Prevalence of resistance to integrase strand-transfer inhibitors (INSTIs) among untreated HIV-1 infected patients in Morocco
Alaoui et al. BMC Res Notes
Prevalence of resistance to integrase strand-transfer inhibitors (INSTIs) among untreated HIV-1 infected patients in Morocco
Najwa Alaoui 0
Moulay Abdelaziz El Alaoui 2
Nadia Touil 0
Hicham El Annaz 0
Marouane Melloul 1
Reda Tagajdid 0
Naoufal Hjira 3
Mohamed Boui 3
El Mostapha El Fahime 2
Saad Mrani 0
0 Faculty of Medicine and Pharmacy, University Mohammed V in Rabat , Av. Mohamed Belarbi El Alaoui, 6203 Rabat , Morocco
1 Laboratory of Physiology, Genetics and Ethnopharmacology, Faculty of Sciences of Oujda, University Mohammed Premier , 60000 Oujda , Morocco
2 Functional Genomic Platform, UATRS, Center for Scientific and Technical Research [CNRST] , 10000 Rabat , Morocco
3 Department of Dermatology and Venereology, Faculty of Medicine and Pharmacy, University Mohammed V in Rabat , Av. Mohamed Belarbi El Alaoui, 6203 Rabat , Morocco
Objective: The integrase strand-transfer inhibitors (INSTIs) are an important class in the arsenal of antiretroviral drugs designed to block the integration of HIV-1 cDNA into the host DNA through the inhibition of DNA strand transfer. In this study for the first time in Morocco, the complete HIV-1 integrase gene was analysed from newly diagnosed patients to evaluate the prevalence of natural polymorphisms and INSTIs resistance-associated mutations in the integrase gene. Results: The 864pb IN coding region was successfully sequenced from plasma sample for 77 among 80 antiretroviral naïve patients. The sequences were interpreted for drug resistance according to the Stanford algorithm. Sixty samples were HIV-1 subtype B (78%), fourteen CRF02_AG (18%), two subtype C and one subtype A. Overall 81 of 288 (28%) amino acid IN positions presented at least one polymorphism each. We found 18 (36.73%), 42 (25.76%) and 21 (27.27%) of polymorphic residues assigned to the N-Terminal Domain, Catalytic Core Domaine and the C-Terminal Domain positions respectively. Primary INSTIs resistance mutation were absent, however secondary mutations L74IM, T97A were detected in four samples (5.2%). These results demonstrate that untreated HIV-1 infected Moroccans will be susceptible to INSTIs.
HIV-1; Integrase; Resistance mutations; INSTIs; Naïve patients; Morocco
Since the introduction of combination therapy (highly
active antiretroviral therapy, HAART) with protease
inhibitors (PIs), nucleoside reverse transcriptase
inhibitors (NRTIs) and non-nucleoside reverse transcriptase
inhibitors (NNRTIs) in Morocco in 1998, the mortality
and morbidity of HIV/AIDS patients has reduced [
These drugs suppress viral replication and reduce HIV
viral RNA loads in the plasma of patients, thus helping
to maintain the immune system, but they do not prevent
escape through the emergence of drug resistant viruses
and subsequent treatment failure.
According to previous Moroccan studies, the
prevalence of resistance to NRTIs, NNRTIs and PIs are
continuously increasing among drug-naïve and treatment
experienced patients [
], thus, developing new drugs
for AIDS treatment would be needed.
INSTIs are the latest antiretroviral (ARV) drugs class
developed for the treatment of HIV-1 infections via
the inhibition of DNA strand transfer [
]. To date three
INSTIs, raltegravir (RAL), elvitegravir (EVG) and
dolutegravir (DTG) are approved for clinical use [
are potent ARV drugs offering more treatment options in
naïve patients as well in pretreated patients with
preexisting drug resistance or treatment complication [
Therefore, they have become an essential component of
HAART used in many countries. Morocco has installed
national programs following World Health Organization
ARV guidelines and newer ARV salvage regimens
including third-line drugs such as INSTIs will been introduced
in the upcoming years. The aim of this study is to analyze,
for the first time in Morocco, integrase (IN) sequence
variability among ARV treatment naïve patients to
determine the frequency of resistance mutations and the
prevalence of natural polymorphisms of the IN gene and in
order to estimate INSTIs efficacy prior to their
introduction into the country.
Study population and samples
Plasma samples were collected for genotypic assay of the
IN gene region from eighty seven HIV-infected,
antiretroviral therapy-naïve patients originating from different
geographic parts of the country enrolling at the
dermatology department of Mohammed V Military
Teaching Hospital in Rabat between the years 2009 and 2015.
Demographic, clinical and laboratory data were collected
for all patients. The quantitative HIV-RNA tests were
performed using Cobas TaqMan HIV-1 Test, version 1.0
(Roche Diagnostics Systems, Germany, P/N: 03542998
190). CD4 cell enumerations were performed using the
FacsCount instrumentation. (FacsCount,
Becton–Dickinson, P/N: 339010). After viral load testing,eighty
samples were used for sequencing assay. The detection of
IN mutations by sequencing was unsuccessful for three
The study was approved by the Ethical Committee of
Biological Research, Faculty of Medicine and Pharmacy–
Rabat, and was conducted with respect to legal aspects.
Written informed consent was obtained from all
participants before any data analysis procedure.
Genetic analysis and drugs resistance
HIV RNA was extracted from plasma using High Pure
Viral RNA Kit (Roche Diagnostics Systems, Germany,
P/N: 11858882001) and the integrase coding region
(867 bp) was amplified by one-step reverse transcriptase
polymerase chain reaction (RT-PCR) using MyTaq
OneStep RT-PCR kit (Bioline, London, UK, P/N: BIO-65049)
and the primer set KVL068 -KVL069 [
NestedPCR assay was carried out using MyFi DNA
Polymerase kit (Bioline, London, UK, P/N: BIO-21118) and the
primer set KVL070 and KVL084 [
reaction was performed using BigDye Terminator v3.1 Ready
Reaction Cycle Sequencing Kit (P/N: 4337455) with an
ABI PRISM 3130XL Genetic Analyzer (Applied
Biosystems) using the POP-7 polymer (P/N: 4393708). Data
were analyzed by sequencing Analysis Software version
5.3.1 (Applied Biosystems, P/N: 4360967).
IN Sequences were assembled and aligned using DNA
Dragon Sequence Assembler version 1.6.0
(SequentixDigital DNA Processing, Germany) and Muscle method
in MEGA 6 software [
], respectively. All sequences
were submitted to GenBank and registered under
accession numbers: KU609274–KU609350.
HIV-1 subtyping and screening of IN polymorphisms
in comparison with the HxB2 HIV-1 clade B consensus
sequence (GenBank accession number K03455.1) were
done using geno2pheno subtyping tools [
Phylogenetic tree was constructed by using Maximum
Likelihood method, and Bootstrap resampling was performed
1.000 times for all sequences with MEGA 6 software
(Fig. 1). INSTIs resistance mutations in IN sequences
were interpreted using the Stanford HIVdb Program
(Version September 23,2016) and screened for the
presence of additional changes in 17 positions (V72I, T112I,
S119PRTG, T124A, T125K, A128T, Q146K, M154I,
K156N, V165I, V201I, I203M,T206S, S230N, D232N,
V249I and C280Y) previously related to INIs resistance
in vitro and frequently reported by different studies [
]. The variability at the D64D116E152 and H12H16C40C43
motifs, the residues that interact with the human lens
epithelium derived growth factor (LEDGF/P75) and the
degree of variability of the three functional areas of IN
were also investigated.
Out of eighty patients, sixty-two (77.5%) were male.
Median age was 36 years old, where patients with an age
between 25 and 44 years old represent 61%. Sixty-five
(81.25%) patients were suspected to have acquired HIV
infection through heterosexual contact, four (5%)
perinatally, and the mode of infection was unknown for 11
patients. The median of CD4+ T cell count and viral
load at the time of sequencing for the available values
were 409 cells/mm3 and 95,800 copies/ml respectively
In 3 samples the sequencing failed and subtypes were
unavailable. The final dataset for the baseline IN
resistance included 77 individuals. IN sequencing was
consistently successful at HIV viral loads higher than 66 copies/
ml. Sixty (77.92%) were of HIV-1 subtype B, fourteen
(18.18%) CRF02_AG, two (2.6%) subtype C and one
(1.3%) subtype A. These data are also summarised in the
IN phylogenetic tree shown in Fig. 1. The screening of
sequences revealed that Overall 81 of 288 (28%) amino
acid IN positions presented at least one polymorphism
each. we found 18 (36.73%), 42 (25.76%) and 21 (27.27%)
of polymorphic residues assigned to the N-Terminal
Domain, Catalytic Core Domaine and the C-Terminal
Domain positions respectively (Fig. 2). As expected, no
polymorphism was found in the HHCC Zn+-binding
motif and in the catalytic triad DDE. Also the amino acid
in IN positions that have been identified as critical for
interaction with LEDGF/P75, H12, L102, A128, A 129,
C130, W131, W132, I161, R166, Q168, E170, H171, T174,
M178 and Q 214 [
] were conserved. The
substitutions detected in more than 95% of samples were D10E,
G123S, R127K and N232D, none of which is ascribed to
INI resistance. None of the primary amino acid
mutations in IN positions 66, 92, 140, 143, 147, 148 and 155
listed in the Stanford resistance algorithm were found
in this study, while secondary mutations L74IM and
T97A associated with drug resistance to RAL and EVG
were observed only in four patients (5.2%) (Additional
file 1). The L74I was observed in two strains, a subtype B
(KU609297) and a subtype A (KU609282). The L74M was
observed in one subtype B strain (KU609337) and T97A
was observed in one CRF02_AG strain (KU609303). No
mutation associated with DTG resistance was observed
among all studied patients. Other mutations in IN
positions not included in Stanford list but frequently reported
a V .5
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t S 2
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c t 2 8
c s s 4
e rsc an 00
ïv e e .0
a D M 4
.5 .5 .25 .75 .25 .75 .25 .25 .5 .57 .5 .52 .5 .5 .52 .5 .75 .25 .57 ruq
7 2 5 5 1 8 1 5 3 5 6 1 7 3 5 7 0 1 2 2 6 2 8 1 8 0 o
7 2 6 2 8 1 3 1 2 2 1 5 2 1 1 3 2 ir
leeaT1obDm iittrrsccaaehC xSe enM eonmW rtycaeeoggA 11< 4121– 4542– 44> iirrtsssaTeuoonnm lrtsxcaeeeuoonH ilrttceoohhdM irtteeeonddNm rttycaaeogCCHD lssaCA lssaBC lssaCC irtteeeonddNm llttcccae4uonCD 490– 9905– 919001– 439002– 035≥ irtteeeonddNm illrrtycaaaeVoodg 249< 9950492– ,290009995– ,,0900099993– ,,000009990001– ,0001000≥ irtteeeonddNm 2,irtscvaaaennSSD
1 10 20 30 40 50
F L D G I D K A Q ED75 DE59 H E RK10 Y H* NTS12 N W KR46 TSA16 M VA1 GNS444 ED43 F N IL6 P P IM1V18 IV6 A K QE1 I IV2 TA1 HCS91 C* ND1 K C* Q QVIL4115 K G E A TLIM742
in regards to INIs resistance in vitro V72I, T112I,
S119PRT, T124A, K156N, V165I, V201I, I203M,T206S
and S230N have been detected with different
frequencies (Fig. 2). Finally there were no significant associations
(p > 0.05) between resistance mutations with exposure
category, viral load, Transmission route and CD4 T cell
Three INSTIs, RAL, EVG and DTG have been approved
for clinical use by the FDA and European Medicines
]. These compounds have proven to be
highly efficient for the treatment of both ARV-naïve
and -experienced individuals even with preexisting
drug resistance or treatment complication [
INIs has rapidly became an important class in the
arsenal of ARV drugs. Eighty HIV-1 untreated patients were
recruited in this study in order to examine the variability
within the IN gene at positions associated with resistance
to INIs. Despite the limited size of the study population,
these results are in good agreement with the current
situation of HIV in Morocco as reported in the national
report on AIDS [
]. The high prevalence of subtype B
among infected Moroccan population has been
previously reported by other studies which suggested that it
was due to the presence of a close relationship between
Morocco and European countries [
phylogenetic analysis of IN sequences showed that all CRF02_
AG isolates were related to strains found in central
Africa and Europe, which agreed with previous reports
that suggested that increasing prevalence of CRF02_AG
might be associated with increasing immigration from
sub-Saharan Africa to Europe via Morocco [
the 288 IN amino acids positions, 81(28.12%) had one or
more variants. This rate (28%) is lower to that reported
by Rhee et al. (39.9%) in different subtypes of group M
integrase sequences obtained from more than 1500
individuals who were INI-naïve, and either ARV-naïve
or ARV-experienced [
]. This digit indicates the
relative conservation of the protein in untreated Moroccan’s
patients. The analysis showed conservation of the HHCC
Zn+-binding motif, the catalytic triad DDE, and several
important IN residues involved in the chemical bond and
hydrophobic contact with LEDGF/P75; which an
essential HIV integration cofactor linking IN to chromatin
]. The conservation of these specific structural
domains is strictly necessary for the correct performance
of HIV-1 IN functions [
Importantly the major resistance mutations with
reduced susceptibility to RAL, EVG and DTG were totally
absent. The absence of such mutations in our study is
consistent with the results of several studies in
treatmentnaïve patients [
19, 20, 31–35
] and with the fact that the
transmission of the drug resistance is unlikely in
populations previously unexposed to INIs treatment [
three secondary drug resistance mutations included in
Stanford list were observed in 4 strains. These mutations
have been previously been described as polymorphic,
occurring in 1–2% of IN sequences, observed in subtypes
A, B, C, D, CRF01_AE and CRF02_AG [
mutations contribute to INI resistance only in the presence
of primary INI resistance mutations [
there were three amino acid substitutions of unknown
significance at position 163 that were encountered in
subtype B and CRF02-A/G strains: G163E, G163T and
G163Q. In IN residues, it is usually considered non
polymorphic in all subtypes except subtype F .
Regarding DTG R263K resistance mutation, no strain from
our study exhibited this mutation, whereas the L101I
and T124A mutations were found in 26 and 12 strains,
respectively. These mutations were previously shown
to be selected in vitro in the presence of DTG and have
shown little impact on virological response to DTG [
]. Conversely, specific additional mutations in amino
acid IN positions 72, 112, 119,156, 165, 201, 203, 206, and
230 occurred with different prevalence in subtype B and
non-B HIV-1 variant were more common. These
mutations have not been described to be associated with RAL
or EGV resistance [
]. In the same way, it has been
reported that in the absence of primary mutation, all
these secondary mutations had little if any effect on drug
susceptibility in vitro, thus suggesting rather a secondary
role for viral fitness rescue and/or increasing resistance
]. Furthermore many previous genotypic studies on
HIV-1 IN in treatment-naïve patients living with
various viral subtypes in different countries: England, Spain,
South Africa, Sub-Saharan Countries, Thailand,
Indonesia and Korea have showed that these differences are
natural polymorphisms [
]. According to
previous studies, and the fact that INIs have not yet been
introduced in Morocco, all secondary and additional
mutations identified in this study are also likely natural
In conclusion, these results demonstrate that untreated
HIV-1 infected Moroccans are likely to benefit from
INSTI-based drug regimens, particularly given the
rising issues related to drug resistance against reverse
transcriptase inhibitors that are currently used in Morocco.
The authors wish to highlight that our limitation
consist on sample size, more participants are necessary
before a large introduction of integrase inhibitors into
Additional file 1. Distribution of IN mutations in subtypes B and non-B in
therapy-naïve patients. Secondary and additional mutations screened in
17 positions (V72I, T112I, S119PRTG, T124A, T125K, A128T, Q146K, M154I,
K156N, V165I, V201I, I203M, T206S, S230N, D232N, V249I and C280Y) using
the Stanford HIV Drug Resistance Program (Version September 23, 2016),
all mutations identified in this study are likely natural polymorphisms.
INSTI: integrase strand-transfer inhibitor; IN: integrase; INI: integrase inhibitor;
CCD: catalytic core domain; HAART: highly active antiretroviral therapy; ARV:
antiretroviral; PI: protease inhibitor; NRTIs: nucleoside reverse transcriptase
inhibitors; NNRTI: non-nucleoside reverse transcriptase inhibitors; RT-PCR:
reverse transcriptase polymerase chain reaction; RAL: raltegravir; EVG:
eviltegravir; DTG: dolutegravir.
AN, EMA, MS: conceived and designed the study; HEA, BM, HN: clinical
studies; EMA, MM: performed the experiments; EEM, TR, and TN: contributed
reagents/materials/analysis tools; AN, EMA: statistical analysis, interpretation
of the results and wrote the paper. All authors read and approved the final
This work was carried out in collaboration between the Military Hospital of
Instruction Mohammed V. of Rabat and the National Center for Scientific and
Technical Research, Morocco. We thank all patients participating in this study
and we thank Pr H. Fleury and his team, Bordeaux University Hospital for their
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article. Nucleotide sequences are available under GenBank accession numbers
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethical Committee of Biological Research,
Faculty of Medicine and Pharmacy—Rabat, and was conducted with respect
to legal aspects. Written informed consent was obtained from all participants
before any data analysis procedure. Consent was received from adult
next-ofkin when the respondent was under 16 years of age.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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