本文選題：肺炎支原體 + 肺炎支原體腦炎�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:肺炎支原體(mycoplasma pneumonia,MP)是引起小兒呼吸道感染的常見病原體之一。MP感染除引發(fā)呼吸系統(tǒng)炎癥外,還幾乎可以累及人體其他全部器官。神經(jīng)系統(tǒng)侵害是MP所導(dǎo)致肺外疾病中常見的類型之一。腦炎是MP感染累及中樞神經(jīng)系統(tǒng)的表現(xiàn)中最為常見的一種[1]。目前,對(duì)于兒童罹患肺炎支原體腦炎(Mycoplasma pneumoniae encephalitis,MPIE)的診斷尚沒有明確標(biāo)準(zhǔn),治療也有很多爭議。目前MP感染的檢測方法包括腦脊液(cerbrospinal fluid,CSF)PCR、腦脊液病原體的培養(yǎng)、血清特異性Ig M和Ig G抗體測定等方法。血清抗體檢測雖方便易行,但僅抗體陽性不能確診,只能作為疑似病例。PCR和病原體培養(yǎng)可以作為確診MPIE的金標(biāo)準(zhǔn),但兩種方法都存在著陽性率極低的缺點(diǎn)。對(duì)于那些有腦炎癥狀而金標(biāo)準(zhǔn)檢測陰性的患者如何確診,是臨床診斷中的難題,急需找到敏感可行的檢測手段,提高檢出率,確定病因,輔助治療。由于實(shí)驗(yàn)技術(shù)手段的日益成熟,傳統(tǒng)的PCR技術(shù)已經(jīng)無法滿足需求,由此而誕生的多重PCR(multiplex PCR)是在同一個(gè)PCR反應(yīng)體系中加上兩對(duì)或更多對(duì)的引物,并能同時(shí)擴(kuò)增出相應(yīng)多個(gè)核酸片段的PCR反應(yīng)。而GeXP多重RT-PCR(GeXP m RT-PCR)即為此原理。本研究預(yù)利用此方法診斷肺炎支原體腦炎,并與傳統(tǒng)的PCR法進(jìn)行比較,分析GeXP多重RT-PCR在診斷肺炎支原體腦炎中的敏感性。方法:1資料收集:2015年5月至2016年8月河北省兒童醫(yī)院神經(jīng)內(nèi)科病區(qū)收治的,初次入院的患兒100例(年齡32日-10歲,男女比例51:49),以發(fā)熱伴頭痛為首發(fā)癥狀,有嗜睡,萎靡,嘔吐癥狀,后繼出現(xiàn)神經(jīng)系統(tǒng)癥狀和體征的疑似腦炎的患兒,每位患兒在入院第一日或第二日采集1份腦脊液,1份靜脈血。2指標(biāo)檢測:對(duì)采集的100份疑似腦炎患者的腦脊液進(jìn)行病原體篩查。(1)對(duì)所有標(biāo)本進(jìn)行常規(guī)腦脊液細(xì)菌培養(yǎng),培養(yǎng)陽性的標(biāo)本可確診細(xì)菌感染性腦炎。(2)排除常見細(xì)菌性腦炎的剩余標(biāo)本進(jìn)行腦脊液抗酸染色檢查,抗酸陽性的標(biāo)本可確診結(jié)核性腦膜炎。(3)還未確診的標(biāo)本進(jìn)行腦脊液行普通涂片染色檢查,檢測新型隱球菌在內(nèi)的微生物,排除其他微生物感染所致腦炎。(4)細(xì)菌檢測陰性的標(biāo)本進(jìn)行病毒學(xué)檢測:GeXP多重RT-PCR法設(shè)計(jì)有甲型流感病毒等多種病毒引物(見表-1),排除病毒性腦炎的病例。同時(shí)亦進(jìn)行EBV血清Ig M抗體檢測與腦脊液EBV DNA檢測。(5)對(duì)以上檢測全部陰性的標(biāo)本進(jìn)行支原體檢測。首先采用被動(dòng)顆粒凝集法進(jìn)行血清中的肺炎支原體Ig M檢測,篩查出血清抗體陽性的疑似支原體腦炎患者,再對(duì)該病人的腦脊液分別采用GeXP多重RT-PCR和普通PCR法檢測,并對(duì)比分析檢測結(jié)果,探尋檢測支原體感染性腦炎的敏感方法。(6)對(duì)所有標(biāo)本進(jìn)行腦脊液生化檢查(項(xiàng)目包括腦脊液葡萄糖含量測定、腦脊液蛋白含量測定、腦脊液氯離子含量測定、腦脊液腺苷脫氨酶測定,其中葡萄糖和蛋白質(zhì)含量作為主要參考指標(biāo))、腦脊液細(xì)胞學(xué)常規(guī)檢查,綜合上述檢測結(jié)果,作出最后的病原學(xué)診斷。(7)最后測定的肺炎支原體陽性標(biāo)本,進(jìn)一步DNA測序鑒定。3分析的指標(biāo):(1)GeXP多重RT-PCR檢測法:標(biāo)本最終經(jīng)過毛細(xì)管電泳呈現(xiàn)圖形顯示,肺炎支原體陽性結(jié)果中除3個(gè)內(nèi)參峰,在核酸片段大小為216.8nt處出現(xiàn)特異的MP陽性峰。(2)抗體顆粒凝集法結(jié)果顯示為(+++)時(shí)為陽性,顯示(-),(+),(++)時(shí)均為陰性。(3)對(duì)兩方法實(shí)驗(yàn)結(jié)果綜合患者各項(xiàng)臨床指標(biāo)和腦脊液實(shí)驗(yàn)室檢查結(jié)果,最后確診是否為肺炎支原體腦炎,并比較檢出率差異。4統(tǒng)計(jì)分析:采用配對(duì)資料χ2檢驗(yàn)查驗(yàn)陽性率有無差異,檢驗(yàn)水準(zhǔn)α=0.05。結(jié)果:1細(xì)菌學(xué)檢測結(jié)果:大腸埃希菌陽性1例,肺炎克雷伯菌陽性1例,金黃色葡萄球菌陽性2例,表皮葡萄球菌陽性1例,人葡萄球菌陽性2例,肺炎鏈球菌陽性7例,結(jié)核分支桿菌陽性4例,白色念珠菌陽性2例(見表-2)。細(xì)菌性腦炎合計(jì)18例,真菌性腦炎2例。2病毒學(xué)檢測結(jié)果:排除上述病原體檢測陽性標(biāo)本20例后,尚未確診的80例標(biāo)本進(jìn)行EBV檢測:結(jié)果顯示EBV Ig M抗體和腦脊液EBV DNA均為陽性的標(biāo)本共2例(見表-3)。3經(jīng)上述檢測后,尚剩余78例標(biāo)本未確診,進(jìn)行支原體檢測:采集的血清經(jīng)抗體顆粒凝集法檢測,19例血清支原體Ig M陽性,視為可疑支原體腦炎病例。隨后將19例可疑腦脊液標(biāo)本經(jīng)GeXP多重RT-PCR分析系統(tǒng)檢測,得到8例陽性標(biāo)本,陽性率為42%。而普通PCR法僅測出2例陽性結(jié)果。經(jīng)x2檢驗(yàn),P0.05,兩種檢測結(jié)果存在統(tǒng)計(jì)學(xué)差異,GeXP多重RT-PCR法優(yōu)于普通PCR法(見表-4)。4最后檢測得到的8例陽性標(biāo)本,由于兩種PCR結(jié)果不一致,故而將PCR產(chǎn)物送生物公司測序檢測,結(jié)果證明這8例均為肺炎支原體陽性結(jié)果(見附圖2)。結(jié)論:1 GeXP多重RT-PCR法在檢測腦脊液肺炎支原體時(shí)優(yōu)于傳統(tǒng)的PCR法;并可同時(shí)排除其他病毒感染。2 GeXP多重RT-PCR檢測法在檢測小兒肺炎支原體方面具有高準(zhǔn)確性,高靈敏度,特異性強(qiáng)且高效等明顯優(yōu)勢,對(duì)臨床醫(yī)生快速準(zhǔn)確的診斷肺炎支原體腦炎具有很高的應(yīng)用價(jià)值,為肺炎支原體腦炎的早期診斷,早期治療提供了實(shí)驗(yàn)室診斷依據(jù)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.9;R725.6

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