黏液卡红复染在诊断艾滋病合并隐球菌脑膜炎中的应用

目的 探讨黏液卡红复染在诊断艾滋病合并隐球菌脑膜炎(cryptococcal meningitis, CM)中的应用价值。方法 采用黏液卡红复染法检测75例艾滋病脑脊液细胞涂片,分析黏液卡红复染法、真菌培养、墨汁染色和隐球菌荚膜抗原检测四种检查方法诊断CM的敏感性和特异性。结果 脑脊液细胞涂片黏液卡红复染背景干净,隐球菌荚膜呈红色,同脱落细胞和其他微生物易鉴别。脑脊液黏液卡红复染法、真菌培养和墨汁染色诊断CM的敏感性分别为94.4%、77.8%和11.1%;特异性均为100%。其中65例的脑脊液隐球菌荚膜抗原检测敏感性为96.2%,特异性为100%。黏液卡红复染法与真菌培养和隐球菌荚膜抗原检测相比,差异无统计学意义(P>0.05);同墨汁染色相比,差异有统计学意义(P<0.005)。结论 脑脊液细胞涂片黏液卡红复染法诊断艾滋病合并CM具有高敏感性和特异性,为临床提供微生物诊断依据。     

新生隐球菌格鲁比变种表型变异菌株的基因及毒力对比研究

新生隐球菌(Cryptococcus neoformans)属于真菌界担子菌门隐球菌属,是临床上的致病真菌,可致器官移植等免疫抑制患者发生新生隐球菌病,与艾滋病有一定相关.宽大的荚膜是其典型形态学特征及临床分离鉴定的重要依据,我们在实验中对获赠自日本千叶大学医学部真菌研究所的新生隐球菌进行传代培养时发现有2株菌(IFM56803和IFM56743)发生了显著的表型变异,出现光滑型(SM)及黏液型(MC)两种不同的菌落,荚膜形态也出现明显差异,墨汁染色发现MC株的隐球菌荚膜较SM株的厚4～5倍.     

肺隐球菌病25例临床病理分析

目的探讨肺隐球菌病(PC)的临床和病理特征及其鉴别诊断。方法对25例PC手术标本进行常规HE染色及淀粉酶消化后过碘酸雪夫(D-PAS)、黏液卡红(MC)和Gorcott六胺银(GMS)特殊染色,观察PC在组织中的形态特点,并结合相关文献进行讨论。结果25例中,男16例,女9例,年龄18～52岁,平均36岁。人类免疫缺陷病毒抗体(HIV-Ab)阳性3例。胸部影像学检查:18例表现为单发或多发肺部结节,7例表现为片状实变影。25例均经胸腔镜活检手术切除,病理检查确诊。HE染色:多核巨细胞内外见大量淡蓝色圆形或卵圆形隐球菌菌体,具有折光性强的胶样荚膜;D-PAS、MC及GMS特殊染色:隐球菌菌体呈紫红色、鲜红色和黑色,更清晰。结论PC多由新型隐球菌引起,其临床及影像学表现缺乏特异性,易误诊和漏诊;组织病理学检查及特殊染色,PC具有典型的形态结构,是确诊本病的重要手段。     

肺隐球菌病17例临床病理分析

目的探讨肺隐球菌病(pulmonary cryptococcosis,PC)的临床病理学特征及鉴别诊断,提高对PC的认识。方法对17例PC进行光镜、组织化学染色及电镜观察,并结合其临床资料进行分析。结果17例患者中男性12例,女性5例,发病年龄5~67岁,主要症状为咳嗽,咳痰,发热,胸痛,头痛等。光镜下,见非干酪性肉芽肿形成,多核巨细胞和巨噬细胞胞质内及间质中可见薄壁圆形或卵圆形空泡状小体,PAS染色呈红色。电镜观察,隐球菌均有荚膜形成,荚膜与细胞壁之间有明显透明带,结构较简单,细胞器不发达。结论PC是由新型隐球菌感染引起的一种非常少见的肺部真菌病,其临床及影像学表现缺乏特异性,诊断主要依靠病理形态学特征、组织化学染色及电镜观察。     

结核性脑膜炎误诊为病毒性脑膜炎一例报告

病例：患儿，女，13岁。发热，体温38℃左右，不欲进食，呕吐，烦躁，易激怒，消瘦，乏力两个月。到哈市某医院治疗根据患儿临床症状脑膜刺激症，做腰穿脑脊液提示外观清亮，蛋白弱阳性，PPD阴性，诊断为病毒性脑炎，但不排除结核性脑膜炎。患儿到传染病医院诊治，根据腰穿结果，脑脊液提示外观清亮，蛋白＋＋，PPD阴性，脑脊液抗酸染色未查到结核菌，墨汁染色未查到新型隐球菌。因患儿病情重，急诊来我院诊治。     

对新型隐球菌的速发型超敏感性

迟发型变态反应是真菌性疾病常见的一种现象。而对类酵母的速发型变态反应的报告很少。本文目的是通过一定量的体内方法评定小鼠的过敏性反应,以进一步研究新型隐球菌主动致敏小鼠的超敏感性及阐明荚膜多糖在这些反应中的作用。实验中所用新型隐球菌B3502(a)为一有毒力的荚膜株(血清型C)和一无荚膜株602,用10~4B3502(a)和8×10~(6-7)602活菌每周一次腹腔致     

HIV感染者的隐球菌病临床表现和实验室检查

检查人类免疫缺陷病毒（ＨＩＶ）感染者１６７例,男１１６例,女５１例,年龄５～５８岁,平均３０３岁。经血清查隐球菌抗原、脑脊液涂片墨汁染色镜检与真菌培养等方法,查出新型隐球菌病１１例,男７例,女４例,年龄２１～５４岁。多为初次机会感染,认为ＨＩＶ感染者出现隐球菌病,是由早期（无症状期）过渡到晚期（艾滋病期）的象征。其中播散性隐球菌病５例（损害脑与脑膜４例）,脑膜炎３例,脑膜炎并菌血症２例,菌血症１例。主要临床表现：１１例均有全身乏力,头痛和精神失常各８例,发热７例,体重减轻、皮肤有丘疹或脓疱疹各…     

原发性肺隐球菌病的病理诊断和超微结构观察

目的探讨原发性肺隐球菌病(PC)的病理诊断和超微结构特点。方法对27例PC的临床病理资料进行回顾性分析、组织化学染色和光镜观察,12例进行透射电镜观察。结果27例中,2例肺穿刺活检明确诊断,25例开胸探查,胶样病变2例,非干酪性肉芽肿病变25例,均检出新型隐球菌(CN)。黏液卡红(MC)、过碘酸雪夫染色(PAS)、奥尔辛蓝(AB)和Grocott六胺银(GMS)染色隐球菌的检出率分别为87．0％(20／23)、100％(27／27)、66．7％(18／27)和100％(23／23)。透射电镜观察,CN均有荚膜形成,大部分结构简单,细胞器不发达,细胞退变;部分隐球菌有细胞核、核仁、线粒体和空泡;CN的检出率为91．7％(11／12)。结论PC的临床表现和影像学缺乏特征性,穿刺或开胸肺活检仍然是PC诊断的主要手段。光镜结合电镜观察以及MC、GMS和PAS组织化学染色,能对PC做出明确诊断及鉴别诊断。     

调衡方多糖增强体外培养巨噬细胞的吞噬能力

目的研究调衡方多糖对巨噬细胞吞噬功能的影响,探讨其多糖对巨噬细胞吞噬作用的免疫调节机制。方法依照中药多糖的制备方法从调衡方中提取粗多糖;从小鼠腹腔分离提取并培养巨噬细胞;(500、200、10)μg/m L调衡方多糖处理巨噬细胞48 h,光学显微镜下观察活细胞的形态,墨汁吞噬实验以及金黄色葡萄球菌吞噬实验观察巨噬细胞的吞噬能力,酶细胞化学染色观察巨噬细胞胞内酸性磷酸酶的变化。结果与对照组比较,(500、200、10)μg/m L调衡方多糖处理巨噬细胞后,巨噬细胞体积显著增大,对墨汁和金黄色葡萄球菌的吞噬能力均显著提高,酸性磷酸酶的活性也明显增强,并与多糖浓度呈正相关。结论调衡方多糖能增强巨噬细胞的吞噬功能,并提高细胞内酸性磷酸酶的活性。     

新生隐球菌荚膜相关基因CAP10启动序列的活性研究

目的 筛选具启动活性的新生隐球菌荚膜相关基因CAP10编码区上游5’侧翼序列（启动子序列），为进一步研究其调控机制打下基础。方法 利用氯霉素乙酰基转移酶（CAT）的编码基因为报告基因，构建了含不同长度的新生隐球菌荚膜相关基因CAP10编码区上游5’侧翼序列和报告基因的重组体，转化酵母，ELISA方法检测各转化子的CAT表达活性，并通过比较找到CAP10启动序列。结果 发现含CAP10编码区上游-303 bp、-390bp、-500bp、-951bp的转化子具有明显的CAT表达活性，而含-202bp、-102bp的转化子无明显的CAT表达活性。结论 CAP10上游5’侧翼端-303bp-0bp是具完整启动活性的最小单位。CAP10上游5’侧翼端-202bp--303bp内含CAP10启动所必需的序列。     

Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine

Summary The inactivation of foot-and-mouth disease virus by formaldehyde was studied under different conditions, both as free virus and (as in routine vaccine production) after adsorption of the virus to aluminium hydroxide gel (alhydrogel). In the latter case infectivity was monitored after elution of the virus from the gel by isopycnic ultracentrifugation of the virusalhydrogel mixture in CsCl. By this method good virus recoveries were obtained. Adsorption of the virus to alhydrogel (without formaldehyde) did not reduce infectivity significantly. Both adsorbed and non-absorbed virus lost infectivity at a rate of about one log₁₀ per day (at pH 8.5, 25° C—no formaldehyde).Kinetics of formaldehyde inactivation of adsorbed and non-adsorbed virus were also identical, with a fast reduction in the initial phase (in case of O₁ and A₁₀-virus approximately one log₁₀/hour). After this initial phase inactivation became linear and rather slow (for O₁ and A₁₀-virus 0.2 log₁₀/hour). No tailing-off was observed. Under standard conditions (0.04 per cent formaldehyde, pH 8.5, 25° C) C_D-virus was inactivated approximately 1.5 times faster than O₁ and A₁₀-virus. At 4° C the inactivation of the three strains continued at about one log₁₀/day.Increased lactalbumin hydrolysate concentrations reduced the inactivation rate, especially at the formaldehyde concentration of 0.02 per cent, which was originally applied. Quaternary amines like Tris strongly inhibited formaldehyde activity. These findings might explain some data of others who observed tailing off.Analysis of formaldehyde inactivated antigen by SDS-PAGE and electrofocusing showed that extensive cross-linking occurs especially of VP₁, probably with other virus proteins but also with non-virus proteins from the medium. VP₂ and VP₃ are less affected. Cross-linking was enhanced when the virus had been adsorbed to alhydrogel during inactivation.Progressive cross-linking was observed during storage of the vaccine at 4° C, which also indicated that inactivation continued at this temperature.These data show that formaldehyde inactivated adsorbate vaccines can be safe.With 7 Figures     

Clinical and mycological features of cryptococcosis]

Cryptococcosis is the third most common deep mycosis in Japan. Cryptococcus neoformans is known to grow in pyres of pigeon feces. Chicken feces in Thailand were tested for whether C. neoformans could be isolated, because there is considerable prevalence of cryptococcal meningitis in patients with HIV in that country. We isolated C. neoformans from chicken feces in as many as at 70 % of the villages tested. Chicken as well as pigeon feces were believed to be an origin of infection. We have studied the relation between in vitro virulence and thickness of polysaccharide capsules. Strains with thicker capsules such as YC-11 or YC-5 showed more resistance to macrophage phagocytosis than strains with thinner capsules like YC-27 or YC-13. This finding was consistent with the cytokine dynamic state in mice cryptococcosis. Th1 was dominant in infections with thinner capsule strains, although Th2 was relatively dominant in those with thick capsules. The clinical features of 104 cases with pulmonary cryptococcosis were summarized. Radiological findings of pulmonary cryptococcosis varied depending on the time course of the disease and on immunological status. There were no specific symptoms and signs except for positive glucronoxylomannan. Those in azole class were the most commonly prescribed antifungals. New generation antifungals voriconazole and intravenous itraconazole showed potent clinical efficacy in pulmonary cryptococcosis.     

发热伴血小板减少综合征病毒热稳定性和灭活条件初步研究

目的 阐明新发危害严重的发热伴血小板减少综合征病毒的稳定性和理化灭活条件.方法 评估细胞培养制备的SFTS病毒在不同温度下的热稳定性,对紫外线、酸性环境、常用消毒剂、有机溶剂敏感性.处理后病毒感染Vero细胞,用间接免疫荧光法确定病毒复制,并采用基于病毒核蛋白的双抗体夹心ELISA方法滴定病毒与无相应处理的对照组,比较分析各种理化条件对病毒感染性的影响.结果 SFTS病毒在37℃能够存活较短时间,感染性下降较快,在4℃能保持相对稳定,1周内感染性无明显下降.对热敏感,60℃30 min能够完全灭活病毒.对紫外线敏感,185 μW/cm2紫外线照射30 min可灭活病毒.对乙醚、氯仿等有机溶剂,β-丙内酯、甲醛和常用有机氯消毒剂敏感,在合适的浓度下可在较短时间内有效灭活病毒,400 mg/L的有效氯灭活病毒需室温放置10 min以上,在pH3.0条件对病毒活力有损害,但不能完全灭活病毒.结论 研究结果初步客观的评价了SFTSV热稳定性和灭活条件,为科学研究和疾病控制中样本采集、病毒灭活、安全防护等工作提供了科学依据.     

Studies of the inactivation of poliomyelitis virus by formaldehyde

Summary Batches of virus suspensions produced on human embryonic tissue and on monkey kidney tissue were inactivated by formaldehyde. The decrease in virus activity was followed by titrations in plasma clot cultures of human embryonic lung and in cultures of trypsinized monkey kidney. No difference could be demonstrated in the rate of inactivation between clarified batches derived from human embryonic tissue and batches produced on monkey kidney tissue and filtered through bacteria retaining filters. The course of inactivation deviated from that of a linear relationship between log. virus activity and time.     

Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization

Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond –50 mV from a holding potential of –100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10–20 s. Rapid inactivation appeared to be due to Ca²⁺ influx since it was slowed markedly when Ba²⁺ was used as the current carrier, while the degree of inactivation mercased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by 20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the –50 to –30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.     

Comparison of two different methods for inactivation of viruses in serum.

In order to compare protocols for inactivation of viruses potentially present in biological specimens, three different model viruses were treated in bovine serum by two different inactivation methods: samples were subjected either to chemical inactivation with ethylenimine (El) at concentrations of 5 and 10 mM at 37 degrees C for periods up to 72 h or to electron-beam irradiation in frozen and liquid form with doses varying between 11 and 46 kGy. The chemical inactivation resulted in nonlinear tailing curves in a semilogarithmic plot of virus titer versus inactivation time showing non-first-order kinetics with respect to virus titer. The time for inactivation of 7 log10 units of porcine parvovirus (PPV) was about 24 h for both El concentrations, whereas 5 log10 units of bovine viral diarrhea virus (BVDV) was inactivated in 2 h for both El concentrations and 6 log10 units of porcine enterovirus (PEV) was inactivated within 3 h. The inactivation with electron-beam irradiation resulted in almost linear curves in a semilogarithmic plot of virus titer versus irradiation dose, reflecting a first-order inactivation. The rate of inactivation was almost twice as fast in the liquid samples compared to the rate in frozen ones, giving values of the doses needed to reduce virus infectivity 1 log10 unit for inactivation of PPV of 11.8 and 7.7 kGy for frozen and liquid samples, respectively, whereas the corresponding values for BVDV were 4.9 and 2.5 kGy, respectively, and those for PEV were 6.4 and 4.4 kGy, respectively. The nonlinear inactivation with El makes it impossible to extrapolate the curves beyond the virus detection limit and thereby predict the necessary time for complete inactivation, i.e., to a level beyond the detection limit, of virus in a given sample. The first-order inactivation obtained with electron-beam irradiation makes such a prediction possible and justifiable. The two methods are discussed with respect to their different kinetics and applicability under different circumstances and criteria for inactivation, and considerations for choice of method are discussed.     

In Vitro Phagocytosis and Intracellular Fate of Variously Encapsulated Strains of Cryptococcus neoformans

Five isolates of Cryptococcus neoformans type A with stable capsular thicknesses were used. Three of the isolates had capsules of medium size, one had a minimal capsule, and the other, a large capsule. Peritoneal exudate cells from Lewis rats were cultured on cover slips in Leighton tubes containing medium 199 and 20% fresh, isologous normal rat serum. Yeast cells were added to the Leighton tube cultures, and, 2 hr later, the extracellular yeasts were rinsed out. Cover slips were removed from some tubes for Wright staining and measurement of both phagocytosis and loss of macrophages. The remaining tubes were reincubated and sampled at 24 or 48 hr. To determine fate of yeast cells after ingestion, washed cover slips were inverted onto agar slide cultures, and specific macrophages were observed in situ for subsequent multiplication of their intracellular yeasts. More than half of the macrophages survived 24 to 48 hr of exposure to different strains of C. neoformans, with small, medium, or large capsules. Phagocytic activity was dependent upon a heat-labile factor in normal rat serum. The number of yeast ingested by macrophages was inversely proportional to the capsular size. Although most of the ingested yeasts were resistant to intracellular killing, the agar culture technique clearly demonstrated that many were unable to multiply, presumably dead. Three of the isolates were more susceptible than the other two, and the fate of these yeasts after engulfment was not correlated with their capsular size.     

灭活SARS病毒的纯化鉴定及初步应用

为制备纯化的灭活SARS病毒,取广东省CDC提供的灭活SARS-CoV病毒培养液进行超滤,收集相对分子质量介于100000至500000之间的组分,并浓缩。对纯化过程中各组分进行抗原活性鉴定和蛋白质含量测定,并加佐剂后免疫动物,测定抗体免疫应答。结果表明,超滤后活性抗原蛋白质回收率7.6%,纯化倍数为9,抗原活性达到1:480。免疫家兔获得的血清ELISA效价达到1:10000以上,中和抗体效价达到1:2000以上。表明该纯化工艺能获得较高纯度的灭活SARS-CoV病毒颗粒。     

Development and Preclinical Evaluation of a Trivalent,Formalin-Inactivated Shigella Whole-Cell Vaccine

Studies were undertaken to manufacture a multivalent Shigella inactivated whole-cell vaccine that is safe, effective, and inexpensive. By using several formalin concentrations, temperatures, and incubation periods, an optimized set of inactivation conditions was established for Shigella flexneri 2a, S. sonnei, and S. flexneri 3a to produce inactivated whole cells expressing a full repertoire of Ipa proteins and lipopolysaccharide (LPS). The inactivation conditions selected were treatment with 0.2% formalin (S. flexneri 2a and 3a) or 0.6% formalin (S. sonnei) for 48 h at 25°C. Vaccine formulations prepared under different inactivation conditions, in different doses (10E5, 10E7, and 10E9 cells), and with or without the inclusion of double-mutant heat-labile toxin (dmLT) were evaluated in mice. Two intranasal immunizations with ≥10E7 inactivated whole cells resulted in high levels of anti-Invaplex and moderate levels of LPS-specific IgG and IgA in serum and in lung and intestinal wash samples. Addition of dmLT to the vaccine formulations did not significantly enhance humoral immunogenicity. Minimal humoral responses for IpaB, IpaC, or IpaD were detected after immunization with inactivated whole Shigella cells regardless of the vaccine inactivation conditions. In guinea pigs, monovalent formulations of S. flexneri 2a of 3a or S. sonnei consisting of 10E8, 10E9, or 10E10 cells were protective in a keratoconjunctivitis assay. A trivalent formulation provided protection against all three serotypes (S. flexneri 2a, P = 0.018; S. flexneri 3a, P = 0.04; S. sonnei, P < 0.0001). The inactivated Shigella whole-cell vaccine approach incorporates an uncomplicated manufacturing process that is compatible with multivalency and the future development of a broadly protective Shigella vaccine.     

A study on the immune response of sheep to foot and mouth disease virus vaccine type 'O' prepared with different inactivants and adjuvants.

Foot and mouth disease virus (FMDV) type 'O' was inactivated either with formaldehyde or binaryethyleneimine (BEI). Vaccines were prepared with inactivated virus incorporating aluminum hydroxide gel or mineral oil as an adjuvant. The antibody response in sheep was monitored by serum neutralization and ELISA test for a period of six months. Significant difference in antibody response was not observed between vaccines inactivated with formaldehyde or BEI. On the other hand significant difference in the antibody response was noticed between alhydrogel and oil vaccines. The high titer of antibodies stimulated by oil adjuvant vaccines persisted longer than those of alhydrogel vaccines within the period of study.