SARS (Severe Acute Respiratory Syndrome)

SARS lab protocol

8 May 2003 - Updated October 2007

There are currently no SARS affected areas. The last known cse of SARS was diagnosed in China in May 2004.
This information is retained on this website to advise clinicians who may have concerns about a particular patient who presents with a history of travel and a clinical presentation which is suggestive of SARS.

Handling of samples from suspected or probable SARS patients within the laboratory

Note: a critical first step is for the clinical assessment of probability of genuine SARS. This protocol refers only to suspected or probable cases. Specimens should not be processed without discussion first with the on-call microbiologist/ID physician.

The range of tests normally requested will be those for any case of pneumonia or travel-related illness.

Summary

1. Samples will be delivered by hand to the laboratory, with co-ordination / supervision by a clinical microbiologist.

2. All manipulations that have the potential to produce aerosols, such as opening tubes after centrifugation, will occur in a safety cabinet.

3. Staff involved in this phase will wear gloves, face masks, eye protection and gowns.

4. Pneumatic tubes must not be used to transport specimens within the hospital: they must be delivered by hand.

5. Normal laboratory safety standards will be sufficient when the samples are going through the biochemical, serological and haematological analysers. However, staff may feel more comfortable if they wear masks in addition to normal laboratory protective clothing (laboratory coat and gloves). As always, good work practice is crucial to prevent contamination of the outside of specimen containers and the general laboratory environment.

Introduction

There are still no definitive tests for SARS at present, and it is essential for patient care that normal laboratory testing is carried out expeditiously: routine biochemistry and haematology as well as general microbiology and serology.
There is a small but finite chance that the laboratory may have to deal with samples from patients whose probable diagnosis is SARS. This protocol must be followed in the event of a probable SARS patient requiring laboratory testing. Respiratory samples are the most infectious. The risk from blood has not been determined, but diagnostic laboratory workers have not been affected, at least to our current knowledge.

NORMAL, SAFE LABORATORY PROCEDURES ARE THE BEST PROTECTION, AND THE RECOMMENDATIONS THAT FOLLOW REFLECT A PRECAUTIONARY APPROACH.

Particular attention should be given to the following:

• All manipulations should be carried out in a certified biological safety cabinet

• Laboratory workers should wear protective equipment including:

• disposable gloves,
• solid front gowns with cuffed sleeves,
• eye protection
• respiratory protection

• Centrifugation should be carried out using sealed centrifuge cups or rotors that are loaded and unloaded in a biological safety cabinet.

Arrival of samples in the laboratory

1. The laboratory will be notified before samples arrive for testing.

2. The samples will be delivered by hand to the laboratory by the clinician caring for the patient. (In most cases this will be a member of the infectious diseases team or the on-call microbiologist.)

3. A pneumatic tube system must not be used to transport specimens.

4. Reception staff will assist with assigning laboratory numbers to OUTSIDE of biohazard bags and process request forms for data-entry as usual.

DO NOT OPEN ANY BIOHAZARD BAGS IN RECEPTION

NOTE: Tube opening and separating should occur in a biosafety cabinet by designated personnel before being transferred to other areas for routine test analyses. A sealed rotor centrifuge should be used whenever possible.

Arrival of samples without warning

The microbiologist on call should be contacted immediately if a sample labelled "SARS" arrives without prior warning or discussion. No further handling of the sample should occur except under advice of the Clinical Microbiologist.

When is it hazardous?

While there is little hard data available yet some initial conclusions have been drawn.

Respiratory samples (sputum, nasopharyngeal and throat swabs in VTM)

These should be treated as highly infectious. Respiratory samples must be processed in a biosafety cabinet with the worker wearing safety equipment as listed above. The sample is considered infectious until:

• nucleic acid extraction has been completed

• the specimen has been fixed for 5 min in alcohol or acetone.

Blood: pre-analytical centrifugation and separation

For blood samples the initial opening of the primary tubes after centrifugation is thought to be the most dangerous step. This is due to the possibility of aerosols being created. For this reason this step should always be performed in a safety cabinet, usually the one in respiratory laboratory.

Separation of blood

Centrifugation must be performed in a sealed bucket rotor centrifuge. Opening of tubes after centrifugation and separation of cells, plasma and serum must be performed in a biosafety cabinet. The registration of the sample, label printing and labelling of tubes (except for the primary one) can occur in the normal manner.

Sample manipulation

1. Staff performing the centrifugation steps will wear protective clothing as described below.
   
   
2. The bag containing patient samples will be opened only in the safety cabinet.
   
   
3. Samples requiring centrifugation will be placed in a sealed centrifuge container.
   
   
4. This sealed container can now be taken out of the safety cabinet and placed in the centrifuge in the centrifuge cabinet.
   
   
5. The sample will be balanced and then spun for 10 minutes at 3500 rpm.
   
   
6. At the end of the centrifugation period the sealed centrifuge container will be removed from the centrifuge and returned to the safety cabinet.
   
   
7. Any separation of primary tubes will then occur in the safety cabinet.
   
   
8. The samples should be transferred to a screw-top tube before being taken to the core laboratory.

Analysis in a core laboratory

1. Evidence to date suggests that the infectivity of serum or blood samples from SARS patients is similar to those samples containing hepatitis and HIV viruses. This may change as further information is gathered.

2. Standard laboratory safety precautions should be taken ie, a laboratory coat worn, gloves worn if considered appropriate, and regular handwashing.

3. Given the uncertain state of knowledge concerning the spread of the virus, it is recommended that staff wear masks while SARS samples are being processed.

Processing of samples for bacteriology

• All samples should be processed in the respiratory laboratory using the biosafety cabinet and personal protective equipment.

• Smears should be air dried in the cabinet and fixed in alcohol for 5 minutes before processing in the normal way for a Gram stain.
• Culture plates should be inoculated in the cabinet, and placed in a bag before incubation.

• Fluids for cell counts should be placed into the counting chamber in the biosafety cabinet and microscopy performed while wearing a mask.

Processing for nucleic acid testing

• Nucleic acid extraction should be conducted in the biosafety cabinet.

• Once extracted and placed in a new tube the specimen is not regarded as infectious. It can then be processed in accordance with IANZ approved methods.

• ESR, Capital & Coast Health, Canterbury Health and Auckland A+ laboratories may have available PCR assay for the putative coronavirus.

Non-automated serological testing

Any step that may produce aerosols, especially pipetting, should be performed in a biosafety cabinet.

As noted above, once spun, separated and placed into a new tube automated serology may be performed in the normal way.

Direct immunofluorescence and direct antigen testing of respiratory samples

• Sample preparation should be performed in a class II biosafety cabinet with the worker wearing protective equipent.

• Once alcohol fixation has been completed, the sample in no longer infectious.

• All steps of antigen testing should be performed in a class II biosafety cabinet with the worker wearing protective equipent.

Storing and packaging of samples to be sent away for other tests

It is important that plasma, serum and respiratory samples be stored for such a time that better diagnostic tests are available. They become of critical importance for contact tracing and epidemiological investigation.

• Respiratory samples should be stored in a screw-cap Nunc tube or similar container in a clean biohazard bag in a box marked '?SARS' in the refrigerator until shipping or long term storage at -70^oC.

• Packaging for shipping should be as an Infectious Substance (Model Regulations and Packing Instruction 602 of the IATA Dangerous Goods Regulations). See Appendix 1.

• The specimen should not be dispatched without the approval of a microbiologist who will discuss the optimal shipping time and method with the receiving laboratory.

Cell culture

It is recommended that standard diagnostic virology laboratories should not attempt cell culture for any virus from specimens of probable SARS cases. This is because of the risk culture-amplifying the pathogen.

• It is recommended that direct antigen detection/immunofluorescence and PCR methods should be used to screen for respiratory viral pathogens.

• Shipping specimens to other laboratories such as Auckland A+ or Canterbury Health Laboratories may be appropriate for nucleic acid testing that is not available locally.

As already noted, testing strategies for suspected/probable SARS cases should be discussed with the relevant microbiologist.

If viral culture from respiratory samples is to be attempted, it must be in the PC3 containment laboratory at Wallaceville (see appendix for shipping details for virology at ESR/Wallaceville).

Disposal of samples

1. After the analysis of samples has been completed they should be placed in sealed plastic bags and held for the appropriate time in a segregated area in a fridge or freezer (as appropriate for sample type).

2. Once the samples have been held for the required duration they should be disposed of after auto-claving.

Protective clothing

EVERY laboratory sample received for ANY test must be opened and prepared in a biological safety cabinet ie, blood, sputum, urine swabs etc.

The donning of PPE (Personal Protective Equipment) is required.

NOTE: Designated staff only and always inside the respiratory laboratory.

Always tie back loose hair and avoid touching any part of face when working in PPE gear.

1. Put on mask as below: NOTE: use mask with eye shield if available.


2. Put on protective eyewear. NOTE: the user must clean reusable safety goggles. Please see instructions below.

3. Put on solid front isolation gown and do it up. NOTE: You must tie both ties firmly.

4. Put on correctly-sized gloves and ensure wrists of gloves are pulled up over wrists of gown sleeves.

5. Sample manipulation may now occur using the Safety Biohazard Cabinets.

Removal and disposal of PPE in a safe manne

It is crucial that PPE gear is removed without accidental contamination.

1. Remain in the Respiratory Laboratory.

2. Untie both ties on gown without touching any unprotected areas.

3. NOTE: It may be necessary to seek the assistance of a colleague who is wearing clean gloves. Alternatively, some recommend the use of double gloves: the first pair are taken off first before removing masks, gowns etc.

4. Pull gown off you so that it turns inside out and immediately place into a foot-operated biosafety bin.

5. Remove gloves by usual technique so that the 'dirty' outside of glove only ever touches the dirty surface of the other dirty glove and 'clean' skin inside of glove only ever touches 'clean' skin of the other hand. Immediately place inside-out gloves into foot-operated biosafety bin.

6. WASH HANDS using the 15 Second ' Standard' Handwash.

7. Take off goggles (if worn separately), handling on the arm and dump immediately into container for washing.

8. NOTE: Goggle washing procedure follows.

9. Take off mask, handling ONLY by its strings and taking great care NOT to touch the body of the mask (the part that goes over the nose and mouth). Immediately dump into biosafety bin.

10. Immediately WASH HANDS thoroughly again using the full 15 Second 'Standard' Handwash.

Reusable eye protection/goggles

• The used goggles should be put immediately into a container (eg, an ice-cream container or equivalent).

• They should be washed in detergent and warm water, preferably in a sink in the Respiratory Laboratory by the following method:

• The staff member must wear gloves and needs to take care not to splash.

• Immerse the goggles (fill the sink first) and clean them under the surface of the water.

• Ensure that all surfaces are wiped with a disposable cleaning cloth.

• When washing is complete, discard the cloth into the yellow waste bag.

• Drain out the washing water.

• Rinse in clean warm water and drain.

• Then remove gloves and wash hands.

• Then dry the (now clean) goggles with a disposable paper towel.

The goggles are then ready for re-use.

Appendix

Information Sheet – Transport of SARS Samples to BSL 3+ facility in NCBID, Wallaceville, Upper Hutt

This section describes a free Courier Service to send SARS samples to the BSL 3+ facility in National Centre for Biosecutiry and InfectiousDisease, Wallaceville, Upper Hutt. (previously known as National Centre for Disease Investigation, NCDI)

Specimens shipped to other laboratories should be packaged and dispatched by someone familiar with the IATA shipping requirements for infectious substances. If unsure, the larger laboratories will be able to help.

When shipping diagnostic specimens and infectious substances to us via this service:

• All the documentation required to comply with land and air transport regulations is provided.

• All the packaging required to comply with land and air transport regulations is provided.

• A courier collection service is set up for you.

• Your samples will arrive at NCBID without delay.

• Your shipper will be promptly returned for re-use.

The shipping materials we have provided for your use consist of:
i. An approved infectious substances shipping container, comprising a plastic screw-cap shipping container (biobottle) with a bubble bag for protecting the specimens inside a cardboard outer box. The box has interchangeable address cards on it in separate pockets, a clear plastic adhesive backed envelope for the "Dangerous Goods" forms, and a reversible "Infectious Substances" label.

ii. "Shipper's Declaration for Dangerous Goods" forms.

iii. A sample completed "Shipper's Declaration for Dangerous Goods" form.

iv. A pre-printed NZ Couriers Charge Label (ESR will be charged for delivery).

v. Human or Non-Human Laboratory Services Request forms can be supplied on request.


How to send your samples:

i. Put adhesive tape round the caps of any containers of liquid samples and wrap each sample individually. Place your samples in the bubblewrap bag and chill pad inside the plastic inner container. The two sachets of liquid-absorbing material are there to soak up any spill.

ii. Place any paperwork you are sending with the samples inside the box but outside the biobottle.

iii. Swap the address cards around on the outer box so that NCBID is in the "To" pocket and your address card is in the "From" pocket. The NCBID address is: National Centre for Biosecurity and Infectious Disease , Wallaceville Research Centre, Ward Street, Upper Hutt.

iv. Complete in duplicate the "Shipper's Declaration for Dangerous Goods" forms (see sample completed form) by:
a. filling in your name and address

b. writing in the weight or volume of the infectious substances

c. signing the declaration.

Fold the two forms and place them in the clear plastic envelope on the box.

i. Fill in the "Sender" box of the NZ Couriers Charge Label and attach in the marked space.

ii. Phone NZ Couriers contact number 0800-800-841 to arrange pickup of your shipment.

iii. Phone or email ESR virology lab (lisa.vanduin@esr.cri.nz or Sue.Huang@esr.cri.nz) to inform the shipment of SARS samples to NCBID. So ESR virology laboratory can prioritise the workload and send the staff to NCBID to do viral isolation and identification for SARS.

Further supplies and inquiries:

If your require more shipping containers or materials, contact our Specimen Reception department. If you have any queries or suggestions please contact Pam Raynel or Sue Huang (e-mail: pamela.raynel@esr.cri.nz or sue.huang@esr.cri.nz)

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Related information:

SARS homepage