Introduction
Since the first patient was diagnosed with acquired immunodeficiency syndrome (AIDS) in Beijing in 1985 [
1], HIV-1 has evolved rapidly in China over 30 years, with an increasing number of infected individuals and increased genotype complexity [
2,
3]. Sexual transmission has contributed greatly to the current HIV-1 epidemic in China. According to relevant reports, in 2017, the proportion of newly discovered HIV/AIDS cases in China through sexual transmission reached more than 90%, 20% of which were attributed to homosexual transmission among MSM [
2‐
7]. Tianjin is one of the four direct-controlled municipalities in China, the gateway to Beijing, an important trading port, and neighbors Beijing and Hebei, with a total population of over 15 million people. Sexual transmission is the historically predominant route of HIV infection in Tianjin. In recent years, MSM transmission, as opposed to heterosexual transmission, has resulted in a tenfold increase in the total number of infections, particularly among young men in Tianjin [
8]. The Tianjin Municipal Health and Family Planning Commission reported that MSM transmission cases accounted for 74.92% of all new diagnoses from January to October 2018, according to statistics from the AIDS prevention and control information management system. Phylogenetic analysis provides insight into the HIV transmission network structure. Typically, clusters are defined by the genetic distance between analyzed sequences and/or statistical support within the inferred tree [
3]. Characterizing populations and forming transmission networks allow for targeted interventions to individuals at risk [
4]. By identifying common clustering among MSM, we examined the HIV molecular transmission network and transmitted drug resistance mutations to determine the characteristics of the HIV epidemic in Tianjin at the provincial level to guide HIV prevention measures.
Discussion
Based on phylogenetic and demographic parameters, we analyzed nucleotide sequences for 436 newly diagnosed patients among MSM to track the characteristics of HIV-1 transmission networks. The results revealed an epidemic characterized by high heterogeneity in the subtypes and high prevalence of recombinant forms of infection (92.7%). From the results of our local MSM cohort, since 2014 CRF55_01B strains were discovered successively and the type and number of recombinant genotypes are increasing, leading the more complicated epidemic trends. Recombination is an important mechanism contributing to the genetic diversity of HIV-1 [
16]. Thus, an increasing number of circulating recombinant forms (CRFs) and URFs have been reported on a global scale [
17,
18]. A total of 102 HIV-1 CRFs are listed in the Los Alamos National Laboratory HIV database (
https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). The emergence of novel recombinant forms may easily occur via co-circulation and dual infection of multiple HIV-1 genotypes among MSM in Tianjin, such as subtype B, CRF01_AE, and CRF07_BC. The recombinant forms are increasing the complexity of the HIV-1 epidemic among the MSM cohort in Tianjin. Therefore, effective HIV-1 molecular epidemiologic investigations are needed to identify the transmission of potential HIV-1 recombinant forms in Tianjin, China [
9].
HIV transmission clusters are most often identified by phylogenetic analysis based on similarities in viral sequences [
19,
20]. Cluster inclusion thresholds tend to be ad hoc, and there is no widely accepted definition [
20]. Comparison of the results from several previous studies can be confounded by varying populations, sampling fraction, individual risk profiles, and varying methods used for cluster identification [
21]. Traditionally, statistical node support for the relationships in a phylogenetic tree is evaluated by bootstrapping [
22]. Different studies have used bootstraps ranging from 70 to 99% in combination with genetic distances of 1% ± 4.5%, more than 90% indicating strong support for a group [
23]. In a study of local and national HIV surveillance in the USA, a
pol genetic distance of two individuals of ≤ 1.5% implies a direct or indirect epidemiological linkage [
24]. Previous studies revealed
pol mean estimated evolutionary rates for CRF01_AE, CRF07_BC, and B of 2.54–2.97 × 10
–3, 1.71–2.03 × 10
–3, and 2.09 × 10
–3 substitutions/site/year in China [
5‐
7]. For this study, we used strict criteria to identify all clusters based on a mean genetic distance of ≤ 1.5% and bootstrap value ≥ 90%, considered the sampling fraction, methodology, and convenience of follow-up. In this analysis, we found that 25.2% of all newly diagnosed cases among MSMs were included in 36 transmission clusters. In the different sized clusters, the CRF55_01B, CRF01_AE, and CRF07_BC (6/18) viruses were mostly in small clusters, whereas B viruses were mostly in large clusters. The clustering characteristics of different subtypes may be related to strain variation and/or lack of linkages because of sample density [
5‐
7]. However, individuals with more linkages may have a higher transmission risk [
5‐
7]. Thus, intra-group concentrated transmission of different subtypes requires further analysis. From 2014 to 2018, the annual trends in the individuals clustered may be associated with current treatment strategies in China and/or lack of some lineages caused by sample selection bias. In 2014, the standard of free antiviral treatment for AIDS in China was adjusted from 350 to 500 CD4-cells/µL. In 2016, the standard was further adjusted to "Discovery is Treatment". Our study in Tianjin also revealed a decreased annual prevalence of DRMs in 2018 after an overall increasing from 2014 to 2017 (Table
2). Whether universal free access to medical care and ART for more patients can reduce further transmission should be evaluated in longer surveillance studies.
In China, the regimen composed of TDF, 3TC, and EFV is currently the most commonly used free first-line therapy [
25]. Our study highlights that DRMs affecting the efficacy of NNRTIs are the most common, followed by those of NRTIs and PIs, which is consistent with results of domestic and foreign studies [
25‐
27]. Related studies in the USA showed that the K103N mutation was the most common, and its generation and transmission were related to the failure of early antiviral therapy caused by patients' long-term and frequent use of the NNRTI drug efavirenz [
26]. In our study, K101E was the most frequently observed mutation in response to NNRTIs, followed by K103N. Whether this is related to region, race or the extensive application of NNRTI drugs and early antiviral failure in antiviral therapy in China should be further evaluated. However, our study showed that the transmission of viruses containing DRMs exhibited the significant increase in large clustered infections (Table
4), and one of the three strains harboring mutations responsible for drug resistance to NRTIs and NNRTIs were confirmed to be clustered in the genetic transmission networks. As early as 2007, a study by Art et al. showed that approximately half of transmitted resistance can be attributed to clustered infections [
28]. Because of the increase in the prevalence of drug resistance and emergence of multi-resistant mutant strains, DRM surveillance is necessary to prevent the spread of HIV.
Since the CCR5 blocker maraviroc was applied clinically for treating patients extensively harboring R5 viruses in Europe and America, studies have focused on HIV-1 tropism [
11]. Nevertheless, current HIV-1 co-receptor usage in China has not been fully characterized. Understanding the co-receptor usage of HIV strains is essential for assessing the candidacy of CCR5 antagonists for treating HIV infection in China [
29,
30]. Simultaneously binding to CD4 and two main co-receptors, CCR5 or CXCR4, is a necessary condition for HIV to infect target cells. Co-receptor selectivity is determined by genetic sequences within the HIV
env “V3” region, which is involved in co-receptor binding [
30]. HIV-1 variants are classified as R5- and X4-tropic viruses according to the ability to use CCR5 or CXCR4, respectively. The CCR5 antagonists, which inhibit HIV-1 binding on the CCR5-coreceptor, are only active on R5-tropic viruses, indicating tropism determination before prescription. Therefore, the determination of tropism is useful in clinical practice [
31]. Several studies have supported the prevalence of tropism associated with HIV disease progression [
32‐
34]. R5-tropic viruses are predominant in the early stages of HIV infection, because of preferentially selective transmission for the viruses as a biological bottleneck inherent to the genital mucosa [
34]. We found that the proportion of R5-tropic virus clustering is slightly higher than that of non-clustering without significant differences. In addition, several studies have shown that X4-tropism for CRF01_AE recombinant is associated with accelerated progression to AIDS [
32‐
34]. We observed a high prevalence of CRF01_AE and CRF55_01B X4 strains. CRF01_AE is the most prevalent in China and has contributed to 84% of HIV infections in Asia [
32‐
34]. Further transmission of X4-tropic CRF01_AE and its second-generation recombinant strains may impede treatment with CCR5 antagonists in the future. Various antagonists are urgently required for effective and targeted treatment in this situation.
By utilizing the information from the inferred molecular HIV transmission network, we combined the demographic, clinical, and molecular data and found that 33.6% of individuals with STDs appeared in the clusters. This is consistent with recent reports on the increasing coincidence of HIV with STDs and high-risk sexual behaviors [
35]. Seventeen of the 36 transmission clusters in the network contained 23 individuals with non-Tianjin permanent register. These individuals were primarily from northern Chinese provinces, including Hebei, Jilin, and Heilongjiang. Therefore, measures of the prevention and control of HIV transmission between Tianjin and major provinces are needed.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.