儿童呼吸道炎症患者人型支原体感染状况研究

为了解我国儿童呼吸道炎症患者人型支原体 (Mh)感染状况 ,我们应用套式PCR(nPCR)对无锡地区 139例患有上呼吸道感染、支气管炎、支气管肺炎和肺炎儿童的咽拭子标本中的Mh特异性核酸进行了检测。139例患儿中检出 10例Mh性者 ,阳性率为 7 2 %。表明我国呼吸道炎症患儿中Mh感染并非罕见。鉴于Mh与其他支原体不同 ,Mh对红霉素、罗红霉素、阿齐霉素天然拮抗 ,仅对交沙霉素敏感。因此 ,对呼吸道炎症患儿进行Mh检测有助于合理用药     

套式聚合酶链式反应（Nested PCR）检测肺炎衣原体

肺炎衣原体（Ｃｈｌａｍｙｄｉａ ｐｎｅｕｍｏｎｉａｅ，ＣＰｎ）可引起人类急性呼吸道炎症，临床表现以肺炎为主，已在世界上许多国家和地区引起流行，本文介绍通过套式ＰＣＲ检测ＣＰｎ，共检测１０９例呼吸道感染者之咽拭标本，检出１３例阳性（阳性率１２％），提示ＣＰｎ的呼吸道感染有我国并不少见。实验结果表明，套式ＰＣＲ用于ＣＰｍ检测方法的建立，在敏感、特异、简便、快速的特点，具有良好的应用前景。     

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。     

应用PCR技术检测小儿呼吸道病原体的研究

本文应用PCR技术检测了1370例患者的肺炎支原体（MP）阳性检出率为12．92％，EB病毒（EBV）阳性检出率为14．52％，腺病毒（AV）阳性检出率为12．35％。这些病原体在小儿呼吸道中感染十分常见，可引起上呼吸道感染、咽炎、肺炎及其他系统炎症。因此早期诊断具有较高的临床意义和流行病学意义。     

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。     

套式（Nested）PCR检测肺炎支原体与肺炎衣原体研究

本文报告根据肺炎支原体（Mp）和肺炎衣原体（Cpn）的16SrRNA基因序列，建立了套式（Nested）PCR检测方法。灵敏度试验表明，Mp套式PCR和Cpn套式PCR灵敏度分别高出Mp普通PCR和Cpn普通PCR二个数量极。     

无锡地区儿童呼吸道炎症者支原体、衣原体感染研究

目的　为了解无锡地区儿童呼吸道炎症者支原体、衣原体感染状况。方法　收集儿童上感、支气管炎、肺炎、哮喘和新生儿肺炎的咽拭子标本进行肺炎支原体 (Mpn)、人型支原体 (Mh)、肺炎衣原体 (Cpn)的联合套式聚合酶链式反应(nestedPCR ,nPCR)检测。结果　无锡地区各种儿童呼吸道炎症组均有Mpn、Mh、Cpn感染者检出。儿童上感、支气管炎、肺炎、哮喘和新生儿肺炎组支原体和衣原体总阳性检出率分别达 1 5 7%、1 5 4 %、2 8 1 %、50 0 %、1 5 2 %。结论　支原体、衣原体已为无锡地区儿童呼吸道感染的重要病原体     

青壮年与中老年社区获得性肺炎病原体的对比分析

目的 通过研究对青壮年及中老年人社区获得性肺炎（CAP）的常见呼吸道病原体分布,为CAP的合理用药治疗提供参考依据。方法 收集2017年6月~2018年6月于我院住院治疗的青壮年及中老年CAP各120例,通过痰细菌学培养、痰液病原体核酸检测、血清呼吸道病原体IgM抗体检测等方法检测病原体。结果 120例青壮年CAP中,通过以上方式检测到病原体感染有52例,病原体最多的为肺炎支原体（31.67%）,其次为流感嗜血杆菌（7.50%）、肺炎链球菌（3.33%）;中老年CAP中,检测到的病原体感染患者有62例,最多的病原体为肺炎支原体（17.50%）,其次为肺炎克雷伯菌（13.33%）、流感嗜血杆菌（12.50%）。非典型病原体感染在青壮年CAP中比例多于中老年人,而细菌感染在中老年人CAP中多于青壮年（P＜0.05）。结论 非典型病原体、细菌感染均是青壮年、中老年人CAP的常见病原体,在青少年CAP中以非典型病原体感染为主,在中老年CAP中以细菌感染为主。     

常熟地区育龄妇女生殖道炎症者NUC感染的套式PCR检测

NUC病原体已是我国育龄妇女生殖道炎症最常见的病原体群.NUC感染并可致不良妊娠结局.为了解江苏常熟地区育龄妇女生殖道炎症者的NUC感染状况.我们应用套式PCR(nPCR)法检测了患者宫颈脱落细胞中的NG、UU、CT的特异性DNA.结果①NUCnPCR检测特异性、正确性较好;②本地区育龄妇女生殖道炎症者中NG、UU、CT的阳性检出率分别高达28.2%、26.7%、13.8%.本文对NUC感染的检测方法和危害性进行了讨论.     

检测肺炎衣原体血清IgM抗体ELISA的建立和应用

特异性抗体测定是目前诊断肺炎衣原体(Chlamydia pneumoniae,C.pn)感染较敏感的方法.主要外膜蛋白(MOMP)是C.pn重要的抗原成分,具有良好的免疫原性,与C.pn的感染密切相关,其特异性抗体具有免疫保护作用[1].机体感染C.pn后体内可出现特异性的IgM抗体,检测该IgM抗体可作为判断C.pn感染的标志.本研究利用特异性的MOMP抗原,建立检测血清中C.pn特异性IgM抗体的ELISA[2],以351例病原学确诊的临床血清标本和11例其他病原体感染的肺炎患者血清标本为对照,对其灵敏度和特异性进行了评价,现报告如下.     

Identification of Chlamydia pneumoniae in intracranial and extracranial arteries in patients with stroke and in controls: combined immunohistochemical and polymerase chain reaction analyses

An association of the obligatory intracellular gram-negative pathogen Chlamydia pneumoniae with coronary artery disease, myocardial infarction, and atherosclerosis was suggested. The presence of C pneumoniae was determined in different arteries (n = 165) from 23 control cases and 10 patients with stroke including coronary arteries, carotid arteries, basilar artery, and middle cerebral arteries of normal controls and patients with stroke using nested polymerase chain reaction (PCR) and immunohistochemistry (IHC). Atherosclerosis was detected in 51.5% of all investigated arteries. No significant differences were detected between controls (59.1% by IHC, 45.5% by nested PCR) and patients with stroke (40% by IHC, 40% by nested PCR). This is the first investigation demonstrating C pneumoniae by IHC and nested PCR in different intracerebral arteries in control persons and patients with stroke. No significant correlation between the presence of chlamydial DNA or antigens in arteries and stroke could be demonstrated. The presence of the C pneumoniae is indicative of a correlation between infection and atherosclerosis, but not of a specific vascular neuropathology such as stroke.     

Development and evaluation of real-time PCR-based fluorescence assays for detection of Chlamydia pneumoniae

Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.     

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.     

Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens

Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae ATCC VR-1310. The proportion positive versus the C. pneumoniae concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested, P < 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median C. pneumoniae concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target C. pneumoniae DNA present.     

Analytical sensitivity, reproducibility of results, and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral blood mononuclear cells

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P<0.001), with the PstI fragment (P<0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P<0.001), and the other 3 assays detected no positive specimens (P<0.001, compared with the ompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect C. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.     

Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.

Chlamydia pneumoniae is an important cause of respiratory disease in humans, but diagnosis of C. pneumoniae is hindered by difficulties in the in vitro growth of the organism. In order to improve detection and identification, we recently developed a polymerase chain reaction (PCR) assay which uses oligonucleotide primers specific for C. pneumoniae. The nucleic acid sequence was determined for the 16S rRNA of C. pneumoniae, and regions in which C. pneumoniae differed from both Chlamydia psittaci and Chlamydia trachomatis were identified. Oligonucleotide primers corresponding to these unique regions were then synthesized and used in a PCR for the detection of C. pneumoniae. The C. pneumoniae-specific primers permitted the identification of six isolates of C. pneumoniae, but no reaction was observed with the 15 serovars of C. trachomatis or two strains of C. psittaci. PCR should prove to be valuable in confirming the identification of C. pneumoniae and in the diagnosis of C. pneumoniae infections.     

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.     

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS--To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS--A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS--PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS--The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.     

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.