GeXP多重RT-PCR在疑似儿童肺炎支原体脑炎诊断中的敏感性研究

发布时间：2018-04-22 15:13

本文选题：肺炎支原体 + 肺炎支原体脑炎　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:肺炎支原体(mycoplasma pneumonia,MP)是引起小儿呼吸道感染的常见病原体之一。MP感染除引发呼吸系统炎症外,还几乎可以累及人体其他全部器官。神经系统侵害是MP所导致肺外疾病中常见的类型之一。脑炎是MP感染累及中枢神经系统的表现中最为常见的一种[1]。目前,对于儿童罹患肺炎支原体脑炎(Mycoplasma pneumoniae encephalitis,MPIE)的诊断尚没有明确标准,治疗也有很多争议。目前MP感染的检测方法包括脑脊液(cerbrospinal fluid,CSF)PCR、脑脊液病原体的培养、血清特异性Ig M和Ig G抗体测定等方法。血清抗体检测虽方便易行,但仅抗体阳性不能确诊,只能作为疑似病例。PCR和病原体培养可以作为确诊MPIE的金标准,但两种方法都存在着阳性率极低的缺点。对于那些有脑炎症状而金标准检测阴性的患者如何确诊,是临床诊断中的难题,急需找到敏感可行的检测手段,提高检出率,确定病因,辅助治疗。由于实验技术手段的日益成熟,传统的PCR技术已经无法满足需求,由此而诞生的多重PCR(multiplex PCR)是在同一个PCR反应体系中加上两对或更多对的引物,并能同时扩增出相应多个核酸片段的PCR反应。而GeXP多重RT-PCR(GeXP m RT-PCR)即为此原理。本研究预利用此方法诊断肺炎支原体脑炎,并与传统的PCR法进行比较,分析GeXP多重RT-PCR在诊断肺炎支原体脑炎中的敏感性。方法:1资料收集:2015年5月至2016年8月河北省儿童医院神经内科病区收治的,初次入院的患儿100例(年龄32日-10岁,男女比例51:49),以发热伴头痛为首发症状,有嗜睡,萎靡,呕吐症状,后继出现神经系统症状和体征的疑似脑炎的患儿,每位患儿在入院第一日或第二日采集1份脑脊液,1份静脉血。2指标检测:对采集的100份疑似脑炎患者的脑脊液进行病原体筛查。(1)对所有标本进行常规脑脊液细菌培养,培养阳性的标本可确诊细菌感染性脑炎。(2)排除常见细菌性脑炎的剩余标本进行脑脊液抗酸染色检查,抗酸阳性的标本可确诊结核性脑膜炎。(3)还未确诊的标本进行脑脊液行普通涂片染色检查,检测新型隐球菌在内的微生物,排除其他微生物感染所致脑炎。(4)细菌检测阴性的标本进行病毒学检测:GeXP多重RT-PCR法设计有甲型流感病毒等多种病毒引物(见表-1),排除病毒性脑炎的病例。同时亦进行EBV血清Ig M抗体检测与脑脊液EBV DNA检测。(5)对以上检测全部阴性的标本进行支原体检测。首先采用被动颗粒凝集法进行血清中的肺炎支原体Ig M检测,筛查出血清抗体阳性的疑似支原体脑炎患者,再对该病人的脑脊液分别采用GeXP多重RT-PCR和普通PCR法检测,并对比分析检测结果,探寻检测支原体感染性脑炎的敏感方法。(6)对所有标本进行脑脊液生化检查(项目包括脑脊液葡萄糖含量测定、脑脊液蛋白含量测定、脑脊液氯离子含量测定、脑脊液腺苷脱氨酶测定,其中葡萄糖和蛋白质含量作为主要参考指标)、脑脊液细胞学常规检查,综合上述检测结果,作出最后的病原学诊断。(7)最后测定的肺炎支原体阳性标本,进一步DNA测序鉴定。3分析的指标:(1)GeXP多重RT-PCR检测法:标本最终经过毛细管电泳呈现图形显示,肺炎支原体阳性结果中除3个内参峰,在核酸片段大小为216.8nt处出现特异的MP阳性峰。(2)抗体颗粒凝集法结果显示为(+++)时为阳性,显示(-),(+),(++)时均为阴性。(3)对两方法实验结果综合患者各项临床指标和脑脊液实验室检查结果,最后确诊是否为肺炎支原体脑炎,并比较检出率差异。4统计分析:采用配对资料χ2检验查验阳性率有无差异,检验水准α=0.05。结果:1细菌学检测结果:大肠埃希菌阳性1例,肺炎克雷伯菌阳性1例,金黄色葡萄球菌阳性2例,表皮葡萄球菌阳性1例,人葡萄球菌阳性2例,肺炎链球菌阳性7例,结核分支杆菌阳性4例,白色念珠菌阳性2例(见表-2)。细菌性脑炎合计18例,真菌性脑炎2例。2病毒学检测结果:排除上述病原体检测阳性标本20例后,尚未确诊的80例标本进行EBV检测:结果显示EBV Ig M抗体和脑脊液EBV DNA均为阳性的标本共2例(见表-3)。3经上述检测后,尚剩余78例标本未确诊,进行支原体检测:采集的血清经抗体颗粒凝集法检测,19例血清支原体Ig M阳性,视为可疑支原体脑炎病例。随后将19例可疑脑脊液标本经GeXP多重RT-PCR分析系统检测,得到8例阳性标本,阳性率为42%。而普通PCR法仅测出2例阳性结果。经x2检验,P0.05,两种检测结果存在统计学差异,GeXP多重RT-PCR法优于普通PCR法(见表-4)。4最后检测得到的8例阳性标本,由于两种PCR结果不一致,故而将PCR产物送生物公司测序检测,结果证明这8例均为肺炎支原体阳性结果(见附图2)。结论:1 GeXP多重RT-PCR法在检测脑脊液肺炎支原体时优于传统的PCR法;并可同时排除其他病毒感染。2 GeXP多重RT-PCR检测法在检测小儿肺炎支原体方面具有高准确性,高灵敏度,特异性强且高效等明显优势,对临床医生快速准确的诊断肺炎支原体脑炎具有很高的应用价值,为肺炎支原体脑炎的早期诊断,早期治疗提供了实验室诊断依据。
[Abstract]:Objective: Mycoplasma pneumonia (MP) is one of the common pathogens causing respiratory tract infection in children..MP infection, in addition to causing respiratory inflammation, can also involve almost all other organs of the human body. The invasion of the nervous system is one of the common types of extrapulmonary diseases caused by MP. Encephalitis is a MP infection involving the central nervous system. One of the most common manifestations of [1]. is that there are no clear criteria for the diagnosis of Mycoplasma pneumoniae encephalitis (MPIE) in children, and there are many disputes in the treatment. Currently, the detection methods of MP infection include cerebrospinal fluid (Cerbrospinal Fluid, CSF) PCR, the culture of the cerebrospinal fluid pathogens, and the serum specificity Ig M. The serological antibody test is easy and easy to detect, but only the antibody positive can not be confirmed. Only the suspected case.PCR and the pathogen culture can be used as the gold standard for the diagnosis of MPIE, but the two methods have the disadvantage of the very low positive rate. For those with the symptoms of encephalitis, how to diagnose the patients with the negative gold standard It is a difficult problem in clinical diagnosis. It is urgent to find sensitive and feasible detection methods, improve the detection rate, determine the etiology, and assist treatment. Because of the growing maturity of the experimental techniques, the traditional PCR technology is unable to meet the needs, and the birth of the multiple PCR (multiplex PCR) is added to the same PCR reaction system with two pairs or more pairs. PCR reaction of multiple nucleic acid fragments can be amplified at the same time. GeXP multiplex RT-PCR (GeXP m RT-PCR) is the principle of this principle. This study was used to diagnose Mycoplasma pneumoniae encephalitis by this method and compared with the traditional PCR method to analyze the sensitivity of GeXP multiple RT-PCR in the diagnosis of pneumonia mycoplasma encephalitis. Methods: 1 data collection: 2015 5 From month to August 2016, 100 children hospitalized in the neurological department of internal medicine, Hebei children's hospital were admitted to the hospital for the first time (age 32 -10 years old, male and female ratio 51:49), with fever accompanied by headache as the first symptom, drowsiness, malaise, vomiting and symptoms of nervous system symptoms and signs of suspected encephalitis, each child was admitted to the first day or second of the hospital. 1 cerebrospinal fluid (CSF) and 1.2 indexes of venous blood were detected on the day: the cerebrospinal fluid of 100 suspected encephalitis patients was screened by pathogens. (1) all specimens were cultured in the routine cerebrospinal fluid, and the positive specimens could be confirmed for bacterial infective encephalitis. (2) the residual specimens of common bacterial encephalitis were excluded from the cerebrospinal fluid anti acid staining. The specimens with positive acid resistance were confirmed to be diagnosed with tuberculous meningitis. (3) the unconfirmed specimens were examined by common smear staining in the cerebrospinal fluid to detect microbes, including Cryptococcus neoformans, to eliminate encephalitis caused by other microbial infections. (4) the specimens with negative bacterial detection were tested for Virology: GeXP multiple RT-PCR method was designed for influenza a disease A variety of virus primers (see table -1), the cases of viral encephalitis were excluded. At the same time, EBV serum Ig M antibody detection and cerebrospinal fluid EBV DNA detection. (5) mycoplasma detection for all negative specimens were detected. First, passive particle agglutination method was used to detect Mycoplasma pneumoniae Ig M in serum, and to screen the positive of haemorrhage antibody positive. In the patients with suspected Mycoplasma encephalitis, GeXP multiple RT-PCR and common PCR were used to detect the cerebrospinal fluid of the patient, and the sensitive methods for detecting mycoplasma infective encephalitis were compared and analyzed. (6) the biochemical examination of cerebrospinal fluid in all specimens (including the determination of cerebrospinal fluid glucose content and the protein content of cerebrospinal fluid) Determination, determination of chlorine ion content in cerebrospinal fluid, determination of adenosine deaminase in cerebrospinal fluid, glucose and protein content as the main reference index, routine cytological examination of cerebrospinal fluid, combined with the above results, to make the final etiological diagnosis. (7) the final determination of Mycoplasma pneumoniae positive specimens, further DNA sequencing and identification of.3 analysis finger Standard: (1) GeXP multiple RT-PCR detection method: the specimen was finally shown by capillary electrophoresis, and the positive results of Mycoplasma pneumoniae showed a specific MP positive peak in the size of nucleic acid fragments at 216.8nt. (2) the result of antibody particle agglutination showed (+ + +) positive, showing (+), (+ +) negative. (3) to two methods The experimental results were combined with the clinical indicators of the patients and the results of the cerebrospinal fluid laboratory examination, and the final diagnosis was the Mycoplasma pneumoniae encephalitis, and the difference of detection rate was analyzed by.4 statistical analysis: there were no difference between the positive rates of the paired data chi 2 test and the test level of alpha =0.05. results: 1 cases of Escherichia coli positive, 1 cases of Escherichia coli, pneumonia gram. 1 cases of Leber bacteria positive, 2 cases of Staphylococcus aureus positive, 1 positive Staphylococcus epidermidis, 2 cases of Staphylococcus positive, 7 cases of Streptococcus pneumoniae positive, 4 cases of Mycobacterium tuberculosis, 2 cases of Candida albicans positive (see table -2), 18 cases of bacterial encephalitis, 2 cases of fungal encephalitis, and 2 cases of.2 virology detection: excluding the above pathogenic examination of Yang After 20 cases of sex specimens, 80 unconfirmed specimens were detected by EBV. The results showed that 2 cases of EBV Ig M antibody and EBV DNA in cerebrospinal fluid were all positive specimens (see table -3).3, after the above detection, the remaining 78 specimens were unconfirmed and carried out mycoplasma detection: the collected serum was detected by antibody particle agglutination, and 19 cases of serum Mycoplasma Ig M positive were considered as the positive. Suspected Mycoplasma encephalitis cases, 19 cases of suspicious cerebrospinal fluid samples were detected by GeXP multiple RT-PCR analysis system, and 8 positive specimens were obtained. The positive rate was 42%. while 2 positive results were detected by ordinary PCR. X2 test, P0.05, two detection results were statistically different, GeXP multiple RT-PCR method was superior to ordinary PCR method (see table -4).4 final examination. The results of the 8 positive specimens, because of the inconsistent results of the two PCR, sent the PCR product to the biological company sequencing test. The results showed that the 8 cases were all Mycoplasma pneumoniae positive results (see Figure 2). Conclusion: 1 GeXP multiple RT-PCR method is superior to the traditional PCR method in the detection of Mycoplasma pneumoniae in cerebrospinal fluid, and the other virus infection of.2 GeX can be excluded at the same time. P multiple RT-PCR detection method has high accuracy, high sensitivity, high specificity and high efficiency in detecting Mycoplasma pneumoniae in children. It has high application value for the rapid and accurate diagnosis of Mycoplasma pneumoniae encephalitis by clinicians. It provides a laboratory diagnosis basis for the early diagnosis of Mycoplasma pneumoniae encephalitis and early treatment.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.9;R725.6

【参考文献】