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Lab Experience with HIV RNA NAAT

Lab Experience with HIV RNA NAAT. Myra Brinson, MT(ASCP) Manager, Virology/Serology North Carolina State Laboratory of Public Health Ph: 919-807-8835 E-mail: Myra.Brinson@ncmail.net. Discussion Topics. History of HIV RNA NAAT at NC State Laboratory

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Lab Experience with HIV RNA NAAT

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  1. Lab Experience with HIV RNA NAAT Myra Brinson, MT(ASCP) Manager, Virology/Serology North Carolina State Laboratory of Public Health Ph: 919-807-8835 E-mail: Myra.Brinson@ncmail.net

  2. Discussion Topics • History of HIV RNA NAAT at NC State Laboratory • GenProbe APTIMA HIV Method Verification • NC State Lab HIV Test Algorithm

  3. Evolution of NCSLPH HIV NAAT: 2001 to 2008 • Utilization of different assays for the detection of HIV-1 RNA • Different pooling algorithms • Different pooling mechanisms • Consistently demonstrated the ability to detect acute HIV-1 infections

  4. Pilot Study 2001 - Design • All consecutive routine HIV tests submitted to the NC State Laboratory of Public Health over 4 weeks from 110 publicly funded counseling and testing sites (CTS) [n=8505] • Initial Ab testing - OT Vironostika HIV-1 Viral Lysate Microelisa (State Lab) • Manual pooling of Ab NR samples (State Lab) • Roche Amplicor HIV-1 Monitor (UNC) – Standard and US

  5. Pilot Study 2001 - Results • Acute infection: 5 per 10,000 • Chronic infection: 44 per 10,000 • Overall specificity: 99.99% • Estimated additional cost per specimen: $2.01 • Estimated total testing costs/additional case diagnosed: $4109 Pilcher CD et al, JAMA, Vol. 288/No. 2, July 10, 2002

  6. Screening and Tracing Active Transmission (STAT) Program – Year 1 • 11/01/2002 to 10/31/2003 - All consecutive routine HIV tests submitted to the State Laboratory from 110 publicly funded counseling and testing sites (CTS) [n=110,890] • Initial Ab testing - bioMerieux Vironostika HIV-1 Viral Lysate Microelisa • Initial manual pooling • NAAT testing – bioMerieux NucliSens HIV-1 Qualitative assay • Automated pooling added-Beckman Coulter bioMek FX

  7. STAT – Pooling Design

  8. STAT Results – Year 1 Pilcher CD et al, New England Journal of Medicine, May 5 2005;352(18):1873-1883.

  9. Screening and Tracing Active Transmission (STAT) Program – Year 2 • Nov 03 to March 05 - All consecutive routine HIV tests submitted to the State Laboratory from 110 publicly funded counseling and testing sites (CTS) [n=118,656] • Initial Ab testing - bioMerieux Vironostika HIV-1 Viral Lysate Microelisa • NAAT testing – GenProbe Procleix HIV-1 Assay • Automated pooling – Hamilton AT Plus • Detected 17 acute HIV cases (1 False Positive)

  10. STAT Overall Results - 2 years • 224,108 EIA negative sera pooled and tested for HIV-1 RNA • 40 True Positive Acute Infections; 3 False Positive RNA tests • Acute infection: 1.8 per 10,000 • Chronic infection: 65.9 per 10,000 • Overall specificity: 99.8% • At least 4% of the HIV-1 infected patients would have been undetected without the use of NAATs

  11. Manual pooling very labor intensive – 30 to 45 minutes/90 samples Must be particularly diligent in pipetting technique to avoid cross contamination of samples 3 of 4 false positives occurred during the period of manual pooling Automated pooling instrumentation, maintenance, and pipet tips can be expensive Much more efficient – 10-15 minutes/90 samples Improved control of process – decreased human error Positive specimen identification - barcodes Manual vs. Automated Pooling

  12. STAT Project Continues • GenProbe Procleix HIV-1 Assay withdrawn from market due to patent dispute with Chiron • March 05 – Nov 07 bioMerieux Nuclisens Easy Q HIV-1 NAAT • EasyMag Automated Extraction/ Real-Time assay • Initial Ab testing - bioMerieux Vironostika HIV-1 Viral Lysate Microelisa • Automated pooling - Hamilton AT Plus • Continued to detect acute HIV cases, although assay sensitivity was somewhat of concern

  13. 2008 – Changes to HIV Testing CATALYSTS • bioMerieux Vironostika HIV-1 Viral Lysate Microelisa discontinued • Contract for HIV NAAT out for bid – opportunity to bring on a more sensitive assay • New LIMS

  14. Increase in HIV Antibody Screens 2001-2007

  15. New HIV Antibody Assay • Bio-Rad Genetic Systems HIV-1/HIV-2 Plus O EIA • Automation - 3 Evolis instruments • More sensitive assay – detects both IgM and IgG • Ability to detect HIV-2 and Group O

  16. New NAAT Assay and Pooling Algorithm • GenProbe APTIMA HIV-1 NAAT RNA assay • Hamilton STARlet robotic pipetting instrument • Reduced pool size (80 samples/pool) • Increased sensitivity for HIV-1 NAAT

  17. New Pooling Strategy

  18. NAAT Questions??? • Cost of NAAT: $37-$50/test, based upon 4,000/year test volume • Two dedicated staff for pooling and NAAT testing – pool daily and test 2-3 times/week • Currently pool all EIA negative samples vs. separating into low and high risk groups • NAAT has also been useful for resolution of EIA reactive/WB negative or indeterminate samples • Increased TAT – 2-3 days to 2-3 weeks

  19. GenProbe APTIMA HIV-1 NAAT Method Verification • Off-label use of an FDA-approved assay * Plasma vs. serum * Waterbaths vs.SB100s • CAP Molecular Verification Checklist *Accuracy *Precision *Reportable Range *Limit of Detection *Analytical Sensitivity *Analytical Specificity *Specificity/Cross Reactivity

  20. Accuracy • 18 Nonreactive and 7 Reactive B Pools previously tested by current method (bioMerieux Nuclisens EasyQ) • 17 of 18 known NR samples tested NR by APTIMA • 7 of 7 known R samples tested R by APTIMA • One discrepant sample QNS to resolve • 96% (24/25)

  21. Precision-Reproducibility • Tested several replicates of pooled NR sera and a R sera (170 copies /ml) over multiple days • 170 copies/ml- prepared by diluting a sample of known copy number from a purchased linearity panel (HIV RNA Linearity Panel, PRD801, BBI Diagnostics) in pooled NR sera • NR Intra-Assay 49% CV; Inter-Assay 51% CV • R Intra-Assay 4% CV; Inter-assay R: 8% CV

  22. Reportable Range • 2,900,000 (2.9 x 106) copy/ml sample from the purchased linearity panel • Serially diluted in pooled NR sera to yield approx. log concentrations of 1 x 106,105,104,103, 102,and10 copies/ml • Observed typical reaction curve for molecular assays • Precipitous drop-off in signal strength from 100 to 10 copies/ml

  23. Serially diluted the 170 copies/ml sample from the purchased linearity panel in pooled NR sera to yield samples of approx. concentration of 85, 43, 21, 11, 5, and 3 copies/ml Tested five replicates of the seven dilutions 100% at ≥43 copies/ml HIV-1 RNA 80% at 5 to 21 copies/ml HIV-1 RNA Limit of Detection

  24. Analytical Sensitivity/Specificity • Calculated by using accuracy sample results • Analytical Sensitivity: 100% 7 of 7 known R samples tested R by APTIMA • Analytical Specificity: 96% 17 of 18 known NR samples tested NR by APTIMA

  25. Specificity/Cross Reactivity • Spiked pooled NR sera with other blood-borne viral pathogens that might be present in tested patient population: HSV-1, HSV-2, CMV, HAV, HBV, and HCV • Required the purchase of HBV viral isolate from ATCC; other viruses obtained locally • Virus concentrations tested were similar to average concentrations of HIV virus expected to be present in patient serum samples • Samples were run twice, on consecutive days • No cross-reactivity or interference with the six blood-borne viral pathogens

  26. Evaluation Questions??? • Duration: Verification required approx. six weeks to complete • Cost: Test kits provided by GenProbe BBI Linearity Panel - $1,380 ATCC HBV - $188 • Availability of NR and R comparison samples - in our study, we were able to use master pool samples run previously by existing assay - may have to purchase additional panels

  27. HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 2 Neg NR x 2 Pos R x 1 NR x 1 Report #1 HIV-1 Western Blot CURRENT NC HIV TEST ALGORITHM HIV-1 (Groups M & O) and HIV-2 antibodies were not detected. HIV-1 RNA not detected.

  28. HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 2 Neg NR x 2 Pos R x 1 NR x 1 HIV-1 Western Blot Report #2 CURRENT NC HIV TEST ALGORITHM HIV-1 RNA detected. Possible Acute HIV Infection. Please consult with Disease Intervention Specialist to arrange for further HIV-1 testing. Recommend clinical evaluation with Infectious Disease Specialist.

  29. HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 2 Neg NR x 2 Pos R x 1 NR x 1 Report #1 HIV-1 Western Blot Report #2 CURRENT NC HIV TEST ALGORITHM

  30. HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 2 Neg NR x 2 Pos* R x 1 NR x 1 Report #1 HIV-1 Western Blot Report #2 CURRENT NC HIV TEST ALGORITHM Red, Green, or Blue Dot Samples* *For individually tested samples, repeat any Pos result: If retest is Pos = Report Pos If retest is Neg, repeat again: 2nd retest Neg = Report Neg 2nd retest Pos = Report Pos

  31. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate Report #3 HIV-1 RNA NAAT Pos Neg CURRENT NC HIV TEST ALGORITHM HIV-1 (Groups M & O) Antibody was detected. Please consult with Disease Intervention Specialist to determine if further testing is warranted to rule out possible co-infection with HIV-2, based upon epidemiological information.

  32. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate Report #3 HIV-1 RNA NAAT CDC for HIV-2 WB or Rapid, by DIS request Pos Neg CURRENT NC HIV TEST ALGORITHM HIV-1 (Groups M & O) Antibody was detected. Please consult with Disease Intervention Specialist to determine if further testing is warranted to rule out possible co-infection with HIV-2, based upon epidemiological information.

  33. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate HIV-1 RNA NAAT Pos Neg Report #4 CURRENT NC HIV TEST ALGORITHM HIV-1 RNA detected. Possible Acute HIV Infection with incomplete seroconversion. Please consult with Disease Intervention Specialist to arrange for further HIV-1 testing. Recommend clinical evaluation with Infectious Disease Specialist.

  34. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate HIV-1 RNA NAAT Pos Neg Report #5 CURRENT NC HIV TEST ALGORITHM HIV-1 RNA detected. Probable Acute HIV Infection with incomplete seroconversion. Please consult with Disease Intervention Specialist to arrange for further HIV-1 testing. Recommend clinical evaluation with Infectious Disease Specialist.

  35. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate HIV-1 RNA NAAT Pos* Neg Report #4 or #5 CURRENT NC HIV TEST ALGORITHM *For individually tested samples, repeat any Pos result: If retest is Pos = Report Pos If retest is Neg, repeat again: 2nd retest Neg = Report Neg 2nd retest Pos = Report Pos

  36. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot R NR Indeterminate Report #3 HIV-1 RNA NAAT CDC for HIV-2 WB or Rapid, by DIS request Pos Neg Report #4 or #5 CURRENT NC HIV TEST ALGORITHM

  37. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot NR or Ind HIV-1 RNA NAAT Neg HIV-1/HIV-2 Rapid NR R HIV-2 Report #6 or #7 CURRENT NC HIV TEST ALGORITHM HIV-1/HIV-2 infection status is Inconclusive. Please submit another sample for further HIV-1/HIV-2 testing.

  38. Repeatedly Reactive HIV-1, HIV-2, Plus O EIA HIV-1 Western Blot NR or Ind HIV-1 RNA NAAT Neg HIV-1/HIV-2 Rapid NR CDC for HIV-2 WB Confirmation R HIV-2 Report #8 or #9 CURRENT NC HIV TEST ALGORITHM HIV-1 (Groups M or O) was not detected. HIV-2 infection status is inconclusive. Specimen referred to CDC for further HIV-2 testing.

  39. Questions???

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