呼吸道感染患者肺炎支原体抗体检测结果分析

目的研究肺炎支原体（MP）感染发病率与患者年龄、性别和季节的关系。方法用被动凝集法检测呼吸道感染患者血清中肺炎支原体抗体（MP-Ab）,并对2010年患者MP-Ab检测结果进行分析。结果5年检测结果阳性率为30．10％;男、女性患者阳性率分别为30．74％、36．12％,差异有统计学意义（P〈0．05）;各年龄组差异有统计学意义（P〈0．001）,3～14岁阳性率最高;季节发病率差异无统计学意义;阳性滴度〉1：640的患者占10．18％。结论MP感染逐年增加,3～14岁儿童为高危人群,女性感染机会高于男性,全年均可发病;大多患者预后良好。     

小儿肺炎支原体感染与年龄、性别的关系探讨

目的通过对呼吸道感染患儿血清中肺炎支原体抗体的检测，调查小儿肺炎支原体抗体感染的状况及与患儿性别、年龄的关系。方法采集患儿静脉血、血清做倍比稀释，用日本富士肺炎支原体诊断试剂盒SERODIA-MYCOⅡ做颗粒凝集实验。结果在5155例呼吸道感染患儿中共检出肺炎支原体抗体阳性病例2831例，阳性率为54．9％，男、女患儿阳性率分别为49．6％和63．3％，男女差别具有统计学意义。1岁以内患儿阳性率明显低于其他年龄段，其他年龄段之间阳性率无明显差异；门诊与住院患儿阳性率分别为65．5％和43．2％。结论肺炎支原体感染好发于1岁以上儿童，女童感染机会高于男童，门诊患儿阳性率高于住院患儿。肺炎支原体抗体检测可作为小儿呼吸道感染的常规检测项目。     

ELISA方法检测MP-IgM与冷凝集试验对小儿肺炎支原体感染诊断价值的比较

目的 通过ELISA法与冷凝集试验两种方法检测小儿肺炎支原体肺炎(MPP)患者的血清,分析两种检验方法在小儿肺炎支原体感染的诊断的价值.方法 选择本院儿科收治确诊的157例肺炎支原体感染患儿,分别采用血清ELISA和冷凝集两种检测方法,分析探讨两组阳性率.结果 ELISA法检测MP-IgM阳性有142例,阳性检出率为90.4％;冷凝集试验方法检测阳性为126例,阳性检出率为80.3％,各年龄段阳性检出率差异具有统计学意义,两种检测方法学差异具有统计学意义(P＜0.05).结论 ELISA法与冷凝集试验相比,敏感性更高,具有良好的实验室诊断价值.     

PCR检测肺炎患儿咽拭子肺炎支原体

本文应用ＰＣＲ技术检测了２７５例肺炎患儿之咽拭子标本中的肺炎支原体特异性ＤＮＡ，结果为７１例患儿之咽拭子阳性，阳性率２５．８％，与国内其他作者的报道相近。本文并就肺炎支原体感染的特异性诊断方法作了复习与讨论。     

反复呼吸道感染患儿肺炎支原体PCR检测及免疫功能研究

本文对152例反复呼吸道感染患儿肺炎支原体PCR及免疫功能进行检测。结果咽拭子肺炎支原体PCR阳性25例，阳性率16．5％。血清IgG、IgA明显降低。CD3＋、CD4＋细胞、CD8＋细胞和CD4＋／CD8＋值均较对照组降低，而可溶性白细胞介素－2受体（SIL－2R）水平明显升高。资料表明反复呼吸道感染患儿存在着免疫功能紊乱。     

小儿肺炎支原体肺炎临床特点（232例）

目的分析小儿肺炎支原体肺炎(MPP)的临床特点、支原体抗体检测最佳时间和糖皮质激素的疗效，为小儿支原体肺炎的诊治提供依据。方法收集我院确诊的232例MPP入院患儿临床资料，回顾性分析临床特征并分组。比较发热组与非发热组住院天数；比较激素组与非激素组住院时间和入院后热退天数；分析发病1w内和1w后肺炎支原体抗体阳性率。结果 MPP主要临床症状为发热及干咳。肺部明显阳性体征仅见于36.6%的患儿。发病1w以后特异性 IgM抗体阳性率（94.05%）明显高于1w内（70.37%）（P<0.01）；激素组热退天数（3.88d）与非激素组（3.27d）无差异（P﹥0.05）。结论小儿支原体肺炎发热咳嗽症状明显而肺部体征相对较少；糖皮质激素可减轻发热症状；发病第2w后查支原体抗体阳性率高。     

小儿肺炎支原体感染肺外器官损害210例临床分析

目的探讨小儿肺炎支原体（MP）感染肺外器官损害的临床表现,提高诊治水平。方法回顾分析有肺外器官损害的210例MP感染患儿临床资料。结果肺炎支原体肺炎（MPP）534例中,210例有肺外器官损害（39．3％）,累及的主要器官有心血管系统69例（32．9％）、消化系统86例（40．9％）、血液系统48例（22．9％）、泌尿系统46例（21．9％）、神经系统8例（3．8％）、皮肤损害37例（14．6％）、肌肉关节损害18例（8．6％）。结论MP感染除引起肺部病变外,可同时有1个或1个以上肺外器官受累。对出现肺外表现或以肺外表现为主要临床症状的MPP患儿,应及早确诊．及时治疗,预后艮好。     

荧光定量PCR对1501例NGU患者病原体基因检测结果分析

目的用实时荧光定量聚合酶链反应（FQ-PCR）技术准确度定量检测江门市区凝有非淋菌性尿道炎患者沙眼衣原体（CT）、解脲支原体（UU）基因,进一步了解UU、CT的感染情况,为临床治疗提供依据。方法运用实时荧光定量PCR法（FQ-PCR）对1501例凝有非淋菌性尿道炎患者同时进行CT、UU检测。结果CT的阳性率是13.1%,UU的阳性率是33.9%,二者之间阳性率比较有显著性差异。结论1.江门市区非淋菌性尿道炎患者病原体CT、UU中占有一定比例,二者之间感染率比较有显著性差异。2.荧光实时定量PCR检测UU、CT具有操作简单、反应时间短、重复性好、结果客观准确、敏感性和特异性好等优点,其结果对临床诊断、治疗有一定指导意义。     

肺炎支原体感染儿童肌红蛋白和肌钙蛋白Ⅰ的分析

目的检测并比较肺炎支原体（MP）感染患儿治疗前后血清中肌红蛋白（Mb）和肌钙蛋白Ⅰ（cTn-I）的含量,以探讨其在肺炎支原体肺炎（MPP）合并心肌损害诊治中的意义。方法抽取肺炎支原体感染并有心肌损伤表现的患儿（46例）急性期和恢复期的静脉血,测定并比较其血清中Mb和cTn-I的含量变化,同时以健康儿童做为对照组。结果 MPP合并心肌损伤组患儿急性期血清中Mb和cTn-I的含量显著高于健康对照组（P〈0.01）。与急性期相比,MPP合并心肌损伤的患儿恢复期血清Mb和cTn-I的含量明显下降（P〈0.01）,但仍高于对照组（P〈0.05）。结论 MPP患儿有部分患者急性期有不同程度的心肌损害。     

呼吸道感染患者肺炎支原体抗体检测结果分析

目的 研究肺炎支原体(MP)感染发病率与患者年龄、性别和季节的关系.方法 用被动凝集法检测呼吸道感染患者血清中肺炎支原体抗体(MP-Ab),并对2010年患者MP-Ab检测结果进行分析.结果 5年检测结果阳性率为30.10%;男、女性患者阳性率分别为30.74%、36.12%,差异有统计学意义(P<0.05);各年龄组差异有统计学意义(P<0.001),3～14岁阳性率最高;季节发病率差异无统计学意义;阳性滴度>1:640的患者占10.18%.结论 MP感染逐年增加,3～14岁儿童为高危人群,女性感染机会高于男性,全年均可发病;大多患者预后良好.     

Clinical use of capillary PCR to diagnose Mycoplasma pneumonia

In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasma antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.     

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.     

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.     

Detection of meticillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and Panton-Valentine leukocidin directly from clinical samples and the development of a multiplex assay using real-time polymerase chain reaction

Meticillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of healthcare-associated infections and isolates containing Panton-Valentine leukocidin (PVL) that cause severe skin infections are emerging as a serious problem. The rapid detection of MRSA would be an invaluable tool in a diagnostic laboratory. The aim of this study is to develop real-time polymerase chain reaction (PCR) assays for the detection of MRSA and PVL directly from clinical samples, and then combining these assays. Individual assays for MRSA (SCCmec) and PVL (lukF and lukS) were optimised and evaluated with screening and wound swabs, respectively. MRSA- and PVL-positive isolates were detected by the assays with an analytical sensitivity of 100 cfu per reaction. No other bacterial species were amplified. Fifty of 402 (12.4%) nasal swabs were positive by culture and PCR. Four of the 402 (1.0%) swabs were PCR-positive/culture-negative. Three of the 402 (0.7%) swabs were PCR-negative/culture-positive. The sensitivity of the MRSA assay is 95% and the specificity is 99% using conventional culture as the gold standard. Five of 240 wound swabs (2.1%) were positive for PVL. Three of the PVL-positive swabs were meticillin-sensitive Staphylococcus aureus (MSSA) and two were MRSA. The MRSA assay is a powerful and sensitive diagnostic tool, giving rapid results and could allow more timely treatment and infection control decisions to be taken. It can also, when combined with the PVL assay, provide valuable epidemiological information.     

Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit

Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus faecalis bacteremia directly in blood samples, without prior cultivation. Whole-blood samples for PCR were obtained simultaneously with blood cultures from patients admitted to the intensive care unit of our hospital. After the extraction of DNA from 200 mul of blood, real-time PCR was performed for the specific detection and quantification of S. aureus and E. faecalis DNA. The sensitivity for bacteremia of the S. aureus PCR was 75% and that of the E. faecalis PCR was 73%, and both tests had high specificity values (93 and 96%, respectively). PCR amplification reactions were positive for S. aureus for 10 (7%) blood samples with negative blood cultures, and 7 (4%) PCR reactions were positive for E. faecalis. The majority of these PCR results were likely (50%) or possibly (42%) related to infection with the specific microorganism, based on clinical data and radiological and microbiological investigations. PCR results were concordant for 95% of paired whole-blood samples, and blood culture results were concordant for 97% of the paired samples. We conclude that the detection of S. aureus and E. faecalis DNA in blood by real-time PCR enables a rapid diagnosis of bacteremia and that a positive DNAemia is related to proven or possible infection with the specific microorganism in the majority of patients with negative blood cultures.     

Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, but it is undoubtedly underdiagnosed. We used a nested PCR assay (targeting the pneumolysin gene) to detect S. pneumoniae DNA in multiple sample types from 474 adults with community-acquired pneumonia and 183 control patients who did not have pneumonia. Plasma or buffy coat samples were PCR positive in only 6 of the 21 patients with positive blood cultures for S. pneumoniae and in 12 other patients (4 of whom had no other laboratory evidence of S. pneumoniae infection). Buffy coat samples from two control patients (neither having evidence of S. pneumoniae infection), but no control plasma samples, were PCR positive. Although pneumococcal antigen was detected in the urine from 120 of 420 (29%) patients, only 4 of 227 (2%) urine samples tested were PCR positive. Overall, 256 of 318 (81%) patients had PCR-positive sputum samples, including 58 of 59 samples from which S. pneumoniae was cultured. Throat swab samples from 229 of 417 (55%) patients were PCR positive and, in those who produced sputum, 96% also had positive PCR results from sputum. Throat swabs from 73 of 126 (58%) control patients were also PCR positive. We conclude that the pneumolysin PCR assay adds little to existing diagnostic tests for S. pneumoniae and is unable to distinguish colonization from infection when respiratory samples are tested.     

Controlled evaluation of the IDI-MRSA assay for detection of colonization by methicillin-resistant Staphylococcus aureus in diverse mucocutaneous specimens

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for the effective control of MRSA transmission in healthcare facilities. The aim of this study was to verify the performance of the IDI-MRSA real-time PCR assay for direct MRSA detection in diverse mucocutaneous swabs from hospitalized patients. Swabs from nares (n = 522) and skin or other superficial sites (n = 478) were prospectively collected for MRSA screening from 466 patients admitted to an 858-bed teaching hospital. Swabs were inoculated onto selective chromogenic MRSA-ID agar, buffer extraction solution for IDI-MRSA assay, and enrichment broth. MRSA was detected by culture in 100 specimens from 47 patients. Compared to enrichment culture, the sensitivity and specificity of the PCR assay were 81.0 and 97.0%, respectively, and its positive and negative predictive values were 75.0 and 97.9%, respectively. The IDI-MRSA assay was more sensitive on swabs from nares (90.6%) than from other body sites (76.5%, P < 0.01). The PCR assay detected MRSA in 42 of 47 patients with culture positive study samples. Of 26 patients with culture-negative but PCR-positive study samples, 11 were probable true MRSA carriers based on patient history and/or positive culture on a new sample. The median turnaround time for PCR results was 19 h versus 3 days for agar culture results and 6 days for enrichment culture results. These data confirm the value of IDI-MRSA assay for rapid screening of MRSA mucocutaneous carriage among hospitalized patients. Cost-effectiveness studies are warranted to evaluate the impact of this assay on infection control procedures in healthcare settings.     

Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults

Respiratory syncytial virus (RSV) accounts for the majority of respiratory virus infections, producing high mortality rates in immunocompromised patients with hematologic malignancies. The available methods for the rapid detection of RSV by antigen detection or PCR either lack sensitivity, require complex laboratory manipulation, or have not been evaluated in this patient population. To assess the applicability of a TaqMan-based real-time PCR technique for the detection of RSV A and B in immunocompromised adults, we developed a rapid, sensitive detection method that simultaneously detects RSV A and B and can be applied in routine diagnostics. The specificity of the assay was assessed using a panel of reference strains of other respiratory viruses and RSV. Electron microscopy-counted stocks of RSV A and B were used to develop a quantitative PCR format. Eleven copies of viral RNA could be detected for RSV A strain Long, and 14 copies could be detected for RSV B strain 9320, corresponding to 50% tissue culture infective doses of 0.86 and 0.34, respectively. The assay was evaluated on 411 combined nose and throat swabs derived from immunocompromised adults with or without signs of respiratory tract infection. The diagnostic efficacy of the TaqMan PCR determined on the clinical samples showed that this real-time PCR technique was substantially more sensitive than the combination of conventional viral culture and shell vial culture. None of the clinical specimens derived from patients without signs of respiratory illness were found to be positive for RSV by real-time TaqMan PCR.     

A comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time PCR

We tested the hypothesis that swabs from the nasopharynx carry a higher viral load than swabs from the oropharynx in patients with real-time polymerase chain reaction (PCR)-confirmed influenza infection. Using flocked swabs, oropharyngeal and nasopharyngeal samples were harvested from hospital-admitted influenza patients no later than 3 days after the initial detection of influenza virus. Comparison of cycle threshold (CT) values was performed to assess differences in viral load in the specimens. Seventeen patients were diagnosed with influenza B, 14 patients with influenza A(H1N1)pdm09, and one patient with influenza A(H3N2). Nasopharyngeal samples were positive at a lower CT value than the oropharyngeal samples [mean difference in CT 5.75, 95 % confidence interval (CI) 3.8–7.7, p?<?0.01], suggesting that, on average, the calculated viral load of the nasopharyngeal samples was 54 times higher (95 % CI 13.7–210.8) than those of the oropharyngeal samples. The corresponding difference in the calculated viral load for influenza A(H1N1)pdm09 virus was 23 times (95 % CI 3.8–136.2, p?<?0.01) and for influenza B virus, it was 80 times (95 % CI 9.3–694.6, p?<?0.01). In patients with acute influenza, nasopharyngeal swabbing was clearly superior to oropharyngeal swabbing in terms of diagnostic yield by real-time PCR.