Genotyping the Human Immunodificiency Virus

Genotyping the Human Immunodificiency Virus

Lee R. Anisman, MD

In the summer of 1996, the world was excited because news from the International AIDS Conference said that triple combination therapy including a protease inhibitor would significantly decrease morbidity and mortality in people living with HIV disease. Initially this seemed to be the case. People on three and four drug combinations saw their viral loads decrease significantly. For about a year now, those of us treating large numbers of patients living with HIV have seen breakthrough resistance occur at an alarming rate. This has resulted in people failing combination therapy and physicians scrambling to find new salvage therapies for their patients. Furthermore, we know HIV is more adaptable to drug therapies than originally thought. To date we can identify 144 mutations within the virus. While some of these mutations are relatively benign (no amino acid changes are seen), some are conservative (amino acid changes are seen, but functional resistance does not increase), many mutations do confer resistance and cross-resistance.

HIV replicates at an enormous rate. With 10 billion virons produced and cleared daily, for someone living with HIV disease, this means the entire virus population is replaced every two to three days. When HIV replicates, it is a sloppy replication process. Many errors in replication occur resulting in mutations, which allow the virus to become resistant to anti-viral drugs. Given the high rate of this viral replication, it does not take long for drug therapy-resistant mutation to develop. HIV has the ability to mutate in all available drugs and develop cross-resistance to other agents. Since virtually all patients are on combination therapy, triple drug treatments do not guarantee such resistance mutations will not occur. In other words, triple drug therapy is not a fail-safe option and does not guarantee the virus will be contained and unable to develop escape mutations.

It is a well-known fact; the dynamics of HIV replication have profound implications for genetic diversity in those living with the disease. At the time of initial infection, virus isolates show little genetic heterogeneity, but over time the population of viruses become increasingly diverse. The rapid replication of kinetics and high mutation rate of HIV, drive this diversity in response to selective pressure from the patient’s immune system and, especially from the drugs taken to control the virus. Therefore, triple therapy without genotyping is a primitive strategy. In the course of the next several years, genotyping with sequential viral loads will be as commonly used as viral load are today, and will become the standard of care for HIV patients.

I believe the ability to accurately detect resistance mutations in the virus will have a major impact on morbidity and mortality in HIV patients over the years ahead. Physicians who are less experienced with the dynamics of HIV are skeptical about genotyping the virus. Some of these same skeptics were not interested in viral loads three years ago. They felt CD4 counts and T4/T8 rations were significant markers of patient progression. We all know viral load is now universally accepted. Genotyping must be done in conjunction with viral load testing and CD4 counts. This combination will result in proper drug management and an enhanced quality of life for the person living with HIV disease.

A major problem with some laboratories which genotype HIV is, the results are variable with unreliable results. The most commonly used techniques to identify mutations in a virus are probe-based. This technique uses dye-labeled probes that bind to and identify specific DNA sequences associated with certain diseases or conditions. Probe-based techniques are fast, relatively inexpensive, and easy to perform. However, probe-based technology is not adequate to test for mutations in HIV. This type of sequencing should only be used when disease mutations are known and constant. Mutations associated with HIV have a high degree of polymorphism, which is variability in the location , and type of mutation. Therefore, probe-based sequencing will miss some mutations.

In contrast, DNA-sequencing which offers a complete map of the sample DNA is much more accurate. Until recently, DNA sequencing has been reserved for conditions in which probes are not available because of the prohibitive cost and time involved with the process. Sequencing DNA involves several major steps. The DNA in question is isolated from a tissue and amplified (multiple copies are generated) before analysis. Analyzing the DNA sequence data can be difficult, expensive, and time consuming. However, patients who have successive DNA sequencing genotypes in combination with viral load testing and appropriate drug management have, consistently feel better while maintaining lower viral loads.

Done with accurate assays, read by experienced clinicians and integrated with information obtained from sequential viral loads and CD4 counts, genotyping HIV by DNA sequencing helps physicians determine why a current regimen is failing. It can also possibly identify an alternative regimen, which can help to lower your viral load while minimizing potential side effects. It is not however, the only test which should be used to monitor your therapy. In addition, it should only be interpreted by a physician who is well versed in the genetics of HIV and very experienced in genotyping the virus. Without these criteria, the information obtained from the test could be more harmful than helpful to a person’s health.