Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light-based photochemical treatment.

BACKGROUND AND OBJECTIVE: Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a member of the coronavirus family. Coronavirus infections in humans are typically associated with respiratory illnesses; however, viral RNA has been isolated in serum from infected patients. Coronaviruses have been identified as a potential low-risk threat to blood safety. The Mirasol Pathogen Reduction Technology (PRT) System utilizes riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious, while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against the pandemic virus SARS-CoV-2 when tested in both plasma and platelets units. MATERIALS AND METHODS: Stock SARS-CoV-2 was grown in Vero cells and inoculated into either plasma or platelet units. Those units were then treated with riboflavin and UV light. The infectious titres of SARS-CoV-2 were determined by plaque assay using Vero cells. A total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study. RESULTS: In both experiments, the measured titre of SARS-CoV-2 was below the limit of detection following treatment with riboflavin and UV light. The mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively. CONCLUSION: Riboflavin and UV light effectively reduced the titre of SARS-CoV-2 in both plasma and platelet products to below the limit of detection in tissue culture. The data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted SARS-CoV-2.

Inactivation of Middle East respiratory syndrome coronavirus (MERS-CoV) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) has been identified as a potential threat to the safety of blood products. The Mirasol Pathogen Reduction Technology System uses riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against MERS-CoV when tested in human plasma. STUDY DESIGN AND METHODS: MERS-CoV (EMC strain) was used to inoculate plasma units that then underwent treatment with riboflavin and UV light. The infectious titers of MERS-CoV in the samples before and after treatment were determined by plaque assay on Vero cells. The treatments were initially performed in triplicate using pooled plasma (n = 3) and then repeated using individual plasma units (n = 6). RESULTS: In both studies, riboflavin and UV light reduced the infectious titer of MERS-CoV below the limit of detection. The mean log reductions in the viral titers were ≥4.07 and ≥4.42 for the pooled and individual donor plasma, respectively. CONCLUSION: Riboflavin and UV light effectively reduced the titer of MERS-CoV in human plasma products to below the limit of detection, suggesting that the treatment process may reduce the risk of transfusion transmission of MERS-CoV.

Treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates Ebola virus in vitro.

BACKGROUND: Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called "convalescent plasma," is an effective treatment for active disease available in endemic areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV+RB) is effective against multiple enveloped, negative-sense, single-stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV+RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of Ebola virus disease (EVD). STUDY DESIGN AND METHODS: Four in vitro experiments were conducted to evaluate effects of UV+RB on green fluorescent protein EBOV (EBOV-GFP), wild-type EBOV in serum, and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1 to 3 were 4.21 log GFP units/mL, 4.96 log infectious units/mL, and 4.23 log plaque-forming units/mL. Conditions tested in the first three experiments included the following: 1-EBOV-GFP plus UV+RB; 2-EBOV-GFP plus RB only; 3-EBOV-GFP plus UV only; 4-EBOV-GFP without RB or UV; 5-virus-free control plus UV only; and 6-virus-free control without RB or UV. RESULTS: UV+RB reduced EBOV titers to nondetectable levels in both nonhuman primate serum (≥2.8- to 3.2-log reduction) and human whole blood (≥3.0-log reduction) without decreasing protective antibody titers in human plasma. CONCLUSION: Our in vitro results demonstrate that the UV+RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV+RB can improve convalescent blood product safety is indicated.

Reduction of Leishmania donovani infectivity in whole blood using riboflavin and ultraviolet light.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by the protozoan parasite Leishmania sp. that is transmitted by sandflies. Travelers to endemic areas, and US military personnel stationed in the Middle East, are at risk for contracting the disease. STUDY DESIGN AND METHODS: Whole blood (WB) units were spiked with human monocytes infected with L. donovani amastigotes to a final concentration of approximately 10(5) infected cells/mL. After riboflavin (RB) addition, units were exposed to 80 J/mLRBCs ultraviolet (UV) light. One pretreatment (collected after RB addition) and one posttreatment sample were collected, serially diluted in culture medium, and incubated at 22°C for up to 5 weeks. Parasite viability was determined by microscopic observation for replicating promastigote forms. RESULTS: Mirasol treatment of 3 units of L. donovani-infected WB with RB and UV light resulted in a parasite reduction of 2.3 ± 0.12 log. CONCLUSIONS: Partial reduction of L. donovani can be achieved in WB using RB and UV light. This technology may be useful when potential donors are exposed to Leishmania sp. during residence, travel, or military deployment to an endemic area.

The effect of riboflavin and ultraviolet light on the infectivity of arboviruses.

BACKGROUND: Arboviruses are an emerging threat to transfusion safety and rates of infection are likely to increase with the increased rainfall associated with climate change. Arboviral infections are common in Australia, where Ross River virus (RRV), Barmah Forest virus (BFV), and Murray Valley encephalitis virus (MVEV), among others, have the potential to cause disease in humans. The use of pathogen reduction technology (PRT) may be an alternative approach for blood services to manage the risk of arboviral transfusion transmission. In this study, the effectiveness of the Mirasol PRT (Terumo BCT) system at inactivating RRV, BFV, and MVEV in buffy coat (BC)-derived platelets (PLTs) was investigated. STUDY DESIGN AND METHODS: BC-derived PLT concentrates in additive solution (SSP+) were spiked with RRV, BFV, or MVEV and then treated with the Mirasol PRT system. The level of infectious virus was determined before and after treatment, and the reduction in viral infectivity was calculated. RESULTS: Treatment with PRT (Mirasol) reduced the amount of infectious virus of all three arboviruses. The greatest level of inactivation was observed for RRV (2.33 log; 99.25%), followed by BFV (1.97 log; 98.68%) and then MVEV (1.83 log; 98.42%). CONCLUSION: Our study demonstrates that treatment of PLT concentrates with PRT (Mirasol) reduces the infectious levels of RRV, BFV, and MVEV. The relevance of the level of reduction required to prevent disease transmission by transfusion has not been fully defined and requires further investigation. In the face of a changing climate, with its associated threat to blood safety, PRT represents a proactive approach for maintaining blood safety.

Riboflavin and ultraviolet light for pathogen reduction of murine cytomegalovirus in blood products.

BACKGROUND: Two studies were performed to test the effectiveness of riboflavin and ultraviolet (UV) light treatment (Mirasol PRT, Terumo BCT) against murine cytomegalovirus (MCMV). The first study utilized immune-compromised mice to measure the reduction of cell-free MCMV. A second study used a murine model to evaluate the ability of Mirasol PRT to prevent transfusion-transmitted (TT)-MCMV infection. STUDY DESIGN AND METHODS: Human plasma was inoculated with MCMV and then treated with Mirasol PRT. The viral titer was measured using an infectious dose 50% assay in nude mice. Mice were euthanized on Day 10 posttransfusion, and their spleens were tested for the presence of MCMV DNA using polymerase chain reaction (PCR). Mirasol PRT was also evaluated to determine its effectiveness in preventing TT-MCMV in platelets (PLTs) stored in PLT additive solution. PLTs were inoculated with either cell-associated MCMV or cell-free MCMV and then treated with Mirasol PRT. Mice were transfused with treated or untreated product and were euthanized 14 days posttransfusion. Blood and spleens were assayed for MCMV DNA by real-time-PCR. RESULTS: Using nude mice to titer MCMV, a modest 2.1-log reduction was observed in plasma products after Mirasol PRT treatment. TT-MCMV was not observed in the mouse transfusion model when either cell-free or cell-associated MCMV was treated with Mirasol PRT; MCMV transmission was uniformly observed in mice transfused with untreated PLTs. CONCLUSIONS: These results suggest that using riboflavin and UV light treatment may be able to reduce the occurrence of transmission of human CMV from infectious PLTs and plasma units.

Inactivation of viruses in platelet and plasma products using a riboflavin-and-UV-based photochemical treatment.

BACKGROUND: Multilayered blood safety programs reduce the risk of transfusion-transmitted diseases; however, there remains a risk of window period transmission of screened viruses and transmission of unscreened and emerging viruses from asymptomatic donors. To reduce this risk, a riboflavin-and-UV-light-based pathogen reduction process was evaluated against eight viral agents. STUDY DESIGN AND METHODS: Riboflavin and UV light was evaluated against the following eight viral agents: encephalomyocarditis virus (EMC), hepatitis A virus (HAV), hepatitis C virus (HCV), influenza A (FLUAV), La Crosse virus (LACV), pseudorabies virus (PRV), sindbis virus (SINV), and vesicular stomatitis virus (VSV). Before treatment, a sample was removed to determine the product's initial viral load. After treatment the product's viral load was reevaluated and the log reduction was calculated. RESULTS: Virus reduction after treatment with riboflavin and UV light is equivalent in platelet (PLT) and plasma units, as demonstrated by a 3.2-log reduction of EMC in plasma, PLTs, and PLT additive solution containing 35% plasma. Additionally, the following viral reductions values were observed: HAV 1.8 log, HCV at least 4.1 log, FLUAV at least 5.0 log, LACV at least 3.5 log, PRV 2.5 log, SINV 3.2 log, and VSV at least 6.3 log. CONCLUSIONS: The results observed in this study suggest that treating PLT and plasma products with a riboflavin-and-UV-light-based pathogen reduction process could potentially eliminate window period transmission of screened viruses and greatly reduce the risk of transfusion transmission of unscreened viruses.

Photochemical inactivation of chikungunya virus in plasma and platelets using the Mirasol pathogen reduction technology system.

BACKGROUND: Chikungunya virus (CHIKV) is a reemerging mosquito-borne virus that has been responsible for a number of large-scale epidemics as well as imported cases covering a wide geographical range. As a blood-borne virus capable of mounting a high-titer viremia in infected humans, CHIKV was included on a list of risk agents for transfusion and organ transplant by the AABB Transfusion-Transmitted Diseases Committee. Therefore, we evaluated the ability of the Mirasol pathogen reduction technology (PRT) system (CaridianBCT Biotechnologies) to inactivate live virus in contaminated plasma and platelet (PLT) samples. STUDY DESIGN AND METHODS: Plasma, PLTs, and phosphate-buffered saline controls were spiked with CHIKV and treated with riboflavin and varying doses of ultraviolet (UV) light using the Mirasol PRT system. Samples were tested before and after treatment for cytotoxicity, interference, and virus titer by titration and quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: A significant reduction in CHIKV titer of greater than 99% was recorded after treatment of plasma or PLTs with the Mirasol PRT system, and the titer reduction was directly proportional to the UV dose delivered to the samples. No cytotoxicity of interference was observed in any sample at any treatment dose. CONCLUSION: These data indicate that the Mirasol PRT system efficiently inactivated live CHIKV in plasma and PLTs and could therefore potentially be used to prevent CHIKV transmission through the blood supply.

Development of a riboflavin and ultraviolet light-based device to treat whole blood.

BACKGROUND: In the United States, blood components are commonly used for patients in need of massive transfusion after blood loss. In combat situations, when severe traumatic injuries occur far from a hospital, fresh whole blood is a valuable transfusion therapy because components may not be available. The risk of infectious or immunological complications from fresh whole blood transfusions could be mitigated by a system that reduces pathogen loads and inactivates white blood cells (WBCs). Such a system is in development and utilizes riboflavin and ultraviolet light to provide pathogen reduction and WBC inactivation. STUDY DESIGN AND METHODS: The system has been tested with in vitro and in vivo animal studies to evaluate WBC inactivation and pathogen reduction, and with in vitro studies to assess the function of the treated blood products. RESULTS: Elimination of viable WBCs with the system is equivalent to gamma-irradiation. Results have been reported for reduction of Babesia microti, Trypanosoma cruzi, HIV, and bacteria, and preliminary results for Babesia divergens are available. Treated whole blood, platelets, and plasma maintain coagulation function. Treated red blood cell components exhibit low hemolysis and high adenosine triphosphate levels at the end of storage. CONCLUSIONS: Treatment with riboflavin and ultraviolet light is a promising alternative to gamma-irradiation. Effectiveness of the system against a variety of pathogens has been established, and further studies are planned. The in vitro studies of function indicate that treated whole blood, as well as components from treated whole blood, will provide acceptable hemostasis and perform well in the next phase of in vivo studies.

Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Photochemical treatment of blood products could help prevent transfusion-transmitted malaria and reduce the need for donor deferrals. In this study we evaluated the effectiveness of riboflavin and ultraviolet (UV) light against both Plasmodium falciparum, which causes the most severe form of human malaria, and Plasmodium yoelii, an in vivo murine model for malaria. STUDY DESIGN AND METHODS: Plasma and platelet (PLT) concentrates were inoculated with either P. falciparum- or P. yoelii-infected red blood cells (RBCs). Aliquots from each unit were collected after inoculation, after addition of riboflavin, and after treatment. In vitro P. falciparum growth was assessed using thin blood films of duplicate samples at 24, 48, 72, and 96 hours. P. yoelii parasitemia was followed in mice for 14 days postinoculation. RESULTS: In the in vitro studies, the mean P. falciparum parasitemia increased 12- to 19-fold in pretreatment samples, both before and after addition of riboflavin, after 96-hour culture. Few parasites were observed in Mirasol-treated units at 24 hours; those that were observed were degenerating. Through the remainder of the 96-hour culture period, cultures of treated samples were negative. In the in vivo study, mouse plasma containing P. yoelii-infected RBCs had a mean starting titer of 4.6 log mouse infectious dose 50%/mL. No infectious parasite was detected in treated samples. CONCLUSION: Treatment with riboflavin and UV light was effective at reducing viable P. falciparum in both PLT and plasma products by at least 3.2 logs. Additionally, an at least 4.4-log reduction was observed with P. yoelii.

Riboflavin and ultraviolet light reduce the infectivity of Babesia microti in whole blood.

BACKGROUND: Babesia microti is the parasite most frequently transmitted by blood transfusion in the United States. Previous work demonstrated the efficacy of riboflavin (RB) and ultraviolet (UV) light to inactivate B.microti in apheresis plasma and platelet units. In this study we investigated the effectiveness of RB and UV light to reduce the levels of B.microti in whole blood (WB). STUDY DESIGN AND METHODS: WB units were spiked with B. microti-infected hamster blood. Spearman-Karber methods were used to calculate infectivity of each sample in terms of hamster infectious dose 50% (HID50 ) value. After RB addition, the units were illuminated with 80 J/mLRBC UV light. Two samples were collected: one before illumination and one after illumination. The samples were serially diluted and dilutions injected into a group of five naive hamsters. Four weeks postinoculation (PI), blood was collected from the animals and evaluated by microscopic observation. RESULTS: One pilot study showed a good dose response in the animals and demonstrated that sample infectivity could be calculated in terms of an HID50 . Three additional replicates were performed in the same manner as the pilot study, but with fewer dilutions. Infectivity values were consistent between the experiments and were used to calculate log reduction. The posttreatment reduction of B. microti for all the experiments was more than 5 log. CONCLUSIONS: The data collected indicate that use of RB and UV is able to decrease the parasite load in WB units thus reducing the risk of transfusion-transmitted B. microti from blood components containing B. microti-infected RBCs.

Evaluating pathogen reduction of Trypanosoma cruzi with riboflavin and ultraviolet light for whole blood.

BACKGROUND: Trypanosoma cruzi, the protozoan parasitic agent of Chagas disease, can be transmitted by blood transfusion. In 2007, most US blood banks started screening blood donations for T. cruzi, but the cost and perceived need of the test have been the subject of ongoing discussion. In this study, we evaluated the ability of the Mirasol System (CaridianBCT), which uses riboflavin (RB) and ultraviolet light to inactivate pathogens, to reduce the levels of infectious T. cruzi in whole blood (WB). STUDY DESIGN AND METHODS: WB units were inoculated with 4, 40, 400, and 4000 trypomastigotes/mL. After addition of RB and illumination at various energy levels, the samples were tested for the presence of live parasites by hemoculture. RESULTS: All preillumination samples exhibited T. cruzi growth in hemoculture, while postillumination samples from units containing 4 and 40 trypomastigotes/mL showed no signs of viable parasites after 16 weeks of culture. In contrast, at both 400 and 4000 parasites/mL, two of the three units were positive for viable parasites. CONCLUSIONS: The total log reduction observed for T. cruzi was 3.5 log or greater, but less than 4.5 log. This level of reduction is likely to be orders of magnitude higher than what would be expected in a tainted blood donation, indicating that the Mirasol System could be effective at preventing transfusion of the causative agent of Chagas disease.

A laboratory comparison of pathogen reduction technology treatment and culture of platelet products for addressing bacterial contamination concerns.

BACKGROUND: Concerns over the risk of bacterial contamination of platelet products have led to implementation of bacteria culture and other screening methods. New approaches for dealing with this issue have also been proposed. STUDY DESIGN AND METHODS: A direct comparison of treatment with riboflavin and ultraviolet (UV) light (Mirasol pathogen reduction technology [PRT] system) versus bacterial culture testing (two-bottle system, 48-hour quarantine) was undertaken to compare their effectiveness. Thirteen clinically relevant bacterial organisms (20 strains) were used in this evaluation. Results were compared with spiking levels at 20 to 100 colony-forming units (CFUs) per product and at less than 20 CFUs per product. RESULTS: At spiking levels of 20 to 100 CFUs per product, the riboflavin and UV light process demonstrated 91% effectiveness against a broad spectrum of bacteria. In comparison, the culture method demonstrated an ability to detect up to 91% of the same contaminants, when used in the two-bottle, 48-hour-to-release configuration. At lower initial titers of contaminating agents (<20 CFUs per product), the effectiveness of PRT increased to 98% whereas the culture method effectiveness decreased to 66%. Effectiveness of the culture method further decreased to 60% when a one-bottle system was used. CONCLUSION: The results from this work suggest that the riboflavin and UV light process may provide up to 98% protection against transfusion of bacterially contaminated units at the most clinically relevant contamination levels (<20 CFUs per product). This compares favorably to the 60% to 66% effectiveness of bacterial culture testing using a 48-hour quarantine period before product release.

Evaluation of Mirasol pathogen reduction system by artificially contaminating platelet concentrates with Staphylococcus epidermidis: A pilot study from India.

BACKGROUND AND OBJECTIVES: This study was conducted to assess the efficacy of Mirasol pathogen reduction system for platelets aimed at preventing bacterial regrowth by spiking buffy coat pooled platelets (BCPP) with clinically relevant load of Staphylococous epidermidis. MATERIALS AND METHODS: BCPP units were prepared using Teruflex BP-kit with Imugard III-S-PL (Terumo BCT, Tokyo, Japan). Two BCPP units were pooled, of which 40 ml of negative control (NC) was removed. The remaining volume of the platelet unit was inoculated with clinically relevant load of bacteria (total of 30 CFU of S. epidermidis in 1 ml); following this the platelet unit was split into two parts. One part served as positive control (PC) and the other part was subjected to pathogen reduction technique (Mirasol PRT, CaridianBCT Biotechnologies, Lakewood, CO, USA). Bacterial detection was performed using BacT/ALERT system, controls after day 1 and day 7 following inoculation of bacteria and on day 7 for Mirasol-treated unit. RESULTS: Of the 32 treatment cycles, 28 were valid and 4 were invalid. No regrowth was observed in 96.4% (27 of 28) after treatment with Mirasol pathogen reduction system. Of four invalid tests, on two instances the NC showed growth, whereas in other 2 no regrowth was detected in 7(th) day PC. Bacterial screening of PCs by BacT/ALERT after 24 h of incubation was 28.6%, whereas the effectiveness increased to 100% when incubated for 7 days. CONCLUSIONS: Mirasol system was effective in inactivating S. epidermidis when it was deliberately inoculated into BCPP at clinically relevant concentrations. Such systems may significantly improve blood safety by inactivating traditional and emerging transfusion-transmitted pathogens.

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 °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 ≥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.

Treatment of Whole Blood With Riboflavin and UV Light: Impact on Malaria Parasite Viability and Whole Blood Storage.

BACKGROUND: Sub-Saharan African countries utilize whole blood (WB) to treat severe anemia secondary to severe blood loss or malaria on an emergency basis. In many areas with high prevalence of transfusion-transmissible agents, blood safety measures are insufficient. Pathogen reduction technology applied to WB might considerably improve blood safety. METHODS: Whole blood from 40 different donors were treated with riboflavin and UV light (pathogen reduction technology) in order to inactivate malaria parasite replication. The extent of parasite inactivation was determined using quantitative polymerase chain reaction methods and was correlated to studies evaluating the replication of malaria parasites in culture. Products were also stored for 21 days at +4°C and monitored for cell quality throughout storage. RESULTS: Plasmodium amplicon was present in 21 samples (>100 copies/mL), doubtful in four (10-100 genome equivalents [gEq]/mL), and negative in 15 U. The majority of asymptomatic parasitemic donors carried low parasite levels, with only six donors above 5,000 copies/mL (15%). After treatment with riboflavin and UV light, these six samples demonstrated a 0.5 to 1.2 log reduction in quantitative polymerase chain reaction amplification. This correlated to equal to or greater than 6.4 log reductions in infectivity. In treated WB units, cell quality parameters remained stable; however, plasma hemoglobin increased to 0.15 g/dL. All markers behaved similarly to published data for stored, untreated WB. CONCLUSIONS: Pathogen reduction technology treatment can inactivate malaria parasites in WB while maintaining adequate blood quality during posttreatment cold storage for 21 days.

Riboflavin and UV-light based pathogen reduction: extent and consequence of DNA damage at the molecular level.

We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.

Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light.

BACKGROUND: A medical device is being developed for the reduction of pathogens in PLT concentrates (PCs). The device uses broadband UV light and the compound riboflavin (vitamin B(2)). STUDY DESIGN AND METHODS: Pathogens were added to single-donor PLTs. After treatment, the infectivity of each pathogen was measured using established biologic assays. In vitro PLT performance was evaluated after treatment and after 5 days of storage using a panel of 10 in-vitro cell quality assays. RESULTS: In studies with viral pathogens, the Pathogen Reduction Technology (PRT) system provided average log reduction factors of 4.46 +/- 0.39 for intracellular HIV, 5.93 +/- 0.20 for cells associated HIV, and 5.19 +/- 0.50 for West Nile virus. For the nonenveloped porcine parvovirus, a reduction factor greater than 5.0 log was observed. Staphylococcus epidermidis and Escherichia coli bacteria were also tested with observed reduction factors to the limits of detection of 4.0 log or greater. PLT cell quality was adequately maintained after treatment and during storage. Although P-selectin expression, glucose consumption, and lactate production increased relative to controls, this was not beyond accepted levels. The pH of treated PCs also decreased slightly relative to control PLTs on Days 1 and 5. CONCLUSION: The data indicate that the device successfully reduced the number of selected pathogens in PCs. Despite the fact that significant differences exist between treated and control in-vitro variables, it is speculated that the clinical effectiveness of both products will not be significantly different, based on comparison to historical data for products in routine clinical use today.