PDF(Pubmed)
Abstract：
Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.
摘要：
肺孢子虫jirovecii,一种导致人类严重肺孢子虫肺炎（PCP）的真菌，长期以来一直被描述为不可文化。迄今为止，仅发表了用P.jirovecii进行的分离的短期实验和涉及动物来源的肺孢子虫物种的少量实验。然而,P.jirovecii培养条件可能与动物源性肺孢子虫的培养条件显着不同，因为它们之间存在主要的基因型和表型差异。建立良好的P.jirovecii培养对于了解PCP及其病理生理过程至关重要。本研究的目的,因此,是为了开发肺孢子虫的无菌培养物。为了确定有希望的种植方法，进行了包括动物来源的肺孢子虫培养物的文献调查。确定的变量，比如孵化时间，pH值,维生素,氨基酸,和其他组件，进行了试验和调整，以找到吉洛维西氏菌培养的最佳条件。这允许我们开发在48天培养后产生42.6倍增加的P.jiroveciiqPCR拷贝数的培养基。最终培养基中活跃生长的肺孢子虫簇的数量和大小在显微镜下证实了生长。DMEM-O3.P.jirovecii倍增时间为8.9天（范围为6.9至13.6天）。总之,我们首次在优化的无细胞条件下在70天的长期培养中成功培养了P.jirovecii。然而,进一步优化这种慢种植者的培养条件是必不可少的。
