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

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

Prevalence, Incidence, and Recent Infections 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 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

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

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…

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

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.

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 >>

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

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

PHIA Results

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

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

Effective Epidemic Control Requires Quick Detection and Response Interrupt further transmission Target prevention Analyze data in real time (who/where/why) Detect Recent Infections

RAPID TESTS FOR RECENT INFECTION (RTRI)

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%

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%

Testing for recent infection 18 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

Algorithm Leverages on Existing Systems 19 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

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

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

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.

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

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

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% (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 Acceptable diagnostic performance characteristics for WHO PQ/USAID waiver Sensitivity: =>99% Specificity: =>98%

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 = 99.31 [98.24-99.81] Specificity = 99.56 [98.89-99.88] PPV = 99.31 NPV = 99.56 Accuracy = 99.47 [98.98-99.27] Kappa = 0.989 [0.981-0.997]

Performance of Asante Rapid Recency Assay LT Line Sedia LAg-Avidity EIA (@2.0 ODn) Asante LT Line (Visual) Recent Long Term Total 80 29 109 Long-Term 18 438 456 98 467 565 % Agreement = 91.68 Kappa = 0.722 (0.648-0.797)

Performance of HIV Swift Recent Infection Assay (RIA) LT Line Sedia LAg-Avidity EIA (@2.0 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 = 0.628 [0.544-0.712]

Comparing the Maxim Swift and Sedia Asante LT Lines – Two Operators 29 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 [0.714-0.848] 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 [0.675-0.817]

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

Preparing for Implementation 31 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

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

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

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