HIV subtypes and molecular transmission characteristics among elderly HIV-infected individuals aged 50 and above: a cross-sectional study in Huzhou City, Eastern China


  • This study constructed propagation clusters within the HIV molecular network in HIV-infected individuals, and used univariate and multivariate logistic regression to analyse the associated factors of molecular transmission in HIV-infected older individuals aged 50 and above.

  • The study’s scope may be constrained by the omission of undiagnosed individuals and high-risk groups, potentially introducing biases in the epidemiological data of HIV-infected patients.

  • Epidemiological investigations have a time lag and cannot provide a dynamic analysis of the changes in transmission clusters.


AIDS is caused by HIV. Since its initial discovery in 1981, AIDS has become a significant global health challenge.1 2 The detrimental effects of AIDS extend beyond viral infections and encompass immune system disorders that weaken the body’s resistance to various diseases and substantially increase the risk of opportunistic infections and mortality.3 With advances in medical technology and the ageing of the population, the risk of HIV infection among individuals aged 50 and above has garnered increasing attention.4 5 The immune systems of older individuals are relatively weak and less equipped to combat HIV, resulting in accelerated disease progression and heightened susceptibility to other illnesses on HIV infection.6 Furthermore, older individuals may exhibit intolerance or poor compliance with drug treatment. Due to their physical condition and physiological changes associated with ageing, they may also be more prone to experiencing the side effects of medications.7 Additionally, AIDS exerts a significant impact on the psychological well-being and social lives of older individuals, fostering potential estrangement from family and society and augmenting their psychological burden.8

HIV is an RNA virus characterised by a highly variable genome that gives rise to a multitude of types worldwide.9 HIV-infected individuals are at risk of coinfection with multiple HIV subtypes, leading to the recombination of genes in host cells and the subsequent emergence of novel circulating recombinant forms (CRFs).10 Furthermore, a small fraction of the viral genome undergoes recombination events that give rise to unique recombinant forms.11 The presence of these diverse types and recombinant gene sequences contributes to the increased genetic diversity of HIV, thereby presenting a greater challenge to the prevention and control of AIDS.

Researchers have continuously developed new tools and techniques for the better prevention and control of HIV/AIDS. HIV molecular transmission network technology is an emerging approach in molecular epidemiology that is primarily grounded in evolutionary theory and sequence analysis; it entails comparing, classifying, tracking and analysing HIV gene sequences in different populations to investigate the transmission patterns and networks of HIV, aiming to enhance the understanding of the virus’s transmission pathways and patterns of spread.12 In this study, the distribution characteristics of HIV-1 epidemic strains from January 2019 to March 2023 were identified through systematic analysis of HIV-1 strain polymerase (pol) gene sequences from newly diagnosed individuals aged 50 and above with HIV/AIDS before antiviral treatment in Huzhou city, encompassing the residential areas of Wuxing District, Nanxun District, Deqing County, Changxing County and Anji County. Furthermore, precise interventions were conducted through the construction of a molecular transmission network to reduce the transmission risk of HIV, providing scientific evidence for preventing the formation of new transmission chains and serving as a reference for developing targeted prevention measures and treatment strategies.

Materials and methods


The participants of this study were defined as 899 newly diagnosed HIV-infected individuals of whom were enrolled in Huzhou City, Zhejiang Province, between January 2019 and March 2023. Blood samples of these individuals were collected before initiating antiviral therapy. A total of 731 blood samples were collected. Out of these, HIV sequences were successfully obtained from 673 individuals, including 274 who were older individuals aged 50 years and above. Anticoagulant-treated samples were obtained by collecting 10 mL of EDTA-treated whole blood during the initial follow-up CD4+T lymphocyte test. The blood samples were promptly inverted, mixed and the plasma was separated and stored at –80°C for future analysis. Subtype analysis was conducted using centralised monthly testing, yielding 274 samples with valid sequences. Basic epidemiological information was retrieved from the Chinese Centres for Disease Prevention and Control (CDC) Information System.


The pol gene was extracted from the amplified RNA in spare plasma and sequenced using bioinformatics software to assemble the qualified sequence. A systematic phylogenetic tree was then constructed for subtype and recombinant subtype determination. We aimed to analyse clusters of HIV molecular transmission and transmission-related drug resistance to characterise the subtype and molecular transmission of HIV-1 in Huzhou City. Sample sources included voluntary counselling and testing (VCT), healthcare provider-initiated HIV testing and counselling (PITC, including testing of prenatal and sexually transmitted infection clinic attendees), preoperative testing, testing of other clinic attendees, testing of voluntary blood donors, testing of detainees, spousal testing, special surveys, testing of entertainment venue personnel, premarital testing and antenatal and postnatal checkups.

RNA/DNA virus extraction

Reagent kits and instruments were purchased from Tianlong Biotechnology. After extracting the RNA template, reverse transcription-polymerase chain reaction (PCR) and nested-polymerase chain reaction (nest-PCR) were performed to amplify the pol gene (including the full-length protease and the first 300 amino acid residues of the reverse transcriptase) to a length of 1316 bp, using designed primers and amplification conditions.13 The second-round PCR products were subjected to 1% agarose gel electrophoresis and verified against a molecular weight marker. Subsequently, qualified products were sent to Shanghai Bojie Medical Technology for purification and sequencing.

Subtype analysis of sequences

The results of the sequencing feedback provided by the company were processed and organised using Sequencher V.5.4.6. Subsequently, the edited sequences were aligned with international reference strain sequences obtained from the US National Laboratory HIV sequence database using the BioEdit V.7.2.0 software. A neighbor-joining phylogenetic tree, MEGA V.6.0 software was utilised with a bootstrap value of 1000. Genetic distances between the sequences were determined. The initial identification of the subtype categories for each sample was performed based on the sample sequences and international reference strains present in the phylogenetic tree. The COMET online analysis tool ( was used to confirm and determine subtype classification.14

Molecular network propagation cluster analysis

Cytoscape V.3.6.0 software was utilised to construct propagation clusters within the HIV molecular network, specifically for sequences having genetic distances of 0.01 or lower. Each molecular cluster comprises a minimum of two samples.

Patient and public involvement


Statistical analysis

The data were organised and analysed using SPSS software (V.19.0). Categorical data were represented using frequency (percentage), while continuous data were presented using mean±SD. Logistic regression was used for both univariate and multivariate analyses. Statistical significance was set at a significance level of p<0.05.


General demographic characteristics of elderly participants

A total of 274 blood samples with valid HIV sequences of HIV-infected older individuals aged 50 and above were obtained. Among this participants, men accounted for 78.5% (215/274); married individuals accounted for 69.3% (90/274); those with at least a junior high school education accounted for 43.4% (119/274); farmers and migrant workers accounted for 58.0% (159/274) and individuals between the ages of 50 and 59 accounted for 58.4% (160/274), with the oldest being 82 years old. Transmission through heterosexual contact accounted for 83.2% (228/274) of infections, while transmission through homosexual contact accounted for 16.8% (46/274). Among the study participants, 44.9% (123/274) had initial CD4 counts below 200 cells/µL and 48.9% (134/274) had progressed to AIDS. Samples obtained through the PITC and VCT accounted for 26.6% (73/274) of the cases. The current addresses of the participants were as follows: 32.8% (90/274) were in the Wuxing District, 19.0% (52/274) were in the Nanxun District, 15.0% (41/274) were in Deqing County, 21.5% (59/274) were in Changxing County and 11.7% (32/274) were in Anji County. The HIV subtypes were CRF01_AE, CRF07_BC, CRF08_BC and other subtypes, accounting for 20.1% (55/274), 55.8% (153/274), 13.5% (37/274) and 10.6% (29/274), respectively.

Analysis of molecular propagation network clusters

Distribution of HIV subtypes

HIV sequences were obtained from all 274 examined cases, which led to the identification of 14 distinct HIV subtypes. Among these subtypes, CRF07_BC accounted for most cases (153 cases; 55.8%). Following the CRF07_BC subtype, the CRF01_AE subtype was the second most prevalent subtype (55 cases; 20.1%), whereas the CRF08_BC subtype was less prevalent (37 cases; 13.5%). The distribution of the HIV subtypes is shown in figure 1.

Figure 1
Figure 1

The HIV subtypes distribution of HIV-infected elderly individuals aged 50 and above. CRF, circulating recombinant forms.

Molecular network transmission clusters

A total of 673 HIV sequences were successfully obtained, including 274 who were older adults. The peak number of molecular clusters was achieved at a genetic distance threshold of 1.0%. In a sequence of 673 cases, 287 cases occur in clusters of molecular transmission network, resulting in a network participation rate of 42.64% (287/673). The molecular transmission network comprising 74 molecular clusters was constructed. The sizes of these clusters varied from 2 to 39 cases each. Two larger clusters were identified, one with 39 cases and another with 35 cases, both of which belonged to the CRF07_BC genotype. The two larger clusters both consisted of cases involving heterosexual transmission and homosexual transmission infections. In one cluster, there were 22 elderly individuals (56.41%, 22/39), while the other cluster comprised 31 elderly individuals (88.57%, 31/35). The elderly individuals in these clusters were primarily infected through heterosexual transmission, with rates of 81.81% (18/22) and 100% (31/31), respectively, and were predominantly male, with proportions of 77.27% (17/22) and 80.65% (25/31), respectively. A total of 150 older individuals aged 50 and above were included in the molecular transmission network, and the proportion of elderly individuals in clustered cases is 52.26% (150/287). In the molecular transmission network of HIV-1 in Huzhou City, the CRF07_BC subtype exhibits the highest enrolment rate among HIV-infected older individuals aged 50 and above. The molecular transmission network clusters are shown in figure 2.

Figure 2
Figure 2

The molecular transmission network diagram. CRF, circulating recombinant forms; MSM, men who had sex with men.

Characteristic analysis of clustered molecular transmission network cases

Among the 150 cases of the clustered molecular transmission network, male individuals accounted for 77.3% (116/150), married individuals accounted for 72.7% (109/150), individuals with primary school education or lower accounted for 59.3% (89/150) and farmers and migrant workers made up 58.0% (87/150). In the 50–59 age group, 50.0% (75/150) of cases were diagnosed. Regarding transmission routes, 86.0% (129/150) of the cases were infected via heterosexual transmission, while 14.0% (21/150) were transmitted through men who had sex with men (MSM). Additionally, 46.0% (69/150) had their first CD4 cell count less than 200 cells/µL, 48.7% (73/150) had disease progression to AIDS and 26.7% (40/150) of the samples were obtained from PITC and VCT. Regarding residential location, 38.0% (57/150), 20.7% (31/150), 10.7% (16/150), 22.6% (34/150) and 8.0% (12/150) were from Wuxing, Nanxun, Deqing, Changxing and Anji counties, respectively. The HIV subtypes of these 150 cases included CRF01_AE (16.7%), CRF07_BC (67.3%), CRF08_BC (10.7%) and others (5.3%). Univariate analysis revealed statistically significant differences (p<0.05) in age group, residential location and HIV subtype between clustered and unclustered cases.

Multivariate regression analysis of clustered molecular transmission network cases

Variables with a p value<0.2 in the univariate analysis were included in the multiple logistic regression analysis. The results indicated that the older age group (60–82 years) (adjusted OR (aOR): 1.801; 95% CI: 1.051 to 3.087) and the CRF07_BC subtype (aOR: 2.791; 95% CI: 1.277 to 6.102) were associated factors for the formation of clusters (ie, with transmission risk). Table 1 presents the results of the study.

Table 1

Univariate and multivariate regression analysis of clustered molecular transmission network cases


HIV, owing to its unique reverse transcription replication method, leads to high genetic variability. This genetic variability results in significant differences in the genotypic diversity of HIV strains across regions, populations and transmission modes. Additionally, different subtypes can affect virus virulence, spread, treatment and vaccine development.15 16 Furthermore, the findings of this study indicated that the main mode of transmission for the 274 participants was heterosexual transmission, which could be attributed to societal behaviour patterns, level of sexual health education and sexual habits. The study found that 44.9% of the participants’ CD4 counts were less than 200 cells/µL at the time of their first CD4 test, suggesting that AIDS had already progressed in its early stages without being easily detected. As the CD4 cell count continues to decline, the disease may progress to the late stage. Of all the participants, 48.9% progressed to AIDS, indicating that the disease progresses rapidly among individuals.

Early HIV diagnosis is essential in extending patients’ lifespan and enhancing their quality of life.17 18 The findings of this study revealed a significant percentage (48.9%) of individuals progressed to AIDS during the advanced stages. This data likely signifies a lack of awareness regarding AIDS among the elderly and a general deficiency in knowledge about HIV testing. It is imperative to heighten the understanding of AIDS risk among the elderly, reinforce sexual education and advocate for safe sexual practices to augment the early diagnosis rate of AIDS and mitigate the adverse effects of late diagnosis.

The findings of this study indicated that disease transmission is significantly influenced by the current residence. HIV cases were distributed among the individuals residing in Wuxing, Nanxun, Deqing, Changxing and Anji, as 32.8%, 19.0%, 15.0%, 21.5% and 11.7%, respectively. This distribution is likely associated with regional economic levels, lifestyle habits, sex education prevalence and distribution of medical resources. Different HIV subtypes, including CRF01_AE, CRF07_BC, CRF08_BC and other subtypes, accounted for 20.1%, 55.8%, 13.5% and 10.6% of the cases, respectively, indicating significant viral variation and the need for targeted prevention and treatment strategies.

Based on the univariate analysis, age, residential location and HIV subtype had a significant impact on case clustering. Furthermore, multiple logistic regression analysis revealed that the 60–82 year age groups and the CRF07_BC subtype were the primary factors influencing case clustering, with the potential for transmission. This suggests the need for specific epidemiological monitoring and interventions to effectively prevent and control this disease. Populations at high-risk should be targeted for these actions.

This study constructed an HIV molecular transmission network by analysing the genetic distance between sequences to assess the level of interconnection, which offers a more comprehensive approach than cluster analysis based on systematic phylogeny.19 The molecular transmission network both provides rapid calculations, and also caters to the needs of epidemiological analysis. Individuals with higher degrees in the network have a greater impact on the transmission of the virus, and those who are more likely to transmit the virus to others are considered super-spreaders.12 The HIV-1 molecular transmission network in Huzhou City, based on newly diagnosed HIV-infected older individuals aged 50 and above, comprised 74 molecular clusters, with the highest level of representation belonging to the CRF07_BC subtype. This study identified molecular clusters involved in both homosexual and heterosexual transmission. This indicates that HIV strains were transmitted through bisexual behaviour and interaction with heterosexual individuals among the older MSM individuals.20

This study has some limitations. First, it employs a retrospective cross-sectional molecular epidemiological study that does not include many individuals who have been infected but are yet to be diagnosed as well as potential high-risk individuals. Therefore, the epidemiological information on HIV-infected patients may be incomplete, resulting in potential distortions. Moreover, epidemiological investigations have a time lag and cannot provide a dynamic analysis of the changes in transmission clusters. Additionally, strengthened monitoring of the HIV molecular network and the integration of epidemiological information should be conducted in a timely manner to intervene in specific geographical areas and target populations. Furthermore, as a sample size calculation of this study was not conducted prior to data collection and analysis, participants for this study were recruited from Huzhou City between January 2019 and March 2023, it is essential to validate whether the sample size is adequate for obtaining precise results.


The presence of adults aged 60 years and older, along with the CRF07_BC subtype, was associated with the clustering of HIV transmission in Huzhou City. Strengthening HIV screening in key areas and target populations in Huzhou City and conducting molecular epidemiological investigations are necessary. Prompt treatment of undiagnosed individuals within the transmission network and implementation of targeted interventions for at-risk, uninfected individuals are crucial for preventing further HIV transmission.

Data availability statement

Data are available upon reasonable request. The data sets used and analysed during the current study are available from the corresponding author upon reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Huzhou Centre for Disease Control and Prevention (approval number: HZ2022012). Written informed consent was obtained from participants. Participants gave informed consent to participate in the study before taking part.


The authors would like to thank all participants for their valuable contributions to this study.

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