FTA-巢式PCR方法鉴定溶组织内阿米巴原虫的初步研究

目的建立一种简便,快速的FTA-巢式PCR方法用于鉴定粪便中溶组织内阿米巴原虫(Entamoeba histolytica, E.h).方法 收集门诊腹泻病人新鲜粪便,用光学显微镜进行初步检查.用FTA卡抽提镜检结果为阳性的粪便DNA,根据溶组织内阿米巴原虫的SSU -rRNA序列设计引物,进行巢式PCR扩增,对PCR产物进行琼脂糖凝胶分析,并对阳性产物进行测序和序列比对分析.结果 根据光学显微镜检测结果,挑选了44例镜检结果为阿米巴原虫阳性的腹泻病人粪便.经FTA-巢式PCR扩增,其中20例样本可扩增出427 bp左右的目的条带,目的条带的测序和序列分析结果表明为溶组织内阿米巴原虫.镜检方法与巢式PCR方法的阳性符合率为45.45%(20/44),将E.h与形态学相似的其他内阿米巴原虫进行了鉴定和区分.结论 本研究建立的FTA-巢式PCR方法具有简单,快速,准确等优点,为临床检验和流行病学调查中E.h的鉴别诊断提供了新的技术方法.     

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy

Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.     

HIV-1C env and gag Variation in the Cerebrospinal Fluid and Plasma of Patients with HIV-Associated Cryptococcal Meningitis in Botswana

HIV-1 compartmentalization in reservoir sites remains a barrier to complete HIV eradication. It is unclear whether there is variation in HIV-1 env and gag between cerebrospinal fluid (CSF) and plasma of individuals with HIV-associated cryptococcal meningitis (CM). We compared HIV-1 env characteristics and the gag cytotoxic T-lymphocyte (CTL) escape mutations from CSF and plasma samples. Employing population-based Sanger sequencing, we sequenced HIV-1 env from CSF of 25 patients and plasma of 26 patients. For gag, 15 CSF and 21 plasma samples were successfully sequenced. Of these, 18 and 9 were paired env and gag CSF/plasma samples, respectively. There was no statistically significant difference in the proportion of CCR5-using strains in the CSF and plasma, (p = 0.50). Discordant CSF/plasma virus co-receptor use was found in 2/18 pairs (11.1%). The polymorphisms in the HIV-1 V3 loop were concordant between the two compartments. From the HIV-1 gag sequences, three pairs had discordant CTL escape mutations in three different epitopes of the nine analyzed. These findings suggest little variation in the HIV-1 env between plasma and CSF and that the CCR5-using strains predominate in both compartments. HIV-1 gag CTL escape mutations also displayed little variation in CSF and plasma suggesting similar CTL selective pressure.     

Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing

There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.     

脑囊尾蚴病患者脑脊液一氧化氮含量分析

目的　探讨脑囊尾蚴病患者脑脊液 (CSF)一氧化氮 (NO)含量以及细胞学成分变化。　方法　采用 Griess法检测 30例脑囊尾蚴病、2 0例结核性脑膜炎 (TBM)及 2 0名健康人 (对照组 ) CSF的 NO水平 ,同时进行其细胞学成分分析。　结果与结论　Griess法能快速、准确地检测 CSF的 NO含量。脑囊尾蚴病组及 TBM组 CSF的 NO水平均明显高于对照组 (P<0 .0 1)。脑囊尾蚴病组 NO水平明显高于 TBM组 (P<0 .0 5 ) ,并以持续的嗜酸性粒细胞增多为主要特点。     

聚合酶链式反应(PCR)扩增基因鉴定蚊胃血血源

目的探寻鉴定蚊胃血来源的实验室快速检测方法。方法对抗人血红蛋白胶体金试纸条(简称试纸条)检测有人血成分的按蚊干血痕标本,依据常见蚊吸血对象(人、牛、猪)的DNA序列的差异,设计特异DNA基因引物,运用聚合酶链式反应(PCR)方法对按蚊胃内干血痕标本进行DNA基因扩增,1.5%琼脂糖电泳检测扩增产物,并对扩增产物进行序列测定后查询其同源性匹配。结果试纸条共检测87只按蚊,血源来自牛和猪的共83只,检出仅含人血痕4份。该4份人血痕蚊标本经PCR扩增检测和基因序列匹配查询,与人基因序列同源性为100%,而无牛和猪基因。结论PCR法鉴定蚊胃血血源快速、灵敏、特异。     

缺氧缺血性脑病患儿其血浆和脑脊液中NO及SOD的含量及意义

目的探讨新生儿缺氧缺血性脑病(HIE)血浆和脑脊液(CSF)中的一氧化氮(NO)和超氧化物歧化酶(SOD)含量变化及其HIE不同时期和不同程度间的相关关系.方法对HIE患儿第3天的CSF和初入院(HIE 2 h内)、第1天、第3天、恢复期的血浆中NO、SOD进行检测,并与正常对照组比较;分析NO和SOD的变化原因和意义.结果HIE患儿血浆中第1天的NO含量最高,而SOD相反;初入院、第1天、第3天的血浆中NO和SOD含量与正常对照组对比均有统计学意义(P＜0.01),而恢复期中NO和SOD含量与正常对照组对比无统计学意义(P＞0.05),血浆和HIE第3天的CSF中NO和SOD水平均呈负相关;病情越重NO浓度越高,SOD越低.结论NO,SOD参与HIE的发病过程,在HIE的发病过程中起着重要作用.     

烟台地区病毒性脑炎病毒分离与病原学分析

目的了解烟台地区病毒性脑炎病原谱及其基因特征。方法采集烟台地区病毒性脑炎患者脑脊液46份及粪便标本10份,通过细胞培养分离病毒,RT-PCR扩增肠道病毒VP1区并测序,进行基因序列分析。结果从46例病毒性脑炎患者脑脊液标本中分离到11株病毒,分离率为23.91%,10份粪便标本中分离病毒5株。16株病毒经鉴定7株为肠道病毒,其中EV71型4株。EV71与其他地区流行株VP1区序列差异较小。结论烟台市病毒性脑炎以肠道病毒为主,有EV71型流行,与其他地区流行株相比,EV71型VP1区基因变异较小。     

单核细胞增生性李斯特菌PCR快速检测方法建立及应用

目的　通过扩增hly基因PCR法建立快速检测单核细胞增生性李斯特氏菌 (Lm )方法。 方法 :用PCR法扩增hly基因来检测标准菌株及临床分离的Lm的纯培养物和模拟污染的生猪肉、水和牛奶 ,并应用于实际食品检验工作中进行验证。结果　 34株Lm的PCR结果呈阳性 ,而其它李斯特菌 ,包括英诺克李斯特菌、绵羊李斯特菌、西尔李斯特菌、威儿李斯特菌和格氏李斯特菌以及非李斯特菌均未出现特异性的片段。表明所扩增的hly基因 3’末段 82 7bp的DNA片段对Lm具有较高的特异性。PCR法对Lm纯培养物的检测限达 10 5CFU/ml,对模拟污染生猪肉、水和牛奶的 30℃ 16h改良LEB增菌培养液用PCR法检测 ,结果检测限达 10CFU/ 2 5 g(ml)。进一步研究表明该PCR扩增体系能检测出 6 .5pg的LmDNA ,整个检测过程只需 2 4h。采用该法对扬州市区 16 9份食品样品检测 ,8份样品Lm呈阳性且结果与传统的生化鉴定方法完全一致。结论　扩增hly基因的PCR法对Lm的鉴定具有特异性强、灵敏度高、速度快和易操作等优点 ,可用于食品的Lm的分离     

巢式PCR筛查早产儿TORCH病原感染的研究

目的了解早产儿巨细胞病毒(HCMV)、弓形虫(TOX)、乙肝病毒(HBV)、单纯疱疹病毒1型(HSV-1)、单纯疱疹病毒2型(HSV-2)、风疹病毒(RV)与柯萨奇病毒B组3型(CVB3)的感染状况。方法采用巢式PCR对122例早产儿和75例正常新生儿进行7种病原体核酸的筛查检测。结果早产儿HCMV感染率为63.9%、TOX感染率为3.3%,HBV感染率为9.8%,HSV-1感染率为4.9%,HSV-2感染率为6.6%,RV感染率为3.3%,CVB3感染率为23.8%。早产儿HCMV、HBV、HSV-2和CVB3感染率与正常新生儿HCMV、HBV、HSV-2、CVB3感染率29.3%、1.3%、0和0比较,差异有统计学意义(P0.05)。结论 HCMV、HBV、HSV-2和CVB3感染是早产儿发生的危险因素之一。     

荧光定量PCR用于按蚊体内疟原虫子孢子检测的研究

目的 建立一种用于按蚊体内疟原虫子孢子定量检测和虫种鉴别的荧光定量PCR方法。方法 采用针对4种人体疟原虫18S rRNA基因属特异性保守区的1对引物, 以疟原虫18S rRNA基因重组质粒与按蚊DNA的混合物为模板, 进行反应体系和反应条件优化, 验证方法的特异性, 并通过熔解曲线分析进行虫种鉴别。应用阴性按蚊DNA稀释的间日疟原虫18S rRNA基因重组质粒为模板制作标准曲线, 并分别以质粒DNA和实验室子孢子感染阳性的按蚊DNA为模板分析建立的荧光定量PCR方法的敏感性。在反应体系中加入不同部位、 不同用量按蚊DNA, 以探讨按蚊DNA对检测效果的影响。结果 该方法对按蚊、 人血DNA均无扩增, 对4种疟原虫DNA均有特异性扩增且扩增产物的熔解温度 （Tm） 易于区分, 三日疟原虫、 恶性疟原虫、 卵形疟原虫和间日疟原虫的Tm值分别为71.0、 72.7、 73.9 ℃和75.9 ℃。标准曲线中循环阈值（Ct值） 与模板浓度具有良好的相关性 （相关系数r = -0.99）。最低可以检出含50拷贝的质粒DNA或32倍稀释的子孢子阳性按蚊DNA样本。按蚊DNA对荧光定量PCR反应具有抑制作用。荧光定量PCR的Ct值在实验内和实验间均具有良好的重现性。结论 新建立的SYBR Green I染料荧光定量PCR方法具有较高的敏感性和特异性, 可用于按蚊体内疟原虫子孢子的检测, 并可同时对4种人体疟原虫进行鉴别。     

脑梗死患者脑脊液和血清中脂蛋白(a)的变化

为观察脑梗死患者脑脊液和血清中脂蛋白 (a)含量的变化 ,选择 80例脑梗死患者和 40例对照者 ,用酶联免疫法测定脂蛋白 (a)在脑脊液及血清中的含量。结果发现 ,脑梗死患者与对照者脑脊液中脂蛋白 (a)含量分别为 2 34± 2 9μg/L及 2 11± 2 1μg/L ,血清中脂蛋白 (a)含量分别为 0 .2 99± 0 .0 2 8g/L及 0 .2 71± 0 .0 2 5g/L ,脑梗死组脑脊液及血清中脂蛋白 (a)含量明显高于对照组 (P <0 .0 1)。脑梗死患者和对照者脑脊液与血清中脂蛋白 (a)水平间均无相关关系 (P >0 .0 5 )。结果提示 ,脑梗死患者脑脊液及血清中脂蛋白 (a)水平明显高于对照者。     

Identification of H. pylori in Saliva by a Nested PCR Assay Derived from a Newly Cloned DNA Probe

A novel probe was developed from genomic DNA ofHelicobacter pylori ATCC type strain 43629. Ithybridized with all 73 H. pylori clinical isolatestested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41different HaeIII-digestion patterns from 57 H. pyloristrains tested. Based on the sequence of the probe, anested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximatelyequivalent to one cell. No PCR products were amplifiedfrom any of 21 non-H. pylori strains tested. Using thisnested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patientswith gastric H. pylori infection. These data suggestthat the probe is useful for typing H. pylori and thatthe nested PCR is a valuable tool for detecting H. pylori DNA in saliva.     

Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype‐specific PCR and deep sequencing

The incidence of mixed genotype hepatitis C virus (HCV) infections in the UK is largely unknown. As the efficacy of direct‐acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR‐based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8%; however, this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (P < .05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR next‐generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the nonquantitative nature of the technique and despite the designation of false‐positive thresholds based on negative controls.     

高血压脑出血病人脑脊液sICAM-1与sIL-6R的对比观察

目的对比观察高血压脑出血急性期不同时间脑脊液(CSF)可溶性细胞间黏附分子(sICAM-1)和可溶性白细胞介素6受体(sIL-6R)及白细胞介素6(IL-6)的变化与继发性脑损伤的关系.方法采用酶联免疫吸附试验(ELISA)测定20例(对照组)和15例高血压脑出血病人CSF中sICAM-1、sIL-6R、IL-6含量变化.结果高血压脑出血后第1天CSF sICAM-1、sIL-6R、IL-6含量升高已达高峰.sICAM-1在出血后5 d～7 d明显下降,但仍高于正常对照,而sIL-6R和IL-6虽有下降,但仍维持在较高水平,两者之间呈正相关(P＜0.001),而sICAM-1与sIL-6R含量变化无明显相关(P＞0.05).结论 sICAM-1、sIL-6R和IL-6参与高血压脑出血的免疫和炎症反应过程,与继发性脑损伤程度有关,sICAM-1与sIL-6R和IL-6调节机制可能不同.     

Use of Direct Oral Anticoagulants in Patients with Sickle Cell Disease and Venous Thromboembolism: A Prospective Cohort Study of 12 Patients

Abstract

Patients with sickle cell disease have an increased risk of venous thromboembolism (VTE) and with a mortality 2-fold higher. The anticoagulation of VTE in a young population is an important question. Indeed, hemorrhagic complications of anticoagulation may occur more frequently than in the general population. The use of a direct oral anticoagulant (DOAC) is not recommended for VTE in patients with sickle cell disease because those patients were not included in the clinical studies. We aimed to study the safety of using DOACs in a prospective cohort of patients with sickle cell disease and VTE. We prospectively followed the cohort of all sickle cell disease patients undergoing recent DOAC treatment for VTE at a sickle cell disease reference center. Twelve patients received rivaroxaban for VTE (eight women and four men). The median age was 27?years (20–45). The sickle cell disease variants included homozygous Hb SS (HBB: c.20A>T) in eight patients, Hb S-β⁺-thalassemia (Hb S-β⁺-thal) in two, Hb S-β⁰-thal in one and Hb S-Hb C (HBB: c.19G>A) in one. The cumulative duration of follow-up was 3134?days under rivaroxaban treatment. There were two thrombotic events, including a patient with a double positivity of antiphospholipid antibodies. No major bleeding was observed, and 6/12 patients presented minor bleeding (epistaxis: n?=?4; anal fissure bleeding: n?=?1; menorrhagia n?=?4). Of these, 3/6 required their treatment to be switched to apixaban, which stopped the bleeding. Direct oral anticoagulants may be an alternative treatment for VTE in patients with sickle cell disease, except for an associated antiphospholipid syndrome.     

Polymerase chain reaction on cerebrospinal fluid cells in the detection of leptomeningeal involvement by B-cell lymphoma and leukaemia: a novel strategy and its implications

In the search of B-cell lymphoma/leukaemia dissemination to cerebrospinal fluid (CSF), we used the highly sensitive semi-nested PCR (snPCR) for the analysis of IgH gene rearrangements. This method detects a rearranged IgH gene from a single B lymphocyte which may or may not represent the neoplastic B-cell population. We therefore performed multiple snPCR (three to five) experiments on the same CSF sample, postulating that the detection of a band of the same size and sequence in different PCR runs was highly indicative of a clonal population. 17 consecutive cases with a differential diagnosis of primary (P cns L) ( n =10) or secondary (S cns L) ( n =7) CNS lymphoma or leukaemia were investigated by the new strategy. The clonal nature of the B-cell population was confirmed in 3/10 of suspected P cns L, and in six other cases the PCR study was indicative of reactive lymphocytosis. One case revealed a clonal B-cell population in the clinical context of an autoimmune disorder. Evidence of clonal B-cell population was found in 4/7 of suspected S cns L. In one of these cases the detected band and its sequence proved identical to that of the primary nodal lymphoma. We believe that the evaluation of B-cell clonality in CSF requires multiple snPCR amplification on the same sample to compare the size of the products and, if necessary, the DNA sequences to ascertain the diagnosis of malignancy in equivocal cytologic and clinical findings.     

ELISA检测脑囊虫病患者脑脊液中囊尾蚴抗原

应用酶联免疫吸附实验(ELISA),以38例脑囊虫病人脑脊液(CSF)和26例脑部其它疾患的CSF为对照,用兔抗囊尾蚴抗血清进行了囊尾蚴抗原检测,28例呈阳性反应,阳性率73.68％;对照组26例全部为阴性。本方法特异性强,重复性和敏感性较好,简单迅速,有希望用于脑囊虫病的临床诊断。     

Diagnostic Value of Detecting Feline Coronavirus RNA and Spike Gene Mutations in Cerebrospinal Fluid to Confirm Feline Infectious Peritonitis

Background: Cats with neurologic feline infectious peritonitis (FIP) are difficult to diagnose. Aim of this study was to evaluate the diagnostic value of detecting feline coronavirus (FCoV) RNA and spike (S) gene mutations in cerebrospinal fluid (CSF). Methods: The study included 30 cats with confirmed FIP (six with neurological signs) and 29 control cats (eleven with neurological signs) with other diseases resulting in similar clinical signs. CSF was tested for FCoV RNA by 7b-RT-qPCR in all cats. In RT-qPCR-positive cases, S-RT-qPCR was additionally performed to identify spike gene mutations. Results: Nine cats with FIP (9/30, 30%), but none of the control cats were positive for FCoV RNA in CSF. Sensitivity of 7b-RT-qPCR in CSF was higher for cats with neurological FIP (83.3%; 95% confidence interval (95% CI) 41.8–98.9) than for cats with non-neurological FIP (16.7%; 95% CI 6.1–36.5). Spike gene mutations were rarely detected. Conclusions: FCoV RNA was frequently present in CSF of cats with neurological FIP, but only rarely in cats with non-neurological FIP. Screening for spike gene mutations did not enhance specificity in this patient group. Larger populations of cats with neurological FIP should be explored in future studies.     

Detection of free immunoglobulin light chains in cerebrospinal fluids of patients with central nervous system lymphomas

Diagnosis of central nervous system (CNS) lymphoma depends on histopathology of brain biopsies, because no reliable disease marker in the cerebrospinal fluid (CSF) has been identified yet. B‐cell lymphomas such as CNS lymphomas are clonally restricted and express either kappa or lambda immunoglobulin light chains. The aim of this study was to find out a potential diagnostic value of free immunoglobulin light chains released into the CSF of CNS lymphoma patients. Kappa (κ) and lambda (λ) free immunoglobulin light chains (FLC) were measured in CSF and serum samples collected from 21 patients with primary and secondary CNS lymphomas and 14 control patients with different neurologic disorders. FLC concentrations and ratios were compared between patient groups and were further analyzed in correlation with clinical, cytopathological, and radiological findings. FLC concentrations for all patients were lower in CSF when compared to serum. In patients with CNS lymphoma, the FLC ratios in CSF were higher (range 392–0.3) compared to control patients (range 3.0–0.3). Irrespective of cytopathological proven lymphomatous meningitis, in 11/21 lymphoma CSF samples the FLC ratios were markedly above 3.0 indicating a clonally restricted B‐cell population. Increased FLC ratios in CSF were found in those patients showing subependymal lymphoma contact as detected in magnetic resonance imaging. In summary, this is the first report demonstrating that a significant proportion of patients with CNS lymphomas display a markedly increased FLC ratio in the CSF.