(AIDSmeds.com) - Researchers at Duke University Medical Center have developed a highly sensitive test to identify drug-resistant HIV strains in the blood, including "minority populations" that may not be detected using standard genotypic or phenotypic assays. Preliminary results from experiments involving the assay were recently reported online and are to published in the February issue of the journal Nature Methods.
Because HIV genes can mutate easily and rapidly, most people who are infected have many different forms of the virus in their bodies. Depending on the number of HIV drugs that have been taken in the past – along with the fact that people can be infected with HIV strains resistant to drugs that they haven't ever used – a number of drug-resistant variants may be present.
Available drug-resistance tests, such as genotypic assays, can analyze the dominant strain of HIV in a blood sample. For example, if someone has a detectable viral load while taking a three-drug combination, the dominant strain of HIV in his or her blood will likely contain mutations conferring resistance to one or more of the drugs being used. However, he or she may also have minority populations of HIV in the blood as well, harboring mutations to other HIV drugs used in the past. These minority strains, if they make up less than 10% to 20% of the HIV population in a collected sample of blood, can be difficult to detect using standard tests.
The new test developed by the Duke team is a parallel allele-specific sequencing (PASS) assay capable of simultaneously analyzing a large number of HIV viral genomes and detecting minor viral populations with drug-resistance mutations.
To assess the test, Duke associated professor of medicine Feng Gao, MD, and his colleagues analyzed blood samples from three different groups of HIV patients: those who had never received antiretroviral treatment, those who had received treatment but were not currently being treated, and those who were receiving treatment but the treatment was not completely successful.
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 sophisticated computer program 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.
"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" Dr. Gao says. "Thus, the assay may permit more accurate prediction of treatment outcomes."
The test also can detect when a virus molecule has more than one mutation, a capability that no commercially available test has achieved, Dr. Gao adds. This capability may prove critical for detecting HIV strains that have become resistant to multiple drugs, a condition that occurs often as many patients are treated with many drugs at the same time.
Dr. Gao hopes that the test may also find broader medical application. He says it has the potential to detect mutations that confer drug resistance in infectious agents that cause other diseases besides HIV, such as hepatitis B, hepatitis C and tuberculosis.
Source:
Cai F, Chen H, Hicks CB, et al. Detection of minor drug-resistant populations by parallel allele-specific sequencing. Nature Meth. Published online: January 7, 2007.
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