类鼻疽伯克霍尔德菌误鉴定1例

正伯克霍尔德菌(Burkholderia)是一种革兰阴性非发酵菌,其中与人类疾病相关的病原菌主要包括洋葱伯克霍尔德菌复合群(B.cepacia complex)、类鼻疽伯克霍尔德菌复合群(B.pseudomallei complex)等。而类鼻疽伯克霍尔德菌复合群可进一步分为类鼻疽伯克霍尔德菌、鼻疽伯克霍尔德菌(B.mallei)、泰国伯克霍尔德菌(B.thailandensis)、俄克拉何马伯克霍尔德菌(B.oklahomensis)和汉普蒂社伯克霍尔德菌(B.humptydooensis)5个种[1],其中除泰国伯克霍尔德菌毒力较     

泰国伯克霍尔德菌错误鉴定1例

目的鉴定1例曾被错误鉴定为类鼻疽伯克霍尔德菌的泰国伯克霍尔德菌。方法对1例肺脓肿合并败血症患者的痰液和血液标本进行细菌分离培养、生化鉴定、分子生物学鉴定和药敏试验鉴定。结果培养出革兰阴性短小杆菌;生化鉴定特征符合泰国伯克霍尔德菌;测序结果显示与泰国伯克霍尔德菌相似度达100%,与类鼻疽伯克霍尔德菌相似度为99%;全基因组测序结果显示该菌与泰国伯克霍尔德菌E444具有最近的亲缘关系。药敏试验结果显示该菌对亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、头孢他啶、甲氧苄啶/磺胺甲噁唑敏感。结论成功鉴定了1例泰国伯克霍尔德菌。     

Vitek 2 Compact GN卡用于不同培养基生长类鼻疽伯克霍尔德菌鉴定结果分析

目的评估不同培养基生长类鼻疽伯克霍尔德菌在Vitek 2 Compact中的鉴定结果。方法收集2010年6月至2017年5月海南省人民医院分离的类鼻疽伯克霍尔德菌127株,分别接种于哥伦比亚羊血平板(CBA)、麦康凯平板(MAC)和流感嗜血巧克力平板(CHA)上,以多位点序列分型(MLST)为金标准,评估Vitek 2 Compact VT2.R7.01版GN卡对不同培养基上生长的类鼻疽伯克霍尔德菌的鉴定准确性。结果 CBA平板上生长的类鼻疽伯克霍尔德菌鉴定准确率最高,为98.4%,2株被错误鉴定为洋葱伯克霍尔德菌;MAC次之,准确率为94.5%,其中4株被错误鉴定为洋葱伯克霍尔德菌,2株为伯克霍尔德菌某种,1株被错误鉴定为铜绿假单胞菌;CHA鉴定准确率最低,为91.3%,其中4株被错误鉴定为洋葱伯克霍尔德菌,2株为伯克霍尔德菌某种,5株无鉴定结果。结论 Vitek 2 Compact GN卡对不同培养基上类鼻疽伯克霍尔德菌鉴定准确率不同,建议优先选择CBA平板上菌落。     

环介导恒温扩增结合纳米生物传感器快速检测类鼻疽伯克霍尔德菌的方法及临床应用

  目的   建立一种环介导恒温扩增（LAMP）结合纳米生物传感器（LFB）快速检测类鼻疽伯克霍尔德菌的快速方法,为临床检测提供依据。  方法   针对类鼻疽伯克霍尔德菌的Ⅲ型分泌系统基因设计LAMP特异性引物,建立LAMP方法,通过纳米生物传感判断结果,并应用临床样本对该方法进行评价。  结果   LAMP-LFB检测的最佳扩增条件为67 ℃反应40 min,对类鼻疽伯克霍尔德菌纯培养物DNA灵敏度达到100 fg,对258株菌株进行检测,包括227株类鼻疽伯克霍尔德菌和31株非类鼻疽伯克霍尔德菌,特异性为100%。 应用于46个临床样本检测时,与传统分离培养方法相比,达到100%的诊断准确率。 LAMP-LFB整个检测过程可在1 h内完成,包括15 min DNA制备,40 min LAMP扩增,2 min的LFB结果判读。  结论   本研究建立的LAMP-LFB技术是一种快速、灵敏、特异的检测类鼻疽伯克霍尔德菌的方法,可作为类鼻疽基础、现场和临床实验室诊断的潜在工具。     

不同培养基上的类鼻疽伯克霍尔德菌Vitek 2 compact GN卡鉴定结果分析

目的 评估不同培养基上类鼻疽伯克霍尔德菌(Burkholderia pseudomallei,BP)Vitek 2 compact的鉴定结果。方法 收集2010年1月-2017年5月间海南省人民医院类鼻疽伯克霍尔德菌127株,分别将其接种于三种不同培养基上,以多位点序列分型(MLST)为金标准,评估不同培养基上生长的类鼻疽伯克霍尔德菌,采用Vitek 2 compact VT2.R7.01版的 GN卡进行鉴定并比较其准确性。结果 哥伦比亚羊血平板(CBA)上的类鼻疽伯克霍尔德菌鉴定准确率最高,为98.4%,2株被鉴定为洋葱伯克霍尔德菌；麦康凯平板(MAC)次之,准确率为94.5%,其中4株被错误鉴定为洋葱伯克霍尔德菌,2株无法鉴定到种,1株被错误鉴定为铜绿假单胞菌；流感嗜血巧克力平板(CHA)鉴定准确率最低,为91.3%,其中4株被错误鉴定为洋葱伯克霍尔德菌,3株无法鉴定到种,4株鉴定不出结果。结论 该研     

哌拉西林他唑巴坦对类鼻疽伯克霍尔德菌的体外抗菌活性评估

目的评估哌拉西林他唑巴坦体外抗类鼻疽伯克霍尔德菌的活性,为临床用药提供理论依据。方法采用E-test法,对临床分离的109株类鼻疽伯克霍尔德菌进行哌拉西林他唑巴坦最低抑菌浓度(MIC)的检测,计算MIC_(50)和MIC_(90),与有CLSI敏感性判断标准的亚胺培南、强力霉素、复方磺胺甲噁唑、头孢他啶、四环素、阿莫西林克拉维酸及有EUCAST敏感性判断标准的美罗培南药敏检测结果进行比较。结果所检测的8种抗菌药物的MIC_(50)分别为哌拉西林他唑巴坦0.25/4μg/mL、亚胺培南0.5μg/mL、美罗培南0.5μg/mL、强力霉素0.5μg/mL、复方磺胺甲噁唑1μg/mL、头孢他啶1μg/mL、四环素2μg/mL、阿莫西林克拉维酸2μg/mL;MIC_(90)分别为哌拉西林他唑巴坦0.5μg/mL、亚胺培南0.5μg/mL、美罗培南1μg/mL、复方磺胺甲噁唑2/38μg/mL、头孢他啶2μg/mL、强力霉素2μg/mL、四环素4μg/mL、阿莫西林克拉维酸4μg/mL。所测有CLSI判断标准的6种抗菌药物敏感性显示,亚胺培南、头孢他啶、阿莫西林克拉维酸的敏感性均为100%,其他药物敏感性依次为强力霉素99.08%,复方磺胺甲噁唑95.41%,四环素94.49%。结论哌拉西林他唑巴坦体外对类鼻疽伯克霍尔德菌的MIC_(50)和MIC_(90)均低于其他7种抗菌药物,表明其在体外对该菌有较强的抗菌活性,结合其体内药代学特点,临床可尝试使用以获取体内对该菌药效学的评估。     

类鼻疽伯克霍尔德菌在青瓜和生菜幼苗中的定植情况研究

  目的  研究类鼻疽伯克霍尔德菌在青瓜、生菜幼苗中的定植情况。  方法  用海南省三亚市人民医院保存的类鼻疽伯克霍尔德菌BP47、BP56、BP66分别感染青瓜、生菜无菌幼苗根部和叶片，从被感染幼苗根部与叶片的研磨液中分离该菌。 采用菌落平板计数检测被感染第1、3、5、7天的幼苗叶片中类鼻疽伯克霍尔德菌的增殖情况，绘制增殖曲线。 通过透射电镜对幼苗叶片中的类鼻疽伯克霍尔德菌进行定位。  结果  从被感染的青瓜、生菜幼苗中分离到了类鼻疽伯克霍尔德菌，而且该菌可以在幼苗中增殖，菌量随着时间推移而增加。 通过透射电镜在青瓜、生菜幼苗叶片中也观察到了该菌定植。  结论  类鼻疽伯克霍尔德菌可以在一些生食类蔬菜（青瓜、生菜）幼苗中定植，为探究其新的感染方式提供了实验依据。     

急性败血症型类鼻疽合并糖尿病病人的护理

<正>类鼻疽(melioidosis)是由类鼻疽伯克霍尔德菌感染引起的一种人畜共患传染病,该病主要分布在热带和亚热带地区。我国类鼻疽疫源地主要位于北纬25°以南的亚热带地区,包括海南、广东、广西、台湾等地均有散发病例报道[1]。类鼻疽伯克霍尔德菌为超级耐药菌,诊治该类病人时,一旦发生医院感染或医院感染暴发,后果不堪设想。我科于2012年11月收治了我院     

鼻疽诺卡菌NFA47650蛋白的鉴定与免疫原性研究

  目的  构建鼻疽诺卡菌NFA47650蛋白原核表达载体,诱导表达及纯化NFA47650蛋白,并分析其免疫原性。  方法  根据鼻疽诺卡菌nfa-47650基因序列设计并合成引物,通过PCR扩增基因片段并构建pET30a（+）表达载体,导入BL21大肠埃希菌诱导表达并纯化。 制备NFA47650小鼠抗血清,通过Western Blot对NFA47650蛋白进行亚细胞定位和免疫原性研究。  结果  成功构建鼻疽诺卡菌NFA47650蛋白的原核重组表达载体,该蛋白在BL21大肠埃希菌中以包涵体的形式高效表达。 Western Blot结果显示,NFA47650蛋白主要存在于菌体培养上清中,能够被鼻疽诺卡菌抗小鼠和兔血清特异性识别。 另外,该蛋白与巴西诺卡菌和盖尔森基兴诺卡菌抗小鼠血清均可发生血清交叉反应。  结论  鼻疽诺卡菌NFA47650蛋白是一种分泌性蛋白,可以与不同种的诺卡菌抗血清发生免疫反应,引起免疫应答,是一种重要的免疫显性蛋白。     

类鼻疽伯克霍尔德菌致血流感染1例

正类鼻疽病(melioidosis)是由类鼻疽伯克霍尔德菌所导致的地方性传染病,主要流行于澳大利亚中部、东南亚等热带和亚热带地区。该菌常存在于疫区水、土壤、粪便及尸体中,可经破损皮肤接触含有致病菌的水或土壤而感染,也可经呼吸道感染[1]。2014年10月,本组从1例高烧患者血液标本中分离出类鼻疽伯克霍尔德菌,报道如下。1资料与方法1.1一般资料患者,男,39岁,因发热、右下肢肿痛入院。     

Particle agglutination assay for sm antibodies using purified sm antigen

Sm antibodies are specific serum markers for the diagnosis of systemic lupus erythematosus (SLE) and are identified by immunodiffusion (ID), counterimmunoelectrophoresis (CIE), and passive hemagglutination (PHA) in the clinical laboratories. These current methods have certain disadvantages of sensitivity, specificity, and complexity. In this report, Sm antigen free from other nuclear antigens was purified. Synthetic particles were coated with Sm antigen and used to test for Sm antibodies in patient's sera. The particle agglutination test was specific for only Sm antibodies among different antinuclear antisera from patients with systemic rheumatic diseases. The particle agglutination test had the same sensitivity and specificity as the enzyme-linked immunoassay (ELISA) test for Sm and RNP antibodies. The correlation coefficient between the ELISA quantitation and the titers obtained by particle agglutination was 0.82. The particle agglutination test can be used for rapid screening and titer determination of anti-Sm antibodies.     

结核分枝杆菌Ag85A/MPT-64融合基因DNA疫苗的构建及免疫研究中ELISPOT技术的应用

目的构建结核分枝杆菌蛋白Ag85A/MPT-64融合基因为基础的DNA疫苗,并利用ELISPOT技术检测其免疫原性。方法利用PCR和基因克隆技术从结核分枝杆菌H37Rv基因组中扩增出Ag85A/MPT-64编码基因,构建真核表达载体,用酶切和双向DNA测序进行鉴定。鉴定正确的阳性克隆重组质粒,肌注免疫小鼠,3周后用ELISPOT法检测抗体滴度。结果 构建到真核载体中的结核分枝杆菌H37Rv株Ag85A/MPT-64融合基因经序列测定证实无突变。ELISPOT法检测几何平均滴度为1∶1 000。结论以Ag85A/MPT-64融合基因为基础的DNA疫苗的成功构建和表达以及ELISPOT技术的应用,为进一步研究其在结核病与肿瘤防治方面的作用奠定了基础。     

Anti-A and anti-B titers in pooled group O platelets are comparable to apheresis platelets

BACKGROUND: Although uncommon, acute hemolytic transfusion reactions (AHTRs) have been reported after transfusion of group O single‐donor apheresis platelets (SDPs) to group A, B, and AB recipients. Current methods for identifying “high‐titer” SDPs include tube and gel methods. The risk of a high‐titer unit is considered low with group O, poststorage, pooled platelet concentrates (PPLTs); however, data regarding anti‐A and anti‐B titers in PPLTs are lacking. STUDY DESIGN AND METHODS: Anti‐A and anti‐B titers were determined in 185 PPLTs by direct agglutination using manual gel and tube methods. PPLTs tested included 124 group O PPLTs, 25 group A PPLTs, 26 group B PPLTs, and 10 PPLTs containing a mix of either groups O plus A or groups O plus B (mixed PPLTs). The reciprocal of the highest dilution giving macroscopic agglutination was considered the agglutinin titer. RESULTS: Mean anti‐A and anti‐B titers in group O PPLTs were, respectively, 16 and 8 by tube and 64 and 32 by gel (p < 0.0001). Gel titers were one to two dilutions higher than tube and sensitive to reagent red cell lots. With the use of at least 64 as a critical titer, 60 percent of group O PPLTs tested by gel would be considered high‐titer. In mixed PPLTs, the addition of one non‐group O PLT significantly decreased or neutralized the corresponding anti‐A or anti‐B (p < 0.0001). CONCLUSION: Anti‐A and anti‐B titers in group O PPLTs are comparable to those reported in group O SDPs and significantly lower than titers reported in AHTR. A critical direct agglutinin titer of 64 for identifying high‐titer units by gel is too low and should be increased to 128 or higher.     

Antibodies to histo-blood group substances A and B: agglutination titers, Ig class, and IgG subclasses in healthy persons of different age categories

Isotypes and IgG subclasses of ABO antibodies from sera of 235 healthy blood donors were determined by an enzyme-linked immunosorbent assay (ELISA). Synthetic A and B trisaccharide-bovine serum albumin glycoconjugates were used for coating and monoclonal antibodies for the detection of heavy chain isotypes. Hemagglutination titers were determined in addition. Blood donors were between 20 and 67 years old, and at least 10 sera per 10-year age category and ABO blood group were included in this study. Antibody concentrations were expressed as a percentage of an internal standard, and sera with subclass-restricted anti-A and/or anti-B (anti-A/B) responses were used to normalize the ELISA values of IgG subclasses. A good correlation between the sum of the four subclasses and the total anti-A/B IgG values (rs = 0.81 for anti-A and 0.84 for anti-B) was obtained. IgG1 and IgG2 were the most predominant subclasses, but were found in various proportions in different individuals. Donor-to-donor variation exceeded age-related changes for all measured parameters. The correlation of anti-A IgM, IgG, IgA, and their sum with the agglutination titers was significant and revealed rs values of 0.70, 0.65, 0.65, and 0.80, respectively. For anti-B as well, the correlation of ELISA values with the agglutination titer was best when all three isotypes were added. We conclude that anti-A/B IgA, together with IgM and IgG, substantially contributes to the agglutination reaction. Potentially autoreactive antibodies were detected in sera of blood groups A, B, and AB.     

Development of a novel ELISA for detection of anti-A and anti-B antibodies in recipients of ABO-incompatible living donor liver grafts

The survival rate in ABO-incompatible (ABO-I) liver transplantation was much lower than that in ABO-compatible recipients for the early experiences. It is therefore essential to develop the precise and fast measurement of anti-A and anti-B antibodies (Abs) to prevent humoral rejection in ABO-I liver transplantation. Agglutination titer has been the standard method to measure these Abs, but the interpretation of the results is subject to bias. Here, we have developed an objective and quantitative enzyme-linked immunosorbent assay (ELISA) to measure anti-A and anti-B Abs. This test requires only a small amount (10 microl) of recipient's serum. We applied the newly developed ELISA to monitor living donor liver transplant recipients and investigated the correlation between ELISA and agglutination titer. The Spearman's correlation coefficient for Abs ranged from 0.461 to 0.812. Moreover, in one case of humoral rejection, the increase of Abs was detected by ELISA one day earlier than by the agglutination titer. In conclusion, our ELISA method proved useful to detect an increase of anti-A and anti-B Abs titers at an early stage, thereby contributing to a prompt treatment of humoral rejection due to ABO-I.     

抗sAPRIL抗体的制备及细胞增殖抑制功能分析

本研究旨在通过免疫小鼠制备抗人可溶性增殖诱导配体(soluble a proliferation-inducing ligand,sAPRIL)的多抗,检测其对人sAPRIL促细胞增殖活性的抑制作用.以丧失天然生物学活性,但保留良好免疫原性的sAPRIL 重组突变体蛋白为免疫原,加上佐剂免疫人化SCID小鼠,6周后取血清,用间接ELISA法测定抗体滴度,Western blot测定抗体特异性,MTT法检测所获抗血清抑制sAPRIL促Raji和Jurkat细胞增殖的作用.结果表明,免疫小鼠,获得特异性的小鼠抗人sAPRIL抗体,抗血清滴度达到1:640.功能实验显示,抗sAPRIL血清可明显降低sARPIL刺激Raji和Jurkat细胞增殖的作用(p<0.05).结论:成功制备了抗人sAPRIL多克隆抗体,该抗体可以在体外抑制sAPRIL促Raji和Jurkat细胞增殖活性.     

Enzyme-linked immunosorbent assay of antibodies in human sera to streptococcal DNase B

An ELISA method for determining the streptococcal anti--DNase B titers in human sera is presented. The details of this technique, a method for converting A493 readings into titers, and a standardization procedure are discussed. Anti--DNase B titers of 20 sera determined by ELISA were compared with titers determined by the microtechnique. A correlation coefficient of 0.96 between the two methods was obtained. The reproducibility of the ELISA method was established by comparing titers obtained from two separate determinations on the 20 sera. Twenty-four pairs of acute and convalescent sera were assayed for anti--DNase B titers by both ELISA and microtechnique to compare the ability of the two techniques to identify significant titer rises. The ELISA was as specific and possibly more sensitive than the microtechnique in identifying significant anti--DNase B titer rises. This ELISA method is simple and rapid and has the potential for automation.     

重组PV-杀白细胞素LukF-PV蛋白多克隆抗体的制备及鉴定

目的制备重组金黄色葡萄球菌PV-杀白细胞素LukF-PV蛋白多克隆抗体并鉴定。方法纯化获得重组蛋白LukF-PV,免疫新西兰白兔,收集多抗血清,ELISA法测定抗体滴度,Western blotting法鉴定免疫活性。结果成功免疫新西兰白兔获得多抗血清,ELISA测定其效价为1∶103,Western blotting鉴定其能识别重组蛋白和金葡菌PVL蛋白。结论成功制备出重组金黄色葡萄球菌PV-杀白细胞素LukF-PV蛋白多克隆抗体,并进行鉴定,为建立产杀白细胞素的金黄色葡萄球菌的快速、廉价的免疫学检测方法奠定基础。     

Stimulation of the immune response by dimethylglycine, a nontoxic metabolite.

The immunomodulating capacities of dimethylglycine (DMG) were examined in a rabbit model. Female New Zealand white rabbits were immunized on day 0 and were given booster inoculations on day 9 with either killed influenza virus or Salmonella typhi vaccine. Experimental animals were force fed 20 mg/kg body weight of DMG daily beginning 14 days prior to the first inoculation and continuing throughout the experiment. Control animals were force fed daily only distilled water. Blood was obtained on day 0, day 9, and day 30. Hemagglutination inhibition assays showed a more than fourfold increase in mean antibody titer to influenza antigen in the DMG-treated animals (p = 0.0006) after the first inoculation, and a fourfold increase in mean titer after the booster inoculation (p = 0.1000). A standard agglutination test for Salmonella typhi O (somatic) and H (flagella) antigens was performed on all sera from animals receiving the typhoid vaccine. Mean antibody titers to the O antigen were significantly higher (more than threefold) after the first inoculation (p = 0.0302) and more than fivefold higher after the booster inoculation (p = 0.0047) in DMG-treated animals. Mean antibody titers to the H antigen were also higher in DMG-treated animals compared with controls after both the first and second inoculation. Lymphocyte transformation assays on cells taken from DMG-treated animals immunized with the influenza vaccine showed a tenfold increase in mean proliferative response (p = 0.0024). Lymphocytes from DMG-treated animals immunized with the typhoid vaccine showed a fourfold increase (mean values) in thymidine uptake (p = 0.0180). No toxicity or adverse effects were observed at any time during the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE QUANTITATIVE DETERMINATION OF INFLUENZA VIRUS AND ANTIBODIES BY MEANS OF RED CELL AGGLUTINATION

1. The agglutination titer for chicken red cells of freshly prepared or carefully stored suspensions of PR8 influenza virus, that is to say virus of maximum pathogenicity, was found to be proportional to the mouse lethal titer of the same preparations. 2. The agglutination titer of infected allantoic fluid procured in a standard way is relatively constant, regardless of the influenza strain used and its pathogenicity for mice. 3. Virus preparations inactivated by heat or storage may retain their agglutinating power. 4. Certain animal sera contain a partially heat-labile factor which, in low dilution, inhibits the agglutination of chicken red cells by influenza A and influenza B viruses. 5. The agglutination inhibition test, using ferret and human sera, gives qualitative data regarding influenza antibodies which are similar to the information obtained on the same sera by means of the virus neutralization test. 6. There is a definite relationship between the agglutination inhibition titer and the virus neutralization titer of a serum. On a logarithmic scale of both variables, this relationship is essentially linear within the range investigated. 7. The agglutination inhibition titer of immune ferret serum is inversely proportional to the amount of virus used in the test.