RESPIRATORY SPECIMEN COLLECTION Royal Center for Disease Control 1

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

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

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

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

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

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

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

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

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

Rapid Diagnostic Test 11

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

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

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

Interpretation of Results 15

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

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

STORAGE/PACKING/TRANSPORTATION 18

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

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

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

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

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”

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

Specimens Transportation Study sites in Bhutan Store specimens at 2-8 °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 -70°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

BIOSAFETY 26

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

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

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

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

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

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

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

THANK YOU 34