【作者】 苏莹珍；

【导师】 俞守义； 陆林；

【作者基本信息】 南方医科大学 ， 流行病与卫生统计， 2011， 博士

【摘要】 研究目的：了解MSM人群对艾滋病预防相关知识及行为特点,HIV感染相关的因素及几种性传播疾病的流行特点,为有针对性地在全省开展艾滋病性病防制提供参考。研究云南省流行的HIV-1毒株的亚型及重组特征,对近年来新发感染率较高的MSM人群中流行的HIV-1毒株进行型特征及分子进化研究。研究方法：本研究采用调查问卷的方法对MSM人群性行为现状及其影响因素的现况调查。EpiData3.1双人录入,对数据进行逻辑纠错。对云南省2006年收集的HIV-1阳性样本进行以州市为单位分层随机抽样；应用巢式PCR分别对扩增HIV-1gag区和env区基因扩增,并将扩增产物测序,对测序结果进行分析整理后应用Mega、Simplot、Entropy、N-GlycoSite、VESPA、Wetcat等程序进行系统树构建、重组分析、氨基酸序列分析、N-糖基化位点分析、V3区特征及病毒辅助受体类型预测。使用SPSS15.0对数据进行统计分析。用非参数检验对不同组的各种特征的构成比进行比较。研究对象：昆明市最近6个月与同性发生过口交或肛交性行为,且同意接受问卷调查和HIV及其它几种传染性疾病血清学检测的成年男性。HIV阳性者纳入MSM HIV-1感染者进行分子特征分析。云南省2006年新检出的未经抗病毒治疗的HIV/AIDS病例为抽样总体,随机抽取样本进行研究。结果：共调查472例男男性接触者,同性恋272人,占57.6%；双性恋192人,占40.7%。年龄最小的18岁,最大69岁,平均年龄28.1±8.6岁,MSM人群年纪较轻,18-24岁及25-35岁的分别占43.2%（204人）和39.8%（188人）。未婚者共328人,占调查人数的69.5%；已婚有婚史及在婚者占19.1%（90/472）。受教育程度较高,高中或中专学历的共有148人,占调查人数的31.4%；大专及以上共有208人,占总调查人数的44.1%。该人群职业分布多元化,商业服务人员占35.4%,学生占16.1%,干部职员占12.7%。该人群收入主要介于1000元至2000元之间,占调查总人数的32.20%。调查对象汉族384人（81.4%）,其余包括白族、彝族、苗族等12个少数民族。该人群预防艾滋病相关知识知晓率较高,平均知晓率为94.9%,知晓率与调查对象学历相关,知晓率最高为大专及以上学历者（98.6%）,最低为小学及文盲73.7%。安全套使用率较低,最近一次与男性发生肛交时安全套使用率、6个月内每次均使用安全套比例分别为72.0%、47.5%。22.5%的人6个月内发生异性性行为,且与异性发生性行为时的安全套使用率低于发生同性性行为时。5.3%的人最近六个月内通过付钱的方式得到过男性性服务,其中83.3%的人每次都使用安全套；有4.6%的人为了钱提供过过男性性服务,仅有66.67%的人每次都使用安全套。MSM人群3.0%的人曾吸毒,但没有共用注射器现象。HIV、梅毒、HCV、HSV-2感染率分别为9.3%、6.6%、1.9%、16.3%；HIV及HSV-2的感染率较高。梅毒、HCV、HSV-2合并HIV感染率分别为29.0%、30%、9.1%,统计分析结果显示,感染梅毒或丙肝的MSM人群相对于没感染梅毒或丙肝的MSM人群更容易感染HIV病毒（p<0.05）。此外有61人在最近一年STI征兆,占调查人数的13%。影响HIV感染率的影响因素主要有：梅毒及丙肝感染状况、性取向、安全套使用频率、同性性行为商业性活动及职业教育状况等。290份样本来源于16个州市。研究对象平均年龄32±11岁,研究对象最小1岁,最大72岁；包括了静脉注射吸毒（IDU）、异性性接触感染者、母婴传播、MSM人群等各类人群,调查数据具有代表性。调查共发现13种亚型或重组型,其中CRF08_BC毒株占39.1%,是云南最主要的流行毒株,其次为CRF01_AE和CRF07_BC毒株,分别占22.4%和18.9%。C亚型和B亚型毒株校正后分别为5.9%和4.5%,在云南已经较少流行。新重组毒株包括BC、01_C、08₀1、C₀1、01_BC、07-01、B₀1、BC₀1为本次调查新发现,合计占9.2%,成为云南第四大流行毒株；重组分析证实云南新重组形式较为复杂,推测新重组毒株为原来暴发流行时的衍生毒株或者在后来漫长的流行过程中由原来的流行毒株重组而成。在IDU人群主要以CRF08_BC和CRF07_BC为主（79%）；CRF01_AE亚型主要在母婴传播感染者及异性接触传播感染者流行。云南HIV-1亚型在地区间的分布特征为：各州市发现的毒株亚型复杂,多样性更加丰富：全省有75%的州市（12/16）均发现有新重组毒株的流行,在西部地区德宏、丽江、怒江、保山和西双版纳所占比例较大,均超过了10%。三种主要亚型在不同性别、民族、年龄层中的分布没有显著差异。MSM人群中流行HIV-1毒株CRF01_AE所占比例最大,其次是CRF07_BC占22%,CRF08_BC仅占5%；MSM人群中HIV-1毒株亚型分布与其它人群相比存在显著差异。与其它传播途径进行系统进化树分析,结果发现而男男性行为人群在系统树上却明显聚在一起,该人群中流行的毒株出现隔离。Gag区基因片段平均遗传距离由大到小分别为B亚型、CRF01_AE、CRF07_BC、BC亚型以及CRF08_BC,推测B亚型是云南最早流行的毒株、其次是CRF01_AE,env区基因片段也得到同样的结论。MSM人群env区及gag区基因片段平均遗传距离均小于其他高危人群中流行毒株,说明MSM人群中HIV的流行时间晚于其他人群,或者说这些毒株是最近进入MSM人群中开始流行的。重组分析结果表明,新重组毒株与云南既往曾经流行的C、B、CRF08_BC, CRF07_BC、CRF01_AE同源,可能是原有毒株在云南的重组。env区基因片段遗传多样性分析表明,同性性传播潜在的PNGS数低于异性性接触人群和注射吸毒人群；CRF07_BC重组型毒株中,注射吸毒人群潜在PNGS频率高于同性传播及异性接触传播人群。V3-V4区序列特征性氨基酸分析结果表明,异性性传播者氨基酸多态性最丰富,其次是MSM人群,均高于IDU人群。云南流行的毒株V3环顶端四肽共有6种类型:GPGR、GPGH、GLGQ、GPGQ、GPGK和GQGR,其中95.0%均为GPGQ型。辅助受体预测结果显示,CRF07_BC、CRF08_BC以及B亚型主要利用CCR5作为辅助受体,在三种流行毒株中占90%以上,CRF01_AE亚型毒株60.9%被预测为能够使用CXCR4辅助受体,但预测的准确性尚需进一步验证。结论：MSM人群年轻化,是壮劳力及性活跃程度较高的人群,HIV感染的风险较大；MSM人群受教育程度普遍较高,但知识与行为分离,安全套的使用率较低,MSM普遍的多性伴、同性及异性性行为共存及吸毒现象等艾滋病相关的危险行为使该人群成为HIV由高危人群向一般人群扩散的“桥梁人群”。该人群中同性商业性行为的广泛存在会加剧艾滋病的流行。MSM人群医疗卫生资源利用率低,如果没有有效的干预措施,随着MSM人群HIV感染率的增加,MSM人群普遍存在的危险行为将加速中国艾滋病的流行；MSM人群HIV感染率较高,且与HSV-2、梅毒、丙肝及其它性传播疾病合并感染现象较普遍,艾滋病的防治同时加强性病及其它血播性疾病的防制。CRF01_AE在性接触传播人群中流行,应注意加强对该人群的干预以降低HIV-1的流行；由于新重组株主要在云南疫情最重的德宏和其邻近地区分布,且有进步向内地扩散的趋势,应加强对德宏邻近地区的防治,以免新型重组毒株流行范围扩大；MSM人群主要流行CRF01_AE,其次是CRF07_BC,如果得不到有效控制,则CRF01_AE和CRF07_BC在该人群大范围扩散,并与其他亚型毒株重组,通过该“桥梁人群”再次播散到普通人群。更多还原

【Abstract】 Objective We designed the study to reveal the characteristics of MSMs’ knowledge on AIDS prevention and their behaviors, factors related to HIV infection and the epidemic characteristics of several sexually transmitted diseases, and to provide evidence for better AIDS and STIs prevention in Yunnan province.Studying on characteristics of HIV-1 subtypes and recombinants circulated come from MSMs and common people in Yunnan province.Methods Characteristics of sexual behavior in MSMs and its influencing factors were investigated by questionnaire. Double input method was employed in EpiData3.1.HIV-1 strains were selected from store centre of Yunnan province by stratified random sampling. Gag and env gene in HIV-1 were amplified by nest PCR, and PCR products were sequenced. We analyzed all sequences by Mega, Simplot, Entropy, N-GlycoSite, VESPA, and Wetcat for phylogenetic tree, bootscanning analysis, amino acid sequences analysis, N-GlycoSite analysis, analysis of V3 loop, and coreceptor forecast.The distribution of HIV-1 subtypes were analyzed by SPSS15.0. Nonparametric tests were performed when analyzing the data. Data were analyzed by SPSS 15.0 and nonparametric tests were performed.Respondents Male adults living in Kunming who have experienced homosexual behavior （oral sex or anal sex） during the past 6 months, would like to participant the investigation and receive serological test for HIV and other infectious diseases. Serum or plasma samples were studied including 64 samples come from MSMs and untreated HIV/AIDS infectors which were new reported in 2006, in Yunnan province at random.Results A total of 472 MSMs aged 18 to 69 （28.1±8.6, mean±standard deviation） years were recruited, of which 272 were gays（57.6%）,192 were bisexuals （40.7%）. It was shown that the respondents are younger than the other high risk behavior people. The respondents who aged 18-24 and 25-35 accounted for 43.2% （204） and 39.8%（188） respectively.69.5%（328） of MSMs are unmarried and 19.1% of MSMs are marriage.31.4% of MSMs （148 cases） have received high school or higher education,208 persons graduated from college, which accounts to 44.1%. There are many kinds of occupations among MSMs,35.4% of which are service workers in commercial company,16.1% are students, and 12.7% are managers or staff.384 cases are Han, and minority includes 12 nationalities such as Bai, Yi, and Miao.The general rate for awareness of knowledge on HIV/AIDS prevention is 94.9%. Those who received college education have the highest awareness level （98.6%）, contrast to primary school （73.7%）.Low condom use rate among MSMs was reported, while this rate in last male anal sexual activity and 100% rate of condoms in the past 6 months are 72% and 47.5% respectively.22.5% of MSMs conduct heterosexual sexual behavior, and condom use rate is lower than those gays. During the last 6 months 5.3% of MSMs get homosexual services by paying and 83.3% of them use condom in all sexual activities.4.6% of them are MB （money boy）, while 66.7% of them conduct 100% condom using.3% of MSMs were drug users, and no one is sharing of syringes.Prevalence rate of HIV, syphilis, HCV and HSV-2 infection is 9.3%,6.6%,1.9% and 16.3% respectively. The results also show that there is high prevalence of HIV and HSV-2 among MSMS. The rate of HIV co-infected with Syphilis, HCV and HSV-2 are 29%,30% and 9.1%. MSM co-infected syphilis or hepatitis C is easier to infect HIV virus （p<0.05）. In addition, there were 61 people （13%） who report they got STI symptoms during the last year. The factors associated with HIV infection in MSMs are:the rate of infection with syphilis or HCV, sexual preference,100% of condom use, homosexual conduct commercial activities and vocational educational background.290 samples of HIV-I infector come from 16 prefectures in Yunnan,whose mean age was 32±11 （from age 1to age 72） years. The transmitting ways of HIV include intravenous drug user （IDU）, heterosexual transmission, mother to child （MTC）, and MSM （men who have sex with men）. The data are of representative Yunnan province.There are 13 subtypes or recombinants of HIV-1 in Yunnan,and CRF08_BC is the most popular subtype accounted for 39.1%, and followed by CRF01_AE and CRF07_BC which accounted for 22.4% and 18.9% respectively. Subtype C and B are account for 5.9% and 4.5% respectively after correction,which were less popular in Yunnan. New recombinants of HIV-1 strain were founded including:BC,01_C, 08₀1, C₀1,01_BC,0701, B₀1, and BC₀1. All of them accounted for 9.2%, and they became the fourth popular strains in Yunnan. Simplot analysis confirmed that new recombinant form become more complex.We concluded that new recombinants are derived of the HIV-1 outbreak in Yunnan, or recombinated in long time transmission in Yunnan.CRF08_BC and CRF07_BC were dominated（79%） in the IDUs; While the strains of CRF01_AE are mainly prevalent in MTCs and people who have heterosexual contact experience. Distribution characteristics of HIV-1 subtypes in different prefectures of Yunnan Province were as follows:The recombinants become more and more complex and diversity in many of prefectures; There were 12 prefectures （75%,12/16） that were found new recombinants in the states and city in the province （twelve-sixteenths prefectures）, especially in the western areas of Yunnan, such as Dehong, Lijiang, Nujiang, Xishuangbanna, and Baoshan, accounted for more than 10% totally. While the HIV-1 subtypes of distribution was no significant differences in gender, ethnicity, and age.The subtype of CRF01_AE strains was the dominated subtype in MSMs, and followed by CRF07_BC accounted for 22%,and CRF08_BC only accounted for 5%. The distribution of subtypes was difference among people with different transmited ways significantly. The phylogeny tree showed that the trains come from MSMs clustered together, and means that the HIV-1 strains were segregated from other population.The average genetic distances of gag gene indicated as follows:subtype B, CRF01_AE, CRF07_BC, BC, and CRF08_BC. We infer that B subtype is earliest strain transmitted into Yunnan, and followed by CRF01_AE. The genetic analysis of env gene also can get same conclusion. The average genetic distances of MSMs of env and gag are lewer than other high risk population. It seems to show that the strains of HIV-1 dominate in MSMs recently.The recombination analysis showed the homology of nucleotide between new recombinants and C, B, CRF08_BC, CRF07_BC, and CRF01_AE strains which had been dominated in Yunnan in the past 20 years. We could infer that it may be the recombination of the past epidemic strains in Yunnan. Env sequences genetic diversity analysis showed that PNGS of homosexual were more than heterosexual contact population and IDUs. The amino acids characteristic of V3-V4 sequence showed there is more diversity of amino acids among heterosexual transmission people, followed by the MSMs, then IDUs.There are 6 types of four peptides in V3 loop:GPGR, GPGH, GLGQ, GPGQ, GPGK and GQGR, and GPGQ account for 95.0%. Major strains of CRF07_BC, CRF08_BC and B may use CCR5 to entry CD4 T lymphocyte, and account for more than 90%. It was about 60.9% of CRF01_AE strains could use CXCR4, but it need to be research further.Conclusions MSMs are young strong labours with high risk of HIV infection. Most of them have good education in general, however, the good awareness of knowledge on HIV and their behavior were separated because of the unprotected sexual activities. The condom use rate is still low. Unprotected anal sex, multiple sex partners, and bisexual behaviors were common in this group. Multiplesexual partners and other high risk behaviors while having sex with men or women among MSMs represents a unique risk group within the population of MSM that may bridged the HIV transmission between HIV high risk group and common people. Few MSMs take good use of health resources. If there are no effective intervention measures, the increased HIV prevalence in MSMs would aggravate AIDS epidemic in China. High prevalence of HIV among MSMs, co-infected with HSV-2, syphilis, hepatitis C and other sexually transmitted diseases are common among MSMs. Other STDs prevention and control should be strengthened in AIDS prevention.The strains of CRF01_AE mainly circulate in such population experienced sexual contacting. We should take good intervention measures to reduce the prevalence of HIV-1 in such high risk of activity. Because of new recombinants distribute in western of Yunnan where lies to Dehong with serious HIV epidemic; and there are some prefectures far away from frontier that have been found new strains of recombination. So government should focus on Dehong and other prefectures nearby to avoid new recombinants spread further. In MSMs, the main circulating strains are CRF01_AE, followed by CRF07_BC. If it is not controlled well, these two strains would recombinate in them. The strains of CRF01_AE, CRF07_BC and new recombinants would widespread into the general people by the "bridge crowd"更多还原