中国部分地区未经治疗HIV-1 感染者耐药基因研究
文献类型：学位论文
| 作者 | 涂源泉 |
| 学位类别 | 博士 |
| 答辩日期 | 2008-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 ; 蒋岩 |
| 关键词 | 未经治疗 HIV-1 耐药 亚型 |
| 其他题名 | HIV-1 genotypic drug resistance among treatment-naïve patients in China |
| 学位专业 | 动物学 |
| 中文摘要 | 高效抗逆转录病毒治疗(HAART)的应用，极大的降低了AIDS发病率和死亡率，延长了HIV感染者的生命。但HIV耐药在很大程度上影响了HAART的疗效，耐药株的产生成为影响抗病毒治疗效果的主要因素。欧洲、美国的耐药监测技术规范均推荐在新感染未经抗病毒药物治疗的患者中进行原发耐药检测。我国政府于2003年底出台了艾滋病治疗的“四免一关怀”政策，陆续在全国范围内开展了大规模的免费抗病毒治疗，监测我国未经抗病毒药物治疗HIV-1感染者中的耐药情况可以为制定合理的用药方案和减少耐药毒株出现提供科学依据。 根据世界卫生组织(WHO)的“HIV耐药监测指南”，无偿献血者中的HIV-1感染者，可以认定为HIV新诊断未治疗人群。分析了云南无偿献血者的血浆和外周血单核细胞(PBMC)，研究云南无偿献血人群的耐药状况。 已有实验室血清学方法识别HIV-1新近感染和长期感染，用BED-CEIA方法，在河南、安徽、山西自愿咨询检测(VCT)人群中检出新近感染人群，进行耐药基因研究, 对照研究了部分长期感染人群。 样品提取核酸后，巢式聚合酶链反应(nested-PCR)扩增pol基因区(含蛋白酶区1～99氨基酸全长和逆转录酶区1～242氨基酸)。PCR产物双脱氧法测序，所得序列与洛斯阿拉莫斯HIV核酸序列库(Los Alamos HIV Database)标准株构建系统进化树分析亚型；用斯坦福大学耐药数据库(Standford HIV Drug Resistance Database)分析耐药。 研究发现，云南省2005～2006年无偿献血者中，有52例为HIV-1阳性，其中49例血浆和相应的PBMC样品病毒基因扩增成功。序列分析表明，HIV病毒的亚型分布为CRF08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%)和B (4.1%)；所有样品均未发现蛋白酶抑制剂（PI）耐药基因位点主要突变，只在6例(11.7%)样品中发现7例次PI次要耐药位点突变；另外，在9例(18.4%)样品中发现10例次核苷类逆转录酶抑制剂(NRTI) 耐药突变，1例(2.0%)发生非核苷类逆转录酶抑制剂(NNRTI) 耐药突变；针对具体药物PI/NRTI/NNRTI均只有1例有潜在的低度耐药，临床仍对药物敏感。PBMC和血浆的病毒耐药没有显著差异。 从河南、安徽、山西27个VCT检测点2006～2007年采集的10310例样品中，通过WB和BED-CEIA检测出新近感染人群63例，分析成功50例血浆样品；河南VCT长期感染样品中随机抽样，分析成功19例样品。分析成功的69例VCT样品中，HIV病毒株的亚型分布分别为B’ (95.7%)，CRF01_AE(2.9%)和C(1.4%)。上诉样品均未检出PI主要耐药相关突变，只在26例(37.7%)样品中存在27例次PI次要耐药相关突变；3例(4.3%)样品出现6例次NRTI 耐药相关突变，7例(10.1%)样品出现8例次NNRTI 耐药相关突变。通过与斯坦福大学耐药数据库比对，没有发现针对PI类药物的临床耐药；但有2例(2.8%)针对NRTI类药物耐药，1例有M184V突变导致对拉米夫定(3TC)和氟代拉米夫定(FTC)高度耐药；1例样品存在T215Y、M41L、L210W三重突变位点，对阿巴卡韦(ABC)、去羟肌苷(ddI)和坦那夫韦(TDF)中度耐药，对齐多夫定(AZT)和司他夫定(d4T)高度耐药；针对NNRTI类药物，有3例(4.3%)毒株有耐药，1例有K103N突变导致对奈韦拉平(NVP)、地拉韦啶(DLV)和依菲韦伦(EFV)的高度耐药；1例有Y188L突变导致对NVP和EFV的高度耐药；1例存在K101E和G190A双重突变，导致对NVP的高度耐药，对DLV、EFV和依曲韦林(ETR)中度耐药。 比较长期感染和新近感染者之间的亚型和耐药，未发现显著差异。 研究结果表明，云南、河南和安徽未经治疗HIV-1感染者中耐药处于低流行状态。亚型分布云南无偿献血者以CRF_BC为主，河南、安徽VCT人群以B’为主。应持续在未经治疗人群中进行耐药监测。 |
| 英文摘要 | The widespread use of highly active antiretroviral therapy (HAART) has led to significant reductions in morbidity and mortality and prolonged life of the person with human immunodeficiency virus (HIV) infection. However, the benefits of HAART have been largely compromised by the development of drug resistance. Widespread dissemination of drug-resistant HIV-1 variants has the serious potential to limit therapeutic effects. U.S and European Drug Resistance Guidelines recommends HIV genotypic drug resistance testing for treatment-naïve persons. Under the “Four Frees and One Care” policy announced in 2003, China’s nationwide free ART has increased. Surveillance on transmission of drug resistance among treatment-naïve HIV-1 infections may guide the selection of treatment regiments and minimize the emergence and transmission of HIV drug resistance variants. According to WHO “Guidelines for surveillance of HIV drug resistance”, HIV-1 infected volunteer blood donors are persons newly diagnosed with HIV and not previously exposed to HIV drugs. Peripheral blood mononuclear cell (PBMC) and plasma samples from Yunnan volunteer blood donors were assayed to understand the drug resistance profiles in Yunnan. There are laboratory-based methods to distinguish recent HIV-1 infection from long-term infection. Recent infected population was distinguished by BED-CEIA assay from Volunteer Counseling and Testing (VCT) population in Henan, Anhui and Shanxi Province. Genotypic drug resistances were characterized between the recent and some long-term infected population. The pol genes (including entire protease codons and 1~242 of the reverse transcriptase codons) were amplified by nested-PCR from plasma RNA and PBMC DNA. The PCR products were sequenced using dideoxynucleoside arrays. Phylogenetic analysis was performed using a set of reference sequences available at the Los Alamos Database to determine subtypes. Edited sequences were analyzed with Stanford HIVdb Drug Resistance Database and drug resistance mutations were determined to particular anti-HIV drugs. From 2005 to 2006, there were 52 HIV-1-positive blood samples from volunteer blood-donors were collected in Yunnan. There were 49 paired samples were successfully analyzed, the circulating strains were CRF08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%) and B (4.1%). No protease inhibitors (PI) major resistance mutation was detected. Six samples (11.7%) displayed 7 PI minor resistance mutations. Nine samples (18.4%) displayed 10 nucleoside reverse transcriptase inhibitors (NRTI) DRMs, and resistance mutation to non-nucleoside reverse transcriptase inhibitors (NNRTI) was present in 1 sample (2.0%). There was just 1 sample (2.0%) in each of the PI/NRTI/NNRTI drugs inferred drug resistance on potential low-level susceptibility. They are still clinically susceptible to the drugs. Difference of the subtypes and drug resistance were not observed between PBMC and plasma. Western Blotting (WB) and BED-CEIA were used to identify 63 recently-infected samples among 10,310 enrolled samples (from 27 VCT sites in Henan, Anhui and Shanxi Provinces in 2006-2007). Fifty recently-infected samples were successfully analyzed. Nineteen samples were successfully analyzed as control by random sampling from Henan long-term infected patients. The circulating strains were B’ (95.7%)，CRF01_AE(2.9%) and C(1.4%). No protease inhibitors (PI) major resistance mutation was detected，26 samples (37.7%) displayed 27 PI minor resistance mutations, 3 samples (4.3%) displayed 6 NRTI DRMs, 7 samples (10.1%) presented 8 NNRTI DRMs. No inferred PI drug resistance was verified. There were 2 samples (2.8%) confer resistance to NRTI drugs, M184V cause high-level in vitro resistance to lamivudine (3TC) and emtricitabine (FTC); M41L in combination with L210W and T215Y, presented intermediate resistance to abacavir (ABC)/didanosine (ddI)/ tenofovir (TDF) and high-level resistance to zidovudine (AZT) and stavudine (d4T). Three (4.3%) samples inferred NNRTI drug resistance，K103N causes high-level resistance to nevirapine (NVP), delavirdine (DLV), and efavirenz (EFV)；Y188L causes high-level resistance to NVP and EFV and low-level resistance to ETR and DLV；1 sample had dual mutations of K101E and G190A, causes high level resistance to NVP, intermediate resistance to DLV, EFV and ETR. Significant difference of the subtypes and drug resistance were not observed between the long-term and recent infected population groups. The prevalence of genotypic drug resistances is low among treatment-naïve HIV-1 infected persons in this study. The subtype CRF_BC was dominant among Yunnan blood donors. Subtype B’ was dominant among Henan and Anhui VCT persons. Surveillance on HIV-1 drug resistance among treatment-naïve patients should be continued in China. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6091]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 涂源泉. 中国部分地区未经治疗HIV-1 感染者耐药基因研究[D]. 北京. 中国科学院研究生院. 2008. |
入库方式： OAI收割
来源：昆明动物研究所
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