Despite substantial progress in antiretroviral (ARV) therapy (ART) coverage, HIV transmission and AIDS-related mortality remain unacceptably high. According to the Joint United Nations Programme on HIV/AIDS, an estimated 1.3 million new HIV infections and 630,000 AIDS-related deaths occurred worldwide in 2024, far exceeding the global targets set for 2025 [1]. The epidemic continues to disproportionately affect low- and middle-income countries and key populations [1,2]. Consequently, HIV infection remains a major public health concern requiring sustained and adaptive interventions [1,2].

The concept of “treatment as prevention” has become a cornerstone of global HIV control strategies. Early initiation of ART effectively suppresses viral replication, reduces onward transmission, and significantly improves survival and quality of life among people living with HIV [2]. Nevertheless, the effectiveness of treatment as prevention is increasingly threatened by the growing prevalence of pretreatment drug resistance (PDR) [3,4]. PDR refers to any drug-resistant virus detected in ARV drug–naive individuals initiating ART or individuals with previous ARV drug exposure initiating or reinitiating first-line ART [5]. PDR can compromise first-line ART regimens, increase the risk of virological failure, and impose substantial economic burdens on health care systems by necessitating the use of more expensive second-line therapies [3,4,6]. Therefore, continuous monitoring of PDR and identification of its associated factors are essential for optimizing treatment strategies and preventing the spread of resistant HIV strains.

In recent years, integrase strand transfer inhibitor (INSTI)–based regimens have become the preferred first-line treatment strategy worldwide [5]. Although the prevalence of INSTI resistance remains relatively low worldwide, the increasing use of INSTIs has been accompanied by the emergence of resistance-associated mutations in both treatment-experienced and, occasionally, treatment-naive populations [2,5,7]. In China, dolutegravir-based regimens have been progressively incorporated into national treatment guidelines; however, population-level surveillance data on INSTI resistance remain limited [8]. Long-acting ARV agents, including capsid inhibitors such as lenacapavir, are expanding the therapeutic landscape. Although resistance to capsid inhibitors remains uncommon, treatment-emergent capsid resistance mutations have been documented [9], highlighting the importance of proactive resistance surveillance as new drug classes are introduced into clinical practice.

Zhejiang Province, located in southeastern China, is an economically developed region characterized by high population mobility. Rapid socioeconomic development and population mobility may facilitate the frequent movement of HIV-positive individuals and ongoing viral recombination, potentially leading to a more complex HIV genotyping distribution and posing challenges to local prevention and control. Several studies have investigated HIV drug resistance in the Zhejiang Province. In Hangzhou, Zhang et al [10] reported a transmitted drug resistance (TDR) prevalence of 4.0% among newly diagnosed individuals in 2013, with CRF01_AE as the dominant subtype. In Ningbo, Hong et al [11] observed a TDR prevalence of 6.1% from 2018 to 2021, with K103N as the most frequent mutation and CRF07_BC as the major genotype. In Wenzhou, Zhang et al [12] reported a TDR prevalence of 5.7% between 2020 and 2023 and identified CRF08_BC as a subtype associated with a higher resistance risk. Recently, in Lishui, Li et al [13] observed a TDR prevalence of 12.1% from 2020 to 2023, with resistance predominantly to nonnucleoside reverse-transcriptase inhibitors (NNRTIs). These studies indicate that HIV-1 genetic diversity and drug resistance are present across Zhejiang but vary by city, period, and study population [10-13]. Therefore, a standardized, population-based study covering the entire province over a recent period is urgently needed to characterize HIV subtype distribution, estimate PDR prevalence using a consistent definition, and identify associated demographic, virological, and prophylaxis-related factors. Moreover, the expanding use of pre-exposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) has raised concerns about the potential selection of drug resistance when these interventions are initiated during undiagnosed HIV infection or when adherence is suboptimal [5]. Nonadherence could allow for breakthrough viremia and the emergence of resistant strains, which might then be transmitted or persist as pretreatment resistance. However, empirical evidence from real-world settings in China remains scarce. Against this background, a systematic analysis integrating viral genetic subtypes, PDR, and associated demographic and behavioral factors is urgently needed.

Therefore, this study aimed to characterize HIV subtype distribution, estimate the prevalence of PDR, and identify associated demographic, virological, and prophylaxis-related factors among newly diagnosed individuals in Zhejiang Province from 2022 to 2024.

This study provided the first representative description of the PDR trend along with HIV-1 variant dynamics over 3 years in Zhejiang Province. This study showed that the most prevalent HIV genotype was CRF07_BC (3913/8798, 44.5%), followed by CRF01_AE (2524/8798, 28.7%) and CRF08_BC (954/8798, 10.8%). Moreover, HIV-1 subtypes showed heterogeneity in distribution across age groups, sexes, transmission routes, and diagnosis years. PDR was prevalent in 5.7% (504/8798) of the study population but presenting an upward trend, with the highest prevalence among NNRTIs (351/8798, 4.0%). In addition, baseline CD4 cell count, diagnosis year, and HIV subtype were independently associated with PDR. Although exposure prophylaxis was not associated with overall PDR, it was significantly associated with pretreatment resistance to FTC and 3TC.

We observed that CRF07_BC and CRF01_AE remained the dominant circulating HIV subtypes, consistent with previous reports from Tianjin, Chongqing, Beijing, Shenzhen, and Xi’an [19-23]. Age-specific differences in subtype composition suggest that distinct transmission dynamics operate across generations. Sex- and transmission route–specific variations in subtype distribution further emphasize the role of social and behavioral structures in shaping HIV epidemics. The statistically significant variation in subtype distribution by diagnosis year suggests that the local HIV epidemic is dynamic rather than static, with ongoing introductions, expansions, or contractions of specific viral lineages. Simultaneously, the complexity and diversity of HIV genotypes may be attributed to population flow and the spread of resistant strains [6]. The observed demographic and temporal heterogeneity in subtype distribution underscores the need to integrate molecular epidemiology into routine surveillance frameworks.

We observed notable geographical heterogeneity in PDR prevalence across Zhejiang Province, with some municipalities (eg, Jinhua and Taizhou) showing PDR prevalence exceeding 10% in 2024 (40/377, 10.6% and 25/248, 10.1%, respectively). This uneven distribution may be explained by the higher rates of CRF01_AE (which is associated with PDR) and clustered transmission of resistant strains [24], which requires further research. PDR has been reported in different regions of China, with a prevalence of 4.8% in Shanghai [25], 23.1% in Shenzhen [19], 7.4% in Guangzhou [17], 10.5% in Chongqing [26], and 18.3% in Xi’an [20]. The varying prevalence of PDR reported in different studies is likely attributed to multifactorial causes, including distinct definition, local differences in HIV subtype distribution, transmission dynamics, and accessibility and quality of ART programs. Therefore, continuous surveillance is essential to detect early warning signals and guide timely policy responses [19].

Notably, higher CD4 cell counts at diagnosis were associated with increased PDR risk. This finding may reflect the predominance of TDR among individuals diagnosed at earlier stages of infection, who are more likely to harbor resistant strains acquired from treated partners and probably no adherence to treatment. In addition, the significantly higher PDR prevalence among individuals diagnosed in 2023 and 2024 suggests an accelerating spread of drug-resistant HIV strains in recent years. These findings underscore the need for comprehensive adherence support interventions, including peer educators, psychological counseling, and ensuring a stable drug supply, to prevent treatment failure and the subsequent spread of resistant strains. We also found that infection with the CRF01_AE subtype was independently associated with a higher risk of PDR compared with CRF07_BC. This observation aligns with previous studies reporting a higher burden of resistance mutations among CRF01_AE strains in other areas [20,27], which supports enhanced resistance surveillance in populations where CRF01_AE is prevalent as it may facilitate a better understanding of local resistance dynamics.

While prior PrEP or PEP use was not associated with overall PDR, drug-specific analyses revealed associations with pretreatment resistance to TDF, FTC, and 3TC—the backbone agents of most prophylaxis regimens. This selective pattern is biologically plausible and warrants careful interpretation. Initiation of PrEP or PEP during undiagnosed acute infection may expose replicating viruses to dual-NRTI pressure, facilitating selection of M184V-, M184I-, or K65R-related resistance pathways [28,29]. In addition, partial adherence to PrEP but not complete nonadherence may favor resistant variant emergence [28]. Alternatively, prophylaxis users may belong to higher-incidence sexual networks where resistant strains are more likely to circulate, reflecting confounding by transmission intensity rather than direct selection pressure. The magnitude of the association observed for TDF resistance was substantial, but the wide CI and inability to adjust for confounders due to the low event count (EPV<1) need to be confirmed in future studies.

Crucially, the potential emergence of drug-specific resistance must be weighed against the substantial epidemiological benefits of PrEP and PEP scale-up. Mathematical modeling and real-world implementation data consistently indicate that the potential risk of increased ARV drug resistance with high levels of PrEP scale-up is offset by decreased incident infections, which alleviates this key concern [30,31]. Consequently, the appropriate response to emerging resistance signals is not restriction of prophylaxis programs but refinement of delivery models, including enhanced diagnostic algorithms to exclude acute infection, adherence reinforcement, and integration of resistance surveillance into routine monitoring frameworks. Our findings, based on a limited number of exposed individuals, underscore the need for larger studies to precisely quantify the risk-benefit balance, but they do not challenge the continued rollout of PrEP and PEP as effective HIV prevention strategies.

Analysis of resistance mutation patterns revealed that M184V, K103N, and M46L were the most common mutations associated with NRTI, NNRTI, and PI resistance, respectively. These mutations have been widely reported in both national and international studies and are known to compromise the efficacy of traditional first-line regimens, particularly NNRTI-based therapies [6]. The M184V and M184I mutations confer high-level resistance to both 3TC and FTC [26]. The K103N mutation is one of the most prevalent NNRTI mutations observed in China. It is a persistent intrahost mutation that confers reduced susceptibility to EFV and NVP [11,19,20]. Moreover, 0.4% (33/8798) of individuals carried PI resistance related to the M46L mutation, which conferred TDR to nelfinavir [11]. Currently, 3TC and EFV are widely used free drugs in first-line ART regimens in China [20]. Long-term use of a limited number of drugs facilitated the development and spread of drug resistance. Therefore, persistent surveillance of drug-resistant strains is required to accurately track and update the prevalence rates of major resistance genes in our region.

Despite the transition toward INSTI-based treatment and the emergence of long-acting ARV agents, continuous surveillance of traditional NRTI and NNRTI resistance remains highly relevant. First, NRTIs continue to serve as the backbone of most first-line regimens, and mutations such as M184V, M184I, and K65R may reduce the effectiveness of companion drugs even when combined with high-barrier INSTIs. Second, historical NRTI and NNRTI resistance profiles remain critical for constructing effective second-line and salvage regimens as archived resistance mutations can influence future treatment strategies. Third, because TDF and FTC constitute the pharmacological foundation of most PrEP programs worldwide, surveillance of resistance to these agents is essential for maintaining the effectiveness of biomedical HIV prevention strategies. Therefore, resistance monitoring should evolve from a single drug class approach to an integrated surveillance framework encompassing INSTI, capsid inhibitor, and traditional reverse-transcriptase inhibitor resistance.

This study has some limitations. First, the cross-sectional design precludes causal inference. Second, behavioral and prophylaxis-related information was self-reported and may be subject to recall and social desirability biases. Third, the small number of individuals with PrEP or PEP use and the low number of drug-specific resistance events limited the precision of our estimates. Although we used Firth logistic regression to reduce bias, the results for FTC and 3TC should be considered exploratory, and the TDF findings could not be adjusted for confounding. Fourth, we used conventional Sanger sequencing, which detects only major drug-resistant variants. However, as a recent study demonstrated, deep sequencing uncovers many low-abundance resistance mutations missed by conventional methods [7]; thus, our PDR prevalence was likely underestimated. Therefore, future studies in Zhejiang Province should adopt next-generation sequencing to capture minority variants and enhance early warning systems. Furthermore, resistance testing for INSTIs was not conducted as part of this investigation. Given the rising use of INSTIs in China, it becomes essential to investigate the impact of HIV-1 drug resistance on INSTIs.

In conclusion, Zhejiang Province currently exhibits an increasing burden of PDR within a genetically diverse HIV-1 epidemic. The association between CRF01_AE infection and elevated PDR risk underscores subtype-specific vulnerability. Although exposure prophylaxis was not linked to overall resistance, its association with FTC- and 3TC-specific resistance highlights the need for enhanced testing strategies and targeted resistance surveillance. Sustained integration of molecular epidemiology, biomedical prevention, and optimized ART selection will be essential to maintain the long-term effectiveness of HIV control efforts.