儿童肺炎肺炎支原体及肺炎衣原体联合基因诊断研究

目的：探讨我国肺炎儿童中肺炎支原体（Mycoplasma pneumoniae,Mpn）和肺炎衣原体（Chlamydia pneumoniae,Cpn）的感染状况和Mpn和Cpn合并感染状况。方法：收集80例健康儿童和134例肺炎儿童之咽拭子标本,应用套式PCR（nested PCR）技术进行检测,并随机抽取Mpn和Cpn和3例套式PCR阳性产物进行全自动荧光DNA测序确诊。结果：肺炎儿童Mpn和Cpn套式PCR阳性检出率分别高达30.0%和35.0%,明显高于健康儿童的2.5%和2 .5%（P均＜0.01）,肺炎儿童中Mpn、Cpn双重阳性者为27例,达20.1%。结论：Mpn和Cpn是我国儿童肺炎的重要病原体,并存在较高比例的Mpn、Cpn双重感染者。     

新生儿和儿童肺炎支原体和衣原体检测研究

为了解新生儿和儿童肺炎者肺炎支原体（Mpn）、解脲脲原体（Uu)、肺炎衣原体（Cpn）、沙眼衣原体（Ct）等支原体、衣原体的感染状况,我们收集了29例足月新生儿肺炎者、24例早产新生儿肺炎者和41例儿童肺炎者的咽拭子标本,就用套式PCR（nPCR）技术进行了检测。结果发观新生儿肺炎者中存在Mpn、Uu、Cpn、Ct4种病原体感染者,阳性率在11．8％－62．2％之间,儿童肺炎者中存在Mpn、Cpn、Ct3种病原体感染者,阳性率分别为19．5％、22．0％、9．8％,无Uu感染者。Mpn、Cpn一直被认为是社区获得性肺炎（CAP）的病原体。本文从新生儿肺炎者的咽部查出Mpn、Cpn进一步支持Mpn、Cpn存在母婴传播之论点。     

母婴健康有关3种衣原体（Cps,Cpn,Ct）通用引物扩增和DNA序列测定方法的建立

目的：建立鹦鹉热衣原体（Cps)，肺炎衣原体（Cpn)和沙眼衣原体（Cr)3种衣原体通用引物扩增和DNA序列测定技术，方法：在衣原体16SrRNA基因序列保守区设计外套引物内套引物，建立Cps,Cpn和Ct3种衣原体通用引物的套式PCR（GnPCR)扩增体系和DNA测序体系,结果：Cps,Cpn和Ct3种衣原体菌体均能被GnPCR扩增出，其中Cps和Cpn的GnPCR产物经DNA测序与标准株序列完全相同，GnPCR灵敏度地较Cpn和Ct种特异传统PCR高，结论：Cps,Cpn和Ct的GnPCR灵敏，特异，因不同的衣原体感染有着相同的疾病谱，因此本方法的建立能降低患者医疗费用的同时使患者在第一时间得到及时有效的治疗。     

新生儿肺炎患儿淋菌套式PCR检测研究

本文用套式PCR技术检测了106例新生儿肺炎患儿及50例正常新生儿鼻咽部分泌物中的淋菌特异性DNA，结果：患儿阳性率17．0％（18／106），正常对照儿阳性率2，0％（1／50），两者有显著差异（P＜0．01）。其中自然分娩儿阳性率23．7％（14／59），剖宫产儿阳性率8．5％（4／47），有显著差异（P＜0．05）。早产、低体重儿、足月小样儿阳性率37．8％（14／37），足月、正常体重儿阳性率5．8％（4／69），两者也有显著差异（P＜0．01）。提示：淋菌感染与新生儿肺炎有关联。用套式PCR技术诊断壮菌感染，对防治围产期及新生儿感染带来的严重危害具有重要价值。     

金标法和冷凝集试验测定肺炎支原体特异性抗体的结果比较

肺炎支原体（Mp）是一种常见病原体，通过呼吸道传播，健康人吸入患者咳嗽、打喷嚏时喷出的口鼻分泌物而受感染。故可引起散发呼吸道感染或小流行。支原体肺炎以儿童及青少年居多，Mp通常存在于纤毛上皮之间，不侵入肺实质，通过细胞膜上神经氨酸受体位点，吸附于宿主呼吸道上皮细胞表面，抑制纤毛活动与破坏上皮细胞而引起肺炎和支气管炎等，肺外病变比较少见。近年国内报道Mp感染儿童较多，成年人较少，无明显季节性，长期以来，Mp感染并不受临床医师所重视，导致漏诊和误诊及抗生素的滥用，     

对儿童肺炎支原体特异性抗体的实验室检测及方法学分析

目的对呼吸道感染患者进行肺炎支原体检测，以期协助临床早诊断早治疗，并对常用检测方法进行分析。方法对1060例呼吸道感染者同时做冷凝集试验并用。结果1060例呼吸道感染者，检出Mp特异性抗体阳性201例，阳性率18、9％。（3岁、-6岁、-14岁、〉14岁4个年龄组Mp特异性抗体阳性率分别为8、7％（28／320）、23．5％（95／402）、33．1％（71／214）、5．6％（7／124）。在201例临床诊为Mp感染的病例中，抗-Mp—IgG阳性131例，敏感性63.5％，抗-Mp—IgM阳性177例，敏感性85．9％例，冷凝集实验阳性99例，敏感性48．1％，抗-Mp—IgM敏感性较冷凝集实验高（P〈0.05）。结论间接免疫荧光法检测血清Mp抗体是目前较稳定可靠的检测方法，可作为早期诊断Mp感染的指标之一。     

关注肺炎支原体与不良妊娠结局研究

肺炎支原体（Mycoplasmapneumoniae，Mpn）是最早被认为与人类疾病相关的支原体。Mpn感染除引起上呼吸道感染、气管炎、肺炎和哮喘等肺部疾病外，并已发现心肌炎、心包炎、溶血性贫血、脑膜炎、关节炎和肾盂肾炎亦与Mpn感染相关近来，国内外学者从成人男女生殖道分离或检出到Mp     

肺炎衣原体的套式（Nested）PCR检测及其临床意义

肺炎衣原体（C_(pn)）可致上、下呼吸道的炎症，并且是非典型性肺炎综合征（APS）的主要病原体之一。C_(pn)已占肺炎病原体的第三或第四位。近年的深入研究表明，C_(pn)还能引起呼吸道之外的脏器炎症。由于C_(pn)为专性细胞内寄生菌，因此培养困难。目前国内尚缺乏C_(pn)感染诊断方法。本文介绍一种灵敏、特异、简便的C_(pn)套式PCR检测方法。应用本法检测55例呼吸道感染者之咽拭子，共检出9例阳性（阳性率16．2％）。表明C_(pn)的呼吸道感染在我国并不少见。C_(pn)套式PCR方法的建立，对于我国的C_(pn)人群感染率和C_(pn)感染的分子流行学研究有着极其重要的意义。     

急性呼吸道感染患儿咽拭子标本的肺炎衣原体套式PCR检测研究

本文应用套式（Nested）PCR技术检测了132例急性呼吸道患儿之咽拭子标本中的肺炎衣原体（CPn）特异性DNA。结果发现．受检标本中有19例检出阳性，即各处呼吸道患儿之咽拭子标本中的CPn－DNA阳性率高达14．4％。提示CPn在我国儿童中有较高的感染率。     

肺炎支原体直接损伤及其免疫学致病机制研究进展

肺炎支原体（ Mycoplasma pneumoniae，Mp）是儿童社区获得性肺炎的主要病原体，20％～40％的儿童呼吸道感染是由Mp感染所致［1］。 Mp除可引起肺炎支原体肺炎（ Mycoplasma pneumoniae pneumonia，MPP）、咽炎等呼吸系统疾病之外，还可导致皮肤损害、免疫性溶血性贫血、支原体脑炎、心肌炎等多系统的肺外并发症。近年来MPP发病率逐年上升，尤其是抗大环内酯类Mp的出现，给临床治疗带来困难［2］。 Mp的致病机制尚未明确，目前认为Mp对宿主直接造成损伤及机体免疫应答紊乱是Mp致病的主要因素［3］，现从这两方面对Mp的致病机制综述如下。     

Decontamination of a Mycoplasma-infected Chlamydia pneumoniae strain by pulmonary passage in SCID mice

We describe a procedure to eliminate contaminating Mycoplasma from Chlamydia pneumoniae (C. pneumoniae) cultures by pulmonary passage in severe combined immunodeficiency mice (SCID). Four weeks after experimental infection only C. pneumoniae could be cultured from the lungs of the infected animals while Mycoplasma could not be detected any longer, as shown by PCR, culture and transmission electron microscopy (TEM).     

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.     

巢式聚合酶链反应诊断肺炎衣原体感染的临床应用探讨

目的 应用并评价巢式聚合酶链反应技术诊断肺炎衣原体（Chlamyolia pueumoniae,Cp)感染。方法 采用以衣原体属及Cp种的16SrRNA特异片段为目的基因。对已知鼻咽拭子Cp培养结果的81例病例进行连续2次扩增,检测Cp基因组DNA,并对其应用予以评价。结果 以培养结果为标准。PCR方法的灵敏度为5/17（29.41%）,特异度为52/64（81.25%),阳性预计值为5/17（29.41%),特异度为52/64（81.25%)。阳性予计值为5/17（29.41%),阴性邓丈夫值为52/64（81.25%)。结论 这种巢式聚合酶链反应技术尚不能用于临床诊断Cp感染,有必要以诊断金标准对其应用进行评价。     

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.     

Role of intracellular pathogens in respiratory tract infections

Recent epidemiologic studies have shown that in patients with acute respiratory tract infection, intracellular pathogens, and particularly Mycoplasma pneumoniae and Chlamydia pneumoniae , might be involved more frequently than was previously assumed. In addition, newer investigations, such as specific culture or polymerase chain reaction (PCR), when systematically performed in patients with asthma, chronic obstructive pulmonary disease and even cystic fibrosis, frequently detect Mycoplasma pneumoniae or Chlamydia pneumoniae in those individuals. The significance of such detection (chronic bronchial infection?) and the role that these intracellular pathogens might play in the natural history of chronic respiratory diseases remain to be evaluated.     

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.     

Demonstration by a nested PCR for Mycoplasma pneumoniae that M. pneumoniae load in the throat is higher in patients hospitalised for M. pneumoniae infection than in non-hospitalised subjects

A nested PCR protocol to detect Mycoplasma pneumoniae DNA in throat specimens was developed. An amplification control (AC) template, which is amplified by the same primers as the M. pneumoniae target sequence, was constructed. The assay allowed highly specific and sensitive detection of M. pneumoniae DNA. In all, 305 throat samples, 62 from hospitalised patients and 243 from non-hospitalised subjects, were analysed by the nested PCR. Inhibition of the PCR was observed in 20% of the samples, but was abolished after a 1 in 10 dilution. Throat samples from 5 (8%) of the hospitalised patients and from 7 (3%) of the non-hospitalised subjects were positive for M. pneumoniae DNA. To investigate the relationship between M. pneumoniae load and the severity of disease, the M. pneumoniae load in 10 throat samples from M. pneumoniae-positive subjects was assessed semi-quantitatively by application of the nested PCR to a series of limiting dilutions of nucleic acid extracted from these throat samples. The calculated M. pneumoniae load varied from 20 to 3830 cfu/ml of throat sample. The mean M. pneumoniae load in samples from the hospitalised patients was significantly higher than that in samples from the non-hospitalised subjects. The nested PCR is a useful tool to detect M. pneumoniae DNA in the throat and to study the relationship between the load of M. pneumoniae in throat samples and severity of disease due to M. pneumoniae infection.     

Evaluation of Chlamydia pneumoniae and Mycoplasma pneumoniae as etiologic agents of persistent cough in adolescents and adults

Chlamydia pneumoniae and Mycoplasma pneumoniae were evaluated as agents of persistent cough in adolescents and adults (n = 491). Tests of 473 respiratory specimens by culture or PCR or both identified four episodes (0.8%) of M. pneumoniae-associated illness and no episodes of C. pneumoniae illness, suggesting that these bacteria do not frequently cause persistent cough.