1. 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

2. Overview Current status of measles control Range of research needs already identified PoCT test for measles IgM Molecular epidemiology – Impact of new sequencing technologies

3. 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

4. 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

5. 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

6. PoCTs available Viruses Bacteria Influenza RSV HIV HBsAg HCV H. pylori Strep pneumoniae Group A Strep Protozoa Biological threats Chlamydia trachomatis Malaria Anthrax Botulism Plague T. pallidum

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

8. Measles IgM PoCT strip: positive control for serum

9. 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.

10. 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)

11. 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)

12. 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%)

13. 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 - + - - - + + + - +

14. Oral fluid specimens investigated for molecular detection and characterisation after POCT

15. 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

16. 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

17. 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 B31 C22 D316 D52 D64 d112 H11 H21 Tree generated using NJ algorithm Based on N 450 only Includes WHO genotype reference sequences

18. Measles Virus Genomic Diversity N P M F H LP Measles virus diversity along the length of the genome. Gene coding regions are shown in red.

19. 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)

20. 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

21. 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

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