566 • CID 2010:50 (15 February) • Jakobsen et al
H I V / A I D SM A J O R A R T I C L E
Transmission of HIV-1 Drug-Resistant Variants:
Prevalence and Effect on Treatment Outcome
Martin R. Jakobsen,1,3Martin Tolstrup,1Ole S. Søgaard,1Louise B. Jørgensen,2Paul R. Gorry,3,4,5Alex Laursen,1
and Lars Østergaard1
1Department of Infectious Diseases, Aarhus University Hospital, Skejby, and
University of Melbourne, Melbourne, Victoria, Australia
2Department of Virology, Statens Serum Institut, Copenhagen,
4Department of Medicine, Monash University, and
3Centre for Virology, Burnet Institute,
5Department of Microbiology and Immunology,
overall success of antiretroviral therapy (ART). Because of the limited sensitivity of commercial assays, transmitted
drug resistance (TDR) may be underestimated; thus, the effect that TDR has on treatment outcome needs to be
investigated. The objective of this study was to investigate the prevalence of TDR in HIV-infected patients and to
evaluate the significance of TDR with respect to treatment outcome by analyzing plasma viral RNA and peripheral
blood mononuclear cell proviral DNA for the presence of drug resistance mutations.
In a prospective study, we investigated the level of TDR in 61 patients by comparing the results of
a sensitive multiplex-primer-extension approach (termed HIV-SNaPshot) that is capable of screeningfor9common
nucleoside reverse-transcriptase inhibitor and nonnucleotide reverse-transcriptase inhibitor mutations with those
of a commercial genotyping kit, ViroSeq (Abbott).
Twenty-two patients were found to carry mutations. More patients with TDR were identified by the
HIV-SNaPshot assay than by ViroSeq analysis (33% vs 13%;
time from initiation of ART to virological suppression between susceptible patients and those carrying low- or
high-level resistance mutations (mean ? standard deviation, 128 ? 59.1vs164.9 ? 120.4;
analyses of CD4 cell counts showed no significant difference between these 2 groups 1 year after the initiation of
ART (mean, 184 vs 219 cells/mL; ).P p .267
We found the prevalence of TDR in recently infected ART-naive patients to be higher than that
estimated by ViroSeq genotyping alone. Follow-up of patients after treatment initiation showed a trend toward
there being more clinical complications for patients carrying TDR, although a significant effect on treatment
outcome could not be demonstrated. Therefore, the clinical relevance of low-abundance resistant quasispecies in
early infection is still in question.
Human immunodeficiency virus type 1 (HIV-1) drug resistance is an important threat to the
). There was no significant difference in theP p .015
).Furthermore,P p .147
Although antiretroviral therapy (ART) has led to de-
creased mortality and morbidity among people with
human immunodeficiency virus type 1 (HIV-1) infec-
tion, failure to suppress viremia still occurs as the result
of the emergence of drug-resistant viral species [1–4].
In recent years, different estimates of the prevalence of
transmitted drug resistance (TDR) in Europe and
North America have been reported, ranging from as
Received 20 July 2009; accepted 24 September 2009; electronically published
19 January 2010.
Reprints or correspondence: Dr Martin R. Jakobsen, Dept of Infectious Diseases,
Aarhus University Hospital, Skejby, Brendstrupgaardvej 100, 8200 Aarhus N, Den-
Clinical Infectious Diseases2010;50:566–73
? 2010 by the Infectious Diseases Society of America. All rights reserved.
low as 4.1% to as much as 23.1% [5–12]. However, the
2 latest European surveillance studies reported the
mean TDR prevalence to be constant, at a level of 9%–
10% from 1996 through 2003 [13, 14]. They observed
that patients primarily carried only 1 TDR mutation
with an increased frequency of nucleoside reverse-tran-
scriptase inhibitor (NRTI) or nonnucleotide reverse-
transcriptase inhibitor (NNRTI) drug resistance mu-
tations, which probably reflects the extensive use of
these drug classes for the past decade.
A problem for many of the studies estimating the
prevalence of TDR is that they are based on interpre-
tations from conventional population sequencing—a
method known to have problems detecting viral qua-
sispecies with a frequency of !20% [15, 16]. Further-
more, previous studies have failed to account for the
archived viral quasispecies that are present within the
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Transmission of HIV-1 Drug Resistance • CID 2010:50 (15 February) • 567
peripheral blood mononuclear cell (PBMC) population. An
approach addressing these 2 factors may provide a more ac-
curate estimate of the frequency of TDR [17–19]; as a conse-
quence of increased sensitivity, the actual prevalence of TDR
may be higher than that provided by current published esti-
However, one important question still needs clarification:
what is the clinical significance of patients carrying TDR when
ART is initiated? Five important studies [20–24] published
within the last 12 months have addressed this question from
different approaches, all using highly sensitive detection meth-
ods for measuring resistance within the plasma compartment.
Only two of these studies showed a significant influence on
treatment response in patients carrying low-abundance TDR
In Denmark, the prevalence of TDR has been reported to
be 4.1%, summarized in a cohort of 690 patients with newly
diagnosed HIV infection from 2000–2006 by means of a stan-
dard genotyping approach . In the present study, we in-
vestigated a subgroup of patients from a cohort attending the
outpatient clinic at the Department of Infectious Diseases, Aar-
hus University Hospital (Skejby, Denmark). Using a highly sen-
sitive point-mutation approach , we investigated the pres-
ence of 9 major NRTI and NNRTI drug resistance mutations
in plasma as well as in proviral DNA from PBMCs and ad-
dressed 2 important questions: is TDR currently underesti-
mated, and does low-abundance TDR affect treatment out-
samples were taken before treatment initiation, and no AIDS
diagnosis was given. Two sets of plasma samples were collected
from each patient at baseline. One set was stored at ?80?C at
the Department of Infectious Diseases, Aarhus University Hos-
pital, and the other was sent to Statens Serum Institute (Co-
penhagen, Denmark) for ViroSeq (Abbott) genotypinganalysis.
Time of infection was estimated on the basis of the patient’s
own statement and the latest negative HIV-1 antibody test re-
sult. All patients provided written informed consent at inclu-
sion. Clinical data on the cohort, includingtreatmentinitiation,
plasma viral load, and CD4 cell count was collected until Feb-
The HIV-SNaPshot assay.
minisequencing technique in which a wild-type or mutant se-
quence is detected using fluorescent single base primer exten-
sion together with capillary electrophoresis . The HIV-
SNaPshot assay is designed to identify mutations in the codons
for 6 NRTI drug resistance mutations—M41L, K65R, K70R,
Q151M, M184V, and T215YF, as well as the revertants T215D/
S/N)—and, in addition, 3 NNRTI drug resistance mutations—
We used the following inclusion criteria: baseline
This assay is a variant of the
K103N, Y181C, and G190ASE. The method has been validated
for subtype variability and has the capability to detect drug
resistance quasispecies that represent only ∼2% of the totalviral
HIV-1 resistance testing of plasma RNA samples.
RNA was extracted from 1 mL of frozen plasma samples from
61 patients. Briefly, the plasma sample was ultracentrifuged for
90 min at 20,000 g, excess volume of plasma was removed
before the sample was resuspended in 560 mL of AVL buffer,
and viral RNA was collected using the QIAamp Viral RNA
Mini kit (Qiagen), in accordance with the manufacturer’s in-
reactions (RT-PCRs) were designed for each patient by means
of an in-house protocol . Following PCR, duplicates were
pooled together, gel purified (GFX column kit; GE Healthcare),
and then used as template for the HIV-SNaPshot reaction.
HIV-1 resistance testing of PBMC samples.
plasma samples used forRNA extraction,only50patientPBMC
samples were available for analysis. Genomic DNA was ex-
tracted using the QIAamp DNA Blood Mini kit (QIAamp).
Gene-specific PCR amplification was conducted on 4 mL of
genomic DNA (∼20–40 ng/mL) together with 10 pmol of each
primer (FW-1 and BW-1 ), 1? high-fidelity buffer, and 1
U of Pfx high-fidelity DNA polymerase (Invitrogen) in a 50-
mL reaction under the following cycling conditions: 10 min at
60?C; 5 min at 94?C; 40 cycles of 15 s at 94?C, 30 s at 60?C,
and 1 min at 68?C; and a final extension step of 10 min at
Investigators were blinded to the resistance profiles from the
ViroSeq genotyping method until comparison analyses were
made with the SNaPshot assay results. The effect of TDR on
treatment outcome was analyzed using a stratification of the
patients according to the susceptibility of their initial ART reg-
imen; this was based on the Stanford HIV drug resistance da-
tabase, in which each individual mutation is categorized as
either being susceptible, having low-level or intermediate re-
sistance, or having high-level resistance.
We used the x2statistic or the Fisher
exact test, as appropriate, to compare baseline categorical var-
iables for individuals who were fully susceptible and those with
low- or high-level resistance against the initial ART regimen.
Continuous variables were compared using the Student t test
and 1-way analysis of variance. Assumptions were checked by
quantile-quantile plots and the Bartlett test. A Kaplan-Meier
plot was used to estimate the time from initiation of ART to
viral suppression (plasma viral load, !50 copies/mL), according
to the level of TDR; the Gehan-Breslow-Wilcoxon method was
applied to test for differences. We used Stata software, version
9.2 (StataCorp) for statistical analyses. The study was approved
by the Danish Data Protection Agency.
Of the 61
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568 • CID 2010:50 (15 February) • Jakobsen et al
Table 1. Baseline Characteristics
Patients, no. (%)
Median age (IOR), years
Treatment initiated, no. (%)
Route of infection
Injection drug use
Self-reported time of infectionh
Acute (?0.5 years)
Recent (10.5 years)
Chronic (12 years)
Mean baseline plasma RNA level (range), log copies/mL
Mean CD4 cell count at treatment initiation (range), cells/mL
38.5 (25.4-48.3) .723b
aPatients were divided into the 2 groups on the basis of Stanford resistance and treatment algorithms.
bPaired t test.
cCombined data from HIV-SNaPshot results (mutations investigated: 41L, 65R, 70R, 103N, 151M, 181C, 184V, 190A, and
215YF/rev) and ViroSeq genotyping results.
dWilcoxon rank-sum test.
eFisher exact test.
gPatients could not tell how and when infection had occurred.
hTime for infection was based on patient interviews and latest negative p24 enzyme-linked immunosorbent assay result.
iData represent only 30 patients because some had not initiated treatment.
HIV-1, human immunodeficiency virus type 1; TDR, transmitted drug resistance.
the study (Table 1). Forty-five patients were fully susceptible,
4 had low-level resistance, and 12 had high-level resistance.
One patient did not receive treatment and was therefore ex-
cluded from the statistical analysis (Table 1). The low- and high-
level resistance groups were merged into one group because of
the small number of observations in the former group. The
groups were comparable with regard to age, sex, and HIV-1
subtype. The route of infection was equally distributed between
homosexual and heterosexual transmission. Only 1 patient re-
ported needle sharing, and 6 could not provide information on
the time or route of infection. Within the susceptible group, 6
Sixty-one patients were included in
patients had different resistance mutations (Table 2) that did not
affect their initial ART regimen (according to Stanford resistance
and treatment algorithms). In the low- or high-level resistance
group, 7 patients carried 11 resistance mutation. The number
of resistance mutations detected (6 vs 16;
cell count at baseline (mean, 400 vs 218 cells/mL;
the only descriptive characteristics that differed significantly be-
tween the susceptible and the low- or high-levelresistancegroup.
HIV-1 drug resistancebyViroSeqgenotyping.
genotyping resistance test was successfully conducted for all 61
patients. The results showed that 8 patients had at least 1 NRTI
or NNRTI mutation. Four patients carried mutations related
to resistance to NRTI regimens, and 2 carried mutations related
) and the CD4
P p .015
P ! .001
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Resistance Mutations and Initial Antiretroviral Treatment Regimen
Sample identifier Subtype
Reverse-transcriptase resistance detected
First-line treatment choice
Low-level resistance High-level resistance
K103N, M184V ABC, LPV-r
M184V, T215Y T215Y
3TC, ABC, EFV
AZT, 3TC, LPV-r
3TC, AZT, LPV-r
K103N, M184V, T215Y
3TC, IDV, RTV
3TC, IDV, RTV
EFV, ABC, 3TC
3TC, AZT, LPV-r
AZT, 3TC, LPV-r
3TC, lamivudine; ABC, abacavir; AZT, zidovudine; EFV, efavirenz; IDV, indinavir; LPV-r, lopinavir-ritonavir; NA, not applicable; RTV, ritonavir.
aMeasured by the HIV-SNaPshot assay.
bMutation is considered to be a normal polymorphism and was not included as a resistance mutation in the analysis.
cTreatment was switched after 3 months to ABC, AZT, and a protease inhibitor.
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570 • CID 2010:50 (15 February) • Jakobsen et al
patients receiving treatment. Patients were divided into 2 groups on the
basis of transmitted drug resistance mutations detected and their rele-
vance to the treatment initiated (according to the Stanford HIV resistance
algorithm). Patients with low- and high-level resistance were grouped
together because of the small number of observations in the low-level
resistance group (). There was no significant difference in then p 4
decline in viral load when extrapolated to the drugs given to the patients
(; Cox proportional hazards model). This indicated that patientsP p .147
who carry resistance mutations and are receiving a 3-drug treatment
regimen do not fail to suppress viremia within the first couple of years
after treatment initiation. However, there might be an effect at later
stages as the level of resistance accumulates, which was indicated for
some of the patients who had high-level resistance mutations (such as
M184V, K103N, and Y181C).
Kaplan-Meier curve illustrating time to viral suppression in
to resistance to NNRTI regimens (Table 2). Furthermore, 2
patients carried natural polymorphisms; however, the Stanford
resistance algorithm did not predict them to interferewithART.
Prevalence of resistance in viral RNA.
scriptase gene was successfully amplified from the correspond-
ing baseline sample for all 61 patients, and the HIV-SNaPshot
assay result was positive for the wild-type and/or mutation
codons at all 9 mutation positions, with the exception of the
Y181C primer in 4 (5%) of 61 patients and the K65R primer
in 1 (2%) of 61 patients.
In total, 16 (26%) of 61 patients harbored 1 or more low-
abundance NRTI or NNRTI resistance mutations in plasma
viral RNA. We found that the most frequently observed mu-
tations were the thymidine-associated mutations T215Y/F (7/
61 [11%]) and K70R (4/61 [6%]), followed by the primary
NNRTI resistance mutation K103N (5/61 [9%]). Furthermore,
we found M184V in 3 patients (5%) and the Y181C mutation
in 1 patient. Of the 16 patients with low-abundance resistance
mutations, only 3 had 11 mutation detected in the plasma
compartment (Table 2).
Prevalence of resistance in proviral DNA.
samples (82%) were available for analysesbytheHIV-SNaPshot
assay. We found that 13 (26%) of 50 patients had detectable
resistance mutations in proviral DNA. The predominant mu-
tation was again T215Y/F (6/50 [12%]), followed by K103N
(5/50 [10%]). Furthermore, we observed K70R in 2 patients
and M184V in 1 patient (Table 2).
Combining the data obtained from all 3 measurements
showed that NRTI mutations (4, 14, and 9 for ViroSeq plasma,
SNaPshot plasma, and SNaPshot PBMCs, respectively) were
found more often than NNRTI mutations (3, 6, and 5 for
ViroSeq plasma, SNaPshot plasma, and SNaPshot PBMCs).
Changes in viral load.
In total, 46 (75%) patients had
started treatment before February 2009. Of these, 21 carried
resistance mutations; 6 carried mutations that werenotrelevant
for the regimen prescribed and were placed in the susceptibility
group, and 15 carried mutations that potentially affected their
treatment and were placed in the low- or high-level resistance
group. The patients were treated with ART regimes that in-
cluded either 2 NRTIs and 1 NNRTI or 2 NRTIs and 1 boosted
protease inhibitor. The most frequently used NRTIs were zi-
dovudine, lamivudine, and abacavir, whereas the NNRTI was
always efavirenz. The mutations detected only by the sensitive
SNaPshot assay were T215Y/F, M184V, and K103N. For 3 pa-
tients (1669, 1846, and 2022), baseline genotype results showed
high-level resistance mutations against lamivudine, butthishad
not been taken into account when the choice of first-line treat-
ment was considered. In these cases, the mean time to reach
a plasma viral load of !50 copies/mL was 280 days (standard
deviation [SD], 197.3 days). Four patients (2022, 1485, 655,
and 729) carried high-level resistance mutations toward efa-
virenz, and this group had a mean time to reach a plasma viral
load of !50 copies/mL of 142 days (SD, 62.8 days). Fivepatients
carried high-level resistance mutations against zidovudine, and
the mean time to a reach plasma viral load of !50 copies/mL
in this group was 64 days (SD, 21.1 days). Only 1 patient(2022)
who had initiated treatment was found to carry 12 primary
drug resistance mutations. These mutations had not been de-
tected by the baseline standard genotyping test, and treatment
that included abacavir, lamivudine, and efavirenz was initiated.
Plasma viral load decreased slowly within the next 3 months
but then increased by 0.23 log10copies/mL. At this time a new
resistance test was conducted, and the presence of both the
K103N and M184V mutations was detected. Subsequently,
treatment was changed to abacavir, zidovudine, and boosted
lopinavir, which was followed by a large drop in plasma viral
load (15 log10copies/mL) within 2months.WhentheSNaPshot
analysis was conducted, these mutations were found in the
baseline sample; this argues for the presence of low-abundance
TDR, which in retrospect was shown to result in early viro-
logical failure of treatment.
When the groups were compared with regard to TDR and
treatment response, we found no significant difference. We an-
alyzed the time to reach a plasma viral load of !50 copies/mL
in the 2 groups by means of a Kaplan-Meier curve (Figure 1)
and found no significant differences between the groups
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Transmission of HIV-1 Drug Resistance • CID 2010:50 (15 February) • 571
who were treatment susceptible are represented by black dots, those
with low-level resistance are represented by gray squares, and those
with high-level resistance are represented by black diamonds. A, Time
to an undetectable viral load. Analysis by the Mann-Whitney U test
showed that there was no significant difference between the groups. B,
Increase in CD4 cell count after 1 year of treatment. Analysis by the
Mann-Whitney U test showed that there was no significant difference
between the groups.
Comparison of clinical data between the 2 groups. Patients
lyzed the difference between the groups in terms of the increase
in CD4 cell count after 1 year of treatment; we found no sig-
nificant differences (P p .267
crease) between patients who were susceptible or had low- or
high-level resistance against the ART regimen (Figure 2).
; Gehan-Breslow-Wilcoxon method). We then ana-P p .147
for mean CD4 cell count in-
This study reports a 36% prevalence of TDR in a representative
sample of a Danish population of patients recently infected
with HIV-1 when a sensitive detection method was used. This
number was significantly higher than the 13% TDR level iden-
tified by paired-sample ViroSeq genotyping analysis.
To date, several studies have shown that low-abundance re-
sistance mutations can be found in chronicallyinfectedpatients
even though standard genotyping results are negative [5, 6, 27,
28]. Our results showed that a significantly higher proportion
of recently infected patients with negative genotype results car-
ried low-abundance resistance mutations, leading us to believe
that the current level of TDR is underestimated.
Our study also justifies the inclusion of proviral DNA from
PMBCs as a valuable compartment for resistance analysis. In
6 patients, we were only able to detect mutations from the
PBMC samples, some of them being high-level resistance mu-
tations. However, the number of mutations in the PBMC com-
partment did not per se exceed the number in the plasma com-
partment, but as expected the inclusion of both strengthened the
overall interpretation of drug resistance level, which is in agree-
ment with previous observations [17, 18]. Thus, the different
observations in the plasma and PBMC compartment is likely
attributed to stochastic effects, given that the detection of mi-
nority variants is at the limit of detection of sensitivity.
The presence of HIV-1 NRTI or NNRTI drug resistance be-
fore ART may lead to suboptimal treatment response [29–33].
However, our results showed no significant difference in time
to viral suppression or CD4 cell count increase between the
susceptibility group and the low- or high-level resistance group
carrying NRTI and/or NNRTI mutations when only1mutation
was detected. However, a trend toward a weaker suppression
of viremia was observed in patients who carried 11 resistance
In comparison, Johnson et al  recently studied the pres-
ence of low-abundance resistance mutations by means of a
highly sensitive allele-specific PCR method and detected 9 dif-
ferent resistance mutations in a large cohort of ART-naive pa-
tients with new diagnoses. They found that 34 of 205 samples
originally tested as wild type contained either NRTI or NNRTI
resistance mutations and that these low-abundance mutations
were significantly associated with virological failure . An-
other study addressed similar questions using the ultradeep
sequencing technique and found no significantly difference for
time to virological failure when the entire cohorts of patients
with and without low-abundance mutations were compared
without taking into account the susceptibility of first-line treat-
ment . However, when the results were stratified into pa-
tients with TDR potentially affecting the individual ART reg-
imens, they found that patients with NNRTI resistance mu-
tations in a NNRTI strategy arm had a significantly higher risk
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572 • CID 2010:50 (15 February) • Jakobsen et al
of virological failure than did patients without detectable re-
sistance. Similarly, a higher risk was present when patients with
any NRTI or NNRTI were grouped and compared with patients
without mutations .
The clinical virological parameter used here (ie, time to viral
suppression) is not directly comparable to virological failure
(defined as detectable viral load after 6 months of treatment);
thus, our data has to be compared with caution to the studies
mentioned above. Although our results imply that low-abun-
dance TDR does not need to be taken into account when de-
signing first-line treatment regimens, they should be interpret-
ed with care. Our study relied on a broad multiplex detection
method with high sensitivity; thus, the relevance of detecting
low-abundance quasispecies may be questionable. The diver-
gent results from this studysupporttheobservationsinKearney
et al , Peuchant et al , and Metzner et al  while
opposing the studies discussed above, but more importantly
they raise the possibility that low-abundance resistance mu-
tations in a recipient may represent random accumulation of
mutations rather than selection of transmitted mutations by
treatment, which is supported by the fact that they seemed to
be of no importance in the present study. The observations in
this study are limited by the numbers of patients with low- or
high-level resistance mutations, which make it difficult to es-
tablish a significant difference despite the fact that a trend to-
ward a higher level of clinical complications in the resistance
group was observed. Another important feature is that half of
the patients in the low- or high-level resistance group were
treated with 2 NRTIs together with a protease inhibitor, re-
sulting in an ART regimen with a highgeneticresistancebarrier.
Hence, the risk of viral failure would be expected to be lower
in the presence of a protease inhibitor, thereby shielding the
clinical effect of NRTI or NNRTI resistance mutations.
The results of this study do justify further studies, mainly a
randomized study where treatment is guided by the resistance
profile determined by the ViroSeq genotyping assay with or
without information from the SNaPshot method. Such a study
would be able to determine more directly whether the presence
and percentage of TDR play a role in the success of first-line
In conclusion, we find that resistance in naive and recently
infected patients can go unnoticed when population-based se-
quencing is used. By comparing results from both the sensitive
SNaPshot and standard ViroSeq genotyping methods, we de-
tected a higher prevalence of drug resistance than previously
reported. Nevertheless, our results do not support the hypoth-
esis that the presence of minority TDR mutations in recently
infected patients has clinical implications in term of early vi-
We are grateful to Jonna Guldberg and Erik Hagen Nielsen for excellent
technical assistance. We also thank the SERO-2000 project group and the
technical support from the Statens Serum Institut for assistance in blood
sample collection and DNA extraction.
The work was partly supported by the Danish AIDS
foundation, the Scandinavian Society for Antimicrobial Chemotherapy
Foundation, and the Augustinus Foundation. M.R.J. is the recipient of the
Danish Alfred Benzon Foundation Research Fellowship. P.R.G. is the re-
cipient of an Australian National Health and Medical Research Council
Biomedical Career Development Award.
Potential conflicts of interest.
All authors: no conflicts.
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