Daniel R. Kuritzkes

Antiretroviral Drug Resistance Testing in Adult HIV-1 Infection: 2008 Recommendations of an International AIDS Society-USA Panel

Resistance to antiretroviral drugs remains an important limitation to successful human immunodeficiency virus type 1 (HIV-1) therapy. Resistance testing can improve treatment outcomes for infected individuals. The availability of new drugs from various classes, standardization of resistance assays, and the development of viral tropism tests necessitate new guidelines for resistance testing. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in drug-resistant HIV-1, drug management, and patient care to review recently published data and presentations at scientific conferences and to provide updated recommendations. Whenever possible, resistance testing is recommended at the time of HIV infection diagnosis as part of the initial comprehensive patient assessment, as well as in all cases of virologic failure. Tropism testing is recommended whenever the use of chemokine receptor 5 antagonists is contemplated. As the roll out of antiretroviral therapy continues in developing countries, drug resistance monitoring for both subtype B and non-subtype B strains of HIV will become increasingly important.

Drug Resistance Mutations for Surveillance of Transmitted HIV-1 Drug-Resistance: 2009 Update

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions.

Antiretroviral Drug Resistance Testing in Adults Infected with Human Immunodeficiency Virus Type 1: 2003 Recommendations of an International AIDS Society-USA Panel

New information about the benefits and limitations of testing for resistance to human immunodeficiency virus (HIV) type 1 (HIV-1) drugs has emerged. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in antiretroviral drug management, HIV-1 drug resistance, and patient care to provide updated recommendations for HIV-1 resistance testing. Published data and presentations at scientific conferences, as well as strength of the evidence, were considered. Properly used resistance testing can improve virological outcome among HIV-infected individuals. Resistance testing is recommended in cases of acute or recent HIV infection, for certain patients who have been infected as long as 2 years or more prior to initiating therapy, in cases of antiretroviral failure, and during pregnancy. Limitations of resistance testing remain, and more study is needed to refine optimal use and interpretation.

Treatment with Lamivudine, Zidovudine, or Both in HIV-Positive Patients with 200 to 500 CD4+ Cells per Cubic Millimeter

BACKGROUND The reverse-transcriptase inhibitor lamivudine has in vitro synergy with zidovudine against the human immunodeficiency virus (HIV). We studied the activity and safety of lamivudine plus zidovudine as compared with either drug alone as treatment for patients with HIV infection, most of whom had not previously received zidovudine. METHODS Three hundred sixty-six patients with 200 to 500 CD4+ cells per cubic millimeter who had received zidovudine for four weeks or less were randomly assigned to treatment with one of four regimens: 300 mg of lamivudine every 12 hours; 200 mg of zidovudine every 8 hours; 150 mg of lamivudine every 12 hours plus zidovudine; or 300 mg of lamivudine every 12 hours plus zidovudine. The study was double-blind and lasted 24 weeks, with an extension phase for another 28 weeks. RESULTS Over the 24-week period, the low-dose and high-dose regimens combining lamivudine and zidovudine were associated with greater increases in the CD4+ cell count (P = 0.002 and P = 0.015, respectively) and the percentage of CD4+ cells (P < 0.001 for both) and with greater decreases in plasma levels of HIV-1 RNA (P < 0.001 for both) than was treatment with zidovudine alone. Combination therapy was also more effective than lamivudine alone in lowering plasma HIV-1 RNA levels and increasing the percentage of CD4+ cells (P < 0.001 for all comparisons), and these advantages persisted through 52 weeks. Adverse events were no more frequent with combination therapy than with zidovudine alone. CONCLUSIONS In HIV-infected patients with little or no prior antiretroviral therapy, treatment with a combination of lamivudine and zidovudine is well tolerated over a one-year period and produces more improvement in immunologic and virologic measures than does treatment with either agent alone.

Workshop on HIV Infection and Aging: What Is Known and Future Research Directions

Highly active antiretroviral treatment has resulted in dramatically increased life expectancy among patients with HIV infection who are now aging while receiving treatment and are at risk of developing chronic diseases associated with advanced age. Similarities between aging and the courses of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome suggest that HIV infection compresses the aging process, perhaps accelerating comorbidities and frailty. In a workshop organized by the Association of Specialty Professors, the Infectious Diseases Society of America, the HIV Medical Association, the National Institute on Aging, and the National Institute on Allergy and Infectious Diseases, researchers in infectious diseases, geriatrics, immunology, and gerontology met to review what is known about HIV infection and aging, to identify research gaps, and to suggest high priority topics for future research. Answers to the questions posed are likely to help prioritize and balance strategies to slow the progression of HIV infection, to address comorbidities and drug toxicity, and to enhance understanding about both HIV infection and aging.

Immunologic Responses Associated with 12 Weeks of Combination Antiretroviral Therapy Consisting of Zidovudine, Lamivudine, and Ritonavir: Results of AIDS Clinical Trials Group Protocol 315

Human immunodeficiency virus (HIV)-1 infection is associated with progressive cell-mediated immune deficiency and abnormal immune activation. Although highly active antiretroviral therapy regimens can increase circulating CD4 T lymphocyte counts and decrease the risk of opportunistic complications, the effects of these treatments on immune reconstitution are not well understood. In 44 persons with moderately advanced HIV-1 infection, after 12 weeks of treatment with zidovudine, lamivudine, and ritonavir, plasma HIV-1 RNA fell a median of 2.3 logs (P < .0001). Circulating numbers of naive and memory CD4 T lymphocytes (P < .001), naive CD8 T lymphocytes (P < .004), and B lymphocytes (P < .001) increased. Improved lymphocyte proliferation to certain antigens and a tendency to improvement in delayed-type hypersensitivity also were seen. Dysregulated immune activation was partially corrected by this regimen; however, the perturbed expression of T cell receptor V regions in the CD4 and CD8 T lymphocyte populations was not significantly affected. Ongoing studies will ascertain if longer durations of virus suppression will permit more complete immune restoration.

HIV-1 protease and reverse transcriptase mutations for drug resistance surveillance

Objectives:Monitoring regional levels of transmitted HIV-1 resistance informs treatment guidelines and provides feedback on the success of HIV-1 prevention efforts. Surveillance programs for estimating the frequency of transmitted resistance are being developed in both industrialized and resource-poor countries. However, such programs will not produce comparable estimates unless a standardized list of drug-resistance mutations is used to define transmitted resistance. Methods:In this paper, we outline considerations for developing a list of drug-resistance mutations for epidemiologic estimates of transmitted resistance. First, the mutations should cause or contribute to drug resistance and should develop in persons receiving antiretroviral therapy. Second, the mutations should not occur as polymorphisms in the absence of therapy. Third, the mutation list should be applicable to all group M subtypes. Fourth, the mutation list should be simple, unambiguous, and parsimonious. Results:Applying these considerations, we developed a list of 31 protease inhibitor-resistance mutations at 14 protease positions, 31 nucleoside reverse transcriptase inhibitor-resistance mutations at 15 reverse transcriptase positions, and 18 non-nucleoside reverse transcriptase inhibitor-resistance mutations at 10 reverse transcriptase positions. Conclusions:This list, which should be updated regularly using the same or similar criteria, can be used for genotypic surveillance of transmitted HIV-1 drug resistance.

Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group.

A standardized antiviral drug susceptibility assay for clinical human immunodeficiency virus type 1 (HIV-1) isolates has been developed for use in clinical trials. The protocol is a two-step procedure that first involves cocultivation of patient infected peripheral blood mononuclear cells (PBMC) with seronegative phytohemagglutinin-stimulated donor PBMC to obtain an HIV-1 stock. The virus stock is titrated for viral infectivity (50% tissue culture infective dose) by use of serial fourfold virus dilutions in donor PBMC. A standardized inoculum of 1,000 50% tissue culture infective doses per 10(6) cells is used in the second step of the procedure to acutely infect seronegative donor PBMC in a 7-day microtiter plate assay with triplicate wells containing zidovudine (ZDV) concentrations ranging from 0 to 5.0 microM. The ZDV 50% inhibitory concentrations (IC50) for reference ZDV-susceptible and ZDV-resistant HIV-1 isolates ranged from 0.002 to 0.113 microM and from 0.15 to > 5.0 microM, respectively. Use of this consensus protocol reduced interlaboratory variability for ZDV IC50 determinations with reference HIV-1 isolates. Among eight laboratories, the coefficient of variation ranged from 0.85 to 1.25 with different PBMC protocols and was reduced to 0.39 to 0.98 with the standardized assay. Among the clinical HIV-1 isolates assayed by the standardized drug susceptibility assay, the median ZDV IC50 increased gradually with more ZDV therapy. This protocol provides an efficient and reproducible means to assess the in vitro susceptibility to antiretroviral agents of virtually all clinical HIV-1 isolates.

Low-Frequency HIV-1 Drug Resistance Mutations and Risk of NNRTI-Based Antiretroviral Treatment Failure A Systematic Review and Pooled Analysis

CONTEXT Presence of low-frequency, or minority, human immunodeficiency virus type 1 (HIV-1) drug resistance mutations may adversely affect response to antiretroviral treatment (ART), but evidence regarding the effects of such mutations on the effectiveness of first-line ART is conflicting. OBJECTIVE To evaluate the association of preexisting drug-resistant HIV-1 minority variants with risk of first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral virologic failure. DATA SOURCES Systematic review of published and unpublished studies in PubMed (1966 through December 2010), EMBASE (1974 through December 2010), conference abstracts, and article references. Authors of all studies were contacted for detailed laboratory, ART, and adherence data. STUDY SELECTION AND DATA ABSTRACTION Studies involving ART-naive participants initiating NNRTI-based regimens were included. Participants were included if all drugs in their ART regimen were fully active by standard HIV drug resistance testing. Cox proportional hazard models using pooled patient-level data were used to estimate the risk of virologic failure based on a Prentice weighted case-cohort analysis stratified by study. DATA SYNTHESIS Individual data from 10 studies and 985 participants were available for the primary analysis. Low-frequency drug resistance mutations were detected in 187 participants, including 117 of 808 patients in the cohort studies. Low-frequency HIV-1 drug resistance mutations were associated with an increased risk of virologic failure (hazard ratio (HR], 2.3 [95% confidence interval {CI}, 1.7-3.3]; P < .001) after controlling for medication adherence, race/ethnicity, baseline CD4 cell count, and plasma HIV-1 RNA levels. Increased risk of virologic failure was most strongly associated with minority variants resistant to NNRTIs (HR, 2.6 [95% CI, 1.9-3.5]; P < .001). Among participants from the cohort studies, 35% of those with detectable minority variants experienced virologic failure compared with 15% of those without minority variants. The presence of minority variants was associated with 2.5 to 3 times the risk of virologic failure at either 95% or greater or less than 95% overall medication adherence. A dose-dependent increased risk of virologic failure was found in participants with a higher proportion or quantity of drug-resistant variants. CONCLUSION In a pooled analysis, low-frequency HIV-1 drug resistance mutations, particularly involving NNRTI resistance, were significantly associated with a dose-dependent increased risk of virologic failure with first-line ART.

Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response

The duration of disease-free survival after infection with human immunodeficiency virus type 1 (HIV-1) varies considerably during antiretroviral therapy. Patients with similar CD4+ lymphocyte counts progress at different rates when they are given the same antiretroviral therapy. Better prediction of risk for progression and its association with viral suppression may help improve antiretroviral management for individual patients and speed the development of new drugs. Higher plasma HIV-1 RNA levels are associated with poorer clinical status and lower CD4+ lymphocyte counts [1-3] and predict subsequent outcome [4-11]. The biological variability of plasma HIV-1 RNA levels in patients receiving stable therapeutic regimens must be quantified to define the magnitude of an antiviral effect that can be reliably detected after antiretroviral treatment is initiated. Determination of infectious HIV-1 titers in mononuclear cells of peripheral blood by quantitative microculture [12, 13] or syncytium-inducing phenotype of an HIV-1 isolate may provide information that is different from or complementary to the information gleaned from measuring plasma HIV-1 RNA levels [14-16]. However, studies have not yet conclusively determined whether measurements of CD4+ lymphocytes in conjunction with any or all of these virological variables should be recommended to optimize prediction or guide antiretroviral treatment more effectively. In this report, we quantify the relative roles of CD4+ lymphocyte counts, plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and the syncytium-inducing viral phenotype as predictors of disease progression during a clinical trial of combination therapy [17]. Our approach was to assess the value of plasma HIV-1 RNA levels and CD4+ lymphocyte count, both of which are readily available to clinicians, and then to assess the additional value of the infectious HIV-1 titer in mononuclear cells of peripheral blood and the syncytium-inducing viral phenotype. We also quantify the variability of plasma HIV-1 RNA levels. Our results suggest guidelines for using these measures in clinical practice for predicting the effectiveness of antiretroviral therapy over 1 year. Methods Study Design We prospectively evaluated virological, immunologic, and clinical data from patients who participated in the intensive virology substudy of ACTG (AIDS Clinical Trials Group) Protocol 241; ACTG Protocol 241 was a multicenter, randomized, double-blind, placebo-controlled trial of 398 patients receiving nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine [17]. All patients at 8 of the 16 AIDS Clinical Trials Units who participated in the main study were enrolled in the substudy (n = 198). For 48 weeks, all 198 patients received open-label zidovudine (600 mg/d) and didanosine (400 mg/d for patients weighing 60 kg and 250 mg/d for patients weighing <60 kg). One hundred of the substudy patients were randomly assigned to receive nevirapine (200 mg/d for the first 2 weeks and 400 mg/d thereafter), and 98 were assigned to receive matching placebo. Participants gave written informed consent, and the protocol was approved by the institutional review board at each participating AIDS Clinical Trials Unit. The study was funded by the ACTG of the National Institute of Allergy and Infectious Diseases; supplemental funding for virology was provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, Connecticut). Study drugs were provided by Glaxo Wellcome (Research Triangle Park, North Carolina), Bristol-Myers Squibb (Princeton, New Jersey), and Boehringer Ingelheim Pharmaceuticals. However, all data were gathered by members of the ACTG and were analyzed and interpreted by the authors, who had sole responsibility for the decision to submit the manuscript for publication. Evaluation of Patients Stable therapy at baseline was defined as the absence of reported change in antiretroviral therapy from 30 days before the preentry visit until the entry visit. All patients were followed prospectively for progression of HIV-related disease. Progression was defined as the development of a new acquired immunodeficiency syndrome (AIDS)-defining event [18]; a newly diagnosed, deep-seated bacterial infection or bacteremia that was not related to injection drug use or an intravascular catheter; pulmonary or extrapulmonary tuberculosis; recurrent Pneumocystis carinii pneumonia; recurrent toxoplasmosis of the central nervous system; or death. Reports of disease progression were reviewed by the study chair; only events that could be confirmed were used in the analysis. We measured CD4+ lymphocyte counts, plasma HIV-1 RNA levels, and infectious HIV-1 titers in mononuclear cells of peripheral blood at the preentry visit (within 14 days of starting study treatment), at the entry visit (before starting study treatment and at least 72 hours after the preentry visit), and at the visits 8 and 48 weeks after the start of study treatment. Specimens could be obtained at any time of day. We used the geometric mean of preentry and entry measurements as the baseline value for each variable. The presence of the syncytium-inducing viral phenotype was determined at the entry visit. Standardized assays were used to determine CD4+ lymphocyte counts [19, 20], infectious HIV-1 titer in mononuclear cells of peripheral blood (in infectious units per million cells) using real-time testing [13, 21], and syncytium-inducing viral phenotype of a virus isolated from mononuclear cells of peripheral blood using MT-2 cells [22]. Plasma samples were frozen at 70C; HIV-1 RNA levels were measured by quantitative reverse transcription polymerase chain reaction assay (Roche Molecular Systems, Alameda, California, and Branchburg, New Jersey) [23]. The lower limit of detection for this assay was 200 HIV-1 RNA copies/mL. Levels of HIV-1 RNA in plasma samples collected from the same patient at the preentry, entry, week 8, and week 48 visits were determined in a single laboratory assay. Statistical Analysis Analysis of plasma HIV-1 RNA levels and infectious HIV-1 titers in mononuclear cells of peripheral blood was done after log10 transformation. Plasma levels of HIV-1 RNA that were below the detectable limit were assigned the value of 200 copies/mL. Infectious HIV-1 titers in mononuclear cells of peripheral blood outside the measurable range (0.22 to 7493 infectious units per million cells) were assigned the value of 0.22 infectious units per million cells if they were below the range and 7493 infectious units per million cells if they were above the range. Linear regression analysis [24] was used to compare the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts according to patient characteristics at baseline and to assess factors associated with the long-term change (from baseline to week 48) in CD4+ lymphocyte counts. Logistic regression analysis [25] was used to assess the association at baseline of the percentage of patients who had AIDS with virological measures and CD4+ lymphocyte counts. The intrapatient SD of plasma HIV-1 RNA levels was estimated using the method of moments for variance components [26]. Spearman correlation coefficients were used to assess the association between preentry and entry measurements. Proportional hazards models [27] were used to assess the association between the risk for disease progression or death and baseline levels and early changes (from baseline to week 8) in plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and log-transformed CD4+ lymphocyte counts as well as baseline syncytium-inducing viral phenotype. These models were stratified by study treatment to control for any differential effects of the two study regimens. Results Patient Characteristics at Study Entry The mean CD4+ lymphocyte count of the 198 patients before treatment was 145 cells/mm3 (range, 1 to 443 cells/mm3). Patients were a median of 39 years of age, predominantly male (81%), predominantly white (76%), and predominantly free of a previous AIDS-defining diagnosis (86%). All but 3 patients had taken zidovudine before study entry, 44% had taken didanosine, and 35% had taken zalcitabine. The median duration of cumulative previous nucleoside therapy was 25 months, and 34% of patients had received therapy for longer than 36 months. Virological Measures at Baseline by Patient Characteristics Table 1 shows the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts at baseline for patients stratified by characteristics that were significantly associated with viral load. We also assessed the associations with age, sex, racial or ethnic group, self-reported homosexuality, and duration of previous nucleoside therapy, but these associations were not significant. Table 1. Plasma HIV-1 RNA Level, Infectious HIV-1 Titer in Mononuclear Cells of Peripheral Blood, and CD4+ Lymphocyte Count at Baseline* Patients with a history of AIDS had a significantly higher mean baseline level of HIV-1 RNA in plasma and a significantly lower mean CD4+ lymphocyte count than did those without such a history (Table 1). More patients with a history of AIDS than those without had baseline HIV-1 isolates with the syncytium-inducing viral phenotype (58% compared with 36%; P = 0.015). However, in a multivariate analysis, only the CD4+ lymphocyte count at baseline was significantly associated with a history of AIDS. Thus, disease status at baseline was explained by CD4+ lymphocyte counts and not by any of the virological measures that were considered. Variability of Virological Measures in Patients Receiving Stable Treatment Variation in plasma HIV-1 RNA levels was evaluated by comparing the preentry and entry measures from the 167 patients who reported no changes in treatment from 30 days