Changing Patterns in HIV Reverse Transcriptase Resistance

HIV/AIDS
BRIEF REPORT
Changing Patterns in HIV Reverse
Transcriptase Resistance Mutations
after Availability of Tenofovir
Carmen de Mendoza,1 Inmaculada Jiménez-Nacher,2 Carolina Garrido,1
Pablo Barreiro,1 Eva Poveda,1 Angélica Corral,1 Natalia Zahonero,1
Juan González-Lahoz,1 and Vincent Soriano1
1
Department of Infectious Diseases and 2Pharmacy Unit, Hospital Carlos III,
Madrid, Spain
Assessment of 1177 human immunodeficiency virus (HIV)
resistance genotypes at an HIV/AIDS clinic showed a decrease in the incidence of the K65R mutation, from 15.2%
of isolates during the period 2002–2004 to 2.7% of isolates
during the period 2005–2006 (P ! .001 ), despite elevated and
stable rates of tenofovir use. A reduction in the rate of coadministration of didanosine (from 41.6% of patients in 2004
to 0.8% of patients in 2006; P ! .001) largely explained this
observation.
Current guidelines recommend the use of tenofovir disoproxil
fumarate (TDF) as one of the preferred nucleoside reversetranscriptase inhibitors (NRTI) for first-line antiretroviral therapy [1]. TDF was marketed in Spain in June 2002, although
the drug had been widely available within an expanded access
program since the middle of 2001.
Three main different patterns of drug resistance mutations
may confer loss of susceptibility to TDF. K65R is the primary
TDF resistance change in drug-naive subjects, and its selection
clearly correlates with an impaired response to the drug [2].
In addition, K65R is known to reduce susceptibility to all other
NRTIs except zidovudine [2]. Not surprisingly, the rate of K65R
rapidly increased after TDF approval [3]. The other resistance
mutations compromising TDF activity are T69S inserts and ⭓3
thymidine-associated mutations (TAMs), including M41L and
L210W [4, 5]. The emergence of the K70E mutation has also
recently been linked to failure of TDF therapy [6]. The aim of
Received 9 July 2007; accepted 28 December 2007; electronically published 21 April 2008.
Presented in part: 5th European HIV Resistance Workshop, Cascais, Portugal, 28–30 March
2007.
Reprints or correspondence: Dr. Carmen de Mendoza, Laboratory of Molecular Biology, Dept.
of Infectious Diseases, Hospital Carlos III, Calle Sinesio Delgado 10, 28029 Madrid, Spain
(cmendoza@teleline.es).
Clinical Infectious Diseases 2008; 46:1782–5
2008 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2008/4611-0023$15.00
DOI: 10.1086/588045
1782 • CID 2008:46 (1 June) • HIV/AIDS
this study was to assess retrospectively trends in the prevalence
of these TDF-associated resistance patterns and determine
which factors are associated with their selection in a relatively
large population of patients treated with TDF.
Patients and methods. All HIV drug resistance tests performed during the period January 2000–December 2006 on
samples from antiretroviral-experienced patients with plasma
HIV RNA levels 11000 copies/mL in a reference laboratory
located in Madrid, Spain, were retrospectively examined. A case
report form was filled out for each sample, in which sex, age,
risk category, viral load, CD4+ cell count, and current and past
antiretroviral therapies were recorded in a computerized database. HIV pol sequences were generated from plasma specimens. Genetic sequence analyses were performed using ViroSeq
(Abbott Molecular Diagnostics). For the purpose of this study,
only mutations associated with reduced susceptibility to TDF,
according to the latest International AIDS Society–USA panel
list, were considered [7].
To compare trends in the emergence of TDF-associated resistance patterns over time with changes in the prescription of
antiretroviral therapy, the proportion of patients receiving antiretroviral therapy—and specifically, the number receiving
TDF—was recorded using clinical charts and the hospital pharmacy registry.
Results. After the approval of TDF in Spain, its use rapidly
increased; at the end of 2002, it was prescribed to 400 individuals (40% of all patients receiving HAART in Spain). The
number further increased to 745 patients (70% of those receiving HAART) in 2003, with relative stabilization since then:
755 patients (59% of those receiving HAART) in 2004, 719
(54%) in 2005, and 736 (54%) in 2006. Trends in TDF prescription in combination with other NRTIs significantly
changed over time. TDF plus didanosine was one of the most
widely used combinations during 2002 and 2003 (used in 36%
of patients receiving TDF). However, by 2006, the coformulated
combination of TDF plus emtricitabine was taken by 180% of
patients receiving TDF (figure 1).
Of a total of 1177 samples examined, obtained from 767
different patients with HIV infection who were experiencing
failure of antiretroviral therapy during the study period and
for whom drug resistance testing results were available, 374
(31.7%) were from patients who experienced failure of treatment with TDF. If the study period was restricted to the time
since TDF was marketed, 996 samples from patients who experienced failure of antiretroviral therapy were available for
analysis; of these, 374 (37.5%) belonged to patients who ex-
Figure 1. A, Absolute number of patients receiving HAART, absolute number of patients receiving tenofovir (TDF), and proportion of patients receiving
HAART who were treated with TDF. B, frequency of TDF coadministration with nucleoside reverse-transcriptase inhibitors. ABC, abacavir; ddI, didanosine;
FTC, emtricitabine; 3TC, lamivudine.
perienced failure of treatment with TDF. As expected, virus
with TDF-associated resistance mutations was significantly
more prevalent among patients who experienced failure of TDF
therapy than among patients experiencing failure of treatment
with other NRTI-containing regimens (table 1). Of note, all
but 1 K70E mutation occurred in isolates from patients with
virus harboring either K65R or ⭓3 TAMs.
The rate of distinct TDF-associated resistance patterns
changed significantly over time. K65R was found in !1% of
isolates before 2001; however, the rate of K65R rapidly increased
to 14% of isolates in 2002, which was coincident with TDF
marketing. It decreased to 2.7% of isolates in the period 2005–
2006 (P ! .001). In contrast, the rate of ⭓3 TAMs steadily increased, from 33% of isolates in the period 2002–2004 to 44.1%
of isolates in the period 2005–2006 (P p .02). The prevalence
of T69S inserts remained fairly stable at ∼1% over the entire
study period (figure 2).
Overall, the decrease in the incidence of K65R was mainly
associated with a reduction in the prescription of the combination of TDF plus didanosine, which accounted for 41.6% of
all TDF-including regimens in 2003 but only 0.8% of such
regimens in 2006 (P ! .001). In the univariate analysis, selection
of K65R in patients receiving TDF regimens was associated with
coadministration of didanosine, compared with administration
of another NRTI (20.3% vs. 5.9%; P ! .001) and compared with
administration of abacavir (26.2% vs. 8.1%; P ! .001). No statistically significant association was found between selection of
K65R and administration of other NRTIs, such as lamivudine
or stavudine. Conversely, a significant inverse correlation was
observed for K65R in patients who were experiencing failure
of TDF therapy and were receiving zidovudine concomitantly,
because none of these patients had virus that developed K65R
(0% vs. 11.8%; P p .042). In the multivariate analysis, the
prescription of TDF, didanosine, abacavir, and M184V were
independently associated with the presence of K65R, whereas
the number of TAMs was inversely associated with the presence
of K65R (table 2).
Discussion. TDF is currently one the most widely used
NRTIs for the treatment of HIV infection. Its efficacy, favorable
toxicity profile, and convenient dosing have made this drug
one of—if not the most—popular for first-line treatment, as
has been recorded in several guidelines [1]. However, the emergence of drug resistance (and, specifically, selection of K65R)
has been a matter of concern in patients who experience failure
of TDF therapy, because K65R may result in broad cross-resistance (it currently confers resistance to all NRTIs except
zidovudine) [7]. In this study, we assessed the prevalence of
TDF-associated resistance mutations in a relatively large group
of patients who experienced failure of antiretroviral therapy
and trends since the marketing of TDF in Spain in 2002.
The overall prevalence of virus with TDF-associated resistance mutations in patients who experienced failure of TDFcontaining regimens was relatively low in this study (!15%),
except for the pattern of ⭓3 TAMs including M41L and/or
L210W, which had an overall prevalence of 36.6%. Our study
shows that selection of K65R is associated with exposure not
only to TDF but also to didanosine and abacavir. In other
studies, K65R was selected in up to 4% of persons experiencing
treatment failure of first-line regimens based on stavudine, lamivudine, and efavirenz [8]. This observation is consistent with
the fact that K65R may compromise the antiviral activity of all
NRTIs other than TDF (except for zidovudine) [2].
HIV/AIDS • CID 2008:46 (1 June) • 1783
Table 1.
Tenofovir (TDF)–associated resistance mutations in the study population.
Mutation
All patients
(n p 1177)
K65R
T69 inserts
K70E
⭓3 TAMs (with M41L or L210W)
50
15
4
350
(4.2)
(1.3)
(0.3)
(29.7)
Patients who
experienced failure of
HAART with TDF
(n p 374)
42
8
4
137
(11.2)
(2.1)
(1.1)
(36.6)
Patients who
experienced failure of
HAART without TDF
(n p 803)
8
7
0
213
(1)
(0.9)
(0)
(26.5)
P
.001
.092
.01
.001
NOTE. Data are no. (%) of patients with virus with the specified mutation. TAM, thymidine-associated mutation.
We and others have previously reported that the rate of K65R
rapidly increased after the introduction of TDF in clinical practice [3]. It is noteworthy that this follow-up study showed a
decrease in the rate of K65R in isolates from patients who
experienced failure of therapy with TDF, from 17% of isolates
in 2004 to 3.6% of isolates in 2005 to 0% of isolates in 2006.
This decrease occurred despite a relatively elevated and stable
rate of prescription of TDF over the course of the study period.
The main determinant of selection of K65R in our study was
the coadministration of didanosine or abacavir. In fact, the high
incidence of K65R in earlier years could be directly linked to
widespread prescription of the combination of TDF plus didanosine at our institution, which peaked in 2003 at ∼35% of
all patients receiving antiretroviral therapy. The coadministration of TDF plus didanosine was popular at that time because
of its simplicity (1 pill each administered once per day), relatively good gastrointestinal tolerance, and expected avoidance
of overlapping resistance. This attractiveness, however, soon
disappeared when unexpected and seemingly parodoxical decreases in CD4+ cell count [9], mitochondrial toxicities (in-
cluding pancreatitis), loss of body weight, and increased risk
of hyperglycemia and diabetes [10] began to be reported. Furthermore, an open-label study was prematurely interrupted
when a high rate of virological failure was noticed in drugnaive patients receiving TDF plus didanosine [11]. All patients
in that study harbored viruses with M184V at the time of viral
rebound, and one-half of those patients had virus with the
K65R mutation. Similar results were obtained using the combination of TDF plus abacavir plus lamivudine in drug-naive
individuals [12]. Both cross-resistance driven by selection of
K65R and intracellular interactions between TDF and either
didanosine and abacavir explained all of these findings. Although this knowledge has altered the use of TDF, there is no
doubt that the availability of the coformulation of TDF with
emtricitabine in a single pill has made the continuous use of
TDF very attractive. In our study, this change in the use of
TDF within the past 2 years has been associated with a dramatic
reduction in the incidence of K65R.
It should be noted that the most prevalent pattern of TDF
resistance in our study was the presence of ⭓3 TAMs including
Figure 2. Proportion of different tenofovir (TDF)–associated resistance mutations in virus obtained from 374 patients who experienced virological
failure of TDF-containing regimens. Ins, insert; TAM, thymidine-associated mutation.
1784 • CID 2008:46 (1 June) • HIV/AIDS
Table 2.
Predictors of K65R in the multivariate analysis.
Variable
Antiretroviral drug
Tenofovir
Didanosine
Abacavir
Lamivudine
Stavudine
Resistance mutation
Acknowledgments
OR (95% CI)
P
23.2 (9.8–54.8)
5.2 (2.6–10.6)
.001
.001
4.1 (2.0–8.6)
1.4 (0.6–3.1)
.001
.419
0.94 (0.3–3.1)
.930
K70E
1.3 (0.1–14.4)
.844
M184V
a
TAM
3.8 (1.9–7.8)
.001
0.4 (0.3–0.5)
.001
NOTE. TAM, thymidine-associated mutation.
a
OR is given per no. of TAMs.
M41L and/or L210W. This is mainly attributable to the fact
that most patients who experienced failure of TDF therapy had
previously been exposed to zidovudine and/or stavudine. An
inverse correlation between the presence of TAMs and K65R
has already been shown [2, 3], and patients taking zidovudine
in combination with TDF generally do not develop infection
with virus that has the K65R mutation. In our study, most
failures of TDF therapy in recent years occurred in subjects
with virus that carried multiple TAMs; therefore, K65R is no
longer a major concern. Other potential changes associated with
TDF resistance, such as inserts at codon 69 or K70E, are very
rare and are generally seen in patients with virus that harbors
multiple TAMs or K65R, respectively.
In summary, selection of virus with K65R in patients with
HIV infection who experience failure of antiretroviral therapy
has significantly decreased in recent years (at present, to !1%
of patients receiving HAART), despite a relatively high and
stable rate of prescription of TDF as part of antiretroviral regimens. A wiser use of TDF and, in particular, avoidance of
coadministration with didanosine seem to explain this
observation.
Financial support. Fundación Investigación y Educación en SIDA, Instituto de Salud Carlos III-Redes Tematicas de Investigacion Cooperativa
(RD06/006), Agencia Lain Entralgo, and Fondo de Investigaciones Sanitarias (projects CP06/00284 and PI06/1826).
Potential conflicts of interest. All authors: no conflicts.
References
1. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1 infected adults and
adolescents. Department of Health and Human Services. December
1st 2007. Available at: http://www.aidsinfo.nih.gov/contentfiles/adult
andadolescentgl.pdf. Accessed December 2007.
2. Miller M. K65R, TAMs and tenofovir. AIDS Rev 2004; 6:22–33.
3. Valer L, Martin-Carbonero L, de Mendoza C, Corral A, Soriano V.
Predictors of selection of K65R: tenofovir use and lack of thymidine
analogue mutations. AIDS 2004; 18:2094–6.
4. Miller M, Margot N, Lu B, et al. Genotypic and phenotypic predictors
of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients. J Infect Dis 2004; 189:
837–46.
5. Barrios A, de Mendoza C, Martin-Carbonero L, et al. Role of baseline
HIV genotype as a predictor of viral response to tenofovir in heavily
pre-treated patients. J Clin Microbiol 2003; 41:4421–3.
6. Delaugerre C, Roudiere L, Peytavin G, Rouzioux C, Virad J, Chaix M.
Selection of a rare resistance profile in an HIV-1 infected patient exhibiting a failure to an antiretroviral regimen including tenofovir DF.
J Clin Virol 2005; 32:241–4.
7. Johnson V, Brun-Vezinet F, Clotet B, et al. Update of the drug resistance
mutations in HIV-1: 2007. Top HIV Med 2007; 15:119–25.
8. Margot M, Lu B, Cheng A, Miller M, and the Study 903 Team. Resistance development over 144 weeks in treatment-naı̈ve patients receiving tenofovir disoproxil fumarate or stavudine with lamivudine
and efavirenz in the study 903. HIV Med 2006; 7:442–50.
9. Barrios A, Rendon A, Negredo E, et al. Paradoxical CD4+ T cell decline
in HIV-infected patients with complete virus suppression taking tenofovir and didanosine. AIDS 2005; 19:569–75.
10. Garcia-Benayas T, Rendon A, Rodriguez-Novoa S, et al. Higher risk
of hyperglycemia in HIV-infected patients treated with didanosine plus
tenofovir. AIDS Res Hum Retroviruses 2006; 22:333–7.
11. Jemsek J, Hutcherson P, Harper E. Poor virological responses and early
emergence of resistance in treatment-naive HIV-infected patients receiving a once daily triple nucleoside regimen of didanosine, lamivudine and tenofovir DF [abstract 51]. In: Program and abstracts of
the 11th Conference on Retroviruses and Opportunistic Infections,
8–11 February 2004 (San Francisco).
12. Gallant J, Rodriguez A, Weinberg W, et al. Early virologic nonresponse
to tenofovir, abacavir, and lamivudine in HIV-infected antiretroviralnaive subjects. J Infect Dis 2005; 192:1921–30.
HIV/AIDS • CID 2008:46 (1 June) • 1785