1. RESPIRATORY SPECIMEN COLLECTION Royal Center for Disease Control1

2. for Specimen Collection and Transport
Supplies Needed
for Specimen Collection and Transport
Viral Transfer Medium (VTM) with sterile swabs in zip-locked bag
Dry ice or Ice or Ice packs (temporary storage and specimen transfer)
Cold box / Styrofoam box
Pre-printed barcode labels
Glove, Mask and Lab gowns
Tongue depressor (optional)
Rapid Diagnostic Test Kit 2

3. Specimen Collection Polyester Fiber-Tipped Swab
Should be synthetic material (e.g. drayon, rayon, or polyester-fiber swabs)
Do not use calcium alginated or cotton swabs nor ones with wooden sticks; they inhibit certain tests (e.g. PCR) 3

4. Specimen Collection Viral Transport Medium (VTM)
Used in the collection of samples for viral isolation and PCR testing
Prevents specimen from drying out
Prevents bacteria and fungi growth
Can be home-made or commercial
Do not use if cloudy in appearance or change in color 4

5. Respiratory Specimen Collection
You will most commonly collect:
Nasal swab
Throat (Oropharyngeal) swab 5

6. 1 nasal swab for Rapid test
Specimen for Laboratory Tests
Throat swab or Nasal swab are technically simple to perform under field conditions.
Up to 3 swabs will be collected from one patient:
Two swabs to be put in a single Viral Transport-Medium (VTM) tube for shipment to AFRIMS laboratory
AND
One nasal swab for on-site Rapid Test (Use the swab that comes with the Rapid Test Kit)
2 throat swabs
1 nasal swab for Rapid test

7. Throat Swab Procedure
Vigorously rub the tonsils, then the posterior pharynx while rotating the swab to collect the specimen on all sides of the swab
Two throat swabs can be performed simultaneously 7

8. Labeling the VTM Tube
Place the swabs into the VTM tube, and break off the tip of the swabs (do not break it too short) so that the top can be closed securely leaving the swabs in place
AFRIMS will send you pre-printed labels with specimen numbers to affix to the VTM tubes. Please do not write patient names on the label 8

9. Pre-printed Labels FLU-Bhutan (WRAIR#1599) Site Code Specimen No.
Study site A
FLU-BTA-00000
Eastern Regional Referral Hospital, Mongar B
FLU-BTB-00000
Jigme Dorji Wanghck National Referral Hospital, Thimphu C
FLU-BTC-00000
Paro Hospital D
FLU-BTD-00000
Punakha Hospital E
FLU-BTE-00000
Trongsa Hospital F
FLU-BTF-00000
Phuentsholing Hospital G
FLU-BTG-00000
Central Regional Referral Hospital, Gelephu H
FLU-BTH-00000
Trashigang Hospital I
FLU-BTI-00000
Damphu Hospital, Tsirang 9

10. Nasal Swab Procedure
Gently insert the swab into one nostril and rub while rotating the swab from a depth of 2-3 centimeters by using the swab provided with the rapid diagnostic test kit 10

11. Rapid Diagnostic Test 11

12. Rapid Test - Basic Facts
The test contains four different antibodies: two for detection of influenza A and two for detection of influenza B
It can give a positive result in 2 to 10 minutes, depending upon the level of virus present in the specimen
The kit is stable for 24 months at room temperature (15º to 30ºC) 12

13. Rapid Diagnostic Test
QuickVue Influenza A+B test (Quidel)Immunochromatography
Sterile nasal swabs in individual pouches
Package insert
and Procedure card
Positive influenza type A, B and Negative control swab
Extraction tubes
Individual packaged strips
Extraction reagent solution 13

14. Rapid Test Procedure
Consult kit package insert for full instructions for use. 14

15. Interpretation of Results15

16. Rapid Test Fast Relatively simple Can be done on-site
Advantages
Fast
Relatively simple
Can be done on-site
May be useful to guide clinical care
Disadvantages
Cannot subtype beyond flu A and B (i.e. cannot distinguish pandemic subtypes from seasonal flu)
Less sensitive than laboratory methods
Low sensitivity/specificity for H5N1
Unknown sensitivity/specificity for pandemic H1N1 (probably somewhat less sensitive than seasonal flu) 16

17. Rapid Test
Reported sensitivity of about 70-75% and specificity of 90-99% for seasonal influenza when compared to virus culture
BUT wide range of sensitivities reported in medical literature from low to moderate
BOTTOM LINE: Negative rapid test does NOT rule out influenza and clinical judgment should guide medical care 17

18. STORAGE/PACKING/TRANSPORTATION18

19. Specimen Storage For specimens in VTM:
Ship specimens to laboratory as soon as possible
Store specimens at 2-8 °C before and during shipment (within 72 hours)
Store specimens at -70 °C beyond 72 hours
Do not store in standard freezer, especially “frost free” freezer – keep on ice or in refrigerator instead 19

20. Specimen Packing and Transportation
2 main goals
Protect environment and carrier
Protect the sample
International Air Transport Association (IATA) –
Provides guidelines for packaging and shipping by air
Categorization of Infectious Substances as “Dangerous Goods” 20

21. Packing Respiratory Samples
for Transportation
Use 3 packaging layers
Primary receptacle holds biological material
i.e. VTM tube wrapped with parafilm
Secondary container
Durable, watertight, leak-proof
Several primary receptacles can go into secondary container
Place absorbent, cushioned material between primary and secondary containers
Outer container
Rigid, durable, insulated
Place itemized list of contents between secondary and outer containers
Place ice/cold packs between secondary and outer containers
Outer container should be properly marked and labeled 21

22. Packing Respiratory Samples
for Transportation (1)
For transferring sample from “Study Site to the Lab”
Seal VTM tube with parafilm
Place VTM within its own watertight ziplock bag made for biological specimens
“this is primary receptacle” 22

23. Packing Respiratory Samples
for Transportation (2)
Place up to 10 single VTM- containing bags within another watertight container (e.g. biohazard bag or sturdy plastic container with lid) along with absorbent material “this is secondary container”

24. Packing Respiratory Samples
for Transportation (3)
Put secondary container into a rigid insulated container (e.g. styrofoam box) with ice/cold packs – this is outer container 24

25. Specimens Transportation
Study sites in Bhutan
Store specimens at °C and send to Laboratory center (PHL) within 72 hours for storing specimens at -70 °C
Specimen can be shipped by ice or icepack if dry-ice is not available.
Laboratory Center
in Bhutan
(PHL)
Store specimens at °C
Badge all specimens from all study sites and ship specimens to AFRIMS Virology on dry ice.
AFRIMS, Bangkok
Receive specimens and perform the laboratory testing (PCR testing)
Specimens
Cold-chain maintenance table form
Specimens
Demographic/Clinical Form
Cold-chain form
Consent form
Enrollment log 25

26. BIOSAFETY 26

27. STANDARD PRECAUTIONS
Wash hands with soap as a cost effective control transmission of infectious by hand
Wash hands immediately with
running water for at least 20 seconds
after gloves are removed
Wash hands immediately with running water
for at least 15 minutes in case of hand
exposure to infectious materials
These are respiratory-based precautions as for other infectious respiratory pathogens.
Hand washing is the single most and effective component for preventing the transmission of infection
Running water and soap with friction should be ideally used for 15 to 20 seconds 27

28. STANDARD PRECAUTIONS Wash Hands :
before and after subject/patient contact
after taking samples
after removing gloves
before eating
before leaving the laboratory working areas

29. Personal Protective Equipment (PPE)
Ensure appropriate personal protective equipment (PPE) is available
Wear PPE at all times (Mask, Gloves, Lab coat, Goggle or face shield)
Properly use of PPE

30. BIOSAFETY FOR LABORATORY
Treat every specimen as if it were an infectious material
Do not eat, drink, smoke, handle contact lenses, apply cosmetics and apply medicine in the area which respiratory specimens are handled
Do not store food, drink, cigarettes, contact lenses, cosmetics and medicine for human consumption in the area which respiratory specimens are handled

31. Materials for a biological spill kit:
Disposable Gloves
Rubber Gloves (utility)
Paper towels
Forceps or big tweezers
Bleach
Mixing / Spray bottle
Biohazard bags
Disposable mask, Goggles or face shield

32. Instruction for Biohazard Spill
Wear Gloves
Place an absorbent towel or gauze on the spill Liberally add a 10% bleach solution to the towel
Leave the area for at least a half an hour
Remove the towel and discard in a biohazard bag
Apply 70% ethanol to the area 2-3 times, wiping the area thoroughly

33. WASTES
Waste Disposal
- Biohazard containers for disposal of contaminated materials
- Double bag system for transport
Adequate and conveniently located biohazard containers should be available for disposal of contaminated materials.
Double bag system for transport should be used. 33

34. THANK YOU 34