2012—2021年北京市海淀区HIV抗体不确定结果分析

目的对2012—2021年北京市海淀区HIV筛查阳性样本的确证试验中HIV抗体不确定结果进行分析。方法对HIV筛查实验室上送的8 454例次筛查阳性样本进行免疫印迹试验(western blot, WB), 分析不确定样本的带型特点和随访情况, 并用SPSS 24.0软件进行统计分析。结果在8 454例次筛查阳性样本中, HIV抗体不确定样本1 895例次, 占比22.4%, 主要来自医疗机构(50.5%), 其次是血液中心(47.8%)。WB条带出现率最高是gag蛋白中的p24(78.7%)和env蛋白中的gp160(17.7%)。在完成随访的211例样本中, 其中35例(16.6%)转为阳性, 79例(37.4%)转为阴性。18～44岁年龄组的男性, 尤其是有男男性行为的转阳率较高。条带越多转阳率越高。结论北京市海淀区HIV抗体不确定结果存在较高假阳性。尽管病例随访率低, 但是仍发现一些急性期阳转病例。应加大随访力度, 做到早发现早诊断早治疗。     

病毒载量检测鉴别诊断HIV早期感染

目的 研究病毒载量检测在鉴别诊断HIV早期感染中的应用.方法 对13份HIV抗体检测结果高度提示为早期感染的样本进行病毒载量检测,并对这些个体进行随访和抗体检测以证实其感染状况.结果 13份样本中,有12份病毒载量阳性,随访确定1例HIV抗体阳性婴幼儿感染者,11例窗口期感染者;1例HIV抗体呈阳性的婴幼儿,病毒载量阴性,随访证实未感染.病毒载量检测结果与最终的感染状况相符.结论 通过病毒载量检测能够有效鉴别诊断早期感染中的婴幼儿感染(18个月以内抗体呈阳性)和窗口期感染者.病毒载量检测可以作为HIV感染早期不确定样本的诊断依据.     

HIV抗体不确定样品的鉴别策略研究

目的探索HIV抗体不确定样品的确证策略。方法对93例HIV抗体不确定样品的抗体检测数据进行分析,并进行病毒载量检测和HIV抗体随访检测。结果复检时两种试剂检测结果不一致的有51例,病毒载量检测结果均为阴性。两种复检试剂检测结果均为阳性的42例样品中,有27例病毒载量检测结果为阳性,其中ELISA S/CO〉6的9例样品病毒载量检测结果均为阳性,ELISA S/CO≤6的33例样品中有18例病毒载量检测结果为阳性。对17例不确定样品进行了HIV抗体随访检测,11例病毒载量为阳性的样品均确证为HIV抗体阳性,6例病毒载量为阴性的样品均排除HIV感染。结论对于HIV抗体不确定样品应当慎重对待,HIV抗体复检结果和病毒载量检测结果有助于鉴别HIV感染状态。     

重组痘苗病毒诱导小鼠产生IgG抗体的动态观察

检测血清中HIV抗原或其相应的抗体是判断HIV感染的间接指标。HIV感染后 ,随着血清的阳转 ,首先出现抗HIV 1gag抗体 ,然后才是抗HIV 1env蛋白及其它蛋白的抗体 ,根据病程的进展HIV 1gag大量产生 ,HIV 1gag抗体被结合 ,抗体滴度逐渐下降。因此 ,血清中gag抗体和抗env编码蛋白的抗体检测仍然是目前最常用的诊断HIV感染的标志 ,它比单纯的CD4 + 细胞计数可提前 3～ 4年预测AIDS的发生。它不仅对HIV感染的诊断 ,而且对病情的发展和预后的判断也有一定的实际意义。本文研究含有HIV 1env、HIV 1gag的重组病毒诱导小鼠产生HIV 1env…     

酶联免疫法筛查HIV抗体在艾滋病诊断中的应用价值

目的 对比在艾滋病检验中HIV抗体ELISA法筛查结果与免疫印迹试验结果,探讨HIV抗体筛查在艾滋病诊断中的临床价值.方法 选取2000年1月-2014年1月于海军总医院门诊或住院部术前或输血前患者79 231例,对所有患者抽血,采用EHSA方法对HIV抗体进行初筛检测,将阳性标本进行ELISA复核检测及免疫印迹试验确证,记录各标本复核结果及确证结果,并利用SPSS软件进行对比分析.结果 初筛结果阳性189例,复核结果双阳性186例,初筛与确证结果符合率为96.77％,其中2例经确证为阴性,4例确证结果为不确定.确证为阳性、阴性及不确定的HIV抗体初筛样本结果S/CO值分别为9.9 ～38(17.02 ±3.19)、1～5.8(5.18 ±0.48)、6～9.9(7.94±2.34).1＜S/CO值＜5.9、6＜S/CO值＜9.9及S/CO值＞9.9的样本确证试验阳性符合率分别为33.33％、85.71％,、98.22％.189例初筛阳性样本确诊结果阳性符合率为95.24％,确证试验共出现11条反应带,出现频率由高到低依次为gp160、gp24、gp120、gp41、p66、p51、p31、p17、p55、p39和p36,前4者出现率为95％以上.结论 通过ELISA方法进行HIV抗体筛查阳性率高,但存在一定的假阳性率,其阳性符合率与S/CO值呈正相关性,虽然高S/CO值不能证明其HIV感染,但它是艾滋病诊断中有效的方法.     

3例急性期HIV-1感染病例实验室检测及病毒亚型分析

目的对艾滋病筛查有反应而确证试验阴性或不确定病例进行随访和HIV-1核酸检测, 分析感染毒株基因亚型。方法对3个艾滋病筛查有反应且免疫印迹试验(western blot, WB)阴性或不确定病例进行HIV-1核酸检测和随访。采用一步法RT-PCR扩增HIV-1pol基因片段, 构建系统进化树分析其基因亚型。结果 3个HIV抗体筛查有反应病例, 经WB检测病例1和3出现p24条、病例2未检测到特异条带。pol基因片段系统进化分析显示病例1和3均感染CRF07_BC毒株, 病例2感染CRF01_AE毒株(第5簇)。对病例1和3进行2周后随访检测, WB条带有进展, HIV-1病毒载量分别为476 385 cp/mL和103 462 cp/mL, CD4+T淋巴细胞计数分别为220个/μl和550个/μl。结论急性期HIV-1感染表现为筛查有反应而WB阴性或不确定, HIV-1核酸检测有助于提高急性期感染的发现和诊断能力。     

人类免疫缺陷病毒基因组结构和基因表达

本文系统地描述了 HIV 的形态、基因组结构及基因表达的特点。HIV 是艾滋病的病原体,由于它的入侵,破坏了人体的免疫系统,使机体丧失对传染病和肿瘤的抵御能力而发病。HIV 是一种逆病毒,其基因组结构和基因表达不仅与其他逆病毒有相似之处,而且具有自身独特之点。gag 和 poi 基因编码病毒颗粒的内部蛋白;sor 基因产物是分子量为2.3KD的蛋白,它对生活在非淋巴细胞中的病毒复制是重要的;env 基因产物是外膜蛋白 gp120和转运膜蛋白 gP41;3′-orf 基因产物可能是 HIV 复制的抑制剂;tat 是蛋白反式激活的基因,其产物作用于转录后水平,它可活化 gag 和 env 基因的转录。     

HIV-2 DNA疫苗免疫小鼠的免疫反应观察

目的：用构建的HIV－2外膜蛋白gp105和核心蛋白gag基因的DNA疫苗免疫小鼠，评价其免疫效果。方法：将HIV－2型gp 105和gag基因克隆到表达载体pIRES1neo中，构建重组DNA疫苗质粒。间接免疫荧光试验证明，构建的DNA疫苗在真核细胞中表达了gp105或／和gag.构建的疫苗免疫小鼠后，用流式细胞仪测定CD4^ 、CD8^ T淋巴细胞亚类数，并用HIV－2抗体ELISA检测试剂盒检测免疫鼠血清中抗HIV－2抗体水平。结果：构建了3种HIV－2 DNA疫苗pIRES1gag、pIRSE1gp105和pIRES1gag-gp105，转染BHK细胞后均能表达抗原蛋白，免疫小鼠后可有效地刺激淋巴细胞增殖并诱导产生抗HIV－2特异性抗体，其中pIRES1gag-gp105免疫鼠中，淋巴细胞增殖最显著，而pIRES1gp105免疫鼠中HIV－2特异性抗体水平最高。结论：构建的DNA疫苗均能诱导机体产生免疫反应，其中pIRES1gp105诱导的体液免疫应答最显著，而pIRES1gag-gp105 诱导的细胞免疫响应最显著。     

深圳地区HIV阳性人群中HPV感染状况研究

为了揭示HIV感染对人乳头瘤病毒(HPV)感染影响的情况,在深圳地区采集了一批病人标本样品,通过逆转录PCR(RT-PCR)和荧光定量PCR对80例筛选标本进行HIV感染者和非HIV感染者的HPV感染情况和病毒载量关系比较.     

IL-18和HIV-1 gag-gp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答

目的 :探讨IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫小鼠的免疫应答。方法 :构建含IL 18的真核表达质粒pVAXIL18,将他与表达HIV 1gag gp12 0嵌合基因的核酸疫苗质粒pVAXGE共同肌注免疫BALB/c小鼠 ,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体滴度。结果 :联合免疫组小鼠脾特异性CTL杀伤活性和血清抗体水平均显著高于单独免疫组 (P <0 .0 5 ) ,空白质粒对照组 (P <0 .0 1)和PBS对照组 (P <0 .0 1)。结论 :IL 18和HIV 1gag gp12 0嵌合基因的DNA疫苗联合免疫可诱导小鼠产生特异性细胞和体液免疫 ,且IL 18发挥了免疫佐剂的作用。     

Evaluation of viral-lysate enzyme-linked immunosorbent assay kits for detecting human immunodeficiency virus (type 1) infections using human sera standardized by quantitative western blotting.

The first generation of proprietary reagents for detecting antibodies to the Human Immunodeficiency Virus Type 1 (HIV-1) by enzyme-linked immunosorbent assay (ELISA) used as antigen partially purified virus from cell culture lysates. These tests, which are still in use, may vary in their antibody measurement capabilities if different proportions of the viral polypeptides are present in the viral lysate mixtures. We determined the quantities of antibodies in the serum of persons infected with HIV-1 by dilution analysis using 3 ELISA kits: Abbott [A], Du Pont [D], Genetic Systems [G]. The proportionate antibody titres of each serum to p24gag and gp160env/120env were established by quantitative Western blotting. Serum antibody titres were high, frequently over 1:10,000, a result observed both by ELISA and Western blot. For Kit D, sera with high proportions of antibody to p24gag produced antibody titration curves with steep slopes whereas shallower slopes were found in sera with high proportions of antibody to gp160env. In contrast, Kit A gave steeper slopes with sera enriched for gp160env antibodies. Kit G gave results with slopes intermediate between Kits A and D. Serum antibody titres differed between kits depending upon the proportion and concentration of antibodies in a given serum to gp160env and p24gag. The findings that both the concentration and proportion of antibodies to specific viral polypeptides in human sera markedly affect the signal intensity produced by proprietary ELISAs suggest the need for several control sera which reflect the diversity of human serum responses. Standardization of human reference sera by quantitative Western blotting will assist in evaluation and quality control of ELISA tests.     

Confirmation of HIV seropositivity: comparison of a novel radioimmunoprecipitation assay to immunoblotting and virus culture

A recently developed radioimmunoprecipitation assay, using 125I-labeled human immunodeficiency virus (HIV) viral proteins enriched for glycoproteins gp120env, gp41env (GRIPA), was compared to the immunoblot assay with respect to sensitivity and specificity for the detection of antibodies to HIV. Longitudinal sets of serum samples of seroconverting homosexual men were studied, as were sera of six blood-bank donors likely to be false-positive in immunoblot. In addition, HIV isolation was attempted from white blood cells of these blood-bank donors and of seropositive and seronegative individuals. In sets of seroconversion samples, the GRIPA appeared at least as sensitive as the immunoblot. Some sera already were clearly positive in the GRIPA at a time when there was only weak reactivity in immunoblot. In contrast, sera from blood-bank donors that were regarded as false-positive in immunoblot were negative in GRIPA. Virus culture from these donors was also negative. It is concluded that reactivity in immunoblot to core proteins only may well be false-positive, whereas antibody reactivity in the radioimmunoprecipitation assay to p24gag solely suggests ongoing seroconversion. This feature, in addition to a sensitivity for anti-gp120env comparable to immunoblotting, makes the GRIPA a useful confirmatory assay in sera that yield conflicting results in other HIV-antibody assays.     

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.     

An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens

An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).     

Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population

Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens. The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6%. Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only [GRO]) (0.4 to 0.5% of donors). Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot. Most GRO sera reacted with p15/p17 bands on HIV immunoblot. Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E. coli-expressed p55gag. This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera. More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.     

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.     

Fine specificity of IgG subclass response to group antigens in HIV-1-infected patients

There is an association between the clinical stage of HIV-1 infection and the presence of antibodies against viral gag proteins (p17 and p24). The IgG subclass (G1 and G3) pattern against these antigens was analysed in stable patients and HIV patients progressing to AIDS. Antibodies were analysed with whole viral or peptide ELISA (using sequentially overlapping peptides) and Western Blots. IgG1 was found to be the dominant anti-HIV-1 IgG subclass and IgG1 antibodies declined in progressing patients against all HIV antigens evaluated in Western blot, including p17, p24, p31, gp41, p64, gp120 and gp160. In contrast IgG3 antibodies, which were found to be predominantly directed against gag proteins, and which could be detected in almost all patients, remained in the circulation during disease progression. By peptide assays distinct immunogenic regions were found in p17 in contrast to more evenly distributed epitopes in p24. A decreased divergence of antibody reactivity to both p17 and p24 peptides in the group of patients who developed AIDS was seen. No reaction to any single gag epitope related to disease progression. The difference between IgG1 and IgG3 anti-gag antibodies in relation to clearance during disease progression may depend on different properties of immune complexes formed by these two IgG subclasses.     

Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.     

Evaluation of a recombinant human T-cell lymphotropic virus type I (HTLV-I) p21E antibody detection enzyme immunoassay as a supplementary test in HTLV-I/II antibody testing algorithms.

To evaluate the usefulness of a human T-cell lymphotropic virus type I (HTLV-I) recombinant p21E immunoassay as a supplementary test in HTLV-I/II serologic testing algorithms, we used this assay to test 378 serum samples previously categorized as positive, indeterminant, or negative for HTLV-I/II antibody, as defined by U.S. Public Health Service guidelines. We found this test to be highly sensitive for detecting antibody to HTLV-I/II env (99.4%) but slightly less specific (96.0%), particularly among samples from intravenous drug users. Our data suggest that this test is most appropriately used to confirm the absence of env antibody in samples which are repeatably reactive in an HTLV-I/II screening assay and gag reactive only by immunoblotting. Because of the high sensitivity of this recombinant p21E test, a negative result in this context could preclude radioimmunoprecipitation testing. However, pending further evaluation of the specificity of this assay, samples testing positive for p21 env antibody may require confirmation by radioimmunoprecipitation, particularly in situations in which the results will be used for diagnostic purposes or blood donor counseling.