OVERVIEW

Researchers have made remarkable strides in HIV care over the past 26 years. Improved HIV treatment has been realized with more effective therapies, but also with a better understanding of how to best use these therapies. An appreciation for HIV drug resistance and its impact on the success of HIV treatment has led to the widespread and rapid assimilation of resistance testing into clinical practice. Combining drug resistance information with patient history is now well recognized as a valuable tool for guiding the success of antiretroviral therapy, so much so that this information is now a routine component of HIV management and therapeutic decision-making. This issue of ResistanceWATCH News© illustrates just how far we have come in our understanding of HIV drug resistance by highlighting past and current knowledge in the field, when and how to best detect drug resistance, and how to optimally use resistance information to guide treatment.

 

TREATMENT AND RESISTANCE: THAT WAS THEN, THIS IS NOW

When the nucleoside reverse transcriptase inhibitor (NRTI), zidovudine, first hit the HIV treatment scene in 1987, clinicians adopted the agent with gusto, glad to finally have some form of treatment for their HIV-infected patients. Although use of the agent in individuals with asymptomatic or early symptomatic HIV infection slowed disease progression, its effectiveness waned with time, and improved survival with long-term use was lacking. Clues began to emerge that the development of select mutations in the HIV reverse transcriptase gene correlated with zidovudine resistance and impending CD4+ cell count decline.[1,2]

 

These findings were complemented by in vitro research showing that HIV replication is a highly error-prone process--and one that can easily select for drug resistance. During the HIV life cycle, creation of HIV DNA from genomic RNA by the HIV reverse transcriptase, which lacks the ability to proofread its work, results in the spontaneous incorporation of an incorrect nucleotide into the growing DNA strand about once in every 10,000-30,000 nucleotides.[3] Because the HIV genome is about 10,000 bases long, this means that every newly replicated viral genome contains about 1 mutation. Ongoing viral replication in the setting of incompletely suppressive therapy (e.g. monotherapy) affords HIV the opportunity to create viral variants with drug resistance mutations that have a survival advantage under drug selection pressure. The extremely large number of viruses and rapid replication rate in HIV-infected individuals affords HIV enormous potential for mutation.

 

Efforts to beef up viral suppression led to the institution of NRTI combination therapy, yet this approach still suffered from waning efficacy with increasing time. Two active drugs were not enough if each could be overcome by a single mutation. It wasn’t until after the introduction of protease inhibitors (PIs) in 1995 that the concept of highly active antiretroviral therapy (HAART) was born--a phenomenon that ushered in a new era of HIV treatment. Combining the use of three or more antiretrovirals, often targeting at least two different pathways in the HIV life cycle, finally appeared to be the key to improved survival and long-term treatment success, as this strategy effectively quashes HIV replication to prevent the emergence of drug resistance. In conjunction, requiring the virus to accumulate at least three new mutations to overcome the antiretroviral regimen is another important strategy for successful therapy. In step with this understanding of HIV resistance, clinicians have learned that salvage regimens for treatment-experienced patients should ideally contain at least two active agents, and at least one of these agents should have a high genetic barrier to resistance in that multiple mutations are needed to confer resistance. Anything less is akin to functional monotherapy or dual therapy--a scenario that we now know invites the development of resistance.

 

HIV DRUG RESISTANCE TODAY

It is clear that effective suppression of viral replication holds the development of resistance at bay. As a corollary, anytime HIV replication is not completely suppressed, drug resistance mutations have the opportunity to emerge. Suboptimal adherence to treatment is one of the most common factors that can render a successful HAART regimen ineffective. A recent meta-analysis found that only 55% of patients achieve at least 95% adherence to HIV treatment, meaning that about 45% of patients are putting themselves at risk for the development of resistance mutations due to suboptimal antiretroviral exposure.[4] In turn, the development of drug resistance ups the risk of treatment failure. Newer drug regimens may not require such high levels of adherence,[5] but patients are encouraged to strive for perfect adherence to be on the safe side.

 

In addition to the possibility of developing drug resistance during suboptimal antiretroviral therapy, those already harboring drug-resistant HIV have the potential to pass their resistant virus along to uninfected individuals. A prospective study of 5 individuals newly infected with drug-resistant HIV found that resistant virus persisted in the blood and semen in all men for nearly a year.[6] Two of the men had resistant virus detectable in the blood and semen for over 2 years and 3 years, respectively. In addition, resistant virus could still be detected in the semen of these 2 men beyond 2-3 years, even though the virus had reverted to wild-type in blood plasma. One of these men infected two sexual partners with the strain of drug-resistant HIV that he was carrying, even though he remained treatment naïve. These findings suggest that long-term persistence of resistant virus, not only in the blood but also in the male and female genital tracts, may fuel horizontal transmission of drug-resistant HIV.

 

Given these factors, it is not surprising that drug resistance is a common feature in populations with ready access to antiretroviral treatment. One population-based study conducted in the United States showed that more than 75% of HIV-infected patients with detectable viremia on treatment had evidence of phenotypic drug resistance.[7] A large prospective cohort study found that 8% of treatment-naïve patients harbored primary drug resistance at baseline.[8] After the 1138 patients started treatment, HIV drug resistance developed in 29% over the first 30 months of therapy. About half (48%) had resistance to just one antiretroviral class, whereas 45% had resistance to two classes and 6% had resistance to all three antiretroviral classes. Emergence of any resistance was associated with a 1.75-fold increased risk of death, and patients with resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) had a 3-fold increased risk of death.

 

Other studies report that the transmission of primary resistance is on the rise and tracks with trends in drug exposure. A recent Canadian study found that the prevalence of primary drug-resistant HIV has undergone a steady increase in recent years, from 4% in 1998 to roughly 14% in 2005.[9] According to antiretroviral class, the largest increases in transmission were observed for NNRTI-resistant HIV, jumping from about 0% in 1998 to about 6% in 2005, which reflects the introduction of these agents in 1997 followed by their widespread use. Primary resistance rates in many U.S. and European countries have stabilized in recent years at around 5%-15%. Given these substantial levels of primary resistance transmission, drug resistance testing is now strongly recommended for all individuals as soon as possible after HIV diagnosis, even if treatment is not imminent. This is because resistant variants can decline in prevalence over time in the absence of selective drug pressure, but will still persist at low levels that often go undetected by standard resistance assays. Early resistance testing provides a record of all resistant variants within a given patient, so that when the individual does decide to initiate therapy, treatment can be designed to account for such viruses.

 

THE UTILITY OF HIV DRUG RESISTANCE TESTING

Clinicians have two types of commercially available HIV drug resistance assays at their disposal: genotype assays and phenotype assays. Genotype tests measure HIV drug resistance via sequencing of the HIV genome and detection of mutation patterns that are known to be associated with drug resistance. Phenotype testing measures laboratory susceptibility of an HIV isolate to a given drug. Genotypic and phenotypic assays are commonly used to help guide treatment choices in patients failing therapy. Genotypic assays are less expensive, quicker to perform, more universally accessible, and preferred for drug-naïve patients. Some people have argued that using both tests in combination in heavily treatment-experienced patients with complex resistance patterns may provide the best information for designing a salvage regimen.[10] Expert interpretation and advice can improve care in advanced and/or complex patients.[11]

 

The value of resistance testing for guiding treatment decisions in HIV-infected individuals has been demonstrated in clinical trials and observational cohort studies. Data supporting the utility of genotypic assays are greater than for phenotypic assays (Table 1). Prospective studies comparing resistance testing versus standard care showed that selecting salvage therapy based on resistance test results produced greater reductions in viral load and enabled more patients to achieve undetectable levels of HIV RNA than selecting treatment based on the patient’s prior treatment history.[11-18] In general, the advantage was moderate and lasted over the short-to-medium term, and not all studies showed a significant benefit. Correct use of resistance testing can also minimize the development of drug resistance and cross-resistance and guide the optimal sequence of antiretroviral agents during a lifetime of therapy.

 

 

 A great challenge for both genotypic and phenotypic assays is clinically-relevant interpretation of the results. Over the years, there has been a large amount of effort dedicated to improving the translation of these laboratory parameters into useful clinical guidance. For genotypic assays, numerous mutations and mutational patterns must be considered in light of cross-resistance and mutational interactions. For phenotype testing, translating fold-changes in susceptibility into lack of virological activity needs to be accurately determined for each and every drug.

 

 Interpretation of genotype has evolved greatly, progressing from attempts to correlate single mutations with specific drugs to clinically derived scores, in which combinations of mutations can predict rates of virological success. Today, there are many systems available to assist HIV providers in interpreting genotypic test results. Examples of commercially available genotypic resistance tests are: HIV-1 TrueGene and ViroSeq, both assays are approved by the FDA. Other genotypic resistance assays such as vircoTYPE HIV-1, GenoSure (Plus) or GeneSeq are established in the laboratories of the respective manufacturers and are used in clinical trials. Two genotypic interpretation systems come with the genotypic resistance testing kits and are approved for use by the U.S. Food and Drug Administration (TRUGENE, ViroSeq). Several academic institutions have their own genotypic interpretation systems (e.g., the HIVdb of the Stanford HIV Drug Resistance Database, the Agencie Nationale Recherche SIDA [ANRS] system, the Rega Institute system), many of which are free and publicly available over the Internet (http://hivdb.stanford.edu/pages/algs/HIValg.html). A number of systems use phenotypic data to predict genotypic assay results. VirtualPhenotype and Geno2pheno interpret genotype test results by comparing a patient’s genotype to a large database of samples with paired genotypic and phenotypic data. The genotypic result is thus translated into a phenotypic result, which can be interpreted based on predetermined cutoffs (similar to a phenotypic assay). Several large reference laboratories also have their own genotype interpretation systems that they use in conjunction with phenotypes. Phenotypic resistance tests include: Antivirogram, PhenoSense and Phenoscript.

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Overcoming the challenges of properly interpreting phenotypic assays has also greatly improved over the years. The rigid, technically-driven cutoffs originally used sometimes lacked clinical correlation, especially for NRTIs where resistance was often underinterpreted.[17] Substantial improvements have come with the determination of clinical cutoffs, which are more meaningful to clinicians since they provide relevant information on whether a virus is fully susceptible, partially susceptible, or not susceptible to each drug. Here, fold-changes in reduced susceptibility are correlated with reductions in viral load for every drug. Large databases and sophisticated statistical analysis have generated clinical cutoffs for many drugs, but clinical cutoffs still remain to be defined for several antiretrovirals in clinical use. Although not feasible in today’s setting, one might expect more impressive results if clinical trials of phenotypic assays were performed today with this improved interpretation system.

 

The role of resistance testing in guiding treatment decisions is continually being redefined. Each time a new drug hits the market, genotype and phenotype resistance scores and cutoffs need to be developed using clinical trials data. While such findings can provide a good general idea of which mutations--and how many mutations--affect susceptibility to a new agent, refinements often emerge with more widespread use and study. For example, the newer PIs--darunavir and tipranavir--were approved with initial genotypic scores and cutoffs, but further study has refined these original data, with current results for darunavir being more consistent than those for tipranavir.[19]

                                                                                                       

With the approval of the first CCR5 antagonist maraviroc comes a new test for determining HIV coreceptor tropism. Since only patients harboring exclusively CCR5-coreceptor–using virus benefit from the drug, a determination of tropism is required. The phenotypic Trofile assay is currently used to screen patients prior to the administration of CCR5 inhibitors to exclude patients with dual/mixed- or CXCR4 (X4)-using virus. The original Trofile assay was shown to be about 85% sensitive at detecting minor X4-using variants present at 5% in a mixed-virus population. Minor variants below this level of detection could be missed. Several modifications have now been made to optimize the Trofile assay. Initial reports suggest that these changes improve its ability to detect minor X4-using variants in a mixed viral population by 10-fold, enable detection of X4-using variants present at only 0.1% of a total mixed viral population, and allow for earlier detection of the emergence of X4-using variants. [20]

 

There are a number of limitations to the Trofile assay: It is expensive, time consuming, and requires clinical samples be shipped to the California facility. Genotypic assays to predict tropism would be faster, less costly, and could be performed locally. Currently, these methods are not sensitive enough for routine clinical use, although intense research continues. [21] Other options are also being developed, including a tropism-testing platform that utilizes both genotypic and phenotypic information, which is in research development. [22]

 

 

CURRENT GUIDELINES FOR HIV DRUG RESISTANCE TESTING

The U.S. Department of Health and Human Services (DHHS) just issued a new set of guidelines for the treatment of HIV-infected adults and adolescents. [23] Two key updates center around drug resistance and the newly added guideline recommendations for the use of tropism testing: (1) Whereas the previous guidelines recommended performing resistance testing before therapy initiation in patients with acute or chronic HIV infection, the current guidelines advocate genotypic drug resistance testing for all treatment-naïve patients entering into clinical care, without regard to the timing of treatment initiation. This provides a snapshot of primary resistance before drug-resistant variants have the opportunity to decay and be replaced with wild-type virus as the dominant species. (2) Tropism testing is now recommended prior to the start of a CCR5 antagonist and should also be considered for patients experiencing failure on a CCR5 antagonist. Current DHHS recommendations for the use of drug resistance testing and tropism testing in clinical practice are shown in Table 2.

 

 

Practice guidelines issued by the British HIV Association (BHIV) and the International AIDS Society (IAS)-USA are generally similar to the DHHS guidelines. [24,25] Whereas the BHIVA also recommends baseline resistance testing in all newly-diagnosed patients, the IAS advocates baseline resistance testing when the prevalence of transmitted HIV drug resistance is greater than 5% or if the transmission of drug resistance is likely. Similar to the DHHS guidelines, both the BHIVA and IAS recommend resistance testing in the setting of virologic failure, ideally when the patient is still taking the failing regimen, and urge consideration of resistance testing if the viral load decline is suboptimal after introduction of a new regimen.

 

SUMMARY OF KEY POINTS

• HIV replication is a highly error-prone process that can easily select for drug resistance.

• Combining the use of three or more antiretrovirals targeting at least two different pathways in the HIV life cycle (i.e., HAART) can improve survival and long-term treatment success by inhibiting HIV replication to prevent the emergence of drug resistance.

• Similarly, salvage regimens for treatment-experienced patients should ideally contain at least two fully active agents to prevent viral replication and hence drug resistance development.

• Poor adherence to treatment is one of the most common factors leading to drug resistance development.

• Resistant virus can persist for long periods of time in blood plasma and the genital tract, which may fuel horizontal transmission of drug-resistant HIV.

• HIV drug resistance testing is a powerful tool that can help clinicians tailor treatment regimens around the specific HIV strain(s) infecting their patients. In turn, this can lead to better virologic control.

• Several systems are available to assist HIV providers in interpreting genotypic and phenotypic test results.

• Current international treatment guidelines recommend genotypic drug resistance testing for all treatment-naïve patients entering into clinical care, regardless of the timing of treatment initiation, so that primary resistance can be detected and recorded.

• HIV coreceptor tropism testing is recommended prior to the start of a CCR5 antagonist to exclude patients with dual/mixed- or X4-using virus who are unlikely to benefit from these agents.

 

 

REFERENCES

1. Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989;246(4934):1155-1158.

2. Kozal MJ, Shafer RW, Winters MA, Katzenstein DA, Merigan TC. A mutation in human immunodeficiency virus reverse transcriptase and decline in CD4 lymphocyte numbers in long-term zidovudine recipients. J Infect Dis. 1993;167(3):526-532.

3. Mansky LM, Temin HM. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol. 1995;69(8):5087-5094.

4. Mills EJ, Nachega JB, Buchan I, et al.  Adherence to antiretroviral therapy in sub-Saharan Africa and North America: a meta-analysis. JAMA. 2006;296(6):679-690.

5. Bangsberg DR, Acosta EP, Gupta R, et al. Adherence-resistance relationships for protease and non-nucleoside reverse transcriptase inhibitors explained by virological fitness. AIDS. 2006;20(2):223-231.

6. Smith DM, Wong JK, Shao H, et al. Long-term persistence of transmitted HIV drug resistance in male genital tract secretions: implications for secondary transmission. J Infect Dis. 2007;196(3):356-360.

7. Richman DD, Morton SC, Wrin T, et al. The prevalence of antiretroviral drug resistance in the United States. AIDS. 2004;18(10):1393-1401.

8. Hogg RS, Bangsberg DR, Lima VD, et al. Emergence of drug resistance is associated with an increased risk of death among patients first starting HAART. PLoS Med. 2006;3(9):e356.

9. Goedhuis N, Jayaraman GC, Brooks J, Pilon R, Sandstrom P, Archibald CP. Primary HIV-1 drug resistance in Canada: updated results from the Canadian HIV Strain and Drug Resistance Surveillance Program. Program and abstracts of the XVI International AIDS Conference; August 13-18, 2006; Toronto, Canada. Abstract THAX0101.

10. Zolopa AR. Incorporating drug-resistance measurements into the clinical management of HIV-1 infection. J Infect Dis. 2006;194:S59-S64.

11. Tural C, Ruiz L, Holtzer C, et al. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS. 2002;16:209-218.

12. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet. 1999;353(9171):2195-2199.

13. Clevenbergh P, Durant J, Halfon P, et al. Persisting long-term benefit of genotype-guided treatment for HIV-infected patients failing HAART. The Viradapt Study: week 48 follow-up. Antivir Ther. 2000;5(1):65-70.

14. Cingolani A, Antinori A, Rizzo MG, et al. Usefulness of monitoring HIV drug resistance and adherence in individuals failing highly active antiretroviral therapy: a randomized study (ARGENTA). AIDS. 2002;16(3):369-379.

15. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS. AIDS. 2000;14(9):F83-F93.

16. Cohen CJ, Hunt S, Sension M, et al. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS. 2002;16(4):579-588.

17. Haubrich R, Kemper C, Hellmann NS. et al. A randomized, prospective study of phenotype susceptibility testing versus standard of care to manage antiviral therapy: CCTG 575. AIDS. 2005;19(3):295-302.

18. Matheron S, Lamotte C, Guiramand S, et al. Phenotypic or genotypic resistance testing for choosing antiretroviral therapy after treatment failure: a randomized trial. AIDS. 2002;16(5):727-736.

19. Mitsuya Y, Liu TF, Rhee SY, Fessel WJ, Shafer RW. Prevalence of darunavir resistance-associated mutations: patterns of occurrence and association with past treatment. J Infect Dis. 2007;196(8):1177-1179.

20. Reeves J, Han D, Hunt P, et al. Enhancements to the Trofile HIV co-receptor tropism assay enable improved detection of CXCR4-using subpopulations and earlier detection of CXCR4-using viruses in sequential patient samples. Program and abstracts of the Targeting HIV Entry: 3rd International Workshop; December 7-9, 2008; Washington, DC. Abstract #11.

21. Low AJ, Dong W, Chan D, et al. Current V3 genotyping algorithms are inadequate for predicting X4 co-receptor usage in clinical isolates. AIDS. 2007;21(14):F17-F24.

22. Van Baelen K, Vandenbroucke I, Rondelez E, Van Eygen V, Vermeiren H, Stuyver LJ. HIV-1 coreceptor usage determination in clinical isolates using clonal and population-based genotypic and phenotypic assays. J Virol Methods. 2007;146(1-2):61-73.

23. U.S. Department of Health and Human Services. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents, December 1, 2007. Available at: http://aidsinfo.nih.gov/contentfiles/AdultandAdolescentGL.pdf. Accessed December 13, 2007.

24. Gazzard B, Bernard AJ, Boffito M, et al. British HIV Association (BHIVA) guidelines for the treatment of HIV-infected adults with antiretroviral therapy (2006). HIV Med. 2006;7(8):487-503.

25. Hammer SM, Saag MS, Schechter M, et al. Treatment for adult HIV infection: 2006 recommendations of the International AIDS Society-USA panel. JAMA. 2006;296(7):827-843