Name: Author Spotlight: Studying Behavior of Acanthamoeba to Develop Targeted Strategies for Preventing Acanthamoeba Keratitis
Uploaded: 2024-09-20T12:40:00
Duration: 1 min 11 s
Description: Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential ...

Long-Term Tracking and Quantification of Acanthamoeba Species Motility

<ol>
	<li>Randag, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rising+incidence+of+Acanthamoeba+keratitis:+A+7-year+nationwide+survey+and+clinical+assessment+of+risk+factors+and+functional+outcomes.">The rising incidence of Acanthamoeba keratitis: A 7-year nationwide survey and clinical assessment of risk factors and functional outcomes.</a> PLoS One. 14 (9), e0222092 (2019).</li><li>Szentmary, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis+-+Clinical+signs,+differential+diagnosis+and+treatment.">Acanthamoeba keratitis - Clinical signs, differential diagnosis and treatment.</a> J Curr Ophthalmol. 31 (1), 16-23 (2019).</li><li>Carnt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+keratitis:+confirmation+of+the+UK+outbreak+and+a+prospective+case-control+study+identifying+contributing+risk+factors.">Acanthamoeba keratitis: confirmation of the UK outbreak and a prospective case-control study identifying contributing risk factors.</a> Br J Ophthalmol. 102 (12), 1621-1628 (2018).</li><li>Verani, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+outbreak+of+Acanthamoeba+keratitis+associated+with+use+of+a+contact+lens+solution,+United+States.">National outbreak of Acanthamoeba keratitis associated with use of a contact lens solution, United States.</a> Emerg Infect Dis. 15 (8), 1236-1242 (2009).</li><li>Tu, E. Y., Joslin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+outbreaks+of+atypical+contact+lens-related+keratitis:+what+have+we+learned.">Recent outbreaks of atypical contact lens-related keratitis: what have we learned.</a> Am J Ophthalmol. 150 (5), 602-608.e2 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+14729:2001/A1.">ISO 14729:2001/A1.</a> Ophthalmic optics-Contact lens care products-Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses. , (2010).</li><li>. ASC Z80, Parent Committee Meeting, February 27 Available from: <a target="_blank" href="https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf">https://www.thevisioncouncil.org/sites/default/files/ASCZ80_ParentCommitteeMinutes_February_27_2018_FINALMar19-2018.pdf</a> (2023)</li><li>Rayamajhee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba,+an+environmental+phagocyte+enhancing+survival+and+transmission+of+human+pathogens.">Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.</a> Trends Parasitol. 38 (11), 975-990 (2022).</li><li>Reverey, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superdiffusion+dominates+intracellular+particle+motion+in+the+supercrowded+cytoplasm+of+pathogenic+Acanthamoeba+castellanii.">Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii.</a> Sci Rep. 5, 11690 (2015).</li><li>Thapa, S., Lukat, N., Selhuber-Unkel, C., Cherstvy, A. G., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+superdiffusion+of+polydisperse+vacuoles+in+highly+motile+amoeboid+cells.">Transient superdiffusion of polydisperse vacuoles in highly motile amoeboid cells.</a> J Chem Phys. 150 (14), 144901 (2019).</li><li>Rudell, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acanthamoeba+migration+in+an+electric+field.">Acanthamoeba migration in an electric field.</a> Invest Ophthalmol Vis Sci. 54 (6), 4225-4233 (2013).</li><li>Fukaya, S., Aoki, K., Kobayashi, M., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+the+motility+of+giant+virus-infected+amoebae+using+phase-contrast+microscopic+images.">Kinetic analysis of the motility of giant virus-infected amoebae using phase-contrast microscopic images.</a> Front Microbiol. 10, 3014 (2019).</li><li>Fukaya, S., Takemura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+Acanthamoeba+castellanii+infected+with+giant+viruses+quantitatively+revealed+process+of+morphological+and+behavioral+changes+in+host+cells.">Kinetic analysis of Acanthamoeba castellanii infected with giant viruses quantitatively revealed process of morphological and behavioral changes in host cells.</a> Microbiol Spectr. 9 (1), e0036821 (2021).</li><li>Cheng, H. J., Hsu, C. H., Hung, C. L., Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+for+cell+and+particle+tracking+on+microscopy+images+using+algorithms+and+deep+learning+technologies.">A review for cell and particle tracking on microscopy images using algorithms and deep learning technologies.</a> Biomed J. 45 (3), 465-471 (2022).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li><li>Svensson, C. M., Medyukhina, A., Belyaev, I., Al-Zaben, N., Figge, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untangling+cell+tracks:+Quantifying+cell+migration+by+time+lapse+image+data+analysis.">Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.</a> Cytometry A. 93 (3), 357-370 (2018).</li><li>Piltti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+time-lapse+imaging+and+single-cell+tracking+of+in+vitro+cultured+neural+stem+cells+-+Tools+for+analyzing+dynamics+of+cell+cycle,+migration,+and+lineage+selection.">Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells - Tools for analyzing dynamics of cell cycle, migration, and lineage selection.</a> Methods. 133, 81-90 (2018).</li><li>Campolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+real-time+motility+analysis+of+Acanthamoeba+reveals+sustained+movement+in+absence+of+nutrients.">Continuous real-time motility analysis of Acanthamoeba reveals sustained movement in absence of nutrients.</a> Pathogens. 10 (8), 995 (2021).</li><li>Campolo, A., Patterson, B., Lara, E., Shannon, P., Crary, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+recovery+of+Acanthamoeba+motility+among+surviving+organisms+after+contact+lens+care+disinfection.">Complete recovery of Acanthamoeba motility among surviving organisms after contact lens care disinfection.</a> Microorganisms. 11 (2), 299 (2023).</li><li>Crary, M. J., Walters, R., Shannon, P., Gabriel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variables+affecting+the+recovery+of+Acanthamoeba+trophozoites.">Variables affecting the recovery of Acanthamoeba trophozoites.</a> Pathogens. 10 (2), 221 (2021).</li><li>. Trackmate Available from: <a target="_blank" href="https://imagej.net/plugins/trackmate/tutorials/getting-started">https://imagej.net/plugins/trackmate/tutorials/getting-started</a> (2024)</li><li>Cherstvy, A. G., Nagel, O., Beta, C., Metzler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Gaussianity,+population+heterogeneity,+and+transient+superdiffusion+in+the+spreading+dynamics+of+amoeboid+cells.">Non-Gaussianity, population heterogeneity, and transient superdiffusion in the spreading dynamics of amoeboid cells.</a> Phys Chem Chem Phys. 20 (35), 23034-23054 (2018).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#188; Ringer&#8217;s solution</td>
			<td>ThermoFisher Scientific</td>
			<td>BR0052G</td>
			<td>Oxoid Ringers Solution Tablets. Follow directions to make one-quarter strength instead of full strength Ringers.&#160;</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette</td>
			<td>Eppendorf Research</td>
			<td>3123000039</td>
			<td>2 &#181;L-20 &#181;L single channel</td>
		</tr>
		<tr>
			<td>10 &#181;L pipette tips</td>
			<td>Neptune Scientific, San Diego, CA, USA</td>
			<td>BT10.N</td>
			<td>10 &#181;L Universal Barrier Tip</td>
		</tr>
		<tr>
			<td>48 well plate</td>
			<td>Millipore Sigma,&#160;</td>
			<td>CLS3548</td>
			<td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td>
		</tr>
		<tr>
			<td>50 mL conical tubes (1 for each sample, 1 for each pass 2 sample)</td>
			<td>Fisher Scientific</td>
			<td>Falcon 352098</td>
			<td>Falcon 50 mL High Clarity Conical Centrifuge Tubes, polypropylene</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Millipore Sigma, Burlington, MA, USA</td>
			<td>CLS3596</td>
			<td>Corning Costar TC-Treated Multiple Well Plate: polystyrene plate, flat bottom wells, sterile, with lid</td>
		</tr>
		<tr>
			<td>Acanthamoeba&#160;</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td>ATCC 30461</td>
			<td>This protocol has been verified for ATCC 50655, 30872, 50702, 30010, 30461, 50370, 50703, 30137, PRA-115, and PRA-411</td>
		</tr>
		<tr>
			<td>Axenic culture media (AC6)</td>
			<td>Made in house</td>
			<td>n/a</td>
			<td>Containing 20 g biosate peptone, 5 g glucose, 0.3 g KH2PO4, 10 &#181;g vitamin B12, and 1 glass5 mg&#160;l-methionine per liter of distilled deionized water. Adjust pH to 6.6-6.95 with 1 M NaOH and autoclave at 121 &#176;C for 20 min, store at room temperature for up to 3 months.&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge and appropriate rotor</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>Sorvall ST 40R</td>
			<td>Any equivalent centrifuge and rotor are acceptable so long as it can spin 50 mL conical tubes at 500 x g for 5 min</td>
		</tr>
		<tr>
			<td>Chemotaxis and Migration Tool</td>
			<td></td>
			<td></td>
			<td>Free software based on ImageJ, available at https://ibidi.com/chemotaxis-analysis/171-chemotaxis-and-migration-tool.html</td>
		</tr>
		<tr>
			<td>Disposable hemocytometer</td>
			<td>Bulldog Bio, Portsmouth, NH, USA</td>
			<td>DHC-N01</td>
			<td>Neubauer Improved 2-Chip Disposable Hemocytometer, Individually packaged, Nonpyrogenic</td>
		</tr>
		<tr>
			<td>ImageJ software with Trackmate plugin (this protocol written with Trackmate version 6.0.2.)</td>
			<td></td>
			<td></td>
			<td>Free software developed by the National Institutes of Health, available at imagej.net. Trackmate plugin available at https://imagej.net/plugins/trackmate/</td>
		</tr>
		<tr>
			<td>Microscope, preferably with automated moveable stage</td>
			<td>Nikon, Tokyo, Japan</td>
			<td></td>
			<td>A Nikon Eclipse Ti-U Microscope was used in this study and the automated moveable stage was utilized to be able to record images in multiple wells at a time.&#160;</td>
		</tr>
		<tr>
			<td>NIS-Elements software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>BrandTech 26331</td>
			<td>BrandTech accu-jet pro Pipet Controller</td>
		</tr>
		<tr>
			<td>Serological pipette tips</td>
			<td>VWR</td>
			<td>5 mL: 76201-710 
			10 mL: 170356 
			25 mL: 89130-900 
			50 mL: 75816-088</td>
			<td>VWR Serological Pipette, Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Sterile aluminum transmission flow cell</td>
			<td>Biosurface Technologies Corporation, Bozeman, MT, USA</td>
			<td>FC 81-AL</td>
			<td>Anodized aluminum single channel transmission flow cell with 96-well plate footprint for use with an inverted microscope</td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>734-2316</td>
			<td>VWR Tissue Culture Flask, 182.5 cm, Surface treated, Plug seal cap, Sterile</td>
		</tr>
	</tbody>
</table>

quantification of acanthamoeba spp. motility

Acanthamoeba research has increased dramatically in recent years due to the increased prevalence of Acanthamoeba keratitis (AK), which is a parasitic infection following the attachment of Acanthamoeba to the cornea1. While outbreaks of AK can be attributed to inappropriate contact lens care or ineffective contact lens care solutions2,3,4,5, there are currently no requirements to demonstrate Acanthamoeba disinfection efficacy for any product on the market. However, there is an ongoing effort in the scientific community and within standards organizations to examine the protocols necessary for quantifying the disinfection efficacy of contact lens care products6,7. Further, due to its resemblance in cellular and functional aspects to human macrophages, Acanthamoeba has been noted as having a significant role in hosting and disseminating other human pathogens8, in addition to the pathogenicity the amoeba itself brings.
Recent techniques have been described that have been able to quantify Acanthamoeba motility - which is generally not highly subject to Brownian movement9,10 - in regard to cytopathic effects or under-exposure to the electric field9,11, as well as advancements in motility analysis in giant virus research using Acanthamoeba as the trackable viral vector12,13. There have also been constant and significant improvements in cell and particle tracking occurring over the last 20 years using new software programs such as the imaging software used here, and new algorithms and deep learning technologies14. However, this is a relatively new and growing field of science regarding benchtop research, clinical application, and industrial standards, and there has been a paucity of data published regarding methods to visualize and track this amoeba, particularly in order to quantify behavioral changes following adherence to contact lenses or during or after contact lens disinfection. Other fields expanding into long-term visual monitoring have supported this effort15,16,17. Due to Acanthamoeba&#39;s inherently challenging nature - including a general resistance to plasmids (which could confer fluorescence), the ability of the amoeba to consume and destroy standard cell dyes, and a unique outer protein makeup - making antibody tagging difficult - methods available to other cells which make them visible in settings other than brightfield imaging have been unusable in this organism. Thus, quantifying the motility of this amoeba has demonstrated a significant addition to the field. Using the method described here, we have been able to ascertain that amoeba remain motile for at least 12 h without nutrients18 and that amoeba that is challenged with a disinfection process and ceases motility during disinfection can recover their motility post-disinfection if they are not fully lysed19.
This protocol details how to visually track and quantify the motility of amoeba microscopically. The primary steps are to record amoeba in brightfield using the appropriate focus and timing between images, transform the images into binary using imaging software, and then use an imaging software&#39;s tracking plugin to set the experimental parameters and follow each amoeba to determine the required measurements such as speed, distance, and confinement. Following this, it is possible to quantify the chemotaxis of an amoeba or an amoeba population in order to define directionality. The key contribution of this method is to visualize and quantify amoeba behavior during different states of nutritional support, surface adherence, disinfection challenge, or other environmental alterations such as cohabitation with mammalian cell culture.

Author Spotlight: Studying Behavior of Acanthamoeba to Develop Targeted Strategies for Preventing Acanthamoeba Keratitis

القياس الكمي للشوكميبة spp. الحركه