HIV-1 CRF01_AE strain is associated with faster HIV/AIDS progression in Jiangsu Province, China

The goal of this study was to assess risk factors associated with HIV/AIDS progression. Between May 2007 and December 2014, 114 subjects were enrolled in Wuxi City and examined every 6 months. The pol gene sequence was amplified to ascertain the HIV-1 subtype. A Cox proportional hazards regression model was used to estimate the factors associated with HIV/AIDS progression. The median follow-up time for all 114 subjects was 26.70 months (IQR: 18.50–41.47), while the median progression time of the 38 progressed subjects was 24.80 months (IQR: 14.13–34.38). Overall, the CRF01_AE subtype was correlated with a significant risk of accelerated progression compared to non-CRF01_AE subtypes (HR = 3.14, 95%CI: 1.39–7.08, P = 0.006). In addition, a lower CD4 count (350–499) at baseline was associated with a risk of accelerated HIV/AIDS progression compared to higher CD4 count (≥500) (HR = 4.38, 95%CI: 1.95–9.82, P < 0.001). Furthermore, interaction analyses showed that HIV-1 subtypes interacted multiplicatively with transmission routes or CD4 count at baseline to contribute to HIV/AIDS progression (P = 0.023 and P < 0.001, respectively). In conclusion, the CRF01_AE subtype and a lower CD4 count at baseline tend to be associated with the faster progression of HIV/AIDS. Understanding the factors affecting HIV/AIDS progression is crucial for developing personalized management and clinical counselling strategies.

(95% CI 40.81-48.30), 18.31% (95% CI 14.71-22.17), 16.45% (95% CI 13.82-19.25), 2.55% (95% CI 1.56-3.73), and 0.37% (95% CI 0.11-0.72), respectively 13 . Notably, the median time from the estimated date of seroconversion to the development of AIDS in the Chinese HIV-1 population was shorter compared with that in other countries [14][15][16] . Thus, understanding the factors that affect HIV/AIDS progression is vitally important for personalized disease management and clinical counselling. However, very few studies have explored the factors associated with HIV/AIDS progression among HIV-1 infected individuals in China. The aim of this study is to explore the natural history and molecular epidemiology of HIV-1 and to evaluate risk factors associated with HIV/AIDS progression.

Results
Demographic characteristics of subjects. Following the application of strict screening criteria, a total of 114 subjects were included in the analysis (Fig. 1). The majority of subjects were male (88.6%) and were of Han ethnicity (99.1%). The age of the subjects ranged from 15-to 74-years-old at the time of HIV-1 infection. The median CD4 count at baseline was 502.00 (IQR: 404.75-614. 25). The median follow-up time was 26.70 months (IQR: 18.50-41.47), and the median progression time was 24.8 months (IQR: 14.13-34.38). The basic demographic data are summarized in Table 1. The distribution of HIV-1 subtypes in Wuxi was as follows: CRF01_AE (53.5%), B (14.9%), CRF07_BC/CRF08_BC (27.2%), and other subtypes (4.4%) (Fig. 2). Considering that the majority of the subjects were infected with CRF01_AE, we then separated the subjects into CRF01_AE and non-CRF01_AE groups. CRF01_AE was more prevalent in the MSM (men who have sex with men) group (65.7%, 44/67) than in the heterosexual group (36.2%, 17/47) (Fig. 2). There were significant differences between the subtypes with regard to gender, route of transmission, age at infection, and education status, all with P values less than 0.05 (Table 1).
Considering the significant differences in gender, route of transmission, age at infection and education between the CRF01_AE and non-CRF01_AE groups (P < 0.05), stratification analyses were performed to explore the association between the 4 factors above and HIV/AIDS progression. We found that HIV/AIDS progressed faster in male subjects infected with CRF01_AE compared to males infected with non-CRF01_AE subtypes (P = 0.046) (Fig. 3). In addition, MSM patients infected with CRF01_AE exhibited a significantly faster progression rate than MSM patients infected with non-CRF01_AE subtypes (P = 0.048) (Fig. 4), while no significant difference between HIV/AIDS progression in CRF01_AE and non-CRF01_AE subtypes was observed in heterosexuals (P = 0.405). Further, no significant association was found between HIV-1 subtype and progression in any of the age groups, nor for either of the education groups. In addition, since lower CD4 count at baseline was a risk factor for fast HIV/AIDS progression, in concordance with the well-known knowledge, we also performed the stratified analysis of CD4 count at baseline (divided into two groups: ≥500 and 350-499) to explore the association between HIV subtypes and progression and found that HIV/AIDS progressed borderline significantly faster in subjects with lower baseline CD4 count (350-499) infected with CRF01_AE compared to those with lower baseline CD4 count (350-499) infected with non-CRF01_AE subtypes (P = 0.055) (Fig. 5), however, no significant association was found between HIV-1 subtype and progression in the higher baseline CD4 count group (≥500).
Considering the significant difference in the route of disease transmission between the CRF01_AE and non-CRF01_AE groups, we investigated whether the effect of HIV-1 subtype on HIV/AIDS progression was modified by transmission route. Interaction analyses showed that HIV-1 subtypes interacted multiplicatively with transmission route to contribute to HIV/AIDS progression (interaction P = 0.023) ( Table 3). In addition, since lower CD4 count at baseline was a risk factor for fast HIV/AIDS progression, in concordance with the well-known knowledge, we also investigated the interaction of HIV-1 subtypes and CD4 count at baseline. As expected, interaction analyses showed that HIV-1 subtypes interacted multiplicatively with CD4 count at baseline to contribute to HIV/AIDS progression (interaction P < 0.001) ( Table 4).

Discussion
In the present study, in order to understand the natural history and disease progression of HIV-1, we studied HIV/ AIDS progression in subjects infected with different subtypes of HIV-1 in Wuxi, Jiangsu by measuring the length of time between the estimated date of seroconversion to the time of a decrease in CD4 count to <350 cells/μL. The subtype distribution in our study was 53.5% CRF01_AE, 14  might be on the rise worldwide, which have already been associated with faster disease progression as seen in Cuba 19 and Brazil 20 . Most importantly, our study demonstrated that CRF01_AE is associated with faster HIV/ AIDS progression, which is in agreement with a previous study conducted in China 14 . In another study, Ng et al. (2011) showed that CRF01_AE-infected seroconverters experienced a faster rate of CD4+ T-cell decline and thus required earlier cART initiation compared to non-CRF01_AE patients 21 . It remains unclear as to why CRF01_AE was associated with rapid CD4 decline. However, some studies have shown that a high proportion of X4 tropism is observed in the CRF01_AE subtype, and numerous studies have demonstrated that X4 tropism is associated with increased rates of CD4 count decline and progression to advanced immunosuppression [22][23][24] . In addition, it has been postulated that the decline of the host immune system after HIV-1 infection may allow X4 viruses to evolve and replicate freely 25 , which may explain the increased rate of disease progression in CRF01_AE patients.  In our study, we found that a lower CD4 count (350-499 cells/μL) at baseline was a risk factor for fast HIV/ AIDS progression, in concordance with previous studies 26 . CD4+ T cells are crucial in forming an immune response to foreign antigens, and they are the primary target cells of the HIV-1 virus. The continuous loss of CD4+ T cells could eventually result in the loss of ability to mount an effective immune response to pathogens, as well as the deaths of subjects in the terminal stage of HIV infection 27 .
The impact of transmission route on disease progression has long been a subject of debate 28 . In our study, no significant relationship between transmission route and HIV-1 progression was observed (P = 0.486); this finding is inconsistent with a previous study conducted in China by Li et al., which revealed that the transmission route in    To the best of our knowledge, although the data are not new, this is the first study of the natural course of HIV-1 infection in antiretroviral-naive subjects in Jiangsu, China. Interestingly, this is the first report of transmission routes interacting multiplicatively with HIV-1 subtypes to contribute to HIV/AIDS progression. Moreover, our study may have important implications for personalized disease management and clinical counselling in other Asian countries since the CRF01_AE strain is dominant in Asia. However, our study has limitations that need to be addressed. First, we defined CD4 < 350 cells/μL as a time-to-event endpoint in HIV/AIDS progression, and we could not exclude the possibility that the standards of baseline CD4 count and progression CD4 count were so approach that the judgment of HIV/AIDS progression might be not specific enough. Further study measuring viral load and the clinical symptoms of AIDS should be conducted. Second, in the current sample size (114 subjects), the statistical power is approximately 49.8% in detecting an effect size of 1.50 with an α-level of 0.05 for the association between subtype and HIV/AIDS progression. Nevertheless, we could not exclude the possibility that a small sample size may result in low statistical power. Therefore, further studies with larger sample sizes are needed to confirm our findings. Third, the differing rates of HIV/AIDS progression in HIV-infected individuals are complex and involve various factors, with viral and host factors being the most impactful; thus, further studies are needed to extensively explore additional factors (e.g., viral load and host genetics) 8,15,29,30 . Fourth, we inferred HIV-1 subtype by sequencing the pol region, which could result in the misclassification of recombinants as pure subtypes. It is also possible that some of the reported recombinants are actually HIV-1 dual/multiple infections, which have been associated with faster disease progression 31,32 . In addition, we only sequenced the pol region and have not sequenced env region at present, thus no data are available about the co-receptor use by HIV-1. Therefore, whole genome sequencing is the ideal method for inferring subtype and performing co-receptor use analysis. Finally, we observed that CRF01_AE is associated with faster HIV/AIDS progression. However, we were unable to elucidate the biological mechanisms underlying this phenomenon; thus, further functional evaluations are necessary.

Conclusions
This study revealed that subjects carrying the CRF01_AE subtype may suffer a faster loss of CD4 cells and earlier HIV/AIDS progression compared with those carrying a non-CRF01_AE subtype. Regular surveillance of HIV-1 subtypes and CD4 count would be beneficial in monitoring HIV/AIDS progression, as well as for improving personalized disease management and clinical counselling. Further studies incorporating subjects with different ethnic backgrounds combined with functional evaluations are warranted to verify the relationship between HIV-1 subtypes and HIV/AIDS progression.

Methods
Ethics Statement. This study was reviewed and approved by the Institutional Review Board at the Human Medical Research Ethics Committee of the Center for Disease Control and Prevention (CDC) in Wuxi, and written informed consent was obtained from every subject. The methods were carried out in accordance with the approved guidelines.

Study population. A total of 231 subjects were enrolled in the study between May 2007 and December 2014
in Wuxi with follow-up intervals of 6 months. CD4 count was determined at each visit. Follow-up started in May 2007, and the last follow-up was concluded in December 2014. Subjects were included if they met the following eligibility criteria: (1) CD4 count was higher than 350 cells/μL at baseline; (2) subject was antiretroviral-naive; (3) HIV was contracted through sexual infection; and (4) the subjects attended subsequent follow-ups from the estimated date of seroconversion (Fig. 1). HIV/AIDS progression was defined as a CD4 count less than 350 cells/μL. Ultimately, a total of 114 individuals were included in this study. Clinical data, including basic demographic information and CD4 counts, were collected from medical charts at the Wuxi CDC. Basic demographic information  Table 4. The interaction between HIV-1 subtypes and CD4 count at baseline on HIV/AIDS progression. a P value of interaction analysis between HIV-1 subtype and CD4 count at baseline on HIV/AIDS progression with adjustment for gender, age at infection and transmission route.
of the subjects included age at HIV-1 infection, gender, ethnicity, marital status, education status and route of HIV-1 transmission.
Viral RNA extraction, PCR amplification and sequencing. All blood samples were processed at the Shanghai Municipal CDC for population-based nucleotide sequencing of plasma HIV-1 RNA encoding regions of reverse transcriptase (RT) and protease (PR) genes. HIV-1 RNA was extracted from 200 µl of stored plasma specimen using the QIAmp Viral RNA Mini kit (Qiagen, Valencia, CA, USA) per the manufacturer's instructions. RT-PCR and nested polymerase chain (nPCR) amplification for the pol region were performed according to previously published methods 17,33 . Subtypes were determined using the Recombinant Identification Program (http:// www.hiv.lanl.gov/content/sequence/RIP/RIP.html).
Statistical analysis. Baseline characteristics were analysed by using the χ 2 test for categorical variables. The Cox proportional hazards regression model was used to estimate risk factors associated with the time of CD4 count decrease to <350 cells/μL. P < 0.05 was considered statistically significant. The SPSS 17.0 statistical package and STATA 13 were used for all analyses.