Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid.
Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid.
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DOI：
10.1186/1471-2334-8-73
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发表时间：
2008-05-29
影响因子：
3.7
通讯作者：
Fredricks, David N.
中科院分区：
文献类型：
--
作者：
Khot, Prasanna D.;Ko, Daisy L.;Hackman, Robert C.;Fredricks, David N.
侵袭性肺曲霉病（IPA）的诊断仍然具有挑战性。支气管肺泡灌洗液（BAL）的培养和组织病理学检查是有用的，但敏感性不佳，在培养的情况下，真菌生长可能需要几天才能明显。采用定量PCR（qPCR）检测BAL液中的曲霉菌DNA，具有早期诊断和提高灵敏度的潜力。重要的是在PCR测定中采用质量控制措施，以解决可能妨碍准确评价诊断性能的假阳性和假阴性。分析了81例患者94次肺炎发作的BAL液。使用真菌病研究组标准，13次发作被归类为已证实或可能的IPA。分别测定BAL的沉淀和上清液部分。用人18 S rRNA基因qPCR确认成功提取。使用基于外源DNA的内部扩增对照（IAC）测量每个qPCR中的抑制。使用靶向真菌18 S rRNA基因的qPCR检测曲霉属中病原体DNA的存在。人18 S rRNA基因qPCR证实所有样品的DNA提取成功。IAC在11个样品中检测到一定程度的初始抑制。培养诊断IPA的敏感性为84.5%，特异性为100%。qPCR受试者操作特性分析表明，以曲霉基因组DNA 13 fg为临界值，其敏感性、特异性、阳性预测值和阴性预测值分别为77%、88%、50%和96%。BAL沉淀和上清液一起分析的灵敏度和特异性与单独BAL沉淀相似。一些患者不符合IPA的标准标准，但通过qPCR检测，BAL液中的曲霉菌DNA水平始终较高。当使用浓缩BAL液沉淀组分进行诊断时，曲霉qPCR检测试剂盒在76.9%的经证实或可能患有IPA的受试者中检出曲霉DNA。分析BAL上清液级分没有益处。提取和扩增对照的使用为解释qPCR结果提供了最佳质量控制，因此可以增加我们对qPCR诊断IPA的真正潜力的理解。
The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance. BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene. Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR. The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.
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影响因子：
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作者：
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