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

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Nucleotide position

Div

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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)