念珠菌血症时甘露聚糖抗原及其IgG抗体检测诊断价值的临床研究

目的评价念珠菌甘露聚糖抗原（M抗原）及甘露聚糖IgG抗体（M-IgG抗体）检测诊断念珠菌血症的价值。方法收集2013年5月～2014年1月我院住院患者及健康体检人群共107例,包括念珠菌血症组（念珠菌血培养阳性患者）13例、危险因素组（临床诊断侵袭性念珠菌病或接受化疗恶性疾病、留置深静脉置管等侵袭性念珠菌病感染高危患者）63例和对照组（健康体检人群）31例。通过ELISA方法检测甘露聚糖抗原及甘露聚糖IgG抗体,比较3组人群检测阳性情况及持续时间,计算两种方法的灵敏度、特异度、阴性预测值、阳性预测值、ROC曲线下面积及Kappa值。结果白念珠菌和光滑念珠菌为念珠菌血症的主要念珠菌病原,均为5例。念珠菌血症的7／14菌株（1例患者合并2种念珠菌感染）来自重症医学科,其次为感染内科（2／14株）。除1例死亡病例外,余12例患者进行了M抗原和M—IgG抗体监测。首次M抗原检测中,4例阳性,1例可疑阳性;首次M-IgG抗体检测中,11例阳性,1例可疑阳性。经抗真菌治疗,监测14d,M-IgG抗体持续阳性时间长于M抗原。甘露聚糖抗原在诊断念珠菌血症的敏感度41．7％,特异度98．8％,阴性预测值92．4％,阳性预测值100％。甘露聚糖IgG抗体在诊断念珠菌血症的敏感度91．7％,特异度52．8％,阴性预测值100％,阳性预测值27．5％。M抗原、M抗原并M—IgG抗体作为念珠菌血症时诊断实验的ROC曲线下面积均为0．708（95％CI：0．517—0．900）,两者的Kappa值分别为0．520和0．559。结论甘露聚糖抗原在诊断念珠菌血症时的特异度较高,甘露聚糖IgG抗体在诊断念珠菌血症的敏感性较高,两者的联合检测可以适当提高检测的敏感度及特异度,有助于念珠菌血症的诊断。     

半乳甘露聚糖用于侵袭性曲霉感染实验诊断的研究现状

曲霉是导致深部真菌感染的重要条件致病菌,临床早期诊断和治疗对预后具有明确意义。ELISA检测血清中半乳甘露聚糖用于诊断曲霉感染具有良好的敏感度(64.5%～76%)和特异度(81%～98.7%),可用于曲霉感染的早期诊断及治疗的监测。     

曲霉菌半乳甘露聚糖单克隆抗体的制备及初步应用

目的:制备抗曲霉菌半乳甘露聚糖的单克隆抗体,并基于获得的抗体建立用于快速准确检测曲霉菌感染的双抗体夹心酶联免疫吸附法(ELISA),以期可用于侵袭性曲霉菌病的临床诊断。方法:提取曲霉菌半乳甘露聚糖后免疫BALB/c小鼠,筛选与制备抗曲霉菌半乳甘露聚糖的单克隆抗体,通过间接ELISA法与Western Blot方法开展单克隆抗体检测性能分析,使用获得的单克隆抗体建立双抗体夹心ELISA方法,并初步应用于曲霉菌感染血清检测。结果:获得抗曲霉菌半乳甘露聚糖单克隆抗体5株,均可特异性识别曲霉菌半乳甘露聚糖,以其中性能最佳的3C9抗体和辣根过氧化物酶标记的3C9抗体配对为基础,建立了双抗体夹心ELISA方法,通过初步评价确定该方法可应用于临床侵袭性曲霉菌病血清检测,并且该方法与现有商品化试剂盒相比检测背景值较低,可更有效区分曲霉菌感染阴阳性血清。结论:本研究筛选获得针对曲霉菌半乳甘露聚糖的特异性单克隆抗体,以该抗体为基础建立双抗体夹心ELISA方法具有潜在转化应用前景,可为侵袭性曲霉菌病的临床诊断提供支持。     

(1,3)-β-D-葡聚糖检测和半乳甘露聚糖抗原检测在侵袭性真菌病诊断中的价值探讨

目的 探讨(1,3)-β-D-葡聚糖检测和半乳甘露聚糖抗原检测在侵袭性真菌病诊断中的价值.方法 回顾性研究北京大学第一医院2008年1月～2010年7月临床疑诊侵袭性真菌病的住院患者.根据诊断标准确定是否诊断侵袭性真菌病.分析非培养诊断方法血清半乳甘露聚糖(GM试验)和血浆(1,3)-β-D-葡聚糖(G试验)的敏感度和特异度,以及二者联合应用后诊断的敏感度和特异度.结果 GM试验灵敏度为70％,特异度为84％;G试验灵敏度为50％,特异度为92％.GM试验和G试验二者联合试验时,其灵敏度和特异度分别为93％和78％.结论 GM试验和G试验均对侵袭性真菌病具有诊断价值,二者联合应用使其应用价值进一步提高.     

槐种子发育中胚乳细胞半乳甘露聚糖积累的研究

槐 ( Sophora japonica L.)开花约 60 d至种子成熟 ,为胚乳半乳甘露聚糖积累期。用组织化学方法 ,对储藏于胚乳细胞壁上的半乳甘露聚糖的形成积累进行了观察 ,结果表明 ,半乳甘露聚糖最先在邻近胚的胚乳细胞的粗面内质网的囊泡腔内形成 ,并通过细胞质膜分泌至细胞壁周围。此后 ,半乳甘露聚糖的积累逐渐向种皮方向扩展 ,及至种子成熟时 ,除糊粉层外 ,所有胚乳细胞几乎全由多糖所填充。此外 ,对半乳甘露聚糖发生部位及其积累过程的消长变化进行了讨论     

GM抗原检测对血液病患者侵袭性曲霉病的诊断价值

目的:探讨血清半乳甘露聚糖(GM)抗原检测对于血液病患者侵袭性曲霉病(invasive aspergillosis,IA)的早期诊断和疗效评价的临床意义。方法:选取137例具有侵袭性真菌病IFD高危因素患者的468份血清标本,进行GM试验,检测抗真菌治疗前后GM抗原水平的变化,同时收集患者的临床资料,进行统计学分析,并评价GM检测对于血液病患者IA的诊断价值。结果:以GM检测单次I≥1.0作为阳性界值时,本试验的敏感性、特异性、阳性预测值和阴性预测值分别为90.91%,95.65%95.24%和91.67%,与试剂盒提供的血清GM试验结果的单次I≥1.5的阳性界值相比敏感性明显提高,而特异性无明显降低,因此能够有效区分临床诊断和拟诊两个IA级别。在其他实验室检测和影像学检查的基础上加入GM试验后,IA临床诊断组的人数明显增加。诊断级别与I值总体均数的分布具有相关性,回顾性确诊IA组、回顾性可疑IA组、回顾性排除IA组的I值呈现明显的由高到低的群落分布,且三个诊断级别的I值分布范围的差异有统计学意义。根据阳性界值标准I≥1.0,GM试验阳性早于痰培养阳性平均7.73±8.71 d,也早于CT影像学证据平均6.89±8.02 d。基于GM值阳性时的抢先抗曲霉治疗组的有效率明显提高(P=0.039)。结论:血清GM抗原检测是早期诊断IA的一种有效方法,将单次I≥1.0作为阳性界值具有较好的敏感性和特异性,在阳性检出率和阳性检出时间方面较主要影像学表现和微生物学证据具有一定优势。在高危血液病伴粒细胞缺乏患者中根据GM试验阳性进行抢先抗曲霉治疗,可提高治疗有效率,监测血清GM浓度的动态变化具有评价疗效的重要价值。该研究成果对临床侵袭性曲霉病的诊断和治疗具有一定指导意义。     

烟曲霉Asp f3和Asp f4线性B细胞抗原表位最小基序鉴定

烟曲霉(Aspergillus fumigatus)是一种广泛存在于自然界中的条件致病菌,其产生的分生孢子被易感人群吸入后定植于肺部,引起3种曲霉病:致咯血的曲霉肿、致肺纤维化的变应性支气管肺曲霉病、致较高死亡率的侵袭性曲霉病。目前临床上用于诊断烟曲霉感染的血清免疫学指标有半乳甘露聚糖和1,3-β-D-葡聚糖,但特异度和灵敏度方面存在一定的局限性。Asp f3和Asp f4为烟曲霉主要抗原,在感染者血清中存在相应的循环抗体。本研究分别用兔抗Asp f3和Asp f4抗血清对其进行抗原表位扫描,鉴定了8个Asp f3和6个Asp f4最小表位基序肽。用所鉴定的最小表位基序与烟曲霉其他抗原的最小表位基序构建嵌合肽,可能有助于提高烟曲霉感染诊断的特异度和灵敏度。     

血清(1,3)-β-D葡聚糖试验与血清半乳甘露聚糖试验对马尔尼菲篮状菌病的诊断价值CSCD

目的探讨血清(1,3)-β-D-葡聚糖检测(G试验)、血清半乳甘露聚糖试验(GM试验)及联合检测对艾滋病(AIDS)患者中合并马尔尼菲篮状菌病(TSM)的诊断价值。方法回顾性研究住院艾滋病患者中有明确病原学检查确诊马尔尼菲篮状菌(TM)感染且有完善G试验及GM试验的患者45例,及同期住院AIDS患者中有完善G试验、GM试验及病原学检查,明确排除TM感染的219例患者,比较各组G试验、GM试验结果、检测时间,通过对比G试验、GM试验及联合检测方法(联合检测一:G试验和GM试验检测均为阳性为联合检测阳性,一种阳性或均阴性视为阴性结果;联合检测二:单独G试验或GM试验检测为阳性或二者均阳性,即为联合检测阳性,均阴性视为阴性结果)的敏感度、特异度、阳性预测值、阴性预测值、误诊率、漏诊率,并绘制ROC曲线。结果G试验及GM试验的敏感度及特异度差异有统计学意义(P<0.05),G试验敏感度为36.36%,特异度为78.14%,ROC曲线下面积AUC为0.602,95%的置信区间为0.539~0.662。GM试验敏感度为55.56%,特异度为96.80%,ROC曲线下面积AUC为0.819,95%CI为0.767~0.864。联合检测一敏感度27.27%,特异度99.53%,ROC曲线下面积AUC为0.634,95%CI为0.572~0.693。联合检测二敏感度65.91%,特异度76.74%,ROC曲线下面积AUC为0.713,95%CI为0.654~0.768,约登指数为0.4265。结论G试验、GM试验是AIDS合并TSM早期辅助性诊断指标,其中GM试验敏感度、特异度均高于G试验,相比病原学检查,有缩短诊断时间的优点,但不是诊断金标准。在G试验、GM试验及联合检测中,联合检测一可以提高特异度,联合检测二可以提高敏感度。     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。     

半乳甘露聚糖胶酶法改性研究进展

由于半乳甘露聚糖的水溶液在低浓度下仍具高黏性以及它的凝胶性质,因此在工业上具有很多重要的应用。半乳甘露聚糖聚糖的酶法改性主要包括脱去支链和切断主链两种方式。相对于化学改性来说,酶法改性具有易控制、反应条件温和等很多优点,因此成为改变半乳甘露聚糖分子结构以获得所需特性的最具潜力的改性方法。α－半乳糖苷酶和 β－甘露聚糖酶是半乳甘露聚糖改性和水解中最常用的酶。简要介绍了有关这两种酶的来源和新型制备菌株的近期研究概况。在医药和食品等工业中,酶法改性后的半乳甘露聚糖具有很广阔的应用前景。     

Immunodiagnosis of Dengue Virus Infection Using Saliva

Keeping in view the complications and the case fatality associated with dengue virus, several serologic tests have been developed. However, the major drawback of these serologic tests is the need for a venous blood sample obtained by invasive venipuncture. As a noninvasive alternative, saliva provides a body fluid that contains antibodies of diagnostic importance. Hence, the detection of DEN-specific IgM and IgG antibodies in serum and saliva from 80 patients was compared. Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases. None of the specimens from the healthy controls showed the presence of IgM or IgG antibodies. The detection of both IgG and IgM antibodies in saliva correlated well with the serum IgG and IgM detection by the ELISA test (r = 0.6322 and r = 0.4227). Detection of salivary IgM antibodies by ELISA showed 100% sensitivity, 70% specificity, 90.9% positive predictive value, and 100% negative predictive value. The detection of IgG in saliva proved to be a promising tool as the sensitivity, specificity, positive predictive value, and negative predictive value were found out to be 93.3%, 100%, 100%, and 83.3%, respectively. Thus, from this study we conclude that the detection of DEN-specific salivary IgG and IgM antibodies are useful markers for dengue infection.     

Usefulness of galactomannan detection in the diagnosis and follow-up of hematological patients with invasive aspergillosis

The usefulness of galactomannan detection using the Platelia Aspergillus test for the diagnosis of invasive aspergillosis was studied in 849 sera from 54 hematological patients with prolonged neutropenia, which were classified according to the risk for invasive aspergillosis. Three patients developed a proven invasive aspergillosis, one a probable invasive aspergillosis and 17 patients a possible invasive aspergillosis. Thirty-three patients showed no evidence of invasive aspergillosis. All patients with proven invasive aspergillosis had a high risk for invasive aspergillosis, while the one having probable invasive aspergillosis had intermediate risk. Detection of galactomannan in this study showed a sensitivity of 66.7% for patients with proven invasive aspergillosis and 50% for patients with proven and probable invasive aspergillosis. The specificity was 98% or higher in all groups studied. The predictive positive and negative values for patients with proven invasive aspergillosis were 66.7% and 98%, respectively. A rise in the concentration of galactomannan was observed in patients who failed to respond to the antifungal treatment. Galactomannan antigenemia preceded post-mortem histological diagnosis of invasive aspergillosis in two patients by 17 and 81 days, respectively. In conclusion, detection of galactomannan by the Platelia Aspergillus test allows for a specific and relatively sensitive diagnosis of invasive aspergillosis in hematological patients with a high and intermediate risk for invasive aspergillosis.     

血浆(1,3)-β-D葡聚糖检测对器官移植患者侵袭性真菌感染诊断价值

目的 探讨血浆(1,3)-β-D葡聚糖(BG)检测(G试验)在诊断器官移植术后合并侵袭性真菌感染(IFI)的诊断价值.方法 回顾性分析2011年1月～2012年12月130例在本院肝、肾移植中心住院疑似IFI的患者的血浆标本,进行G实验检测.其中64例最终确诊或临床诊断为IFI患者,设为IFI组,余66例为非IFI组.应用MB-80微生物动态快速检测系统和GKT-5M Set动态真菌检测试剂盒,血浆BG浓度≥10 pg/mL判定G试验阳性.采用四格表计算G试验诊断IFI的敏感度、特异度、阳性预测值和阴性预测值.对G试验结果进行受试者特征工作曲线(ROC曲线)分析,并计算曲线下面积.结果 130例患者中,IFI组中G试验阳性57/64,阳性率89.1％;阴性7例,假阴性率10.9％.非IFI组G试验阳性15/66,假阳性率22.7％.G试验阳性诊断IFI的敏感度、特异度、阳性预值和阴性预测值分别为89.1％,77.3％,79.2％和87.9％.根据G试验结果绘制ROC曲线,曲线下面积为0.875(95％ CI:0.813～0.937).结论 G试验对器官移植患者IFI具有中等诊断价值.适当提高诊断界值及重复检测可较大程度地消除假阳性.     

Biomarkers for Diagnosis of Candidal Infections

Candidemia and invasive candidiasis are associated with significant mortality, in part because of inadequate diagnostic tests. The current gold standard is blood culture, which is negative in 50% of cases. As such, development of novel diagnostics is a top priority. In Europe, studies of combined mannan antigen and anti-mannan antibody detection assays have demonstrated sensitivity of 71–100% and specificity of 86–93%. In the United States, there is more experience with (1 → 3)-β-D-glucan detection as a broad-spectrum assay for fungal pathogens. Sensitivity and specificity for diagnosing invasive candidiasis have ranged widely in studies (47–95% and 76–100%, respectively). Nucleic acid detection assays such as polymerase chain reaction (PCR) are potentially powerful tools that are now approaching the clinic. Overall, optimized PCR assays have sensitivities and specificities for candidemia and proven or probable invasive candidiasis that exceed 90%. Studies to determine the precise clinical roles of non–culture-based diagnostics are in progress.     

国产血清半乳甘露聚糖检测试剂对侵袭性肺曲霉菌病的诊断价值评估

目的：评估国产血清半乳甘露聚糖（Galactomannan,GM）检测试剂对侵袭性肺曲霉菌病的诊断价值。方法根据血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则（第四次修订版）[1]收集临床确诊侵袭性肺曲霉菌病（inva-sive pulmonary aspergillosis,IPA）、临床诊断IPA、拟诊IPA、排除IPA四组病例。采用天津贻诺琦公司酶联免疫吸附法（ELISA）试剂检测纳入的86例患者血清标本的GM浓度,分析其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）。结果86例病例中,临床诊断27例、拟诊12例、排除47例。在3种不同的阳性判断标准下,敏感性：9444%、9630%、6296%;特异性：5625%、4576%、6441%;PPV：4474%、4483%、4474%;NPV：9643%、9643%、7917%。统计学分析证实标准1（即血清GM值〉095μg/L为阳性,〈075μg/L为阴性,075~095μg/L为灰区,未将灰区加入计算）在3种判断标准中最优,故选择其为最终判断标准。结论该血清GM检测试剂盒诊断性能较好,可以用于侵袭性肺曲霉菌病的辅助诊断。     

Biomarkers for Diagnosis and Follow-Up of Invasive Candidiasis: A Brief Review of the ECIL Recommendations

The main biomarkers for rapid and non-invasive diagnosis of invasive candidiasis include 1,3-beta-D-glucan (BG), mannan antigen (Mn), anti-mannan antibodies (A-Mn) and various molecular methods. BG, Mn and A-Mn assays are standardized, and data on BG are the most extensive. For haematology and oncology population, its performance is characterised by suboptimal sensitivity but excellent specificity. For these populations, the experts from the third European Conference on Infections in Leukemia (ECIL-3) recommended BG for detection of invasive fungal infections with grading BII, and combined Mn/A-Mn detection for the diagnosis of candidemia and hepatosplenic candidiasis, with grading CII and BIII, respectively. While very promising, PCR lacks standardization and thus could not be formally recommended. There is limited and contradictory data on the performance of BG for the monitoring of outcome in patients with invasive candidiasis, although a trend of decline or increase of BG levels was found to be associated with, respectively, a successful response or failure.     

Non–Culture-Based Methods for the Diagnosis of Invasive Candidiasis

Given the limitations of current fungal diagnostics, the use of non–culture-based methods for the diagnosis of invasive candidiasis (IC) is highly warranted. The implementation of molecular diagnostic strategies could permit the timely onset of appropriate therapy and may be expected to pave the way for improved clinical outcome of IC. Polymerase chain reaction (PCR) may have higher sensitivity for the diagnosis of IC than conventional blood cultures. The detection of fungal antigens generally requires a large fungal burden, and the presence of fungus-specific antibodies may not correlate with the underlying diseases. Therefore, the combined mannan and anti-mannan antibody testing is recommended. No single test has been shown convincingly to compensate for all the limitations of culture. Real-time PCR coupled with fungal culture and/or antigen detection will likely be required to significantly ameliorate the diagnostic problems in IC.     

Standardization of ELISA IgM and IgA for immunodiagnosis of human trichinosis

An ELISA test for trichinosis using as antigen a larvae soluble fraction from Trichinella spiralis was carried out for the detection of IgM and IgA specific antibodies in 45 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. All the patients had positive serology detected by precipitin test, bentonite floculation test, indirect hemagglutination test and ELISA IgG test. The cut-off value was determined using two criteria. Criterion A was determined in each plate, using three positive controls and two negative ones; the average of the negative controls and the weakest positive control, multiplied by a 1.2 factor was, considered the cut-off value. Criterion B was determined using the average plus three standard deviations from 64 apparently healthy persons serum samples. In both cases, three serum dilutions (1:10, 1:100 and 1:500) were used. The sensitivity of ELISA IgM was 100.0, 93.3 and 82.2% using serum dilutions of 1:10, 1:100 and 1:500 respectively (criterion A) and 100.0, 97.8 and 95.6% for the same dilutions (criterion B), whereas the values for ELISA IgA were: 100.0, 91.1 and 86.7% (criterion A) and 100.0, 100.0 and 91.1% (criterion B). In order to find out the specificity of ELISA IgM and ELISA IgA, additional 118 serum samples from individuals with other parasitoses, such as cysticercosis (18) hydatidosis (39), fascioliasis (12), toxocariasis (30), Chagas' disease (12) and individuals with non-specific eosinophilia (7), were also tested. ELISA IgM presented a specificity of 92.3, 93.4 and 97.3% (criterion A) and 96.2, 97.8 and 97.8% (criterion B) whereas the results for ELISA IgA were 97.8, 98.9 and 99.4% (criterion A) and 98.4% for the 1:10 and 1:100 dilutions and 100.0% for the 1:500 dilution (criterion B). The positive predictive values of ELISA IgM were 76.3, 77.8 and 88.1% (criterion A) and 86.5, 91.7 and 91.5% (criterion B) whereas the negative ones were 100.0, 98.3 and 95.7% (criterion A) and 100.0, 99.4 and 98.9% (criterion B). The positive predictive values of ELISA IgA were 91.8, 95.3 and 97.5% (criterion A) and 93.8, 93.8 and 100.0% (criterion B) whereas the negatives ones were: 100.0, 97.8 and 96.8% (criterion A) and 100.0, 100.0 and 97.8% (criterion B). The use of ELISA IgM and ELISA IgA in the immunodiagnosis of trichinosis is discussed.     

Glycosylphosphatidylinositol-anchored fungal polysaccharide in Aspergillus fumigatus

Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C18-phytosphingosine and monohydroxylated lignoceric acid (2OH-C(24:0) fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN. The galactomannan polymer is linked to the GPI structure through the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.