儿童马尔尼菲青霉菌感染的鉴定及该菌酵母相的体外药敏分析

Identification of Penicillium marneffei in Children and Antifungal Susceptibility of Yeast form of Penicillium marneffei

目的确定患儿体内所感染的病原体为马尔尼菲青霉菌(PM),并用E-test法测定该菌酵母相的药物敏感性,指导临床合理用药。方法取患儿血液、骨髓涂片染色镜检和真菌双相培养,观察真菌生长情况及菌落形态,显微镜下观察菌体特征。并采用M27-p方案中的E-test法测定6株PM的酵母相(yeast)对伊曲康哇、酮康哇、5-氟胞嘧啶、氟康唑、两性霉素B的MIC值。结果PM为双相性真菌,于25℃为青霉相,于37℃为酵母相,并均有典型的菌落形态特征。瑞氏染色可见菌体呈圆形、椭圆型或腊肠样,大小不一,直径2～8μm,胞壁染紫色且清楚连续,在腊状的细胞内可见一明显的横隔。该菌37℃酵母相时伊曲康哇、酮康哇、5-氟胞嘧啶、氟康唑、两性霉素B的MIC范围分别为0.002～0.016μg/mL、0.012～0.125μg/mL、0.032～0.380μg/mL、1.500～6.000μg/mL、0.047～2.000μg/mL,对酮康唑、5-氟胞嘧啶出现耐药株各1株,对两性霉素B耐药株2株。结论马尔尼菲青霉菌的特征性菌落形态和骨髓及外周血发现的真菌孢子对该菌有诊断价值,而药敏结果显示该菌对伊曲康唑敏感性最强,其次为5-氟胞嘧啶、氟康唑、酮康唑,两性霉素B敏感性最弱。

Objective To identify the Penicillium marneffei （PM） pathogen isolated from the children and determine the antifungal susceptibilities of the yeast form of PeniciUium marneffei by E-test method and guide to select suitable antifungal agents for clinic treatment. Methods The blood or bone marrow samples were examined directly and incubated at 25℃ and 37℃, respectively for culturing the fungal pathogens. The growth and colonial morphology of PM was inspected and the characteristics of PM smear were observed under microscope. According to the E-test method NCCLS M27-p document, the MICs of yeast form of the 6 PeniciUium marneffei to the 5 antifungel agents （itraconazole, ketoconazole, 5-fluorocytosine, fluconazole, amphotericin B） were determined. Results PM is a thermally dimorphic PeniciUium. Under 25℃ it is mycelial form. Under 37℃ it is yeast form. The two forms have its typical colonial morphological characteristics. The spore is round or ellipsoidal or sausage-like. And the size is about 2-8μm. In the Wright＇s staining, cell wall of PM is violet, distinct and continuous. There are transparent transverses septums in sausage-like spore. The MIC ranges of itraconazole, ketoconazole, 5-fluorocytosine, fluconazole, amphotericin B on yeast form were determined as 0.002-0.016 μg/mL, 0.012-0.125 μg/mL, 0.032-0.380 μg/mL, 1.500-6.000 μg/ mL and 0.047-2.000 μg/mL, respectively. There were 1 stain resistant to ketoconazole and 5-fluoroeytosine and 2 stains resistant to amphotericin B. Conclusion PM＇s typical colonial morphology and fungal spore in bone marrow and peripheral blood are important features for identification. The pathogen is more susceptibile to itraeonazole than to the other 4 antifungal agents.