Increased Risk of Q151M and K65R Mutations in Patients Failing Stavudine-Containing First-Line Antiretroviral Therapy in Cambodia

Abstract

Multi-nucleos(t)ide resistance (MNR) mutations including Q151M, K65R mutations, and insertion at codon 69 of HIV-1 reverse transcriptase coding region may confer resistance to all molecules of nucleos(t)ide reverse transcriptase inhibitors (NRTI). The presence of these mutations is an emerging problem compromising non-nucleoside reverse transcriptase inhibitors and protease inhibitors-based therapies. Furthermore, factors associated with selection of these mutations are still not well defined. The current study aimed to evaluate the frequency and to characterize factors associated with the occurrence of multi-nucleos(t)ide resistance mutations among HIV-1 infected patients failing recommended first-line antiretroviral regimens in Cambodia.
This is a retrospective analysis of HIV-1 drug resistance genotyping of 520 HIV-1 infected patients in virological failure (viral load > 250 copies/mL) while on first-line antiretroviral therapy in Cambodia with at least one reverse transcriptase inhibitor resistance associated mutation. Among these 520 patients, a total of 66 subjects (66/520, 12.7%) presented ≥1 MNR mutation, including Q151M, K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%) patients, respectively. In multivariate analysis, both Q151M (p = 0.039) and K65R (p = 0.029) mutations were independently associated with current stavudine- compared to zidovudine-use.
Such selection of mutations by stavudine drastically limits the choice of antiretroviral molecules available for second-line therapy in resource-limited settings. This finding supports the World Health Organization's recommendation for stavudine phase-out.

Full text

Increased Risk of Q151M and K65R Mutations in Patients
Failing Stavudine-Containing First-Line Antiretroviral
Therapy in Cambodia
Janin Nouhin
1,2
*, Yoann Madec
3
, Nicole Ngo-Giang-Huong
4,5,6
, Laurent Ferradini
7
, Eric Nerrienet
1,8
1HIV/Hepatitis Unit, Institut Pasteur in Cambodia, Phnom Penh, Cambodia, 2Universite
´Paris Diderot, Sorbonne Paris Cite
´, Paris, France, 3Emerging Disease Epidemiology
Unit, Department of Infection and Epidemiology, Institut Pasteur, Paris, France, 4Institut de Recherche pour le De
´veloppement (IRD UMI 174), Paris, France, 5Faculty of
Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand, 6Harvard School of Public Health, Boston, Massachusetts, United States of America, 7FHI 360,
Phnom Penh, Cambodia, 8Regulation of Retroviral Infection Unit, Department of Virology, Institut Pasteur, Paris, France
Abstract
Background:
Multi-nucleos(t)ide resistance (MNR) mutations including Q151M, K65R mutations, and insertion at codon 69
of HIV-1 reverse transcriptase coding region may confer resistance to all molecules of nucleos(t)ide reverse transcriptase
inhibitors (NRTI). The presence of these mutations is an emerging problem compromising non-nucleoside reverse
transcriptase inhibitors and protease inhibitors-based therapies. Furthermore, factors associated with selection of these
mutations are still not well defined. The current study aimed to evaluate the frequency and to characterize factors
associated with the occurrence of multi-nucleos(t)ide resistance mutations among HIV-1 infected patients failing
recommended first-line antiretroviral regimens in Cambodia.
Methodology/Principal Finding:
This is a retrospective analysis of HIV-1 drug resistance genotyping of 520 HIV-1 infected
patients in virological failure (viral load .250 copies/mL) while on first-line antiretroviral therapy in Cambodia with at least
one reverse transcriptase inhibitor resistance associated mutation. Among these 520 patients, a total of 66 subjects (66/520,
12.7%) presented $1 MNR mutation, including Q151M, K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%) patients,
respectively. In multivariate analysis, both Q151M (p = 0.039) and K65R (p = 0.029) mutations were independently associated
with current stavudine- compared to zidovudine-use.
Conclusion:
Such selection of mutations by stavudine drastically limits the choice of antiretroviral molecules available for
second-line therapy in resource-limited settings. This finding supports the World Health Organization’s recommendation for
stavudine phase-out.
Citation: Nouhin J, Madec Y, Ngo-Giang-Huong N, Ferradini L, Nerrienet E (2013) Increased Risk of Q151M and K65R Mutations in Patients Failing Stavudine-
Containing First-Line Antiretroviral Therapy in Cambodia. PLoS ONE 8(8): e73744. doi:10.1371/journal.pone.0073744
Editor: Nicolas Sluis-Cremer, University of Pittsburgh, United States of America
Received May 7, 2013; Accepted July 19, 2013; Published August 28, 2013
Copyright: 2013 Nouhin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: njanin@pasteur-kh.org
Introduction
Q151M, K65R substitutions, and insertions at codon 69
(Insert69) in the reverse transcriptase encoding region of HIV-1
genome confer resistance to a large range of nucleos(t)ide reverse
transcriptase inhibitors (NRTIs) and are thus called multi-
nucleos(t)ide resistance (MNR) mutations. Q151M confers resis-
tance to all NRTIs, except tenofovir (TDF) [1]. K65R confers
resistance to stavudine (d4T) and TDF and possibly to
lamivudine/emtricitabine (3TC/FTC), didanosine (ddI) and
abacavir (ABC) [2,3,4,5], while Insert69 confers resistance to all
NRTIs available today [6,7,8]. These mutations dramatically
compromise subsequent antiretroviral (ARV) regimens.
Factors associated with selection of these mutations are still not
well defined. d4T/ddI dual therapy has been associated with the
occurrence of Q151M and K65R [9,10,11], as well as young age,
low CD4
+
T-cell count, high HIV RNA viral load (VL) and
experience of more than two ARV regimens before resistance
genotyping testing [12,13]. However, these studies mostly enrolled
patients under dual therapies. Few data on Highly Active
Antiretroviral Therapy (HAART) failure in resources limited
setting are available [5,13]. Our study aimed to evaluate the
frequency and to identify factors associated with the occurrence of
MNR mutations among HIV-1 CRF01_AE infected patients
failing recommended first-line HAART in Cambodia.
Methods
Ethics statement
The current study was a retrospective and anonymous analysis
of data collected in the context of routine care of HIV infected
patients. Data was stored in a database of HIV/Hepatitis
Laboratory, Institut Pasteur in Cambodia situated in Phnom
Penh (the capital city of Cambodia). This kind of analysis complies
with the Cambodian ethical guidelines for exemption from ethical
approval requirement [14].
PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e73744

Study design
We conducted a retrospective and observational study using
data routinely collected between December 2004 and January
2011 from 9018 HIV-1 infected patients, on first-line ARV
regimen, and in the context of routine care. All samples of these
9018 individuals were referred from many parts of Cambodia
(including the capital city and 7 provinces).
In agreement with the World Health Organization (WHO)
recommendation, the Cambodian national guidelines have
recommended d4T, 3TC, and nevirapine (NVP) as the standard
first-line ARV regimen and zidovudine (AZT), TDF, ABC,
efavirenz (EFV), and/or protease inhibitors in case of drug side-
effect or interaction. Laboratory monitoring included CD4
+
T-cell
count every six months whereas HIV-1 RNA VL and drug
resistance genotyping were carried out to confirm clinical and/or
immunological failure [15,16].
All specimens were assessed for HIV-1 RNA VL testing and
drug resistance genotyping in HIV/Hepatitis Laboratory of
Institute Pasteur in Cambodia which was (at the time of the
study) centralizing all virological tests for HAART monitoring.
HIV-1 RNA VL was performed on stored (280uC) plasma
specimens using the G2 Generic HIV-1 VL ANRS kit (Biocentric,
Bandol, France) [17]. If the HIV-1 RNA VL is above the
threshold (VL .250 copies/mL), the reverse transcriptase
inhibitor (RTI) resistance genotyping test was performed using
bulk sequencing of HIV-1 RT coding region according to
complete sequencing procedures and primer sequences described
by ANRS (Agence Nationale de Recherche sur le SIDA et les
hepatites virale – French national Agency for research on AIDS
and viral hepatitis) working group [18].
Results and Discussion
Of 9018 patients referred to our laboratory, 8304 (92.1%) had
undetectable HIV-1 RNA VL. This rate represented a good
virological outcome and was consistent with studies previously
conducted in Cambodia amongst HIV-1 infected patients after
one to three years of first-line ARV regimen [19,20,21,22]. Seven
hundred and fourteen patients had detectable HIV-1 RNA VL.
Among these, 194 patients presented no resistance associated
mutation (RAM) and were excluded from the present analysis as
we assumed they were not adherent to the ARV treatment [23].
Finally, 520 subjects infected with HIV-1 CRF01_AE strains and
presenting at least one RTI RAM were included in this analysis.
Their median age was 28 years [Inter Quartile Range (IQR): 9-
38] at the time of sampling, and 233 (44.8%) patients were
children (#15 years) followed-up at the National Pediatric
Hospital (Table 1). Median duration on current first-line regimen
was 30 months [IQR: 18-44], and median HIV-RNA VL was
4.5log
10
copies/mL [IQR: 4.0-5.1]. Three hundred and twenty
eight patients (63%) (212 children and 116 adults) were receiving
d4T-containing regimens and the remaining was under an AZT-
containing regimen.
According to the ANRS algorithm (version 19) [18], 498
(95.8%) patients were resistant to at least 3 RTIs and 339 (65.2%)
to 4 RTIs. The most frequently detected mutations were: M184I/
V (92.3%), Y181C/I/V (47.1%), T215I/C/F/N/S (38.8%),
D67E/G/N (37.3%), K103N/R/S (33.9%), and G190A/Q/S
(32.5%). Regarding MNR mutations, a total of 66 subjects (66/
520, 12.7%) presented $1 MNR mutation, including Q151M,
K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%)
patients, respectively.
To identify factors associated with the presence of Q151M and
K65R mutations, univariate and multivariate logistic regression
analysis were performed using the following variables: gender, age
group (#or .15 years), HIV-1 RNA VL level (#or .4.5log
10
copies/mL), current first-line regimen (d4T- or AZT-containing)
and duration (#or .30 months) at sampling time.
In univariate analysis, Q151M was significantly more frequent
in patients on d4T-containing regimens (14.3%) than in those on
AZT-containing regimens (6.3%) (p = 0.005). A high HIV-1 RNA
level was also significantly associated with Q151M (p,0.0001).
Q151M mutation tended to be associated with age #15 years
(p = 0.069) and duration .30 months under current regimen
(p = 0.071). In multivariate analysis, independent risk factors for
Q151M were d4T-containing regimen (Odds ratio (OR) [95%
confidence interval (CI)]: 2.21 [1.0524.65]), high HIV-1 RNA
level (OR [95% CI]: 4.02 [2.1327.59]), and duration .30 months
under current first-line regimen (OR [95% CI]: 2.11 [1.1823.76])
(table 1).
The occurrence of K65R was significantly associated with d4T-
containing regimens (p = 0.017) and high HIV-1 RNA level
(p = 0.003), while there was a trend with duration .30 months
under current first-line regimen (p = 0.054). In multivariate
analysis, independent risk factors for K65R were d4T-containing
regimen (OR [95% CI]: 3.02 [1.1228.13]), high HIV-RNA level
(OR [95% CI]: 3.97 [1.6429.61]) and duration .30 months
under current first-line regimen (OR [95% CI]: 2.6 [1.1625.83])
(table 1).
In multivariate analysis, besides higher HIV-1 RNA VL level,
probably associated with longer duration of failure, we found that
d4T-use remained independently associated with the presence of
MNR mutations (either Q151M or K65R). Finally, the Q151M
and K65R mutations were positively associated to each other.
Indeed, K65R was detected in 12/59 (20.3%) patients harboring
Q151M compared to 3.9% of patients without Q151M (x
2
test,
p,0.001). This result is consistent with previous study conducted
in Italy [24]. In our study, other NRTI-RAMs known to be
associated with Q151M-mediated multi-nucleoside resistance
including A62V, V75I, F77L, and F116Y [12], were also
significantly more common in patients harboring the Q151M
mutation (p,0.001).
Other studies have reported a link between the use of d4T/ddI
combination and the selection of MNR mutations, in particular
among patients infected by HIV-1 non B genotype
[9,10,11,13,25]. However, in our study, no patients received ddI
at the time of genotyping. Indeed, children could not be prescribed
ddI as first-line regimen under Cambodian protocol. Thus, our
data strongly suggests that d4T per se is directly associated with the
occurrence of both Q151M and K65R mutations.
Our retrospective study has some limitations. First, history of
ARV treatment was incomplete for all patients included in the
study. The initial first-line regimen recommended in Cambodia
was based on d4T and it is very likely that a significant proportion
of adults under AZT at the time of genotyping had initiated a prior
d4T-based first-line regimen which was then changed to AZT due
to the occurrence of adverse events. This might explain the
presence of Q151M mutation among some patients under AZT
regimen (6.3%) at the time of genotyping. As reported recently
(2012) by Phan et al., in a follow-up conducted between 2003 and
2010 in the Sihanouk-Hospital-Center-of-Hope in Phnom Penh,
Cambodia, d4T was discontinued and switched to AZT in around
48% of patients due to d4T-toxicity [26] while no AZT to d4T
switch was reported. Due to similar setting, it is very likely that the
proportion of d4T to AZT (and AZT to d4T) switches in our study
population are very close to those found by Phan et al. As
consequence, we may have underestimated the risk of MNR
mutations due to d4T-use since some have been erroneously
Factors Associated with MNR Mutations in Cambodia
PLOS ONE | www.plosone.org 2 August 2013 | Volume 8 | Issue 8 | e73744

attributed to AZT-use. Second, we were not able to document
duration of treatment failure, and adherence levels to ARV drugs.
It is thus possible that other factors not analyzed herein were not
taken into account with the occurrence of MNR mutations.
Conclusion
In conclusion, our data suggests that the administration of d4T
per se is associated with the emergence of both Q151M and K65R
MNR mutations which confer resistance to a large range of
NRTIs. It is important to note that the effect of d4T is observed
even after adjusting for HIV-1 RNA VL. Selection of such
mutations is a critical issue in resources-limited settings where
NRTI molecules available for second-line regimen are still limited.
Although the primary reasons for the 2010 WHO’s recommen-
dation to reduce or abandon the use of d4T as part of first-line
ARV regimen in resource-limited settings were related to excessive
toxicity [27], our findings further support this guidance due to
association with mutations which render the virus resistant to most
or all of NRTI. However, the transition to alternative regimens
still appears difficult and d4T remains widely used in such settings
because of its affordability as a low-cost generic fixed-dose
combinations. Recently, the Cambodian HIV National Program
committed to move out d4T from its recommended first-line ARV
regimen in the national guideline.
Nucleotide accession number
Five hundred and twenty nucleotide sequence of CRF01_AE
HIV-1 reverse transcriptase coding region included in the current
analysis have been submitted to Genkbank under the accession
numbers KF292310 to KF292829.
Acknowledgments
We thank Kerya Phon, Sreymom Ken, and Sopheak Ngin for their
technical assistances. We are grateful to Dr. Franc¸ois Rouet for his critical
review of the manuscript.
Author Contributions
Conceived and designed the experiments: JN LF EN. Performed the
experiments: JN. Analyzed the data: YM. Wrote the paper: JN YM NN LF
EN.
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Table 1. Multivariate analysis of factors associated with the occurrence of Q151M and K65R mutations.
Q151M K65R
All subjects
(n = 520)
Patients
(n = 59)
Adjusted OR
{
[95% CI]
`
p value
Patients
(n = 29)
Adjusted OR
{
[95% CI]
`
p value
Male gender, n (%) 330 (63.5) 42 (71.2) 17 (58.6)
Age group, n (%) 0.32
Children (#15 years) 233 (44.8) 33 (55.9) 1 17 (58.6)
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HIV RNA VL level, n(%) ,0.001 0.002
#4.5log
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copies/mL 264 (50.8) 15 (25.4) 1 7 (24.1) 1
.4.5log
10
copies/mL 256 (49.3) 44 (74.6) 4.02 [2.13 – 7.59] 22 (75.9) 3.97 [1.64 – 9.61]
Current 1
st
line regimen, n (%) 0.039 0.029
D4T containing 328 (63.1) 47 (79.7) 2.21 [1.05 – 4.65] 24 (82.8) 3.02 [1.12 – 8.13]
AZT containing 192 (36.9) 12 (20.3) 1 5 (17.2) 1
Duration under current 1
st
line, n (%) 0.012 0.020
#30 months 269 (51.7) 24 (40.7) 1 19 (65.6) 1
.30 months 251 (48.3) 35 (59.3) 2.11 [1.18 – 3.76] 10 (34.4) 2.60 [1.16 – 5.83]
{
Odd ratio estimated using logistic analysis.
`
95% Confident interval.
doi:10.1371/journal.pone.0073744.t001
Factors Associated with MNR Mutations in Cambodia
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