分离自蛇体的侵入内阿米巴滋养体的培养与应用

将分离自蛇体的侵入内阿米巴(Entamoeba invadens)滋养体接种于营养琼脂血清盐水培养基,22℃恒温培养,每周转种1次。转种后的培养液内含大量滋养体,制成玻片标本,显微镜下观察虫体形态。侵入内阿米巴滋养体的形态变化特点、运动方式、生殖周期和侵袭力等与感染人的溶组织内阿米巴滋养体基本相同。以侵入内阿米巴滋养体代替溶组织内阿米巴滋养体,学生能观察到活体阿米巴滋养体。     

三苯甲咪唑对阿米巴培养中的白色念珠菌的作用

白色念珠菌对溶组织内阿米巴的培养不利,吖啶黄虽可清除人芽生菌,但对白色念球菌无清除作用。我们通过实验发现,三苯甲咪唑可清除白色念珠菌,从而促进溶组织内阿米巴的生长繁殖。 三苯甲咪唑悬液配制 三苯甲咪唑片剂(Clotr mazole)系上海黄河制药厂产品,批号760901,每片含三苯甲咪唑25mg,将500mg投入10ml溶组织内阿米巴双相培养基覆盖液内,于蒸气高压下制成悬液。于4～8℃冰箱内可保效5年以上。当发现标     

试管滤纸培养法分离痢疾阿米巴滋养体的实验观察

痢疾阿米巴滋养体可随粪便排出体外,常用的生理盐水涂片法检查慢性病人粪便,只在含粘液血便中较易查到。至于不含粘液血便中有否痢疾阿米巴滋养体,滋养体在外界环境中的形态活力的情况,分离出的痢疾阿米巴滋养体的形态特征与其它非致病的阿米巴滋养体的区别等问题国内尚未见报道。我们用检出率较高的试管滤纸培养法检查慢性病人粪便中的滋养体,详细观察其分布、形态与活力的变化情况。一、内容与方法 (一)检查混有粘液和血而成形的粪便确诊为慢性痢疾阿米巴病人,排便在清洁的托盘上成各部分不相     

棘阿米巴角膜炎的实验诊断

[目的 ]寻找棘阿米巴角膜炎快速诊断和棘阿米巴快速鉴定的方法。 [方法 ]10 %氢氧化钾 (KOH)湿封片镜检、棘阿米巴培养、倒置显微镜观察、病理切片H .E .染色和SPA染色检查。 [结果 ]角膜刮片及手术切除的角膜材料 ,经 10 %氢氧化钾湿封片镜检 ,前者检出 7例棘阿米巴角膜炎病例 ,后者确诊 5例 ;手术切除的角膜材料经培养 ,分离出 6株棘阿米巴 ;应用倒置显微镜直接观察 ,检出棘阿米巴的包囊、滋养体和棘刺。 [结论 ]应用 10 %KOH湿封片镜检可对棘阿米巴角膜炎作出快速诊断 ;通过倒置显微镜直接观察也可对棘阿米巴角膜炎在 2 0h内作出诊断 ;倒置显微镜可直接观察和鉴定棘阿米巴 ,方法简便、无污染、快速及实用。     

胆固醇对溶组织内阿米巴体积的影响

用长期培养于LES培养基中的4株溶组织内阿米巴滋养体,各分成两个管序,一个管序加超声粉碎的胆固醇悬液1滴,作为试验管;另一管序则不加胆固醇,作为对照管。在37℃下培养,每48小时转种1次,每次转种前随机抽样测量50个阿米巴的体积,测得的数据按两组资料的比较作秩和检验     

吉曼尼兹(Gimenez)染色法快速筛选阿米巴滋养体内嗜肺军团菌

目的寻找简便、有效的染色方法,用于快速筛选阿米巴滋养体内嗜肺军团菌。方法嗜肺军团菌(Legionella pneumophila)与多噬棘阿米巴(Acanthamoeba polyphaga)共培养,取不同时点的共培养物制作涂片,采用革兰氏染色、吉曼尼兹(Gimenez)染色、姬姆萨(Giemsa)染色、免疫荧光染色等多种方法,用光学显微镜及荧光显微镜观察鉴别阿米巴滋养体与其胞内嗜肺军团菌,并比较这些染色方法的效果。结果Gimenez染色方法效果较好。虫体呈蓝色、嗜肺军团菌呈红色,两色分明。在共培养初期即可观察到阿米巴滋养体胞内少量的杆状嗜肺军团菌。本法灵敏度高,省时、耗材少,操作简便。结论Gimenez染色对阿米巴滋养体胞内嗜肺军团菌形态学检测具有实用价值,适合于实验室检测和临床诊断应用。     

营养缺乏对自由生活阿米巴自噬的影响

目的研究在营养缺乏的环境中自由生活阿米巴自噬的变化和自噬结构的形态学特征。方法对照组自由生活阿米巴用涂有大肠埃希菌的琼脂培养基培养。实验组将在大肠埃希菌中培养的阿米巴转移至不含大肠埃希菌的琼脂培养基上培养12 h。扫描电镜下观察不提供细菌的培养环境中阿米巴的形态学变化,透射电镜下观察阿米巴自噬的变化及自噬前体、自噬体和自噬溶酶体的结构特点,图像分析仪测量虫体内自噬结构与细胞质的断面面积。单丹磺酰尸胺(MDC)染色法标记自由生活阿米巴虫体内的自噬体,在共聚焦激光扫描显微镜下观察和定量分析。结果对照组中自由生活阿米巴均为滋养体形式;实验组中,滋养体逐渐向包囊转变。对照组阿米巴虫体内充满细菌碎片,只发生轻微的自噬,自噬结构数目较少。与对照组比较,实验组阿米巴自噬水平显著提高,自噬结构数目增多,自噬前体、自噬体和自噬溶酶体与细胞质的断面面积比增大(P0.05或0.01);部分阿米巴虫体内残存未消化的细菌碎片。结论在缺乏营养的环境中,自由生活阿米巴由滋养体向包囊转变,虫体内自噬功能显著增强。     

溶组织内阿米巴滋养体在不同培养基内的完整性、活力和吸附力

无菌培养的阿米巴滋养体 HK 9株和HM1株,经40℃ 5分钟后,以90g 离心3分钟,用培养液冲洗一次,制成1仍~10,/ml的滋养体悬液,将此悬液接种在9种培养基里,并观察在TP和TY12种基础培养基(TP:含有10形或0.2%马血清     

溶组织内阿米巴5例的粪便鉴定

目的探讨溶组织内阿米巴的粪便鉴定,并就溶组织内阿米巴包囊及滋养体的镜检要点及常见漏检原因进行讨论。方法用显微镜法对5例外院漏诊的肠阿米巴病患者粪便标本进行检验,分别用生理盐水涂片法、碘染色和苏木素染色法,在显微镜下进行观察鉴定。结果溶组织内阿米巴包囊及滋养体在生理盐水涂片、碘染色涂片和苏木素染色涂片中有典型的形态学特点。结论对于可疑肠阿米巴病患者,应多次及时送标本进行检验,采用几种不同方法进行鉴定,并注意将溶组织内阿米巴与吞噬细胞及迪斯帕内阿米巴相鉴别。     

H_2O_2体外抗棘阿米巴作用研究

目的检测H_2O_2的抗棘阿米巴(Acanthamoebaspp.)作用。方法自角膜炎患者角膜刮片分离获得棘阿米巴复合体(AcanthamoebaLugdunensis-Acanthamoebaquina)。于培养基(PYG)培养传代。实验前将棘阿米巴用新鲜的PYG培养1d使其活化,配成2.5×10~6/ml细胞悬液加入细胞培养板,实验组各孔分别加入不同浓度H_2O_2,对照组加等量PYG,28℃24h后实验组更换新鲜PYG继续培养3d。取细胞悬液滴片,瑞氏染色,观察细胞形态变化。用定量培养法作棘阿米巴生长曲线,观察其增殖速率。用四甲基偶氮唑盐(MTT)比色法,观察H_2O_2对棘阿米巴成活率的影响。用乳酸脱氢酶(LDH)测定法测定H_2O_2对棘阿米巴的损伤。结果棘阿米巴滋养体在0.125％H_2O_2作用下,不可逆转地成为包囊,20～120h增殖率为0;1％H_2O_2可使其破裂。结论H_2O_2具有较强的抗棘阿米巴作用,有可能成为预防棘阿米巴角膜炎的理想药物。     

Intestinal colonization with Candida albicans and mucosal immunity

AIM: To observe the relationship between intestinal lumen colonization with Candida albicans and mucosal secretory IgA (sIgA).METHODS: A total of 82 specific-pathogen-free mice were divided randomly into control and colonization groups. After Candida albicans were inoculated into specific-pathogen-free mice, the number of Candida albicans adhering to cecum and mucosal membrane was counted. The lymphocyte proliferation in Peyer‘s patch and in lamina propria was shown by BrdU incorporation, while mucosal sIgA (surface membrane) isotype switch in Peyer‘s patch was investigated. IgA plasma cells in lamina propria were observed by immunohistochemical staining. Specific IgA antibodies to Candida albicans were measured with ELISA.RESULTS: From d 3 to d 14 after Candida albicansgavaging to mice, the number of Candida albicans colonizing in lumen and adhering to mucosal membrane was sharply reduced.Candida albicans translocation to mesenteric lymph nodes occurred at early time points following gavage administration and disappeared at later time points. Meanwhile, the content of specific IgA was increased obviously. Proliferation and differentiation of lymphocytes in lamina propria were also increased.CONCLUSION: Lymphocytes in lamina propria play an important role in intestinal mucosal immunity of specific-pathogen-free mice when they are first inoculated with Candida albicans. The decreasing number of Candida albicans in intestine is related to the increased level of specific IgA antibodies in the intestinal mucus.     

Is endoscopic diagnosis of Candida albicans esophagitis reliable? Correlations with pathology and mycology

AIM OF STUDY: To assess the reliability of endoscopic diagnosis of Candida albicans esophagitis.PATIENTS AND METHODS: A case - control prospective study was carried out from November 1997 to July 1998 at the Campus Teaching Hospital of Lome, in patients with esophagitis macroscopically suggestive of Candida albicans origin at upper digestive endoscopy. Fifteen subjects with normal endoscopy served as controls. Esophageal biopsies for mycologic and pathological examination were performed, as well as HIV serology.RESULTS: During the study period, 26 of the 850 endoscopies performed in our Unit revealed an esophagitis suggestive of Candida albicans origin. Mycology confirmed the presence of filamentous form of Candida albicans in 23 patients and pathology showed non-specific lesions of esophagitis, 20 with intramucous hyphae. HIV serology was positive in 19/23 patients (82.6%) and in 1/15 controls (6.6%). Sensitivity and specificity of upper GI endoscopy for the diagnosis of Candida albicans were 100 and 83.3% respectively; positive and negative predictive values were 88.5 and 100%, respectively.CONCLUSION: Upper digestive endoscopy is a reliable method for the diagnosis of Candida albicans esophagitis. However, mycological confirmation is warranted.     

呼吸道白色念珠菌耐药基因CDR1、CaMDR1的研究

目的分析白色念珠菌菌株耐药性与耐药基因的表达规律。提供念珠菌临床用药及预后监测的参考。分析白色念珠菌致病性与耐药基因表达的关系。方法抽提白色念珠菌总RNA,通过半定量RT-PCR检测耐药基因CDR1与CaMDR1的相对表达量,分析比较白色念珠菌耐药性与耐药基因的关系。结果耐药性高的菌株其耐药基因CaMDR1的表达量增加;唑类药物诱导耐药菌株后未见耐药基因CDR1及CaMDR1表达增加。结论白色念珠菌的耐药基因CaMDR1的表达与耐药性有关,即耐药性高的菌株其耐药基因CaMDR1的表达量增加;白色念珠菌耐药菌株经唑类抗真菌药诱导后,其耐药基因CDR1及CaMDR1的表达未见明显增加。     

In vitro effects of ethanol on the phagocytic and microbial killing activities of normal human monocytes and monocyte-derived macrophages.

Human blood monocytes were cultured within the wells of chamber slides in growth medium to which 0, 1, 2, or 3 mg ethanol per ml was added at the start of the culture. After incubation for 1 h, 1 day and 7 days, their ability to phagocytoze IgG-sensitized red cells and to phagocytoze and kill non-opsonized Candida albicans was assessed. In all alcohol-containing wells, the concentration of alcohol in the growth medium fell progressively, reaching negligible values after 3 days. When compared with control cells, monocytes incubated with 1, 2 or 3 mg ethanol/mg for 1 h showed impaired phagocytosis of IgG-sensitized RBC and non-opsonized C. albicans and those incubated with 1 or 2 mg ethanol/ml for 1 h showed impaired killing of Candida. After incubation for 7 days, the monocyte-derived macrophages in wells initially containing 1, 2 or 3 mg ethanol/ml showed increased phagocytic activity towards C. albicans but not towards sensitized RBC. In addition, in 4 of 5 experiments, the percentage of phagocytozed organisms killed was increased in wells initially containing 1 mg ethanol/ml. The results support the view that the susceptibility of chronic alcoholics to certain infections may be partly dependent on an ethanol-induced depression of the phagocytic and killing functions of macrophages. They also suggest that a few days after a brief period of exposure to ethanol there may be stimulation of certain but not all effector functions of macrophages.     

法尼醇在促氟康唑耐药白念珠菌凋亡中的作用机制

目的:初步探讨法尼醇促进氟康唑耐药白念珠菌凋亡的可能机制。方法:采用药物浓度梯度法构建氟康唑耐药的白念珠菌株,经鉴定后设为氟康唑耐药组;氟康唑敏感的白念珠菌株作为对照组。在2组中加入终浓度为200μmol/L的法尼醇,孵育24 h后采用四甲基偶氮唑盐(methylthiazolyl tetrazolium,MTT法)比色法检测加入法尼醇后氟康唑耐药与敏感白念珠菌的增殖情况。应用流式细胞仪、激光共聚焦显微镜等,检测并比较2组菌株的胱天蛋白酶3(caspase 3)的活化程度、菌株内还原型谷胱甘肽的含量及细胞内活性氧类(reactive oxygen species,ROS)含量,进而比较不同组菌株在细胞凋亡、细胞内氧化还原及氧化应激水平上的差异。结果:与对照组比较,加入法尼醇后,氟康唑耐药的白念珠菌株增殖下降,其差异具有统计学意义(P=0.0057)。氟康唑耐药组白念珠菌内的还原型谷胱甘肽的含量下降,细胞内ROS含量及胱天蛋白酶3活性上升,且差异均具有统计学意义(分别P=0.011、P=0.023、P=0.036)。结论:法尼醇通过减少耐药菌株细胞内还原型谷胱甘肽的含量,增加胞内ROS含量,增强胱天蛋白酶3活性,使耐药白念珠菌抗氧化能力下降并促使真菌发生细胞凋亡。     

Candida albicans aggravates duodenal ulcer perforation induced by administration of cysteamine in rats

BACKGROUND: Candida sp are frequently isolated from the ascitic fluid of patients with perforated ulcers. The present study was performed to examine whether Candida infection may be involved in the process of ulcer perforation. METHODS: Male Wistar rats were divided into a saline group (n = 15) and a Candida group (n = 17). Cysteamine-HCl (Sigma; 31 mg/100 g) was administered thrice on day 1 to both groups of animals. Candida albicans at a density of 10(8) in 0.5 mL of saline was administered 1 h before, and 12 h and 24 h after the first administration of cysteamine in the Candida group. RESULTS: Perforated duodenal ulcers were observed in 94.1% of the rats in the Candida group, but only 26.7% of the rats in the saline group (P < 0.01). The area of the duodenal ulcers in the Candida group was 40.89 +/- 33.07 mm2, whereas that in the saline group was 16.53 +/- 20.4 mm2 (P < 0.05). The mortality rate was significantly higher in the Candida group than in the saline group. In the Candida group, colonization by C. albicans was recognized at the ulcer base, surrounded by marked granulocytic infiltration. The number of eosinophils infiltrating the ulcer base was also significantly greater in the Candida group than in the saline group. Immunohistochemical analysis revealed the expression of secretory aspartyl protease (SAP) in the region of the ulcer showing colonization by C. albicans in the Candida group. CONCLUSION: Candida albicans aggravates duodenal ulcer perforation in the experimental model of cysteamine-induced duodenal ulcer perforation. The present findings suggest that SAP and host-parasite relationships, including granulocyte-dependent mechanisms, may be involved in the aggravation of ulcer perforation by C. albicans.     

白念珠菌的生物形态分型研究

本文报道了首先建立的白念珠菌（白念）的生物形态分型法－型别由５个数字组成的编码表示。１７５株临床分离的白念被分为８５个型,以编码００００２为最常见（占７．４％）,分辨指数为０．９８２。并分析了型别与临床的关系,初步表明菌落条纹与毒力可能有一定关系。该法具有简单、经济、重复性好和分辨率高等优点,适于病原学研究和流行病学调查。     

Diagnosis of normal and abnormal delayed hypersensitivity to Candida albicans. Importance of evaluating lymphocyte activation by flow cytometry

Delayed type hypersensitivity (DTH) to Candida albicans is commonly observed in human. Abnormal DTH has already been described but its diagnosis is difficult to ascertain. We present now a clinical and biological study in 60 patients with a clear distinction between these two kind of Candida albicans DTH. Clinical abnormal Candida albicans DTH was characterized by a syndromic reaction 24 to 48 hours after intradermal injection. This reaction was characterized by an exacerbation of clinical symptoms. In vitro, activation of whole blood with Candida albicans antigen was detected by using flow cytometry after staining for activating markers. CD 25 positive T cells were detected in a 7 days culture in all patients. Percentage of CD 25 positive T cells was correlated to the intensity of the local cutaneous DTH reaction. CD 69 positive T cells were detected after a one day culture only in patient who presented a syndromic reaction to intradermal injection.     

Alternative Pathway of Complement Activation by Candida Albicans

Summary: Alternative pathway of complement activation by Candida albicans. Y. H. Thong and A. Ferrante, Aust. N.Z. J. Med. 1978, 8 , pp. 620–622.
Activation of the alternative pathway of complement by Candida albicans was examined using a chemotactic assay. Two serologically defined strains and eight clinical isolates of Candida albicans were used in these experiments. The results showed that all ten strains of Candida albicans were capable of alternative complement pathway activation. These findings may provide an insight into host resistance to this infection.     

Early diagnosis of candidiasis in non-neutropenic critically ill patients

OBJECTIVE: To determine a method for the early diagnosis of candidiasis in non-neutropenic critically ill patients in order to reduce mortality. METHODS: A prospective study in non-neutropenic critically patients in whom Candida spp. were detected, was made in an intensive care unit (ICU) during an 8-year period from 3389 patients admitted. A diagnostic and therapeutic protocol was designed. Invasive candidiasis was defined according to dissemination and multifocality. RESULTS: Candida spp. were found in 145 cases (4.3%): 120 (83%) were considered as invasive candidiasis and 25 as colonisation (17%). The hospital mortality was 46% (67/145). A post-mortem study was carried out in 54% (36/67) of hospital deaths. Candida albicans was the most frequently isolated species (87%), followed by Candida glabrata (18%). There were 24 candidemias and three cases of endophtalmitis. Digestive and respiratory samples and non-C. albicans yeasts were risk factors for invasive candidiasis. The mortality rate was related statistically to invasive candidiasis and inversely to the appropriate antifungal treatment. CONCLUSIONS: Invasive candidiasis is related to digestive and respiratory samples and to the presence of non-C. albicans species. A simpler definition of invasive candidiasis in non-neutropenic critically ill patients will permit more rapid and accurate specific antifungal therapy.