体外青蒿素衍生物抗卡氏肺孢子虫作用的PCR-SSCP分析

目的　研究双氢青蒿素、青蒿琥酯对卡氏肺孢子虫胸腺嘧啶合成酶基因 (TS)、二氢叶酸还原酶基因 (DHFR)的影响。方法　用不同浓度的双氢青蒿素、青蒿琥酯作用于体外培养的卡氏肺孢子虫。用药 7天后收集培养液提取卡氏肺孢子虫DNA ,分别进行PCR扩增 ,将扩增产物作DNA的单链构象多态性分析 (SSCP)。结果　卡氏肺孢子虫TS基因和DHFR基因的扩增产物分别为 40 3和 2 73bp。经青蒿素衍生物作用后TS基因、DHFR基因在溴化乙锭染色上的强度均有明显减少。继续作SSCP分析发现 ,用药组与不用药组对比DHFR基因DNA条带无明显差别 ,而用TS基因DNA条带中有一条带的位置发生改变 ,提示该基因可能出现变异。结论　SSCP分析结果提示青蒿素衍生物对卡氏肺孢子虫的DHFR基因结构无影响 ,而TS基因则可能出现部分变异。提示TS基因可能是该类药物抗卡氏肺孢子虫的机理之一     

肺孢子虫感染大鼠模型的建立及ITS巢式PCR检测敏感性研究

目的 建立肺孢子虫感染大鼠模型并探讨ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫DNA的敏感性. 方法 大鼠分为实验组和对照组,实验组从第1周开始用每周2次每次每只皮下注射地塞米松1 mg的方法诱导,共8周,并分别在首次注射后第2、4、6、8周解剖大鼠制作肺印片、肺组织匀浆液及支气管肺泡灌洗液(BAL)涂片,并进行六亚甲基四胺银(Gomori's methenamine silver,GMS)染色镜检;对照组不作激素注射,并分别在0周和第10周剖杀染色检查.同时分别提取大鼠BAL和肺组织的肺孢子虫DNA进行巢式PCR扩增,比较GMS法和ITS巢式PCR检测的敏感性. 结果 实验组从第6周开始染色镜检,可见少量肺孢子虫包囊,第8周肺印片、肺组织匀浆液均检测到包囊,检出率为100%(10/10),BAL的检出率为80%(8/10),对照组则均未检出.用ITS1-5.8S rDNA-ITS2巢式PCR法均能检测出实验组第8周大鼠BAL和肺组织卡氏肺孢子虫DNA,阳性率为100%(10/10),对照组均为阴性.比较BAL标本、肺印片和肺组织匀浆液GMS染色法的检出率,BAL最低,肺组织匀浆液最高.其中20%(2/10)大鼠的BAL标本用GMS染色法未能检出,但用巢式PCR方法均能成功扩增.结论成功用地塞米松诱导法建立了肺孢子虫大鼠感染模型;ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫敏感性高、特异性强,可推广应用于临床诊断肺孢子虫肺炎.     

PCR技术对大鼠卡氏肺孢子虫DNA的检测

目的　探讨诊断卡氏肺孢子虫感染的最佳方法。方法　建立大鼠动物模型,肺涂片用吉氏染色法查肺孢子虫包囊,支气管灌洗液、肺组织和血液标本,用ＰＣＲ技术进行卡氏肺孢子虫ＤＮＡ的检测,共计实验组大鼠１２ 只及对照组８ 只。结果　实验组大鼠,吉氏染色法卡氏肺孢子虫包囊检出率为６６－７％ ,ＰＣＲ技术支气管灌洗液、肺组织和血液标本,卡氏肺孢子虫ＤＮＡ检出率分别为８１－８ ％ 、５０％ 和５０％ 。比肺印片提前２ 周检出。对照组大鼠,吉氏染色法未检出卡氏肺孢子虫包囊,在支气管灌洗液和血液标本中,ＰＣＲ技术测出了卡氏肺孢子虫ＤＮＡ。结论　在血液标本中ＰＣＲ的成功应用,为卡氏肺孢子虫感染的流行病学调查及人类卡氏肺孢子虫肺炎的诊断,提供了一种有用的、非创伤性的方法。     

PCR检测大鼠卡氏肺孢子虫的研究

目的探讨PCR技术检测大鼠卡氏肺孢子虫的应用价值。方法SD大鼠和Wistar大鼠均随机分成实验组和对照组,实验组每周两次皮下注射醋酸可的松,诱导产生卡氏肺孢子虫;8week后,收集肺组织和支气管肺泡灌洗液(BALF),用PCR技术检测卡氏肺孢子虫DNA,并与Giemsa染色法比较。结果实验组两种大鼠肺组织卡氏肺孢子虫DNA阳性率分别为9643%和100%,BALF阳性率亦分别为9643%和100%,它们之间均无显著性差异(P>005);BALF的PCR阳性检出率显著高于Giemsa病原染色法,肺组织的两种方法检出率无显著性差异。结论PCR是一种检出率较高的方法,可作为早期诊断PCP的常规方法,特别适用于BALF检测卡氏肺孢子虫DNA。     

99例AIDS患者痰液卡氏肺孢子虫PCR检测结果分析

目的 采用巢式PCR检测对AIDS患者痰液标本进行卡氏肺孢子虫检测,为AIDS患者合并卡氏肺孢子虫肺炎的临床诊断提供参考价值.方法 将我院感染科2008年1月至10月收治的具有呼吸道症状的99例AIDS患者痰液标本进行收集,将痰标本进行镜检,巢式PCR扩增卡氏肺孢子虫基因,并且对99例患者进行临床诊断和实验室诊断.结果 99例AIDS患者中,临床诊断卡氏肺孢子虫仅2例;油镜下观察到卡氏肺孢子虫4例;PCR痰阳性共43例,取其中4例成功扩增出耶氏肺孢子菌基因.结论 采用巢式PCR对AIDS患者痰液标本进行检测,可以早期诊断卡氏肺孢子虫,减少卡氏肺孢子虫肺炎患者的漏诊率.     

大鼠卡氏肺孢子虫的三个基因序列分析

目的 分析我国大鼠源卡氏肺孢子虫的基因序列特征。方法：以Wistar大鼠建立肺孢子虫肺炎动物模型,收集鼠肺,抽提DNA,以PCR方法扩增卡氏肺孢子虫的5SrRNA基因、线粒体rRNA大亚基基因（mtLSU rRNA）、胸腺嘧啶核苷酸合成酶基因（TS）。纯化扩增产物,直接测序。检索基因库（GenBank）,进行序列比较。结果 三个基因的PCR扩增呈阳性。感染Wistar大鼠的卡氏肺孢子虫5S rRNA基因与gb|S78185|S78185、gb|M28193|PMCRAA两个序列同源性分别为93.3%和91.7%;mtLSU rRNA基因与gb|U20173|PCU20173 、gb|U20169|PCU20169两个序列同源性分别为76.2%和55.2%;TS基因与gb|M25415|PMCTHYSY、gb|S77510|S77510两个序列同源性均为90.9%。结论 Wistar大鼠感染的卡氏肺孢子虫的三个基因与GenBank内的相应基因序列同源性较高。     

聚合酶链反应方法检测诱导排痰标本对卡氏肺孢子虫肺炎的诊断价值

目的 评价聚合酶链反应方法检测诱导排痰标本中卡氏肺孢子虫DNA对卡氏肺孢子虫肺炎（PCP）的诊断意义。方法 分别用姬姆萨和六胺银（GMS）两种染色方法和mt-rRNA-PCR方法检测痰液中的卡氏肺孢子虫。结果 化学染色法检测16例临床拟诊PCP的患者痰标本。结果 8例阳性,20例非PCP患者痰标本均为阴性,化学染色方法的敏感性和特异性分别为50％和100％。PCR方法检测16例临床拟诊PCP患者痰液中卡氏肺孢子虫,14例阳性,20例非PCP患者痰标本均为阴性,mt-rRNA-PCR方法的敏感性和特异性分别为88％和100％。结论 姬姆萨和GMS两种细胞化学染色方法联合检测痰标本卡氏肺孢子虫,特异性高,但敏感性偏低。mt-rRNA-PCR检测痰标本中卡氏肺孢子虫DNA方法敏感性高于化学染色法且特异性高,更适于临床应用。     

双氢青蒿素对患卡氏肺孢子虫肺炎大鼠血清和肺泡巨噬细胞上清液IL-1水平的影响

为研究双氢青蒿素对患卡氏肺孢子虫肺炎（Pneumocystis carinii pneumonia,PCP）大鼠血清和肺泡巨噬细胞培养上清液IL－1水平的影响，以醋酸可的松皮下注射Wistar大鼠建立卡氏肺孢子虫肺炎动物模型，用60mg/kg双氢青蒿素治疗实验大鼠，杀鼠取肺，用胶原酶消化法分离大鼠肺泡巨噬细胞，用LPS刺激培养72h，同时设有感染对照组和正常对照，用IL－1β试剂盒分别检测血清和培养上清液IL－1β的水平，结果显示感染组和治疗组大鼠IL－1β水平显著高于正常对照，治疗组大鼠IL－1β水平则低于感染组，说明卡氏肺孢子虫感染可能引起大鼠肺泡巨噬发泌高水平IL－1，经双氢青蒿素治疗PCP后大鼠肺泡巨噬细胞产生IL－1水平降低。     

快速DNA提取法在检测卡氏肺孢子虫PCR中的应用

为寻求一种适用于临床诊断的PCR方法。方法用快速PCR模板DNA制备方法提取实验大鼠肺组织,支气管肿泡灌洗液和血液中的卡氏肺孢子虫DNA,进行PCR扩增,并与传统的DNA提取法进行对比。结果两种方法收到一致的理想结果。结论快速法提取的DNA适用于卡氏肺孢子虫的PCR检测。将此法试用于临床病例检测,也收到如期结果,初步显示其推广应用前景。     

卡氏肺孢子虫感染裸小鼠的实验研究

用醋酸可的松诱导Wistar大鼠获得卡氏肺孢子虫感染,取大鼠肺组织含虫匀装直接接种于NCS系裸小鼠两侧肺0.05ml,以及接种保存于-70℃低温中5月以上的含虫肺匀奖和分离纯化后的虫体,均使裸小鼠获得感染,发生卡氏肺孢子虫肺炎。在国内首先建立起裸小鼠卡氏肺孢子虫肺炎实验模型。     

Identification of Pneumocystis carinii DNA in oropharyngeal mouth washes from AIDS children dying of respiratory illnesses

Polymerase chain reaction (PCR) using Pneumocystis carinii-specific primers pAZ 102-H(5'-GTGTACGTTGCAAAGTACTC-3') and pAZ 102-E(5'-GATGGCTGTTTCCAAGCCCA-3') was performed on oropharyngeal washes obtained at autopsy from 22 AIDS children with histologically confirmed P. carinii pneumonia (PCP), and 48 control AIDS children who died from other infections. Fifteen of 22 (68%) PCP samples and none of 48 (0%) control samples had detectable P. carinii DNA (sensitivity 68%; specificity 100%; positive predictive value 100%; negative predictive value 87%). This method requires further validation in clinical practice.     

Emerging importance of Pneumocystis carinii among indian immunosuppressed patients

Pneumocystis carinii is known to cause both significant morbidity and mortality in immunosuppressed individuals. In a six and a half year period starting with 1992, a total of 204 samples including bronchial aspirates, induced sputum and suction catheter tips were examined for P. carinii by direct microscopic examination of Giemsa's and toluidine 'O' stained smears. At a later stage of the investigation, immunofluorescent staining using monoclonal reagent (Meriflour, USA) was also used for examination of the specimens. In all 24 (11.8%) of 204 samples were positive for P. carinii. In addition, Pneumocystis carinii pneumonia (PCP) was diagnosed in five (33%) of 15 patients of acquired immunodeficiency syndrome (AIDS) with opportunistic infections. All these 15 patients had CD4 T cell counts of less than 500 T lymphocyte equivalent (TLE) /ml as measured by a Trax ELISA assay. Laboratories in India have to be better equipped for an early and correct diagnosis of PCP that is bound to rise with the increase in the number and variety of immunosuppressed patients.     

蒿甲醚治疗实验大鼠肺孢子虫肺炎的电镜观察

用皮下注射醋酸可的松６周建立实验大鼠肺孢子虫肺炎动物模型，并用国产蒿甲醚进行试验治疗。治后大鼠肺组织超薄切片透射电镜观察，发现蒿甲醚可导致卡氏肺孢子虫产生以下３种超微结构改变：（１）胞浆内出现大量空泡；（２）线粒体肿胀；（３）核膜破裂。以上改变与戊烷脒对照组相似。     

Detection of Pneumocystis carinii by DNA amplification in patients with connective tissue diseases: re-evaluation of clinical features of P. carinii pneumonia in rheumatic diseases

OBJECTIVES: We evaluated the polymerase chain reaction (PCR) detection of Pneumocystis carinii DNA in induced sputum of patients with connective tissue diseases and assessed the clinical features of patients positive for P. carinii. METHODS: Sputum was induced by nebulization in 29 in-patients with various connective tissue diseases who presented with symptoms suggestive of P. carinii pneumonia (PCP), and was examined by PCR. RESULTS: Detection of P. carinii DNA by PCR was significantly more sensitive than cytology; 54.5% patients were positive by PCR and only 4.5% by cytology. The prevalence of PCP was higher than previously considered and was especially high in patients receiving > 30 mg/day prednisolone with or without other immunosuppressants. P. carinii-positive patients had significant lymphocytopenia and a low serum IgG level compared with P. carinii-negative patients. P. carinii disappeared within 7-10 days after therapy with trimethoprim/sulfamethoxazole. CONCLUSION: We propose that the use of PCR for detection of P. carinii using induced sputum is a useful and non-invasive method that has high sensitivity and specificity for the early diagnosis of PCP.     

Role of multiple induced sputum examination in the diagnosis of Pneumocystis carinii pneumonia

We evaluated the usefulness of repeated nebulized saline induced sputum examinations among 60 HIV infected patients clinically suspected to have Pneumocystis carinii pneumonia (PCP). We found that the first sample was positive for 15 episodes (21.4%); the second sample was positive in 33 episodes (47.1%); the third sample was positive in 22 episodes (31.4%). Repeated nebulized saline induced sputum examination imporved the yield of Pneumocystis carinii and enhanced the sensitivity of a positive result. This technique is simple, cost-effective, non-invasive, and reliable. We recommend the examination of multiple induced samples of nebulized saline induced sputum in all HIV infected patients with suspected PCP. This recommendation may decrease the need for invasive procedures.     

从大鼠肺灌洗液中分离纯化培养卡氏肺孢子虫

目的 建立卡氏肺孢子虫（Pneumocystis carinii, P.c）纯培养株。 方法　 从肺孢子虫肺炎（PCP）大鼠模型离体肺脏的灌洗液中分离P.c，用改良IMDM培养基进行体外培养；以四胺银染色计数法观察虫体增殖情况；用PCR扩增培养物中P.c线粒体大亚基rRNA基因，进行基因鉴定；用透射电镜观察培养虫体的超微结构。 结果 用添加ｓ-腺苷甲硫氨酸（SAM）等辅助剂的IMDM培养基从8只PCP大鼠的肺灌洗液中分离出5个P.c纯培养株（P.c Rat1~5）。分离培养的P.c可进行冷冻保存和复苏培养。培养72 h的P.c包囊可增殖19~22倍。形态、超微结构及基因序列分析证实从大鼠肺灌洗液中分离出的培养物是大鼠源性肺孢子虫。 结论 从大鼠肺灌洗液中分离培养出5株大鼠源卡氏肺孢子虫。     

The high burden of Pneumocystis carinii pneumonia in African HIV-1-infected children hospitalized for severe pneumonia.

OBJECTIVE: To evaluate the burden of Pneumocystis carinii pneumonia (PCP) and the usefulness of induced sputum and nasopharyngeal aspirates (NPA) in diagnosing PCP in African children in whom the use of bronchoalveolar lavage is unavailable. DESIGN: Children aged 2-24 months who were either known or suspected of being HIV-1 infected and who were hospitalized for severe pneumonia were investigated for P. carinii using induced sputum and NPA. P. carinii identification was performed using a direct monoclonal antibody immunofluorescent stain. A group of children who subsequently died also had lung biopsies performed. RESULTS: P. carinii cysts were identified in 51 out of 105 (48.6%) children either from induced sputum (37/105, 35.2%) or NPA (26/101, 25.7%) samples, or from both. Neither clinical nor laboratory tests were useful in distinguishing between HIV-1-infected children with and without PCP. Twenty-eight per cent (14/51) of HIV-1-infected children who developed PCP had a history of being on cotrimoxazole prophylaxis at the time of their illness. Mortality rates of HIV-1-infected children with and without PCP were equally high (27.5 and 27.8%, respectively). Histological evidence of PCP and cytomegalovirus pneumonia was observed on post-mortem lung biopsy in eight out of 18 (44.4%) children each. Using post-mortem lung histology as a reference, the sensitivity and specificity for induced sputum and NPA in diagnosing PCP were 75 and 80%, respectively. CONCLUSION: Strategies to reduce the high burden of PCP, which can successfully be diagnosed using NPA and induced sputum, in HIV-1-infected children hospitalized with severe pneumonia are urgently warranted in Africa.     

Nested PCR in the diagnosis of Pneumocystis carinii pneumonia (PCP) in AIDS patients

The studies were undertaken to evaluate the usefulness of a nested PCR assay in the diagnosis of Pneumocystis carinii pneumonia (PCP) in AIDS patients. To achieve the end, 51 excretions samples from the respiratory tract were collected from HIV-infected patients with respiratory symptoms, and examined for the presence of specific DNA. A portion of the mitochondrial large-subunit rRNA gene of Pneumocystis carinii was amplified with outer primers pair pAZ 102E, pAZ 102H and internal primers pAZ 102X, pAZ 102Y. Positive nested PCR results were obtained with 36 out of 51 examined samples. Some 52.8% of the positive results were obtained with samples collected from patients without clinical diagnosis of PCP. It was concluded, that the nested PCR method, being too sensitive, is not suitable for the routine diagnosis of PCP in AIDS patients.