Confirming HIV infectiondepts.washington.edu/hivtcg/presentations/uploads/52/confirming_hiv... ·...

December 5th, 2016

Bernard M. Branson, M.D.Scientific Affairs, LLC

Atlanta, Georgia

Confirming HIV infection:

What’s New?

Disclosure of Financial Relationships

• Dr Branson has served as consultant to Gilead

Sciences, Inc, and Caldwell-Everson. He has received

compensation from Siemens Healthcare for development

of a presentation on HIV diagnostics at an educational

symposium.

Confirmatory: Use of Two Tests in Sequence

• After a positive screening test, conduct a

second test on persons who test positive.

• Decision rule: Must be positive on - both

tests -

either test

• Positive on both tests = reduces false

positives

– Increases specificity, but reduces sensitivity

• Net Specificity = Spec1 + Spec 2 – (Spec1

x Spec2)

• HIV Example: Spec1 = .996, Spec2 = .998

– Spec1 + Spec2 = 1.994

– Spec1 x Spec2 = .994

– Net Specificity = 1.00

Net Specificity, Sequential Tests (Both Tests Positive)

- Albritton et al, J Inf Dis 1996

HIV-1 Antigens and RNA

gp 120

gp 41

p24

RNA

5

p 31

gp 160

(Precursor)

Western Blot

6

• Antigens from lysate of HIV grown in cell

culture, separated by molecular weight

• HIV-1 positive requires presence of at

least 2:

– p24, gp41, gp120, gp160

• Indeterminate:

– Presence of bands insufficient to meet criteria,

or

– Presence only of other viral or non-viral bands

• No FDA-approved HIV-2 Western blot .

HIV-2 Infection

• Remains uncommon in U.S., but

– Does not respond to NNRTIs, some PIs (first line

therapy)

– Undetectable by HIV-1 viral load tests

• Misclassified by HIV-1 Western blot:– 54/58 (93%) HIV-2 patients tested had positive HIV-1 WB result (NYC)1

– 97/163 (60%) HIV-2 cases reported had positive HIV-1 WB result (CDC)2

1Torian et al, Clinical Infectious Disease 20102MMWR July 2011

7

HIV-2 Antigens and RNA

gp 105

gp 36

p26

RNA

8

p 31

gp 140

(Precursor)

HIV-1/2 Ag/Ab combo immunoassay

HIV-1/HIV-2 antibody differentiation immunoassay

(-)(+)

HIV-1 (+)

HIV-2 (-)

HIV-1 antibodies

detected

HIV-1 (-)

HIV-2 (+)

HIV-2 antibodies

detected

HIV-1 (-) or indeterminate

HIV-2 (-)

RNA

RNA (+) Acute HIV-1 infection

RNA (-)Negative for HIV-1

Negative for HIV-1 and HIV-2 antibodies and p24 Ag

HIV-1 (+)

HIV-2 (+)

HIV antibodies

detected

June 27, 2014

FDA approved March 2013

Serum ControlHIV-1 Recombinant gp41

HIV-2 Peptide gp36 HIV-1 Peptide gp41

Multispot HIV-1/HIV-2

10

HIV-1/HIV-2 Differentiation Assays


FDA approved March 2013

Serum ControlHIV-1 Recombinant gp41



11

Initial assays all test for HIV-

1 (gp41), HIV-2 (gp 36)

some test for more:

gp120 or gp160

Positive 2nd result confirms

>99% of reactive Ag/Ab

combo results on same

specimen

Negative or indeterminate

(only 1 spot) RNA

FDA approved, March 2013

Serum Control HIV-1 Recombinant gp41



FDA approved, Oct. 2014

Geenius HIV-/HIV-212


12

GeeniusTM HIV-1/2 Lines

HIV-1 & HIV-2 Associated Lines

13

Dual Path Platform Technology

Strip 1

Strip 2

Conjugate

Specimen

Dual Path Platform:

Add

5 µL serum/plasma

Or

15 µL whole blood

to specimen

well

15

Add

5 drops buffer

To buffer well.

16

Wait min 15-20 min (max 30 min) for results

17

Insert test cassette in

reader for automated

interpretation

18

Geenius Results: New Interpretations

1

2

3

20

20


1456

2

3

21

21


1456

2

3

7

8

22

22

250 HIV-1 Established Infections

Combo IA r/r, Multispot HIV-1 reactive

n = 250

Multispot HIV-1/HIV-2 n Geenius TM HIV-1/HIV-2

HIV-1 reactive, HIV-2 nonreactive (neat)

243 HIV-1 Positive, HIV-2 Negative (225)HIV-1 Positive, HIV-2 Indeterminate (16)(8/16 resolved as HIV-2 Negative on repeat)

HIV Positive – Untypable (2)(1/2 resolved as HIV-1 Positive, HIV-2 Negative on repeat)

HIV-1 reactive, HIV-2 nonreactive (1:10)

7 HIV-1 Positive, HIV-2 Negative (2)HIV-1 Positive, HIV-2 Indeterminate (3)(1/3 resolved as HIV-2 Negative on repeat)

HIV Positive – Untypable (2)(1/2 resolved as HIV-1 Positive, HIV-2 Indeterminate on repeat)

• 100% concordance for HIV positivity (includes Untypable results)

• 7 (2.8%) initial Multispot results required dilution testing per package insert

• 23 (9.2%) initial Geenius results required repeat testing per package insert

• Geenius repeat testing resolved 10/23 (43%) as HIV-1 Pos/HIV-2 Neg

23

60 HIV-1/2 Combo False Positives

Combo IA r/r, Multispot HIV-1 nonreactive or indeterminate,

HIV-1 RNA nonreactive

n = 60


HIV-1 nonreactive, HIV-2 nonreactive (neat)

57 HIV-1 Negative, HIV-2 Negative (54)HIV-2 Indeterminate, HIV-1 Negative (2)(2/2 remained HIV-2 Indeterminate on repeat, both HIV-2 RNA Not Detected)

HIV-1 Indeterminate, HIV-2 Negative (1)

HIV-1 indeterminate, HIV-2 nonreactive (neat)

3 HIV-1 Negative, HIV-2 Negative (1)HIV-2 Indeterminate, HIV-1 Negative (2)(2/2 resolved as HIV-2 Negative on repeat)

• 59 HIV-1 negative Geenius results vs. 57 HIV-1 nonreactive Multispot results

• All Geenius HIV-2 Indeterminate results were resolved as HIV-2 Negative by repeat testing or HIV-2 RNA.

• All Geenius and Multispot results indicate the need for reflex HIV-1 RNA testing. 24

- Fordan S, 2016 HIV Diagnostics Conference

HIV-1 Early Infections

Combo IA r/r, Multispot HIV-1 indeterminate,

HIV-1 RNA reactive

n = 10


HIV-1 indeterminate, HIV-2 nonreactive (neat)

10 HIV-1 Negative, HIV-2 Negative (1)

HIV-1 Indeterminate, HIV-2 Negative (3)

HIV-1 Positive, HIV-2 Indeterminate (3)(2/3 resolved as HIV-2 Negative on repeat)

HIV-1 Positive, HIV-2 Negative (3)

• 6/10 (60%) were HIV-1 Positive by Geenius and therefore reclassified as

HIV-1 established infections per study design.

- Fordan S, 2016 HIV Diagnostics Conference

25

Geenius results not generated previouslyCDC Technical Update August 2016

1. HIV-2 with HIV-1 cross-reactivity

– Consider HIV-2 Positive

26https://stacks.cdc.gov/view/cdc/40790



2. HIV-2 indeterminate

– Repeat; negative on repeat, report as

negative

– Repeatedly reactive:

• Conduct HIV-1 NAT

• Refer for validated HIV-2 antibody or NAT, or

• Repeat testing in 2-4 weeks

27https://stacks.cdc.gov/view/cdc/40790


2. HIV-2 indeterminate

3. HIV indeterminate

– Repeat; negative on repeat, report as

negative

– Repeatedly reactive:

• Conduct HIV-1 NAT

• Refer for validated HIV-2 antibody or NAT, or

• Repeat testing in 2-4 weeks

https://stacks.cdc.gov/view/cdc/40790


HIV Nucleic Acid Test (NAT) for Diagnosis:

Qualitative RNA vs Viral Load

• APTIMA HIV-1 qualitative RNA assay is

the only NAT FDA-approved for diagnosis

• Under FDA and CLIA regulations,

clinicians can order HIV-1 RNA viral load

tests, but labs cannot use them as a reflex

part of the algorithm


HIV-1/HIV-2 antibody differentiation immunoassay

(-)(+)

HIV-1 (+)

HIV-2 (-)

HIV-1 antibodies

detected

HIV-1 (-)

HIV-2 (+)

HIV-2 antibodies

detected

HIV-1 (-) or indeterminate

HIV-2 (-)

RNA

RNA (+) Acute HIV-1 infection

RNA (-)Negative for HIV-1


HIV-1 (+)

HIV-2 (+)

HIV antibodies

detected

June 27, 2014

HIV-1 RNA viral load


HIV-1 RNA viral load

(-)(+)

HIV-1/HIV-2 antibody

differentiation assayVL detectable HIV-1 infection

VL (-)


HIV-1+

(Viral suppression)HIV-2+

HIV-2 infection

Negative(False positive

combo assay)

How about…

Useful clinical information

“Point-of-Care” HIV Nucleic Acid Tests

GeneXpert

Not available in U.S.

Alere q

HIV-1/2

Detect

Cobas LIAT

PCR

Summary

• The new HIV 1/2 differentiation assay,

Geenius, generates more indeterminate

results than Multispot

• Additional NAT and HIV-2 testing may be

necessary

• HIV RNA viral load will play an

increasingly important role in diagnosis33

Next to Consider

• Antibodies may fail to develop or may decline

when ART is initiated during acute HIV infection

• Antibody response may be attenuated in

persons who become infected while on PrEP

34

deSouza et al, Clin Inf Dis 2016

Grossman et al, R4P Conference 2016

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