Prevalence of HIV-1 Drug Resistance, Distribution of Subtypes and Drug resistance-associated mutations among Treatment-experienced Individuals in Chengdu, Southwest China, 2014-2016

Background: The National Free Antiretroviral therapy (ART) Program in China has initiated to provide ART to HIV-1 patients, which may cause problems with drug resistance (DR). The number of HIV/AIDS patients in Chengdu ranks first in the national capital city. However, there is little data on the prevalence of HIV-1 DR in this region. Therefore, epidemiological surveillance was conducted on HIV-1 DR among patients receiving ART in Chengdu. Methods: From 2014 to 2016, HIV/AIDS patients (15 years and older) who had received first-line ART for at least six months were enrolled in this study. Demographic, behavioral information and medical history were recorded, and blood samples were collected for viral loads and immune cell count analyses. HIV-1 pol was amplified, sequenced, and analyzed for HIV-1 subtypes and drug resistance-associated mutations (DRMs). Results: A total of 13,782 individuals were enrolled and 653 cases were considered treatment failure after 6 months of ART. 481 (481/653) samples were amplified and sequenced successfully for subtypes and drug resistance analysis. Six subtypes were identified, among which CRF01_AE (54.3%) and CRF07_BC (41.6%) were the dominant subtypes, and CRF55_01B was detected in Chengdu for the first time. The overall prevalence of HIV-1 DR was 1.8%, with 1.2% to nucleoside reverse transcriptase inhibitors (NRTIs), 1.7% to non-NRTIs (NNRTIs) and 0.14% to protease inhibitors (PIs). The leading DRMs observed in the study were M184I/V (59.59%) against NRTIs and K103N (37.55%) against NNRTIs. Conclusions: This study focused on the HIV-1 molecular surveillance among treatment-experienced individuals in Chengdu. The overall prevalence of DR was relatively low among treated patients. These findings were believed to be contributed to an understanding of HIV-1 subtypes, DR prevalence and DRMs profiles in Chengdu, and


Introduction
and cryopreserved for analyses. Patients who provided written informed consent participated in the study. Demographic, behavioral information and medical history were recorded using a standard questionnaire.

Immune cells count and HIV-1 viral loads assay
To assess immune response, CD4 + T cells count was measured by flow cytometer BD FACSCount TM System (Becton Dickinson, Franklin Lakes, N. J., USA). HIV-1 RNA viral load (VL) was quantified with Abbott RealTime HIV-1 Amplification Reagent Kit (ABBOTT Molecular, Chicago, USA) and NUCLISENS EASYQ HIV-1 2.0 (BioMérieux, France) according to the manufacturer's instructions. VL more than 1000 copies/ml were considered as virologic failures and HIV-1 pol Gene were analyzed.
Amplification of HIV-1 pol gene HIV viral RNA was extracted from plasma using Abbott RealTime HIV-1 Amplification Reagent Kit and then was synthesized into cDNA by using AccessQuick™ RT-PCR System (Promega, Madison, Wisconsin, USA). The pol gene fragments, containing the entire protease gene and partial reverse transcriptase gene (codons 1±300) were amplified by nested PCR (2×Pfu PCR MasterMix, Tiangen, Beijing, China). Primers and cycling conditions were previously described [18]. The DNA fragments were identified by 1.0% agarose gel electrophoresis. Then the products were purified and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

Drug resistance-associated mutations analysis
The obtained nucleotide sequences were assembled, edited and aligned using ChromasProl.33 and Bioedit 7.0. HIV-1 subtyping was performed by constructing the HIV-1 pol phylogenetic tree (MEGA5.0). The sequences were then submitted to the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu) at Stanford University for the determination of DRMs. Drug resistance was divided into five levels: sensitive, potentially resistant, low resistant, moderate resistant and high resistant.

Statistical analysis
Statistical analysis was performed using SPSS Statistics version 22.0. Categorical variables were described in numbers and proportions. Possible associations of HIV-1 subtypes, HIV-1 drug resistance with demographic, exposure category and clinical variables were analyzed by using the Chi-square test or Fisher's exact test and logistic regression. All tests were two-sided with statistical significance at p<0.05.

Results
Demographic characteristics of study participants and the prevalence of  E138A/G/R/K/Q was found in this case, and was predicted to have low/potential resistance to RPV and ETR. PIs-associated mutations only resided in CRF01_AE, CRF07_BC. The distribution of mutations in the PIs coding region was significantly different in the two recombinant subtypes (P 0.05). One patient infected with CRF07_BC recombinant subtype showed M46I, I47A and I50V 3 primary mutations, which was predicted to be resistant to all PIs. The overall prevalence of DR studied subjects in 2014-2016 was 1.8% in Chengdu. The rate was relatively lower than that in other areas of China, such as Xinjiang, Yunnan and Jiangsu, and also lower than average rate of Sichuan [22,23,29,30]. However, the rate varies a lot from previous reports in Chengdu, which reported that 17% (27/159)  Although FTC and DDI were not used in clinical settings, significant cross-resistance was observed. The structure, mechanism and efficacy of FTC are similar to 3TC, and FTC was predicted to have the same TDR profiles with 3TC. So is DDI with ABC. Under the 3TCbased medication regimen, a high proportion of drug resistance was observed in 3TC, most of which showed high and moderate resistance. Besides, the number of cases resistant to AZT is the lowest, most of which were low and have potential resistance. Thus, of 3TC+AZT is the best choice in the current NRTIs regimen, which is consistent with other reports [34].

Discussion
In our study, M184I/V (59.59%) was the most prevalent mutation associated with NRTIs resistance in our study and was also frequently found in Europe, Africa and other regions in China [35,36]. It alone causes high resistance to 3TC and FTC, low resistance to ABC and potential resistance to DDI. K 65R is one of the mutations with broad-spectrum resistance, and was found in more than one-quarter of the cases. Nine mutations associated with DR were observed in large proportions and resistant to many NNRTIs. K101 E / H / P, Y181 C / V, and G190 A / E / K / Q / S / V are broad spectrum general mutations resistant to all NNRTIs. The L100I and M230L mutations (4.08%) had low incidence and moderate or High resistance to RPV and ETR but no resistance to the firstline drugs EFV and NVP. These two mutations were found in CRF_01AE, CRF_07BC and subtype C, and patients with these two mutations were undergoing 3TC+TDF+EFV/NVP, indicating that this regimen may be related to these mutations. Three primary mutations M46I, I47A and I50V resistant to PIs were detected in one patient, whose regimen was 3TC+AZT+NVP then switched to LPV/r + 3TC+TDF. This patient showed decreased treatment adherence, and thus resulted in reduced effectiveness of treatment, indicating that clinicians should pay attention to compliance education to avoid similar cases and the transmitting of "super" resistant strains.
Patients infected with the CRF01_AE are associated with DR. All 14 NRTIs-associated mutations and 16 NNRTIs-associated mutations were all found in CRF01_AE. Both of the two cases identified with CRF55_01B both show DR, suggesting that this newly discovered CRF may likely develop DRMs. NRTIs-associated DRMs M184I/V and K65R and NNRTIsassociated DRMs with extensively drug resistance K101E/H/P, V179I/D/E/T, Y181C/V and G190A/E/K/Q/S/V were detected in CRF55_01B. PIs-associated mutations were found in 97 cases, most of which were secondary mutations like L10I/V, A71I/T/V and K20I/R, so, there were relatively few cases of DR. In this study, PI-associated DR mutations between CRF01_AE and CRF07_BC, K20I/R and T74S were mainly found in CRF01_AE while A71I/T/V, Q58E and V82I were frequently observed in CRF07_BC, indicates that different mutations may vary among different subtypes.