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New HIV Immunoassay With Boosted Sensitivity May Support Earlier Screening Using Oral Fluid

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NEW YORK (360Dx) – A new immunoassay being developed by researchers at Stanford University and spinout company Enable Biosciences offers an improved ability to detect a person's immune response to HIV using oral fluid samples, potentially making it amenable for large-scale population screening.

According to a study published in PNAS this week led by Carolyn Bertozzi, a chemist at Stanford, the test is 1,000 to 10,000 times more sensitive than the HIV enzyme-linked immunoassays currently used in clinics.

The test was multiplexed to simultaneously detect antibodies against the three standard antigens used for clinical HIV antibody testing, and was shown on a set of clinical samples to have 100 percent sensitivity and specificity for detecting antibodies to HIV in oral fluid samples. It was also able to reclassify a small set of samples that had shown indeterminate results using a commercially available oral fluid test that involved Avioq HIV-1 Microelisa as a first-tier assay with positive samples confirmed using a Western blot from OraSure Technologies. 

The study authors also noted that the recent discontinuation of the US Food and Drug Administration-approved Western blot test previously used to confirm positive immunoassay results means there may be a need in the HIV screening community for a new confirmatory test.

In general, HIV testing can use blood samples or oral fluid, with the former requiring a blood draw or finger stick and the latter involving a swab of a patient's gums.

Blood-based tests can detect the virus itself or they can detect a patient's antibodies to the virus. Saliva, meanwhile, contains enzymes and inhibitors that inactivate HIV, but antibodies against the virus are present in oral fluids. In both types of samples, concentration of antibodies varies with time after infection.   

In the seven- to 10-day post-infection window, viral loads are incredibly high, and the virus is extremely transmissible. But, the host immune response has yet to be fully activated, so antibody-based blood tests are typically negative.

Tests that use PCR to detect viral RNA in patient blood samples are very sensitive and can give a positive signal within days of infection, but they tend to be expensive and the requirement for a blood draw makes them not so useful as screening tests. 

However, some researchers are attempting to bring down the cost so PCR might be used for screening for acute infections, particularly in high-risk populations. For example, a campaign in Vancouver used pooled samples and PCR to screen a population of men who were at higher risk, while research conducted in a community in San Diego with a high incidence of HIV showed that offering screening using the Hologic Procleix Ultrio nucleic acid blood donation screening test reduced transmission.

Immunoassays, on the other hand, detect the patient's immune response to the virus, which becomes elevated in blood around 15 days after infection but is not fully elevated in oral fluid until around 40 days. 

The Stanford oral fluid test, however, has detected positive cases as early as 30 days, Bertozzi said in an interview.

Her team is hoping to increase the sensitivity still further, potentially making the test competitive with blood-based immunoassays that can detect positivity by around 15 days.

The test boosts sensitivity by using antibody detection by agglutination-PCR (ADAP), as previously described. This method essentially uses the patient's antibodies to force antigen-conjugated DNA strands close enough together for ligation and quantitative PCR amplification.  

As it stands now, the assay can be run by any lab that has a thermal cycler, Bertozzi said. It is a lab-based test, not a rapid point-of-care assay, so would take about two hours from sample collection to results reporting, she said.

However, Bertozzi emphasized the value of screening using oral fluid as compared to blood. "If you're trying to do testing on the public health scale, it's hard to do that with blood draw or finger prick – it's much easier to do with oral fluid, and it's also safer for the healthcare workers," she said, adding that oral fluid is considered to be not infectious.

The ADAP test "can achieve sensitivity approaching a blood test, but with oral fluid," she said.

Bertozzi is a co-founder of Enable Biosciences, a spinout set to commercialize the ADAP method. The company has not yet determined for certain whether it will take its tests through the US Food and Drug Administration regulatory process or establish them as lab-developed tests, but it has mapped out a plan for completing the FDA submission process within two years if further clinical data is supportive of that move.

The PNAS study was also done in collaboration with the Alameda County Public Health Department.

Discontinued test may open the market

Mark Pandori, the public health lab director and co-author on the PNAS study, noted that there is a large gap in the use of oral fluid testing because the FDA-cleared OraSure HIV-1 Western Blot Kit that had been used for confirmatory testing has gone off the market.

A representative at OraSure confirmed that the kit was discontinued in 2017 and is no longer being manufactured by the firm but declined to say why or whether it will be manufactured by another firm. OraSure also manufactures the OraQuick Rapid HIV-1/2 Antibody Test, an FDA-approved and CLIA-waived test for HIV antibodies in oral fluid, as well as an assay approved for testing at home.

The Western blot test had been used for confirmation of immunoassay blood tests, but guidance from the US Centers for Disease Control and Prevention and the Association of Public Health Laboratories published in June 2014 said the test should no longer be used because of the inability to detect acute infection and the potential to misclassify HIV-2 infection as an HIV-1 infection.

Instead, the guidance suggested confirming results of an HIV-1/2 antigen and antibody combination test by using additional immunoassays or nucleic acid amplification tests.

The discontinuation of the Western blot test "is a bad situation because there are still a lot of testing populations where phlebotomy is impossible or very difficult, and those populations need HIV testing," Pandori said.

He noted that he is aware of some laboratories validating the BioRad Geenius — an FDA-approved test for confirming blood HIV tests that are reactive — using a modified protocol for oral fluid test confirmation.

The Geenius HIV 1/2 Supplemental Assay is a single-use immunochromatographic assay for the confirmation and differentiation of individual antibodies to HIV types 1 and 2 in fingerstick whole blood, venous whole blood, serum, or plasma samples, according to the Bio-Rad website.

However, Pandori said, "The ADAP method is special, and potentially useful, in that like the Geenius or the Western blot, it can detect and distinguish antibodies to different parts of the HIV particle, [and] in that way, it theoretically could function as a sensitive Western blot."

In addition to the HIV oral fluid assay, Bertozzi and Enable Biosciences are also developing a test using the ADAP method for early detection of type 1 diabetes. Enable is also interested in partnering with other firms in the industry and has interacted with a few diagnostics companies already, Bertozzi said. "We're trying to get on the radar screen," she added.