Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis:: Demonstration of efficacy of caspofungin acetate
Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis:: Demonstration of efficacy of caspofungin acetate
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DOI：
10.1128/aac.45.12.3474-3481.2001
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发表时间：
2001-12-01
影响因子：
4.9
通讯作者：
Douglas, CM
中科院分区：
文献类型：
--
作者：
Bowman, JC;Abruzzo, GK;Douglas, CM
卡泊芬净醋酸酯（MK-0991）是一种抗真菌抗生素，可抑制1，3-β-D-葡聚糖的合成，1，3-β-D-葡聚糖是几种致病真菌细胞壁的重要成分。醋酸卡泊芬净最近被批准用于治疗对其他疗法难治或不耐受的侵袭性曲霉病患者。在肺部或播散性疾病的动物模型中，通过使用生存期延长或组织CFU减少作为测定终点，评价了1，3-β-D-葡聚糖合成抑制剂对烟曲霉的活性。由于这些方法的灵敏度有限或与真菌生长的相关性较差，因此我们开发了一种基于定量PCR（qPCR）（TaqMan）的检测方法来监测疾病进展并测量药物疗效。A.将烟曲霉作为未萌发的分生孢子或小胚加入到未感染的肾脏中，产生超过至少4个数量级的线性qPCR响应。在播散性曲霉病小鼠模型中，A.在感染后4天，通过qPCR检测在5个不同器官中的每一个中检测到烟曲霉，并且这些器官中的平均真菌负荷比通过CFU测量确定的平均值高1.2至3.5 log（10）单位。当用于监测感染小鼠的疾病进展时，qPCR测定法检测到感染后第1天和第4天之间肾脏的分生孢子当量增加了近4 log（10）/g，真菌负荷峰值与显著死亡率的发生一致。传统的CFU方法仅检测到相同组织中真菌负荷的边际增加。相比之下，当小鼠感染白色念珠菌（其在组织中不形成真正的菌丝体）时，随着感染的进展，通过qPCR和CFU测定法对肾脏负荷的定量是强烈相关的。最后，用卡泊芬净或阿替霉素B治疗诱发播散性曲霉病的小鼠可降低A。感染肾脏中的烟曲霉菌负荷达到qPCR测定的检测限。由于其更大的动态范围，qPCR测定法在监测播散性曲霉病的进展和评价抗真菌抗生素对曲霉菌的活性方面优于传统的CFU测定法上级。烟熏。
Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-beta -D-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-beta -D-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naive, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log(10) units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log(10) conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.