集合HIV RNA RT-PCR方法验证及其在IDUs中窗口期检测的应用

目的 对已建立的集合艾滋病病毒（HIV）RNA逆转录-聚合酶链反应（RT-PCR）方法进行实验室验证，并将其应用于静脉吸毒人群中窗口期的检测。方法 采用50：1、10：1、1：1三级集合HIVRNA RT-PCR方法进行盲样验证及可重复性验证。结果（1）集合HIVRNART．PCR方法盲法验证的结果和预期值一致，重复性验证中9个一级集合的病毒载量对数值的变异系数为6％，单因素方差分析P〉0．05，方法的重复性良好。（2）检测3570份吸毒人群的HIV抗体阴性血清或血浆样本。对发现的RNA阳性感染者进行追踪采样，发现2例HIVRNA窗口期阳性。追踪随访，HIV-1抗体阳转。结论 集合HIVRNART-PCR方法具有良好的实验性能，可应用于HIV高危人群窗口期检测。     

用HIV抗原/抗体联合试剂筛查吸毒者中窗口期HIV感染者

目的筛查窗口期艾滋病病毒(HIV)感染者,并验证HIV抗原/抗体联合试剂(第四代试剂)的检测性能。方法用HIV抗原/抗体联合试剂对经HIV抗体筛查呈阴性反应的吸毒者样本进行检测,并与HIV抗原检测及HIV-1 RNA检测的结果作比较,以证实其为窗口期感染样本。结果1 934份HIV抗体阴性样本中发现3例窗口期HIV感染者;所用第四代试剂检出窗口期样本的敏感性为100%,特异性为99.28%,假阳性率为0.77%。结论用第四代试剂检测虽然可以在某种程度上缩短检测窗口期,但该试剂有一定的假阳性结果,尤其是在S/CO值较低时其假阳性比率增高,故检测时应注意S/CO值的高低,如有条件可进一步做HIV-1 RNA检测。     

南昌地区无偿献血人群HIV-1感染状况及特征分析

目的调查近5年南昌地区无偿献血人群1型艾滋病病毒(HIV-1)感染状况,描述感染者特点,为本地区降低HIV经输血传播风险,制定有效献血者招募及艾滋病防控策略提供数据支持。方法无偿献血者血液标本采用酶联免疫吸附试验(ELISA)方法进行抗-HIV初复检筛查,ELISA初筛阴性标本用核酸检测(NAT)方法检测艾滋病病毒核糖核酸(HIV RNA),抗-HIV或HIV RNA反应性标本用蛋白印迹试验(WB)方法进行确证。统计2014-2018年南昌地区献血者HIV抗体检出率,整理分析HIV-1感染者人口学特征及流行病学资料。结果 2014-2018年南昌地区无偿献血者HIV-1型抗体确证阳性57例,HIV抗体不确定30例,抗体检出率0.018%。ELISA-/NAT+1例,WB鉴定为HIV抗体不确定。57例感染者中,男性占98.25%;21～30岁青年人占40.35%;未婚占64.91%;本科文化占33.33%;本市(县区)户籍占43.86%;高校学生占40.35%;初次献血占75.44%,捐献全血占96.49%;男男同性性传播占52.63%,异性性传播占43.86%;合并梅毒感染占14.04%。HIV-1型抗体阳性标本出现WB特异性条带gp160、gp120、p24均占100%,p66占96.49%,gp41占91.23%,HIV抗体不确定标本出现条带p24占66.67%,gp160占36.67%。结论近5年南昌地区无偿献血人群中HIV-1感染者以初次捐献全血的高校青年男男性行为者(MSM)为主。应采用先进的检测方法和试剂开展HIV筛查,保证血液安全。     

集合PCR在检测MSMHIV急性期感染中的应用

目的应用集合聚合酶链反应（PCR）方法,发现广州市男男性行为人群（MSM）中艾滋病病毒（HIV）急性感染者,并估计该人群HIV感染的发病率。方法采用10∶1、5∶1、1∶1三级集合HIV-1核糖核酸（RNA）反转录PCR（RT-PCR）方法进行检测。结果 2008年,检测1 250份MSM HIV-1抗体阴性血浆,发现HIV-1RNA阳性3例,追踪随访,2例HIV-1抗体阳转,1例失访;初步估计HIV-1年发病率约为8.50%[95%可信区间（CI）：1.75%～24.86%]。2009年,检测1 002份MSM HIV-1抗体阴性血浆,发现HIV-1RNA阳性4例,追踪随访,4例均HIV-1抗体阳转;估计HIV-1年发病率约为14.17%（95%CI：3.85%～36.21%）。结论集合HIV-1RNART-PCR是发现急性感染者的有效方法,并可估计人群HIV发病率。     

HIV-1 WB试验不确定样本的HIV-1 RNA定量检测结果分析

目的探讨1型艾滋病病毒(HIV-1)核糖核酸(RNA)定量检测,在HIV-1抗体不确定样本诊断中的应用,为类似样本的诊断提供参考。方法对31例HIV-1抗体不确定的样本进行HIV-1RNA定量检测,通过随访并结合流行病学资料确定其感染状况。结果 31例HIV-1蛋白印迹试验(WB)不确定样本中,HIV-1RNA定量检测结果高于检测限的18例,低于检测限的13例。HIV-1RNA定量检测结果高于检测限的18例中,随访15例,占83.3%;其中11例抗体阳转,4例WB带型无进展,但复查HIV-1RNA定量检测结果仍高于检测限,判定为HIV感染。HIV-1RNA定量检测结果低于检出限的13例中,随访4例,占30.8%;其中2例WB带型消失(p24),2例带型无进展(p24 1例,p24、gp160 1例),其中1例(p24和gp160)复查,HIV-1RNA定量检测结果仍低于检测限,结合流行病学资料排除HIV感染。结论 HIV-1RNA定量检测是WB试验的有效补充,在HIV-1抗体不确定时,尽快采用HIV-1RNA定量检测并结合流行病学资料分析,有助于更快对个体HIV感染状态进行诊断。     

ELISA检测HIV感染者尿液标本中抗HIV-1抗体结果分析

目的利用尿液标本检测抗HIV-1抗体.方法通过ELISA方法检测50例抗HIV-1抗体阳性感染者及100例抗HIV-1抗体阴性对照组尿液中抗HIV-1抗体.结果显示在50例HIV感染者中有49例尿液抗HIV-1抗体阳性,1例阴性;对照组中尿液中均未检测出抗HIV-1抗体,以血液标本为标准,检测尿液抗HIV-1抗体的灵敏性为98.0%,特异性为100%.尿液标本与血液标本检测的一致性达99.3%.结论本实验提示在采集血液标本不便的情况下,可通过尿液抗HIV-1抗体的检测对高危人群的HIV感染情况进行监测和筛查.     

HIV-1 RNA定量检测在HIV感染诊断中的应用

目的 通过采用HIV-1 RNA定量检测,对HIV筛查试验有反应但WB结果为阴性或不确定的样本进行诊断分析,探讨HIV-1 RNA定量检测在实际HIV感染诊断临床验证工作中的应用。方法 回顾性地对2020年江西省确证实验室收集的172例WB阴性和138例WB不确定的样本进行HIV-1 RNA定量检测,以HIV-1 RNA定量结果>5 000拷贝/mL为HIV感染诊断标准,将核酸诊断结果与病例追踪复检的WB确证结果进行比较分析。结果 WB阴性的样本有4.65%(8/172)检出HIV-1 RNA阳性,WB不确定的样本有64.49%(89/138)检出HIV-1 RNA阳性。其中96例（8例WB阴性、88例WB不确定）样本HIV-1 RNA定量结果>5 000拷贝/mL,另有1例WB不确定样本HIV-1RNA定量结果在20～5 000拷贝/mL之间,该97例样本随访的WB结果均为阳性;HIV-1 RNA定量结果低于检出限者213例（164例阴性、49例不确定）,随访WB结果均为阴性。以5 000拷贝/mL为阳性诊断阈值,本研究中HIV-1 RNA定量检测应用于HIV感染诊断的敏...     

尿液HIV-1抗体检测的临床应用评价

目的通过对尿液艾滋病病毒1型(HIV-1)抗体检测在不同人群临床应用中性能的评价,探讨尿液HIV-1抗体检测作为艾滋病实验室检测指标的可行性。方法自2014年3月21日至2015年3月1日,采集不同地区、不同人群尿液样本1310例,采用酶联免疫吸附试验(ELISA)检测尿液HIV-1抗体,并将其结果与HIV抗体诊断(WB)结果进行比较,分析尿液HIV-1检测的灵敏度、特异性,以及与血液诊断的符合率。结果尿液HIV抗体检测灵敏度为97.28%(250/257),特异性为99.06%(947/956),与血液HIV抗体诊断符合率为98.68%。不同人群尿液HIV抗体检测的灵敏度为94.34%~100%,特异性为95.39%~100%,其中吸毒人群最低。对于HIV抗体阳性正在接受抗病毒治疗的人群,尿液检测的灵敏度为88.66%(86/97),显著低于其他人群。结论尿液HIV抗体检测可作为HIV抗体检测指标,但不能用于HIV抗体阳性接受治疗人群。     

重庆市男男性行为人群2009年HIV-1 BED发病率调查

目的采用连续横断面调查和BED-捕获酶免疫试验(BED-capture enzyme immunoassay,BED-CEIA)估算重庆市男男性行为人群(MSM)2009年艾滋病病毒Ⅰ型(HIV-1)的发病率,分析流行趋势,讨论该方法应用于MSM人群的效率。方法利用"滚雪球"和同伴推动抽样方法(RDS),采用标准化问卷收集行为信息并采集血样,对HIV抗体确认阳性者采用BED-CEIA方法检测HIV-1新近感染,根据标准公式和2种校正公式,对该人群2009的发病率按不同年龄、寻找性伴方式、学历、收入状况等进行分层计算,与历年结果进行比较分析。结果共招募MSM志愿者942名,平均年龄为(26.32±7.11)岁,大专及以上学历者598人(63.48%);共发现HIV感染者142人,BED检测为新近感染者62人。重庆市MSM人群2009年HIV-1年发病率为15.43%(95%CI:11.59～19.28),较2008年增长了64.49%,与过去3年有显著性差异。21～30岁年龄组年发病率为15.03%(95%CI:10.42～19.42),通过网络寻找性伴组的年发病率为14.69%(95%CI:10.14～19.24),大专及以上学历组年发病率为13.74%(95%CI:9.25～18.23)。结论重庆市MSM人群HIV-1发病率呈快速上升趋势,2009年发病率校正之后的绝对值与前三年比较,结果有显著性差异;不同的校正公式得到的结果没有显著性差异,但与估计感染率之间存在令人深思的矛盾现象。     

绍兴市HIV-1毒株的耐药变异研究

目的了解绍兴市1型艾滋病病毒(HIV-1)株的耐药变异情况。方法分析2013-2014年绍兴地区新确认未经抗病毒治疗的130例HIV-1感染者,以及2014年前抗病毒治疗满一年且病毒未抑制的34例感染者样本,采用反转录-聚合酶链式反应(RT-PCR)和巢式-聚合酶链式反应(Nested-PCR)方法扩增HIV-1的pol基因区全长,参照美国斯坦福大学HIV耐药数据库,确定HIV亚型及耐药突变位点。结果 34例新诊断HIV感染者及33例病毒未抑制感染者的样品检测到耐药突变,主要亚型为CRF01_AE和CRF07_BC。仅在病毒未抑制感染者中检测到针对核苷类反转录酶抑制剂(NRTIs)的耐药突变,主要类型有M184V(42.42%)、K70R(15.15%)、D67N(12.12%)。在新确认感染者和病毒未抑制感染者两组人群中,均检测到针对非核苷类反转录酶抑制剂(NNRTIs)耐药的突变,主要类型有V179D/E/T(10.00%和18.18%)、K103N(2.22%和21.21%);在病毒未抑制感染者中还有Y181CY(21.21%)、V90I(12.12%)、G190A(15.15%)。在反转录酶(RT)区和整合酶(IN)区仅检测到少量次要耐药突变。结论绍兴市HIV经抗病毒治疗后易产生耐药性,部分可在新感染人群中传播,应对HIV耐药变异加强监测。     

Strong correlation between the complement-mediated antibody-dependent enhancement of HIV-1 infection and plasma viral load.

OBJECTIVE: We have previously demonstrated that complement-mediated antibody-dependent enhancement (C-ADE) of HIV-1 infection correlates with accelerated immunosuppression and disease progression in HIV-1-infected individuals. In the present work the relationship between C-ADE and plasma HIV-1 RNA concentrations was studied to determine the effect of C-ADE on viral replication. METHODS: Three studies were performed: (a) C-ADE and HIV-1 RNA concentrations were determined in the serum and plasma aliquots taken at the same time from 98 HIV patients, mostly in the advanced stage of the disease; (b) the above two parameters as well as HIV enzyme-linked immunosorbent assay (ELISA)-reactive antibodies (Abbott HIV 1/2 test), and p24 antigen levels (Abbott antigen test; Abbott, Delkenheim, Germany) were determined in four seroconversion panels purchased from the Boston Biomedica firm; (c) changes of HIV-1 RNA concentration and C-ADE during a 17 month follow-up period were determined in 18 HIV-infected patients. C-ADE was measured by the method previously established in our laboratories. The results were expressed by an enhancement/neutralization index (E/NI). HIV-1 RNA levels were determined with the Amplicor monitor kit (Roche, Basel, Switzerland), and in some experiments with the nucleic acid sequence based amplification (Organon Teknika, Turnhout, Belgium) kits. RESULTS: (a) We found a highly significant (P<0.0001) positive correlation between E/NI values reflecting the extent of HIV-1 infection enhancement and plasma HIV-1 RNA levels. Both E/NI and HIV-1 RNA levels negatively correlated to the CD4 cell counts. (b) C-ADE was first detected just before, or concomitantly with, seroconversion in 4/4 seroconversion panels. (c) Both E/NI values and HIV-1 RNA levels significantly (P<0.001) increased during a 17 month observation period in 18 HIV-infected patients. CONCLUSION: We found strong association between the extent of the complement-mediated antibody-dependent enhancement of HIV-1 infection and the plasma viral load in HIV patients. On the basis of these findings, C-ADE correlates with HIV replication in vivo, and potentially contributes to the progression of HIV disease.     

两种HIV-1核酸定量检测试剂盒v2．0的对比研究

目的 比较罗氏COMBAS AmpliPrep／COMBAS TaqMan HIV—1 Test version 2．0（简称TagMan v2．0试剂）和生物梅里埃NucliSENS EasyQ HIV-1 v2．0（简称EasyQ v2．0试剂）两种试剂,检测艾滋病病毒Ⅰ型（HIV-1）病毒载量间的相关性和一致性。方法 对40份血浆样本采用TaqMan EasyQ v2．0试剂和EasyQ v2．0试剂分别检测HIV-1病毒载量。统计学处理采用配对t检验、回归分析和Bland—Altman分析。结果TaqMan v2．0和EasyQ v2．0试剂测得的病毒载量均值分别为（4．41±0．72）log10拷贝／mL和（3．75±0．75）log10拷贝／mL,差异有统计学意义（t=10．441,P〈0．001）。对两种试剂的检测结果进行回归分析表明,两种试剂有较强的相关性（R2=0．817）。用Bland—Altman分析比较两种试剂检测结果的差异均值,结果具有较好的一致性。结论TaqMan v2．0和EasyQ v2．0两种试剂盒在检测HIV-1病毒载量时具有较好的相关性和一致性。     

Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays

The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.     

Evaluation of a new fourth-generation microwell enzyme-linked immunosorbent assay for detection of HIV-1 subtype B and E antibodies

The recent fourth-generation enzyme-immunoassays have been used to increase the sensitivity for detecting HIV-1 antibodies and reduce the window period of HIV infection. The HIV antigens utilized in those assays were prepared from HIV-1 clade B which is different from HIV-1 subtypes circulating in Thailand. We evaluated 323 HIV-1 seropositives either B or E subtype to determine whether they were detected with the new combined anti-HIV and the p24 Ag assay. Under evaluation we found that this enzyme immunoassay manufactured by Organon Teknika showed the high sensitivity and specificity with a greater delta (delta) value with B than E subtypes samples (+15.29 vs +5.73).     

HIV-1 infection in HIV-1 enzyme-linked immunoassay seronegative patients in Kinshasa, Zaire

Serum samples of 62 African patients who had clinical manifestations of HIV-1 infection but were seronegative for HIV-1 by ELISA (Organon) were subsequently further tested by another HIV-1 ELISA test (Wellcozyme), HIV-1 IgG Western blot, HIV-1 antigen detection and HIV-2 ELISA. Patients' lymphocytes were cultured for HIV-1 and 2. Because of limited quantities of serum available all tests were not performed on all samples. Seven (26%) of 27 sera of patients meeting the WHO clinical case definition of AIDS were Western-blot-positive. In contrast, of 35 patients' sera with possible HIV related disease, only one (3%) was Western blot positive (P = 0.02) and none of 75 sera from HIV-1 ELISA (Organon) seronegative blood donors (P less than 0.01) were Western blot positive. Of 30 HIV-1 ELISA (Organon) seronegative patients tested with the HIV-1 ELISA Wellcozyme assay only one was seropositive (this patient's serum was also Western blot positive). Of 17 HIV-1 ELISA (Organon) seronegative patients tested, HIV-1 antigen was found in 1 case (6%) (this patient's serum was Western blot negative). None of the 34 patients tested by HIV-2 serology was HIV-2 seropositive. HIV-1 was isolated by culture in 3 (21%) of 14 HIV-1 ELISA seronegative patients (sera of the 3 patients were Western blot negative). In total, 12 (19%) of 62 HIV-1 ELISA (Organon) seronegative patients were found to be positive for HIV, either by Western blot HIV antigen testing or viral culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors of intermittent HIV-1 excretion in semen and efficiency of sperm processing in obtaining spermatozoa without HIV-1 genomes

OBJECTIVE: To study the risk factors for HIV-1 in semen according to the localization of HIV-1 in sperm cell fractions and to assess the efficiency of sperm processing in obtaining spermatozoa without HIV-1 genomes. METHODS: Ninety-four HIV-infected patients provided 281 paired blood and semen samples. Sperm cell separation was performed using two successive methods. HIV-1 RNA was quantified in blood and seminal plasma. HIV-1 RNA and DNA were detected in cell fractions. RESULTS: HIV-1 RNA was found in 14% of seminal plasma samples and up to 8.7% of native semen cells were positive for HIV-1 RNA and DNA. Ten seminal plasma samples had detectable RNA although blood viral load was undetectable. Antiretroviral treatment reduced the likelihood of RNA detection in seminal plasma. For semen with polynuclear cells and HIV-1 RNA in seminal plasma, the likelihood of detecting HIV-1 genomes in semen cells was increased fourfold and sixfold, respectively. In 25% of patients, HIV-1 excretion was intermittent. In the group of patients with systematic negative seminal plasma, HIV-1 genomes were detected in up to 10% of sperm cell samples. Our method of sperm processing always enabled us to obtain spermatozoa without detectable HIV-1 genomes. CONCLUSIONS: Polynuclear cells in semen are a risk factor for seminal HIV-1 excretion. Blood viral load was the only predictive factor for the intermittence of HIV-1 excretion in semen over time. Sperm processing using two successive methods was effective in obtaining spermatozoa without detectable HIV-1 genomes regardless of the viral load level in native semen.     

Comparison of the Abbott m2000 HIV-1 Real-Time and Roche AMPLICOR Monitor v1.5 HIV-1 assays on plasma specimens from Rakai, Uganda

The need for viral load (VL) monitoring of HIV patients receiving antiretroviral therapy (ART) in resource-limited settings (RLS) has become apparent with studies showing the limitations of immunological monitoring. We compared the Abbott m2000 Real-Time (Abbott) HIV-1 assay with the Roche AMPLICOR Monitor v1.5 (Roche) HIV-1 assay over a range of VL concentrations. Three hundred and eleven plasma samples were tested, including 164 samples from patients on ART ≥ six months and 147 from ART-na?ve patients. The Roche assay detected ≥400 copies/mL in 158 (50.8%) samples. Of these, Abbott produced 145 (91.8%) detectable results ≥400 copies/mL; 13 (8.2%) samples produced discrepant results. Concordance between the assays for detecting HIV-1 RNA ≥400 copies/mL was 95.8% (298/311). The sensitivity, specificity, positive predictive value and negative predictive value of Abbott to detect HIV-1 RNA ≥400 copies/mL were 91.8%, 100%, 100% and 92.2%, respectively. For the 151 samples with HIV-1 RNA ≥400 copies/mL for both assays, a good linear correlation was found (r = 0.81, P < 0.0001; mean difference, 0.05). The limits of agreement were -0.97 and 1.07 log(10) copies/mL (mean ± 2 SD). The Abbott assay performed well in our setting, offering an alternative methodology for HIV-1 VL for laboratories with realtime polymerase chain reaction (PCR) capacity.