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 GenProbe APTIMA HIV Method Verification NC State Lab HIV Test Algorithm

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

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

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

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

STAT – Pooling Design

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

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)

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

Manual vs. Automated Pooling 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

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

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

Increase in HIV Antibody Screens 2001-2007 This graph illustrates the reason why we at the State Lab felt the need to move to a fully automated platform for initial HIV antibody screens. The last few years have shown a dramatic increase in our volume of HIV testing, from around 92,000 in 2001 to a projected test volume by the end of this year of over 180,000. We have doubled our test volume over seven years and our current semi-automated test platform with existing personnel is maxed out. The Evolis automation will allow us to increase our test capacity without additional personnel costs.

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

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

New Pooling Strategy

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

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

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)

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

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, and 10 copies/ml Observed typical reaction curve for molecular assays Precipitous drop-off in signal strength from 100 to 10 copies/ml

Limit of Detection 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

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

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

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

CURRENT NC HIV TEST ALGORITHM HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 1 NR x 1 NR x 2 Neg Pos R x 2 HIV-1 Western Blot Report #1 HIV-1 (Groups M & O) and HIV-2 antibodies were not detected. HIV-1 RNA not detected. So now I would like to go over our new test HIV test algorithm. I will be presenting this algorithm in a piecemeal fashion due to it’s relative complexity (it wouldn’t fit on one slide) and you can follow along with the complete algorithm on the last page of the handout.

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. HIV-1, HIV-2, Plus O EIA NR R HIV-1 RNA NAAT Repeat EIA x 2 R x 1 NR x 1 NR x 2 Neg Pos Report #2 R x 2 HIV-1 Western Blot So now I would like to go over our new test HIV test algorithm. I will be presenting this algorithm in a piecemeal fashion due to it’s relative complexity (it wouldn’t fit on one slide) and you can follow along with the complete algorithm on the last page of the handout.

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

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

CURRENT NC HIV TEST ALGORITHM Repeatedly Reactive HIV-1, HIV-2, Plus O EIA NR HIV-1 RNA NAAT Pos Indeterminate R Report #3 HIV-1 Western Blot Neg 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.

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

CURRENT NC HIV TEST ALGORITHM Repeatedly Reactive HIV-1, HIV-2, Plus O EIA NR HIV-1 RNA NAAT Pos Indeterminate R HIV-1 Western Blot Report #4 Neg 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.

CURRENT NC HIV TEST ALGORITHM Repeatedly Reactive HIV-1, HIV-2, Plus O EIA NR HIV-1 RNA NAAT Pos Indeterminate R HIV-1 Western Blot Report #5 Neg 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.

CURRENT NC HIV TEST ALGORITHM Repeatedly Reactive HIV-1, HIV-2, Plus O EIA NR HIV-1 RNA NAAT Pos* Indeterminate R HIV-1 Western Blot Report #4 or #5 Neg *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

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

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

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

Questions???