这项工作得到了美国国际开发署，外援救助办公室（AID-OF-A-15-00026）的支持。 Marlene Wolfe得到了国家科学基金会的资助（授予0966093）。

伦理声明:这里描述的研究（Phi6和大肠杆菌为埃博拉病毒的代理人）由塔夫茨医学中心和塔夫茨大学健康科学园区（＃12018）批准。哈佛大学向塔夫茨机构审查委员会提交审查。 注意:在开始此协议之前，必须完成两个步骤。首先，必须确定并选择要对人类受试者安全使用的待研究的病原体的生物安全1级（BSL-1）代用品或非传染性版本。本协议对BSL-1替代或非感染性病原体是必需的，因为该生物体将用于接种人类志愿者的裸手。其次，在招募志愿者或开始实验之前，必须获得地方机构审查委员会对人类科目进行研究的批准。该协议的许多方面可以进行调整，以满足这一点具体需要的研究问题的兴趣。 招募符合条件的人类学科<ol><li>招募志愿者，在公告板上张贴纸张传单，并向可能有兴趣参加的会员发送电子邮件。这些公告应包括学习目的，联系信息和资格标准。 </li><li>与志愿者会面，评估资格。确认志愿者健康，年龄在18至65岁之间，目前尚未怀孕或服用抗生素，并且没有皮肤损伤/疾病，已知的洗手剂过敏症或与卫生有关的精神卫生问题史。 </li><li>有资格的志愿者阅读同意书。回答他们提出的任何问题，并让志愿者和调查员签署两份表格。保留一份表格，并向志愿者提供一份。 </li><li>管理基线调查，包括有关人口信息，人物的问题皮肤病史，以及近期洗手行为的信息。检查手部皮炎，皮肤损伤或基线皮肤异常迹象31 。 </li><li> 安排志愿者为感兴趣的每个生物进行两次测试（一次用于土壤负荷测试，一次用于测试）。指导志愿者在测试前避免抗菌产品进行为期七天的清洗期，以避免个人用品的混淆。 <ol><li>为志愿者提供抗菌产品（洗发水，护发素和肥皂）以代替其常用产品。提供重型乙烯手套，并在使用诸如房屋清洁产品的产品时指导受试者佩戴。 </li></ol></li></ol> 2.准备应急响应中常用的洗手液（肥皂，ABHS，0.05％HTH，NaDCC和NaOCl溶液） 注意:氯溶液可以制备<html>在实验前提前12小时，如果储存&gt; 12小时会降解。 <ol><li>选择和购买与执行测试的上下文相关的肥皂。 注意:在大多数情况下，对于发展中国家的传染病紧急情况，这将是一大堆肥皂。 </li><li>选择和购买与执行测试的上下文相关的ABHS解决方案。 注意:所选择的ABHS应具有大于或等于70％的乙醇环境，以确保疗效。 </li><li> 通过向超纯水中加入颗粒状Ca（ClO） 2粉末，制备0.05％次氯酸钙（Ca（ClO） 2 ）溶液。根据被测试对象的数量确定需要的解决方案。 <ol><li>使用以下等式，使用给定百分比的可用氯来确定在给定体积的水中制备所需量的溶液所需的粉末量: /files/ftp_upload/55604/55604eq1.jpg"/&gt; 注意:Ca（ClO） 2粉末通常具有60-80％的可用氯。 </li></ol></li><li> 通过向超纯水中加入粒状NaDCC粉末，制备0.05％二氯异氰脲酸钠（NaDCC）溶液。 <ol><li>使用以下等式，使用给定百分比的可用氯来确定在给定体积的水中制备所需量的溶液所需的粉末量: <img alt="方程" src="/files/ftp_upload/55604/55604eq2.jpg" /> 注意:NaDCC粉末通常具有约50％的可用氯。 </li></ol></li><li> 通过向超纯水中加入次氯酸钠溶液来制备稳定的0.05％NaOCl溶液。 <ol><li>根据制造商的说明书（ 例如碘量滴定法），使用滴定测试方法确认NaOCl储备溶液的浓度（可能为5-8％）;参见"Su"的材料清单ggested试剂盒）。 </li><li>使用测试方法的结果，使用以下公式计算添加到水中的溶液量: <img alt="方程" src="/files/ftp_upload/55604/55604eq3.jpg" /></li></ol></li><li> 通过将使用电氯离子，超纯水和实验室级氯化钠（NaCl）制备的次氯酸钠溶液加入到超纯水中，制备稳定的0.05％NaOCl溶液。 <ol><li>根据制造商的说明书，使用电氯离子，用超纯水和NaCl准备1％的氯溶液。 </li><li>使用滴定测试方法（ 如碘滴定）来确认NaOCl储备溶液32的浓度。 </li><li>使用测试结果，使用以下公式计算添加到水中的溶液量: <img alt="方程" src="/files/ftp_upload/55604/55604eq3.jpg" /></li></ol></li><li>确认c使用滴定法（ 例如碘滴定法）对每种氯洗涤溶液进行浓缩，并通过加入水或氯源粉末/溶液调节溶液，直到它们在目标浓度（0.045-0.055％）的10％误差内。 </li></ol> 3.准备生物和土壤负荷并结合生产接种注意:在以下小节中， 大肠杆菌和Phi6用作方法描述的样品细菌和病毒生物。 <ol><li> 准备用于细菌浓度大于10×10 8 CFU / mL的试验用的生物体，病毒大于10×10 7 PFU / mL。 <ol><li>为了制备大肠杆菌 ，将非致病性大肠杆菌菌株连接到Luria-Bertani（LB）琼脂平板上，并在37℃下孵育24小时以获得单个菌落。储存于4°C。 注意:这可以完成在实验前几天。 <ol><li>在实验开始前一天，从板上挑一个菌落，用无菌环接种10 mL LB肉汤。在37℃下振荡孵育过夜。 </li><li>在实验的早晨，通过将1mL的过夜培养物加入到20mL新鲜的LB肉汤中来开始新鲜的培养。孵育约2.5小时以达到细胞密度大于10 8 CFU / mL。 </li><li>使用分光光度计来估计培养物的浓度。 注意:使用来自所用大肠杆菌菌株的标准曲线的先前确定的转化因子，确保浓度大于10 8 CFU / mL 33 。如果细胞密度不够高，请将培养基返回培养箱，然后再次测试直至准备好。 </li></ol></li><li>使用膜过滤确认浓度34 。 注意:执行连续稀释of在磷酸盐缓冲盐水（PBS）中的培养物，使得过滤的溶液将在板上产生可数数量的菌落（精确数目将取决于所用培养基）。 <ol><li>用无菌过滤漏斗和真空接头安装火焰和过滤歧管。用乙醇燃烧镊子来灭菌镊子。使用它们在过滤歧管上放置一个0.45μm的过滤器，同时网格朝上。用少量无菌PBS湿过滤器。 </li><li>将漏斗放在基座上，并通过移液或直接倒在过滤器上添加要处理的样品溶液。吸入真空直到整个样品通过膜。用无菌PBS冲洗漏斗的两侧，再次吸入真空。 注意:样品应至少100μL，最多可达100 mL。如果样品少于10 mL，在过滤之前，将大约20 mL的PBS加入过滤漏斗，以确保均质过滤乐。 </li><li>取出漏斗，对镊子进行火焰灭菌，并从基座提起过滤器。将过滤器轻轻放在培养皿中的LB琼脂上，网格朝上，确保过滤器与表面齐平。倒置板并在37℃下孵育24小时。 </li><li> 24小时后，从培养箱中取出板并计数大肠杆菌菌落。使用此数据和已知的稀释因子和溶液体积计算过滤溶液的浓度，单位为CFU / mL。 </li></ol></li><li>使用双重琼脂覆盖法在Pseudomonas syringae宿主中传播Phi6。 <ol><li>将100μLPhi6储备悬浮液和100μL 丁香球菌过夜培养直接加入6mL营养肉汤酵母（NBY）软琼脂（0.3％）中。将其倒入含有NBY硬琼脂（1.5％）的板上，并在26℃下孵育过夜。准备足够的板材以产生足够的接种物以供实验估计每板约4mL病毒悬浮液的产量。 </li><li>第二天，在软琼脂层的顶部加入5mL PBS。在室温下放置4 h，用移液管取回，并用0.45μm过滤器过滤。储存于4°C。 注意:该溶液将作为病毒接种。 </li></ol></li><li>使用斑块测定法确认浓度大于10 7 PFU / mL 35 。在PBS中进行病毒悬浮液的连续稀释，使得100μL在板上产生可数量的斑块。 <ol><li>将100μL适当的稀释样品和100μL过夜宿主培养物直接吸入含有6mL NBY软琼脂的管中。将软琼脂倒入NBY硬琼脂上，并在26℃孵育24小时。 </li><li>第二天，从培养箱中取出板块，并计算每块板块的数量。使用此数据和已知稀释度ctor和溶液体积，以计算PFU / mL中过滤溶液的浓度。 </li></ol></li></ol></li><li> 准备三份土壤负荷，旨在模拟人体血清。 <ol><li>结合7.80mg / mL牛血清白蛋白，10.92mg / mL胰蛋白胨和2.52mg / mL牛粘蛋白以产生所需体积的土壤负荷。混合土壤负荷后，将其过滤通过0.22μm过滤器进行灭菌。储存于4°C直至使用。不要加热灭菌，因为蛋白质会变性。 </li></ol></li><li>准备一个0.9％的NaCl溶液，用于混合接种条件，无土壤负荷。 </li><li>在测试之前，准备由68％细菌或病毒悬浮液和32％土壤负荷组成的接种物。例如，使用1.02mL来自步骤3.1.1.2或3.1.3.2的细菌或病毒悬浮液和0.48mL的土壤负荷（步骤3.2.1）或0.9％NaCl溶液（步骤3.3）。旋转或旋转轻轻混合。 注意:1.5 mL在每个条件下，每个志愿者将使用接种物，因此确保所制备的接种物的总体积足以达到预期的测试次数。 </li></ol> 4.准备志愿者进行实验注意:确定当天要测试的有机体和土壤负荷状况。同样的志愿者可以用于测试多个条件，但每个志愿者只能在48小时内进行一轮测试。 <ol><li>在开始测试之前，确认志愿者通过口头验证他们是否遵守7天抗菌清洗期，并通过目视确认他们的皮肤没有发生任何休息或异常，仍然有资格。 </li><li>使用随机数字生成器，分配每个志愿者使用他们的右手或左手进行抽样测试。分配执行洗手条件的顺序。 注意:例如，ABHS可以被分配为＃3并将被执行第三。 </li><li> 在测试开始时进行一次"清洁洗涤"，以剥离皮肤的污垢和油，以便每个后续测试在相同的条件下进行。 <ol><li>要进行清洁洗涤，通过实验的每个步骤（下面的第5部分），使用空白接种（LB肉汤或PBS），并在不进行洗手的情况下取样。 </li></ol></li></ol> 5.实验程序<ol><li>为了测试每名志愿者的皮肤pH值（以控制变异），将平头皮肤pH探针置于手掌表面皮肤和指针和中指之间的腹板空间。确保电极与皮肤平齐。记录pH读数。 </li><li> 踩着手。 <ol><li>让志愿者双手合身。通过小心地将750μL移液到每个手掌中缓慢滴入1.5mL的接种物。 </li><li>使志愿者轻轻地将手握在一起，直到手的所有表面涂上接种物，同时尽可能少地摩擦手。 </li><li>志愿者将手静止，远离身体30秒，以使接种干燥。接种可能不会完全干燥。 </li></ol></li><li> 洗手 <ol><li>对于所有以下洗涤步骤，在大型样品收集袋中从手中捕获漂洗水。向袋中加入4.5 mL 12％硫代硫酸钠溶液，以在2小时内中和接触和过程中的氯。 注意:硫代硫酸钠应加入所有样品（即使没有氯的样品），以控制其对生物的任何影响。 </li><li>接种后（5.2节），用指定的顺序用下一个方法洗手。 <ol><li>对于控制A，不要执行洗手步骤并直接移动到步骤5.5。 </li><li>对于对照B，通过具有已知流速的漏斗在室温（约21℃）下用纯超纯水清洗手。 注意:这里使用1.5L / m的流量和500mL的水。 </li><li>用肥皂洗手，用10毫升超纯水湿手。让志愿者用肥皂泡上双手，然后再将手揉在另外20秒钟。通过漏斗以1.5 L / m的流速在室温下倒入500 mL超纯水冲洗手。 </li><li>对于所有氯溶液（ 例如 ABHS，HTH，NaDCC和NaOCl），以1.5L / m的流速通过漏斗倒入200mL氯溶液，并让志愿者彻底擦拭手。 </li></ol></li></ol></li><li> 用手工清洗手套进行手工清洗。 <ol><li>洗手后，立即放置每个志愿者的手（ 即手（右或左）为睾丸选择在步骤4.2）中将其装入含有75mL洗脱液（ 例如 PBS）的样品袋中，直到手腕。把手的顶部紧紧握在手腕上。 注意:使用含有足够的硫代硫酸钠的洗脱液进行取样以中和用于洗手的任何氯。 PBS是常用的适用于许多生物体的洗脱液。 </li><li>志愿者在解决方案中轻轻擦手30秒，注意手指和指甲之间的距离。轻轻按摩手袋30秒，以确保整个手在洗脱液中彻底冲洗，一直到手腕。 </li><li>密封袋子，并按照第6节所述的适当测定法，在2小时内处理。 </li></ol></li><li> 去污。 <ol><li>在每次洗手方法重复过程之前，志愿者用肥皂和温水彻底洗手。喷雾志愿者用70％乙醇洗手，直到两面涂上。让他们干燥</li><li>对于每个洗手条件，重复第5节中的所有步骤，仅使用在步骤4.2（ 图1 ）中随机选择的手。 </li></ol></li></ol>量化<ol><li>执行适合所选生物体的测定（ 例如，细菌的膜过滤或病毒的斑块测定，分别如3.1.2节和3.1.4节）。 </li><li>计数板后，记录每次测试的CFU / mL或PFU / mL（分别为3.1.2和3.1.4）。 </li></ol> 7.分析<ol><li> 使用步骤6.2的结果，计算生物在手上，每种生物和土壤负荷状态以及每种受试者和洗手方法的对数减少值。 <ol><li>对于洗手功效，比较每种细菌/病毒的浓度<html>手洗样品控制A（无洗手）。对于冲洗水持久性，比较每个冲洗水样品以控制B（仅用水洗涤）。使用以下标准公式: 减少记录（洗手 ）= <img alt="方程" src="/files/ftp_upload/55604/55604eq4.jpg" /><img alt="方程" src="/files/ftp_upload/55604/55604eq5.jpg" /> 减量（冲洗水 ）= <img alt="方程" src="/files/ftp_upload/55604/55604eq4.jpg" /><img alt="方程" src="/files/ftp_upload/55604/55604eq6.jpg" /> 注意:减少日志也可以表示为log 10 （无手洗） - log 10 （带洗手） </li></ol></li><li> 使用单向重复测量方差分析（ANOVA）来评估洗手方法和事后Tukey HSD检验中计算的对数减少值之间的显着差异，以显着模型进行成对评估显着性差异（p &lt;0.05）ef"&gt; 36。 <ol><li>在运行方差分析之前，先评估每个数据集的球形度（ 例如使用Bartlett测试）。当测试表明球形度被侵犯时，应用校正（ 例如，温室 - Geisser校正）。 </li></ol></li></ol>

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UNDP Medical Waste Experts Assessment and Recommendations Regarding Management of Ebola-Contaminated Waste Available from: <a target="_blank" href="https://noharm-global.org/sites/default/files/documents-files/3127/Report%20to%20WHO%20WASH%20and%20Geneva%20on%20Ebola%20final.pdf">https://noharm-global.org/sites/default/files/documents-files/3127/Report%20to%20WHO%20WASH%20and%20Geneva%20on%20Ebola%20final.pdf</a> (2015)</li><li>Hopman, J., Kubilay, Z., Allen, T., Edrees, H., Pittet, D., Allegranzi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+chlorine+solutions+used+for+hand+hygiene+and+gloves+disinfection+in+Ebola+settings:+a+systematic+review.">Efficacy of chlorine solutions used for hand hygiene and gloves disinfection in Ebola settings: a systematic review.</a> Antimicrob Resist Infect Control. 4 (1), 1 (2015).</li><li>Lowbury, E. J. L., Lilly, H. A., Bull, J. 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作者宣称他们没有竞争的经济利益。

The method described here provides an approach for testing handwashing efficacy in a controlled laboratory setting. This method highlights the use of human volunteers and surrogate, non-infectious organisms. Using the method, it was possible to demonstrate differences in: 1) the efficacy of handwashing methods and 2) organism persistence in rinse water. The purpose of presenting this protocol is to provide a general framework that can be adapted to test a wide range of surrogate organisms and handwashing methods relevant...

广泛推荐洗手，以防止疾病的传播，特别是那些通过粪便口服或空气传播的途径，包括腹泻和呼吸系统疾病1 。令人惊讶的是，洗手方法的功效，例如用肥皂和水（HWWS）洗手和用酒精洗手液（ABHS）进行洗手的手段上的生物体的功效几乎没有相似的证据。初步研究发现，与洗手方法相反，洗手的机械作用可能占大多数生物体的去除2,3 。此外，几乎没有哪个洗手方法最有效的比较证据。在非正式文献综述中，确定了14项比较肥皂和洗手液对生物体去除效果的研究。在这些研究中，五个发现ABHS更有效4 ， </在5,6,7,8中，七个发现HWWS更有效9,10,11,12,13,14,15，两个发现方法16,17之间没有显着差异。这些发现是不一致的，不能解决洗手后冲洗水中有机体持续存在的疾病风险。总的来说，洗手方法对除感染性疾病致病菌的比较功效的证据是有限的。 这种有限的证据导致了在疫点设置中哪些方法最适合的不确定性。例如在2013年至2016年在西非的埃博拉病毒（EVD）病毒爆发期间，几个大型国际响应者对HWWS，ABHS或0.05％氯解决方案提出了矛盾的建议。无国界医生组织（MSF）建议使用0.05％氯溶液进行洗手，而世界卫生组织（WHO）建议使用HWWS或ABHS（如果手不明显污染）。世界卫生组织甚至指出氯不应该被使用，除非没有其他选择可用，因为由于皮肤18,19,20,21,22所需的氯需求，其效果不如其他方法。此外，氯溶液通常由四种不同的氯化合物生产，包括高试验次氯酸盐（HTH），局部产生的和稳定的次氯酸钠（NaOCl）和草酸二氯异氰脲酸钠（NaDCC）。世卫组织委托西非EVD疫情作出的系统评估报告最近发现，只有四项研究调查洗手氯比较的效果23 。这些研究也产生了相互矛盾的结果，这些研究中没有一个使用推荐的氯浓度为0.05％的手洗或调查的微生物，与埃博拉病毒10,24,25,26,27相似。因此，这些建议没有被证明是循证的，目前还不清楚哪些建议是最有效的。 需要进一步的研究来比较洗手方法，以防止感染性病原体的传播，因为洗手干预措施是预防流行病传播的重要工具。这些h洗礼建议必须以证据为依据。因此，开发了用替代物或非感染性病原体进行洗手功效和冲洗水持久性的方法，2，28，29。在这里给出了使用Phi6作为埃博拉病毒替代品并使用大肠杆菌作为常见指示生物的样品结果。在该方案中，提出洗手功效和冲洗水持久性测试。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Soap bar</td>
			<td>Dove</td>
			<td colspan="2">White Beauty Bar soap</td>
		</tr>
		<tr>
			<td>Alcohol-based hand sanitizer</td>
			<td>Purell</td>
			<td colspan="2">Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol</td>
		</tr>
		<tr>
			<td>HTH Powder</td>
			<td>Acros Organics</td>
			<td>300340010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaDCC Powder</td>
			<td>Medentech</td>
			<td>Klorsept granules</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOCl Solution</td>
			<td>Acros Organics</td>
			<td>419550010</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrochlorinator</td>
			<td>AquaChlor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodometric titrator</td>
			<td>Hach</td>
			<td>1690001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedicals</td>
			<td>NC0117242</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Mucin</td>
			<td>EMD Milipore</td>
			<td>49-964-3500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>EMD Milipore</td>
			<td>GVWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin pH probe</td>
			<td>Hanna Instruments</td>
			<td>H199181</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Whirlpak Sample Bag</td>
			<td>Nasco</td>
			<td>B01447WA</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Whirlpak Sample Bag</td>
			<td>Nasco</td>
			<td>B01323WA</td>
			<td></td>
		</tr>
		<tr>
			<td>Funnel bottle</td>
			<td>Thermo Scientific</td>
			<td>3120850001</td>
			<td>You may drill an appropriately sized hole in the lid of a bottle to form a funnel that will dispense water at the appropriate flow rate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoScientific</td>
			<td>615090010</td>
			<td>Mix with water to produce 70% ethanol</td>
		</tr>
		<tr>
			<td>Spray bottle</td>
			<td>Qorpak</td>
			<td>PLC06934</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>ATCC</td>
			<td>25922</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher BioReagents</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Globe Scientific</td>
			<td>22-170-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi6</td>
			<td>HER</td>
			<td>102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient broth</td>
			<td>BD Difco</td>
			<td>BD 247110</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneQuant 100 Spectrophotometer</td>
			<td>General Electric</td>
			<td>28-9182-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>Fisher Chemical</td>
			<td>S445-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane filter (47mm, 0.45 &#181;m)</td>
			<td>EMD Millipore</td>
			<td>HAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>m-ColiBlue24 broth media</td>
			<td>EMD Millipore</td>
			<td>M00PMCB24</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish with pad (47mm)</td>
			<td>Fisherbrand</td>
			<td>09-720-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Manifold</td>
			<td>Thermo Scientific/Nalgene</td>
			<td>09-752-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter funnels</td>
			<td>Thermo Scientific/Nalgene</td>
			<td>09-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas syringae</td>
			<td>HER</td>
			<td>1102</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Thermo Scientific</td>
			<td>10010031</td>
			<td>Solution may also be mixed from source compounds according to any basic recipe</td>
		</tr>
	</tbody>
</table>

a method to test the efficacy of handwashing for the removal of emerging infectious pathogens

手洗被广泛用于预防传染病传播。然而，一般来说，洗手方法的效果几乎没有可比证据。此外，很少有证据表明比较洗手方法，以确定哪些最有效地去除感染性病原体。需要进行研究，为传染病爆发期间可能使用的不同洗手方法提供证据。在这里，描述了一种用于评估洗手方法从手中除去微生物及其在漂洗水中的持续性的功效的实验室方法。志愿者的手首先加入测试生物体，然后用各种手洗方法进行洗涤。通常，替代微生物用于保护人类受试者免受疾病。使用改进的"手套汁"方法测试洗涤后志愿者手上剩余的生物体数量:将手放置在带有elu的手套中洗涤并悬浮微生物并通过膜过滤（细菌）或噬斑测定（病毒/噬菌体）使其可用于分析。直接收集从洗手水中冲洗的水分析。手洗效果通过比较洗手后取样样本之间的对数减少值进行量化，不用洗手。通过将来自各种洗手方法的冲洗水样品与仅用水洗手洗后收集的样品进行比较来定量冲洗水持久性。虽然这种方法受到需要使用替代生物来保护人类志愿者的安全性的限制，但它捕获了在体外研究中难以复制的洗手方面，并填补了洗手效力和感染生物在冲洗中持续存在的研究空白水。

在这里，协议（ 图1 ）完成了18名志愿者，他们分别使用大肠杆菌和Phi6进行测试。在具有和不具有土壤负荷的大肠杆菌和具有土壤负荷的Phi6之间的洗手结果之间发现显着的差异（ 图2和图3 ）。对于没有土壤负荷的大肠杆菌 ，使用HTH，NaDCC和稳定的NaOCl进行洗手都比仅用水洗手更大（F?...

Watch this Scientific Journal Video about A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens at JoVE.com

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

手洗被广泛用于预防传染病传播。然而，几乎没有证据显示洗手方法在去除传染病病原体方面是最有效的。我们开发了一种评估洗手方法去除微生物的功效的方法。

一种测试洗手方法去除新发感染性病原体的方法

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

a-method-to-test-efficacy-handwashing-for-removal-emerging-infectious

Research

JoVE Journal

Immunology and Infection

18.9K 次观看.  Tufts University. 手洗被广泛用于预防传染病传播。然而，几乎没有证据显示洗手方法在去除传染病病原体方面是最有效的。我们开发了一种评估洗手方法去除微生物的功效的方法。 

视频: A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Xpert山地车/ RIF测试与诊断肺结核

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia.

费希尔344大鼠气管内接种土 tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

一种测试洗手方法去除新发感染性病原体的方法

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