Integrase Strand Transfer Inhibitor Resistance Mutations in Antiretroviral Therapy-Naive and Treatment-Experienced HIV Patients in South Korea

Abstract

Updated guidelines for the treatment of antiretroviral therapy (ART)-negative patients with HIV recommend integrase strand transfer inhibitor (INSTI)-based regimens. HIV patients are tested for resistance to antiretrovirals, and the reported prevalence of transmitted INSTI resistance remains rare worldwide. However, no data related to INSTI resistance in Korean HIV patients have been reported. We aimed to determine the prevalence of INSTI resistance-related mutations in South Korea. We subjected both ART-naive (n = 58) and ART-experienced Korean HIV patients (n = 41) to genotypic resistance analysis and determined a prevalence of INSTI major resistance mutations of 3.4% (n = 2) among ART-naive patients and 22.0% (n = 9) among ART-experienced patients. In the former group, both major INSTI resistance cases involved the nonpolymorphic E92Q mutation in the integrase strand. Our findings suggest that INSTI resistance testing should be included in the standard resistance screening protocols for Korean HIV patients.

Both clinical trials and real-world data have demonstrated the excellent efficacy, safety, and tolerability profiles of integrase strand transfer inhibitor (INSTI)-based regimens for HIV-infected patients.¹ Since the release of raltegravir (RAL) in 2007, treatment guidelines published by the U.S. Department of Health and Human Services (DHHS), International Antiviral Society, and other organizations have recommended the administration of INSTI-based regimens to both antiretroviral therapy (ART)-naive and -experienced patients.^2,3 Accordingly, in 2015, INSTIs accounted for 25% of the approximately $24.23 billion global HIV drug market.⁴

Currently, the DHHS recommends that all ART-naive patients undergo standard genotypic HIV drug resistance testing to evaluate resistance to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-NRTIs (NNRTIs), and protease inhibitors (PIs). Supplementary INSTI resistance testing should be included for patients with suspected resistance to these drugs or who experience virologic failure while receiving INSTI-based therapies.²

Worldwide, transmitted INSTI resistance remains nonexistent or rare, according to recent reports from developed countries such as the United States and in Europe.^5
–7 However, the prevalence of transmitted INSTI resistance may increase in future with increasing use of INSTI-containing ART.⁸ No data on INSTI resistance among Korean HIV patients are currently available. Therefore, this study aimed to identify INSTI resistance mutations ART-naive and -experienced HIV-infected patients in South Korea.

A total of 99 HIV-1-infected patients (58 ART-naive and 41 ART-experienced patients) who visited a tertiary care hospital in Seoul, Korea, and underwent genotypic drug resistance testing between October 2014 and April 2017 were enrolled. The initial test results were recorded for patients who underwent multiple examinations, and three patients with changes in resistance were included as duplicates. Nested polymerase chain reaction and population-based sequencings were performed to determine sequences of the integrase gene, according to the French National Agency for AIDS Research (ANRS) consensus techniques, which are described at www.hivfrenchresistance.org (accessed January 15, 2017). The outer primers used were INPS1 (5′-TAGTAGCCAGCTGTGATAAATGTC-3′) and INPR8 (5′-TTCCATGTTCTAATCCTCATCCTG-3′), and inner primers were INPS3 (5′-GAAGCCATGCATGGACAAG-3′) and INPR9 (5′-ATCCTCATCCTGTCTACTTGCC-3′). The amplification condition was 45 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, and a final extension at 72°C for 7 min. The protease (PR) and reverse transcriptase (RT) sequences were obtained using the ViroSeq HIV-1 Genotyping System 2.0 (Abbott Molecular, Des Plaines, IL).The sequences were assembled, aligned to HXB2 consensus, and manually edited using Bioedit v7.1.3. HIV subtypes were identified using the REGA subtyping tool v3.0 (http://dbpartners.stanford.edu:8080/RegaSubtyping/stanford-hiv/typingtool/, accessed January 18, 2018). Antiretroviral resistance mutations were identified using the Stanford HIV Drug Resistance Database 8.4 (https://hivdb.stanford.edu/hivdb/by-mutations/, accessed January 18, 2018). Eligible patients' sequences were submitted to GenBank (accession nos.: MG763765–MG763863). INSTI major resistance mutation such as T66A/I/K, E92Q, E138K/A/T, G140A/C/S, Y143C/H/R, S147G, Q148H/K/R, N155H, and R263K in integrase were analyzed. INSTI minor resistance mutations such as H51Y, L74M, Q95K, T97A, S153Y/F, E157Q, G163R/K, and S230R were also tested. In addition, drug resistance mutations for surveillance of transmitted drug resistance mutation (SDRM) according to the World Health Organization (WHO) 2009 definition were evaluated.⁹

Of the 99 included patients, most were male (95/99, 96.0%), and 88.9% (88/99) harbored subtype B viruses. Six of 58 (10.3%) ART-naive patients harbored major resistance mutations for any single antiretroviral drug class, including 2 (3.4%) with major INSTI resistance mutations. Five patients (8.6%) harbored minor INSTI resistance mutations (Table 1). Both cases of major INSTI resistance among ART-naive patients involved the E92Q mutation. The minor INSTI resistance mutations included E157Q (n = 3), S230RS (n = 1) and G163R (n = 1). Identified major NNRTI resistance among naive patients were the K103N (n = 2) and P225HP (n = 1), and M46IM (n = 1) was PI resistance mutation identified. No patients harbored major mutations for NRTI resistance (Table 2). Among the 41 ART-experienced patients, 9 (22.0%) and 6 (14.6%) harbored major and minor INSTI mutations, respectively. All experienced patients with INSTI mutations were using INSTI-based regimens. The characteristics and the regimen at the time of resistance testing are described in Table 3.

Table 1.

Baseline Characteristics of Patients

	Naive (n = 58)	Experienced (n = 41)
Male, n (%)	54 (93.1)	41 (100)
Age, years (IQR)	32 (27–43)	40 (32–45)
Transmission route, n (%)
Homosexual	38 (65.5)	25 (61.0)
Heterosexual	10 (17.2)	11 (26.8)
No record	10 (17.2)	5 (12.2)
CD4+ T cell (IQR)	248 (166–364)	188 (55–321)
Plasma viral load, log₁₀ (IQR)	4.73 (4.32–5.15)	4.71 (4.16–5.14)
Subtype, n (%)
B	48 (82.8)	40 (97.6)
Non-B	10 (17.2)	1 (2.4)
Mutation, n (%)
INSTI major	2 (3.4)	9 (22.0)
INSTI minor	5 (8.6)	6 (14.6)
NRTI	0/56 (0)	18/38 (47.4)
NNRTI	3 (5.2)	12 (31.6)
PI major	1 (1.8)	2 (5.3)
PI minor	1 (1.8)	1 (2.6)
Patients with any one class of major mutation, n (%)	6 (10.3)	23 (56.1)

INSTI, integrase strand transfer inhibitor; IQR, interquartile range; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside/nucleotide reverse transcriptase inhibitor; PI, protease inhibitor.

Table 2.

List of Integrase Strand Transfer Inhibitor Resistance Mutations and Surveillance of Transmitted Drug Resistance Mutations

	INSTI major mutation	INSTI minor mutation	SDRM_NRTI	SDRM_NNRTI	SDRM_PI
Treatment-naive patients	E92Q (n = 2)	E157Q (n = 3) S230RS (n = 1) G163R (n = 1)		K103N (n = 2)P225HP (n = 1)	M46IM (n = 1)
Treatment-experienced patients	Y143C (n = 3)E92Q + N155H (n = 2)E92Q (n = 1)Q148R + G140C (n = 2)E138EK + Y143R (n = 1)	E157Q (n = 2)T97A + E157Q (n = 1)T97A + S230R (n = 1)G163K + S230R (n = 1)T97A (n = 1)	M184V/I (n = 9)M184V + K219E/N (n = 2)M184V + K65R (n = 1)M184V + K70R + K219E (n = 1)M184V + K65R + K70E (n = 1)M184V + M41L + Y115F (n = 1)M184V + K70E + Y115F (n = 1)M184V + M41L + L210W + T215F (n = 1)M41L + T215DY (n = 1)	L100IL (n = 1)K101E (n = 1)K103N/S (n = 5)Y181C (n = 1)Y188L (n = 1)G190A (n = 1)K101E + Y181I (n = 1)K101P + K103N/S (n = 1)	V82IL (n = 1)M46IM + I47IV + I54ILV + I84IV (n = 1)

SDRM, surveillance of transmitted drug resistance mutation.

Table 3.

Characteristics of Patients Who Have HIV Integrase Strand Transfer Inhibitor Major Mutations

Patient	Gender	Age	Transmission route	Subtype	CD4+ T cell	Plasma viral load	INSTI major mutation	Experienced INSTI	Regimen at the resistance test
A	M	44	Homosexual	B	193	17,600	Y143C	RAL	TDF/FTC/RAL
B	M	56	Unknown	B	338	10,300	Y143C	RAL/EVG	TDF/FTC/COBI/EVG
C	M	35	Unknown	B	17	238,000	Y143C	RAL	TDF/FTC/RAL
D	M	30	Homosexual	B	35	225,000	Q148R, G140C	EVG	TDF/FTC/COBI/EVG
E	M	42	Heterosexual	B	28	53,500	E92Q, N155H	RAL/EVG	TDF/FTC/COBI/EVG
F	M	45	Homosexual	CRF06_cpx	30	51,000	E92Q, N155H	RAL/EVG	ABA/3TC/RAL
G	M	37	Homosexual	B	85	9,660	Q148R, G140C	EVG	TDF/FTC/COBI/EVG
H	M	37	Heterosexual	B	275	29,500	E92Q	EVG	TDF/FTC/COBI/EVG
I	M	21	Homosexual	B	318	5,750	E92Q	Naive	None
J	M	25	Homosexual	B	261	1,930,000	E92Q	Naive	None
K	M	32	Unknown	B	129	97,800	E138K, Y143R	RAL	TDF/FTC/RAL

3TC, lamivudine; ABA, abacavir; COBI, cobicistat; EVG, elvitegravir; FTC, emtricitabine; RAL, raltegravir; TDF, tenofovir disoproxil fumarate.

This study provides the first data of INSTI resistance mutations among HIV patients in Korea. Notably, the prevalence of major INSTI resistance mutations among ART-naive patients was low at 3.4%, compared with 22.0% of ART-experienced patients. These data suggest a low rate of transmitted INSTI resistance mutations but a high frequency of acquired resistance mutations among ART-experienced and especially INSTI-experienced patients. These results suggest the need for pretreatment resistance testing among Korean HIV-naive patients. Currently, drug resistance testing is considered cost-effective if the transmitted drug resistance level is >5% in ART-naive patients.¹⁰ Although the prevalence of major INSTI resistance mutations was 3.4% among ART-naive patients in this study, the inclusion of INSTI in standard resistance screening of ART-naive Korean HIV patients should be considered. Previously, intervals of 3–5 years were observed between the approval of new NRTI, NNRTI, PI, and entry inhibitor drugs and the emergence of transmitted resistance mutations.¹¹ Similarly, a transmitted INSTI resistance mutation was first reported in 2010, 3 years after the 2007 release,¹¹ and the prevalence of transmitted INSTI resistance may soon increase with the increasing administration of INSTI base regimens.⁸

E92Q, a nonpolymorphic INSTI mutation, reduces susceptibility to elvitegravir (EVG) by >30-fold, to RAL by >5-fold, to dolutegravir (DTG) by ∼1.5-fold, although these effects may vary. E92Q is often associated with EVG-based treatment failure,¹² and two ART-naive patients with E92Q mutations did not harbor transmitted resistance mutations for other drug classes. Although minor INSTI mutations such as E157Q, S230R, G163R, and L74I may further reduce susceptibility, they are not sufficient in the absence of other mutations.⁸ In this study, the resistance patterns differed between ART-experienced and -naive patients. Many of the 41 ART-experienced patients harbored resistance mutations, including 9 (22.0%) with INSTI major mutations; all of the latter were INSTI-experienced patients and harbored mutations such as E138EK, G140C, Y143C, Q148R, and N155H, as well as E92Q pathway. Most of these patients experienced virologic failure during EVG or RAL use and switched to other ART classes (e.g., PI, NNRTI) after INSTI resistance testing. These results reaffirm the guideline recommendation of INSTI resistance mutation testing for INSTI-experienced patients to determine the subsequent use of drugs from this class.²

DTG is associated with relatively higher genetic barriers to resistance²; accordingly, EVG- or RAL-resistant viruses often remain susceptible to DTG.¹³ For such strains, twice-daily DTG plus two susceptible NRTIs may be feasible.² However, patients experiencing virologic failure with INSTI may exhibit low-to-intermediate DTG resistance through acquired mutations. In this study, two patients exposed to EVG harbored Q148R and G140C mutations and exhibited intermediate DTG resistance.

In 2013, we investigated resistance rates among Korean HIV patients and determined an SDRM (2009 WHO list) of 0% among ART-naive patients and a total drug resistance mutation frequency of 14%.¹⁴ Another report showed that the overall prevalences of transmitted resistance mutations were 2.5%–8.8% among ART-naive Korean HIV patients.¹⁵ Here, we identified SDRM in four ART-naive patients (7.1%; K103N, 2 cases; P225HP, 1 case; M46IM, 1 case). This rate would increase with the inclusion of other mutations. The prevalence of transmitted resistance mutations seems to be increasing among Korean ART-naive patients.

This study was limited by the single-center setting and small sample size. In addition, we could not amplify the RT and PR lesions of the pol genes to test NRTI, NNRTI, and PI resistance mutations from five subjects. And, only partial sequence for testing INSTI resistance could be used in a patient whose full integrase lesion sequence could not be obtained. Finally, we could not distinguish between acute and chronic infection in ART-naive patients.

In conclusion, we have reported the INSTI resistance mutation status in both ART-naive and -experienced patients in South Korea. The respective prevalence rates of INSTI major resistance mutation in these populations were 3.4% and 22.0%. We recommend the inclusion of INSTI resistance testing when making decisions regarding treatment regimens for Korean HIV patients.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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