HIV Resistance Assays: Part 1 of a primer on resistance testing written by Johnathan Schapiro, MD, well known in resistance and clinical research of HIV. Dr Schapiro is writing this specifically for the NATAP web site.
Part 1: Types of Resistance Assays- Jonathan M Schapiro, MD
Great strides have been made in our understanding of how HIV becomes resistant to antiretroviral agents. Together with our understanding of resistance, technologies to study this phenomenon have been developed and improved.
Two types of resistance assays are currently used to determine HIV sensitivity or resistance to individual antiretroviral drugs. Genotypic assays determine the changes in the genetic code of the viral enzymes targeted by the drugs. By determining the changes, or mutations, in the genetic code of the reverse transcriptase gene, we can learn about possible resistance to the drugs targeting this enzyme, NRTI's and NNRTI's. Likewise, mutations in the protease gene (and possibly its cleavage sites) effect the protease inhibitors. Changes in the genetic code result in the replacement of a "standard" or "wild type" amino acid in the protease chain of the enzyme with a "mutant" amino acid, thus changing the protein and reducing the effect of the drug designed against it. When multiple amino acids change, the effect on the enzyme may be greater and resistance higher. Genotypic resistance assays detect these changes in the genetic code and report the specific position and name of the amino acid change. For example, the protease enzyme has 99 amino acids in each of its two identical protean chains. If the amino acid at position 90 (the 90's amino acid in the chain) changes from the normal Leucine (L) to a Methionine (M), it would be reported by the assay as protease mutation L90M. This is in fact a common mutation seen with protease inhibitor treatment.
Drugs are designed to inhibit specific viral enzymes. This design is based on our precise knowledge of the protein and the drug interacts with specific amino acids thus inhibiting the enzyme's action. If these amino acids change, or are even effected by changes in amino acids near by, the drugs ability to inhibit the enzyme may be reduced or eliminated entirely. Since not all drugs of a class interact with all the same enzymatic amino acids (although some are often shared), mutations in specific amino acids may effect the action of different members of the class to a different degree.
Individual mutations or clusters of mutations are often associated with resistance to a specific drug. In many cases a mutation may have an effect on more than one drug in that class, although not always to the same degree. An understanding of the precise degree to which a mutation confers resistance to each of the drugs of the class is crucial for understanding of the significance of the genotypic assay results. This is further complicated by the fact that often multiple mutations are present. These complexities often make the understanding of genotypic assay results a challenging task. This may be addressed by consulting an expert in the field, or by systems that provide an interpretation of the results. In the past, tables and lists have assisted clinicians and patients in correlating specific mutations to drugs. Currently, computer systems are performing this function either directly as part of the assay report form, or as a freestanding system.
Phenotypic resistance assays test the degree to which the virus from a patient is inhibited by a specific drug. Drug is added to a culture of replicating virus. If the virus is still sensitive to the drug, replication will be inhibited. If resistance to the drug has developed, the virus will continue replicating. As the virus grows more resistant to the drug, greater concentrations of the drug will be required to inhibit replication. The degree of phenotypic resistance is reported as the fold change in the concentration of drug required to inhibit virus. The assay can be repeated for all available drugs, producing a result that will reflect the specific virus' resistance to each of the drugs.
The dynamic range (difference seen between sensitive and resistant virus) differs greatly between drugs, as does our experience performing the assay. The report of a phenotypic assay is quite straightforward. Often a threshold of a 4-fold change is used to segregate between sensitive and resistant virus.
Genotypic assays are less expensive, easier to perform and have a shorter turnaround time than phenotypic assays. On the other hand, the interpretation of phenotypic assays is more direct and easier to understand. In future issues we will discuss some of the tradeoffs between the assays and their use in clinical practice.