An HIV resistance test which can detect one drug resistant virus in a sample of 1,000 virions has been developed by scientists at Duke University, North Carolina. The test may be able to identify drug resistance that has been `archived` and which does not show up on standard resistance tests, allowing doctors to refine the choice of regimen for treatment-experienced patients. It could also allow the detection of drug resistance that was acquired at the time of HIV transmission but which had faded away to a low level by the time that treatment was begun.
The study, published in the journal Nature Methods, shows how the research group identified a short genetic fragment from HIV’s pol gene that contains the sites of all the major drug resistance mutations in the reverse transcriptase and protease genes. They used this fragment as the basis for their investigations.
After processing the blood samples and isolating the genetic material in each of them, the researchers added tiny fluorescent tags designed to stick to HIV genes in particular ways. Tags designed to stick to mutated gene locations known to produce drug resistance were labeled to appear green, while tags designed to stick to the same gene locations but where the genes had not mutated were labeled to appear red.
The researchers used a computer programme to count the number of molecules with green or red fluorescent tags in each sample. The test proved sensitive enough to detect a single mutated virus out of 10,000 nonmutated viruses in the patient samples, Gao said.
"This level of sensitivity makes the assay about 1,000 times more sensitive than the most widely used assays on the market for detecting drug-resistant HIV viruses" said Dr Feng Gao, one of the research team. "Thus, the assay may permit more accurate prediction of treatment outcomes."
The assay appeared to produce comparable results across different viral subtypes and recombinant forms of HIV, and was sensitive down to the level of six viral genomes, which means that the assay can be used on very small samples of blood.
Fagping Cai et al. Detection of minor drugresistant populations by parallel allele-specific sequencing. Nature Methods (advance online publication, January 7 2007).