Remote Laboratory Management: Respiratory Virus Diagnostics

1. Installation

NOTE: Only 2-4 people are needed to deploy the "Lego-like" laboratory unit. Optimally, 4 individuals would be used to deploy, but it is possible with only 2.

1. Utilize a fork lift truck (Figure 1A,B) or other suitable lifting device to handle the container. Use a fork lift truck with at least seven tons of lifting capacity to handle the two types of containers²⁴.
2. To set up a laboratory unit, select a patch of approximately 90 x 60 square feet (27.4 x 18.3 m²) on a flat landscape to ensure no obstructions hinder the proper layout. Ensure that the site has well-drained soil to assure site drainage as this may cause potential problems with water dissipation after rain fall. Use grounds that have been previously leveled and that are hard compacted soil of a minimal compressive strength of 10 kN/dm². The surrounding area should permit access to equipment needed for unloading the unit from its conveyance device and accommodate the support equipment necessary to accomplish the task.
3. Place the unit or units in its 'Transport Mode' in the center of selected site and adjust the level. Each unit is equipped with four leveling jacks to allow deployment on a site that has a maximum grade of 6.5% (~4 degrees). The minimum elevation for the containers is ~ 6 inches to ensure that the floor drains and discharge pipes work properly. Do not extend jacks more than 12 inches. Attach the support bracket to the arm of the jack. Ensure that the container is level by placing a bubble leveler at the center of each bottom rail. Do not expand the unit until it has been properly positioned!
4. Expand the unit by opening the panels for full functionality. First locate the two-piece support pole. Connect the support pole so that its height is almost as high as the container unit. The pole allows the user to open the panel and support the weight of the roof panel as the side doors are opened. There is a safety clip that acts as a pin to keep the panels locked. Remove the safety clip first, then lift and pull the cam lock pin from the hole. Place the pin behind the lever and out of the way of the cam lock lever located on the bottom (expandable) sides of the container.
5. Raise the roof Panel 1 considering that this panel has gas struts and once the panel doors are unlocked, the struts will release. This will allow for the user to raise the roof (Panel 1) using the two-piece support pole. Move the support pole's tip under the roof panel to temporarily support it (Figure 4A).
   1. While holding up the roof panel with the support poke, find the safety chain, located on top left side of the container. With the assistance of 2-3 individuals, carefully drag down Panel 2 until the safety chain is straight, holding the weight of the panel 2 and has engaged.
   2. Connect the winch strap belt to the lug mount by hand by wrapping it around the exterior of the gas strut. Note if there is no winch tool, the previous step can be manually completed with at least two people on each side of the panel manually holding and lowering it.
      CAUTION: The weight of the panel is 260 lb!
6. Ensure there are no people or items in the way of Panel 2 and using the winch and drill, proceed to lower the panel (Figure 4B). When Panel 2 is completely lowered, disconnect the winch strap and reel it back into the winch. Remove the winch and place it on the opposite side of the container in preparation for use. Both sides of the unit are identical and follow the same steps above for the other side of the unit.
7. To complete deployment of the first side, reach down to the Panel (which is currently the floor) and with at least two people on each side, manually lift Panel 3 upward into place as the door and front wall of the side (Figure 4C). The two people will remain to hold Panel 3, while the third person removes the support pole.
   CAUTION: No one should be inside the unit or under the roof until the Panel 3 wall is in place!
8. From the inside of Panel 3, locate the two latches and lock them into place using the safety strap. Make sure the black roof panel rubber gaskets are pulled out to face the inside of the units. This must be done to prevent rain and other water penetration into the unit.
   1. From the inside of the container, unlock Panel 4. Once unlocked, push out Panel 4 (Figure 4D) so that it swings open like a door. Lock the two safety latches on inside wall. Unlock Panel 5 and repeat the same steps for Panel 4. Secure this Panel with same internal safety latches. Once the entire unit interior has been locked, re-tighten the turnbuckle until the floor and end walls are sealed.
9. Once both sides of the container are safely expanded, check the jacks and make any necessary adjustments from shifting that may have occurred. Check the shelter to be level weekly. After extreme weather (rain or wind) inspect jacks on containers and adjust accordingly.
10. Expand the second container if the connectable laboratory modules are planned for use (Figure 5).
11. Connect the units to power source and water supply. A detailed instruction on installation of a diesel generator connected to the units through a breaker box can be found in the according manual²⁴.
    NOTE: The remote laboratory unit is now deployed. The inner non-collapsible volume of the unit allows to store minimum of the equipment and laboratory supply necessary for particular diagnostic tests. Installation of pressurization system for BSL-3 module is described in detail²⁴ and requires additional quality control.²⁴

2. Checklist for personal protection and basic laboratory workflow

NOTE: An error in any of the general safety and laboratory testing requirement phases may invalidate the results of the entire testing process.

1. Before preparing to enter the installed laboratory unit, ensure that all BSL-2 or BSL-3 safety requirements are accounted for: dressing with proper personnel protective equipment (PPE), washing hands, wearing gloves, and decontaminating any workspaces that are to be used.
2. Follow the checklist in Table 1 that contains safety requirements for personal protection during tests run in the lab BSL-2 and the BSL-3 module (the assembled glove box room – negative pressure and PCR room – positive pressure).
3. Decontaminate all work space and supplies in the laboratory. If planning to use sodium hypochlorite solution (0.5%), also known as liquid bleach, to decontaminate the workspace and supplies, also use 70% ethanol to clean all areas exposed to bleach, as bleach can mix with other chemicals in the workspace to create toxic fumes. Dispose all bleach products into their own designated waste bin.
4. Before beginning to work in the laboratory unit, become familiar with its arrangement and layout. Strict rules apply for processing samples in the glove box (GB) room. The GB room is negative pressure! To operate a glove box, check the manufacturer's instruction. Multiple sources provide detailed tutorials on glove box operation²⁵.
   NOTE: The purpose of this assay is to extract and purify ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), if present, from specimens. The extracted RNA/DNA will be tested by a real-time RT-PCR to detect the presence or absence of targeted viral pathogens – influenza (INF).

3. Rapid influenza virus diagnostics by RT-PCR in a BSL-2 laboratory facility

1. Receive and register
   1. Apply the checklist for PPE (see Section 2). INF is a Class 2 agent requiring BSL-2 practice. PPE appropriate for BSL-2 practice is required. Additionally, wear safety glasses and cuffed sleeve lab coats to minimize potential skin exposure.
   2. According to World Health Organization (WHO) recommendations²⁶, use sterile Dacron or rayon swabs with plastic shafts for sampling from the respiratory tract.
      NOTE: Cotton or calcium alginate swabs, or swabs with wooden sticks may contain compounds that inactivate some viruses and inhibit PCR testing^26,27.
   3. As sample swabs are taken from patients, transport them to the laboratory facility from the field or clinic. Transfer samples via the pass-through window; this window cannot be opened from both sides.
   4. In the pass-through window, spray tubes containing samples with bleach for 1 min followed by 70% ethanol and wipe dry in order to provide adequate decontamination before they enter the laboratory unit. Following the submersion, the lab technician inside the unit will open the pass-through window and collect the samples from the bleach container to be registered. Usually, the person passing the sample and the person extracting the sample are not the same.
   5. Open the pass-through window and collect the samples to be registered. Wipe down any samples that were disinfected in bleach and ethanol. Wipe down the interior of the pass-through window with bleach followed by 70% ethanol solution. Register a sample within interactive tablet-based system or a laptop. Identify a sample with the following information:
      – Collection date
      - Onset date
      - Patient age and sex
      - Specimen type (e.g., nasal swab)
      - Unique identifiers
      - Other pertinent information
   6. Use barcodes for labeling tubes. Add barcode to each sample tube and four empty tubes designated to aliquots. Move the samples to the vent hood. Scan a barcode on each tube and make sure that the proper sample identification information appears on the tablet-based system or laptop screen. If barcodes are not available, use an alcohol resistant marker. Always mark the vial itself, never the cap as this can get switched during handling! Complete the registration process.
2. Sample aliquot
   1. Once test tubes have been labelled, use a certified Class 2 biosafety cabinet to handle specimens and make aliquots of samples. One aliquot can be used for immediate testing and the others retained for reference purpose or retesting.
   2. As specimens arrive with nasal swab tip in the viral transport medium, stir up the swab tip in the medium for 30 s and squeeze it against the side of the vial. Then remove it from the medium and dispose of it with a biohazardous waste protocol (appropriately discard, autoclave or hang into 1:100 chlorine solution).
      NOTE: The smallest volume of medium that should be stored is 0.5 mL. Thus, a 3 mL sample can be divided into six aliquots (sub-samples). Use 1 mL cryo-storage tube for each sample of 0.5 mL to provide extra volume for frozen medium.
   3. When taking aliquots, use fresh sterile or disposable pipettes for each sample and discard them into biohazardous waste containers in order to avoid cross-contamination. Make sure every tube is tightly sealed and closed.
   4. Utilize one aliquot per specimen for immediate extraction and store any other aliquots in the freezer (at -80 °C as it is a respiratory specimen) for future use.
   5. Before moving to the work station, clean all workspace surfaces and equipment with bleach followed by 70% ethanol solution.
3. Extraction and purification
   1. To assure quality of testing, move the barcoded PCR sample aliquots from specimen handling area to the work station (BSL-2 safety cabinet) designated for extraction. This safety cabinet has a separate set of pipettes for handling of the sample.
   2. Use the viral RNA mini kit for extraction of RNA samples. Follow the manufacturer's instructions for purification of viral RNA by spin protocol. Prepare master mix according to the number of samples that need to be extracted. Keep the sample and lysis buffer master mix at room temperature.
   3. Prepare the number of samples that need to be extracted. Label the 1.5 mL micro-centrifuge tubes with the barcode numbers or unique identifier.
   4. Set the pipette to 560 µL. Apply a clean pipette tip. Add 560 µL of the lysis buffer to each labelled tube. Discard tip. Apply a clean pipette tip. Add 140 µL of the sample. Discard old and apply a clean pipette tip. Repeat with the other sample. Apply a clean pipette tip. Add 140 µL buffer to negative control tube. Close each tube securely.
   5. Pulse-vortex sample aliquot 1 with the lysis buffer for 15 s. Repeat with sample aliquot 2 and the control tube. Micro-centrifuge each sample for 5 s. Incubate the samples for 10 min at room temperature.
   6. After 10 min of incubation, re-centrifuge the tubes to remove any drops from inside of each tube lid. Add 560 µL of ethanol solution to sample. Change the pipette tip. Repeat with any remaining or additional samples. Close each sample tube securely and pulse-vortex each sample for 15 s. Micro-centrifuge the samples for 5 s.
   7. Apply the mixture to the spin column. Obtain clean 2 mL collection tubes. Add spin columns, and label them to match the samples. Transfer 630 µL of sample to matching column, accordingly.
   8. Secure caps and move to the centrifuge. Evenly distribute the samples in centrifuge. Centrifuge at 6,000 × g for 1 min to remove the lysis buffer. Return to the work station. Replace collection tubes. Add the remaining lysis buffer and repeat the centrifugation step. Discard the original aliquot sample tubes.
   9. Dispose the eluate and wash the spin column with two buffers. Apply 500 µL of Buffer AW1. Repeat with every sample. Secure caps of every sample and centrifuge at 6,000 × g for 1 minute. Repeat with the second Buffer AW2 and centrifuge at 20,000 × g for 3 min.
   10. Finally elute the RNA by adding elution buffer. Place the column into a clean 1.5 mL tube, open the column and add 60 µL of Buffer AVE. Incubate at room temperature for 1 min and centrifuge at 6,000 x g for 1 min. The samples are now ready for PCR analysis.
4. PCR amplification and detection
   1. Perform the PCR amplification in a separate area designated for PCR. Carry out the PCR amplification of the viral target using PCR protocol for one step procedure according to the manufacturer's instructions. Note, a master mix is made using viral specific primers, probes, 2x RT-PCR buffer and RT-PCR enzyme. Add the master mix to plates or tubes and then add and mix individual samples (see Section 4.4).
   2. Transfer the plate to the PCR machine and run according to the viral target amplification conditions. Once samples are loaded onto the PCR instrument it takes approximately 90 minutes to complete the run.
5. Maintenance after the equipment usage
   1. Perform regular and periodic maintenance after the equipment usage according to the Table 2.

4. Rapid influenza virus diagnostics by RT-PCR in a BSL-3 laboratory facility

NOTE: Under BSL-3 conditions, the experimental protocol will stay the same, but safety measures will take precedence above anything else. Before entering the BSL-3 laboratory, look through the transparent window to be sure that negative pressure has been established in the glove box unit. It will be evident that negative pressure has been established when a pink ball in a wall is visible.

1. Receive and register
   1. Once negative pressure has been established, open the door and enter the unit. Immediately wash hands and then proceed with the PPE. Apply the checklist for PPE and workflow BSL-3 (see Section 2).
   2. Proceed to putting on PPE in the following order: under gloves, gown, shoe covers, mask, face shield, second pair of gloves. Wearing a full gown that protects the whole body is recommended.
   3. Turn on the machine and allow pressure in the glove box to stabilize. Use a bleach spray solution to decontaminate all areas and supplies to be used inside and outside the glove box. Dispose of bleach waste products into a bleach only container. Use 70% ethanol solution to clean off any areas for bleach has been used.
   4. Transfer the sample via the pass-through window.
      NOTE: Prior to being dropped at pass through window, disinfect tubes containing samples by submerging in a hypochlorite bath for 1-2 min. Particularly, spray samples with a bleach solution and leave alone for at least 1 min and pass-through before receiving them inside the BSL-3 unit. The person passing the sample from outside and the person receiving, extracting the sample inside BSL-3 laboratory should not be the same.
   5. Receive the samples inside the BSL-3 unit and clean them before proceeding to the registering and labeling steps.
   6. Register a sample with the interactive tablet-based system or a laptop. Identify a sample with the following information (see Section 3.1.5).
   7. Use barcodes for labeling tubes. If barcodes are not available, use an alcohol resistant marker.
      NOTE: Always mark the vial itself, never the cap as this can get switched during handling!
2. Sample aliquot
   1. Once samples are registered and tubes has been labeled, place the samples into the certified glove box via the air locked tray to retrieve the samples from inside the glove box. Close the door. Then open the other door through the glove box to retrieve the samples. Do not open both doors at once. Open and close each door in two different steps for safety precautions. Turn on UV-C lighting.
   2. Once samples have been safely moved to the glove box interior, follow the steps previously described for creating sample aliquots in a glove box. One aliquot of sample can be used for immediate testing and the others retained for reference purpose or retesting. Specimens arrive with nasal swab tip in the viral transport medium. Take specimens and put one into each vial. Stir the swab tip in the medium for 30 s and squeeze it against the side of the vial before removing it from the medium and disposing it utilizing a biohazardous waste protocol (appropriately discard, autoclave or hang into 1:100 chlorine solution).
      NOTE: The smallest volume of medium that should be stored is 0.5 mL, use 1 mL vials for this purpose. Thus, a 3 mL sample can be divided into six aliquots (sub-samples).
   3. Use fresh sterile or disposable pipettes for each sample and discard them as biohazardous waste. Once samples are aliquoted, move the samples in the airtight container. Close vials with protection and remove from the glove box.
   4. Within the glove box, close the biohazard waste bag and prepare a new waste bag to avoid cross contamination. Decontaminate the glove box workspace applying bleach for 5 min and 70% ethanol solution afterwards.
   5. Utilize one aliquot per specimen for immediate analysis and store others in freezer at -80 °C as it is a respiratory specimen. After decontamination retain one aliquot for testing, move all the samples out of glove box for storage in -80 °C freezer.
3. Extraction and purification
   1. After decontamination, move the barcoded sample aliquots needed for PCR analysis from the specimen handling area back into the glove box.
   2. In the glove box, perform all the lysis steps for the extraction procedure. Use the viral RNA mini kit for extraction of RNA samples according to the manufacturer's instructions. Follow the step-by-step instructions for purification of viral RNA by spin protocol in the manufacturer's instructions.
   3. Prepare the number of samples that need to be extracted. Label the 1.5 mL micro centrifuge tubes with the barcode numbers or unique identifier.
   4. Add 560 µL of the lysis buffer to 140 µL of the sample and pulse vortex. Incubate for 10 minutes at room temperature.
      NOTE: The inactivation procedure can vary depending on the type of BSL-3 pathogen that is being extracted and in some cases further inactivation may be necessary.
   5. After the lysis step, tightly seal the caps on each sample and place into the pressurized air locked passage (if the centrifuge is placed outside the glove box). Transfer the lysed samples into biosafety cabinet for the remainder of the procedure.
      NOTE: The procedure for extraction and purification of RNA can be completed in the glove box or in a glove box followed by bio-safety cabinet depending on the BSL-3 pathogen and protocols required.
   6. Make sure to decontaminate the workspace and supplies in the glove box again using bleach, and 70% ethanol solution to clean all areas exposed previously to bleach.
   7. Follow steps in Section 3.3.6 – 3.3.10 for washing and purification of the RNA.
   8. After extraction, transfer the samples to the pass-through window for PCR analysis.
   9. After samples has been removed and transferred, decontaminate the laboratory workspace outside the glove box, according to Section 2.
   10. Before removing PPE wait until air circulation in the unit has safely reached the proper number of filtering cycles before beginning to remove the PPE. Immediately after removing and disposing of all PPE into the PPE waste container, proceed to washing hands in the laboratory with soap and water before exiting the unit, according to Section 2.
4. PCR amplification and detection
   NOTE: The PCR amplification is performed in a separate area designated for PCR and connected with the glove box area via pass-through window. The workspace and supplies have to be decontaminated prior to a test.
   1. Remove the extracted RNA samples from the pass-through window.
   2. Perform the PCR amplification of the viral target using one-step PCR protocol. Prepare a master mix using viral specific primers, probes, 2x RT-PCR buffer and RT-PCR enzyme. Use a 1.5 mL tube with the following component for each targeted assay: water, primers and probes, 2x buffer and RT-PCR enzyme. Vortex and spin the master mix.
   3. Aliquot the master mix into each of the strip tubes. Return the PCR kit to storage at the recommended temperature once the master mix has been prepared.
   4. Add the individual samples to each of strip tubes using a separate tip between each strip tube. Spin the sample plate or tubes at 1,500 rpm for 1 min. The samples are ready to be loaded onto the real-time PCR unit.
   5. Transfer the plate with samples to the PCR instrument and run the machine according to the viral target amplification conditions. It takes approximately 90 min to complete one run.
   6. Before collecting the results and leaving the laboratory, remove PPE and adequately decontaminate each workstation and preparation for the next diagnostic test, according to Section 2.
5. Maintenance after the equipment usage
   1. Perform regular and periodic maintenance after the equipment usage according to the Table 2.
      NOTE: The overall diagnostic turnaround time is approximately 4 hours. Extraction time and PCR set up time can vary depending on the number of samples, and the diagnostic test can take 4-5 hours or more, correspondingly.