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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
