1 Specimen Collection Field Diagnostics and
Learning Objectives • Choose appropriate diagnostic tests for various respiratory outbreak settings • Select appropriate specimens for diagnostic tests • Demonstrate specimen collection • Demonstrate use of rapid influenza tests 2
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI outbreak Mild No No in community ILI outbreak Mild with at No No in least 1 community severe ILI outbreak Generally No No in moderate to community severe 3
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI outbreak Mild No No Rapid test in Viral culture community ILI outbreak Mild with at No No Rapid test in least 1 PCR? community severe Viral culture ILI outbreak Generally No No Rapid Test in moderate to PCR community severe Viral culture 4
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI outbreak Mild Yes No in community ILI outbreak Mild No Yes in community ILI in Severe No No hospitalized patient 5
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI outbreak Mild Yes No Rapid test in PCR community Viral culture ILI outbreak Mild No Yes Rapid test in PCR community Viral culture ILI in Severe No No Rapid test? hospitalized PCR patient Viral culture 6
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI in Severe Yes No hospitalized patient ILI in Severe No Yes hospitalized patient ILI in Severe Yes Yes hospitalized patient 7
Selecting Testing Methods Setting Severity of Concurrent Epi Links to Appropriate Disease Poultry Human Tests Epidemic Cases ILI in Severe Yes No PCR hospitalized Viral culture patient ILI in Severe No Yes PCR hospitalized Viral culture patient ILI in Severe Yes Yes PCR hospitalized Viral culture patient 8
Right specimen for right test • For rapid influenza tests, check manufacturer’s requirements 9
Right specimen for right test • For culture and PCR, include nasopharyngeal and posterior pharyngeal swabs (higher sensitivity for H5) in same viral transport medium • Lower respiratory tract specimens may be very useful, if available 10
Materials for Viral Culture • Viral transport medium (VTM) • Cold chain • Appropriate swabs 11
How to choose VTM • Different types of VTM: – For collection of animal specimens – For viral isolation – For molecular testing (Do not use phosphate-based media) • If VTM is not available, 100% ethanol can be used for molecular testing 12
Cold Chain • Vials can be stored in a freezer at -20 ºC until use • Vials can be stored for short periods of time at 4 - 6 ºC • False negatives will occur if the cold chain is violated 13
Specimen Collection Kit • Collection vials with • Secondary container VTM • Ice packs • Polyester fiber-tipped • Items for blood applicators collection • Sterile saline • Personal protective (0.85% NaCl) equipment • Tongue depressors • Field collection forms • Specimen collection • A pen or marker for cups or Petri dishes labeling samples • Disinfectant 14
Polyester Fiber-Tipped Applicator • Should be dacron, rayon, or polyester- fiber swabs • Do not use calcium alginated or cotton swabs nor ones with wooden sticks; they inhibit PCR 15
Personal Protective Equipment • Gloves • Mask • Gown • Eye protection 16
How to Manage Kits • Store specimen collection kits in a dry, cool place • Store specimen collection kit where it will be accessible after hours and on weekends 17
What to Collect Respiratory specimens • Posterior pharyngeal swabs* (Culture, PCR, some rapid tests) • Nasal swabs (some rapid tests) • Nasopharyngeal swabs (Culture, PCR) • Nasopharyngeal aspirate (not practical for field) • Nasal washing (not practical for field) • Lower respiratory tract specimens 18
When to Collect Respiratory Specimens: Idealized Guidelines • Within 7 days of onset • Before antiviral medications are administered • If no improvement in symptoms, late specimens viable 19
What to Collect Serologic samples • Acute serum (within 2-7 days of symptom onset) • Convalescent serum (2-4 weeks after acute serum) 20
21 Rapid Influenza Tests
I nfluenza Types Rapid Test Acceptable Specim ens Detected Directigen Flu A7 (Becton- NP swab,2 throat swab, nasal A Dickinson) wash, nasal aspirate Directigen Flu A+ B7, 9 NP swab,2 throat swab, nasal A and B (Becton-Dickinson) wash, nasal aspirate NP swab,2 throat swab, nasal FLU OIA7 (Thermo Electron) A and B4 aspirate, sputum FLU OIA A/ B7, 9 (Thermo NP swab,2 throat swab, nasal A and B Electron) aspirate, sputum Nasal wash, NP swab,2 XPECT Flu A/ B7, 9 (Remel) A and B throat swab NOW Flu A Test7, 9 NOW Flu Nasal wash, NP swab2 Nasal A B B Test7, 9 (Binax) wash, NP swab2 QuickVue Influenza Test8 NP swab,2 nasal wash, nasal A and B4 (Quidel) aspirate QuickVue Influenza A+ B NP swab,2 nasal wash, nasal A and B9 Test8 (Quidel) aspirate AS Influenza A7, 9 SAS NP wash,2 NP aspirate2 NP A B Influenza B7, 9 wash,2 NP aspirate2 ZstatFlu8 (ZymeTx) A and B4 throat swab 22
Rapid Influenza Tests: Components • Expiration Date • Package Insert • Test tubes with lyophilized buffer 23
Rapid Influenza Tests: Components • Sterile foam swabs • Reagent • Box used as test tube holder 24
Rapid Influenza Tests: Components • Test strips • Positive and or negative control 25
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27 Check expiration date
Steps of Rapid Test 1. Understand test 2. Add reagent to test tube 3. Place swab in tube and roll/push material into tube 4. Add test strip 5. Time 6. Document results 28
Label Samples Use pre-printed barcode labels: – On the specimen container – On the field data collection form – On the log book Label each specimen with: – Subject’s name – Subject’s unique identification number 29
Serological Samples Paired serum samples are most useful Acute sample Within 7 days after symptom onset Convalescent sample More than 12 days after symptom onset 30
31 How to Collect Specimens
Posterior Pharyngeal Swab • Ask the subject to open his or her mouth • Depress the tongue • Swab the posterior pharynx AND tonsils 32
Nasal Swab 1. Examine each of the nares (nostrils) 2. Insert swab into the nostril with most fluid 3. Leave in place for a few seconds. 4. Withdraw with a rotating motion 33
Nasopharyngeal Swab 1. Insert dry swab into nostril and back to nasopharynx 2. Leave in place for a few seconds 3. Slowly remove swab while slightly rotating it 34
Nasopharyngeal Swab continued 4. Use a different swab for the other nostril (if obtaining 2 specimens; this is not common) 5. Put tip of swab into vial containing VTM, breaking/cutting applicator’s stick 35
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37 Specimen Handling, Storage, and Transportation
How to Store Specimens For specimens in VTM: • Transport to laboratory as soon as possible • Store specimens at 4 °C before and during transportation within 48 hours • Store specimens at -70 °C beyond 48 hours • Do not store in standard freezer – keep on ice or in refrigerator 38
How to Store Specimens For sera: • Store specimen at 4 °C for up to 1 week • Freeze at -20 °C beyond 1 week • For both VTM specimens and sera, avoid repeated freeze-thaw cycles 39
Packing Specimens for Transportation • Keep specimens at 4 ºC – Fill a cooler with ice packs – Double-bag specimens if you use dry ice • Include an itemized list of specimens with identification numbers and laboratory instructions 40
Veterinary Specimens • Feces • Cloacal swabs • Oronasal swabs • Tissue samples 41
42 Specimen Collection Demonstration
43 Questions
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