Ozer Sinem, Yücesoy Mine
Dokuz Eylül Universitesi Tip Fakültesi, Mikrobiyoloji ve Klinik Mikrobiyoloji Anabilim Dali, Izmir.
Mikrobiyol Bul. 2007 Jul;41(3):419-28.
Although blood culture method is accepted as gold standard in the laboratory diagnosis of invasive candidiasis seen in immunocompromised patients, cultivation of blood is considered as not a reliable and rapid method for the diagnosis of candidemia, since it may be negative in approximately half of the patients, slow growth rate of Candida in routine culture media and requirement of large amounts of blood for the isolation. The aim of this study was to detect Candida DNA in simulated blood samples by using polymerase chain reaction (PCR). Simulated samples were prepared by using blood samples of healthy volunteers. These samples were inoculated into tubes with EDTA and BACTEC 9240 blood culture bottles in which no growth was detected and with standard strains of C. albicans, C. tropicalis, C. parapsilosis, C. krusei, Escherichia coli and Staphylococcus aureus together with the clinical isolates of Aspergillus fumigatus, C. kefyr, C. glabrata, C. lusitaniae, C. guilliermondii and Rhodotorula sp. Additionally, blood culture samples of 23 cases whose blood culture bottles signaled as positive and revealed growth of Candida in agar plates were examined. DNA extraction of all samples were performed according to the standard procedure proposed by the MN Nucleospin Tissue Kit (Macherey-Nagel, Germany) for tissue samples; following the pre-treatment with erythrocyte, leukocyte and fungus cell wall lysis buffers. DNAs were amplified with PCR, using primers specific for the 5S rDNA region (PCon 1 and PCon 2 primers) and PCR products were obtained by electrophoresis in 2% agarose gel. Presence of a 105 base pair (bp) product was considered as positive. The lowest detection limit of PCR has been determined as 10(2)-10(3) cfu/ml Candida for our simulated samples. The presence of a 105 bp band has been observed in samples prepared with all Candida strains included in the study. Blood samples spiked with E. coli, S. aureus, A. fumigatus and Rhodotorula sp. and negative blood samples has been found negative in terms of Candida DNA. The same 105 bp product has been observed for blood culture samples with Candida growth. The PCR method applied in this study takes approximately seven hours and the cost was calculated approximately 6.00 U.S. dollars per patient. As a result, it has been determined that Candida DNA can be detected in a shorter time by PCR using specific primers for 5S rDNA gene from simulated samples prepared with either blood or BACTEC blood culture bottles.
尽管血培养方法被公认为免疫功能低下患者侵袭性念珠菌病实验室诊断的金标准,但血液培养被认为不是诊断念珠菌血症的可靠快速方法,因为大约一半的患者血培养可能呈阴性,念珠菌在常规培养基中生长缓慢,且分离需要大量血液。本研究的目的是通过聚合酶链反应(PCR)检测模拟血样中的念珠菌DNA。模拟样本采用健康志愿者的血样制备。将这些样本接种到含有乙二胺四乙酸(EDTA)的试管和未检测到生长的BACTEC 9240血培养瓶中,并与白色念珠菌、热带念珠菌、近平滑念珠菌、克柔念珠菌、大肠杆菌、金黄色葡萄球菌的标准菌株以及烟曲霉、解脂念珠菌、光滑念珠菌、葡萄牙念珠菌、季也蒙念珠菌和红酵母的临床分离株一起接种。此外,对23例血培养瓶显示阳性且在琼脂平板上发现念珠菌生长的病例的血培养样本进行了检查。所有样本的DNA提取均按照德国Macherey-Nagel公司MN Nucleospin Tissue Kit试剂盒针对组织样本提出的标准程序进行;先用红细胞、白细胞和真菌细胞壁裂解缓冲液进行预处理。使用针对5S rDNA区域的特异性引物(PCon 1和PCon 2引物)通过PCR扩增DNA,并通过在2%琼脂糖凝胶中电泳获得PCR产物。出现105碱基对(bp)的产物被视为阳性。对于我们制备的模拟样本,PCR的最低检测限已确定为每毫升10²-10³cfu念珠菌。在本研究纳入的所有念珠菌菌株制备的样本中均观察到105 bp条带的存在。添加了大肠杆菌、金黄色葡萄球菌、烟曲霉和红酵母的血样以及阴性血样在念珠菌DNA方面均为阴性。对于有念珠菌生长的血培养样本也观察到了相同的105 bp产物。本研究中应用的PCR方法大约需要7小时,每位患者的成本计算约为6.00美元。结果表明,使用针对5S rDNA基因的特异性引物,通过PCR可以在更短的时间内从用血或BACTEC血培养瓶制备的模拟样本中检测到念珠菌DNA。