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Havlir DV, Hellmann NS, Petropoulos CJ, et al. Drug Susceptibility in HIV Infection After Viral Rebound in Patients Receiving Indinavir-Containing Regimens. JAMA. 2000;283(2):229–234. doi:10.1001/jama.283.2.229
Author Affiliations: University of California, San Diego (Drs Havlir and Richman), San Diego Veterans Affairs Medical Center (Dr Richman); ViroLogic Inc, San Francisco, Calif (Drs Hellmann, Petropoulos, and Whitcomb); Harvard Medical School, Boston, Mass (Dr Hirsch); University of Washington School of Medicine, Seattle (Dr Collier); Washington University, St Louis, Mo (Dr Tebas); and the University of Alabama, Birmingham (Dr Sommadossi).
Context Loss of viral suppression in patients infected with human immunodeficiency
virus (HIV), who are receiving potent antiretroviral therapy, has been attributed
to outgrowth of drug-resistant virus; however, resistance patterns are not
well characterized in patients whose protease inhibitor combination therapy
fails after achieving viral suppression.
Objective To characterize drug susceptibility of virus from HIV-infected patients
who are failing to sustain suppression while taking an indinavir-containing
Design and Setting Substudy of the AIDS Clinical Trials Group 343, a multicenter clinical
research trial conducted between February 1997 and October 1998.
Patients Twenty-six subjects who experienced rebound (HIV RNA level ≥200 copies/mL)
during indinavir monotherapy (n = 9) or triple-drug therapy (indinavir, lamivudine,
and zidovudine; n = 17) after initially achieving suppression while receiving
all 3 drugs, and 10 control subjects who had viral suppression while receiving
Main Outcome Measure Drug susceptibility, determined by a phenotypic assay and genotypic
evidence of resistance assessed by nucleotide sequencing of protease and reverse
transcriptase, compared among the 3 patient groups.
Results Indinavir resistance was not detected in the 9 subjects with viral rebound
during indinavir monotherapy or in the 17 subjects with rebound during triple-drug
therapy, despite plasma HIV RNA levels ranging from 102 to 105 copies/mL. In contrast, lamivudine resistance was detected by phenotypic
assay in rebound isolates from 14 of 17 subjects receiving triple-drug therapy,
and genotypic analyses showed changes at codon 184 of reverse transcriptase
in these 14 isolates. Mean random plasma indinavir concentrations in the 2
groups with rebound were similar to those of a control group with sustained
viral suppression, although levels below 50 ng/mL were more frequent in the
triple-drug group than in the control group (P =
Conclusions Loss of viral suppression may be due to suboptimal antiviral potency,
and selection of a predominantly indinavir-resistant virus population may
be delayed for months even in the presence of ongoing indinavir therapy. The
results suggest possible value in assessing strategies using drug components
of failing regimens evaluated with resistance testing.
Complete and prolonged suppression of human immunodeficiency virus (HIV)
replication is a primary objective of antiretroviral therapy.1
Rates of viral suppression achieved by potent combination therapies exceed
90% in select clinical trial groups, but these rates are less with the same
regimens outside research settings.2-4
Rebound of plasma viremia also may occur after having suppression below level
of detectability. Major factors contributing to loss of suppression include
suboptimal drug potency, inadequate drug exposure, and insufficient regimen
adherence. A large increase in CD4 cells with therapy, providing more target
cells for virus replication, has been proposed5
and observed6 to contribute to loss of suppression.
Resistance to protease inhibitor (PI) monotherapy is characterized by
sequential acquisition of mutations conferring stepwise reductions in drug
susceptibility.7,8 Early mutant
virus appears to be fitness-disadvantaged vs wild-type virus, but later mutations
in protease and gag cleavage sites appear to compensate for this.9-12 Early
reports of PI resistance featured patients failing monotherapy or having a
PI added to their regimen. Multiple protease-resistance mutations were present
in virus isolated from these patients.7,8
However, these patients' regimens had not suppressed the virus fully, thus
providing opportunity for selection of virus with resistance mutations. Resistance
patterns in patients failing PI combination therapy following suppression
are less well characterized.
We describe drug susceptibility in 26 trial participants achieving suppression
with indinavir, zidovudine, and lamivudine followed by loss of suppression.
Nine patients were receiving only indinavir when rebound was observed.
Subjects were a subset of the AIDS Clinical Trials Group 343 (ACTG 343)
participants (for whom eligibility criteria were CD4 cells ≥200 ×
106/L, HIV RNA level ≥1000 copies/mL, limited treatment [<7
days] with PIs, and no prior treatment with lamivudine or abacavir).6 The goal for ACTG 343 was to assess whether suppression
achieved by potent triple-drug therapy could be sustained with less intensive
therapy. Subjects (n = 509) were prescribed 6 months of open-label induction
therapy with indinavir, 800 mg every 8 hours, lamivudine, 150 mg twice daily,
and zidovudine, 300 mg twice daily. Levels of HIV RNA were assayed at 4-week
intervals. Treatment discontinuation was recorded but detailed adherence studies
were not performed. Subjects with HIV RNA levels less than 200 copies/mL after
16, 20, and 24 weeks of induction therapy were randomized (blinded) in the
maintenance phase to receive indinavir monotherapy (n = 100), zidovudine plus
lamivudine (n = 104), or all 3 drugs (n = 105). Loss of suppression (plasma
HIV RNA levels of ≥200 copies/mL) was the primary study end point. Subjects
reaching a study end point had the option to resume triple-drug therapy. Of
those receiving indinavir and those receiving zidovudine plus lamivudine,
23% in each arm had rebound early during maintenance vs 3% of those continuing
triple-drug therapy.6 The first available specimens
were assessed, as per resource constraints, from 9 of 23 subjects (indinavir
group) with rebound after switching to indinavir, 17 of 75 subjects (triple-drug
therapy group) with at least 1 HIV RNA level of less than 200 copies/mL during
induction and 10 of 178 subjects (control group) receiving triple therapy
with sustained suppression throughout the trial. Subjects provided written
informed consent. Given the expectation that more than 95% of subjects would
have indinavir-resistant virus at virologic rebound, there was a greater than
99.9% probability that at least 1 subject in the group of 9 patients and 99.9%
probability that at least 1 subject in the group of 17 would have indinavir-resistant
virus at rebound.
Resistance was evaluated using a phenotypic assay for drug susceptibility
(PhenoSense, ViroLogic Inc, San Francisco, Calif) on baseline and follow-up
plasma samples from all patients in the indinavir, triple-drug, and control
groups as previously reported.13 Drug susceptibility
was quantified by determining the 50% inhibitory concentration (IC50) of drug assayed in vitro of a recombinant test strain incorporating
protease and reverse transcriptase gene segments from patient isolates in
the presence of protease and reverse transcriptase inhibitors compared with
a control (NL4-3) strain. The IC50 values greater than 2.5-fold
those of the drug-susceptible reference strain indicated reduced susceptibility
based on assay validation studies.14
Sequence analysis of drug-resistance mutations in reverse transcriptase
and protease genes was done using population-based sequence analysis (PE Biosystems,
Foster City, Calif) on all resistance test-vector plasmid pools evaluated
for evidence of resistance by phenotypic assay. Amino acid substitutions identified
via comparison with NL4-3 were reported. As with the PhenoSense assay, the
sequencing results represent the majority species of the HIV RNA amplified
from plasma, except in 1 case involving 1 subject. In a prior study evaluating
the sensitivity of this method for detecting mixtures of virus pools with
5 HIV polymerase gene polymorphisms, we found that the majority population
was readily detectable.15 Minority species
may not be uniformly detected by this method. Resistance mutations were classified
as primary or secondary based on recent consensus guidelines.16
Indinavir concentrations were measured in a central laboratory using
high-pressure liquid chromatography on plasma from 29 subjects with available
banked plasma samples from time points coinciding with ACTG 343 protocol visits.
After extracting indinavir with ethyl t-butyl ether, indinavir and an internal
standard were back-extracted from the organic layer following acidification.
Repeat extraction of indinavir and the internal standard with methyl t-butyl
ether was performed after basification and the final organic was decanted
and evaporated. The residue was dissolved with a phosphate buffer and acetonitrile
mixture, and the extract was analyzed using high-pressure liquid chromatography
with column switching. Chromatograph peaks were monitored by assessing absorbance
at 210 nm.
The standard curve range for the indinavir assay ranged from 5 to 500
ng/mL. Precision and inaccuracy were 5.0% and 5.8%, respectively, at the low
standard, and 1.6% and 0.7% at the high standard. The indinavir concentration
was weighted by number of indinavir measures (range, 4-8) for each subject.
Mean and median of indinavir values for each subject were used to generate
weighted mean and median indinavir concentrations for each group. These values
were compared using Kruskall-Wallis analysis of variance. The proportion of
subjects with at least 1 indinavir value of less than 50 ng/mL was compared
among the 3 groups using Fisher exact test.
During induction of triple-drug therapy, suppression below a plasma
HIV RNA level of 50 copies/mL was achieved in the 9 subjects subsequently
randomized to indinavir maintenance monotherapy (mean baseline HIV RNA level,
46,109 copies/mL). Four subjects had HIV RNA levels of less than 50 copies/mL
by 8 weeks, 2 by 12 weeks, and 3 by 16 weeks. Rebound was detected 2 to 8
weeks after subjects switched to maintenance therapy. Peak HIV RNA levels
during rebound ranged from 103 to 105 copies/mL.
Viral isolates were assayed for drug susceptibility and drug-resistance
mutations 3 to 14 weeks after the switch to indinavir monotherapy, and for
3 subjects were assessed at 2 sequential time points (Table 1). Levels of HIV RNA ranged from 102 to 105 copies/mL in the samples collected at the same time points used for
drug susceptibility testing. Viral isolates at baseline and during rebound
showed no reduction in susceptibility to indinavir or to PIs nelfinavir, ritonavir,
Nucleotide sequencing detected no primary mutations known to be associated
with indinavir resistance (codons 46 and 82). Changes at codons 10, 20, 24,
32, 54, 63, 71, 73, and 90 were reported in patients with indinavir resistance
and classified as secondary mutations.16 Persons
infected with HIV may have changes at these codons prior to therapy. Subjects
4 and 7 had L63P at baseline, and subjects 1 and 2 had this substitution identified
in rebound isolates. Subject 3 had L10I at baseline.
After loss of suppression was confirmed, subjects were encouraged to
change therapy. Five of the 9 subjects discontinued study participation after
rebound was detected. Four subjects resumed open-label, triple-drug therapy
with indinavir, zidovudine, and lamivudine. At the time zidovudine and lamivudine
were added back to the indinavir monotherapy regimen, the viral loads had
been greater than 200 copies/mL for 2 to 8 weeks. Suppression was achieved
by 4 weeks in 3 subjects and sustained for 7 to 10 months. Initial suppression
was lost 4 months after all 3 drugs were resumed in the fourth subject.
No significant changes in indinavir susceptibility were detected during
rebound in the 17 patients receiving triple-drug therapy despite peak HIV
RNA levels during rebound of 1864 to 138,989 copies/mL (Table 2) (a representative subject's experience is illustrated
in Figure 1). The primary indinavir-resistance
mutation M46L was identified in subject 24 at week 35 but not week 41. Antiretroviral
therapy interruption with reduction of selective pressure shortly after the
week 35 visit may explain the reappearance of wild-type virus at week 41.
Secondary-resistance mutations were present at baseline at codon 63 (9 subjects),
codon 10 (5 subjects), and codon 71 (2 subjects), but no new secondary indinavir-resistance
mutations appeared in any rebound isolate. Duration of observation during
rebound (mean, 6 months; range, 1-12 months) was longer in patients failing
triple-drug therapy vs those with rebound when receiving indinavir maintenance
therapy (mean, 1 month; range, 0.5-2.5). Although encouraged to switch to
alternative antiretroviral regimens, patients chose to continue taking this
triple-drug therapy due in part to the limited number of other regimens available
at that time.
In 14 of the 17 subjects, lamivudine resistance was detected with the
phenotypic assay in viral isolates obtained during rebound. Sequencing confirmed
that the methionine to valine substitution at codon 184 of reverse transcriptase,
known to confer high-level resistance to lamivudine, was present in all 14
isolates. In 13 of the 14 subjects with lamivudine resistance, lamivudine
susceptibility decreased by more than 100-fold at rebound vs the control isolate.
In 1 of the 14 subjects, a mixture of isolates with methionine and valine
were present, and susceptibility to lamivudine was 7-fold less than that in
the control group. Rebound isolates were sensitive to lamivudine in 3 subjects.
In analyses from a separate pharmacokinetic study (J-P.S., unpublished data,
1999), indinavir concentrations were undetectable at weeks 12, 20, and 35
in subject 24, who had no resistance to lamivudine, suggesting prescribed
medications were not taken.
Detectable indinavir concentrations were present in 98% of samples from
the indinavir group, 72% from the triple-drug group, and 82% from the control
group. At least 4 samples per patient were assessed (mean, 5.4 per patient).
Indinavir levels were obtained both during suppression and rebound in 7 patients
in the triple-therapy group and 2 patients in the indinavir group. Levels
were obtained during suppression for the other subjects. Weighted mean indinavir
concentrations were 1486, 1429, and 1627 ng/mL for indinavir, triple-drug,
and control groups, respectively, and were not significantly different (Table 3). In the triple-therapy group,
mean indinavir concentration was 990 ng/mL during suppression and 1280 ng/mL
during rebound (P = .74). Although weighted mean
indinavir concentrations did not differ significantly among groups, the proportion
of patients with at least 1 indinavir level below 50 ng/mL was higher in the
group failing triple therapy vs the control group (P
= .03; Table 3).
In earlier studies of PI resistance in patients receiving combination
antiretroviral therapy, patients received sequential therapy and plasma HIV
RNA levels were only partially suppressed.17
Under these conditions, PI-resistant virus emerged rapidly. These observations
and similar ones involving PI monotherapy18
led to the generally held assumption that when suppression failure occurs
with a regimen containing a PI, PI-resistant virus accounts for HIV RNA rebound.
Failure to detect resistance in some patients was attributed to regimen nonadherence.19,20 The results from this study and others
challenge this view and suggest that suboptimal antiviral potency permits
rebound, and that selection of a predominantly PI-resistant virus population
may be delayed for months.21,22
The patients in this study had suppressed viral load to below 50 copies/mL
while taking triple-drug therapy. Suppression was then lost either when continuing
triple therapy or when switching to indinavir maintenance therapy. In both
groups, indinavir levels were detectable in most samples tested and indinavir-sensitive
virus was the predominant population identified during rebound. In most patients
continuing to receive lamivudine as part of triple-drug therapy, virus was
lamivudine-resistant phenotypically and genotypically at the time of rebound.
Outgrowth of indinavir-sensitive, lamivudine-resistant virus with continuing
treatment pressure may be explained by viral fitness and antiviral potency.
By definition, the predominant virus replicating under a set of selective
pressures is the most fit. For lamivudine or non–nucleoside reverse
transcriptase inhibitors such as nevirapine or efavirenz, a single nucleotide
change can confer a 20- to 1000-fold reduction in susceptibility.23-26 In
the presence of drugs, the mutant virus is so much more fit that it will predominate.
Clinical data confirm that when antiviral potency of a regimen containing
one of these drugs is insufficient to suppress replication, drug-resistant
virus rapidly emerges.27-29
Most patients failing triple therapy herein had lamivudine resistance. In
a study of isolates from patients with rebound when taking an efavirenz and
indinavir combination regimen, most isolates were resistant to efavirenz.30
Why did indinavir-sensitive virus appear in patients continuing therapy?
Possible factors include impaired fitness of early indinavir-resistant mutant
virus, reduced antiviral potency, and an increase in target cells. In contrast
to lamivudine and non–nucleoside reverse transcriptase inhibitors, development
of high-level resistance to PIs and zidovudine requires the accumulation of
For PIs, the first mutation confers only limited reduction in susceptibility,
usually less than 10-fold.33 Also, the first
mutations adversely affect protease function and virus replication.9-11,34 Thus,
a virus with 1 or 2 mutations is less fit than wild type, even in the presence
In those receiving indinavir maintenance therapy, reduction in antiviral
potency (ie, discontinuation of zidovudine and lamivudine) allowed increased
viral replication. Because the wild-type virus had a fitness advantage over
early mutant virus, it was the predominant population for months. Replication
may also have been enhanced by an increase in target cells. In patients randomized
to maintenance therapy in ACTG 343, loss of suppression was most likely in
those with the greatest increment in CD4 cell number,6
supporting predator-prey models proposed to explain viral dynamics in patients
receiving zidovudine.35 The models were later
extended to induction-maintenance treatment strategies.5
In these models, increased numbers of target cells resulting from treatment
provide better conditions for the virus when suppression is incomplete.
Based on prior studies of indinavir monotherapy, one would expect that
had patients failing indinavir maintenance therapy not been switched back
to more potent regimens, indinavir-resistant virus would have become the predominant
population. Continuing growth of the breakthrough virus in the presence of
drug selects for an accumulation of mutations conferring both reduced susceptibility
and compensation for the adverse impact of resistance mutations on protease
function and virus replication. Compensatory mutations have been well characterized
both in protease outside the substrate binding site and in protease cleavage
sites in gag.9-11,34
The maximum period of observation of indinavir maintenance failures was 3
months. It is probable that selection of early indinavir-resistant mutant
virus occurred, but that the prevalence remained below the limit of detection
of the assay used to assess drug susceptibility.
In patients failing triple-drug therapy, diminished antiviral potency
(as a result of suboptimal adherence or drug delivery) undoubtedly contributed
to rebound. Although the specimen collection schedule was not designed to
assess indinavir exposure, evaluation of random samples for indinavir levels
suggested that patients taking triple-drug therapy that was failing had more
dosing interruptions than the indinavir maintenance group (data not shown).
Brief periods of low or undetectable drug levels may have allowed unabated
replication and the fitness disadvantage of early indinavir-resistant mutant
virus may have allowed sensitive virus to predominate for months.
In terms of alternative hypotheses to explain outgrowth of virus wild
type in protease with indinavir, the presence of p7/p1 or p1/p6 gag cleavage-site
mutations were ruled out by the sequencing, which also excluded the theoretical
possibility of a gag-pol frameshift mutation resulting in increased expression
of protease. Also, drug efflux transporters could have diminished indinavir's
effect and not been detected via measure of indinavir levels. This possibility
is supported by the recent recognition of P glycoprotein transporters that
can serve as protease efflux pumps in vitro.36,37
Our findings have several clinical implications. First, in patients
failing suppressive antiretroviral combination regimens, the predominant virus
population may be resistant to 1 (ie, lamivudine), but not all (ie, PI) components
of the regimen. Second, not all drugs in a failing regimen (defined as a rebound
in HIV RNA levels) may be lost options. Third, these data suggest that drug-resistance
testing early after loss of suppression may be useful in identifying components
of a failing regimen that might be useful in a new combination regimen. These
results suggest value in assessing strategies using drug components of a failing
combination evaluated by resistance testing. However, systematic studies are
needed to address concerns that retaining part of a regimen that appears sensitive
on resistance testing could lead to selection of resistant minority species
that may contribute to virologic failure of the new regimen and reduced treatment
options. Finally, it must be acknowledged that PI-sensitive virus in patients
taking a failing regimen is not necessarily evidence of nonadherence.
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