The detection of HIV-1 variants harboring mutations associated with decreased susceptibility to antiretroviral agents is common among antiretroviral-experienced patients [1–4], and they are increasingly found among antiretroviral-naive newly infected individuals as transmitted drug-resistant strains [5,6]. Depending on the specific mutations and their pattern, antiretroviral drug resistance may reduce the likelihood of achieving virological suppression . Moreover, as a result of existing antiretroviral class cross-resistance [8,9], constructing a new antiretroviral combination regimen with at least two active agents  may be substantially compromised, particularly in the presence of triple-class antiretroviral drug resistance (TC-DR).
Although TC-DR has considerable relevance to HIV clinical management and the development of new efficacious antiretroviral agents, relatively little is known about the overall prevalence and evolution of TC-DR in routine HIV medical care. Earlier research in North America and Europe suggests that 4–13% of HIV-1-infected antiretroviral experienced patients harbor TC-DR [1,4,11,12]. In this study, we identified patients in one large clinical cohort with evidence of TC-DR, defined here as the presence of at least one resistance mutation to each of the three widely used antiretroviral drug classes: nucleos(t)ide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), and protease inhibitors (PI). We estimated the prevalence and incidence of TC-DR, and identified factors predicting the acquisition of TC-DR.
All HIV-1-infected patients at least 18 years of age and participating in the University of North Carolina, Center for AIDS Research, HIV Cohort Study were included. This observational clinical cohort initiated in January 2000, with ongoing enrollment, has been described previously . Briefly, all HIV-1-infected patients receiving primary HIV care at the University of North Carolina HIV Clinic are eligible to participate. Cohort data include all institutionally available electronic patient records and periodic standardized medical record reviews are completed.
Genotypic antiretroviral drug resistance
For these analyses we identified all available genotype reports for each cohort participant. Genotypes were obtained as part of routine clinical care and indicated when a patient on antiretroviral therapy (ART) had at least two sequential HIV-RNA levels above 1000 copies/ml to ensure sufficient polymerase chain reaction product for analysis. Genotypic sequencing was performed using commercially available assays, with over 95% of assays using HIV Genosure or Genosure Plus (LabCorp., Research Triangle Park, North Carolina, USA). The HIV Genosure and HIV Genosure Plus sequence codons 1–99 of the protease gene and 1–400 of the reverse transcriptase gene on an ABI 3700 capillary sequencer (Applied Biosystems, Foster City, California, USA). Mutations associated with reduced antiretroviral drug susceptibility were based on the International AIDS Society – USA Panel Guidelines . In particular, mutations associated with reduced susceptibility to NRTI were defined as mutations at reverse transcriptase gene positions M41L, E44D, A62V, K65R, D67N, 69 insertion, K70R, L74V, V75I, F77L, Y115F, F116Y, V118I, Q151M, M184I/V, L210W, T215F/Y, and K219E/Q. NNRTI-associated mutations included amino acid substitutions at reverse transcriptase gene positions L100I, K103N, V106A/M, V108I, Y181C/I, Y188C/H/L, G190A/S, P225H, M230L, and P236L. Major PI mutations included amino acid changes at protease gene positions D30N, V32I, L33F, M46I/L, I47V/A, G48V, I50L/V, V82A/F/L/T/S, I84V, N88S, and L90M.
TC-DR was defined as having at least one of the above mutations to each of the three main antiretroviral drug classes (NRTI, NNRTI, and PI), excluding minor PI mutations. We estimated a cumulative genotype for each patient at each resistance test date, such that mutations represented both current and previously detected mutations . The first genotypic evidence of TC-DR, based on the calculated cumulative genotype, was defined as the date of acquiring TC-DR.
We also estimated antiretroviral resistance by relying on an online publicly available database, the Stanford HIV Drug Resistance Database, which includes a genotypic resistance interpretation algorithm . We considered Stanford inferred drug resistance scores of intermediate or high-level resistance as indicating resistance to a particular antiretroviral agent.
Monotherapy was defined as a regimen that included only one NRTI agent. HAART was defined as a combination of at least three antiretroviral agents with at least one being an NNRTI or a PI. Non-HAART combination therapy was defined as any combination antiretroviral regimen that did not meet the criteria of HAART. For all analyses we considered patients who initiated triple nucleoside therapy with zidovudine, lamivudine and abacavir, and patients who initiated with an NNRTI and a PI but no NRTI, as having initiated therapy with non-HAART combination therapy; however, we also assessed these patients separately.
All patients were followed from ART initiation until the first genotypic evidence of TC-DR, the most recent genotype, or last clinic visit, whichever occurred earlier. All patient characteristics were measured before this timepoint, including patients' nadir CD4 cell count, peak HIV-RNA level, time on ART, and the number of antiretroviral drugs received.
We estimated the prevalence of TC-DR and calculated 95% confidence intervals (CI) relying on the exact binomial distribution. To compare patients with TC-DR to patients with genotypes but without evidence of TC-DR, patients without genotypes, and all patients without TC-DR, we relied on standard hypothesis tests. This included the two-sample t-test for normally distributed variables, the Wilcoxon Mann–Whitney and Kruskal–Wallis tests for non-normally distributed variables, and the Pearson's chi-square test for categorical variables. To identify independent predictors of TC-DR we fit multivariable models using logistic regression. First we fit a full model including all patient characteristics associated with TC-DR with a P value less than 0.10 in stratified analyses. We then relied on a backwards elimination procedure to arrive at a final model including only factors predictive of TC-DR with P values less than 0.05. We fit a number of models based on different patient subgroups, including all available patients, patients with previous NRTI, NNRTI and PI exposure, and patients with available genotypes.
As HAART provision is the standard of care, we repeated the above analyses restricting to patients whose first antiretroviral regimen was HAART. In addition to control for the duration of antiretroviral drug exposure, we estimated incidence rates of TC-DR by taking the number of patients with TC-DR and dividing by the person-time of ART exposure, expressed as the number of cases of TC-DR per 1000 person-years. CI around incidence rates were calculated relying on an exact Poisson distribution. All statistical analyses were conducted using the SAS statistical package (version 9.1; SAS Institute, Inc., Cary, North Carolina, USA). This study was approved by the University of North Carolina Chapel Hill Committee on the Protection of the Rights of Human Subjects.
Of all patients participating in the University of North Carolina, Center for AIDS Research, HIV Cohort Study as of 1 January 2006 (N = 1724), 8% (N = 137) were ART naive and were excluded from further analyses. Among the remaining patients (N = 1587), one-third were women (N = 501, 32%), three-fifths were African American (N = 943, 59%), and one-third were white (N = 532, 34%). The median age was 42 years [interquartile range (IQR) 36, 48]. One-third were men who have sex with men (N = 507, 32%), and 15% had ever injected drugs (N = 244). The median nadir CD4 cell count was 131 cells/μl (IQR 30, 294), the median peak HIV-RNA level was 5.0 log10 copies/ml (IQR 4.4, 5.5), and 39% (N = 619) of patients had a previous AIDS-defining clinical condition.
Thirty-eight per cent of patients (N = 607) had at least one available genotype after initiating ART. Essentially all patients were infected with HIV-1 subtype B (N = 596, 98%), with the remainder having subtype C (N = 4), subtype H (N = 1), subtype A1 (N = 1), recombinant forms CRF01_AE (N = 1) and CRF02_AG (N = 2), and multiple recombinants (N = 2). Thirty-nine patients had genotypes available before ART initiation and these genotypes were excluded from the analyses. Only two patients had evidence of resistance before therapy initiation (one patient had a V118I and one patient a V118I and K103N) and neither of these two patients had a repeat genotype. Of 980 patients on therapy without a genotype, 38% had at most one HIV-RNA level greater than 1000 copies/ml during the analysis period, and in comparison with patients with genotypes had lower average HIV-RNA levels (median 3.3 log10 copies/ml; IQR 2.3, 4.2, versus 4.4; IQR 3.8, 4.9, P < 0.001).
Antiretroviral drug exposure
Just over one-fifth of patients initiated ART before 1995 (N = 357, 22%), 28% (N = 442) between 1995 and 1997, 28% (N = 450) between 1998 and 2000, and 21% (N = 338) between 2001 and 2005. Half of patients initiated ART with HAART (N = 789, 50%), 20% (N = 320) with non-HAART combination therapy, and 30% (N = 478) with one nucleoside. The median length of time on ART was 5.7 years (IQR 2.9, 8.6), for the entire cohort; however, this differed by the type of initial therapy, with 9.6 years (IQR 7.2, 12.0), 5.8 years (IQR 3.3, 8.3), and 3.8 years (IQR 1.9, 5.9) among patients initiating with monotherapy, non-HAART combination therapy, and HAART, respectively, P < 0.001. Overall, the median number of antiretroviral drugs patients had been exposed to was six (IQR 4, 8), and this was also different by the type of initial ART received, with seven (IQR 5, 10), six (IQR 4, 8), and four (IQR 3, 7) among patients initiating with monotherapy, non-HAART combination therapy, and HAART, respectively, P < 0.001. Virtually all treated patients received at least one NRTI (N = 1581), 66% (N = 1041) an NNRTI, 77% (N = 1215) a PI, and 47% (N = 752) had been exposed to at least one antiretroviral drug in each of the three main antiretroviral drug classes (NRTI, NNRTI and PI).
Triple-class antiretroviral drug resistance prevalence
Eight per cent of patients (N = 121) had evidence of TC-DR, with at least one NRTI, NNRTI and major PI mutation (95% CI 6%, 9%), with 3% prevalence (95% CI 2%, 4%; N = 24) among HAART initiators. Irrespective of the assumptions made about the patients who did not have genotypes available, the estimated prevalence of TC-DR was highest among patients who initiated therapy with mono or non-HAART combination therapy and among patients exposed to all three antiretroviral drug classes (NRTI, NNRTI and PI; Fig. 1). Including only patients with genotype data (N = 607), the estimated prevalence of TC-DR was 20% (95% CI 17%, 23%); however, this was substantially different among non-HAART initiators in contrast to HAART initiators; 26% (95% CI 21%, 31%) and 10% (95% CI 7%, 15%), respectively, P < 0.001. Only two TC-DR patients had not been exposed to all three antiretroviral drug classes, with both patients never having received an NNRTI (one patient had an Y188L and one patient a V108I mutation).
Triple-class antiretroviral drug resistance predictors
In bivariate analyses, sex, race, age and mode of HIV exposure were not associated with TC-DR acquisition. The patient characteristics associated with TC-DR included lower nadir CD4 cell count, higher peak HIV-RNA level, having a previous AIDS-defining clinical condition, earlier calendar year of ART initiation, non-HAART initial therapy, and receiving a greater number of antiretroviral drugs (Table 1). These factors were consistent whether comparing patients with TC-DR with all other patients with genotypes but without TC-DR, with patients without genotypes, or with all patients without TC-DR. Within the two groups defined by initial ART (non-HAART and HAART), the detection of TC-DR was not related to the duration of ART among patients with available genotypes.
In multivariable analyses, the number of previous antiretroviral drugs, but not the duration of therapy, was consistently a statistically significant independent predictor of TC-DR in all models fit (Table 2). For example, in a model among all patients with available genotypes (N = 607), the odds ratio for each additional antiretroviral agent a patient had been exposed to was 1.5 (95% CI 1.4, 1.7). Receiving a non-HAART regimen as initial ART, in contrast to a HAART regimen, was also predictive of TC-DR in all models [odds ratios (OR) ranging from 1.7 to 1.9].
Triple-class antiretroviral drug resistance among HAART initiators
Of the 121 patients with TC-DR, only 24 (20%) initiated ART with HAART. The majority of patients with TC-DR and HAART as a first regimen initiated ART with an unboosted PI (N = 21, 88%); nelfinavir (N = 15, 63%), saquinavir (N = 3, 13%), indinavir (N = 2, 8%), and full dose ritonavir (N = 1, 4%; Table 1). In analyses restricted to patients who initiated therapy with HAART, we identified identical predictors of TC-DR in bivariate analyses as those obtained among all patients. In addition, the type of initial HAART was strongly associated with the acquisition of TC-DR. In particular, patients whose initial HAART included a single, non-ritonavir-boosted PI were at especially high risk of acquiring TC-DR. Among HAART initiators, 42% (N = 329/789) started therapy with a single PI-based regimen, and of these 6% (N = 21/329) acquired TC-DR, in contrast to 1% of other HAART initiators (N = 3/460). The length of time on therapy was similar between these two groups (1591 person-years versus 1531 person-years, respectively).
These findings were confirmed in multivariable analyses in which the use of a single PI-based regimen increased the odds of acquiring TC-DR approximately fourfold in all models fit (Table 2). In addition, in multivariable models a greater number of previous antiretroviral drugs received also increased the odds of acquiring TC-DR (OR ranging from 1.3 to 1.6). A higher peak HIV-RNA level was independently predictive of acquiring TC-DR in models that included all HAART-initiating patients (i.e. assuming patients without an available genotype were without TC-DR).
Triple-class antiretroviral drug resistance incidence
The overall time contributed by the 1587 patients from ART initiation until the first genotypic evidence of TC-DR, the last genotype date, or last clinic visit, whichever occurred earlier, was 9592 person-years. Therefore, the overall incidence rate of TC-DR was 13 TC-DR cases per 1000 person-years of ART exposure (95% CI 10, 15). This differed by type of ART initiated, however, with TC-DR incidence rates per 1000 person-years of 15 (95% CI 11, 19), 16 (95% CI 11, 23), and eight (95% CI 5, 11) among patients initiating with monotherapy (N = 478), non-HAART combination therapy (N = 320), and HAART (N = 789), respectively. When limiting the analyses to only patients with available genotypes the TC-DR incidence rates per 1000 person-years were obviously higher given the smaller denominators at 31 (95% CI 24, 39), 41 (95% CI 29, 59), and 28 (95% CI 19, 42) among patients initiating with monotherapy (N = 242), non-HAART combination therapy (N = 134), and HAART (N = 231), respectively.
Among patients who initiated ART with HAART, incidence rates differed substantially by the type of initial regimen (Table 3). The highest incidence rates were observed among patients who initiated HAART with a single PI-based regimen. Patients initiating with a single PI-based regimen had incidence rates of TC-DR approaching those of patients who initiated ART with a non-HAART regimen.
Triple-class antiretroviral drug resistance mutations
The most common mutations among patients with TC-DR were observed in the reverse transcriptase gene at positions M184V/I (N = 95, 79%), T215Y/F (N = 66, 55%), and K103N (N = 70, 58%), and in the protease gene at position L90M (N = 71, 59%). TC-DR patients who initiated ART with non-HAART in comparison with HAART had similar distributions of mutations, except at a higher frequency, especially to some NRTI and PI, including M41L, L210W, T215Y/F, L10F/I/R/V, V82A/F/L/S/T, and L90M (Fig. 2a). The median number of mutations among patients with TC-DR who initiated therapy with non-HAART regimens was eight (IQR 5, 10), which was higher than that observed among patients with TC-DR who initiated therapy with HAART, which was six (IQR 4, 7), P value less than 0.001 (Fig. 2b).
Based on an online publicly available genotype interpretation system, TC-DR patients who initiated with non-HAART had lost susceptibility to a greater number of antiretroviral drugs than HAART initiators (Fig. 2c). Although the majority of TC-DR patients who initiated with HAART lost susceptibility to all three currently available NNRTI (N = 19, 79%), few had resistance to three or more PI (N = 8, 33%), and one-half had resistance to three or more NRTI (N = 12, 50%).
In all analyses patients who initiated ART with zidovudine, lamivudine and abacavir, or with one NNRTI and one PI, were considered as initiating with non-HAART combination therapy. Of the triple-nucleoside initiators, 39% (N = 19/49) had an available genotype, with most (N = 14) having only NRTI mutations and none having TC-DR. Of the dual-antiretroviral NNRTI and PI initiators (N = 9), none had a genotype. Excluding these patients or including them as HAART initiators did not affect the overall results (data not shown).
Antiretroviral drug resistance to each of the three commonly used antiretroviral drug classes may reduce the likelihood of subsequent full suppression of HIV-1 replication with currently available antiretroviral drugs, which is the goal of HIV therapy even in the most highly experienced patients [8,9,17,18]. An antiretroviral regimen with less than two active agents may not adequately suppress HIV-1 replication , leading to inferior immune reconstitution, and thereby also to adverse clinical outcomes, including mortality . Therefore, TC-DR may be associated with an increased risk of clinical progression and mortality [20,21]. If HIV-1 replication is not fully suppressed with subsequent therapy, or if therapy is not switched because of limited treatment options, patients with TC-DR may also be at further risk of acquiring additional antiretroviral drug mutations , leading to further challenges in constructing tolerable regimens able to control HIV-1 replication. Finally, as multidrug-resistant HIV-1 variants may be transmitted to others , TC-DR may affect the treatment and clinical outcomes of newly HIV-1-infected individuals [5,6].
In this study, we estimated an overall TC-DR prevalence of 8% among antiretroviral-experienced patients, and 3% among those initiating therapy with HAART. Although other studies have relied on alternative TC-DR measurements and definitions, among different patient populations, our findings are similar to recent prevalence estimates from the United Kingdom (4%) , Canada (7%) , and Italy (4%) , and are slightly lower than the HIV Cost and Services Utilization Study results representing US patients between 1996 and 1998 (13%) .
The main explanatory factor for TC-DR acquisition was a patient's antiretroviral treatment history. Nearly all TC-DR patients initiated ART with non-HAART regimens (80%) or with a regimen composed of an unboosted PI and two NRTI (17%). On average, TC-DR patients were exposed to 12 different antiretroviral agents over an average of 6 years before TC-DR was identified. In all multivariable models, non-HAART versus HAART as a first regimen increased a patient's risk of acquiring TC-DR, a logical result given the superior virological and resistance outcomes of HAART in comparison with non-HAART [23–25]. Our data suggest that although TC-DR is a prevalent and clinically important problem, the incidence of patients with TC-DR is declining as all patients with access to antiretroviral drugs should begin therapy with HAART. The proportion of patients developing TC-DR virus are likely to decline further as we observed that very few patients initiating therapy with NNRTI and none initiating with a boosted PI developed TC-DR virus, despite an average of 3.8 years of observation per patient.
Furthermore, a longer time on ART was not associated with TC-DR acquisition, suggesting that the evolution of TC-DR is not ‘just a matter of time’. Among both HAART and non-HAART initiators, a greater risk of TC-DR acquisition occurred among patients with a shorter duration on a specific HAART regimen and a greater number of regimens received. Patients who were at greatest risk of TC-DR were those who did not achieve HIV-RNA suppression predominantly because of less potent therapy (either initial non-HAART or initiation with an unboosted PI) [26–30], and were subsequently switched to new antiretroviral regimens to which they also did not have a sustained virological response. Patients with TC-DR who initiated therapy with a non-HAART regimen also had more complex resistance patterns, greater numbers of thymidine analogue and major PI mutations, and fewer agents to which their virus was susceptible.
Our findings underscore the importance of the initial response to a first antiretroviral regimen for longer-term resistance evolution outcomes. We were unable to detect a difference in TC-DR risk when comparing patients who initiated HAART with efavirenz versus a ritonavir-boosted PI. One case of TC-DR occurred among 227 patients initiated on efavirenz over 713 person-years of follow-up, and no cases of TC-DR occurred among 116 patients who initiated a ritonavir-boosted PI over 258 person-years of follow-up. Long-term follow-up of patients initiating with newer HAART combinations in clinical cohorts is obviously very important.
We may have underestimated TC-DR in the entire cohort if patients with TC-DR did not have an available genotype or if minority quasi-species with resistance mutations were not detected. Reassuringly, irrespective of the assumptions we made about TC-DR prevalence among patients without genotypes, the risk factors for TC-DR acquisition remained consistent across models. In addition, patients on therapy without a genotype had low average HIV-RNA levels (median 2000 copies/ml), with 38% having at most one HIV-RNA level greater than 1000 copies/ml, supporting the inclusion of models in which patients without genotypes were assumed not to have TC-DR.
In conclusion, a small but clinically relevant group of patients have acquired resistance to antiretroviral agents in all three of the commonly used antiretroviral drug classes. At first evidence of TC-DR, most patients are candidates for subsequent regimens with sufficient potency to suppress HIV-1 replication. Moreover, the risk of acquiring highly resistant HIV-1 variants appears to have substantially decreased with newer and more efficacious initial HAART regimens. Whereas mortality rates in patients with TC-DR may be higher than those with more sensitive virus, these rates fortunately remain low. Therefore, even as incidence falls there will be a substantial subpopulation with TC-DR virus in need of new antiretroviral agents, either in existing or new classes, with activity against multidrug-resistant variants.
Sponsorship: This research was supported by the University of North Carolina at Chapel Hill, Center for AIDS Research, National Institutes of Health funded program P30 AI50410, the University of North Carolina, General Clinical Research Center, National Institutes of Health funded program RR00046, and the University of North Carolina, Training in Sexually Transmitted Diseases and AIDS, the United States Department of Health and Human Services and National Institutes of Health funded program T32 AI07001.
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