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???