Name: a method to test the efficacy of handwashing for the removal of emerging infectious pathogens
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Bu çalışma, Birleşik Devletler Uluslararası Kalkınma Ajansı, Dış Afet Yardım Ofisi (AID-OFDA-A-15-00026) tarafından desteklenmiştir. Marlene Wolfe, Ulusal Bilim Vakfı tarafından desteklendi (hibe 0966093).

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	<li>Kampf, G., Kramer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+Background+of+Hand+Hygiene+and+Evaluation+of+the+Most+Important+Agents+for+Scrubs+and+Rubs.">Epidemiologic Background of Hand Hygiene and Evaluation of the Most Important Agents for Scrubs and Rubs.</a> Clin Microbiol Rev. 17 (4), 863-893 (2004).</li><li>Miller, T., Patrick, D., Ormrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+decontamination:+influence+of+common+variables+on+hand-washing+efficiency.">Hand decontamination: influence of common variables on hand-washing efficiency.</a> Healthc Infect. 16 (1), 18 (2013).</li><li>Jensen, D. A., Danyluk, M. D., Harris, L. J., Schaffner, D. 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Yazarlar, rekabet eden mali çıkarlarının olmadığını beyan ettiler.

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 to infectious disease.
During the use of the method, two key data quality recommendations were noted as important. First, the inoculate must be applied both as similarly as possible across the rounds of testing and in a manner, that minimizes loss. This is to ensure that sufficient inoculate is applied to the hands to allow for statistically significant results. Second, be sure to complete the "cleansing wash" step, in which the protocol is performed without handwashing prior to testing, as previous work has shown that there are likely to be significant differences between a first wash and subsequent washes, but not between subsequent washes performed after a cleansing round29. Additionally, this step clears residual hand contamination, which would interfere with results. 
The main limitation of this protocol is that it can be uncomfortable for volunteers. During each round of testing, which lasted about 2 h, volunteers' hands became cold. Some volunteers reported mild pain from their hands being constantly wet. Additionally, after a few rounds of testing, volunteers' hands became supersaturated, no longer fully drying between rounds. Although the randomization of the order of handwashing methods for each volunteer accounted for supersaturation, it is possible that the supersaturation could act as a confounding or modifying factor in this type of testing. To address this limitation, it is recommended that volunteers are appraised of this risk during consent disclosures and are reminded of their right to drop out of the study at any time. Volunteers should not undergo testing for more than 2 h per day to allow time for the hands to return to a baseline state and to minimize discomfort. A second limitation is the need to use a surrogate organism or non-infectious variant of a pathogenic organism to protect the health of volunteers. This might cause concern about the generalizability of results. However, for some pathogenic organisms (such as the Ebola virus), this limitation cannot be ethically overcome. Care must be taken during surrogate organism selection. Lastly, this is a laboratory study on efficacy. Results may only translate to effective disease prevention in real-word contexts where handwashing methods are made accessible to those in need and are used properly and consistently.
This protocol draws on previous work on handwashing efficacy but attempts to streamline methods and emphasizes the use of human hands (rather than surrogate surfaces) for testing. Additionally, rinse water is a transmission risk that had previously not been assessed. Existing studies on handwashing efficacy vary in methodology, leading to non-comparable data. We hope that standardized protocols for conducting handwashing method comparisons will encourage comparable and replicable results. Previous work has demonstrated that in vitro testing on surrogate surfaces such as pig skin, where, for example, the actual Ebola virus could be used, produced results that do not match those found after testing on human hands38. Therefore, a method using human hands and surrogate or non-infectious organisms is currently the best available approach to estimate handwashing efficacy and rinse water persistence for infectious microorganisms.
Handwashing is critical to prevent disease transmission. However, there is a lack of evidence on the comparative efficacy of handwashing methods that are commonly recommended. This protocol can be used to generate evidence about handwashing efficacy and rinse water persistence. This is especially important for infectious diseases with the potential to cause large outbreaks, such as the Ebola virus. We hope that other researchers will find this protocol useful to generate much-needed additional evidence on handwashing method efficacy and rinse water persistence that will assist in developing recommendations to reduce the transmission of infectious diseases.

El yıkama, hastalığın yayılmasını önlemek için yaygın olarak önerilir, özellikle diyare ve solunum yolu hastalıkları da dahil olmak üzere dışkı-oral veya hava yolundan bulaşan hastalıklar 1 . Şaşırtıcı bir şekilde, sabun ve suyla (HWWS) el yıkama ve alkollü el temizleyicisi (ABHS) gibi el yıkama yöntemlerinin etkinlikleri hakkında, organizmaların ellerden uzaklaştırılmasına ilişkin çok az kanıt bulunmaktadır. İlk araştırmalar, el yıkama yönteminin aksine, el yıkamasının mekanik etkisinin çoğu organizmanın giderilmesini 2 , 3 hesaba kattığını bulmuştur. Buna ek olarak, el yıkama yönteminin en etkili olduğuna dair çok az kanıt bulunmaktadır. Gayrı resmi bir literatür taramasında, sabun ve el dezenfektörünün organizmaların çıkarılması üzerindeki etkinliğini karşılaştıran 14 çalışma tespit edilmiştir. Bu çalışmaların beşinde ABHS&#39;nin daha etkili olduğu bulunmuştur4 , </9 , 10 , 11 , 12 , 13 , 14 , 15 ve daha fazla olmak üzere HWWS&#39;nin , 16 , 17 numaralı yöntemler arasında anlamlı bir fark bulamadığını bulmuşlardır. Bu bulgular tutarsızdır ve el yıkama sonrasında durulama suyunda organizmaların sürdüğü sürecin hastalık riskini ele almamaktadır. Genel olarak, bulaşıcı hastalıklara neden olan patojenlerden arındırma için el yıkama yöntemlerinin karşılaştırmalı etkinliği hakkında kanıt sınırlıdır. Bu kısıtlı kanıt salgın ayarlarında hangi yöntemlerin en uygun olduğuna ilişkin belirsizliğe neden olmuştur. Örneğin, Batı Afrika&#39;daki 2013&#39;ten 2016&#39;ya kadar olan Ebola Virüs Hastalığı (EVD) salgını sırasında, birkaç büyük uluslararası müdahale uzmanı, HWWS, ABHS veya% 0.05 klorlu solüsyonlar için çelişkili tavsiyeler verdi. Dünya Sağlık Örgütü (WHO), HWWS veya ABHS (eğer eller görünür şekilde kirli değilse) tavsiye ederken, Médecins Sans Frontieres (MSF), el yıkama için% 0.05 klor solüsyonu kullanılmasını önerir. DSÖ, başka seçenek mevcut olmadığı sürece klorun kullanılmaması gerektiğini ileri sürmektedir; zira cildin uyguladığı klor talebi nedeniyle diğer yöntemlerden daha az etkili 18 , 19 , 20 , 21 , 22 . Buna ek olarak, klor çözeltileri, yaygın olarak, yüksek test hipoklorit (HTH), yerel olarak üretilen ve stabilize edilmiş sodyum hipoklorit (NaOCl) ve çimDikloroizosiyanürat (NaDCC). Dünya Sağlık Örgütü&#39;nün Batı Afrika&#39;daki EVD salgınına tepki olarak sistemli bir inceleme yapan elle yıkamanın klor ile karşılaştırma etkinliğini araştıran sadece dört çalışma 23 bulundu . Bu çalışmalar aynı zamanda çelişkili sonuçlar vermiştir ve bu çalışmaların hiçbirinde el yıkama veya% 10 , 24 , 25 , 26 , 27 Ebola virüsüne benzer mikroorganizmalar için önerilen% 0.05 klor konsantrasyonu kullanılmamıştır. Bu nedenle, tavsiyeler kanıta dayalı olarak bulunmadı ve hangi önerilerin en etkili olduğu belirsizdi. Bulaşıcı patojenlerin yayılmasını önlemek için el yıkama yaklaşımlarını karşılaştırmak için ek araştırmalara ihtiyaç vardır çünkü el yıkama müdahaleleri salgın hastalık bulaşmasını önlemede önemli bir araçtır. Bu hVe yıkama tavsiyeleri kanıtlara dayanmalıdır. Böylece, 2 , 28 , 29 , vekil veya bulaşıcı olmayan patojenlerle yapılan el yıkama etkinliğini ve durulama suyu devamlılığını test etmek için bir yöntem geliştirildi. Phi6&#39;yı Ebola virüsü için vekil olarak kullanan ve Escherichia coli&#39;yi ortak bir indikatör organizma olarak kullanan örnek sonuçlar burada sunulmuştur. Bu protokolde el yıkama etkinliği ve durulama suyu devamlılık testleri sunulmaktadır.

<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

Bulaşıcı hastalık bulaşmasını önlemek için el yıkama yaygın olarak önerilir. Bununla birlikte, el yıkama yöntemlerinin genel olarak etkililiği hakkında az kıyaslanabilir kanıt bulunmaktadır. Ek olarak bulaşıcı patojenlerden hangisinin daha etkili olduğunu belirlemek için el yıkama yöntemlerini karşılaştıran çok az kanıt bulunmaktadır. Bulaşıcı hastalık salgınlarında istihdam edilebilir el yıkama için farklı yaklaşımlar için kanıt sağlamak için araştırma gerekmektedir. Burada, el yıkama yöntemlerinin ellerdeki mikroorganizmaların yok edilmesinde etkinliğini ve durulama suyundaki ısrarını değerlendirmek için bir laboratuar yöntemi anlatılmıştır. Gönüllülerin elleri ilk olarak test organizması ile çakıştırılır ve sonra her bir el yıkama yöntemiyle yıkanır. Genellikle, vekil mikroorganizmalar, insanları hastalıktan korumak için kullanılır. Yıkadıktan sonra gönüllülerin elinde kalan organizmaların sayısı değiştirilmiş bir &quot;eldiven suyu&quot; yöntemi kullanılarak test edilir: eller elu ile eldivenlere yerleştirilirMikroorganizmaları süspansiyon haline getirmek ve zar filtreleme (bakteri) veya plak tahlili (virüs / bakteriyofaj) ile analiz için kullanılabilir hale getirmek üzere yıkanır. Elde yıkamadan üretilen durulama suyu analiz için doğrudan toplanır. El yıkama etkinliği, elle yıkamadan sonra alınan numuneler arasındaki günlük azaltma değerinin, el yıkama yapılmayan numuneler ile karşılaştırılması ile nicelleştirilir. Durulama suyu persistansı, çeşitli elle yıkama yöntemlerinden elde edilen durulama suyu örneklerini, sadece su ile el yıkama işleminden sonra toplanan örneklerle karşılaştırarak nicelleştirilir. Bu yöntem insan gönüllülerinin güvenliğini korumak için yedek organizmaları kullanma ihtiyacı ile sınırlı olmakla birlikte, bir in vitro çalışmada çoğaltılması zor olan el yıkama yönlerini yakalar ve yıkama etkinliğinde araştırma boşluklarını ve bulaşık organizmalarının yıkamadaki ısrarını doldurur. Su.

Burada, protokol ( Şekil 1 ) hem E. coli hem de Phi6 kullanılarak test edilen 18 gönüllü ile tamamlandı. Elde yıkama sonuçları arasında hem toprak yüklü hem de topraksız E. coli ile Phi6 toprak yükü arasında anlamlı fark bulundu ( Şekil 2 ve Şekil 3 ). Toprak yükü olmayan E. coli için, HTH, NaDCC ve stabilize NaOCl ile yapılan el yıkama işlemi, sadece elle yıkamaya göre (F (6,102) = 2,72, p = 0,034) log azalmalarına önemli ölçüde neden oldu. Toprak yükü ile HTH E. coli&#39;nin sadece suya, HWWS&#39;ye ve ABHS&#39;ye göre log indirgenmesine neden olmuştur (F (6,102) = 3.94, p &lt;0.001). Phi6 için toprak yükü olmayan yöntemler arasında anlamlı bir fark yoktu (F (6,66) = 2.04, p = 0.073). Bununla birlikte, toprak yükü olan Phi6 için (F (6,102) = 7.01, p &lt;0.001), su tek başına bir gre ile sonuçlandıABHS veya stabilize NaOCl&#39;den daha az log indirgemesi ve ABHS, stabilize NaOCl&#39;den daha büyük bir log redüksiyonunda HWWS ve üretilen NaOCl. HTH&#39;nin ayrıca ABHS ve stabilize NaOCl&#39;ye göre daha büyük bir log indirgemesi vardı ve NaDCC, stabilize NaOCl ve ABHS&#39;den daha büyük bir log indirgemesine neden oldu. HTH, istikrarsız olarak en iyi koşulları yerine getirirken, bir çok güven aralığı büyük olduğu için önemli sonuçların aşırı yorumlanmasına karşı uyarıda bulunacağız, pek çok durumda 0.5 logun altında ve 1.5 log azalmasına kadar değişir. Durulama suyunda klor, durulama suyunda devam eden E. coli&#39;nin HWWS&#39;den (toprak yükü olmaksızın F (4,68) = 331.7, p &lt;0.001, toprak yükü F (4,68 ) = 162.44, p &lt;0.001) ( Şekil 4 ). Aynı şekil, Phi6&#39;da toprak yükü olmaksızın bulundu ((F (4,43) = 8.95, P &lt;0.001), tüm klor çözeltileri,Durulama suyundaki Phi6&#39;nun HWWS&#39;den belirgin derecede daha fazla azaltılması. Durulama suyunda Phi6 ve zemin yükü ile persistanlık arasında anlamlı bir farklılık yoktu (F (4,67) = 3.35, p = 0.071) ( Şekil 5 ). <img alt="Şekil 1" class="xfigimg" src="/files/ftp_upload/55604/55604fig1.jpg" /> Şekil 1: Denetime genel bakış. El yıkama turlarının her biri için yapılan beş adım şunları içerir: 1) pH testi, 2) ellerin inoküle edilmesi, 3) el yıkama, 4) ellerin durulanması ve 5) test edilen sekiz koşulun her biri için ellerin arındırılması. <a href="http://ecsource.jove.com/files/ftp_upload/55604/55604fig1large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> <img alt="şekil 2" class="xfigimg" src="/files/ftp_upload/55604/55604fig2.jpg" /> Şekil 2: E. coli el yıkama çözeltisi<html> ka bul etmez. Elde yıkama yöntemine kıyasla, elle yıkama yöntemleri karşılaştırıldığında, test edilen el yıkama yöntemleri E. coli&#39;de topraktaki ortalama yük kaybı 1.94-3.01 ve zemin yükü ile 2.18-3.34 arasında ortalama bir log düşüşü ile sonuçlanmıştır. Su ile el yıkama, her iki koşulda E.coli&#39;de en düşük azalmayı göstermiştir (1.94 ve 2.18 log). NaDCC ile el yıkama, toprak yükü olmaksızın en büyük azalmaya neden olmuştur (3.01) ve HTH, toprağa yükle (3.34) en büyük azalmaya neden olmuştur. Çizelgelerdeki çizgi, organizmalarda yüzde düşüşü temsil eder ve hata çubukları log indirgemesinin standart hatasını temsil eder. Ctrl B, kontrol B; HWWS, sabunla el yıkama; ABHS, alkol bazlı el temizleyicisi; HTH, yüksek test hipoklorit; NaDCC, sodyum dikloroizosiyanürat; St NaOCl, stabilize sodyum hipoklorit; Gen NaOCl, sodyum hipoklorit üretti. <a href="http://ecsource.jove.com/files/ftp_upload/55604/55604fig2large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> Ve_content &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Şekil 3" class="xfigimg" src="/files/ftp_upload/55604/55604fig3.jpg" /> Şekil 3: Phi6 el yıkama sonuçları. Elde yıkama yöntemine kıyasla, elle yıkama yöntemine kıyasla, test edilen el yıkama yöntemleri Phi6&#39;da, zemin yükü olmaksızın 2.44-3.06 ve zemin yükü ile 2.71-3.69 arasında ortalama bir günlük düşüş ile sonuçlanmıştır. Sabunla el yıkama işlemi, Phi6&#39;da az miktarda azalma (2.44) ve stabilize NaOCl ile el yıkama işlemi toprak yüküyle en düşük azalma (2.71) ile sonuçlandı. Elde edilen NaOCl ile el yıkama, toprak yükü olmaksızın en büyük azalmaya (3.06) neden oldu ve sabunla el yıkama işlemi, zemin yükü (3.69) ile en büyük azalmaya neden oldu. Çizelgelerdeki çizgi, organizmalarda yüzde düşüşü temsil eder ve hata çubukları log indirgemesinin standart hatasını temsil eder. Ctrl B, kontrol B; HWWS, sabunla el yıkama; ABHS, alkol bazlı el temizleyicisi; HTH, yüksek test hipoklorit; NaDCC, sodyum dikloroizosiyanürat;St NaOCl, stabilize sodyum hipoklorit; Gen NaOCl, sodyum hipoklorit üretti. <a href="http://ecsource.jove.com/files/ftp_upload/55604/55604fig3large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> <img alt="Şekil 4" class="xfigimg" src="/files/ftp_upload/55604/55604fig4.jpg" /> Şekil 4: E. coli el yıkama sonuçları. Sadece el ile yıkama suyuna kıyasla, durulama suyunda kalan E. coli&#39;nin ortalama log azalması toprak yükü olmaksızın 0.28-4.77 ve toprak yükü ile 0.21-4.49 idi. Hem toprak yüklü hem de topraksız, en düşük azalma sabunlu elle yıkamada (0.28 ve 0.21) bulundu. En büyük azalmalar, toprak yükü olmaksızın stabilize edilmiş ve üretilmiş NaOCl (her ikisi de 4.77) ve HTH ile ve toprak yükü ile oluşturulan NaOCl ile gözlenmiştir. Çizelgelerdeki çizgi, organizmalarda yüzde düşüşü temsil eder ve hata çubukları,Log redüksiyonunun tandard hatası. HWWS, sabunla el yıkama; ABHS, alkol bazlı el temizleyicisi; HTH, yüksek test hipoklorit; NaDCC, sodyum dikloroizosiyanürat; St NaOCl, stabilize sodyum hipoklorit; Gen NaOCl, sodyum hipoklorit üretti. <a href="http://ecsource.jove.com/files/ftp_upload/55604/55604fig4large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a> <img alt="Şekil 5" class="xfigimg" src="/files/ftp_upload/55604/55604fig5.jpg" /> Şekil 5: Phi6 el yıkama sonuçları. Sadece el ile yıkama suyuna kıyasla, durulama suyunda kalan Phi6&#39;nın ortalama log düşüşü toprak yükü olmaksızın 1.26-2.02 ve toprak yükü ile 1.30-2.20 idi. Toprak yükü ile sabunla yapılan el yıkamada en düşük azalma bulundu (1.26). Toprak yükü olmaksızın, HTH en düşük redüksiyonla sonuçlandı (2.02). En büyük azalmalar, NaDCC ile toprak yükü olsun olmasın gözlemlendi(2.02 ve 2.20). Çizelgelerdeki çizgi, organizmalarda yüzde düşüşü temsil eder ve hata çubukları log indirgemesinin standart hatasını temsil eder. HWWS, sabunla el yıkama; ABHS, alkol bazlı el temizleyicisi; HTH, yüksek test hipoklorit; NaDCC, sodyum dikloroizosiyanürat; St NaOCl, stabilize sodyum hipoklorit; Gen NaOCl, sodyum hipoklorit üretti. <a href="http://ecsource.jove.com/files/ftp_upload/55604/55604fig5large.jpg" target="_blank">Bu rakamın daha büyük bir versiyonunu görmek için lütfen tıklayınız.</a>

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

Bulaşıcı hastalık bulaşmasını önlemek için el yıkama yaygın olarak önerilir. Bununla birlikte, bulaşıcı hastalık patojenlerinin çıkarılmasında hangi yıkama yöntemlerinin en etkili olduğuna dair çok az kanıt bulunmaktadır. Mikroorganizmaların yok edilmesinde el yıkama yöntemlerinin etkinliğini değerlendiren bir yöntem geliştirdik.

Yükselen Bulaşıcı Patojenlerin Giderilmesi İçin El Yıkama Etkinliğini Test Etmenin Bir Metodu

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.0K Görüntüleme.  Tufts University.  Bulaşıcı hastalık bulaşmasını önlemek için el yıkama yaygın olarak önerilir. Bununla birlikte, bulaşıcı hastalık patojenlerinin çıkarılmasında hangi yıkama yöntemlerinin en etkili olduğuna dair çok az kanıt bulunmaktadır. Mikroorganizmaların yok edilmesinde el yıkama yöntemlerinin etkinliğini değerlendiren bir yöntem geliştirdik. 

Video: 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.

Enterovirus Kültür ve RT-qPCR Su Norovirüs Oluşum EPA Metot 1615 Ölçüm. Virüs Örneklerinin I. Koleksiyonu

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

Çevre

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.

Sıcak Su Aklamanın Viral Dezenfeksiyon Etkinliğinin Belirlenmesi

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

Mühendislik

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.

Yüzeyler İnsan Norovirus Tespiti sürüntü Örnekleme Yöntemi

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

İmmünoloji ve Enfeksiyon

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.

Antimikrobiyal sinerji mürekkep püskürtmeli yazıcı yoluyla tarafından test otomatik Dama Tahtası dizi ve el ile zaman-kill yöntem

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.

VirWaTest, su örnekleri virüslerin tespiti için bir nokta-of-kullanım yöntemi

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.

Prion Güvenlik Laboratuvarı Kaydırma Testi

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.

Kentsel Çevrenin Taranması - Çevre Rezervuarlarından SARS-CoV-2'nin Örneklemesi ve Tespiti için Bir Boru Hattı

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

Biyoloji

Nano Bitki Kaplı Kumaşların Antimikrobiyal Etkinliği

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.

Yazar Spotlight: Esansiyel Yağların Nano-Bitkisel Kapsüllemesini Kullanan Antimikrobiyal Bir Kumaş  

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.

Uçucu yağların nano-bitkisel kapsüllemesini kullanan bir antimikrobiyal kumaş

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

Yükselen Bulaşıcı Patojenlerin Giderilmesi İçin El Yıkama Etkinliğini Test Etmenin Bir Metodu

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Yüzeyler İnsan Norovirus Tespiti sürüntü Örnekleme Yöntemi

Antimikrobiyal sinerji mürekkep püskürtmeli yazıcı yoluyla tarafından test otomatik Dama Tahtası dizi ve el ile zaman-kill yöntem

VirWaTest, su örnekleri virüslerin tespiti için bir nokta-of-kullanım yöntemi

Prion Güvenlik Laboratuvarı Kaydırma Testi

Kentsel Çevrenin Taranması - Çevre Rezervuarlarından SARS-CoV-2'nin Örneklemesi ve Tespiti için Bir Boru Hattı

Uçucu yağların nano-bitkisel kapsüllemesini kullanan bir antimikrobiyal kumaş

Uçucu yağların nano-bitkisel kapsüllemesini kullanan bir antimikrobiyal kumaş

Uçucu yağların nano-bitkisel kapsüllemesini kullanan bir antimikrobiyal kumaş