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.