疟原虫检查的微量浓集法

传统疟原虫检查法 ,薄血膜虫体形态完整 ,易辩认 ,但虫量太少 ,易漏检 ,且检查费时 ,厚血膜则溶血时间太长 ,虫体形态有所改变 ,很难辩认。而传统浓集法虽显著提高了检出率 ,有利于虫种鉴别 ,但需抽取静脉血 ,操作较繁琐 ,不适于大规模普查 ,且其镜检范围只限于离心后的表层红细胞 ,易漏诊。微量浓集法则克服上述缺点 ,该法根据受间日疟侵袭的红细胞比正常红细胞轻 ,而受恶性疟、三日疟、卵形疟侵袭的红细胞则比正常红细胞略重的原理 ,用毛细管采血 ,通过高速离心分层 ,分别取离心后的最底层血和与白细胞交界的表层红细胞 ,涂片染色镜检。现…     

采用离心浓集法提高乙型肝炎病毒表面抗原检出率的可行性研究

目的 探讨离心浓集法预处理血清标本对提高乙型肝炎血清学标志物乙型肝炎表面抗原(HBsAg)检出率的可行性研究.方法 收集广州市内各大医院2009年52例HBsAg阳性者和疑似乙型肝炎感染者血清标本,分别用酶联免疫吸附试验(ELISA) 和化学发光免疫分析法(CLIA)测定HBsAg.再以PEG6000为沉淀剂,将标本进行高速离心浓集(30 000 r/min)25 ℃,40 min,取沉淀用TE Buffer溶解,采用上述相同方法分别进行测定,比较离心前后其HBsAg 的S/CO值与HBsAg定量值的上升情况,用SPSS统计学软件对数据进行非参数检验,分析其差别是否有统计学意义.结果 健康对照组与空白对照经高速离心处理前后的HBsAg 检测结果差异均无统计学意义(P>0.05),而疑似HBV感染组和HBV感染组患者血清经高速离心预处理后的测定结果均明显高于处理前(P<0.01).结论 离心浓集法预处理对于血清中有低水平乙型肝炎病毒颗粒的感染者有效,血清中的病毒颗粒可通过高速离心处理后得到浓缩,提高临床实验室常规诊断手段的灵敏度的范围,使隐匿型乙型肝炎的检出率有所提高.     

介绍一种疟原虫的血液浓集法

介绍一种疟原虫的血液浓集法朱卫中（龙泉市人民医院，浙江龙泉３２３７００）血膜涂片查找疟原虫是疟疾确诊的方法和依据，通常采用薄血膜法和厚血膜法。薄血膜法的血膜经染色后原虫形态结构完整、清晰，可根据各发育阶段的形态特征辨别原虫类型，适用于临床疟疾类型的诊...     

用简易低渗浓集法检查胸腹水中癌细胞

临床上送来检查胸水、腹水等穿刺液中的癌细胞时,经常会遇到血性穿刺液。因为大量红细胞的存在,不但影响了癌细胞的检出,而且涂片不易制作,常致涂片在固定及染色水洗过程中脱片,使观察结果受到限制和影响。几年来,我们利用癌细胞和红细胞耐低渗能力的显著性差异。自制了一种低渗溶液进行低渗溶血浓集后制片。此法不但消除了,红细胞的影响,又达到了浓集的目的,从而提高了癌细胞的检出率。     

快速浓集法与直接涂片法抗酸杆菌检测结果的比较

痰结核分枝杆菌检查不仅是确诊肺结核的重要手段,也是制订治疗方案、考核疗效的主要依据。我们对1 014例患者的痰标本分别用直接涂片法和快速浓集法检测痰结核分枝杆菌,用结核病病原学诊断的“金标准”结核分枝杆菌的分离培养作对照,对结果进行了比较分析。一、材料和方法1.病例2005年1月至2006年6月来我中心就诊的肺结核患者1 014例,其中男698例,女316例,年龄11~80岁。2.标本收集留痰时采用蜡纸盒为容器,每份痰量不少于3 mL。每位患者先取即时痰(就诊时深呼吸后咳出的痰液)作涂片检查,如阴性,再嘱患者留取晨痰(患者晨起立即用清水嗽口后咳…     

快速浓集法与直接涂片法抗酸杆菌检测结果的比较

痰结核分枝杆菌检查不仅是确诊肺结核的重要手段,也是制订治疗方案、考核疗效的主要依据。我们对1014例患者的痰标本分别用直接涂片法和快速浓集法检测痰结核分枝杆菌,用结核病病原学诊断的“金标准”结核分枝杆菌的分离培养作对照,对结果进行了比较分析。     

浓集法检查痰液含铁血黄素

浓集法检查痰液含铁血黄素褚丽林九玲马玉臻侯云霞（山东益都中心医院，山东青州２６２５００）关键词含铁血黄素α－糜蛋白酶浓集法痰液痰液含铁血黄素的检查，原法是玻片法，将痰液直接涂片染色镜检。但由于痰液粘稠度高，阳性率较低。本文介绍的浓集法，明显提高了阳性...     

浓集法检查人血中疟原虫

本文作者采用Percoll连续密度梯度离心法浓集血中疟原虫.其方法为取一份1.5M NaCl加9份Percoll即成100%Percoll混合液,密度近似1.1300g/ml.取8.5份100% Percoll液加1.5份生理盐水混合即成85%(V/V)实验溶液.用一支无菌滴管从含EDTA或肝素抗凝管内吸取1.0ml含疟原虫全血放入含有85%Percoll 9ml 聚乙烯试管内(内径16mm)使成一血液层,30000g10C离心40分钟.离心后血液被分成6层,从上而下分别为S_1(血     

疟原虫血液浓集法的改进

疟原虫血液浓集法的改进韦林（广东省恩平市圣堂中心卫生院，广东恩平５２９４４１）《临床检验杂志》１９９４年第３期“介绍一种疟原虫的血液浓集法”（以下简称原法）一文觉得还不够完善。原法用抗凝血离心后只取红细胞表层做镜检是不够的，这仅适用于红细胞体积显著增...     

浓集法检查人血中疟原虫

本文作者采用Percoll连续密度梯度高心法浓集血中疟原虫。其方法为取一份1．smol／LNaCI加9份Percoll即成100％Percll混合液，密度近似1．13009／ml，取8．5份100％Percoll液加1．5份生理盐水混合即成85％（V／V）实验溶液。用一支无菌滴管从含EryTA或肝素抗凝管内吸取1．0ml含疟原虫全血放人含有85％Percollgml聚乙烯试管内（内经16mm）使成一血液层，3000910oC离心40分钟，离心后血液被分成6层，从上而下分别为SI（血浆和血小板）、S：（白细胞）、S3（网织红细胞）、S‘（间日疟感染红细胞）、S3（恶性疟感染成熟红细胞）、s‘…     

多重巢式PCR检测恶性疟和间日疟原虫

目的建立鉴别诊断恶性疟原虫(P.f)和间日疟原虫(P.v)的多重巢式PCR法。方法针对P.f、P.v 18S rRNA基因设计外引物和内引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重巢式PCR,并检测54例疑似疟疾临床标本,以镜检法为金标准评价敏感性和特异性等指标。结果该方法可扩增出162 bp(P.f)和112 bp(P.v)基因片段,并能检出混合感染。该方法检测P.f,敏感性为87.50%、特异性为63.33%;检测P.v,敏感性为69.23%、特异性为68.29%。结论所建立的多重巢式PCR方法能可靠诊断疟疾并鉴别虫种,敏感性高,在混合感染的诊断方面具有优越性。     

复合PCR系统检测间日疟原虫的应用研究

应用一种简易、快速的复合 PCR扩增系统检测间日疟原虫 (P.v)及混合感染。方法 :以间日疟原虫和恶性疟原虫 (P.f)小亚单位核糖体核糖核酸基因 (SSUr DNA)特定片段为靶基因 ,设计并合成引物 ,建立复合 PCR扩增系统。采用煮沸法快速制备 DNA模板研究该系统的敏感性和特异性 ,并用于临床血样的检测。结果 :从间日疟原虫和恶性疟原虫感染血样中分别扩增出分子量大小为 70 5bp和 575bp的特定扩增带。而食蟹猴疟原虫、诺氏疟原虫及正常人血样均无扩增带出现。单独检测 P.v敏感度达到 0 .1 5× 1 0 -5(约 7个原虫 /μl全血 ) ,在 P.f存在的情况下检测 P.v敏感度为 0 .1 5× 1 0 -4 。检测 1 32例疟区门诊病人冻存血标本 ,1 0 4份与镜检法结果相同。并发现 1 4份混合感染和 5份为镜检虫种鉴别失误。结论 :该系统敏感性高 ,特异性强 ,操作简单 ,并可在一次扩增中同时检出间日疟原虫和恶性疟原虫 ,具有一定的推广应用价值。     

High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax

Background: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger‐prick blood and venipunctured frozen liquid blood. Methods: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa‐stained thin and thick smears from each individual to determine the malaria‐positive or ‐negative specimens, spotting two to three drops of finger‐prick blood onto the DBC filter paper, and collecting a 2‐ml venous blood sample into EDTA‐contained test tube from each individual. A semi‐nested Multiplex PCRtechnique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. Results: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM‐PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. Conclusions: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger‐prick blood samples is a trustable and useful facilitator particularly in remote malaria‐endemic areas. J. Clin. Lab. Anal. 25:185–190, 2011. © 2011 Wiley‐Liss, Inc.     

Management of relapsing Plasmodium vivax malaria

Introduction: Relapses are important contributors to illness and morbidity in Plasmodium vivax and P. ovale infections. Relapse prevention (radical cure) with primaquine is required for optimal management, control and ultimately elimination of Plasmodium vivax malaria. A review was conducted with publications in English, French, Portuguese and Spanish using the search terms ‘P. vivax’ and ‘relapse’.

Areas covered: Hypnozoites causing relapses may be activated weeks or months after initial infection. Incidence and temporal patterns of relapse varies geographically. Relapses derive from parasites either genetically similar or different from the primary infection indicating that some derive from previous infections. Malaria illness itself may activate relapse. Primaquine is the only widely available treatment for radical cure. However, it is often not given because of uncertainty over the risks of primaquine induced haemolysis when G6PD deficiency testing is unavailable. Recommended dosing of primaquine for radical cure in East Asia and Oceania is 0.5 mg base/kg/day and elsewhere is 0.25 mg base/kg/day. Alternative treatments are under investigation.

Expert commentary: Geographic heterogeneity in relapse patterns and chloroquine susceptibility of P. vivax, and G6PD deficiency epidemiology mean that radical treatment should be given much more than it is today. G6PD testing should be made widely available so primaquine can be given more safely.     

间日疟原虫乳酸脱氢酶GST融合表达载体的构建及表达

目的构建间日疟原虫乳酸脱氢酶（PvLDH）融合表达载体,并表达PvLDH的GST融合蛋白。方法将PvLDH基因片段克隆到表达载体pGEX-4T-1中,构建PvLDH/pGEX-4T-1融合表达载体,IPTG诱导表达目的基因,SDS-PAGE电泳分析表达产物,Western blot检测其抗原性。结果成功构建了PvLDH/pGEX-4T-1融合表达系统,在大肠杆菌BL21中以包涵体形式高效表达,表达产物能与恶性疟原虫和间日疟原虫感染患者血清反应,而不与正常人血清反应。结论间日疟原虫LDH蛋白在大肠杆菌中获得高效表达,表达产物具有良好的抗原性。     

The mechanisms of resistance to antimalarial drugs in Plasmodium falciparum

Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia. It causes severe fever or anaemia that leads to more than a million deaths each year. The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions. The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs. Plasmodium falciparum drug-resistant malaria originates from chromosome mutations. Analysis by molecular, genetic and biochemical approaches has shown that (i). impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii). one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii). the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv). treatment with sulphadoxine-pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD). The mechanisms of resistance for amino-alcohols (quinine, mefloquine and halofantrine) are still unclear. Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries. Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug-resistance encountered in the forests of South-east Asia and South America. In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria. Nevertheless, resistance levels may differ according to place and time. In vivo and in vitro tests do not provide an adequate accurate map of resistance. Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.     

Adjunctive therapy for cerebral malaria and other severe forms of Plasmodium falciparum malaria

Severe malaria due to Plasmodium falciparum causes more than 800,000 deaths every year. Primary therapy with quinine or artesunate is generally effective in controlling P. falciparum parasitemia, but mortality from cerebral malaria and other forms of severe malaria remains unacceptably high. Long-term cognitive impairment is also common in children with cerebral malaria. Of the numerous adjunctive therapies for cerebral malaria and severe malaria studied over the past five decades, only one (albumin) was associated with a reduction in mortality. In this article, we review past and ongoing studies of adjunctive therapy, and examine the evidence of efficacy for newer therapies, including inhibitors of cytoadherence (e.g., levamisole), immune modulators (e.g., rosiglitazone), agents that increase nitric oxide levels (e.g., arginine) and neuroprotective agents (e.g., erythropoietin).     

Erythrocytapheresis for Plasmodium falciparum infection complicated by cerebral malaria and hyperparasitemia

In malaria due to Plasmodium falciparum, life-threatening complications are in part related to the degree of parasitemia. Whole blood exchange and red blood cell exchange (RCE) have been used for the rapid removal of parasites from the circulation of patients with a high parasite load complicated by cerebral, pulmonary, and renal dysfunction. We have treated three 5-45-year-old patients with hyperparasitemia and end-organ dysfunction with red cell exchange by automated apheresis as an adjunct to specific anti-malarial chemotherapy. Parasitemia dropped more than 80% in all three patients immediately after the exchange, and all patients had an uneventful and full recovery. In combination with effective anti-malarial chemotherapy, apheresis RCE is a safe and rapid approach to treat complicated malaria due to P. falciparum.     

彩超新方法检测肝癌

目的 本文设计一种新彩色多普勒影像处理方法以突出展现肝细胞癌（ＨＣＣ）的异常涡流。方法 计算两项新（双－向,低峰）指数检测ＨＣＣ特征血流。结果 在实验资料,由狭窄形成的涡流其两项指数较高。在临床方法向,ＨＣＣＲ 两项指数显著高于慢性肝病患者门脉及肝静脉内的相应测值。７７％ＨＣＣ２可检出该涡流,而很少见于门脉或肝静脉。结论该项新可用来ＨＣＣ涡流。     

In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression

Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/collections before and after exposure to P. falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite, and blinded serologic studies were completed. CD35 (complement receptor 1), CD55 (decay-accelerating factor), CD59 (membrane inhibitor of reactive lysis) and CD47 (integrin-associated protein) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa-stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty-three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.