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

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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.
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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

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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 ?

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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 EIAs and found
  8 specimens of 457 tested that were confirmed
  positive for HIV-2 antibodies
  (J.AIDS 1992.5417-423)
• 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

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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

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HIV-1 Genetic Diversity In Maryland
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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)

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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 PBMCs)

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HIV-2 () Misdiagnosis Cross Reaction on HIV-1
Western Blot
HIV-1 Western Blot
SIV Western Blot
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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)
• gt2.0 HIV-2 , lt0.5 HIV-1 andgt0.5 to lt2.0 dual
  HIV-1and HIV-2 reactivity dilute specimen to
  determine predominant reactivity
• The SelectHIV 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

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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)

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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 EIAs
• 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

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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

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Differential HIV-1 LTR and HIV-2 LTR Proviral
DNA PCRs

• 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 Size199 bp

HIV-1
HIV-2
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Notes 8 of 8 Confirmed positive for HIV-2
antibodies



3 of 30 Confirmed positive for HIV-2
antibodies
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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 gt2.0 HIV-2()
• All 8 strongly HIV-2 reactive specimens were
  confirmed as HIV-2() by MutispotHIV-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 PBMCs

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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

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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 MutispotHIV-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 (lt0.5) that indicated HIV-1 infections
• 7 specimens had HIV-1 binding ratios after
  dilution and were also Multispot HIV-1() after
  dilution

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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 EIAs 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
  EIAs and were HIV-1 WB positive

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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 EIAs if HIV-2
  infections occur in your testing population
• Recognize the need for manufacturers to develop
  cost effective HIV-1/HIV-2 discriminatory assays

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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