RESPONSIBILITIES

OPERATORS

Operators will proceed with the RNA extraction protocol using the GeneJET Viral DNA and RNA Purification Kit, in the BSC. Operators are responsible to set up BSC. All essential reagents, materials and equipment are provided in a checklist:

a. Waste bottle with 4% deconnex, b. Column Preparation Liquid (red cap), c. 100% EtOH (ice cold), in RNAse free water, d. Buffer NI (supplied with the kit), e. Digestion Mix (for each sample prepare a mixture of 10 μL of DNase I (reconstituted) and 90 μL of Digestion buffer ( supplied with the kit). Mix by gentle pipetting. Store this mixture on ice, f. Buffer NWR1 ( supplied with the kit), g. Buffer NWR2 ( supplied with the kit), with absolute EtOH added, h. RNase-free Water ( supplied with the kit), i. Collection tubes ( supplied with the kit), j. Eluent (white cap) (preheated to 56°C).

OVERSEER

The overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and be ready to help in any emergency. The overseer must also:

a. add information of the working team to the file “Sample ID form” previously filled-in by the team present in the BSL3- Virus Inactivation room; b. print the labels according to the information received from the BSL3 - Virus Inactivation Room and bring them to the RNA Extraction room; c. supplement lysis solution with the Carrier RNA (supplied with the kit) and mix by pulse-vortexing (always prepare fresh) and deliver it to the antechamber; d. pick up Rack 3 with samples from the antechamber of the BSL3 - Virus Inactivation Room, once a phone call is received from the BSL3 Virus Inactivation Room announcing that samples are ready; e. deliver Rack 3 to the operator of BSC A or BSC B.

E3. 1 B2 DETAILED PROCEDURES FOR RNA EXTRACTION – GeneJET Viral DNA and RNA Purification Kit (ThermoScientific)

MATERIAL PREPARATION 1. Prepare a rack with 5 rows of Spin Columns preassembled within the wash tube (number of tubes per row equal to the number of samples to be extracted) and label the lid with internal ICVS ID. 2. Add 50 µL of Column Preparation Liquid to the center of Spin Column membrane so that the membrane is entirely moistened to maximize binding of the nucleic acids to the membrane, resulting in more consistent yields. DO NOT centrifuge and store at room temperature until used for sample processing. 3. Preheat the eluent to 56°C using the thermoblock presented in bench. 4. Ready to receive the samples in Rack 3 by the overseer.

RNA EXTRACTION PROTOCOL

Following the instructions of the GeneJET Viral DNA and RNA Isolation Kit, from ThermoScientific.

5. Centrifuge tubes for 3-5 seconds at full speed to collect any sample solution from the inside of the lid. 6. Add 300 µL of EtOH (96-100%) and mix by vortexing. 7. Incubate the sample at room temperature for 3 minutes. 8. Centrifuge for 3-5 seconds at full speed to collect drops from the inside of the lid. 9. Transfer the lysate to the prepared Spin Column preassembled within the wash tube. 10. Centrifuge the column for 1 minute at 6,000 g. 11. Discard the Wash Tube containing flow-through. 12. Place the Spin Column into a new 2 mL Wash Tube. 13. Add 700 µL of Wash Buffer 1 supplemented with EtOH to the Spin Column. 14. Centrifuge the column for 1 minute at 6,000 g. 15. Discard the Wash Tube containing flow-through. 16. Place the Spin Column into a new 2 mL Wash Tube. 17. Add 500 µL of Wash Buffer 2 supplemented with EtOH to the Spin Column. 18. Centrifuge the column for 1 minute at 6,000 g. 19. Discard the Wash Tube containing flow-through. 20. Place the Spin Column into a new 2 mL Wash Tube. 21. Add 500 µL of Wash Buffer 2 supplemented with EtOH to the Spin Column. 22. Centrifuge the column for 1 minute at 6,000 g. 23. Discard the Wash Tube containing flow-through. 24. Place the Spin Column into a new 2 mL Wash Tube. 25. Centrifuge the column for 3 minutes at 16,000 g. 26. Discard the Wash Tube containing remaining flow-through. 27. Place the Spin Column into a new 1.5 mL elution tube. 28. Add 40 µL of Eluent preheated to 56°C to the center of Spin Column membrane. 29. Incubate for 2 minutes at room temperature. 30. Centrifuge the column for 1 minute at 13,000 g. 31. Discard the Spin Column. 32. Keep the elution tube containing pure viral nucleic acids. 33. Label each RNA sample with the labels brought to the room by the overseer. Store the RNA on ice in a styrofoam box. 34. Overseer will transport the RNA sample styrofoam box to the first floor to proceed to RT-qPCR.

E3.2 – VIRUS INACTIVATION AND RNA EXTRACTION USING NZYTech Total RNA Isolation Kit E3.2 A – VIRUS INACTIVATION ROOM

PPE: Room: ICVS 3rd floor, room I3γ04 a. coveralls (BSL3) b. FFP2 or N95 face mask c. hair cover Virus Inactivation Station: d. two pairs of gloves BSC 1: Virus Inactivation station 1 e. clogs BSC 2: Virus Inactivation station 2 f. two pairs of shoe covers g. sleeve covers/disposable gowns h. safety glasses/face visor

Team: - One operator and one overseer for BSC 1 - One operator and one overseer for BSC 2

- An extra person may come to the ante-chamber temporarily to drop or pick-up samples, but never enter the virus room.

Maximum capacity with 2 BSC running: 4 people Minimum capacity 1 BSC working: 2 people

E3.2 A1 – VIRUS INACTIVATION TEAM: RESPONSIBILITIES

OPERATORS

Operators will proceed with the inactivation of the virus with the lysis protocol in the BSC. Operators are also responsible for setting up the Biosafety cabinet (BSC). All essential reagents, materials and equipment are provided in a checklist:

a. Benchcoat b. Vortex c. Solid waste bag d. Liquid waste boat with 4% deconnex e. 1000 uL pipette tip Box f. 1000 uL pipette g. Disposable/sterile pasteur pipette h. Disinfectant wipes i. Disinfectant squirt bottle j. EtOH squirt bottle k. Rack 1 and Rack 2 for eppendorfs l. Rack A and Rack B for swab falcon tubes m. RNAse-free eppendorfs n. Parafilm strips o. Buffer NR with 1% 2-mercaptoethanol - this is to be prepared by the RNA Extraction Team Overseer p. Rack 3 and Rack C on adjacent table

OVERSEER

The overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and must be ready to help in any emergency. In addition, the overseer must:

a. pick up samples from the antechamber of the BSL3-Virus Inactivation Room; b. fill in the “Sample ID form” with internal ICVS ID; c. place Rack 3 with inactivated samples in the clean area of the antechamber and call the RNA Extraction Room for sample pick up.

E3.2 A2 DETAILED PROCEDURES FOR VIRUS INACTIVATION – NZY Total RNA Isolation kit

MATERIAL PREPARATION 1. Inside the BSC, prepare eppendorf tubes, label tubes with internal ICVS ID (ICVS- XXX) and place them on Rack 1 (only prepare the necessary number to match the number of samples to inactivate - this information is given by the overeer). 2. Close flask of eppendorf tubes and move it to the side to be immediately removed from BSC by overseer. 3. Load eppendorf tubes with 353.5 µ L of Buffer NR with 1% 2-mercaptoethanol. 4. Close eppendorf tubes in Rack 1 and move Rack 1 to the back of the benchcoat area.

SAMPLE CHECK-IN

5. Samples arrive at the antechamber in a screwtop, leakproof container, containing the swab falcon tubes in viral transport medium. 6. Overseer collects the samples from antechamber and moves the sample container into the working area (onto the benchcoat). 7. Open the container and remove the swab falcon tubes. 8. Wipe the swab falcon tubes with bleach wipes, check that the lid is tightly screwed on, and place tubes onto Rack A. 9. Discard any plastic bag(s) in solid waste bag. 10. Overseer collects Rack A and brings samples to check-in area. 11. Overseer registers check-in team details: a. the person who deposits the sample container in the antechamber room; b. BSL3 operators’ and overseers’ name. 12. In parallel for all samples (one-by-one): a. Operator reads the Hospital ID on the swab falcon tube - each tube should be removed from the Rack to be annotated, to avoid errors. b. Overseer registers a corresponding ICVS ID to the Hospital ID already filled in the spreadsheet. c. Operator labels the swab falcon tube with the internal ICVS ID.

SAMPLE TRANSFER (SWAB FALCON TUBE TO EPPENDORF) AND SAMPLE LYSIS 13. Overseer brings Rack A to the BSC. 14. Vortex each swab falcon tube for 10 seconds and return to Rack A. 15. For all samples one-by-one: a. Pick-up plastic Pasteur pipette. Unwrap and discard wrap in solid waste, b. Open the eppendorf tube with the Buffer NR (w/ 1% of 2- mercaptoethanol) and leave it on Rack 1, c. Open the swab falcon tube with the same ICVS ID, holding the swab falcon tube at an elevated position so that the operator is protected by the BSC sash, d. Transfer 100 µL of the sample from the swab tube to the eppendorf tube, e. Return the swab falcon tube to Rack A, f. Discard plastic Pasteur pipette in liquid waste container, g. Close the swab falcon tube, h. Close the eppendorf tube. 16. Move Rack A with the swab falcon tubes to the back of the BSC but leave it in the working area. 17. Close the liquid waste bottle and the tip box. 18. Vortex each eppendorf tube for 10 seconds and return to the same position. 19. Wipe the swab falcon tubes, one-by-one, with bleach wipes and move to Rack B. 20. Put parafilm on swab tubes. 21. Wipe eppendorf tubes, one-by-one, with bleach wipes and move to Rack 2. 22. When all the swab falcon tubes are disinfected and parafilmed and in Rack B and all eppendorf tubes are disinfected, remove outer gloves and place in the liquid waste boat with 4% deconnex. 23. Put on new gloves.

EXIT OF INACTIVATED MATERIAL AND CLEANING PROCEDURES: END OF SHIFT OR END OF DAY

Same as E2. 1 A2

E3.2 B RNA EXTRACTION ROOM

PPE: a. labcoat Room: ICVS 3rd floor, room I3α05 (BSL2) b. gloves

c. surgical mask

Team: RNA Extraction Station: - One operator for BSC A

- One operator for BSC B BSC A: RNA extraction station A - One overseer for BSC A and BSC B BSC B: RNA extraction station B

Maximum capacity with 2 Hoods running: 3 people

Minimum capacity 1 Hood working: 2 people

E3.2 B1 RNA EXTRACTION TEAM - RESPONSIBILITIES

OPERATORS

Operators will proceed with the RNA extraction protocol, using NZY Total RNA isolation kit from NZYTech, in the BSCs. Operators are responsible to set up BSC with all essential reagents and materials:

a. Waste bottle with 4% deconnex b. 70% EtOH (ice cold), in RNAse free water c. Buffer NI (provided with the NZYTech kit) d. Digestion Mix (for each sample prepare a mixture of 10 μL of DNase I (reconstituted) and 90 μL of Digestion buffer (provided with the NZYTech kit). Mix by gentle pipetting. Store this mixture on ice. e. Buffer NWR1 (provided with the NZYTech kit) f. Buffer NWR2 (provided with the NZYTech kit), with absolute EtOH added g. RNase-free Water (provided with the NZYTech kit) h. Collection tubes (provided with the NZYTech kit)

OVERSEER

Overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and be ready to help in any emergency. The overseer must also:

a. Prepare Buffer NR NR with 1% 2-mercaptoethanol. b. Print the labels according to the information received from the BSL3 - Virus Inactivation Room and bring them to the BSL2 - RNA Extraction room. c. Pick up the Rack 3 with samples from the antechamber of the BSL3 - Virus Inactivation Room, once a phone call is received from the BSL3 - Virus Inactivation Room announcing that the samples are ready. d. Add information of the working team to the file “Sample ID form” previously filled-in by the team present in the BSL3 - Virus Inactivation Room; e. Deliver the samples to the first floor to the RT-qPCR Team.

E3.2 B2 DETAILED PROCEDURES FOR RNA EXTRACTION - NZY Total RNA isolation kit

MATERIAL PREPARATION

1. In the BSC A and/or BSC B, label the NZYSpin Binding column (blue ring) with the internal ICVS ID . 2. Prepare a rack with 5 rows of collection tubes (number of collection tubes per row equal to the number of samples to be extracted) 3. Label 2 sets of 1.5 mL RNAse-free eppendorf tubes for RNA elution with the internal ICVS ID (ICVS-xxx) written on the lid. 4. Ready to receive the samples in Rack 3 by the overseer.

RNA EXTRACTION PROTOCOL

Following the instructions of the NZY Total RNA isolation kit from NZYTech.

5. Centrifuge the sample tubes at 11.000 g for 1 minute to remove drops from the lid. 6. One-by-one, add 350 μL of 70% ethanol (ice cold) and mix immediately by pipetting up and down. Do not centrifuge. 7. Pipette the lysate onto the NZYSpin Binding column (blue ring). Centrifuge at 11.000 g for 30 seconds. Discard the flow-through and place the column into a new collection tube. 8. Pipette 350 μL of Buffer NI into each NZYSpin Binding column (blue ring). Centrifuge at 11.000 g for 30 seconds. Discard the flow-through and place the column back into the collection tube. 9. Apply 95 μL of the Digestion Mix directly into the centre of the silica membrane of NZYSpin Binding column (blue ring) and incubate at room temperature for 15 minutes. 10. Add 200 μL of Buffer NWR1 and centrifuge at 11.000 g for 1 minute. Discard the flow-through and place the column in a new collection tube. 11. Add 600 μL of Buffer NWR2 and centrifuge at 11.000 g for 1 minute. Discard the flow-through and place the column back in the collection tube. 12. Add 250 μL of Buffer NWR2 into each column. Centrifuge at 11.000 g for 1 minute. 13. Do not remove the samples from the centrifuge and centrifuge again at 11.000 g for 1 minute, to dry the membrane. 14. Place the NZYSpin Binding Column in a clean 1.5 mL RNase-free microcentrifuge tube. Add 40 μL RNase-free water directly to the column membrane. Incubate for 1 minute and centrifuge at 11.000 g for 1 min to elute the RNA. Store the RNA at -20°C for short-term or at -70°C for long-term. 15. Centrifuge at 11.000 g for 2 minutes to elute the RNA. Discard the column and close the Eppendorf tubes (OPTIONAL?). 16. Label each RNA sample with the labels brought to the room by the overseer. Store the RNA on ice on a styrofoam box. 17. Overseer will transport the RNA sample styrofoam box to the first floor to proceed to RT-qPCR.

E3. 3 – VIRUS INACTIVATION AND RNA EXTRACTION USING THE TRIZOL (NZYTechh) METHOD

E2. 3 A VIRUS INACTIVATION ROOM

Team: - One operator and one overseer for BSC 1 - One operator and one overseer for BSC 2

- An extra person may come to the ante-chamber temporarily to drop or pick-up samples, but never enter the virus room.

Maximum capacity with 2 BSC running: 4 people Minimum capacity 1 BSC working: 2 people

E2. 3 A1 VIRUS INACTIVATION TEAM: RESPONSIBILITIES

OPERATORS

a. Benchcoat b. Vortex c. Solid waste bag d. Liquid waste boat with 4% deconnex e. 1000 uL pipette tip Box f. 1000 uL pipette g. Disposable/sterile pasteur pipette h. Disinfectant wipes i. Disinfectant squirt bottle j. Ethanol squirt bottle k. Rack 1 and Rack 2 for Eppendorf tubes l. Rack A and Rack B for swab tubes m. 1x number of samples of RNA-free eppendorfs n. Parafilm strips o. RNase-free water p. Trizol at 4° q. Rack 3 and Rack C on adjacent table.

OVERSEER

The overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and must be ready to help in any emergency. In addition, the overseer must:

a. pick up samples from the antechamber of the BSL3 - Virus Inactivation Room; b. fill in the “Sample ID form” with internal ICVS ID c. place Rack 3 with inactivated samples in the clean area of the antechamber and all the RNA Extraction Room for sample pick up.

E3. 3 A2 DETAILED PROCEDURES FOR VIRUS INACTIVATION – TRIZOL

MATERIAL PREPARATION

1. Inside the BSC, prepare eppendorf tubes, label tubes with internal ICVS ID (ICVS- XXX) using a pencil and place them on Rack 1 (only prepare the necessary number to match the number of samples to inactivate - this information is given by the overeer). 2. Close flask of eppendorf tubes and move it to the side to be immediately removed from BSC by overseer. 3. Load eppendorf tubes with 700 ul of Trizol and place in Rack 3. 4. Close eppendorf tubes in Rack 1 and move Rack 1 to the back of the benchcoat area.

SAMPLE CHECK-IN

5. Samples arrive at the antechamber in a screwtop, leakproof container, containing the swab falcon tubes in viral transport medium. 6. Overseer collects samples from antechamber and moves sample container into the working area (onto the benchcoat). 7. Open the container and remove the swab falcon tubes. 8. Wipe the the swab falcon tubes with bleach wipes, check that the lid is tightly screwed on, and place tubes onto Rack A. 9. Discard any plastic bag(s) in solid waste bag. 10. Overseer collects Rack A and brings samples to check-in area. 11. Overseer registers check-in team details: a. the person who deposits the sample container in the antechamber room; b. BSL3 operators’ and overseers’ name. 12. In parallel for all samples (one –by-one): a. Operator reads the Hospital ID on the swab falcon tube - each tube should be removed from the Rack to be annotated, to avoid errors. b. Overseer registers a corresponding ICVS ID to the Hospital ID already filled in the spreadsheet. c. Operator labels the swab falcon tube with the internal ICVS ID.

SAMPLE TRANSFER (SWAB TUBE TO EPPENDORF) AND SAMPLE LYSIS

13. Overseer Brings Rack A to the BSC. 14. Vortex each swab tube for 10 seconds and return to Rack A 15. For all samples one-by-one: a. Pick-up plastic Pasteur pipette. Unwrap and discard wrap in solid waste, b. Open eppendorf tube (number XXX) and leave it on Rack 1. c. Open the swab tube from Rack A,holding the tube at an elevated position so that the operator is protected by the biosafety cabinet sash, and transfer 300 μL of the sample to Eppendorf tube from rack 1, d. Mix the Eppendorf tube by turning the tube up and down by hand 5 times. (sample Lysis), e. Discard plastic Pasteur pipette in liquid waste container, 16. Incubate eppendorf sample tubes for 5 minutes at room temperature (this step inactivates de virus). There is no need to incubate the samples for these 5 minutes inside the BSC. The samples MUST get into BSL within 10 minutes. Wipe the swab tubes, one-by-one, with wipes and move to Rack B. Put Parafilm on swab tubes 17. Wipe eppendorf tubes, one-by-one, with wipe and move to Rack 2 18. When all swab tubes are disinfected and parafilmed and in Rack B and all eppendorf tubes are disinfected, remove outer gloves and place in the liquid waste boat with 4% deconnex 19. Put on new gloves.

EXIT OF INACTIVATED MATERIAL AND CLEANING PROCEDURES: END OF SHIFT OR END OF DAY

Same as E2. 1 A1

E3. 3 B RNA EXTRACTION ROOM - BSL2

PPE: a. labcoat Room: ICVS 3rd floor, room I3α05 (BSL2) b. gloves

c. surgical mask

Team: RNA Extraction Station: - One operator for BSC A

- One operator for BSC B BSC A: RNA extraction station A - One overseer for BSC A and BSC B BSC B: RNA extraction station B

Maximum capacity with 2 Hoods running: 3 people

Minimum capacity 1 Hood working: 2 people

E3. 3 B1 RNA EXTRACTION TEAM – RESPONSIBILITIES

OPERATORS

Operators will proceed with the RNA extraction protocol, using Trizol, in the BSCs. Operators are responsible to set up BSC with all essential reagents and materials:

a. Waste bottle with 4% deconnex b. Chloroform c. Isopropyl alcohol (ice cold) d. 75% ethanol (ice cold) e. RNase-free water f. 1.5mL microcentrifuge tubes (1x the number of samples) g. 200 uL Micropipette h. 1000 uL Micropipete i. 200 Tips for micropipettes j. 1000 Tips for micropipettes k. Ensure a rack is placed in the -20°C freezer l. Ensure microcentrifuge is at 4°C

OVERSEER

Overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and be ready to help in any emergency. The overseer must also:

a. print the labels according to the information received from the BSL3 - Virus Inactivation Room and bring them to the BSL2 - RNA Extraction room. b. Pick up the Rack 3 with samples from the antechamber of the BSL3 - Virus Inactivation Room, once a phone call is received from the BSL3 - Virus Inactivation Room announcing that the samples are ready. c. Add information of the working team to the file “Sample ID form” previously filled-in by the team present in the BSL3 - Virus Inactivation Room; d. Deliver the samples to the first floor to the RT-qPCR Team.

E2. 3 B2 DETAILED PROCEDURES FOR RNA EXTRACTION - TRIZOL

MATERIAL PREPARATION

1. In the BSC A and/or BSC B, label the NZYSpin Binding column (blue ring) with the internal ICVS ID (ICVS-xxx). 2. Prepare a rack with 5 rows of collection tubes (number of collection tubes per row equal to the number of samples to be extracted) 3. Label 2 sets of 1.5mL RNAse-free eppendorf tubes for RNA elution with the internal ICVS ID written on the lid. 4. Ready to receive the samples in Rack 3 by the overseer.

RNA EXTRACTION PROTOCOL

5. In the BSC A and/or BSC B, add 200µL of chloroform to the samples present in the Rack 3 brought by the overseer from ante-chamber room. 6. Cap tubes securely and shake vigorously by hand for 15 seconds. 7. Incubate samples for 2-3 minutes at room temperature. 8. Centrifuge samples at 12.000 g for 15 minutes at 4 °C.

Note: The sample will separate into a lower green phenol-chloroform phase, an interphase, and a colourless upper aqueous phase that contains the RNA.

9. During the 15 minutes centrifugation, prepare new 1.5mL centriguge tubes on a new rack. Label the eppendorf tubes with printed labels prepared by the overseer. These will be the final RNA sample tubes. 10. Take the tubes from the centrifuge and transfer 550 µL of the aqueous phase VERY carefully, without disturbing the interphase or organic layer, to the tube labeled with the printed labels. 11. Precipitate the RNA by mixing with ice cold isopropyl alcohol. 12. Add 500 µL of ice cold isopropyl alcohol to the aqueous phase and mix by gently inverting the tube (this step will precipitate the RNA). 13. Incubate samples for 10 minutes in -20°C freezer inside the rack present there. 14. Centrifuge at 12.000 g for 10 minutes at 4°C. Discard the supernatant with a micropipette.

Note: Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.

15. Resuspend the pellet in 1 mL of ice cold 75% ethanol. 16. Mix samples briefly, and then centrifuge at 12.000 g for 5 minutes at 4 °C. Discard the supernatant carefully with a micropipette. 17. Air dry the pellet for 5-10 minutes. 18. Gently resuspend the pellet in 40 μL of RNase-free water by pipetting the solution up and down. (Incubate for 10 minutes at 55-60°C if necessary.) 19. Place the RNA tubes on a styrofoam box with ice. 20. The overseer will transport the RNA sample box to the first floor to proceed to RT-qPCR.

E4. RT-qPCR RUN

E4. 1 RT-qPCR USING SensiFAST Probe One-Step kit, no ROX (BioLine) (This section of the document is still being updated)

E4. 1 A RT-qPCR STATION

Room: ICVS 1st floor, rooms I1.β02, PPE: a. lab coat I1.01 and I1.α06 b. gloves

c. surgical mask RT-qPCR Room: Team:

- One operator Pre-PCR room: assay and mix preparation - One overseer Maximum capacity with 3 Real-Time PCR running: 2 people Assembling station: Plate loading and sealing. - One operator for results’ analysis Minimum capacity 1 Hood working: 2 people PCR Station: experiment design and RT-qPCR run.

E4. 1 A1 RT-qPCR TEAM – RESPONSABILITIES

OPERATORS

Operators will proceed with the RT-qPCR protocol. Operators are responsible turn ON the RT-qPCR equipment and to set up the different stations for RT-qPCR Run protocol (pre-PCR station, Assembling Workstation and PCR station) with all essential reagents and materials:

a. qPCR primers/probe sets b. Positive template control c. Nuclease-free PCR grade 1.5ml tubes d. SensiFAST Probe One-Step kit, no ROX e. Molecular grade water, nuclease-free f. One full set of micropipettes (P10 to P1000) (pre-PCR room) g. One vortex per station (Pre-PCR Station and Assembling Workstation) h. A P10 pipette and an 8-channel P10 pipette (Sample/No-Template Control (NTC) and Positive Control (PC) Loading – in this exact order) i. P2/P10, P20, P100, P300 and P1000 aerosol barrier tips to be distributed in each hood and never interchanged. j. RT-qPCR Equipment: 2x ABI 7500 Fast Real Time PCR system, 1x Bio-Rad CFX96. k. 96-well real-time PCR reaction plates and optical seals compatible with each equipment l. Surface decontaminants: RNase away, 10% Bleach and 70% EtOH

OVERSEER

Overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and be ready to help in any emergency. The overseer must also:

a. fill in the “Sample ID form” with ICVS internal ID, using the 96-well template form; b. Select PC to be used on that day, by accessing shared Excel file and choosing a previously ran sample with low Ct value for all genes of interest; c. Assist during plate loading.

E4. 1 A2 DETAILED PROCEDURES FOR RT-qPCR PROTOCOL

IMPORTANT INFORMATION - PRIMERS AND PROBES

Primers and probes specific for SARS-CoV-2 refer to those published by Victor M. Corman from Charité Virology and approved by World Health Organization (WHO), with an extra set of primers and probe for a human control gene - Ribonucleoprotein (RNP)

1. https://eurosurveillance.org/content/10.2807/1560- 7917.ES.2020.25.3.2000045 2. https://www.who.int/docs/default-source/coronaviruse/protocol-v2- 1.pdf?sfvrsn=a9ef618c_2

The set includes:

a. 2 primers +1 probe set for RdRP gene region [one probe specific for SARS-CoV-2 (s-SARS-CoV-2)]. b. 2 primers + 1 probe set for E gene. c. 2 primers + 1 probe set for RNP gene.

Primers and Probes MUST NEVER go to the sample preparation area (Virus Inactivation Room) or the plate loading area (Assembling Workstation).

MATERIAL PREPARATION

1. PRE - PCR ROOM - PREPARATION OF PRIMER AND PROBES STOCK AND WORKING SOLUTIONS a. Clean and decontaminate all work surfaces, pipettes, centrifuges and other equipment prior to use, using RNase away followed by 70% EtOH. b. In the Mix Preparation Hood, resuspend the primers and probes with RNase-free water to a final concentration of 100µM. Do aliquots of 100µL at 10µM final concentration for each primer and 30µL at 10µM final concentration for each probe. Store at –20ºC in the freezer at the Pre- PCR room.

2. PRE - PCR ROOM - PREPARATION OF REACTION MIXES FOR RT-qPCR a. Clean and decontaminate all work surfaces, pipetts, centrifuges and other equipment prior to use, using RNase away, followed by 70% EtOH. b. In the Preparation Hood, thaw SensiFAST Probe No-ROX One-Step mix, primers and probes aliquots, on ice. c. Label one 1.5 mL eppendorf for each primers/probe set mix.

3. AMPLIFICATION REACTION MIXES PREPARATION

Plate is to be run with 29 samples, a No-Template Control (NTC), an Internal Extraction Control (IEC) and Positive Control (PC) to be analyzed per plate. However, plate set-up configuration can vary with the number of specimens and work day.

A) REACTION MIX FOR E Gene AMPLIFICATION

Mix/sample 32 Reactions+10% (μL)

Nuclease free 2.6 91.52 H2O

SensiFAST Probe No-ROX One- 10 352 Step Mix (2x)

10μM E gene 0.8 28.2 primer Forward 10μM E gene 0.8 28.2 primer Reverse

10 μM Probe E Gene SARS-CoV- 0.2 7.04 2

RiboSafe RNase Inhibitor 0.4 14.08

Reverse transcriptase 0.2 7.04

B) REACTION MIX FOR Gene RdRP AMPLIFICATION

Mix/sample (μL) 32 Reactions+10%

Nuclease free 2.6 91.52

H2O

SensiFAST 10 352 Probe No-ROX One-Step Mix (2x)

10μM RdRP 0.8 28.2 primer Forward

10μM RdRP 0.8 28.2 primer Reverse

10 μM Probe 0.2 7.04 RdRP SARS-CoV- 2 RiboSafe RNase 0.4 14.08 Inhibitor

Reverse Transcriptase 0.2 7.04

C) REACTION MIX FOR RNP Gene (control gene) AMPLIFICATION

Mix/sample (μL) 32 Reactions+10%

Nuclease free 2.6 91.52 H2O

SensiFAST 10 352 Probe No-ROX One-Step Mix (2x)

10μM RNP 0.8 28.2 primer Forward

10μM RNP 0.8 28.2 primer Reverse

10 μM Probe 0.2 7.04 RNP

RiboSafe RNase 0.4 14.08 Inhibitor

Reverse transcriptase 0.2 7.04

a. Pipette reaction mix reagents into the respective labelled 1.5 mL eppendorf tube, according the following order: i. Nuclease-free Water; ii. SensiFAST Probe No-ROX One-Step Mix (2x), iii. primers, iv. probe(s), v. RiboSafe RNase Inhibitor and Reverse Transcriptase. b. Mix by pipetting up and down and close the tube. Do not vortex. c. Transport all the reaction mixes to the ASSEMBLING WORKSTATION.

RT-qPCR PROTOCOL

4. SET UP THE REACTION PLATE – ASSEMBLING WORKSTATION a. On the BSC, set up a reaction plate in a 96-well rack, on ice. Turn off the BSC light (to protect probes’ fluorescence). Mix by pipetting up and down. Attention: Use 96-well plates compatible with equiment (Applied Biosystems and Bio-Rad) b. Centrifuge for 5 seconds at maximum speed to collect contents at the bottom of the tube. Place the eppendorf tube on ice. c. Pipette 15 µL of each mix into the appropriate wells. d. Turn off flow hood ventilation and introduce samples inside the workstation. A total of 5µL per sample, per assay, is used. Overseer will assist the operator directing samples’ loading order according to the physic template file, he designed. e. Add 5µL of nuclease free water into the NTC wells. f. Add 5µL of IEC to its respective wells. NOTE: internal extraction control is a RNase/DNase free water, used during RNA extraction process. This sample should not exhibit a Ct in any gene being analyzed. g. Add 5µL of the PC to the respective wells. Note: positive control will always be a positive patient sample (previously diagnosed using the protocol here described). h. Seal the plate, using the appropriate optical seal and the plate sealer. Protect from the light using aluminum foil.

5. RT-qPCR EQUIPMENT SET UP AND RUN – PCR ROOM a. Transfer plate into PCR room, on ice. b. Name the run using the code: SARS-Cov-2 Diagnostics – ICVS ID (ICVS_xxx)_ICVS_zzz dd-mm-yy. Note: Use ONLY the symbols “_” or “-”. c. Set up plate run and cycling parameters on the RT-qPCR system i. Detector (FAM); ii. Quencher (None); iii. Passive Reference: (None); iv. Run Mode: (Fast); v. Sample volume: 20µL; vi. Cycling parameters

45ºC – 10’

95°C – 2’

95°C – 5’’

58°C – 30’’

Repeat last two steps 44 times

Note: fluorescence acquisition selected to occur during the annealing/extension step

d. Insert plate into the designated RT-qPCR System; e. Start the run. It takes about one hour to be completed. f. When PCR run is finished, Save raw data .eds file to the server and discard plate into the appropriate waste bin.

E4. 2 RT-qPCR TEAM USING – One-Step NZYSpeedy RT-qPCR Probe kit, no ROX (NZYTech)

E4. 2 A RT-qPCR STATION

Room: ICVS 1st floor, rooms I1.β02, PPE: a. lab coat I1.01 and I1.α06 b. gloves

c. surgical mask RT-qPCR Room: Team:

E4. 2 A1 RT-qPCR TEAM – RESPONSABILITIES

OPERATORS

Operators will proceed with the RT-qPCR protocol. Operators are responsible to set up the different stations for RT-qPCR Run protocol (pre-PCR station, Assembling Workstation and PCR station) with all essential reagents and materials and turn ON the RT-qPCR equipment, following the checklist:

a. qPCR primers/probe sets b. Positive template control c. Nuclease-free PCR grade 1.5ml tubes d. One-step NZYSpeedy RT-qPCR Probe kit, no ROX e. Molecular grade water, nuclease-free f. One full set of micropipettes (P10 to P1000) (pre-PCR room) g. One vortex per division (Pre-PCR room and Assembling Workstation) h. A P10 pipette and an 8-channel P10 pipette (Sample/No-Template Control (NTC) and Positive Control (PC) Loading – in this exact order) i. P2/P10, P20, P100, P300 and P1000 aerosol barrier tips to be distributed in each hood and never interchanged. j. PCR Machines: 2x ABI 7500 Fast Real Time PCR system, 1x Bio-Rad CFX96. k. 96-well real-time PCR reaction plates and optical seals compatible with equipment l. Surface decontaminants: RNase away, 10% Bleach and 70% EtOH

OVERSEER

Overseer must stand behind the operator during the procedure to ensure the operator follows all the procedures and be ready to help in any emergency. The overseer must also:

a. fill in the “Sample ID form” with ICVS ID, using the 96-well template form; b. Assist the plate loading.

E3.2.1 DETAILED PROCEDURES FOR RT-qPCR PROTOCOL

IMPORTANT INFORMATION - PRIMERS AND PROBES Primers and probes specific for SARS-CoV-2 refer to those published by Victor M. Corman from Charité Virology and approved by World Health Organization (WHO), with an extra set of primers and probe for a human control gene - Ribonucleoprotein (RNP)

1. https://eurosurveillance.org/content/10.2807/1560- 7917.ES.2020.25.3.2000045 2. https://www.who.int/docs/default-source/coronaviruse/protocol-v2- 1.pdf?sfvrsn=a9ef618c_2

The set includes:

a. 2 primers +1 probe set for RdRP gene region [one probe specific for SARS-CoV-2 (s-SARS-CoV-2)]. b. 2 primers + 1 probe set for E gene. c. 2 primers + 1 probe set for RNP gene.

Primers and Probes MUST NEVER go to the sample preparation area (Virus Inactivation Room) or the plate loading area (Assembling Workstation).

MATERIAL PREPARATION

1. PRE - PCR ROOM - PREPARATION OF PRIMER AND PROBES STOCK AND WORKING SOLUTIONS a. Clean and decontaminate all work surfaces, pipettes, centrifuges and other equipment prior to use, using RNase away followed by 70% EtOH. b. In the Mix Preparation Hood, resuspend the primers and probes with RNase-free water to a final concentration of 100µM. Do aliquots of 100µL at 10µM final concentration for each primer and 30µL at 10µM final concentration for each probe. c. Store at –20ºC in the freezer present at the Pre-PCR room.

2. PRE - PCR ROOM - PREPARATION OF REACTION MIXES FOR RT-qPCR a. Clean and decontaminate all work surfaces, pipetts, centrifuges and other equipment prior to use, using RNase away, followed by 70% EtOH. b. In the Preparation Hood, thaw NZYSpeedy qPCR Probe master mix, primers and probes aliquots, on ice. c. Label one 1.5 mL eppendorf for each primers/probe set mix.

3. AMPLIFICATION REACTION MIXES PREPARATION Plate is to be run with 29 samples, a NTC an internal extraction control (IEC) and a Positive Control to be analyzed per plate. However, plate set-up configuration can vary with the number of specimens and work day.

A) REACTION MIX FOR E Gene AMPLIFICATION

Mix/sample (μL) 32 Reactions+10%

Nuclease free H2O 2.4 84.48

One-step NZYSpeedy qPCR 10 352 Probe

master mix (2x)

10μM E gene primer Forward 0.8 28.2

10μM E gene primer Reverse 0.8 28.2

10 μM Probe E Gene SARS- 0.2 7.04 CoV-2

NZYRT mix 0.8 28.2

B) REACTION MIX FOR Gene RdRP AMPLIFICATION

Mix/sample (μL) 32 Reactions+10%

Nuclease free H2O 2.4 84.48

One-step NZYSpeedy qPCR Probe 10 352 master mix (2x)

10μM RdRP primer Forward 0.8 28.2

10μM RdRP primer Reverse 0.8 28.2

10 μM Probe RdRP SARS-CoV-2 0.2 7.04

NZYRT mix 0.8 28.2

C) REACTION MIX FOR RNP Gene (control gene) AMPLIFICATION

Mix/sample (μL) 32 Reactions+10%

Nuclease free H2O 2.4 84.48

One-step NZYSpeedy qPCR 10 352 Probe

master mix (2x)

10μM RNP primer Forward 0.8 28.2

10μM RNP primer Reverse 0.8 28.2

10 μM Probe RNP 0.2 7.04

NZYRT mix 0.8 28.2

a. Pipette reaction mix reagents into the respective labelled 1.5 mL eppendorf tube, according the following order: i. Nuclease-free Water; ii. One-step NZYSpeedy qPCR Probe master mix (2x); iii. primers, probe(s); iv. NZYRT mix. a. Mix by pipetting up and down and close the tube. Do not vortex. b. Transport all the reaction mixes to the ASSEMBLING WORKSTATION.

RT-qPCR PROTOCOL

4. SET UP THE REACTION PLATE – ASSEMBLING WORKSTATION a. On the BSC, set up a reaction plate in a 96-well rack, on ice. Turn off the BSC light (to protect probes’s fluorescence). Mix by pipetting up and down. Attention: Use 96-well plates compatible with equiment (Applied Biosystems and Bio-Rad). b. Centrifuge for 5 seconds at maximum speed to collect contents at the bottom of the tube. Place the eppendorf tube on ice. c. Pipette 15μL of each mix into the appropriate wells. d. Turn off flow hood ventilation and introduce samples inside the workstation. A total of 5µL per sample, per assay, is used. Overseer will assist the operator directing samples’ loading order according to the physic template file, he designed. e. Add 5µL of nuclease free water into the NTC well. f. Add 5µL of IEC to its respective wells. NOTE: internal extraction control is a RNase/DNase free water, used during RNA extraction process. This sample should not exhibit a Ct in any gene being analyzed. g. Add 5µL of the Positive Control to the respective well. Note: positive control will always be a positive patient sample (previously diagnosed using the protocol here described). h. Seal the plate, using the appropriate optical seal and the plate sealer. Protect from the light using aluminum foil.

5. RT-qPCR EQUIPMENT SET UP AND RUN – PCR ROOM a. Transfer plate into PCR room, on ice. b. Name the run using the code: SARS-Cov-2 Diagnostics – ICVS ID (ICVS_xxx) – ICVS_zzz dd-mm-yy Note: Use ONLY the symbols “_” or “- ”. c. Set up plate run and cycling parameters on the RT-qPCR system i. Detector (FAM); ii. Quencher (None); iii. Passive Reference: (None); iv. Run Mode: (Fast); v. Sample volume: 20µL; vi. Cycling parameters

50ºC – 15’

95°C – 5’

95°C – 5’’

58°C – 30’’

Repeat last two steps 44 times

Note: fluorescence acquisition selected to occur during the annealing/extension step

d. Insert plate into the designated RT-qPCR System. e. Start the run. It takes about 1 hour and 15 minutes to be completed. f. When PCR run is finished, Save raw data .eds file to the server and discard plate into the appropriate waste bin.

6. RESULTS AND ANALYSIS INTERPRETATION a. Operators will open .eds file using corresponding software and adjust the threshold above the noise to fall on the exponential part of the curves, for each gene. Use preferentially automatic setup of threshold. b. Export results to an excel file using the code SARS-Cov-2 Diagnostics – ICVS_xxx – ICVS_zzz_dd-mm-yy_”gene” c. Save the amplification plot for each gene analyzed using the code SARS- Cov-2 Diagnostics – ICVS_xxx – ICVS_zzz_dd-mm-yy_”gene” d. Update common excel file, only using PCR sheet. e. Repeat previous steps for each plate analyzed. f. Prepare the report file, for each diagnostic. The reports must including the general amplification plot for each and a table with the results interpretation (as follows).

Notes Sample ID/ E Gene (Ct) Specific SARS- RNP gene (Ct) Conclusion Controls CoV-2 RdRP assay (Ct)

Ixxx

NTC

Guidelines for results interpretation

Positive All 3 assays present a Ct value below 35;

NTC Ct is undetermined;

PC presents a Ct value below 35.

Negative All assays present a Ct value above 35;

NTC Ct is undetermined; PC presents a Ct value below 35.

Inconclusive At least one of the following occurs:.

Either gene E or RdRP shows a Ct value above 35;

NTC shows a Ct below 35;

PC shows a Ct value above 35.

Invalid RNP gene shown no amplification or invalid curve. Analysis mus be repeated.

IEC or NTC shows amplification.

7. RESULTS VALIDATION

The analysis operator will, independently analyse each report, and validate the results.

E4. RESULTS COMMUNICATION At the end of the day, diagnostics will be sent, via e-mail, from [email protected], to the health care entity that provided the sample on a .pdf file containing a table with the original ID of each sample, conclusion of the test including details of Ct value for each gene and the general amplification plot for each gene.

Additional information

Plate template

Plate distribution example

Primers and Probes

Table 1 - Primers and probes, real-time RT-PCR for 2019 novel coronavirus.

Assay/use Oligonucleotide Sequence a

RdRP_SARSr-F GTGARATGGTCATGTGTGGCGG

RdRP gene RdRP_SARSr-P2 FAM-CAGGTGGAACCTCATCAGGAGATGC-BHQ

RdRP_SARSr-R CARATGTTAAASACACTATTAGCATA

E_Sarbeco_F ACAGGTACGTTAATAGTTAATAGCGT

E gene

E_Sarbeco_P1 FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ E_Sarbeco_R ATATTGCAGCAGTACGCACACA

RNP_F AGATTTGGACCTGCGAGCG

RNP gene RNP_R GAGCGGCTGTCTCCACAAGT

RNP_P FAM-TTCTGACCTGAAGGCTCTGCGCG- BHQ

aW is A/T; R is G/A; M is A/C; S is G/C. FAM: 6-carboxyfluorescein; BHQ: Black Hole Quencher.

SECTION F - RISK ASSESSMENT AND CONTINGENCY MEASURES

The risk assessment described below is based on the orientations of the document “Laboratory Biosafety Guidance related to coronavirus disease 2019 (COVID-19), Interim Guidance, 19th March 2020” from the WHO and the UK Guidance ”COVID-19: Safe Handling and Processing for samples in laboratories", 28 march 2020”.

Before starting the procedures, everyone must be trained on the procedure that will participate and then sign the responsibility term.

While in the Building of the ICVS/School of Medicine of the University of Minho and through all the procedures, you must follow the COVID-19’s prevention rules: social distance (especially if someone is sick - more than 1 meter); wash hands frequently or disinfect them; do not touch eyes, mouth and nose with unclean hands.

OTHER IMPORTANT CONSIDERATIONS 1. DISINFECTANTS TO BE USED (following WHO guidelines) • 70% ethanol; 0.5% hydrogen peroxide; quaternary ammonium compounds; and phenolic compounds, If used according to the manufacturer’s recommendations. • Other biocidal agents such as 0.05–0.2% benzalkonium chloride or 0.02% chlorhexidine digluconate can be less effective.

Particular attention should be paid not only to the selection of the disinfectant but also to the contact time (e.g., 10 minutes), dilution, and expiry date after the working solution is prepared.

2. RISK OF PPE DEPLETION

In the eventual cenario of a risk of PPE depletion, there is a need to conserve supplies, namely masks/respirators. Measures described in the document from the CDC “Recommended Guidance for Extended Use and Limited Reuse of N95 Filtering Facepiece Respirators in Healthcare Settings”, from 28 March 2018, will be implemented.

F1. RISK DESCRIPTION AND RESPECTIVE CONTINGENCY MEASURES

RISK DESCRIPTION CONTINGENCY MEASURES

Biological Hazard - Maintain, whenever possible, a safe social distance.

Picking up samples in a health care entity

- The sample must be transported in a triple container, following the WHO-category B (UN 3373) recommendations.

- The sample tube and transport box must be decontaminated by the personnel from the provider, before delivering it.

- Do not accept a sample box in bad shape, dirty or with leakage signs.

If the person needs to enter the sample provider facilities:

- Use adequate PPE: lab coat, gloves and a surgical mask (optional).

- Before entering the ICVS, remove the mask and one glove to open doors and push elevator buttons.

- Do not touch the mask or eyes with gloves or unclean hands.

- Wash your hands after delivery of the samples.

Biological Hazard - Work must be performed under BSL3 conditions.

Virus Inactivation Room COVID-19 - Medium risk

- Use a validated BSC.

- Follow good practices when working in the BSC (ex: slow movements; only the essential material inside, without covering the BSC grids).

- Use absorbent material to cover the surface of the BSC, where contaminated material is going to be used.

- Use of PPE: coveralls, double gloves, safety goggles, disposable safety mask (FFP2/ FFP3), cap, sleeve covers, clogs and shoe covers.

- Follow the donning and removal procedures, in the following order:

Correct donning of PPE upon entry:

- Register the name, date, and time in the logbook located in the locker room;

- Put on and zip up the coveralls;

- Put on your personal clogs at the entry of the common corridor;

- Put on the headcover, making sure that all hair is tucked in and the forehead is covered;

- Put on a facemask;

- Put on two pairs of gloves;

- Secure with tape the coveralls and the first pair of gloves;

- Put on two pairs of shoe covers;

- Put on sleeve covers and safety glasses; - Once you have double-checked that all PPE is correctly worn (use mirror located within the locker room), enter the BSL‑3, Virus Inactivation Room.

Correct removal of PPE upon exit:

Before you exit the first experimental room:

- Remove the outer pair of shoe covers;

- Remove the cover sleeves;

- Remove the outer pair of gloves;

- Remove the safety glasses, disinfect and store in an appropriate container;

- Dispose of coveralls in the dedicated container to be autoclaved;

- Remove the face mask, hair cover and gloves while holding your breath. Dispose of in the appropriate bin;

- Open the door and step into the common corridor;

- Make sure you close the door behind you;

- Wash and disinfect your hands.

- All contaminated or possibly contaminated material must be disposed of at the end of the procedure.

- Clean and decontaminate work area at the end of the procedure.

- The person that is working in the BSC must always be overseen by someone, to help him/her in case of need/emergency.

- Before leaving the BSC, all tubes with inactivated samples must be decontaminated and transferred to a clean rack.

Chemical Hazard - Work must be performed in BSL2.

RNA Extraction Room - Low Risk

- Use a validated BSC.

- Follow good practices when working with BSC (ex: slow movements; only the essential material inside, without covering the BSC grids).

- Use of PPE: lab coat, wrist-length gloves, surgical mask. - Disposable PPE material must be disposed of in a white waste bag.

- All material must be disposed at the end of the procedure.

- Clean and decontaminate work area at the end of the procedure.

- Wash your hands at the end of the procedure.

Chemical Hazard - Use a validated BSC.

Prepare Lysis Buffer - Medium risk

- Follow good practices when working with BSC (ex: slow movements; only the essential material inside, without covering the BSC grids).

- Use of PPE: lab coat, gloves, surgicval mask.

- Wash hands at the end of the procedure.

Generation of aerosols and droplets - Aerosol production must be avoided.

- All practices that may produce aerosols must be performed inside the BSC (ex.: vortexing).

- The use of cleaning sprays is forbidden; use only wipes with disinfectant.

- In the Virus Inactivation Room wear FFP2/FFP3 masks.

F2. EMERGENCY PROCEDURES (EP)

INCIDENT EMERGENCY PROCEDURE

Biological spill/leaking sample - If a spill occurs inside the BSC:

- Cover with paper towels and soak with disinfectant.

- Remove the absorbent benchcoat in the BSC and put it in the waste bag inside the BSC. - Close the bag.

- Discard contaminated wrist gloves and put new ones on.

- Decontaminate all the materials and the work surface of the BSC with wipes soaked in disinfectant. - Discard contaminated wrist gloves and put new ones on. - Place a new absorbent benchcoat on the work area of the BSC and continue to work. - Spill Kit is available near the BSC in Virus Inactivation Room: - It contains a box of wipes soaked in disinfectant and a bottle of 4% deconnex. - Should never be used except for spills.

- Prepare fresh every 7 days!!

- The person handling the spill must be fully equipped with the PPE mandatory for the room.

Active biological material in contact with - Undress PPE. skin, eyes, mouth or nose - Clean the affected area with plenty of water and soap.

- Contact the overseeing person to follow the ICVS/EM/UMinho contingency plan for COVID-19. - The overseeing person must inform the Safety and Health Compliance.

Chemical/inactivated biological material in - Clean the affected area with water and soap. contact with skin, eyes, mouth or nose

- Follow the instructions in the respective Safety Data Sheet.

ATTACHED DOCUMENTS

1. Statement of Responsability EN version. 2. UMinho COVID-19 Contingency Plan.

Plano de Contingência – Vol. 1: Azurém e Gualtar Versão 1.1

5 de março de 2020

1. Introdução

O presente Plano descreve os procedimentos a adotar perante docentes, estudantes, investigadores, “trabalhadores, técnicos, administrativos e de gestão” e aqueles que, por motivos profissionais ou outros, se desloquem às instalações da Universidade do Minho – doravante designados genericamente por Trabalhador com Sintomas (caso suspeito de infeção pelo novo Coronavírus SARS-CoV-2, agente causal da COVID-19). Este Plano pode ser atualizado a qualquer momento, tendo em conta a evolução do quadro epidemiológico da COVID-19. As situações não previstas neste Plano devem ser avaliadas caso a caso pela Comissão de Elaboração e Gestão do Plano de Contingência Interno COVID-19 da Universidade do Minho, nomeada pelo Despacho RT-21/2020. A definição apresentada na tabela 1 é baseada na informação disponível, à data, no Centro Europeu de Prevenção e Controlo de Doença Transmissíveis (ECDC), e é a adotada pela Universidade do Minho.

Tabela 1. Critérios clínicos e critérios epidemiológicos.

Critérios clínicos Critérios epidemiológicos

História de viagem para áreas com transmissão comunitária ativa1 nos 14 dias antes do Infeção respiratória início de sintomas. aguda (febre ou ou tosse ou dificuldade e Contacto com caso confirmado ou provável de infeção por SARS-CoV-2/COVID-19, nos 14 respiratória) dias antes do início dos sintomas. requerendo ou não ou hospitalização. Profissional de saúde ou pessoa que tenha estado numa instituição de saúde onde são tratados doentes com COVID-19.

Caso apareça algum dos sintomas referidos (no próprio ou nos seus conviventes), não se desloque à Universidade do Minho nem aos serviços de saúde, mas ligue para a linha Saúde 24 (808 24 24 24). Siga as orientações que lhe forem transmitidas e informe a sua chefia direta (ver Anexo I).

2. Procedimentos específicos

1 Consulte a informação atualizada das áreas afetadas pelo COVID-19 em https://www.dgs.pt/coronavirus.aspx

Este plano define os seguintes procedimentos: − Procedimentos perante um Caso Suspeito (ponto 8); − Procedimentos perante um Caso Suspeito Validado (ponto 9); − Procedimento de Vigilância de Contactos Próximos (ponto 10); − Processo de Alerta e Comunicação Interna (ponto 11); − Processo de Registo de Contactos com o Caso Suspeito (ponto 12).

3. Responsabilidades

Principais responsabilidades inerentes a este plano:

− Todos os trabalhadores devem reportar à sua chefia direta (ver Anexo I) uma situação de doença enquadrada como trabalhador com sintomas e ligação epidemiológica compatíveis com a definição de caso possível de COVID-19 (Trabalhador com Sintomas). Em caso de impedimento por isolamento ou internamento de algum elemento de chefia direta o Anexo I será devidamente atualizado identificando essa exceção; − Sempre que for reportada uma situação de Trabalhador com Sintomas, a chefia direta do trabalhador informa, de imediato, a Linha COVID-19 - UMinho (253 601 601) e a Segurança do respetivo campus (Azurém: 253 510 603; Gualtar 253 604 135); − A segurança informa qual a área de isolamento mais próxima disponível bem como o respetivo circuito para a ela aceder e acompanha o Trabalhador com Sintomas no percurso. Deverá isolar a área, e oportunamente, se necessário, encaminhar e acompanhar o INEM até à área de isolamento; − A chefia direta indica um trabalhador que preste assistência telefónica ao Trabalhador com Sintomas durante o período de isolamento. Por defeito considerar-se-á o trabalhador indicado no Anexo II.

4. Áreas de “isolamento” e circuitos até às mesmas

A colocação de um Trabalhador com Sintomas numa área de “isolamento” visa impedir que outros trabalhadores possam ser expostos e infetados. Tem como principal objetivo evitar a propagação da doença transmissível na Universidade do Minho e na comunidade. As áreas de “isolamento” têm como finalidade evitar ou restringir o contacto direto dos trabalhadores com o trabalhador doente (com sinais e sintomas e ligação epidemiológica compatíveis com a definição de caso suspeito, critérios referidos no ponto 1) e permitir um distanciamento social deste, relativamente aos restantes trabalhadores. As áreas de “isolamento” têm ventilação natural, ou sistemas de ventilação mecânica, e possuem revestimentos lisos e laváveis. Estas áreas estão equipadas com: telefone; cadeira ou marquesa (para descanso e conforto do Trabalhador com Sintomas, enquanto aguarda a validação de caso e o eventual transporte pelo INEM); kit com água e alguns alimentos não perecíveis; contentor de resíduos (com abertura não manual e saco de plástico); solução antisséptica de base alcoólica (disponível no interior e à entrada desta área); toalhetes de papel; máscara(s) cirúrgica(s); luvas descartáveis; termómetro. Nestas áreas, ou próximo destas, deve existir uma instalação sanitária devidamente equipada, nomeadamente com doseador de sabão e toalhetes de papel, para a utilização exclusiva do Trabalhador com Sintomas. No Anexo III apresenta-se a localização das áreas de isolamento. Os seguranças conhecerão os circuitos a privilegiar quando um Trabalhador com Sintomas se dirige para uma área de “isolamento”. Na deslocação do Trabalhador com Sintomas, devem ser evitados os locais de maior aglomeração de pessoas/trabalhadores nas instalações.

5. Disponibilização de equipamentos e produtos

A Universidade do Minho compromete-se a disponibilizar os seguintes equipamentos e produtos:

− Solução antisséptica de base alcoólica em sítios estratégicos (ex. zona de refeições, registo biométrico, áreas de “isolamento”), conjuntamente com informação sobre os procedimentos de higienização das mãos; − Máscaras cirúrgicas para utilização do Trabalhador com Sintomas (caso suspeito); − Máscaras cirúrgicas e luvas descartáveis, a utilizar, enquanto medida de precaução, pelo(s) segurança(s) que acompanhe(m); − Toalhetes de papel para secagem das mãos, nas instalações sanitárias e noutros locais onde seja possível a higienização das mãos; − Contentor de resíduos com abertura não manual e saco plástico.

6. Informação e formação

A Universidade do Minho compromete-se a:

− Divulgar o Plano de Contingência específico a todos os trabalhadores, nomeadamente na página https://www.uminho.pt/PT/viver/COVID-19/; − Esclarecer os trabalhadores, mediante informação precisa e clara, sobre a COVID-19 de forma a, por um lado, evitar o medo e a ansiedade e, por outro, estes terem conhecimento das medidas de prevenção que devem instituir; − Informar e formar os trabalhadores quanto aos procedimentos específicos a adotar perante um caso suspeito.

7. Diligências a efetuar na presença de trabalhadores suspeitos de infeção por SARS-CoV-2

A Universidade do Minho compromete-se a:

− Acionar o Plano de Contingência para COVID-19; − Confirmar a efetiva implementação dos procedimentos específicos previstos no Plano de Contingência para COVID-19; − Procurar manter atualizada a informação sobre COVID-19, na página https://www.uminho.pt/PT/viver/COVID-19/, de acordo com o disponibilizado pela Direção-Geral da Saúde, Autoridade de Saúde Local e meios de comunicação oficiais.

8. Procedimentos num Caso Suspeito

No Anexo IV apresenta-se o fluxograma a seguir numa situação de Trabalhador com sintomas de COVID-19. Neste ponto descreve-se os passos a seguir. Qualquer trabalhador com sinais e sintomas de COVID-19 e ligação epidemiológica, ou que identifique um trabalhador com critérios compatíveis com a definição de caso suspeito, informa preferencialmente por via telefónica a chefia direta (ver Anexo I). A chefia direta deve contactar, de imediato, a Linha COVID-19 - UMinho (253 601 601) e a Segurança do respetivo campus (Azurém: 253 510 603; Gualtar 253 604 135). A chefia direta indicará um trabalhador que preste assistência telefónica ao Trabalhador com Sintomas durante o período de isolamento. Por defeito considerar-se-á o trabalhador indicado no Anexo II. A segurança informa qual a área de isolamento mais próxima disponível bem como o respetivo circuito para a ela aceder e acompanha o Trabalhador com Sintomas no percurso. Sempre que possível deve-se assegurar a distância de segurança (superior a 1 metro) do doente. Deverá isolar a área e perante um caso suspeito validado deverá encaminhar e acompanhar o INEM até à área de isolamento. O(s) segurança(s) que acompanha(m)/presta(m) assistência ao Trabalhador com sintomas, deve(m) colocar, momentos antes de se iniciar esta assistência, uma máscara cirúrgica e luvas descartáveis, para além do cumprimento das precauções básicas de controlo de infeção quanto à higiene das mãos, após contacto com o Trabalhador doente. O Trabalhador doente (caso suspeito de COVID-19) já na área de “isolamento”, contacta o SNS 24 (808 24 24 24). Este trabalhador deve usar uma máscara cirúrgica, se a sua condição clínica o permitir. A máscara deverá ser colocada pelo próprio trabalhador. Deve ser verificado se a máscara se encontra bem ajustada (ou seja: ajustamento da máscara à face, de modo a permitir a oclusão completa do nariz, boca e áreas laterais da face. Em homens com barba, poderá ser feita uma adaptação a esta medida - máscara cirúrgica complementada com um lenço de papel). Sempre que a máscara estiver húmida, o trabalhador deve substituí-la por outra. O profissional de saúde do SNS 24 questiona o Trabalhador doente quanto a sinais e sintomas e ligação epidemiológica compatíveis com um caso suspeito de COVID-19. Após avaliação, o SNS 24 informa o Trabalhador: − Se não se tratar de caso suspeito de COVID-19: define os procedimentos adequados à situação clínica do trabalhador; − Se se tratar de caso suspeito de COVID-19: o SNS 24 contacta a Linha de Apoio ao Médico, da Direção-Geral da Saúde, para validação da suspeição. Desta validação o resultado poderá ser: − Caso Suspeito Não Validado, este fica encerrado para COVID-19. O SNS 24 define os procedimentos habituais e adequados à situação clínica do trabalhador. O trabalhador informa a chefia direta da não validação. − Caso Suspeito Validado, a DGS ativa o INEM, o INSA e Autoridade de Saúde Regional, iniciando-se a investigação epidemiológica e a gestão de contactos. A chefia direta do Trabalhador informa o empregador da existência de um caso suspeito validado na Universidade do Minho. Na situação de Caso suspeito validado: − O trabalhador doente deverá permanecer na área de “isolamento” (com máscara cirúrgica, desde que a sua condição clínica o permita), até à chegada da equipa do Instituto Nacional de Emergência Médica (INEM), ativada pela DGS, que assegura o transporte para o Hospital de referência, onde serão colhidas as amostras biológicas para testes laboratoriais; − O acesso dos outros trabalhadores à área de “isolamento” fica interditado, exceto aos trabalhadores designados para prestar assistência (ver anexo II); − A Universidade do Minho colabora com a Autoridade de Saúde Local na identificação dos contactos próximos do doente (Caso suspeito validado); − A Universidade do Minho informa os restantes trabalhadores da existência de Caso suspeito validado, a aguardar resultados de testes laboratoriais, mediante os procedimentos de comunicação estabelecidos no Plano de Contingência. O Caso suspeito validado deve permanecer na área de “isolamento” até à chegada da equipa do INEM ativada pela DGS, de forma a restringir, ao mínimo indispensável, o contacto deste trabalhador com outro(s) trabalhador(es). Devem-se evitar deslocações adicionais do Caso suspeito validado nas instalações da Universidade do Minho.

9. Procedimentos perante um Caso Suspeito Validado

A DGS informa a Autoridade de Saúde Regional dos resultados laboratoriais, que por sua vez informa a Autoridade de Saúde Local. A Autoridade de Saúde Local informa a Universidade do Minho dos resultados dos testes laboratoriais e: − Se o Caso for infirmado, este fica encerrado para COVID-19, sendo aplicados os procedimentos habituais da Universidade do Minho, incluindo de limpeza e desinfeção; − Se o Caso for confirmado, a área de “isolamento” deve ficar interditada até à validação da descontaminação (limpeza e desinfeção) pela Autoridade de Saúde Local. Esta interdição só poderá ser levantada pela Autoridade de Saúde.

Na situação de Caso confirmado: − A Universidade do Minho deve:

Providenciar a limpeza e desinfeção (descontaminação) da área de “isolamento”;

Reforçar a limpeza e desinfeção, principalmente nas superfícies frequentemente manuseadas e mais utilizadas pelo doente confirmado, com maior probabilidade de estarem contaminadas. Dar especial atenção à limpeza e desinfeção do posto de trabalho do doente confirmado (incluindo materiais e equipamentos utilizados por este);

Armazenar os resíduos do Caso Confirmado em saco de plástico (com espessura de 50 ou 70 mícron) que, após ser fechado (ex. com abraçadeira), deve ser segregado e enviado para operador licenciado para a gestão de resíduos hospitalares com risco biológico.

A Autoridade de Saúde Local, em estreita articulação com o médico do trabalho, comunica à DGS informações sobre as medidas implementadas na Universidade do Minho, e sobre o estado de saúde dos contactos próximos do doente.

10. Procedimento de vigilância de contactos próximos

Considera-se “contacto próximo” um trabalhador que não apresenta sintomas no momento, mas que teve ou pode ter tido contacto com um caso confirmado de COVID-19. O tipo de exposição do contacto próximo, determinará o tipo de vigilância (Anexo V). O contacto próximo com caso confirmado de COVID-19 pode ser de: − “Alto risco de exposição”, é definido como: Trabalhador do mesmo posto de trabalho (gabinete, sala, secção, zona até 2 metros) do Caso;

Trabalhador que esteve face-a-face com o Caso Confirmado ou que esteve com este em espaço fechado;

Trabalhador que partilhou com o Caso Confirmado loiça (pratos, copos, talheres), toalhas ou outros objetos ou equipamentos que possam estar contaminados com expetoração, sangue, gotículas respiratórias. − “Baixo risco de exposição” (casual), é definido como:

Trabalhador que teve contacto esporádico (momentâneo) com o Caso Confirmado (ex. em movimento/circulação durante o qual houve exposição a gotículas/secreções respiratórias através de conversa face-a-face superior a 15 minutos, tosse ou espirro).

Trabalhador(es) que prestou(aram) assistência ao Caso Confirmado, desde que tenha(m) seguido as medidas de prevenção (ex. utilização adequada da máscara e luvas; etiqueta respiratória; higiene das mãos). Perante um Caso Confirmado por COVID-19, além do referido anteriormente, deverão ser ativados os procedimentos de vigilância ativa dos contactos próximos, relativamente ao início de sintomatologia. Para efeitos de gestão dos contactos a Autoridade de Saúde Local, em estreita articulação com a Universidade do Minho, deve: − Identificar, listar e classificar os contactos próximos (incluindo os casuais); − Proceder ao necessário acompanhamento dos contactos (telefonar diariamente, informar, aconselhar e referenciar, se necessário). O período de incubação estimado da COVID-19 é de 2 a 12 dias. Como medida de precaução, a vigilância ativa dos contactos próximos decorre durante 14 dias desde a data da última exposição a caso confirmado. A vigilância de contactos próximos deve ser a apresentada na tabela 2.

Tabela 2. Vigilância de contactos próximos.

“Alto risco de exposição” “Baixo risco de exposição” − Monitorização ativa pela Autoridade de Saúde Local durante 14 dias desde a última exposição; − Auto monitorização diária dos − Auto monitorização diária dos sintomas da COVID-19, sintomas da COVID-19, incluindo incluindo febre, tosse ou dificuldade em respirar; febre, tosse ou dificuldade em − Restringir o contacto social ao indispensável; respirar; − Evitar viajar; − Acompanhamento da situação pelo médico do trabalho. − Estar contactável para monitorização ativa durante os 14 dias desde a data da última exposição.

De referir que: − A auto monitorização diária, feita pelo próprio trabalhador, visa a avaliação da febre (medir a temperatura corporal duas vezes por dia e registar o valor e a hora de medição) e a verificação de tosse ou dificuldade em respirar; − Se se verificarem sintomas da COVID-19 e o trabalhador estiver na Universidade do Minho, devem-se iniciar os “Procedimentos num Caso Suspeito”, estabelecidos no ponto 8; − Se nenhum sintoma surgir nos 14 dias decorrentes da última exposição, a situação fica encerrada para COVID-19.

11. Processo de alerta e comunicação interna

Quaisquer novas instruções aplicáveis à Administração Pública, em geral, ou às Instituições de Ensino Superior Publico e à Universidade do Minho, em particular, serão imediatamente comunicadas à comunidade académica, nomeadamente através da página https://www.uminho.pt/PT/viver/COVID-19/.

12. Processo de registo de contactos com o Caso Suspeito

O registo de contactos com o Caso Suspeito deverão ser efetuados no formulário que se apresenta no Anexo VI.

Anexo I

Chefias Diretas

Trabalhador Investigador Docente Estudante Reitoria Reitor Serviços Diretor Serviços de Ação Social Administrador Unidades de Interface Diretor Centros de Investigação Diretor Diretor Presidente do Unidades Orgânicas sem Presidente Presidente Presidente Conselho Departamentos Pedagógico Unidades Orgânicas com Diretor Diretor Diretor Diretor de Curso Departamentos

Anexo II

Trabalhadores que prestam apoio a Trabalhadores com Sintomas

Trabalhador Investigador Docente Estudante Reitoria Chefe de Gabinete Serviços Chefe de Divisão Diretor de Serviços de Ação Social Departamento Unidades de Interface Secretário Secretário de Secretário de Centros de Investigação Escola Escola Unidades Orgânicas sem Secretário de Secretário de Secretário de Secretário de Departamentos Escola Escola Escola Escola Unidades Orgânicas com Secretário do Secretário do Secretário do Secretário do Departamentos Departamento Departamento Departamento Departamento

Anexo III

Áreas de Isolamento

Campus de Azurém

Campus de Gualtar

Anexo IV

Fluxograma de situação de Trabalhador com sintomas de COVID-19

Trabalhador com sintomas

Trabalhador informa chefia direta

Chefia direta contacta o 253 601 601 e a Segurança do Campus

Segurança identifica a área de “isolamento” e circuito

Segurança acompanha o trabalhador com sintomas à área de “isolamento

Trabalhador contacta SNS 24 (808 24 24 24)

SNS 24 questiona o trabalhador

Caso não suspeito Caso suspeito

SNS 24 adota o SNS 24 contacta Linha procedimento de acordo Apoio ao Médico com a situação clínica

Caso Suspeito Não Caso Suspeito Validado Validado

Trabalhador informa a INEM transporta Chefia direta informa 253 601 chefia direta Trabalhador para Hospital 601 do caso validado de referência

Chefia direta informa o Colheita de amostras biológicas no Hospital de 253 601 601 referência

Universidade: Processo encerrado Caso Caso − Veda acesso à área de Infirmado Confirmado para COVID-19 isolamento − Colabora com Autoridade de Saúde Local de contactos SNS 24 define os Autoridade de próximos do procedimentos adequados Saúde Local Autoridade de trabalhador à situação clínica do informa a Saúde Local − Informa os Trabalhador Universidade informa a trabalhadores dos dos resultados Universidade laboratoriais dos resultados negativos laboratoriais positivos e procede à gestão de Processo contactos encerrado para COVID-19

Universidade providencia a limpeza e desinfeção da área de “isolamento”

Autoridade de Saúde Local levanta interdição após descontaminação

Autoridade de Saúde Local informa a DGS das medidas implementadas Anexo V

Fluxograma de monitorização dos contactos próximos (trabalhadores assintomáticos) de um

Caso confirmado de COVID-19 (trabalhador)

Trabalhador assintomático

Tipo de exposição

Baixo risco de Alto risco de exposição exposição

Auto − Monitorização ativa pela monitorização Autoridade de Saúde Local diária dos sintomas durante 14 dias desde última de COVID-19, exposição. incluindo febre, − Auto monitorização diária dos tosse ou sintomas da COVID-19, incluindo febre, tosse ou dificuldade em dificuldade em respirar. respirar. − Restringir o contacto social ao indispensável. − Evitar viajar. − Estar contactável para monitorização ativa durante 14 dias desde a exposição.

Tem sintomas de COVID-19?

Sim Não

Procedimentos de “Caso Situação encerrada Suspeito” para COVID-19

Anexo VI

Formulário de registo de contactos com o Caso Suspeito

REGISTO DOS TRABALHADORES EXPOSTOS COM EQUIPAMENTO DE PROTEÇÃO INDIVIDUAL ADEQUADO

Serviço / Unidade: ______Data: ___/_____/______

Nome N.º Mec. Procedimentos Realizados

IDENTIFICAÇÃO DOS TRABALHADORES EXPOSTOS SEM EQUIPAMENTO DE PROTEÇÃO INDIVIDUAL ADEQUADO

Serviço / Unidade: ______Data: ___/_____/______

Categoria Data do Hora do Nome N.º Mec. Morada Telefone Profissional Contacto Contacto

___/___/___

Anexo VII

Serviços Imprescindíveis na Escola de Medicina

Atendendo à especificidade de algumas das atividades desenvolvidas na Escola de Medicina e no seu laboratório de investigação (ICVS) definem-se as funções que têm que ser asseguradas em situação de elevação do nível do Plano de Contingência e encerramento total do edifício.

Assim, definem-se como serviços imprescindíveis:

Unidade Serviços Número de Trabalhadores (Horas/dia por trabalhador) Unidade de 1. Colocação de água e dieta 5 trabalhadores (5 horas) Biotério 2. Higiene de gaiolas e jaulas (incluindo esterilização) 3. Vigilância de bem-estar animal 4. Desmame de colónias de roedores 5. Reparação de equipamentos essenciais Unidade 1. Receção de encomendas 1 trabalhador (1 hora) Laboratorial imprescindíveis para a manutenção da cultura de células e amostras biológicas e outro material imprescindível ao funcionamento do Biotério 2. Monitorização da qualidade da coleção de amostras biológicas humanas não substituíveis 3. Reparação de equipamentos essenciais