Collection of Specimen for Lab Diagnosis of SARSCoV-2

Zeleke Mekonnen(PhD) Professor, Institute of Health

Jimma University, Jimma, EthiopiaOrganized by: ORHB, Universities in Oromia, ORHB AC, OPA,

IOHPA, EEHA, EEA

Structure of the virus & Range of Hosts

• Coronaviruses1. 229E (alpha coronavirus)2. NL63 (alpha coronavirus)3. OC43 (beta coronavirus)4. HKU1 (beta coronavirus)5. MERS-CoV (the beta coronavirus)6. SARS-CoV (the beta coronavirus) ?

7. 2019 Novel Coronavirus (COVID-19 virus)

Background: Specimen Collection § Please review guidelines recommended by the CDC

to ensure the appropriate IPC precautions are in place

§ One should immediately implement recommended IPC practices if a patient is suspected of having COVID-19

§ For initial diagnostic testing for COVID-19, CDC/WHO recommends upper respiratory tract specimens (nasopharyngeal swab)

§ EOCs shall be reached @ xxxxx for assistance with obtaining, storing,& shipping specimens to EPHI or other test centres, including after hours or on weekends/ holidays

§ Increasing testing capacity will allow clinicians to consider COVID-19 testing for a wider group of symptomatic Pts

• Sampling from respiratory tract is very hazardous as the operator is very close to the patient and the process could generate aerosol sprays and droplets

• Take necessary infection prevention measurements • During communication with suspected case• During sample collection • During transportation• During opening of specimen container

Safety procedures during Sample Mgt

Droplet dispersal by sneezing - approx 20,000 particles

From Medical Microbiology, Pelczar and Chan

Safety procedures during Sample Mgt

Personal Protective Equipment (PPE)

Recommended• Mask: N-95 respirator• Gloves• Disposable long sleeved

cuffed gown • Goggles/protective eyewear

Optional• Cape (high risk, suctioning)• Plastic apron (splashing of

blood, secretions, excretions)

Instructor Demonstration

• Sterile rayon or dacron-tipped applicator swabs• Tongue depressor, wooden• Cryogenic vials or containers with VTM or UTM • PPE (glove, respirator, gown, alcohol…)• Sterile Scissors• Paper towels• Autoclavable Biohazard bags• Secondary and tertiary container with ice packs

to keep sample cold• Formats

Sample Collection Materials

Sample Collection Materials Kit contents:

1. Tube of Universal Transport Media (UTM)

2. Nasopharyngeal swab (smaller swab, flexible shaft) (CDC recommended)

3. Ziploc specimen bag containing absorbent pad

4. RI State Health Laboratories Test Requisition form

5. Ice pack (keep in a freezer until ready to package & transport specimens)

– Essential for virus survival

– Prevents specimen from drying out = virus death

– Prevents bacteria and fungi growth

• Storage of VTM

– Aliquot 2-3mls into a sterile vial and store at -20°C

– Can be stored at 4-8°C for short periods (days)

• UTM Universal transport media is a suitable

alternative

VTM Viral transport media

https://www.cdc.gov/coronavirus/2019-nCoV/guidelines-clinical-specimens.html https://www.cdc.gov/coronavirus/2019-ncov/hcp/caring-for-patients.html

Visit the following for more Collection Instructions:

• Upper Respiratory Tractü Nasopharyngeal swab üThroat swab

• Lower Respiratory Tractü Sputumüendotracheal secretions üPleural fluidübronchoalveolar lavage

• Serum(both acute and convalescent samples)• Stool• urine

Type of Samples

Currently acceptable specimen types 1. Upper Respiratory Specimens

i. Nasopharyngeal (NP) swab only in viral transport media (VTM) or Universal Transport Media (UTM) OR

ii. Combined NP swab and Oropharyngeal (OP) swab in VTM or UTM.

ü Once the NP swab is collected, place it in VTM/UTM,

swirl it in the media for a few seconds and break off the

shaft.

ü Next, collect the OP swab, place it in the same VTM as

the NP swab, swirl it for a few seconds and break off

the shaft. Please screw of the lid of the vial tightly.

Notes: follow the specific instruction in the collections kits!

When to Collect Specimens

(Prof F.Hayden, personal communication)

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Virus detectable in throatswabAntibody in blood

Possible RNAaemia

Onset of symptoms

• Once cases are identified, timing of specimen collection is critical• The solid line shows RNAemia (presence of viral RNA in

respiratory secretions or blood• RNA from patients can be detected about 1 day before the onset

of symptoms

• Viraemic phase remains high up to first 3 days after onset of symptoms (at this time there is a high likelihood of finding RNA in the patient’s specimen).

ü NP specimen is the preferred choice for swab-based SARS-CoV-2 testing

ü NP swabs can be used for testing asymptomatic persons

ü At this time anterior nares &mid-turbinate specimen are only appropriate for symptomatic Pts

ü Both nares should be swabbed. ü If both NP and OP swabs are

collected, they should be combined in a single tube to maximize test sensitivity and limit use of testing resources.

Procedure for Collection of nasopharyngeal swab

1. A swab is inserted into the nostril and back to the nasopharynx and left in place for a few seconds.

2. It is then slowly withdrawn with a rotating motion.

3. Put swab into a vial containing 2-3 mL of VTM or UTM & place on ice immediately

4. Place on ice or refrigerate at 2-8°C and ship as soon as possible to the laboratory.

Procedure for Collection of nasopharyngeal swab

https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html

Please: For update and details refer

§ When collection of a NP swab is not possible, the following are acceptable alternatives:

– An oropharyngeal (OP) specimen collected by a healthcare professional, or

– A nasal mid-turbinate (NMT) swab collected by a healthcare professional or by onsite self-collection (using a flocked tapered swab), or

– An anterior nares (nasal swab; NS) specimen collected by a healthcare professional or by onsite self-collection (using a flocked or spun polyester swab)

– Nasopharyngeal wash/aspirate or nasal aspirate (NA) specimen collected by a healthcare professional

Procedure for Collection of Throat swab

1. Have the pt open his/her mouth wide open

2. Hold tongue down with tongue depressor and swab posterior pharynx by rotating tip of swab vigorously

3. Insert swab into the posterior pharynx & tonsillar areas.

4. Rub swab over both tonsillar pillars &the tongue, teeth, and gums.

5. The patient should try to resist gagging & closing the mouth while the swab touches the back of the throat near the tonsils

6. Put swab into a vial containing 2-3 mL of VTM or UTM & mix and place on ice immediately

7. Place on ice or refrigerate at 2-8°C and ship as soon as possible to the laboratory.

Procedure for Collection of Throat swab

ü Use only synthetic fiber swabs with plastic shafts.

ü Do not use calcium alginate swabs or swabs with wooden shafts,

ü As they may contain substances that inactivate some viruses and inhibit PCR testing.

ü Place swabs immediately into sterile tubes containing 2-3 mL of VTM.

ü In general CDC is now recommending collecting only the NP swab. If both swabs are used, NP and OP specimens should be combined at collection into a single vial.

ü OP swabs remain an acceptable specimen type

Precautions during Collection of NP/OP

2. Lower respiratory tract§ Bronchoalveolar lavage, tracheal aspirate, pleural fluid, lung

biopsy

ü BAL: Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container.

Due to increased technical skill & equipment needs, collection of specimens other than sputum from lower respiratory tract may be limited

§ Sputumü Educate the patient about the difference between sputum

and oral secretions (saliva).

ü Have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile, leak-proof, screw-cap collection cup or sterile dry container.

Note: Additional specimens may be requested on a case by case basis.

Labeling of Specimen & Documentation

• Specimen labelingü Patient nameü Patient ID numberü Specimen type(s)ü Date collected

• Patient Information / documentationüCase based reporting form üLab request and report forms

ü Proper collection of specimens is the most important step in the laboratory diagnosis of infectious disease.

ü A specimen that is not collected correctly may lead to false negative test results.

ü All specimens must be refrigerated/stored at (2-8ᵒC) promptly upto 72 hours after collection

ü And generally couriered/shipped on cold packs within 72 hours. ü If a delay in testing or shipping is expected, store specimens at -

70°C or below and couriered/shipped on dry ice.

Specimen Storage & handling

ü Any specimen being sent must have the Important paperwork accommodating the specimen

ü Ensure that all specimens submitted and their respective test requisition form has the following information on it:

• Full name of patient • Date of birth • Unique patient identifier (e.g., medical record number,

patient ID, PUI #) • Date and time of specimen collection

ü Incorrectly labeled requisitions and specimens will result in testing delays.

Specimen Labelling & paper work

Important points to consider……………………………during specimen collections• Viruses are usually intracellular

• To detect a virus in a specimen you need to collect cells• Specimens of choice for coronavirus infection• Respiratory material

– In ambulatory patients• nasopharyngeal and oropharyngeal• sputum (if produced)

– Patient with severe disease • endotracheal aspirate or • bronchoalveolar lavage

• Serum for serological testing, acute sample and convalescent sample.

Does not necessarily mean the pathogen is not present i.e. is just probably not present in that specimen?

• Specimen collected from incorrect site

• Cells not present – require cells as viruses are in cells

• Specimen collected from patient too late – viral shedding has ceased

• Specimen size is too small (not enough for detection)/too large (pathogen diluted out)

• Specimen integrity compromised

• Pathogen viability compromised due to inappropriate processing, handling or storage

• Agent is an emerging pathogen not detectable by current laboratory methods (primer sites).

Important points to consider………………………………Negative results – why?

Infectious substances

• Infectious substances (Category A) An infectious substance which is transported in a form that, when

exposure to it occurs, is capable of causing permanent disability, life threatening or fatal disease in otherwise healthy humans animals.

UN2814 – humans, E.G. specimens from an Ebola patient, culturs

UN2900 – animals

• Biological substances (Category B) Infectious substance which does not meet the criteria for category A and has not

been determined by a medical professional to have a minimum likelihood that pathogens are present.

UN 3373

Infectious substances are classified into different categories

Packing and Shipping of samples

• Follow shipping regulations

• Triple package has 2 main goals:

– Protect the environment and the carrier

– Protect the sample arrives in good condition to be

analyzed

Principles of Sample Packaging

3

2

Absorbant & protection

1

General models of Sample Packaging

Category B – Biological Substances

• Wrap the container containing the specimen with absorbent paper.

• Place the wrapped specimen container in a leak-proof secondary container.

General models of Sample Packaging

• Place the wrapped primary container in a leak-proof secondary container and close tightly.

General models of Sample Packaging

• Place ice-packs inside a cool box • Put the secondary container inside the cool box

• The secondary container should be placed upright.

General models of Sample Packaging

• Close the shipping box tightly

• Place shipper and consignee details on outside of the shipping box.

• Make sure box is labeled correctly• Clean the external body of the container

General models of Sample Packaging

Specimen Rejection criteria:

• Specimens not kept at 2-4°C (≤3 days) or frozen at

-70°C or below.

• Incomplete specimen labeling or documentation.

• Inappropriate specimen type.

• Insufficient specimen volume

• Wrong transport media

Key Pointsü Proper sample collection and handing is basic for Laboratory

diagnosis of any disease!ü Appropriate IPC precautions/practices shall be in place all the

time and shall be implemented as recommended

• Use standard PPE in the right way and place

• Protect yourself, others, and the environment• Limit opportunities for “touch contamination”• Avoid touching:

– your face or mask or adjusting PPE with contaminated gloves– environmental surfaces except as necessary during patient

care

ü Ensure shipper label information for your facility is completed in full and affixed to the outer package

Resources for references• https://www.cdc.gov/coronavirus/2019-ncov/downloads/COVID-19-Specimen-

Submission-Guidance.pdf

• https://www.cdc.gov/coronavirus/2019-nCoV/guidelines-clinical-specimens.html • https://www.cdc.gov/coronavirus/2019-ncov/hcp/caring-for-patients.html

• https://www.youtube.com/watch?v=DVJNWefmHjE

• https://www.sciencedirect.com/science/article/pii/S2352013220300521

Thank you