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

•

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

Ideal features of PoCT

• 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

PoCTs available

Influenza RSV HIV HBsAg HCV

Viruses Protozoa Bacteria

T. pallidum H. pylori Strep pneumoniae

Group A Strep

Chlamydia trachomatis

Biological threats

Anthrax Botulism Plague Malaria

Principle of the IgM capture near patient test

Direction of reagent flow Oral fluid IgM rNP Nitrocellulose membrane Test line: anti-human IgM Control line: Anti-mouse IgG Cotton linter paper wick Glass fibre Conjugate release pad Anti-NP gold conjugate Plastic backing card

Measles IgM PoCT strip: positive control for serum

Serum measles IgM PoCT protocol Step1 Step 2 Step 3 Step 4 Step 5 Dilute serum 1/100 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 Sensitivity : 90.8% (95% CI 81.9 – 96.2) Specificity : 93.6% (95% CI 86.6 – 97.6) - ppv 92.0 (95% CI 83.4 – 97.0) - npv 92.4 (95% CI 85.4 – 96.9)

Measles IgM PoCT: 10 Sera (# 69 – 78, incubation: 10 minutes) 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

+ + + + + +/- +/-

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 MEOF1 MEOF2 MEOF5 MEOF7 MEOF8 MEOF10 MEOF11 MEOF12 MEOF16 MEOF17 MEOF19 MEOF21 MEOF22 MEOF6 MEOF23 MEOF20 MEOF3 MEOF4 MEOF9 MEOF13 MEOF14 MEOF15 MEOF18 MEOF24

H- geme real time PCR

Oral Fluid (Ct) 36.43

30.72

POCT strip (Ct) 35.88

29.48

25.16

34.89

34.23

33.20

32.40

26.85

30.91

27.62

35.01

35.12

33.28

33.53

27.91

28.26

31.88

32.51

31.23

34.07

30.69

37.32

nd 36.64

nd nd nd nd nd nd nd 31.76

33.32

31.86

33.99

32.24

34.34

nd nd nd nd nd nd nd nd nd

Genotype

N-gene D4 D4 D4 D4 D4 D4 D4 D4 D4 D4 D4 D4 D4 D9 A D4 POS POS POS POS NEG POS POS POCT POS POS POS POS POS POS POS POS POS NEG NEG POS POS NEG NEG NEG POS

Measles Igm results

Microimmune T/N Result 19.57 POS 30.09 POS 5.54 POS 25.4 POS 23.63 POS 8.32 POS 19.57 POS 1.09 POS 9.74 POS 10.78 POS 27.74 POS 15.67 POS 17.14 POS 17.52 POS 23.5 POS 19.4 POS 0.41 NEG 0.57 NEG 1.22 POS 0.99 EQV 0.58 NEG 0.76 NEG 0.72 NEG 1.11 POS

Measles Virus Genomic Structure

Manual for the laboratory diagnosis of measles and rubella virus infection WHO – EPI, WHO/IVB/07.01: 2 nd 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 B3 C2 D3 D5 D6 d11 H1 H2 34 1 2 16 2 4 2 1 1

Tree generated using NJ algorithm Based on N 450 only Includes WHO genotype reference sequences

DQ345722.1

DQ345721.1

DQ345723.1

AF266286.1

DI059890.1

AY486083.1

AF266288.2

refseqid_1_gtype_A_name_Edmonston-wt.USA/54 BD137590.1

AY486084.1

BD137596.1

E04903.1

BD137597.1

FJ416068.1

EF033071.1

DI045579.1

Z66517.1

AF266290.1

S58435.1

DI021106.1

AB046218.1

AY730614.1

DI062293.1

BD137595.1

FJ211590.1

FJ211583.1

AF266291.1

AF266287.1

FJ211589.1

DI010567.1

BD137594.1

AF266289.1

K01711.1

EU435017.1

FJ416067.1

refseqid_3_gtype_B2_name_Libreville.GAB/84 refseqid_2_gtype_B1_name_Yaounde.CAE/12.83

refseqid_5_gtype_B3_name_New_York.USA/94 refseqid_4_gtype_B3_name_Ibadan.Nie/97/1 HM439386.1

refseqid_22_gtype_F_name_MVs/Madrid.SPA/94_SSPE refseqid_21_gtype_E_name_Goettingen.DEU/71 refseqid_6_gtype_C1_name_Tokyo.JPN/84/K refseqid_7_gtype_C2_name_Erlangen.DEU/90 refseqid_8_gtype_C2_name_Maryland.USA/77 DI052604.1

BD137591.1

refseqid_24_gtype_G2_name_Amsterdam.NET/49.97

refseqid_25_gtype_G3_name_MVi/Gresik.INO/17.02

refseqid_23_gtype_G1_name_Berkley.USA/83 refseqid_27_gtype_H2_name_Beijing.CHN/94/1 DQ211902.1

refseqid_26_gtype_H1_name_Hunan.CHN/93/7 FJ161211.1

refseqid_10_gtype_D10_name_MVi/Kampala.UGA/51.00/1 refseqid_11_gtype_D2_name_Johannesburg.SOA/88/1 refseqid_16_gtype_D6_name_New_Jersey.USA/94/1 DQ227320.1

DQ227319.1

DQ227321.1

DQ227318.1

refseqid 9 gtype D1 name Bristol.UNK/74 (MVP) refseqid_17_gtype_D7_name_Illinois.USA/50.99

refseqid_18_gtype_D7_name_Victoria.AUS/16.85

refseqid_55_gtype_d11_name_MVi/Menglian.Yunnan.CHN/47.09

DI083540.1

BD137592.1

refseqid_19_gtype_D8_name_Manchester.UNK/30.94

refseqid_14_gtype_D5_name_Bangkok.THA/93/1 refseqid_13_gtype_D4_name_Montreal.CAN/89 AB012949.1

AB012948.1

refseqid_15_gtype_D5_name_Palau.BLN/93 refseqid_20_gtype_D9_name_MVi/Victoria.AUS/12.99

AB481088.1

AB481087.1

AB032167.1

AB016162.1

DD461937.1

NC_001498.1

EU293551.1

EU293552.1

EU293549.1

EU293548.1

EU293550.1

refseqid_12_gtype_D3_name_Illinois.USA/89/1 DI006056.1

BD137593.1

GQ376027.1

GQ376026.1

AB254456.1

0.0070

Measles Virus Genomic Diversity

8 6 4 14 12 10 2 0 N P M F H LP

Nucleotide position

Measles virus diversity along the length of the genome. Gene coding regions are shown in red.

The next steps

Full-length measles genome sequencing would provide:

Greater level of genetic variation Improved lineage stratification of temporally related strains Mutation rate:Measles:1:6x10 4 Polio:1:1x10 2 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 Goals by WHO Region, June 2010

Americas, Europe, E. Mediterranean, W. Pacific, Africa have elimination goals 2000 GIVS Goal: by 2010 (vs. 2000) 2010 90% reduction in deaths 2010 2020 2012 2010 SEAR RC Endorsed WHA 2015 targets Target date for elimination TBD