巢式PCR检测石蜡包埋组织中着色霉、孢子丝菌和马尔尼菲青霉

Detection of Fonsecaea, Sporothrix and Penicillium marneffei in paraffin wax embedded tissues bynested PCR

目的评价巢式PCR对不同深部真菌感染组织石蜡包埋标本检测的价值。方法收集着色芽生菌病、孢子丝菌病、马尔尼菲青霉病及其他深部真菌病石蜡组织标本共44份，行组织病理观察并提取石蜡组织标本中DNA。使用针对着色霉、孢子丝菌及马尔尼菲青霉核糖体DNA特定区域的特异性巢式PCR引物，分别对所提取的真菌DNA进行扩增。分析巢式PCR对这3种病原真菌扩增的敏感性和特异性，并与组织病理检查方法比较。结果20例着色芽生菌病组织蜡块中8例扩增阳性，10例孢子丝菌病组织蜡块中7例扩增阳性，10例马尔尼菲青霉病组织蜡块均扩增阳性，其余对照深部真菌病组织蜡块扩增均为阴性，巢式PCR检测3种真菌的敏感性分别为40％、70％和100％，特异性均达到100％。组织病理检查3种真菌的阳性率分别为95％、70％、80％。结论巢式PCR扩增石蜡包埋组织中的真菌DNA是诊断深部真菌病的一种方法，尤其适用于诊断马尔尼菲青霉病。

Objective To evaluate the performance of nested PCR in the detection of different fungi in paraffin wax embedded tissues. Methods Forty-four tissue samples were resected from rats infected with Fonsecaea monopbora, patients with chromoblastomycosis, sporotrichosis or penicilliposis marneffei followed by preparation of paraffin wax embedded tissue sections for pathological examination and DNA extraction. Nested PCR was performed by using specific primers targeting the ribosomal DNA of Fonsecaea, Sporothnx and PeniciHium marnelTei, respectively. The sensitivity and specificity of nested PCR were analyzed and compared with those of pathological examination. Results The nested PCR showed positive results in 8 of 20 samples from rats with chromoblastomycosis, 7 of 10 samples from patients with sporotrichosis and all of the 10 sam- ples from patients with penicilliposis marneffei, but not in the control samples. In the detection of Fonsecaea, Sporo^rix schenki and Penicillium matneffei, the sensitivity was 40% ,70% and 100%, respectively, and the specificity was consistently 100%, for the nested PCR. Pathological examination revealed fungal elements in 95%, 70% and 80% of the corresponding samples, respectively. Conclusion Detection of fungal DNA in paraffin wax embedded tissue by nested PCR can be applied to the diagnosis of deep mycosis, especially to the diagnosis of penicilliposis marneffei.