Abstract
Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.
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A total of 66 peripheral blood samples were collected from drug-treated, HIV-1-infected patients at a clinic in Surabaya. We randomly recruited patients who had received ART for more than 24 months regardless of the viral load or ART failure. All patients received a first-line ART regimen: a combination of two nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and a nonnucleoside reverse transcriptase inhibitor (NNRTI). Information including the components of the ART regimen, duration of ART, side effects, opportunistic infections, clinical conditions of the patients, and possible routes of HIV-1 transmission was collected from the patient medical record database. This study was approved by the Institutional Ethics Committees of Airlangga University and Kobe University Graduate School of Medicine. Informed consent was obtained from all patients prior to sample collection.
Ten milliliters of ethylenediaminetetraacetic acid (EDTA) anticoagulated peripheral blood was collected from each participant. Plasma was then isolated from peripheral blood samples by centrifugation for 10 min at 2,000 rpm. Peripheral blood mononuclear cells (PBMCs) were also isolated by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). DNA was extracted from PBMCs using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich).
The HIV-1 pol gene encoding protease (PR gene) and reverse transcriptase (RT gene) was then amplified from DNA extracted from PBMCs by nested polymerase chain reaction (PCR) using Ex Taq (Takara Bio, Shiga, Japan) and primer sets as follows. To amplify the viral PR gene fragment, the primers DRPRO5 [5′-AGACAGGYTAATTTTTTAGGGA-3′; corresponding to nucleotides (nt) 2074 to 2095 of an HIV-1 reference strain, HXB2 (GenBank accession no. K03455)] and DRPRO2L (5′-TATGGATTTTCAGGCCCAATTTTTGA-3′; nt 2716 to 2691) were used for the first PCR, and the primers DRPRO1M (5′-AGAGCCAACAGCCCCACCAG-3′; nt 2148 to 2167) and DRPRO6 (5′-ACTTTTGGGCCATCCATTCC-3′; nt 2611 to 2592) were used for the nested PCR. In addition, to amplify the viral RT gene, RT1L (5′-ATGATAGGGGGAATTGGAGGTTT-3′; nt 2388 to 2410) and GPR2M (5′-GGACTACAGTCYACTTGTCCATG-3′; nt 4402 to 4380) were used for the first PCR and RT7L (5′-GACCTACACCTGTCAACATAATTGG-3′; nt 2485 to 2509) and GPR3L (5′-TTAAAATCACTARCCATTGYTCTCC-3′; nt 4309 to 4285) were used for the nested PCR. PCR products amplified at the end-point dilution of DNA templates were subjected to sequencing analysis to examine the genomic fragment of the major viral population in a sample.
Sequencing analysis of the amplified HIV-1 PR and RT genes was carried out using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM3500xL genetic analyzer (Applied Biosystems, Foster City, CA), and data were assembled using Genetyx version 10 software (Genetyx, Tokyo, Japan). The full length of the PR gene (297 bp; nt 2253 to 2549) and the N-terminus of the RT gene (762 bp; nt 2550 to 3311) were sequenced and subjected to subsequent analyses. HIV-1 subtyping was carried out using the Recombinant Identification Program (RIP) available on the HIV sequence database website (
Of the 66 samples derived from drug-treated patients, 58 PR genes and 53 RT genes were successfully sequenced. The demographic characteristics of the study participants are shown in Table 1; the majority were male (75.8%) with a mean age of 39.9 years. The most prevalent transmission route among the study participants was heterosexual intercourse (83.3%). All participants had received a first-line ART regimen based on the WHO recommendations. Most participants [45.5% (30/66)] were receiving ART with a combination of 3TC, AZT, and NVP. The median duration of ART was 47 months (interquartile range 33–58). Furthermore, 72.7% (48/66) of the participants had no opportunistic infection.
ART, antiretroviral therapy; 3TC, lamivudine; AZT, zidovudine; NVP, nevirapine; EFV, efavirenz; TDF, tenofavir; d4T, stavudine; IQR, interquartile range.
HIV subtyping has been an important molecular tool for monitoring geographic changes in the worldwide AIDS epidemic. 8 Based on the results of RIP and phylogenetic tree analyses for the PR and RT genes (Fig. 1), 60 samples were CRF01_AE (90.9%; 60/66), two samples (TD17 and TD64) were subtype B (3.0%), one sample (TD2) was subtype D (1.5%), two samples (TD60 and TD67) were the recombinant of CRF01_AE and subtype B, CRF33_01B (3.0%), and one sample (TD3) was subtype G or CRF02_AG (1.5%). CRF01_AE has been identified as the predominant HIV-1 subtype, similar to the epidemics in Malaysia, Thailand, and Taiwan. 9 –11 CRF01_AE is also the main epidemic strain in Jakarta, Surabaya, and Bali; however, subtype B and unique recombinant forms are also prevalent. 12,13 Our results were consistent with previous findings. The detection of various subtypes and recombinants in this study indicated the high diversity of the prevalence of HIV-1 in Surabaya, Indonesia.
Phylogenetic analysis of HIV-1 protease (PR) and reverse transcriptase (RT) gene sequences. Phylogenetic trees were generated for newly sequenced HIV-1 PR
An examination of drug resistance mutations revealed no major mutations in the PR genes. However, most samples contained the minor mutations M36I (77.6%), H69K (77.6%), and L89M (75.9%), which conferred resistance to ritonavir-boosted indinavir (IDV/r), atazanavir (ATV/r), and tipranavir (TPV/r). 7 Based on the results of the QuickAlign analysis on the CRF01_AE PR genes registered on the HIV-1 sequence database, the mutations M36I (99.7%), H69K (97.6%), and L89M (97.9%) were highly prevalent among the CRF01_AE PR genes; therefore, these minor mutations may be present as natural polymorphisms. 7
In contrast to the PR gene, 37.7% (20/53) of RT genes contained one or more mutations (Table 2). These mutations were commonly detected in patients on ART with the combination of two NRTIs and NNRTI, which are widely used as first-line ART in Indonesia. 14 The most frequently detected mutations were M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs), including M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E (20.8%). These mutations were categorized as drug resistance-associated major mutations that conferred resistance to the NRTIs, abacavir (ABC), emtricitabine (FTC), 3TC, AZT, and d4T, as well as the NNRTIs, EFV and NVP. 7 TAMs have been shown to confer high-level resistance to the thymidine analogue NRTIs, AZT and d4T, and low-level resistance to other NRTIs. 7 The appearance pattern of drug resistance mutations in RT genes was similar to previous findings, in which the mutations M184V (35.3%), Y181C (23.5%), K103N (11.7%), and TAMs (11.7%) were detected among ART-failed patients in Bandung, Indonesia. 15 TAMs were more frequently detected in our study subjects than those in Bandung. In contrast, Y181C, which confers resistance to NNRTI, was not detected in Surabaya, whereas its prevalence in Bandung was 23.5%. These findings indicated that resistance to NRTI appeared rapidly, and NNRTI was still more effective in Surabaya than in Bandung.
This subtype was assigned based on The Recombinant Identification Program (RIP) and phylogenetic analyses.
The determination of drug resistance mutations was based on the guidelines published by the International AIDS Society United States (IAS-USA). 7 Drug resistance-associated major mutations are shown in bold.
ART, antiretroviral therapy; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; 3TC, lamivudine; AZT, zidovudine; NVP, nevirapine; EFV, efavirenz; TDF, tenofovir; d4T, stavudine; ddI, didanosine; ABC, abacavir; FTC, emtricitabine; ETR, etravirine; RPV, rilpivirine.
In conclusion, we herein report the high prevalence of CRF01_AE as the predominant strain among HIV-1-infected patients on ART in Surabaya, Indonesia. We also reveal the high prevalence of drug resistance mutations to NRTIs and NNRTIs among HIV-1 patients who were receiving the first-line regimen of ART, especially mutations that confer resistance to 3TC, AZT, d4T, EVP, and NVP. Our results indicate that ART programs and the monitoring system for the effectiveness of ART need to be improved in Indonesia, and other classes of ART drugs such as protease and integrase inhibitors also need to be included in the regimen for ART-failed patients who were treated with the first-line regimen.
Sequence Data
Nucleotide sequences are available under GenBank accession numbers KM212829–KM212939.
Footnotes
Acknowledgments
This work was supported by the program of the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID); by the Ministry of Education, Culture, Sports, Science and Technology of Japan; and the Institute of Tropical Disease as the Center of Excellence (COE) program by the Ministry for Research and Technology (RISTEK) of Indonesia.
Author Disclosure Statement
No competing financial interests exist.