Integrase Strand Transfer Inhibitors (INSTIs) Resistance Mutations in HIV-1 Infected Turkish Patients

Abstract

Objectives: Integrase strand transfer inhibitor (INSTI) is a new class of antiretroviral (ARV) drugs designed to block the action of the integrase viral enzyme, which is responsible for insertation of the HIV-1 genome into the host DNA. The aim of this study was to evaluate for the first time INSTI resistance mutations in Turkish patients.

Methods: This study was conducted in Turkey, between April 2013 and April 2015 using 169 HIV-1-infected patients (78 ARV naive patients and 91 ARV-experienced patients). Laboratory and clinical characteristics of ARV naive and ARV-experienced patients were as follows: gender (M/F): 71/7 and 80/11, median age: 38 and 38.4; median CD4⁺ T-cell: 236 and 216 cells/mm³, median HIV-1 RNA: 4.95+E5 and 1.08E+6 copies/ml. Population-based seqeunces of the reverse transcriptase, protease, and integrase domains of the HIV-1 pol gene were used to detect HIV-1 drug resistance mutations.

Result: INSTI resistance mutations were not found in recently diagnosed HIV-1-infected patients. However, ARV-experienced patients had major resistance mutations associated with raltegravir and elvitegravir; the following results were generated:F121Y, Y143R, Q148R and E157Q (6/91 – 6.6%).

Conclusions: The prevalence of INSTI resistant mutations in ART-experienced patients suggested that resistance testing must be incorporated as an integral part of HIV management with INSTI therapies.

Keywords:

• HIV-1 integrase
• Integrase inhibitors
• Raltegravir
• Elvitegravir
• Dolutegravir
• Drug resistance
• DNA sequencing

Introduction

The HIV-1 integrase enzyme is responsible for the integration of the pro-viral DNA into the host DNA, it also provides stabilization of the pre-integration complex. Integrase strand transfer inhibitor (INSTI) is a new class of antiretroviral (ARV) drugs designed to block the action of the HIV-1 integrase enzyme. To date, raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) have all been approved as INSTIs to be used in the treatment of HIV-1 infections.^1,2

Several polymorphic and non-polymorphic mutations have been identified within the HIV-1 integrase gene, some of which are listed as follows: Y143C/H/R, Q148H/K/R, and N155H/S; these examples are of high-level resistance mutations either as genotypically or phenotypically associated with RAL. However, cross resistance between RAL and EVG is possible and both drugs are known to have low genetic barriers which forms the development of resistance with a single mutation.^1–4

To assist in the treatment management of HIV-1 infections (96% recommendation level), in 2004 the European HIV Drug Resistance Guiedlines Panel recommended the use of initial HIV-1 drug resistant testing.⁵ In addition, all other current guiedlines also recommend HIV-1 drug resistance testing for all HIV-1-infected patients prior to therapy initiation.^6–8 The World Health Organization (WHO) is constantly tracking toward the global surveillance of transmitted HIV-1 drug resistance, by making use of their own laboratories.⁹ In a population, genotypic drug resistance testing is considered as cost-effective if the level of transmitted drug resistance is >5% in HIV-1 infections.¹⁰ However, all of the above listed recommendations are yet to include INSTI resistance mutation testing.

According to the Turkish Ministry of Health of HIV/AIDS surveillance data, 1767 HIV-1-infected new patients have been diagnosed in 2014. However, in the period between 1985 and 2014, there are 9379 cumulative HIV/AIDS cases found in Turkey. By the end of 2014, the cumulative increase in HIV-1 patients was 38%.¹¹ Only a limited number of studies demonstrate that recently diagnosed HIV-1-infected Turkish patients, also transmitted drug resistance mutation (TDRM); the prevalence range was between 6.2 and 10.1%.^12,13 INSTIs are available to be prescribed in Turkey, although there is no available data on the pre-existing or transmitted INSTI resistance mutations in HIV-1-infected Turkish patients.

The aim of this study was to determine the prevalence of INSTI resistance mutations and to understand the effectiveness of resistance mutation testing before INSTI treatment is administered to HIV-1-infected patients.

Material and Methods

Patient population

The present study was conducted between April 2013 and April 2015, and included 169 HIV-1-infected patients [78 ARV naive patients and 91 ARV experienced patients]. Clinic and laboratory characteristics of the patients are presented in Table 1. The study was approved by the local ethics committee (Clinical Research Ethics Committee of Kocaeli University), and a written consent was obtained from each patient. All of the patients were categorized as HIV carriers according to European AIDS Clinical Society guidelines.⁶ The US Centers for Disease Control and Prevention classification system was used on the HIV infection stage in patients.¹⁴ Blood samples with K2EDTA were separated by centrifugation immediately, aliquoted and then kept at −80 °C until testing. Anti-HIV-1/2 antibody was tested using commercially available microparticle enzyme immunoassay kits (Axsym; Abbott Laboratories, Abbott Park, IL, USA and Elecsys, Roche Diagnostics, Mannheim, Germany). All of the samples that are anti-HIV positive by ELISA were also confirmed at least twice for anti-HIV positivity by Western blot testing (DIA PRO, HIV-1 LIA, Diagnostic Bioprobes Srl, Milano, Italy) at Istanbul Veneral Diseases Hospital in Turkey. To maintain subject confidentiality, a unique identification number was assigned to each sample.

Table 1 Clinical and laboratory characteristics of the patients studied

Characteristic	Antiretroviral naive	Antiretroviral experienced
Patient, no.	78	91
Gender, M/F (%)	71/7 (91%/9%)	80/11 (88%/12%)
Age, median years (range)	38 (23–61)	38.4 (2.5–66)
CD4^+ T-cell count, median mm^3 (range)	236 (6–626)	216 ( 3–884)
HIV-1 RNA load, median, copies/ml (range)	4.95E+5 (3.06E+2 – 3.33E+6)	1.08E+6 (1.48E+2 – 1.57E+7)
Acquisition route, n (%)
Heterosexual contact	52 (67%)	62 (68%)
MSM	24 (31%)	20 (22%)
Bisexuel contact	2 (2%)	4 (5%)
Blood transfusion	–	2 (2%)
Dental surgery	–	1 (1%)
Vertical transmission	–	2 (2%)
HIV-1 subtip, n (%)
Subtype B	48 (62%)	55 (60)
Non-subtype B	20 (26%)	17 (19)
A1	11 (14%)	5 (6%)
C	2 (3%)	3 (3%)
D	1 (1%)	1 (1%)
F1	5 (7%)	6 (7%)
G	1 (1%)	2 (2%)
Circulating recombinant form CRF01_AE	10 (13%)	19 (21%)
CRF02_AG	4 (5%)	12 (13%)
CRF03_AB	4 (5%)	4 (5%)
CRF07_BC	–	1 (1%)
CRF06_cpx	–	1 (1%)
CRF13_cpx	2 (3%)	–
	–	1 (1%)
Coinfection status, n (%)
Hepatitis B	3 (4%)	1 (1%)
Hepatitis C	–	4 (4%)
Hepatitis D	1 (1%)	–
P.jirovecii pneumonia	1 (1%)	2 (2%)
Oral candidiasis	2 (3%)	3 (3%)
Pulmoner tuberculosis	1 (1%)	6 (7%)
CMV infection	–	1 (1%)
Cerebral toxoplasmosis	1 (1%)	2 (2%)
Treatment status, n (%)
	–	Lopinavir/r combination; 38 (42%)
		Efavirenz combination; 31 (34%)
		Lamivudin + zidovudin comb; 17 (19%)
		Prezista/r combination; 2 (2%)
		Raltegravir combination; 2 (2%)
		Abacavir combination; 1 (1%)

HIV-1 RNA detection

HIV-1 RNA was detected and quantified by commercial real-time PCR assays—QIAsypmhony + Rotorgene Q/artus HIV-1 QS-RGQ v1 (Qiagen GmBH, Hilden, Germany) COBAS Ampliprep/COBAS TaqMan HIV-1 Test (Roche Molecular Systems, Inc. Pleasanton, CA, USA) and Abbott M2000 SP/Abbott RealTime HIV-1 Amplification Kit (Abbott Molecular Inc. Des Plaines, IL, USA).

Viral population sequencing

The genotypic test was performed by population sequencing of the viral protease, part of RT and integrase domains were used as an in-house method. Classical TDRM and INSTI resistance mutations were determined according to ANRS AC11 Resistance Study Group PCR and sequencing procedures: HIV-1 version January 2015 (www.hivfrenchresistance.org). Conditions of the study were applied as previously described.¹²

Drug resistance mutation detection

HIV-1 classical drug resistance mutations were detected by population-based sequencing of the RT (codon 41–238) and protease (codon 1–99) domains of the pol gene of HIV-1, and analyzed according to the criteria established by the WHO 2009 list of surveillance drug resistance mutations.⁹ In addition, the integrase (codon 62–182) domain of HIV-1 pol gene was used for the investgation of INSTI resistance mutations and interpreted according to the French ANRS; National Agency for AIDS Research, AC11 resistance group PCR and sequencing algoritm (www.hivfrenchresistance.org).

HIV-1 subtyping

HIV-1 subtypes were determined using the HIVdb-Stanford University and geno2pheno (http://coreceptor.bioinf.mpi-inf.mpg.de) subtyping tools. The collected data were then compared to a consensus subtype B reference sequence, and the differences were used as query parameters to interrogate the HIV database which were rapid computer-assisted virtualphenotyping.

The nucleotide sequences provided from study patients were submitted to the GenBank (BankIt1841284 and BankIt1842343) and they are under processing.

Statistical analysis

Differences between two proportions were measured using Fisher’s exact test. P ≤ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS for Windows statistical software (SPSS Inc., Chicago, Ill., USA).

Results

INSTIs resistance mutations were not found in the treatment of naive HIV-1-infected patients (n = 78). However, in ARV-experienced patients F121Y, Y143R, Q148R, and E157Q were detected as major resistance mutations associated with raltegravir and elvitegravir. The overall prevalence of INSTI resistance mutations in ARV-experienced patients is 6.6% (6/91) (Table 2). Furthermore, treatment status of these patients are tenofovir + emtricitabine + lopinavir/r (n = 3), tenofovir + emtricitabine + raltegravir (n = 2) and tenofovir + emtricitabine + efavirenz (n = 1). Difference of the prevalences between two groups were significant (The Fisher’s exact test; p = 0.04).

Table 2 Integrase strand transfer inhibitors (INSTIs) resistance analysis in Turkish patients infected with HIV-1

Drug class	Resistance mutation pattern
	Antiretroviral naïve, (n = 78)	n	%	Antiretroviral experienced, (n = 91)	n	%
INI	–	–	–	INSTI – naive, (n = 89)
				F121Y, Q148R, E157Q	4	4.4
				INSTI – experienced, (n = 2)
				Y143R, E157Q	2	2.2
				Total	6	6.6

In addition, the patients of the current study were also analyzed fo classical HIV-1 genotypic resistance mutations. Two different primary drug resistance patterns (K65R, M184 V) for NRTI (±Thymidine analogue—associated mutations profile), 10 different patterns for NNRTI (L100I, K101E, K103 N/S, V106A/M, Y188L, Y181C, G190A/S), and 9 different patterns for PI drug classes (L24I, M46I, I54 V, L76 V, V82A/T, N83D, N88D, L90 M) were determined in ARV-experienced patients (Table 3). The ARV-experienced patients displayed multi-class drug resistance. In 13 patients (14%) NRTI + NNRTI + PI, in 29 patients (32%) NRTI + NNRTI, and in 4 patients (4%) NRTI + PI resistance mutations were determined. However, one patient had both M184 V + K103 N and one patient had K103 N on its own, while two patients had M46L in ARV-naive study group (Table 3).

Table 3 Resistance mutation patterns on the revers transcriptase and protease domains of HIV-1 in the patients studied

Drug class	Drug resistance mutation pattern
	Treatment naive (n = 78)	n (%)	Antiretroviral experienced (n = 91)	n (%)
NRTI	M184V	1 (1.2)	K65R, M184V ± TAM1, TAM2 or TAM1 + TAM2	39 (43)
NNRTI	K103N	2 (2.5)	L100I, K101E, K103N/S, V106A/M, Y188L, Y181C, G190A/S	46 (51)
PI	M46L	2 (2.5)	L24I, M46I, I54V, L76V, V82A/T, N83D, N88D, L90M	12 (13)
Total		4 (5)*		56 (62)

NRTI; nucleoside revers transcriptase inhibitors, NNRTI; non- nucleoside revers transcriptase inhibitors, PI; protease inhibitors, ARV; antiretroviral, TAM; thymidine analogue - associated mutation, TAM1; M41L, L210W, T215Y, TAM2; D67N, K70R, K219E/Q/N/R, T215F, T215C/D/S, TAM1 + TAM2; M41L + K219N, M41L + T215C/D/S.

* One patient had M184V + K103N patterns.

HIV-1 subtyping results were shown in three categories (subtype B, non-subtype B, and circulating recombinant form; CRF). According to the results, subtype B was predominantly found in 62% and 60% in naive and ARV-experinced patients, respectively. Hence, non-subtype B and CRF subtypes were found in 26% and 19, 13, and 21%, respectively (Table 1). Differences of the prevalences between HIV-1 subtypes categories were not significant (p ≥ 0.05).

Discussion

In this study, we have investigated the INSTIs resistance mutations in HIV-1-infected Turkish patients. Our results demonstrate that naturally occuring mutations or substitutions known to confer primary resistance to INSTIs are not common. According to recently published data (The SPEARD Programme of Europe), no INSTI-resistant variants were circulating in Europe before the introduction of INSTIs therapies.¹⁵ Therefore, INSTIs resistance testing prior to the beginning of INSTIs as first-line therapy may not be required. However, INSTIs resistance testing could be performed prior to switch therapies, because the major RAL and EVG resistance mutation (F121Y, Y143R, Q148R, and E157Q) rates were 6.6% in ARV-experienced patients by population sequencing (Table 2). Actually, there is no sufficient data associated with the INSTIs resistance mutations. In the limited number of recent reports, detected transmitted major resistances were as follows; E92Q, Y143H, Q148H, and E157Q for INSTI with different prevalence ranges of 0%—8.3% in Germany, Spain, Italy, Brasil, South Africa, and USA.^16–21 Monitoring of INSTIs resistance mutations in regional levels in HIV-1 strains may have produced a remarkable amount of results for the ARV treatment guidelines. However, for a global perspective similar reports may be require more.

Our findings show that the classical HIV-1 genotypic resistance rate is high (total amount 62%) in the ARV-experienced patients and included many different patterns for NRTI, NNRTI, and PI drug classes (Table 3). However, there is multi-class drug resistance in all of the ARV drug class options. Drug resistance reduces therapeutic options and our results may suggest the requirement and importance of a new drug class for the switched therapies of ARV-experinced patients. Our resistance testing results indicated that the integrase inhibitor which has a high genetic barrier may be a strong option among the limited ARV drugs available in the management of HIV-1-infected Turkish patients.

Our study indicates subtype B is prevalent among HIV-1-infected patients in Turkey (Table 1). Furthermore, this indicates consistency with our previous studies.^12,13 However, there is a high level of heterogeneity in the HIV-1 diversity, specific geographic localization of Turkey may assist in explaining this phenomenon. Extraodinary mass population movements have occured in Turkey over the last couple of years largely as a consequence of refugees from Syria and Africa seeking asylum in Turkey. There are also an estimated 100,000 asylum-seekers from Iraq, the Islamic Republic of Iran and Afghanistan. Subsequently, human trafficking for suspected sexual exploitation is also on the rise.^22–24 In light of these onging events, HIV-1 subtype diversity will continue to be a major challenge in the global control of HIV-1. Therefore, regular monitoring of HIV-1 subtypes and also drug resistance mutations in Turkey may be a critical extension for effective global control.

In conclusion, the prevalence of major INSTIs resistance mutations in ARV-experienced patients indicate that INSTIs resistance testing should be incorporated as an integral part of HIV-1 infection management, and the choice of a second-line therapy regime should be guided by the genotypic resistance results. Hence, INSTIs may be a strong option for the initial or switch ARV therapies in HIV-1-infected inidividuals. Alternatively, the new HIV-1 resistance guidelines should also include the INSTIs resistance mutation analysis.

Funding

This study was not funded by any organization.

Disclosure statement

No conflict of interest to declare.

Contributors

MS served as lead investigator. He designed the study and obtained funding and ethics approval, analyzed the data, wrote the article in whole, drafted the manuscript, and revised the article. All authors collected clinic data, participated in revising the manuscript critically for important intellectual content, and approved the final version for publication.

Ethical approval

KOU KAEK 201345