Incorporation of the Aptima® HIV-1 RNAAssay into Serodiagnostic and Rapid Test

Confirmation Testing Algorithms to Resolve Discordant Serological Results

M. Parker1, R. Boromisa1, T. Sullivan1, M. San Antonio-Gaddy2, A. Richardson-Moore2

New York State Department of Health1Wadsworth Center, 2AIDS Institute

Issue

• Confirmatory testing of EIA RR and Rapid test reactive specimens

• For neg. or indeterminate WB results: Follow-up specimens are requested

• In some cases, no follow-up specimen is sent, or HIV infection status cannot be resolved by follow-up specimen

NAAT Supplemental Test

• Testing strategies 1/2 addressed

• Two lab-based algorithms evaluated:– Serodiagnostic (EIA screening)– Rapid test referral for confirmation

• Data from 2006 and 2007 presented

Screening Assays

• A1 Serodiagnostic algorithm:– Genetic Systems HIV-1/HIV-2 + O (Bio-Rad)

• A1 Rapid test referral algorithm:– OraQuick Rapid Test (OraSure)– Uni-Gold Rapid Test (Trinity Biotech)

• Additional screening test: Multispot

Supplemental Tests

• B1 HIV-1 Western Blot– Genetic Systems HIV-1 WB (Bio-Rad)– Cambridge Biotech HIV-1 WB (Maxim)

• B2 Individual HIV-1 NAAT– Procleix HIV-1 RNA Qualitative Assay (’06)– Aptima HIV-1 RNA Qualitative Assay (’07)

(both Gen-Probe)

Serodiagnostic Test Data

Serodiagnostic Algorithm: Initial

RequestFollow-up

EIA - -Report:

Negative

Report:Positive

EIA +

EIA ++/+-

Multispot

ReactiveNeg or Ind WB

Not detected Detected

NAAT

ReactiveReport:Positive

Neg or Ind WB

Revised Algorithm: NAAT Added

Report: RNA –Follow-up only if risk

Report: RNA +Possible AHS

Serodiagnostic Testing

2006 2007 Total

Specimens tested 52,665 10,901 63,556

EIA RR 565 149 714

WB POS 467 (82.7%) 129 (86.6%) 596 (83.5%)

WB IND 20 (3.5%) 6 (4.0%) 26 (3.6%)

WB NEG 78 (13.8%) 14 (9.4%) 92 (12.9%)

Data needs

• 7: # and % of false positive supplemental tests for HIV-1 as confirmed by additional follow-up testing of original or follow-up specimens

• 8: # and % of false-negative and false indeterminate supplemental tests for HIV-1 as confirmed by additional follow-up testing (including NAAT) of original or follow-up specimens

Serodiagnostic Testing

2006 2007 Total

Specimens tested 52,665 10,901 63,556

EIA RR 565 149 714

WB POS 467 (82.7%) 129 (86.6%) 596 (83.5%)

WB IND 20 (3.5%) 6 (4.0%) 26 (3.6%)

IND NAAT tested 18 6 24

WB NEG 78 (13.8%) 14 (9.4%) 92 (12.9%)

NEG NAAT tested 72 14 86

Serodiagnostic/NAAT Data

2006 2007 TOTAL

RNA + 7 (38.9%) 0 (0%) 7 (29.2%)

RNA - 10 (55.6%) 6 (100%) 16 (66.7%)

Invalid 1 (5.6%) 0 (0%) 1 (4.2%)

TOTAL 18 6 24

WB Indeterminate

WB Indeterminate/NAAT Positive

• 7 RNA reactive:– EIA s/co mean 7.75 (range = 3.1-10.0; sd 2.12)– 6 MS HIV-1 +– 1MS neg (EIA = 9.0, WB bands = 24+/-)– RNA NR : EIA s/co mean 1.56 (1.1-3.1; sd 0.5)

2006 2007 TOTAL

RNA + 7 (38.9%) 0 (0%) 7 (29.2%)

RNA - 10 (55.6%) 6 (100%) 16 (66.7%)

Invalid 1 (5.6%) 0 (0%) 1 (4.2%)

TOTAL 18 6 24

Serodiagnostic/NAAT Data

WB Negative

2006 2007 TOTAL

RNA + 1 (1.4%) 0 (0%) 1 (1.2%)

RNA - 65 (90.3%) 13 (92.9%) 78 (90.7%)

Invalid 6 (8.3%) 1 (7.1%) 7 (8.1%)

TOTAL 72 14 86

WB False Negative or RNA False Positive?

• 1 RNA detected:– EIA s/co 1.4; MS neg; No bands– Specimen 30 days prior: EIA +, MS -, WB -, RNA –– Follow-up rapid test referral: MS -, WB neg

2006 2007 TOTAL

RNA + 1 (1.4%) 0 (0%) 1 (1.2%)

RNA - 65 (90.3%) 13 (92.9%) 78 (90.7%)

Invalid 6 (8.3%) 1 (7.1%) 7 (8.1%)

TOTAL 72 14 86

Rapid Test Referral Data

Rapid test referral algorithm: 2006

Report:Positive

Report:Positive

ReactiveReactive HIV-1 Reactive

HIV-1 WBHIV-1 WB Multispot

Neg or IndNeg or Ind

RequestFollow-up

RequestFollow-up

HIV-1 R/NRHIV-1 R/NR

Rapid test referral algorithm: 2007

EIA MultispotWB

Follow-up onlyif risk factors

Non-reactive

Report:Positive

Reactive

Rapid reactive

Reactive Neg or Ind

RNA testscheduled

R or NR

Not detected Detected

NAAT

ReactiveReport:Positive

Neg or Ind WB

Revised Algorithm: NAAT Added

Report: RNA –Follow-up only if risk

Report: RNA +Possible AHS

Rapid Test Referral Data

2006 2007 Total

Referrals 253 291 544

WB POS 162 (64.0%) 202 (69.4%) 364 (66.9%)

WB IND 14 (5.5%) 10 (3.4%) 24 (4.4%)

WB NEG 77 (30.4%) 79 (27.1%) 156 (28.7%)

Rapid Test Referral Data

2006 2007 Total

Referrals 253 291 544

WB POS 162 (64.0%) 202 (69.4%) 364 (66.9%)

WB IND 14 (5.5%) 10 (3.4%) 24 (4.4%)

IND NAAT tested 11 7 18

WB NEG 77 (30.4%) *79 (27.1%) 156 (28.7%)

NEG NAAT tested 48 10 58

*In 2007, EIA test addedOnly EIA reactives rec’d NAAT (10 of 79)

Rapid Test Referral Data

2006 2007 TOTAL

RNA + 7 (63.6%) 7 (100%) 14 (77.8%)

RNA - 3 (27.3%) 0 (0%) 3 (16.7%)

Invalid 1 (9.2%) 0 (0%) 1 (5.6%)

TOTAL 11 7 18

WB Indeterminate

WB Indeterminate/NAAT Positive

2006 2007 TOTAL

RNA + 7 (63.6%) 7 (100%) 14 (77.8%)

RNA - 3 (27.3%) 0 (0%) 3 (16.7%)

Invalid 1 (9.2%) 0 (0%) 1 (5.6%)

TOTAL 11 7 18

• 7 RNA detected in 2007:– EIA mean s/co 10.0 (range 9.4-10.1; sd 0.45) 7 MS +

• 7 RNA detected in 2006; EIA on 4 retrospectively– Mean s/co 9.9 (sd = 0) 7 MS +

Rapid Test Referral Data

WB Negative

2006 2007 TOTAL

RNA + 1 (2.1%) 1 (10.0%) 2 (3.5%)

RNA - 45 (93.7%) 9 (90.0%) 54 (93.1%)

Invalid 2 (4.2%) 0 (0%) 2 (3.5%)

TOTAL 48 10 58

WB False Negative or RNA False Positive?

• 1 RNA detected in 2007:– EIA s/co 10.7; MS + (no further follow-up)

• 1 RNA detected in 2006– EIA s/co 0.227; MS – (no further follow-up)

2006 2007 TOTAL

RNA + 1 (2.1%) 1 (10.0%) 2 (3.5%)

RNA - 45 (93.7%) 9 (90.0%) 54 (93.1%)

Invalid 2 (4.2) 0 (0%) 2 (3.5%)

TOTAL 48 10 58

Considerations

• Expense of the RNA test (Aptima™) – Consistent specimen load from ongoing PHI

study ensured frequent Aptima™ runs

• Plasma specimens required for Aptima™

• EIA screening of rapid reactive specimens greatly reduced # of RNA tests run due to negative or indeterminate WB

Concluding Remarks

• NAAT is being used to resolve discordant serological test results at NYSDOH– More data are needed to assess the

costs/benefits of NAAT supplemental testing

• Of the RNA reactive results we obtained, at least one was a probable false positive– More data are needed to fully assess the

specificity of NAAT in this setting

Acknowledgements

Diagnostic HIV Lab• Judith Wethers• Joe Schwendemann• The technical staff

• CDC/PHI Study Team– Pragna Patel– Duncan Mackellar – Steve Etheridge

Western Blot

• WB interpretation:– Consortium for Retrovirus Serology

Standardization

• 2 bands: – p24 or p31, plus – gp41 or gp120/gp160

Description of Multispot (BioRad) HIV-1/HIV-2 Reactive Control Spot and Test Spots

1. Procedural control: Anti-human IgG (goat)2. HIV-2 Peptide: Peptide representing the HIV-2

virus gp36 3. Recombinant HIV-1: Recombinant gp41

(gp41rDNA)4. HIV-1 Peptide: Peptide representing the HIV-1

virus gp41