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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 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 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