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SoGAT and HIV NAT Standards - 2 nd International Standard for HIV-1 RNA

SoGAT and HIV NAT Standards - 2 nd International Standard for HIV-1 RNA. Harvey Holmes*, Clare Davis* and Alan Heath** Divisions of Retrovirology* and Informatics** National Institute for Biological Standards and Control (NIBSC) UK. SoGAT and NAT Standards available from NIBSC.

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SoGAT and HIV NAT Standards - 2 nd International Standard for HIV-1 RNA

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  1. SoGAT and HIV NAT Standards -2nd International Standard for HIV-1 RNA Harvey Holmes*, Clare Davis* and Alan Heath** Divisions of Retrovirology* and Informatics** National Institute for Biological Standards and Control (NIBSC) UK XVIII SoGAT Washington 24 May 2005

  2. SoGAT and NAT Standards available from NIBSC • SoGAT and NIBSC have collaborated in establishing: International Standards: • 1997: 1st International Standard for HCV RNA (96/790) • 1999: 1st International Standard for HIV-1 RNA (97/656) • 1999: 1st international Standard for HBV DNA (97/746) • 2000: 1st International Standard for B19 DNA (99/800) • 2002: 1st International Standard for HAV RNA (00/560) • 2003: 1st International Reference Panel for HIV-1 Genotypes (01/466) – genotypes A-H • 2003: 2nd International Standard for HCV RNA (96/798) Working Reagents: • HIV-1 RNA PWS-1, PWS-2, PWS-3 • HCV RNA • HBV DNA • HAV RNA • B19 DNA • Multiplex (HCV, HBV, HIV, HAV, B19) • HCV Genotype Panel (genotypes 1 -6) XVIII SoGAT Washington 24 May 2005

  3. Retrovirology Standards and Working Reagents XVIII SoGAT Washington 24 May 2005

  4. Replacement of 1st International Standard for HIV-1 RNA (NIBSC Code 97/656) Sample YY established as 1st International Standard (code 97/656) in 1999 Assigned a concentration of 100,000 IU per vial/ml Standard made from HIV-1 PCR-positive, antibody-negative, plasmapheresis donation diluted in defibrinated plasma 2300 vials freeze-dried and stored at -200C SoGAT 2003 - agreement to replace standard due to: low Stocks (~ 450) presence of HBV DNA – may affect suitability for multiplex assays Interim report on collaborative study presented by Clare Davis at SoGAT 2004 XVIII SoGAT Washington 24 May 2005

  5. Candidate 2nd International Standard • SoGAT recommended that sample XX from first collaborative study be re-evaluated as candidate 2nd International Standard (IS) • Characteristics: • HIV-1 subtype B low passage field isolate (CCR5/NSI) from Edinburgh (P Simmonds) • Propagated on human PBMCs - stock stored under LN • Virus diluted in human cryosupernatant to suitable concentration • 2200 vials of material freeze-dried • Fill CV = 0.2% XVIII SoGAT Washington 24 May 2005

  6. International Collaborative Study to Establish 2nd International Standard for HIV-1 RNA • Aims of study:- • Calibrate XX against 1st International Standard (1st IS) • Confirm stability of XX • Determine values in current assays • 10 laboratories participated • Candidate XX and the 1st International Standard recoded (samples 1 and 2 respectively) • Participants asked to test samples in 3 independent assays. • Initially at 10-fold dilutions • Subsequently at half log dilutions around the end point • 8 data sets received, 5 from quantitative assays and 3 from qualitative assays • NIBSC collated and analysed data XVIII SoGAT Washington 24 May 2005

  7. 10 9 10 28 NU 8 9 22 5 M M 8 7 24 2 M M 7 6 2 18 4 M M NU 6 18 5 17 5 34 M M M NU 5 17 46 4 16 6 28 M M IH IH NU 4 34 10 22 3 NU IH M 44 47 10 46 3 M B3 IH M 43 43 48 6 2 IH MQ B3 IH 41 14 48 24 2 IH B3 B3 M 41 44 47 14 1 IH M B3 B3 43 43 3 19 33 45 9 1 16 3 19 33 45 9 IH MQ IH AS B3 A M IH IH AS B3 A M 0 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 0 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 Estimated RNA Copies (log10/ml) Estimated RNA Copies (log10/ml) Monitor assays Nuclisens bDNA 3 Abbott LCx In-House Ampliscreen Nuclisens bDNA 3 Abbott LCx In-House Ampliscreen Monitor assays Original Collaborative Study 1st International Standard for HIV-1 RNA (YY) Sample XX XVIII SoGAT Washington 24 May 2005

  8. 10 9 9 M 8 44 M 7 2 M 6 4 5 18 NU M M 5 34 46 17 NU M M 4 28 3 6 NU IH IH 3 24 10 48 43 M IH B3 MQ 2 41 33 47 22 IH B3 B3 M 1 16 19 45 14 43 IH AS A B3 IH 0 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 Estimated IU (log10/ml) Monitor assays Nuclisens bDNA 3 Abbott LCx In-House Ampliscreen Original Collaborative Study Sample XX relative to1st IS in IU XVIII SoGAT Washington 24 May 2005

  9. 10 10 9 9 8 8 7 7 6 6 5 5 4 4 7 72 P T 3 3 4 72 NU P 2 2 4 71 NU IH 1 1 70 58 7 22 73 71 58 70 22 73 IH B3 T M A IH B3 IH M A 0 0 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 Estimated RNA Copies (log10/ml) Estimated RNA Copies (log10/ml) Monitor assays Nuclisens bDNA 3 Procilex In-House Amplicore TMA Monitor assays Nuclisens bDNA 3 Procilex In-House Amplicore TMA Current Collaborative Study 1st IS (coded Sample 2) Sample XX in Current Study (coded Sample 1) XVIII SoGAT Washington 24 May 2005

  10. 10 9 8 7 6 5 4 3 72 P 2 4 22 NU M 1 70 7 58 73 71 IH T B3 A IH 0 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 Estimated IU (log10/ml) Monitor assays Nuclisens bDNA 3 Procilex In-House Amplicore TMA Current Collaborative StudyPotency of XX (Sample 1) relative to 1st IS (IU) XVIII SoGAT Washington 24 May 2005

  11. Current Study: Relative Potency of XX to 1st IS – Log10 IU/ml XVIII SoGAT Washington 24 May 2005

  12. Relative Potency of XX to 1st IS Original Study (Log10 IU/ml) XVIII SoGAT Washington 24 May 2005

  13. 2nd International Standard for HIV-1 RNA • No evidence of any drift in values between the two studies - no stability issues • Four options for assigning value to XX: • 1: Use results from original study (5.60 log10 IU/ml) • 2: Use results from current study (5.44 log10 IU/ml) • 3: Pool data from both studies (5.56 log10 IU/ml) • 4: Determine overall mean for each method and obtain compromise between methods – pragmatic rather than statistically based • Option 3 favoured by NIBSC statistician XVIII SoGAT Washington 24 May 2005

  14. International Collaborative Study to Establish 2nd International Standard for HIV-1 RNAParticipants • T Cuypers Sanquin – CLB, The Netherlands • M Chudy Paul Ehrlich Institute, Germany • J Docker Gen Probe, USA • K Fransen Institute of Tropical Medicine, Belgium • I Hewlett CBER, FDA, USA • F Intelmann Baxter, Austria • D Jardine National Reference Laboratory, Australia • J Turczyn Bayer Testing lab, USA • Richard Smith NGI, USA • C Davis/H Holmes NIBSC, UK XVIII SoGAT Washington 24 May 2005

  15. HIV-1 Variability – Genotype panels • WHO 1st International Reference Panel for HIV-1 Genotypes (Code 01/466) established in 2003 • Contains 11 members: • Group M – Subtypes A - G, and AG-GH recombinant • Group N and O representative • HIV diversity increasing, with greater frequency of unusual subtypes, outlier groups and circulating recombinant forms (CRFs) being encountered especially in cities eg London, Paris • Greater diversity presents continuing challenge to NAT technology • Would a second genotype panel be of value containing: • More diverse subtypes, group N, group O representatives? • More CRFs? • HIV-2 representatives? XVIII SoGAT Washington 24 May 2005

  16. Conclusions • Report on candidate 2nd International Standard for HIV-1 RNA to WHO ECBS • Recommendation on unitage – option 3?? • Need for 2nd HIV Genotype Panel containing more diverse field isolates?? XVIII SoGAT Washington 24 May 2005

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