Laboratory training course:

Proper selection, handling and shipping of samples for diagnosis of

influenza and other respiratory viruses

SESSIÓN 4: SELECTION, HANDLING AND CORRECT SHIPPING OF SAMPLES

http://www.livescience.com/16382-viruses-raw-sewage.html

• Know criteria for selection and characterization of samples

• Review the guidelines for sending samples to WHO CC

• Review basic concepts of packing and shipping samples

• Know the requiered documentation for sending samples

Objectives

What Information is Important about Influenza Viruses?

• Emergence of novel subtypes• Mutations among circulating viruses• Increasing incidence/emergence of antiviral

resistance among circulating viruses• Comparison of circulating viruses to viruses in

current seasonal vaccine

How can we resolve these questions?

To answer these questions it is necessary to have different laboratory tests and tools for identification. Some are available at the NICs and others are performed the WHO CC, among which are the following:

• Inmunofluorescence• Viral Isolation (MDCK and Embryonated Eggs)• Titration of HA and IHA (Antigenic Characterization)• Microneutralization• Typing and subtyping by qRT-PCR • Pyrosequencing and Sequencing• Bioinformatics analyzes

National Influenza Centers

http://www.who.int/influenza/gisn_laboratory/national_influenza_centres/terms_of_reference_for_national_influenza_centres.pdf

WHY, WHERE AND FOR WHAT SEND SAMPLES?

Because of the capacity of transmission and evolution of

influenza viruses, it is necessary to complement monitoring by more

detailed characterization of circulating viruses

Why?

Regional Labs to NICs

NICs to WHO CC

WHO CC shares information and viruses with the Global Influenza Surveillance Network )

Where?

Selection of strains for vaccine formulation

Antiviral Resistance

Novel Virus Mutation detection

WHO-CC

For What?

CRITERIA FOR SELECTION OF SAMPLES

Samples for the WHO-CC

Objective How to Select Samples

When to Send Samples

Funding Source to Ship Samples

Detect novel subtype Any unsubtypeable influenza A virus

Immediately

WHO Global Shipping Project,

PAHO, CDC, Country Resources

Detect anti-viral resistance

Cases receiving anti-viral therapy, immunosuppressed cases Routine

(2-4 times each year)

Number of samples is according to

epidemiological presentation

Number of samples is between 15 to 20

Detect mutations Random

Contribute to vaccine composition

Random

Based upon influenza season and by August and January 15

Random Selection

• All circulating virus types/subtypes• Different age groups• Varying geographic regions• Ambulatory and hospitalized cases• Deaths• Outbreaks• Recipients of antiviral therapy

- 5 to 10 representative samples of unusual outbreaks (e.g. outside the expected seasonal)

- Any sample corresponding to unusual cases with inconclusive results

Sending at any time

Other criteria

Additional Principles in Selecting Samples to Submit

• Type of sample to send isolates preferred, also send clinical material

• Time period to select from recent viruses (1-3 months old)

Objective 1: Detect Novel Subtype

• Which samples to send: any influenza A virus which is unable to be subtyped

• When to send: immediately • Public health significance

– Emergence of novel subtype with pandemic potential

– Introduction of H5 or H7 in the Americas Region

Objective 2: Detect Anti-viral Resistance

• Which samples to send– Prioritize

• Cases receiving anti-viral therapy• Cases who are immunosuppressed

– Part of random selection of 15-20 samples

• When to send– Routinely (2-4 times each year)

• Public health significance– Emergence of anti-viral resistant strain– Fewer options for treatment/chemoprophylaxis

Objective 3: Detect Mutations

• Which samples to send– Part of random selection of 15-20 samples

• When to send– Routinely (2-4 times each year)

• Public health significance– Poor vaccine match

Objective 4: Contribute to Vaccine Composition Development

• Which samples to send– Part of random selection of 15-20 samples

• When to send– Routinely (2-4 times each year)– Before strain selection meeting (February,

September)

• Public health significance– Data needed to determine which three/four strains

to include in the seasonal influenza vaccine

Seasonal Influenza Vaccine

• Trivalent or quadrivalent vaccine– Influenza A (H1)– Influenza A (H3)– Influenza B (Yamagata and/or Victoria)

• Southern Hemisphere and Northern Hemisphere composition– Southern Hemisphere composition determined in

September– Northern Hemisphere composition determined in

February– Recommendations match ~66% of the time

When to Send for Routine Submission

JAN FEB MAR APR MAY JUN JUL AUG SEPT

OCT

NOV DEC

NH INFLUENZA SEASON

NH INFLUENZA SEASON

SH INFLUENZA SEASON

NH STRAIN SELECTIONMEETING

SH STRAIN SELECTIONMEETING

TROPICAL COUNTRIES INFLUENZA SEASON

When to Send for Routine Submission- Southern Hemisphere

JAN FEB MAR APR MAY JUN JUL AUG SEPT

OCT

NOV DEC

SH INFLUENZA SEASON

NH STRAIN SELECTIONMEETING

SH STRAIN SELECTIONMEETING

Off-season

Early- season

Mid- season

Late- season

When to Send for Routine Submission- Tropical Countries

JAN FEB MAR APR MAY JUN JUL AUG SEPT

OCT

NOV DEC

NH STRAIN SELECTIONMEETING

SH STRAIN SELECTIONMEETING

Off-season

Late- season

Early- season

Mid- season

Santa Cruz, Bolivia

Chile

Sep FebOctAgoJul

Jun

Nov Dic Ene Mar Abr M

ay

Selection and sending

Meeting for the formulation of the vaccine

SH NH

When to Send for Routine Submission

Muestras enviadas a los CC OMS para caracterización adicional por fecha recibida y por país, 2014

January 15, 2014 January 16, 2014 January 21, 2014 February 14, 2014 February 21, 2014

May 12, 2014 May 16, 2014 June 4, 2014 June 9, 2014 June 11, 2014July 3, 2014 July 18, 2014 July 28, 2014

August 6, 2014 August 8, 2014 August 13, 2014 August 14, 2014 August 22, 2014 August 25, 2014 August 26, 2014September 5, 2014 September 8, 2014

September 18, 2014 September 19, 2014October 20, 2014

0

10

20

30

40

50

60

70

80

90

100

Number of samples

10

0

10

4

15

49

77

13

16

40

20

20

40

40

50

20

20

20

40

20

10

10

74

14

24

24

34

52

32

11

76

4 2

Source: WHO CC data.Note: Each color represents one country.

NH SH

Samples sent to WHO CC for additional characterization by date received and by country, 2014

Samples sent to WHO CC for additional characterization by Region and month received, 2014

Source: WHO CC data.

SENDING SAMPLES

Transport of Dangerous Goods

International Regulations

UN Committee of Experts on the Transport of Dangerous Goods (UNCETDG)

Transport of dangerous goods

International Regulations

• The International Air Transport Association (IATA)Air

• Regulations concerning the International Carriage of Dangerous Goods by Rail (RID)

Rail

• The European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR)

Road

• The International Maritime Dangerous Goods Code published by the International Maritime Organization (IMO)

Sea

Difficulting finding

transport

Exposure to infectious

substances

Conse-quences

Panic incident

Shipment detained

by customs

Civil or criminal lawsuits

Errors and delays in delivery

Consequences of improper packaging

Sender responsibilities

Complete documentation

Coordinate with carrier

and receiver

EnsureResponsibility of the sender Label

PackagingClasification of the substance

-Categorization

-appropriate packaging

-appropriate labeling

-Documentation Required

-Format Air Shipping

-Statement of dangerous goods (A)

-Certificate Training

Good to know…

Categorization

• Class 2: Gases

• Class 3: Flammable liquids

• Class 6: Toxic and infectious substances

Division 6.1: Toxic substances

Division 6.2: Infectious substances

• Class 9: various dangerous goods and articles, including environmental substances

Classes of Dangerous Goods

1. Infectious substances (Category A)

2. Biological substances (Category B)

3. Biological products

4. Genetically modified organisms

5. Clinical or Medical wastes

6. Live and infected animals

7. Human samples/animal samples exemptions

8. Other exemptions

Substances class 6

Class 6: Toxic and Infectious substancesDivision 6.2: Infectious substances

CATEGORY A

• Any infectious substance that could generated disability, life threatening or fatal disease in healthy humans or animals

• Proper shipping name:• UN 2814 "INFECTIOUS

SUBSTANCE, AFFECTING HUMANS"

• UN 2900 "INFECTIOUS SUBSTANCE, AFFECTING ANIMALS ONLY"

CATEGORY B

• Any biological substance that does not meet the criteria for Category A.

• Proper shipping name :• UN 3373: "BIOLOGICAL

SUBSTANCE, CATEGORY B"

• A 3291: "CLINICAL WASTE, UNSPECIFIED"

Categorization (Division 6.2)

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Flow for the classification of infectious substances

• Category A (Infectious Substances)

• Category B (Biological Substances)

• Exemptions:

1. Samples without infectious components or with low probability of causing disease in humans or animals

2. Substances with non-pathogen agents for humans or animals

3. Neutralized or inactivated substances

Categorization (division 6.2)

• Category A (Infectious Substances)

Highly pathogenic avian influenza virus (viral culture)

• Category B (Biological Substances)

Highly pathogenic avian influenza virus (patient sample)

• Exemptions:

1. Samples without infectious components or with low probability of causing disease in humans or animals

2. Substances with non-pathogen agents for humans or animals

3. Neutralized or inactivated substances

Categorization (division 6.2)

Some infectious goods included in category A

Packaging

• Use three layers of packaging• The first container must be resistant to seepage• Use absorbent material in each container

Packaging

Primary container

Secondary container Outer container

http://upload.wikimedia.org/wikipedia/commons/2/28/Tripe_embalaje.png

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Packaging Category A

• All Category A infectious substances must comply with the instructions for packaging the United Nations (UN), P620 .

• This guarantees the packaging has passed stringent stress tests (pressure, stacking, punching, etc.)

• The outer container must indicate the sign of the United Nations, ensuring quality packaging to officialshttp://apps.who.int/iris/bitstream/10665/78075/1/

WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Packaging Category A

Labeling Category A

Information on the outer container :

• Name and address of the sender• Telephone number responsible for

shipping• Name and address of consignee• Proper shipping number (UN 2814

or UN 2900)• Temperature requirements (optional)• If dry ice or liquid nitrogen is used:

technical name, UN number and total amount.

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Labeling Category A

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Labeling Category A

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Labeled Category A

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Labeled Category A

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Packaging Category B

Each shipment must have the following information :

• Airway: name, phone number and address of the sender

• Phone of person responsible for emergency contact

• Name, telephone number and address of the recipient .

• Proper name, visible: (“BIOLOGICAL SUBSTANCE, CATEGORY B")

• Optional: Special requirements for temperature

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Labeled Category B

• A packing list format or the address of the consignee, the number of packages to send, weight and detail of the content.

• Declaration or import/export permit (if required).

• Sending Format (air waybill) where the content is detailed

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Air waybill:

Documentation of Category B

• Declaration of Dangerous Goods

• A packing list format or the address of the consignee, the number of packets to send, weight and detail of the content.

• Declaration or permit import/export (if required).

• Appropriate documents (waybill) for shipments by rail, road or sea.

http://apps.who.int/iris/bitstream/10665/78075/1/WHO_HSE_GCR_2012.12_eng.pdf?ua=1

Declaration of Dangerous Goods These documents must be prepared

and signed by the sender:

Documentation of Category B

Declaration of Dangerous Goods

XDocumentation of Category B

• Ensure cold chain.• Include details of sending

samples, identification number and epidemiological data

Final recomendations…

Blanton,L., Brammer,L., et al.(2011) Influenza. Manual for the surveillance of vaccine-preventable diseases. 5th Edition. Available online at: http://www.cdc.gov/vaccines/pubs/surv-manual/chpt06-influenza.pdf

World Health Organization (WHO). (2006) Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1) virus infection. Guide for field operations. Available online at: http://www.who.int/csr/resources/publications/surveillance/CDS_EPR_ARO_2006_1.pdf?ua=1

World Health Organization (WHO). (2004) Guidelines for the collection of clinical specimens during field investigation of outbreaks. Available online at: http://www.who.int/csr/resources/publications/surveillance/whocdscsredc2004.pdf

World Health Organization (WHO).(2009) Instructions for storage and transport of suspected or confirmed human and animal specimens and virus isolates of pandemic (H1N1). Available online at: http://www.who.int/influenza/gisrs_laboratory/logistic_activities/transport_storage_specimens_isolates/en/

References

World Health Organization (WHO). Laboratory biosafety manual, Biosafety guidelines. Third Edition (2004). Available online at: http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf?ua=1

World Health Organization (WHO). Manual for the laboratory diagnosis and virological surveillance of influenza. Available online at: http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf?ua=1

World Health Organization (WHO). Nasopharyngeal and Oropharyngeal Swabs. Available online at: http://www.youtube.com/watch?v=mfZYAMDpGNk

World Health Organization (WHO). Selection of clinical specimens for RT-PCR and virus isolation and of viruses for shipment from National Influenza Centres to WHO Collaborating Centres. Available online at: http://www.flu.mn/mgl/images/stories/Sudalgaa_shinjilgee/GISN2010/suppl.03.%20Selection%20on%20clinical%20specimens%20for%20PCR,%20virus%20isolation%20and%20shipment%20for%20NICs.pdf

World Health Organization (WHO). WHO information for molecular diagnosis of influenza virus in humans – update (2012). Available online at: http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201211.pdf

World Health Organization (WHO). Personel Protective Equipment (PPE). Available at: World Health Organization (WHO).

References (2)

Thank you!