HIV Testing Technologies

Barbara G. Werner, PhD Infectious Disease Consultant

MA Department of Pubic Health December 5, 2013

Disclosures

I, Barbara G. Werner, PhD, have no relevant financial, professional or personal relationship to disclose.

Learning Objectives

• Discuss current HIV diagnostic tests

• Identify limitations of the current HIV diagnostic algorithm

• Describe revised HIV diagnostic algorithm

• Describe the use of the new algorithm to identify acute HIV infections

Acknowledgement

Thanks to Bernard M. Branson, MD for sharing a number of slides used in this presentation.

1st and 2nd Generation EIA Plasma/serum (1 h/37o C)

Plasma/serum

Color

reagent

Antigen

1st - Viral lysate

2nd – Recombinant proteins or

synthetic peptides

IgG HIV antibody

enzyme

anti-human IgG

Detects HIV

IgG

Enzyme-

detection

Available 1st & 2nd Generation Assays

• HIV-1 Western blot

• HIV-1 IFA

• HIV-1 EIA

• Six rapid HIV antibody tests

EIA

Western blot

IFA

Uni-Gold Recombigen

OraQuick Advance Clearview Complete

Clearview Stat Pak INSTI

CLIA Waived POC Rapid Tests

Multispot HIV-1/HIV-2 Rapid Test

• CLIA moderate complexity with serum, plasma

• Perform test in 15 minutes

• Shelf life: 1 year refrigerated, 3 months room temperature

• FDA-approved for use in algorithm March 28, 2013

FDA-approved HIV-1/HIV-2 Antibody Differentiation Assay

Peptide HIV-2

Recombinant HIV-1

Peptide HIV-1

Reactive Control

3rd Generation “Sandwich” EIA

Antigen:

Recombinant proteins or

synthetic peptides Enzyme-

detection

Color

reagent

Plasma/serum HIV antibody

enzyme

HIV antigen

Detects HIV

IgM or IgG

IgG IgM

Available 3rd Generation Assays

• One HIV-1/HIV-2 EIA

– GS HIV-1 HIV-2 Plus O

• Two HIV-1/HIV-2 CIAs

– Advia Centaur 1/O/2

– Ortho Vitros HIV1+2

4th Generation Combo Assay

Color

reagent

Enzyme-

detection

HIV antigen

p24 antibody

Plasma/serum HIV antibodies

p24 antigen

HIV antigen

p24 antibody

Detects IgM

or IgG

antibody or

p24 antigen

Available 4th Generation Assays

• One HIV-1/HIV-2 antigen/antibody EIA

• One HIV-1/HIV-2 antigen/antibody CIA

• And most recently, an HIV-1/HIV-2 antigen/antibody rapid test which distinguishes antigen from antibody

• Chemiluminescent immunoassay

• Detects p24 antigen and HIV antibody

• Time to result: 29 minutes

• FDA-approved June 22, 2010

Abbott Architect 4th Generation Ag/Ab

Combo Assay

Bio-Rad GS HIV Combo Ag/Ab EIA

• Microwell plate EIA

• 3rd generation Ab format:

- HIV-1: gp160

- HIV-2: gp36

- Group O

• p24 antigen

• FDA-approved July 25, 2011

Determine Combo Rapid HIV 1/2 Ag/Ab Test

• CLIA moderate complexity

• Distinguishes Ag from Ab

• Whole blood, serum plasma

• FDA-approved August 2013

Antibody

Antigen

Control

APTIMA Qualitative HIV-1 RNA Assay Aid to HIV-1 diagnosis

Diagnosis of acute HIV-1 infection in antibody-negative persons

Confirmation of HIV-1 infection in antibody-positive persons when RNA is detected

FDA-approved July 2006

Laboratory-based Algorithm

• From late 1980s

• Initial screening test, generally EIA

• Supplemental test, WB or IFA, performed following repeatedly reactive EIA

• Results reported as Positive, Negative, or Indeterminate

Limitations of the Current Algorithm

• Antibody tests do not detect infection in ~10% of infected persons at highest risk of transmission---miss acute infections

• Western blot confirmation is less sensitive during early infection than many widely used screening tests---indeterminate or false negative results

Further Limitation

• HIV-2 infections may be miss-classified as HIV-1 on Western blot – 54/58 (93%) HIV-2 patients tested had positive

HIV-1 WB (NYC)*

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

• HIV-2 remains uncommon in U.S., but – Does not respond to NNRTIs, some PIs (first line

therapy) – Undetectable by HIV-1 viral load tests

*Torian et al.,CID 2010; **MMWR July 2011

Sequence of Test Positivity Relative to WB (serum)

166 specimens, 17 Seroconverters - 50 % Positive Cumulative Frequency

Modified from Masciotra et al, J Clin Virol 2011

Days before WB positive

WB

po

sit

ive

AP

TIM

A (

-26

)

GS

1/2

+O

(-

12

)

Mu

lti-

Sp

ot

(-7

) R

eve

al G

3, (-

6)

Vir

on

os

tka

(+2

)

Ora

Qu

ick

(-1

)

Un

igo

ld (

-2)

25

20

10

5

0

15

Arc

hit

ec

t A

g/A

b C

om

bo

(-

20

)

CO

MP

LE

TE

HIV

-1/2

(-

5)

H

IV-1

/2 S

TA

T-P

AK

(-

5)

Ad

via

(-

14

) V

itro

s (-

13

)

INS

TI (

-9)

Bio

-Ra

d A

g/A

b C

om

bo

(-

19

)

and Owen et al, J Clin Micro 2008

Avio

q

De

term

ine

Ag

/Ab

Co

mb

o (-

15

)

DP

P (

-8)

HIV RNA (plasma)

HIV Antibody

0 10 20 30 40 50 60 70 80 9

0

100

HIV p24 Ag

22

1st

gen

2nd

gen

3rd

gen

Days

Modified after Busch et al. Am J Med. 1997

Infection 4th

gen

Acute HIV Infection

HIV Infection and Laboratory Markers

New HIV Diagnostic Algorithm for serum and plasma

Some Alternatives and Limitatons 1. If 3rd gen HIV-1/2 IA as initial test: perform subsequent

testing specified in the algorithm. Will miss some acute infections

2. If alternative 2nd Ab test is used (e.g., WB or IFA): If

negative or indeterminate, perform HIV-1 NAT; if HIV-1 NAT is negative, perform Ab IA for HIV-2

Might miss-classify HIV-2 infections; requires more tests; increases TAT

3. HIV-1 NAT as 2nd test: if positive, HIV-1 infection; if

negative, perform HIV-1/HIV-2 Ab differentiation assay. Won’t distinguish acute from established infections; increases TAT and costs

HIV

RN

A in

Sem

en

(Lo

g 10 c

op

ies/

ml)

2

3

4

5

1/1000 - 1/10,000

1/500 - 1/2000

1/100- 1/1000

1/30- 1/200

Increased Risk of Sexual Transmission of

HIV

Cohen & Pilcher, J Infect Dis. 2005

Virus 75-750 times more infectious

Detecting Acute HIV Infections • Sensitivity among frequently-tested MSM in

Seattle

– 192 infected with HIV

• 23 (12%) detected only by RNA

– (15/16 tested detected by Ag/Ab immunoassay)

• 169 (88%) detected by serum Ab immunoassay

• 153 (80%) detected by oral fluid rapid test

- Stekler et al, Clin Inf Dis 2009

Phoenix ED Screening July 2011 through February 2013

• 4th gen screening of patients who had blood drawn – 15% of patients declined testing

– 13,014 patients tested

– 37 (0.3%) new HIV infections • 12 (32.4%) had Acute HIV Infection (antibody negative)

• Median viral load: – Patients with acute infections: 6 million

– Patients with established infections: 25,000

-MMWR June 21, 2013

RNA vs. 4th Generation Ag/Ab Assay

• RNA+/ 3rd gen-negative specimens detected by 4th generation EIA: – 38 of 46 (83%) – Australia* – 10 of 14 (71%) – CDC AHI study** – 51 of 61 (84%) – CDC panel***

– 4 days after RNA – 9 seroconversion panels***

* Cunningham P, HIV Diagnostics Conf 2007 ** Patel P, CROI 2009 *** Owen M, CROI 2009

4th generation HIV-1/2 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 (-)

NAT

NAT (+) Acute HIV-1 infection

NAT (-) Negative for HIV-1

Negative for HIV-1 and HIV-2

antibodies and p24 Ag

HIV-1 (+)

HIV-2 (+)

HIV antibodies

detected*

*Additional testing required to rule out dual infection

Use of the New Algorithm MA State PHL- 1st year results with 4th gen

• 7984 sera tested (BioRad “Combo”)

• 258 (3.2%) positive for HIV-1

• 1 positive for HIV-2

• 16 4th gen EIA RR and MS negative

– 8 had detectable HIV RNA, i.e., acute infections

– 8 had no detectable HIV RNA, i.e., likely false positive on initial screen

Goodhue etal. 2013 JClinVirol

Acute HIV Infections in MA Retrospective analysis of 8 specimens from acutely infected persons:

5 were negative on a 3rd generation test and would have been reported as negative

3 reactive specimens were run on Western Blot

None were positive

2 were indeterminate and 1 was negative

At best, confirmation of infection would have been delayed until a follow up specimen was obtained.

Acute infections in MA

• 8 individuals were given accurate, timely information about their infection status and linked to clinical care.

With a collaborative effort of lab, program, clinical providers, DIS and others, transmission can be impacted if acutely infected can be identified

Contacts were identified

Characteristics of 8 Persons with Acute Infection

• Substantial sexual risk, primarily MSM

• Certain vulnerabilities, such as homelessness, limited English proficiency, recent immigration

Resources • Criteria for Laboratory Testing and Diagnosis of Human

Immunodeficiency Virus Infection. Approved guideline. CLSI document M53-A. Wayne, PA: Clinical and Laboratory

Standards Institute: 2011. • Update on HIV Diagnostic Testing Algorithms. Journal of Clinical

Virology – open access 2011 and 2013 http://www.journalofclinicalvirology.com/supplements# • CDC/APHL Document. Updated Recommendations for Laboratory

Testing for Diagnosis of HIV Infection. To be published in 2014. • CDC and APHL and HIVtestingconference websites

Contact Information

• Barbara G. Werner, PhD

• Barbara.werner@state.ma.us