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• 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

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 ?

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

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

Maryland DHMH Laboratories:HIV-1 pol Genotypes Ryan White Patients 2005-06

n=278

2

198

24

2

1

4

35 1

2

2

2

1

3

1

9

M_(A/CRF01_AE)

M_(B)

M_(C)

M_(CRF01_A/AE)

M_(CRF01_AE)

M_(CRF01_AE/A)

M_(CRF02_AG)

M_(CRF02_AG)/M_(CRF01_AE)M_(CRF02_AG/F)

M_(CRF02_AG/G)

M_(D)

M_(D/B)

M_(G)

M_(J/G)

HIV-1 Genetic Diversity In Maryland

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)

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)

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

SIV Western BlotHIV-1 Western Blot

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

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)

HIV-2 AssaysHIV-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

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

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

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

HIV-1(-) & HIV-2 (-) HIV-1(+) HIV-2(+)

HIV-1(+) & HIV-2 (+)

HIV-1(-) & HIV-2 (-) HIV-1(+) HIV-2(+)

HIV-1(+) & HIV-2 (+)

865 68 8* 1 0 663 0 30**

Maryland Department of Health Laboratories Select HIV-1/HIV-2 Discriminatory EIA Results 10/01/04- 9/30/07 (n=1636)

HIV-1 Western Blot Positive Specimens From Two Maryland Metro DC Counties

Routine Screening EIA: Reactive or Grey-zone HIV-1 Western Blot: (Indeterminate or

Negative) Specimens

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

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

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

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

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

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