Transplantation-Oncology Infectious Diseases Program, Weill Medical College of Cornell University, 1300 York Ave., Rm. A-421, New York, NY 10065, USA.
J Clin Microbiol. 2011 Dec;49(12):4150-7. doi: 10.1128/JCM.00570-11. Epub 2011 Oct 5.
Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.
诊断侵袭性肺曲霉病（IPA）仍然是临床微生物学实验室面临的主要挑战。我们利用 TaqMan 技术开发了用于曲霉属和种特异性鉴定的快速灵敏定量 PCR（qPCR）检测方法。为了验证这些检测方法并了解其潜在的诊断效用，我们随后对具有四种重要医学意义的曲霉属物种的 IPA 特征明确的模型进行了支气管肺泡灌洗（BAL）液标本的盲法研究。通过利用独特的 ITS1 区域，为四种最常见的具有重要医学意义的曲霉属物种（烟曲霉、黄曲霉、黑曲霉和土曲霉）开发了一套实时 qPCR 引物和探针。未经处理（P < 0.0007）和处理（P ≤ 0.008）动物的 IPA 中，BAL 液中的泛曲霉属和种特异性 qPCR 比培养法更敏感，可检测到烟曲霉所致 IPA。对于土曲霉和黑曲霉引起的感染，未经处理和处理动物的 BAL 液培养和 PCR 扩增产生了相似的敏感性。在治疗动物中，泛曲霉属 PCR 比培养法更敏感，可检测到黄曲霉（P = 0.002）。BAL 液泛曲霉属和种特异性 PCR 的敏感性与 BAL 液半乳甘露聚糖（GM）检测相当。qPCR 检测方法的拷贝数与定量培养相关，可确定肺部组织中残留的真菌负荷。泛曲霉属和种特异性 qPCR 检测方法可能有助于提高免疫功能低下患者 IPA 的快速准确诊断。
