PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia
PCR-RFLP detection and species identification of fungal pathogens in patients with febrile neutropenia
复制标题
DOI：
10.1111/j.1469-0691.2003.00719.x
复制
发表时间：
2003-12-01
影响因子：
14.2
通讯作者：
Benedík, J
中科院分区：
文献类型：
--
作者：
Dendis, M;Horváth, R;Benedík, J
目的探讨聚合酶链反应（PCR）对免疫功能低下患者真菌感染的诊断价值。方法设计了一种快速、灵敏的真菌病原菌pcr检测方法，并对该方法在癌症合并发热性中性粒细胞减少症（FN）患儿中的适用性进行了研究。结果分析了40种真菌病原菌的ITS2序列及其邻近区域，设计了检测真菌的引物。扩增产物长度多态性（APLP）和限制性片段长度多态性（RFLP）产生属或种特异性模式。该方法的灵敏度约为每1ml血液中3个白色念珠菌细胞。样品采集后8小时内可得到结果。对24例癌症合并发热性中性粒细胞减少症（FN）患儿的53份血液样本和1份肺活检样本进行了试验。PCR检测了10例患者25份临床标本的真菌DNA。只有五个样本的血培养呈阳性，而另外两个血培养阴性的患者的咽拭子培养呈阳性。其余14例患者均为培养阴性和pcr阴性。培养分离菌株与PCR-APLP-RFLP鉴定结果完全吻合。通过扩增产物的直接测序证实了真菌的种类。结论PCR-APLP-RFLP检测可用于诊断免疫功能低下患者的真菌感染。
Objective To assess the usefulness of polymerase chain reaction (PCR) assays in the diagnosis of fungal infections in immunocompromised patients.Methods A rapid and sensitive PCR-based assay for the detection and identification of fungal pathogens was designed and applicability of this method was investigated in a group of children with cancer and febrile neutropenia (FN).Results The ITS2 sequences and adjacent regions of 40 fungal pathogens were analyzed and primers for detection of all analyzed fungal species were designed. Amplification product length polymorphism (APLP) and restriction fragment length polymorphism (RFLP) generated genus- or species-specific patterns. The sensitivity of the method was approximately three cells of Candida albicans per 1 mL of blood. The results were available within 8 h after sample collection. The method was tested on 53 blood samples and one lung biopsy sample from 24 children with cancer and febrile neutropenia (FN). The PCR assay detected fungal DNA in 25 clinical samples from ten patients. Blood cultures were positive in only five samples, while another two blood-culture negative patients had positive cultures from throat swabs. The remaining 14 patients were both culture- and PCR-negative. Culture-isolated strains matched completely those obtained by PCR-APLP-RFLP identification. The identity of fungal species was confirmed by direct sequencing of amplified products.Conclusion Our results suggest that PCR-APLP-RFLP assays can be useful in the diagnosis of fungal infections in immunocompromised patients.