Prevalence of Transmitted HIV drug resistance in antiretroviral treatment naïve newly diagnosed individuals in China

To investigate the prevalence and temporal trend of transmitted drug resistance (TDR), a nationwide cross-sectional survey was conducted among 5627 ART naïve newly diagnosed HIV-infected individuals in 2015 in China. Totally 4704 partial pol sequences were obtained. Among them, the most common HIV-1 circulating recombinant form (CRF) or subtype was CRF01_AE (39.0%), followed by CRF07_BC (35.6%), CRF08_BC (8.9%), and subtype B (5.5%). TDR mutations were found in 3.6% of the cases, with 1.1% harboring TDR to protease inhibitors (PIs), 1.3% having TDR to nucleoside reverse transcriptase inhibitors (NRTIs), and 1.6% to non-nucleoside reverse transcriptase inhibitors (NNRTIs). No significant difference was found in the prevalence of TDR, as compared with the results of another nationwide survey performed among ART naïve HIV-infected people in between 2004 and 2005, except in the 16–25 year-old group. In addition, four drug-resistant transmission clusters were identified in phylogenetic trees, accounting for 6.2% (9/145) of the individuals with TDR. Although the rate of TDR remained relatively low in the past 10 years in China, surveillance is still needed to monitor the trend of TDR and to optimize the first-line regimens.

TDR in transmission routes, subtypes and age groups. The prevalence of TDR in heterosexuals, MSMs and IDUs was 4.1%, 3.1% and 1.7%, respectively. The prevalence of TDR of CRF01_AE, CRF07_BC, CRF08_BC and subtype B was 4.0%, 2.8%, 3.1% and 4.6%, respectively. There was no significant association   DRMs in the clusters were M46L (cluster A), K103N (cluster B), K101E (cluster C), and G190A (cluster D). The presence of drug-resistant strains within drug-resistant transmission clusters accounted for 6.2% (9/145) of the total drug-resistant strains in this study.

Drug resistance-associated transmission network analysis.
To explore the effect of TDR on viral transmission based on the transmission network, the 4704 sequences were divided into two groups: drug-resistant (167) and drug-sensitive (4537). The overall rate of clustering was 68.7%. The rate of clustering in drug-resistant strains was 60.5%, lower than in drug-sensitive strains (69.0%), P = 0.02. In addition, the degree was significantly lower in drug-resistant strains (P < 0.0003, Table 3).

Discussion
In this study, we investigated TDR among ART-naïve HIV-infected individuals in China who were newly diagnosed in 2015. The overall prevalence of TDR was 3.6%. Except a significant decreasing trend of TDR in the 16-25 year-old group, there was no significant difference in the rates of TDR in the whole study populations, or in any specific transmission routes and subtypes between the surveys conducted in 2004-2005 and 2015. In general,   21 . The main transmitted DRMs were different between China and countries in Europe and North America. The most frequent NRTI-associated DRM found in our study was M184V, whereas were T215rev (revertant mutation) and M41L in Europe and North America [18][19][20][22][23][24][25][26][27][28] . The most frequent PI-associated mutation in our study was M46I/L, which is related to the high prevalence of CRF01_AE in China 29 . The most frequent PI-associated mutations were L90M and M46I/L in Europe and North America [18][19][20][22][23][24][25][26][27][28] . Consistent with that of Europe and North America, the most frequent NNRTI-associated DRM in China was K103N/S. The differences of the main types of DRM can be explained by the different ART regimens used and subtype distribution between regions. Firstly, zidovudine (AZT), lamivudine (3TC), tenofovir (TDF), abacavir (ABC), efavirenz (EFV), nevirapine (NVP), ritonavir-boosted lopinavir (LPV/r) were provided through the NFATP in China. Not all PIs are available and none of the integrase strand transfer inhibitors (INSTIs) are provided in China compared to Europe and North America. Secondly, the major subtypes in China are CRF01_AE and CRF07_BC, whereas it is subtype B in Europe and North America.
Four clusters containing HIV strains sharing the same DRM were found in the present study. The presence of drug-resistant strains within transmission clusters accounted for only 6.2% (9/145). This revealed that the prevalence of TDR was not concentrated in study populations. This finding is consistent with the low prevalence of TDR in China.
The degree and rate of clustering in transmission networks were significantly lower in drug-resistant strains. This may be explained by the lower replication capacity of the resistant virus. The resistant virus was transmitted only approximately 20% as frequently as expected according to a previous study 30 .
As with other observational studies, our study has limitations. Firstly, a proportion of the studied individuals might not be recently infected, and the drug-resistant strains in plasma become minor quasi-species after a period of infection. The Sanger sequencing method may underestimate the prevalence of TDR. Secondly, there would be a biased sampling since only 10% of individuals diagnosed in the second quarter of 2015 were randomly enrolled in this study.
In conclusion, the overall prevalence of TDR among recently diagnosed individuals in China remained at a low level in the recent 10 years. The prevalence of TDR was not concentrated in 2015. We suggest that effective measures are still needed to strengthen monitoring and guide ART usage.

Methods
Study population. We conducted a cross-sectional survey in 31 provinces, autonomous regions and municipalities of mainland China. Inclusion criteria for the subjects (1) were over 16 years old, (2) were permanent residents, (3) were diagnosed as HIV seropositive from April 2015 to June 2015, and (4) were ART naïve. Individuals who met the criteria were stratified by random sampling. The sampling ratio of each province was determined by the average number of HIV-infected individuals reported in 2011-2013. For provinces with fewer reported cases, higher sampling ratios were used to assure statistical confidence. The sampling ratios for provinces with >2000, 1200-2000, 800-1200, 250-800, and <250 cases were 5%, 10%, 12.5%, 15% and 20%, respectively. All patients provided written informed consent for participation in this study.
To explore the changes of TDR in the recent 10-year period in China, the data of a cross-sectional survey conducted in 28 provinces from September 2004 to October 2005 was introduced into the analysis. The data contained sequences and basic information of 676 ART-naïve individuals 12 .

Identification of transmitted drug resistance. Whole blood samples were collected at local Centers for
Disease Control and Prevention (CDCs). For plasma samples, which were separated by centrifugation, HIV drug resistant test was carried out. The HIV pol region (protease 1-99 amino acids and reverse transcriptase 1-250 amino acids) was amplified by an in-house polymerase chain reaction protocol 31   Statistical analyses. Categorical variables were compared using the χ 2 test, Fisher exact tests, or logistic regression analysis. The statistical significance was defined as P < 0.05. All statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC).

Ethics statement. Institutional review board (IRB) approval was granted by National Center for AIDS/STD
Control and Prevention (NCAIDS), Chinese Center for Disease Control and Prevention (China CDC). All experimental protocols were approved by IRB at NCAIDS, China CDC, according to the international and Chinese ethical guidelines, and the methods were carried out in accordance with the approved guidelines. All patients were willing to provide informed consent for this research.