Molecular Diagnosis of pathogenic fungi

聚合酶链式反应彻底改变了传染病的诊断。通过微生物的DNA或RNA检测和识别微生物可能更敏感、更特异和更准确，与大多数生物的表型测试相比，它几乎可以立即检测到。挑剔的生物体，可能比我们意识到的要多得多，基本上是不能培养的，或者需要丰富的培养基和过多的孵化时间。尽管有这种潜力，但临床实验室中基于聚合酶链式反应的真菌检测落后于病毒和细菌。缺乏广泛使用的分子工具来识别真菌是一种耻辱，因为系统性真菌感染的高死亡率往往被诊断得太晚。真菌感染的检测对基于PCR的方法来说是一个挑战，因为采样点的细胞数量较少，而且细胞壁坚硬，使有效的细胞裂解变得复杂。文献回顾强调，在PCR用于烟曲霉和其他真菌的可靠诊断之前，还需要更多的优化。我们的长期目标是开发快速、定量和灵敏的分析方法，直接从临床样本中检测和鉴定真菌种类，并最终确定它们的抗真菌敏感性。为了实现这一目标，我们的具体目标是：1.优化样本处理，以提供来自任何临床来源的单细胞检测。2.修改目前真菌细胞裂解的方法，以实现在存在大量过剩的人类细胞的情况下从单个真菌细胞中回收DNA，以有利于高通量处理的方式，以最少的技术人员技能。3.探索不同的物种特异性引物或杂交探针的制备方法，并与其他实验室研制的引物进行比较。4.将目前对白色念珠菌和烟曲霉的实时荧光定量聚合酶链式反应技术推广到其他真菌物种。5.对确诊或可能存在真菌感染的患者的临床标本进行验证。我们预计，这里为真菌制定的方案和策略将扩展到其他不可培养的生物体。

PCR has revolutionized the diagnosis of infectious diseases. Detecting and identifying microorganisms by their DNA or RNA is potentially more sensitive, more specific and accurate, and allows nearly immediate detection compared to phenotypic tests for most organisms. Fastidious organisms, perhaps many more than we are aware of, are essentially unculturable, or require enriched culture medium and excessive incubation times. Despite this potential, PCR-based testing of fungi in the clinical lab is lagging behind virus and bacteria. Lack of widely used molecular tools for identifying fungi is a shame, given the high mortality rates associated with systemic fungal infections that are often diagnosed too late. Detection of fungal infections is a challenge for PCR-based methods, due to the low numbers of cells at sampling sites, and the hard cell wall that complicates efficient cell lysis. Reviews of the literature underscore that more optimization is needed before PCR will be reliable for diagnosis of Aspergillus fumigatus and other fungi. Our long term objective is to develop rapid, quantitative and sensitive assays for detection and identification of fungal species, and ultimately their antifungal susceptibilities, directly from clinical samples. Toward this goal, our specific aims are: 1. To optimize sample processing to provide single cell detection from any clinical source. 2. To modify current methods of fungal cell lysis to achieve DNA recovery from single fungal cells in the presence of a vast excess of human cells, in a manner conducive to high-throughput processing, with minimal technician skill. 3. To explore different methods of generating species-specific primers or hybridization probes, and to compare our primers with those developed in other laboratories. 4. To extend current real time PCR capabilities for Candida albicans and Aspergillus fumigatus to other fungal species. 5. To validate the new assays on clinical samples from patients with definite or probable fungal infection. We expect that the protocols and strategies developed here for fungi will extend to other, non-culturable organisms.