人芽囊原虫在不同培养基中生长状况的观察

目的筛选培养人芽囊原虫的最适培养基。方法将同一株人芽囊原虫阳性粪便标本以2×105细胞/管接种至RPMI1640、199和LES培养基中,加入20%小牛血清及青、链霉素,pH值为7.5,放置厌氧罐中于37℃恒温培养,每24h计数,每6d转种1次。观察人芽囊原虫在3种培养基中的存活时间、虫体密度和虫体形态。结果人芽囊原虫在RPMI1640培养基中存活时间最长、虫体密度最高,虫体以空泡型多见;在LES培养基中存活时间最短、虫体密度最低,但虫体形态清晰、规则;在199培养基中存活时间和虫体密度均介于前两者之间。结论RPMI1640培养基适宜人芽囊原虫的生长繁殖,为人芽囊原虫体外培养的首选培养基;LES培养基中虫体形态清晰、规则,可用于人芽囊原虫的形态学研究;199培养基也可用于人芽囊原虫的体外培养,但不作为首选。     

3种培养基体外培养人芽囊原虫的效果观察

目的　观察人芽囊原虫在 3种培养基中的生长繁殖情况 ,以探讨最佳的培养方法。　方法　从大学生粪便中分离人芽囊原虫 ,转种至 3种不同培养基 ,定时观察人芽囊原虫的生长繁殖情况 ,根据原虫密度评价培养效果。　结果培养 96h后 ,Locke’s鸡蛋血清 (LES)双相培养基中的人芽囊原虫密度最高 ,改良Jones单相液体培养基次之 ,Locke’s琼脂血清 (LAS)双相培养基原虫密度最低 ,差异有显著性 (P <0 .0 5 ) ;观察 3种培养基中的原虫密度高峰持续时间分别为 3 60h、96h和 72h ,最长存活时间分别为 5 5 2h、3 3 6h和 168h。　结论　Locke’s鸡蛋血清双相培养基较适用于人芽囊原虫体外增殖培养。     

人芽囊原虫体外纯培养法的改良及形态观察

以琼脂取代蛋白对传统的洛克氏液-鸡蛋-血清(Locke’s egg serum,LES)双相培养基进行改良,联合采用LES双相培养基、改良LES双相培养基(m LES双相培养基)、IMDM液相培养基和IMDM固相培养基建立人芽囊原虫的体外纯培养的标准流程,并观察虫体在不同培养基中的形态特征和生长情况。结果显示,人芽囊原虫在4种培养基中均以空泡型和颗粒型的形态为主,培养后镜下未见细菌生长。在mLES双相培养基中的生长速度最快,培养后第2天原虫数为3.09×10~6,达到中对数期。原虫在LES双相培养基、mLES双相培养基和IMDM液相培养基中的生长对数期的分裂时间分别为(49.72±1.35)、(24.16±2.53)和(36.3±1.5)h。提示联合采用LES双相培养基、mLES双相培养基、IMDM液相培养基和IMDM固相培养基可建立人芽囊原虫的无菌纯培养体系。     

2型糖尿病患者并发人芽囊原虫感染3例

目的 描述并分析3例2型糖尿病患者检出的人芽囊原虫各期虫体形态学特点、诊治经过及临床表现,为糖尿病并发人芽囊原虫感染的诊治提供经验依据。方法 采集3例2型糖尿病患者静脉血,行血常规检查;用生理盐水涂片法联合涂片瑞吉染色法检查患者粪便,用光学显微镜观察虫体形态,分析鉴定虫种,描述并分析患者诊治经过及临床表现。结果 3例患者经过相关检查及虫种鉴定,确诊为2型糖尿病并发人芽囊原虫感染,口服甲硝唑驱虫治疗后,患者康复出院,随访患者身体良好,未见复发。结论 患有糖尿病等使人体免疫功能低下的疾病时,要警惕人芽囊原虫的感染,粪便检出人芽囊原虫可确诊。用生理盐水涂片法联合涂片瑞吉染色法检查粪便,可观察到虫体的运动情况和看清虫体内部结构,准确识别虫体形态,有效避免人芽囊原虫病的漏诊和误诊。     

阴道毛滴虫在两种培养基中的生长与形态观察

目的 通过对改良肝浸汤培养液及RPMI1640培养基内阴道毛滴虫生长情况的观察,选择适合教学和科研的培养基。 方法 用临床分离的阴道毛滴虫标本,分别接种至改良肝浸液及RPMI1640两种培养基内进行培养,pH值为5.6~5.8,通过计数了解生长情况并观察阴道毛滴虫的形态。 结果 肝浸汤培养液中培养48 h,阴道毛滴虫达到生长高峰,虫体呈现出多分裂相,虫体变大、呈圆形,内含4~12个细胞核,细胞膜外缘可见数丛鞭毛。RPMI1640培养基中阴道毛滴虫72 h达到生长高峰,虫体密度约为肝浸液高峰期的2／3,未见多分裂现象,虫体与生理盐水涂片中阴道毛滴虫形态、大小相近。 结论 改良的肝浸液培养基适于阴道毛滴虫的药物试验及其他实验项目的研究,RPMI1640培养基更适用于教学、标本的制作及实验室保种工作。     

中药牛至油、鸦胆子对人芽囊原虫的体外杀灭作用

目的通过观察牛至油、鸦胆子对人芽囊原虫的体外杀灭作用,筛选治疗人芽囊原虫感染有效药物及浓度。方法从腹泻患者新鲜粪便中分离人芽囊原虫,分别用含牛至油和鸦胆子1640培养基培养,药物设200、400、800、1600、3200和6400μg/ml组。于加药24h和72h作虫体计数,评价药物效果,试验设甲硝唑药物对照组和不加药空白对照组。结果牛至油1600、3200μg/ml作用72h、6400μg/ml作用24h,虫体全部死亡,最适杀虫体浓度为800μg/ml;鸦胆子6400μg/ml24h虫体全部死亡,最适杀虫浓度为1600～3200μg/ml;甲硝唑40μg/ml72h、≥80μg/ml24h虫体全部死亡。结论牛至油和鸦胆子具有体外杀灭人芽囊原虫作用。且牛至油的杀虫效果强于鸦胆子。     

感染人芽囊原虫昆明鼠肠粘膜的病理改变

目的通过观察昆明鼠感染人芽囊原虫后肠粘膜病理变化,探讨人芽囊原虫的致病机制。方法从腹泻患者粪便中分离培养人芽囊原虫。15只昆明鼠分为3组:A组为接受免疫抑制剂处理组,B组为非免疫抑制剂处理组,经口感染人芽囊原虫1 .6×105个/鼠,观察小鼠感染情况及肠粘膜的病理变化;C组为健康对照组。结果实验鼠人芽囊原虫检出率90 %(9/10) ,虫体主要寄生在小鼠回盲部肠腔和肠粘膜表面,个别虫体侵入肠粘膜上皮,部分肠粘膜充血水肿,腺体增生,粘液分泌增强,可见有炎症细胞浸润,偶见局灶性肠粘膜坏死。病变程度免疫抑制剂处理组比未接种免疫抑制剂处理组重。结论感染人芽囊原虫的昆明鼠回盲部肠粘膜发生病理损害,病变程度受宿主机体免疫状况的影响。     

不同环境中人芽囊原虫包囊的结构观察

目的　观察人芽囊原虫包囊在不同环境中的光学和超微结构的形态特点。　方法　从粪便分离人芽囊原虫 ,转种至改良的Jones培养基培养 72h后 ,再转种至混合纤维素脂微孔滤膜成囊培养基和Suresh成囊培养基中培养。采用光学和电子显微镜对培养基中与粪便中的包囊进行光学和超微结构的观察。　结果　光镜下观察 ,新鲜粪便中的包囊微小圆球形 ,具有折光性 ,不同的个体大小悬殊 ,HE染色可显示囊壁有 3层。用Suresh成囊培养基培养的包囊圆形或椭圆形 ,中心体色淡 ,内有大小不等的孢子体。混合纤维素脂微孔滤膜培养的包囊均较大 ,有空泡型、颗粒型、薄壁与厚壁包囊之分。透射电镜下观察 ,无纤维层的裸露包囊形态大都是圆形或卵圆形。包囊直径为 14 μm。包囊呈典型的人芽囊原虫核 ,其线粒体较多 ,胞质中糖原呈大小不一块状。　结论　包囊在不同环境中的形态基本相同、大小不一、结构多型。     

mLES法培养人芽囊原虫的实验观察

目的建立一种简便、灵敏的可用于人芽囊原虫(Blastocystis hominis,Bh)感染诊断实验室方法。方法比较改良洛氏-鸡蛋-血清双相培养法(modified Locke-egg serum medium culture method,mLES培养法)与碘液直接涂片法对Bh的检出率;比较普通洛氏-鸡蛋-血清双相培养基(Locke-egg serum medium culture method,LES培养法)与mLES培养法中Bh形态、最低检出限、生长曲线及厌氧条件形成时间。结果检查397份临床粪便标本,mLES培养法Bh阳性率为5.54%(22/397),碘染法阳性率为1.01%(4/397),差异有统计学意义(P0.01)。中央空泡型是Bh存在两种培养基中的主要形态。mLES培养法48hBh最低检出限(10~2/5ml)优于LES培养法(10~3/5ml),重复测量方差分析表明组内Bh密度在不同时间点有差异、时间与分组交互作用及培养基种类的差异有统计学意义(P0.05);除了第6d,其余各时间点两种培养基中人芽囊原虫生长密度差异均有统计学意义(P0.05),mLES培养法前3dBh密度高于LES培养法。mLES培养法先于普通培养基达到厌氧环境。结论 mLES培养法简便、灵敏,可用于诊断Bh的体外培养检查。     

不同环境中人芽囊原虫包囊的结构观察

目的 观察人芽囊原虫包囊在不同环境中的光学和超微结构的形态特点。方法 从粪便分离人芽囊原虫，转种至改良的Jones培养基培养72h后，再转种至混合纤维素脂微孔滤膜成囊培养基和Suresh成囊培养基中培养。采用光学和电子显微镜对培养基中与粪便中的包囊进行光学和超微结构的观察。结果 光镜下观察，新鲜粪便中的包囊微小圆球形，具有折光性，不同的个体大小悬殊，HE染色可显示囊壁有3层。用Suresh成囊培养基培养的包囊圆形或椭圆形，中心体色淡，内有大小不等的孢子体。混合纤维素脂微孔滤膜培养的包囊均较大，有空泡型、颗粒型、薄壁与厚壁包囊之分。透射电镜下观察，无纤维层的裸露包囊形态大都是圆形或卵圆形。包囊直径为14μm。包囊呈典型的人芽囊原虫核，其线粒体较多，胞质中糖原呈大小不一块状。结论 包囊在不同环境中的形态基本相同、大小不一、结构多型。     

Simple and efficient method for clinical isolation of Bordetella pertussis

Commercially available Amies transport medium with charcoal and three isolation media were tested to assess their efficiency in the clinical isolation of B. pertussis. First, the isolation rates of B. pertussis were compared between direct inoculation of nasopharyngeal specimens and inoculation after stored in Amies transport medium for 8 hours or less. The comparative isolation rates were 81% both (35 of 43 specimens from 29 patients) for direct inoculation and transport medium. Second, nasopharyngeal specimens were incubated for 5 days at 35 degrees C on the three media; Bordet-Gengou Medium (BG) with 5 micrograms of CEX per ml, Cyclodextrin Solid Medium (CSM) with 5 micrograms of CEX per ml, and Charcoal Agar with 40 micrograms of CEX per ml. The organism was detected from 44 nasopharyngeal specimens from 20 patients on at least one of the three tested media. The comparative isolation rates were 91% (40 of 44) on BG with 5 micrograms of CEX per ml, 93% (41 of 44) on CSM with 5 micrograms of CEX per ml, and 91% (40 of 44) on CA with 40 micrograms of CEX per ml. Although no differences in the isolation rates were observed among the three media, the appearance of the colonies was earlier by one day on BG than the rest of the two media. The detection of B. pertussis was occasionally easier on CA than the rest of the two because of its higher suppression for nasopharyngeal flora. CSM has its advantage in that it does not need any blood and can be prepared at anytime. Also, the shelf life of these three media proved to be at least one month when stored at 10 degrees C. We conclude that the clinical isolation of B. pertussis was highly successful with the following simple procedure: nasopharyngeal specimens stored in Amies transport medium and inoculated on one of the three media, BG, CSM, or CA, and then incubated for 5 days at 35 degrees C.     

Molecular epidemiology of malaria in Cameroon. XXIII. Experimental studies on serum substitutes and alternative culture media for in vitro drug sensitivity assays using clinical isolates of Plasmodium falciparum

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.     

Frequency of recovery of Blastocystis hominis in clinical practice

We examined the frequency of isolation of Blastocystis hominis from stools of patients seen in an indigent-care teaching hospital. Over a 2-year period, 2,744 stool specimens were examined prospectively. B. hominis was found in 262 stools (9.5% of all stool specimens and 53.5% of the positive specimens). Clinical data were obtained from 80 patients with stools positive for B. hominis. B. hominis was the only parasite isolated in 39 of 47 (83%) of the adults, compared with 17 of 33 (52%) of the children (p = 0.006). All but 2 of 52 patients without concomitant parasitic infection or bacterial pathogens in stool had gastrointestinal symptoms (41 abdominal pain, 26 diarrhea, and 5 vomiting), but no association was seen with fever, peripheral leukocytosis, stool occult blood, fecal leukocytes, or endoscopic or radiologic evidence of colitis. Therefore, B. hominis was frequently recovered from stools examined in a hospital clinical parasitology laboratory. The clinical presentations of patients in our series did not suggest that B. hominis was invasive. Most patients with B. hominis probably do not require treatment since they will either have spontaneous resolution of symptoms or will be found to have an alternative explanation for their problem.     

依赖利福平结核分支杆菌在无利福平条件下的生长情况研究

目的 探讨依赖利福平结核分支杆菌（依R菌）在无R条件下的生长情况。方法 取初步研究的典型依R菌株，挑取最佳R浓度管中的单个菌落，制备10^-4mg/ml菌液，分别接种于L－J和12B液体培养基，37℃培养；对比观察不同试验管细菌的生长速度、特点以及光镜、电镜下的菌体形态。结果 L－J培养有R管2周可见菌落，无R管3周才见到细小菌落；12B液体培养，前者9天获得阳性指数，后者需13天。有R管中菌落生长粗大、凸起、花菜样、易刮取和乳化；无R管中菌落生长细小、扁平、荷包蛋样、不易刮取和乳化。光镜下有R管菌体粗大、深染；无R管菌体细小、浅染，且大小不等。透射电镜下有R管菌体细胞壁完整，无R管菌体细胞壁缺陷。结论 依R菌在无R条件下可呈细胞L型样改变。     

Blastocystis hominis in Hospital Employees

Several reports have appeared that either support or deny the importance of the protozoan Blastocystis hominis as an intestinal pathogen in humans. In this report, we describe the clinical characteristics of B. hominis and its response to therapy in hospital employees found to have the parasite on routine screening of stools. During the study, 49 patients with B. hominis were identified, and 413 stools were examined from these patients. Twenty-nine patients were asymptomatic (59%), and 20 had symptoms of bloating, flatulence, soft/loose stools, or constipation. Of these 20 patients, 10 had symptoms that correlated with the presence or absence of B. hominis, four had symptoms that were independent of B. homonis, and six had other intestinal parasites that could account for their symptoms. Nineteen percent of patients without treatment had eradication of B. hominis from stool on follow-up examination. Metronidazole did not increase this rate. Iodoquinol treatment eradicated the organism in 41% of patients (p less than 0.05), and resulted in the reduction or eradication of the parasite in 62%, as determined by follow-up examination.     

不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响

目的：通过探讨不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响，了解它们对成纤维细胞体外生存和生长的情况。方法：采用体外细胞培养技术进行小鼠成纤维细胞L929细胞，并采用磺基罗丹明B（sulforhodamineB，SRB）酶标仪法（A490nm波长下）测量各孔的吸光值（A值）并转换为小鼠成纤维细胞L929细胞系生长曲线；同时，分别采用HE（苏木精-伊红）染色法、、吖啶橙荧光染色和Hoechst33342荧光染色法进行小鼠成纤维细胞L929细胞系形态学观察。结果：小鼠成纤维细胞L929细胞系不同浓度的血清分别在3种不同的介质（RPMI1640培养基、0．9％生理盐水和5％葡萄糖生理盐水）中表现出不同的生长和生存情况及细胞形态学改变。其中，在不同血清浓度（0％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％）的同一介质中，血清浓度为0％和100％浓度组的小鼠成纤维细胞L929细胞生长明显抑制及吸光度（A）值呈逐步减少的趋势，而除此之外的其它血清浓度组对小鼠成纤维细胞L929细胞的影响，基本表现为随着血清浓度的升高，吸光度（A）值呈逐步增加及促进细胞生长的趋势。而某些相同血清浓度在不同介质中对小鼠成纤维细胞L929细胞系生长和生存及形态学的有利影响，则基本表现为RP—M11640培养基作为介质的细胞培养效果优于0．9％生理盐水，而0．9％生理盐水作为介质的细胞培养效果又优于5％葡萄糖生理盐水。结论：不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长和生存及细胞学形态均有明显影响。提示在进行体外细胞培养时应充分考虑细胞培养介质及所添加血清的浓度。     

几种药物对体外培养日本血吸虫成虫毒性作用的研究

目的观察金诺芬、顺铂、阿霉素及化合物4N、H、B、O对体外培养日本血吸虫的毒性作用,以及对硫氧还蛋白谷胱甘肽还原酶（TGR）活性的抑制作用。方法取日本血吸虫成虫置于RPMI 1640培养基中,培养30～60 min后,加入不同浓度药物,分别于6、24、48、72 h观察虫体活力、形态改变及死亡情况。随后换无药物的新鲜培养基,观察虫体活力的恢复情况,并计算药物半数致死剂量（LD50）。测定药物处理后的成虫匀浆上清液中日本血吸虫TGR的硫氧还蛋白原酶（TrxR）和谷胱苷肽还原酶（GR）的活性。结果 5μg/ml金诺芬作用24 h、20μg/ml 4N作用72 h、60μg/ml H作用72 h、80μg/ml顺铂作用72 h对日本血吸虫成虫致死率分别为100%、60%、66.7%、100%,LD50值分别为2.56、17.59、54.14μg/ml和52.87μg/ml,其他药物对日本血吸虫无作用。金诺芬、4N、顺铂、H对成虫的毒性作用具有不可逆性;金诺芬、顺铂对日本血吸虫的TGR酶活性有抑制作用,其他药物对TGR无作用。5～30μg/ml金诺芬、20～30μg/ml 4N、70～150μg/ml顺铂及60～150μg/ml H作用24 h均可使虫体形态发生改变。结论金诺芬、顺铂、化合物4N和H对体外培养的日本血吸虫成虫具有杀伤作用,其中金诺芬、顺铂杀伤日本血吸虫成虫的作用可能与其抑制TGR活性有关。     

Ethanol Modulates Metastatic Potential of B16BL6 Melanoma and Host Responses

The experimental metastatic potential (lung-colonizing ability) of B16BL6 melanoma cells was examined in C57BL/6 mice after exposure to ethanol in vitro and in vivo. In vitro, tumor cells were cultured with ethanol (0.3% v/v), or medium alone, for three passages at 5-day intervals. In vivo, B16BL6 melanoma was exposed to ethanol by administering ethanol (10% or 20% w/v) to mice following subcutaneous inoculation of tumor cells into the dorsal hip. All tumor cells were subsequently inoculated intravenously into the lateral tail vein of water-drinking mice to assess changes in metastatic phenotype. Tumor cells cocultured in vivo with ethanol produced significantly higher numbers of superficial lung colonies, compared with tumor cells cultured in control medium. Experimental metastasis of tumor cells obtained from 20% w/v ethanol-consuming mice was also significantly increased, compared with cells obtained from water-drinking mice. Metastasis of B16BL6 melanoma cells previously obtained from mice consuming 10% w/v ethanol did not differ from controls. In other experiments, water-drinking and ethanol-consuming (2.5%, 10%, and 20% w/v) mice were inoculated subcutaneously into the dorsal hip with B16BL6 melanoma cells, and monitored for tumor growth rate and survival time. In these experiments, survival times were significantly shorter in mice consuming 20% ethanol, compared with all other groups. Subcutaneous tumor growth rate was unaffected by ethanol consumption. Lung metastasis resulting from subcutaneous tumor implantation of B16BL6 melanoma was respectively inhibited, or absent, in 10% and 20% ethanol-consuming groups. Thus, tumor growth rate and incidence of lung metastases were not apparent determinants of decreased survival in 20% ethanol-consuming mice. The results of this study indicate that the experimental metastatic potential of B16BL6 melanoma is increased during exposure to ethanol; however, metastasis from subcutaneous tumor-bearing mice is suppressed. This latter finding is consistent with previous results in which spontaneous metastasis was also suppressed after inoculation of the tumor into the pinna of the ear. Although ethanol increases the ability of B16BL6 melanoma to colonize the lung after intravenous inoculation, this effect is abated in the presence of host factors in ethanol-consuming mice.     

Blastocystis hominis: an organism in search of a disease

Blastocystis hominis is a protozoan organism frequently found in the human intestinal tract. Eleven consecutive patients with symptoms of enteritis and having B. hominis as the sole enteropathogen were studied in an attempt to define the association of blastocystosis with clinical disease. B. hominis could not be implicated as the etiologic agent of enteritis in any of these patients. All eleven had alternative (and usually noninfectious) explanations for their intestinal symptoms. There was no correlation between resolution of symptoms and either antiprotozoal therapy or disappearance of B. hominis from the stools. All prior reports associating B. hominis with human disease have been reviewed and provide no convincing proof of a causal relation. B. hominis is rarely, if ever, a human pathogen, and treatment directed at the eradication of B. hominis is not indicated.