How Does HIV Genetic Resistance Testing Work?

Even for people with optimal adherence to therapy, some degree of HIV drug resistance is expected to develop over time due to natural mutations of the virus. In other cases, resistance can develop rapidly when suboptimal adherence allows for resistant HIV populations to thrive, leading eventually to treatment failure.

When treatment failure does occur, alternative drug combinations must be selected in order to suppress this new population of resistant virus. Genetic resistance testing helps facilitate this by identifying the types of resistant mutations in a person’s "viral pool," while ascertaining how susceptible those viruses are to possible antiretroviral agents.

Two primary tools are used for genetic resistance testing in HIV: the HIV genotypic assay and the HIV phenotypic assay.

What Is a Genotype and a Phenotype?

By definition, a genotype is simply the genetic makeup of an organism, while a phenotype is the observable characteristics or traits of that organism.

Genotypic assays (or genotyping) function by identifying the inherited instructions within a cell’s genetic coding, or DNA. Phenotypic assays (or phenotyping) confirm the expression of those instructions under the influence of different environmental conditions.

While the association between genotype and phenotype is not absolute, genotyping can often be predictive of phenotype, particularly when changes in the genetic code confer to expected changes in traits or characteristics—as in the case of developing drug resistance.

Phenotyping, on the other hand, confirms the "here-and-now." It aims to assess an organism’s reaction to specific changes in the environmental pressure—such as when HIV is exposed to different medications and/or drug concentrations.

Explaining HIV Genotyping

HIV genotyping is generally the most common technology used for resistance testing. The goal of the assay is to detect specific genetic mutations in the gag-pol region of the virus' genome (or genetic code). This is the region where reverse transcriptase, protease and integrase enzymes—the targets of most antiretroviral drugs—are encoded on the DNA chain.

By first amplifying the HIV genome using polymerase chain reaction (PCR) technology, lab technicians can sequence (or "map") the genetics of the virus using various mutation detection technologies.

Theses mutations (or accumulation of mutations) are interpreted by technicians who analyze the relationship between the identified mutations and the virus' expected susceptibility to different antiretroviral drugs. Online databases can assist by comparing the test sequence to that of a prototype "wild-type" virus (i.e., HIV that contains no resistant mutations).

The interpretation of these tests is used to determine drug susceptibility, with the greater number of key mutations conferring to higher levels of drug resistance.

Explaining HIV Phenoytyping

HIV phenotyping assesses the growth of person's HIV in the presence of a drug, then compares that to growth of a control, wild-type virus in the same drug.

As with genotypic assays, phenotypic tests amplify the gag-pol region of the HIV genome. This section of the genetic code is then "grafted" onto a wild-type clone using recombinant DNA technology. The resulting recombinant virus is used to infect mammalian cells in vitro (in the lab).

The viral sample is then exposed to increasing concentrations of different antiretroviral drugs until 50% and 90% viral suppression is achieved. The concentrations are then compared to results from the control, wild-type sample.

The relative "fold" changes provide the value range by which drug susceptibility is determined. A four-fold change simply means that four times the amount of drug was needed to achieve viral suppression compared to that of the wild type. The greater the fold value, the less susceptible the virus is to a specific medication.

These values are then placed within lower-clinical and upper-clinical ranges, with upper values conferring to higher levels of drug resistance.

When Is a Genetic Resistance Test Performed?

In the U.S., genetic resistance testing is traditionally performed on treatment naïve patients to determine if they have any "acquired" drug resistance. Studies in the U.S. suggest that between 6% and 16% of transmitted virus will be resistant to at least one antiretroviral drug, while nearly 5% will be resistant to more than one class of drug.

Genetic resistance testing is also used when drug resistance is suspected in individuals on therapy. Testing is performed while the patient either taking the failing regimen or within four weeks of treatment discontinuation if the viral load is greater than 500 copies/mL. Genotypic testing is generally preferred in these instances as they cost less, have a faster turnaround time, and offer greater sensitivity for detecting mixtures of wild-type and resistant virus.

A combination of phenotypic and genotypic testing is generally preferred for persons with complex, multi-drug resistance, particularly for those exposed to protease inhibitors.