HIV Testing in 2013 – New Tests, New Questions, Few Answers

1. HIV Testing in 2013 – New Tests, New Questions, Few Answers Sheldon Campbell M.D., Ph.D.

2. Overview • HIV Epidemiology in the US and the Islamic World • Diagnosing HIV: Traditional and Current Test Formats • Managing Evolving Tests: Models and Algorithms for HIV Testing • A Practical Example; VA New England Healthcare HIV Testing

3. HIV Infections Continue to Occur CDC Data

4. HIV in the Islamic World • Low Prevalence Overall • UNAIDS 2011 data indicates that the Middle East / North African region is one of the two with thefastest-growing AIDS epidemic. • I chose a few countries from this WHO-defined region as examples, trying for variations in size, location, wealth, civil stability, and presence of outsiders; each nation in the region, and different regions within a nation, do vary: • Jordan • Iran • Morocco • UAE • Afghanistan

5. HIV in Jordan • Low Prevalence • Total HIV positive cases (1986-2011): 847 (29% Jordanians and 71% foreigners) • Total HIV positive cases registered in 2010 and 2011 is 36 (78% males and 22% females) • By December, 2011, 99 Jordanian PLHIV had died of AIDS. • 56% of transmission is heterosexual. • Risks • Youth: 57% of population <30y/o • Non-Jordanian workers 13.1% of workforce; many Jordanian men work outside the country. • Women vulnerable in male-dominated workplaces. • Stigma related to HIV diagnosis, and some legal restrictions, are disincentives to case-finding. • Data are likely very incomplete.

6. HIV in Iran • In the ‘concentrated’ phase; with high prevalence in vulnerable populations. • 15% of IDU are infected. • Prevalence of 4.5% in female sex workers. • 23,497 PLWH identified in Iran by September 21, 2011 • 91.3% men and 8.7% women • 3168 have AIDS, 4419 dead • 46.4% are in the 25-34 age range • Models of detection and prevalence suggest that this is <1/3 of the real number. • Decrease in newly-identified cases may reflect limitations of case-finding mechanisms rather than actual decrease in transmission.

7. HIV in Morocco (I Don’t Read French!) • By December 2011, 6453 cases • 4169 with AIDS, 2284 asymptomatic HIV • 65% identified 2005-2011. • 71% between 25 and 44y/o. • Roughly 50% women. • Complex modes of spread

8. HIV in the UAE • 1980s till the end of 2011 cumulative total of 726 HIV still-alive cases • 2010-2011 93 new HIV cases reported among UAE nationals • No estimates in high-risk groups • Risks • massive labour migration • influx of tourists • changing sexual norms and practices among young people • "Those who suspect they may have been exposed to HIV – e.g. through sexual relationships or injecting drug use – may avoid the existing screening programmes."

9. HIV in Afghanistan • High risk due to: • 30y of armed conflicts. • Displaced populations. • Poppy cultivation and injection drug use. • Unsafe blood supply and injection practices. • Challenges • Security and economic problems. • Lack of infrastructure. • High stigma • Prevalence data scarce to non-existent. • Likely high levels in IDU and other high-risk populations. • Struggling to develop effective responses.

10. Assessment • HIV is underdiagnosed in the WHO North African-Middle East region; this is not unique; HIV is underdiagnosed everywhere. • In general, knowledge of HIV transmission and prevention is limited. • The potential for continued and increased spread is present. • Diagnostic capacity is essential to limit new infections. • That would be us.

11. ART prevents HIV

12. Treatment=Prevention • Effect of early vs. delayed treatment on transmission of HIV to partners Prevention of HIV-1 Infection with Early Antiretroviral Therapy. N Engl J Med 2011; 365:493-505 August 11, 2011

13. HIV in the US • According to CDC, 1.2 million people in the United States are living with HIV infection and 1 in 5 are unaware of their infection. • Current recommendations (2009-onward) are to broadly expand HIV testing to try and test everyone at risk, even without clinical suspicion.

14. Current diagnostic algorithms for HIV infection • Diagnosis of HIV infection is still primarily serological; detecting HIV antibodies. • As with all laboratory tests, false-positive and false-negative tests occur. The usual pre-analytical causes, plus: • False-negatives • Hypogammaglobulinemia, immunosuppression, • ‘Window period’; varies with assay • Unusual viral types • HIV-2 • Unusual types of HIV-1 • Improved Technology • False-positives • Cross-reacting antibodies • Pregnancy, multiple transfusions, hypergammaglobulinemia, hemodialysis, autoantibodies associated with autoimmune disease, recent vaccinations and viral infections • Confirmatory Testing

15. Timing of Diagnostic Events in HIV Infection From Branson B: J Acquir Immune DeficSyndr 2010;55:S102–S105

16. The Standard Algorithm From Griffith, BP, Campbell S and Mayo, DR (2007) ‘Human Immunodeficiency Viruses’ in Murray PR et al (eds) Manual of Clinical Microbiology, 9th Edition

17. The ‘Generations’ of HIV Screening Tests • 1st Generation: viral lysates • 2nd Generation: recombinant antigens, ↑ specificity • As recently as 2006, 70% of public health labs used 1st or 2nd gen assays. • 3rd Generation: recombinant antigens, sandwich format with improved IgM detection, ↑ sensitivity in early infection. • 4th Generation: includes antigen detection capability for even earlier detection • Abbot 4th-gen test recently approved in US, but available in EU x years; others submitted

18. Screening Test Formats • Plate or tube EIA • Semi-automated; being phased out. • Automated chemiluminescent tests • 3rd or 4th generation; highly automated on random-access immunochemical systems. • Rapid / Point-of-care tests • Typically perform like 2nd or 3rd gen assays. • Rapid 4th gen tests newly introduced.

19. HIV Confirmatory Tests • FDA-approved • Western blot • IFA (rarely used, but still done by a few labs) • Bio-Rad Multispot: a rapid flow-through assay that differentiates HIV-1 and HIV-2. • GenProbe HIV RNA • Not FDA-approved • HIV viral load assays used for management

20. Western Blot 1 2 3 4 • HIV culturelysate gel → membrane • Subjective; CDC interpretive criteria require reactivity to 2/3 of the gp120/160, gp41, and p24 antigens. • Indeterminate results vary by population tested • Evolving reactions, HIV-2 infection, other viral infections • 6% of Western blots performed at VA CT since 2007 • Insensitive in window period relative to current screening tests • Not automated, labor-intensive. gp 160 gp 120 gp 41 p 24

21. Bio-RadMultispot Control spot HIV-2 Spot HIV-1 Spots • Discriminatory test for HIV-1 and HIV-2 • Rapid; simple, though not automated • Unclear how much specificity it adds apart from detection of (currently very rare) HIV-2 infection.

22. Gen-Probe HIV RNA • Qualitative test for detection of HIV RNA. • TMA amplification, chemiluminescent detection • Not used for viral load testing, so in a routine lab would only be used for HIV confirm. Uneconomical for most labs. • Sensitive down to 33 IU/ml • Positive median 12d sooner than HIV-1 Ab and 6d sooner than HIV-1 Ag on seroconversion panels. • Concern: do automated serology instruments have molecular carryover problems? • Unknown. Separate sample needed for confirmation?

23. Other HIV Viral Load Tests • Not FDA-approved for diagnostic use. • Extensive validation would be required. • Reimbursement? • Optimized for very-low-end sensitivity; not well-studied for confirmatory use. • Borderline (e.g. ‘detected <48 copies/ml’) results very frequent in HIV-infected patients on HAART; unclear how many would occur in screening environment, and what they’d mean.

24. Relationship of Confirmatory Methods to Window Period From Branson B: J Acquir Immune DeficSyndr 2010;55:S102–S105; highlights added

25. Proposed Algorithms • Sources • CDC / APHL • CLSI • Types • Lab-Based vsRapid • Based on Different Screening Tests (e.g. 4th gen vs others) • Population/Patient-Based (Risk level, acute HIV) • Generate different levels of diagnostic certainty • Require various resources

26. Proposed Algorithm – CDC/APHL • Primary test with 4th Gen Assay • Confirm (with Multispot) to differentiate HIV-1 and HIV-2 • Refer positives to care (with viral load) • Accept possible non-specificity, treating entire serological process as a screening test. • Low volume of RNA tests; few labs can maintain this. • Sensitive for acute HIV infection. From Branson B: J Acquir Immune DeficSyndr 2010;55:S102–S105

27. CLSI Lab-based Algorithms • From M-53-A: Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus Infection • Algorithm 1: Single Initial 4th-Gen Assay • Algorithm 2: Initial HIV-1/2 Antibody Assay with Additional HIV-1 tests • Algorithm 3: Sequential HIV Ab Assay for Presumptive Diagnosis • Algorithm 4, 5: 4=Oral-fluid confirmatory test; 5 begins with an HIV-1 Ag/Ab discriminatory test; no US-approved versions • Algorithm 6: Algorithm for Acute HIV Infection

28. CLSI 1 • Essentially equivalent to the CDC/APHL algorithm.

29. CLSI 2 • The standard/current algorithm, but allowing for use of NAT as a confirmatory test.

30. CLSI 3 • An even more radical version of the CDC/APHL algorithm; uses a second EIA/CIA as a confirmatory test instead of the Multispot. • Produces a presumptive positive result. • Why doesn’t Algorithm 1 do the same? • Analogous to suggested multiple-rapid-test algorithms.

31. CLSI 6 • Specifically aimed at acute HIV infection; college students, active drug-use communities. • Individual or pooled NAT used on seronegative samples. • Extremely expensive if pooled testing not used. • Ag/Ab combination assays detect ~85% of Ab-negative, NAT-positive specimens during acute infection. • Positive NAT should be repeated for confirmation if a low level of viral RNA (<5000 copies) is detected.

32. A Practical Example: HIV Testing in VA VISN 1 • VISN 1 includes all the New England facilities • 11 Major facilities with labs • West Haven and Newington (CT), Northampton, West Roxbury, Jamaica Plain, Bedford, Broxton (MA), Providence (RI), White River Junction (VT), Manchester (NH), Togus (ME) • Virology testing, including HIV serology, has been centralized to Virology Reference Lab in West Haven for many years.

33. Mandated HIV Screening • In early 2010, VA Central Office mandated routine HIV testing for all VA patients, reflecting the 2009 CDC recommendations. • Reinforced by automated clinicial reminders via the VA CPRS EMR system.

34. Impact 2010 • Nearly a 6-fold increase in HIV screening volume. • A lesser increase in screen-positive samples • As expected screening lower-risk persons • Hard to say if more cases being found. • Continued into early 2011

35. A Change In Testing Models • VISN 1 clinical laboratories do major instrument acquisitions as a network. • In 2010-11 the general and immunochemistry systems went to bid, and Abbot got the contract. • Facilities sending hundreds of HCV and HIV tests/week to West Haven said (roughly): ‘forget all the packing and shipping and tracking, we’re bringing this in-house’

36. Decentralized Testing • VISN moved from centralized testing for HIV on Ortho Eci (3rd generation) to dispersed testing on Abbot Architect (4th generation). • Western blot and HIV viral load testing still done at VA CT VRL. • How to confirm positive screens?

37. Options • Have everyone do Multispot locally (algorithm 1) • Major validation headache. Very small number of tests in each facility. • Implement a NAT confirmation assay • Very small volume for Genprobe; tremendous validation problem on another method. • Need for second specimen on hundreds of negatives for each positive, or validation of carryover from screening instruments. • Maintain near-status-quo (algorithm 2). • Send positives to WH for Western blot; if positive, call HIV positive, if negative request additional specimen for NAT. • Discuss!! VISN 1 certainly did.

38. VISN 1 Algorithm Impressive VRL Newsletter courtesy Dr. David Peaper

39. Result interpretations

40. Comments on Ab/Ag screen results • Still evaluating algorithm (essentially a variant of CLSI algorithm #2). • How often will HIV VL be ordered in a reasonable time-frame? • Use of S/CO ratio to optimize algorithms? • Compromise between sensitivity, over-calling positivity, and laboratory practicality.

41. Impact 2011

42. Current European Practice – After Years w/ 4th Gen Assays You’d think that with years of experience with 4th gen assays this would’ve been worked out. But no.

43. Lessons • To prevent HIV, you have to look for it and treat it. • You (and your ordering providers) must know the properties of your screening test. • When testing low-incidence populations, confirmatory testing is essential; be aware of the properties of your confirmatory tests as well.

44. Acknowledgements • Background: Four Horsemen of Apocalypse, by Viktor Vasnetsov. Painted in 1887. From left to right, they are Death/Plague on the pale horse, Famine on the black, War on the red, and a rider whose identity is unclear in the Revelation text on the white. • VA Algorithm and much collegial discussion provided by Dr. David Peaper. • The staff of the VA CT Virology Reference Laboratory.

46. Transmembrane Envglycoprotein (gp41) Capsid protein (p24) Surface Envglycoprotein (gp120) Nucleocapsid protein (p7) Integrase (p32) Matrix protein (p17) Viral single-strandedRNA genome Reverse transcriptase (p66/p51) Lipid membrane Protease (p11)

47. HIV in Oman • 1984-2011, 2,164 Omani HIV cases reported • 1,371 (63.4%) still alive at the end of 2011 • 72.2% males, 28.8 percent females • Routes of transmission • heterosexual 50.2% • homosexual or bisexual 14.1% • mother to child 5.5% • injecting drug users 4.2% • blood transfusion 3.3%. • July 2009 HIV screening offered for all pregnant women (near 100% implementation). • No VCT services; limited access to at-risk groups.