1. Can Sequentially Reactive EIA’s Replace HIV-1 Western Blot Testing? Robert A. Myers Ph.D.

2. Presentation Overview The Maryland DHMH Public Health Laboratory uses a unique HIV testing algorithm that incorporates several testing strategies or features of the proposed testing algorithms Data from our laboratory’s routine testing activities illustrate the potential utility of implementing proposed testing Strategy #3 that utilizes an second EIA or rapid test positive in lieu of conventional western blot or IFA testing to confirm or refute the positive HIV antibody reactivity detected in the initial screening assay

3. Presentation Overview Our findings are primarily based on the rate of HIV-1 western blot confirmation of dually HIV reactive specimens that were generated using third generation synthetic peptide based EIA and discontinued first generation viral lysate based EIA. Although our results using the dual EIA reactivity are promising, we also would to point- out some of the potential pitfalls in utilizing this testing strategy.

6. Maryland DHMH Laboratory HIV Testing Algorithm Utilizes two HIV Screening EIAs that are performed simultaneously on all diagnostic specimens HIV-1 viral lysate and HIV-1 recombinant protein or HIV- 1/2 synthetic peptide EIAs have been performed in tandem since 1992 Supplemental testing to detect possible HIV-1 seroconversions and possible HIV-2 infections: All HIV EIA reactive(one or both EIAs ) or Gray-Zone Negative specimens that are not confirmed by HIV-1 WB are tested further using: HIV-1 NAAT (in house validated real-time HIV LTR PCR) and a research use HIV-1(gp41)/HIV-2(gp36) synthetic peptide EIA (Select HIV) that discriminates between HIV-1 or HIV- 2 antibody reactions

7. Reasons for Performing two HIV Screening EIAs Takes advantage of the performance characteristics of each HIV assay type Recombinant/Synthetic Peptide Assays (Second or Third Generation) Immuno-dominate epitopes: earlier detection of HIV-1 sero- conversion and specific detection of HIV-2 or HIV-1(O) infections Viral Lysate Assays (First Generation) Broader array of HIV epitopes : more likely to detect antibodies directed against newly emerging HIV viruses Quality Assurance Reduces turnaround time on HIV(+)specimens Reduces the possibility of technician errors Ability to continue to perform HIV testing if a single manufacturer cannot supply their product

8. Supplemental HIV-2 Specific Testing We internally use a differential HIV-1(gp41) /HIV-2 (gp36) synthetic peptide EIA (Select HIV) to initially identify HIV-2(+)’s from HIV-1/HIV-2 screening EIA(+)’s not confirmable as HIV-1 (+) by WB If HIV-2 infection is suspected [Select EIA: HIV-2 (+)] reflex to perform: HIV-2 EIA ( Bio-Rad) reportable Multi-spot (Bio-Rad) reportable SIV WB ( Gene Labs) research use In-house conventional proviral HIV-1/HIV-2 (LTR) DNA PCR (requires fresh EDTA blood for PBMC’s)

9. Supplemental Tests: HIV-1 NAAT In-house real-time PCR targeting LTR region of HIV-1 Sensitivity: 1,000-2,000 RNA copies /ml 100ul of diagnostic sample used (usually serum) Initial NAAT positives confirmed by bDNA viral load and /or secondary real-time primer/probe set (gag) Individual NAAT’s using serum specimen submitted for HIV antibody diagnostic testing EIA reactive not confirmed by WB [Negative or indeterminate] (cadaveric specimens not tested) Rapid test (+), EIA(-), WB (-) or WB (indeterminate) HIV-1 NAAT (+) results are use not the sole means of diagnosing a HIV infection. The results are used to identify possible acute infections and to request aggressive follow-up testing by the healthcare provider to quickly resolve the patients’ HIV status

10. HIV EIA Comparison Bio-Rad (HIV-1/HIV-2 )+O Peptide vs. BioMuerieux HIV-1 Viral Lysate (Vironostika) Non-Cadaveric Specimens 10/1/04 to 08/31/07 [N=133,601] HIV EIAPeptidePositive(+) Grey Zone(+) ResultsViral LysatePositive (+)Negative(-)Grey Zone(+)Negative HIV-1 W.B. Positive(+) 4864*000 Negative(-) 431313 Indeterminate 421340 HIV EIAPeptideGrey Zone(+) Negative(-)Negative ResultsViral LysatePositive(+)Grey Zone(+)Positive(+)Grey Zone(+) HIV-1 W.B. Positive(+) 0000 Negative(-) 0096 Indeterminate 00110 Total EIA Reactive or Grey-zone Reactive not confirmed by HIV-1 WB: 406 Note* Includes three specimens from one individual that were later confirmed as HIV-2 (+ )

11. Dual HIV EIA Reactive Specimens 4864 of 4915 Dually EIA reactive or reactive/grey zone reactive (0.9X cut-off)specimens were HIV-1 Western Blot Positive 98.96 % Confirmation rate for specimens reactive or reactive/grey-zone reactive in both EIA’s Three HIV-1 WB(+) specimens were from one HIV-2 infected HIV- individual whose HIV-2 antibodies cross– reacted extensively with HIV-1 antigens on the WB All HIV-1 WB confirmed specimens demonstrated reactivity in both EIA’s Only 51 dually reactive or reactive/grey zone reactive specimens were not confirmed by HIV-1 WB

13. Breakdown of Dual EIA Reactive or Reactive/Grey-zone Reactive Specimens Not Confirmed Positive by HIV-1 WB 46 HIV-1Western Blot Indeterminate Specimens 6 Confirmed HIV-2 (+) Specimens : from 3 individuals 27 HIV-1 NAAT(+) 17 specimens (14 individuals) confirmed HIV-1 Seroconversions by testing follow-up specimens 7 specimens (6 individuals) suspected HIV-1Seroconversions no follow-up specimens 3 specimens (2 individuals) End Stage AIDS 13 HIV-1 NAAT (-) 1 Confirmed HIV-1Seroconversion by testing follow-up specimens 1 waning maternal HIV-1 antibodies in 1yr. old baby 1 no change in HIV antibody reactivity after follow-up 10 lost to follow-up

14. Breakdown of Dual EIA Reactive or Reactive/Grey-zone Reactive Specimens Not Confirmed Positive by HIV-1 WB 5 HIV-1Western Blot Negative Specimens 5 HIV-1 NAAT (-) 2 no change in HIV antibody reactivity after follow-up (both specimens from the same individual) 3 lost to follow-up

15. Specimens with Unresolved HIV Status Only 16 of 4915 specimens (0.33%) with dual EIA reactivity (excluding cadaveric specimens) had no other evidence of HIV infectivity : [WB(+), NAAT (+),HIV-2 antibodies, later HIV-1 seroconversion, or maternal antibodies] 13 of the 16 unresolved dually reactive EIA specimens are from anonymous patients or have been lost to follow-up 3 specimens (2 individuals) demonstrated no change in EIA/WB reactivity upon follow-up [HIV-1 NAAT(-)]

16. HIV EIA Comparison Bio-Rad (HIV-1/HIV-2 )+O Peptide vs. BioMuerieux HIV-1 Viral Lysate (Vironostika) Non-Cadaveric Specimens 10/1/04 to 08/31/07 [N=133,601] HIV EIAPeptidePositive(+) Grey Zone(+) ResultsViral LysatePositive (+)Negative(-)Grey Zone(+)Negative HIV-1 W.B. Positive(+) 4864*000 Negative(-) 431313 Indeterminate 421340 HIV EIAPeptideGrey Zone(+) Negative(-)Negative ResultsViral LysatePositive(+)Grey Zone(+)Positive(+)Grey Zone(+) HIV-1 W.B. Positive(+) 0000 Negative(-) 0096 Indeterminate 00110 Total EIA Reactive or Grey-zone Reactive not confirmed by HIV-1 WB: 406 Note* Includes three specimens from one individual that were later confirmed as HIV-2 (+ )

17. Potiential Pitfalls when using Two Sequentially reactive EIA’s to replace HIV-1 Western Blot Testing? When using a second EIA in lieu of WB to confirm a screening EIA reactive it is important to use the most sensitive assay for screening and not confirmation If the less sensitive Viral Lysate based EIA only was used for initial screening and HIV-1 NAAT screening of EIA(-)’s was not performed: 5 HIV confirmed HIV-1 serconversions and 2 suspected seroconversions would have been missed [7 NAAT(+) positive specimens] 2 HIV-2 cases would not have detected Recommend to use the more sensitive assay (EIA or rapid) for screening and follow-up discordant EIA results with supplemental HIV-1 NAAT or HIV-2 specific testing (if applicable)

18. Potiential Pitfalls when using Two Sequentially reactive EIA’s to replace HIV- 1 Western Blot Testing? Sequential EIA’s should be selected so that the populations of false positives they individually generate do not significantly overlap 102 of 355(28.7%) of the discordantly EIA reactive/NAAT(-) specimens in this study had subsequent follow-up testing preformed. None of these patients seroconverted to HIV-1 or HIV-2 upon follow-up The number of EIA falsely reactive specimens will vary by testing population. The positive predictive value dual EIA reactive results will likely be lower in populations were the prevalence of HIV infections are lower. Recommend that each laboratory evaluate the relative sensitivity and specificity of each EIA with specimens from their testing population prior to implementing two sequential EIA’s to replace WB testing

19. Potential Pitfalls when using Sequentially reactive EIA’s to replace HIV-1 Western Blot Testing? Assume the worst case scenario using data in the study generated with our testing population: Two third generation hypothetical EIA’s to be used sequentially have identical overlapping populations of 336 unresolved [Dual EIA(+)/ HIV-1 NAAT(-)/HIV-2(-)] positives that were found in the study using a third generation EIA Assume none of these 336 dual EIA(+) specimens will result in later HIV-1 sero-conversion “true false positives” Assume “ True Dual EIA(+) Positives” are: [ HIV- 1WB(+) or, HIV-1 NAAT(+) or HIV-2(+), or later developed HIV-1 seroconversion or maternal HIV antibodies]

20. Predicative Value of Dual Reactive HIV EIA’s Predicative value of dual EIA’s(+): True Positives* / True Positives + False Positives X 100% * True Positives =[Total(+)- False(+)] Study Results (3 rd. generation EIA then a 1 st. generation EIA) : 4899/4915 X100%= 99.67% Hypothetically (3 rd. generation EIA then another 3 rd. generation EIA with complete overlap of EIA false positives : 4908/5244X100%= 93.59%

21. Concluding Remarks Our dual EIA testing data demonstrates the feasibility of using two sequential EIA (+)’s in lieu of HIV-1WB confirmation testing Recommend to use the more HIV-1/HIV-2 sensitive assay for screening and follow-up discordant EIA results with supplemental HIV-1 NAAT or discriminatory HIV-2 specific testing if HIV-2 infections occur in your testing populations

22. Concluding Remarks (cont.) Recommend that each laboratory evaluate the relative sensitivity and specificity of each EIA or other immunoassay with specimens from their testing population prior to implementing two sequential EIA’s to replacing WB testing or refer to published studies using testing Strategy #3 in populations with a HIV prevalence to comparable to the HIV prevalence in your testing population

23. Acknowledgements: The staff of Maryland DHMH Retrovirology and Molecular Diagnostics, and Molecular Epidemiology Laboratories The staff of Maryland DHMH AIDS Administration The organizers of the APHL meeting