The human immunodeficiency virus affected more than 37 million individuals worldwide, and there were approximately 0.85 million people living with HIV/AIDS (PLWH) in China as of 2018. The number of PLWH in Sichuan has been increasing dramatically in recent years, and a total of 161,456 PLWH cases were reported in 2018. Liangshan Yi Autonomous Prefecture (Liangshan), the largest traditional settlement of the Yi minority people in China, has the highest prevalence of HIV. Butuo county in Liangshan is one of the areas with the most serious HIV epidemic in China (the prevalence of HIV was over 1% in recent years) (1–3). Effective interventions are needed to reduce HIV transmissions in this area.

Antiretroviral therapy (ART) is effective in suppressing HIV, preventing the disease progression, and secondary HIV transmission (4, 5). However, antiretroviral resistance remains to be one of the barriers to sustaining HIV suppression during ART (6, 7). According to the origin of drug-resistant virus strains in vivo, resistance can be divided into transmitted drug resistance (TDR) and acquired drug resistance (ADR). ADR is defined as drug resistance that develops when HIV mutations emerge due to viral replication in individuals receiving antiretroviral drugs (8). TDR occurs when previously uninfected individuals are infected with drug-resistant HIV strains, and TDR is detected in PLWH with no history of antiretroviral drug exposure (8). Severe TDR is likely to occur in non-recently contaminated patients (9, 10), and the policy of treating all PLWH decreases the risk of transmission of resistant strains. The diversity of HIV-1 subtypes and recombinants is thought to contribute to the TDR levels (11). TDR may not only increase the risk of virologic failure on ART but also lead to the spread of drug-resistant strains, bringing a challenge in eliminating HIV. Therefore, TDR should be an intensive focus in HIV surveillance to prevent the spread of drug-resistant strains.

Genetic transmission networks are mainly focused on similar viruses with similarities and connections. Genetic distance-based methods describe a putative transmission link to any pair of viral sequences that are within a predetermined genetic distance threshold (12–14). The genetic relatedness of HIV-1 could partly reflect the unobserved relationship between PLWH in combination with an epidemiologic investigation, and the spread of TDR among them, based on which the potential genetic transmission networks can be referred (15, 16). Analysis of the genetic transmission networks among newly diagnosed PLWH is useful to understand TDR and provide information for the first-line ART strategy (15, 16). However, there is a dearth of studies investigating molecular epidemiology of TDR in China, and no study was conducted among PLWH in Liangshan (the prevalence of HIV in five counties was over 1%).

To fill the knowledge gap, we analyzed the molecular epidemiology of TDR and genetic transmission networks and evaluated the TDR-associated mutations in genetic transmission networks among newly diagnosed PLWH identified by all population-based HIV screening methods in Liangshan. The results would be useful for understanding TDR and improving HIV surveillance and public health intervention activities especially in areas seriously affected by HIV.

When HIV replicates under a non-adapted or not well-taken ART, HIV drug resistance emerges. TDR of HIV strains is associated with a suboptimal virologic response to initial ART, and the TDR test is beneficial to guide the regimen selection to optimize the treatment combination and improve the efficiency of ART in a population with a high mortality rate (22). The prevalence of TDR among PLWH in Liangshan was 8.12%, which was higher than other epidemic areas in China (3.7–6.12%) (23–25) and higher than six sub-Sahara African countries with a similar period of ART roll-out as China (26). Since the stage of AIDS and history of frequent needle sharing were risk factors for TDR, strengthening the monitor of TDR among AIDS patients and HIV-infected drug users in Liangshan is perhaps important than ever before.

The TDR to NRTIs was not common in our study. The results are similar to the findings of other studies (27, 28). The main reason might be the use of a TDF- or AZT-based regimen in place of the D4T-based regimen that is associated with a higher adverse drug reaction and drug resistance (29). In the past few years, China's National Free Antiretroviral Treatment Program (NFATP) expanded the use of TDF according to the WHO guideline and gradually phased out D4T in favor of TDF for the first-line ART (30). TDF and initiated TDF-based regimens are associated with lower drug resistance and lower TDR, as newer ART agents are more effective and safer than older nucleoside reverse transcriptase inhibitor agents. The prevalence of TDR to current first- and second-line regimens was 0.91–6.30% and 1.67%, indicating a high sensitivity to the first-line regimen among most newly diagnosed PLWH. Therefore, even if the drug-resistant test cannot become a routine test, there is no need to expand the second-line treatment for PLWH. These findings are of great significance for both the clinical practice of HIV treatment and public health policy-making.

Our study found that the two NRTI mutations (M184MI/V and T215F/L) had a higher prevalence than other TDR mutations in this population. Since 2016, all newly diagnosed PLWH are required to receive ART in China, and the massive scale-up of ART that could not ensure easy access to well-tolerated antiretroviral drugs to any PLWHA has inevitably increased the threat of TDR (28). One point of concern is the free NRTI drug 3TC, which had been listed and widely used in the first- and second-line ART regimens for the past 20 years in China. It is reported that the M184MI/V mutation showed a high- and intermediate-level resistance to 3TC that reduces the replicative capacity of reverse transcriptase (31, 32). Although it is still debatable whether continued use of 3TC provides actual benefit, it is still lacking a fully active multitherapy in the presence of the M184V mutation, which would lead to a greater risk for virologic failure and may be related to higher TDR in newly diagnosed PLWH (33). Previous studies in China also observed a high percentage of M184 MI/V in ART-naive individuals across the country, indicating the importance of modifying the regimen accordingly (28). The resistance mutations to 3TC should be closely monitored to prevent the threat of TDR.

In the genetic transmission network analysis, some certain demographic and transmission characteristics are worthy of attention. First, a large number of transmission clusters were identified in the network, with 43.78% of the analyzed samples. This is not uncommon in HIV epidemics as a smaller population is often part of wide transmission networks (25, 34), suggesting that many individuals were infected with HIV in this area. Large clusters mostly comprising more than two sequences were dominant over small clusters, which is consistent with heterosexual contact being the main route of transmission in newly diagnosed PLWH. Second, the majority of PLWH in the clusters were from different villages or towns. Unlike other minority groups in China, the Yi minority in Liangshan still maintains cultural traditions and values that condone casual sex (3, 35, 36). Casual sex among people of the Yi minority usually occurs during social events/gatherings (wedding, marketing date, Yi festivals, etc.), which would not limit their sexual partners to their place of residence (35, 36). Third, 50% (18/36) of the recently infected individuals who were involved in the network were infected through heterosexual contact. Further effective and tailor-made programs are needed to reduce sexual transmission among sexually active individuals.

In terms of TDR-associated mutations in transmission clusters, 37.38% of individuals carrying TDR were involved in the network. Specifically, 16 transmission clusters that contained individuals carrying TDR had a transmission relationship with each other; the remaining 17 clusters, involving individuals carrying TDR in the network, were independent, suggesting the serious spread of TDR-associated mutations. K103N (42.5%, 17/40) were the most frequent TDR-associated mutations and were in five clusters in which they linked with those carrying the same mutations. K103N mutations might have been in circulation for a while and were unsuspectingly propagated onward in the community as new infections. This finding was also observed in other undeveloped (37) and developed settings (38, 39). Routine TDR testing is needed to reduce TDR-associated mutations.

The proportion of recent infections (within 130 days) among all newly diagnosed PLWH was only 2.73% in this area, which also deserves concern. The proportion of recent infections was lower than that in other areas with a high rate of HIV in China (40, 41) and those in other countries (40, 42), indicating that most of the newly diagnosed PLWH were not detected in time. HIV might be spread by many individuals unaware of their infection, leading to a widespread of HIV in this area. Early detection of HIV should be advocated in this area, so as to effectively control the spread and prevalence of HIV.

Several limitations should be highlighted in this study. First, this study was a cross-sectional survey; time-based sequences and cause-effect relationships among these variables cannot be established. Second, patients in this study were newly diagnosed, but we cannot confirm the time of initial infection, so it is hard to determine the time sequence in the genetic transmission networks. Third, to fully understand the determinants of the genetic transmission network, more PLWH in this area should be enrolled, rather than newly diagnosed PLWH. Fourth, our study was conducted in areas with a high HIV prevalence, so caution should be taken when generalizing the findings to other areas or contexts. Despite the aforementioned limitations, the main strength of this study is the use of samples collected from the HIV screening samples, which represents the area with a high rate of HIV in China.

To our knowledge, this is the first effort to investigate the prevalence and molecular epidemiology of TDR on the basis of whole-population screening. Our data suggest that a concentrated epidemic is characterized by a predominant HIV-1 CRF07_BC, a relatively high level of TDR, and many transmission clusters. The high prevalence of long-term HIV-1-infected individuals and a large proportion of individuals within the genetically transmitted networks suggest that HIV-1 may be propagated by HIV-1-infected individuals who were unaware of their HIV-1 status. Therefore, targeted HIV-1 prevention, early identification, and monitoring of resistance are warranted to reduce TDR and prevent HIV-1 transmission in areas with a high rate of HIV.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study protocol was approved by the Ethics Committee of Sichuan Center for Disease Prevention and Control. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

DY, BY, SY, PJ, and SL had taken a principal role in the conception of ideas, writing the protocol, developing methodologies, analyses and write-up of the article, and drafted the manuscript. ZW and PJ contributed to the proposal writing and made a critical revision to the paper and manuscript. PJ, SY, and ML contributed to the write-up of the study protocol and revised the paper. YL, LY, YH, LS, YZ, LA, MC, CZ, LL, and LZ supervised the study and edited the manuscript. ZW revised the paper. All authors read and approved the final manuscript.

This research was funded by the National Natural Science Foundation of China (81703279); Sichuan Science and Technology Program (2019YJ0148); National Major Science and Technology Projects of China: Study on the assessment technology and prediction model of AIDS epidemic in China (2017ZX10201101), Science and Technology Project of Sichuan Provincial Health Committee (20PJ121), and the International Institute of Spatial Lifecourse Epidemiology (ISLE). PJ and SY, Co-directors of the ISLE, thank the West China School of Public Health and West China Fourth Hospital in Sichuan University, the Chinese Center for Disease Control and Prevention, the Netherlands Organization for Scientific Research, and the Royal Netherlands Academy of Arts and Sciences for funding the ISLE and supporting ISLE's research activities.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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