BY THE END OF 2007, there were approximately 700,000 persons living with HIV/AIDS in China, among whom an estimated 50,000 persons were newly infected during 2007.1 Among the estimated 700,000 HIV infected individuals, 11.0% were infected through male homosexual transmission.1 Furthermore, 12.2% new HIV cases in 2007 were also caused by male homosexual transmission.1 The proportion of HIV infection via male homosexual transmission was higher in the 2007 estimate than in the 2005 estimate (7.3%),2 implying that China is facing an increasing HIV-1 epidemic among men who have sex with men (MSM).3
MSM are highly vulnerable to HIV infection because of unprotected anal intercourse and multiple sexual partners. They play a bridging role in the transmission of HIV and other sexually transmitted diseases (STDs) to heterosexual women.3–5 Two previous cross-sectional studies demonstrated the increases of HIV prevalence among MSM population in Beijing, the capital of China, from 2004 to 20066 and in Harbin, the capital of Heilongjiang province, China, from 2002 to 2006.7 Furthermore, another study conducted in Bangkok, Thailand, also demonstrated a rapidly increasing HIV-1 prevalence among MSM from 17.3% in 2003 to 28.3% in 2005.8 The increase in HIV prevalence among MSM implies that more concern should be focused on MSM population.4,9
Jiangsu province that borders Shanghai on the West is located at the Yangtze River Delta, one of the most developed economic areas of China. As a developed area and an important transportation hub in eastern China, Jiangsu has a large migrant population that may increase the spread of HIV/AIDS.10 By the end of 2007, a total of 2336 HIV cases including 517 AIDS patients had been reported in Jiangsu since the first case was detected in 1991. However, a previous cross-sectional study (n = 144) conducted in 5 cities (including Nanjing and Yangzhou) of Jiangsu in 2003 showed that there was high prevalence of STDs, but no HIV prevalence among MSM.11 After 2 years, another study (n = 296) conducted in Nanjing showed that there was a HIV prevalence of 4.73% among MSM,12 suggesting a rapid increase in HIV-1 infection. Therefore, there is an urgent need to investigate the current status of HIV epidemic among MSM in Jiangsu.
Currently, a growing concern in China has focused on the survey of HIV prevalence and risk behaviors of MSM population.5 However, data about the molecular epidemiology of HIV-1 among MSM are very limited.5,9 To our knowledge, up to now, only 2 HIV-1 molecular epidemiology studies conducted in Beijing focused on MSM, and showed that HIV-1 subtype B dominated the AIDS epidemic among this population.13,14 Except in Beijing, there is no report on HIV-1 subtype characterization among MSM in other regions (including Hong Kong and Taiwan) of China.1,9 In Jiangsu, very preliminary and limited data showed that there were 5 HIV-1 subtypes, A, B (including Thai-B′), C, D, and CRF01_AE circulating among intravenous drug users (IDUs), commercial blood donors, and sex workers and their clients.15 However, the detailed subtype characterization of HIV circulating among MSM remains unknown. In the current study, we explored the molecular epidemiology of HIV-1 strains circulating among MSM in 2 cities of Jiangsu, and found a rapidly increasing HIV prevalence and complex HIV-1 subtypes circulating among this population, implying an urgent need of effective prevention strategies.
Materials and Methods
The baths are the one of the most favorite venues of MSM to have sex in Jiangsu province.12 With the help of the key informants, 2 baths were ascertained to conduct the cross-sectional surveys on local MSM population. We held a forum with key informants and the volunteers of MSM to discuss about how to prevent and control the spread of HIV/AIDS and STDs among MSM population, and to let them know the objective and design of our study before every survey. After obtaining approval of the medical ethics committee of Jiangsu Center for Disease Control and Prevention, and agreements from the owners/managers of baths, 2 surveys of MSM were conducted from September 15 to October 1, 2006 in Nanjing city, and from July 7 to September 9, 2007 in Yangzhou city, respectively (Fig. 1).
To better protect their privacy, MSM who agreed to participate in the study were asked to complete a short anonymous, self-administered questionnaire in a separate chat room after obtaining the written informed consents. At the same time, the health consultation related to the transmission of HIV/AIDS and STDs was supplied for them. All participants were assigned a unique study identification number, and were given a card that included the telephone numbers of our staff and voluntary counseling and testing clinic, by which they could know their own test results in 2 weeks and voluntary counseling and testing results at any time using their identification number.
Sample Collection and Laboratory Testing
Whole blood samples (about 5 mL) were collected from these MSM using sterile ethylenediaminetetraacetic acid tubes, and transferred to the laboratory for testing of HIV, HBV, HCV, and syphilis infection within 6 hours. All assays were performed in HIV Confirmatory Center Laboratory at Jiangsu Center for Disease Control and Prevention according to the manufacturer’s instructions. HIV infection was tested by enzyme immunoassay) kits (BioMerieux Ltd., France), and positive results were further confirmed by Western blot assay (Genelabs Diagnostics Pte Ltd., Singapore). The presence of hepatitis B surface antigen and anti-HCV antibody was detected by enzyme immunoassay kits (Shanghai Kehua Biotechnology Ltd., Shanghai, China). Syphilis infection was first detected by Treponema pallidum particle agglutination assay (TPPA; Fujirebio Diagnostics Inc., Tokyo, Japan), and then active infection was further tested by rapid plasma reagin (RPR, Shanghai Kehua Biotechnology Ltd, Shanghai, China). Double seropositive individuals determined by TPPA and RPR were referred to as syphilis infection in this study.
RNA Extraction and Amplification of HIV-1 Gene Fragments by RT-PCR
Separated plasma samples were stored at −80 °C. From July 2007 to March 2008, HIV-1 RNA was extracted from 300 μL plasma using MagAttract viral RNA M48 kit (Qiagen, Inc.) on Biorobot M48. HIV-1 segments (p17 and C2V3) were amplified using RT-nested PCR. The first PCR reactions were performed using SuperScriptTM III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen) with outer primer pair GAG-F2/GAG-E2 (p17) or ED5/ED12 (C2V3). The second PCR reactions were performed using TaKaRa ExTaq kit (TaKaRa Biotechnology Co. Ltd., Dalian, China) with inner primer pair c-gag/306 (p17) or E7-1/E8-1 (C2V3). The sequences of primers used in this study have been previously described in detail.14 Amplified PCR products were separated on an agarose gel, and purified with the QiaQuick gel extraction kit (Qiagen, Inc.). Purified products were subjected to direct DNA sequencing using an automated ABI 3730/3730xl DNA sequencer in Shanghai GeneCore BioTechnologies Co., Ltd.
Phylogenetic Analysis of HIV-1 Sequences
To avoid potential laboratory errors, all the nucleotide sequences obtained in this study were screened using online HIV BLAST Search tool (http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html) to search for sequence similarities to previously reported sequences in the HIV database. Nucleotide sequences were aligned with the reference strains and HIV-1 B′ strain RL42,16 using ClustalW program implemented in MEGA 4.0,17 and were further adjusted manually. The subtype references and B subtype sequences from other regions of China were retrieved from HIV-1 sequences database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Phylogenetic trees of p17 and C2V3 segments were constructed with MEGA 4.0 by using the neighbor-joining method.17 The stability of the nodes was assessed by using maximum likelihood with a bootstrap value of 1000 replications.
All statistical comparison between any 2 different groups in this study was conducted using two-sided Fisher exact test [GraphPad Prism version 4.03 (Windows demo)].
Nucleotide Sequence Accession Numbers
The GenBank accession numbers of the HIV-1 sequences reported in this article are EU676124-EU676148.
Prevalence of HIV, HBV, HCV, and Syphilis Among MSM in Nanjing and Yangzhou Cities
A total of 433 MSM, including 296 MSM from Nanjing and 137 MSM from Yangzhou, were recruited into this study (Fig. 1). Table 1 shows the prevalence of the 4 infectious diseases. Among Nanjing MSM, the prevalence of HIV-1 HIV, HBV, HCV, and syphilis infections was 4.7%, 10.8%, 1.0%, and 26.7%, respectively. Among Yangzhou MSM, the prevalence of 4 infections was 8.0% for HIV-1, 11.7% for HBV, 29.9% for syphilis, but no infection with HCV. Of 14 HIV-infected MSM in Nanjing, 8 (2.7%) and 1 (0.3%) were coinfected with syphilis and HBV, respectively. In Yangzhou, the numbers of HIV-1 coinfection with syphilis and HBV were 8 (5.8%) and 3 (2.2%), respectively. Interestingly, there was no HIV-1 coinfection with HCV among MSM regardless of whether they were in Nanjing or in Yangzhou, quite distinct from those among IDUs.18 The prevalence of HIV, HBV, HCV, syphilis, and their corresponding coinfections were not significantly different between Nanjing and Yangzhou cities (Table 1).
Jiangsu Has a Rapidly Increasing HIV Prevalence, Higher Than Other Regions of China
A cross-sectional study conducted in July 2003 showed no HIV infection among 144 MSM in Jiangsu.11 In comparison with the 2003 results, prevalence of both HIV (5.8%, 25/433) (P = 0.0013) and syphilis (27.7%, 120/433) (P ≤0.0001) among MSM was significantly higher in the current study (from September 2006 to July 2007) (Table 2). In contrast, there was no significant difference in HBV and HCV prevalence in Jiangsu between present studies and those conducted in 2003 (P >0.05) (Table 2). These results suggest that Jiangsu had a rapidly increasing HIV and syphilis prevalence from 2003 to the present. When comparing with other regions of China (including Beijing, Shanghai, Jinan, and Harbin) (Fig. 1),7,19–23 Jiangsu seemed to have significantly higher HIV (P ≤0.003) and syphilis (P <0.0001) prevalence among MSM than other regions (Table 3).
Prevalent HIV-1 Subtypes Among Jiangsu MSM
A total of 25 HIV-seropositive samples were detected among MSM in Jiangsu. From them, 14 gag fragments (p17 gene, about 616 bp) and 11 C2V3 fragments (about 505 bp) were amplified. However, 7 HIV-1 positive samples failed to amplify regardless of gag or C2V3 fragments, possibly because of low viral loads. Phylogenetic trees of 14 p17 and 11 C2V3 sequences are shown in Figures 2 A, B, respectively. Among p17 sequences, 35.7% (5/14) belonged to HIV-1 subtype B, 35.7% to CRF01_AE, and the remaining 28.6% (4/14) to CRF07_BC. For C2V3 sequences, the subtype distribution was 45.5% (5/11) for subtype B, 36.4% (4/11) for CRF01_AE, and the remaining 2 sequences (18.2%) for China BC recombinants. HIV-1 subtypes circulating among MSM in Jiangsu were significantly different from those in Beijing (gag sequences: 71.1% for B, 24.4% for CRF01, and 4.4% for CRF07; env sequences: 89.7% for B, 3.4% for CRF01, and 6.9% for CRF07) (P <0.05).14
HIV-1 B Subtypes Among Jiangsu MSM Have a Phylogenetic Origin From B Strains Circulating in Beijing, Different From Majority of Subtype B Strains in China
In the phylogenetic tree of p17 sequences, 1 HIV-1 subtype B strain (06JS-NJ173) clusters with B subtype reference sequences (including RL42, a China representative B′ strain). However, another 4 subtype B sequences (07JS-YZ67, 06JS-NJ231, 06JS-NJ245, and 06JS-NJ279) form an independent subcluster with a bootstrap support of 99%, separating from all subtype B reference sequences (Fig. 2A). In C2V3 tree, a similar independent B subtype subcluster was also observed in the 4 Jiangsu B strains (Fig. 2B). These results suggest that except strain 06JS-NJ173, all HIV-1 B subtypes isolated from Jiangsu MSM had an independent evolutionary origin.
To determine the evolutionary origin, another 2 phylogenetic trees of Jiangsu B sequences were constructed with 75 p17 and 66 C2V3 B sequences from other regions of China (including Beijing, Henan, Hebei, Hubei, Yunnan, Liaoning, and Heilongjiang), respectively (Fig. 3). In p17 tree, all HIV-1 B sequences from Jiangsu MSM except strain 06JS-NJ173 cluster closely with B sequences from Beijing,24 forming an independent evolutionary clade with a high bootstrap support of 94%, quite distinct from 71 other B sequences (including RL42) from other regions of China, which form another independent lineage (Fig. 3A). In C2V3 tree, Jiangsu B subtype subcluster clusters closely with 1 Beijing B sequence,25 and also forms an independent evolutionary clade together with another Beijing B sequence and 2 Heilongjiang B sequences (bootstrap value: 84%) (Fig. 3B). The Heilongjiang strain CNHLJM065058 seemed to be an earlier ancestor of this independent subtype B clade (Fig. 3B). The close phylogenetic relationship with Beijing and Heilongjiang B strains strongly suggests that distinct from the majority of subtype B′ strains circulating in other regions of China, HIV-1 B subtypes circulating among Jiangsu MSM were derived from B subtype circulating in Beijing,24,25 which might earlier originate from few strains circulating in Heilongjiang province (Fig. 3).
MSM play an important role in bridging the transmission of HIV and other STDs from their high-risk male sexual partners to their low-risk female partners.4,26 In this study, we found rapidly increasing prevalence of HIV and syphilis among MSM in Jiangsu from 0% and 6.9% in 2003 to 5.8% and 27.7% in 2006–2007 (Table 2). However, the prevalence of HBV, HCV, and their coinfections with HIV showed no significant difference between 2003 and 2006–2007 (Table 2). Previous studies demonstrated that increased risk of HIV infection was closely associated with increasing syphilis prevalence.5,19,21,22 The high and rapidly increasing syphilis prevalence might imply increasing risk behavior of unprotected anal sex among Jiangsu MSM, which resulted in the rapidly increasing HIV prevalence among MSM. In addition, it is noteworthy that Jiangsu had significantly higher syphilis prevalence among MSM than other regions of China (4.9%–13.5%) (Table 3). Therefore, it might seem easy to understand why Jiangsu had a significantly higher HIV prevalence among MSM than other regions (e.g., Beijing, Shanghai, Jinan, and Harbin) (0.7%–2.6%) (Table 3). In China, gay bars, saunas, and baths are the major places for MSM to meet. In this study, for convenience sampling, 433 MSM willing to participate were recruited only from 2 gay baths in 2 cities. The sampling strategy might result in a selection bias, and as a consequence, the results of our study might not be generalized to the total MSM population in Jiangsu. Therefore, current and future prevalence of STDs (including HIV and syphilis) in Jiangsu and other regions merit further investigation.
The phylogenetic analyses of HIV-1 p17 and C2V3 sequences showed that 3 subtypes including B, CRF01_AE, B/C intersubtype recombinants (especially CRF07_BC) were circulating among MSM in Jiangsu (Fig. 2). The subtype distribution among Jiangsu MSM was significantly different from that in Beijing, where HIV-1 subtype B (>71.1%) dominated among MSM.13,14 HIV-1 B/C recombinants (including CRF07_BC and CRF08_BC) originated from Yunnan, and often were detected among almost all IDUs in China (especially in Yunnan, Guangxi, and Xinjiang) (Fig. 1).27–29 The bootscan analyses of 14 sequences of 2.6 gag-RT regions from IDUs in Jiangsu showed that 90.9% (20/22) sequences belong to CRF07_BC (Guo H et al., unpublished data). Several studies had reported that few MSM in China were also IDUs7,23 and to obtain drug, few IDUs were also likely to trade sex for money18 (Guo H et al., unpublished data), which may increase the opportunity of HIV-1 transmission from IDUs to MSM via heterosexual transmissions (from female IDUs to MSM) or homosexual intercourses (from male IDUs to MSM).26 Therefore, B/C recombinants were likely to be introduced into MSM through injection drug use, which resulted in the emergence of high proportion (in p17: 28.6%; in C2V3: 18.2%) of B/C recombinants (especially CRF07_BC) among MSM in Jiangsu during last 3 years. About CRF01_AE, it might be introduced into MSM via heterosexual transmissions.
Further phylogenetic analyses showed that B subtype sequences isolated from Jiangsu MSM form a subcluster, closely clustering with B strains identified from Beijing and Heilongjiang during 2002 (Fig. 3).24,25 The close phylogenetic relationship between Jiangsu B strains and Beijing B strains suggests that the ancestor of majority of Jiangsu B viruses circulating among MSM was derived from Beijing and was introduced into Jiangsu after 2002. Given no HIV infection in the 2003 cross-sectional study, 25 HIV-positive individuals detected in current study should be newly infected after July 2003, consistent in time with the results of phylogenetic analyses. On the other hand, because MSM in Jiangsu and Beijing are geographically separated from each other, the introduction of B strains from Beijing into Jiangsu might be ascribed to a certain HIV-carrying migrant MSM. Therefore, the migrant population may be a potential reason for the rapid increase of HIV prevalence in Jiangsu. In addition, the large migrant population in Jiangsu may accelerate the introduction of geographically separated HIV-1 strains,10,30 which may increase the HIV-1 subtype diversity in Jiangsu and result in future further intersubtype recombination in this region.
Jiangsu borders Shanghai on the west, Shandong on the south, Anhui and Henan on the east, and Zhejiang on the north (Fig. 1). HIV-1 prevalence among MSM had been reported in its neighboring provinces, Shandong and Shanghai. However, in Zhejiang, especially in Henan and Anhui, where many former plasma donors were infected by HIV-1 subtype B′,24 the information of HIV-1 prevalence among MSM is still very limited. The rapidly increasing HIV prevalence and complex HIV-1 subtypes circulating among MSM in 2 cities of Jiangsu suggest that there is an urgent need to assess the true severity of HIV-1 infection and to investigate viral subtype characterization among MSM population in other cities of Jiangsu and its neighboring provinces, which will provide very valuable information for the development of effective prevention and intervention strategies.
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