HIV论文HIV 抗体 艾滋病血清学诊断 确证试验.doc

HIV论文：HIV 抗体 艾滋病血清学诊断 确证试验【中文摘要】我国的艾滋病检测工作已经建立起一套较为完善的程序。依据全国艾滋病检测技术规范(2009年修订版),HIV抗体检测的常规程序分为筛查试验(包括初筛和复检)和确证试验两个步骤,对于筛查试验呈阳性反应的样品,需再做确证试验。确证试验结果阳性,报告HIV抗体阳性；结果阴性,报告HIV抗体阴性；结果不确定,报告HIV抗体不确定,并建议随访检测。目前经国家食品药品监督管理局批准的HIV抗体确证试剂有3种产品(1种进口、2种国产),它们的原理均为免疫印迹试验(western blot, WB).随着HIV抗体确证试验的广泛开展,WB试剂在某些情况下易产生不确定结果的缺点凸显出来。例如,2007-2010年全国抗体确证检测数分别为51894、73735、88498和91928,结果不确定数分别为2459、4680、5437和5653,所占比例分别为4.7%、6.3%、6.1%和6.1%。这些不确定结果产生的原因有2种：窗口期感染和非特异性反应,其中后者比例更大,大量非特异性不确定结果的产生给受检者本人和后续的随访工作带来了很多负担。新一代基于重组蛋白和(或)合成肽的线性免疫试验(line immuno assays, LIA)可以大大减少非特异性不确定结果的产生,国外已有商品化的试剂盒,如INNO-LIA、Pepti-Lav和RIBA(recombinant immunoblot assay),国内也在积极研发此类新型试剂。研究对国产人类免疫缺陷病毒1+2型抗体检测试剂盒(重组免疫印迹法,北京万泰生物技术有限公司等研发)进行临床评价研究,评价其检测人类血浆样本的临床性能及检测HIV早期感染的能力。研究方法：1.本实验室收集的常规临床血浆样本510份作为研究样本,包括400份单一HIV-1抗体阳性、110份HIV抗体阴性。分别用万泰RIBA试剂盒和新加坡MP-WB试剂盒检测所有样本并对检测结果进行比较,对于两种试剂检测结果不一致的样本,重复检测,复检结果仍不一致用第三方试剂INNO-LIA试剂检测,评价万泰RIBA试剂盒与新加坡MP-WB试剂盒检测相同样本的符合情况。2.本实验室购买的HIV-2抗体阳性样本5份作为研究样本。分别用万泰RIBA试剂盒和新加坡MP-WB试剂盒检测,了解它们对HIV-2的抗体检测能力。3.5份HIV-1抗体阳性样本分别用同一份1份HIV抗体阴性样本进行10倍系列稀释,最高稀释10万倍作为研究样本。分别用万泰RIBA试剂、新加坡MP-WB试剂和比利时INNO-LIA试剂检测,比较3种试剂盒的分析灵敏度。4.本实验室收集的HIV-1阳转血清盘(10套)、从BBI购买的阳转血清盘(1套)作为研究样本。分别用万泰RIBA试剂、新加坡MP-WB试剂和比利时INNO-LIA试剂检测所有样本,比较3种试剂盒检测HIV早期感染的性能。结果：1.万泰RIBA试剂检测性能评价：(1)已知单一HIV-1抗体阳性样本400份：MP-WB试剂检测结果阳性的400份,敏感性为100%,万泰RIBA试剂检测阳性400份,敏感性为100%,阳性符合率为100%；(2)HIV抗体阴性样本110份：MP-WB试剂检测结果阴性的91份,特异性82.7%,万泰RIBA试剂检测阴性90份,特异性为98.2%,阴性符合率为98.90%,万泰；在MP-WB试剂检测结果为不确定的19份样本中,万泰RIBA试剂检测结果为阴性18份,不确定1份；用第三方试剂INNO-LIA检测两试剂检测结果不一致的19份样本,万泰RIBA试剂与INNO-LIA试剂对其中17份样本的检测结果一致。万泰RIBA试剂与MP-WB试剂的总符合率为96.27%,Kappa值0.89,一致性强度为最强。2.万泰RIBA试剂对HIV-2抗体的检出能力：(1)在对已知背景为单一HIV-1抗体阳性的400份样本检测过程中,16份(占4%)样本用参考试剂MP-WB检测出现了HIV-2指示带(gp36),而万泰RIBA试剂检测均没有非特异性HIV-2指示带检出；(2)对5份已知HIV-2抗体阳性样本,万泰RIBA和MP-WB试剂均能检出HIV-2指示带(gp36),它们对HIV-2抗体检测无漏检。3.三种试剂盒的分析灵敏度比较：5套系列稀释样品的检测结果显示,在3套系列稀释样品中,当MP-WB试剂盒和INNO-LIA试剂盒结果从阳性转变为不确定甚至阴性时,万泰RIBA试剂盒仍显示阳性结果；在另外2套系列稀释样品中,INNO-LIA最先转变为不确定,而MP-WB试剂盒转变最晚。万泰RIBA的分析灵敏度不亚于MP-WB和INNO-LIA。4.三种试剂盒检测早期感染性能比较：在ELISA试验呈阳性反应的48份HIV抗体阳转血清盘样品中,万泰RIBA、MP-WB和INNO-LIA的确证阳性率分别为97.92%(47/48)、81.25%(39/48)和91.67%(44/48)。万泰RIBA与MP-WB之间差异有统计学意义(X2=6.13,P0.05)。对于含有结果不确定样品的6套阳转血清盘,用万泰RIBA、MP-WB和INNO-LIA检测的平均阳转时间分别为0.7、13.3、3.7 d。结论：1.万泰RIBA试剂“人类免疫缺陷病毒1+2型抗体检测试剂盒(重组免疫印迹法)”检测人血浆样本的临床性能良好,敏感性100%、特异性98.2%、阳性符合率为100%、阴性符合率为98.90%、总符合率为96.27%。2.与我国目前常用的新加坡MP-WB试剂相比,万泰RIBA试剂可以大幅度减少非特异性不确定结果的产生,在实际工作中可以大大减少不必要的随访检测。3.对HIV-1阳转血清盘的检测结果显示,万泰RIBA试剂和INNO-LIA可以缩短HIV抗体确证的窗口期。4.万泰RIBA试剂对HIV-2抗体检测性能良好,可以通过HIV-2指示带反应提示HIV-2感染。5.新一代基于重组蛋白和(或)合成肽的确证试剂有望成为我国今后HIV抗体确证试剂的发展方向。【英文摘要】BackgroundA normative procedures have been developed for the diagnosis of HIV infection in China, according to the national technical regulation for HIV detection (2009). Serological tests for detecting antibodies to HIV are generally classified as screening assays and confirmatory assays. Screening assays can provide the presumptive identification of antibody-positive specimens, and confirmatory assays are used to confirm whether specimens found reactive with a particular screening assay contain antibodies specific to HIV and/or HIV antigen. If confirmatory tests result is positive, reporting antibody positive; if result is negative, reporting antibody negative; if result is indeterminate, reporting antibody indeterminate and suggesting follow-up detecting. Approved by China FDA, there are three confirmatory assay kits (one kit is made in Singapore and two kits in China) on the basis of western blot(WB). With the widely used of WB kits, under some conditions, it can produce a relatively large number of indeterminate results. From 2007 to 2010, the total test numbers of HIV antibody confirmatory assays in China are 51894,73735,88498 and 91928, respectively, and indeterminate test numbers are 2459,4680,5437 and 5653, respectively. The reasons of indeterminate results are early infection and/or non-specific reaction, the later one is the main reason. Similar assay kits, based on recombinant proteins and/or synthetic peptides capable of detecting antibodies to specific HIV-1 and/or HIV-2 proteins, have been developed abroad. Examples of this technology include the INNO-LIA, Pepti-LAV and RIBA assays. In general, these assays produce fewer indeterminate results as compared to WB. Recently, a domestic diagnostic kit for antibody to HIV-1 and HIV-2 (recombinant immunoblot assay, RIBA) was developed in China.To evaluate the performance of diagnostic kit for antibody to HIV-1 and HIV-2 (recombinant immunoblot assay, RIBA), produced by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Methods1. Collected 510 plasma specimens (including 400 HIV-1 antibody positive and 110 HIV antibody negative) by NARL as research specimens, detecting these samples by using two kinds of kits (including Wantai-RIBA (Recombinant immunoblot assay, Beijing Wantai Biological Pharmacy, China), MP-WB (HIV Blot 2.2 WB, MP Biomedicals Asia Pacific Pte. Ltd., Singapore) respectively. If the results are no the same, repeating the experiments, use the INNO-LIA(INNO-LIATM HIV/Score, Innogenetics N.V., Belgium) to detect the specimens which the results of two kits are still no the same. Then compare the results to evaluate Wantai RIBA kit consistency with other antibody confirmatory assay kits.2. Purchasing five HIV-2 antibody positive plasmas as research specimens, then using Wantai RIBA kit and MP-WB kit to detect. Compare the results to evaluate the performance for detecting HIV-2 specific band of Wantai RIBA kit.3. Five HIV-1 antibody positive plasma samples were ten fold serially diluted with the same HIV antibody negative specimen(the maximum diluted fold was 100,000) and then detected by ELISA. The above diluted positive specimens were detected with the three kits to analyze their sensitivity.4. Compare the results by detecting the eleven sets seroconversion panels with three antibody confirmatory assay kits to evaluate the Wantai RIBA kitsperformance of detecting early infection.Results1. Evaluate the performance of Wantai RIBA kit:1)400 HIV antibody positive samples, Wantai RIBA kit detected 400 positive samples, sensitivity is 100%, positive accordance rate is 100%.2) 110 HIV antibody negative samples, Wantai RIBA kit detected 90 negative, specificity is 98.2%, negative accordance rate is 98.90%, total accordance rate is 96.27%, Kappa value is 0.89.2. The performance of Wantai RIBA kit in detecting HIV-2:1) 16 of 400 HIV-1 antibody positive specimens were detected HIV-2 specific band gp36 by MP-WB kit, but Wantai RIBA kit did not do.2) To HIV-2 antibody positive, Wantai RIBA kit and MP-WB kit all can detect HIV-2 specific band gp36.3. To the five sets diluted specimens, when the results of MP-WB kit and INNO-LIA kit turned indeterminate or negative, but Wantai was still positive in three sets specimens. In the other two sets, INNO-LIA kit became indeterminate at first, MP-WB at last. The analytical sensitivity of Wantai RIBA kit was no less than MP-WB kit and INNO-LIA kit.4. Among the fouty-eight ELISA reactive specimens from eleven HIV seroconversion panels, the confirmatory HIV antibody positive percents of Wantai-RIBA, MP-WB and INNO-LIA were 97.92%(47/48),81.25%(39/48) and 91.67%(44/48) respectively. There was a statistically significant difference between the confirmatory results of Wantai-RIBA and MP-WB (2=6.13, P0.05). For 6 HIV seroconversion panels containing indeterminate specimens, the average seroconversion time by using Wantai-RIBA, MP-WB and INNO-LIA were 0.7,13.3 and 3.7 days, respectively.Conclusion1. Wantai HIV 1+2 antibody confirmatory assay kit(Recombinant immunoblot assay) has a good performance of detecting plasma specimens, sensitivity is 100%, positive accordance rate is 100%, specificity is 98.2%, negative accordance rate is 98.90% and total accordance rate is 96.27%.2. Compare with MP-WB kit (made in Singapore), which is the most widely used in China, Wantai RIBA kit can reduce a