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Robert A. Myers Ph.D.

Identifying HIV-2 Infections Using Differential Serological Assays HIV-1 (gp41)/HIV-2(gp36) (Select HIV or MultiSpot) by Testing HIV EIA Reactive Specimens Unconfirmed HIV-1 Antibody by HIV-1 Western Blot. Robert A. Myers Ph.D. Presentation Overview.

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Robert A. Myers Ph.D.

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  1. Identifying HIV-2 Infections Using Differential Serological Assays HIV-1 (gp41)/HIV-2(gp36) (Select HIV or MultiSpot) by Testing HIV EIA Reactive Specimens Unconfirmed HIV-1 Antibody by HIV-1 Western Blot Robert A. Myers Ph.D.

  2. Presentation Overview • The key feature of proposed testing Strategy #5 is the use of a HIV-1/HIV-2 discriminatory assay to quickly identify presumptive HIV-2 infections by testing HIV EIA reactive specimens that cannot be conclusively confirmed positive for HIV-1 antibodies • For over 15 years our laboratory has successfully used HIV-1/HIV-2 discriminatory EIA and/or HIV-1/HIV-2 rapid test to routinely identify presumptive HIV-2 cases that sporadically appear in our testing population

  3. Presentation Overview • Why do we need to routinely perform HIV-2 screening in Maryland? • What assays are used in our HIV-2 testing strategy? • What have we found using HIV-1/HIV-2 discriminatory assays as proposed in testing strategy #5 ?

  4. Why do we routinely test for HIV-2 In Maryland? • In in 1991 we conducted a retrospective study that re-tested HIV-1 WB indeterminate sera using HIV-2 specific synthetic peptide EIA’s and found 8 specimens of 457 tested that were confirmed positive for HIV-2 antibodies (J.AIDS 1992.5:417-423) • These specimens were from 4 HIV-2 infected individuals who were identified using available demographic information as West African expatriates living in the MD suburbs of Washington DC

  5. Maryland in the Shadow of the National Capitol • Washington DC is an International City associated with extensive international travel and immigration into the region • Significant HIV diversity has been documented in our testing populations in the DC metro area • All HIV-2 cases documented to date in our testing populations were from two MD Counties in the DC metro area

  6. HIV-1 Genetic Diversity In Maryland

  7. HIV-2 Cases in Maryland • Using HIV-1/HIV-2 discriminatory assays as proposed in testing strategy #5 on average we have found one to two new HIV-2 infected individuals in our testing population each year since 1991 for a total of 30 documented HIV-2 cases to date • 5 of the 30 HIV-2(+) patients were negative in HIV-1 viral lysate based assays • 9 of 18 HIV-2(+) patients were negative in HIV-1 recombinant protein based EIA (Recombigen) • All HIV-2 cases had antibodies that cross reacted with gag and/or pol antigens on HIV-1 western blots • Cross reactions to HIV-1 env antigens were less pronounced ( in one case complete cross reactions gag pol and env HIV-1 antigens was documented)

  8. HIV-2 Testing Strategy • 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 • We also test all HIV-1 WB(+) specimens from two Maryland Counties adjacent to Washington DC where the majority of HIV-2 infections routinely are found in Maryland • If HIV-2 infection is suspected [Select EIA: HIV-2 (+)] we perform: • HIV-2 EIA ( Bio-Rad) reportable • Multi-spot (Bio-Rad) reportable • SIV WB ( Gene Labs) • In-house conventional proviral HIV-1/HIV-2 (LTR) DNA PCR (requires fresh EDTA blood for PBMC’s)

  9. HIV-2 (+) Misdiagnosis: Cross Reaction on HIV-1 Western Blot HIV-1 Western Blot SIV Western Blot

  10. HIV-1/HIV-2 Discriminatory Assays HIV-1 /HIV-2 EIA [Select- HIV Adaltis Inc.] • Individual microwells coated with either HIV-1 or HIV-2 transmembrane synthetic peptide antigens • EIA binding ratio determines HIV specific reactivity: • Binding ratio: HIV-2 (O.D. signal)/HIV-1 (O.D. signal) >2.0 HIV-2 , <0.5 HIV-1 and>0.5 to <2.0 dual HIV-1and HIV-2 reactivity dilute specimen to determine predominant reactivity • The Select–HIV EIA is not FDA approved therefore it is only used as supplemental test inconjunction with other HIV-2 assays in our HIV-2 testing algorithm

  11. HIV-1/HIV-2 Discriminatory Assays Multispot HIV-1/HIV-2 Rapid test [BioRad] • Incorporates highly conserved HIV-1 and HIV-2 recombinant or synthetic peptide transmembrane antigens coated on microscopic particles immobilized membrane in individual test cartridge • Interpretation of individual spotted antigens determines HIV specific reactivity • Dilution procedure for specimens demonstrating dual HIV-1and HIV-2 reactivity at screening • FDA approved for in vitro diagnostic use but is not approved to screen blood plasma , cell or tissue products • We primarily use this assay as a supplemental test to verify HIV-1 or HIV-2 reactivity that has been demonstrated in other assays (i.e., Select-HIV EIA or Genetic Systems HIV-2 EIA)

  12. HIV-2 Assays HIV-2 Viral Lystate EIA (Genetic Systems Bio-Rad) • Non-discriminatory: extensive cross reactivity with HIV-1 antibodies and the non-specific reactions associated with 1st generation EIA’s • FDA Approved Assay: Generates Reportable Results • When HIV-2 testing is specifically requested • When discriminatory assays are reactive for HIV-2 afterre-testing HIV screening EIA reactive specimens that cannot be confirmed as HIV-1 positive • All specimens that were exclusively HIV-2 reactive specimens in the discriminatory assays were HIV-2(+) reactive in viral lystate EIA

  13. SIV Western Blot (ZeptoMetrix) • SIV extensive Cross reactivity with HIV-2 antibodies • Used as a supplemental test to test HIV-2 EIA reactive specimens • Interpretation not standardized • Some cross reactivity to HIV-1 antibodies can be observed primarily to gag and pol antigens

  14. Differential HIV-1 LTR and HIV-2 LTR Proviral DNA PCR’s • Requires PBMC separated from a fresh whole blood (EDTA) follow-up specimen • Useful to resolve possible dual HIV-1/HIV-2 infection • Conventional PCR : Sensitivity 10 copies/ PCR rxn. • HIV-1 LTRIII & LTR IV primers (Refn.: J.Virology 1991; 65 :2816-2828) Product Size: 255 bp • HIV-2 LTRC & LTR D primers (Refn. : J.Virology 68 7433-7447) Product Size:199 bp HIV-1 HIV-2

  15. Notes: * 8 of 8 Confirmed positive for HIV-2 antibodies ** 3 of 30 Confirmed positive for HIV-2 antibodies

  16. HIV-1 Western Blot Indeterminate Specimens With HIV-2 Reactivity • 8 specimens from 5 individuals demonstrated strong HIV-2 reactivity in the Select HIV EIA (signal/cut off values(17.05-21.05) and had undiluted HIV-1/HIV-2 binding ratios of [325 to14.2 :>2.0= HIV-2(+)] • All 8 strongly HIV-2 reactive specimens were confirmed as HIV-2(+) by Mutispot:HIV-2(+), Viral Lysate HIV-2 EIA(+) and SIV WB(+) • In two of the 5 individuals follow-up proviral HIV-2 LTR PCR testing demonstrated HIV-2 DNA in the patients PBMC’s

  17. HIV-1 Western Blot Negative Specimen With HIV-1/HIV-2 Reactivity • One hemolized specimen demonstrated weak HIV-1 /HIV-2 reactivity in Select HIV EIA for HIV-1 (signal/cutoff: 1.14) and HIV-2 (signal/cutoff: 1.89) Binding ratio (1.74: undifferentiated at screening dilution) • This specimen was initially only reactive in the HIV-1/HIV-2 Plus O EIA (signal/cutoff: 4.95) ,was HIV-1 WB(-) and HIV-1 NAAT(-) • The Select HIV EIA HIV-2 reactivity could not be verified by Multispot(-) and SIV WB(-) • Patient was negative in both EIA screening assays and both HIV-1/HIV-2 discriminatory assays upon follow-up

  18. HIV-1 Western Blot Positive Specimens with HIV-1 & HIV-2 Reactivity • 3 specimens from the same individual had HIV-2 (+)binding ratios( avg. 21.45) at the screening dilution . The HIV-2 (+) status was confirmed by Mutispot:HIV-2(+) by dilution, SIV WB(+) and proviral HIV-2 LTR (+) by PCR • 20 of 30 specimens dually reactive for HIV-1 & HIV-2 in the Select HIV EIA had limited HIV-2 cross reactivity that was resolved at the screening dilution by the HIV-2/HIV-1 binding ratios (<0.5) that indicated HIV-1 infections • 7 specimens had HIV-1 binding ratios after dilution and were also Multispot HIV-1(+) after dilution

  19. HIV-2 (+) Specimens Detected (10/01/04 - 09/30/07) • 11 specimens were confirmed HIV-2(+) from 6 individuals • 4 Specimens (2 individuals) were reactive in both EIA’s and were HIV-1 WB indeterminate • 2 Specimens (2 individuals) were were reactive in the HIV-1/HIV-2 +O EIA and negative in the viral lysate EIA and were HIV-1WB indeterminate • 2 Specimen (1 individual) was reactive in the HIV-1 /HIV-2 +O EIA and grey-zone reactive in the viral lysate EIA and was HIV-1WB indeterminate • 3 specimens (1individual )were reactive in both EIA’s and were HIV-1 WB positive

  20. Concluding Remarks • Our data has demonstrated the utility of using discriminatory HIV-1/HIV-2 assays as proposed in testing strategy #5 to quickly and accurately identify HIV-2 infections in our testing population • We strongly recommend the routine use of a differential HIV-1/HIV-2 serology tests to properly evaluate reactive results from HIV-1/HIV-2 combination screening EIA’s if HIV-2 infections occur in your testing population • Recognize the need for manufacturers to develop cost effective HIV-1/HIV-2 discriminatory assays

  21. Acknowledgements: • The staff of Maryland DHMH Retrovirology, Molecular Diagnostics, and Molecular Epidemiology Laboratories • The staff of Maryland DHMH AIDS Administration • The organizers of the CDC/APHL HIV Diagnostics Conference

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