HIV Testing in 2013 – New Tests, New Questions, Few Answers
Sheldon Campbell M.D., Ph.D.
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
HIV Infections Continue to OccurCDC Data
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 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:– Jordan– Iran– Morocco– UAE– Afghanistan
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.
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.
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
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."
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.
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.
ART prevents HIV
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
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.
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
Timing of Diagnostic Events in HIV Infection
From Branson B: J Acquir Immune Defic Syndr 2010;55:S102–S105
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
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
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.
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
Western Blot• HIV culture lysate 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.
Bio-Rad Multispot
• 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.
Control spot
HIV-2 Spot
HIV-1 Spots
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?
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.
Relationship of Confirmatory Methods to Window Period
From Branson B: J Acquir Immune Defic Syndr 2010;55:S102–S105; highlights added
Proposed Algorithms
• Sources– CDC / APHL– CLSI
• Types– Lab-Based vs Rapid– 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
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 Defic Syndr 2010;55:S102–S105
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
CLSI 1
• Essentially equivalent to the CDC/APHL algorithm.
CLSI 2
• The standard/current algorithm, but allowing for use of NAT as a confirmatory test.
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.
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.
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.
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.
Impact 2010
Jan FebMarAprMayJun Jul AugSep OctNovDec0
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# CIA Positive # WB PositiveHIV CIA Volume
• 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
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’
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?
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.
VISN 1 Algorithm
Impressive VRL Newsletter courtesy Dr. David Peaper
Result interpretations
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.
Impact 2011
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec0
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HIV WB WB Pos HIV CIA Tests
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.
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.
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.
Transmembrane Envglycoprotein (gp41)
Surface Envglycoprotein (gp120)
Matrix protein (p17)
Lipid membrane
Capsid protein (p24)
Protease (p11)
Viral single-strandedRNA genome
Nucleocapsid protein (p7)
Reverse transcriptase (p66/p51)
Integrase (p32)
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.