1. Good afternoon, everyone.
- Annual STD Update conference –
April 2, 2014
Cherry Hill, NJ
4th Generation HIV Diagnostics & the new CDC/APHL Confirmatory Algorithm
Eugene G. Martin, Ph.D.
Professor of Pathology & Laboratory Medicine
Rutgers – Robert Wood Johnson Medical School
New Brunswick, NJ
&
Co-Director, NJ HIV
Rapid HIV Test Support
NJ Department of Health
Division of HIV, STD & TB Services
Good afternoon, everyone.
I’m Gene Martin, I’m a Professor of Pathology & Laboratory Medicine at Rutgers University and the Co-Director of NJ HIV, the Rapid HIV Test Support Group that works with the Department of Health in NJ overseeing rapid testing at 140 sites throughout the state.
I’m pleased to be with you this evening and will do my best to provide you with an overview of Fourth Generation HIV Testing.

2. Potential Conflicts of Interest
Disclosures of Financial Relationships
Research support from:
Abbott Diagnostics
Alere Diagnostic Systems
Gratefully acknowledged

3. Learning Objectives
At the end of this presentation, you should be able to
Understand differences between antibody, antigen and RNA in identifying HIV infection
Understand “Diagnostic window of Detection”
Appreciate advantages and disadvantages of current lab-based and rapid testing technologies
Understand why and how we are focusing on earlier stages and dynamics of an HIV infection
Appreciate the evolution of testing and role of various biomarkers and the importance of confirmatory testing
Fourth Generation HIV Screening – Lab-based and POC
The proposed CDC/APHL HIV Confirmatory Algorithm
Fourth Generation HIV Diagnostics has changed the playing field. These are more sensitive tests, that alert us early on to acute HIV infections; but this increased sensitivity has come with a price – it has disrupted the traditional confirmatory algorithm which has been in place since 1989.
This evening we are hoping to help you:
Understand why we are focusing on earlier stages of an HIV infection
Review the dynamics of an HIV infection
Understand the evolution of HIV testing and the appearance and timing of various biomarkers
Understand the historic role and importance of confirmatory testing
We’d like to describe some of the differences between Lab-based and POCT-based 4th generation HIV screening
Talk about the recently proposed CDC HIV Confirmatory Algorithm

4. Learning Objectives
At the end of this presentation, you should be able to:
Understand why and how we are focusing on earlier stages of an HIV infection
Be able to review the dynamics of an HIV infection
The evolution of HIV testing and the role of various biomarkers
The role and importance of confirmatory testing
Fourth Generation HIV Screening
The differences between laboratory-based and POCT-based 4th generation HIV screening
The proposed CDC/APHL HIV Confirmatory Algorithm
Fourth Generation HIV Diagnostics has changed the playing field. These are more sensitive tests, that alert us early on to acute HIV infections; but this increased sensitivity has come with a price – it has disrupted the traditional confirmatory algorithm which has been in place since 1989.
This evening we are hoping to help you:
Understand why we are focusing on earlier stages of an HIV infection
Review the dynamics of an HIV infection
Understand the evolution of HIV testing and the appearance and timing of various biomarkers
Understand the historic role and importance of confirmatory testing
We’d like to describe some of the differences between Lab-based and POCT-based 4th generation HIV screening
Talk about the recently proposed CDC HIV Confirmatory Algorithm

5. Take Home Messages
There are a wide variety of HIV screening tests in the US market
They vary in how early they recognize an HIV infection
HIV confirmatory algorithms have existed since 1987
Current approaches to HIV Confirmation include:
HIV Western blot (The original ‘Gold Standard’)
The growing use of orthogonal (independent) assays (So-called ‘Rapid-Rapid’)
A newly introduced CDC/APHL confirmatory algorithm
HIV transmission occurs at a variable rate over the lifetime of an infection. Maximal infectivity occurs BEFORE HIV antibodies can be detected!
Fourth generation assays recognize both HIV Antibodies AND p24 Antigen and are sensitive to HIV infection earlier than previous assays
Early treatment improves immune preservation AND may reduce the likelihood of transmission in individuals effectively treated with anti-retrovirals

6. HIV HISTORY…. LESSONS

7. HIV Diagnostics in the US – Slow to the Table
Many technologies such as 4th generation HIV testing have been in active use outside this country for several years.
The US Food and Drug Administration regulates and provides guidance, for marketing in vitro diagnostic devices (IVD). 
Diagnostic product approval in the US is expensive!
POCT HIV testing devices found a home initially in resource constrained environments.
Many technologies such as the newly introduced 4th generation HIV testing have been in active use outside this country for several years. The roots of this delay are tied to the fact that the US Food and Drug Administration regulates and provides guidance, for marketing in vitro diagnostic devices (IVD) and Diagnostic product approval in the US is an expensive, time-consuming proposition! It’s interesting to recognize that POCT HIV testing devices found a home, initially in resource constrained environments, where they were in active use long before they came to the United States.

8. HIV ANTIBODY TESTING
Historical Driving Force: Protection of the blood supply –
First Generation Tests – 1985 – Viral lysate
Problem - false positives among low risk donors 
Introduction of Western Blot Confirmation
Second Generation Tests – Recombinant proteins and synthetic peptides. Improved specificity
Third Generation Tests – Early 1990’s - Sandwich ELISAs use labeled antigen as conjugate. Increased sensitivity
Fourth Generation Tests – Antigen and Antibody
The screening for HIV infection was originally based upon the detection of HIV antibodies. Supplemental tests were used to confirm whether specimens found reactive truly contained antibodies specific to HIV.
The most widely used screening tests have traditionally been Enzyme-Linked Immuno Assays. At the infancy of the HIV epidemic in the United States these assays were found in laboratory settings and used to screen blood donations, as well as high-risk individuals.
The earliest assays used purified HIV lysates (1st generation), and lacked sensitivity and specificity.
Improved assays based on recombinant proteins and/or synthetic peptides, which also enabled the production of combined HIV-1/HIV-2 assays became available (2nd generation) in 1987, but persistent issues of false positive results led to the introduction of a secondary step, the HIV Western blot confirmation to improve the liklihood that a positive test the result of a true HIV infection.
In the early 1990’s, a so-called 3rd generation or sandwich ELISAs, which use labeled antigen as conjugate, with improved sensitivity and a reduced period of time before it indicated infection.
To further reduce the window period, enhanced ELISA assays have been developed that detect both HIV antibody and antigen (4th generation assays).

9. HIV Antibody Testing
Antibodies are proteins produced by the immune system to neutralize infections
Following sexual transmission, most people develop detectable HIV antibodies 2-8 weeks after infection (avg. 25 days)
Many labs currently employ HIV testing algorithm based upon 2nd or 3rd gen. EIA confirmed by the old ‘Gold Standard’ the HIV Western blot.

10. Established CDC Testing Algorithm
HIV-1/HIV-2 EIA
If repeatedly reactive
HIV-1 Western Blot or IFA
HIV-1 POSITIVE
HIV-1 NEGATIVE
HIV-1 INDETERMINATE
POSSIBLE HIV-2 Infection – Perform HIV-2 EIA if identifiable risk factors
HIV-2 EIA
If repeatedly reactive
HIV-2 Supplemental
HIV-2 POSITIVE
NEGATIVE
INDETERMINATE

11. Early Generation HIV Assays
1992
Abbott
HIV-1/HIV-2
EIA
1994
Vironostika
Oral fluid
EIA
1987
Vironostika
HIV-1 EIA
1992
Fluorognost HIV
IFA
The current HIV Testing algorithm originated in 1989, but the same principle of using a second, independent assay to confirm a screening assay is at play in the new, proposed HIV Testing Algorithm.
As Bernie Branson, the Associate Director of the CDC Laboratory likes to point out …
When the HIV algorithm was first introduced in 1989:
The telephone booth was still a common sight
A “portable” computer was the size of a small suitcase
While the algorithm had much to recommend it, for some of us, it leaves much to be desired. As the sensitivity of the initial screening tools have improved, there have become more and more instances where confirmation has become a complicated process.
The first-generation immunoassay tests employed HIV lysate as the source of antigen to capture antibody present in the sample. This generation of tests suffered from many problems associated with the antigen preparation. These included a large number of false-positive results caused by contamination of the antigen with proteins from the cells used to culture the virus, and batch-to-batch variation in the lysates employed. As a result, the specificity of the first-generation tests fell well short of that required by blood-service laboratories for screening blood donations.
The introduction of the first HIV testing algorithm was designed to solve a problem – too many people were being falsely identified as HIV positive. The Western blot improved our ability to reliably identify those truly infected, but it wasn’t an easy process. At one point or another there were at least 7 different sets of criteria for interpreting a Western blot. Highly complex, open to interpretation, labor intensive, and costly the Western blot was performed in only limited numbers of high complexity laboratories usually as a batched process which resulted in delays identifying and linking the infected into care.
Over the past 30 years, the FDA has approved ~ 30 tests to detect HIV-1. Broadly speaking, we categorize these HIV assays into HIV generations. Each step forward improved on the performance of the previous generation of test.
But it is important to recognize is that the FDA while approving new tests, does not disapprove tests. A product survives as long as it retains market share. As a result, there are examples of each generation of HIV testing available somewhere in the country. Virtually all of these tests are useful in detecting long-standing infection, but only the newest, 4th generation test is useful in identifying very recent infection.
There have been improvements in testing HIV screening assays over the years, but the role of the Western blot as a ‘Gold Standard’ for infection continues today even though its usefulness has become a debatable issue.
Since 1985, when commercial immunoassays for HIV detection first became available, there have been four successive generations of these tests used for screening and diagnosis. These four generations differ in nature and performance, as summarized inTable 2 . Each new generation of HIV immunoassays achieved a reduction in the window period and, therefore, improved detection of early infection. Enzyme immunoassays (EIAs) have remained central to HIV diagnostics, although advances have been progressively made in the basic immunoassay technology.
The first-generation immunoassay tests employed HIV lysate as the source of antigen to capture antibody present in the sample. This generation of tests suffered from many problems associated with the antigen preparation. These included a high number of false-positive results caused by contamination of the antigen with proteins from the cells used to culture the virus, and batch-to-batch variation in the lysates employed.[3] As a result, the specificity of the first-generation tests fell well short of that required by blood-service laboratories for screening blood donations.
April 1987 saw the introduction of second-generation anti-HIV-1 immunoassays into blood-bank screening. This led to a reduction of the mean infectious window period from 56 to 42 days.[3] Second-generation tests initially relied on recombinant Escherichia coli-derived antigen preparations, and were made possible by the complete nucleotide sequencing of the virus. False-reactive results were still often encountered as a result of contaminating bacterial proteins. Second-generation assays are still in use in some laboratories in the USA; reactive results are confirmed using western blots
Third-generation assays displayed a significant change in format and were introduced in the early 1990s. Whereas second-generation tests (indirect immunoassays) used an anti-IgG conjugate for detecting bound anti-HIV antibody, third-generation tests used a 'sandwich' technique, employing conjugated antigen. Since the antigen recognized all immunoglobulin classes, and because the appearance of IgM antibody tends to precede that of IgG antibody in early infection, sensitivity was improved while specificity was maintained.[13] The window period was reduced to an estimated 20–25 days.
1992
Murex
SUDS
1994
Orasure
HIV-Specimen
Collection
Device
1985
Abbott
HIV 1 EIA
1991
Cambridge
HIV-Western blot
1989 – PHS introduces Western blot confirmation

12. BACKGROUND & PErspective
It’s not just a question of how sensitive the test… it’s a question of how effectively we link and retain in care…
While the focus of our conversation today is about HIV Diagnostics, it is essential to place this into a historic context with an understanding that the goal of an HIV screen is to identify those who have been infected as early as possible and to effectively link these individuals into care.
BACKGROUND & PErspective

13. People refuse confirmatory tests
Unanticipated Consequence to the need for HIV Western blot confirmation
People refuse confirmatory tests
In NJ, 7.1% of positives could not be confirmed because specimens were not even collected!
Many don’t return to get their final results
New Jersey: 25 – 30% fail to return for a second testing-related visit.
Los Angeles: 35-40% fail to return
Other urban environments – similar story sometimes even worse
Bottom line:
ONLY ~ 70 % actually get their confirmed POSITIVE result!!
Impact  Linkage to Care is 
Delayed – Sometimes for years!
As laboratorians we strive for perfection; but sometimes there are unanticipated consequences.
Take the traditional confirmatory HIV Western blot and put into the context of public health HIV screening. The psychological assault of a tentative diagnosis is often the excuse for procrastination and hiding. In NJ, 7.1% simply refused to go on with a testing process and allow blood to be drawn. Many didn’t return to receive their final results. In NJ the number was somewhere between 25-30% . In Los Angeles it was between 35-40%. Other urban environments – similar stories. The BOTTOM LINE …. About 70% of our HIV + clients actually received their Western blot confirmed result. When we looked at the impact on linkage to care was that this delay often translated into a multi-year delay.

14. Returning is a Problem.
NJ Statewide Data
Problem
Preliminary Positive clients fail to return to receive “Confirmed” results (21.8%)
NAP succeeds ONLY 20% of the time in locating these clients
Solution
Confirmatory testing on-site, same day
In 2005, we described a problem that we thought was unique – Clients not getting linked to care because they failed to return to receive their final, Western blot HIV results. Now there could be several reasons for this:
Some might already know their status?
Some might pursue HIV confirmation independently at an alternate location?
Some might have difficulty working up their courage  and any delay becomes an excuse for additional delay
When I presented this data at the 2006 annual APHA meeting in San Diego, I learned that New Jersey was not alone. Los Angeles reported that up to 45% of individuals failed to return and get linked to care. Others in public health reported similar difficulties.
We know that:
Identifying an infected individual and linking them to care immediately  increases the likelihood for treatment
Consistently identifying truly infected individuals improves overall program credibility
Testing and Linking to Care in a single visit gives us an improved opportunity to engage and retain clients 14

15. LIMITATIONS OF RAPID TESTING
Rapid HIV Tests if used as a part of a RTA no longer require a CONFIRMATORY TEST – Western blot! “Presumptive Diagnosis”
Current FDA CLIA-waived rapid HIV tests measure
Antibodies to HIV
DOES NOT Measure?
HIV virus/RNA or DNA
NOT YET CLIA-waived - Determine Combo – HIV1/2 Ab plus p24 Ag
How Sensitive Is It?
At least as sensitive as some of the older EIA used in some hospitals and laboratories.
In some cases it is more sensitive than the HIV Western blot, the ‘Gold Standard’
This slide is from a talk I gave early in It points out that CDC had changed their surveillance definition to accommodate the use of a rapid testing algorithm that relied upon two DIFFERENT HIV tests. At the time, it pointed that an RTA allowed presumptive identification of an HIV infection using two rapid antibody screening tests. It also pointed out that these were not as sensitive as other technologies including RNA/DNA assays for HIV. As you can see I wrote that in some cases rapid HIV antibody tests were more sensitive than the HIV Western blot. This was a theme that has become increasingly important as testing technologies have improved. We’ll talk more about that later.

16. HIV Screening IS NOT a Clinical Workup!
A successful rapid HIV screen sets the stage for a successful clinical workup and follow-up by a provider
An Accurate HIV test screen
Effective counseling – risk behaviors, prevention and otherwise
Credible referrals
Expeditious Linkage to Clinical Care
4th gen. testing identifies infected individuals sooner
‘Test to treat’ means initiating ARV sooner
We lose large numbers before care is even initiated…
A ‘Presumptive Diagnosis’ is a diagnosis with a requirement for ‘physician follow-up’!
The story IS NOT simply what is the most sensitive test available to us, but rather HOW CAN we IDENTIFY, LINK and PROVIDE TIMELY TREATMENT.
Fourth generation testing along with the availability of improved anti-retrovirals means that care strategies will push us towards linking individuals to care sooner. The growing realization of the benefits of early treatment and an understanding of the transmission dynamics increasing call upon us to not only Identify an infected individual, but Link them to a knowledgeable care giver, AND start treatment with effective ARVs as soon as possible.

17. Continuum of Engagement to HIV Care
21% UNDIAGNOSED
31% NOT LINKED or DELAYED
41% NOT RETAINED
19-29% vl<200/ul
How well are we doing diagnosing, linking, retaining and suppressing HIV nationally? Simple answer --- Not so well. We currently fail to diagnose OR failed to link in a timely fashion more than half those infected with HIV in the US.
The hope is that a new generation of HIV tests and new algorithms for confirming infection will lead to better outcomes and potentially long term cost savings in managing HIV across the nation, but it’s important to recognize the strengths and the weakness of these new technologies.
Gardner et al, The Spectrum of Engagement in HIV Care and its Relevance to Test-and-Treat Strategies for Prevention of HIV Infection
Clin Infect Dis. (2011) 52 (6): doi:  /cid/ciq243
CDC. MMWR. 2011;60:

18. A NEW GENERATION OF HIV DIAGNOSTICS
New Possibilities – New Directions
A NEW GENERATION OF HIV DIAGNOSTICS
HIV diagnostics in the United States is in a state of flux. There are new, recently approved tests and confirmatory algorithms with increased sensitivity and simplicity that clearly allow us to detect earlier stages in an HIV infection.

19. This slide shows examples of each generation of HIV test along with a typical phone from it’s era.
1st generation HIV test
Detects IgG Ab (HIV1) by viral lysate as target antigen. The Vironostika HIV-1 assay shown on the previous slide is a great example. These early assays were associated with false positive screening results partly because of the use of viral lysates in their production.
2nd generation HIV test
Detects IgG Ab (HIV1 & 2) by using recombinant peptides representing HIV capsid and envelope. These assays were less likely to incorrectly identify someone as infected who was not. Examples of a second generation assay are the two rapid HIV tests: the Clearview ® StatPak or the OraQuick Advance ®.
3rd generation HIV test
Detects IgM as well as IgG Abs. (antigen sandwich method). IgM antibodies are formed when an infection occurs for the first time and are present in much lower amounts than IgG antibodies. Detects HIV 1 group O and HIV2
Examples of 3rd Generation HIV tests include the Trinity Unigold Rapid Test, and ADVIA ® Centaur HIV 1/O/2
4th generation HIV test
Detects IgM and IgG Abs, HIV1 group O and HIV2, as well as detecting the HIV1 viral capsid antigen p24

20. Stages of the infection process:
Acute HIV Infection
Stages of the infection process:
ENTRY, ECLIPSE PHASE, VIRAL RAMP-UP, AHI
The virus enters through the mucosa, travels to lymph nodes, and is initially not detectable. Then after some time begins a phase referred to as viral ramp-up. At a certain point this ramp-up is detectable by nucleic acid assays. As the viremia accelerates, and the immune system begins to respond, the individual may experience an acute viral syndrome consisting of a number of very non-specific symptoms: fever, rash, diarrhea, fatigue and/or headache. The individual becomes infectious prior to the appearance of antibodies!
These non-specific symptoms present an opportunity to identify an evolving HIV infection, but are often missed
Screening with tools that identify p24 Ag or RNA associated with the HIV virus allow detection of individuals who have been most recently infected.
Catherine Brennan, Ph.D, Abbott Diagnostics

21. HIV p24 Antigen Core protein of the virus
2005
HIV p24 Antigen
Core protein of the virus
p24 antigen is present before antibody can be detected
Detected 2 to 3 weeks after HIV infection
Detected about 6 days before antibody tests become reactive
Historically used for:
Diagnosis of pediatric HIV-1 infections
Blood bank safety (high incidence countries)
Historically used in the US until 1990s when NAAT entered widespread use
P24 antigen testing can detect HIV infection earlier than any antibody tests
Detected 2 to 3 weeks after HIV infection
p24Ag detection recognizes HIV infection days before antibody tests become positive
They were developed to shorten the window period and decrease the risk of transmission of HIV through blood transfusion.
Module 3: Overview of HIV Testing Technologies

22. Laboratory Markers of HIV Infection
This slide shows the ability of the various generations of HIV assays to pickup an infection. Early assays typically could identify a sexually transmitted infection about 55 days after infection. The fourth generation assays will typically identify individuals days after infection. It is important to note that there is an even more sensitive assays available, these are the so-called nucleic acid amplification assays or NAAT which are sensitive infection several days earlier.
Appearance of markers for HIV infection vary over time
Viral RNA is first detectable marker
Antibodies do not appear during early infection
Antigen appears early (alone) and later is complexed with antibodies
Graph was modified from data Fiebig et al.  Fiebig EW, Wright DJ, Rawal BD, et al. Dynamics of HIV viremia and antibodyseroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS. 2003;17(13):

23. NAAT Testing of 2nd gen. Rapid HIV Negative Individuals
When compared against current rapid HIV tests, NAAT tells us we’re missing between 6-8% of those infected when we screen for antibodies using one of the traditional rapid HIV tests
Those with the highest risk of infecting others are the one’s that are being missed!!
The same issues with patient return and process completion occur with NAAT that occur with traditional testing!!!
Solution: A test that picks up p24 Ag COULD identify a substantial proportion of the same population. A POCT device could increase the pickup without losing the ability to link patients to care.
When compared against antibody ONLY rapid HIV tests, NAAT tells us we’re missing between 6-8% of those infected when we screen for antibodies using one of the traditional rapid HIV tests Those with the highest risk of infecting others are the one’s that are being missed!!
Solution: A test that picks up p24 Ag COULD identify a substantial proportion of the same population. A POCT device could increase the pickup without losing the ability to link patients to care.
The same issues with patient return and process completion occur with NAAT that occur with traditional antibody testing!!! In our study in Newark, half of the patients offered a more sensitive assay, simply refused to be tested. The ultimate solution is a point-of-care test of great sensitivity and no delays.
E.G. Martin et al. / Journal of Clinical Virology 58S (2013) e24–e28

24. NAAT Testing of Antibody Negative Blood
Program
Dates
Description
Rapid Tested
NAAT Tested
AHI
HIV Ab+
% HIV Ab +
% Inc in Yield
% Yield AHI
Maryland
6/06-3/08
HIV Ab neg adults seen at two STD clinics (6/06--3/08); multiple venues 7/07-3/08)
58,925 7
1,709
2.90%
0.41%
0.01%
North Carolina
11/02-10/03
HIV Ab neg persons in North Carolina seeking HIV testing at 110 publicly funded sites (n = 109,250)
108,667 23
583
0.54%
3.95%
0.02%
Los Angeles
2/04-4/04
HIV Ab neg men seeking HIV testing at three STD clinics (n = 1712)
1,698 1 14
0.82%
7.14%
0.06%
NEWARK, NJ
2/10 to 1/12
HIV Ab neg adults receiving testing and counseling at two high risk urban hospitals in Newark, NJ
12,390
6,785 8
116
0.94%
6.90%
0.12%
Seattle King County
9/03-1/05
HIV Ab neg MSM seeking HIV testing through Seattle-King County (n = 3525)
3,439 5 81
2.36%
6.17%
0.15%
Atlanta
10/02-1/04
2202 adults receiving HIV testing and counseling at three high risk urban sites in Atlanta, Georgia
2,136 4 66
3.09%
6.06%
0.19%
San Francisco
10/03-7/04
HIV Ab neg persons seeking HIV testing at San Francisco Municipal STD clinic (n = 3075)
2,722 11
105
3.86%
10.48%
0.40%
When clients are screened with the traditional HIV tests looking for HIV antibodies, they miss a proportion of the HIV infected. This slide shows 7 different studies conducted between 2003 and With the exception of a study performed in Maryland, all the other studies found that by using the most sensitive assay – a nucleic acid amplification assay, one could increased the yield of individuals identified as infected by, on average, about 7%. What is important to note is that the individuals who were missed were highly significant in the transmission of the infection.

25. Why is this Important? 5
Risk of Transmission Male to Female - Blue
Reflects Genital Viral Burden – Yellow
Effect of ART – Theoretical - Red
(1/30-1/200)
HIV RNA in Semen
(Log10 copies/ml) 4
(1/100-
1/1000) 3
(1/500 -
1/2000)
(1/1000 –
1/10,000)
I mentioned that a person is most infectious during the Acute phase of infection. This graph from Myron Cohen and Chris Pilcher shows the risk of male-to-female transmission at different phases of HIV. The yellow is the viral burden in genital secretions throughout infection and the ratios show the probability of male-to-female HIV transmission per coital act, as a function of HIV disease stage.
There is an 8-10 fold increase in the risk of transmission during acute HIV.
Red is the theoretical effect of a biological intervention designed to reduce viral excretion; dashed line, a potential threshold for HIV transmission. 2
Acute Infection
Asymptomatic Infection
HIV Progression
AIDS
Cohen and Pilcher, JID 191:1391, 2005

26. Fourth generation diagnostics

27. FDA Approval – 4th Generation Assays: LABORATORY-BASED ASSAYS
18 June 2010 – Abbott Architect HIV Ag/Ab Combo Assay
22 July GS HIV Combo Ag/Ab EIA, (Bio-Rad Laboratories)
Both assays allow simultaneous qualitative detection of Human Immunodeficiency Virus (HIV) p24 antigen and antibodies to HIV Type 1 (HIV-1 groups M and O) and HIV Type 2 (HIV-2) in human serum or plasma
IMPORTANT: Neither test distinguishes between HIV-1 p24 antigen, HIV-1 antibody, or HIV-2 antibody in a sample, but they are sensitive to the presence of p24Ag.
IMPORTANT: Testing sequence – Initial Test followed by duplicate repeat tests. If positive  independent confirmatory assay:
Wblot, IFA, Aptima, Differentiation assay – Biorad Multi-Spot
At the moment the FDA has approved two 4th generation assays: the Abbott Architect HIV Ag/Ab Comb and the Biorad GS HIV Combo Ag/Ab EIA. Both assays are CLIA Moderate Complexity systems and both allow simultaneous detection of p24 Ag, as well HIV-1 groups M & O and HIV 2 in serum or plasma. There are other assays in the pipeline

28. HIV Tests have come a long ways
Both 4th generation assays turn positive approximately 20 days before a Western blot turns positive. The most sensitive 3rd generation antibody test turns positive nearly two weeks before a Western blot, but significantly more sensitive assays that rely upon the presence of antigen or nucleic acids. Even the most sensitive assay does become positive for 9-11 days after the infection.

29. 4th Gen. Performance
GS HIV Combo Ag/Ab
Abbott Architect Ag/Ab Comb
AHI: 48 AHI specimens (83%) were detected
Based upon initial ARCHITECT test
Sensitivity %
Specificity %
Repeat testing - resulted in corrected specificity of 99.50%
AHI: Two studies - Identified 95.2% (20/21) and 86.4% (38/44).
Sensitivity -100% (1603/1603) in known antibody positive samples.
HIV-1 seroconversion panel: detection improved by a range of 0–20 days compared to a 3rd generation HIV test.
Specificity:
99.9% (5989/5996) in low risk,
99.9% (959/960) in high risk; and
100% (100/100) in pediatric populations
Chavez et al. Evaluation of the performance of the Abbott ARCHITECT HIV Ag/Ab Combo Assay.
J. Clin Virol 2011 Dec;52 Suppl 1:S51-5. doi: /j.jcv Epub 2011 Oct 7.
BOTH 4th Generation Lab-based assays are very sensitive. They will identify MOST individuals at the stage we call Acute HIV infection. Data from several studies suggest that they pickup 85-90% of AHI,
Bentsen et al. Performance evaluation of the Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA, a 4th generation HIV assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.
J Clin Virol. 2011 Dec;52 Suppl 1:S doi: /j.jcv Epub 2011 Oct 12.

30. The Upside of Lab-based 4th Gen. HIV Tests
Substantially more sensitive than 3rd Generation HIV assays, earlier generation rapid HIV tests, and confirmatory assays
Somewhat more sensitive than POC-based 4th Generation rapid HIV assay (Alere Determine Combo assay)
They identify a significant proportion of acutely infected individuals (~90%)
May be used in the diagnosis of HIV-1/HIV-2 infection in pediatric subjects (i.e. children as young as 2 years of age) and in pregnant woman
When combined with the new proposed CDC Confirmatory algorithm, they have the potential of providing a complete laboratory workup without the need for a send out test.
The upside of lab-based fourth generations assays are that they are more sensitive than all previous HIV antibody assays. They are somewhat more sensitive than POC-based 4th Generation rapid HIV assay (Alere Determine Combo assay) and they identify a significant proportion of acutely infected individuals (~90%).
When they are combined with the new proposed CDC Confirmatory algorithm, they have the potential of providing a complete laboratory workup without the need for a send out test.

31. The Downside of Lab-based 4th Gen HIV Assays:
Less Sensitive than NAAT tests – (Individual or Pooled); therefore MISSING some cases of AHI.
Although the manufacturer claims it is ~ 35 minutes to an initial result, the reality is that in many laboratories the average time to an initial single result is much longer
Both FDA-approved Lab-based assays report a single combined specimen result, so neither can differentiate initially between recent and established HIV infections.
When used in conjunction with the new confirmatory algorithm they provide identification of individuals who have HIV antibodies, but require an additional NAAT test to ‘rule in’ AHI.
As of today, the only available 4th generation test that can on a preliminary basis identify recent infection is the standalone rapid test: The Determine Combo.
Lab-based Fourth Generation HIV assays miss some cases of AHI because of the delayed appearance of p24 Antigen. In addition, the majority of fourth-generation assays generate a single, numerical result which does not discriminate whether the test is positive owing to the detection of HIV antibodies or to the detection of p24 antigen. Subsequently, in the absence of the symptoms of acute retroviral syndrome and their recognition by clinicians, acutely infected individuals with positive results will not be distinguished from patients with established HIV infection.

32. Consider Turn Around Time to a Confirmed Result!
Architect package Insert:
Fully-automated, random-access (no Control brackets)
 Stat capability
 HIV Combo assay:
 29 minute time to first result
 >150 tests per hour on i2000SR
 >50 tests per hour on i1000SR
Automated equipment can be impressive. The Abbott Architect has the ability to run between HIV Combo tests per hour (depending on the instrumental model used) and the package insert claims that the first result is available within 29 minutes. The problem is that an initial reactive result needs to be retested in duplicate before concluding that it is HIV Positive. And if you then add the differentiation assay, and the possibility of a NAAT test, the inherent delays can prove troubling delays particularly in a CBO setting.
The question we’ve asked is how long would it take in a real world setting?
Manutac et al. JCV. 58S (2013) e44-47

33. Avg.: 57.7 min
In the Point-of-Care environment, such as occurs in Community Based Screening (CBOs) sites, clients are often intolerant of the need to return for results or for prolonged waits to receive results.
The Abbott package insert indicates that the expected TAT for a single assay is 29 minutes. In the real world environment of a functioning laboratory such as our Lady of Lourdes (Camden, NJ) the TAT for a single NEG result actually averages min. Granted there are occasional examples of TAT approaching 29 minutes, but these are few and far between. On a worst case scenario, examples of delays that approached 3 hrs. do occur. This is not surprising in a busy hospital laboratory where urgent situations have a way of asserting their own priorities.

34. So…. Delay in some settings is a problem.
CDC Algorithm :Diagnostic Laboratory Testing for HIV Infection in the United States
Biorad MultiSpot
These delays are compounded by the need for secondary confirmation. This slide shows you the recently proposed CDC Algorithm. Basically it begins with a 4th generation immunoassay and if positive proceeds to confirmation by a HIV1/2 antibody differentiation assay, the (Biorad MultiSpot) serves as the second test with an ability to independently confirm the presence of either HIV-1 or HIV-2 antibodies. Those cases that are not confirmed require RNA confirmation before an HIV infection is ruled out.
Unfortunately, in many settings the initial step of determining whether the IA is repeatedly reactive may exceed 3 hours even before the MultiSpot has been employed. In a hospital setting with hospitalized clients, this will not be a problem, but it is possible that clients will be lost to follow-up in many public health settings.
If the MultiSpot is negative the algorithm calls for an individual NAAT to be run. Currently the only approved assay is the Aptima.
So…. Delay in some settings is a problem.
Aptima

35. MultiSpot Improves Turn Around Time Sometimes
WHAT IT CAN DO:
CONFIRM reactive EIA results due to the presence of either HIV1 or HIV 2 ANTIBODY
Identify false-positive EIA results;
Shorten reporting time by a median of two days compared to Western blot
Differentiates HIV1 & HIV2
WHAT IT CAN’T DO IS:
It cannot confirm an Ag ONLY (AHI) result
Necessary to perform a NAAT assay
The MultiSpot assay is shown in this slide. It uses two HIV-1 indicators: a recombinant HIV-1 and an HIV-1 peptide; and a second HIV-2 peptide to identify potential HIV-2 infections. Compared to the Western blot it shortens reporting time by days and has the ability to differentiate an HIV-1 from an HIV-2 infection. What it cannot do is confirm the presence of HIV p24 Ag, so in some instances it is simply one more step without a conclusion.
J Dragavon et al., CDC/APHL 2010 Diagnostics Conference

36. 4th Gen. Point –of-Care HIV Screening
Lot number
Name of Test
Patient Identification
Alere Determine Ag/Ab Combo
Tests for the simultaneous and separate qualitative detection of free HIV-1 p24 antigen and antibodies to HIV-1 and HIV-2.
It is intended for use as a point-of-care test to aid in the diagnosis of infection with HIV-1 and HIV-2, including an acute HIV-1 infection, and may distinguish acute HIV-1 infection from established HIV-1 infection when the specimen is positive for HIV-1 p24 antigen and negative for anti-HIV-1 and anti-HIV-2 antibodies.
Control Line
p24 Antigen Result
Antibodies Result
As we’ve discussed, the majority of fourth-generation assays have only a positive or negative result. They do not discriminate whether the test is positive owing to the detection of HIV antibodies or p24 antigen. Subsequently, in the absence of the symptoms of acute retroviral syndrome and its recognition by a clinician, acutely infected individuals with positive results will not be distinguished from patients with established HIV infection.
The fourth-generation Determine HIV 1/2 Ag/Ab Combo assay provides separate results for the antibody and p24 antigen components. This may have important consequences in screening, but there are also important limitations we need to discuss.
Sample Pad
Alere Determine™ HIV-1/2 Ag/Ab Combo Package Insert Rev: /09

37. Panel AS PRB943 (BBI, Seracare)
Seroconversion panels
Determine HIV-1/2 (3rd gen) Ab
Day:
Day:
Determine Combo (4th gen) Ab Ag
We can see on the top, that the 3rd gen test became positive on day 19. On the lower part of the screen you can see that the Determine Combo detected the antibodies on the same day, (19) but in addition, Ag was positive already on day 12, and continued to be positive for additional nine days.
when looking at the entire panel it is easy to draw conclusions about the stage of infection but this is not available in reality.
Earlier detection
Panel AS PRB943 (BBI, Seracare)

38. Seroconversion panels
Ab Nonreactive. Dismissed.
Determine HIV-1/2 Ab
Ag Reactive. Presumably Recent infection.
Determine Combo
In reality, this individual will be tested at best only once a year, Lets assume it happened to be on day …. the result on the Combo is positive and indicate recent infection while on the 3rd generation it is negative and this person probably have been dismissed. Ag Ab
Day: 12
Panel AS PRB943 (BBI, Seracare)

39. Seroconversion panels
Comparison Table – Days to Positivity
Bleeding day
Test name
Marker 5 7 12 14 19 21
Roche Amplicor
RNA
NEG
POS
Abbott Architect 4th gen
Ag/Ab
Determine Combo 4th gen Ag Ab
Determine HIV-1/2 3rd gen
Abbott HIV Prism 3rd gen
Siemens Advia Centaur 3rd gen
Here we can see the performance of the Determine Combo in comparison to a number of other assays on the same panel. We can see that the Determine combo is less sensitive than the Abbott Architect 4th generation test and both of them are less sensitive than RNA test. But the Combo rapid test is more sensitive than the Ab only tests, either the Determine 3rd generation or the lab based 3rd generation EIA.
Panel AS PRB943 BBI (Seracare)

40. Number of Early HIV Infections Identified
Patel et al. Rapid HIV Screening: Missed Opportunities for HIV Diagnosis and Prevention. JCV. May, 2012
This population study from Pragna Patel et al at the CDC evaluated the sensitivity of rapid and fourth-generation tests for the detection of recent HIV infection. They screened ~100K samples of which 62 met the criteria of early HIV infection based on positive NAT and indeterminate or negative Ab. Of the 62 cases there was sufficient quantity to test 33 samples. Five FDA approved Rapid tests demonstrated 22-33% sensitivity. The third-generation lab based assay demonstrated 57%. Determine combo identified 25 specimens (76%) of which 17 specimens were Ag-only positive providing diagnosis of early infection. These findings suggest that current FDA-approved rapid tests fall short in detecting early HIV infections. And therefore miss the opportunity to interrupt onward HIV transmission especially when using these tests in high-risk populations.
Number of Early HIV Infections Identified
and Confirmed by NAAT

41. Seroconversion panels: FDA approved assays
NABC 2013
Seroconversion panels: FDA approved assays
4th Generation Lab Assays
Days Before Western Blot positive
I showed you this slide earlier as a bit of a setup. We’ve talked about the increasing disconnect between HIV Western blot recognition of infection and various generation of HIV screening tests. And I showed you earlier how the 4th Generation Lab-Based assays have turned positive at much as 20 days before the Western blot. On this slide, I am adding our current 4th generation point-of-care assay, which turns positive approximately 15.5 days before a Western blot will be interpreted as positive.
S. Masciotra (CDC), Journal of Clinical Virology, July 2013 demonstrates the sensitivity of FDA approved tests compared to Western Blot.
This is the first study to evaluate the 2011 version of Alere Determine™ HIV 1/2 Ag/Ab Combo with samples collected in early stage infection.
Determine Combo RT is reactive 15.5 days before WB, which is between 3rd & 4th generation lab based tests
The Ab component of DC detects HIV 7 days before WB, sooner than some lateral flow tests and similar to flow-through RTs
The data shows that DC Ab component performs as well or better than Ab-only RTs currently in use in the U.S.market
The Ag component confers an advantage over other RTs in detecting early HIV infection
* Modified from Silvina M, et al. Performance of the Alere DetermineTM HIV ½ Ag/Ab Combo Rapid Test with specimens from HIV-1 serocoverters from the US and HIV-2 Infected individuals from Ivory Coast. J Clin Virol 2013: Published Online 05 August DOI: /j.jcv

42. Summary Declining order of sensitivity to acute HIV infection:
Individual NAAT – Aptima
Pooled NAAT
4th Generation Tests
Lab-based – Architect / Biorad
POCT – Determine Combo
More than half of HIV transmission is thought to occur during the earliest phase of infection
Weighing the potential benefit of slightly improved sensitivity versus the immediacy of the result is a decision that needs to be driven by a careful assessment of the circumstances involved in screening.

43. Thanks!
Thanks for your attention. I’d be happy to answer any questions.
THE END