HIV表型耐药及检测方法的研究进展

高效抗逆转录病毒治疗（HAART）的应用有效地控制了艾滋病患的病情，延长了患的生命，大大地降低了HIV／AIDS相关的发病率和死亡率。但是耐药性的出现却极大地限制了HIV抗病毒治疗效果。选择一个能最大抑制HIV-1复制的方案成为一个极具挑战性的任务。对于接受过抗病毒治疗的病人，不但可能对使用的药物产生耐药，对其它药物亦会产生潜在的交叉耐药，因此耐药性的产生对救援治疗方案的效力亦具有很大的影响。即使未经抗病毒治疗的患，耐药病毒的感染也会限制初期联合抗病毒方案的病毒学和免疫学效力。研究证明根据耐药检测结果选择抗病毒治疗方案可有效地降低病毒载量，升高CD4细胞，从而极大地改善HIV一1感染抗病毒治疗的效果，因此许多专家组织，如IAS—USA、DHHS及EuroGroup已提供耐药检测的推荐和指南。目前HIV耐药性检测方法主要有3种：基因型耐药检测、表型耐药检测和虚拟表型耐药检测。[第一段]     

吗啡对拉米夫定抗HIV-1药效影响的体外MT2细胞实验研究

目的 探讨吗啡是否影响拉米夫定(3TC)抗HIV-1的抗病毒效果.方法 MT2细胞随机分为吗啡+3TC、吗啡+纳洛酮+3TC、纳洛酮+3TC处理组及3TC对照组和病毒对照组;用纳洛酮处理相应组的MT2细胞0.5 h后,加入吗啡处理细胞24 h,然后每组细胞加入等量的HIV-1ⅢB病毒和3TC溶液;病毒感染细胞第3、4、5和6天,取培养上清,用ELISA法检测培养上清的HIV-1 p24抗原,根据p24抗原表达量,计算各个处理组的3TC抗HIV-1 p24抗原抑制率.结果 HIV-1感染细胞第3和第4天,吗啡+3TC处理组的3TC抗HIV-1 p24抗原抑制率最低,与3TC对照组相比差异有统计学意义(P<0.05);吗啡+纳洛酮+3TC处理组和纳洛酮+3TC处理组的3TC抗HIV-1 p24抗原抑制率相当,两组差异无统计学意义(P>0.05),这两组分别与3TC对照组相比,差异无统计学意义(P>0.05);病毒感染细胞第5和第6天,各处理组的3TC抗HIV-1 p24抗原抑制率与3TC对照组相比,差异无统计学意义(P>0.05);各个处理组的3TC抗HIV-1 p24抗原抑制率随感染时间延长而降低,呈现时间一效应关系.结论 吗啡在病毒感染初期能降低3TC 的抗HIV-1药效;纳洛酮能够阻滞吗啡降低3TC的抗HIV-1药效作用.

Abstract：

Objective To determine whether morphine having the ability to influence the antiviral effect of lamivudine(3TC)in vitro study.Methods MT2 cells were randomly assigned into morphine+3TC treatment group,morphine+naloxone+3TC treatment group,naloxone+3TC treatment group.Both 3TC and virus control groups were set up.The corresponding MT2 cells were treated with opiates antagonist(naloxone)for 0.5 hours before the 24-hours morphine treatment program was implemented while all of the groups were then infected with equal amounts of cell-free HIV-1 ⅢB strain and 3TC.HIV-1 p24 antigen in culture supernatants collected at days 3,4,5 and 6after infection status was tested and the inhibition of 3TC anti-HIV-1 p24 antigen of various treatment groups calculated.Results Inhibition of 3TC anti-HIV-1 p24 antigen of Morphine+3TC treatment group was the lowest when HIV-1 infected cells at 3rd and 4th day and showed significant difierence (P<0.05)when compared to the 3TC control.However,there was no statistically significant difference among them(P>0.05),when virus was infected the cells at 5th and 6th day.The difference of 3TC anti-HIV-1 p24 antigen inhibition between the morphine+naloxone+3TC treatment group and the naloxone+3TC treatment group was not significant(P>0.05).Similar results were obtained when these two groups were compared to the 3TC control group(P>0.05),respectively.The 3TC anti-HIV-1 p24 antigen inhibition of each treatment group reduced as the time of infection prolonged,showing a significant and time-course effbct.Conclusion The 3TC antiviral effect was reduced by morphine in the early stage of infection,and could be blocked by naloxone.     

对in-house HIV-1基因型耐药性检测方法的评价

目的 评价in-house HIV-1基因型耐药检测方法的敏感性与准确性。方法 收集2004年4月至2008年10月全军艾滋病检测中心检测的来自河南、广西130份血浆标本。以美国FDA批准的HIV-1基因型耐药性检测系统(ViroSeqTM v2.0)为参考方法,并与建立起的基因型耐药性检测方法(in-house)平行检测待检样本,比较二者在扩增效率、耐药突变位点检测以及耐药报告等方面的一致性。结果已知的14 850个耐药突变位点中,2种方法可同时对99.3％(14 752/14 850)的耐药突变位点准确检出;在对不同突变位点的检测中,2种方法对蛋白酶抑制剂耐药突变位点、逆转录酶抑制剂耐药突变位点及两类抑制剂耐药突变位点检测一致率分别为99.7％、99.0％和99.3％（Kappa值分别为0.909 9、0.952 1和0.948 8,P值均＜0.01）;2种方法出具的两类药物的耐药结果报告一致率94.6％（Kappa=0.637 4,P＜0.01）。本研究共检测到34个ViroSeqTM数据库(ViroSeqTM software v2.7)未收录位点,其中2个突变位点对耐药的影响较大。结论in-house基因型耐药性检测方法与ViroSeqTM基因型耐药性检测系统在耐药突变位点检测以及评价上具有高度一致性,是一种快速准确、性价比高的HIV-1基因型耐药检测方法,同时在耐药数据库方面具有一定优势。     

河南某农村接受抗病毒治疗的艾滋病患者HIV-1耐药发生的六年队列分析

Objective To analyze the occurring rules of human immunodeficiency virus (HIV)drug resistance under an unique therapy model among HIV-1 infected individuals on antiretroviral therapy (ART) in rural areas of Henan, China. Methods A cohort of 75 individuals on an ART regimen of zidovudine (ZDV) , dideoxyinosine (ddI) and nevirapine (NVP) was established in March 2003. A total of 12 surveillances were conducted and 788 person-times were studied until 2010. The parameters of CD4 cell count and viral load (VL) were tested in each survey. And genotypic resistance testing was performed in patients with a failure of viral suppression. Survival analysis was used to estimate the occurrence time of resistance. Results The cumulative mortality rate was 16% (12/75) in the cohort. And the cumulative resistance rate was 88% (66/75) from 2004 to 2010. The rate of resistance reached 54. 7% and the probability from susceptibility to drugs developing resistance decreased drastically from 100% to 45. 3% within the first 1 year of initiation. The occurrence time of resistance for half of individuals in the cohort was at 12.0 months(95% CI 8. 6 - 17. 0)after initiation, 25. 1 months(95%C/19.0-33. 3)in those whose VL was less than 4. 0 lgU/ml and 4. 8 months (95% CI 4.1 - 5. 6) at VL ＞ 4. 0 lgU/ml during the first investigation. The individuals with an early occurrence of resistance within 12 months carried high risks for afailure of viral suppression and a decrease of CD4 counts. Conclusion The occurrence of resistance rises with the course of therapy. And the greatest probability for resistance is within the first 1 year of initial therapy. A high level of VL has a significant impact on the development of resistance. Preventing the occurrence of resistance during the initial therapy remains a key goal.     

HIV抗体初筛实验室质控考评结果分析

目的：评价艾滋病毒检测初筛实验室的检测质量,促进质控水平的提高。方法：用包括ＨＩＶ抗体阳性、弱阳性和阴性共２０例考证血清对５３个医疗防疫单位的艾滋病毒检测初筛实验室进行现场测试。结果：３５个实验室检测结果完全正确,１７个实验室出现了不同程度的阴性结果,近１／３的实验室检测弱阳性标本的Ｓ／ＣＯ值低于质控标准的９５％可信限以下。结论：敏感性达不到标定的范围是ＨＩＶ检测存在的主要问题。未严格遵守艾滋病毒     

灭活前后HIV-1生物学性状改变的研究

S/D法及低 p H法是常用的血制品灭活病毒的有效方法。在评价 S/D法及低 p H法灭活 HIV试验中 ,发现不病变样品经盲目传代可以传出病变 ,说明 HIV在灭活因素作用下发生变异。 HIV具有高度的变异性 ,不同地区不同机体 ,同一机体不同病程 ,甚至同一机体一次取样均可分离到不同基因型不同生物学性状的 HIV株 [1 ]。我们曾在 MT4细胞长期自然传代培养 HIV,分析 HIV包膜基因变异及生物学性状改变 ,获得体外传代 HIV包膜基因变异不显著 ;病毒感染毒力及病毒产量上升 ;亚型不变 ,生物学性状相对稳定的结论。为了进一步了解灭活作用下 H…     

人免疫缺陷病毒HB-J株的分离和生物学特性研究

目的：分离和鉴定我国流行的人免疫缺陷病毒（ｈｕｍａｎ ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ,ＨＩＶ）毒株,研究我国ＨＩＶ毒株的生物学特性。方法：周外用血单个核细胞（ｐｅｒｉｐｈｅｒａｌ ｂｌｏｏｄ ｍｏｎｏｎｕｃｌｅａｒ ｃｅｌｌｓ,ＰＢＭＣ）共培养的方法分离ＨＩＶ,用观察细胞病变、检测Ｐ２４抗原和前病毒ＤＮＡ的方法监测病毒生长的情况。结果：在建立共培养的第１２天观察到明显的细胞病变,主要表面为大泡样细胞和多核巨细胞。接种第９天细胞培养上清液的Ｐ２４抗原由阴性转为阳性,并且浓度迅速增加,接种第１２天浓度即达到１７８０ｐｇ／ｍｌ,第１４天达到峰值,为２３２０ｐｇ／ｍｌ。但该株病毒的ＰＢＭＣ培养上清液转种于ＭＴ４细胞后,培养上清液Ｐ２４抗原和前病毒ＤＮＡ始终为阴性且不出现细胞病变。结论：用ＰＢＭＣ共培养的方     

部队医务人员掌握艾滋病知识情况调查

目的 了解医务人员掌握艾滋病知识的程度,为制订部队医务人员艾滋病教育和训练规划提供依据。方法 于１９９９年５～６月试卷考核的方法对部队个医疗单位的８７０名医务人员进行了艾滋病知识调查。结果 （１）对艾滋病的一般知识,如ＨＩＶ／ＡＩＤＳ的名称,三种传播途径等掌握较好,回答的正确率部在９０％以上。（２）对专业性较强的艾滋病知识如ＨＩＶ的抵抗力、窗口期、有效的预防措施等掌握稍差,回答的正确率是６０％～７     

河南某农村接受抗病毒治疗的艾滋病患者HIV-1耐药发生的六年队列分析

Objective To analyze the occurring rules of human immunodeficiency virus (HIV)drug resistance under an unique therapy model among HIV-1 infected individuals on antiretroviral therapy (ART) in rural areas of Henan, China. Methods A cohort of 75 individuals on an ART regimen of zidovudine (ZDV) , dideoxyinosine (ddI) and nevirapine (NVP) was established in March 2003. A total of 12 surveillances were conducted and 788 person-times were studied until 2010. The parameters of CD4 cell count and viral load (VL) were tested in each survey. And genotypic resistance testing was performed in patients with a failure of viral suppression. Survival analysis was used to estimate the occurrence time of resistance. Results The cumulative mortality rate was 16% (12/75) in the cohort. And the cumulative resistance rate was 88% (66/75) from 2004 to 2010. The rate of resistance reached 54. 7% and the probability from susceptibility to drugs developing resistance decreased drastically from 100% to 45. 3% within the first 1 year of initiation. The occurrence time of resistance for half of individuals in the cohort was at 12.0 months(95% CI 8. 6 - 17. 0)after initiation, 25. 1 months(95%C/19.0-33. 3)in those whose VL was less than 4. 0 lgU/ml and 4. 8 months (95% CI 4.1 - 5. 6) at VL ＞ 4. 0 lgU/ml during the first investigation. The individuals with an early occurrence of resistance within 12 months carried high risks for afailure of viral suppression and a decrease of CD4 counts. Conclusion The occurrence of resistance rises with the course of therapy. And the greatest probability for resistance is within the first 1 year of initial therapy. A high level of VL has a significant impact on the development of resistance. Preventing the occurrence of resistance during the initial therapy remains a key goal.     

静脉注射人免疫球蛋白酸性孵放法灭活艾滋病病毒的效果研究

目的研究静脉注射人免疫球蛋白在生产过程中酸性孵放法灭活艾滋病病毒的效果。方法采用细胞培养法,对含有艾滋病病毒的静脉注射人免疫球蛋酸性孵化法灭活效果进行了实验室检测。结果将含有艾滋病病毒的静脉注射人免疫球蛋白酸度调整为pH4,于21～25℃条件下放置4d,其中艾滋病病毒的滴度(TCID50)由9.50log下降至2.00log以下,下降值达到7.00log以上。相同的样品在pH值7.0条件下放置14d,其中艾滋病病毒滴度仍在3.3log以上;经放置21d,所有样品中艾滋病病毒滴度均在2.0log以下。经过对3批样品重复测定,均未出现细胞病变;经过将样品盲传三代仍未出现细胞病变。结论采用酸性孵放法常温下放置21d,可使静脉注射人免疫球蛋白中艾滋病病毒完全失去活性。