February 5, 2018 Kim Krisberg 0Comment

Public health workers have two main tools for HIV screening: a blood test that detects HIV a couple weeks after infection or a saliva test that detects the virus more than a month after infection. With a trick of chemistry, however, scientists at Stanford University have combined the best attributes of both — and the result could mean a serious boost for HIV prevention.

In a study published last month in the Proceedings of the National Academy of Sciences (PNAS), the scientists found that their new screening tool not only correctly identified HIV antibodies (an indicator of HIV infection) in oral fluid samples, it could do so at an earlier stage of infection than the oral HIV tests already on the market. In other words, the new test can detect HIV infections that the current oral test would otherwise miss. Carolyn Bertozzi, a co-author of the study and a professor of chemical and systems biology and radiology at Stanford, said the breakthrough could offer public health screening programs a new advantage in reaching at-risk populations.

“In terms of public health management (of HIV), the most cost-effective strategy is testing so we can immediately intervene with care and behavioral interventions,” Bertozzi told me. “The more people who get tested, the better job we can do at containing the threat of this virus.”

The tricky part, of course, is getting people tested early. From a public health perspective, a blood test is the “gold standard,” Bertozzi said — that testing method is highly sensitive and can pick up HIV antibodies as early as two weeks after a person has been exposed to the virus. That’s important because knowing one’s HIV status is associated with behavioral changes that reduce the risk of transmission, while early antiretroviral treatment is linked with a host of improved health outcomes for people living with HIV. Early detection is also key when trying to get ahead of an HIV outbreak.

But the blood test has its drawbacks: taking and storing blood samples can be difficult to do out in the field and on a large scale, drawing blood puts workers at risk of infection, and people just don’t like getting poked with needles. On the other hand, getting a mouth swab is easier and less painful than a needle, oral fluid doesn’t expose workers to HIV risks, and oral samples store better than blood. The drawback is that the current oral fluid test isn’t able to detect HIV antibodies — these are the proteins your body produces to fight off invaders such as bacteria and viruses — until at least 40 days after exposure.

“There are a lot of benefits in switching to the oral test, but the big disadvantage is that it’s not nearly as sensitive,” Bertozzi told me.

The screening problem came to Bertozzi’s attention about three years ago when she was at the University of California-Berkeley. It was at Berkeley that Bertozzi and colleagues connected with Mark Pandori, a professor at the University of California-San Francisco and director of the Alameda County Public Health Laboratory, who put the challenges of HIV testing into real-life perspective. In particular, Pandori — a co-author on the PNAS study — highlighted the difficulty of encouraging adolescents and young people to get screened for HIV. Young people make up about 22 percent of new HIV diagnoses, report low rates of HIV testing and don’t typically seek out HIV testing on their own. A solution is to bring HIV testing to young people and make it as easy as a quick swab of the inner cheek. In an ideal world, however, that pain-free oral HIV test would detect HIV as early as the blood test does.

At the time, Bertozzi and her colleagues were already immersed in research to improve the sensitivity of antibody testing. After connecting with Pandori, they decided to see if they could make a better HIV test, eventually bringing the research with them to Stanford.

“There was a need for a better oral fluid test,” said Bertozzi, also an investigator at the Howard Hughes Medical Institute. “And we can fulfill that public health need right now.”

The secret to the breakthrough was discovering a new way to detect antibodies. Currently, the oral HIV test doesn’t work during the early weeks of exposure because HIV antibodies just don’t show up in the same concentrations or accumulate as quickly in saliva as they do in a person’s blood. Researchers needed to find a way to detect small amounts of HIV antibodies. So, instead of trying to find the actual antibody, they devised a test based on the behavior of an HIV antibody.

To understand how the new test works, first you have to understand how the antibody behaves. Bertozzi told me to imagine an antibody like the letter “Y” — the top part of the “Y” represents the antibody’s arms, which latch on to the invading HIV virus. With that in mind, Bertozzi and colleagues created a synthetic molecule made of bits of HIV and one half of a strand of DNA. The synthetic molecules are then added to an oral fluid sample; if HIV antibodies are present in the sample, they’ll use their “arms” to grab ahold of the molecules, essentially bringing two halves of a DNA strand close together. Scientists then add an enzyme — known as a DNA ligase — that facilitates the joining of two DNA halves together to form a continuous strand.

The synthetic molecule acts like an adapter that converts the attacking antibody proteins into a complete DNA strand. Scientists can then use polymerase chain reaction or PCR testing — a common technology in clinical and public health labs — to detect the DNA strand. If a complete strand is detected, the test is positive for HIV.

According to the PNAS study, the scientists used their new testing method to analyze oral fluid samples from the Alameda County Public Health Lab’s HIV screening program, confirming previous HIV diagnoses with 100 percent accuracy. They also tested eight oral fluid samples that were previously ruled “indeterminate” and found six were positive for HIV. One of those additional oral fluid samples was confirmed as HIV-positive via a blood test. The new test, known as Antibody Detection by Agglutination-PCR (ADAP), was 1,000 to 10,000 times more sensitive than the standard oral fluid HIV testing technique. And most importantly, the new test could effectively detect HIV antibodies earlier than the oral fluid test now in use, at 30 days post exposure versus 40 days.

And that’s just the first generation of the new saliva test, Bertozzi noted. She and study co-authors Cheng-ting Tsai, Peter Robinson, Felipe de Jesus Cortez, Maria Elma, David Seftel, Narges Pourmandi and Mark Pandori write that “ADAP presents the unique potential to detect early HIV infection using easily acquired (oral fluid) samples on a standard qPCR machine. ADAP meets the pressing public health need to identify HIV and treat it as early as possible to both improve interruption of disease transmission and enhance patient outcomes.”

“Right now, it’s already better than the existing oral test,” Bertozzi told me. “If we can eventually get the same outcome with oral fluid that we can get with blood, that will be a big win.”

Bertozzi and her colleagues plan to continue improving and testing their new HIV assay, with an eventual goal of obtaining official approval from the U.S. Food and Drug Administration. In fact, she said the technology behind the test could be used for all kinds of public health problems. For example, her group now has funding to develop an oral fluid test to screen for population gaps in measles immunization coverage. The oral test, which would detect the antibodies produced by the measles vaccine, could be especially useful in global settings where immunization records aren’t easily accessible.

On the HIV front, she said the new test could be a significant boost for early detection and prevention efforts. But it’s still only one part of what’s needed to turn back the HIV epidemic.

“The goal of eradicating HIV will require a multipronged approach and I think we’re a small part of that,” she said. “Having a really good, low-cost, population-scale screening test is really important, but only if it’s part of much larger, parallel efforts to stop HIV.”

Bertozzi is looking for new public health partners interested in using and testing out the HIV assay in their own prevention efforts — those interested can reach out to her at bertozzi@stanford.edu or through the company she co-founded, Enable Biosciences. To request a full copy of the new study, visit PNAS.

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