• Guide selection of antiretroviral drugs

• Determine eligibility for CCR5 antagonist therapy in patients infected with human immunodeficiency virus 1 (HIV-1)

Numerous antiretroviral drugs belonging to 7 different classes are available to treat HIV-1 infection. Entry inhibitors, the newest class of antiretroviral drugs, target the process by which HIV-1 enters the host cell. This process requires the use of the CD4 receptor and 1 of 2 chemokine coreceptors, CCR5 or CXCR4, located on the host cell surface membrane. HIV-1 virions that use CCR5 are called R5-tropic and those using CXCR4 are called X4-tropic. Viral mixtures using both coreceptors are called dual or mixed-tropic (DM). R5-tropic viruses are more common in treatment-naive patients. In contrast, X4- tropic viruses are found in only about 13% of recent seroconverters¹ but in up to 50% of treatment-experienced patients and those with advanced disease.²

Maraviroc (MVC; Selzentry^®) was one of the first entry inhibitors to obtain FDA approval. MVC binds to the CCR5 coreceptor, thereby inhibiting coreceptor binding of R5-tropic HIV-1 virions. Clinical trials using a phenotypic assay to determine HIV-1 tropism showed that MVC is not effective against X4- or DM HIV-1.³ Thus, tropism testing should be performed before initiating MVC therapy and may also be considered for patients who exhibit virologic failure while taking a CCR5 inhibitor.⁴ In addition to phenotypic testing, genotypic tropism testing is now clinically available. Although phenotypic tropism testing is generally preferred because of a greater weight of supporting evidence, genotypic tropism testing is considered an alternative because of lower cost and faster analytical times.⁴

The Quest Diagnostics HIV-1 coreceptor tropism test begins with standard Sanger sequencing of the third variable (V3) loop of the HIV-1 gene, the primary determinant of viral tropism. To enhance sensitivity for detection of minority X4 virus in DM viral populations, next-generation DNA sequencing (ultradeep sequencing [UDS]) is performed if standard sequencing detects only R5 virus. UDS performance is comparable to phenotypic tropism tests for predicting clinical response to MVC therapy.^5,6 The positive and negative predictive values of the Quest Diagnostics test for distinguishing virologic responders from non-responders are similar to those of a high-sensitivity phenotypic test.⁷

• Patients being considered for treatment with CCR5 antagonist therapy

• Patients who exhibit virologic failure while taking a CCR5 antagonist

• Reverse transcription and PCR amplification of the HIV-1 envelope V3 loop in triplicate

• Triplicate Sanger DNA sequencing of codons 1-35 of the V3 loop and bioinformatic analysis

–   Reflex to UDS if Sanger sequencing detects CCR5 (R5) (with an additional charge and CPT code)

• Result reported:

–   Population sequencing CXCR4 (X4): detected or not detected

–   Ultradeep sequencing X4: detected or not detected, based on a clinically-validated cutpoint of 2%^5-7

–   Net tropism assessment: R5 or DM/X4

–   MVC activity anticipated: yes or no

• Sensitivity

–   Technical: limit of detection (LOD₉₅) of UDS instrument is 0.5% X4.⁷

–   Analytical (biological): LOD₉₅ of UDS assay in dual/mixed mimicked clinical samples is 12%

 X4 virus at 25,000 copies/mL and 5% X4 virus at 100,000 copies/mL.⁷

• HIV-1 subtype specificity: HIV-1 subtypes A, B, C, D, AG, and H are successfully amplified, but subtypes AE, F, and G are not.

• Aliases: human immunodeficiency virus, CCR5, CXCR4, R5, X4

CXCR4 (X4) not detected

CCR5 antagonists such as MVC are not recommended for patients infected with X4-tropic HIV-1.³ Thus, alternative therapy should be considered if the net tropism assessment result is “DM/X4.” A net tropism assessment of “R5” is consistent with eligibility for treatment with a CCR5 antagonist.

Individuals infected with X4-tropic or dual/mixed-tropic HIV-1 are more likely to have a lower CD4 cell count, higher viral load, and more rapid progression to AIDS than individuals without an X4-tropic population.^8,9

Because fewer tropism data are available for non-B subtypes, tropism determination for these subtypes may have a greater degree of uncertainty.