CN109477820A - 用于诊断尿路感染的装置和液体组合物 - Google Patents
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Abstract
一种用于尿路感染的比色诊断的装置,其特征在于,其包括至少一个小瓶,其中容纳有包含水部分和有机部分的液体分析混合物,所述有机部分与水部分不混溶且密度低于水部分。所述水部分由试剂混合物组成,所述试剂混合物含有:(a)营养物质,其包含选自肉浸液或蛋白胨、植物蛋白胨、酪蛋白水解物、胰蛋白
Description
技术领域
本发明涉及一种用于诊断尿路感染的装置和相关的试剂组合物。
背景技术
尿路感染是目前最常见的细菌感染形式之一,并且是国家医疗保健支出的重要支出项目。
通常通过与在合适的医疗设施中进行的抗菌谱相关的尿液培养试验来诊断尿路感染。目前的测试方法涉及约48小时的响应时间并且需要专业的人员和设施。
或者,尿路感染的快速测试可以通过尿液测试条(也称为浸渍检查法(dipsticktest))进行,其在浸入尿液样品后改变颜色,从而提供相对的结果。具体地,尿液测试条包含一系列试剂,其能够与尿液中存在的化合物反应,产生与可能存在的尿路感染相关的特征颜色。尽管该技术具有非常简单且能够非常快速地(大约在120秒内)提供结果的优点,但它不能单独解决问题并且需要与其他临床测试相结合。此外,与尿液培养技术的比较表明,浸渍检查法经常导致假阴性和假阳性结果。
基于血清生物标记物的快速诊断感染的其他方法,例如C-反应蛋白、降钙素原等,尽管能够在几分钟内提供结果,但如果用于诊断尿路感染,则不能确保所要求的可靠性。
最后,应指出的是,一些国际结构(例如世界卫生组织和亚特兰大疾病控制和预防中心(美国,乔治亚州))确定,为了可靠地诊断尿路感染,测试必须检测到每毫升尿液中存在的细菌数量大于100,000个。
因此,需要一种可靠的技术,其能够比尿液培养试验更快地提供尿路感染的诊断结果,同时,还能够突出显示有效对抗所检测到的细菌的抗生素,重要的是,其无需专门的设施进行测试。
本发明的发明人设计了一种技术,其除了满足上述要求外,还具有非常经济有效的优点。
发明内容
本发明的主题是一种用于诊断尿路感染的装置,其主要特征在权利要求1中阐述,并且其优选和/或附加特征在权利要求2和3中阐述。
本发明的另一主题是一种用于尿路感染的比色诊断的液体分析混合物,其主要特征在权利要求4中阐述,并且其优选和/或附加特征在权利要求5和6中阐述。
具体实施方式
以下为一些说明性和非限制性的实例。
本发明的装置由至少一个小瓶构成,该小瓶包含由水部分和有机部分组成的液体分析混合物,所述有机部分与水部分不混溶且密度低于水部分。所述水部分包含试剂混合物,所述试剂混合物转而包含用于可能的细菌培养的营养物质、电位在-100至+500mV范围内的氧化还原指示剂和能够将pH维持在6.0至8.0范围内的缓冲液体系。
特别地,所述试剂混合物包含94.0至99.9重量%的营养物质和0.1至6.0重量%的氧化还原指示剂。
以上表明本发明涉及包含以特定重量比存在的物质的试剂混合物,然而,在不脱离本发明范围的情况下,其也可包含其他物质。
所述营养物质包括选自肉浸液或蛋白胨、植物蛋白胨、酪蛋白水解物、胰蛋白胰蛋白胨和酵母提取物的氨基酸源,优选与碳水化合物源组合,所述碳水化合物源选自可被微生物代谢的低聚碳水化合物或单体碳水化合物或其代谢物,例如葡萄糖和乳糖或其中间代谢物。
所述氧化还原指示剂具有测量细菌代谢的作用。在存在细菌的情况下,在与细菌浓度成比例的时间内,氧化还原指示剂将从氧化态变为还原态,因此根据所存在的活细菌的数量而改变含有所述指示剂的溶液的颜色。因此,具有高的活细菌含量的尿液将导致指示剂的氧化还原态快速改变,且因此含有试剂的水溶液的颜色将快速改变。另一方面,具有低的活细菌含量或完全没有细菌的尿液不会导致指示剂的氧化还原态改变,或者——如果导致改变——其仅在很长时间内改变,因此在观察期间将不会看到含有试剂的水溶液的颜色变化。
氧化还原指示剂可以是亚甲蓝或另一种电位在-100至+500mv范围内的氧化还原指示剂,例如:二氯苯酚-靛酚、邻甲酚-靛酚、硫堇(“劳氏紫”)、亚甲蓝、靛蓝四磺酸、靛蓝三磺酸、靛蓝胭脂红(靛蓝二磺酸)、靛蓝单磺酸、酚藏花红(phenosafranin)、番红精T(safranin T)、中性红5、四甲基苯二胺,或其组合。
本发明的实施需要缓冲液体系。实际上,为了突出显示细菌代谢,本发明中使用的大多数氧化还原指示剂具有取决于pH的氧化还原电位。反应介质的pH变化会改变指示剂的氧化还原态的变化,因此会使颜色变化失真,从而妨碍准确检测尿液中存在的细菌数量。
有机相的存在对于实施本发明也是必要的。实际上,有机相在含有待测尿液并且含有任何细菌和上述试剂的水溶液上方自发分层,因而防止了大气氧在所述水溶液中扩散。实际上,任何到达水相的大气氧都会与氧化还原指示剂相互作用,再氧化可能已被细菌代谢还原的任何物质,因此,将不再存在与待测尿液样品中存在的活细菌数量成比例的颜色变化。
有机部分的实例是凡士林油。
本发明的小瓶可以包括盖子,该盖子具有由易碎壁界定并容纳消毒物质的槽。这样,一旦测试结束,可以通过破坏限定槽的壁来对小瓶进行消毒。上述具有槽的盖子是已知的,因此,将不再详细描述。
表I示出了在10ml蒸馏水中的十六种试剂混合物(1-16)的组成。
表I
通过检查pH为7.0来制备试剂混合物,从而制备本发明的液体分析混合物的水部分。
通过加入1.5ml凡士林油作为有机部分,使用表I的试剂混合物之一而制备各自的液体分析混合物。
采用尿液样品测试上述每个小瓶,所述尿液已被两种不同浓度(102和107CFU/ml)的致病菌人工地污染了,所述致病菌为例如:大肠杆菌(Escherichia coli)(ATCC 25992)、粪肠球菌(Enterococcus faecalis)(ATCC 29212)、绿脓杆菌(Pseudomonas aeruginosa)(ATCC 27853)、金黄色葡萄球菌(Staphylococcus aureus)(ATCC 12600)。所述致病菌是最常见的引起尿路感染的细菌。具体地,微生物的添加浓度等于尿路感染中通常检测到的浓度(>106CFU/ml)或小于尿路感染中通常检测到的浓度(<103CFU/ml)。
对于每个小瓶,测试包括添加0.5ml如上所述的人工污染的尿液。将由此制备的小瓶在37℃(+0.5℃)的温度下温育,并以定时的时间间隔进行观察,记录颜色变化所需的时间。
采用本发明的装置的比色诊断试验可以使用0.5μl至10ml的尿液样品进行。
表II和表III示出了所进行的每个测试的以小时计的值。
表II
表III
表II和III中所示的值证明了,本发明的技术确保了对尿液样品中存在的细菌负荷的快速且有效的响应。
观察颜色变化所需的时间的变化性可部分归因于相关试剂混合物的组成差异,部分归因于所用的不同细菌的培养和代谢差异。
应强调的是,为了实施本发明,归因于由细菌代谢引起的氧化还原指示剂的氧化还原态的变化,一定能够仅在液体(即在具有透明壁的小瓶内所含的水溶液)中观察到颜色变化,因此本发明不能用在微生物学所用的普通固体琼脂培养基上。
比色响应可以通过使用能够读取小瓶的颜色变化并将其转换为信号和/或值的读取器设备——其为已知的,因此未详细描述——来支持。
本发明的装置的一些小瓶还含有抗生素,用于测试其对待测尿液样品中存在的可能的细菌负荷的有效性。
如果不含抗生素的小瓶检测到存在细菌负荷,则包含不同抗生素的其他小瓶突出显示其中哪一个有效。实际上,在确定了存在细菌负荷之后,不显示任何颜色变化的含有抗生素的小瓶必然是含有有效抗生素的小瓶。
Claims (6)
1.一种用于尿路感染的比色诊断的装置,其特征在于,其包括至少一个小瓶,其中容纳有包含水部分和有机部分的液体分析混合物,所述有机部分与水部分不混溶且密度低于水部分,所述水部分包含试剂混合物,所述试剂混合物含有:(a)营养物质,其包含选自肉浸液或蛋白胨、植物蛋白胨、酪蛋白水解物、胰蛋白、胰蛋白胨和酵母提取物的氨基酸源;(b)至少一种电位在-100至+500mV范围内的氧化还原指示剂;和(c)能够将pH维持在6.0至8.0范围内的缓冲液体系;所述试剂混合物包含94.0至99.9重量%的营养物质和0.1至6.0重量%的氧化还原指示剂。
2.根据权利要求1所述的装置,其特征在于,所述营养物质包含选自低聚碳水化合物或单体碳水化合物或其代谢物的碳水化合物源,其可被微生物代谢。
3.根据权利要求1或2所述的装置,其特征在于,所述试剂混合物包含抗生素。
4.一种用于尿路感染的比色诊断的液体分析混合物,所述液体分析混合物的特征在于,其包含水部分和有机部分,所述有机部分与水部分不混溶且密度低于水部分,所述水部分包含试剂混合物,所述试剂混合物含有:(a)营养物质,其包含选自肉浸液或蛋白胨、植物蛋白胨、酪蛋白水解物、胰蛋白、胰蛋白胨和酵母提取物的氨基酸源;(b)至少一种电位在-100至+500mV范围内的氧化还原指示剂;和(c)能够将pH维持在6.0至8.0范围内的缓冲液体系;所述试剂混合物包含94.0至99.9重量%的营养物质和0.1至6.0重量%的氧化还原指示剂。
5.根据权利要求4所述的液体分析混合物,其特征在于,所述营养物质包含选自低聚碳水化合物或单体碳水化合物或其代谢物的碳水化合物源,其可被微生物代谢。
6.根据权利要求4或5之一所述的液体分析混合物,其特征在于,所述试剂混合物包含抗生素。
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EP0611001A1 (en) * | 1993-02-11 | 1994-08-17 | Gist-Brocades N.V. | Unit for the detection of residues of antibacterial compounds in liquids |
CN1849399A (zh) * | 2003-09-11 | 2006-10-18 | 帝斯曼知识产权资产管理有限公司 | 血液和尿液检验方法 |
WO2008007206A2 (en) * | 2006-07-11 | 2008-01-17 | Universita' Degli Studi Roma Tre | Colorimetric method and relative device for bacterial load detection |
CN103842816A (zh) * | 2011-09-30 | 2014-06-04 | 狮王株式会社 | 测定氧化还原指示剂的色调变化的方法 |
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WO2013171158A1 (en) * | 2012-05-14 | 2013-11-21 | Sanofi | Methods for the activation of silent genes in a microorganism |
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EP0611001A1 (en) * | 1993-02-11 | 1994-08-17 | Gist-Brocades N.V. | Unit for the detection of residues of antibacterial compounds in liquids |
CN1849399A (zh) * | 2003-09-11 | 2006-10-18 | 帝斯曼知识产权资产管理有限公司 | 血液和尿液检验方法 |
WO2008007206A2 (en) * | 2006-07-11 | 2008-01-17 | Universita' Degli Studi Roma Tre | Colorimetric method and relative device for bacterial load detection |
CN103842816A (zh) * | 2011-09-30 | 2014-06-04 | 狮王株式会社 | 测定氧化还原指示剂的色调变化的方法 |
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