Specimen Collection Field Diagnostics and Learning Objectives - - PDF document

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Specimen Collection and Field Diagnostics

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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

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI outbreak in community Mild No No ILI outbreak in community Mild with at least 1 severe No No ILI outbreak in community Generally moderate to severe No No

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI outbreak in community Mild No No Rapid test Viral culture ILI outbreak in community Mild with at least 1 severe No No Rapid test PCR? Viral culture ILI outbreak in community Generally moderate to severe No No Rapid Test PCR Viral culture

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI outbreak in community Mild Yes No ILI outbreak in community Mild No Yes ILI in hospitalized patient Severe No No

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI outbreak in community Mild Yes No Rapid test PCR Viral culture ILI outbreak in community Mild No Yes Rapid test PCR Viral culture ILI in hospitalized patient Severe No No Rapid test? PCR Viral culture

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI in hospitalized patient Severe Yes No ILI in hospitalized patient Severe No Yes ILI in hospitalized patient Severe Yes Yes

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Selecting Testing Methods

Setting Severity of Disease Concurrent Poultry Epidemic Epi Links to Human Cases Appropriate Tests ILI in hospitalized patient Severe Yes No PCR Viral culture ILI in hospitalized patient Severe No Yes PCR Viral culture ILI in hospitalized patient Severe Yes Yes PCR Viral culture

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Right specimen for right test

• For rapid influenza

tests, check manufacturer’s requirements

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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

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Materials for Viral Culture

• Viral transport medium (VTM)
• Cold chain
• Appropriate swabs

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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

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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

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Specimen Collection Kit

• Collection vials with

VTM

• Polyester fiber-tipped

applicators

• Sterile saline

(0.85% NaCl)

• Tongue depressors
• Specimen collection

cups or Petri dishes

• Secondary container
• Ice packs
• Items for blood

collection

• Personal protective

equipment

• Field collection forms
• A pen or marker for

labeling samples

• Disinfectant

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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

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Personal Protective Equipment

• Gloves
• Mask
• Gown
• Eye protection

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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

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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

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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

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What to Collect

Serologic samples

• Acute serum (within 2-7 days of

symptom onset)

• Convalescent serum (2-4 weeks after

acute serum)

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Rapid Influenza Tests

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Rapid Test I nfluenza Types Detected Acceptable Specim ens

Directigen Flu A7 (Becton- Dickinson) A NP swab,2 throat swab, nasal wash, nasal aspirate Directigen Flu A+ B7, 9 (Becton-Dickinson) A and B NP swab,2 throat swab, nasal wash, nasal aspirate FLU OIA7 (Thermo Electron) A and B4 NP swab,2 throat swab, nasal aspirate, sputum FLU OIA A/ B7, 9 (Thermo Electron) A and B NP swab,2 throat swab, nasal aspirate, sputum XPECT Flu A/ B7, 9 (Remel) A and B Nasal wash, NP swab,2 throat swab NOW Flu A Test7, 9 NOW Flu B Test7, 9 (Binax) A B Nasal wash, NP swab2 Nasal wash, NP swab2 QuickVue Influenza Test8 (Quidel) A and B4 NP swab,2 nasal wash, nasal aspirate QuickVue Influenza A+ B Test8 (Quidel) A and B9 NP swab,2 nasal wash, nasal aspirate AS Influenza A7, 9 SAS Influenza B7, 9 A B NP wash,2 NP aspirate2 NP wash,2 NP aspirate2 ZstatFlu8 (ZymeTx) A and B4 throat swab

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Rapid Influenza Tests: Components

• Expiration Date
• Package Insert
• Test tubes with

lyophilized buffer

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Rapid Influenza Tests: Components

• Sterile foam swabs
• Reagent
• Box used as test

tube holder

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Rapid Influenza Tests: Components

• Test strips
• Positive and or

negative control

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Check expiration date

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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

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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

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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

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How to Collect Specimens

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Posterior Pharyngeal Swab

• Ask the subject to
• pen his or her mouth
• Depress the tongue
• Swab the posterior

pharynx AND tonsils

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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

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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

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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

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Specimen Handling, Storage, and Transportation

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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

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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

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• 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

Packing Specimens for Transportation

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Veterinary Specimens

• Feces
• Cloacal swabs
• Oronasal swabs
• Tissue samples

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Specimen Collection Demonstration

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Questions