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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
P150 P90 C E2 E1 PolyA
Nonstructural 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
P150 P90 C E2 E1 PolyA
Nonstructural Proteins
Cap
Structural Proteins
P150 P90 C E2 E1 PolyA
Nonstructural 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
Peru 1C oral fluid 0 478 nd Positive Positive
Ghana 1G TS na 500 nd Positive Positive
Ghana 1G TS na 500 Positive Positive Positive
Peru 1C oral fluid 1 1080 nd Positive Positive
Peru 1C oral fluid 0 1250 nd 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.
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.
1E, 1G, and 2B, had a widegeographic distribution andwere frequently found.
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, 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
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 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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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%**
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
< 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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
• 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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
• 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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
Summary of 2010 Measles/Rubella Lab testingSummary of 2010 Measles/Rubella Lab testing
D9
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
Monitoring of Annual Accreditation
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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)
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
Day 4, ThursdayDay 4, Thursday
• Session 5: Practical - Sequence analysis for measles virus genotyping
Country reports
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
Measles outbreaks and Measles outbreaks and
importations in 2009importations in 2009--2010 2010
Vietnam, Philippines Vietnam, Philippines
New Zealand, AustraliaNew Zealand, Australia……..
8
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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.
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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)
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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.
World Health Organization • Western Pacific Regional Office • Expanded Programme on ImmunizationWorld Health Organization • Western Pacific Regional Office • Expanded Programme on Immunization
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)
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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)
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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
191919 WHO Vaccine Preventable Disease Lab Network
202020 WHO Vaccine Preventable Disease Lab Network
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
222222 WHO Vaccine Preventable Disease Lab Network
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.
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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
WHO Vaccine Preventable Disease Lab Network
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
� Avoid taking things into & out of 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
� Avoid taking things into & out of Room 817
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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