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 HIV Epidemiology in the US and the Islamic World Diagnosing HIV: Traditional and Current Test Formats Diagnosing HIV: Traditional and Current Test Formats Managing Evolving Tests: Models and Algorithms for HIV Testing Managing Evolving Tests: Models and Algorithms for HIV Testing A Practical Example; VA New England Healthcare 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 Low Prevalence Overall UNAIDS 2011 data indicates that the Middle East / North African region is one of the two with the fastest-growing AIDS epidemic. UNAIDS 2011 data indicates that the Middle East / North African region is one of the two with the fastest-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: 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 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 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. Data are likely very incomplete.

6. HIV in Iran In the ‘concentrated’ phase; with high prevalence in vulnerable populations. 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 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. 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 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 Complex modes of spread

8. HIV in the UAE 1980s till the end of 2011 cumulative total of 726 HIV still-alive cases 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 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." "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: High risk due to: – 30y of armed conflicts. – Displaced populations. – Poppy cultivation and injection drug use. – Unsafe blood supply and injection practices. Challenges Challenges – Security and economic problems. – Lack of infrastructure. – High stigma Prevalence data scarce to non- existent. Prevalence data scarce to non- existent. Likely high levels in IDU and other high-risk populations. Likely high levels in IDU and other high-risk populations. Struggling to develop effective responses. 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. 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. In general, knowledge of HIV transmission and prevention is limited. The potential for continued and increased spread is present. The potential for continued and increased spread is present. Diagnostic capacity is essential to limit new infections. Diagnostic capacity is essential to limit new infections. That would be us. That would be us.

11. ART prevents HIV

12. Treatment=Prevention Effect of early vs. delayed treatment on transmission of HIV to partners 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. 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. 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. 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: As with all laboratory tests, false-positive and false-negative tests occur. The usual pre-analytical causes, plus: False-negatives False-negatives – Hypogammaglobulinemia, immunosuppression, – ‘Window period’; varies with assay – Unusual viral types HIV-2 HIV-2 Unusual types of HIV-1 Unusual types of HIV-1 – Improved Technology False-positives False-positives – Cross-reacting antibodies Pregnancy, multiple transfusions, hypergammaglobulinemia, hemodialysis, autoantibodies associated with autoimmune disease, recent vaccinations and viral infections 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 Defic Syndr 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, 9 th Edition

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

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

19. HIV Confirmatory Tests FDA-approved 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 Not FDA-approved – HIV viral load assays used for management

20. Western Blot HIV culture lysate gel → membrane HIV culture lysate gel → membrane Subjective; CDC interpretive criteria require reactivity to 2/3 of the gp120/160, gp41, and p24 antigens. Subjective; CDC interpretive criteria require reactivity to 2/3 of the gp120/160, gp41, and p24 antigens. Indeterminate results vary by population tested 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 Insensitive in window period relative to current screening tests Not automated, labor-intensive. Not automated, labor-intensive.

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

22. Gen-Probe HIV RNA Qualitative test for detection of HIV RNA. Qualitative test for detection of HIV RNA. TMA amplification, chemiluminescent detection 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. 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? 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. Not FDA-approved for diagnostic use. – Extensive validation would be required. – Reimbursement? Optimized for very-low-end sensitivity; not well-studied for confirmatory use. 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 Defic Syndr 2010;55:S102–S105; highlights added

25. Proposed Algorithms Sources Sources – CDC / APHL – CLSI Types Types – Lab-Based vs Rapid – Based on Different Screening Tests (e.g. 4 th 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 4 th Gen Assay Primary test with 4 th Gen Assay Confirm (with Multispot) to differentiate HIV-1 and HIV-2 Confirm (with Multispot) to differentiate HIV-1 and HIV-2 Refer positives to care (with viral load) 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. Low volume of RNA tests; few labs can maintain this. Sensitive for acute HIV infection. Sensitive for acute HIV infection. From Branson B: J Acquir Immune Defic Syndr 2010;55:S102–S105

27. CLSI Lab-based Algorithms From M-53-A: Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus Infection From M-53-A: Criteria for Laboratory Testing and Diagnosis of Human Immunodeficiency Virus Infection Algorithm 1: Single Initial 4 th -Gen Assay Algorithm 1: Single Initial 4 th -Gen Assay Algorithm 2: Initial HIV-1/2 Antibody Assay with Additional HIV-1 tests Algorithm 2: Initial HIV-1/2 Antibody Assay with Additional HIV-1 tests Algorithm 3: Sequential HIV Ab Assay for Presumptive Diagnosis 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 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 Algorithm 6: Algorithm for Acute HIV Infection

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

29. CLSI 2 The standard/current algorithm, but allowing for use of NAT as a confirmatory test. 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. 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. Produces a presumptive positive result. – Why doesn’t Algorithm 1 do the same? Analogous to suggested multiple- rapid-test algorithms. Analogous to suggested multiple- rapid-test algorithms.

31. CLSI 6 Specifically aimed at acute HIV infection; college students, active drug-use communities. Specifically aimed at acute HIV infection; college students, active drug-use communities. Individual or pooled NAT used on seronegative samples. Individual or pooled NAT used on seronegative samples. Extremely expensive if pooled testing not used. 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 VISN 1 includes all the New England facilities 11 Major facilities with labs 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. 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. 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. 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. 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): 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 (3 rd generation) to dispersed testing on Abbot Architect (4 th generation). VISN moved from centralized testing for HIV on Ortho Eci (3 rd generation) to dispersed testing on Abbot Architect (4 th generation). Western blot and HIV viral load testing still done at VA CT VRL. Western blot and HIV viral load testing still done at VA CT VRL. How to confirm positive screens? How to confirm positive screens?

37. Options Have everyone do Multispot locally (algorithm 1) Have everyone do Multispot locally (algorithm 1) – Major validation headache. Very small number of tests in each facility. Implement a NAT confirmation assay 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). 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. 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). 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. Compromise between sensitivity, over-calling positivity, and laboratory practicality.

41. Impact 2011

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

43. Lessons To prevent HIV, you have to look for it and treat it. 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. 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. 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. 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. VA Algorithm and much collegial discussion provided by Dr. David Peaper. The staff of the VA CT Virology Reference Laboratory. The staff of the VA CT Virology Reference Laboratory.

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

47. HIV in Oman 1984-2011, 2,164 Omani HIV cases reported 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 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). July 2009 HIV screening offered for all pregnant women (near 100% implementation). No VCT services; limited access to at-risk groups. No VCT services; limited access to at-risk groups.