肝移植后播散性隐球菌病1例及其实验研究

A case of disseminated cryptococcosis after liver transplantation:clinical and laboratory investigation

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。

Objective To report a case of disseminated cryptocoecosis after liver transplantation and related laboratory investigation. Methods One 47-year-old female lady was admitted due to a coma with multiple ulcers on the face, shoulders and four limbs after liver transplantation. We confirmed the diagnosis by CT, tissue biopsy, PAS stain, tissue culture, urease test, API test, PCR and sequence analysis, laser capture microdissection, subsequenee PCR and sequence analysis. Results Skin tissue biopsy shows plenty of fungus components which were PAS stain positive. Both blood and cerebrospinal fluid （CSF） were negative by direct microscope examination. Tissue culture produced yeast-like colonies, which was positive by urease test. The strain was identified as Cryptococcus neoformans by API test, further identification as Cryptococcus neoformans var. grubii , by sequence analysis of ribosomal internal transcribed spacer region. The same result was acquired by microdissection, subsequence PCR and sequence analysis. Cryptoeoeeal specific antigen test showed：CSF （ ＋ ＋ ） , serum （ ＋ ＋ ＋ ＋ ）. Brain CT showed multi high-density regions. She was dignosed as disseminated cryptococcosis caused by Cryptococcus neoformans vat. grubii. Conclusions Study on this case will assist diagnosis of disseminated cryptococcosis and associated lab research. It was confirmed that microdissection and sequence analysis of ITS region will be a promising approach to diagnosis of skin fungal infections.