An investigational test for HIV drug resistance has the potential to provide results on phenotypic susceptibility within a few hours at a low cost, according to research presented at the annual meeting of AIDS 2026 in Rio de Janeiro, Brazil.

“We set out to answer if an enzyme activity assay could achieve necessary sensitivity for simultaneous HIV viral load and drug resistance testing,” Dorothy Mims, MS, bioengineering PhD student and STD AIDS Research Fellow at the University of Washington in Seattle, told attendees. “With our assay, we showed detection down to 10 HIV RT [reverse transcriptase] molecules, corresponding to a constant viral load of approximately 25 copies of RNA/mL, and then also phenotypic detection of resistance to all classes of RT inhibitors, and we believe that this rapid and inexpensive approach could significantly increase access to drug-resistant screening.”

Currently available drug resistance testing options are complex, expensive, and time-consuming, Mims said, which limits their routine use, particularly in point-of-care settings. Genotypic testing looks at HIV genetic sequences to find mutations associated with resistance but takes multiple days or weeks to run and cannot directly link a particular mutation with loss of susceptibility to specific drugs, she explained. Phenotypic tests, on the other hand, can assess susceptibility to specific drugs but require “weeks of user-intensive cell culture to assess HIV replication in the presence of different inhibitors,” she said.

Hence, her team turned to enzyme activity assays because they do not require cell culture and take only a few hours. The product-enhanced RT (PERT) assay measures RT activity based on its ability to produce DNA.

“With a drug-susceptible HIV RT enzyme, minimal DNA will be produced in the presence of an RT inhibitor, but then, with a drug-resistant RT enzyme, ample DNA will be produced despite the presence of this inhibitor,” Mims said.

The PERT is a modified version of the typical RT quantitative polymerase chain reaction that’s often used for RNA-based HIV viral load testing, she said, except they switch out the HIV RNA for a non-HIV RNA and switch out the RT enzyme for HIV RT.

“At baseline, this gives us a measurement of HIV viral load based on HIV RT quantity, but then we can also add the RT inhibitor into the cDNA [complementary DNA] synthesis reaction, and alongside this, get a measurement of susceptibility to various RT inhibitors,” she said.

While these RT enzyme activity assays have existed for over three decades, currently commercially available ones are only used for screening of retroviral contamination in viral vector systems. The researchers therefore aimed to develop an optimized, updated version and assess its use for HIV viral load and drug resistance testing.

She provided several examples of the assay’s capability for some specific mutations.

“Using the M184V mutation, we can distinguish between resistant and susceptible HIV RT using lamivudine (3TC) and emtricitabine triphosphate (FTC-TP),” she said. “Using K65R, we can distinguish resistance with tenofovir diphosphate, and then using relevant resistance mutations for NNRTIs [non-nucleoside RT translocation inhibitor], we can also distinguish between resistant and susceptible with doravirine and rilpivirine. And finally, with the newly approved first-in-class NRTTI [nucleoside RT translocation inhibitor] islatravir triphosphate, we can also distinguish between resistant and susceptible HIV RT using the M184V mutation, which confers low-level resistance to islatravir.” She also showed results for the Y188L and K101P mutations.

The World Health Organization recommends that products be able to detect up to 20% resistance at viral loads of 1,000-5,000 copies RNA/mL. All of these tests of the PERT assay detected resistance to RT inhibitors at a viral load of approximately 2500 copies, and they were able to show distinction down to 10% resistant enzyme fraction. The cost of reagents per test is approximately $4, Mims said, and the assay runtime is approximately 2 hours.

“The advantage of this resistance assay is that it doesn’t use expensive sequencing technology but, instead, uses enzymatic assays to find resistance to reverse transcriptase inhibitors, which allows for real-world testing,” Monica Gandhi, MD, MPH, professor of medicine in HIV at the University of California, San Francisco, told Medscape Medical News.

The 2-hour turnaround is nearly point of care, she noted, and the low cost is another plus, though she added that more testing than traditional sequencing platforms will be necessary before this assay would gain approval.

“However, a major limitation of this assay should be addressed now, which is that this novel assay will provide data on reduced susceptibility to reverse transcriptase inhibitors only,” Gandhi said.

“The HIV community is more worried about integrase inhibitor resistance, as resistance to the first-line integrase inhibitor — dolutegravir — is rising worldwide. Dolutegravir can usually be used with tenofovir and lamivudine (TLD) even with reverse transcriptase inhibitor resistance, so the true utility of this assay to clinicians remains to be seen.”

The research was funded by the Washington Entrepreneurial Research Evaluation and Commercialization Hub (WE-REACH) and the Arnold and Mabel Beckman Foundation. Mims had no disclosures. Senior author Ayokunle Olánrewájú has ownership in Ireti Biosciences, Inc., and he and a coauthor are inventors on a patent filed based on this assay. Gandhi had no disclosures. 

Tara Haelle has covered science and medicine for nearly two decades and is the author of Vaccination Investigation and The Informed Parent: A Science-Based Resource for Your Child’s First Four Years. She is based in Dallas.