Upload
odelia
View
37
Download
7
Tags:
Embed Size (px)
DESCRIPTION
Innovation and new Technologies for measles eradication. Dr. David Brown Virus Reference Department HPA Microbiology services Global Measles and Rubella Management Meeting 15 th March 2011. Overview. Current status of measles control Range of research needs already identified - PowerPoint PPT Presentation
Citation preview
Innovation and new Technologies for measles eradication
Dr. David Brown
Virus Reference Department
HPA Microbiology services
Global Measles and Rubella Management Meeting
15th March 2011
Overview
• Current status of measles control
• Range of research needs already identified
• PoCT test for measles IgM
• Molecular epidemiology – Impact of new sequencing technologies
Measles R&D needs Identified
• Measles elimination: Indicators for elimination/performance of surveillance programme Risk factors for outbreaks
Waning Immunity? 2° infections HIV and measles
Immunization Strategies
Changing patterns of susceptibility Outbreak response immunization Effectiveness of different immunization strategies
Laboratory Activities Vaccine development Antivirals Diagnostics/Molecular epidemiology
Laboratory tools for Surveillance of Measles:
• IgM assays and RT-PCR for confirmation of acute infections
• IgG assays to guide vaccination, serosurveys
• IgG avidity to confirm primary infections
• RT-PCR/sequencing for Molecular epidemiology
– Track epidemiological pathways
– Surveillance of virus diversity
– Investigation of potential adverse events
• Highly sensitive and specific
• Rapid (15 – 30 minutes)
• No sample preparation
• Simple, with few operator dependent steps
• Inexpensive
• Clear and stable end-point result
• Storage at room temperature
• Results improve treatment or public health response
Ideal features of PoCT
PoCTs available
Viruses
Bacteria
InfluenzaRSVHIV
HBsAg
HCV
H. pylori
Strep pneumoniae
Group A Strep
ProtozoaBiological threats
Chlamydia trachomatis
Malaria
AnthraxBotulismPlague
T. pallidum
Anti-NP gold conjugate
Test line:anti-human IgM
Control line:Anti-mouse IgG
Cotton linter paper wick
Plastic backing card
Nitrocellulosemembrane
Glass fibreConjugate release pad
Principle of the IgM capture near patient test
Direction of reagent flow
Oral fluid IgM
rNP
Measles IgM PoCT strip: positive control for serum
Serum measles IgM PoCT protocol
Step1 Step 2 Step 3 Step 4 Step 5
Dilute serum1/100
Add 5µl antigen to reaction tube.
Mix diluted serum and antigen.
Insert test strip.Incubate room temp.
Read result.
Evaluation of measles PoCT for serum samples:
Serum Samples:
100 sera collected during measles elimination programme in Malaysia in 2004.
62 sera identified as rubella IgM positive during measles surveillance in Ethiopia 2003-2004.8 cases of rubella IgM positive sera from Russia
PoCT vs Siemens IgM on serum samples
ppv 92.0 (95% CI 83.4 – 97.0) npv 92.4 (95% CI 85.4 – 96.9) Sensitivity : 90.8% (95% CI 81.9 – 96.2)
Specificity : 93.6% (95% CI 86.6 – 97.6)
EIA results
- - - - - + + + + + + +T/N ratios: 0.16 0.13 0.24 0.22 0.12 4.06 1.54 1.68 2.93 1.03 PC PC
PoCT results
- - - - - + + + + + +/- +/-
Measles IgM PoCT: 10 Sera (# 69 – 78, incubation: 10 minutes)
Evaluation of measles PoCT for OF samples:
Oral fluid Samples:
A total of 282 OF specimens received in the UK during 2008 as part of surveillance programme. 232 received for measles investigation, 39 for rubella investigation, 11 for measles and rubella investigation.
PoCT performance vs MicroImmune on OF samples.
Sensitivity: 90.0% (95CI 80.5 – 95.8%)
Specificity: 96.2% (95% CI 92.6 – 98.3%)
ppv 88.7% (95%CI 79.0 – 95.0%)
npv 96.6% (95%CI 93.2 – 98.6%)
Measles IgM PoCT: 10 Oral fluid specimens (#165 – 174, incubation: 20 minutes)
EIA results - + - - - + + + - + T/N ratios:
0.25 19.25 0.18 0.17 0.21 2.66 3.65 1.33 0.13 5.65
PoCT results - + - - - + + + - +
Oral fluid specimens investigated for molecular detection and characterisation after POCT
H- geme real time PCROral Fluid POCT strip Genotype
(Ct) (Ct) N-gene POCT T/N ResultMEOF1 36.43 35.88 D4 POS 19.57 POSMEOF2 30.72 29.48 D4 POS 30.09 POSMEOF5 25.16 27.62 D4 POS 5.54 POSMEOF7 34.89 35.01 D4 POS 25.4 POSMEOF8 34.23 35.12 D4 POS 23.63 POSMEOF10 33.20 33.28 D4 POS 8.32 POSMEOF11 32.40 33.53 D4 POS 19.57 POSMEOF12 26.85 27.91 D4 POS 1.09 POSMEOF16 30.91 28.26 D4 POS 9.74 POSMEOF17 31.88 31.76 D4 POS 10.78 POSMEOF19 32.51 33.32 D4 NEG 27.74 POSMEOF21 31.23 31.86 D4 POS 15.67 POSMEOF22 34.07 33.99 D4 POS 17.14 POSMEOF6 30.69 32.24 D9 POS 17.52 POSMEOF23 37.32 34.34 A POS 23.5 POSMEOF20 nd nd D4 POS 19.4 POSMEOF3 36.64 nd NEG 0.41 NEGMEOF4 nd nd NEG 0.57 NEGMEOF9 nd nd POS 1.22 POSMEOF13 nd nd POS 0.99 EQVMEOF14 nd nd NEG 0.58 NEGMEOF15 nd nd NEG 0.76 NEGMEOF18 nd nd NEG 0.72 NEGMEOF24 nd nd POS 1.11 POS
Measles Igm resultsMicroimmune
Measles Virus Genomic Structure
Manual for the laboratory diagnosis of measles and rubella virus infectionWHO – EPI, WHO/IVB/07.01: 2nd edition 2007
High-Throughput Sequencing
Amplification methods now available to generate 100,000 clones from single reaction, either multiple or single samples.
Requires high-throughput bioinformatics
Operational Issues:
• Cost: £100 – 1000, further reductions in price likely
• Turnaround Times >7days
From 454 life sciences website
Measles Genome Sequences
Increasing the amount of genetic information used in phylogenetic analysis may improve the quality of inferences.
There are 63 full-length measles sequences in GenBank
Genotype distribution:
A 34
B3 1
C2 2
D3 16
D5 2
D6 4
d11 2
H1 1
H2 1
Tree generated using NJ algorithm
Based on N 450 only
Includes WHO genotype reference sequences0.0070
refseqid_23_gtype_G1_name_Berkley.USA/83
EU293550.1refseqid_12_gtype_D3_name_Illinois.USA/89/1
AF266289.1
refseqid_2_gtype_B1_name_Yaounde.CAE/12.83
NC_001498.1
BD137596.1
FJ161211.1
AY486084.1
BD137594.1
refseqid_24_gtype_G2_name_Amsterdam.NET/49.97
EU293551.1
refseqid_8_gtype_C2_name_Maryland.USA/77
FJ211583.1
EU293548.1
AF266288.2
refseqid_21_gtype_E_name_Goettingen.DEU/71
DI045579.1
DI006056.1
DQ227321.1
refseqid_10_gtype_D10_name_MVi/Kampala.UGA/51.00/1
refseqid_14_gtype_D5_name_Bangkok.THA/93/1
BD137592.1
AB254456.1
DI059890.1
EU435017.1
refseqid_16_gtype_D6_name_New_Jersey.USA/94/1DQ227320.1
refseqid_15_gtype_D5_name_Palau.BLN/93
DI021106.1
DQ345723.1
refseqid_1_gtype_A_name_Edmonston-wt.USA/54
GQ376026.1
AF266291.1
AB016162.1
BD137593.1
refseqid 9 gtype D1 name Bristol.UNK/74 (MVP)
DD461937.1
refseqid_18_gtype_D7_name_Victoria.AUS/16.85
FJ211589.1
FJ416067.1
refseqid_26_gtype_H1_name_Hunan.CHN/93/7
AF266286.1
refseqid_13_gtype_D4_name_Montreal.CAN/89
EF033071.1
DI010567.1
DQ345721.1
EU293552.1
K01711.1
BD137597.1
refseqid_20_gtype_D9_name_MVi/Victoria.AUS/12.99
BD137595.1
DI083540.1
AY730614.1
DQ211902.1
Z66517.1
DQ227319.1
GQ376027.1
AB481088.1
refseqid_22_gtype_F_name_MVs/Madrid.SPA/94_SSPE
DI062293.1
FJ211590.1
refseqid_5_gtype_B3_name_New_York.USA/94
DQ345722.1
AB012949.1
BD137591.1
refseqid_3_gtype_B2_name_Libreville.GAB/84
refseqid_6_gtype_C1_name_Tokyo.JPN/84/K
AY486083.1
AB046218.1
AF266287.1
refseqid_25_gtype_G3_name_MVi/Gresik.INO/17.02
AB481087.1
refseqid_19_gtype_D8_name_Manchester.UNK/30.94
AB032167.1
E04903.1
S58435.1
HM439386.1
FJ416068.1
DI052604.1
refseqid_11_gtype_D2_name_Johannesburg.SOA/88/1
refseqid_7_gtype_C2_name_Erlangen.DEU/90
refseqid_17_gtype_D7_name_Illinois.USA/50.99
EU293549.1
BD137590.1
refseqid_4_gtype_B3_name_Ibadan.Nie/97/1
AF266290.1
AB012948.1
DQ227318.1
refseqid_27_gtype_H2_name_Beijing.CHN/94/1
refseqid_55_gtype_d11_name_MVi/Menglian.Yunnan.CHN/47.09
Measles Virus Genomic Diversity
0
2
4
6
8
10
12
14
0 500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9500
10000
10500
11000
11500
12000
12500
13000
13500
14000
14500
15000
15500
16000
Nucleotide position
Div
ersi
ty
N P M F H LP
Measles virus diversity along the length of the genome. Gene coding regions are shown in red.
The next stepsFull-length measles genome sequencing would provide:
Greater level of genetic variation
Improved lineage stratification of temporally related strains
Mutation rate:Measles:1:6x104
Polio:1:1x102 Substitutions per site per year.
Polio mutation rate 600x measles
Robust phylogenies of clusters (D4 Enfield) that are not resolved by 450 nt of nucleocapsid
Sequence studies need to be supported by models of:
Sampling framework
Susceptible population (vaccine uptake)
Discussion points
• Measles PoCT evaluated for IgM and Virus detection/genotyping on serum and OF samples. IgM PoCT has appropriate sens/spec for field use. RT- PCR on OF extracted strips sensitive and stable (4 weeks at 22C)
• Further evaluation of PoCT with WHO AFRO gave similar results using 100 sera and OF collected in Zimbabwe surveillance programme.
. PoCTs have potential to make a significant contribution to Measles surveillance
Challenges-how to make widely available? ( is there a market? what cost)
Acknowledgement: Lennesha Warender, Dhan Samuel
Discussion points (2)
Technological developments have provided a straightforward way to investigate the value of finer characterisation of virus strains.
Will it be useful for tracking chains of transmission.
Or for discriminating between multiple chains of transmissionwith closely related strains.
Acknowledgement: Richard Myers
Measles Control Goalsby WHO Region, June 2010
20002000
20102010
20102010 20122012
Americas, Europe, E. Mediterranean, W. Pacific, Africa have elimination goals
20202020
2010 SEAR RCEndorsed WHA 2015 targets Target date for elimination TBD
GIVS Goal: 90% reduction in deaths by 2010 (vs. 2000)