The findings and conclusions in this presentation are those of the author(s) and do not - PowerPoint Presentation

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The findings and conclusions in this presentation are those of the author(s) and do not - PPT Presentation

CDC RealTime RTPCR Support Strategies and Updates Stephen Lindstrom PhD Diagnostics Development Team Virus Surveillance and Diagnosis Branch Influenza Division Centers for Disease Control and Prevention ID: 933191

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Slide1

The findings and conclusions in this presentation are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention

CDC Real-Time RT-PCR

Support Strategies and Updates

Stephen Lindstrom, Ph.D.

Diagnostics Development TeamVirus Surveillance and Diagnosis BranchInfluenza DivisionCenters for Disease Control and Prevention

Third Annual SARInet MeetingMay 9-11, 2016San Juan, Puerto Rico

National Center for Immunization and Respiratory Diseases

Influenza Division

Slide2

Overview

Testing strategies & algorithms for surveillance and diagnostic testingCDC

rRT-PCR assays for detection and characterization of influenzaSupport strategies for

rRT-PCR testingCLSIS – protocols/proceduresIRR - Reagent kits, control materialsA(H1N1)pdm09 reactivity and assay update

Slide3

Influenza

Viruses in Humans - 2016

Potential

Pandemic

strains

Avian

Avian H5

Avian H9

Type A

Seasonal

Strains

Type

B/Yam

B/Vic

1918

1957

1968

1977

1997

1998/9

2003

2009

1940

H1v

2016

H3v

Avian

Avian H10

Slide4

Routine Surveillance

No Known risk for avian influenza

Perform RT-PCR testing for seasonal human influenza A and influenza B viruses

InfA (+)

InfA

unsubtypable

– possible novel influenza

Based on risk assessment, test for H5, H7 and/or other novel strains w/RT-PCR

InfA

inconclusive samples with possible SOIV

Inconclusive samples with possible

coinfection

/LAIV

InfA

(+)

H3 or

pdm

H1 (+)

InfB (+)

Test for YAM, VIC

Report Results

Test for H3,

pdm

H1,

pdmInfA

Negative

Report Results

Routine Influenza Testing Algorithm

InfB

(+)

YAM or VIC (+)

Report Results

Slide5

Influenza

Detection Algorithm and Sample Flow

Slide6

CDC Influenza Virus Real-time RT-PCR Reagent Kits

Influenza A H5 (Asian lineage) Subtyping Panel

(

InfA

, H5a, H5b, RP)

Influenza A

Subtyping

Panel

(

InfA

, H1, H3,

pdmInfA

, pdmH1)

Influenza A/B Typing Panel

(

InfA

Inf

B, RP)

Influenza

B genotyping Panel

(

InfB

, Yam, Vic, RP)

Influenza A

EuH7 Assay

(EuH7)

Slide7

Interpretation of

Real-time RT-PCR Results

InfA

pdm

InfA

pdmH1

Result

+/-

A/H3

+/-

A/H1

+/-

A/H1pdm

+/-

Negative

Inconclusive

Unsubtypable

Potential novel influenza

Slide8

Interpretation of

Real-time RT-PCR Results

Pigs

Poultry

H3N2v, H1N1v, H1N2v

H5N1/H5Nx, H7N9

Slide9

Slide10

Slide11

Slide12

Wild bird infection with I

nfluenza A

/H5Nx (Clade 2.3.4.4) viruses

in the U

. - 2015

Slide13

Slide14

H5 ASSAY UPDATES

A/gyrfalcon/Washington/41088-6/2014

(H5N8)

Titer

(ID50/mL

tested)InfA

H5a RUO (V2

)

(current)

H5a H5Nx

Combined H5a (0.5 uL)

6.5

20.41

20.45

20.56

26.84

26.69

25.86

21.37

20.68

20.61

22.06

21.1

21.09

4.5

27.51

27.79

28.05

34.01

33.91

33.8128.46

28.2828.37

28.5928.89

28.89

10 2.5

35.5334.81

34.55

43.4841.7139.97

34.4

37.06

35.2435.35

36.21

35.82

H5VC25.64

32.46

31.28

30.36

Titer (ID50/mL tested)

InfA

H5b RUO (V2

)

(current)

H5b H5Nx

Combined H5b (0.5 uL)

10 6.5

20.4120.45

20.56

23.2523.2

23.22

20.0220.21

20.35

20.1120.35

20.2610

4.527.51

27.7928.05

30.1

29.7130.34

26.6527.56

27.62

27.3327.7

27.42

10 2.535.53

34.81

34.5537.17

38.03

3734.67

35.17

35.9634.43

35.59

35.22H5VC

25.64

31.13

34.33

31.76

Slide15

Recommendations for H5Nx influenza testingThe recommendations for testing for suspect novel influenza cases, including suspect human infection

with clade 2.3.4.4 HPAI viruses such as H5N2 and H5N8, remain the same as in the current guidance outlined in the CDC Flu Dx Panel package insert. Testing should be considered in suspect human cases with a recent history of contact with poultry (e.g. poultry farm, household raising poultry, or bird market) either in the United States or during travel to a country with documented HPAI avian influenza in poultry and/or humans

Slide16

Confirmed Human H7N9 Cases

as of April 20, 2016 (FAO)

Slide17

Number of confirmed cases*

779Number of fatal confirmed cases 300

Human cases of avian

influenza A (H7N9)

* As of

April, 2016

Slide18

Strain Designation

sub-type

Lineage

Invitrogen

Quanta

InfA

EuH7

InfA

EuH7

A/ANHUI/1/2013

H7N9

A/DUCK/VIETNAM/NCVD-197/2009

H7N3

A/TURKEY/ITALY/5425/07

H7N3

Shoveler

/Egypt/00017-NAMRU3/2007

H7N3

A/Mallard/Netherlands/12/2000

H7N3

A/Chicken/Italy/5074/1999

H7N1

A/MEXICO/7218/2012

H7N3

N.Am.

Undet

Eu = Eurasian lineage;

N.Am. = North American lineage.

Evaluation of CDC EuH7 Assay

Slide19

Human cases of

“variant” Influenza

infection in the United States

Slide20

S Lindstrom, et al. Emerg. Infect. Dis. 2012 May

Evolutionary Relationships Among Influenza A (H3) Hemagglutinin (HA) Genes

Slide21

InfA

pdm InfA

pdmH1

Result Interpretation

Influenza A detected;

Presumptive positive for Influenza A(H3N2) variant virus

H3N2v Results Interpretation

Slide22

Human cases of infection with

SOIV

the U

. 2009-2015

Year

H3N2v/state

cH1N1v/state

H1N2v/state

Total

States

2009/2010

7/5

2011

12/5

1/1

2012

309/12

2013

19/4

2014

2/1

2015

2/2

Slide23

H3N2v

H1N1v

H1N2v

Total

Detected

Total cases

354

379

Total states

Detected U.S. Infections

with Variant

Influenza Viruses by State since December

2005 - 2015

Slide24

Canine

nfluenza A

/H3N8 and A/H3N2

viruses

in the U

.S.

Dogs

H3N8

H3N2

Dogs

Asian Avian origin

Equine origin

Slide25

Reactivity of CDC Real-time

RT-PCR Panel with Canine H3N2/H3N8

InfA

pdm

InfA

pdmH1

Result

Unsubtypable

Potential novel influenza

Slide26

Support Strategies for Public Health and Research Laboratories

Slide27

Reagent ordering - IRR

www.influenzareagentresource.org

Slide28

Real-Time

RTPCR Kits

Catalogue#

Shipped

FluRUO-01 A/B TypingFR-198

418

FluRUO-04 A Subtyping

FR-929

244

FluRUO-05 Influenza B Lineage

FR-1209

170

FluRUO-06 A/H5 Subtyping (assay update)

FR-1229

132

FluRUO-07 A/H7 (Eurasian Lineage) Subtyping

FR-1258

VA2714 Human Specimen Control (HSC)

FR-174

146

Total kits

distributed

1207

Control

Material

Catalogue#

Shipped

VA2715 Influenza A/H5N1Positive Control (H5VC)

FR-176

130

VA2716 Pooled Influenza Positive Control (PIPC)

FR-895

289

VA2733 Influenza B Positive Control (IBPC)

FR-1273

133

KK0817 A/H7

(Eurasian Lineage) Positive Control (EuH7PC)

FR-1257

Total Controls distributed

630

International Distribution of

rRT-PCR kits and control materials FY2014/FY2015

Slide29

Global Distribution of

rRTPCR

Reagents in FY14 & FY15

111 Countries

Slide30

CDC rRT-PCR Influenza Kit Distribution to PAHO countries

Real-time RT-PCR Panels

Catalogue

Shipped 2014

Shipped 2015Shipped 2016

CDC Influenza VirusReal-time RT-PCR

FluRUO-01

CDC Influenza Virus Real-time RT-PCR Influenza A (H1/H3/H1 pdm09) Subtyping Panel (RUO)

FluRUO-04

CDC Influenza B Lineage Genotyping Panel (RUO)

FluRUO-05

CDC Real-time RT-

PCRInfluenza

Virus A/H5

FluRUO-06

CDC Influenza A/H7 (Eurasian Lineage) Assay

FluRUO-07

109

Total 103

9426

as of May 201627 laboratories in

21 countries

Slide31

CDC rRT-PCR Influenza Kit Distribution to PAHO countries

Control Material

Catalogue#

Shipped 2014

Shipped 2015

Shipped 2016Pooled Influenza Positive Control (RUO)

VA271630

Influenza B Positive Control (RUO)

VA2733

Influenza A/H5N1 (

Asian Lineage

) Virus

(RUO) PC

VA2715

Influenza A/H7 (

Eurasian Lineage

) (RUO) PC

KK0818

116

Human Specimen Control

(RUO)

VA27146

Total 74

5816

as of May 2016 27 laboratories in

21 countries

Slide32

PAHO countries receiving reagents from IRR - 2016

21 countries

Slide33

CDC

Sharepoint

Site for Laboratory Support for Influenza Surveillance

Provide public health and research laboratories access to multiple assays, procedures and methods depending on need (equipment, chemistry, etc.)

Allow for coordinated communication with registered laboratories

Provide timely notification of assay updates

www.cdc.gov/flu/clsis

>260 users

Slide34

Current CLSIS Users

The findings and conclusions in this presentation are those of the author(s) and do not - PowerPoint Presentation

The findings and conclusions in this presentation are those of the author(s) and do not - PPT Presentation

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