一种测试洗手方法去除新发感染性病原体的方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Soap bar | Dove | White Beauty Bar soap | |
| Alcohol-based hand sanitizer | Purell | Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol | |
| HTH Powder | Acros Organics | 300340010 | |
| NaDCC Powder | Medentech | Klorsept granules | |
| NaOCl Solution | Acros Organics | 419550010 | |
| Electrochlorinator | AquaChlor | ||
| Iodometric titrator | Hach | 1690001 | |
| Bovine serum albumin | MP Biomedicals | NC0117242 | |
| Tryptone | Fisher | BP1421-100 | |
| Bovine Mucin | EMD Milipore | 49-964-3500MG | |
| 0.22 µm Filter | EMD Milipore | GVWP04700 | |
| NaCl | Fisher | BP358-1 | |
| Skin pH probe | Hanna Instruments | H199181 | |
| Large Whirlpak Sample Bag | Nasco | B01447WA | |
| Small Whirlpak Sample Bag | Nasco | B01323WA | |
| Funnel bottle | Thermo Scientific | 3120850001 | 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 |
| Ethanol | ThermoScientific | 615090010 | Mix with water to produce 70% ethanol |
| Spray bottle | Qorpak | PLC06934 | |
| E. coli | ATCC | 25922 | |
| LB Broth | Fisher BioReagents | BP1426-2 | |
| LB Agar | Fisher BioReagents | BP1425-500 | |
| Sterile loop | Globe Scientific | 22-170-204 | |
| Phi6 | HER | 102 | |
| Nutrient broth | BD Difco | BD 247110 | |
| GeneQuant 100 Spectrophotometer | General Electric | 28-9182-04 | |
| Sodium thiosulfate | Fisher Chemical | S445-3 | |
| Membrane filter (47mm, 0.45 µm) | EMD Millipore | HAWP04700 | |
| m-ColiBlue24 broth media | EMD Millipore | M00PMCB24 | |
| Petri dish with pad (47mm) | Fisherbrand | 09-720-500 | |
| Vacuum Manifold | Thermo Scientific/Nalgene | 09-752-5 | |
| Filter funnels | Thermo Scientific/Nalgene | 09-747 | |
| Pseudomonas syringae | HER | 1102 | |
| Phosphate Buffered Saline | Thermo Scientific | 10010031 | Solution may also be mixed from source compounds according to any basic recipe |
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Cite This Article
Wolfe, M. K., Lantagne, D. S. A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens. J. Vis. Exp. (124), e55604, doi:10.3791/55604 (2017).