这项研究得到了美国国立卫生研究院（NIH）国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases）校内研究计划的支持。我们感谢 Linden T. 胡、Sam R. Telford III、Kenneth Dardick、Carla Williams、Erin Chung 和 Christina Brandeburg 参与制定程序。

让志愿者暴露于蜱虫叮咬是一种实验方法，必须根据相关监管机构批准的临床研究方案进行。临床研究（NCT01143558、NCT02446626 和 NCT05036707）由各自的机构审查委员会批准，在美国食品和药物管理局授予的研究性器械豁免下进行，并根据良好临床实践指南进行。此外，这些研究已在 ClinicalTrials.gov 注册，并获得了所有参与者的书面知情同意书。
1. 密闭敷料准备
<ol><li>要创建测量模板，请沿测量线的一侧复印 3“ x 3” 水胶体密封敷料（图 1A 和 材料表）。</li>
	<li>沿 1 英寸线切割复印模板。然后，沿着 2 英寸的线切割，创建一个 2 英寸的圆，中心为 1 英寸（图 1B）。</li>
	<li>使用此模板，切割非粘性泡沫敷料，创建一个 2 英寸的圆圈，中心为 1 英寸（图 2A、B 和 材料表）。</li>
	<li>使用相同的模板，在 4“ x 4” 超薄水胶体敷料的中心切一个 2 英寸的圆圈（图 2C，D）。</li>
	<li>在 11/2“ 线上切割 3” x 3“ 密闭敷料的水胶体层。</li>
	<li>拉回安全壳敷料的水胶体层的纸衬垫，露出粘合剂。将切割的泡沫敷料放入粘合剂的 2 英寸圆内（图 3A）。这将确保泡沫敷料延伸到安全壳敷料的开口之外。将纸衬垫更换到敷料的水胶体层上（图3B）。 注：这些测量值用于涉及多达 10 个 幼虫 I. scapularis 蜱的研究。对于涉及多达 30 只 肩胛蜱 幼虫的研究，泡沫中心的模板在 1 英寸和 11/2 英寸标记之间切割，以留出更大的放置区域。密闭敷料的测量值可以根据研究设计进行调整。</li>
</ol>2. 刻度放置
<ol><li>即时报价存储<ol><li>将 肩胛蜱 幼虫储存在医疗级冰箱中，温度设置为 9-10 °C。</li>
		<li>将蜱虫存放在覆盖有网帽的小瓶中，以允许足够的气流。用轻轻湿润的海绵将一小瓶蜱虫包装在可密封的塑料袋中，以保持湿度并防止干燥。</li>
	</ol></li>
	<li>放置刻度<ol><li>从冰箱中取出蜱虫，让它们加热到室温。蜱虫可以在冰箱外短短 15 分钟内变得活跃。</li>
		<li>在放置区域周围放置一张白纸，以便轻松看到任何无意中掉落的刻度。</li>
		<li>用清水或生理盐水弄湿 4 英寸 x 4 英寸的纱布垫，然后用它来清洁放置部位的皮肤。该区域应拍干或让其风干。 注意：在选择放置蜱虫的身体部位时，请考虑不限制志愿者日常活动的区域，并且参与者可以轻松监测敷料的完整性。不要将蜱虫放置在可能因直接压力而被压碎的区域（例如背部）。评估参与者的体毛，如有必要，使用剪刀在粘合剂部位小心修剪过多的体毛，避免对皮肤造成伤害。避免使用剃须刀脱毛，因为它可能会增加皮肤受伤和感染的风险。</li>
		<li>一旦皮肤干燥，从准备好的敷料的水胶体侧取下粘性衬垫，并将其牢固地固定在放置部位的皮肤上，确保网状层保持开放。</li>
		<li>将木棉签（见 材料表）对半掰开，露出锯齿状的末端。</li>
		<li>小心地打开一小瓶蜱虫。</li>
		<li>使用木棉签的锯齿状端，从小瓶中转移活性蜱虫，并将它们放在收容敷料内的皮肤上（图4A）。让额外的团队成员监控蜱虫放置部位，以确保蜱虫留在收容敷料内。在此过程中，使用胶带捕获从小瓶中逸出的蜱虫。 注意： 打破木签并使用锯齿状的一端可以有效地操作和转移蜱虫，而不会对蜱虫造成损坏。带有浅色柔软刷毛的小刷子也可用于将蜱虫从容器转移到皮肤部位。</li>
		<li>放置研究的指定数量的刻度后，从密闭敷料的网状层上取下粘性衬垫，并将其封闭在开口处。</li>
		<li>在确保粘合剂牢固附着后，将准备好的 4“ x 4” 超薄水胶体敷料涂在密闭敷料上以增加安全性（图 4B）。 注：有关无病原体肩胛胛蜱菌落和引用研究中使用的蜱暴露程序持续时间的更多信息，可在已发表研究19,22 的方法部分找到。</li>
	</ol></li>
</ol>3. 现场护理
<ol><li>为研究参与者提供防水罩和 2 英寸低过敏性胶带（参见 材料表）。</li>
	<li>提供护理蜱虫遏制部位的说明。<ol><li>定期检查敷料并监测边缘是否牢固地粘附在皮肤上。根据需要使用额外的胶带来加固敷料边缘。</li>
		<li>在研究期间，请勿刮擦敷料部位，并避免打开或取下敷料。</li>
		<li>避免洗澡或浸泡在水中。此外，避免热水淋浴并限制淋浴的时间。淋浴前，用提供的防水罩保护安全壳敷料，并在完成淋浴后将其取下。轻轻拍干敷料，并检查它以确保它保持牢固。</li>
		<li>避免可能导致大量出汗和损害敷料完整性的活动（例如，有氧运动、长时间徒步旅行）。</li>
	</ol></li>
</ol>4. 去除蜱虫
<ol><li>收集瓶制备<ol><li>在蜱虫收集瓶上贴上所需的标识符（例如，研究代码、日期）。</li>
		<li>如果需要保存活蜱虫，用20G针在小瓶盖上穿孔。使用一层尼龙或聚酯网以及瓶盖密封小瓶。将一小块湿海绵与一小瓶蜱虫一起放入可密封的袋子中以保持湿度。</li>
	</ol></li>
	<li>去除蜱虫<ol><li>在绷带部位周围铺上白色床单，并确保收集盆触手可及。</li>
		<li>使用酒精湿巾，小心地从皮肤上取下绷带。</li>
		<li>完全取下后，将绷带放入收集盆中，并检查是否存在脱落的蜱虫。</li>
		<li>评估放置地点是否有附着的蜱虫。已经补充的蜱虫将分离（图5A）。对于剩余的附着的蜱虫，使用细尖镊子去除（图5B）。</li>
		<li>用酒精湿巾或肥皂和水清洁皮肤。</li>
	</ol></li>
</ol>

人类接触幼虫蜱虫用于蜱传疾病研究

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Adriana Marques 博士拥有美国专利 8,926,989 B2;并且是全球莱姆病联盟和美国莱姆病基金会的无薪科学顾问。Siu Ping Turk 和 Aleah Eschman 没有可能构成利益冲突的关联。本出版物的内容并不一定反映美国卫生与公众服务部的观点或政策，提及商品名称、商业产品或组织也不意味着美国政府的认可。

虽然涉及暴露于蜱虫的动物研究 4,5,6,7,21 对于提高我们对宿主对蜱传疾病和蜱虫叮咬反应的理解非常宝贵，但这些模型在预测人类宿主反应的程度上存在局限性。该模型描述了以可控方式将人类暴露于蜱虫叮咬的方法，可以很容易地适应回答不同的研究问题，并扩展了在人?...

硬蜱（硬蜱科：Acari）是全球范围内发生的专性血液摄食性体外寄生虫，能够传播广泛的病原体，包括细菌、病毒和寄生虫，具有重要的医学和兽医意义。硬蜱蜱必须与宿主保持附着数天才能完成血粉，它们有能力保持附着在皮肤上，同时避免识别，防止局部血液凝固，并促进长期摄食1,2,3。动物研究表明，不宽容的宿主会通过反复接触蜱虫叮咬获得对蜱虫叮咬的抵抗力，这可能导致传播病原体的能力降低，而蜱虫可以反复寄生于宽容的宿主。获得性蜱耐药性取决于宿主免疫反应的性质 4,5,6,7。
蜱传疾病在美国是一个日益严重的威胁，2004 年至 2016 年间报告的病例数增加了一倍多 8,9。由于气候变化，不同蜱虫的地理范围继续扩大10,11。美国主要的蜱传疾病包括莱姆病、无形体病、埃立克体病、斑疹热立克次体病、巴贝虫病、土拉菌病和波瓦桑病毒病8。莱姆病由伯氏疏螺旋体感染引起，是美国和欧洲最常见的蜱传疾病12.美国每年约有 476,000 人被诊断患有莱姆病，这给个人和社会带来了公共卫生和经济负担 13,14,15。
肩胛硬蜱 （黑腿蜱或鹿蜱）是莱姆病的主要传播媒介，在美国也是无形体病、巴贝虫病、 宫本疏螺旋体 病和波瓦桑病毒病的主要传播媒介。在美国，其他具有医学重要性的蜱种包括 Amblyomma americanum （孤星蜱）、 Dermacentor variabilis （美国狗蜱）、 太平洋硬蜱 （西部黑腿蜱）、 Dermacentor andersni （落基山木蜱）、 Cookei硬蜱 （土拨鼠蜱）、 Dermacentor occidentalis （太平洋沿岸蜱）、 Rhipicephalus sanguineus （棕色狗蜱）和 Amblyomma maculatum （墨西哥湾沿岸蜱）16。
开发一种使研究志愿者暴露于蜱虫叮咬的方法支持使用自然载体寻找感染证据的研究，这一过程称为异种诊断 17,18,19,20,21，并更多地了解暴露于蜱虫引起的免疫力，这有助于发现抗蜱虫疫苗 5,6,7.本文描述的程序是在2014年发表的第一项人类研究中开发和使用的，该研究使用实验室饲养的肩胛硬蜱幼虫在抗生素治疗（NCT01143558）后对伯氏疏螺旋体感染进行异种诊断19。该系统已成功用于一项 2 期研究，该研究调查了阳性异种诊断是否与莱姆病 （NCT02446626） 抗生素治疗后症状的持续存在相关，以及一项探索宿主对蜱虫叮咬反应的研究 （NCT05036707）。
该程序协议描述了创建收容敷料、蜱虫放置程序和蜱虫清除程序的过程，以及维持收容敷料所需的现场护理。关于上述研究中使用的无病原体肩胛胛蜱菌落和蜱暴露程序的详细信息之前已经描述过19,22。这种方法提供了一种灵活的研究工具，可用于研究人类宿主对蜱虫叮咬的反应、蜱虫预防药物的有效性以及莱姆病和其他蜱传疾病的不同方面。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 G needle</td>
			<td>Any brand</td>
			<td></td>
			<td>For puncturing the vial cap.</td>
		</tr>
		<tr>
			<td>3&#34; x 3&#34; containment dressing</td>
			<td>Monarch Labs Names</td>
			<td>LeFlap</td>
			<td>https://www.monarchlabs.com/ordering</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; extra-thin hydrocolloid dressing</td>
			<td>ConvaTec</td>
			<td>DuoDerm</td>
			<td>https://www.convatec.com/products/advanced-wound-care/brand-names/pc-wound-duoderm-granluflex/duoderm-extra-thin-dressing/</td>
		</tr>
		<tr>
			<td>4&#34; x 4&#34; gauze</td>
			<td>Monarch Labs Names</td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Clean water or saline</td>
			<td></td>
			<td></td>
			<td>For cleaning skin</td>
		</tr>
		<tr>
			<td>Moisture barrier (e.g. 7&#34; x 7&#34;)</td>
			<td>AquaGuard</td>
			<td>TIDI</td>
			<td>For showering, ttps://www.tidiproducts.com/product-listing/aquaguard-shower-cover-sheets&#160;</td>
		</tr>
		<tr>
			<td>Non-adhesive foam dressing</td>
			<td>Coloplast</td>
			<td>Biatain</td>
			<td>https://www.coloplast.us/biatain-non-adhesive-en-us.aspx</td>
		</tr>
		<tr>
			<td>Roll of 2&#34; hypoallergenic tape</td>
			<td>Monarch Labs Names</td>
			<td>Durapore</td>
			<td>For reinforcing containment dressing.</td>
		</tr>
		<tr>
			<td>Roll of adhesive tape</td>
			<td></td>
			<td></td>
			<td>For trapping ticks</td>
		</tr>
		<tr>
			<td>Vials for collection (e.g. cryovials)</td>
			<td>Ependorf</td>
			<td>ECC200</td>
			<td></td>
		</tr>
	</tbody>
</table>

a model for experimental exposure of humans to larval ixodes scapularis ticks

蜱传疾病是美国和全世界的一个重大公共卫生问题。蜱虫是专性吸血节肢动物;硬蜱必须保持附着在宿主的皮肤上，并完成其为期多天的摄食过程以获取其血粉。将动物暴露于蜱虫是研究宿主对蜱虫叮咬和蜱传疾病反应的常见做法。我们开发了该程序，进行了第一项人体研究，并发表了关于将人类志愿者暴露于未感染的 肩胛硬蜱 幼虫的研究结果。本文介绍了用于构建收容敷料的方法，如何将蜱虫应用并固定到宿主身上，如何维护敷料，以及如何从宿主中清除蜱虫。让志愿者暴露于蜱虫叮咬是一种实验程序，必须根据相关监管机构批准的临床研究方案进行。这种方法允许转化研究，以更好地了解人类对蜱虫叮咬的反应，并促进蜱传疾病的诊断、预防和治疗的发展。

Hard ticks (Ixodidae: Acari) are obligate blood-feeding ectoparasites that occur worldwide and are capable of transmitting a broad range of pathogens, including bacteria, viruses, and parasites, of major medical and veterinary importance. Ixodid ticks must remain attached to the host for days to complete a blood meal, and they have the capacity to stay attached to the skin while avoiding recognition, preventing local blood coagulation, and facilitating long-term feeding1,2,3. Animal studies have demonstrated that non-permissive hosts acquire resistance to tick bites with repeated tick exposures, which can lead to a decreased ability to transmit a pathogen, while ticks can repeatedly parasitize permissive hosts. Acquired tick resistance is dependent on the nature of the host immune response4,5,6,7.
Tickborne diseases are an increasing threat in the United States (US), with the number of reported cases more than doubling between 2004 and 20168,9. Due to climate change, the geographic ranges of different ticks continue to expand10,11. The leading tickborne diseases in the US include Lyme disease, anaplasmosis, ehrlichiosis, spotted fever rickettsiosis, babesiosis, tularemia, and Powassan virus disease8. Lyme disease, caused by infection with Borrelia burgdorferi sensu lato, is the most common tickborne disease in the US and Europe12. With approximately 476,000 individuals diagnosed with Lyme disease annually in the US, there is both a public health and economic burden to individuals and to society13,14,15.
Ixodes scapularis (the black-legged or deer tick) is the primary vector for Lyme disease, as well as anaplasmosis, babesiosis, Borrelia miyamotoi disease, and Powassan virus disease in the US. Other medically important tick species in the US include Amblyomma americanum (Lone star tick), Dermacentor variabilis (American dog tick), Ixodes pacificus (Western black-legged tick), Dermacentor andersoni (Rocky Mountain wood tick), Ixodes cookei (Groundhog tick), Dermacentor occidentalis (Pacific Coast tick), Rhipicephalus sanguineus (brown dog tick), and Amblyomma maculatum (Gulf Coast tick)16.
Developing a method to expose research volunteers to tick bites supports studies using the natural vector to search for evidence of infection, a procedure known as xenodiagnosis17,18,19,20,21, and to learn more about immunity induced by exposure to ticks, which can contribute to the discovery of an anti-tick vaccine5,6,7. The procedure described here was developed and used in the first human research study using laboratory-reared Ixodes scapularis larva for xenodiagnosis of B. burgdorferi infection after antibiotic therapy (NCT01143558), published in 201419. The system has been successfully used in a phase 2 study investigating whether a positive xenodiagnosis correlated with the persistence of symptoms after antibiotic treatment of Lyme disease (NCT02446626) and in a study exploring the host response to tick bites (NCT05036707).
This procedure protocol describes the process for creating the containment dressing, the tick placement procedure, and the tick removal procedure, as well as the site care needed to maintain the containment dressing. The details regarding the pathogen-free I. scapularis tick colony and tick exposure procedures used for the above-cited studies have been previously described19,22. This methodology offers a flexible research tool that can be adapted to studying different aspects of the human host response to tick bites, the effectiveness of tick prevention medications, as well as Lyme disease and other tickborne illnesses.

作者聚焦：用于蜱虫研究和莱姆病研究的受控人类暴露模型

人类接触 肩胛蜱 幼虫的实验暴露模型

This protocol offers an effective method for a controlled exposure of humans to ticks. For research, the protocol can be used to study the human immune response tick bites, as well as the development of diagnostics and preventions against tick-borne diseases. There were no published procedures for placing ticks on human research volunteers.The challenge was to develop a system that allowed the ticks to feed for days while also being comfortable for the participants. This model contains the ticks for the duration of the feeding and allow the research participants to proceed with their daily activities with minor lifestyle adjustments.

该研究表明，该手术是安全且耐受性良好的，主要不良事件是叮咬部位的轻度瘙痒，在 58% 的手术中观察到。当使用干净的实验室饲养的肩 胛蜱幼 虫时，没有发生与该程序相关的严重不良事件19.在进行的 43 个程序中，与放置的蜱虫相比，附着的蜱虫的平均恢复百分比为 45% ± 27% （SD），中位数百分比为 40%（图 6）。
肩胛蜱</e...

Watch this Scientific Journal Video about A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks at JoVE.com

本文介绍了将人类暴露于 肩胛硬蜱 幼虫以进行临床研究的方法。该技术比较简单，研究志愿者可以忍受，并且可以根据实验需要进行修改。此类涉及人类受试者的研究必须根据适当监管机构批准的临床研究方案进行。

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整。

a-model-for-experimental-exposure-humans-to-larval-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

332 次观看.  National Institutes of Health. 该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整?...