Drug resistance determination in HIV patients

Last updated on 26-1-2023 by Marinka Vangenck

Guidelines for drug resistance determination in HIV patients, by the seven AIDS Reference Laboratories (ARL).

Purpose of the test

To detect drug resistance in HIV patients, with the aim of investigating the cause of virological failure under antiretroviral therapy and predicting the response under antiretroviral therapy.

Description of the test

Drug resistance is estimated by genotyping assays that amplify and sequence either HIV-1 or HIV-2 RNA molecules using HIV-1 or HIV-2 specific primers. The genotype refers to the nucleotide sequence of the viral proteins that are directly or indirectly targeted by the antiviral drugs. The drugs that currently are available target either the viral proteins reverse transcriptase, protease, integrase, envelope, capsid or the cellular receptors CD4 or CCR5. The genotypic drug resistance tests that currently are available, are confined to the genes that encode for protease, reverse transcriptase, integrase and/or the envelope V3 loop. 

The viral nucleic acid sequence is determined and translated into an amino acid sequence, which is compared to reference consensus sequences to enlist mutations that are subsequently interpreted to a resistance profile by specialized software packages.

Viruses with an amino acid sequence comparable to those of viruses found in untreated patients are generally considered to be wild-type and susceptible to antiviral drugs. Genetic differences (mutations) leading to amino acid changes are considered to be associated with drug resistance when they reduce the antiviral efficacy of a particular drug. During the genotypic interpretation, the observed amino acid changes that are expected to have a biological and clinical significance are enlisted for each antiviral drug and converted into a resistance score for each specific drug.

The HIV subtype can be deduced from the viral nucleic acid sequence  which might be relevant for certain drug classes.

Prescription of the test

HIV drug resistance testing can only be performed during the follow-up of patients infected with HIV-1 and/or HIV-2, as confirmed by one of the ARL, and preferentially accompanied by a viral load request or known viral load. The results of drug resistance tests are complex and require expert interpretation if they are to be used successfully in therapy guidance. Therefore, they will only be performed on patients who are registered in an HIV Reference Centre (HRC) or who are followed by a physician closely collaborating with an HRC or ARL

Indications of the test

Drug naive patients:

  • Testing for resistance against protease, reverse transcriptase and integrase inhibitors is recommended at diagnosis, to detect the presence of transmitted drug resistance and to better understand the genetic changes occurring at subsequent therapeutic failure.
  • Unless there is a pre-existing high risk for resistance, the drug resistance results should not delay the initiation of first-line therapy. It is important to test the earliest sample available for this baseline drug resistance test.
  • If testing was not done at diagnosis, it is still recommended to do it before initiating therapy.
  • ARL provide genotypic drug resistance testing against protease, reverse transcriptase and integrase inhibitors for all drug naïve patients in Belgium, as part of our diagnostic service to HIV patients but also as part of our surveillance duties as a reference laboratory.

Drug-exposed patients:

  • Genotypic drug resistance testing against protease, reverse transcriptase and integrase inhibitors is indicated in case of therapy failure.
  • Testing for co-receptor use is recommended before starting a CCR5 antagonist.

Other exceptional circumstances:

  • Contact your ARL for further information. 

Limitations of the test

Resistance tests are characterized by a higher detection limit than the quantification limit of viral load assays. Therefore, it might be possible that no results are obtained at low viral loads, or that the obtained results are less representative for the virus population.

HIV exists in infected individuals as a complex quasispecies in which many different subpopulations of wild type and drug-resistant variants can co-exist.

  • If the sample is collected in the absence of drug selective pressure, the chances are high that the wild-type variants will be detected instead of mutant variants as the latter often display a reduced replication capacity in comparison to wild-type variants and therefore they are rapidly overgrown. An important limitation of drug resistance tests, especially the Sanger method, is their unreliability to detect minority variants. When testing is performed when drug selective pressure is not present (e.g. a few weeks after infection or therapy interruption), some resistant variants decay and it is more likely to detect wild type virus and to score the virus susceptible to some or all antiviral drugs. However, the resistant variants remain as a minority within the viral or proviral population and they can rapidly re-emerge in a subsequent regime, causing therapy failure.
  • This limitation can complicate the interpretation of resistance tests particularly in patients with complicated treatment histories. The complexity of this quasispecies may influence the success of therapy in ways that cannot be predicted by any single drug resistance test. Therefore treatment history, viral load evolution and resistance history all have to be taken into account.

Drug resistance testing is limited in its predictive value.

  • Drug resistance is not the only cause of treatment failure. Non-adherence, the use of insufficiently potent treatment regimens, pharmacokinetic factors that decrease the levels of one or more drugs in a treatment regimen, and potentially other factors may contribute to treatment failure.
  • The relationship between drug resistance mutations and clinical response remains complex and difficult to interpret. An antiviral drug may have some beneficial effect on the viral load, even in the setting of resistance, because of some, although limited, residual antiviral effect and because many drug-resistant variants have a reduced replication capacity compared to drug-susceptible variants.

Practical instructions and guidance

To perform a test for drug resistance in HIV patients:

  1. Collect blood in an EDTA-tube by venipuncture (10 ml).
  2. Request an HIV drug resistance test. 
  3. Store mother-tube at room temperature and send as soon as possible to any clinical laboratory. Plasma should be isolated within one day and stored at -20°C.

Although EDTA plasma is the most appropriate specimen for drug resistance testing, other specimens such as CSF or serum can be used for some indications. Please contact your ARL for further information.

HIV-2 drug resistance testing is available but requires dedicated assays (blood sampling is however the same). Please contact your ARL for further information.

Test results

The identified mutations leading to amino acid changes that are expected to have a biological and clinical significance are enlisted for each antiviral drug and converted into a resistance score for each specific drug. This resistance prediction profile is provided to the HIV-specialist of the patient.

HIV-1 subtyping based on these sequences is also relevant for later use when for instance injectable drugs are considered.

Treatment modification can only be prescribed by an HIV-specialist and should not only be based on the resistance profile obtained for the investigated sample. Treatment history, viral load evolution and resistance history all have to be taken into account.

For more details, contact the ARL of your choice.


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