实验性卡氏肺孢子虫肺炎的病理学研究

Ｗｉｓｔａｒ大鼠皮下注射醋酸考的松诱发卡氏肺孢子虫肺炎，自第６～１２周，每周解剖病鼠２只观察，全部给药鼠均发病。肺部病变表现为：（１）肺印片查见卡氏肺孢子虫包囊和滋养体。（２）组织病理学改变：第６～８周ＨＥ染色切片呈间质性肺炎伴中度淋巴细胞浸润，肺泡内缺乏泡沫样渗出物，第９～１２周肺泡内出现泡沫样渗出物，ＰＡＳ染色该渗出物呈阳性反应，ＧＭＳ染色查见染成黑色的包囊。（３）停用醋酸考的松后４～６周，肺部炎症明显好转，提示大鼠卡氏肺孢子虫肺炎有自愈可能。电镜显示包囊、滋养体及受损肺泡上皮细胞的超微结构。     

卡氏肺孢子虫病的诊断及其进展

卡氏肺孢子虫病诊断方面的研究表明，辅助诊断方法缺乏特异性，在确定病人是否有肺感染，感染的程度及疗效的考核上都有一定作用。但ＰＣ感染的最后确诊主要靠病原体检测。取材方法：对于肺感染者应以导痰为首选，其次是ＢＡＬ和ＴＢＬＢ。儿童可采用经皮穿刺肺吸引的方法取材。在前述方法无法实施的紧急情况下，才考虑开胸肺活检。肺外感染者可在病变局部穿刺取材。制片法：可用穿刺物或肺组织等做直接涂片，印片，或悬液滴片等简捷方法，也可采用组织切片。染色方法：常用Ｇｉｅｍｓａ、初检、ＴＢＯ或ＧＭＳ法进一步确诊。ＩＦ可提高检出率。ＰＣＲ技术检测孢子虫病更敏感和特异，有操作简便，易于推广应用等优点。     

应用巢式PCR方法诊断微小隐孢子虫感染的实验研究

目的应用巢式PCR扩增方法诊断微小隐孢子虫感染。方法用昆明种小鼠通过免疫抑制方法建立动物模型，通过不连续蔗糖密度梯度离心法分离、纯化隐孢子虫卵囊。抽提DNA后，用巢式PCR扩增隐孢子虫的18S核糖体DNA，电泳、割胶回收后测序。结果成功建立隐孢子虫的小鼠免疫抑制动物模型，纯化后的隐孢子虫卵囊形状均一，呈圆形或椭圆形，大小在3-6um左右。经巢式PCR扩增，在840bp左右有一条特异性条带。通过测序和生物信息学分析，证实该基因序列与微小隐孢子虫18S具有高度同源性。结论巢式PCR方法扩增18S基因是诊断微小隐孢子虫感染的敏感方法。     

卡氏肺孢子虫肺炎大、小鼠低死亡率动物模型的建立

为建立低死亡率卡氏肺孢子虫肺炎 (PCP)SD大鼠和ICR小鼠动物模型 ,本试验将雌性SD大鼠和ICR小鼠分别随机分为实验组和对照组 ,实验组采用按体重定量皮下注射地塞米松的方法 ,免疫抑制诱导建立PCP动物模型 ,对照组注射与地塞米松等体积的生理盐水。分别制作肺印片 ,经瑞 姬氏复合染色后 ,检查卡氏肺孢子虫包囊。制作肺组织病理切片 ,经HE染色后观察肺组织病理变化。用地塞米松诱导后 ,实验组SD大鼠和ICR小鼠死亡率均为 0 ,肺印片阳性率均为 76 7% (2 3 30和 2 3 30 )。肺组织出现典型的病理变化 ,并可观察到Pc包囊。实验组SD大鼠体重下降明显 ,与对照组体重比较具有极显著性差异 (P <0 0 1)。ICR小鼠经诱导后 ,体重变化不显著。采用按体重定量皮下注射地塞米松的方法可建立低死亡率PCP动物模型     

双氢青蒿素对卡氏肺孢子虫肺炎大鼠TNF-α水平的影响

目的研究双氢青蒿素治疗对卡氏肺孢子虫肺炎大鼠血清和肺泡巨噬细胞培养上清液 TNF- α水平的影响。方法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 60 m g/ 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肺泡巨噬细胞 ,用 L PS刺激培养 72 h,同时设有感染组和正常对照。用 TNF- α试剂盒分别检测血清和培养上清液 TNF- α的水平。结果感染组和治疗组 TNF- α水平均高于正常对照 ,治疗组 TNF- α水平则低于感染组。结论卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞分泌高水平的 TNF- α,但双氢青蒿素治疗后 PCP大鼠肺泡巨噬细胞产生 TNF- α水平降低。     

双氢青蒿素对卡氏肺孢子虫肺炎大鼠肺泡巨噬细胞凋亡的影响

目的：检测双氢青蒿素对卡氏肺孢子虫肺炎（PCP）大鼠肺泡巨噬细胞凋亡的影响。方法：以醋酸可的松皮下洲Wistar大鼠建立PCP动物模型，对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肺泡巨噬细胞，可PI和TUNEL法检测其凋亡，同时设有正常大鼠对照组。结果：感染组和治疗组大鼠肺泡巨噬．细胞凋亡率显著高于正常对照组，治疗组大鼠肺泡巨噬细胞凋亡率明显低于感染组。结论：卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞发生凋亡，经双氢青蒿素治疗后PCP大鼠肺泡巨噬细胞凋亡降低。     

环介导等温扩增技术检测卡氏肺孢子虫的研究

目的 环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法 醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论 检测Pc的LAMP方法敏感、特异及简便.     

巢式PCR检测日本血吸虫感染的研究

本研究通过设计2对巢式引物扩增日本血吸虫高拷贝的Sjα1基因片段,建立检测日本血吸虫感染的巢式PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的Sjα1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检测日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子生物学检测技术. PCR技术,并对感染小鼠血清、全血样本以及实验和现场钉螺样本进行检测.建立的巢式PCR方法特异性扩增日本血吸虫420 bp的sja1片段,和曼氏血吸虫没有交叉,基因组DNA作为模板时最低检测量为0.1fg.小鼠感染日本血吸虫后2周的血清样本中即能检测出特异性DNA,建立的方法能同时检测血清和全血标本.钉螺实验感染4 h后能检测到日本血吸虫DNA,现场采集钉螺的检测结果显示比传统的镜检方法敏感性高.建立的巢式PCR检 日本血吸虫感染具有较高的敏感性和特异性,为疾病诊断和媒介调查提供了新的分子     

卡氏肺孢子虫感染大鼠血清中蛋白及血液中嗜酸粒细胞的变化分析

卡氏肺孢子虫肺炎（pneumocystis carinii pneumonia,PCP）是由卡氏肺孢子虫（pneumocystis carinii,PC）引起的一种呼吸系统机会性感染,多见于免疫功能低下患者。据报道,未经药物预防的爱滋病（AIDS）患者约80％感染PCP。     

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia

Objective   To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii .
Methods   The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR.
Results   Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample.
Conclusions   IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii .     

Enteroviral polymerase chain reaction in the investigation of aseptic meningitis

We used a nested polymerase chain reaction (nPCR) to seek evidence for enteroviruses in clinical samples from patients with symptoms of aseptic meningitis. When compared with conventional virus isolation methods on a total of 366 samples collected during 1994–1995, an increase in positivity from 6% to 27% was shown. The results indicate that nPCR would be a valuable aid to the laboratory diagnosis of enteroviral infections as it can detect those enteroviruses that cannot be identified by current isolation methods. © 1996 Wiley-Liss, Inc.     

A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens

Objective: Because presently used methods for diagnosis of Legionella pneumonia lack sufficient sensitivity and sometimes specificity and rapidity, the detection of Legionella spp. by amplification of nucleic acids might be valuable. However, performing polymerase chain reaction (PCR) on clinical samples such as sputum is difficult because of the presence of extraneous DNA and inhibitors of the reaction. An attempt to circumvent these problems was made.
Method: A nested PCR method was devised using primers from the mip gene of Legionella pneumophila. This PCR was tested on pure cultures of legionellae and clinical isolates of other bacteria. Clinical samples (bronchoalveolar lavage fluid, bronchial aspirate and sputum) from patients who suffered from legionellosis and samples from patients who suffered from other causes of pneumonia were also tested.
Results: The PCR was specific for L. pneumophila and no non- Legionella bacteria reacted. Ten to 50 colony forming units of Legionella in the sample could be detected. Twenty-two of 25 clinical samples were positive among patients suffering from pneumonia proven to be due to L. pneumophila serogroups 1, 3, 4, 5 and 6. Two of the three negative samples were from patients who had been treated with adequate therapy for at least 2 days and were culture negative. However, nine other culture-negative samples were PCR positive, of which seven came from patients who had been treated for 3–7 days. All pneumonia patients in the control group proved negative in PCR. A commercial kit for DNA preparation from clinical samples, based on absorption of nucleic acids to silica gel, was superior to the traditional phenol/chloroform extraction and increased the rapidity, simplicity and sensitivity of the procedure.
Conclusions: A nested, simplified and rapid PCR method using mip primers proved to be more sensitive than culture and as sensitive and specific as other PCR procedures previously reported.     

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.     

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples

Purpose: To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods: A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results: Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion: Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).     

Sensitive detection and differentiation of Sapporo virus, a member of the family Caliciviridae, by standard and booster nested polymerase chain reaction

Norwalk virus and Sapporo virus (SV) were approved as type species of the genus Norwalk-like viruses and the genus Sapporo-like viruses, respectively, in the family Caliciviridae. Nested polymerase chain reaction (PCR), using newly designed primers in the RNA-dependent RNA polymerase region, was developed to detect and differentiate viruses in the three genetic groups of SV based on the relative size of the PCR products obtained. In addition, a booster nested PCR that performs nested PCR in a single tube was introduced to reduce the chance of contamination during the procedure of standard nested PCR. The specificity of the newly developed PCR was confirmed by testing 77 stool specimens and 16 tissue culture fluids derived from growth of unrelated viruses. The sensitivity of the nested PCR was compared with the conventional PCR using Sapp35/Sapp36 primer pair by testing the three cDNA clones obtained from viruses in the SV/SV82, the SV/London92, and the SV/Parkville virus, respectively. This assay can detect SV in a more sensitive way than the conventional PCR and Southern hybridization. Sensitive and suitable methods to detect and differentiate SV are required to obtain accurate epidemiological data on these viruses and the standard and booster nested PCR should be a very useful tool for this purpose.     

Detection of adenoviruses in stools from healthy persons and patients with diarrhea by two-step polymerase chain reaction.

The use of the polymerase chain reaction (PCR) for detection of human adenoviruses in diluted stool samples was investigated. Two sets of nested primers, including primers specific for the hexon-coding region and for the E1B region of enteric adenoviruses (EAd), were assessed by two-step amplification. The primers constitute two different PCR systems designed for the detection of adenoviruses belonging to all six subgenera (A-F), and the two EAds Ad40 and Ad41, respectively. In a two-step PCR mediated amplification a single virus particle was detected when the two sets of general hexon primers or EAd specific primers were used. Earlier results from PCR detection of adenoviruses in stool from children suffering from diarrhea gave indications that adenovirus particles are commonly shed in stools without being identified as the cause of illness [Allard et al.: Journal of Clinical Microbiology 28:2659-2667, 1990]. Therefore, the general and the EAd specific PCR assays were assessed on 150 stool specimens from three groups including 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea. When the two sets of general hexon primers were used, 25 of the 50 specimens from the healthy children (mean age 21 months) were found positive by two-step PCR amplification. Nine of the 50 specimens from the healthy adults (mean age 32 years) were found positive whereas 12 of the 50 specimens from sick adults (mean age 31 years) gave amplification products, using the two sets of general hexon primers in a nested fashion. None of the 150 specimens were found to be positive by two-step PCR amplification using the two sets of EAd-specific primers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples

Objective: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S–1-specific probe.
Methods: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella bumetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method.
Results: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S–1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test.
Conclusions: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.     

Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I,II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic virus (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-IPCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples. J. Med. Virol. 52:1–7, 1997.© 1997 Wiley-Liss, Inc.