Primary resistance to integrase strand transfer inhibitors in Spain using ultrasensitive HIV-1 genotyping

Abstract

Background:
Transmission of resistance mutations to integrase strand transfer inhibitors (INSTIs) in HIV-infected patients may compromise the efficacy of first-line antiretroviral regimens currently recommended worldwide. Continued surveillance of transmitted drug resistance (TDR) is thus warranted.

Objectives:
We evaluated the rates and effects on virological outcomes of TDR in a 96 week prospective multicentre cohort study of ART-naive HIV-1-infected subjects initiating INSTI-based ART in Spain between April 2015 and December 2016.

Methods:
Pre-ART plasma samples were genotyped for integrase, protease and reverse transcriptase resistance using Sanger population sequencing or MiSeq™ using a ≥ 20% mutant sensitivity cut-off. Those present at 1%-19% of the virus population were considered to be low-frequency variants.

Results:
From a total of 214 available samples, 173 (80.8%), 210 (98.1%) and 214 (100.0%) were successfully amplified for integrase, reverse transcriptase and protease genes, respectively. Using a Sanger-like cut-off, the overall prevalence of any TDR, INSTI-, NRTI-, NNRTI- and protease inhibitor (PI)-associated mutations was 13.1%, 1.7%, 3.8%, 7.1% and 0.9%, respectively. Only three (1.7%) subjects had INSTI TDR (R263K, E138K and G163R), while minority variants with integrase TDR were detected in 9.6% of subjects. There were no virological failures during 96 weeks of follow-up in subjects harbouring TDR as majority variants.

Conclusions:
Transmitted INSTI resistance remains rare in Spain and, to date, is not associated with virological failure to first-line INSTI-based regimens.

Full text

Primary resistance to integrase strand transfer inhibitors in Spain
using ultrasensitive HIV-1 genotyping
M. Casadella`
1
†, J. R. Santos
2
*†, M. Noguera-Julian
1
, R. Mica´n-Rivera
3
, P. Domingo
4
, A. Antela
5
, J. Portilla
6
,
J. Sanz
7
, M. Montero-Alonso
8
, J. Navarro
9
, M. Masia´
10
, N. Valcarce-Pardeiro
11
, A. Ocampo
12
,
L. Pe´rez-Martı´nez
13
, J. Pasquau
14
, M. J. Vivancos
15
,A.Imaz
16
, P. Carmona-Oyaga
17
,L.Mu
~
noz-Medina
18
,
J. Villar-Garcı´a
19
, P. Barrufet
20
and R. Paredes
1,2
on behalf of the INSTINCT Study Group‡
1
IrsiCaixa AIDS Research Institute, Badalona, Catalonia, Spain;
2
Lluita contra la SIDA Foundation, Hospital Universitari Germans Trias i
Pujol, Badalona, Spain;
3
University Hospital La Paz, Madrid, Spain;
4
Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau,
Barcelona, Spain;
5
Infectious Diseases Unit, Santiago de Compostela Clinical University Hospital, Santiago de Compostela, Spain;
6
Hospital General Universitario de Alicante, Alicante, Spain;
7
University Hospital de La Princesa, Madrid, Spain;
8
Infectious Diseases
Unit, La Fe University and Polytechnic Hospital, Valencia, Spain;
9
Infectious Diseases Department, Hospital Universitari Vall d’Hebron,
Barcelona, Spain;
10
Infectious Diseases Unit, Elche University General Hospital, Elche, Spain;
11
Infectious Diseases Unit, Hospital
Arquitecto Marcide, Ferrol, Spain;
12
HIV Unit, Hospital A
´lvaro Cunqueiro, Vigo, Spain;
13
Infectious Diseases Area, Hospital San Pedro-
CIBIR, Logro~
no, Spain;
14
University Hospital Virgen de las Nieves, Granada, Spain;
15
Infectious Diseases Unit, Ramo´ n y Cajal Hospital,
Madrid, Spain;
16
HIV and STI Unit, Infectious Diseases Department, Bellvitge University Hospital, Bellvitge Biomedical Research
Institute (IDIBELL), Hospitalet de Llobregat, Spain;
17
Infectious Diseases Unit, Donostia University Hospital, San Sebastia´ n, Spain;
18
University Hospital San Cecilio, Granada, Spain;
19
Infectious Diseases Department, Hospital del Mar – IMIM, Barcelona, Spain;
20
Infectious Diseases Unit, Mataro´ Hospital, Mataro´ , Spain
*Corresponding author. E-mail: jrsantos@flsida.org
†These authors contributed equally.
‡Members are listed in the Acknowledgements section.
Received 2 April 2020; accepted 3 July 2020
Background: Transmission of resistance mutations to integrase strand transfer inhibitors (INSTIs) in HIV-
infected patients may compromise the efficacy of first-line antiretroviral regimens currently recommended
worldwide. Continued surveillance of transmitted drug resistance (TDR) is thus warranted.
Objectives: We evaluated the rates and effects on virological outcomes of TDR in a 96 week prospective multi-
centre cohort study of ART-naive HIV-1-infected subjects initiating INSTI-based ART in Spain between April 2015
and December 2016.
Methods: Pre-ART plasma samples were genotyped for integrase, protease and reverse transcriptase resistance
using Sanger population sequencing or MiSeq
TM
using a 20% mutant sensitivity cut-off. Those present at
1%–19% of the virus population were considered to be low-frequency variants.
Results: From a total of 214 available samples, 173 (80.8%), 210 (98.1%) and 214 (100.0%) were successfully
amplified for integrase, reverse transcriptase and protease genes, respectively. Using a Sanger-like cut-off, the
overall prevalence of any TDR, INSTI-, NRTI-, NNRTI- and protease inhibitor (PI)-associated mutations was
13.1%, 1.7%, 3.8%, 7.1% and 0.9%, respectively. Only three (1.7%) subjects had INSTI TDR (R263K, E138K and
G163R), while minority variants with integrase TDR were detected in 9.6% of subjects. There were no virological
failures during 96 weeks of follow-up in subjects harbouring TDR as majority variants.
Conclusions: Transmitted INSTI resistance remains rare in Spain and, to date, is not associated with virological
failure to first-line INSTI-based regimens.
Introduction
Integrase strand transfer inhibitors (INSTIs) are the current key-
stones of ART due to their high efficacy, safety and tolerability.
1–3
The first-generation INSTIs, raltegravir and elvitegravir, were the
preferred drugs for ART initiation until 2017.
4,5
Dolutegravir and,
more recently, bictegravir have replaced them as the preferred
drugs for naive patients,
2,3
and are also suitable options for salvage
V
CThe Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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therapy, even in subjects with previous failure or some degree of
resistance to raltegravir or elvitegravir failure.
6–9
Virological failure (VF) to first-generation INSTI-based regimens
is often associated with selection of INSTI-resistant HIV-1, which
can be transmitted and potentially impair the clinical and virologic-
al outcomes of INSTI-based ART in drug-naive individuals.
10–12
However, although anecdotal cases of INSTI resistance transmis-
sion have been reported,
13,14
the prevalence of INSTI-associated
mutations in ART-naive subjects has remained low to date.
15–19
Given the increasing global use of INSTI-containing regimens,
including raltegravir, elvitegravir, dolutegravir, bictegravir and soon
cabotegravir, and the significant degree of cross-resistance among
different INSTIs, it is essential to perform continuous surveillance
of integrase transmitted drug resistance (TDR) as well as to moni-
tor its clinical impact.
In this study, we evaluated the prevalence of integrase-
associated mutations in naive patients from clinical practice start-
ing INSTI-based regimens in Spain between 2015 and 2016, and
the rate of VF in patients with transmitted INSTI mutations.
Materials and methods
Study design and data collection
The INSTINCT cohort study is a prospective, 96 week observational study
involving a cohort of995 HIV-positive adult subjects who initiated raltegra-
vir, elvitegravir or dolutegravir between 1 April 2015 and 5 October 2016 in
19 HIV care centres across Spain. Patients were included if they satisfied
the inclusion criteria for one of the following groups: (i) ART-naive, i.e. previ-
ously untreated subjects at the time of INSTI initiation; (ii) ART switch, i.e.
ART-experienced individuals initiating the new INSTI with a viral load (VL)
50 copies/mL; and (iii) ARTsalvage, i.e. ART-experienced patientsinitiating
the new INSTI with a VL >200 copies/mL, including patients who were on a
voluntary ART interruption. Data were collected according to a pre-defined
schedule on the date of raltegravir, elvitegravir or dolutegravir initiation
(baseline) and every 6 months thereafter up to 2 years, in accordance with
the study protocol. The study collected information on demographic char-
acteristics, AIDS stage at the time of INSTI initiation, HCV and HBV coinfec-
tions, HIV-1 RNA levels, CD4 cell counts, CD4 count nadir at baseline and
genotypic test results performed before raltegravir, elvitegravir and dolute-
gravir initiation(when available) and at VF.
In the current analysis, we evaluated the percentage of TDR to INSTIs
and other drug classes in ART-naive patients as well as the rates of VF in
subjects with TDR to INSTIs. VF was defined as two consecutive HIV-1 RNA
measurements 200 copies/mL after 6 months while receiving raltegravir,
elvitegravir or dolutegravir, and at least 3 monthsafter INSTI initiation.
Ethics
Prior approval was given by the Ethics Committee (EPA-15-006) of each
participating centre for the study, which was performed following the stipu-
lations of the Declaration of Helsinki (Brazil, 2013). All patients provided
their written informedconsent at study enrolment.
Virological analyses
Baseline plasma samples of subjects from 13 different Spanish hospitals
were shipped to the IrsiCaixa laboratory (Badalona, Spain) for ultrasensitive
genotyping using next-generation sequencing (NGS). Local performance of
Sanger population sequencing was allowed in cases where there were
issues of storage or shipping.
Given the current absence of a consensus list of transmitted INSTI
mutations, we considered as INSTI resistance mutations those included in
the IAS-USA 2019 list
20
or in the Stanford University HIV drug resistance
database (HIVdb, version 8.9 available at: https://hivdb.stanford.edu/)
with a scoreof >14 to at least one INSTI. For protease (PR) and reverse tran-
scriptase (RT) mutations, the WHO 2009 list was used to determine the
prevalence of primaryresistance.
21
We defined two viral population sensitivity thresholds: mutations
detected by Sanger sequencing or present at 20% of the virus population
by NGS (Sanger-like sequencing) were regarded as high-frequency variants;
those present at 1%–19% of the virus population were considered low-
frequency variants. High-frequency variants were considered in order to
evaluate the prevalence of TDR mutations in our study.
In addition, the number of ‘active’ drugs prescribed at baseline was
evaluated by means of the HIVdb Genotypic Susceptibility Score (GSS),
considering a score of 0–9 as susceptible (1 point), 10–59 as intermediate
(0.5 points) and 60 as resistant (0 points).
For NGS, amplification and sequencing steps were performed as
previously described by Inzaule et al.
22
In brief, viral RNA was extracted
from plasma samples using the QIAmp Viral RNA Mini Kit (Qiagen Inc.,
Chatsworth, CA, USA), following the manufacturer’s protocol. RNA was
thereafter retro-transcribed and amplified, generating an amplicon of the
HIV-1 pol gene by a one-step reaction using the SuperScript
TM
III One-Step
RT–PCR System with Platinum Taq DNA Polymerase (ThermoFisher
Scientific Inc., Waltham, MA, USA), followed by a nested PCR using
Platinum
TM
Taq DNA Polymerase HighFidelity (ThermoFisher Scientific Inc.).
Once amplified, DNA was purified using AMPure XP Beads (Beckman Coulter
Inc., Brea, CA, USA) and quantified in duplicate using the Quant-iT
TM
PicoGree0060045
TM
dsDNA Assay Kit (ThermoFisher Scientific Inc.). It was
then diluted and prepared for sequencing using the Nextera XT DNA
Sample Preparation Kit and Nextera XT Index Kit (Illumina, San Diego, CA,
USA). Finally, samples were pooled and sequenced with a 500 cycle MiSeq
ReagentKit v0.2 (Illumina).
MiSeq sequences (FastQ files) were downloaded from Basespace
(Illumina) and analysed using PASeq v1.4 (https://paseq.org) (IrsiCaixa,
Barcelona, Spain). Briefly, data were quality filtered using Trimmomatic
(v0.30),
23
with a Q25/5bp minimum sliding window and a 70 bp minimum
length. Non-viral contamination was filtered out using BBsplit (v35.76).
Filtered reads were then merged with PEAR (v0.9.6) aligned to reference
sequence using Bowtie2 (v2.1.0).
24
Amino acid variants were then called at
the codon level using Perl code, and these data were used to query the
Stanford HIVdb for resistance interpretation. A consensus sequence was
built using major nucleotide readoutat each position.
Results
A total of 241 naive subjects were included in the study. Of these,a
genotype was requested in 232. Reliable results by NGS were
obtained for 146 samples, whereas Sanger sequencing was locally
available for 68 subjects. In Sanger-sequenced samples, the inte-
grase gene was analysed in only 27 (39.7%), while the RT and PR
genes were evaluated in 64 (94.1%) and 68 (100.0%) samples, re-
spectively. In summary, out of a total of 214 available genotypes,
173 (80.8%), 210 (98.1%) and 214 (100.0%) contained informa-
tion for the integrase, RT and PR genes, respectively (a flow chart of
genotyped samples is detailed in Figure 1). Subtype B was the
most common HIV-1 subtype found (158/214, 73.8% of subjects).
Subjects’ characteristics are summarized in Table 1.
Considering only high-frequency variants, the overall rate of
TDR was 13.1% (28/214), if we only considered subjects with com-
plete genotypic information on integrase, RT and PR. Respectively,
INSTI-, NRTI-, NNRTI- and PI-associated resistance mutations
were detected in 3/173 (1.7%), 8/210 (3.8%), 15/210 (7.1%) and
2/214 (0.9%) subjects (Figure 2a). In addition, 37/146 (25.3%)
Casadella` et al.
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samples evaluated by NGS had low-frequency TDR mutations
(Figure 2a). The mutations found are listed inTable S1 (available as
Supplementary data at JAC Online).
INSTI resistance
Only 3/173 (1.7%) individuals had transmitted mutations with
expected impact on INSTI susceptibility, E138K, R263K and
G163R, respectively (Figure 2a). Polymorphic mutations were
found in another 5/173 (2.8%) subjects [T97A in three samples;
L74I and L74M in another two (Table S1)]. All subjects carried
only one INSTI mutation, except for one individual who simul-
taneously revealed T97A (>99% of viruses) and R263K (1.9% of
viruses) mutations.
Low-frequency INSTI-associated mutations were found in
14/146 (9.6%) subjects (Figure 2a). Only two subjects (1.4%)
with low-frequency Q148H and R263K variants, respectively,
were detected (Figure 2b). No patients with N155H or Y143R
were found. Combinations of majority and minority INSTI muta-
tions were found in three (2.1%) individuals analysed by NGS,
including G163R (2.3%) !Q95K (2.3%), E138K (2.8%) !S230R
(1.0%) and the previously described T97A (>99%) !R263K
(1.9%).
NNRTI resistance
K103N was the most frequently detected NNRTI mutation, being
found as the majority variant in 10/210 (4.8%) samples. All the
other NNRTI-associated mutations only accounted for 2.4% of
transmitted NNRTI mutations (Figure 2b).
A total of 6/146 (4.1%) samples had low-frequency NNRTI-
associated mutations, two of which had K103S and Y188C
mutations in >5%–20% of their viral population while four had
Figure 1. Flow chart of baseline samples for HIV genotypes. NGS, next-
generation sequencing; INI, integrase; RT, reverse transcriptase; PR, pro-
tease. This figure appears in colour in the online version of JAC and in
black and white in the print version of JAC.
Table 1. Patient characteristics (n= 241)
RAL (n= 11) EVG (n= 80) DTG (n= 150) Total (n= 241)
Age (years) 42.0 (28.5–57.5) 33.5 (29.0–41.5) 35.0 (30.0–43.0) 35.0 (29.0–43.0)
HIV-1 RNA (copies/mL) 78 311 (37 060–301 444) 34 669 (7617–127 837) 56063 (17 700–180 068) 51 500 (15 043–174 500)
Gender, n(%)
female 1 (9.1) 8 (10.0) 17 (11.3) 26 (10.8)
male 10 (90.9) 72 (90.0) 133 (88.7) 215 (89.2)
Mode of transmission, n(%)
MSM 4 (36.4) 60 (75.0) 103 (68.7) 167 (69.3)
MSW 4 (36.4) 15 (18.8) 31 (20.7) 50 (20.7)
IDU 1 (9.1) 3 (3.8) 6 (4.0) 10 (4.1)
other/unknown 2 (18.2) 2 (2.5) 10 (6.7) 14 (5.8)
Baseline CD4 cell count, cells/mm
3
454.0 (117.5–515.0) 415.00 (273.0–548.0) 429.0 (295.8–614.5) 429.0 (285.3–599.0)
Nadir CD4 cell count, cells/mm
3
451.0 (123.0–467.0) 374.0 (286.0–520.0) 384.5 (288.3–551.8) 384.0 (281.5–542.5)
C stage of AIDS at diagnosis, n(%) 3 (27.3) 4 (5.0) 9 (6.0) 16 (6.6)
Hepatitis coinfection, n(%)
no/unknown 0 2 (2.5) 0 2 (0.8)
HCV 0 3 (3.8) 2 (1.3) 5 (2.1)
HCV 2 (18.2) 6 (7.5) 9 (6.0) 17 (7.1)
Nucleoside pair, n(%)
TDF or TAF/FTC 5 (83.3) 50 (98) 15 (16.5) 70 (47.3)
ABC/3TC 1 (16.7) 1 (2) 76 (83.5) 78 (52.7)
RAL, raltegravir; EVG, elvitegravir; DTG, dolutegravir; MSM, men who have sex with men; MSW, men who have sex with women; IDU, intravenous drug
use; HCV, hepatitis C virus; HBV, hepatitis B virus; TDF, tenofovir disoproxil fumarate; TAF/FTC, tenofovir alafenamide plus emtricitabine; ABC/3TC, aba-
cavir plus lamivudine.
Data are presented as median (IQR) unless otherwise stated.
Primary INSTI resistance in Spain JAC
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other NNRTI-associated mutations in 1%–5% of their viral popula-
tion (Figure 2b).
NRTI resistance
NRTI-associated mutations were detected as majority variants
in 8/210 (3.8%) samples, and in another 8/146 (5.5%) subjects
as low-frequency variants (Figure 2b). The most frequent muta-
tion was M41L, which was detected as the majority variant in 3/
210 (1.4%) individuals. Other NRTI-associated mutations found
as majority and low-frequency variants are detailed in
Figure 2b.
PI resistance
Only 2/214 (0.9%) subjects had transmitted PI mutations
(L90M and M46L, respectively) as majority variants. Additional
low-frequency PI-resistant mutants included in the WHO 2009
surveillance mutation list were found in 10/146 (6.8%) subjects
(Figure 2b).
GSSs
Considering only subjects with the integrase gene successfully
analysed and mutations found as majority variants, virtually all
viruses showed full susceptibility to INSTI-based regimens. Only
one subject harbouring L74I (GSS = 2 points) started raltegravir !
abacavir/lamivudine (Figure 3). Of subjects undergoing dolutegra-
vir/lamivudine dual therapy, only 2/173 (1.15%) had a GSS 1.5
points (each harbouring mutations E138K and R263K in 99.8% of
their viral population, respectively).
Virological outcomes of TDR
TDR did not affect the outcomes of first-line INSTI-based ART, since
none of the subjects harbouring transmitted INSTI-, NNRTI-, NRTI-
or PI-associated mutations experienced VF during the 96 weeks
of follow-up.
Discussion
This study found a very low rate of transmitted INSTI resistance
mutations in naive patients starting INSTI-based regimens in
Figure 2. Patients with transmitted HIV mutations according to viral population. (a) Percentage of patients with transmitted HIV mutations according
to antiretroviral classes. This represents the percentage of patients with TDR detected according to the viral population and the antiretroviral class
affected. (b) Number of patients with mutations in the integrase, protease and reverse transcriptase genes. For integrase, those mutations included
in the IAS-USA 2019 list or in the Stanford University HIV drug resistance database (HIVdb, version 8.9, available at: https://hivdb.stanford.edu/) with
a score >14 for at least one INSTI were considered. For reverse transcriptase and protease, those mutations included in the WHO 2009 surveillance
mutation list were considered. WHO 2009 non-listed mutations were not represented. Percentages of patients harbouring mutations were calculated
based on the number of successfully amplified samples for integrase, reverse transcriptase and protease genes. This figure appears in colour in the
online version of JAC and in black and white in the print version of JAC.
Casadella` et al.
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Spain. Moreover, TDR had absolutely no impact on the effective-
ness of first-line INSTI ART, since all subjects with TDR remained
virologically suppressed at 96 weeks.
As in other Spanish and European cohorts,
15,16,18,19,25,26
the
overall rate of TDR in this study was 13.1%, with a predominance
of NNRTI mutations and very low rates of INSTI- and PI-associated
mutations. In our study, only 1.7% of subjects had INSTI-
associated mutations as majority variants, although this rate
increased to 9.6% when low-frequency variants were also
considered.
INSTI-associated low-frequency resistance mutations have been
analysed in several studies,
27–31
but there is no consensus regarding
their clinical relevance. Although they may affect INSTI susceptibility
in INSTI-experienced subjects,
31
studies have failed to show a dis-
cernible impact of low-frequency INSTI mutations on virological out-
comes of first-line INSTI-based ART.
28,30,32
Concordantly with these
data, in our study, none of the subjects with transmitted INSTI- or
NRTI-associated mutations, either as high- or low-frequency var-
iants, developed VF. In fact, virtually all subjects retained an HIV-1
fully susceptible to all INSTI-based regimens.
As far as we are aware, this is the first study demonstrating that
R263K transmission is possible in clinical settings and, in this case,
it was not associated with any significant effect on virological re-
sponse. Interestingly, we found one sample with transmitted
R263K alone as the majority variant, while in another sample it
was a low-frequency variant, together with the T97A mutation as
a majority variant. To date, the mutation R263K had only been
reported in clinical samples in patients developingVF to dolutegra-
vir.
33–35
This mutation confers low-level resistance to dolutegravir
and bictegravir as a single mutation, does not seem to be
associated with compensatory substitutions that might increase
levels of resistance
36,37
and has a negative impact on viral replica-
tive fitness.
37,38
In fact, some studies have hypothesized that
R263K leads HIV into an evolutionary dead-end,
39
although this
does not seem to happen in vivo.
In our study, mutation E138K was detected as a majority vari-
ant in only one case, and in combination with other INSTI minority
variants, including one subject with Q148H who did not experience
VF during follow-up. By itself, the E138K mutation does not reduce
INSTI susceptibility, but in combination with Q148 mutations it
confers cross-resistance to all INSTIs.
36,40
Q148H/R/K mutations
can be selected in patients receiving raltegravir, elvitegravir and
cabotegravir, and also have minimal effects on dolutegravir sus-
ceptibility when they are detected as a single mutation, but confer
a high-level cross-resistance to all INSTIs when other secondary
resistance substitutions are present.
40
Nevertheless, our study
suggests that Q148H does not seem tohave any impact on the ef-
ficacy of first-line INSTI-based ART when it is present as a minority
variant.
Integrase polymorphisms, and in particular E157Q, may have a
negative impact on INSTI-based regimens and have been found to
be relatively prevalent in some subpopulations of HIV-infected
patients.
41–43
Nevertheless, the studies where they have been
reported have included a limited number of subjects.
41–43
In our
study, we did not find viruses harbouring E157Q, although we
found other integrase mutations, such as G163R, detected as both
majority and minority variants, and minority viruses harbouring
some polymorphic and non-polymorphic mutations (T97A, L74I/
M, H51Y, V151A/L and S230R). Alone, however, they had little, if
any, effect on INSTI susceptibility.
38
The main strengths of this study are that, unlike most epi-
demiological surveillance studies performed in Europe in which the
transmitted resistance is usually evaluated by Sanger sequencing,
the analysis of TDR was mainly performed by means of NGS.
In addition, the clinical outcomes of TDR could be evaluated thanks
to the prospective follow-up of the patients. Nevertheless, our
study is subject to a number of limitations. Genotyping tests at
baseline were performed by means of NGS and Sanger population
sequencing. Therefore, the rate of minority variants and their
effects on virological outcomes may have been underestimated.
However, this reflects the observational design of our study and
the reality of HIV care centres in which Sanger sequencing is still
widely used. In addition, there is no consensus list of integrase
mutations for surveillance. Although integrase resistance
Figure 3. Susceptibility for INSTI based on regimens according to GSS in naive patients starting first-line ART and with an available genotyping test
analysed by NGS or Sanger population sequencing (n= 173). The potential susceptibility of the main INSTI-based regimens currently available was
evaluated by means of the GSS corresponding to the number of ‘active’ drugs prescribed at baseline. According to the GSS, susceptibility of regimens
was classified in: 2.5–3.0; 2; and <1.5 points. HIVdb was used to calculate the GSS, considering a score of 0–9 as susceptible (1 point), 10–59 as inter-
mediate (0.5 points) and 60 points as resistant (0 points). RAL, raltegravir; EVG, elvitegravir; DTG, dolutegravir; 3TC, lamivudine; ABC, abacavir; FTC,
emtricitabine; TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide; BIK, bictegravir. This figure appears in colour in the online version of JAC
and in black and white in the print version of JAC.
Primary INSTI resistance in Spain JAC
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information was obtained taking into account majority variants
included in validated resistance algorithms (IAS and HIVdB), there
are differences between these algorithms that could make it diffi-
cult to evaluate the rate of mutations. In particular, the inclusion
of polymorphic integrase positions in our evaluation could have led
to an overestimation of the already low rates of transmitted INSTI
resistance. This reflects the need to develop a consensus list for
the purpose of epidemiological surveillance, which would make it
possible to perform larger and more precise epidemiological stud-
ies. Automated analysis pipelines for NGS could be useful for this
purpose.
44,45
However, to date, there is no evidence that lowering
the mutant detection threshold below 15%–20% (e.g. the Sanger
sequencing equivalent) provides any clinical or public health value.
Taken together, however, our data confirm that the prevalence
of transmitted INSTI-associated mutations is still very low in naive
patients and such mutations have a minimal impact, if any, on the
susceptibility of currently available INSTIs. The low frequency of
TDR mutations conferring resistance to dolutegravir, and the more
recently available bictegravir, is concordant with the idea that
genotyping tests are not mandatory when triple INSTI-based
regimens are considered for starting first-line ART.
46
Conversely,
pre-ART resistance testing probably continues to provide value in
subjects initiating dolutegravir !lamivudine dual therapy.
47
Although transmission of INSTI mutations is rare, transmission
of M184V mutants is frequent in newly diagnosed subjects with
previous prophylaxis pre-exposition exposure.
48
In conclusion, the rate of INSTI resistance mutations in naive
subjects remains very low in Spain and thus far has not been
associated with VF. Pre-ART genotyping tests are not needed to
guide INSTI-based initiation as first-line ART, although periodic sur-
veillance of INSTI TDR is warranted.
Acknowledgements
We are grateful to Ne´stor Sanchez and Nuria Pe´rez-A
´lvarez for support
with data management and the statistical analysis, Miryam Soler for
help with coordinating and recording all data, and Michael Kennedy-
Scanlon for proofreading assistance.
Members of the INSTINCT Study Group
Jose´ R. Santos, Isabel Bravo, Anna Chamorro, Cristina Miranda (Lluita
contra la SIDA Foundation, Hospital Universitari Germans Trias i Pujol,
Badalona); Rafael Mica´n Rivera, Juan Gonza´lez (University Hospital La
Paz, Madrid); Antonio Antela (Infectious Diseases Unit, Santiago de
Compostela Clinical University Hospital); Marcos Diez, Irene Portilla,
Melissa Carreres, Livia Giner, Vicente Boix, Sergio Reus, Esperanza Merino,
Diego Torru´s, Joaquı´n Portilla (General University Hospital, Alicante);
Jesu´s Sanz, A
´ngela Gutie´rrez Liarte, Ana Go´mez Berrocal (University
Hospital de La Princesa, Madrid); Pere Domingo, Marı´a del Mar Gutie´rrez,
Marı´a Gracia Mateo, Je`ssica Mu~
noz Rodrı´guez (Infectious Diseases Unit,
Hospital de la Santa Creu i Sant Pau, Barcelona); Marta Montero-Alonso
(Infectious Diseases Unit, La Fe University and Polytechnic Hospital,
Valencia); Adria` Curran, Ariadna Torrella, Bibiana Planas, Jordi Navarro
(Infectious Diseases Department, Hospital Universitari Vall d’Hebron,
Barcelona); Mar Masia´, Sergio Padilla, Catalina Robledano, Araceli Adsuar,
Fernando Montolio, Fe´lix Gutie´rrez (Infectious Diseases Unit, Elche
University General Hospital, Elche); Nieves Valcarce Pardeiro, Hortensia
A
´lvarez, Ana Mari~
no (Infectious Diseases Unit, Hospital Arquitecto
Marcide, Ferrol); Antonio Ocampo, Alfredo Rodrı´guez, Celia Miralles (HIV
Unit, Hospital A
´lvaro Cunqueiro, Vigo); Laura Pe´rez-Martı´nez, Jose´ Ramo´n
Blanco (Infectious Diseases Area, Hospital San Pedro-CIBIR, Logro~
no);
Coral Garcı´a Vallecillos, Juan Pasquau (University Hospital Virgen de las
Nieves, Granada); Marı´a Je´ sus Pe´rez-Elı´as, Fernando Dronda, Marı´a Jesu´s
Vivancos, Santiago Moreno (Infectious Diseases Unit, Ramo´n y Cajal
Hospital, Madrid); Arkaitz Imaz, Daniel Podzamczer [HIV and STI Unit,
Infectious Diseases Department, Bellvitge University Hospital, Bellvitge
Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat];
Maialen Ibarguren, Xabier Kortajarena, Marı´a Pilar Carmona-Oyaga,
Josean A. Iribarren (Infectious Diseases Unit, Donostia University
Hospital, San Sebastia´n); Leopoldo Mu~
noz Moreno, Jose´ Herna´ndez
Quero (University Hospital San Cecilio, Granada); Judit Villar-Garcı´a,
Hernando Knobel (Infectious Diseases Service, Hospital del Mar,
Barcelona); Pilar Barrufet, Lluı´s Force (Infectious Diseases Unit, Mataro´
Hospital, Mataro´); Maria Casadella`, Roger Paredes, Marc Noguera-Julian
(IrsiCaixa AIDS Reseach Institute, Hospital Universitai Germans Trias i
Pujol, Universitat Auto`noma de Barcelona, Badalona).
Funding
This study was supported by ViiV Healthcare and, in part, by grants from
the Lluita contra la SIDA Foundation (Barcelona, Spain), the Spanish AIDS
Network ‘Red Tema´tica Cooperativa de Investigacio´n en SIDA’ [RIS,
RD16/0025/0041, NEAT (European AIDS Treatment Network)], the
Ministerio de Economı´a y Competitividad of Spain (MINECO/FEDER,
MTM2015-64465-C2-1-R) and GRBIO (Grup de Recerca en Bioestadı´stica i
Bioinforma`tica; 2017 SGR 622). The funders had no role in the study de-
sign, data collection and analysis, the decision to publish or drafting of
the manuscript.
Transparency declarations
J.R.S., A.A. and J. Pasquau have received research funding, consultancy
fees and lecture sponsorships from and have served on advisory boards
for Gilead Sciences, Janssen-Cilag, Merck Sharp & Dohme and ViiV
Healthcare. J. Portilla, J.S., R.P. and A.O. have received research funding
and consultancy fees from and have served on advisory boards for
Gilead Sciences, Merck Sharp & Dohme and ViiV Healthcare. P.D. has
received honoraria for speeches, advisory boards, slide sets and research
grants from Gilead Sciences, Merck Sharp & Dohme, ViiV Healthcare and
Janssen-Cilag. M.M. has received research funding, consultancy fees
and lecture sponsorships from and has served on advisory boards for
Gilead Sciences, Janssen-Cilag, Merck Sharp & Dohme, Pfizer and ViiV
Healthcare. M.M.-A. has received consultancy fees and lecture sponsor-
ships from and has served on advisory boards for Abbvie, Janssen-Cilag,
ViiV Healthcare and Merck Sharp & Dohme. J.N. has received honoraria
and/or speakers’ fees from Abbvie, Gilead, Janssen-Cilag, Merck Sharp &
Dohme and ViiV Healthcare outside of the submitted work. M.J.V. has
received research funding, consultancy fees and lecture sponsorships
from and has served on advisory boards for Gilead and ViiV Healthcare.
A.I. has received research funding, financial compensation for lectures
and educational activities, or has served on advisory boards for Gilead
Sciences, Janssen-Cilag, Merck Sharp & Dohme and ViiV Healthcare.
P.B. has received consultancy fees from and has served on advisory
boards for Gilead Sciences, Merck Sharp & Dohme, Janssen-Cilag and
ViiV Healthcare. M.C., N.V.-P., R.M.-R., L.M.-M., J.V.-G., P.C.-O., L.P.-M. and
M.N.-J. declare no competing interests.
Author contributions
J.R.S. and R.P. designed the INSTINCT study. M.C., J.R.S. and R.P. wrote the
manuscript, which was reviewed and approved by all the authors. M.C.
performed the laboratory and sequencing data analyses. The remaining
Casadella` et al.
3522
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authors contributed clinical and Sanger sequencing data (when required)
and followed the study subjects.
Supplementary data
Table S1 is available as Supplementary data at JAC Online.
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