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Lab Experience with HIV RNA NAAT
Myra Brinson, MT(ASCP) Manager, Virology/Serology
North Carolina State Laboratory of Public HealthPh: 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
Building The BSL-3 Facility At TheNC State Laboratory Of Public Health
Or … Stop Bugging Me!
Bath Building 306 North Wilmington Street Raleigh, NC
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
Master Pool (1:90)
Intermediate Pools (1:10)
Figure 1: Schema for pooling Ab-negative specimens
Individual Specimens Master Pool (1:90)
Intermediate Pools (1:10)
Figure 1: Schema for pooling Ab-negative specimens
Individual Specimens
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
Figure 2 : Disposition of Specimens
8505 Total Specimens
38 EIA Repeat Reactive Sera
37 Newly Diagnosed WB+ Chronic Infections 1 WB Negative
v 8298 EIA negative & 5 EIA Unconfirmed Pos.
8005 Ab Neg. Specimens Pooled 5 RNA Positive
1 False + RNA Test299 Specimens
Insufficient Vol. 4 True Positive Acute Infections
12 Previously Tested HIV+
8341 Persons at Risk
152 with Data Incomplete or Questionable
2 HIV+, Status Unknown
Figure 2 : Disposition of Specimens
8505 Total Specimens
38 EIA Repeat Reactive Sera
37 Newly Diagnosed WB+ Chronic Infections 1 WB Negative
v 8298 EIA negative & 5 EIA Unconfirmed Pos.
8005 Ab Neg. Specimens Pooled 5 RNA Positive
1 False + RNA Test299 Specimens
Insufficient Vol. 4 True Positive Acute Infections
12 Previously Tested HIV+
8341 Persons at Risk
152 with Data Incomplete or Questionable
2 HIV+, Status Unknown
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
Master Pool 96 sera
Intermediate Pools 8-12 sera
Figure 1: Schema for pooling Ab-negative specimens
Individual Specimens Master Pool 96 sera
Intermediate Pools 8-12 sera
Figure 1: Schema for pooling Ab-negative specimens
Individual Specimens
STAT Results – Year 1
110,890 requested VCT
Testing Population
109,250 successfully tested
Population at risk
583 EIA + WB+
Long term HIV infected
213 Previously HIV +
108,667 EIA -, WB – or indeterminate RNA tested
25 individual specimens
RNA positive
1,427 Insufficient sample
23 individuals confirmed HIV positive
Acutely HIV infected
2 individuals EIA – RNA – through wk 12
False RNA positive; HIV negative
108,642 negative RNA screen
HIV negative
107 Less sensitive EIA – by STARHS
Likely recent infection
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
20012002
20032004
20052006
2007
91,668 99,551112,716 112,904
134,241146,116
180,000
0
20,000
40,000
60,000
80,000
100,000
120,000
140,000
160,000
180,000
# o
f T
ests
Year
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 Strategy80 HIV-1/HIV-2 Plus O Nonreactive Samples
1 B Pool (containing 120 µl x 10 A Pools) = 1200 µl 10 A Pools (containing 20 µl x 8 samples) = 160 µl
X X X X X X X X
X X X X X X X X
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 OEIA
NR
R
HIV-1 RNA NAAT
Repeat EIAx 2
R x 1NR 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.
CURRENT NC HIV TEST ALGORITHM
HIV-1, HIV-2, Plus OEIA
NR
R
HIV-1 RNA NAAT
Repeat EIAx 2
R x 1NR x
1
NR x 2
Neg
Pos
Report#2
R x 2
HIV-1 Western
Blot
HIV-1 RNA detected.
Possible Acute HIV Infection.Please consult with DiseaseIntervention Specialist to
arrangefor further HIV-1
testing.Recommend
clinical evaluation
with Infectious Disease
Specialist.
CURRENT NC HIV TEST ALGORITHM
HIV-1, HIV-2, Plus OEIA
NR
R
HIV-1 RNA NAAT
Repeat EIAx 2
R x 1NR x
1
NR x 2
Neg
Pos
Report#2
R x 2
HIV-1 Western
Blot
Report#1
CURRENT NC HIV TEST ALGORITHM
HIV-1, HIV-2, Plus OEIA
NR
R
HIV-1 RNA NAAT
Repeat EIAx 2
R x 1NR x
1
NR x 2
Neg
Pos*
Report#2
R x 2
HIV-1 Western
Blot
Report#1
Red, Green,or Blue Dot
Samples*
*For individually testedsamples, repeat any Pos result: If retest is Pos = Report PosIf retest is Neg, repeat again:2nd retest Neg = Report Neg2nd 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 todetermine if further testing
iswarranted to rule out
possibleco-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 DiseaseIntervention Specialist to
determine if further testing iswarranted 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
Infectionwith incomplete seroconversion.
Please consult with Disease
Intervention Specialist to arrange
for further HIV-1 testing.Recommend clinical
evaluationwith 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
Infectionwith incomplete seroconversion.
Please consult with Disease
Intervention Specialist to arrange
for further HIV-1 testing.Recommend clinical
evaluationwith 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 testedsamples, repeat any Pos result: If retest is Pos = Report PosIf retest is Neg, repeat again:2nd retest Neg = Report Neg2nd 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
Report#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
RHIV-2
HIV-1 Western
BlotNR or Ind
Report#6 or #7
HIV-1/HIV-2 Rapid
HIV-1/HIV-2 infectionstatus is Inconclusive.
Please submit another samplefor 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
RHIV-2
Report#8 or #9
HIV-1 Western
BlotNR 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 isinconclusive.
Specimen referred to CDCfor further
HIV-2 testing.
Questions???
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