A rapid assay capable of simultaneously measuring HIV viral load and phenotypic drug resistance could offer a simpler alternative to existing resistance testing methods, according to new research.
Researchers optimised a product-enhanced reverse transcriptase (PERT) assay that quantifies the activity of HIV reverse transcriptase (HIV-RT), an enzyme essential for viral replication. The approach demonstrated high analytical sensitivity while detecting resistance to several established and emerging antiretroviral drugs.
Drug resistance testing can help guide effective HIV treatment, but existing approaches present practical challenges. Genotypic testing requires interpretation of an expanding number of resistance-associated mutations, while conventional phenotypic testing relies on viral culture, making it comparatively slow and labour intensive.
The researchers therefore developed an enzyme-based phenotypic approach designed to provide more direct information about drug susceptibility.
PERT combines complementary DNA synthesis by HIV-RT with quantitative PCR amplification and detection. The optimised assay detected as few as 10 HIV-RT molecules in a 25 µL sample, corresponding to an estimated viral load of approximately 10 HIV RNA copies/mL.
To investigate its potential for phenotypic resistance testing, researchers initially evaluated lamivudine-5′-triphosphate (3TC-TP). The HIV M184V mutation is associated with high-level resistance to lamivudine, allowing the team to determine whether PERT could distinguish resistant enzyme activity.
The assay successfully differentiated mixtures containing a 10% fraction of M184V HIV-RT among 1,000 total HIV-RT molecules, corresponding to an estimated viral load of approximately 2,500 RNA copies/mL.
According to the researchers, this surpassed the minimum analytical sensitivity requirement of 20% low-abundance variant detection specified in the 2023 WHO Target Product Profile for HIV drug resistance testing.
The team also demonstrated the assay’s ability to detect resistance to other reverse transcriptase inhibitors, including tenofovir-diphosphate, doravirine, and islatravir-triphosphate.
A key potential advantage of PERT is its speed and compatibility with existing laboratory infrastructure. The assay takes approximately 2 hours and could potentially make use of RT-qPCR instruments already employed for HIV viral load testing.
This could help reduce some of the complexity associated with current resistance testing and potentially support more decentralised surveillance and treatment selection.
The findings demonstrate the analytical potential of the assay, but further clinical validation will be needed to establish its performance using diverse patient samples and determine whether it can meet the requirements for routine HIV drug resistance testing.
Reference
Mims DK et al. 0. An optimized product-enhanced reverse transcriptase assay for sensitive and quantitative detection of HIV viral load and phenotypic drug resistance. J Clin Microbiol. 2026;DOI: 10.1128/jcm.01838-25.
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