抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体的研制及初步应用

本文采用杂交瘤技术,制备了７ 株抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体（ｍ Ａｂ） 的杂交瘤细胞株。对３ 株ｍＡｂ（２Ｃ８、２Ｅ６ 和５Ｄ７） 进行了鉴定,ｍＡｂ ２Ｃ８ 和５Ｄ７ 为ＩｇＭ,２Ｅ６ 为ＩｇＧ１（κ）亚类,可分别识别不同的抗原表位,均具有很强的特异性,即只与甲型副伤寒沙门氏菌及其鞭毛抗原起反应,而与相关肠道细菌：尼亚里木沙门氏菌、达卡沙门氏菌及伤寒沙门氏菌、乙型和丙型副伤寒杆菌、猪霍乱沙门氏菌、纽波特沙门氏菌等均无交叉反应。免疫印迹显示,３ 株ｍＡｂ 只能与甲型副伤寒沙门氏菌鞭毛抗原Ｍｒ 为５２ ０００ 的蛋白结合。用ｍＡｂ ２Ｅ６ 和５Ｄ７ 建立的双ｍＡｂ 夹心ＥＬＩＳＡ 法,检测了１３ 例血培养阳性的副伤寒患者血清中的甲型副伤寒沙门氏菌鞭毛抗原,１２ 例呈阳性,阳性符合率为９２－３％ 。     

实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。     

伤寒杆菌鞭毛抗原酶联免疫分析的建立及初步应用

作者利用纯化的伤寒杆菌鞭毛抗原，制备出抗伤寒直菌鞭毛抗原单克隆抗体。实验发现，四株单抗体与伤塞杆菌鞭毛抗原和伤寒杆菌发生免疫反应，与甲、乙、丙副伤寒杆菌、大肠杆菌及部分沙门氏菌无交叉反应。选用其中２株单抗分别作为包被抗体和标记抗体，建立检测伤寒杆菌鞭毛抗原的夹心ＥＬＩＳＡ。３０名正常人和４２例非伤寒发热待查患者血清标本经验测为阴性，１２例途塞患者血清标本为阳性。本文方法可直接用血清进行检测，具有早     

关于产科麻醉与分娩镇痛方法的探讨

目的：观察并研究革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用方法与结果。方法选取我院自2011年9月~2013年9月所收集的血培养阳性标本共计100份作为研究对象,所有标本均为该期间内分离的非重复性标本。基于VITEK方法对血培养阳性标本进行直接细菌鉴定。同时以API检出结果为金标准,对VITEK直接细菌鉴定于药敏应用结果的符合率情况进行观察分析。结果 VITEK直接细菌鉴定方法下细菌鉴定总符合率为98.00%(98/100),药敏鉴定结果总符合率为98.37%(964/980),与API金标准方法检出结果对比无明显差异,P>0.05,不具有统计学意义。结论基于VITEK方法对血培养阳性标本进行直接细菌鉴定与药敏分析检出结果可靠,操作简便,可为临床及时修正用药提供依据,值得进一步推广。     

BACT/Alert120血培养联合白细胞计数快速诊断伤寒

目的为准确和快速诊断伤寒,以便尽快获得病原菌和控制伤寒流行.方法通过 BACT/Alert120进行血培养和肠道拭子培养,同时用Sysmex-K-4500血细胞分析仪进行白细胞计数,并利用伤寒沙门菌某些固有特点项目进行快速诊断.结果儿童伤寒组白细胞计数与正常对照儿童组比较无显著差异(p>0.05);成人伤寒组白细胞计数低于成人对照组(p<0.01);伤寒患者早期血培养阳性率88.9%,而肠道拭子阳性率仅为5.3%.结论成人伤寒白细胞明显下降,而儿童则变化不大,利用早期第1周血培养高阳性率和伤寒沙门氏菌诊断特点可以在24～48小时内快速做出诊断.     

单克隆抗体鉴定鼠伤寒沙门氏菌的实验研究

本文应用沙门氏菌单克隆抗体建立了ELISA检测鼠伤寒沙门氏菌(STM)的方法,并与协同凝集方法对临床分离的162株STM进行对比鉴定研究,结果表明ELISA其方法灵敏高度,可检出5×10~2菌/ml,特异性强,且结果准确客观,对鼠伤寒沙门氏菌0-4抗原的检出率为100％,抗原H-i的检出率为96.3％,同时结果表明所用沙门氏菌McAb特异性和敏感性都达到了目前所用的沙门氏菌诊断因子血清的标准,具有推广应用的前景。     

儿童脓毒症39例病原菌及临床分析

目的：了解近3年我科儿童脓毒症发生情况及临床特点。方法对2010~2012年我院儿科血培养阳性病例39例进行临床相关因素分析。结果①本组病例男性多于女性;②1岁内儿童脓毒症发生率较高;③临床上多以发热及呼吸道感染为主要症状;④本组血培养病原菌阳性检出率为1.26%;⑤本组血培养阳性病例中检出革兰阳性菌29株(占79.6%)、革兰阴性菌10株(占20.4%)。结论儿童脓毒症以1岁内多见;病原菌以葡萄球菌居首位。     

BACT／Alert120血培养联合白细胞计数快速诊断伤寒

目的　为准确和快速诊断伤寒 ,以便尽快获得病原菌和控制伤寒流行 .方法　通过BACT/Alert12 0进行血培养和肠道拭子培养 ,同时用Sysmex -K - 4 5 0 0血细胞分析仪进行白细胞计数 ,并利用伤寒沙门菌某些固有特点项目进行快速诊断 .结果　儿童伤寒组白细胞计数与正常对照儿童组比较无显著差异 (p >0 .0 5 ) ;成人伤寒组白细胞计数低于成人对照组 (p <0 .0 1) ;伤寒患者早期血培养阳性率 88.9% ,而肠道拭子阳性率仅为 5 .3% .结论　成人伤寒白细胞明显下降 ,而儿童则变化不大 ,利用早期第 1周血培养高阳性率和伤寒沙门氏菌诊断特点可以在 2 4～ 4 8小时内快速做出诊断     

抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体的研制及 …

本文采用杂交瘤技术，制备了７株抗甲型副伤寒沙门氏菌鞭毛抗原单克隆抗体的杂交瘤细胞株。对３株ｍＡｂ（２Ｃ８，２Ｅ６和５Ｄ７０进行了鉴定，ｍＡｂ ２Ｃ８和５Ｄ７为ＩｇＭ，２Ｅ６为ＩｇＧ１亚类，可分别识别不同的抗原表位，均具有很强的特异性，即只与甲型副伤寒沙门氏菌及其鞭毛抗原起反应，而与相关肠道细菌：尼亚里木沙门氏菌，达卡沙门氏菌及伤寒沙门氏菌，乙型和丙型副伤寒杆菌，猪霍乱沙门氏菌，纽波特沙门氏菌等均无     

超声心动图对感染性心内膜炎的诊断价值

目的评价超声心动图对感染性心内膜炎的诊断价值.方法对感染性心内膜炎的患儿23例临床资料分析.结果血培养阳性率为38%,连续2次血培养阳性且为同一种细菌者仅为1例.超声心动图检出赘生物共17例(73.9%).结论超声心动图对感染性心内膜炎的诊断有重要意义.     

Evaluation of passive bacterial agglutination for the diagnosis of typhoid fever.

We evaluated the reliability of a passive bacterial agglutination test to detect Salmonella typhi somatic antigen(s) in the sera of patients with typhoid fever. It was positive in 32 of 33 bacteriologically proven typhoid fever cases. Among 13 patients with a presumptive diagnosis of typhoid fever, 11 were positive by passive bacterial agglutination. The serum of one patient with paratyphoid A was also positive. Among 50 febrile patients without typhoid fever, one was persistently positive during the course of illness; 49 were negative. The sensitivity, specificity, and accuracy indices of the passive bacterial agglutination test were over 95%. The positive and negative predictive values were 94 and 98%, respectively.     

Enteric Fever and Other Extraintestinal Salmonellosis in University Hospital, Nottingham, UK, Between 1980 and 1997

 The clinical spectrum of extraintestinal salmonellosis comprises enteric fever (typhoid and paratyphoid) and invasive infections due to nontyphoidal salmonellae. This study describes the clinical spectrum, management and outcome of all confirmed cases of extraintestinal salmonellosis in patients admitted to University Hospital, Nottingham, UK, between 1980 and 1997. There were 142 cases (children, 42; adults, 100) of extraintestinal salmonellosis, of which 38 (children, 20; adults, 18) were enteric fever, consisting of 21 cases of typhoid, 12 of paratyphoid A and five of paratyphoid B. All patients with typhoid and paratyphoid A fever were from Indian or Pakistani families and, except for two adults, all were considered to be previously fit. The outcome in patients with enteric fever was excellent, and there were no complications. Of the 104 patients (children, 22; adults, 82) with nontyphoidal salmonellosis, 69 were bacteraemic secondary to gastroenteritis, 10 were bacteraemic without an obvious focus of infection and 25 had focal infections. The three major sites of focal infections were meningitis in five infants, osteomyelitis in two children and three adults, and arterial infections in ten adults. The three most frequently isolated organisms were Salmonella enteritidis (40%), Salmonella typhimurium (25%) and Salmonella virchow (14%). Sixty-seven percent of these patients had underlying disease(s)/risk factors. In contrast to the outcome of enteric fever, there were 19 deaths (children, 2; adults, 17) in patients with nontyphoidal salmonellosis. Sixteen of the 17 adults who died were over the age of 60 years. Eight (25%) of 32 males over the age of 60 years with nontyphoidal Salmonella bacteraemia had arterial infections. In some patients, the diagnosis of Salmonella arterial infection is likely to be delayed or missed altogether if blood cultures are not obtained. Mortality in patients over the age of 60 years with nontyphoidal Salmonella infections was 28%.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Detection of urinary Vi antigen as a diagnostic test for typhoid fever

Since Vi antigen is limited primarily to Salmonella typhi, it has been thought that detection of the antigen may be a useful method for diagnosing acute typhoid fever. The slide coagglutination method and enzyme-linked immunosorbent assay have recently been suggested as ways to detect small quantities of Vi antigen in urine. In Santiago, Chile, we compared the results of these two methods in patients with acute typhoid fever, paratyphoid fever, and other febrile illnesses and in afebrile control subjects. Using a cut-off value that maximally separated typhoid patients from controls, the enzyme-linked immunosorbent assay was positive in 62.4% of 141 patients with culture-proven typhoid infections and in 13.2% of 159 afebrile control subjects. The enzyme-linked immunosorbent assay was false positive in 64.7% of 34 culture-proven paratyphoid A or B patients and 47.1% of 21 patients with other nontyphoidal febrile illnesses. The coagglutination test was positive in 34% of typhoid patients, 14% of afebrile control subjects, and 46% of febrile control subjects. We conclude that these tests when performed with the Vi antibodies employed in this study are of little value for the diagnosis of typhoid fever in this setting.     

昆明市官渡区2008~2017年伤寒、副伤寒流行特征分析

目的 分析2008~2017年官渡区伤寒、副伤寒流行特征,为制定有效的防控策略提供科学依据。方法 统计“中国疾病预防控制信息系统”中 2008~2017年官渡区伤寒、副伤寒病例数据,对其进行描述流行病学分析。结果 2008~2017年官渡区共报告伤寒、副伤寒1407例,年均发病率为15.92/10万,主要以伤寒为主,伤寒、副伤寒构成比为1.41∶1,发病季节性明显,每年3月份发病开始上升,5~10月份为高发季节,9月份达高峰,11月份开始下降,官渡区伤寒、副伤寒各个街道均有病例报告,关上街道发病居官渡区第1位,金马街道和小板桥街道发病交错排位第2位和第3位。各年龄组均有发病,主要集中于10~40岁组人群,男性发病多于女性,男女之比为1.12∶1。发病人数最多的职业是学生,其次是家务及待业和农民,每年占总发病数的37%~68%。结论 近10年官渡区伤寒、副伤寒发病仍维持较高的发病水平,发病呈散发状态,高危人群为学生、家务及待业、农民,应加强疫情监测。针对性采取综合措施,进一步降低伤寒、副伤寒的发病率。     

Standardisation of polymerase chain reaction for the detection of Salmonella typhi in typhoid fever.

To improve the diagnosis of Salmonella typhi infection, a polymerase chain reaction (PCR) assay was developed for the amplification of the dH flagellin gene of S typhi. Primers were designed from dH flagellin gene sequence which will give an amplification product of 486 base pairs. In tests to study the specificity of the assay, no amplification was seen in non-salmonella strains or salmonella strains with flagellar gene other than "d". Sensitivity tests determined that 28 pg of S typhi target DNA or 3 x 10(2) target bacteria could be detected by the PCR assay. Subsequently, the PCR technique was used for detection of S typhi in blood or clot cultures from 84 patients clinically suspected of having typhoid fever, and from 20 healthy control subjects. Twenty five of 84 samples from clinically suspected cases were positive by PCR; four of which were culture negative. No amplification was seen in samples from patients who were culture positive for organisms other than S typhi or from controls. The time taken for each sample for PCR analysis was less than 48 hours compared with three to five days for blood or clot culture. PCR appeared to be a promising diagnostic test for typhoid fever.     

Value of a Single-Tube Widal Test in Diagnosis of Typhoid Fever in Vietnam

The diagnostic value of an acute-phase single-tube Widal test for suspected typhoid fever was evaluated with 2,000 Vietnamese patients admitted to an infectious disease referral hospital between 1993 and 1998. Test patients had suspected typhoid fever and a blood culture positive for Salmonella typhi (n= 1,400) or Salmonella paratyphi A (n = 45). Control patients had a febrile illness for which another cause was confirmed (malaria [n = 103], dengue [n = 76], or bacteremia due to another microorganism [n = 156] or tetanus (n = 265). An O-agglutinin titer of >/=100 was found in 18% of the febrile controls and 7% of the tetanus patients. Corresponding values for H agglutinins were 8 and 1%, respectively. The O-agglutinin titer was >/=100 in 83% of the blood culture-positive typhoid fever cases, and the H-agglutinin titer was >/=100 in 67%. The disease prevalence in investigated patients in this hospital was 30.8% (95% confidence interval, 26.8 to 35.1%); at this prevalence, an elevated level of H agglutinins gave better positive predictive values for typhoid fever than did O agglutinins. With a cutoff titer of >/=200 for O agglutinin or >/=100 for H agglutinin, the Widal test would diagnose correctly 74% of the blood culture-positive cases of typhoid fever. However, 14% of the positive results would be false-positive, and 10% of the negative results would be false-negative. The Widal test can be helpful in the laboratory diagnosis of typhoid fever in Vietnam if interpreted with care.     

Detection of Salmonella typhi protein antigen in serum and urine: a value for diagnosis of typhoid fever in an endemic area

Using haemoculture as the gold standard, a double antibody sandwich ELISA for the detection of Salmonella typhi Barber protein antigen (BP) was compared with the Widal test. Specimens used were serum and urine obtained from normal healthy individuals and from patients with typhoid fever, paratyphoid fever, pyrexia caused by other bacteria and pyrexia with negative haemoculture. The ELISA for antigenuria gave a significantly higher sensitivity, specificity, accuracy and positive predictive value than the Widal test (p less than 0.05). The ELISA for antigenaemia gave a significantly higher sensitivity and positive predictive value only. All other values were not significantly different. The timing of specimen collection was critical for sensitivity in the ELISA for antigenaemia and antigenuria, and the best results could be obtained by carrying out both assays simultaneously. The clearance of BP from serum into urine occurred around 16 days after the onset of fever in one patient. In two patients, BP could be detected in sera up to 3 weeks after the onset of fever. In two patients, serum BP could still be detected although haemoculture was negative.     

Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR

We quantified the gene copies from Salmonella enterica serovar Typhi (S. Typhi) in the blood of patients suspected of having typhoid fever by using TaqMan-based real-time PCR (TaqMan assay) to target the S. Typhi flagellin gene in genomic DNAs isolated from blood samples. Of 55 blood samples taken from suspected typhoid fever patients, eight blood samples with a positive blood culture had S. Typhi loads ranging from 1.01 x 10(3) to 4.35 x 10(4) copies/ml blood, and from 47 blood samples with negative blood culture, there were 40 (85.1%) TaqMan assay-positive samples with loads ranging from 3.9 to 9.9 x 10(2) copies/ml blood. In the present study, the TaqMan assay detected more than 10(3) copies/ml blood of S. Typhi in all of the blood culture-positive samples, whereas less than 10(3) copies/ml blood of S. Typhi were detected in the blood culture-negative samples. Our findings suggest that a TaqMan assay may be useful for assessing S. Typhi loads, especially in cases of suspected typhoid fever with negative results from the standard blood culture test.     

Evaluation of a simple and rapid dipstick assay for the diagnosis of typhoid fever in Indonesia

To support the clinical diagnosis of typhoid fever in Indonesia, where most hospitals and health centres have no facilities for culture, a rapid dipstick assay for the detection of Salmonella typhi-specific IgM antibodies was evaluated on serum samples from 127 patients clinically suspected of having typhoid fever. In a single blood sample collected on admission to hospital, the sensitivity of the dipstick assay was 69.8% when compared with bone marrow culture and 86.5% when compared with blood culture. The specificity as calculated for the group of patients with suspected typhoid fever but a negative culture result was calculated to be 88.9%. Of 80 patients with febrile illnesses other than typhoid fever, reactivity was observed in only three patients with dengue haemorrhagic fever. The assay uses stabilised components that can be stored outside the refrigerator, does not require special equipment, and may be of use in remote health facilities that have no culture facilities.