BACKGROUND:
The role of minor (low frequency) drug-resistant variants in failure of
antiretroviral therapy is not defined. In ACTG 398, 212 non-nucleoside
reverse transcriptase inhibitor (NNRTI)-experienced and 269 NNRTI-naive
patients were randomized to efavirenz, abacavir, adefovir and amprenavir
with a second protease inhibitor (PI) or placebo. This study provided the
opportunity to examine relations between NNRTI experience, baseline NNRTI
resistance, virological response and the emergence of efavirenz resistance.
METHODS:
Genotypes of baseline plasma were obtained in 452 of 481 patients by a
standard method [ViroSeq version 2.0 kit (Applied Biosystems)].Mutations
were classified according to the IAS-USA table. Minor NNRTI-resistant
variants were sought with two methods: single genome RT-PCR and sequencing (SGS)
and a yeast-based chimeric Ty1/HIV-1 RT retrotransposon system that measures
the frequency of efavirenz resistance. Phylogenetic analyses were performed
with the neighbour joining method (PHYLIPv3.573c).
RESULTS:
Virological failure (confirmed HIV RNA >200 copies/ml) was associated with
NNRTI experience (P<0.001), baseline NNRTI mutations (P<0.001),
and development of efavirenz resistance (P<0.001). Standard
genotyping did not detect NNRTI mutations in baseline samples from 50 of 216
(23%) NNRTI-experienced patients. Virological outcome in this group,
however, was not better than in the group (n=166) with baseline NNRTI
mutations. By contrast, among all patients with negative baseline genotypes
for NNRTI mutations, virological outcome was significantly better at weeks
24 (P=0.015) and 48 (P=0.02) in NNRTI-naive patients (n=237)
compared with NNRTI-experienced patients (n=50). These findings
suggested that standard genotyping may not have adequately detected
NNRTI-resistant variants. Baseline plasma from a random sample of 10
NNRTI-experienced and eight NNRTI-naive patients who had virological failure
despite a negative baseline genotype for NNRTI mutations were tested for
minor NNRTI-resistant variants. Variants encoding NNRTI-resistance mutations
were identified by SGS in six of 10 NNRTI-experienced patients with the
following frequencies per positive patient: 181C and 190A (5 of 15
sequences); 181C (3 of 19); 181C (3 of 22); 108I (2 of 35); 103N (1 of 33);
and 103N (1 of 34). By comparison, NNRTI-resistant variants were found in
only one of eight NNRTI-naive patients: 100I (1 of 33 sequences). The
Ty1/HIV-1 RT assay detected efavirenz-resistant yeast colonies in 8 of 10
NNRTI-experienced patients with the following frequencies: 10.9, 6.7, 6.4,
3.3, 2.0, 1.6, 1.3 and 0.8%. In NNRTI-naive patents, resistant colonies were
found in two of eight patients with frequencies of 0.6 and 0.3%.
Phylogenetic analysis showed close clustering of baseline NNRTI-resistant
variants identified by SGS with the genotype at virological failure in five
of six NNRTI-experienced patients. In the NNRTI-naive patient, the L100I
mutant was not evident at failure and did not cluster with the failure
genotype.
CONCLUSIONS:
Prior NNRTI experience selects minor NNRTI-resistant variants that are
often missed by standard genotyping and can lead to failure of
efavirenz-based regimens.
