HIV Testing — Genotypic Technology in Tropism and Drug Resistance Testing

Li, Jonathan Z., MD
Physician, Brigham and Women's Hospital
Instructor in Medicine, Harvard Medical School
Boston, MA Also by this Author 

HIV tropism is defined as the cell type that HIV is able to enter and infect.  The identification that HIV frequently uses the CCR5 co-receptor for cell entry led to the introduction of a class of HIV drugs called the CCR5 coreceptor antagonists. An HIV-1 tropism test is performed before initiating coreceptor antagonist therapy to help determine patient eligibility.1

Dr. Jonathan Li, Infectious Disease Physician and Researcher, Brigham and Women’s Hospital, discusses how advances in genotypic testing technology offer physicians a new option for tropism screening and enable an accurate prediction of virologic response to therapy.

HIV Tropism and Coreceptor Antagonist Therapy

The HIV-1 virus uses the CD4 cell receptor, together with a coreceptor – either CCR5 or CXCR4 - to enter a cell.2 The virus using CCR5 is often referred to as R5-tropic, and that using CXCR4 as X4-tropic. When there’s a mixed population or when the virus uses both receptors it’s called dual-tropic virus. “At the beginning of HIV infection the majority of patients have R5-tropic virus,” notes Dr. Li, “while about 10-15% of patients have X4-tropic virus. As their disease progresses, an increasing number of patients have detectable X4-tropic virus.”

A CCR5 antagonist is a small molecule that binds to the CCR5 receptor and blocks virus entry. One drug within this class, maraviroc, is approved by the FDA.3 “Resistance to maraviroc and other CCR5 antagonists occurs by one of several mechanisms,” explains Dr. Li.  “Either by the adaption of HIV to use the drug-bound receptor, or through the use of CXCR4 as a co-receptor instead of CCR5. Before maraviroc or other CCR5 antagonists can be prescribed, clinicians have to be sure the patient’s virus only uses the CCR5 receptor since maraviroc is unable to prevent HIV entry in patients who harbor CXCR4 using variants - that’s the role of tropism testing.”

Resistance and Tropism Testing

“As with HIV drug resistance testing, there are two main ways to perform tropism testing. First, there’s phenotypic testing in which you directly measure the effect of patient-derived HIV genes on viral growth rates. For phenotypic tropism testing, a portion of the HIV envelope is inserted into laboratory strains of HIV and the ability of the virus to replicate in the presence of maraviroc is compared with that of wild-type virus under the same condition.”

“Then there is genotypic testing, where the sequence of the HIV genes is scrutinized. For HIV drug resistance testing, we evaluate the reverse transcriptase protease, and integrase genes, while for tropism testing, the key determinant is the V3 region of the HIV envelope.”

“The phenotypic approach for resistance or tropism testing is more intuitive than genotypic technique, and is akin to how we perform bacterial resistance testing.  However, it’s also more expensive, takes longer to perform, and is less likely to be available internationally.”4, 5

In the U.S., phenotypic testing has been the standard method for tropism testing. A version of this test, the Trofile™ assay, was the method used to screen for the CCR5 antagonist clinical trials and a more sensitive test, Trofile™ Enhanced Sensitivity (TF-ES), is now available.6 Outside the U.S. genotypic testing is the most commonly used methodology.5

Ultra-deep Sequencing (UDS) - A New Approach to Genotypic Testing

One of the newer approaches to HIV resistance and tropism testing involves the use of ultra-deep sequencing. “Deep sequencing, or next generation sequencing, is a category of novel high-throughput sequencing techniques and methodologies,” says Dr. Li. “The general principle underlying the most commonly used platforms is the clonal amplification of millions of individual fragments of HIV that can then be sequenced in parallel.”

In the past, standard sequencing has not been shown to be sensitive enough to detect minor non-R5 variants. Ultra deep sequencing has addressed this shortcoming through clonal amplification and sequencing of thousands of individual variants for each sample.7 This technology provides greater sensitivity than conventional population sequencing to detect minor populations of HIV-1 variants.7 If standard sequencing detects only R5-tropic virus, UDS may still detect minority X4-tropic virus or dual-tropic viral populations.

“The genotypic assay with deep-sequencing has similar test characteristics to the enhanced phenotypic assay, but is currently faster to perform and more cost-effective,” notes Dr. Li.

Application of Deep Sequencing in Clinical Practice

Dr. Li believes that deep-sequencing techniques offer the potential for broader application in clinical HIV care. “There’s increasing evidence that deep sequencing can be useful for drug-resistance testing overall, even outside of tropism testing,” he says. “This approach for drug-resistance testing will be more cost-efficient than traditional Sanger sequencing, especially for high-throughput centers.”

With better bioinformatics support and technical familiarity, Dr. Li sees deep-sequencing moving from the research arena to that of clinical care: “The key from a research perspective is to help physicians interpret results and to give them a sense of which minority variants are likely to cause a significantly increased risk of treatment failure. There are already areas where we know that minority variants  - present below about 15-20% of viral population, and not detectable by conventional Sanger sequencing - may have a significant effect on the risk of treatment failure. This has been shown in two main areas: the use of CCR5 antagonists like maraviroc, and the use of non-nucleoside reverse transcriptase inhibitors (NNRTIs), both in women who have previously received single-dose nevirapine in Africa as well as in treatment-naïve individuals in developed countries.”

Developments in HIV Patient Care

Dr Li foresees that evolving areas in HIV will present clinicians with some significant challenges. “The first key topic in patient management is determining when to start therapy,” he says. “There are downsides to starting therapy earlier, including toxicity of long term treatment, pill fatigue and drug-resistance, but there are also many benefits, including preserved immune function, decreased risk of HIV transmission, decreased inflammation and avoiding the long-term effects of chronic inflammation. There’s increasing evidence that earlier treatment, even when CD4 cells are relatively high, can improve mortality and morbidity.”

“Another challenge is going to be the clinical manifestation of accelerated ageing. By 2014 it’s estimated that half of HIV patients in the United States will be 50 years or older, and HIV clinicians are now faced with a growing number of chronic, age-related conditions. A number of recent studies have described higher rates of cardiovascular disease, renal disease, bone fractures, diabetes and other age-related conditions in HIV patients compared to similarly matched HIV negative controls.”

Finally, the challenge remains of reducing the proportion of those with HIV who are undiagnosed. “There’s been an effort by the CDC to increase testing of HIV,8 especially in those who are at high risk, and to try to decrease the stigma surrounding HIV testing. But even with these policy changes and educational campaigns, the number of undiagnosed individuals in the U.S. is still staggeringly high – over a quarter of a million.9 That’s a major challenge if this epidemic is going to be brought under control.”

References


  1. 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; 1–167.
    Available at http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf . Accessed 1/22/13
  2. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease.
    Berger EA et al. Annu Rev Immunol1999;17:657-700.\
  3. Approval of Maraviroc, CCR-5 co-receptor antagonist for treatment of HIV. U.S. Food and Drug Administration. Aug 6, 2007.
    www.fda.gov/ForConsumers/ByAudience/ForPatientAdvocates/HIVandAIDSActivities/ucm124198.htm
  4. Prosperi et al. Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping. Retrovirology 2010, 7:56
    http://www.retrovirology.com/content/7/1/5
  5. Vandekerckhove L et al. European guidelines on the clinical management of HIV-1 tropism testing. The Lancet Published Online March 22, 2011 DOI:10.1016/S1473- 3099(10)70319-4 www.kostrikislab.com/files/shared/Vandekerckhove__Lancet_2011.pdf
  6. Monogram Biosciences. http://www.trofileassay.com/
  7. Kagan R, Johnson E, Siaw M, et al. (2012) A Genotypic Test for HIV-1 Tropism Combining Sanger Sequencing with Ultradeep Sequencing Predicts Virologic Response in Treatment-Experienced Patients. PLoS ONE 7(9): e46334. doi:10.1371/journal.pone.0046334
  8. Revised Recommendations for HIV Testing of Adults, Adolescents, and Pregnant Women in Health-Care Settings. MMWR September 22, 2006 / 55(RR14);1-17. Centers for Disease Control and Prevention. www.cdc.gov/mmwr/preview/mmwrhtml/rr5514a1.htm. Accessed 1/22/13
  9. HIV Surveillance --- United States, 1981--2008June 3, 2011 / 60(21);689-693.Centers for Disease Control and Prevention
    www.cdc.gov/mmwr/preview/mmwrhtml/mm6021a2.htm. Accessed 1/22/13