High throughput identification and quantification of fungi using high resolution

描述（由申请人提供）：真菌分子诊断仍然落后于细菌和病毒的对应物，有几个很好的原因。真菌滴度在感染样品中较低，真菌具有难降解的细胞壁，目标基因与竞争和数字优势的人类序列具有更多的同源性。此外，威胁生命的感染的发生率很低，这就要求分子分析必须非常经济。尽管如此，真菌感染往往危及生命，其早期发现、物种鉴定和耐药性突变对成功的干预至关重要。个别真菌物种对一些不断增长的抗真菌药物具有抗性，或者获得抗性突变，使特定药物无效或不那么有效。基于培养的真菌、菌种或敏感性鉴定是不够的，因为培养时间超过了有效治疗的机会之窗，而且许多感染患者的样本培养阴性。我们需要的是一种基于PCR的高通量方法，这种方法不依赖于培养，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，并且能够检测物种和抗性突变。这个目标要求很高，这解释了为什么缺乏可靠的商业工具。缺乏商业上可用的PCR诊断方式一直是临床医生的主要失望，揭示了诊断能力的重大缺陷。我们建议测试和优化使用一种新的工具，高分辨率熔体分析，结合更有效的泛真菌引物，以满足这些需求。早期迹象表明，HRM分析可以直接从PCR反应中区分所有具有重要临床意义的主要真菌种类，而无需任何额外的动手工作，成本或延迟。多项研究证实，诊断的延迟，特别是准确诊断的延迟，是发病率和死亡率增加的直接原因。迫切需要新的方法。公共卫生相关性：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、量化和鉴定真菌种类，这是一项尚未完成的临床任务。真菌感染在高危患者群体中仍然很常见，其死亡率高得惊人。早期、症状前的检测和识别对于降低这些比率至关重要。鉴于传统的霉菌感染检测方法存在特异性低和生存能力差的问题，未来将有更多的霉菌种类被PCR鉴定为具有传染性，而且这些被错误鉴定的物种可能对抗真菌药物反应不佳，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。这些感染的后勤工作（低发病率但高死亡率；对最高风险患者的不精确识别）需要既精确又非常经济的检测。目前的所有检测都需要多次检测或昂贵的后续检测来确定菌种和耐药性。我们建议测试高分辨率熔融曲线分析（HRM），结合使用泛真菌引物的定量PCR，是否能解决这一长期存在的临床需求。

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.