GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估

目的对GeneXpert实时荧光定量聚合酶链反应(PCR)在快速检测临床粪便标本中艰难梭菌的应用进行评估。方法采用双拭子蘸取临床未成形粪便标本,一支拭子用于GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因tcdB,另一支用于常规厌氧菌培养检测;对GeneXpert实时荧光定量PCR检测结果与常规厌氧菌培养结果的一致性进行统计学分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。结果临床收集到141例未成形粪便标本,GeneXpert实时荧光定量PCR检出艰难梭菌毒素基因tcdB阳性42例,其中常规厌氧菌培养阳性34例,两者一致性较好(Kappa=0.775 0,P0.01),GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。结论 GeneXpert实时荧光定量PCR直接检测粪便标本中的艰难梭菌具有检测快速、操作简便等优点,有重要的临床应用价值。     

他汀类调脂药物对脓毒症凝血-炎症网络的影响

目的脓毒症是由于感染引起的全身系统性炎症反应,可发展为严重脓毒症和感染性休克。尽管抗感染治疗和器官功能支持技术取得了长足的进步,脓毒症的病死率仍高达30％～70％。近年来的研究表明,脓毒症发生时,脂质代谢同样发生紊乱。他汀类药物因为其能有效地降低血脂,改善动脉粥样硬化情况而被大家所认识。随着对该药物的进一步研究发现,他汀类药物降脂以外的作用越来越受到重视,如改善内皮功能、抑制血管炎症、改善凝血功能、减少血栓形成和改善总体血管功能等。本文就目前他汀类药物治疗脓毒症时抗炎、抗凝影响的研究做一简述。     

利用实时荧光定量聚合酶链式反应快速检测人及动物粪便中的沙门菌

目的为了快速检测、鉴定沙门菌属细菌,提高食源性疾病暴发应对能力,本研究建立了针对沙门菌属的实时荧光定量聚合酶链式反应(qPCR)检测方法,并对其特异性、敏感性和检测下限进行评价。方法筛选针对沙门菌的属特异基因,并建立该基因的qPCR检测体系,利用肠道不同种属细菌、不同亚种及血清型沙门菌属细菌、动物及人粪便样本评价该体系的特异度、灵敏度及检测下限。结果获得沙门菌的属特异基因ttrA,建立基于该基因的qPCR检测方法。发现该方法对纯DNA的最低检测下限为2拷贝/反应。对沙门菌属以外的肠道致病菌无扩增,对1 100株不同亚种及血清型的沙门菌的扩增结果均为阳性,对150份沙门菌致腹泻患者的粪便增菌液和210份动物带菌粪便增菌液均检测阳性。结论本研究建立的基于单一基因的沙门菌属快速分子检测方法具有特异度高、灵敏度高的特点,可用于快速筛查、鉴定沙门菌及由其引起的感染性腹泻。     

全身炎症反应综合征患者血脂变化与炎症反应的相关性研究

目的观察全身炎症反应综合征(SIRS)患者血浆高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、总胆固醇(TC)、三酰甘油(TG)和肿瘤坏死因子(TNF-α)水平的变化,探讨血脂变化在炎症反应中的意义。方法符合SIRS诊断48例,分别在入院后第1、3和第5天清晨空腹抽肘静脉血3mL,测定HDL、LDL、TC、TG和TNF-α,并设对照组。结果SIRS患者血浆HDL、LDL、TC、TG入院后各监测点均比对照组显著下降(P<0.01,P<0.05),HDL入院第3天为最低,并且降低更为显著(P<0.01)。SIRS患者TNF-α入院后各监测点均比对照组显著升高(P<0.05,P<0.01)。HDL、LDL、TC、TG与TNF-α具有相关性(P<0.05,P<0.01)。结论机体过度炎症反应时,HDL、LDL、TC、TG水平下降,并且与炎症反应的程度密切相关,可作为评估预后的一个参考指标;在治疗原发病的基础上,提高血脂和脂蛋白水平,有助于缓解病情,改善预后。     

全身炎症反应综合征患者凝血功能的研究

目的对全身炎症反应综合征(SIRS)患者凝血功能的变化进行研究,以期为临床诊治提供新思路.方法将入住急诊监护病房的患者分为SIRS组50例和非SIRS组30例,另选健康对照组30例,分别测其血小板计数(PLT)、D-二聚体(DD)、凝血酶原时间(PT)、凝血酶时间(IT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FBG)、血栓调节蛋白(TM)、蛋白C(PC)、蛋白S(PS)、组织纤溶酶原激活物(TPA)、纤溶酶原激活物抑制物(PAI-1)11项与凝血功能有关的实验室指标.结果 SIRS组中TT、APTT、PT、DD、TM和PAI-1均高于非SIRS组及对照组(P＜0.05);PC、PS、TPA、PLT、FBG均低于非SIRS组及对照组(P＜0.05).结论①SIRS时存在着凝血功能紊乱.②SIRS时凝血系统功能紊乱主要特征表现为各种促凝物质增加,机体抗凝物质减少,纤溶系统被抑制,而纤溶系统抑制在SIRS发生、发展过程中可能起到更加重要的作用.     

2009年至2013年萍乡市流感病例样本real-time RT-PCR结果分析

目的通过对萍乡市历年流感样病例的病毒核酸检测结果分析,了解当地流感疫情状况,分析当地流感发病趋势,为预防和控制流感流行提供科学论证凭据。方法萍乡市人民医院为国家流感样病例监测哨点医院,对符合流感样病例定义的病例收集相关信息,采集咽拭子标本,采用实时荧光逆转录聚合酶链反应法（real-time RT-PCR）对标本进行流感病毒核酸检测,并进行统计分析。结果萍乡市流感病毒核酸阳性率22.09%,其中2009年阳性率59.75%最高,之后呈下降趋势。新甲型H1N1流感病毒和乙型流感病毒阳性数接近,构成比分为33.74%和30.89%。新甲型H1N1共检出阳性166例,其中2009年9-12月检出阳性143例,2009年11月为最高峰,阳性率达到（75/143）52.45%,2010年以后有少量病例发现。乙型和季H3流感检出阳性数逐年增加。男、女流感核酸阳性检出率分别为23.19%（291/1255）和20.68%（201/972）。年龄分5个组统计分析,阳性率分别是37.25%、30.59%、19.40%、18.69%和13.00%。结论萍乡市流感病例核酸阳性以新甲型H1N1和乙型为主;季H3流感病例核酸阳性有所增加,且新甲型H1N1在2009年12月后得到有效控制,流感病毒易感染人群以学龄儿童和青少年为主,性别无差异。     

Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR

We aimed to detect causative pathogens in cerebrospinal fluid (CSF) collected from patients diagnosed with bacterial meningitis by real-time polymerase chain reaction (PCR). In addition to Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae described previously, five other pathogens, Neisseria meningitidis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes, were targeted, based on a large-scale surveillance in Japan. Results in CSF from neonates and children (n = 150), and from adults (n = 18) analyzed by real-time PCR with molecular beacon probes were compared with those of conventional culturing. The total time from DNA extraction from CSF to PCR analysis was 1.5 h. The limit of detection for these pathogens ranged from 5 copies to 28 copies per tube. Nonspecific positive reactions were not recognized for 37 microorganisms in clinical isolates as a negative control. The pathogens were detected in 72.0% of the samples by real-time PCR, but in only 48.2% by culture, although the microorganisms were completely concordant. With the real-time PCR, the detection rate of H. influenzae from CSF was high, at 45.2%, followed by S. pneumoniae (21.4%), S. agalactiae (2.4%), E. coli (1.8%), L. monocytogenes (0.6%), and M. pneumoniae (0.6%). The detection rate with PCR was significantly better than that with cultures in patients with antibiotic administration (χ² = 18.3182; P = 0.0000). In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.     

实时荧光定量PCR在快速检测耐甲氧西林金黄色葡萄球菌中的评价与应用

目的评价实时荧光定量聚合酶链反应(PCR)在快速检测耐甲氧西林金黄色葡萄球菌(MRSA)中的临床应用价值。方法经培养鉴定的70例已知细菌中22例为MRSA,48例为非MRSA,对已知细菌用实时荧光定量PCR进行检测,了解该方法的特异性和敏感性。对临床88例医护人员和患者的鼻拭子进行实时荧光定量PCR检测,了解医院内MRSA的携带情况。结果培养为MRSA,实时荧光定量PCR检测结果均为MRSA,敏感性为100%;培养为非MRSA,实时荧光定量PCR检测结果均为非MRSA,特异性为100%。实时荧光定量PCR检测MRSA的敏感性可确定为1×103cfu/mL。48例患者的鼻拭子mecA耐药基因阳性26例,金黄色葡萄球菌阳性28例,两者同时阳性(即为MRSA)20例,MRSA阳性检出率为41.6%;40例医护人员鼻拭子mecA耐药基因阳性12例,金黄色葡萄球菌阳性13例,两者同时阳性(即为MRSA)9例,MRSA阳性检出率为22.5%。结论实时荧光定量PCR可用于临床对MRSA的快速检测。     

Plasma mitochondrial DNA levels in patients with trauma and severe sepsis: Time course and the association with clinical status

Purpose

This study aimed to investigate the serial changes in plasma levels of mitochondrial DNA (mtDNA) in patients with trauma and severe sepsis and the mechanism of increase in mtDNA levels and the association between the levels and severity.

Materials and Methods

We conducted a prospective observational study of patients with trauma having injuries with an Abbreviated Injury Scale score of 3 or higher (n = 37) and patients with severe sepsis (n = 23). The mtDNA concentrations in clarified plasma were measured using real-time quantitative polymerase chain reaction.

Results

Concentrations of mtDNA peaked on the day of admission (day 1) in patients with trauma, whereas they increased on day 1 and remained constant until day 5 in patients with sepsis. The mtDNA levels on day 1 correlated with the maximal levels of creatinine phosphokinase in patients with trauma (R² = 0.463, P < .05) but not in patients with sepsis (R² = 0.028, P = .43). The mtDNA levels on day 1 were significantly higher in nonsurvivors compared with survivors of trauma (P < .05) but not sepsis.

Conclusions

The levels of mtDNA were elevated during traumatic injury and severe sepsis, although time course and prognostic significance differed between the groups, suggesting that the mechanisms of mtDNA release into plasma differ.     

产毒型霍乱弧菌实时定量三重TaqMan PCR快速检测方法的建立

应霍乱弧菌产生霍乱毒素的能力.     

浙江省衢州市应用聚合酶链反应检测钩端螺旋体效果评价

目的 建立聚合酶链反应(PCR)检测钩端螺旋体的快速方法,应用于宿主动物钩端螺旋体的快速检测,同时比较PCR方法和常规检测的效果.方法 根据中国疾病预防控制中心提供的引物,以致病性大肠杆菌、产毒性大肠杆菌、溶藻弧菌、沙门菌、霍乱弧菌、痢疾志贺菌、非O1、O139霍乱弧菌、副溶血性弧菌、肠出血性大肠杆菌O157:H7为对照,建立PCR检测钩端螺旋体的快速方法,用于钩端螺旋体的快速检测.结果 衢州市2006年分离钩体菌株进行PCR鉴定结果与血清学鉴定结果相符.100只鼠和100只蛙肝、脾、肾标本进行PCR检测,PCR检测阳性率10%,钩体菌株培养分离率仅为1%,二者差异有统计学意义(2=16.05,P0.005).结论 PCR检测钩端螺旋体灵敏度高、特异性强,可用于钩端螺旋体的快速检测.     

血小板计数及动态变化在瓣膜置换术后SIRS中的意义

目的　监测心脏瓣膜置换术后血小板计数的动态变化 ,探讨血小板计数与病情及预后的关系。方法　 87例心脏瓣膜置换术患者 ,从术后 6h开始动态监测血小板值的变化 ,根据有无全身炎症反应综合征 (SIRS)分成非SIRS组及SIRS组 ,观察术后恢复情况与血小板下降程度、回升趋势的关系。结果　非SIRS组手术结束 6h血小板计数为术前的 (72± 2 1) % ,术后第 3天开始回升 ,术后第 7天开始超过术前水平。SIRS组手术结束 6h血小板计数为术前的 (4 8± 16 ) % ,恢复到术前水平约需 9～ 17d。两组相比有统计学差异 (P <0 0 5 )。结论　心脏瓣膜置换术后血小板值下降程度与变化趋势能较正确、敏感地反映病情和预后 ,可作为术后能否获得良好恢复的预警指标。     

The usefulness of multiplex PCR for the identification of bacteria in joint infection

Background: The diagnosis of septic arthritis (SA) relies on synovial analysis and conventional culture. But, these methods lack of sensitivity and culture is time consuming to establish a definite diagnosis. This study evaluated a new multiplex PCR assay which entailed screening PCR for Gram typing and identification PCR for species identification using two primer mixes. Methods: A total of 80 synovial fluid samples from patients with suspected SA were collected. Culture, multiplex PCR, and 16S rRNA gene PCR were performed. Results: The analytical sensitivity of multiplex PCR assay was 10¹ CFU/ml for each type of bacteria. There was no cross‐reactivity with common bacterial pathogens. Bacteria were detected in 20, 25, and 26 of 80 samples for culture, multiplex PCR, and 16S rRNA gene PCR, respectively. Nineteen (95%) of 20 culture‐positivesamples and 6 (10%) of 60 culture‐negative samples were positive for the multiplex PCR. Five of six samples which were positive only from multiplex PCR were also positive in 16S rRNA gene PCR. The multiplex PCR showed 2 false‐negative in 27 true‐positive samples but no false‐positive. The sensitivity and specificity of the multiplex PCR were 92.6 and 100%, and the agreement with culture and 16S rRNA gene PCR were 91.3 and 96.3%, respectively. The time to detection for multiplex PCR was a maximum of 6 hr. Conclusion: This multiplex PCR assay offers high sensitivity and improved detection speed relative to culture. The appropriate combination of this new multiplex PCR assay with culture may contribute to the accurate and rapid diagnosis of SA. J. Clin. Lab. Anal. 24:175–181, 2010. © 2010 Wiley‐Liss, Inc.     

基于芯片点样技术的RT-PCR平台建立及其在HCV、HIV-1快速检测中的应用

目的：建立基于芯片点样技术的荧光定量PCR（RT‐PCR）平台并将其用于HIV‐1和HCVRNA载量的检测。方法选择HIV‐1和HCV的保守区域设计引物,制备基因芯片管,建立RT‐PCR测定HIV‐1和HCV的反应体系,利用溶解曲线区分病毒种类。对该方法的灵敏度和特异度进行评价,并用临床标本验证其检测性能。结果该方法的特异度较好,其检测HCV、HIV‐1的最低检测限为1×103copy/mL,对RNA载量为1×103～1×106copy/mL的临床标本能准确检测。结论该方法为检测HCV、HIV‐1提供了新思路。     

布鲁菌PCR快速检测方法的建立及应用

目的建立布鲁菌聚合酶链反应(PCR)方法,对其进行应用评价。方法针对布鲁菌外膜蛋白编码基因(omp-2)设计PCR引物,建立PCR方法,利用19种常见细菌和布鲁菌株DNA分别对其进行特异性和敏感度评价,对3株疑似布鲁菌株进行核酸检测,并与分离培养法进行结果比对。结果本研究建立的布鲁菌PCR检测方法仅能检出布鲁菌阳性菌株,对照菌DNA未出现目的条带;敏感度为100拷贝/反应;PCR方法对3株疑似布鲁菌株检出omp-2基因条带,鉴定结果为布鲁菌,与分离培养法结果相同。结论本研究建立了一种布鲁菌PCR检测方法,与分离培养法相比,布鲁菌PCR方法准确、快速,适合布鲁菌病疫情的快速检测。     

快速细菌学诊断在全身性炎症反应综合征中的应用

目的探讨快速细菌学诊断(聚合酶链反应,PCR)与普通血培养的差异性及对ICU全身性炎症反应综合征(SIRS)患者治疗的指导意义.方法抽取37例40例次SIRS患者的外周血标本,同时做普通血培养和PCR检测血中细菌,并根据PCR检测结果指导临床使用抗生素,观察1周的疗效.结果两种检测方法有明显的差异性(χ2=5.142,P<0.025),PCR法较灵敏.根据PCR结果将病例分为2组,阳性组11例更换了抗生素或使用了高效、广谱抗生素,1周后90.91%的病例SIRS状态得到有效控制;阴性组26例未更换抗生素或加用抗生素,2例1周内因原发病恶化死亡(7.69%),余24例中1周后19例SIRS状态没有恶化(73.08%),5例SIRS符合标准率上升(19.23%).结论PCR检测法较普通血培养法灵敏,对SIRS的病因分类和临床抗生素治疗有指导意义.     

基于DPO的四重荧光PCR检测常见分枝杆菌方法的建立与应用

目的 利用多重荧光PCR技术,建立一种快速、准确、特异的检测常见分枝杆菌的方法.方法 以分枝杆菌16S rRNA作为检测靶基因,设计双启动寡核苷酸(DPO)引物和TaqMan探针,探针的5’端分别用FAM 、ROX、HEX和JOE进行荧光标记,3’端标记淬灭荧光.通过对4种分枝杆菌标准株基因组DNA的扩增,评价多重荧光PCR方法的特异性和灵敏度.采用建立的多重荧光PCR方法,采用该方法检测2010年6至12月杭州市红十字会医院68例结核科住院患者清晨痰液标本,结果与细菌培养和抗酸染色镜检进行比较.采用x2检验比较3种方法的阳性率.结果 该方法可准确、特异地鉴定结核分枝杆菌和3种常见非结核分枝杆菌,检测灵敏度均为1 ×101 cfu/ml.对68份痰液标本进行检测,结果显示31份检测结果阳性,阳性率45.6％,明显高于涂片染色镜检(阳性率14.7％),差异具有统计学意义(x2=15.4,P ＜0.05),其中28例为结核分枝杆菌,1例为鸟分枝杆菌,2例为胞内分枝杆菌,与细菌培养鉴定方法一致.1例培养阳性而PCR检测结果阴性标本经16SrRNA扩增测序鉴定为龟分枝杆菌.结论 本研究建立的多重荧光PCR方法敏感、特异、快速,能有效检测结核分枝杆菌和常见非结核分枝杆菌,可用于分枝杆菌感染者的实验室快速诊断.     

PCR methodology and applications for the detection of human fungal pathogens

Introduction: Polymerase chain reaction (PCR) has emerged as a promising technology for the rapid and reliable detection and identification of medical mycoses. Recent technological advancements – including microarray, multiplex PCR with magnetic resonance, and beacon probes – have mitigated the technical difficulties of performing nucleic amplification in fungi, thereby improving the sensitivity and specificity of PCR-based assays. In this paper, we examine current applications of PCR in the diagnosis of human fungal infections and look ahead to emerging techniques that may play a larger role in molecular diagnostics in the future.

Areas covered: This review includes a brief overview of the advantages and disadvantages of PCR using various clinical specimens, manual versus automated DNA extraction procedures, panfungal versus specific targets, and spectrum of pathogens detected. This is followed by a brief synopsis of species-specific PCR approaches and a more in-depth look at the obstacles to widespread implementation.

Expert commentary: The review concludes with a short perspective for the next five years, including the hurdles to standardization and validation, as well as the role of PCR coupled with electrospray-ionization mass spectrometry (PCR/ESI-MS) or nuclear magnetic resonance for the diagnosis of medical mycoses.     

Leukocyte activation in sepsis; correlations with disease state and mortality

Objective: The immune response in sepsis shows a bimodal pattern consisting of an early, frequently exaggerated inflammatory response followed by a state of hyporesponsiveness often referred to as the compensatory anti-inflammatory response syndrome (CARS). Insight into the disease state may be helpful in deciding whether to choose immune stimulatory or anti-inflammatory therapy in these patients and may determine clinical outcome. We hypothesized that poor outcome in patients with sepsis is related to the severity of CARS, as reflected in the degree of leukocyte activation. Design: Prospective study. Setting: Intensive and respiratory care unit at a university hospital. Patients: Twenty consecutive patients with sepsis and 20 healthy age-matched volunteers. Interventions: None. Measurements and results: Analysis of surface expression of HLA-DR, CD11 b, ICAM-1, CD66 b, CD63 and CD64 on neutrophils and monocytes by flow cytometry and determination of plasma concentrations of lactoferrin, interleukin 6 and neopterin by ELISA at the time of diagnosis. Patient data were related to those of controls; moreover patient data between survivors and non-survivors were compared. Increased expression of all markers, except HLA-DR, was observed on both neutrophils and monocytes from patients compared to healthy controls. HLA-DR expression on monocytes was significantly decreased in patients with sepsis (p < 0.01). Expression of CD11 b and HLE on neutrophils, and ICAM-1 on monocytes, were lower in patients who died compared to those who survived (p < 0.05). Conclusion: In sepsis, both neutrophils and monocytes are activated compared to healthy controls. Poor prognosis is associated with a lower expression of activation markers on monocytes and neutrophils, suggesting that poor outcome in these patients may be due to the compensatory anti-inflammatory response. Received: 4 October 1999/Final revision received: 29 February 2000/Accepted: 4 April 2000