HIV Coreceptor Tropism With Reflex to Ultradeep Sequencing
A Genotypic Test for HIV-1 Tropism Combining Sanger Sequencing with Ultradeep Sequencing Predicts Virologic Response in Treatment-Experienced Patients - (10/09/12) |
PLOS One Sept 27 2012. Ron M. Kagan1*, Erik P. Johnson1, Martin Siaw1, Pinaki Biswas2, Douglass S. Chapman3, Zhaohui Su4, Jamie L. Platt1, Rick L. Pesano1
HIV-1 Coreceptor Tropism with Reflex to Ultradeep Sequencing
· Guide selection of antiretroviral drugs
· Determine eligibility for CCR5 antagonist therapy in patients infected with human immunodeficiency virus 1 (HIV-1)
As of 2011, 26 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. 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 dual/mixed-tropic 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
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 dual/mixed (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 Quest Diagnostics test is also comparable to a high-sensitivity phenotypic test in distinguishing between virologic responders and nonresponders.7
Individuals Suitable for Testing
· 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
- 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 maraviroc 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
1. Poveda E, Briz V, de Mendoza C, et al. Prevalence of X4 tropic HIV-1 variants in patients with differences in disease stage and exposure to antiretroviral therapy. J Med Virol. 2007;79:1040-1046.
2. Wilkin TJ, Su W, Kuritzkes DR, et al. HIV type 1 chemokine coreceptor use among antiretroviral-experienced patients screened for a clinical trial of a CCR5 inhibitor: AIDS Clinical Trial Group A5211. Clin Infect Dis. 2007;44:591-595.
3. Selzentry® (maraviroc) tablets [prescribing information]. Research Triangle Park, NC:ViiV Healthcare; July 2011. Available at: http://www.viivhealthcare.com/products/~/media/Files/G/GlaxoSmithKlinePlc/Attachments/
pdfs/products/us_selzentry_jul2011.pdf. Accessed February 23, 2012.
4. Panel on antiretroviral guidelines for adults and adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. October 14, 2011. Available at:
http://www.aidsinfo.nih.gov/contentfiles/adultandadolescentgl.pdf. Accessed February 23, 2012.
5. Swenson LC, Mo T, Dong WW, et al. Deep sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of maraviroc in treatment-experienced patients. J Infect Dis. 2011;203:237-245.
6. Swenson LC, Mo T, Dong WW, et al. Deep V3 sequencing for HIV type 1 tropism in treatment-naive patients: a reanalysis of the MERIT trial of maraviroc. Clin Infect Dis. 2011:53:732-742.
7. Data on file at Quest Diagnostics Nichols Institute, San Juan Capistrano, CA.
8. Daar ES, Kesler KL, Petropoulos CJ, et al. Baseline HIV type 1 coreceptor tropism predicts disease progression. Clin Infect Dis. 2007;45:643-649.
9. Waters L, Mandalia S, Randell P, et al. The impact of HIV tropism on decreases in CD4 cell count, clinical progression, and subsequent response to a first antiretroviral therapy regimen. Clin Infect Dis. 2008;46:1617-1623.
This test was developed and performance characteristics have been determined by Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical performance of the test.