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目的播散性隐球菌病临床及实验研究。方法患者女,47岁,肝移植术后2 d,面部、肩部、四肢皮肤出现多发溃疡,伴昏迷。通过脑计算机断层扫描、皮损组织病理检查、PAS染色、皮损组织真菌培养及激光俘获显微切割结合PCR扩增序列分析确诊,并对获得菌株进行尿素酶试验、API试验、PCR扩增测序等实验研究。结果皮损组织病理可见大量圆形和椭圆形菌体,PAS染色阳性。血液和脑脊液真菌镜检均为阴性。皮损组织真菌培养可见酵母样菌落生长,菌株尿素酶试验阳性,API试验鉴定为新生隐球菌。ITS区序列分析鉴定为新生隐球菌grub ii变种。激光俘获显微切割结合PCR扩增,序列分析与培养获得的菌株直接PCR扩增后序列分析结果一致。脑脊液特异性隐球菌抗原(++),血液特异性隐球菌抗原(++++)。脑CT显示为多发结节灶。依据临床及实验室检查确诊为播散性隐球菌病,致病菌为新生隐球菌grubii变种。结论通过对该病例的深入研究,为临床明确诊断播散性隐球菌病奠定基础,确立了显微切割技术在皮肤真菌感染中的应用价值。
Objectives Disseminated cryptococcosis clinical and experimental study. Methods The patient, 47 years old, 2 days after liver transplantation, had multiple ulcers on the face, shoulders and extremities with coma. Pathological examination of the lesions, pathological examination of the lesions, PAS staining, fungal culture of the skin lesions and laser capture microdissection combined with PCR amplification confirmed the sequence analysis. The obtained strains were subjected to urease test, API test, PCR amplification and sequencing Other experimental research. Results A large number of round and oval cells were found in pathological lesions, PAS staining was positive. Fungus microscopic examination of blood and cerebrospinal fluid were negative. Yeast-like colony growth was observed in the fungal cultures of skin lesions. The urease test of the strains was positive. The Cryptococcus neoformans was identified by the API test. ITS sequence analysis identified as C. neoformans grub ii variant. Laser capture microdissection combined with PCR amplification, sequence analysis and culture obtained by direct PCR amplification of the sequence analysis of the results. Cerebrospinal fluid-specific cryptococcal antigen (++), blood-specific cryptococcal antigen (++++). Brain CT showed multiple nodules. According to clinical and laboratory tests diagnosed as disseminated cryptococcosis, pathogens of C. neoformans grubii variants. Conclusion Through in-depth study of this case, lay the foundation for the clinical diagnosis of disseminated cryptococcosis and establish the value of microdissection in the skin fungal infection.