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Rubella Virus RNA Assays:

Rubella Diagnostic RT-PCR Kit

(conventional RT-PCR)

Rubella Virus Genotyping Kit

(conventional RT-PCR, 2 Fragments)

Joseph P. IcenogleInter-country Hands-on Training on the Molecular Epidemiology of Measles and Rubella Laboratory

WHO Western Pacific Region

Public Health Laboratory Centre

Hong Kong

SEQUENCE WINDOW OF RUBELLA VIRAL RNA

FOR MOLECULAR EPIDEMIOLOGY

Plotsimilarity 100 nt window

C E2 E1 97006512 82587412

900 846 1443

Positive strand RNA---9762 nucleotides

601

8869 9469

TYPICAL SEQUENCE WINDOWFOR MOLECULAR EPI.

Incr

easi

ng

Var

iab

ilit

yB

etw

een

Vir

use

s

P150 P90 C E2 E1 PolyA

Nonstructural Proteins

Cap

Structural Proteins



Cap

Structural Proteins

Plotsimilarity 100 nt window

C E2 E1 97006512 82587412

900 846 1443

Positive strand RNA---9762 nucleotides

601

8869 9469

TYPICAL SEQUENCE WINDOWFOR MOLECULAR EPI.

Incr

easi

ng

Var

iab

ilit

yB

etw

een

Vir

use

s



Cap

Structural Proteins



Cap

Structural Proteins

SEQUENCE

REGION (739 NT)

8731 9469

Structural protein coding region(3192 nucleotides) Referenceviruses

Accepted Genotyping window(739 nucleotides in E1 coding region) Reference viruses

Basic Nomenclature for Wild-Type Rubella Viruses

Basic Nomenclature for Wild-Type Rubella Viruses

Genetic characterization has identified 2 clades which differ by 8-10% at the nucleotide level. Clade 1 is divided into 10 genotypes (1a, 1B, 1C, 1D, 1E, 1F, 1G, 1h, 1i, and 1j), of which6 are recognized and 4 are provisional (designated by lower case letters). Clade 2 contains 3 genotypes (2A, 2B, and 2C).

A Method for Detection of Rubella virus RNA by conventional RT-PCR

• Starts with a two primer set from Bosma, et al. in Journal of Clinical Microbiology, 1995.

• Produces a 185 nucleotide amplicon in the E1 protein coding region.

• Sensitivity has been shown to be approximately 100 copies of RNA.

RV11

RV12

8807 8991

Original CDC Conventional PCR for Rubella Virus RNA Detection

E1

739bp

RV12 Primer and Modifications

• The primer RV12 binds to a region of the genome

where there is variation, especially between Clade 1

and Clade 2 viruses.

• RV12: CCA CAA GCC GCG AGC AGT CA

• RV12-2: CCA CGA GCC GCG AAC AGT CG

A modified RV12 primer was made containing

the Clade 2 majority sequence.

RV11

RV12

8807 8991

Current 3 Primer Conventional PCR for Rubella Virus RNA Detection

E1

739bp

RV12-2

Rubella Virus Synthetic RNA Positive Control Construct

Diagnostic RT-PCR Kit

1 2 3 4 5 6

1 – Ladder

2 – Mock RNA

3 – Detection fragment

4 – Genotyping Fragment 1

5 – Genotyping Fragment 2

6 – Positive control (detection

primers)

Lanes 3-5 contain products made

with wild-type rubella RNA.

Lane 6 contains a product made

using synthetic RNA with a 30 bp

Insertion.

Day of sample collection

0 1 2 3 4

Per

cent

Pos

itive

10

20

30

40

50

60

70

80

90

Comparison of RT-PCR, serum IgM by 2 tests and OF IgM

Analysis of specimens from 225 persons with suspected rubella

Percent of suspected rubella cases confirmed by RT-PCR (combination of conventional and real time results) using oral fluid (•―•)serum IgM using Dade Behring (o…..o) serum IgM using Microimmune ( )oral fluid IgM using Microimmune (∆-..-..∆ )

Challenges for sequencing rubella

viruses directly from sample

• High GC content : kit selection

• Amplicon/sequence window size

• Primer design: sequence conservation

• Copy number in clinical samples

GC Content

• Rubella virus has a high GC content

– 69.6% for the whole genome

– 73% for the C coding region

– 71% for the E2 coding region

– 66.5% for the E1 coding region

• High GC content RNAs often contain stable

intrinsic secondary structures that can inhibit

reverse transcriptase and/or primer annealing

• Q Solution

Amplicon/Sequence Window Size

• Rubella sequence window is 739 nts in the E1

coding region (nts 8731-9469)

• RT-PCR amplicon needs to be larger (e.g. 945

nts) to allow for primer binding sites and low

quality sequence close to the primer sites

• In general, the larger the amplicon, the higher

the minimum copy number required for

template production

Sensitivity of 739 “sequence window size “fragments using the Qiagen kit

8656/9549 (894 bp) 8633/9577 (945 bp)

Template is F-Therien RNA Order: Mock, 1x104, 1x103, 1x102 copies

Sensitivity of both primer sets is approximately 1000 copies of RNA.3/9/10

8633

6512

Poly A TailC E2 E1

Single large amplicon

2 overlapping amplicons

Sequenced Region 8731 9469739bp

8633

9577

945bp

480bp

9577

633bp9112

8945

9762

Objective: Design a 2 Fragment System to Increase

Sensitivity

2 Fragment System for Rubella Genotyping: Qiagen Kit Sensitivity

Template is F-Therien RNA, 1X103 – 1x100 copies

M 3 2 1 0 M 3 2 1 0

8633F/9112R4

480 bp

8945F/9577R

633 bp

2/24/10Sensitivity is approximately 100 copies of RNA for both fragments.

Qiagen RT-PCRs on rubella viruses with changes in at least 1 primer binding

site

1: 8633/9112 8 viruses2: 8945/9577 6 viruses

All reactions were positive for both fragment 1 and fragment 2.

Place Genotype Sample type Day of coll Copy #/2.5ul Single (945nt) Frag 1 (480nt) Frag 2(633nt)

MI 2B TS 0 >10 Negative Negative Negative

Ghana 1G TS na >10 Negative Negative Negative

WI 2B ur 7 >10 Negative Positive Negative

Ghana 1G TS na 15 Negative Negative Negative

AZ 1G NP-2 6 20 Negative Negative Negative

MI 2B TS 4 21 Negative Negative Negative

Ghana 1G TS na 26 Negative Weak Positive Negative

MI 2B TS 1 30 nd Positive Positive

Ghana 1G TS na 34 Negative Negative Negative

ND 2B ur 2 34 Negative Positive Positive

WA 1E ur 1 47 Negative Negative Negative

Yemen 2B swab 2 49 Negative Negative Negative

WA 1E NP 1 57 Negative Positive Weak Positive

Yemen 2B swab na 58 Negative Negative Negative

Yemen 1E swab 1 69 Negative Postitive Negative

Peru 1C oral fluid 0 86 nd Postitive pos

Yemen 2B swab 4 88 Negative Negative Negative

MN 2B NP 1 143 Negative Positive Positive

CA 2B TS 3 mo (CRS) 267 Negative Positive Positive

Peru 1C oral fluid 0 267 nd Positive Positive


Ghana 1G TS na 500 nd Positive Positive

Ghana 1G TS na 500 Positive Positive Positive



Yemen 1E swab 1 1686 Weak Positive Positive Positive

WI 2B NP 7 2000 Weak Positive Positive Positive

AZ 1G NP-1 1 4536 Positive Positive Positive

ME 1E TS 1 50,000 Positive Positive Positive

Rubella RNA copy numbers in 29 clinical samples

20% were

positive for

single amplicon

100% over 100

copies were

positive for both

smaller amplicons

Rubella Genotyping Procedure

• Extract RNA from throat swab, oral fluid, or

urine using Qiagen Viral RNA kit or equivalent

• Screen for rubella RNA using real-time RT-PCR

or high sensitivity conventional RT-PCR (e.g.

RV11 RV12 RV12-2): 185 bp amplicon)

Rubella Genotyping Procedure, cont.

• If either real-time or conventional RT-PCR is

positive, set up 2 conventional RT-PCR

reactions using primers 8633/9112 (480 bp)

and 8945/9577 (633 bp).

• Analyze the products on a gel and, if positive,

clean the RT-PCR reactions and perform

sequencing reactions.

8633

9762

cMyc (30 nts)

cMyc (30 nts) 84 nts

84 nts

8812 8996

8864

9093 9176

RV E1 wt

E1∆28

E1cMyc+∆28

E1cMyc

8731 9469

RT-PCR fragment for 739-nt window

185-nt (diagnostic RT-PCR)

Rubella Virus Synthetic RNA Positive Control Constructs

Works with

both 3’ and 5’

fragments.

9577

Deletion

Insertion

12

Two Fragments for Rubella

Genotyping

NC wt PC

Fragment 1

NC wt PC

Fragment 2

The positive control (PC) contains a 30 nt insertion in fragment 1 and an 84 nt deletion in

fragment 2

Table 1. Global Distribution of Reported Rubella Genotypes#, 2005-2010@

1a 1B 1C 1E 1G 1h 1j 2B 2C

Cambodia 09 S.Africa 07,08 Chile05 Belarus05,06 Algeria07 Belarus 05,06 Brazil 05^ Argentina 08 Russia 05

Japan 08 Peru 05 China 05,06,07,08~,09,10~ Belarus 05 Kazakhstan 08~ Philippines 10~ Bangladesh 09

Kazakhstan 06 China HK SAR 08,09,10 Ghana 05,08 Kyrgystan 09 Spain 05^ Bosnia Herz 09,10

France 05~ Côte d'Ivoire 05*,08 Russia 05,06,07,08,09,10 UK 06^ Brazil 06,07,08,09

Kazakhstan 06 Kenya 05,10* USA 10^ Chile 07

Laos 09 Libya 09 China 08

Malaysia 05* Netherlands 05~ China HK SAR 08,09

Mongolia 10 Russia 05*,06,08 Dubai 09*

Poland 07,08* Sudan 05 Egypt 07*

Russia 05,06,07,08,10 Uganda 07* France 09

South Africa 08 UK 07 India 05,07,08*,10*

Sri Lanka 08 Ukraine 09 Italy 08*

Sudan 05 Japan 07^

Thailand 05, 09 Kazakhstan 08^*

Tunisia 08 Mexico 08*

UK 08 Nepal 08,09,10

Ukraine 07 Russia 09^

USA 08^ South Africa 07,08

Viet Nam 07* Spain 09

Yemen 08 Sri Lanka 08

Sudan 06

UK 06,07,08,10

Ukraine 10

USA 07^,09^,10^

Viet Nam 06*,09

Yemen 08

.

# Genotypes 1D, 1F, 1i, and 2A were inactive during this period.@ Country(ies) and year(s) of report are indicated.^ Probable import, but links unknown* Exported virus, importation countries are shown later.


1a 1B 1C 1E 1G 1h 1j 2B 2C













Sudan 05 Japan 07^



UK 08 Nepal 08,09,10





Sudan 06

UK 06,07,08,10

Ukraine 10

USA 07^,09^,10^

Viet Nam 06*,09

Yemen 08

.


1E, 1G, and 2B, had a widegeographic distribution andwere frequently found.


1a 1B 1C 1E 1G 1h 1j 2B 2C













Sudan 05 Japan 07^



UK 08 Nepal 08,09,10





Sudan 06

UK 06,07,08,10

Ukraine 10

USA 07^,09^,10^

Viet Nam 06*,09

Yemen 08

.


1a, 1B, 1C, 1h, 1j, and 2C werereported sporadically or ingeographically restricted regions.

Rubella virus genotypes reported sporadicallyor in geographically restricted regions.

(Probable imports with unknown links not shown)

1a

1B

1C

1h

1j

2C


1a 1B 1C 1E 1G 1h 1j 2B 2C













Sudan 05 Japan 07^



UK 08 Nepal 08,09,10





Sudan 06

UK 06,07,08,10

Ukraine 10

USA 07^,09^,10^

Viet Nam 06*,09

Yemen 08

.

# Genotypes 1D, 1F, 1i, and 2A were inactive during this period.@ Country(ies) and year(s) of report are indicated.^ Probable import, but links unknown* Exported virus, importation countries are shown in Table 2.

140 µl

0.5 ml

remainder

Store at -70 ° C

Extract RNA

Advantage: quick (1-2

days)

Disadvantages: limited

amount of RNA

RT-PCR and sequencing

can be difficult.

Infect cells

Disadvantage: slow (1-3

weeks)

Advantages: unlimited

amounts of RNA

RT-PCR and sequencing is

usually easy.

Sample processing for RNA extraction and virus isolation

AcknowledgementsCDC MMRHLB

• Emily Abernathy

• Dr. Min-hsin Chen

• Dr. Ludmila Perelygina

• Ada Ogee-Nwanko

• Lijuan Hao

• Reference: Confirmation of Rubella within 4

Days of Rash Onset, Journal of Clinical

Microbiology, 47(1), 2009.

39

Rubella Virus RNA Assays:

Rubella Diagnostic RT-PCR Kit

(conventional RT-PCR)

Rubella Virus Genotyping Kit

(conventional RT-PCR, 2 Fragments)

Joseph P. IcenogleInter-country Hands-on Training on the Molecular Epidemiology of Measles and Rubella Laboratory

WHO Western Pacific Region

Public Health Laboratory Centre

Hong Kong

Protocol information---Monday

a.Rubella Virus Genotyping Kit (We learned in preparing for this course that dried control RNA has instability in the Fragment 1 (5’, c-myc) region. (We are working on this!!!)

i. So, we will use the Rubella Diagnostic RT-PCR Kit control for Fragment 1ii.Positive controls

1.Fragment 12.USE 1ul of positive control of master stock from Diagnostic RT-PCR kit3.Fragment 24.Resuspend Rubella Virus Genotyping Kit positive control in 100 ul of nuclease free-water.5.USE 1 ul as positive control for rubella virus genotyping kit.6.NOTE: in your home labs, you should made 10 ul aliquots and store at – 70 C. Remove 1ul from one of these aliquots.

iii.Wild-type rubella virus1.USE 5 ul of redissolved RNA (made in Rubella Diagnostic RT-PCR kit procedure).

•We will carry through the RNA from rubella virus infected cells and control1. Templates volumes and identity are;

a.Diagnostici.5 ul negative control extracted RNAii.5 ul virus positive extracted RNAiii.5 ul control RNA (wt-rubella RNA) from CDCiv.1 ul Diagnostic Positive control from CDC (insert)

+ 4 ul nuclease free waterb.Sequencing Fragment 1

i.5 ul negative control extracted RNAii.5 ul virus positive extracted RNAiii.5 ul control RNA (wt-rubella RNA) from CDCiv.1 ul of master stock from Diagnostic RT-PCR kit

+ 4 ul nuclease free waterc.Sequencing Fragment 2

i. 5 ul negative control extracted RNAii.5 ul positive extracted RNAiii.5 ul control RNA (wt-rubella RNA) from CDCiv.1 ul of Positive Control RNA from Sequencing kit

+ 4 ul nuclease free water

1

World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

Updates of WPRO measles and rubella laboratory network

Youngmee Jee (M.D. Ph.D.)

Expanded Programme on Immunization

Western Pacific Regional Office

World Health Organization


OutlineOutline

• Regional EPI goals

• Measles and rubella incidence and vaccination, challenges and plans

• Achievements and Monitoring of performances of WPR measles labnet

• Genotyping data in WPR

• Training, meetings and plans

• Objectives of the training


Regional goalsRegional goals

• 2003: RC resolution WPR/RC54.R3: established regional goals of measles elimination, Hep B control and maintaining polio-free status

– Urged Member States to “use measles elimination and hepatitis B control strategies to strengthen EPI and other public health programmes, such as prevention of congenital rubella syndrome”

• 2005: RC resolution WPR/RC56.R8 established 2012 as the target date to achieve the “twin goals”: agreed by all member states– Achieve measles elimination

– Reduce chronic hepatitis B infection rates to < 2% in 5 year old children as an interim milestone towards final goal of < 1%

• 2010: to achieve and maintain control of rubella and prevention of congenital Rubella Syndrome in the WPR by 2015 (19th TAG)

– Rubella: ≤ 10 / 1 million population, excluding imported cases

– CRS: ≤ 10 / 1 million LBs, excluding imported casesWorld Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

Measles Elimination Progress

Measles Drops 58% in the Western Pacific Region!

Measles Down an Incredible 93% Two Years after Japan Launches Its Measles Elimination Plan!

China’s SIA Intensification Yields a Decrease of60% in Measles Cases and 64% in Deaths!

Nationwide SIA in 2010

25 Countries and Areas in the Western PacificMay Already Have Eliminated Measles!

WHO Announces Regional Verification Commissionfor Measles Elimination to be Formed!

Technical Consultation on Verification of Measles Elimination


Measles Incidence*Measles Incidence*Western Pacific Region, 2009Western Pacific Region, 2009

< 1.0 (22)

1.0 – 4.9 (6)

50.0 – 99.9 (3)

LEGEND

10.0 – 49.9 (3)

5.0 – 9.9 (2)

Source: WPRO surveillance database, 2009

* per million population


Measles Cases, by Month, China, 2005Measles Cases, by Month, China, 2005--2010*2010*

Total cases per year: 107,538 118,031 131,441136,594

C 4 8 10½ 16 26½ 31

F 3 5 26

52,461

Cumulative no. provinces conducting catch-up or follow-up (F) SIAs

40,198*Data as of 19 August 2010

**Comparison in number of cases in January-July 20010 with the same period 2009

39.0

88.8

98.4

81.4

104.0Incidence rates (per million)

0

5000

10000

15000

20000

25000

30000

35000

2005 2006 2007 2008 2009 2010

Number of cases

60% 60% 18%** 18%**

2


0

500

1000

1500

2000

2500

Jan Feb MarApr MayJun Jul Aug Sep Oct NovDec Jan FebMarAprMayJun Jul Aug Sep Oct NovDec Jan Feb Mar AprMayJun Jul

2008 2009 2010

num

ber

of c

ases

Measles Cases by Month Measles Cases by Month

Japan, 2008Japan, 2008--2010*2010*

SIA – 13 & 18 y

SIA - 13 & 18 y SIA - 13 & 18 y

*Data as of 19 August 2010

**Comparison in number of cases in January-July 20010 with the same period 2009

10,944 705Total cases By year: 312

94% 94% 56%** 56%**


Reported MCV1 and MCV2 Coverage Reported MCV1 and MCV2 Coverage

by Country/Area, Western Pacific Region 2009by Country/Area, Western Pacific Region 2009

0

10

20

30

40

50

60

70

80

90

100

BRUNIU

TOKTON

CHNVTN

HOKM

AANRU

FRPNEC

SINKOR

MOG

WAF*

JPN

AUSCAM

MACTUV

NEZPHL

CNMIM

ICAM

S*KIR

GUM*VAN

MSI

COKBLA FIJ

SOLLA

OPNG

SMA

Country

Cov

erag

e (%

)

MCV1 MCV2

* For five countries with 2009 data not available, historic coverage data were used, including AMS (2008), WAF (2007), GUM (MCV1 in 2005,

MCV2 in 2008) Source: WHO/UNICEF JRFs, 2009

A=8 B=7 C= 21

MCV1 > 95% either MCV1 or MCV2 MCV1 < 95%

MCV2 > 95% > 95% MCV2 < 95%

World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationSource: WHO/UNICEF JRFs, 2008-2009

School entry requirement (16)

No School entry requirement but

school entry check in practice (6)

No data (1)

LEGEND

No school entry requirements and vaccination check (13)

School-entry Immunization

Requirements for Measles

Western Pacific Region, 2009


Measles “Catch-Up” SIAs

Western Pacific Region,

1994-2010

2008

2003 – 2007

2009

2010

LEGEND:

None

1994 – 2002

1990-1995: measles control1996-2002: accelerated measles control2003-2012: measles elimination Source: country SIAs reports


< 1.0 (21)

1.0 – 9.9 (8)

10.0 – 19.9 (3)

LEGEND:

20-99.9 (4)

Rubella Incidence*Rubella Incidence*Western Pacific Region, 2009Western Pacific Region, 2009

Source: WHO-UNICEF Joint Reporting Forms (data for 2009); National surveillance data submitted to WPRO in 2009

* per million population


Rubella Vaccination History, WPRRubella Vaccination History, WPR

• Long standing RCV programs, protecting F & M (≥ 20 yrs) (16)

• HOK, KOR, MAC, NEZ, SIN, 11 PICs

• Long standing programs, protecting F (≥ 20 yrs) (5):

• AUS, JPN, 2 PICs (COK, FIJ), possibly Brunei Darussalam

• Using RCV > 10 years, protecting F and M up to 15 but < 20

years of age (4)

• 4 PICs (FRP, NEC, NIU, WAF)

• Recent introduction of RCV (no protection for F and M up to 15

yrs) (N=5)

• 2 PICs (TOK, NRU), CHN, MOG, PHL

• Yet to introduce RCV (N=6)

CAM, VTN (MCV1 > 80%)

LAO, PNG, SOL, VAN

Average incidence in 2007-2009: 2.3/million

Average incidence in 2007-2009: 59/million

3


Rubella Routine Immunization

Western Pacific Region, 2009

Not Offering (6)

MR (7)

MMR (23)

Type of vaccine:

30 (83%) already use RCV

MR at 8 months

MMR at 18-24 months


0

2,000

4,000

6,000

8,000

10,000

12,000

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

2008

2009

2010

AUS BRU CAM HOK JPN KOR LAO MAA MAC MOG NEZ PHL PIC PNG SIN VTN

Suspected measles cases

Discarded Clinically confirmed Lab-confirmed Epi-linked Pending

Suspected Measles Case Classification by CountrySuspected Measles Case Classification by CountryWestern Pacific Region, 2008 Western Pacific Region, 2008 -- 2010*2010*

* Data for January – August 2010


Challenges to Measles EliminationChallenges to Measles Elimination

• Coverage and surveillance performance is not homogenously high across the countries and within countries– Low sensitivity of surveillance at sub-national levels

– Accumulation of susceptibles

– Large proportion of clinically confirmed cases in some countries

• Obtaining accurate epidemiologic data– Incomplete case investigation data

– Discrepancies between lab reports and national reports

• Changing epidemiology and unexpected outbreaks

– Age distribution shifting to infants and adults

– Increased risk in densely populated areas • Importations within the WPR and from other regions, floating

populations • Resource mobilization (funding for SIAs) and political commitment

• Monitoring genotype changes

• Complexity of case classification


Plans for Measles Elimination in the Plans for Measles Elimination in the

Western Pacific Region by 2012Western Pacific Region by 2012

• SIAs in CHN, PHL, PNG, VTN in 2010; CAM & LAO in 2011

• Optimize routine schedules and approaches to minimize immunity gaps and maximize coverage

• Strengthen epidemiologic and laboratory surveillance and communication

• Engage partners and political leaders• Establish regional and national verification

committees for measles elimination by early 2011

• Advocacy for high level political commitment to mobilize human and financial resources


1

-

VIDRL,

Austral

ia

HBV

2

-

Australi

a

- Korea

CDC

IBD

9

IgM ELISA

(Panbio and in

house assays)

PRNT, HI

RT-PCR, virus

culture

Some

provincial

labs in

China(?)

6

2

-China CDC

-Korea CDC

1

NIID, Japan

JE

2

Ag capture

ELISA and

RT-PCR

sequencing

2

-Royal

Children’s

Hospital,

Australia

- Korea

CDC

Rota

393 31 provincial + 331

prefectural

laboratories in China

31

provincial

laboratories

in China

Sub

national

Molecular

method

Various

DNA typing

IgM ELISA (Siemes etc)

RT-PCR

Sequencing

Virus culture

Virus culture/

Neutralization,

ITD PCR,

sequencing

Real time PCR

Technique

s used

441238243Total No.

3116

Including 4 PIC labs in

Fiji, FP, NC, Guam

9National

142

-NIID

Japan

- Royal

Women's

Hospital,

Australia

3

- VIDRL, Australia

- CDC, China

- Public Health

Laboratory Center,

HK

2

-VIDRL,

Australia

- CDC, China

Regional

referenc

e

31

- NIID, Japan

1

- NIID, Japan

Global

specializ

ed

Total

No.

HPVMeasles/rubellaPolio (AFP)

EPI Laboratory Network in WPR

ba


WPR Measles and Rubella Laboratory Network

+331 prefectural labs

1 GSL

3 RRLs16 National (13 fully functional)31 provincial

+ 331 prefectural

Accreditation48 labs

382 laboratoires

4


IgMIgM testing Workload (except China and Japan)testing Workload (except China and Japan)

based on lab reports: 2006based on lab reports: 2006--2010*2010*

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

2006 2007 2008 2009 2010*

No. of specimens

Pending/Unknown

Positive

Equivocal

Negative

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

2006 2007 2008 2009 2010*

No. of specimens

Pending/Unknown

Positive

Equivocal

Negative

Measles IgM Rubella IgM

* Data for January – October 2010China2008: 96,664 samples tested for measles from sporadic cases

2009: 39,459 samples2010; 18,217 samples (Jan-May)

Vietnam outbreak

Philippines outbreak


• Accreditation status

All GSL and RRLs, 10/13 national labs including Fiji excluding

three PIC labs, all 31 provincial labs in China are accredited as of Nov 22, 2010

• Monthly case-based laboratory reporting

Introduced in 2008, As of Nov 2010, 15/17 labs except China

and Japan

• Confirmatory testing in all NMLs: twice a year from 2010,

concordance rates in 2009: >90%-100%

• All 49 laboratories receiving WHO global PT samples passed

WHO proficiency test. Most lab scored 100% (100% for 16/18+31/31=47/49 labs, 95% two labs for both measles and

rubella)

• Genotyping data on recent measles virus strains available from

most countries except PNG, Fiji and other PICs

WPR Measles/rubella WPR Measles/rubella LabNetLabNet

UpdatesUpdates


• Regional Laboratory Training/Hands-on workshop in October 2009 for priority countries :ELISA/DBS, virus culture, moleculardetection

• Financial support to cover operational costs (TSA) and kits and some equipment support for the NMLs in priority countries from 2008

• China labnet

– implemented excellent QA programme including PT and confirmatory testing for prefectural labs as well as for provincial labs :WHO global PT samples to 31 provincial labs from 2009 and all scored 100%

– Most provincial labs perform virus isolation using Vero/SLAM cells and RT-PCR

– In 2009-2010, all provincial labs implemented real time PCR for measles, rubella and mumps

– Annual meetings and hands on training courses for 31 provincial labs organized by China CDC

– Tibet has been on site reviewed in 2010 for the first time in 2010-> all 31 provincial labs in China has been accredited!

WPR Measles/rubella WPR Measles/rubella LabNetLabNet

UpdatesUpdates


Summary of 2010 Measles/Rubella Lab testingSummary of 2010 Measles/Rubella Lab testing

D9


Completeness and Timeliness* of Measles Lab ReportingCompleteness and Timeliness* of Measles Lab Reporting

Western Pacific Region, 2008 Western Pacific Region, 2008 –– 2010**2010**

* Deadline for submission is on the 10th of the following month** Data for January – October 2010, as of Nov 16 2010


1j(2) VI samples

1E (2010)

21

1E

22B2B

22

2

Genotypes

2007: 11/12 (91.67%)2008: 20/22 (95.23%)

2009:14/15(93.3%)

2008: /1616 (100%)

2010: 80/80(100%)2009:29/30(96.7%)2008: 45/45 (100%)2007: 9/10 (90%)2006: 3/4 (75%)

2009: 15/16 (93.75%)2008: 6/6 (100%)2007: 9/9 (100%)2006: 7/7 (100%)

2007: 56/57 (98.24%)2008: 15/15 (100%)2009: 9/10 (90%)

2008-2010: 65 (100%)

2007: 30/30 (100%)2009: 10/10 (100%0

2005-2006: 9/10 (90%)

2007: 17/18 (94.44%)2008: 56/62 (90.32%)

2009: 50/50(100%)2010: 60/60(100%)

2007: 7/15 (46.67%)2008: 8/15(53.34%)2009: 61/61(100%)

2009-2010: 29/31(93.5%)

2009:18/20(90%)

2006: 12/15 (80%)2008: 75/80 (93.75%)2009: 60/60 (100%)

2009-2010:70/70 (100%)

2006: 14/20 (70%)2008: 80/80(100%)

2009: 68/70(97.1%)

2006: 12/14 (85.71%)2009:35/37 (90%)

2007: 45/50 (90%)2008: 77/86 (89.53%)2008-9: 65/65 (100%)

2009-2010: 156/156(100%)

Measles

D9(2)D4 (2), D9(1)

G3(1)

D9, H1

D9,, G3

H1, D4D9G3

H1(2008 and 2009)

D9(2) VI samples

G3(1) VI samplesG3

H1

D9H1

H1H1

H1H1

H1

D9

Genotypes

HK2008: 35/35(100%) Macao

2007: 14/21 (66.67%)2008: 42/51 (82.35%)

2009(100%)

2010:60/60(100%) 2009: 30/30(100%)

2008: 44/45 (97.78%)2007: 8/10 (80%)

2006: 4/6 (66/67%)

2007: 7/7 (100%)2006: 19/19 (100%)

2007: 32/48 (66.67%)2008: 15/20 (75%)

2009: 10/10%2008-2010:82(97.6%)

2007: 10/10 (100%)2009: 10/10 (100%)

2007: 20/20 (100%)2008: 57/60 (95%)

2009:47/48 (98%)

2010”60/60(100%)

2007: 7/8 (87.5%)2008: 7/8 (87.5%)2009: 62/65 (97%)

2009-2010:58/58(100%)

2009:17/20(85%)

2006: 9/15 (60%)2008: 62/70 (88.57%)

2009: 72/72 (100%)2009-2010:50/50(100%)

2006: 11/15 (73.33%)2008: 72/80(90%) 2009: 48/50(96%)

2006: 13/14 (92.86%)2009: 37/37(100%)

2007: 39/45 (86.67%)2008: 102/110 (92.72%)2008-9: 68/70(97.1%)

2009-2010: 64/70 (91.4%)

Rubella

HK

HKAustralia

Australia

HK

JapanHK

Australia

Australia

HK

HK

Australia

HK

HK

Australia

HK

RRL

Singapore

Philippines

New Zealand

Mongolia

Korea

Malaysia

Laos

PNG

Vietnam HCM city

Vietnam Hanoi

Fiji

Cambodia

Confirmatory testing

5


China

H1; 232

D9; 1

D4; 1

D11; 1

From France: Shanxi

From Thailand: Sichuan Korea

H1; 1B3; 1

Mongolia

H1, 1

JapanH1, 1

D5, 1

Hong Kong

A; 4H1; 5

D9; 1

D8; 1

B3; 1

Vietnam

H1. 20

Laos

D9, 6

Philippines

D9, 9

G3, 2

Cambodia

D9; 3

H1; 1

Malaysia

D9, 2

G3, 1

Singapore

H1, 2

G3, 3D8,

1

D9, 8 Australia

A; 3

D4; 9

D8; 18

D9; 1

H1; 12

New Zealand

D8; 1

H1; 3

D4; 2

H1 Mongolia (2008-9)

B3 importation from Lybia 2009, 2010 H1

Japan D5, H1, D9 (2010)

HK D9 (Philippines), D8, B3 (South Africa) in 2010

Philippines D9 strains (9)in 2009-2010, G3 (2) from 2009 samples

Cambodia-one H1 imported from Vietnam (1)

Malaysia 2 D9 and 1 G3 (2009)

Singapore G3(1),D9(1), D8(1), H1(1) in 2009

G3(2), D9 (7), H1 (1) in 2010

D4, H1, B3 and D8

AB3D4D5D8D9D11H1G3

Genotypic Distribution of Measles viruses in WPR (2009-10) World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

PT onlyNLGuam

not participating NL

New Caledonia

PT onlyNLFrench Polynesia

Yes(Hk)Yes2B2BH1H1H1AccreditNLVietnam, HCMC

Yes (HK)Yes222H1H1H1H1AccreditNLVietnam, Hanoi

YesYesD9, G3, H1D9, D8, H1, G3D4, D9D5, D9 AccreditNLSingapore

Yes(HK)Yes1jD9D9, G3D9, G3AccreditNLPhilippines

NA Yes (started in

2010)Pending NLPNG

Partly(Aus)YesD8

D4, B3,

H1D9AccreditNL

New

Zealand

Yes (HK)Yes1EH1H1AccreditNLMongolia

Yes (starting)YesG3D9, G3D9AccreditNLMalaysia

Yes (HK)YesH1, D9AccreditNLMacao

Yes(HK)Yes (started in

2010)1E2H1D9H1Pending NLLaos

Yes (starting)YesH1B3H1, D5AccreditNLKorea

Yes(Aus)YesAccreditNLFiji

Yes (HK)Yes (stopped &restarted )1a2D9D9, H1PendingNLCambodia

YesYes1E1E1ED9, H1, D8,

B3, D5

H1, (D9,

D8, B3)H1, D9H1, D9AccreditedRRLHK (PHLC)

YesNo1E1EH1, d11, D9 H1, (D4, D9, d11)H1H1AccreditedRRLChina (CDC)

YesYes

B3, D4,D8, H1, D9

D8, D4, D9, H1

D4, D5, D8, D9, H1

D4, D5, D8AccreditedRRL

Australia (VIDRL)

NIID receives data from prefecturesNo2B

D5, H1, D9, D8

D5, D9, D8D5, D4D5 AccreditedGSL

Japan (NIID)

20102009200820072010200920082007

Genotype

reporting

Monthlylab

Reporting

RubellaGenotypesMeaslesGenotypesAccreditati

on in 2010 Country

Unpublished data, Not for Circulation


Summary of Summary of VirologicVirologic Surveillance and Surveillance and

genotyping of Measles in WPRgenotyping of Measles in WPR

• Recent genotype information is available for most countries in the region and HK, China, Singapore, Australia and Japan share genotype information with WHO, Genotype information for other countries (Cambodia, Laos, Mongolia, Philippines, Vietnam) reported by Hong Kong RRL

• Many national and China sub-national labs as well as RRL, GSL have laboratory capacities to perform PCR, sequence analysis and virus isolation

• HK RRL conducted genotyping/sequencing of measles and rubella viruses using confirmatory serum samples for Vietnam, Cambodia, Laos, Philippines, Mongolia, Malaysia and Macao.

• Endemic transmission of H1 strains in China and Vietnam• D9 strains detected in many countries including Malayisa, Singapore,

Philippines, Cambodia and Laos in 2009-2010

• Measles outbreaks in 2009-2010: H1 from 2009-2010 outbreaks in Vietnam and D9 from 2010 outbreaks in Philippines


Monitoring of Annual Accreditation


33rdrd Regional Hands on training for Regional Hands on training for

measles and rubella labs in Oct 2009measles and rubella labs in Oct 2009

• Laboratory Hands on training course in HK China in October 2009 for priority countries

– Participants from China, Cambodia, Laos, Malaysia, Mongolia, Philippines, Vietnam, Fiji

– ELISA/DBS, cell culture, molecular detection of measles and rubella viruses

– Facilitators from US CDC, NIID and WHO


Measles Rubella Session during 2Measles Rubella Session during 2ndnd

VPD VPD LabnetLabnet meetingmeeting

• Two day meeting in Feb 25-26 2010

• All NML, RRL, GSL attended: participants from 17 network laboratories and National surveillance/EPI officers

• Discussed how to strengthen the quality of the performances of measles/rubella laboratory networks and to enhance the regional capacity for virus isolation and genotyping

6


3) Molecular surveillance

As recommended by the Global Measles Laboratory Network Meetings (Sep 2008 and Oct 2009), laboratories are encouraged to submit representative genotype/sequence information on their measles and rubella viruses to the WHO genotype and MeaNS databases, preferably on a “real-time” basis, but at least by the end of the month in which the genotyping wascompleted. A copy of the information should be sent also to the regional laboratory coordinator.

a. Laboratories which participated in the regional hands on laboratory training in 2009 and other sequencing laboratories, are encouraged to perform virus isolation and/or molecular detection of measles and rubella viruses to identify the genotype and obtain sequence information on circulating measles and rubella viruses.

b. Efforts should be made to collect sequence data from all chains of infection, especially in those countries where no baseline sequence data exists.

c. The LabNet should utilize the well validated tools and samples available for enhancing molecular surveillance where appropriate, such as; i. Oral fluid, throat swabs, urine and PBMC as samples for virus detectionii. Detection of viral RNA in archival sera

iii. Standardized PCR methods including the use of a validated, unique PCR control.

Recommendations from 2nd Recommendations from 2nd

LabnetLabnet meeting, 2010 Febmeeting, 2010 Feb


Priority indicators for components of the verification process Priority indicators for components of the verification process

for measles elimination in WPR: Laboratoryfor measles elimination in WPR: Laboratory

√Timely sharing laboratory testing data

with WPRO and genotype data within

2 months with WPRO & WHO HQ

√Baseline virologic (genotype) surveillance

data available

√100% concurrence of lab-confirmed cases

between surveillance and lab units

√>80% outbreaks with virologic and

genotypic analysis

√>80% cases confirmed at WHO accredited

labs

√All WHO measles labs accredited

ComplementaryCoreCriterion

Technical Consultation meeting for verifying measles eliminationTechnical Consultation meeting for verifying measles elimination --June 2010June 2010


Plans for Plans for LabnetLabnet in 2010in 2010--2011 2011 • Building up further capacity for virologic surveillance in the region:

Follow up Hands on training in Nov 22-27, 2010

• Encourage collection of virus isolation samples from all chains of transmission, both sporadic and outbreaks

• Regular collection of measles (and rubella) genotypic information from all countries in the region by 2010

• Collection of measles virus genotype information from remaining countries: baseline genotype information using stored serum samples

• Review and accreditation of 3 pending laboratories by early 2011

• Quality assurance: confirmatory testing at least twice a year

• Reporting, communication, information sharing– WPRO Measles/rubella bulletin to include laboratory testing data

including genotype data

– Improve completeness and timeliness (10th) of case based laboratory reporting and genotype reporting for measles/rubella viruses

– Meeting and training workshopWorld Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

MeaslesGlobal SpecializedNational Institute of Infectious Diseases, Japan

Regional ReferenceVictorian Infectious Diseases Reference Laboratory, AustraliaCDC, ChinaPublic Health Laboratory Center, HK

National1. National Centre for Communicable Diseases (NCCD), Mongolia2. National Institute of Health, Korea CDC, Republic of Korea3. National Institute of Hygiene and Epidemiology, Hanoi, Vietnam4. Pasteur Institute, Ho Chi Minh City, Vietnam5. National Public Health Laboratory, Malaysia6. National Institute of Public Health, Cambodia7. National Center for Laboratory and Epidemiology, Laos8. Singapore General Hospital, Singapore9. Research Institute for Tropical Medicine, Philippines10. Canterbury Health Laboratories, New Zealand11. Laboratory de Saude Publica, Macao12. Central Public Health Laboratory, PNG 13. Public Health Laboratory-Mataika House, Fiji 14. Institute Louis Malarde, French Polynesia15. Department of Public Health and Social Sciences, Guam16. Institute Pasteur de Nouvelle-Caledonie, New Caledonia& 31 Provincial and 331 prefectural labs in China

Acknowledgements

WHO HQ David Featherstone

US CDC Paul Rota

US CDC Joe Icenogle

Xu Wenbo and CCDC team

Wilina Lim and HK PHL team

WPRO EPI Measles teamWHO country EPI officers


Objectives of trainingObjectives of training

• To enhance knowledge and skills in:(a) molecular detection and genotyping of measles and rubella viruses (RT-PCR and sequencing)(b) sequence analysis of measles and rubella viruses

• To discuss the regional data management using the new laboratory reporting format and sharing and reporting genotype and sequence data


Day 1, MondayDay 1, Monday

• Session 1: Lectures on Global and regional update on measles, rubella and molecular epidemiology

Lunch

• Session 2: two practicals - 1) RNA extraction and 2) RT-PCR for rubella detection and genotyping

7


Day 2, TuesdayDay 2, Tuesday

• Session 3: Practicals1) RT-PCR for measles genotyping and 2) gel electrophoresis of rubella detection &

genotyping RT-PCR 3) gel electrophoresis of RT-PCR for measles

genotyping4) PCR purification of RT-PCR for measles and

rubella genotyping 5) gel electrophoresis of purified PCR products

(measles and rubella)

Country reports (China, Japan, Malaysia, Mongolia, New Zealand) -Can be moved to Thursday


Day 3, WednesdayDay 3, Wednesday

• Session 4: Practicals- Cycle sequencing, purification of cycle sequencing products and demonstration of sequencing run

Country reports (Philippines, Korea, Singapore, Viet Nam NIHE and PI)


Day 4, ThursdayDay 4, Thursday

• Session 5: Practical - Sequence analysis for measles virus genotyping

Country reports


Day 5, FridayDay 5, Friday

Session 6: Data management and reporting- Monthly laboratory data to Western Pacific Regional Office (WPRO)

- Genotype and sequence data submission to WHO HQ and Means

Session 7: Practical: Sequence analysis for rubella virus genotyping


Day 6, SaturdayDay 6, Saturday

• Course assessment and quiz

• Next steps, summary of assessment and quiz

• Distribution of MR IgM ELISA proficiency test panel samples (reporting within 14 days) and filter paper proficiency test panel (reporting within 2 months)

• USB with all presentation files and reference materials

• Closing and presentation of certificates


Measles outbreaks and Measles outbreaks and

importations in 2009importations in 2009--2010 2010

Vietnam, Philippines Vietnam, Philippines

New Zealand, AustraliaNew Zealand, Australia……..

8


Measles Cases by Month Measles Cases by Month

2007 2007 –– 2010*2010*

*Data as of 19 August 2010.

Source: Measles cases refer to laboratory or epi-linked confirmed cases.

0

100

200

300

400

500

600

700

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ≥

40

age in years

nu

mb

er

of

case

s

0

100

200

300

400

500

600

700

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ≥

40

age in years

nu

mb

er

of

case

s Measles Cases by AgeMeasles Cases by Age

Oct 2008 Oct 2008 –– Jul 2010**Jul 2010**

Viet Nam

0

500

1,000

1,500

2,000

2,500

Jan

Feb

Mar Apr

May Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar Apr

May Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar Apr

May Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar Apr

May Jun

Jul

2007 (N=26) 2008 (N=113) 2009 (N= 6473 ) 2010 (N= 1034 )

num

ber

of ca

ses


Measles Cases in Viet Nam Measles Cases in Viet Nam

Oct 2008 Oct 2008 –– July 2010*July 2010*Oct-Dec08 Jan-Jun09 Jul-Dec09 Jan-Jul10

North

South * Measles cases refer to laboratory or epi-linked confirmed cases. Data as of 20 August 2010.


Measles Measles IgMIgM Positive CasesPositive Casesby Epidemiologic Week, Viet Nam, 2009by Epidemiologic Week, Viet Nam, 2009 --2010*2010*

Source: Measles-Rubella Lab Reports * Data received as of 21 June 2010

0

50

100

150

200

250

300

350

400

450

1 2 3 4 5 6 7 8 910

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53 1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

2009 2010

Number of cases

North SouthWorld Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

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 26 27 28 29 ≥30

age in years

Num

ber

of Case

s

0

50

100

150

200

250

300

350

400

450

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

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53 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

26

27

28

29

2009 2010

epidemiologic week

num

ber o

f cas

es

Laboratory Epi-linked Clinically

Confirmed Measles Cases, by Week of Rash Onset

(2009-2010) Philippines 2010*

Lab Confirmed and Epi-linked Measles Cases, by Age

1 dot = 1 case

ORI

* Data as of 12 August 2010


0

200

400

600

800

1000

1200

1400

1600

J F M A M J J A S O N D J F M A M J J A S O N D J F M AM J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M

Year and Month

num

ber

of c

ases

National SIA, Feb and Mar 04 (9m-7yr) National SIA, Oct -Dec 07

(9m-4yr)

2004 2006 2007 2008 200920052003 2010

* Reports through 2 June 2010

Confirmed Measles Cases, by Month of Rash Onset, Philippines 2003 - 2010*

D3

D9, G3

D9

D9

Reintroduction


0

10

20

30

40

50

60

70

80

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Apr

2007 (N=7) 2008 (N=8) 2009 (N=190) 2010

(N=25)

num

ber o

f cas

es

† Data as of 11 June 2010* Lab confirmed and epidemiologically linked

Measles Cases,* by Month of Onset, New Zealand 2007 - 2010†

Unpublished Data from MOH, New Zealand (David Wansbrough)

imported

imported

9


Genotyping Results of Measles viruses in 2009: Genotyping Results of Measles viruses in 2009:

New ZealandNew Zealand

Weak positive on Realtime PCRPCR NegativeND24.09.09P8106Nasal

CHCH outbreak strain1/10/2009D421.09.09P8053NP

Most likely Edmonston (vaccine) strain16/09/2009A09.09.09P7820TS+NP

Weak positive on Realtime PCRPCR Negative ND09.09.09P7816NP

Most likely Edmonston (vaccine) strain9/09/2009A25.08.09P7435Urine

Weak positive on Realtime PCRPCR Negative ND22.08.09P7377NP

Most likely Edmonston (vaccine) strain27/08/2009A21.08.09P7364NP

ChCh outbreak25/08/2009D419.08.09P7292NP

Invercargill (unable to type)PCR Negative ND10.08.09P7178NP

Most likely Edmonston (vaccine) strain25/08/2009A12.08.09P7168Nasal

Auckland20/08/2009D408.08.09P7084NP+Throat

ChCh outbreak5/08/2009D425.07.09P6808Nasal + Throat

ChCh outbreak5/08/2009D421.07.09P6695NP + Throat

ChCh outbreak5/08/2009D419.07.09P6645Nasal

ChCh outbreak5/08/2009D415.07.09P6601NP

ChCh outbreak5/08/2009D408.06.09P5943Pernasal

ChCh5/08/2009D810.03.09P4811NP

Dunedin outbreak5/08/2009H112.02.09P4504Throat

Dunedin outbreak5/08/2009H128.01.09P4325Urine

CommentsSequence runGenotypeDateVirus No.Sample

103 measles IgM positive cases in 2009, 19 genotyped

Courtesy Dr. Anja Werno and New Zealand Crown Public Health World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

Measles Virus Importation, Australia 2008-2009

WA

NT

Qld

NSW

Vic

Tas

SA

India

D8 ,D4, D4*

D9, D8, D4, H1

D8, H1

Iran

H1

??

VietnamH1, H1* Thailand

D4, D5

NEZ

MalaysiaG3, D9

PhilippinesD9

UKD4D8

ChinaH1

USAD8

IndonesiaD9

H1, D4

D9

D9, D5

H1

Virus has entered Australia from at least 13 countries in all 6 Regions

S. Africa


Measles Cases, Australia 2009*

Imported cases Import-related cases* Data as of 11 June 2010

9

19

24

1

1

3

2

1

1

0

5

10

15

20

25

30

D4 D8 D9 H1 Unknown

India

India

New Zealand

India

UK

UK

Philippines Iran

Viet Nam

Viet Nam

New Zealand

China

France

India

Indonesia

Indonesia/Korea

Philippines

S. Africa

India

Thailand

US

Viet Nam

Viet Nam

104 cases = 4.9/million-34 importations from 13 countries in 6 WHO Regions70 cases = 3.3/million

-62 import-related cases

8 sporadic cases = 0.4/million


Measles Cases, by Month of Rash Onset and Method of Confirmation, Cambodia 2003 – 2010*

* Data through May

Expected suspected measles cases: > 296No. AFR cases reported: 4,779 in 2009

Expected suspected measles cases: > 296No. AFR cases reported: 4,779 in 2009

Reporting AFR cases

Discarded measles

rate: 26.4 per

100,000 population

86.5% measles cases

confirmed clinically

2009

Phased national SIAs

Dec 2000-May 2004

(9m – 14yr)


2010 Cambodia 2010 Cambodia

13228057235023195037722522350

December

November

191110741111October

5701388077118888September

422932361019432138236August

31836192316623192192July

02556261322137261261June

03152317723080317317May

02282230016169230230April

03939402034062402402March

038810398037523398398February

02105215017936215215JanuaryCambodia

Rubella IgM

equivo

cal

Rubella IgM

(-)

Rubella IgM

(+)

Sample Tested

for

Rubella

IgM

Measles

equiv

ocal

Measles IgM

(-)

Measles

IgM

(+)

Samples tested

for

measles

IgM

Total Number of

samples

receivedMonthCountry


Atypical Measles outbreak (H1) among students Atypical Measles outbreak (H1) among students

with high vaccination coverage in one middle with high vaccination coverage in one middle

school in 2010 in school in 2010 in IncheonIncheon, Korea , Korea

69 vaccinated out of 71 lab confirmed cases

Unpublished Data from Korea CDC

10


2.3**2.81.62.5> 2 per 100 000National reporting of discarded measles cases

47%25%48%83%<10%Proportion of clinically confirmed measles cases

94%

96%

72%

43%

43%

34.0

2009

NA

NA

70%

27%

31%

43.5**

2010*

88%

93%

62%

47%

27%

81.6

2008

92%> 95%National MCV1 coverage

86%> 95%National MCV2 coverage

High Population Immunity

65%> 80% % of suspected cases with adequate blood specimens

27%> 80%% of suspected cases with adequate investigation

31%> 80%% of districts reporting ≥ 1/100 000 discarded measles cases

High Quality Surveillance

73.7Confirmed measles cases (confirmed by lab, epidemiologic linkage or clinically)

Incidence (per million population)

2007TargetCategory

Progress Towards Measles Elimination

Western Pacific Region 2007-2010*

* Data as of 19 August 2010. ** Annualized rates

Source: WPRO surveillance databaseWorld Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization

Technical Consultation on Technical Consultation on

Verification of Measles EliminationVerification of Measles Elimination• Recommendations from this consultation referred to the

19th TAG in Aug 2010

• TAG requested RD to seek Regional Committee endorsement of

– Renewed commitments by Member States to achieve measles elimination by 2012

– Establishment of regional and national verification committees for measles elimination

– Use of measles elimination activities to accelerate rubella control and CRS prevention


51%

19%

61%

78%

47%

81%85%

94%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

2007 2008 2009 2010 2007 2008 2009 2010

Completeness Timeliness

Completeness and Timeliness of Country Reporting

to the Western Pacific Regional Office, 2007-2010*

•Data as of 19 August 2010. Source: WPR surveillance database


Recommendations from 2Recommendations from 2ndnd LabnetLabnet

meeting, 2010 Febmeeting, 2010 Feb

1) Data reportingCase based measles and rubella laboratory data reporting should be fully implemented in all network laboratories. Data should be reported to the regional office on a monthly basis by the 10th day of the month. The new data reporting format in MS Access discussed at the meeting should be adopted by all network laboratories. . Countries where case based reporting is not feasible should also make every effort to share aggregated data with WPRO in a format and frequency agreed upon with the regional office.

2) Confirmatory testingThe confirmatory testing mechanism of serum samples established in the region should be maintained and it is recommended that national laboratories send a representative 10% of samples or a minimum of 15 samples to the designated regional reference or global specialized laboratories, at least annually but preferably twice a year. A table including a linelist of the samples and the raw data (OD readings) obtained by the national laboratory should be included with the shipment. Before sending samples, the national laboratories should notify and consult with the regional laboratory coordinator to confirm the number and selection of samples to be sent.


Recommendations from 2nd Recommendations from 2nd

LabnetLabnet meeting, 2010 Febmeeting, 2010 Feb

4) Quality assurance of commercial laboratories

In countries where most measles and rubella IgM testing is performed in private/commercial laboratories, it is vital that the performance of private/commercial laboratories be monitored. Performance of these laboratories may be assessed through an external quality assurance programme and pre-existing quality assurance data should be assessed by the national laboratories, where possible.

5) Communications

Laboratory and immunization/surveillance colleagues are encouraged to have regular interactions and meetings to ensure classification of suspected cases and to harmonize laboratory and surveillance data.

6) TrainingsFollow-up hands on training courses focusing on molecular

detection of measles and rubella viruses are encouraged in the region.

1

Importance of Molecular Epidemiology Importance of Molecular Epidemiology

and Timely Reporting of Genotype and Timely Reporting of Genotype

InformationInformation

The 4th Regional HandsThe 4th Regional Hands--On Training Workshop On The On Training Workshop On The

Laboratory Diagnosis Of Measles And Rubella Focusing On Laboratory Diagnosis Of Measles And Rubella Focusing On

Molecular DiagnosisMolecular Diagnosis

WHO Vaccine Preventable Disease Lab Network

David Featherstone

EPI / IVB WHO/HQ

22 to 27 November 2010Public Health Laboratory Centre, Hong Kong (China)

Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance Power of molecular surveillance

Sequence information, in combination with Sequence information, in combination with epidemiological data can:epidemiological data can:

–– Allow mapping of transmission pathways Allow mapping of transmission pathways

–– Identify possible source of virus and determine Identify possible source of virus and determine whether it is indigenous or from an imported whether it is indigenous or from an imported sourcesource

–– Determine whether suspected case due to Determine whether suspected case due to vaccine or wild virusvaccine or wild virus

–– Assist with confirmation of true positives Assist with confirmation of true positives

–– Improve diagnostic resolution in first few days Improve diagnostic resolution in first few days after rash onset in combination with IgM after rash onset in combination with IgM (especially for rubella)(especially for rubella)


Capacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasingCapacity for Molecular testing increasing

Molecular capacity has become accessible to more Molecular capacity has become accessible to more labs labs –– National and even some subNational and even some sub--national labs have PCR national labs have PCR equipm'tequipm't

–– RealReal--time and conventional PCR time and conventional PCR

Specific training workshops focusing on molecular Specific training workshops focusing on molecular techniques held in all WHO regionstechniques held in all WHO regions

Kit based RTKit based RT--PCR systems increase ease of usePCR systems increase ease of use

Oral fluid samples may permit detection of measles Oral fluid samples may permit detection of measles and rubella RNA up to several weeks after disease and rubella RNA up to several weeks after disease onsetonset


Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and Identifying the geographical origin and

tracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virustracing the transmission pathways of a virus

Building comprehensive knowledge of global Building comprehensive knowledge of global distribution of virus molecular datadistribution of virus molecular data–– Source of virus in new outbreaks may be determinedSource of virus in new outbreaks may be determined

Sequence information alone may not be sufficient Sequence information alone may not be sufficient to identify source or transmission pathwaysto identify source or transmission pathways–– Epidemiological investigations can also identify possible Epidemiological investigations can also identify possible source of origin and/or transmission pathways source of origin and/or transmission pathways

Combination of epidemiological and molecular data Combination of epidemiological and molecular data idealideal–– Outbreak investigation with comprehensive laboratory Outbreak investigation with comprehensive laboratory analysisanalysis


The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2008. All rights reserved

Measles D4 (Enfield strain) distribution March 2007 Measles D4 (Enfield strain) distribution March 2007 Measles D4 (Enfield strain) distribution March 2007 Measles D4 (Enfield strain) distribution March 2007 –––– June 2010June 2010June 2010June 2010

(Stars may not represent exact location of the case)(Stars may not represent exact location of the case)(Stars may not represent exact location of the case)(Stars may not represent exact location of the case)

Acknowledgements:

WHO Measles LabNet espHPA, Lux, RKI & CDC

Measles Surveillance Programmes

As of 23 July 2010

Mar 07-June10

Oct 08

Apr 07Apr 07

Jan 08Jan 08

Aug 07

Feb 08

Mar-May 08

Aug 08

Jun-Jul 08

Aug 08

Oct 08

Jun-Jul 08

Jun-Jul 08 Mar-May 08

Nov 07

XX 08 (JAM)

Jun 08 (CA)

Feb 08

Apr 08 x3

Dec 08

Jan 09 (CA)

Dec 08 (MA)

Dec 08 (IL)

May 2010

Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination* Indicators for determining Elimination*

DEFINITIONS:DEFINITIONS:DEFINITIONS:DEFINITIONS:DEFINITIONS:DEFINITIONS:DEFINITIONS:DEFINITIONS:

Measles eradication. Measles eradication. Measles eradication. Measles eradication. Measles eradication. Measles eradication. Measles eradication. Measles eradication. Worldwide interruption of measles Worldwide interruption of measles transmission in the presence of a verified, transmission in the presence of a verified, wellwell--performing surveillance systemperforming surveillance system..

Measles elimination. Measles elimination. Measles elimination. Measles elimination. Measles elimination. Measles elimination. Measles elimination. Measles elimination. The absence of endemic measles The absence of endemic measles transmission in a defined geographical area (e.g. transmission in a defined geographical area (e.g. region) for a period of at least 12 months or more, in the region) for a period of at least 12 months or more, in the presence of a presence of a wellwell--performing surveillance systemperforming surveillance system. .

Endemic measles transmissionEndemic measles transmissionEndemic measles transmissionEndemic measles transmissionEndemic measles transmissionEndemic measles transmissionEndemic measles transmissionEndemic measles transmission. The existence of . The existence of continuous transmission of indigenous or imported continuous transmission of indigenous or imported measles virus measles virus that persists for that persists for a period of 12 months or a period of 12 months or moremore in any defined geographical area.in any defined geographical area.

* WER 3 Dec 2010

2

Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination Indicators for determining Elimination

ReReReReReReReRe--------establishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmissionestablishment of endemic transmission. Re. Re--establishment of establishment of endemic measles transmission is a situation in which endemic measles transmission is a situation in which epidemiological andepidemiological and laboratory evidence indicates laboratory evidence indicates the the presence of a presence of a chain of transmission of a virus strainchain of transmission of a virus strain22 that that continues uninterrupted for a period of twelve months or more continues uninterrupted for a period of twelve months or more in a defined geographical area where measles was previously in a defined geographical area where measles was previously eliminated. eliminated.

Measles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goalMeasles outbreak in countries with an elimination goal. When . When two or more confirmed cases are temporallytwo or more confirmed cases are temporally--related (with 7related (with 7--21 21 days between dates of rash onset), and are epidemiologically days between dates of rash onset), and are epidemiologically and/or and/or virologicallyvirologically linkedlinked..

A measles imported case.A measles imported case.A measles imported case.A measles imported case.A measles imported case.A measles imported case.A measles imported case.A measles imported case. Is a case exposed outside the Is a case exposed outside the region/country during the 7 to 21 days prior to rash onset, as region/country during the 7 to 21 days prior to rash onset, as supportedsupported by epidemiological and/or virological evidence.by epidemiological and/or virological evidence.

22 Viruses with N gene (450) sequences that are at least 99.7% ideViruses with N gene (450) sequences that are at least 99.7% identical (1 nt change)ntical (1 nt change)


Rationale behind development of Rationale behind development of Rationale behind development of Rationale behind development of Rationale behind development of Rationale behind development of Rationale behind development of Rationale behind development of

genotype/sequence databasesgenotype/sequence databasesgenotype/sequence databasesgenotype/sequence databasesgenotype/sequence databasesgenotype/sequence databasesgenotype/sequence databasesgenotype/sequence databases

Labs with sequence data not always aware of Labs with sequence data not always aware of importance of sharing with surveillance programmeimportance of sharing with surveillance programme

–– Data sometimes released months or years after sample Data sometimes released months or years after sample collected collected

–– Data sometimes kept until publication finalizedData sometimes kept until publication finalized

–– Countries and regions at different stages of Countries and regions at different stages of control/eliminationcontrol/elimination

�� Elimination countries need to know whether cases detected Elimination countries need to know whether cases detected are due to imported or indigenous virus ASAPare due to imported or indigenous virus ASAP

�� Control countries need to know their baseline sequence dataControl countries need to know their baseline sequence data

–– Centralised databases will allow ready access to all Centralised databases will allow ready access to all reported viruses reported viruses

Decision to develop database for Decision to develop database for Decision to develop database for Decision to develop database for Decision to develop database for Decision to develop database for Decision to develop database for Decision to develop database for

Sequences/GenotypesSequences/GenotypesSequences/GenotypesSequences/GenotypesSequences/GenotypesSequences/GenotypesSequences/GenotypesSequences/Genotypes

Open AccessOpen AccessRestricted Restricted

Access Access Restricted AccessRestricted Access

Sequence Sequence

datadataSequence and epi Sequence and epi

datadataGenotype and epi Genotype and epi

data data

GenBank access GenBank access

No.No.

GenBankGenBank2008 MeaNS 2008 MeaNS

Sequence Sequence

Database (HPA) Database (HPA)

2006 WHO 2006 WHO

Genotype Genotype

DatabaseDatabase

DatabasesDatabasesDatabasesDatabasesDatabasesDatabasesDatabasesDatabases

Decision for two databases at 2006 Global meeting:Decision for two databases at 2006 Global meeting:–– Genotype database WHO database Genotype database WHO database –– Simple, Excel basedSimple, Excel based

–– Sequence database MeaNS Sequence database MeaNS

Issues Issues –– Accessibility to sequence database Accessibility to sequence database

–– Accessibility to nonAccessibility to non--public domain sequence datapublic domain sequence data

–– Frequency of updating data Frequency of updating data

�� More timely = more useful for programmatic application, More timely = more useful for programmatic application,

�� Preferably real time accessPreferably real time access


Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype Variables for WHO Genotype

DatabaseDatabaseDatabaseDatabaseDatabaseDatabaseDatabaseDatabase

ISO country code and country name ISO country code and country name

Date of case: (month, year)Date of case: (month, year)

WHO name of virus: e.g. WHO name of virus: e.g. MVi/Guizhou.CHN/47.09/1[H1] MVi/Guizhou.CHN/47.09/1[H1]

GenotypeGenotype

Epi link (if known)Epi link (if known)

Submission data (who, when)Submission data (who, when)

GenBank accession number GenBank accession number ––access to sequence data access to sequence data


3

Viruses contributed to WHO database or GenBank

WHO region ISO3_countryCode Country NameEMR AFG AfghanistanEMR DJI DjiboutiEMR EGY EgyptEMR IRN Iran (Islamic Republic of)EMR IRQ IraqEMR JOR JordanEMR BHR Kingdom of BahrainEMR KWT KuwaitEMR LBN LebanonEMR LBY Libyan Arab JamahiriyaEMR MAR MoroccoEMR OMN OmanEMR PAK PakistanEMR QAT QatarEMR SAU Saudi ArabiaEMR SOM SomaliaEMR SDN SudanEMR SYR Syrian Arab RepublicEMR TUN TunisiaEMR ARE United Arab EmiratesEMR YEM Yemen

181818 WHO Vaccine Preventable Disease Lab Network

WHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current StatusWHO Global genotype database: Current Status

Viruses submitted dating from 1954 to 2010 Viruses submitted dating from 1954 to 2010

66666666

66666666

WHO WHO WHO WHO RegionsRegionsRegionsRegions

36% 36% 36% 36% 36% 36% 36% 36%

37%37%37%37%37%37%37%37%

Proportion Proportion Proportion Proportion with GenBank with GenBank with GenBank with GenBank

entriesentriesentriesentries

749749749749749749749749

85538553855385538553855385538553

Number of Number of Number of Number of virusesvirusesvirusesviruses

1111111111111111

2323232323232323

Genotypes Genotypes Genotypes Genotypes Countries Countries Countries Countries

4040404040404040

118118118118118118118118

RubellaRubellaRubellaRubellaRubellaRubellaRubellaRubella

Measles Measles Measles Measles Measles Measles Measles Measles

Data as of 21 Nov 2010

18

4



Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999Measles Viruses by Year of Detection 1999--------20102010201020102010201020102010

0

200

400

600

800

1000

1200

1400

1600

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Year of Virus Detection

No.

of v

iruse

s su

bmitt

ed

14

18

23

26

30

2939

57*

55

53

48

36

* = No. of countries* = No. of countries* = No. of countries* = No. of countries

124 Countries in total

As of Sept 2010

212121

28411579116590391328629631611688Grand Total

42933Viet Nam

272416122Singapore

39372Republic of Korea

22615Philippines

1616Papua New Guinea

22Palau

541New Zealand

22Mongolia

33Marshall Islands

817Malaysia

1138Lao

26422122176388Japan

422China, Macao SAR

10197211China, Hong Kong SAR

2032519863518166China

761372Cambodia

187133521935122426311167Australia

Grand TotalH2H1G3G2D9D8D7D5D4D3D2d11D1C2C1B3Country Name

WPR Country Submissions to Measles Databases


NRVanuatuWPR

NRTuvaluWPR

NRTongaWPR

NRSolomon IslandsWPR

NRSamoaWPR

NRNiueWPR

NRNauruWPR

NRMicronesia (Federated States of)WPR

NRKiribatiWPR

4FijiWPR

NRCook IslandsWPR

2Brunei DarussalamWPR

Laboratory confirmed cases 2010

Sequence Info but

no ReportNo ReportRegion

Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype Countries With No Measles Genotype

Information ReportedInformation ReportedInformation ReportedInformation ReportedInformation ReportedInformation ReportedInformation ReportedInformation Reported

MeaNS Measles Sequence Database Current status of database

Data manually submitted or automatic downloads from GenBank

• N - 450 – 4900 sequences

• N – full – 5 sequence

• H – full – 486 sequences

Ability to sort if large number of identical hits

Mapping feature – not available/working

5

Submission to GenBank - 450 or 456 ntall fields automatically filled for you (you can edit if wish)e-mail confirmation etc sent to you (not MeaNS)

MeaNS database

http://www.who-measles.org – live

Practice site – for meetinghttp://www.hpa-bioinformatics.org.uk/gnani/Measles/ Public/Web_Front/main.php

User name: labnet1……labnet10Password: labnet99

Acknowledgments:Richard Myers, S (Gnani) Gnaneshan, Jon Green, David Brown,

All those who have submitted data

Recommendations from Recommendations from Recommendations from Recommendations from Recommendations from Recommendations from Recommendations from Recommendations from

Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010Global LabNet Meeting 2010

Timely submission of sequences to MeaNS allows all Timely submission of sequences to MeaNS allows all reporting criteria to be met in one action as these reporting criteria to be met in one action as these data are automatically submitted to the WHO data are automatically submitted to the WHO database and optionally to GenBank. MeaNS also database and optionally to GenBank. MeaNS also assists with characterization and QC of the virus assists with characterization and QC of the virus sequence. sequence.

At a minimum, measles virus genotypes are to be At a minimum, measles virus genotypes are to be submitted to the WHO database, either through submitted to the WHO database, either through MeaNS or directly. MeaNS or directly.

For all rubella virus genetic information, the WHO For all rubella virus genetic information, the WHO database should be used with sequence data database should be used with sequence data preferably also submitted to GenBank. preferably also submitted to GenBank.


Summary & Recommendations Summary & Recommendations Summary & Recommendations Summary & Recommendations Summary & Recommendations Summary & Recommendations Summary & Recommendations Summary & Recommendations Reporting of timely sequence information critical for Reporting of timely sequence information critical for

programme monitoring and determining eliminationprogramme monitoring and determining elimination

Need to identify sequences from all outbreaks and Need to identify sequences from all outbreaks and

chains of infection chains of infection

Timeliness of genetic information reporting monitored Timeliness of genetic information reporting monitored

through Accreditation checklist through Accreditation checklist

Recommended that measles viruses are deposited in Recommended that measles viruses are deposited in

MeaNS database (with sequence)MeaNS database (with sequence)

Rubella viruses should continue to be deposited in Rubella viruses should continue to be deposited in

WHO databaseWHO database


AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

HPA and CDCHPA and CDC

All labs who have contributed timely data All labs who have contributed timely data

Youngmee JYoungmee Jee ee for following up on labs for following up on labs


Thank You !!

Day 1, 22 November

Grouping arrangement for practicals

� Group A (China x 2)

� Group B (Philippines x 2)

� Group C (Vietnam x 2)

� Group D (Malaysia & Singapore)

� Group E (Korea & New Zealand)

� Group F (Japan & China)

� Group G (Mongolia & Hong Kong)

Practical: RNA extraction for measles and rubella� Each group will be given 5 samples

� Measles: 2 urine samples and 1 culture fluid

� Rubella: 1 urine sample and 1 culture fluid

� Qiagen QIAamp Viral RNA Mini kit� Reconstitute carrier RNA with buffer AVE (310µl)

� Prepare buffer AVL by adding reconstituted carrier RNA (100µl reconstituted carrier RNA to 10ml buffer AVL-provided)

� Absolute ethanol and buffers AW1 & AW2 (with ethanol added), 1.5ml snap cap vials (caps cut away by scissors) will be provided

� Participants will take turns to add samples to buffer AVL in safety cabinet (Rooms 906 & 907)-white gown� Subsequent extraction steps will be performed in Room 907

� After elution, keep RNA on ice and go to Room 809� Take off white gown before leaving Room 809

Practical: Rubella detection & genotyping RT-PCR� Separate rooms will be used

� Room 811: Master mix preparation (blue gown)

� Room 809: RNA template addition (white gown)

� Room 817: PCR product handling (yellow gown)

� Participants will take turns to prepare master mix in safety cabinet or PCR workstation in Room 811-blue gown

� Qiagen One-Step RT-PCR kit + RNase inhibitor (ABI)

� Primers in working concentration (20µM) will be provided

� 3 sets of PCR� Rubella detection: 3 primers (RV11, RV12 & RV12-2)

� Rubella genotyping: 2 sets of primers (8633F & 9112R; 8945F & 9577R)

� RNA samples to be tested: 2 extracted RNA + 1 wild type RNA from CDC; all three add 5µl to PCR tubes

� Keep PCR master mixes on ice and move to Room 809

� Take off blue gown before leaving Room 811

� Put on white gown in Room 809

� Three rubella RNA controls will be provided for� Detection RT-PCR: add 1 µl to PCR tube

� Genotyping fragment 1 RT-PCR: add 1 µl to PCR tube

� Genotyping fragment 2 RT-PCR: add 1 µl to PCR tube

� Keep the PCR tubes on ice

� Carry the PCR tubes to Room 817

� Take off white gown before leaving Room 809

� Two groups will share one ABI 9700 thermal cycler

� Both detection and genotyping RT-PCR share the same PCR conditions

Day 2, 23 NovemberPractical: Measles genotyping RT-PCR� Take turns to prepare master mix in Room 811-blue gown� Qiagen One-Step RT-PCR kit + RNase inhibitor (ABI)� Primers in working concentration (20µM) will be provided

� Two primers (MeV216 and MeV214)

� Keep PCR master mix on ice� Take off blue gown before leaving Room 811� Put on another blue gown in Room 809� Measles control RNA will be provided: add 1µl� Keep the PCR tubes on ice� Take off blue gown before leaving Room 809� Put on yellow gown in Room 817� Two groups will share one ABI 9700 thermal cycler

Practical: Gel electrophoresis for rubella RT-PCR� 2% gels will be provided (one gel tank per group)

� 96-well plate for mixing samples and loading dye

� Samples: 2µl 6X loading dye + 5µl samples

� Marker: 2µl 6X loading dye + 2µl 123bp ladder + 8µl 1X TBE

� Load mixtures to the gel (Remember the sample order!)

� Power supply will be shared (140V, 25-30min)

� Avoid taking things into & out of Room 817

� Take off yellow gown and wash hands before leaving Room 817

Practical: Gel electrophoresis for measles RT-PCR� Similar to gel electrophoresis for rubella RT-PCR

Practical: PCR purification for measles & rubella� Qiagen QIAquick PCR Purification kit

� Buffer PE (with ethanol added) and 1.5ml snap cap vials (caps cut away by scissors) will be provided

� Purify all positive samples, including wild type RNA from CDC

� Put on yellow gown in Room 817


� Take off yellow gown and wash hands before leaving Room 817

Practical: Gel electrophoresis of purified PCR products� Similar to previous gel electrophoreses

� Determine amount of DNA templates to be added for cycle sequencing the next day

Day 3, 24 NovemberPractical: Cycle sequencing� Prepare cycle sequencing master mix in Room 811

� BigDye Terminator v3.1, sequencing primers (3.2µM) and water will be provided

� Keep cycle sequencing mix on ice

� Add the sequencing primers in Room 817

� Two groups will share one ABI 9700 thermal cycler


Practical: Purification of cycle sequencing products� Qiagen DyeEx 2.0 Spin kit

� Put on yellow gown in Room 817

� Speedvac (~1hr)

Practical: Sequencing run� Fill in sample ID in worksheet and computer of sequencers

� Add Hi-Di formamide in safety cabinet (Rooms 839 & 840)

� Separate sequencers will be used for measles and rubella

BigDye +5X sequencing buffer +H2O

1. Prepare cycle sequencing master mix

……

2. Aliquot 18µl into 0.2ml PCR tubes

……

……

3. Add 1µl of sequencing primer to each tube according to worksheet

4. Add 1µl of corresponding purified PCR product to each tube

……

……

4th REGIONAL HANDS4th REGIONAL HANDS --ON TRAINING WORKSHOP ON TRAINING WORKSHOP ON THE LABORATORY DIAGNOSIS OF MEASLES ON THE LABORATORY DIAGNOSIS OF MEASLES

AND RUBELLA FOCUSING ON MOLECULAR AND RUBELLA FOCUSING ON MOLECULAR DIAGNOSIS DIAGNOSIS

Public Health Laboratory Centre, Hong Kong Public Health Laboratory Centre, Hong Kong (China)(China)

22 to 27 November 2010 22 to 27 November 2010

DEPARTMENT OF HEALTH AND HUMAN SERVICESCENTERS FOR DISEASE CONTROL AND PREVENTION

Introduction to Molecular Introduction to Molecular Techniques for Detection of Techniques for Detection of

Measles Virus and Genotyping by Measles Virus and Genotyping by RTRT--PCR and RealPCR and Real --Time RTTime RT--PCRPCR

Paul A. Rota, Ph.D.Measles, Mumps, Rubella and Herpes Viruses Laborator y

Branch

Division of Viral Diseases,

Molecular techniques can provide a valuable tool to:

• Differentiate between ongoing transmission of endemic virus from new, imported source of virus

• Aid in the classification of unusual or severe cases

• Confirm suspected vaccine reactions

• Molecular techniques have an increasing roles case confirmation especially in low incidence settings and/or when serologic results are difficult to interpret

Importance of Virologic Importance of Virologic Surveillance and Viral DetectionSurveillance and Viral Detection

•Molecular epidemiologic studies are a key component of verification of measles elimination

•One indicator for verification of elimination will be absence of an endemic genotype for one year

•Genetic data can be used to track transmission patterns and identify sources of infection

•There is rapid global transmission of measles viruses. For example, viruses associated with outbreaks in Africa are soon detected in association with imported cases in the Americas

•It is impossible to predict how/when a virus will spread from an endemic area to other parts of the world

•Vaccination programs frequently interrupt transmission of measles lineages, but reintroduction of measles is a problem (sometimes with an apparent switch in genotype)

Lessons Learned from Nearly 20 Years of Lessons Learned from Nearly 20 Years of Virologic Surveillance for Measles VirusVirologic Surveillance for Measles Virus --11

•All measles vaccines are in genotype A

•Vaccination has been effective globally despite the presence of different endemic genotypes (provided the vaccine is administered properly)

•Molecular epidemiolgic data clearly show that it is possible to maintain elimination of measles transmission despite constant importation of multiple genotypes of virus

•Sporadic cases and small outbreaks will continue to occur depending on the distribution of susceptible individuals

•Rapid confirmation of vaccine reactions (through molecular techniques) is important in elimination settings in cases where there was vaccination in response to a recent exposure or potential exposure

Lessons Learned from Nearly 20 Years of Lessons Learned from Nearly 20 Years of Virologic Surveillance for Measles VirusVirologic Surveillance for Measles Virus --22

Transmission patternsTransmission patterns

•Endemic: Multiple transmission pathways lead to mul tiple lineages within endemic genotype(s)

•Elimination: Few cases and multiple imported genoty pes with no endemic genotype

•Reintroduction: Rapid spread of genetically homogen ous viruses

What Virologic Surveillance Data Tell Us About Meas les What Virologic Surveillance Data Tell Us About Meas les Transmission PatternsTransmission Patterns

•Endemic transmission of measles: Multiple transmission pathways lead to multiple lineages within endemic genotype(s)

•Example: China

•Elimination of measles: Few cases and multiple imported genotypes with no endemic genotype

•Examples: Western Hemisphere, Australia, New Zealand, Hong Kong

•Reintroduction following interruption: Rapid spread of genetically homogenous viruses

•Example: Burkina Faso, Philippines

Limitations of Virologic SurveillanceLimitations of Virologic Surveillance

•Molecular studies can confirm independent sources of infection if different genotypes or clearly distinct lineages are detected.

•Molecular studies alone cannot differentiate between continuous circulation of virus and multiple introductions from the same source.

•Therefore, it is extremely important to conduct high quality case investigations and to classify cases after all of the epidemiologic and laboratory data have been reviewed.

Global Status of Laboratory Support Virologic Surve illance for Global Status of Laboratory Support Virologic Surve illance for WildWild --type Measles Virusestype Measles Viruses

•All regions and most countries have conducted baseline virologicsurveillance for measles

•Most Regional Reference Labs in the WHO LabNet are capable of PCR and sequence analysis

•Most LabNet labs use Vero/hSLAM for isolation

•PCR and viral isolation are included in all WHO intercountry training courses

•Timely reporting of sequence information (within 2 months) is anaccreditation requirement in all regions

•Two global databases have been established•WHO (genotypes only)•HPA-MeaNS (approaching 8000 sequences) enable almost real time analysis

Requirements for Virologic SurveillanceRequirements for Virologic Surveillance --11

•Testing should be performed in an accredited laboratory

•In a pre or post elimination settings, the goal is to obtain genetic information from every chain of transmission

•This can be difficult with sporadic cases, usually because of failure to collect adequate samples in a timely manner•Typically, there is greater success with obtaining adequate samples from outbreaks

•Specimens must be taken at first contact with suspected case•Good specimens for virus detection or virus isolation: throat swab, nasal wash or aspirate, oral fluid, white blood cells (notrecommended for safety reasons)• Other samples in which viral RNA can be detected at lower frequencies: dried blood spots, IgM positive serum

Requirements for Virologic SurveillanceRequirements for Virologic Surveillance --22

•An important pre-requisite is to conduct baseline virologic surveillance in all countries to document endemic genotype(s) that are present before the accelerated control measures required forelimination are initiated

•Testing archival samples may be useful if these specimens are available

N P/C/V M F H L

Standard Methods for Measles Molecular Standard Methods for Measles Molecular Epidemiology of MeaslesEpidemiology of Measles

RT-PCR and Sequence targets:

•N and H genes are the most variable regions on the measles genome.

•Amplify and sequence the 450 nt. Coding for the COOH terminal 150 amino acids of the N protein from all specimens (Minimum amount of sequence needed for reporting genotype).

•Amplify and sequence the entire coding region of the H gene fromselected isolates

Genotype Status Reference strains (MVi) H gene N gene

A Active Edmonston-wt.USA/54 U03669 U01987

B1 Inactive Yaounde.CAE/12.83 “Y-14” AF079552 U01998

B2 Active Libreville.GAB/84 “R-96” AF079551 U01994

B3 Active New York.USA/94 L46752 L46753

C1 Active Tokyo.JPN/84/K AY047365 AY043459

C2 Active Maryland.USA/77 “JM” M81898 M89921

D1 Inactive Bristol.UNK/74 (MVP) Z80805 D01005

D2 Active Johannesburg.SOA/88/1 AF085198 U64582

D3 Active Illinois.USA/89/1 “Chicago-1” M81895 U01977

D4 Active Montreal.CAN/89 AF079554 U01976

D5 Active Palau.BLA/93 L46757 L46758

D6 Active New Jersey.USA/94/1 L46749 L46750

D7 Active Victoria.AUS/16.85 AF247202 AF243450

D8 Active Manchester.UNK/30.94 U29285 AF280803

D9 Active Victoria.AUS/12.99 AY127853 AF481485

D10 Active Kampala.UGA/50.00-1 AY923213) AY923185

d11 Active Menglian.Yunnan.CHN/47.09/1 GU440572.1 GU440576

E Inactive Goettingen.DEU/71 “Braxator” Z80797 X84879

F Inactive MVs/Madrid.SPA/94 SSPE Z80830 X84865

G1 Inactive Berkeley.USA/83 AF079553 U01974

G2 Active Amsterdam.NET/49.97 AF171231 AF171232

G3 Active Gresik.INO/17.02 AY184218 AY184217

H1 Active Hunan.CHN/93/7 AF045201 AF045212

H2 Active Beijing.CHN/94/1 AF045203 AF045217

WHO Measles Reference Sequences and Accession Numbe rsWHO Measles Reference Sequences and Accession Numbe rs

Thanks to Paul Chenowith and David Featherstone

Year Number of cases

Number of chains

Number chains with

genotype (%)

Number of outbreaks

Number of outbreaks with genotype (%)

2002 44 26 8 (31) 3 1

2003 56 29 7 (24) 3 2

2004 37 21 6 (28) 2 2

2005 66 25 12 (48) 3 2

2006 55 31 14 (45) 4 2

2007 43 27 7 (26) 4 3

2008 141 39 15 (39) 9 9

2009* 71 33 19 (57) 8 7

2010 51 41 14 (34) 4 3

Total 561 272 102 (38) 40 31 (77)

Virologic Surveillance for Measles, USA: 2002Virologic Surveillance for Measles, USA: 2002 --2010 (9/1/10)2010 (9/1/10)

*In 2009, 2 outbreaks genotyped by nested PCR only

Measles Cases with Genotypes USA: 2009Measles Cases with Genotypes USA: 2009 --2010 (YTD2010 (YTD))

COUNTRY Year Month WHONAME GENOTYPE SOURCE ACCESSIONUSA 2009 1 MVi/New York.USA/2.09/2 D8 UK FJ848879USA 2009 1 MVi/New York.USA/2.09/1 D8 UK FJ848879USA 2009 1 MVi/California.USA/4.09 D4 UK FJ904784USA 2009 2 MVi/Pennsylvania.USA/7.09 D8 India FJ848880USA 2009 2 MVi/California.USA/6.09 D4 UK FJ904784USA 2009 4 MVi/Virginia.USA/15.09 D8 unknown GQ337691USA 2009 5 MVi/New York.USA/22.09 D4 unknown GQ337695USA 2009 5 MVi/Pennsylvania.USA/20.09 H1 unknown GQ337693USA 2009 5 MVi/Florida.USA/19.09 D4 UK GQ369953USA 2009 6 MVi/New York.USA/23.09 D4 unknown GQ369954USA 2009 6 MVi/West Virginia.USA/23.09 D4 unknown GQ369955USA 2009 6 MVi/New York.USA/25.09 D4 unknown GQ369956USA 2009 6 MVi/New York.USA/26.09/3 D4 unknown GQ369956USA 2009 6 MVi/Massachusetts.USA/26.09 B3 South Africa GQ903686USA 2009 7 MVi/Arizona.USA/31.09 D4 France GQ903687USA 2009 8 MVs/Georgia.USA/32.09 H1 unknown GQ903688USA 2009 12 MVi/California.USA/52.09 D4 unknown HM044306

USA 2010 1 MVs/Virgina.USA/04.10 [SSPE] D3 N/A HM044305USA 2010 2 MVs/Arizona.USA/06.10 B3 Ethiopia HM044304USA 2010 2 MVs/Washington.USA/7.10 D4 India HM044303USA 2010 2 MVs/California.USA/6.10 D9 unknown pendingUSA 2010 3 MVi/California.USA/11.10/1 D8 India HM367087USA 2010 3 MVs/California.USA/11.10/2 D8 unknown HM367086USA 2010 3 MVs/California.USA/11.10/3 H1 Canada pendingUSA 2010 4 MVi/Kentucky.USA/14.10 D8 India HM362784USA 2010 4 MVs/California.USA/17.10/1 B3 Germany pendingUSA 2010 5 MVs/Nebraska.USA/20.10 D9 unknown pendingUSA 2010 5 MVs/California.USA/17.10 D9 Philippines pendingUSA 2010 6 MVs/Massachusetts.USA/22.10 H1 Japan pendingUSA 2010 7 MVs/Connecticut.USA/27.10 D4 unknown pending

Pending: submitted via MeaNS

MVs/California.USA/11.10/2MVi/California.USA/11.10/1

manches.unk 94 d8MVi/Kentucky.USA/14.10

vic.aus 85 d7illin.usa 99 d7

chicago.usa 89 d3montreal.can 89 d4

MVs/Washington.USA/7.10MVs/Massachusetts.USA/27.10MVs/Connecticut.USA/27.10

palau.bla 93 d5bangkok.tha 93 d5

vic.aus 99 D9MVs/California.USA/17.10

MVs/California.USA/6.10MVs/Nebraska.USA/20.10

nj.usa 94 d6mvp.uk 74 d1

johann.soa 88 d2uganda-01-d10

madrid.spa 94 fed-wt a

jm.usa 77 c2wtf.deu 90 c2

tokyo.jpn 84k c1braxator.deu 71 e

libreville.gab 84 b2yaounde.cae 83 b1

ny.usa 94 b3MVs/California.USA/17.10/1

MVs/Arizona.USA/06.10ibadan.nie 97 b3

amster.net 97 g2gresik.ino 02 G3

berkeley.usa 83 g1beijing.chn 94 h2

hunan.chn 93 h1MVs/Massachusetts.USA/22.10

MVs/California.USA/11.10/3

5

Measles Cases Measles Cases with Genotypes with Genotypes USA, 2010USA, 2010

D8

D8

D4

D9

B3

H1

MVs/Chester.GBR/3.09/ nucleoproteinMVi/Barka.OMN/21.08/1MVi/Villupuram.Ind/03.07MVi/Villupuram.Ind/07.07MVs/Imphal.IND/19.09California-11.10/1MVs/Milton Keynes.GBR/20.07/MVs/Mirbat.OMN/21.08/1MVs/Kalahandi.IND/04.08/2California-11.10/2

MANCHES.UNK 94 D8MVs-BritishColumbia-CAN-13-10-2

MVs/Papumpare.Ind/51.07/4/Kentucky-14.10

MVs/Papumpare.IND/51.07/4MVs/Patna.IND/51.05MVs/Purulia.Ind/39.06

Measles Cases with Genotype D8 USA, 2010Measles Cases with Genotype D8 USA, 2010

Thanks to colleagues at SEAR Laboratory Network

California 2/16/2010

Nebraska 5/17/2010

MVs/HongKong.CHN/07.10 from PHL

M03/10-NCR-PHL

MVs/HongKong.CHN/28.07/5 from PHL

California 5/12/2010 (CA MV-2897)

M05/09-Region11-PHL

M06/09-Region11-PHL

VIC.AUS 99 D9

Measles Cases with Genotype D9 USA, 2010Measles Cases with Genotype D9 USA, 2010

Thanks to colleagues at PHL, Hong Kong, RITM, Manila, and WPRO

H1MVi/Kaohsiung.TWN/53.08

AMSTER.NET 97 G2GRESIK.INO 02 G3

BERKELEY.USA 83 G1BEIJING.CHN 94 H2

HUNAN.CHN 93 H1CA.USA/11.10/3CA.USA/11.10/3MVs-BC-CAN-12-10-1

MVs/Sari.IRA/30.2009(1)MVi/Taipei.TWN/30.07

MVs/Taoyuan.TWN/29.08MVi/Sichuan.PCR/28.04/1MViSh705MVs/New York.USA/29.06MVi/Jiangsu.PRC/23.05/3MVs/Tainan.TWN/33.07MVi/Guangdong.PRC/10.05/2MVi/Hebei.PRC/09.05/1MVi/Chongqing.PRC/20.04/1

5

Measles Cases with Genotype H1 USA, 2010Measles Cases with Genotype H1 USA, 2010

Thanks to colleagues at Health Canada

Measles Genotypes in Latin Measles Genotypes in Latin America: 2010 America: 2010

2010: B3, Argentina from SA

2010: D4, Brazil (Para) from ?

2010: B3, Brazil from ARG

Thanks to colleagues at FioCRUZ and Inst. Carlos Malbran

Use of conventional and real time Use of conventional and real time PCR for case classification and PCR for case classification and

molecular surveillancemolecular surveillance

Serum SampleSerum SampleThroat or Throat or Nasal Nasal

Swab, UrineSwab, Urine

Oral Fluid, Oral Fluid, Dried Blood Dried Blood

SpotSpot

Measles and Rubella IgM Measles and Rubella IgM

Measles IgGMeasles IgG

AvidityAvidity

RNA extraction, RNA extraction, RTRT--PCRPCR

Virus IsolationVirus Isolation

Sequence Analysis for GenotypeSequence Analysis for Genotype

Testing Scheme for Confirmation of Measles Infectio nTesting Scheme for Confirmation of Measles Infectio n

Vaccine or WildVaccine or Wild--typetype

Classification Clinical IgM IgG Avidity & PCR

Vaccine reaction Fever/Rash PP or N Low Positive and

Sequence indicates vaccine strain

Vaccine reactionFever/Rash

N P High

MeaslesMeets CCD* P

P or N

Low can confirm IgM Positive PCR can confirm IgM

Measles (but recently

vaccinated)

Modified#P P

Measles-Primary Vaccine Failure

Meets CCD*P

P or N

Secondary vaccine Failure

Modified# P or NP High

Positive PCR can confirm case

Utility of New Assays in the Measles Diagnostics T ool BoxUtility of New Assays in the Measles Diagnostics T ool Box

*=clinical case definition (CCD)

#=modified or mild presentation does not meet CCD

&=Collection of sample is ≤ 3 weeks after rash onset

Real time RTReal time RT--PCRPCR

Sequence Analysis for GenotypeSequence Analysis for Genotype

Testing Scheme for Measles RTTesting Scheme for Measles RT --PCRPCR

Vaccine or WildVaccine or Wild--typetype

Standard RTStandard RT--PCRPCR

RNA extractionRNA extraction

Standard RTStandard RT--PCRPCR Nested RTNested RT--PCR*PCR*High Ct (>36)Low Ct (<36)

*nested PCR recommended only for experienced Global or Regional Labs

Or

Low incidence settings

Location of primer binding sites relative to Location of primer binding sites relative to sequencing windowsequencing window

Improving Primers for Measles Genotyping PCRImproving Primers for Measles Genotyping PCR

Results and ConclusionsResults and Conclusions

• The primer sets were tested against RNA from 11 different genotypes of measles. RNA concentrations were determined by realtime RT-PCR

• The MV 60 and MV 63.3 primer pair amplified all of the virus genotypes tested at 106 copies.

• The MeV 214-216 forward and the reverse primer pair was the most sensitive set for detecting measles RNA and detected all genotypes tested at 104 copies of viral RNA.

New primers amplify 10New primers amplify 10 44 copies of RNA templatecopies of RNA template

1 9 10 11 12 13 144 5 6 7 82 3

1 100 bp marker2 Water control3 RNA from uninfected

cells4 Genotype A5 Genotype B36 Genotype D27 Genotype D38 Genotype D49 Genotype D510 Genotype D611 Genotype D812 Genotype G213 Genotype G314 Genotype H1

600 bp

RT-PCR was performed with MeV216 and MeV214

Version 2 of the Measles Genotyping Kit: Control RNA

854 nt

634 nt

900 nt800 nt700 nt600 nt500 nt

1 2 3 4

Version 2 of the Measles Genotyping Kit: Results

•Lane 1: MW ladder•Lane 2: Pos control•Lane 3: Pos sample•Lane 4: Negative (water) control

1633 GGC TCA GAC ACG GAC ACC CCT ATA GTG TAC AAT GAC AGA AAT CTT CTA 1681↓↓↓↓ end of window

��MV63 ��MeV2141681 GAC TAG GTG CGA GAG GCC GAG GGC CAG AAC AAC ATC CGC CTA CCC TCC 1728

1729 ATC ATT GTT ATA AAA AA

Sequence of the 3Sequence of the 3 ’’ terminus of the terminus of the measles N gene mRNA (3measles N gene mRNA (3 ’’ terminus of terminus of

““ sequencing windowsequencing window ”” ))

Since the previous RNA control did not contain the entire 3’non-coding region of the MeV mRNA, a new positive control RNA was synthesized to be used with MeV 214-216.

Version 2.0 of the Measles Genotyping kit:Version 2.0 of the Measles Genotyping kit:•In this version, the forward and reverse primers have been modified to increase sensitivity and reduce background and the synthetic positive control RNA has been modified. The reaction conditions, primer and template concentrations, and cycling parameters are the same as those used with the previous version of the kit •This new primer set will not amplify the synthetic RNA control supplied in the previous version of the kit. Amplification from infected cell RNA will not be affected. •Measles RNA for RT-PCR can be extracted from either infected cells or clinical samples. •Primers MeV214 and MeV216 are designed to amplify a 634 nucleotide region coding for the 3’ terminus of the nucleoprotein (N) gene in a standard RT-PCR reaction. The positive control RNA, MeV-N3in included in this kit, is a synthetic RNA transcript of the N gene of measles and it is provided to serve as a positive internal control in the RT-PCR reactions. The control RNA has been modified so that the RT-PCR reaction will produce a larger PCR product (854 nucleotides) than the PCR product produced from measles RNA, thereby providing a means to identify laboratory contamination of the RT-PCR reaction

Kit Contents: •Forward primer: MeV216, 25ul Stock Solution 200uM•Reverse primer: MeV214, 25ul Stock Solution 200uM•Dried Positive Control RNA, MeV-N3in, 1011 copies•1ml of 1X nuclease-free TE

The sequences of the primers are:Forward primer: MeV 216, 5’-TGG AGC TAT GCC ATG GGA GT-3’Reverse primer: MeV 214, 5’-TAA CAA TGA TGG AGG GTA GG-3’

To make working stocks of primers MeV214 and MeV216 for RT-PCR: •Add 90ul nuclease-free water to 10ul of MeV214•Add 90ul nuclease-free water to 10ul of MeV216•Concentration = 20uM•Store at -20ºC•Use 0.5 – 1ul per RT-PCR reaction as per RT-PCR protocol.

Rehydration of measles positive control RNA, MeV-N3in To make master stock:•Add 100ul nuclease-free TE and vortex tube.•Place at 55ºC for 10 minutes and vortex, again.•Make 10ul aliquots.•Concentration = 109 copies/ul•Store at-70ºC

To make working stock:•Add 90ul nuclease-free TE to the 10ul master stock.•Concentration = 108copies/ul•Store at -20ºC for short-term and at -70ºC for long-term.•Use 1ul per RT-PCR reaction.

Background: TaqMan Real Background: TaqMan Real time RTtime RT --PCRPCR

•Probe contains 5’fluorophore and 3’ quencher

•Forward and reverse primers are not modified and are the same as those used in standard RT-PCR

•Relatively short amplicons (typically less than 100nt)

•Probe is degraded by 5’ to 3’ exonuclease activity of the Taq polymerase

•Consumption of probe results in fluorescence

•Fluorescence is measure at each cycle (typically 40)

•Threshold cycle (Ct) is cycle at which fluorescence crosses the threshold.

Detection of Measles RNA by Real Time RTDetection of Measles RNA by Real Time RT --PCRPCR

•Real-time assays can detect 10-100 copies of viral mRNA/sample in a high throughput format, results in 3-4 hours

•Can help to confirm a case when serologic results are inconclusive

•Negative results do not rule out a case

•Standard RT-PCR is less sensitive than real time RT-PCR.

•Sequence information from the standard PCR product required for genotype and confirmation of vaccine reactions.

Controls Positive Sample

MeV N GeneMake a 15 µM stock of each forward and reverse primer; final concentration is 300 nM.Make a 12.5 µM stock of the probe; final concentration is 250 nM.

•Forward Primer (MVN1139F): 5’ TGG CAT CTG AAC TCG GTA TCA C 3’•Reverse Primer (MVN1213R): 5’ TGT CCT CAG TAG TAT GCA TTG CAA 3’•Probe (MVNP1163P): 5’ FAM CCG AGG ATG CAA GGC TTG TTT CAG A BHQ 3’

Human RNase P Gene (Reference gene)Make a 15 µM stock of each forward and reverse primer; final concentration is 300 nM.Make a 5 µM stock of the probe; final concentration is 100 nM.

•Forward Primer (HURNASE-P-F): 5’ AGA TTT GGA CCT GCG AGC G 3’•Reverse Primer (HURNASE-P-R): 5’ GAG CGG CTG TCT CCA CAA GT 3’•Probe (BHQ1HURNASE-P): 5’ FAM TTC TGA CCT GAA GGC TCT GCG CG BHQ 3’

Primers and Probes for Measles Real time RTPrimers and Probes for Measles Real time RT --PCR and for PCR and for Reference Gene ControlReference Gene Control

Measles Real time RTMeasles Real time RT --PCR on ABI 7500PCR on ABI 7500

Measles StandardsMeasles Standards RNaseP StandardsRNaseP Standards

Measles Real time RTMeasles Real time RT --PCR on ABI 7500: Standard CurvePCR on ABI 7500: Standard Curve

Viral gene (Ct) RNase P (Ct) Result

Ct < 40 Ct < 40 PositiveCt < 40 Undetermined PositiveUndetermined Ct < 40 NegativeUndetermined Undetermined Indeterminate

Measles Real time RTMeasles Real time RT --PCR: InterpretationPCR: Interpretation

•Ct is average Ct from 2 or 3 replicates/sample•Positive if 2/3 replicates are <40•If testing in duplicate, repeat if one Ct is <40 and the other is Und.•Repeat Indeterminate samples with fresh RNA extraction if possible

Samples from Suspected Measles Cases, Samples from Suspected Measles Cases, USA, 2009USA, 2009--2010 (through Sept 10)2010 (through Sept 10)

• Samples for real time RT-PCR: 266 (US only)

• 79 positive (29%)

• 8 indeterminate

• 180 negative

• Virus isolation attempts: 271 (includes international)• 2009: 36 positive from 210 samples (17%)• 2010: 7 positive from 61 samples (11%)

Drying cells infected with measles or rubella viruses onto specimen paper

Purpose: To submit measles or rubella isolates to the Regional or Global Specialized Laboratory for sequence analysis and genotyping. Once the samples are dry, the samples can be stored at 4 C. Samples can be shipped at room temperature.

May also be used to transport proficiency panels for RT-PCR assays

Important: Both measles and rubella viruses have been recovered from specimen paper. Treat samples as infectious material.

CDC is testing inactivation protocols as well as the FTA cards that are similar to those used by the polio labs.

Shipping viral isolates on Shipping viral isolates on filter paperfilter paper

What about infectivity?What about infectivity?

A non-infectious samples would be useful a number of purposes including shipment of PT panels and control RNA for RT-PCR

Whatman FTA elute micro cardWhatman FTA elute micro card

•Chemically treated to lyse cells and denature proteins on contact.•Nucleic acids are protected from microbial and fungal attack.•Samples are not infectious.•Samples can be stored and shipped at room temperature.•Extract RNA using Qiagen Viral RNA Mini kit.

day 1 1 week RT

1 week 37 °C

wat

er

pos

con

trol

600 bp

2 week sRT

wat

er

pos

cont

rol

1 week RT1 week 37 °C

600 bp

4 week sRT

1 monthRT

3 weeks RT1 week 37 °C

wat

er

wat

er

pos

cont

rol

pos

cont

rol

1 month RTwith 1 week 37 °C

Measles RNA is stable for at least one month on FTA cardsMeasles RNA is stable for at least one month on FTA cards PCR Products are Stable for at least 1 monthPCR Products are Stable for at least 1 month

Day 0Day 0 1 Month1 Month

•PCR reactions were incubated at, RT, 4C and 37 C for up to 1 month with no degradation of PCR product

•PCR reactions can be shipped to sequencing lab at 4C or RT

•Place thin-walled tube in sturdy container (e.g. 1.5 ml Eppendorf)

Thank YouThank You

•Measles, Mumps, Rubella and Herpesviruses Laboratory Branch, DVD/CDC

•William Bellini, Jennifer Rota, Yuan Tian, Luis Lowe, Elena Lopareva, Bettina Bankamp, Lauren, Rebecca, McNall Byrd, Joe Icenogle, Emily Abernathy, Lijuan Hao

•Epidemiology Branch, DVD/CDC

•Kathy Gallagher, Susan Redd, Al Barskey

•Global Immunization Division/CDC

•National Microbiology Laboratory, Canada

•PAHO LabNet: Carlos Castillo, Christina Marsigli

•FioCRUZ: Marilda Siqueira

•Inst. Carlos Malbran: Elsa Baumeister

•WHO/HQ, RRL and NLs

•State and Local Public Health Labs, USA

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