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Author Affiliation: Divisions of Clinical Pharmacology and Infectious Diseases, Departments of Medicine and Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Md.
Fourteen antiretroviral drugs are now Food and Drug Administration (FDA)–approved
for the treatment of human immunodeficiency virus (HIV) infection. Patients
must take combination therapy perpetually because rapid virus turnover and
high mutation rates promote drug resistance. The increasing prevalence of
resistance has prompted development of drug resistance assays, and high-throughput
techniques are now commercially available.
This issue of THE JOURNAL contains recommendations, developed by an
international panel of experts, for the use and interpretation of anti-HIV
drug resistance assays.1 Two major analytic
approaches are discussed: genotypic assays that identify particular mutations,
usually point mutations, associated with resistance; and phenotypic assays
that measure the ability of the patient's virus to grow in the presence of
known concentrations of anti-HIV drugs. Although these techniques are scientifically
sophisticated and exciting, they present dilemmas for the clinician and patient.
First, these assays are expensive. Despite automation, charges for a
single genotypic assay are in the range of $400 to $550; for a phenotypic
assay, $700 to $1000. Most third-party payers do not cover these assays. If
an insurer denies payment, the patient is charged, or, in a large academic
referral center, the center often bears the cost.
Turnaround time is slow for both assays—usually 2 to 4 weeks from
the time blood is drawn until results reach the physician. This is too slow
to have an impact in many clinical settings; information is needed quickly
for managing occupational needlestick exposures and primary, or acute, HIV
infection. But the problems of cost and time are overshadowed by the reliability
of these tests.
Both genotypic and phenotypic assays have fundamental problems with
sensitivity and specificity. Genotypic assays generally provide better sensitivity
than phenotypic assays, with the capacity to detect a virus that constitutes
as little as 10% of the patient's circulating virus population. But this depends
on plasma HIV RNA concentration (viral load) at the time of sequencing and
may miss some mutants that constitute up to 50% of the patient's viral pool.2,3 Because these assays amplify sequences
from a mixture of HIV virus, only the predominant variants, or subspecies,
within an individual are amplified.
Not surprisingly, academic laboratories frequently underestimate genotypic
resistance.4 In large part, this reflects an
"ongoing need for adequate standardization and clinical validation" of the
assays.1 Questions also exist about the relative
difficulty in amplifying DNA from virus of different HIV-1 clades.
Both resistance assays require a plasma HIV concentration of 500 to
1000 RNA copies/mL to generate sufficient polymerase chain reaction product
for analysis. Viral load assays used for monitoring patients now have sensitivities
down to at least 50 copies/mL. Resistance assays are therefore not reliable
in patients with viral loads of 50 to 1000 copies/mL, many of whom are now
considered to have failed therapy.
Phenotypic assays also produce false-negative results; samples can contain
a mixture of drug-sensitive and drug-resistant virus, thus underestimating
resistance. Patients who have had treatment failure with multiple drugs may
harbor low levels of drug-resistant virus in quiescent CD4+ lymphocytes,
even if such mutant strains are not detectable in the plasma.5,6
Because a drug-resistant virus can grow out rapidly and become the dominant
virus within a few weeks under pressure of drug treatment, it is important
to identify these minority populations. Therefore, a negative resistance assay
provides little reassurance to the clinician or patient.
To avoid false-negative results, the International AIDS Society–USA
(IAS-USA) recommendations indicate that blood for resistance testing should
be drawn while the patient is still taking a failing regimen.1
Some physicians may find this impractical, even unethical.
Low specificity is also a concern with both types of assays. In the
2 commercially available phenotypic tests, resistance is defined as a 2.5-
to 4-fold increase in the concentration of drug required to inhibit viral
replication by 50% (IC50).1 By comparison,
pneumococcal resistance to penicillin is defined as at least a 33-fold increase
in the concentration of drug required to inhibit bacterial growth.7 If HIV drug resistance phenotyping standards were
applied to penicillin, many patients would be denied a safe, effective, and
inexpensive antibiotic. A more biologically rational definition of resistance
incorporates the relationship between drug sensitivity and drug concentration
in the patient.
Point mutations affect HIV drug sensitivity differently for different
drug classes. A single point mutation may increase the IC50 1000-fold
for nonnucleoside reverse transcriptase inhibitors (NNRTIs),8
while single point mutations generally increase the IC50 for protease
inhibitors only 3- to 5-fold.9 To use the penicillin
analogy, if in vitro sensitivity changes by only a few fold, then the drug
should still inhibit replication of the organism in the patient if drug concentrations
are many fold higher than the IC50 of mutants with low- or intermediate-level
False-positive results also can occur with genotypic drug resistance
testing. Although 20% of patients in 1 report had baseline mutations associated
with some degree of NNRTI resistance, initial response to efavirenz-containing
regimens was as good as that in similar patients who lacked these mutations,10 thus calling into question the clinical relevance
and predictive value of the test.
The complexity of interpreting genotypic results limits their value
for the nonexpert. For example, some mutations, such as M184V in reverse transcriptase,
reduce susceptibility to lamivudine but increase susceptibility to zidovudine.
High-level resistance to protease inhibitors may require accumulation of 3
or more key mutations, and drug sensitivity may be affected by mutations outside
the normal genetic "hot spots." Some gag and gag-pol mutations involving protein cleavage sites are
outside the protease gene and can confer resistance to protease inhibitors.11 Genotypic assays do not allow examination of such
sequences. The 2 phenotypic assays amplify the protease and reverse transcriptase
genes from virus in patient plasma and insert them into a recombinant vector
system in which other HIV genes are derived from a standard laboratory strain,
which misses resistance conferred by mutations outside the target genes. A
"virtual" phenotype can be created by reporting the average drug sensitivity
of virus containing a given set of mutations.12
However, to be complete such analyses become complex: a gene as simple as
that for HIV-1 protease, which contains only 99 amino acid codons, produces
4297 possible genotypes and nearly as many phenotypes.
Incorporating these assays into the management of HIV-infected patients
may seem difficult. Several retrospective studies found an association between
genotype or phenotype and virologic response to therapy, as described by Hirsch
et al.1 It is no surprise that drug resistance
is a risk factor for treatment failure; the important question is whether
access to genotype or phenotype data benefits the patient. To date, only 3
prospective controlled studies of the clinical utility of resistance testing
have been reported,13-15
and, of these, just 1 was published in a peer-reviewed journal.13
None of these studies was blinded, and, for 1, only preliminary data were
presented.15 The positive and negative predictive
values of resistance testing were not calculated in these studies, although
all reported better short-term virologic outcome with access to resistance
In the VIRADAPT Study,13 treatment-experienced
patients for whom genotype data were available had a slightly better mean
viral load decrease after 6 months (1.15 vs 0.67 log10 copies/mL)
and were about twice as likely to have a viral load less than 200 copies/mL
(32% vs 14%) than patients who received standard care without genotyping.
In the Genotypic Antiretroviral Resistance Testing (GART) Trial,14
patients were randomized to treatment recommendation by an expert panel with
access to resistance data or to standard care. Short-term results were similar
to those of the VIRADAPT study, with a modest improvement in mean viral load
decline (1.17 vs 0.62 log10 copies/mL) in the tested group after
8 weeks. However, since the test group was the recipient of regimen advice
from an expert panel and the control group was not, it is possible that clinical
benefit was the consequence of expert opinion rather than genotype data.
In preliminary analysis of outcomes in 127 treatment-experienced patients
in a trial to examine the benefit of phenotypic resistance testing (VIRA3001),
a greater mean viral load decrement at week 16 (0.87 vs 0.43 log10
copies/mL by intention to treat) and more patients with a viral load of less
than 400 copies/mL (62% vs 33%) were observed in the group with access to
However, the 2 genotyping trials (VIRADAPT and GART) were conducted
in patients with baseline viral loads greater than 10,000 and 26,000 copies/mL,
respectively. Whether these results will apply to patients with lower viral
loads, or to treatment-naive subjects, is unknown.
In the IAS-USA recommendations published in this issue of THE JOURNAL
resistance testing is advocated for all patients who have failed a treatment
regimen and for pregnant HIV-infected women. The IAS-USA group also suggests
consideration of resistance testing for patients with primary infection and
prior to starting treatment in patients with established infection. This covers
every HIV-infected patient except those who refuse therapy or are already
fully suppressed on an established regimen. The firmest grounds for recommending
resistance testing—as an adjunct for patients failing their first or
subsequent multiple-drug regimen—are weakened by the fact that other
important causes of treatment failure, such as nonadherence, intermittent
inadequate drug concentrations, or both, may be difficult, if not impossible,
to exclude with assurance.
To recommend testing all HIV-infected pregnant women may be premature
because assessment of the mother's treatment history and treatment response
may provide sufficient information to avoid initiating inactive drugs. Some
drugs may be active in preventing vertical transmission of HIV even if the
maternal HIV appears to be genotypically or phenotypically resistant. The
fetus should receive drugs with a track record and known safety profile rather
than untested antiretroviral combinations.
A somewhat different classification scheme distinguishes primary from
secondary resistance. In primary resistance, the patient is infected by a
drug-resistant virus variant. In secondary resistance, the patient acquires
drug resistance mutations during therapy. These have very different treatment
implications. Primary resistance for most approved antiretroviral drugs is
uncommon at present but may be increasing in prevalence.16,17
It is in this setting that resistance testing is likely to have its greatest
impact. Secondary resistance is a common cause of treatment failure, but studies
and models suggest that this is most likely to be caused by noncompliance.18 Intervention for secondary resistance should focus
more on improving adherence and regimen tolerance and less on picking effective
agents from a computer printout.
Cost-effectiveness is also an important consideration. As the authors
of the recommendations point out, resistance testing should not be used as
"the principal criterion for deciding when to initiate or change therapy."1 How much, then, should patients and third-party payers
be willing to spend on such a diagnostic adjunct? If genotypic or phenotypic
testing is ordered several times a year for the same patient, charges could
exceed $3000. For some patients with secondary treatment failure, it seems
that those same monies might be more effectively spent, for example, on directly
observed therapy managed by a visiting home nurse.
Of note, many of the shortcomings of resistance testing outlined herein
were pointed out in the HIV treatment guidelines published by the IAS-USA
group in January 2000.19 The recommendations
presented in this issue of THE JOURNAL endorse the value of resistance testing
in several clinical settings despite being unable to approach the issues in
a traditional evidence-based manner, a limitation alluded to by the authors
themselves. An HIV-specialist panel assembled by the US Department of Health
and Human Services, in guidelines published electronically at about the same
time, also supports resistance testing.20
To paraphrase the IAS-USA recommendations, when determining antiretroviral
treatment, practitioners must consider treatment history, viral load, tolerance,
adherence, and concomitant medications and disease.1
However, despite the great promise of these technologies, it is still unclear
how much resistance testing adds beyond the careful consideration of these
other important factors. Only controlled, prospective studies can define the
magnitude of benefit. Adequate evidence may never exist if resistance testing
becomes standard practice now.
Flexner C. HIV Genotype and Phenotype—Arresting Resistance? JAMA. 2000;283(18):2442–2444. doi:10.1001/jama.283.18.2442
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