Name: a method to test the efficacy of handwashing for the removal of emerging infectious pathogens
Uploaded: 2017-06-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens at JoVE.com

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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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><tbody><tr><td>Soap bar</td><td>Dove</td><td>White Beauty Bar soap</td><td></td></tr><tr><td>Alcohol-based hand sanitizer</td><td>Purell</td><td>Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol</td><td></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 &amp;micro;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>&lt;em&gt;E. coli&lt;/em&gt;</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 (47 mm, 0.45 &amp;micro;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 (47 mm)</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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

19.0K 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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

Determining Viral Disinfection Efficacy of Hot Water Laundering

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the coronavirus disease 2019 (COVID-19) pandemic, it is intended to be a framework, adaptable to other virus disinfection studies; it demonstrates the steps for preparing the test virus, inoculating the test material, assessing visual and integrity changes to the washed items due to the laundering process, and quantifying the reduction in viral load. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the personal protective equipment (PPE) items after multiple laundering cycles. The representative results presented with the protocol use the Phi6 bacteriophage inoculated onto cotton scrub, denim, and cotton face-covering materials and indicate that the hot water laundering and drying process achieved over a 3-log (99.9%) reduction in viral load for all samples (a 3-log reduction is the disinfectant performance metric in U.S. Environmental Protection Agency's Product Performance Test Guideline 810.2200). The reduction in viral load was uniform across different locations on the PPE items. The results of this viral disinfection efficacy testing protocol should help the scientific community explore the effectiveness of home laundering for other types of test viruses and laundering procedures.

In response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a laboratory protocol was developed to test the viral disinfection efficacy of hot water laundering of cloth face coverings, cotton scrubs, and denim pants. The Phi6 virus (bacteriophage) was used as the organism to test disinfection efficacy.

确定热水洗涤的病毒消毒效果

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol ...

工程学

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

免疫与感染

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

病毒测试,水样中检测病毒的一种使用点方法

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

朊病毒安全实验室滑动测试

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

生物学

纳米草本涂层织物的抗菌功效

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

作者聚焦：使用精油纳米草药封装的抗菌织物  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

一种使用纳米草本封装精油的抗菌织物

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

一种测试洗手方法去除新发感染性病原体的方法

摘要

探索更多视频

此视频中的章节

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

确定热水洗涤的病毒消毒效果

人类诺沃克病毒的表面上检测拭子采样方法

喷墨打印机辅助自动跳棋阵列和手动消时法的抗菌协同试验

病毒测试,水样中检测病毒的一种使用点方法

朊病毒安全实验室滑动测试

城市环境 -环境水库SARS-CoV-2采样和检测管道

一种使用纳米草本封装精油的抗菌织物

一种使用纳米草本封装精油的抗菌织物

一种使用纳米草本封装精油的抗菌织物