1. OVERVIEW OF RTRI: ASSAY PRINCIPLE AND
TEST PERFORMANCE
HIV-1 Rapid Test for Recent
Infection
International Laboratory Branch
Division of Global HIV/AIDS & Tuberculosis
Centers for Disease Control and Prevention
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2. Outline
• Introduction
• Describe HIV infection and progression
• Explain the assay principle
• Test Performance
• Review the evaluation and validation data
• Discuss the advantages and limitations of rapid
recency testing
• Preparing for implementation
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3. Prevalence, Incidence, and Recent Infections
Prevalence: Estimates % HIV positives in the tested
population at a given time and gives an indication of the
relative burden of HIV/AIDS in that population
Incidence: Estimates the rate of new HIV infections in a
given time per 100 persons, usually expressed as xx
per 100 Person Year, and indicates the level of new HIV
transmission
Recent HIV Infection: Newly or recently acquired HIV
infection as detected by a given laboratory method or
incidence assay
4. Recent HIV Infections
• High viral load
• Immature, weak immune response
• Continued high risk behavior
• High probability of ongoing transmission (40%-60%
of transmissions)
• Opportunity for interruption of transmission
• Recent contacts are likely known – contact tracing
possible
• Likely increase yield of HIV testing by partner
testing (HIV-positive persons)
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5. Different Approaches for Measuring HIV
Incidence
• Direct observational cohort: Follow
seronegative persons in longitudinal cohort until
they seroconvert
• Mathematical modeling: Deduced from multiple
rounds of prevalence
• Laboratory based tests: Can be applied to
cross-sectional specimens
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6. Bio-marker Dynamics During HIV infection
Maturation of HIV
antibodies:
• HIV Antibody titers
• Antibodies to different
proteins or epitopes
• Antibody avidity
• Antibody isotype
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7. Detecting Recent Infection Using Antibody Avidity
• Antibody avidity = binding
strength of antibody
• Functional property of
maturing antibodies
• Antibody avidity increases
over time after
seroconversion
• Surrogate marker of time
since infection
• Can be used to detect and
distinguish recently infected
persons from long-term
infections
Time >>
Antibody
Avidity
>>
Time >>
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8. RTRI Assay Principle:
Limiting Antigen and Antibody Avidity
Excess Antigen:
• Both low avidity and high avidity
antibodies bind
Limiting Antigen:
• Only high avidity antibodies bind
(Long-term infections)
• Low avidity antibodies do not bind
(recent infections)
Low High
Ag
Ag
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11. PHIA outcomes
• Prevalence (national and subnational)
• VL suppression/ART coverage
• Status of 90-90-90 cascade
• Incidence (national)
• About 30-40 true recent HIV infections (LAg+VL) per
survey
• 25,000 to 30,000 participants
• Small number of recent infections limit further
detailed analysis
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12. Cross-Sectional Surveys Take A Long
Time to Complete
Planning
• Protocol
development
• House-hold
listing
Training
• Laboratory
training
• Interviewer
trainings
Logistics
• Procurement
• Supply chain
management
Data
Collection
• House-hold
visits
• Sample
collection
• Sample
processing
and storage
Laboratory
Testing
• HIV
Diagnostic
Testing
• LAg testing
• VL testing
• ARV
Analysis
• Data review
and QC
• Data
Analysis
>12 months
Long time, high cost, and few recent infections
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13. Rapid Recent Infection Assay
• Example Country M:
• Key notes
• Use of recency in the context of CBS (Importance of unique ID)
• Deduplication and identifying those 1) with prior diagnosis and on
ART and 2) AIDS
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1.2 M Tested
120,000 HIV+
60,000 new
diagnosis
3,000 to 6,000
recent infections
10% yield
50% repeat testers
5-10 %
recent infection
14. Effective Epidemic Control Requires
Quick Detection and Response
Interrupt
further
transmission
Target
prevention
Analyze data
in real time
(who/where/
why)
Detect
Recent
Infections
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16. Available RTRI test on market
There are three manufacturers currently providing or
developing commercialized RTRI test.
1. Asanté™ HIV-1 Rapid Recency® Assay by Sedia
Bioscience – available and being implemented in ongoing
recency surveillance
2. Swift™ HIV Recent Infection Assay by Maxim
Biomedical – under CDC validation before releasing.
3. Sure Status HIV-1 Rapid Recency Card Test by Premier
Medical – under CDC validation before releasing.
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17. Schematics of The Sedia Asanté Assay
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C
V
LT
Asanté Strip Antigen(s) Role
C
(Control Line)
Goat anti-
human IgG
• Internal control which binds
to human IgG
• Indicates if the test result is
valid
V
(Positive
Verification Line)
HIV-1: p24-
gp41
HIV-2: gp36
• Binds to HIV-1/2 Ab
• Verify the presence of HIV-
1/2 Ab
LT
(Long-Term
Line)
HIV-1: limited
amount of rIDR-
M
• Limited amount of rIDR-M
antigen can only capture
HIV-1 Ab with strong avidity
but not low avidity Ab.
• Indicates if HIV-1 positive
diagnosis is long-term
infection
18. Algorithm Leverages on Existing Systems
HTS client
Rapid Test 1
Non Reactive Reactive
Rapid Test 2
INDETERMINATE
(Follow country guidelines)
HIV-Positive
Non Reactive
Viral load test
Perform RTRI on
New Diagnosis
Report NEGATIVE
Tested
Recent
Tested
Long term
RITA recent
(Tested recent + VL≥1,000 copies/mL)
Report POSITIVE
National HIV
testing algorithm
Supplementary test for
recent infection
among newly
diagnosed
Routine case
finding strategies
National
surveillance
systems
National M&E
systems
Report RECENT
19. RTRI Testing Cascade
HIV positive
Clients who visit HTS
clinics for HIV testing
Test on national
algorithm
• Newly diagnosed but not
AIDS
• Adults (e.g., aged ≥15 years)
• Consent for RTRI testing
RTRI recent
HIV negative
Test on RTRI
HIV positive but not
eligible/consent for RTRI
testing
Test on VL
Long-term
infection
RITA recent
Long-term
infection
20. Specimen Types for RTRI
• Do not use lipemic, hemolysed or contaminated specimens
• Frozen samples must be brought to room temperature before testing
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Serum Plasma
Whole
blood
(Venous
draw or
FingerPrick)
21. Advantages of the Test
Assay:
• Easy to use
• Minimum technical skill required
• No equipment required
• Rapid results for same-day counseling
Surveillance:
• Data can be analyzed in real-time to identify hotspots to
improve intervention program planning
• Large number of persons being tested, coupled with index
testing, can yield more # of recent infections
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22. Limitations of the Test
• Rapid Tests for Recent Infection are not yet approved for HIV
diagnosis, hence only confirmed HIV positive specimens (by
national algorithm) should be tested by RTRI
• Rapid tests for recent infection detects antibodies to both
HIV -1 and HIV-2 on the diagnostic line; however, recency
assays cannot distinguish between HIV-1 recent infections
and HIV-2 infections. Therefore, if known, HIV-2 samples
should be excluded from this testing
• Recency assays are configured only for the specimen types
listed (blood, serum and plasma). It is not to be used with
saliva, urine, or DBS.
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24. CDC Evaluation of the Rapid Recent
Infection Assay
Approach
• Performance of diagnostic
verification line (HIV status)
• Performance of LT line
(recent/LT)
• Ease of use
• Ease of interpretation
• Reproducibility
• Lot consistency
Specimen Panel
• Well-characterized world-wide
panel of specimens, N=1500
• HIV positive, N=580, HIV neg = 920
• HIV status determined by EIA
followed by confirmatory Western
blot testing
• Reference recency testing done by
LAg-Avidity EIA for comparison
• Additional testing using longitudinal
seroconversion panels
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25. Performance of Asante Rapid Recency
Assay Verification Line
Acceptable diagnostic performance
characteristics for WHO PQ/USAID waiver
Sensitivity: =>99%
Specificity: =>98%
EIA/WB Algorithm
Asante
VL
Visual
HIV pos HIV neg Total
HIV Pos 575 10 585
HIV Neg 5 910 915
Total 580 920 1500
Sensitivity = 99.14% (98-99.72)
Specificity = 98.91% (98.01-99.48)
% Accuracy= 99% (98.36-99.44)
Kappa = 0.979 (0.968-0.990)
NPV = 99.45
PPV = 98.29
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26. Performance of Asante Rapid Recency
Assay LT Line
Sedia LAg-Avidity EIA (@2.0 ODn)
Asante
LT
Line
(Visual)
Recent Long Term Total
Recent 80 29 109
Long-Term
18 438 456
Total 98 467 565
% Agreement = 91.68
Kappa = 0.722 (0.648-0.797)
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27. 0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
0 200 400 600 800
LOG
Long
Term
Line
Days Since SC
Asante antibody maturation
Longitudinal Seroconversion Panels (N=9)
Recent
infections
Long-term
infections
Interpretation
Recent HIV infection
Results indicate that HIV
infection was likely
acquired within last one
year
Long-term HIV infection
Results indicate that HIV
infection is long-term and
was likely acquired more
than a year ago
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28. Preparing for Implementation
• Training
• Training materials; presentations, SOPs, job aids etc.
• Preparation of training/competency panels; mixture of
known Long-term, Recent and Negative specimens
• Hands on training
• Certification of tester
• Testing
• Administered as an additional test to National Algorithm
• Similar to most rapid tests with additional information
• Recommend routine run of QC specimens (monthly
depending on the setting)
• Negative, Recent and Long-term
• Record management
29. Key points
• Performance of test
• Robust QA system in place for the rapid recency
assay
• Lot QC performed by manufacturer and verified at
CDC
• Lots not meeting CDC criteria are rejected
• Field implementation
• Hands on training with standardized panel
• Standardized data collection forms
• Ongoing data review to ensure excellent performance
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30. Review
• How does HIV infection progress?
• Why use the rapid test for recent infection (RTRI)?
• What is the assay principle for RTRI?
• What are the possible specimen types recommended
for use with RTRI?
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What are some characteristics of Recent HIV infections?
When you first become infected with HIV, there is a rapid burst of viral replication. Your body will not recognize it at first, so there will be a weak and immature or nonspecific immune response. Some people may not be aware they have the infection, and therefore may continue their high-risk behavior, resulting in a high rate of transmission.
These indiv in this are considered drivers of HIV transmission, accounting for 40-60% of transmissions. However, if we can identify these individuals early on at this stage, this presents an opportunity to interrupt transmission as recent contacts are likely to be known. This makes contact tracing easier and will likely increase the yield of partner/index case testing.
In direct observational cohort, seronegative persons are recruited and followed over time until they seroconvert. While this is the best method as it provides incidence directly, it suffers from recruitment bias and the Hawthorne effect (enrollment counseling may reduce observed incidence).
On the other hand, mathematical modeling can also be used and measures incidence deductively from multiple rounds of prevalence. This is based on a number of assumptions on mortality/survival, ARV coverage, etc. and is retrospective in time, while at the same time is Limited in sub-group analysis.
In recent years, laboratory-based tests have been developed and applied to cross-sectional specimens to estimate incidence and is an unbiased method for detection of incidence
Laboratory based tests
Can be applied to cross-sectional specimens
Unbiased detection of incident infection
May have subtype or population specific biases
Misclassification, ART
To better understand how this test was designed, lets review over the host virus dynamics in the early phase of infection. This slide is a schematic taken from Stephane’s review article and shows the 3 different biomarkers which can be used to detect new infections.
On the x-axis, you have the days since infection, which starts with initial contact at timepoint 0, up to 250 days (~8 months). On the y-axis, we are measuring 3 different biomarkers, with increasing concentrations starting from low to high as you move up the scale.
Upon entry into the host, there is a rapid burst of viral replication and shedding, as measured by both the RNA and the p24 Agic components of the virus. This is represented by the black and light blue curves.
The dark blue curve is the host response, as measured by the Ab. The response is a little delayed, about 2-3 weeks after the initial infection. Note that it is not as steep as the other 2 curves because it takes a while for the Abs to mature and develop into more HIV- specific Abs. However, it does increase over time, and when this happens, you see a decline in both viral markers, because the Abs are maturing and are more effective at neutralizing the virus.
There are several ways to measure maturation of antibodies. As shown here in the box on the right, you can measure the Ab titers, detect Abs to different proteins or epitopes, measure Ab avidity, and lastly, detect the various isotypes.
The RTRI assay uses the principle of avidity which I will go into more detail in the next slide.
Basically, antibody avidity looks at the functional aspects of maturing antibodies, which is the binding strength.
The illustration on the right, shows that antibody avidity increases over time after seroconversion
This allows us to use this information to serve as a marker of time, and distinguish between a recent HIV-1 infection, where there are more weak Abs present, from long-term infections, where there are more mature and stronger, antibodies present.
One additional note I would like to add is that early ART treatment and elite controllers can affect Ab development. With early ART, you can see a blunted, immature humoral response, and with elite controllers, you don’t really see full maturation, due to a lack of viral stimulation.
Since Asante is the only test currently available. This section will be focusing on Asante.
rIDR-M : recombinant protein which contains major variants of gp41 immunodominant regions among the HIV-1 group M virus
Evaluation results of Asante diagnostic line with a panel of 1500 specimens is summarized in this 2x2 table.
The data demonstrated that sensitivity of Asante was >99% while specificity was 98.9%.
Overall agreement with reference testing was close to 99% with high kappa value.
Asante results of testing longitudinal seroconversion panels are shown on this slide, demonstrating that recent infections within first few months change over to LT infections within 6-12 months.
Horizontal red line represents cutoff.
