The authors have no acknowledgements.

1. Bacteria Suspension Preparation:
<ol>
	<li>From a streaked culture plate, aseptically inoculate a single colony into a 250 ml&#160;sterile bottle containing 100 ml&#160;of the appropriate growth media (Table 1) by way of sterile inoculating loop.</li>
	<li>Place the bottle into an orbital shaking incubator at the appropriate conditions (Table 1) for 1-2 days, checking for growth periodically. Set the shaker speed to 140 RPM.</li>
	<li>Aseptically transfer the bacterial suspension into two sterile 50 ml&#160;conical tubes and centrifuge the tubes at 3,000 x g for 20 min at 23 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend each bacterial pellet with 15 ml&#160;of sterile peptone water. Vortex as necessary. Combine the suspended bacteria culture into one tube/container.</li>
	<li>Titer the stock culture in triplicate per section 4 to determine the stock culture titer.</li>
	<li>Refrigerate for up to 2 weeks at 4 &#177; 2 &#176;C until needed. Re-titer the culture at the time of use, see step 4.8.</li>
</ol>
2. Platelet Product Preparation:
<ol>
	<li>Collect a standard apheresis human platelet product at an accredited blood bank. The platelet product must meet the following collection specifications: 90&#8211;360 ml&#160;volume and 0.8 &#8211;3.8 x 106 platelets/&#181;l&#160;concentration.
	<ol>
		<li>Ensure that the platelet product has rested for a minimum of 2 hr&#160;prior to processing for pathogen reduction. Store the products at 22 &#177; 2 &#176;C in a platelet incubator without agitation for these 2 hr. After two hours ensure the platelet product is agitated at 22 &#177; 2 &#176;C in a platelet incubator. Use the product for up to 22 hr&#160;post collection.</li>
	</ol>
	</li>
	<li>Using a 3 ml&#160;syringe remove a 2 ml&#160;sample to measure the pH22C of the platelet product. Use a blood gas analyzer to ensure the product has a pH &#62;6.6. Use the same sample to measure the platelet concentration of the platelet product using a hematological analyzer. Ensure the product meets the 0.80&#8211;3.8 x 106 platelets/&#181;l&#160;collection concentration requirement.</li>
	<li>Visually evaluate the swirl of the platelet product. If the product has a &#8220;0&#8221; swirl the product will be discarded.</li>
	<li>Hold the platelet bag up under a direct white-light source and squeeze the platelet bag briefly. Visually observe the swirling pattern of the platelet sample. Score the product using the following scale. 
	NOTE: 2 &#8211; Positive Swirl: Swirling inhomogeneity visible throughout the entire bag with strong contrast. 1 &#8211; Intermediate Swirl: Some inhomogeneity visible, but only in a few places and with poor contrast. 0 &#8211; Negative Swirl: Turbid homogeneity remaining before and after squeezing the platelet bag</li>
</ol>
3. Riboflavin and UV Light Process:
<ol>
	<li>Transfer the platelet product to a riboflavin and UV light illumination and storage bag using a sterile tubing docking device. After transfer remove the empty platelet unit collection bag using a tubing sealer and discard the empty bag as biohazardous waste.</li>
	<li>Remove a 35 ml&#160;pouch of 500 &#956;M riboflavin stock solution from its light protective outer wrap. Sterile dock the riboflavin kit onto the inlet line of the treatment and storage bag. Allow the contents to drain into the platelet unit.</li>
	<li>Hang the riboflavin pouch / treatment and storage bag set from the riboflavin pouch and gently express residual air out of the treatment and storage bag and into the riboflavin bag. Ensure a small amount of platelet product is also expressed into the riboflavin pouch to rinse any residual riboflavin into the platelet product. Allow the riboflavin and platelet solution drain back into the treatment and storage bag and then clamp the inlet line. Remove the empty riboflavin bag using a tubing sealer and discard the empty bag as biohazardous waste.</li>
	<li>Sterile dock a 6&#8221; tubing line with female syringe connector onto the inlet line of the illumination and storage bag. Attach a 10 ml&#160;syringe barrel (remove the plunger) onto the female syringe connector.</li>
	<li>Aseptically add 5 ml&#160;of stock bacteria into the syringe barrel via a pipette. Open the clamp on the inlet line and allow the bacteria to drain into platelet product. Rinse the syringe barrel by allowing some platelet product to flow back into the 10 ml&#160;syringe barrel. Remove the 10 ml&#160;syringe barrel.</li>
	<li>Aseptically attach a 30 ml&#160;syringe and flush the inlet port a minimum of two times to ensure the stock bacteria is mixed into the platelet product. After the product is well mixed, use a clean 5-10 ml&#160;syringe and remove a 2 ml&#160;pre-treatment sample. Using a new syringe ensure any air introduced from the inoculation step or sampling step is removed.
	<ol>
		<li>Titer the pre-treatment sample per section 4.</li>
	</ol>
	</li>
	<li>Remove the inlet line from the treatment and storage bag and weigh the product.</li>
	<li>Place the product into the illuminator and follow the onscreen commands to start a standard platelet treatment.
	<ol>
		<li>Enter in Operator ID.</li>
		<li>Secure and mount the bag to the Illuminator platen.</li>
		<li>Enter the Sample ID into the Illuminator using either the barcode reader or the manual entry keypad.</li>
		<li>Scan product type.</li>
		<li>Close the quartz clamp.</li>
		<li>The illumination device will automatically deliver 6.24 J/ml&#160;of energy. The typical treatment time takes 5-10 min.</li>
	</ol>
	</li>
	<li>Following treatment sterile dock a 6&#8221; tubing line with female syringe connector onto the sample bulb line of treatment and storage bag. Ensure the product is well mixed and then attach a 5 ml syringe and remove a 2 ml&#160;post-treatment sample.
	<ol>
		<li>Titer the post-treatment sample per section 4.</li>
	</ol>
	</li>
</ol>
4. Bacterial Titer
<ol>
	<li>Estimate the titer of the pre-treatment and post-treatment samples and prepare the appropriate number of 5 ml&#160;dilution tubes needed to perform an end point serial dilution of each sample. Aseptically fill the required tubes with 1.8 ml&#160;of peptone water.</li>
	<li>Titer each sample using a 10-fold serial dilution scheme. Aseptically transfer 0.2 ml&#160;from the undiluted sample to the first dilution tube (10-1). Vortex this dilution tube and transfer 0.2 ml&#160;to the next dilution tube. Continue the serial dilution through the required number of dilutions.
	<ol>
		<li>Aseptically transfer 100 &#181;l of each dilution to be plated to an appropriate agar plate (Table 1).</li>
	</ol>
	</li>
	<li>Using a sterile cell spreader, evenly distribute the sample onto each agar plate.</li>
	<li>Invert and incubate the plates at the appropriate conditions (Table 1) for 1-2 days checking for growth periodically.</li>
	<li>Count plates with 30&#8211;300 colonies. Plates containing greater than 300 colonies are considered too numerous to count (TNTC). Plates containing less than 30 colonies are considered to be below the limit of quantification (LOQ).</li>
	<li>Calculate the titer of each test sample and record the results as CFU/ml.
	<ol>
		<li>Use the following equation to calculate CFU/ml: 
		<img alt="Equation 1" fo:content-width="2.5in" src="/files/ftp_upload/52820/52820eq1.jpg" width="250" /></li>
		<li>If there are no colonies on the lowest dilution plate, use the following equation to calculate the Limit of Detection (LOD): 
		<img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq2.jpg" width="100" /> 
		and 
		<img alt="Equation 3" fo:content-width="1in" src="/files/ftp_upload/52820/52820eq3.jpg" width="100" /> 
		Where: 
		N =lowest number of particles (CFU) in product that can be detected with confidence. 
		P = probability that a microorganism will be undetected; for a 95% confidence of detecting a microorganism, P = .05. 
		V = total product volume in ml 
		v =volume plated (ml) x dilution level of plate</li>
	</ol>
	</li>
	<li>For Negative Control, plate 0.5 ml&#160;of the peptone water that is used for the serial dilutions onto each of two agar plates. Use the same plate type as the samples to be tested. Invert and incubate the plate at the appropriate conditions for 1-2 days checking for growth periodically.
	<ol>
		<li>If growth is observed on either agar plate, the bottle of peptone water used must be discarded; the results of the study are also invalidated.</li>
	</ol>
	</li>
	<li>For Positive Control, re-titer the stock culture of bacteria that was used. Invert and incubate the plates at the appropriate conditions for 1-2 days checking for growth periodically. The titer shall be &#177; 1.0 log CFU/ml&#160;of the recorded stock titer (See section 1.5) for the study to be considered valid.</li>
</ol>

<ol>
	<li>Brecher, M. E., Hay, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contamination+of+blood+components.">Bacterial contamination of blood components.</a> Clin. Microbiol. Rev. 18 (1), 195-204 (2005).</li><li>Brecher, M. E., Holland, P. V., Pineda, A. A., Tegtmeier, G. E., Yomtovian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+bacteria+in+inoculated+platelets:+implications+for+bacteria+detection+and+the+extension+of+platelet+storage.">Growth of bacteria in inoculated platelets: implications for bacteria detection and the extension of platelet storage.</a> Transfusion. 40 (11), 1308-1312 (2000).</li><li>Cardo, L. J., 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=Pathogen+inactivation+of+Leishmania+donovani+infantum+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Vox Sang. 90 (2), 85-91 (2006).</li><li>Cardo, L. J., Salata, J., Mendez, J., Reddy, H., Goodrich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+inactivation+of+Trypanosoma+cruzi+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Transfus. Apher. Sci. 37 (2), 131-137 (2007).</li><li>Dodd, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+viral+risks+of+blood+and+blood+products.">Current viral risks of blood and blood products.</a> Ann. Med. 32 (7), 469-474 (2000).</li><li>Dodd, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+contamination+and+transfusion+safety:+experience+in+the+United+States.">Bacterial contamination and transfusion safety: experience in the United States.</a> Transfus. Clin. Biol. 10 (1), 6-9 (2003).</li><li>Dumont, L. J., 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=Screening+of+single-donor+apheresis+platelets+for+bacterial+contamination:+the+PASSPORT+study+results.">Screening of single-donor apheresis platelets for bacterial contamination: the PASSPORT study results.</a> Transfusion. 50 (3), 589-599 (2010).</li><li>Goodrich, R. P., Gilmour, D., Hovenga, N., Keil, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+comparison+of+pathogen+reduction+technology+treatment+and+culture+of+platelet+products+for+addressing+bacterial+contamination+concerns.">A laboratory comparison of pathogen reduction technology treatment and culture of platelet products for addressing bacterial contamination concerns.</a> Transfusion. 49 (6), 1205-1216 (2009).</li><li>Greco-Stewart, V. S., 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=Serratia+marcescens+strains+implicated+in+adverse+transfusion+reactions+form+biofilms+in+platelet+concentrates+and+demonstrate+reduced+detection+by+automated+culture.">Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture.</a> Vox Sang. 102 (3), 212-220 (2012).</li><li>Keil, S. D., 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=Inactivation+of+Plasmodium+spp.+in+plasma+and+platelet+concentrates+using+riboflavin+and+ultraviolet+light.">Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light.</a> Transfusion. 53 (10), 2278-2286 (2013).</li><li>Kleinman, S., Chan, P., Robillard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+associated+with+transfusion+of+cellular+blood+components+in+Canada.">Risks associated with transfusion of cellular blood components in Canada.</a> Transfus. Med Rev. 17 (2), 120-162 (2003).</li><li>Larsen, C. P., Ezligini, F., Hermansen, N. O., Kjeldsen-Kragh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+years'+experience+of+using+the+BacT/ALERT+system+to+screen+all+platelet+concentrates,+and+additional+testing+of+outdated+platelet+concentrates+to+estimate+the+frequency+of+false-negative+results.">Six years' experience of using the BacT/ALERT system to screen all platelet concentrates, and additional testing of outdated platelet concentrates to estimate the frequency of false-negative results.</a> Vox Sang. 88 (2), 93-97 (2005).</li><li>Mundt, J. M., Rouse, L., Van den Bossche, J., Goodrich, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+Biological+Mechanisms+of+Pathogen+Reduction+Technologies.">Chemical and Biological Mechanisms of Pathogen Reduction Technologies.</a> Photochem. Photobiol. , (2014).</li><li>Murphy, W. G., 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=Screening+platelet+concentrates+for+bacterial+contamination:+low+numbers+of+bacteria+and+slow+growth+in+contaminated+units+mandate+an+alternative+approach+to+product+safety.">Screening platelet concentrates for bacterial contamination: low numbers of bacteria and slow growth in contaminated units mandate an alternative approach to product safety.</a> Vox Sang. 95 (1), 13-19 (2008).</li><li>Brien, S. 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=Current+incidence+and+residual+risk+of+HIV,+HBV+and+HCV+at+Canadian+Blood+Services.">Current incidence and residual risk of HIV, HBV and HCV at Canadian Blood Services.</a> Vox Sang. 103 (1), 83-86 (2012).</li><li>Pearce, S., Rowe, G. P., Field, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+platelets+for+bacterial+contamination+at+the+Welsh+Blood+Service.">Screening of platelets for bacterial contamination at the Welsh Blood Service.</a> Transfus. Med. 21 (1), 25-32 (2011).</li><li>Ramirez-Arcos, S., Kou, Y., Perkins, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+universal+point-of-issue+assay+for+bacterial+detection+in+buffy+coat+platelet+components.">Evaluation of a universal point-of-issue assay for bacterial detection in buffy coat platelet components.</a> Vox Sang. 107 (2), 192-195 (2014).</li><li>Reddy, H. L., 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=Toxicity+testing+of+a+novel+riboflavin-based+technology+for+pathogen+reduction+and+white+blood+cell+inactivation.">Toxicity testing of a novel riboflavin-based technology for pathogen reduction and white blood cell inactivation.</a> Transfus. Med. Rev. 22 (2), 133-153 (2008).</li><li>Ruane, P. H., 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=Photochemical+inactivation+of+selected+viruses+and+bacteria+in+platelet+concentrates+using+riboflavin+and+light.">Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light.</a> Transfusion. 44 (6), 877-885 (2004).</li><li>Tonnetti, L., Proctor, M. C., Reddy, H. L., Goodrich, R. P., Leiby, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Mirasol+pathogen+reduction+technology+system+against+Babesia+microti+in+apheresis+platelets+and+plasma.">Evaluation of the Mirasol pathogen reduction technology system against Babesia microti in apheresis platelets and plasma.</a> Transfusion. 50 (5), 1019-1027 (2010).</li><li>Vanlandingham, D. L., 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=Photochemical+inactivation+of+chikungunya+virus+in+plasma+and+platelets+using+the+Mirasol+pathogen+reduction+technology+system.">Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system.</a> Transfusion. 53 (2), 284-290 (2013).</li><li>Walther-Wenke, G., 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=Monitoring+bacterial+contamination+of+blood+components+in+Germany:+effect+of+contamination+reduction+measures.">Monitoring bacterial contamination of blood components in Germany: effect of contamination reduction measures.</a> Vox Sang. 100 (4), 359-366 (2011).</li></ol>

All authors of this paper are currently employed by Terumo BCT, developer and manufacturer of the Mirasol PRT system.&#160; Publication fees for this video-article have been paid by Terumo BCT.

Bacterial screening has become common practice in blood banking institutions and for many locations it serves as the primary method to prevent transfusion of bacterially contaminated platelets. Although bacterial screening typically interdicts the majority of fast growing Gram negative bacteria like S. marcescens, E. coli and E. cloacae, it struggles to detect slow growing Gram positive bacteria like S. aureus, Streptococcus spp. and S. epidermidis8. Unfortunately, these three Gram positive bacteria are implicated in upwards of 50% of all contaminated platelet units and transfusion of platelet products containing these three organisms has lead to patient mortality1.
Bacterial screening relies on the bacterial titer in a platelet product to be sufficiently high enough so that at least one organism is contained within the typical 4-8 ml screening sample. If the bacterial titer is not sufficiently high for a bacterium to be captured in the 4-8 ml sample volume, the unit is incorrectly flagged as negative and it is released for transfusion. An alternative approach to bacterial screening could be to employ a proactive process that can be performed on all platelet units and that could render the contaminating organisms as non-functional.
In this study, high titers of bacteria were purposely added to platelet units to test the total reduction capacity of the riboflavin and UV light pathogen reduction process. Although the bacterial loads tested in this study far exceed those normally found in blood products at the time of donation, this study helps to demonstrate that the riboflavin and UV light process is able to significantly reduce the titer of viable bacteria in a contaminated platelet unit by 2.6 to 5.4 log (99.7-99.9996% reduction of contaminating bacteria) (Figure 2). To obtain an accurate log reduction value, it is critical that the operator have good aseptic technique along with precise pipetting skills when performing the bacterial titer steps. Imprecise pipetting when performing serial dilutions can lead to an accumulation of errors, thus an inaccurate titer measurement.
The data collected in this study supports previous work with riboflavin and UV light, which challenged the system against low level, clinically relevant bacteremia (&#60;20 CFU/unit). The previous study was designed to mimic an actual clinical contamination event and the results showed riboflavin and UV light was effective in preventing bacterial growth over the 7-day platelet storage period. Modeling based on the data from the previous study suggests that the current risk estimates of bacterial contamination of platelet products could be reduced by as much as 98% from current levels8. The riboflavin and UV light process compares favorably to bacterial screening, which only demonstrated a 66% effectiveness to detect bacterial contaminants in platelet units when challenged with a clinically relevant bacterial load8.
As with all technologies, PRT treatment of platelet products to prevent complications associated with bacterially contaminated platelet units has its limitations. PRT systems are not effective against bacterial spores and do not inactivate endotoxin, thus even if a PRT system can inactivate high titers of Gram negative bacteria the remaining endotoxin can still cause septic shock in the recipient. PRT is best performed early during storage before the bacteria can propagate to high titers.
In conclusion, as demonstrated by the combination of these two studies, the riboflavin and UV light process is effective at reducing the bacterial loads of both Gram negative and Gram positive organisms and may offer a viable alternative to bacterial screening. Routinely treating platelet products with riboflavin and UV light also has the added benefit of reducing the potential transmission of other blood borne agents like viruses and parasites.

Transfusion of platelets, plasma, packed RBCs and whole blood products play a major role in modern medicine and can be used to treat various medical conditions, replace vital fluids and ultimately save lives. Platelets are a cellular product that are either isolated from whole blood and pooled into a transfusable dose or are collected through the process of platelet apheresis. The main role of platelets in the body is to stop bleeding at wound sites and to help maintain hemostasis. Patients suffering from a low platelet cell count (thrombocytopenia) are susceptible to spontaneous bleeding events and are transfused with platelets to bring their platelet cell count back into a normal range. Collected platelets are stored for a maximum of 5-7 days and are stored at 22 &#177; 2 &#176;C while under constant agitation.
Despite the life saving properties of platelet transfusions there remains a slight risk to transfusion recipients due to contamination of these products by parasites, bacteria and viruses11. The implementation of viral nucleic acid testing (NAT) has significantly decreased the risk of viral transmission of major blood borne agents, such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immuno-deficiency virus (HIV)5. A recent publication from the Canadian Blood Services estimates the residual risk for these agents to be 1 per 8 million donations for HIV, 1 per 6.7 million donations for HCV and 1 per 1.7 million donations for HBV15.
Although bacteria typically garner less attention in the general public, the frequency of bacterial contamination in platelet products has been estimated to be as high as 1:1,0007 and because millions of platelet products are transfused each year many recipients are exposed to potentially life threatening complications like sepsis6. Research suggests that the bacterial load at the time of contamination is low, &#60;100 colony forming units (CFU)/product2,16, however the nutrient rich environment and room temperature storage allows contaminating bacteria to proliferate to dangerously high titers prior to transfusion. Currently, the only approved methods available to prevent bacterially contaminated products from reaching a platelet recipient is through the use of culture based systems and rapid point of care testing. Briefly, for culture based systems platelet products are stored on an agitator at 22 &#176;C for 12-24 hr to allow bacteria to proliferate in the product, upon which a 4-8 ml sample is removed from the platelet product and inoculated into a bottle containing nutrient media. The bottle is placed into an instrument which monitors the bottle for bacterial growth. If the instrument detects bacterial growth in the bottle it is flagged and the corresponding platelet unit is discarded. While this process is reasonably successful at detecting fast growing organisms, many of the slow growing species do not grow to a high enough titer to be detected, thus creating the potential for false-negative units to be released for transfusion7,12,14,16,22. Unlike culture based detection systems, rapid point of care testing is typically performed later in the platelet storage period when the bacterial load has increased significantly. The higher titers are required since point of care tests are less sensitive than the culture based systems, and only reliably detect bacteria once they reach a titer &#8805;1 x 103 CFU/ml17. However such tests can provide results within 1 hour of sampling. Variability in the performance of these tests have led to the release of a false negative product, causing a fatal septic reaction in the recipient9.
An alternative way to combat the issue of bacterial contamination of platelet products is through the routine use of a pathogen reduction process that can inactivate contaminating bacteria instead of try to detect them. Using riboflavin as a photosensitizer, in combination with UV light, has been shown to reduce the infectivity of a broad range of pathogenic blood-borne contaminants, including bacteria3,4,8,10,19-21.The use of riboflavin and UV light for pathogen reduction is non-toxic and non-mutagenic, and riboflavin and UV light-treated components have been shown to be safe for transfusion recipients as well as for those handling blood products18. Briefly, riboflavin molecules can associate with the nucleic acids (DNA and RNA) of bacteria, parasites, viruses and any nucleated cell (e.g. white blood cells). Exposure to UV light activates riboflavin, causing a chemical alteration to functional groups of the nucleic acids (primarily guanine bases), thus preventing replication and/or transcription of the nucleic acids and leaving the organism inactivated13. Anucleated cells like platelets and red blood cells are not affected by the riboflavin chemistry due to the lack of nucleic acid.
Previous work with riboflavin and UV light technology evaluated a select group of bacteria using an experimental design intended to mimic a platelet product contaminated with a clinically relevant bacterial load (&#60;20 CFU/product)8. The goal of this study was to evaluate the riboflavin and UV light process against high titer bacterial contamination (&#62;1.0 x 105 CFU/ml) in platelet products treated in plasma in order measure the total bacterial reduction capacity of the system. Based on data collected from hemovigilance studies1, a panel of commonly occurring gram negative and gram positive organisms was selected for evaluation in this study and included the following species: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus mitis, Streptococcus pyogenes, Serratia marcescens, Yersinia enterocolitica, Brucella neotomae, Bacillus cereus (vegetative form), Esherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. All organisms, except B. cereus, were obtained from ATCC and a priority was placed on obtaining bacterial strains that had been identified as being isolated from blood components. The B. cereus tested in this study is a clinical strain isolated internally from a contaminated platelet product.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mirasol Illuminator</td>
			<td>Terumo BCT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirasol Treatment/Storage Bag</td>
			<td>Terumo BCT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematology Analyzer</td>
			<td>Beckman-Coulter</td>
			<td>Ac&#8226;T diff&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Gas Analyzer</td>
			<td>Siemens</td>
			<td>RapidLab 1265&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Docking Device</td>
			<td>Terumo BCT</td>
			<td>TSCD</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Incubator</td>
			<td>Helmer</td>
			<td>PC2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Incubator Shaker</td>
			<td>Lab-Line</td>
			<td align="right">4628</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Incubator</td>
			<td>Fisher</td>
			<td>Iso-temp</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II A/B3 Biosafety Cabinet</td>
			<td>Thermo-forma</td>
			<td align="right">1286</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Sealer</td>
			<td>Sebra</td>
			<td>Smart Sealer II</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Centrifuge</td>
			<td>IEC</td>
			<td>Centra GP8R</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>BD</td>
			<td align="right">309604</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL Syringe</td>
			<td>BD</td>
			<td align="right">309650</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Dilution Tube</td>
			<td>VWR</td>
			<td>211-0058</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Corning</td>
			<td align="right">430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Gilson</td>
			<td>P-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Rainin</td>
			<td>R-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeat Pipette</td>
			<td>Eppendorf</td>
			<td align="right">4780</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L Filter Tip</td>
			<td>Costar</td>
			<td align="right">4810</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Disposable Serrological Pipette</td>
			<td>Costar</td>
			<td align="right">4489</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Disposable Serrological Pipette</td>
			<td>Costar</td>
			<td align="right">4487</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mL 500 &#181;M Riboflavin</td>
			<td>Terumo BCT</td>
			<td>35 mL 500 &#181;M&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Brucella Agar with 5% Horse Blood</td>
			<td>BBL</td>
			<td align="right">221547</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Teknova</td>
			<td>B9993</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone Water</td>
			<td>BD</td>
			<td align="right">257087</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase Soy Broth</td>
			<td>BD</td>
			<td align="right">299113</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar</td>
			<td>Remel</td>
			<td>R01917</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Horse Serum</td>
			<td>Gibco</td>
			<td align="right">26050</td>
			<td></td>
		</tr>
	</tbody>
</table>

treatment of platelet products with riboflavin and uv light: effectiveness against high titer bacterial contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The reduction of eleven different bacterial species was evaluated using the riboflavin and UV light based PRT process. These agents represent some of the more common contaminants of platelet products and include five Gram positive and six Gram negative organisms. At least six platelet products were evaluated per organism and each product was inoculated with enough bacteria to yield &#62;1.0 x 105 CFU/ml&#160;final titer of bacteria in the platelet product. Prior to treatment a sample was removed to determine the pre-treatment titer, after which the platelet product was promptly treated with the riboflavin and UV process. Following treatment of the platelet products the following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805; 5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%) (Figure 2). All units met the system specifications as stated above for the riboflavin and UV process. Additional reduction testing was performed with S. pyogenes using buffy coat derived platelets. No significant difference was observed in the reduction of S. pyogenes in buffy coat derived platelets versus apheresis platelets (data not shown).
<img alt="Table 1" src="/files/ftp_upload/52820/52820table1.jpg" /> 
Table 1: Growth conditions for bacterial species evaluated using riboflavin and UV light. A panel of both Gram negative and Gram positive bacteria that have been shown to contaminate platelet products were selected for this study. The growth conditions (temperature and media type) used to propagate and titer the organisms are shown.
<img alt="Figure 1" src="/files/ftp_upload/52820/52820fig1.jpg" /> 
Figure 1: Riboflavin and UV light pathogen reduction process. Riboflavin was dissolved in 0.9% saline at a nominal concentration of 500 &#181;M. The solution was sterile connected and drained into each platelet product. Bacteria was added aseptically to each platelet unit and then treated with 6.2 J/ml&#160;of UV energy (approximately 6&#8211;10 min). In this in vitro study the post-treatment platelet unit was sampled to determine the final bacterial titer.
<img alt="Figure 2" src="/files/ftp_upload/52820/52820fig2.jpg" /> 
Figure 2: Reduction of different bacterial species when treated with riboflavin and UV light.&#160;Riboflavin and UV light was used to reduce the bacterial loads of common bacterial contaminants of platelet products. The log10 reduction is the difference between the average pre-treatment titer and the average post-treatment titer. <a href="https://www.jove.com/files/ftp_upload/52820/52820fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination at JoVE.com

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

treatment-platelet-products-with-riboflavin-uv-light-effectiveness

Research

JoVE Journal

Immunology and Infection

13.4K ビュー.  Scientific Affairs. The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

ビデオ: Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...