BLOOD COMPONENTS BRIEF REPORT Reduction of Leishmania donovani infectivity in whole blood using riboflavin and ultraviolet light Laura Tonnetti,1 Aaron M. Thorp,1 Heather L. Reddy,2 Shawn D. Keil,2 Suzann K. Doane,2 Raymond P. Goodrich,2 and David A. Leiby1

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 105 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.

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eishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania that are endemic to areas of the tropics, subtropics, and southern Europe.1 The parasite is naturally transmitted to humans through the bite of an infected female hematophageous sandfly with the geographic distribution of the parasite limited by the distribution of the sandfly.2 To date, the relatively few cases of Leishmania transmitted through blood transfusion (n = 15) have been reported primarily in endemic areas and attributed to asymptomatic infections.3 Pathogen reduction technologies using riboflavin (RB) and ultraviolet (UV) light have been shown to reduce the parasite load up to 7 log in platelets (PLTs) and plasma;4 this study explores reduction of L. donovani induced in whole blood (WB).

ABBREVIATIONS: dPBS = Dulbecco’s phosphate-buffered saline; ΔFBS = heat-inactivated fetal bovine serum; RB = riboflavin; SDM = Schneider’s Drosophila medium; WB = whole blood. From the 1Transmissible Diseases Department, American Red Cross Holland Laboratory, Rockville, Maryland; and 2Terumo BCT, Lakewood, CO. Address reprint requests to: Laura Tonnetti, PhD, Transmissible Diseases Department, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855; e-mail: [email protected] Supported by Terumo BCT, DOD Grant W81XWH-09-20100. The US Army Medical Research Acquisition Activity was the awarding and administering acquisition office. The content of the information contained herein does not necessarily reflect the position or the policy of the government and no official endorsement should be inferred. Received for publication January 29, 2014; revision received June 26, 2014, and accepted June 30, 2014. doi: 10.1111/trf.12820 © 2014 AABB TRANSFUSION 2015;55:326–329. TRANSFUSION **;**:**-**.

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MATERIALS AND METHODS Maintenance of L. donovani cultures Leishmania promastigotes were cultured at 22°C in Schneider’s Drosophila medium (SDM; Gibco, Grand Island, NY) containing 20% heat-inactivated fetal bovine serum (ΔFBS; Gibco). Stationary-phase promastigotes, used to infect the monocytes, were obtained after 7 days of culture at a parasite concentration of 2 × 107 to 5 × 107/mL.

Research Blood Program. This protocol was approved by the local institutional review board and by the US Army Medical Research and Materiel Command’s Office of Research Protections, Human Research Protections Office. Blood units were collected in Fenwal (Fenwal Inc., Lake Zurich, IL) sets containing 70 mL of CPD and were not leuokoreduced. Each unit was collected from a single donor on the day of the experiment.

Monocyte isolation and infection with L. donovani

Treatment with the Mirasol System

Peripheral blood mononuclear cells (PBMNCs) were collected from research PLT donors that met all FDA and AABB criteria for regular donation (i.e., weight, hematocrit [Hct], PLT counts, pathogen testing, blood component donation intervals). PBMNCs were recovered from the contents of a discarded Trima set and isolated by density sedimentation in Ficoll solution at Terumo BCT and then shipped overnight to Holland Laboratory in 50-mL conical tubes containing RPMI and 10% FBS. The tubes were centrifuged at 200 × g for 15 minutes at 4°C. After the supernatant was discarded, each pellet was resuspended in 15 mL of Hanks’ balanced salt solution (Gibco) and cells were centrifuged at 200 × g for 15 minutes at room temperature. The supernatant was discarded and each tube was resuspended in 30 mL of RPMI containing 2% ΔFBS, 1% l-glutamine, and 0.4% gentamicin. The contents of each tube were transferred to a 150-cm2 flask and incubated at 37°C, 5% CO2 for 4 to 6 hours to allow the monocytes to adhere to the flask. After incubation, the medium containing nonadherent monocytes was discarded and replaced with 30 mL of fresh medium. The total number of monocytes per flask was estimated based on cell counts performed at the time of collection. Each flask was inoculated with 1 mL of resuspended promastigotes at a multiplicity of infection of 4:1 (parasites/monocytes) and incubated overnight at 37°C, 5% CO2. The following morning, the medium was discarded to remove free promastigotes and flasks were washed with 5 mL of sterile Dulbecco’s phosphate-buffered saline (dPBS). To collect adherent cells, 5 mL 0.25% trypsin-EDTA (Gibco) was added to each flask and cells were removed using a cell scraper and gentle mechanical disruption. To quench the trypsin, 20 mL of RPMI containing 5% ΔFBS was added and the medium was collected in a 50-mL conical tube. Infected monocytes were centrifuged at 200 × g for 10 minutes. All pellets were combined and resuspended in 8 mL of sterile dPBS. Recovered monocytes were counted using a hemocytometer, and the percent infected was determined by microscopic examination of Giemsastained cells.

The Mirasol System requires the transfer of WB to a Mirasol illumination bag, the addition of 35 mL of 500 μmol/L RB solution, and the delivery of the UV energy dose. After the Hct was measured manually, the WB units were inoculated with 8 mL of L. donovani–infected human monocytes, to a final concentration ranging between 104 and 105 infected monocytes/mL. The WB units were gently and thoroughly mixed and then attached to the Mirasol illumination bag using a sterile docking device and the WB was allowed to flow until the total volume of 520 mL had been reached. The weight necessary to provide a 520-mL volume was calculated as

Collection of WB WB units were collected under an approved human use protocol by the American Red Cross Holland Laboratory 2

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Volume (mL ) × Density (g mL ) = Weight (g ) , where the density was calculated using the previously measured Hct value. After 35 ± 5 mL of RB was added and the residual air was removed, the WB was gently mixed and a 20-mL sample was removed (before illumination) for testing and manual measurement of Hct. Once the illumination set and the inlet line were sealed, the set was placed in the illuminator and illuminated with an UV energy dose of 80 J/mLRBCs. After the treatment finished, the set was removed from the illuminator and a 40-mL sample was collected (after illumination). Both pre- and postillumination samples were washed twice in PBS before culturing and resuspended in culture medium. The mean illumination time was 44 minutes.

Sampling, parasite detection, and statistical analysis For all experiments, each WB unit had a 20-mL preillumination sample and a 40-mL postillumination sample removed to determine the 50% infection dose. The preillumination sample was collected after the addition of the RB, whereas the postillumination sample was collected upon completion of the delivery of the UV energy dose. The samples collected before and after the Mirasol treatment were washed twice with dPBS and resuspended in SDM before placing in culture at 22°C. Samples were serially diluted 1:10 in SDM to a relative concentration of 10−5 for the preillumination samples and 10−3 for the postillumination samples. Each sample was cultured in Volume 55, February 2015 TRANSFUSION 327

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Fig. 1. Parasite titers and the log reduction measured for each unit.

replicates of either n = 5 (before illumination) or n = 10 (after illumination) and observed weekly for the presence of motile promastigotes under an inverted microscope at 20× for up to 5 weeks. The 50% infection dose was calculated by the Spearman-Karber method as previously described.5

RESULTS Six L. donovani–infected WB units were treated with RB and UV light. Data from 3 units were used to optimize the experimental conditions (data not shown), while the other 3 units were used to determine reduction of L. donovani. The mean illumination volume was 527 ± 6 mL and the mean Hct for illumination was 37% ± 2%. The units were spiked with L. donovani–infected monocytes to an average final concentration of 5.36 × 104 infected cells/ mL, with each monocyte infected with one to six parasites. Figure 1 lists the parasite titers and the log reduction measured for each unit. The mean log reduction of L. donovani for the 3 units was 2.3 ± 0.12.

DISCUSSION Transmission of the Leishmania parasite through blood transfusion has not been reported in the United States. 328 TRANSFUSION Volume 55, February 2015

However, cases of transfusion-transmitted L. donovani have been described in several countries.3,6-9 Most of the reports occurred before 1997 and do not contain specific manufacturing details about the transfused blood, but it is unlikely that the blood was leukoreduced. The widespread use of leukoreduction filters at the present time may explain the relatively low number of recently reported cases.10 However, one case from India was attributed to PLTs, suggesting that extracellular parasites, or residual white blood cells, could be responsible for transmitting the parasite by transfusion.11 Visceral infection with L. donovani and L. infantum is asymptomatic in more than 95% of the cases, which creates the dual effect of increasing the risk of transmission by transfusion, while underestimating the actual number of transfusion cases. In addition, the potential risk of Leishmania transmission is further validated by the survivability of the parasite under standard blood bank storage conditions.12 The absence of a viable donor screening test coupled with cost–benefit considerations related in part to the paucity of transfusion cases suggests that blood bank screening for Leishmania is unlikely to be implemented, even though the parasite is present in peripheral blood, capable of surviving blood storage conditions and remaining infectives. Still, transfusion of infected blood Volume **, ** **

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carrying the parasite poses a risk to blood transfusion recipients, and for this reason, Leishmania parasites are of interest to the US military with respect to the pathogen reduction of blood products. The goal of this study was to measure the reduction achieved by the Mirasol System for WB against L. donovani when treated with a UV energy dose of 80 J/ mLRBCs. We dedicated particular attention to the preparation of the inoculum to infect the blood units. To ensure that the correct life cycle stage of the parasite was used to infect the blood units, L. donovani promastigotes were kept in culture until they reached the stationary phase, the stage when the parasites more efficiently infect monocytes. To achieve the maximum infection rate, we tested different incubation times and eventually chose an overnight incubation, which demonstrated a monocyte infection rate between 68 and 78%. We also washed any free promastigotes from the culture, ensuring that only the intracellular amastigote form of the parasite, which is the life cycle stage present in the blood of an infected donor, was injected into the blood units. Data collected from three experiments showed a mean reduction of 2.3 log. However, RB and UV light seem to perform better in reducing L. donovani in plasma and PLTs than in WB. This could be due to the fact that most of the parasites were intracellular at the time of treatment and not easily accessible by the reducing factors. Still, we were able to show reduction of the parasite in WB and the study provides a proof of concept for the efficacy of this process against Leishmania in WB. CONFLICT OF INTEREST HLR, SDK, and RPG are employed by Terumo BCT, the company that developed and sells the Mirasol Pathogen Reduction Tech-

2. Herwaldt BL. Leishmaniasis. Lancet 1999;354:1191-9. 3. Dey A, Singh S. Transfusion transmitted leishmaniasis: a case report and review of literature. Indian J Med Microbiol 2006;24:165-70. 4. Cardo LJ, Rentas FJ, Ketchum L, et al. Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light. Vox Sang 2006;90:85-91. 5. Tonnetti L, Thorp AM, Reddy HL, et al. Riboflavin and ultraviolet light reduce the infectivity of Babesia microti in whole blood. Transfusion 2013;53:860-7. 6. Andre R, Brunt L, Dreytus B, et al. Leishmaniose cutanee. Leishmaniose cutanee-ganglionnare et kala-azar transfusional. Trop Dis Bull 1958;55:379-81. 7. Cummins D, Amin S, Halil O, et al. Visceral leishmaniasis after cardiac surgery. Arch Dis Child 1995;72:235-6. 8. Mpaka MA, Daniil Z, Kyriakou DS, et al. Septic shock due to visceral leishmaniasis, probably transmitted from blood transfusion. J Infect Dev Ctries 2009;3:479-83. 9. Singh S, Chaudhry VP, Wali JP. Transfusion-transmitted kala-azar in India. Transfusion 1996;36:848-9. 10. Kyriakou DS, Alexandrakis MG, Passam FH, et al. Quick detection of Leishmania in peripheral blood by flow cytometry. Is prestorage leucodepletion necessary for leishmaniasis prevention in endemic areas? Transfus Med 2003;13:59-62. 11. Mathur P, Samantaray JC. The first probable case of platelet transfusion-transmitted visceral leishmaniasis. Transfus Med 2004;14:319-21. 12. Grogl M, Daugirda JL, Hoover DL, et al. Survivability and infectivity of viscerotropic Leishmania tropica from Operation Desert Storm participants in human blood products maintained under blood bank conditions. Am J Trop Med Hyg 1993;49:308-15.

nology (PRT) System for Platelets and Plasma. LT, AMT, and DAL have disclosed no conflict of interest.

REFERENCES 1. van Griensven J, Balasegaram M, Meheus F, et al. Combination therapy for visceral leishmaniasis. Lancet Infect Dis 2010;10:184-94.

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

Leishmaniasis is a vector-borne disease caused by the protozoan parasite Leishmania sp. that is transmitted by sandflies. Travelers to endemic areas, ...
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