多重聚合酶链反应快速检测脑膜炎中的病原体

目的 建立脑膜炎多重PCR检测系统,以在一次扩增中快速、特异地同时检出新型隐球菌、结核杆菌、脑膜炎双球菌,用于脑膜炎病原体感染的快速诊断。方法 根据新型隐球菌URA保守序列、脑膜炎双球菌基因组中特定的H.8外膜蛋白基因序列、结核分枝杆菌基因组中的IS986插入基因序列片段,分别设计出3对特异性寡聚核苷酸引物,采用多重PCR技术,同时检出脑脊液中的新型隐球菌、结核杆菌、脑膜炎双球菌。结果 应用多重PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果：我重PCR扩增的预期结果为：单一菌感染出现一条特异性扩增区带;混合感染应出现2条或3条特异性扩增区带,经实验达到预期的扩增结果;对15份已明确诊断的脑脊液标本,分法均能扩增出预期的目的片段。符合率达100%,对20例临床脑脊液标本的PCR扩增结果分别为：新型隐球菌15%（3/20）、结核肝菌20%（5/20）、脑膜炎双球菌10%（2/20）。结论 多重PCR有效地为临床病原体的混合感染,以及原因不明的脑膜炎患的快速诊断,提供了快速、准确的诊断手段。有效地减少了脑膜炎的误诊和漏诊,更提高了检验效率。     

溶菌酶对酵母型真菌抗菌作用的实验观察

目的：探讨溶菌酶抗真菌作用。方法：在涂布接种白色念珠菌,新生陷球菌的沙保琼脂平板滴加５０－２０００μｇ的溶菌酶,于３７℃培养进行抑菌试验,在低倍显微镜下观察抑菌圈内实验菌的形态大小及细小菌落的形态特征,并涂片染色镜检,以加酶部位形成抑菌圈及抑菌圈内溶菌酶作用后的实验菌大小、形态结构及染色反应色镜检,以加酶部位形成抑菌圈及抑菌圈内溶菌酶作用后的实验菌大小、形态结构及染色反应判定溶菌酶的抗真菌作用。结     

Antifungal efficacy of ten selected South African plant species against Cryptococcus neoformans

Fungal infections are a major threat to public health care. Cryptococcosis in humans and animals, caused by Cryptococcus neoformans, is a life-threatening disease. In a random antifungal screening of acetone leaf extracts of 400 tree species against Cryptococcus neoformans, the following plant species had good activity: Zanthoxylum capense (Thunb.) Harv. (Rutaceae), Morusmesozygia Stapf (Moraceae), Calodendrum capense (L.f.) Thunb. (Rutaceae), Catha transvaalensis Codd (Celastraceae), Cussonia zuluensis Strey (Araliaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae), Croton sylvaticus Hochst. ex C. Krauss (Euphorbiaceae), Maytenus undata (Thunb.) Blakelock (Celastraceae), Celtis africana Burm.f. (Ulmaceae), and Cassine aethiopica Thunb. (Celastraceae). Hexane, dichloromethane, acetone, and methanol extracts of these 10 plants were tested against Cryptococcus neoformans using bioautography and microdilution assays. Acetone extracted the highest quantity of plant material. Dichloromethane and hexane extracts of Maytenus undata showed clear bands in bioautography while the other species did not produce good results in bioautography. Maytenus undata extracts had promising antifungal activity against C. neoformans, with average minimum inhibitory concentration (MIC) of 0.09?mg/mL after 24?h and 0.18?mg/mL after 48?h incubation. Croton sylvaticus and Catha transvaalensis extracts also had good activity, with average MIC values of 0.07?mg/mL and 0.09?mg/mL, respectively. Because of the clear bands on bioautograms and low MIC values compared to the other plant species investigated, M. undata was identified as a good candidate for further studies.     

Immunomodulator tuftsin increases the susceptibility of Cryptococcus neoformans to liposomal amphotericin B in immunocompetent BALB/c mice

The co-administration of immunomodulators and antibiotics has been proved very successful for treatment of opportunistic infectious diseases. In the present study, we evaluated the combination of liposomal amphotericin B (lip-Amp B) and immunomodulator tuftsin to cure Cryptococcus neoformans infection in BALB/c mice. Mice infected with C. neoformans were treated with Amp B deoxycholate and tuftsin free or tuftsin-loaded Amp B liposomes. The results of the present study demonstrated higher efficacy of tuftsin-loaded Amp B liposomes against experimental murine cryptococcosis, in terms of enhanced survival rate and reduced fungal burden in organs (lungs and brain) of the treated mice. Interestingly, pre-treatment of mice with liposomal tuftsin before challenging them with the C. neoformans infection resulted in 100% survival of the treated animals followed by treatment with lip-Amp B. Immunomodulator-based therapy seems likely to be more beneficial for treatment of fungal infectious diseases.     

Autochthonous and Dormant Cryptococcus gattii Infections in Europe

Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe (57 from 40 human patients, 22 from the environment, and 21 from animals) with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.     

真菌的中药干预研究近5年进展

近年来真菌感染率逐年上升,传统抗真菌药物易产生耐药性,而中药在防治真菌感染方面具有一定的优势。本文就近5年来中药对白念珠菌、皮肤癣菌、曲霉菌、马拉色菌、串珠镰孢菌、申克孢子丝菌、新生隐球菌及真菌生物膜的干预研究进展进行综述。     

Evaluation of Cryptococcus neoformans galactoxylomannan-protein conjugate as vaccine candidate against murine cryptococcosis

Galactoxylomannan (GalXM) is a complex polysaccharide produced by the human pathogenic fungus Cryptococcus neoformans that mediates profound immunological derangements in murine models. GalXM is essentially non-immunogenic and produces immune paralysis in mice. Previous studies have attempted to enhance immunogenicity by conjugating GalXM to a protein carrier, but only transient antibody responses were elicited. Here we report the generation of two GalXM conjugates with bovine serum albumin (BSA) and protective antigen (PA) of Bacillus anthracis, respectively, using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the cyanylating reagent. Both conjugates induced potent and sustained antibody responses as detected by both cross antigen-based and CovaLink direct ELISAs. We confirmed the specificity of the response to GalXM by inhibition ELISA and immunofluorescence. The isotype composition analysis revealed that IgG and IgM were abundant in the immune sera against GalXM, consistent with the induction of a T cell-dependent response. IgG1 was the predominant IgG subclass against GalXM, while immunization with Quil A as adjuvant elicited a significantly higher production of IgG2a than with Freund's adjuvant. Immune sera were not opsonic for C. neoformans and there was no survival difference between immune and non-immune mice challenged with C. neoformans. These results demonstrated the effectiveness of the GalXM-protein conjugate to induce robust immune responses although no evidence was obtained that such responses contributed to host defense.     

Microcomputer system for automatic identification of theCryptococcus neoformans and its clinical application

Summary In this study, microcomputer image processing and pattern recognitiontechnology, and the knowledge of morphology and optical characteristics ofCryptococcus neoformans were used for identification ofCryptococcus neoformans. Four groups of mice were lethally infected with standard strain, Wuhan strain, American B-2643 strain and Var. Shanghainesis of theCryptococcus neoformans. The samples collected included mice brain, lung, kidney, liver, small intestine tissue and were observed under a light microscope. More than 600 images of the fungus were input into a microcomputer. A system of computer for automatic identification of theCryptococcus neoformans was developed. The technique involved image preprocessing, image segmenting, coding of line-length on the edge, curve fitting, extracting of image feature, building of image library and feature data bank etc. And then, 768 images of the clinical samples and other fungus samples whose morphological features tend to be confused withCryptococcus neoformans were input into microcomputer and subjected to automatic identification. TheCryptococcus neoformans was accurately identified within 15 min, and the consistence rate with results of routine culture was 98 %.     

原发性肺部隐球酵母菌性肺炎误诊为结节病临床分析

目的将原发性肺部隐球酵母菌性肺炎误诊为结节病的临床病理和影像学表现,加以分析报道。方法应用CT扫描,病理发现结节病并随诊。结果半年后患者出现发热、头痛及肺部阴影,脑脊液培养为新型隐球酵母菌,重新阅读病理确诊为肺新型隐球酵母菌性肺炎。结论肺部隐球酵母菌性肺炎临床及影像学检查特异性少,常常误诊。