Background
HIV has been a leading cause of infectious diseases and death globally over the last three decades. Since highly active antiretroviral therapy (HAART) became available, the HIV-infected patients’ life expectancy and health outcomes have been drastic improved. However, the widespread use of HARRT has led to drug resistance (DR) to ARVs, which undermines their effectiveness [
1,
2]. Drug resistance can be categorized into ADR mutations in treatment-experienced patients and transmitted drug resistance (TDR) mutations in treatment-naïve individuals, both of which could cause the failure of the antiretroviral therapy (ART). Ignoring TDR can result in treatment failure of antiretroviral regimens, and ADR is often associated with virological failure (VF) and can increase the burden of treatment [
3,
4]. While TDR has been well documented, fewer studies report on the rates of ADR. A systematic review from resource limited settings reports on rates of ADR steadily increased with time on ARV [
5].. M184V is the most commonly occurring NRTI ADR mutation. In vitro, it causes high-level resistance to lamivudine (3TC). The next most common ADR mutation is the K103N mutation (associated with non-nucleoside reverse transcriptase inhibitors; NNRTIs) which cause high-level resistance to nevirapine (NVP) and variable resistance to efavirenz (EFV) [
6].
Since the first case of HIV infection was found in 1992, there were 29,002 people living with HIV (PLWH) to the end of 2017 in Hunan province, which is in south central China, and has a population of 68 million. More than 25,530 HIV/AIDS patients had received free highly active antiretroviral therapy (HAART) and were still in treatment which is supported by the government by the end of 2017. Although stavudine (D4T) and zidovudine (AZT) are not recommended as first-line therapy in the US and Europe, in much of Asia and other resource-limited settings, the most common first-line regimens for HIV-infected patients still contain either D4T or AZT, which are less costly than tenofovir (TDF) [
7‐
9]. The first line regimen of HAART given to patients in Hunan Province China is a triple therapy choosing from d4T, AZT, TDF, 3TC, Efavirenz (EFV) and nevirapine (NVP), all these generic drugs produced in China. Lopinavir/ritonavir (LPV/r), produce outside China, is the second-line drug. A recent Hunan Province molecular epidemiology survey showed that 4 HIV-1 subtypes in Hunan province reported were CRF_01AE, CRF07_BC, B and C. CRF_01AE was the dominant subtype strain (more than 70%) [
10‐
12].
The emerging ADR and subsequent treatment failure pose a major concern for HIV ART in resource-limited settings where treatment options are limited. The objective of this study is to determine the prevalence and DRMs of HIV-1 virologic failure and acquired drug resistance among ART-experienced adults in Hunan Province, China.
Discussion
Our study aimed to elucidate the prevalence of HIV ADRs in ART VF subjects, since ADR is one major problem with the efficacy of ART as it affects the clinical outcomes of treatment. In this study, the annual ADR rate was lower than the threshold low-incidence rate defined by WHO of 5% [
17] (from 2012 to 2017: 2.76, 2.44, 2.98, 2.68, 2.24 and 2.19%). This situation is mainly due to the expansion of National Free Antiretroviral Treatment Program (NFATP) in Hunan Province in recent years. Early antiviral treatment can control virus replication as early as possible, and reduce the possibility of drug resistance [
13,
18]. The result showed that CRF01 AE is still the most dominance subtype in Hunan province (64.14%), followed by subtype BC,B and C, the finding corresponding to the last years of the Hunan Province molecular epidemiology survey (2009–2015) results [
10‐
12,
19].. The prevalence of HIV-1 subtype B was significantly higher in Zhangjiajie City compared with other areas in Hunan province. Most of the HIV infected people were older people, who lived same area. Previous studies have had similar findings [
20]. A HIV epidemic outbreak of subtype B occurred in former plasma donors (FPDs) in central China in 1990s [
21,
22]. Some of the FPDs came from Zhangjiajie city, and as a result the B strains were introduced into Zhangjiajie. Due to the economic backwardness of the region, traffic congestion, limited emigration, and open sex, the B strain became a localized epidemic.
In addition to the common subtypes, other subtypes including combined subtype CRF06cpx, CRF09cpx, CRF18cpx and CRF45cpx were found in the last 2 years (2016 and 2017). This may indicate a more complicated and diverse trend of HIV-1 epidemiology emerging in Hunan province.
The most frequently identified ADR present in NRTI was M184V/I (61.27%), followed by K65R (19.96%). The elevated presence of M184V/I is expected and arises as a consequence of the use of 3TC as part of all the first-line regimens in China [
23]. Although M184V/I causes high-level in vitro resistance to 3TC, M184V/I is not a contraindication to continued treatment with 3TC because it increases susceptibility to AZT, and in addition, it is associated with clinically significant reductions in HIV-1 replication [
14,
24,
25]. Similar to our study finding, a high prevalence of M184 V/I mutation has been reported in Asia, Sub-Saharan Africa and Latin America, but a lower prevalence in western Europe [
26‐
31]. This difference can be explained by the more frequent use of 3TC in low- and middle-income countries than in West European countries. K65R is the signature mutation associated with TDF resistance [
32,
33]. This study showed that K65R codon mutation had a significant increase from 2014 (71.4, 6.74, 16.42, 22.73%, 20.48 and 30.91%, respectively, from 2012 to 2017), due to the widespread use of TDF replacing AZT and D4T as a part of the first-line treatment from 2014 in China following WHO recommendations [
34].
NNRTIs are notorious for rapidly triggering the emergence of drug-resistant HIV-1 variants. One NNRTI drug mutation can lead to multiple and high-level NNRTI-associated drug resistance [
35]. The prevalence of NNRTIs DRMs (91.91%) was higher than NRTIs (74.00%) and PIs (7.44%) in this study. This is due to the fact that NNRTIs have a low genetic barrier to resistance and a single major mutation was often leads to multiple and high-level resistance to NNRTIs drugs [
30,
35,
36], which is why the second-line therapy did not include NNRTIs. K103N/S (41.32%) was the most frequently observed resistance mutation in NNRTIs, followed by Y181C(27.83%) and G190A(26.21%), This is a consequence of the frequent use of NNRTI-based (EFV/NVP) first-line therapy for more than a decade in China, and these results are similar to the data from low- and middle-income countries [
27,
37‐
39]. K103N/S was strongly associated with failure against NVP and EVP. Y181C reduces susceptibility to NVP by > 50-fold and to EFV by 2-fold. G190A caused high-level resistance to NVP and intermediate resistance to EFV and could result in attenuation of the resistance that occurs with K103N alone, and G190A had a higher frequency (83.47%) of drug resistance in the HIV-1 CRF 01AE subtype in this study.
The overall rate of resistance to PIs was low (7.44%) in our study. This might be explained by the low PI coverage in China as a second-line regimen; and since PIs have a much higher barrier to resistance, patients receiving PI-based regimens are less likely to develop resistance. Our study results showed increasing trends in PIs mutations year by year from 2012 to 2017(from 0 to 8.31%), associated with the increasing use of second-line regimens in recent years. Therefore, it is advisable to include surveillance of PIs mutation in the coming years.
Our study results revealed that the patients’ low-level CD4 count (≤200 cells/mm3), clinical symptoms and bad medication compliance (missing drugs) could more easily lead to HIV-1 drug- resistance mutation among participants experiencing treatment failure. These phenomena are a reminder that monitoring resistant strains using CD4 counts and clinical symptoms in the course of ART as well as evaluating patients’ therapy compliance are very important. TDF has been commonly used as the first-line regimen since 2014, and LPV/r has always been used as the second-line regimen. The regimens containing TDF or LPV/r resulted in less drug resistance compared with AZT-contained ARVs because they were not used as much as AZT in Hunan Province. But this study result showed that the PI related mutation had an tendency to increase year by year from 2012 to 2017. This means that in the future we should pay more attention to second-line regimens drug resistance surveillance.
Several limitations of this research should be noted. First, in this study we focused on PI- and RT-associated mutations as the most commonly used ARV classes in Hunan province up to 2017. However, some patients have used integrase inhibitor drugs at their own expense in recent years, and the picture of integrase-associated DRMs remains blank and warrants further study. A second deficiency in our research is that a moderate proportion of subjects did not return for cd4 count or viral load monitoring, and the surveillance frequency of HIV virus load (VL) was relatively low (free VL testing once a year) due to local government policy, all of which may have reduced our power to detect associations between ADR and virologic outcome.
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