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Update on CBER HIV-1 Subtype panel. Indira Hewlett, Ph.D CBER/FDA. CBER HIV-1 subtype panel. 20 specimens representing subtypes A-G of group M, N and O 2 isolates of each subtype of group M, 1 of group N and 5 of group O Dilutions 10 4 , 10 3 , 10 2 per ml for all subtypes

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update on cber hiv 1 subtype panel

Update on CBER HIV-1 Subtype panel

Indira Hewlett, Ph.D

CBER/FDA

cber hiv 1 subtype panel
CBER HIV-1 subtype panel
  • 20 specimens representing subtypes A-G of group M, N and O
  • 2 isolates of each subtype of group M, 1 of group N and 5 of group O
  • Dilutions 104, 103, 102 per ml for all subtypes
  • Samples tested by five manufacturers
prototype panel composition
Prototype Panel Composition
  • Each panel was comprised of 2 isolates
  • Seven members including one negative control

per panel; Panel O has nineteen members containing five distinct isolates and four negative controls.

  • Diluted to targeted ranges of Log 2 to Log 4 (copies/ml).
  • Tested by 5 different manufacturer’s assays
expected final formulation for panel
Expected final formulation for panel
  • One isolate of each subtype representing subtypes A-G of group M, N and O
  • Four members including one negative control per panel
  • Diluted to targeted ranges of Log 2 to Log 4 (copies/ml).
summary
Summary
  • CBER has developed a well characterized panel for qualitative and quantitative detection of HIV-1 subtype RNA
  • Panel may be used 1) to establish LOD and 2) for lot release of HIV NAT assays for blood and plasma donor screening
background

Background

The 2002 US outbreak of WNV identified transmission by Blood transfusion, Transplantation, Breast-feeding, Transplacental and Occupational by percutaneus injury

During Aug 28, 2002-March 1, 2003, 61 possible Transfusion-Transmitted cases reported

21 confirmed from 14 blood donations

19 are not transfusion related,

21 inconclusive due to incomplete donor follow-up

wnv testing
WNV testing
  • All reported cases of WNV transmission by blood transfusion have occurred during the acute, viremic phase
  • NAT may be the most appropriate strategy to interdict infectious donations
  • Virus titer in blood is low compared to other transmissible viruses (~1-5x103 copies/ml) and the viremia is transient.
  • Impact of pooling on sensitivity of WNV NAT assays is of concern
analytical sensitivity
Analytical sensitivity
  • FDA’s current standard for WNV NAT assays is 100 copies/ml for the individual donation
  • Standard may be revised as assay sensitivity improves and additional data on viremia and infectivity become available in future studies
fda panel development efforts
FDA Panel Development Efforts
  • Lot release panel for licensure and post-market surveillance of NAT tests
  • Qualification panel for evaluation of relative sensitivities of investigational NAT assays
fda nat panels
FDA NAT Panels
  • FDA NY99 and FDA-Hu2002 isolates characterized by genetic sequencing
  • Viral infectivity determination
    • PFU determined at both FDA and NY Dept. of Health Laboratories, and by cytopathic assays at FDA
  • RNA concentration measurements
    • Fluorescence and Optical density determination
    • TaqMan
  • Final panel specifications are being established through collaborative studies
pfu results on fda isolates
PFU Results on FDA Isolates
  • At FDA
    • NY99 (CDC Flamingo Isolate) 108/mL
    • HuWNV2002 108/mL
  • At NY State Dept. of Health
    • NY99 (CDC Flamingo Isolate) 5.5 x107/mL
    • HuWNV2002 9.5 x106/mL
fda plan for qualification panel
FDA Plan for Qualification Panel
  • At least 100 pedigreed clinical specimens
    • RNA positive only
    • IgM positive only
    • Dual RNA and IgM positive
    • Panel will be evaluated in collaborative studies using various candidate NAT assays
  • Specifications for NAT panel will be established based on results of collaborative studies
summary19
Summary
  • Both NY99 and FDA-Hu2002 stocks have a viral titer of 1010 copies/mL
  • PFU titers determined at NY State Dept of Health Laboratory and at FDA were three logs lower than copy numbers
  • Heat treatment of virus resulted in loss of infectivity and 2 to 3 log reduction in copy numbers as determined by TaqMan
  • Prototype panel is being formulated to evaluate performance of panel in a larger collaborative study
acknowledgements
Acknowledgements

CBER

  • Owen Wood
  • Sherwin Lee Nelson Michael, DoD
  • Rolf Taffs Harvey Holmes, NIBSC
  • Jinjie Hu
  • Ana Machuca Robert Lanciotti, CDC
  • Maria Rios Laura Kramer, NYDOH
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