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Incidence and types of HIV-1 drug resistance mutation among patients failing first-line antiretroviral therapy
Journal of Pharmacological Sciences 139 (2019) 275e279
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Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
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Incidence and types of HIV-1 drug resistance mutation among patients failing first-line antiretroviral therapy Xiao-lu Luo a, Li-da Mo a, *, Guo-sheng Su a, Jin-ping Huang a, Jing-yu Wu b, Han-zhen Su a, Wan-hong Huang a, Shun-da Luo a, Zu-yan Ni a a
The 4th People's Hospital of Nanning, The Infectious Disease Hospital of Nanning Affiliated to Guangxi Medical University, and the AIDS Clinical Treatment Center of Guangxi (Nanning), Nanning, 530023, China The Disease Control and Prevention Center of Xingning District of Nanning, Nanning 530023, Guangxi, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2018 Received in revised form 7 November 2018 Accepted 9 November 2018 Available online 17 January 2019
Objective: This study aims to investigate the prevalence and types of drug resistance mutations among patients failing first-line antiretroviral therapy (ART). Methods: Plasma samples from 112 patients with human immunodeficiency virus-1 (HIV-1) were collected for virus RNA extract and gene amplification. The mutations related to drug resistance were detected and the incidence was statistically analyzed, and the drug resistance rate against common drugs was also evaluated. Results: 103 cases were successfully amplified, and the main drug resistance mutations in the reverse transcriptase (RT) region were M184V (50.49%), K103N (28.16%), Y181C (25.24%), and K65R (27.18%), while no drug main resistance mutation was found in the protease (PR) region. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial ART regimens and viral load. The drug resistance rate to the common drugs was assessed, including Efavirenz (EFV, 71.84%), Nevirapine (NVP, 74.76%), Lamivudine (3TC, 66.02%), Zidovudine (AZT, 4.85%), Stavudine (D4T, 16.51%), and Tenofovir (TDF, 21.36%). Conclusion: The drug resistance mutations to NRTIs and NNRTIs are complex and highly prevalent, which was the leading cause of first-line ART failure. This study provides significant theoretical support for developing the second-line and third-line therapeutic schemes. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: HIV-1 Drug resistance mutations First-line ART Drug resistance rate NRTIs NNRTIs
1. Introduction Due to high variability of retrovirus, human immunodeficiency virus (HIV) produces plenty of subtypes in the spreading process. HIV-1 pol gene differs from the other different subtypes,1 and it encodes the reverse-transcriptase (RT), protease (PR) and integrase enzymes that are the major targets of antiretroviral therapy.2,3 Drugs targeting PR are defined as the protease inhibitors (PIs); and drugs targeting RT are classified into the nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).4 By effectively inhibiting the virus
* Corresponding author. Changgang Road Erli No. 1, Nanning 530023, Guangxi, China. E-mail address: [email protected] (L.-d. Mo). Peer review under responsibility of Japanese Pharmacological Society.
replication, the highly active antiretroviral therapy (HAART) decreases the morbidity and mortality of HIV-1 infection, making the Acquired Immune Deficiency Syndrome (ADIS) a chronic and remediable disease.5 However, increasing use of antiretroviral treatment leads to continuous selective pressure of drugs and high variability of HIV, which results in drug resistance mutations and declined therapeutic effect.6 Drug resistance mutation is the leading cause of antiretroviral therapy (ART) failure, becoming a challenge in AIDS treatment. With HIV-1 drug resistance mutations development, the original antiretroviral drugs are unable to inhibit the resistant virus and the viral load may be sharply raised.7 Subsequently, with the immune system undermined and the lymphocyte CD4þT cell counts dramatically decreased, patients are more prone to be infected, which is manifested as clinical therapy failure.8,9 Worse still, patients failed in first-line antiretroviral drugs may also likely to fail with the second-line drugs, which results in severe public health
https://doi.org/10.1016/j.jphs.2018.11.016 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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problems. The World Health Organization (WHO) recommends that drug resistance monitoring should be carried out not only for long-term effectiveness of first-line ART in patients, but also for switching therapies following initial treatment failure.10 Researches in recent years have claimed that HIV-1 resistant genotype was different among individuals, which leads to individual difference in effectiveness and toxic reactions to ART.11 It has been widely demonstrated that HIV-1 drug resistance mutations were mainly caused by selective pressure of drugs, high variability of virus, poor drug compliance, interaction among drugs and pharmacokinetic factors.12 These factors may also have varying impact among different areas and patient populations, and further influence the prevalence and characteristic of drug resistance mutations.13 Guangxi is a high prevalence area of AIDS, locating at the junction of tropics and subtropics with minority (including Zhuang and Yao nationalities) concentrated.14 Distribution of HIV-1 subtype and prevalence of HIV-1 drug resistance in Liuzhou and Nanning (Guangxi) have been investigated,15 in which the prevalence of HIV-1 drug resistance mutations in antiretroviral-naive patients is 11.9%.16 Nonetheless, the data were collected within small scope and have not involved in the whole Guangxi. We undertake this study to investigate HIV-1 drug resistance mutations and individual difference among patients failing first-line ART in Guangxi, which is crucial and profound for clarifying drug resistance rate and improving drug treatment regimen, and providing theoretical support for developing the second-line or third-line therapeutic schemes. 2. Materials and methods 2.1. Research subjects From June 2014 to December 2016, 112 patients with AIDS who were admitted to the AIDS clinical treatment center of Guangxi and failing with the first-line ART were enrolled in this study (Fig. 1). The specific inclusion criteria for patients were as follows, (1) Patients were treated with first-line ART for more than 9 months; (2) Patients had a viral load less than 500 copies/ml in plasma after 24 weeks of first-line ART, while they had a viral load higher than 1000 copies/ml with the treatment prolonged, which was detected and verified for two times.
2.2. Samples collection 10 ml venous blood of the patients was taken and kept in EDTA anticoagulative tube, and the plasma was isolated by centrifugation and stored at 80 C for HIV-RNA load detection and drug resistance mutation detection. All the patients agreed and signed the documented informed consent for this study before sample collection. This research was approved by the local ethics committee and performed in accord with the Helsinki Declaration. 2.3. The original ART regimens The main regimens used in the patients are as follows. Azidothymidine (AZT) regimen: AZT þ 3TC þ EFV/NVP; Stavudine (D4T) regimen: D4T þ 3TC þ EFV/NVP; Tenofovir (TDF) regimen: TDF þ 3TC þ EFV/NVP. Drugs such as 3TC, AZT, D4T, and TDF are belonging to the NRT1 drugs, while NNRT1 drugs include EFV and NVP. 2.4. Detection of viral load The CAP/CTM48 thermo-cycler (Roche) and the COBAS Taqman 48 (Roche) were used to determine the HIV-1 viral load in plasma, according to the manufacturer's instructions. The viral load higher than 1000 copies/ml was considered failure for ART. 2.5. Genotypic resistance analysis HIV-1 RNA was extracted from the plasma using miRNeasy Plasma Kit (Invitrogen), and the cDNA was synthesized using the cDNA Reverse Transcription Kit (Applied Biosystems). The RTnested-PCR was used to amplify the PR and RT target DNA fragment of HIV-1 pol, and the Contig Express software was used to splice the origin nucleotide sequence. The BioEdit software was used to complete the assembly, editing and sequence alignment. Then sequences were submitted to the Stanford Database (http:// hivdb.stanford.edu/index.html) for HIV-1 drug resistance mutations analysis. 2.6. Drug resistance mutations detected Three kinds of drug resistance mutations were detected in the present study. Mutations to PI mainly including L10I and A71T were paid attention. Mutations to NRT1 were primarily focused on, such as K65R, M184I, and D67N. Mutations to NNRT1 were also determined, including K103N, Y181C, and V179D. 2.7. Statistical analysis All statistical analyses in this study were carried out with SPSS 22.0 software package (SPSS Inc.). The enumeration data was expressed with percentage (%) and difference between two groups was assessed with X2-test. Difference among more than two groups was analyzed by Tukey's Wholly Significant Difference, after X2test. A value of P < 0.05 was taken as significant. 3. Results 3.1. Basis clinical data of the patients
Fig. 1. Patient flowchart. 112 patients underwent the first-line ART for more than 9 months and with a HIV-1 viral load higher than 1000 copies/ml were enrolled. 103 plasma samples were successfully amplified and used for drug resistance mutations analysis.
Samples of 103 cases were successfully amplified among 112 patients. Table 1 displayed the basic data of patients with drug resistance mutations to NRT1, including their sex ratio, ages, nationalities, marital status, routes of infection, duration of treatment, initial ART regimens, and viral load. The clinical data of patients
X.-l. Luo et al. / Journal of Pharmacological Sciences 139 (2019) 275e279
277
Table 1 Clinical data of patients with drug resistance mutations to NRTIs. Program
Cases (n)
Mutations to NRTIs K65R
Genders (Male/Female) Ages (30/31e50/>50) Nationality (Han/Zhuang/Yao) Marital status (Married/Unmarried/Divorced or widowed) Route of infection (IV drug use/Sexual contact) Years since treatment (0.75e1/1e2/2e3/3e4/>4) Initial ART regimens (AZT/D4T/TDF) Viral load (lg) (3e4/4e5/>5)
77/26 29/61/13 63/38/2 53/37/13 21/82 24/26/25/9/19 36/25/42 18/54/31
with drug resistance mutations to NNRT1 were shown in Table 2. Among the 103 cases, males were in the majority, and patients were mainly aged 31e50, with different nationalities and marital status. Sexual contact is the main route of infection. Most of them had been treated within three years and had a viral load at 4e5 Ig copies/ml. The original ART regimens were AZT (36 cases), D4T (25 cases) and TDF (42 cases) (Tables 1 and 2), among which EFV was used in 75 cases, and NVP was used in 28 cases (data no shown). During treatment, the initial ART fist-line regiments were changed in 31 patients, such as AZT was replaced by TDF (16 cases), AZT was replaced by D4T (3 cases), D4T was replaced by TDF (9 cases), and TDF was replaced by AZT (3 cases) (data no shown). 3.2. Individual difference in incidence of drug resistance mutations Individual difference was noted in drug resistance mutations among patients failing the first line ART. The K65R and K103N mutations were more likely to occur in patients who aged over 50 than these younger than 30; the M184I mutation was more likely to be found in patients aged over 50 than these between 30 and 50. The K103N mutation was more likely to be observed in married patients compared with unmarried patients. The Y181C mutation was more likely to appear in patients infected through sexual contact compared with these through intravenous drug use. The K65R mutation was more likely to appear in patients treated for 0.75e1 year than these treated for 2e3 years and over 4 years; the D67N mutation was more likely to be noted in patients treated for 2e3 years than these treated for over 4 years; the K103N mutation was more likely to be found in patients treated for 1e2 years and over 4 years than these treated for 2e3 years; the Y181C mutation was more likely to occur in patients treated for 3e4 years than these treated for 1e2 years. The K65R mutation was more likely to be observed in patients treated with TDF regimen than these with AZT regimen, and the D67N mutation was more likely to take place in patients treated with TDF and D4T regimens compared with AZT
M184I
D67N
n
P
n
P
n
P
20/8 3/19/6 16/11/1 15/9/4 6/22 12/9/4/1/2 5/6/17 2/11/15
0.636 0.031 0.712 0.875 0.783 0.043 0.030 0.005
14/4 4/8/6 12/6/0 7/8/3 3/15 6/4/5/2/1 5/5/8 2/9/7
0.747 0.015 0.741 0.502 0.378 0.701 0.779 0.583
7/4 5/5/1 9/2/0 4/5/2 2/9 2/1/7/1/0 0/3/8 1/6/4
0.371 0.405 0.326 0.564 0.465 0.029 0.025 0.719
regimen. The K65R mutation was more likely to occur in patients with a viral load higher than 5 Ig copies/ml than these with a viral load at 4e5 Ig copies/ml. No significant difference was found in the common drug resistance mutations among genders and nationalities (Tables 1 and 2). 3.3. The incidence of drug resistance mutations No main mutation was found in the PR region among patients. The mutations relate to NRTIs were M184V (50.49%) and K65R (27.18%); and mutations relate to NNRTIs were K103N (28.16%) and Y181C (25.24%) (Table 3). 3.4. Analysis of NRTIs and NNRTIs drug resistance The incidence of drug resistance mutations to NRTIs, NNRTIs and PIs was 70.87%, 81.55% and 0.00%, separately, among the 103 cases (Table 4). The EFV and NVP are the NNRTIs that mainly used in the 103 cases, and the incidence of drug resistance mutations to them was 71.84% and 74.76%, respectively; 3TC, AZT, D4T and TDF are the NRTIs whose incidence of drug resistance mutations was 66.02%, 4.85%, 16.51%, and 21.36%, separately (Table 5). 4. Discussion In this study, we demonstrated that drug resistance mutations relate to NRTIs and NNRTIs were complex and highly prevalent among patients failing first-line ART in Guangxi. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial therapeutic schedules and viral load. The individual difference of drug resistance mutations among patients failing first-line ART were highlighted, providing reference for optimizing drug treatment regimen.
Table 2 Clinical data of patients with drug resistance mutations to NNRTIs. Program
Cases (n)
Mutations to NNRTIs K103N
Genders (Male/Female) Ages (30/31e50/>50) Nationality (Han/Zhuang/Yao) Marital status (Married/Unmarried/Divorced or widowed) Route of infection (IV drug use/Sexual contact) Years since treatment (0.75e1/1e2/2e3/3e4/>4) Initial ART regimens (AZT/D4T/TDF) Viral load (lg) (3e4/4e5/>5)
77/26 29/61/13 63/38/2 53/37/13 21/82 24/26/25/9/19 36/25/42 18/54/31
Y181C
n
P
n
P
23/6 3/20/6 22/7/0 20/5/4 7/22 10/6/2/1/10 11/7/11 2/15/12
0.508 0.027 0.139 0.043 0.887 0.023 0.913 0.119
18/8 7/17/2 16/10/0 13/7/6 2/24 10/2/8/3/3 6/7/13 2/13/11
0.455 0.637 0.707 0.152 0.027 0.020 0.332 0.163
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Table 3 The incidence of different drug resistance mutations in HIV-1 among 103 patients. Mutations Mutations to PIs L10I Other PI related mutations Mutations to NRTIs M184V K65R M184I D67N Y115F K70E K70R V75M Other NRTIs related mutations Mutations to NNRTIs K103N Y181C V179D G190A K101E V106M K103R V179E G190AG L100I A98G Other NNRTIs related mutations
Mutant cases
Ratio (%)
Drugs
8 8
7.77 7.77
52 28 18 11 12 11 5 11 63
50.49 27.18 17.48 10.68 11.65 10.68 4.85 10.68 61.17
NRTIs 3TC ABC AZT D4T DDI FTC TDF NNRTIs DLV EFV ETR NVP
29 26 22 18 14 12 11 5 4 5 4 60
28.16 25.24 21.36 17.48 13.59 11.65 10.68 4.85 3.88 4.85 3.88 58.25
Among the drug resistance mutations related to NRTIs, the incidence of M184V was 50.49%, which was similar with the previous report, and it might be associated with the combined use of 3TC.17 The drug resistance mutations of K103N (28.16%), Y181C (25.24%) and G190A (17.48%) were found in this study, while it has been reported that the incidence of G190A and Y181C was about 40% and that of K103N was less than 20%.18 The difference might be attributed to the combined use of EFV in our study, while NVP was rarely used.19 M41L (6.9%) and T215Y (3.45%) mutations was found in our study, and coincidence of the both has been claimed to have the greatest impact on AZT regimen.20 9 minor mutations to PIs were noted in the present study, but they made no difference on drug resistance, regardless of individual or joint occurrence.21,22 No drug resistance mutation was found in the PR region in this study, implying that combination of PIs can be popularized among patients failing first-line ART in Guangxi. The efficacy of ART drugs and drug resistance mutation were all differed among individuals. K65R, K103N and M184I were more likely to occur in the elder patients, which may be related to the difference in pharmacokinetics and body immunity among different age groups.23,24 In addition, the elder usually knew little about AIDS and had poor drug compliance,25 which all contributed to the occurrence of resistance. K103N is a mutation that conferred high-level resistance to all of the first-line NNRTIs,26 and was more frequently to be noted in married patients in our study. The Y181C mutation was more frequently to appear in patients infected Table 4 The drug resistance to different kinds of ART drugs among 103 patients. Types of drug resistance
Cases of drug resistance
Incidence of drug resistance mutations (%)
Resistant Resistant Resistant Resistant Resistant Resistant Resistant
88 73 84 0 69 4 15
85.44 70.87 81.55 0 66.99 3.88 14.56
to to to to to to to
some drugs NRTIs NNRTIs PIs NNRTIs and NRTIs NRTIs only NNRTIs only
Table 5 The drug resistance to ART drugs among 103 patients. Cases
Incidence of drug resistance mutations (%)
68 48 5 17 26 67 22
66.02 46.62 4.85 16.51 25.24 65.05 21.36
74 74 22 77
71.84 71.84 21.36 74.76
through sexual contact compared with these through intravenous drug users, which might be associated with impacts of drug on HIV replication and ART metabolism.14 HIV-1 showed higher adaptability for replication as the AZT regimen was used, leading to rapid development of D67N and K70R mutations.27 But the K65R mutation may develop rapidly in C subtype with TDF regimen.28 The K65R mutation was more frequently in patients with higher viral load and it was also considered to be associated with moderate resistance to TDF and 3TC, low resistance to D4T, and susceptibility to AZT.28,29 Cross resistance and multi-drug resistance were liable to occur against NNRTI drugs,30 and our study demonstrated that the incidence of drug resistance to NNRTIs reached 81.55%, which was close to the incidence of all the mutations (85.44%). Analysis of individual difference in drug resistance mutations contributes to optimization of the regimen and improving condition of AIDS patients. It was suggested that the PIs drugs could be dominated in the future ART regimen in Guangxi. Some limitations exist in the present study. Due to the underdevelopment of Guangxi, most patients failing first-line ART were not included for analysis of drug resistance mutation, which resulted in small sample size in the study. Moreover, with 70% of the subjects came from the south of Guangxi and only 30% of them came from the north, the influence of regional difference on development of drug resistance mutation should be taken into consideration in larger sample size in the future. In addition, the drug compliance of the enrolled objects needs further investigation for more comprehensive analyses. With much research funding, more samples and larger survey region, overall conclusion may be expected with much further researches carried out in the future. To summarize, the drug resistance mutations to NRTIs and NNRTIs were complex and highly prevalent, which was the leading cause of first-line ART failure. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, treatment duration, initial ART regimens and viral load. Our study provides theoretical support for developing the second-line and third-line therapeutic schemes to decrease mortality and medical resources of AIDS. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by the program of Scientific Research and Technological Development of Xingning District of Nanning, China (No. 2015A15).
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Full Paper
Incidence and types of HIV-1 drug resistance mutation among patients failing first-line antiretroviral therapy Xiao-lu Luo a, Li-da Mo a, *, Guo-sheng Su a, Jin-ping Huang a, Jing-yu Wu b, Han-zhen Su a, Wan-hong Huang a, Shun-da Luo a, Zu-yan Ni a a
The 4th People's Hospital of Nanning, The Infectious Disease Hospital of Nanning Affiliated to Guangxi Medical University, and the AIDS Clinical Treatment Center of Guangxi (Nanning), Nanning, 530023, China The Disease Control and Prevention Center of Xingning District of Nanning, Nanning 530023, Guangxi, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 August 2018 Received in revised form 7 November 2018 Accepted 9 November 2018 Available online 17 January 2019
Objective: This study aims to investigate the prevalence and types of drug resistance mutations among patients failing first-line antiretroviral therapy (ART). Methods: Plasma samples from 112 patients with human immunodeficiency virus-1 (HIV-1) were collected for virus RNA extract and gene amplification. The mutations related to drug resistance were detected and the incidence was statistically analyzed, and the drug resistance rate against common drugs was also evaluated. Results: 103 cases were successfully amplified, and the main drug resistance mutations in the reverse transcriptase (RT) region were M184V (50.49%), K103N (28.16%), Y181C (25.24%), and K65R (27.18%), while no drug main resistance mutation was found in the protease (PR) region. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial ART regimens and viral load. The drug resistance rate to the common drugs was assessed, including Efavirenz (EFV, 71.84%), Nevirapine (NVP, 74.76%), Lamivudine (3TC, 66.02%), Zidovudine (AZT, 4.85%), Stavudine (D4T, 16.51%), and Tenofovir (TDF, 21.36%). Conclusion: The drug resistance mutations to NRTIs and NNRTIs are complex and highly prevalent, which was the leading cause of first-line ART failure. This study provides significant theoretical support for developing the second-line and third-line therapeutic schemes. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: HIV-1 Drug resistance mutations First-line ART Drug resistance rate NRTIs NNRTIs
1. Introduction Due to high variability of retrovirus, human immunodeficiency virus (HIV) produces plenty of subtypes in the spreading process. HIV-1 pol gene differs from the other different subtypes,1 and it encodes the reverse-transcriptase (RT), protease (PR) and integrase enzymes that are the major targets of antiretroviral therapy.2,3 Drugs targeting PR are defined as the protease inhibitors (PIs); and drugs targeting RT are classified into the nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).4 By effectively inhibiting the virus
* Corresponding author. Changgang Road Erli No. 1, Nanning 530023, Guangxi, China. E-mail address: [email protected] (L.-d. Mo). Peer review under responsibility of Japanese Pharmacological Society.
replication, the highly active antiretroviral therapy (HAART) decreases the morbidity and mortality of HIV-1 infection, making the Acquired Immune Deficiency Syndrome (ADIS) a chronic and remediable disease.5 However, increasing use of antiretroviral treatment leads to continuous selective pressure of drugs and high variability of HIV, which results in drug resistance mutations and declined therapeutic effect.6 Drug resistance mutation is the leading cause of antiretroviral therapy (ART) failure, becoming a challenge in AIDS treatment. With HIV-1 drug resistance mutations development, the original antiretroviral drugs are unable to inhibit the resistant virus and the viral load may be sharply raised.7 Subsequently, with the immune system undermined and the lymphocyte CD4þT cell counts dramatically decreased, patients are more prone to be infected, which is manifested as clinical therapy failure.8,9 Worse still, patients failed in first-line antiretroviral drugs may also likely to fail with the second-line drugs, which results in severe public health
https://doi.org/10.1016/j.jphs.2018.11.016 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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problems. The World Health Organization (WHO) recommends that drug resistance monitoring should be carried out not only for long-term effectiveness of first-line ART in patients, but also for switching therapies following initial treatment failure.10 Researches in recent years have claimed that HIV-1 resistant genotype was different among individuals, which leads to individual difference in effectiveness and toxic reactions to ART.11 It has been widely demonstrated that HIV-1 drug resistance mutations were mainly caused by selective pressure of drugs, high variability of virus, poor drug compliance, interaction among drugs and pharmacokinetic factors.12 These factors may also have varying impact among different areas and patient populations, and further influence the prevalence and characteristic of drug resistance mutations.13 Guangxi is a high prevalence area of AIDS, locating at the junction of tropics and subtropics with minority (including Zhuang and Yao nationalities) concentrated.14 Distribution of HIV-1 subtype and prevalence of HIV-1 drug resistance in Liuzhou and Nanning (Guangxi) have been investigated,15 in which the prevalence of HIV-1 drug resistance mutations in antiretroviral-naive patients is 11.9%.16 Nonetheless, the data were collected within small scope and have not involved in the whole Guangxi. We undertake this study to investigate HIV-1 drug resistance mutations and individual difference among patients failing first-line ART in Guangxi, which is crucial and profound for clarifying drug resistance rate and improving drug treatment regimen, and providing theoretical support for developing the second-line or third-line therapeutic schemes. 2. Materials and methods 2.1. Research subjects From June 2014 to December 2016, 112 patients with AIDS who were admitted to the AIDS clinical treatment center of Guangxi and failing with the first-line ART were enrolled in this study (Fig. 1). The specific inclusion criteria for patients were as follows, (1) Patients were treated with first-line ART for more than 9 months; (2) Patients had a viral load less than 500 copies/ml in plasma after 24 weeks of first-line ART, while they had a viral load higher than 1000 copies/ml with the treatment prolonged, which was detected and verified for two times.
2.2. Samples collection 10 ml venous blood of the patients was taken and kept in EDTA anticoagulative tube, and the plasma was isolated by centrifugation and stored at 80 C for HIV-RNA load detection and drug resistance mutation detection. All the patients agreed and signed the documented informed consent for this study before sample collection. This research was approved by the local ethics committee and performed in accord with the Helsinki Declaration. 2.3. The original ART regimens The main regimens used in the patients are as follows. Azidothymidine (AZT) regimen: AZT þ 3TC þ EFV/NVP; Stavudine (D4T) regimen: D4T þ 3TC þ EFV/NVP; Tenofovir (TDF) regimen: TDF þ 3TC þ EFV/NVP. Drugs such as 3TC, AZT, D4T, and TDF are belonging to the NRT1 drugs, while NNRT1 drugs include EFV and NVP. 2.4. Detection of viral load The CAP/CTM48 thermo-cycler (Roche) and the COBAS Taqman 48 (Roche) were used to determine the HIV-1 viral load in plasma, according to the manufacturer's instructions. The viral load higher than 1000 copies/ml was considered failure for ART. 2.5. Genotypic resistance analysis HIV-1 RNA was extracted from the plasma using miRNeasy Plasma Kit (Invitrogen), and the cDNA was synthesized using the cDNA Reverse Transcription Kit (Applied Biosystems). The RTnested-PCR was used to amplify the PR and RT target DNA fragment of HIV-1 pol, and the Contig Express software was used to splice the origin nucleotide sequence. The BioEdit software was used to complete the assembly, editing and sequence alignment. Then sequences were submitted to the Stanford Database (http:// hivdb.stanford.edu/index.html) for HIV-1 drug resistance mutations analysis. 2.6. Drug resistance mutations detected Three kinds of drug resistance mutations were detected in the present study. Mutations to PI mainly including L10I and A71T were paid attention. Mutations to NRT1 were primarily focused on, such as K65R, M184I, and D67N. Mutations to NNRT1 were also determined, including K103N, Y181C, and V179D. 2.7. Statistical analysis All statistical analyses in this study were carried out with SPSS 22.0 software package (SPSS Inc.). The enumeration data was expressed with percentage (%) and difference between two groups was assessed with X2-test. Difference among more than two groups was analyzed by Tukey's Wholly Significant Difference, after X2test. A value of P < 0.05 was taken as significant. 3. Results 3.1. Basis clinical data of the patients
Fig. 1. Patient flowchart. 112 patients underwent the first-line ART for more than 9 months and with a HIV-1 viral load higher than 1000 copies/ml were enrolled. 103 plasma samples were successfully amplified and used for drug resistance mutations analysis.
Samples of 103 cases were successfully amplified among 112 patients. Table 1 displayed the basic data of patients with drug resistance mutations to NRT1, including their sex ratio, ages, nationalities, marital status, routes of infection, duration of treatment, initial ART regimens, and viral load. The clinical data of patients
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277
Table 1 Clinical data of patients with drug resistance mutations to NRTIs. Program
Cases (n)
Mutations to NRTIs K65R
Genders (Male/Female) Ages (30/31e50/>50) Nationality (Han/Zhuang/Yao) Marital status (Married/Unmarried/Divorced or widowed) Route of infection (IV drug use/Sexual contact) Years since treatment (0.75e1/1e2/2e3/3e4/>4) Initial ART regimens (AZT/D4T/TDF) Viral load (lg) (3e4/4e5/>5)
77/26 29/61/13 63/38/2 53/37/13 21/82 24/26/25/9/19 36/25/42 18/54/31
with drug resistance mutations to NNRT1 were shown in Table 2. Among the 103 cases, males were in the majority, and patients were mainly aged 31e50, with different nationalities and marital status. Sexual contact is the main route of infection. Most of them had been treated within three years and had a viral load at 4e5 Ig copies/ml. The original ART regimens were AZT (36 cases), D4T (25 cases) and TDF (42 cases) (Tables 1 and 2), among which EFV was used in 75 cases, and NVP was used in 28 cases (data no shown). During treatment, the initial ART fist-line regiments were changed in 31 patients, such as AZT was replaced by TDF (16 cases), AZT was replaced by D4T (3 cases), D4T was replaced by TDF (9 cases), and TDF was replaced by AZT (3 cases) (data no shown). 3.2. Individual difference in incidence of drug resistance mutations Individual difference was noted in drug resistance mutations among patients failing the first line ART. The K65R and K103N mutations were more likely to occur in patients who aged over 50 than these younger than 30; the M184I mutation was more likely to be found in patients aged over 50 than these between 30 and 50. The K103N mutation was more likely to be observed in married patients compared with unmarried patients. The Y181C mutation was more likely to appear in patients infected through sexual contact compared with these through intravenous drug use. The K65R mutation was more likely to appear in patients treated for 0.75e1 year than these treated for 2e3 years and over 4 years; the D67N mutation was more likely to be noted in patients treated for 2e3 years than these treated for over 4 years; the K103N mutation was more likely to be found in patients treated for 1e2 years and over 4 years than these treated for 2e3 years; the Y181C mutation was more likely to occur in patients treated for 3e4 years than these treated for 1e2 years. The K65R mutation was more likely to be observed in patients treated with TDF regimen than these with AZT regimen, and the D67N mutation was more likely to take place in patients treated with TDF and D4T regimens compared with AZT
M184I
D67N
n
P
n
P
n
P
20/8 3/19/6 16/11/1 15/9/4 6/22 12/9/4/1/2 5/6/17 2/11/15
0.636 0.031 0.712 0.875 0.783 0.043 0.030 0.005
14/4 4/8/6 12/6/0 7/8/3 3/15 6/4/5/2/1 5/5/8 2/9/7
0.747 0.015 0.741 0.502 0.378 0.701 0.779 0.583
7/4 5/5/1 9/2/0 4/5/2 2/9 2/1/7/1/0 0/3/8 1/6/4
0.371 0.405 0.326 0.564 0.465 0.029 0.025 0.719
regimen. The K65R mutation was more likely to occur in patients with a viral load higher than 5 Ig copies/ml than these with a viral load at 4e5 Ig copies/ml. No significant difference was found in the common drug resistance mutations among genders and nationalities (Tables 1 and 2). 3.3. The incidence of drug resistance mutations No main mutation was found in the PR region among patients. The mutations relate to NRTIs were M184V (50.49%) and K65R (27.18%); and mutations relate to NNRTIs were K103N (28.16%) and Y181C (25.24%) (Table 3). 3.4. Analysis of NRTIs and NNRTIs drug resistance The incidence of drug resistance mutations to NRTIs, NNRTIs and PIs was 70.87%, 81.55% and 0.00%, separately, among the 103 cases (Table 4). The EFV and NVP are the NNRTIs that mainly used in the 103 cases, and the incidence of drug resistance mutations to them was 71.84% and 74.76%, respectively; 3TC, AZT, D4T and TDF are the NRTIs whose incidence of drug resistance mutations was 66.02%, 4.85%, 16.51%, and 21.36%, separately (Table 5). 4. Discussion In this study, we demonstrated that drug resistance mutations relate to NRTIs and NNRTIs were complex and highly prevalent among patients failing first-line ART in Guangxi. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial therapeutic schedules and viral load. The individual difference of drug resistance mutations among patients failing first-line ART were highlighted, providing reference for optimizing drug treatment regimen.
Table 2 Clinical data of patients with drug resistance mutations to NNRTIs. Program
Cases (n)
Mutations to NNRTIs K103N
Genders (Male/Female) Ages (30/31e50/>50) Nationality (Han/Zhuang/Yao) Marital status (Married/Unmarried/Divorced or widowed) Route of infection (IV drug use/Sexual contact) Years since treatment (0.75e1/1e2/2e3/3e4/>4) Initial ART regimens (AZT/D4T/TDF) Viral load (lg) (3e4/4e5/>5)
77/26 29/61/13 63/38/2 53/37/13 21/82 24/26/25/9/19 36/25/42 18/54/31
Y181C
n
P
n
P
23/6 3/20/6 22/7/0 20/5/4 7/22 10/6/2/1/10 11/7/11 2/15/12
0.508 0.027 0.139 0.043 0.887 0.023 0.913 0.119
18/8 7/17/2 16/10/0 13/7/6 2/24 10/2/8/3/3 6/7/13 2/13/11
0.455 0.637 0.707 0.152 0.027 0.020 0.332 0.163
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Table 3 The incidence of different drug resistance mutations in HIV-1 among 103 patients. Mutations Mutations to PIs L10I Other PI related mutations Mutations to NRTIs M184V K65R M184I D67N Y115F K70E K70R V75M Other NRTIs related mutations Mutations to NNRTIs K103N Y181C V179D G190A K101E V106M K103R V179E G190AG L100I A98G Other NNRTIs related mutations
Mutant cases
Ratio (%)
Drugs
8 8
7.77 7.77
52 28 18 11 12 11 5 11 63
50.49 27.18 17.48 10.68 11.65 10.68 4.85 10.68 61.17
NRTIs 3TC ABC AZT D4T DDI FTC TDF NNRTIs DLV EFV ETR NVP
29 26 22 18 14 12 11 5 4 5 4 60
28.16 25.24 21.36 17.48 13.59 11.65 10.68 4.85 3.88 4.85 3.88 58.25
Among the drug resistance mutations related to NRTIs, the incidence of M184V was 50.49%, which was similar with the previous report, and it might be associated with the combined use of 3TC.17 The drug resistance mutations of K103N (28.16%), Y181C (25.24%) and G190A (17.48%) were found in this study, while it has been reported that the incidence of G190A and Y181C was about 40% and that of K103N was less than 20%.18 The difference might be attributed to the combined use of EFV in our study, while NVP was rarely used.19 M41L (6.9%) and T215Y (3.45%) mutations was found in our study, and coincidence of the both has been claimed to have the greatest impact on AZT regimen.20 9 minor mutations to PIs were noted in the present study, but they made no difference on drug resistance, regardless of individual or joint occurrence.21,22 No drug resistance mutation was found in the PR region in this study, implying that combination of PIs can be popularized among patients failing first-line ART in Guangxi. The efficacy of ART drugs and drug resistance mutation were all differed among individuals. K65R, K103N and M184I were more likely to occur in the elder patients, which may be related to the difference in pharmacokinetics and body immunity among different age groups.23,24 In addition, the elder usually knew little about AIDS and had poor drug compliance,25 which all contributed to the occurrence of resistance. K103N is a mutation that conferred high-level resistance to all of the first-line NNRTIs,26 and was more frequently to be noted in married patients in our study. The Y181C mutation was more frequently to appear in patients infected Table 4 The drug resistance to different kinds of ART drugs among 103 patients. Types of drug resistance
Cases of drug resistance
Incidence of drug resistance mutations (%)
Resistant Resistant Resistant Resistant Resistant Resistant Resistant
88 73 84 0 69 4 15
85.44 70.87 81.55 0 66.99 3.88 14.56
to to to to to to to
some drugs NRTIs NNRTIs PIs NNRTIs and NRTIs NRTIs only NNRTIs only
Table 5 The drug resistance to ART drugs among 103 patients. Cases
Incidence of drug resistance mutations (%)
68 48 5 17 26 67 22
66.02 46.62 4.85 16.51 25.24 65.05 21.36
74 74 22 77
71.84 71.84 21.36 74.76
through sexual contact compared with these through intravenous drug users, which might be associated with impacts of drug on HIV replication and ART metabolism.14 HIV-1 showed higher adaptability for replication as the AZT regimen was used, leading to rapid development of D67N and K70R mutations.27 But the K65R mutation may develop rapidly in C subtype with TDF regimen.28 The K65R mutation was more frequently in patients with higher viral load and it was also considered to be associated with moderate resistance to TDF and 3TC, low resistance to D4T, and susceptibility to AZT.28,29 Cross resistance and multi-drug resistance were liable to occur against NNRTI drugs,30 and our study demonstrated that the incidence of drug resistance to NNRTIs reached 81.55%, which was close to the incidence of all the mutations (85.44%). Analysis of individual difference in drug resistance mutations contributes to optimization of the regimen and improving condition of AIDS patients. It was suggested that the PIs drugs could be dominated in the future ART regimen in Guangxi. Some limitations exist in the present study. Due to the underdevelopment of Guangxi, most patients failing first-line ART were not included for analysis of drug resistance mutation, which resulted in small sample size in the study. Moreover, with 70% of the subjects came from the south of Guangxi and only 30% of them came from the north, the influence of regional difference on development of drug resistance mutation should be taken into consideration in larger sample size in the future. In addition, the drug compliance of the enrolled objects needs further investigation for more comprehensive analyses. With much research funding, more samples and larger survey region, overall conclusion may be expected with much further researches carried out in the future. To summarize, the drug resistance mutations to NRTIs and NNRTIs were complex and highly prevalent, which was the leading cause of first-line ART failure. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, treatment duration, initial ART regimens and viral load. Our study provides theoretical support for developing the second-line and third-line therapeutic schemes to decrease mortality and medical resources of AIDS. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by the program of Scientific Research and Technological Development of Xingning District of Nanning, China (No. 2015A15).
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