1. Lab Experience with HIV RNA NAAT
Myra Brinson, MT(ASCP)
Manager, Virology/Serology
North Carolina State Laboratory of Public Health
Ph:

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 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/ 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 ;352(18):

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 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 – minutes/90 samples
Improved control of process – decreased human error
Positive specimen identification - barcodes

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
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.

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

23. 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

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. 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.

28. 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.

29. 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

30. 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

31. 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.

32. 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.

33. 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.

34. 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.

35. 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

36. 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

37. 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.

38. 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.

39. Questions???