Lateral flow assay for detecting colonization by Streptococcus agalactiae

描述(申请人提供)：无乳链球菌，又名。B群链球菌(GBS)是导致新生儿发病和死亡的主要原因之一。我们建议使用我们专有的解旋酶依赖扩增(HDA)和嵌入在封闭反应容器的设备中的横向流动条来开发GBS的分子分析，以防止扩增物对实验室的污染。HDA与聚合酶链式反应(PCR)的相似之处在于它使用两个引物来指数地扩增核酸。与聚合酶链式反应一样，HDA检测也可以使用竞争性的内部对照；即，将已知浓度的模板DNA添加到原始样品中，该模板DNA可以用与分析物相同的引物进行扩增，但要单独检测。与PCR不同，HDA是完全等温的，因此不需要昂贵的热循环器。事实上，我们经常使用水浴进行化验。第一阶段的目标是：1)用一组来自邻近菌株以及采样点常见菌株的DNA样本验证该检测的敏感性和特异性。2)使用迈阿密大学医学中心(UMMC)Gary Procope博士的临床标本，评估体内提出的过滤样品制备方法的性能。3)制定临床计划&供食品和药物管理局(FDA)审查的前IDE，用于多地点临床研究，以寻求监管部门批准销售用于人类诊断的分析方法。这一阶段的里程碑是向FDA交付预IDE。FDA的意见将被用于设计第二阶段的临床研究。我们相信我们建议的检测系统可以获得FDA的中等复杂程度的指定。事实上，要获得这样的称号，系统制造商在回答7个问题时必须获得12分以下的分数，这些问题包括：用户知识、用户培训、试剂制备、操作步骤、测试材料、故障排除和结果解释。这7个问题的答案可以得到1或3分。我们估计我们建议的系统可能会得到11分。考虑到我们的检测系统将不需要昂贵的仪器和塑料一次性用品，我们相信我们可以提供一个有用的Xpert GBS(Tm)的替代品，如果我们的测试能够获得FDA的批准的话。NIAID的支持对于实现这一目标至关重要。公共卫生相关性：新生儿的B组链球菌(GBS)感染通常发生在产道，可导致败血症、肺炎和脑膜炎。新生儿感染GBS估计要花费大约3亿美元的额外医疗费用。目前的“护理标准”是对怀孕35至37周的所有妇女进行筛查，并根除殖民妇女中的GBS。尽管采取了这种积极的方法，侵袭性早发性疾病在美国的发病率仅下降了34%。新生儿早发性侵袭性GBS的危险因素有：分娩前18小时以上胎膜破裂、分娩时母亲发热、早产和既往分娩时有GBS病史。目前的做法是向具有上述风险特征的母亲开预防性抗生素，即使在没有证据表明GBS殖民的情况下也是如此。胎膜早破(PROM)发生在大约三分之一的早产儿中，它增加了围产期感染的可能性，因为它与胎膜破裂和分娩之间的短暂潜伏期有关。早产只占新生儿的7%到11%，但占早发性GBS疾病的32%到38%，因为母亲在分娩前没有进行测试。美国疾病控制与预防中心估计，美国有50万名早产婴儿，其中14.3万名母亲没有接受产前护理。当在分娩时进行培养时，很少能提供足够快的周转时间来影响是否开始抗菌治疗的决定。显然，在分娩前对女性进行快速检测是必要的。不幸的是，当前的快速测试选项不敏感、太慢或太昂贵。一些以培养为基础的检测可以相对快速地检测到大量殖民的妇女中的GBS定植。例如，FDA批准的StrepB胡萝卜肉汤“(Hardy Diagnostics，Santa Maria，CA)可以在接种培养物后短短6小时内检测到重度GBS定植。FDA批准的基于探针的核酸测试也可用于检测培养物中生长的GBS。不幸的是，在许多情况下，6个小时太长了。造父变种的GeneXpert系统已经获得了FDA的许可，可以在实验室中使用，获得许可进行“中等复杂程度”的测试。这种测试可以在不到1小时的时间内得出结果，但它的成本比速度较慢的替代方法要高得多。一些医生质疑这项测试的经济合理性。我们建议提供一种成本较低的分子检测方法，中间检测时间为~1-2小时，也应符合FDA将其归类为中等复杂性检测的条件。

DESCRIPTION (provided by applicant): Streptococcus agalactiae, a.k.a. group B streptococcus (GBS), is one of the leading causes of morbidity, and mortality among newborns. We propose to develop a molecular assay for GBS using our proprietary helicase dependent amplification (HDA), and a lateral flow strip embedded in a device that encloses the reaction vessel to prevent contamination of the laboratory by amplicons. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. Like PCR, HDA assays can use a competitive internal control; i.e., a template DNA of known concentration spiked into the raw sample that can be amplified by the same primers as the analyte, but detected separately. Unlike PCR, HDA is entirely isothermal, and thus does not require costly thermocyclers. In fact we routinely use water baths to perform assays. The objectives for Phase I are to: 1) Validate the sensitivity and specificity of the assay with a panel of DNA samples from near neighbor strains, as well as strains commonly found in the sampling sites. 2) Evaluate the performance of the filtration sample preparation method proposed in the body with clinical specimens from Dr. Gary Procop, University of Miami Medical Center (UMMC). 3) Develop a clinical plan & a pre-IDE for review by the Food and Drug Administration (FDA) for a multi-site clinical study to seek regulatory clearance for sale of the assay for human diagnostics. The milestone for this Phase I is to deliver a pre-IDE to FDA. Comments from FDA will be used to design a clinical study during Phase II. We believe our proposed assay system can obtained the moderate degree of complexity designation from the FDA. Indeed, to obtain such a designation, a system manufacturer must obtain a score of 12 of less when answering 7 questions covering: user knowledge, user training, reagent preparation, operational steps, testing materials, troubleshooting, and result interpretation. The answer to these 7 questions can receive a score of 1 or 3. We estimate our proposed system could get a score of 11. Considering our assay system will not require costly instrumentation and plastic disposables, we believe we can offer a useful alternative to the Xpert GBS(tm), if we can obtain FDA clearance for our test. Support from NIAID is essential to reaching this goal. PUBLIC HEALTH RELEVANCE: Group B streptococcus (GBS) infection of newborns usually takes place in the birth canal and can lead to sepsis, pneumonia, and meningitis. GBS infection of neonates is estimated to cost roughly $300 million in additional healthcare costs. The current "standard of care" is to screen all women at 35 to 37 weeks of gestation, and to eradicate GBS in colonized women. Despite this aggressive approach, invasive early-onset disease incidence has only declined by 34% in the US. Risk factors for early-onset invasive GBS in the newborns are: rupture of membranes more than 18 h before delivery, a febrile mother during labor, preterm delivery, and a history of GBS disease in a previous delivery. Current practice it to prescribe prophylactic antibiotics to mothers with the aforementioned risk profile, even in the absence of evidence of colonization by GBS. Premature rupture of membranes (PROM) occurs in approximately one third of preterm births and it increases the potential for perinatal infection because of its association with brief latency between membrane rupture, and delivery. Preterm pregnancies only represent 7% to 11% of births, but account for 32% to 38% of early-onset GBS disease because the mother was not tested prior to labor. CDC estimates that there are 500,000 pre-term births, and 143,000 births from mothers that received no pre-natal care in the United States. When culture is performed at the time of delivery, it seldom provides a sufficiently rapid turnaround time to influence the decision of whether or not to initiate antimicrobial therapy. A rapid test is clearly needed to test women just before delivery. Unfortunately, current rapid test options are insensitive, too slow, or too costly. Some culture-based tests allow for relatively rapid detection of GBS colonization in heavily colonized women. For example, the FDA cleared StrepB Carrot Broth" (Hardy Diagnostics, Santa Maria, CA) can detect heavy GBS colonization in as little as 6 hours after inoculation of the culture. FDA cleared probe based nucleic acid tests to detect GBS grown in cluture are also commercially available. Unfortunately, 6 hours is too long in many situations. Cepheid's GeneXpert System has obtained FDA clearance for use in laboratories cleared for performing test with "a moderate degree of complexity". This test can yield results in less than 1 hour, but it is far more costly than the slower alternatives. Some physicians have questioned the economic justification for the test. We propose to offer a lower cost molecular assay with an intermediate assay time of ~1 to 2 hours that should also be eligible for being categorized as a moderate degree of complexity assay by FDA.