High throughput identification and quantification of fungi using high resolution

描述(申请人提供)：真菌分子诊断仍然落后于细菌和病毒同行，有几个很好的原因。在被感染的样本中，真菌的滴度很低，真菌有难耐的细胞壁，目标基因与竞争和数字显性的人类序列有更多的同源性。此外，威胁生命的感染发生率很低，这就要求分子检测必须非常经济。然而，真菌感染往往危及生命，他们的早期检测、物种鉴定和耐药性突变对于成功的干预至关重要。个别真菌物种对越来越多的抗真菌药物中的一些产生抗药性，或者获得了使特定药物无效或不那么有效的抗药性突变。基于培养的真菌、物种或敏感性鉴定是不够的，因为孵化时间超过了有效治疗的机会窗口，而且许多来自感染患者的样本是培养阴性的。所需要的是一种高通量的基于PCR的方法，该方法独立于培养，对每毫升血液或其他组织类型中一个真菌细胞的水平敏感，并能够检测物种和耐药性突变。这一目标要求非常高，这解释了为什么缺乏可靠的商业工具。缺乏商业化的聚合酶链式反应诊断模式一直是临床医生的一大失望，揭示了诊断能力的重大缺陷。我们建议测试和优化一种新工具的使用，高分辨率熔融分析，与更有效的全真菌底物相结合，以满足这些需求。早期迹象表明，HRM分析可以直接将所有具有重大临床意义的主要真菌物种与PCR反应区分开来，而不需要任何额外的实际操作、成本或延迟。多项研究证实，延误诊断，特别是延误准确诊断，直接导致发病率和死亡率的增加。迫切需要新的方法。与公共卫生相关：有一项尚未完成的临床任务，即建立和实施敏感、快速和特定的诊断工具，直接从临床样本中检测、量化和识别真菌种类。真菌感染在高危患者人群中仍然很常见，其归因于死亡率高得令人震惊。早期、症状前的检测和识别是降低这些发生率的关键。考虑到霉菌感染的传统检测方法存在的特异性低和生存能力差的问题，未来将有更多的霉菌物种被聚合酶链式反应鉴定为有感染性的，并且这些被错误识别的物种对抗真菌药物的反应可能很差，这应该不会让人感到惊讶。此外，随着新的抗真菌药物的开发和测试，PCR检测和鉴定将成为选择最合适的药物和跟踪治疗进展的关键工具。这些感染的后勤保障(低发病率但高死亡率；对最高风险患者的识别不准确)需要既精确又非常经济的测试。目前的所有检测都需要多次化验或昂贵的后续测试来确定品种和抗药性。我们建议测试高分辨率熔融曲线分析(HRM分析)，结合使用全真菌引物的定量PCR，是否为这一长期的临床需求提供了解决方案。

DESCRIPTION (provided by applicant): Fungal molecular diagnostics still lags behind bacterial and viral counterparts for several good reasons. Fungal titers are low in infected samples, fungi have refractory cell walls, and target genes have much more homology to competing and numerically dominant human sequences. In addition, the incidence of life-threatening infections is low, imposing a requirement that the molecular assays need to be very economical. Nevertheless, fungal infections are often life-threatening and their early detection, identification of species and resistance mutations, is crucial to successful intervention. Individual fungal species are resistant to some of the growing list of antifungal agents, or have acquired resistance mutations that make specific agents ineffective or less effective. Culture based identification of fungi, species, or susceptibilities are not adequate, since incubation times exceed the window of opportunity for effective therapy, and since many samples from infected patients are cultures negative. What is needed is a high throughput PCR based method that is independent of culture, sensitive to the level of one fungal cell per ml of blood or other tissue types, and capable of detecting species and resistance mutations. This goal is very demanding, explaining the lack of a reliable commercial tool. The absence of a commercially available PCR diagnostic modality has been a major disappointment to clinicians, revealing a major deficiency in diagnostic capability. We propose to test and optimize the use of a new tool, high resolution melt analysis, in combination with more effective panfungal primers, to address these needs. Early indications suggest that HRM analysis can distinguish all major fungal species of major clinical significance directly from the PCR reaction without any additional hands-on efforts, costs, or delays. Multiple studies confirm that DELAY in diagnosis particularly accurate diagnosis is directly responsible for increased morbidity and mortality. New methods are urgently needed. PUBLIC HEALTH RELEVANCE: There is an unfulfilled clinical mandate to establish and implement sensitive, rapid, and specific diagnostic tools for detecting, quantifying, and identifying fungal species directly from clinical samples. Fungal infections are still frequent among high risk patient populations and their attributed mortality rates are alarmingly high. Early, presymptomatic detection and identification is crucial to reduce those rates. It should surprise no one, given problems with low specificity and poor viability in conventional assays for mold infections, that many more species of molds will be identified in the future by PCR as infectious, and that these misidentified species may respond poorly to antifungals. Furthermore, as new antifungals are being developed and tested, PCR detection and identification will be a crucial tool in selecting the most appropriate drug, and in following the progression of therapy. Logistics of these infections (low incidence but high mortality; imprecise identification of highest risk patients) demands tests that are both precise and very economical. All current tests require multiple assays or expensive follow-up tests to identify species and resistance. We propose to test whether high resolution melt curve analysis (HRM analysis), coupled with quantitative PCR using panfungal primers, offers a solution to this long-standing clinical need.