HIV Testing Algorithms – A Long Journey – Eugene G. Martin, Ph.D .

1. HIV Testing Algorithms – A Long Journey – Eugene G. Martin, Ph.D. Professor – Pathology & Laboratory Medicine UMDNJ – Robert Wood Johnson Medical School Co-Director, NJ HIV

2. AGENDA • Background – HIV Testing 1985 -2013 • Overview of Current  HIV Testing Algorithms • Testing performed at public sites – LICENSURE DETERMINES OPTIONS • Testing preformed in hospital laboratories – MIXTURE OF LAB-BASED & POCT-BASE • Testing performed in national laboratories – GAMUT with use of reflex testing driven by results • CDC updates and revisions over the past few years: • CDC/APHL DIAGNOSTIC CONFERENCES 2004-2012 • CDC TASKFORCES: HIV LAB & POCT TESTING, AHI DEFN • DRAFT HIV DIAGNOSTIC GUIDELINES 2013 • Testing Results • Result and Interpretation.     • THE connection: • Linkage to Care

3. THE CONTEXT BACKGROUND

4. CDC estimates • 1.2 million people (US) are living with HIV • One in five (20%) are unaware of their infection • While relatively stable for several years the rate of ‘new’ HIV infection rate is substantial  • About 50,000 become infected each year • Prevalence is increasing because of anti-retroviral therapy. • The problem infectivity is largely a function of viral load and risk encounters

5. 19%-29% VL<50 c/mL 41% Not retained 21% Undiagnosed 31% Not linked/delayed Gardner et al. Clin Infect Dis 2011;52; Marks et al. AIDS 2010;24

6. Problem Preliminary Positive clients fail to return for results (25.2%) NAP succeeds ONLY 20% of the time in locating these clients Solution Confirmatory testing on-site, same day In use, high prevalence areas worldwide Why Rapid Testing Algorithms are Need in Public Health?

7. Key Questions • What strategies will get more people to learn their HIV status? • How do we get more infected individuals into care AND encourage earlier treatment? • How does improved ART impact efforts to reduce transmission?

8. Recently Large Change in Focus. Why? • 40% of HIV infections occur in the earliest stages of the disease • New 4th generation HIV Tests are allowing us to identify infected individuals when they are most infectious! • Earlier treatment preserves immune function and improves morbidity • LINKAGE TO CARE – Underpins prevention & treatment ... • Test to Treat • Treatment as Prevention

9. Transmission is a function of viral load! 5 Risk of Transmission Male to Female - Blue Reflects Genital Viral Burden – Yellow Effect of ART – Theoretical - Red Evolving Opportunity! HIV RNA in Semen (Log10 copies/ml) 4 (1/100- 1/1000) (1/30-1/200) 3 (1/500 - 1/2000) (1/1000 – 1/10,000) HIV Screening before 2012 2 Acute Infection Asymptomatic Infection HIV Progression AIDS Cohen and Pilcher, JID 191:1391, 2005

10. AHI – Acute HIV Infection • 70-80% symptomatic, 3-12 weeks after exposure • Surge in viral RNA copies to >1 million • Recently we had one 10 million copies!! • CD4 count drop to 300-400 w/ rebound • Recovery in 7-14 days • Because individuals with AHI are highly infectious, have engaged in high risk behaviors, and are often unaware of their status they contribute substantially to the spread of HIV. • Although AHI is short (typically 3-4 weeks), studies have consistently shown that 40-50% of new HIV transmissions are caused by onward transmission from an individual with AHI. • SYMPTOMS - ACUTE HIV INFECTION • Rash &/or fever(s), possibly in combination with: • Malaise • Loss of Appetite • Weight loss • Sore Throat • Mouth Sores • Joint Pain • Muscle Pain • Swollen lymph nodes • Diarrhea • Fatigue • Night sweats • Nausea/vomiting • Headache • Genital Sores

11. HIV Testing 1983  Present Day 1980s -T-cell assays 1985 – HIV Antibody testing – Lab-based – Enzyme Immunoassays: 1st Gen 1987 – HIV Western Blot criteria – Why? 1991 – Improved EIA: 2nd Gen 1996 – Oral mucosal transudate testing- OraSure 2003 – Rapid testing (blood and then oral transudate) Current: Rapid 3rd gen assays and laboratory 4th gen assays with available nucleic acid amplification testing (NAAT) Current: Rapid 4th gen assays with both antibody and antigen p24 testing (Determine, FDA approved) Future: Rapid CD4/CD8 assays and rapid viral load assays

12. HIV Infection Symptoms Antibody by 1st gen EIA Antibody by Western Blot Antibody by 3rd gen EIA Antigen RNA / NAAT Acute Infection Silent Infection AIDS Weeks after infection 5-10 years 1-3 years

13. THE CONTEXT Serologic markers DURING HIV-1 infection

14. Assay Reactivity during Early HIV

15. Typical HIV Serologic Profile

16. Viremia During Early HIV Infection • Ramp-up ViremiaDoublingTime = 21.5 hrs • Peak Viremia106 – 108 gEq/mL • Viral set-point102 – 105 gEq/mL • WINDOW • Antibody – 22 Days • Antigen – 16 Days • Pooled NAT – 14 Days • Individual NAT – 11 Days HIV Antibody – 3rd Generation 22 Days P24 Ag 16 Days PooledNAAT 14 Days Individual NAAT 11 Days 0 10 16 22 DAYS ANTIBODY WINDOW

17. HIV Tests are NOT all equal

18. BACKGROUND • Testing 1985-2003 • CLIA - Waived Rapid HIV antibody tests: • Orasure • Oral • Fingerstick • Clearview • Trinity • Insti (2011) – 1 Minute to Read • 2010  4th generation testing • Laboratory-based (CLIA – MOD. COMPLEXITY) : • Abbott Architect Combo HIV1/2 Ag/Ab • Biorad • Rapid HIV Antigen/Antibody tests (2013) (PENDING CLIA WAIVER) • Alere Determine (HIV1/2 Ag/Ab)

19. What’s it all about? • SCREENING versus DIAGNOSIS • SCREENING FOR HIV  Focus on ‘LINKAGE TO CARE’ • Orthogonal Confirmation • “Presumptive Diagnosis” – Pending additional testing: • CD4 • NAAT Testing – Aptima • LAB-BASED DIAGNOSIS: • Manufacturer’s Package Insert couple with a confirmatory step:

20. MMWR September 22, 2006 / 55(RR14);1-17Revised Recommendations for HIV Testing of Adults, Adolescents, and Pregnant Women in Health-Care SettingsBernard M. Branson, MD1 H. Hunter Handsfield, MD2Margaret A. Lampe, MPH1Robert S. Janssen, MD1Allan W. Taylor, MD1Sheryl B. Lyss, MD1Jill E. Clark, MPH3 1Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (proposed) 2Division of STD Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (proposed) and University of Washington, Seattle, Washington 3Northrup Grumman Information Technology (contractor with CDC) Routine HIV testing for adolescents and adults in health-care settings Test everybody unless specifically denied Screen for HIV regardless of prevalence (as effective in very low prevalence as in high prevalence areas). High-risk individuals at least annually, recommended every 6 months Drug users are by definition high-risk Addiction treatment centers Methadone programs Needle exchange programs …strange advantage – patients keep returning to the center, so counseling, linkage to care or additional tests can be performed

21. To Date: FDA Has Approved 2 4thgen. Lab Based Assays

22. FDA Approval – 4th gen. Lab Based Assays: • 18 June 2010 – Abbott Architect HIV Ag/Ab Combo Assay • First diagnostic test approved by FDA for use in children as young as 2 years of age, and pregnant women. • Specific for the detection of the HIV-1 p24 antigen , as well as antibodies to HIV-1 groups M and O, and as antibodies to HIV-2. • 22 July 2011 - GS HIV Combo Ag/Ab EIA, (Bio-Rad Laboratories) • 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. • “Patients … who identify a specific risk occurring more that 4 weeks previously, should not be made to wait three months (12 weeks) before HIV testing. They should be offered a 4th generation laboratory HIV test and advised that a negative result at 4 weeks post exposure is very reassuring/highly likely to exclude HIV infection. An additional HIV test should be offered to all persons at three months (12 weeks) to definitively exclude HIV infection. Patients at lower risk may opt to wait until three months to avoid the need for HIV testing twice.

23. Study design • 9150 samples at four U.S. clinical trial sites, using three kit lots. Unlinked samples were from routine testing, repositories or purchased from vendors. Results • GS HIV Combo Ag/Ab EIA detection in samples from individuals in two separate populations with acute HIV infection was 95.2% (20/21) and 86.4% (38/44). Sensitivity was 100% (1603/1603) in known antibody positive [HIV-1 Groups M and O, and HIV-2] samples. • HIV-1 seroconversion panel detection improved by a range of 0–20 days compared to a 3rd generation HIV test. Specificity was 99.9% (5989/5996) in low risk, 99.9% (959/960) in high risk and 100% (100/100) in pediatric populations.

24. NJ Facilities with 4th Gen. HIV testing – Oct. 2013 VA East Orange 385 Tremont Ave. East Orange NJ Hackensack University Medical Center 30 prospect ave. Hackensack, NJ 07601 Manhattan Labs 25 riverside Dr. Pine Brook NJ Our Lady Of Lourdes Medical Center 1600 Haddon Ave. Camden NJ Shore Memorial Hospital 1 E New York Ave. Somers Point, NJ 08244 RWJ Hamilton 1440 Lower Ferry Rd. Ewing twp, Nj St, Josephs regional Medical center 703 Main Street. Paterson, NJ 07503 UMDNJ 150 Bergen stHackettstown Medical Center 651 Willow Grove St. Hackettstown, NJ 07840 Holy Name Medical center 718 Teaneck Rd. Teaneck, NJ 07666 CentraState Medical Center 901 West Main Street, Freehold TWP NJ 07728 Jersey Shore University Medical Center 1945 New Jersey 33 Neptune, NJ St. Barnabas Medical Center 94 Old Short Hills Rd. Livingston, NJ 07039 Newark Beth Israel Medical Center 201 Lyons Ave. Newark, NJ 07112 St. Peter’s Medical Center 254 Easton Ave. New Brunswick. NJ 08901 St.Francis Medical Center 601 Hamilton Ave. Trenton, NJ

26. HIV Rapid Screening Tests CLIA-waived Complexity Clearview StatPak Clearview HIV1/2 Complete Trinity Uni-Gold Oraquick Rapid MedMira Reveal

28. Test develops in 20-40 minutes

29. Rapid HIV Testing Results Trinity Unigold Orasure Oraquick

30. 3.5  4th Gen – Point-of-Care Test

31. Even though there is a 64% improvement over a third generation (Ab only) POCT, health care professionals should still be aware that the Determine HIV-1/2 Ag/Ab Combo is not as sensitive as 4th generation Lab-based EIAs in diagnosing primary HIV-1 infections!! • All 7 false positive p24 Ag sera were correctly identified by the Determine Combo test as negative. • 5/14 of the p24 Ag true positive sera (early seroconversion) were missed by the Determine Combo test and tested negative for both p24 Ag and antibodies

32. ALGORITHMS • Laboratory-based • Point-of-Care based

33. NJ RAPIDTESTING ALGORITHM ORTHOGONAL

34. “PRESUMPTIVE DIAGNOSIS” • When Rapid HIV Tests are used as a part of an RTA, a diagnosis can be made with a CONFIRMATORY Western blot; OR by a second (but different manufacturer’s) rapid test. • If the diagnosis is made by a second rapid: • “Presumptive Diagnosis“ – and requires further testing at the treatment site as a part of staging the infection.

35. Dear Colleagues:Thank you for joining us on last week’s HICSB Quarterly Call.  Attached is the letter discussed during the call regarding the new HIV testing algorithms guidance issued by the Clinical Laboratory and Standards Institute (CLSI).  The letter affirms that these new algorithms meet the current HIV case definition and provides instructions for recording a case diagnosed using the new algorithms in eHARS.We recognize these new algorithms represent a shift in surveillance practices.  To help states address these changes, HICSB is creating a list of Frequently Asked Questions (FAQs).  Please send your questions to Adria Prosser at ahp8@cdc.gov and cc your surveillance program’s CDC epidemiology consultant.Best regards,H Irene Hall, PhD, FACE November, 2011RTA MEETS CDC HIV CASE DEFINITION

36. Review of HIV-1 Confirmation testing WB/Aptima • While Western blot (WB) is still widely considered a ‘gold standard’ • No longer suitable, more sensitive assays in use already • Issue aggravated by potential availability of Ag/Ab Combo rapid assays • Cost. Also, cost dependent on TAT requirements i.e. if rapid TAT, cost increases (kit-based assay) • Serum sample • Aptima • approved for diagnosis of HIV-1 (early AHI/ primary HIV, no antibodies yet) • Approved for confirmation of HIV-1 if antibody screen is positive • Lab based method, sensitivity similar to FDA approved viral load assays • Plasma sample (or conversely, Whole Blood if spun adequately)

37. Possible HIV CONFIRMATORY pathways: • On-site RAPID3 with On-site RAPID3 verification (current RR algorithm) • On-site RAPID3 with remote EIA3 or EIA4 • EIA can serve as an orthogonal assay • On-site RAPID3 with remote RAPID3 • On-site RAPID4 Antigen ONLY with remote Aptima • On-site RAPID4 Antibody/Antigen (Lab-based or POCT) with an ON-SITE RAPID3 • Discordant results will be handled by same procedures by NJHIV staff/ docs • Still need sample collected for discordant resolution • If remote EIA/ rapid, need to get client back to site • Delay referral • Delay entry into care • Refuse confirmation possible for all remote tests • If on-site verification, referral to care faster, eliminates non-returners, blood draw refuse

38. Summary of Interpretation of HIV-1 Specimen Results * The individual should be referred for medical follow-up and additional testing. ** Antibody results should be confirmed with Western blot or IFA. *** A nonreactive test result does not preclude the possibility of exposure to or infection with HIV-1. Sample requirements for Aptima (studies with alternative specimens, good results available): • 1.6 mL frozen plasma (EDTA, lavender-top tube); 0.6 mL minimum • Alternatively, frozen PPT-potassium EDTA plasma (white-top tube) may be submitted. • Centrifuge blood, transfer plasma to a plastic screw-capped tube, and freeze within 6 hours of collection.

39. But an important question remains • How often do we miss an early infection? • How often do we screen an individual and tell them they’re negative, when, in fact, they are most likely to infect others?

40. NAAT TESTING: • Screening for Acute HIV Infection in Newark, NJ Eugene Martin1*, Debbie Mohammed2, Gratian Salaru1, Joanne Corbo1, Michael Jaker2, Joan Dragavon4, Robert Coombs4, Sindy Paul3, and Evan Cadoff1 • 1 UMDNJ – Robert Wood Johnson Medical School, Somerset, NJ 088732 UMDNJ – New Jersey Medical School3 New Jersey State Department of Health and Senior Services, Trenton, NJ 4 University of Washington, Seattle, WA • Use of rapid HIV in conjunction with pooled NAAT allows assessment of the burden of acute HIV infection (AHI) in a particular locale. • Clients offered NAAT testing after rapid HIV testing. Of those accepted (~50%), specimens collected shipped to Univ. of Washington where NAAT was performed. • 8 AHI’s identified in 6785 specimens tested. Approximately 6.9% increase in yield over AB + only

41. Reminder: 10 -14 Days Ramp-Up Phase – Rapid Viral Replication

42. NAAT Testing of Antibody Negative Blood : Results Nationwide

43. HIV Tests have come a long ways

44. Conclusions: • NAAT tells us we’re missing of 6-8% of those infected when we screen for antibodies! • Those with the highest risk of infecting others are the one’s being missed!! • The same issues with patient return and process completion occur with NAAT that occur with traditional testing!!! • Solution: EIA’s that pickup p24 Ag COULD pickup a substantial proportion of the same population. A POCT device could increase the pickup without losing the ability to link patients to care.

45. Recommendations 2013 - CDC Diagnostics Recommendations • Initiate screening with a 4th generation Ag/Ab combination immunoassay (IA) • Reactive (repeatedly reactive) specimens should be tested with a 2nd generation Ab IA that differentiates HIV-1 from HIV-2 antibodies. (MULTISPOT) • Persons whose specimens are positive on the initial IA and antibody differentiation IA should be considered positive for HIV-1 or HIV-2 antibodies and initiate medical care that includes laboratory tests such as viral load, CD4, and antiretroviral resistance assays. • Specimens reactive on the initial IA and negative on the HIV-1/HIV-2 Ab differentiation IA should be tested for HIV-1 RNA. A reactive result indicates Acute HIV-1 infection. • Follow this same testing algorithm (beginning with 4th generation IA) for specimens with a previous reactive rapid HIV test result.

46. Alternatives: • If 3rd gen HIV-1/2 IA as initial test: perform subsequent testing specified in the algorithm. • 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 • HIV-1 NAT as 2nd test: if positive, HIV-1 infection; if negative, perform HIV-1/HIV-2 Ab differentiation assay.

47. Supersedes: • Recommendations for Use of Western Blot (1989) • Recommendations for HIV-2 Antibody Testing (1992) • Protocols for confirmation of reactive rapid tests (2004) • Screens for both virologic and serologic markers of HIV infection • Incorporates NAT to resolve discordant IA results • Identifies acute HIV-1 infection • Reduces indeterminate test results • All IA-positive specimens tested for HIV-2 • Emphasizes sensitivity • For initial testing • During supplemental testing • Rare false-positive antibody test results might occur • False-positive results would be discovered during subsequent laboratory testing recommended as part of initial clinical evaluation

48. THE END

49. NJHIV – WHO WE ARE Rapid HIV testing support group Composed of laboratorians MD, PhD, MT, RN Department of Pathology and Laboratory Medicine at Rutgers Robert Wood Johnson Medical School Built upon an existing Rutgers Robert Wood Johnson Medical School, multi-facility, point-of-care-testing program Develop a centralized quality assurance process Management by board certified Pathologists, experienced laboratory professionals, RNs and medical technologists Supervisory control through site coordinators

50. New Jersey Rapid Testing RWJ Sites: 97 Non RWJ Sites: 64