Genotypic determination of HIV-1 CCR5/CXCR4 tropism

HIV-1 uses one of two coreceptors for entry into cells: CCR5 and CXCR4. CCR5 inhibitors, block the CCR5 receptor, but not the CXCR4 coreceptor, reducing viral entry, replication and therefore plasma viral load. CCR5 inhibitors, of which maraviroc is the only one licensed, are only effective if a patient’s HIV uses CCR5 as its coreceptor.[34] [35] If any proportion uses CXCR4, there will be no meaningful response to maraviroc. Therefore, the patient must be documented as having HIV that only uses CCR5 within the 3 months before the commencement of maraviroc.

Viral coreceptor usage is determined primarily by sequencing the section of the envelope gene encoding the V3 loop of the HIV-1 gp120 viral envelope protein. Previously, viral tropism has been determined by a phenotypic assay (Trofile®) which was expensive, required a significant blood volume and had a lengthy turn-around time. More recently, genotypic assays that use viral RNA from plasma or viral DNA from white blood cells have been developed and validated. These assays work like a genotypic resistance assay. The viral RNA or DNA is extracted, amplified and sequenced. This sequence is then examined by the Geno2Pheno (G2P) algorithm.

The G2P algorithm provides a statistical estimate of the likelihood that the virus binds to CXCR4. This process generates a false-positive rate, which is reported along with the tropism result. The lower the false-positive rate the more likely the patient’s HIV uses CXCR4. The higher the false-positive rate, the more likely the virus uses CCR5. Currently, the consensus position is to use a false-positive rate of 20% as the break-point for determining whether a virus binds CCR5 or CXCR4. As even a minor proportion of HIV using CXCR4 will compromise the activity of any CCR5 inhibitor such as maraviroc, the test is performed in triplicate and the result with the lowest false-positive rate determines the result reported. That is, for a virus to be reported as CCR5-using each of the three results must have a false-positive rate above 20. If any result is below 20, then the virus is reported as being CXCR4-using.

The plasma viral RNA G2P assay has undergone the most rigorous validation and is the test of choice for any viraemic patient. The pro-viral DNA G2P assay should be used only in patients who have viral loads of less than 2000 copies/mL because there is substantially less clinical experience with this test and the results generated are more variable.
Ethylenediaminetetraacetic acid (EDTA) anticoagulant treated whole blood is required. If a RNA-based tropism is ordered, a HIV viral load test must also be ordered on the same sample, as the test only works in patients with HIV RNA levels  of 2000 copies/mL or above, and will not be performed when the viral load is lower.

The turn-around time for results is approximately 2 weeks, similar to that for genotypic resistance assays. The report will give the overall result as either:

  • Analysis of sequences of V3 loop of the HIV-1 envelope gene does not detect the presence of a substantial sub population of CXCR4 using virus. CCR5 inhibitors are likely to be effective
  • Analysis of sequences of V3 loop of the HIV-1 envelope gene suggests the presence of CXCR4 using virus. CCR5 inhibitors are unlikely to be effective

The report will include the G2P results for the triplicate assays, including the false-positive rate and the interpretation of each of the triplicates performed.

Once a CXCR4-using virus has been detected, the test should not be ordered again. It is not appropriate to start a CCR5 inhibitor in a patient with a history of a CXCR4-using virus. Therefore, as the virus only evolves from CCR5 to CXCR4 use, once a virus has been labelled CXCR4-using, it will always be CXCR4-using, and a CCR5 inhibitor is not recommended. To start maraviroc a patient must have carriage of a CCR5-using virus within 3 months of the start date.