Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 1999, p. 264-270, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Rapid Non-Culture-Based Assay for Clinical Monitoring of
Phenotypic Resistance of Human Immunodeficiency Virus Type 1 to
Lamivudine (3TC)
J. Gerardo
García
Lerma,1
Raymond F.
Schinazi,2
Amy S.
Juodawlkis,2
Vincent
Soriano,3
Yulin
Lin,2
Kathleen
Tatti,2
David
Rimland,2
Thomas M.
Folks,1 and
Walid
Heneine1,*
HIV and Retrovirology Branch, Division of
AIDS, STD, and TB Laboratory Research, Centers for Disease Control
and Prevention, Atlanta, Georgia1;
Georgia Veterans Affairs Research Center for AIDS and HIV
Infections, and Department of Pediatrics and Department of Medicine,
Emory University, Decatur, Georgia2; and
Servicio de Enfermedades Infecciosas, Hospital Carlos III,
Instituto de Salud Carlos III, Madrid, Spain3
Received 6 July 1998/Returned for modification 29 August
1998/Accepted 31 October 1998
 |
ABSTRACT |
Monitoring for lamivudine (3TC) resistance is important both for
the clinical management of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with 3TC and for surveillance of
transmission of 3TC-resistant HIV-1. We developed a novel
non-culture-based assay for the rapid analysis of phenotypic
resistance to 3TC of HIV-1 in plasma. The assay measures the
susceptibility of HIV-1 reverse transcriptase (RT) activity to 3TC
triphosphate (3TC-TP) in plasma. RT detection was done by the Amp-RT
assay, an ultrasensitive PCR-based RT assay. Under our
assay conditions, we found that 5 µM 3TC-TP inhibited RT activity
from wild-type (WT), zidovudine-resistant, or nevirapine-resistant
HIV-1 but not from HIV-1 carrying either the M184V mutation or
multidrug (MD) resistance mutations (77L/116Y/151M or
62V/75I/77L/116Y/151M). Mixing experiments showed a detection threshold
of 10% 3TC-resistant virus (M184V) in a background of WT
HIV-1. To validate the assay for the detection of phenotypic resistance of HIV-1 to 3TC in plasma samples, HIV-1 RT in 30 plasma specimens collected from 15 patients before and during therapy with 3TC
was tested for evidence of phenotypic resistance by the Amp-RT
assay. The results were compared with those of genotypic analysis. The RT in 12 samples was found to be 3TC sensitive, while the
RT in 18 samples had evidence of phenotypic resistance. All 12 samples
with 3TC-sensitive RT had WT genotypes at codon 184 and were retrieved
before treatment with 3TC. In contrast, all 18 specimens with
3TC-resistant RT were posttherapy samples. This assay provides a
simple, rapid, and reliable method for the detection of phenotypic
resistance of HIV-1 to 3TC in plasma.
| |
INTRODUCTION |
Lamivudine [3TC;
(
)-
-2',3'-dideoxy-3'-thiacytidine] is one of several
nucleoside analogs that are currently approved for the treatment of
human immunodeficiency virus type 1 (HIV-1) infections (5).
3TC has potent anti-HIV-1 activity and minimal toxicity, and its
triphosphate (3TC-TP) inhibits HIV-1 reverse transcriptase (RT) by
acting as a competitive inhibitor of 2'-deoxycytidine-5'-triphosphate (dCTP) and as a chain terminator (1). 3TC is one of the most commonly used drugs in combination therapy as first-line treatment for
HIV-1-infected patients (4, 5). 3TC administered in combination with zidovudine (AZT) and protease inhibitors slows the
progression of HIV-1 disease and reduces levels of HIV-1 RNA to less
than 500 copies per ml in 90% of patients for as long as 1 year
(13).
The use of 3TC in both monotherapy or combination therapy, however, has
resulted in the emergence of 3TC-resistant variants of HIV-1 (13,
21, 33, 40). This resistance is conferred by mutations at codon
184 of the HIV-1 RT gene, in which the wild-type (WT) methionine (M;
ATG) residue is replaced with either a valine (V; GTG) or an isoleucine
(I; ATA) residue (3, 31, 38). The presence of the M184V
mutation has been associated with a >500-fold resistance to 3TC and
with the loss of the antiretroviral and clinical benefits of 3TC
(41).
It is therefore important to monitor HIV-1 for 3TC resistance in
patients treated with 3TC. Phenotypic assays provide definitive information on resistance to 3TC and are well suited for assessments of
the complex resistance patterns that may arise from combination therapy. However, most phenotypic assays developed to date are based on
virus isolation and culture and are therefore labor intensive, costly,
and unsuitable for rapid clinical monitoring or surveillance of drug
resistance. In addition, these assays are fraught with biologic
variabilities, including those related to viral isolation and tropism
(23, 25). To circumvent the problem of virus isolation and
tropism, recombinant virus assays in which an infectious
virus is generated by recombination of patient-derived RT
sequences with an RT-deleted HIV-1 backbone were developed
(16, 22). However, these improved assays still require
2 to 3 weeks and may not be easily adapted to clinical laboratories.
In the absence of rapid phenotypic assays, several genotypic tests are
being used to monitor for the presence of resistance mediated by the
M184V mutation (21, 33, 37). However, clinical monitoring of
3TC resistance by genotypic testing may not detect resistance mediated
by unrecognized mutations. In addition, genotypic testing cannot detect
potential synergistic or antagonistic effects of complex mutation
patterns arising from combination therapy with different RT inhibitors.
The transient suppression of phenotypic resistance to AZT conferred by
the M184V or the L74V mutation illustrates the effect that combinations
of mutations may have in a given phenotype (26, 36, 38).
In this report, we describe the development and application of a rapid
non-culture-based assay for the analysis of phenotypic resistance of
HIV-1 to 3TC in plasma samples. The assay is based on the direct
analysis of the susceptibility of plasma HIV-1 RT to inhibition by
3TC-TP. We describe the ability of the assay to successfully detect the
phenotypic resistance of HIV-1 to 3TC in plasma samples from
3TC-treated persons. We also identify resistance to 3TC in HIV-1 RT
carrying mutations associated with resistance to multiple
nucleoside analogs (multidrug [MD] resistance).
| |
MATERIALS AND METHODS |
Principle of the phenotypic analysis of 3TC resistance.
The
phenotypic assay is based on the analysis of the susceptibility of the
RT activity of HIV-1 from plasma to inhibition by 3TC-TP. RT activity
in plasma is detected by the Amp-RT assay, an ultrasensitive
PCR-based RT assay (12, 14, 43). The susceptibility of the
RT activity in plasma to 3TC-TP is determined on the basis of the level
of inhibition produced by 3TC-TP and is measured by running
quantitative Amp-RT reactions in the presence and absence of
3TC-TP.
RT analysis by the Amp-RT assay.
Amp-RT detects RT
activity by using a known nonretroviral heteropolymeric RNA template
and a complementary DNA oligonucleotide primer. The cDNA is detected by
PCR amplification and probing with an internal oligonucleotide
(12, 14, 43).
For detection of RT activity in culture supernatant, 10 µl of the
supernatant was used directly in the Amp-RT assay. For testing of
plasma, a volume of 100 µl was clarified by centrifugation at
10,000 × g for 5 min and was then ultracentrifuged at
a fixed angle at 99,000 × g for 1 h at 4°C. The
viral pellet was resuspended in 100 µl of RT buffer (50 mM Tris-HCl,
50 mM KCl, 10 mM MgCl2), and aliquots of 2 to 10 µl were
used for analysis by the Amp-RT assay.
RT levels were quantitated by enzyme-linked immunosorbent assay,
with a standard curve generated with known units of RT activity
from a
reference HIV-1 stock (
12). The Amp-RT signals
were expressed
as units of RT activity per milliliter and reflect the
average
of duplicate or triplicate results. Qualitative detection of
the
Amp-RT products was done by Southern blot hybridization as
described
previously (
12).
Analysis of phenotypic resistance to 3TC by the Amp-RT
assay.
For RT detection and analysis of phenotypic resistance to
3TC, samples (10 µl of culture supernatant or virus pellets from 2 to
10 µl of plasma) were applied to an RT buffer containing 10 ng of
encephalomyocarditis virus RNA template, 10 U of RNasin, 100 ng of
5'-biotin-labeled EMCR2 antisense primer, 20 µM (each) dATP, dGTP,
and dTTP, and 5 µM dCTP (12). To determine the
susceptibility of the RT to 3TC-TP, an additional Amp-RT
reaction was done in the presence of 3TC-TP. Reactions were
incubated at 37°C for 2 h and then heated at 95°C for 5 min to
destroy RT activity. PCR amplification of encephalomyocarditis virus
cDNA was made as described previously (12) after the
addition of each deoxynucleoside triphosphate at a concentration of 200 µM. Quantitative detection of the products obtained by the Amp-RT
assay was done as described above.
Susceptibility of HIV-1 RT to 3TC-TP was determined from the level of
inhibition of RT activity by 3TC-TP. We calculated the
percentage of
inhibition by using the ratio of the RT level obtained
in the
Amp-RT reactions containing 3TC-TP to the RT level seen
in
Amp-RT reactions done in the absence of 3TC-TP multiplying
that
ratio by 100. The drug concentrations resulting in 50 and
90%
inhibition (IC
50 and IC
90, respectively) were
also determined
by testing RT in the presence of several 3TC-TP
concentrations.
These values were calculated by the method of Chou and
Talalay
(
6).
Detection of 3TC resistance mutations.
Mutations at codon
184 of HIV-1 RT were detected by using the HIV-1 Line probe assay
(LiPA), which detects both WT Met and mutant Val (37).
Study population.
A total of 30 EDTA-anticoagulated plasma
samples from 15 HIV-1-infected patients from the Veteran Affairs
Medical Center in Decatur, Ga., were studied. The samples were
collected from patients before and during antiretroviral therapy with
3TC. The antiretroviral therapy histories for all patients are
presented in Table 1. The
Amp-RT-based phenotypic assay was done under code with respect to
the date of serial bleeding and RT genotype. One plasma specimen from a
blood donor who tested antibody negative for HIV-1, HIV-2, human T-cell
leukemia virus type I (HTLV-I), and HTLV-II was used in the assay as a
negative control.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Direct analysis of phenotypic resistance of HIV-1 RT
activity to 3TC in plasma from 15 HIV-1-infected persons by
determination of levels of RT inhibition
by 3TC-TPa
|
|
Viruses and 3TC-TP.
For assay development and validation,
HIV-1 molecular infectious clones xxBRUpitt and
M184Vpitt were used as WT and 3TC-resistant (M184V
mutation) HIV-1 reference strains, respectively (31). Other
reference viruses included M184V/Y181CEU,
181Y/CEU, and HIV-1RTMC/MT-2, which represented
3TC- and nevirapine-resistant (M184V/Y181C), nevirapine-resistant
(Y181C), and AZT-resistant (D67N/K70R/T215F/K219Q) HIV-1 isolates,
respectively (24). WT HIV-1SUM9 and multiple
dideoxynucleoside-resistant isolates HIV-1SUM8 (Q151M
mutation), HIV-1SUM12 (F77L/F116Y/Q151M), and
HIV-1SUM13 (A62V/V75I/F77L/F116Y/Q151M) were kindly
provided by H. Mitsuya (35).
3TC-TP was synthesized by the method of Ludwig and Eckstein
(
28). The crude 3TC-TP was purified by fast-performance
liquid
chromatography with HiLoad 26/10, a Q Sepharose Fast Flow
Pharmacia
column, and a gradient of TEAB (triazol ammonium bicarbonate)
buffer (pH 7.0). The compound was characterized by UV, proton,
and
phosphorous nuclear magnetic resonance imaging, mass spectroscopy,
and
high-pressure chromatography. The concentration of 3TC-TP
resulting in
50% inhibition of incorporation of [
3H]dCTP into an
(rI)
n-dC
12-18 template primer by
recombinant p66/p51 HIV-1 RT (Biotechnology General, Rehovot,
Israel)
was 1.3 µM, as determined by the decrease in the formation
of
acid-insoluble product compared to the amount formed by the
untreated
control (
10,
30).
| |
RESULTS |
Test conditions that differentiate WT and 3TC-resistant HIV-1
RTs.
To determine the optimal ratio of 3TC-TP and dCTP needed to
inhibit WT but not 3TC-resistant RT, we tested RT activity from WT
(xxBRUpitt) and 3TC-resistant (M184Vpitt)
HIV-1 molecular clones in the presence of increasing concentrations of
3TC-TP (from 0.1 to 10 µM) and a fixed concentration of 5 µM dCTP.
Since previous reports have shown that augmented chain termination by
3TC-TP is observed with decreasing concentrations of both 3TC-TP and dCTP, we used a low concentration of dCTP in the RT step
(2). We selected the dCTP concentration of 5 µM because it
was found to be the lowest concentration that did not compromise the
performance of the Amp-RT assay (data not shown), which previously
was done with excess dCTP (12).
Figure
1A illustrates the inhibition seen
with 10
7 U of RT activity, in which complete inhibition
of RT activity from WT but
not from 3TC-resistant virus was
accomplished with 5 µM 3TC-TP.
This concentration of 3TC-TP resulted
in the inhibition of 10
7 and 10
8 U of RT
activity from WT HIV-1, which are equivalent to 10
5 and
10
4 HIV-1 particles of the reference virus per ml,
respectively (Fig.
1B) (
12). With a higher input of RT
(10
6 U of RT activity; the equivalent to 10
6
HIV-1 particles of the reference virus per ml), these conditions
did
not result in complete inhibition. The residual RT activity
in the
Amp-RT reaction containing 3TC-TP was found to be 0.4%
of the
Amp-RT signal from the control reaction that had no 3TC-TP.
This
reduction in RT signal is equivalent to a 2.35 log
10
drop
in virus load as detected by the Amp-RT assay. No
significant
inhibition of the 3TC-resistant HIV-1 was seen when
it was tested
with either a high or a low input of RT, demonstrating
the ability
of the assay to distinguish between WT and 3TC-resistant
RTs within
a wide range of RT levels (Fig.
1B). On the basis of these
results,
we adopted as a primary screening assay in all subsequent
tests
for the detection of 3TC resistance the Amp-RT assay
conditions
in which 5 µM 3TC-TP and 5 µm dCTP were used,
unless otherwise
indicated.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
Detection of phenotypic resistance to 3TC by measuring
levels of RT inhibition by the Amp-RT assay. (A) Inhibition of
107 U of RT activity from WT (xxBRUpitt) and
3TC-resistant (M184Vpitt) HIV-1 by different concentrations
of 3TC-TP. (B) Inhibition of RT activity (from 106 to
108 U) from WT (xxBRUpitt) and 3TC-resistant
(M184Vpitt) HIV-1 by 5 µM 3TC-TP.
|
|
To demonstrate that the Amp-RT-based phenotypic assay is specific
for the detection of 3TC resistance, we tested several HIV-1
reference
clones with well-characterized phenotypic resistance
to nucleoside and
nonnucleoside RT inhibitors. Figure
2
shows
that resistance to 3TC was seen only with RTs carrying the M184V
mutation. As expected, HIV-1 RTs carrying AZT (D67N, K70R, T215F,
K219Q) or nevirapine (Y181C) resistance mutations were all found
to be
susceptible to 3TC-TP. These results confirm that the assay
was
specific for viruses with phenotypic resistance to 3TC and
indicate
that the presence of other mutations associated with
AZT and nevirapine
resistance does not affect the inhibition of
RT activity by 3TC-TP.
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 2.
Analysis of the specificity of the Amp-RT-based
phenotypic assay for detection of 3TC resistance by testing RTs from
several HIV-1 reference clones in the presence and absence of 5 µM
3TC-TP. Lane 1, WT clone xxBRUpitt; lane 2, 3TC-resistant
clone M184Vpitt; lane 3, nevirapine-resistant clone
181C/YEU; lane 4, 3TC- and nevirapine-resistant clone
M184V/Y181CEU; lane 5, AZT-resistant clone
HIV-1RTMC/MT-2; lane 6, water control (Neg, negative).
|
|
Detection threshold of 3TC resistance in mixtures of WT and
3TC-resistant viruses.
To determine the detection threshold of the
Amp-RT-based assay, we tested mixtures of WT
(xxBRUpitt) and 3TC-resistant (M184Vpitt) virus
for evidence of 3TC resistance. The reference viruses were adjusted so
that they had similar levels of RT activity before virus mixtures were
prepared. We compared the level of inhibition of RT activity by 3TC-TP
observed in each mixture with the proportion of 3TC-resistant virus
used. The assay detection threshold was found to be 10% 3TC-resistant
viruses in a background of WT HIV-1 (Fig.
3A). A good correlation between the
proportion of virus carrying the M184V mutation and the level of
inhibition was also observed. For instance, in mixtures containing 25 or 75% 3TC-resistant virus, the observed inhibition was 87 and 26%,
respectively, which very likely represents the signals from the
3TC-resistant RT and therefore suggests that only WT RT activity was
inhibited.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 3.
Evaluation of the detection threshold of 3TC resistance
by the Amp-RT assay and comparison with the genotypic analysis at
codon 184 by the HIV-1 LiPA. (A) Levels of RT inhibition by 3TC-TP (5 µM) in mixtures of WT clone xxBRUpitt and 3TC-resistant
HIV-1 clone M184Vpitt. (B) Genotypic detection of the M184V
mutation in the same mixtures by the HIV-1 LiPA. Conj. control,
conjugate control.
|
|
We also used the same mixtures to compare the detection threshold of
the Amp-RT-based phenotypic assay with the genotypic
detection
threshold for viruses carrying the M184V mutation by
the HIV-1 LiPA
(Fig.
3B). The detection threshold for 3TC-resistant
virus by the LiPA
was also 10%, indicating that both assays can
reliably detect low
levels of either genotypic or phenotypic resistance
to 3TC. However,
the signal intensities in mixtures containing
50% WT and 50%
3TC-resistant viruses were not similar in the LiPA
(Fig.
3B). This may
be due to different levels of HIV-1 RNA in
both reference viruses
resulting from adjustment of virus RT activity
rather than adjustment
of RNA levels or to different efficiencies
in the hybridization of the
WT- or 184V-specific
probes.
MD resistance mutations confer phenotypic resistance to 3TC.
To determine if mutations other than 184V confer resistance to 3TC, we
analyzed HIV-1 clones containing mutations associated with resistance
to several dideoxynucleoside analogs. The IC50s and
IC90s of 3TC for viruses containing one (Q151M;
HIV-1SUM 8), three (F77L/F116Y/Q151M;
HIV-1SUM 12), and all five
(A62V/V75I/F77L/F116Y/Q151M; HIV-1SUM 13) mutations
associated with MD resistance were determined by the Amp-RT assay.
Control WT HIV-1 RTs were also tested.
The RT from HIV-1 carrying the Q151M mutation had a slightly reduced
susceptibility to 3TC, with IC
50s and IC
90s
approximately
twofold higher than those for WT reference viruses (Fig.
4 and
Table
2). However, the presence of additional
MD resistance mutations
resulted in higher levels of resistance to 3TC,
with an increase
in IC
50s of about six- and eightfold for
virus with three or all
five MD resistance mutations, respectively,
compared to the IC
50s
and IC
90s for WT viruses.
These results suggest that these MD
resistance mutations in HIV-1 RT
confer phenotypic resistance
to 3TC.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 4.
Determination by the Amp-RT assay of the
IC50s and IC90s of 3TC-TP for HIV-1 RT carrying
MD resistance mutations in the presence of increasing concentrations of
3TC-TP. HIV-1SUM9 and xxBRUpitt are WT;
Y181CEU is nevirapine resistant, M184Vpitt is
3TC resistant, and HIV-1SUM8, HIV-1SUM12, and
HIV-1SUM13 are MD-resistant HIV-1 clones.
|
|
Analysis of phenotypic resistance to 3TC in plasma HIV-1 RT and
correlation with mutations at codon 184.
We evaluated the
performance of the Amp-RT-based phenotypic assay with plasma
samples by testing 30 specimens collected from 15 HIV-1-infected
patients before and during treatment with 3TC. The results are shown in
Table 1. Plasma HIV-1 RT in all pretreatment samples
(n = 12) had WT phenotype, with RT inhibition values of >95%. The observed inhibition of the RT activity in these samples ranged from 95.9 to 100% (mean, 98.7% ± 1.8%; median, 99.8%). Of
these samples, 11 had WT viral genotypes at codon 184 and one had a
mixture of viruses of the WT and mutant (M184I) genotypes (sample 7A).
Interestingly, mutations at codon 69, which are associated with
resistance to dideoxycytosine (ddC) (11, 32), were observed in viruses in samples from the two 3TC-naive individuals whose RT had
lower susceptibilities to 3TC-TP (samples 13A and 15A; RT inhibition
values, 95.9 and 95.5%, respectively).
In contrast, values of RT inhibition of
95% were seen only for
viruses in samples obtained from patients after 1 to 60 weeks
of
antiretroviral therapy with 3TC (
n = 18). By LiPA,
viruses
in 12 of these samples had the M184V mutation, viruses in 4 samples
had mixtures of WT and M184V genotypes, and viruses in 2 samples
(samples 10A and 14A) had only WT genotypes. The mean
inhibition
in the samples with viruses with evidence of only the 184V
mutation
was 30.8% (median, 24.9%), reflecting the high frequency of
3TC-resistant
viruses. The mean inhibition of RT activity in samples
with mixtures
of viruses with WT and resistant genotypes was 49.3%
(median,
52.4%), indicating lower levels of resistance, which was
expected
since the viruses in these samples have higher proportions of
WT RT. The lowest level of resistance to 3TC among viruses from
posttherapy samples was seen in two specimens collected after
1 and 4 weeks of therapy (samples 14A and 10A; RT inhibition values,
94.7 and
87.8%, respectively). The viruses in both samples had
the WT genotype
at codon 184, and the virus in sample 10A had
ddC and AZT resistance
mutations (D69 and L41/R70/Y215, respectively).
The inability to detect
the M184V mutation among the viruses in
these samples by LiPA may
likely be due to difficulty in detecting
the M184V mutation at the
detection threshold of the LiPA. Both
patients had evidence of the
M184V viral genotype and had high
levels of resistance to 3TC after 12 and 44 weeks of 3TC treatment
(Table
1). Figure
5 illustrates representative results for
plasma
from three patients and shows the presence of phenotypic
differences
of RT activity from specimens collected before and during
antiretroviral
therapy with 3TC.
View larger version (32K):
[in this window]
[in a new window]
|
FIG. 5.
Detection of phenotypic resistance to 3TC of HIV-1 in
plasma by the Amp-RT assay. Results of duplicate tests with plasma
samples from three HIV-1-infected patients (patients 6, 8, and 14)
obtained at different time points before therapy (basal and
pre-therapy) and during antiretroviral therapy (1 to 44 weeks) with 3TC
are shown. SC, HIV-1-, HIV-2-, HTLV-I-, and HTLV-II-seronegative
control; w, water control.
|
|
| |
DISCUSSION |
The efficacy of antiretroviral therapy with 3TC is strongly
limited by the emergence of 3TC-resistant HIV-1 variants. We have developed a rapid non-culture-based assay for the analysis of phenotypic resistance of HIV-1 RT to 3TC in plasma samples. The assay
uses a small volume of plasma, and the HIV-1 RT phenotype for 3TC is
determined on the basis of the level of RT inhibition by a single
3TC-TP concentration. Compared to standard culture-based phenotypic
assays, this novel approach has several advantages. First, test results
are obtained in 1 to 2 days, providing rapid information on resistance
to 3TC that is of clinical relevance to treatment decisions and patient
management. Second, testing is done directly with virion-associated RT
from plasma, and therefore, unlike culture-based methods, the assay
does not select for particular viral phenotypes (23,
27). Third, the assay has a low detection threshold for
the presence of 3TC-resistant viruses and may be useful for the
early detection of 3TC resistance.
Our data demonstrate that the assay can be used to successfully monitor
resistance to 3TC mediated by mutations at codon 184. Decreased RT inhibition by 3TC-TP occurred in samples obtained from
persons after treatment with 3TC and coincided with the emergence of
resistant genotypes. In addition to providing phenotypic information on
resistance to 3TC, the Amp-RT reaction done without 3TC-TP provides
information on the level of virus in plasma on the basis of the RT
level and therefore may be used to simultaneously monitor the virologic
response to treatment with 3TC (12).
In this study, our primary objective was to validate the assay to
monitor resistance mediated by mutations at codon 184, since resistance
to 3TC has mainly been associated with mutations in this position of
the HIV-1 RT gene (3, 31, 38). However, we
also examined the effect that other resistance mutations may have
on the susceptibility of RT to 3TC-TP. We provide evidence of
resistance to 3TC in RTs carrying MD resistance mutations, thus
expanding the pattern of MD resistance conferred by these mutations to
3TC. Our results obtained with an RT-based assay confirm the low level
of resistance to 3TC observed in some culture-based assays (18,
19). The alteration of the RT's substrate recognition caused by
these mutations has been implicated in resistance to multiple
dideoxynucleosides and may likely explain the observed resistance to
3TC-TP (29, 35).
The association between MD resistance mutations and phenotypic
resistance to 3TC is of special importance. These mutations have been
found in viruses from 8 to 16% of patients receiving combination
therapy with AZT and dideoxyinosine (ddI) or AZT and ddC (20,
34) and may likely compromise future treatment with 3TC, despite
the lower level of resistance of viruses with these mutations compared
to that of viruses with the M184V mutation. Similar levels of
resistance to ddI or ddC mediated by other mutations have been
associated with drug failures (8). The finding of phenotypic
resistance to 3TC mediated by MD resistance mutations supports the use
of the present phenotypic assay for the monitoring of 3TC resistance
conferred by non-M184V mutations.
Our assay was designed for the rapid evaluation of resistance to 3TC
and included tests with an optimal concentration of 3TC-TP. Using this
assay format, we found an interesting association between mutations at
codon 69 and borderline susceptibility to 3TC-TP, suggesting that this
mutation may confer some level of resistance to 3TC, a finding which is
also consistent with recent observations (42). However, to
confirm the role of mutations at codon 69 in the observed borderline
susceptibility to 3TC, additional phenotypic testing by culture-based
assays is required in these samples.
In addition to clinical monitoring of 3TC resistance, the present
assay may also be used as a rapid method for surveillance of
transmission of 3TC resistance among persons with newly diagnosed HIV-1
infections. Transmission of drug-resistant HIV-1 raises public health
concerns because of its potential to compromise the efficacy of
antiretroviral therapy both in the initial treatment of HIV-1-infected
persons and in therapy for the prevention of perinatal transmission.
While transmission of HIV strains with resistance to AZT and nevirapine
has been documented (7, 9, 17, 39), little is known
about the transmission of 3TC-resistant HIV-1. However, because
of the wide use of 3TC, the risk of transmission of 3TC-resistant
viruses is likely increasing. Therefore, the present assay may be used
as a tool for the rapid detection of resistance to 3TC in 3TC-naive
HIV-1-infected patients.
In conclusion, in contrast to culture-based methods, this RT-based
phenotypic assay is a rapid and simple method for the direct analysis
of phenotypic resistance of HIV-1 to 3TC mediated by mutations at codon
184 and at other positions such as those associated with MD resistance.
The use of small volumes of plasma, the rapidity of testing, and the
lack of selection bias all make the Amp-RT-based phenotypic assay a
useful method for clinical monitoring and management of HIV-1
resistance to 3TC. This testing approach may also be expanded to
analysis of resistance to nonnucleoside RT inhibitors and other
nucleoside analogs such as ddC, ddI, and AZT (Q151M-mediated resistance) (15).
| |
ACKNOWLEDGMENTS |
We thank Hiroaki Mitsuya and John Mellors for providing molecular
infectious clones. HIV-1RTMC/MT-2 was obtained from Brendan Larder through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases.
This work was supported in part by the Georgia Veterans Affairs
Research Center for AIDS and HIV infections (to R.F.S. and D.R.)
and by NIH grant 1-R01-AI41980 (to R.F.S.). Work at ISCIII was supported in part by grants from CAM, ISCII, and AIS (to
V.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: HIV and
Retrovirology Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, MS G-19, Atlanta, GA 30333. Phone: (404) 639-0218. Fax: (404) 639-1174. E-mail: WMH2{at}cdc.gov.
| |
REFERENCES |
| 1.
|
Arts, E. J., and M. A. Wainberg.
1996.
Mechanisms of nucleoside analog antiviral activity and resistance during human immunodeficiency virus reverse transcription.
Antimicrob. Agents Chemother.
40:527-540[Medline].
|
| 2.
|
Arts, E. J.,
J. Marois,
Z. Gu,
S. F. J. Le Grice, and M. A. Wainberg.
1996.
Effects of 3'-deoxynucleoside 5'-triphosphate concentrations on chain termination by nucleoside analogs during human immunodeficiency virus type 1 reverse transcription of minus-strand strong-stop DNA.
J. Virol.
70:712-720[Abstract].
|
| 3.
|
Boucher, C. A. B.,
N. Cammack,
P. Schipper,
R. Schuurman,
P. Rouse,
M. A. Wainberg, and J. M. Cameron.
1993.
High level resistance to () enantiomeric 2'-deoxy-3'-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcriptase.
Antimicrob. Agents Chemother.
37:2231-2234[Abstract/Free Full Text].
|
| 4.
|
Caroline, M. P., and D. Faulds.
1997.
Lamivudine: a review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in the management of HIV infection.
Drugs
53:657-680[Medline].
|
| 5.
|
Carpenter, C. C.,
M. A. Fischl,
S. M. Hammer,
M. S. Hirsch,
D. M. Jacobsen,
D. A. Katzenstein,
J. S. Montaner,
D. D. Richman,
M. S. Saag,
R. T. Schooley,
M. A. Thompson,
S. Vella,
P. G. Yeni, and P. A. Volberding.
1997.
Antiviral therapy for HIV infection in 1997: updated recommendations of the International AIDS Society USA panel.
JAMA
277:1962-1969[Abstract/Free Full Text].
|
| 6.
|
Chou, T. C., and P. Talalay.
1984.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.
Adv. Enzyme Regul.
22:27-55[Medline].
|
| 7.
|
Conlon, C. P.,
P. Klenerman,
A. Edwards,
B. A. Larder, and R. E. Phillips.
1994.
Heterosexual transmission of human immunodeficiency virus type 1 variants associated with zidovudine resistance.
J. Infect. Dis.
169:411-415[Medline].
|
| 8.
|
D'Aquila, R. T.
1996.
Nucleosides and foscarnet: clinical aspects, p. 191-224.
In
D. D. Richman (ed.), Antiviral drug resistance. John Wiley & Sons, Inc., New York, N.Y.
|
| 9.
|
Erice, A.,
D. L. Mayers,
D. G. Strike,
K. J. Sannerud,
F. E. McCutchan,
K. Henry, and H. H. Balfour.
1993.
Brief report: primary infection with zidovudine-resistant human immunodeficiency virus type 1.
N. Engl. J. Med.
328:1163-1165[Free Full Text].
|
| 10.
|
Eriksson, B. F. H.,
C. K. Chu, and R. F. Schinazi.
1989.
Phosphorylation of 3'-azido-2',3'-dideoxyuridine and preferential inhibition of human and simian immunodeficiency virus reverse transcriptases by its 5'-triphosphate.
Antimicrob. Agents Chemother.
33:1729-1734[Abstract/Free Full Text].
|
| 11.
|
Fitzgibbon, J. E.,
R. M. Howell,
C. A. Haberzettl,
S. J. Sperber,
D. J. Gockle, and D. T. Dubin.
1992.
Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.
Antimicrob. Agents Chemother.
36:153-157[Abstract/Free Full Text].
|
| 12.
|
Garcia Lerma, J. G.,
S. Yamamoto,
M. Gomez-Cano,
V. Soriano,
T. A. Green,
M. P. Busch,
T. M. Folks, and W. Heneine.
1998.
Measurement of human immunodeficiency virus type 1 plasma virus load based on reverse transcriptase (RT) activity: evidence of variabilities in levels of virion-associated RT.
J. Infect. Dis.
177:1221-1229[Medline].
|
| 13.
|
Gulick, R. M.,
J. W. Mellors,
D. Havlir,
J. E. Eron,
C. Gonzalez,
D. McMahon,
D. D. Richman,
F. T. Valentine,
L. Jonas,
A. Meibohm,
E. A. Emini, and J. A. Chodakewitz.
1997.
Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy.
N. Engl. J. Med.
337:734-739[Abstract/Free Full Text].
|
| 14.
|
Heneine, W.,
S. Yamamoto,
W. M. Switzer,
T. J. Spira, and T. M. Folks.
1995.
Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type 1.
J. Infect. Dis.
171:1210-1216[Medline].
|
| 15.
|
Heneine, W.,
J. G. Garcia Lerma,
J. G. Vazquez-Rosales,
S. Qari,
A. Juodawlkis,
D. Havlir,
R. F. Schinazi,
D. D. Richman, and T. M. Folks.
1998.
A novel non culture-based approach for the rapid analysis of phenotypic resistance to reverse transcriptase inhibitors of HIV-1 from plasma, abstr. 63, p. 42.
In
Program and abstracts of the 2nd International Workshop on Drug Resistance and Treatment Strategies.
|
| 16.
|
Hertogs, K.,
M. de Bethune,
V. Miller,
T. Ivens,
P. Schel,
A. Van Cauwenberge,
C. Van den Eynde,
V. Van Gerwen,
H. Azijn,
M. Van Houtte,
F. Peeters,
S. Staszewski,
M. Conant,
S. Bloor,
S. Kemp,
B. Larder, and R. Pauwels.
1998.
A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs.
Antimicrob. Agents Chemother.
42:269-276[Abstract/Free Full Text].
|
| 17.
|
Imrie, A.,
A. Beveridge,
W. Genn,
J. Vizzard,
D. A. Cooper, and the Sydney Primary HIV Infection Study Group.
1997.
Transmission of human immunodeficiency virus type 1 resistant to nevirapine and zidovudine.
J. Infect. Dis.
175:1502-1506[Medline].
|
| 18.
|
Iversen, A. K. N.,
R. W. Shafer,
K. Wehrly,
M. A. Winters,
J. I. Mullins,
B. Chesebro, and T. C. Merigan.
1996.
Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy.
J. Virol.
70:1086-1090[Abstract].
|
| 19.
|
Jellinger, R. M.,
R. W. Shafer, and T. C. Merigan.
1997.
A novel approach to assessing the drug susceptibility and replication of human immunodeficiency virus type 1 isolates.
J. Infect. Dis.
175:561-566[Medline].
|
| 20.
|
Kavlik, M. F.,
K. Wyvill,
R. Yarchoan, and H. Mitsuya.
1998.
Emergence of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 variants, viral sequence variation, and disease progression in patients receiving antiretroviral chemotherapy.
J. Infect. Dis.
177:1506-1513[Medline].
|
| 21.
|
Kavlick, M. F.,
T. Shirasaka,
E. Kojima,
J. M. Pluda,
F. Hui,
R. Yarchoan, and H. Mitsuya.
1995.
Genotypic and phenotypic characterization of HIV-1 isolated from patients receiving ()-2',3'-dideoxy-3'-thiacytidine.
Antivir. Res.
28:133-146[Medline].
|
| 22.
|
Kellam, P., and B. A. Larder.
1994.
Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.
Antimicrob. Agents Chemother.
38:23-30[Abstract/Free Full Text].
|
| 23.
|
Kusumi, K.,
B. Conway,
S. Cunningham,
A. Berson,
C. Evans,
A. K. Iversen,
D. Colvin,
M. V. Gallo,
S. Coutre, and E. G. Shpaer.
1992.
Human immunodeficiency virus type 1 envelope gene structure and diversity in vivo and after cocultivation in vitro.
J. Virol.
66:875-885[Abstract/Free Full Text].
|
| 24.
|
Larder, B. A., and S. D. Kemp.
1989.
Multiple mutations in HIV-1 reverse transcriptase confer high level resistance to zidovudine (AZT).
Science
246:1155-1158[Abstract/Free Full Text].
|
| 25.
|
Larder, B. A.,
G. Darby, and D. D. Richman.
1989.
HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy.
Science
243:1731-1734[Abstract/Free Full Text].
|
| 26.
|
Larder, B. A.,
A. P. Kemp, and P. R. Harrigan.
1995.
Potential mechanism for sustained antiretroviral efficacy of AZT+3TC combination therapy.
Science
269:696-699[Abstract/Free Full Text].
|
| 27.
|
Li, Y.,
J. C. Kappes,
J. A. Conway,
R. W. Price,
G. M. Shaw, and B. H. Hahn.
1991.
Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes.
J. Virol.
65:3973-3985[Abstract/Free Full Text].
|
| 28.
|
Ludwig, J., and F. Eckstein.
1989.
Rapid and efficient synthesis of nucleoside 5'-O-(1-thiotriphosphates), 5'-triphosphates and 2'3'-cyclophosphorothioates using 2-chlore-4H-1,2,3-benzodioxaphosphorin-4-one.
J. Org. Chem.
54:631-635.
|
| 29.
|
Sarafinos, S. G.,
V. N. Pandey,
N. Kaushik, and M. J. Modak.
1995.
Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.
Biochemistry
34:7207-7216[Medline].
|
| 30.
|
Schinazi, R. F.,
A. McMillan,
D. Cannon,
R. Mathis,
R. M. Lloyd,
A. Peck,
J Sommadossi,
M. St. Clair,
J. Wilson,
P. A. Furman,
G. Painter,
W. Choi, and D. C. Liotta.
1992.
Selective inhibition of human immunodeficiency viruses by racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
Antimicrob. Agents Chemother.
36:2423-2431[Abstract/Free Full Text].
|
| 31.
|
Schinazi, R. F.,
R. M. Lloyd,
M. H. Nguyen,
D. L. Cannon,
A. McMillan,
N. Ilksoy,
C. K. Chu,
D. C. Liotta,
H. Z. Bazmi, and J. W. Mellors.
1993.
Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides.
Antimicrob. Agents Chemother.
37:875-881[Abstract/Free Full Text].
|
| 32.
|
Schinazi, R. F.,
B. A. Larder, and J. W. Mellors.
1997.
Mutations in retroviral genes associated with drug resistance.
Int. Antivir. News
5:129-142.
|
| 33.
|
Schuurman, R.,
M. Nijhuis,
R. van Leeuwen,
P. Schipper,
D. de Jong,
P. Collis,
S. A. Danner,
J. Mulder,
C. Loveday,
C. Christopherson,
S. Kwok,
J. Sninsky, and C. A. Boucher.
1995.
Rapid changes in HIV-1 RNA load and appearance of drug-resistant virus population in persons treated with lamivudine (3TC).
J. Infect. Dis.
171:1411-1419[Medline].
|
| 34.
|
Shafer, R. W.,
A. K. N. Iversen,
M. A. Winters,
E. Aguiniga,
D. A. Katzenstein,
T. C. Merigan, and the AIDS Clinical Trials Group 143 Virology Team.
1995.
Drug resistance and heterogeneous long-term virologic responses of human immunodeficiency virus type 1-infected subjects to zidovudine and didanosine combination therapy.
J. Infect. Dis.
172:70-78[Medline].
|
| 35.
|
Shirasaka, T.,
M. F. Kavlick,
T. Ueno,
W. Gao,
E. Kojima,
M. L. Alcaide,
S. Chokekijchai,
B. M. Roy,
E. Arnold,
R. Yarchoan, and H. Mitsuya.
1995.
Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.
Proc. Natl. Acad. Sci. USA
92:2398-2402[Abstract/Free Full Text].
|
| 36.
|
St. Clair, M. H.,
J. L. Martin,
G. Tudor-Williams,
M. C. Bach,
C. L. Vavro,
D. M. King,
P. Kellam,
S. D. Kemp, and B. A. Larder.
1991.
Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.
Science
253:1557-1559[Abstract/Free Full Text].
|
| 37.
|
Stuyver, L.,
A. Wyseur,
A. Rombout,
J. Louwagie,
T. Scarcez,
C. Verhofstede,
D. Rimland,
R. F. Schinazi, and R. Rossau.
1997.
Line probe assay for the rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene.
Antimicrob. Agents Chemother.
41:284-291[Abstract].
|
| 38.
|
Tisdale, N.,
S. D. Kemp,
N. R. Parry, and B. A. Larder.
1993.
Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase.
Proc. Natl. Acad. Sci. USA
90:5653-5656[Abstract/Free Full Text].
|
| 39.
|
Veenstra, J.,
R. Schuurman,
M. Cornelissen,
A. B. van't Wout,
C. A. B. Boucher,
H. Schuitemaker,
J. Goudsmit, and R. A. Coutinho.
1995.
Transmission of zidovudine-resistant human immunodeficiency virus type 1 variants following deliberate injection of blood from a patient with AIDS: characteristics and natural history of the virus.
J. Infect. Dis.
21:556-560.
|
| 40.
|
Wainberg, M. A.,
H. Salomon,
Z. Gu,
J. S. G. Montaner,
T. P. Cooley,
R. MaCaffrey,
J. Ruedy,
H. M. Hirst,
N. Cammack,
J. Cameron, and W. Nicholson.
1995.
Development of HIV-1 resistance to () 2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex.
AIDS
9:351-357[Medline].
|
| 41.
|
Wainberg, M. A.,
M. Hsu,
Z. Gu,
G. Borkow, and M. A. Parniak.
1996.
Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.
AIDS
10:S3-S10.
|
| 42.
|
Wainberg, M. A.,
M. D. Miller,
Y. Quan,
H. Salomon, and J. Cherrington.
1998.
The M184V substitution in reverse transcriptase moderately increases sensitivity of HIV-1 to PMPA, abstr. 680, p. 207.
In
Program and abstracts of the 5th Conference on Retroviruses and Opportunistic Infections.
|
| 43.
|
Yamamoto, S.,
T. M. Folks, and W. Heneine.
1996.
Highly sensitive qualitative and quantitative detection of reverse transcriptase activity: optimization, validation, and comparative analysis with other detection systems.
J. Virol. Methods
61:135-143[Medline].
|
Antimicrobial Agents and Chemotherapy, February 1999, p. 264-270, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Garcia-Lerma, J. G., Heneine, W.
(2002). Rapid biochemical assays for phenotypic drug resistance testing of HIV-1. J Antimicrob Chemother
50: 771-774
[Full Text]
-
Qari, S. H., Magre, S., García-Lerma, J. G., Hussain, A. I., Takeuchi, Y., Patience, C., Weiss, R. A., Heneine, W.
(2001). Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors. J. Virol.
75: 1048-1053
[Abstract]
[Full Text]
-
García-Lerma, J. G., Gerrish, P. J., Wright, A. C., Qari, S. H., Heneine, W.
(2000). Evidence of a Role for the Q151L Mutation and the Viral Background in Development of Multiple Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1. J. Virol.
74: 9339-9346
[Abstract]
[Full Text]
-
Lerma, J. G. G., Soriano, V., Mas, A., Quiñones-Mateu, M. E., Arts, E. J., Heneine, W.
(2000). Quantitation of Human Immunodeficiency Virus Type 1 Group O Load in Plasma by Measuring Reverse Transcriptase Activity. J. Clin. Microbiol.
38: 402-405
[Abstract]
[Full Text]
Top
Abstract
Introduction
