with Mark W. Pandori, PhD  
Alameda County Public Health Laboratory,
Oakland, California
 

Medical Lab Management: When the Centers for Disease Control and Prevention (CDC) published a revised laboratory HIV testing algorithm in June 2014,¹ the Western blot test was not included for the first time since 1989. How is this recommendation changing laboratory practice?

Mark W. Pandori: The supplemental test recommended in the latest algorithm is an HIV-1/HIV-2 antibody differentiation immunoassay. Because these tests are less expensive than the Western blot, have faster turnaround times (TAT), and are easy to perform, they are changing dramatically the amount of time and money labs spend on HIV confirmatory testing. Confirming one specimen by Western blot can consume $120 to $150 worth of reagents, whereas the only FDA-approved HIV-1/2 antibody differentiation test on the market as of this writing costs about $20 to $40 per immunoassay. Another antibody differentiation immunoassay by the same manufacturer is expected to be available soon, having received FDA premarket application approval in October 2014.² The new test is intended for use as an additional, more specific test to confirm the presence of antibodies to HIV-1 and HIV-2 for specimens found to be repeatedly reactive by diagnostic screening procedures.³ 

In terms of TAT, Western blot tests require several hours to set up and run, so labs tend to batch them to run once per day. Conversely, the HIV-1/2 antibody differentiation test can be performed in approximately 20 minutes, making it possible for labs to confirm a positive result almost immediately or, at a minimum, on the same day the specimen is received.

MLM: Do the new guidelines mean other changes for labs?
Pandori: Yes. For first-line screening, the CDC algorithm recommends that labs transition to the most sensitive screening tests available, which are HIV-1/2 antigen/antibody combination immunoassays (see FIGURE 1).⁴ These fourth-generation diagnostic assays are highly sensitive when used to screen for HIV infection and are particularly adept at identifying recent HIV infection. This is because they detect not only HIV-1 and HIV-2 antibodies, but also the HIV-1 p24 antigen; HIV p24 is detectable before seroconversion. Studies show that detecting HIV during the acute phase (2 to 4 weeks after infection occurs and before the body produces detectable levels of antibodies) is vital not only because of the benefits to the patient of starting antiretroviral treatment earlier in the course of disease, but also because the acute phase is marked by extremely high viral load and contributes disproportionately to transmission.⁵ The traditional approach to HIV testing (ie, a third-generation immunoassay followed by the Western blot) could not detect acute infection or differentiate between HIV-1 and HIV-2. 



The downside is that fourth-generation assays are about two to five times more costly than earlier ELISA screening methods, so laboratory costs per test may rise. In addition, some of these assays require specialized equipment and trained technicians to conduct testing.⁶

The other change recommended by the CDC guidelines involves the addition of RNA testing in select circumstances. A reactive result on a fourth-generation immunoassay may indicate only that a person’s blood has either antibody or antigen from HIV infection. The test does not indicate whether it has detected antigen or antibody; it indicates only that it is positive for one or both. If a fourth-generation test is positive because antibody is present, the next step recommended by the CDC—an HIV-1/2 antibody differentiation test—can reveal whether the person has antibody to HIV-1 or HIV-2. If a person is positive on a fourth-generation test because of the presence of HIV antigen (or perhaps, IgM) due to recent or acute HIV infection, the lab may not be able to confirm a positive result with an HIV-1/2 antibody differentiation immunoassay. 

The CDC recommends resolving this situation (ie, specimens that are reactive on an initial fourth-generation antigen/antibody combination screening immunoassay and nonreactive or indeterminate on an HIV-1/2 antibody differentiation test) by running a test that detects HIV-1 RNA. However, only one RNA test is FDA-approved as a supplemental HIV screening test. Currently, none of the RNA tests used to determine viral load are approved for diagnostic purposes.

MLM: So, in essence, another layer of testing has been added where one did not exist before?
Pandori: Precisely, and that layer facilitates the detection and differentiation of recent or acute HIV infection. A sample that is discordant upon screening, but positive for RNA on an HIV-1 nucleic acid testing (NAT) suggests that an individual has an acute infection and has not completely seroconverted. 

Fourth-generation assays are negative across the board in the first (eclipse) phase of HIV infection. Then the normal progression within the blood of an HIV-infected individual is a positive result for RNA, then a positive result for RNA and antigen, followed by a positive result for RNA, antigen, and IgM, and, finally, a positive result for RNA, antigen, IgM, and IgG. A discordant result can occur because a sample is positive for antigen but not antibody, or positive for antigen and IgM without IgG. 

MLM: How will this extra layer of testing affect the bottom line? 
Pandori: Screening tests drive the cost of HIV lab testing, and costs will be higher with a fourth-generation screening assay, but reactive results represent a small minority of screening tests performed. The vast majority of HIV screening tests are nonreactive and require no further testing. So, although costs will be higher for discordant cases, the odds of finding true acute infection are quite low.

The CDC recommends that labs be prepared to deal with discordant cases, so labs now need to choose one of three options: 

• Procure an RNA test that is FDA-approved for diagnostic purposes and perform it onsite or in a core facility
• Send discordant specimens out to another laboratory for resolution with an RNA test
• Validate an RNA viral load test that is not FDA-approved for resolving discordant cases

Keep in mind that to appropriately challenge an RNA test, a validation should include at least 50 to 100 positive, negative, and discordant specimens (ie, positive for virus and negative for antibody), hopefully using specimens from patients with recent or acute HIV infection. Finding acute infection can require testing a high number of high-risk individuals. Success in this endeavor is likely to vary considerably by community. Our facility is transitioning to the algorithm and plans to validate an RNA viral load test to resolve discordance. 

MLM: Are there any other advantages or challenges associated with the new algorithm? 
Pandori: The algorithm is heavily dependent on a handful of products. It recommends a fourth-generation antigen-antibody screening test, yet only two lab-based immunoassays and one rapid test currently fit that description. Only one company makes the supplemental test, and only one company makes an FDA-approved RNA diagnostic test. Thus, at this point, the number of options is somewhat limited.

However, from a public health perspective, the algorithm has tremendous potential. HIV transmission and infection data indicate that recent or acute infections may be driving the AIDS epidemic.^5,7 By detecting acute infection, the new recommendations facilitate diagnosis of individuals when they are the most infectious.

MLM: What about rapid and point-of-care (POC) testing for HIV? 
Pandori: Many rapid tests are available, but they vary widely in sensitivity and specificity. Only one POC test is FDA-approved to screen both blood and oral fluid. Although oral fluid testing is popular among patients, oral fluid is the least sensitive means of screening for HIV.⁸ Rapid testing facilitates screening a greater number of individuals, but does not necessarily target those who are the most infectious.

The introduction of POC testing for HIV severely eroded lab-based testing, but the method appeals to patients, and greater acceptability means more testing can be accomplished. As a result, much HIV testing was pushed out of labs and into offices, clinics, and emergency rooms. 

At many clinics frequented by individuals at high risk for HIV, everyone receives an HIV POC antibody test. If the test is positive, HIV infection is conclusively identified. If the test is negative, the patient receives an RNA test—a validated RT-PCR viral load test—to detect HIV that may have been missed by the POC test. To reduce costs, many labs pool the negative antibody screening tests and test the pool with the RNA test. When a positive pool is identified, the lab then breaks down the pool and identifies the infected individual(s) for follow-up care. 

RNA tests performed on individual specimens can detect viral loads as low as 40 copies of HIV-1 RNA/mL, although sensitivity with pooling is lower; for example, a pool of ten might lower sensitivity to detection of 400 copies/mL.⁹ In contrast, for a sample to be positive for antigen on a fourth-generation screening assay, the viral load needs to approach 50,000 copies/mL.^10,11 Facilities located in communities with a large number of high-risk individuals should consider screening all patients with an RNA test to maximize identification of recent or acute infection. 

MLM: Why is RNA testing not recommended as first-line HIV screening for everyone?
Pandori: There is no question that screening everyone for HIV with an RNA test would impact public health significantly. RNA tests are routinely used in clinics and hospitals to test for chlamydia and gonorrhea, but not for HIV. Patients who are missed with antibody testing tend to have high viral loads, while those who are missed using RNA testing tend to have low viral load, and, therefore, less of an ability to transmit the infection. Clinics in cities, such as New York, San Francisco, and Seattle, that have a high concentration of high-risk individuals, are screening for HIV with RNA tests because the burden of missing patients with acute or recent HIV infection is much greater. In a long-term study we conducted in San Francisco, fourth-generation assays identified 80% of acute infections.¹⁰ That means 20% of recently infected individuals were missed, but would have been identified with an RNA test. On the other hand, first-line RNA screening of every presenting patient would be quite costly. This is why sample pooling often is used in communities where RNA screening is being performed. 

Certainly there is more that can be done, but the most recent CDC algorithm has shifted the medical establishment from being incapable of detecting recent and acute HIV infection to having the ability to identify the subgroup of people that potentially has the greatest influence on transmission moving forward.

Mark W. Pandori, PhD, is the director of the Alameda County Public Health Laboratory, Oakland, California, and an associate clinical professor in the Department of Laboratory Medicine at the University of California San Francisco School of Medicine. 

References

1. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014. Accessed April 20, 2015.
2. US Food and Drug Administration. October 24, 2014 Approval Letter - Geenius HIV 1/2 Supplemental Assay. http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/ucm420751.htm. Accessed April 28, 2015. 
3. US Food and Drug Administration. Geenius HIV 1/2 Supplemental Assay. http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/ucm420713.htm. Accessed May 12, 2015.
4. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Quick Reference Guide—Laboratory Testing for the Diagnosis of HIV Infection. Updated Recommendations. Published June 27, 2014. http://www.cdc.gov/hiv/pdf/guidelines_testing_recommendedLabTestingAlgorithm.pdf. Accessed April 20, 2015.
5. 5. Pilcher CD, Tien HC, Eron JJ, et al. Brief but efficient: acute HIV infection and the sexual transmission of HIV. J Infect Dis. 2004;189:1785-1792. 
6. Centers for Disease Control and Prevention. Advantages and disadvantages of FDA-approved HIV immunoassays used for screening by generation and platform. Updated 12/16/2014. http://www.cdc.gov/hiv/pdf/testing_AdvDisadvHIVtesting.pdf. Accessed April 28, 2015.
7. Gray RH, Wawer MJ, Brookmeyer R, et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet. 2001;357(9263):1149-1153.
8. Pilcher CD, Louie B, Facente S, et al. Performance of rapid point-of-care and laboratory tests for acute and established HIV infection in San Francisco. PLoS One. 2013 Dec 12;8(12):e80629. http://www.ncbi.nlm.nih.gov/pubmed/24349007. Accessed April 22, 2015.
9. Abbott Realtime HIV-1 Package Insert. http://www.fda.gov/downloads/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/UCM091193.pdf. Accessed May 13, 2015.
10. Pandori MW, Hackett J, Louis B, et al. Assessment of the ability of a fourth-generation immunoassay for human immunodeficiency virus (HIV) antibody and p24 antigen to detect both acute and recent HIV infections in a high-risk setting. J Clin Microbiol. 2009;47(8):2639-2642.
11. Brennan CA, Yamaguchi J, Vallari A, et al. Architect^® HIV Ag/Ab combo assay: Correlation of HIV-1 p24 antigen sensitivity and RNA viral load using genetically diverse virus isolates. J Clin Virol. 2013;57(2):169-172.