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Case 1: Discussion

Testing for HIV resistance to antiretroviral medications has become an important component of the clinical care of HIV-infected persons. Resistance assays can assist the clinician in selecting a maximally effective HAART regimen by identifying the level of resistance, if any, to antiretroviral agents. The December 2007 DHHS guidelines for the use of antiretroviral therapy guidelines in adults and adolescents provided specific recommendations regarding HIV drug resistance testing (Figure 1Resistance Testing RecommendationsRating Scheme for Clinical PracticeAcknowledgments)[1]. The January 2008 DHHS antiretroviral therapy guidelines did not make any changes in the 2007 recommendations for resistance testing.

Resistance Testing for Virologic Failure

The December 2007 DHHS antiretroviral therapy guidelines recommend resistance testing in the setting of virologic failure or suboptimal viral suppression in patients receiving HAART[1]. The term "virologic failure" refers to the development of a rising HIV RNA level in a patient on HAART who previously had excellent virologic control (HIV RNA levels less than 50 copies/ml) (Figure 2). Unless the patient has simply stopped taking his or her medications, this clinical scenario suggests the development of resistance. Several randomized controlled trials have demonstrated the clinical benefit of resistance testing among patients with virologic failure[2,3,4]. "Suboptimal viral suppression" or "failure to suppress" describes the patient who initially shows a favorable initial response to HAART with a decrease in HIV RNA level, but is not able to drive the HIV RNA level below the level of detection (less than 50 copies/ml) (Figure 3). Suboptimal viral suppression can result from the rapid development of resistance, inadequate potency of the regimen, or both. As disccussed below, resistance testing in the setting of virologic failure is usually not recommended when the HIV RNA level is less than 1,000 copies/ml, mainly because it is hard to obtain an accurate result at very low levels of viremia.

Resistance Testing with Acute HIV Infection

In the setting of acute HIV infection, the 2007 DHHS antiretroviral therapy guidelines recommend obtaining resistance testing, regardless of whether the decision is made to initiate antiretroviral therapy[1]. The recommendation to obtain resistance testing even when the decision in made to defer therapy is based on the greater likelihood of detecting transmitted resistant early in the course of HIV infection. If therapy is deferred, the guidelines suggest considering repeat resistance testing prior to initiating antiretroviral therapy, since the patient may possibly acquire resistant HIV in the interim[1]. In a retrospective study that involved 10 North American cities and 377 patients with acute HIV infection, investigators reported that high-level phenotypic resistance increased in each of the major antiretroviral drug classes when comparing isolates obtained during 1995 to 1998 with those from 1999 to 2000 (Figure 4)[5] . Moreover, the frequency of multi-drug resistance in these patients increased from 1.1-6.2% between these two time periods. More recent studies suggest that 6-16% of newly infected patients have acquired HIV with resistance to at least one drug[1]. Among patients infected with a drug-resistant strain of HIV, time to viral suppression after initiation of therapy was longer and time to virologic failure shorter when compared with patients who did not have evidence of having resistant HIV. In a separate study that involved 225 patients diagnosed with acute HIV infection in the San Francisco Bay Area from 1996 to 2001, drug resistance among newly HIV-infected individuals increased, predominantly in the non-nucleoside reverse transcriptase class (Figure 5)[6] . This study also found patients who acquired resistant strains of HIV had a longer time to virologic suppression when treated with HAART compared with those individuals without evidence of resistant HIV (12 weeks versus 5 weeks).Taken together, these studies show a significant proportion of new HIV infections involve drug-resistant virus, the resistance rates have increased since the mid-1990's, and acquisition of resistant HIV has an impact on response to antiretroviral therapy[1,5,6].

Resistance Testing with Chronic Infection

Because most HIV-infected individuals are diagnosed with HIV long after their acute HIV illness, the first opportunity for resistance testing typically occurs years after initial HIV infection. The December 2007 DHHS antiretroviral therapy guidelines recommend performing resistance testing in the setting of chronic HIV infection at the time a patient enters into care for their HIV disease[1] . Some controversy exists regarding whether or not resistant HIV strains acquired during initial infection will persist at a high enough level to be detected if resistance testing is performed in a chronically HIV-infected person who has never received antiretroviral therapy. Among those with acute HIV infection who acquire resistant HIV strains and who do not receive antiretroviral therapy, several possible scenarios exist regarding subsequent detection of resistant HIV strains. First, in the absence of any antiretroviral selective pressure, those resistant strains of HIV may back-mutate to more fit wild-type strains, or be overgrown by wild-type virus (Figure 6). Alternatively, if particular resistant stains of HIV do not cause a loss in viral fitness, the strains could persist indefinitely and this could involve multiple distinct resistant mutations (Figure 7). Last, it is possible that some resistant mutations will revert to more fit wild-type whereas others that do not cause a significant loss in fitness could persist (Figure 8). One study that involved relatively few patients found that some multi-drug resistant strains acquired during initial infection persisted for up to 5 years as the dominant quasispecies, despite these strains being less fit than wild type strains. Regardless of whether or not resistance testing will detect transmitted strains of resistant HIV, most experts would agree that these transmitted drug-resistant variants persist in cellular reservoirs, such as latently infected CD4 cells, and thus could reemerge upon the selective pressure of antiretroviral therapy.

Resistance Testing after Discontinuation of Therapy

The December 2007 DHHS antiretroviral therapy guidelines state that drug resistant assays are usually not recommended when patients who have taken antiretroviral agents in the past, but have been off therapy for more than 4 weeks[1]. Among chronically-infected persons who develop resistance in response to antiretroviral therapy, populations of wild-type and resistant strains of HIV may co-exist, but upon discontinuation of antiretroviral therapy, replication of the more fit wild type strain generally outpaces that of the resistant strain (Figure 9). Accordingly, the resistant strain will likely become a minority species in the overall viral population. Because currently available resistance assays do not reliably identify strains of HIV that constitute a low percentage (generally defined as less than 10-20%) of the overall viral population, minority resistant strains often evade detection by resistance assays in chronically infected persons who discontinue therapy. The speed at which resistant strains become overgrown by wild type strains will likely vary from patient to patient, but available data suggest most wild-type strains predominate over resistance strains within 12 weeks of stopping antiretroviral therapy (Figure 10)[8,9] . Although in this setting resistant strains may not be evident on resistance testing, they remain archived in the host[8] and thus would presumably play a role if the patient reinitiated antiretroviral therapy that included medications to which the patient had previously developed resistance.

Resistance Testing with Low HIV RNA Levels

The 2007 DHHS antiretroviral therapy guidelines do not recommend resistance testing in the setting of a patient experiencing apparent virologic breakthrough with an HIV RNA level less than 1,000 copies/ml[1]. This recommendation exists because of technical aspects of most currently available resistance assays that require HIV viral levels of approximately 1,000 copies/ml or more to accurately identify resistant HIV. As the technology of resistance assays improves, these tests may become more accurate at detecting resistance at HIV RNA levels significantly less than 1,000 copies/ml.

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    Figure 1: 2007 DHHS Recommendations for the Use of Drug Resistance Assays Image 1. Resistance Testing Recommendations
    Figure 1
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    Figure 2. Virologic Failure on HAART

    This graph illustrates the concept of virologic failure while receiving HAART as shown by gradual increases in HIV RNA levels after a period of sustained optimal HIV RNA suppression.


    Figure 2
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    Figure 3. Suboptimal Viral Suppression after Starting HAART

    This graph illustrates the concept of failing to optimally suppress HIV RNA levels after initiating HAART. Although initally the HIV RNA levels substantially decrease, the levels never decrease to a level considered as optimal viral suppression (HIV RNA levels less than 50 copies/ml).


    Figure 3
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    Figure 4. Drug Resistance Rates in Patients Recently Infected with HIV in 10 North American Cities

    Abbreviations: NRTI = Nucleoside Reverse Transcriptase Inhibitor; NNRTI = Non-Nucleoside Reverse Transcriptase Inhibitor; PI = Protease Inhibitor; Multi = Multi-Drug-Resistant Isolates

    This graph shows the frequency of antiretroviral drug resistance among individuals recently infected with HIV who have not received antiretroviral therapy prior to drug resistance testing. The patients participated in the study in one of 10 major North American cities. As shown, the frequency of antiretroviral drug resistance represents high-level phenoyptic resistance defined by a value of at least 10 times the ratio of the 50% inhibitory concentration (IC50) when comparing the patient's HIV isolate with a known drug-sensitive reference strain. Resistance rates increased from the time period 1995-1998 to 1999-2000 in all drug classes, as well as in multi-drug resistant isolates. This figure is based on data from Little SJ, Holte S, Routy JP, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med. 2002;347:385-94.


    Figure 4
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    Figure 5. Drug Resistance Rates in Patients Recently Infected with HIV in the San Francisco Bay Area

    Abbreviations: NRTI = Nucleoside Reverse Transcriptase Inhibitor; NNRTI = Non-Nucleoside Reverse Transcriptase Inhibitor; PI = Protease Inhibitor; Multi = Multi-Drug-Resistant Isolates

    Antiretroviral drug resistance rates among individuals recently infected with HIV who had not received antiretroviral therapy declined from the time period 1996-1997 to 1998-1999, but then increased overall to the highest levels in 2000-20001; the nucleoside reverse transcriptase inhibitor class resistance rates were highest in the time period 1996-1997. As shown, the frequency of antiretroviral drug resistance represents high-level phenoyptic resistance defined by a value of at least 10 times the ratio of the 50% inhibitory concentration (IC50) when comparing the patient's HIV isolate with a known drug-sensitive reference strain. This figure is based on data from Grant RM, Hecht FM, Warmerdam M, et al. Time trends in primary HIV-1 drug resistance among recently infected persons. JAMA. 2002;288:181-8.


    Figure 5
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    Figure 6. Reversion to Predominant Wild-Type Virus After Primary HIV Infection

    Over time, without selective pressure from antiretroviral therapy, patients who have acquired resistant HIV during primary (acute) HIV infection can have a gradual decrease in the proportion of resistant isolates in the overall composition of plasma HIV. This process of diminishing HIV resistance after acute HIV infection can occur because of back mutations of resistant strains to wild-type strains, or by overgrowth of the more fit (and faster growing) wild-type virus.


    Figure 6
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    Figure 7. Persistence of Resistance Strains after Primary HIV Infection

    Despite the absence of antiretroviral selective pressure, some patients who have acquired resistant HIV during primary (acute) HIV infection can have persistence of these resistant isolates. Presumably, these resistant isolates have maintained strong virologic fitness.


    Figure 7
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    Figure 8. Persistence of Some Resistance Strains after Primary HIV Infection

    Following simultaneous acquisition of multiple resistant isolates of HIV during acute HIV infection, it is possible that over time some of the resistant isolates may decrease to the point where there are no longer detectable in plasma, whereas other isolates may persist.


    Figure 8
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    Figure 9. Reversion to Predominant Wild-Type Virus After Discontinuing HAART

    This illustration shows virologic breakthrough and the emergence of multiple resistant strains of HIV in the setting of selective pressure from HAART. After temporarily discontinuing HAART, wild type virus becomes predominant.


    Figure 9
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    Figure 10. Timing of Reversion to Predominant Wild-Type Virus After Discontinuing HAART

    These graphs show four patients who had virologic breakthrough and then discontinued their antiretroviral therapy. The graphs represent changes in drug-resistance ratio when comparing HIV isolates from the patients with known wild-type reference strains. This figure is reproduced from Deeks SG, Wrin T, Liegler T et al. Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia. N Engl J Med 2001;344:472-480. Reproduced with permission from the Massachusetts Medical Society. Copyright © 2001 Massachusetts Medical Society. All rights reserved.


    Figure 10