1. Overview of RTRI: Assay Principle and Test Performance
HIV-1 Rapid Test for Recent Infection
Overview of RTRI: Assay Principle and Test Performance
International Laboratory Branch
Division of Global HIV/AIDS & Tuberculosis
Centers for Disease Control and Prevention

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

3. Prevalence, Incidence, and Recent Infections3
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 100PY, 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. Rapid Recent Infection Assay
Example Country M:
Key issues
Use of recency in the context of CBS (Importance of unique ID)
Deduplication and identifying those 1) with prior diagnosis and 2) on ART
1.2 M Tested
120,000 HIV+
60,000 new diagnosis
3,000 to 6,000 recent infections
10% prevalence
50% incidence
5-10 %
recent infection

5. 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)
New HIV cases are defined as infections that occur within 6 months. New infections are characterized by…

6. 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
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.
Laboratory based tests
Can be applied to cross-sectional specimens
Unbiased detection of incident infection
May have subtype or population specific biases
Misclassification, ART

7. Host-virus dynamics during HIV infection
Maturation of HIV antibodies:
HIV Antibody titers
Antibodies to different proteins or epitopes
Antibody avidity
Antibody isotype
This slide is a a schematic taken from Stephane’s review article, shows simplified host virus dynamics in early phase of infection and depicts three different markers that can be used to detect recent HIV-1 infections.
First two are during pre-seroconversion period and are based on detection of nucleic acid or p24 prior to antibody development.
The third is during the post-seroconversion period defined by the antibody based assays.

8. 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
Antibody Avidity >>
Time >>
(how strongly HIV antibodies bind to HIV)
weak antibodies) from those with long-term infections (strong antibodies)
Time >>

9. 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

10. Laboratory-based Limiting Antigen (LAg) Avidity Enzyme Immuno-assay

11. PHIA Results

12. PHIA outcomes Prevalence (national and subnational)
VL suppression/ART coverage
Status of cascade
Incidence (national)
About true recent HIV infections (LAg+VL) per survey
25,000 to 30,000 participants
Small number of recent infections limit further detailed analysis

13. 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

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

15. RAPID TESTS FOR RECENT INFECTION (RTRI)

16. Sedia Asante Rapid Recency Assay
HIV Rapid Tests for Recent Infection
Sedia Asante Rapid Recency Assay
Negative
Recent
Long Term
Test is undergoing WHO PQ
CDC performance data, positive Verification line:
Sensitivity = 99.1%
Specificity = 98.9%
Acceptable diagnostic performance characteristics for WHO PQ
Sensitivity: =>99%
Specificity: =>98%

17. HIV Rapid Tests for Recent Infection
Maxim Rapid Test for Recent Infection
Long Term
Recent
Negative
LT T C
Swift
RIA
Test is undergoing WHO PQ
CDC performance data, Test line:
Sensitivity = 99.3% Specificity = 99.6%

18. Testing for recent infection18
Relationship with HTS
Facility or community where testing for recent infection is performed
Newly diagnosed AND recent infection
HTS_RECENT
(numerator)
HTS_TST
Testing for recent infection
HTS_TST_POS (≥15 years)
Newly diagnosed HIV positive
NOTE: Only persons who are newly diagnosed HIV-positive should be tested for recent infection. Persons who have been diagnosed HIV positive and are retesting or persons who are on ART should not receive testing for recent infection. Each country may use different methods for determining who is truly newly diagnosed HIV positive (e.g., additional post-test counseling to discuss prior diagnosis or ART history, review of registers to check whether a client is a silent transfer, and viral load testing to help eliminate those who test recent and are on ART and virally suppressed).
HTS_RECENT
(denominator)
Newly diagnosed AND long-term infection
Newly diagnosed HIV-positive persons aged ≥15 years who tested for recent infection and have a result
HIV negative
HIV Recency Study Health Facility Training Guide

19. Algorithm Leverages on Existing Systems19
Algorithm Leverages on Existing Systems
HTS client
National HIV testing algorithm
Routine case finding strategies
National surveillance systems
National M&E systems
Rapid Test 1
Report NEGATIVE
Non Reactive
Reactive
Rapid Test 2
INDETERMINATE
(Follow country guidelines)
Non Reactive
HIV-Positive
Report POSITIVE
Test for recent
Infection of New Diagnosis
Supplementary test for recent infection among newly diagnosed
Tested
recent
Tested
Long term
Viral load test
If feasible
Confirmed recent
(Tested recent + VL≥1,000 copies/mL)
Report RECENT

20. Specimen Types for RTRI
Serum
Plasma
Whole blood
(Venous draw or FingerPrick)
Do not use lipemic, hemolysed or contaminated specimens
Frozen samples must be brought to room temperature before testing

21. Advantages of the Test Assay: Surveillance: 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

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, these assays cannot distinguish between HIV-1 and HIV-2 specimens. Therefore HIV-2 positive specimens, if known, should be excluded for 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.

23. Rapid Tests for Recent Infection Assays Evaluation/Validation: Sedia Asante and Maxim Swift Assays

24. CDC Evaluation of the Rapid Recent Infection Assay
Approach
Specimen Panel
Performance of diagnostic verification line (HIV status)
Performance of LT line (recent/LT)
Ease of use
Ease of interpretation
Reproducibility
Lot consistency
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

25. Performance of Asante Rapid Recency Assay Verification Line
EIA/WB Algorithm
Asante VL Visual
HIV pos
HIV neg
Total
HIV Pos
575 10
585
HIV Neg 5
910
915
580
920
1500
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.
Sensitivity = 99.14% ( )
Specificity = 98.91% ( )
% Accuracy= 99% ( )
Kappa = ( )
NPV = 99.45
PPV = 98.29
Acceptable diagnostic performance characteristics for WHO PQ/USAID waiver
Sensitivity: =>99%
Specificity: =>98%

26. Performance of HIV Swift Recent Infection Assay (RIA) Test Line
EIA/WB Algorithm
Maxim Swift Test Line, Visual
HIV pos
HIV neg
Total
HIV Pos
576 4
580
HIV Neg
914
918
1498
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.
Acceptable diagnostic performance characteristics for WHO PQ/USAID waiver
Sensitivity: =>99%
Specificity: =>98%
Sensitivity = [ ]
Specificity = [ ]
PPV = 99.31
NPV = 99.56
Accuracy = [ ]
Kappa = [ ]

27. Performance of Asante Rapid Recency Assay LT Line
Sedia LAg-Avidity EIA ODn)
Asante LT Line (Visual)
Recent
Long Term
Total 80 29
109
Long-Term 18
438
456 98
467
565
% Agreement = 91.68
Kappa = ( )

28. Performance of HIV Swift Recent Infection Assay (RIA) LT Line
Sedia LAg-Avidity EIA ODn)
Maxim Swift LT Line (Visual)
Recent
Long Term
Total 72 35
107
Long-Term 28
432
460
100
467
567
% Agreement = 88.89
Kappa = [ ]

29. Comparing the Maxim Swift and Sedia Asante LT Lines – Two Operators29
Comparing the Maxim Swift and Sedia Asante LT Lines – Two Operators
Asante LT Line Visual
Recent LT
Total 88 17
105 21
439
460
109
456
565
%agreement =93.27
Maxim LT Line Visual Operator 1
Kappa=0.781 [ ]
Asante LT Line Visual
Recent LT
Total 85 20
105 24
436
460
109
456
565
%agreement =92.21
Sedia Asante visual LT correlates well with Maxim Swift visual LT
Maxim LT Line Visual Operator 2
Kappa=0.746 [ ]

30. Longitudinal Seroconversion Panels (N=9)
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
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.
Recent
infections

31. Preparing for Implementation31
Preparing for Implementation
Training
Training materials; presentations, SOPs, job aids etc.
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

32. Key points Performance of test
Robust QA system in place for the rapid recency assay
Lot QC performed in Sedia and verified in 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

33. Review How does HIV infection progress?
2008
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?
Overview of HIV Rapid Testing

34. Thank You