Pathogen Inactivation Methods Research Articles

Inactivation of parvovirus B19 in human platelet concentrates by treatment with amotosalen and ultraviolet A illumination

The human erythrovirus B19 (B19) is a small (18- to 26-nm) nonenveloped virus with a single-stranded DNA genome of 5.6 kb. B19 is clinically significant and is also generally resistant to pathogen inactivation methods. Photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) inactivates viruses, bacteria, and protozoa in platelets (PLTs) and plasma prepared for transfusion. In this study, the capacity of PCT to inactivate B19 in human PLT concentrates was evaluated. B19 inactivation was measured by a novel enzyme-linked immunosorbent spot (ELISPOT) erythroid progenitor cell infectivity assay and by inhibition of long-range (up to 4.3 kb) polymerase chain reaction (PCR), under conditions where the whole coding region of the viral genome was amplified. B19-infected plasma was used to test whether incubation of amotosalen with virus before PCT enhanced inactivation compared to immediate PCT. Inactivation of up to 5.8 log of B19 as measured by the infectivity assay, or up to 6 logs as measured by PCR inhibition can be achieved under non-limiting conditions. Inactivation efficacy was found to increase with incubation prior to UVA illumination. Without incubation prior to illumination 2.1 +0.4 log was inactivated as determined by infectivity assay. When measured by PCR inhibition, inactivation varied inversely with amplicon size. When primers that spanned the entire coding region of the B19 genome were used, maximum inhibition of PCR amplification was demonstrated. Under defined conditions, PCT with amotosalen combined with UVA light can be used to inactivate B19, a clinically significant virus that can be transmitted through blood transfusion, and heretofore has been demonstrated to be refractory to inactivation.

The Pathogen Inactivation of Platelets: Studies of the Possible Mechanisms of the Platelet Dysfunction Seen Following Photochemical Treatment.

Background: The platelet mitochondrial genome has many similarities to bacterial and viral genomes. These include the lack of DNA redundancy, the lack of introns, no protective histone proteins, and inefficient DNA repair mechanisms; all of which are important characteristics that likely contribute to the increased rate of mutations occurring with mitochondrial DNA (mtDNA) compared to that seen with nuclear genomic DNA. Recent studies have shown that the use of pathogen inactivation involving UVA phototherapy using the combination of ultraviolet A (UVA) light and either the photoactive psoralen 4′-aminomethyltrioxsalen hydrocholoride (AMT/UVA, known to induce cross-linking of DNA) or the B-vitamin riboflavin (RF/UVA, known to produce the oxidized guanine moiety 8-OH-dG) are effective approaches to reduce and/or inactivate the bacterial and viral contaminants often present in human blood products. Such treatments however have been shown to be associated with impaired in vivo platelet recovery, survival and hemostatic function.Methods and Results: In the current study, rabbit and human platelets were treated with the two photochemical pathogen inactivation methods, (AMT/UVA or RF/UVA), using various UVA exposure, and the resultant platelets compared to control platelets with regard to their in vivo recovery, survival, and hemostatic function, as well as evaluating evidence of damage to platelet mtDNA. Rabbit platelets treated with UVA (1 to 5 joules/cm2) alone, RF alone, or AMT alone, showed minimal or no effect on in vivo platelet function; without evidence of damage to mtDNA. In contrast, the photochemical treatment of rabbit platelets with either AMT/UVA or RF/UVA showed a UVA-dose-dependent effect on in vivo platelet function as well as on mtDNA damage. Platelets treated with AMT/UVA(1 to 5 joules/cm2) revealed a 10–30% UVA-dose-dependent reduction in platelet recovery, survival, and hemostatic function, as well as a widespread but a non-specific effect upon platelet mtDNA structure. The treatment of rabbit platelets with RF/UVA(1 to 5 joules/cm2) showed considerably greater impact on platelet recovery, survival, and hemostatic function than that seen with AMT/UVA, at equivalent UVA doses. This was associated with a reproducible impairment of the amplification of a 3.8kb fragment within the platelet mtDNA genome. This mtDNA fragment is specifically associated with the coding for the NADH dehydrogenase subunit 1. A threshold dose of 1.0 joule/cm2 was noted for RF/UVA treatment, below which damage to mtDNA was found to be UVA-dose-dependent. Using HPLC to determine the concentration of 8-OH-dG within photochemically treated human platelets resulted in the demonstration of a UVA-dose-dependent increase of 8-OH-dG production for RF/UVA treated platelets. Control (no treatment), UVA alone, or AMT/UVA treated platelets produced no increase in 8-OHdG levels, even at UVA doses of 5 joules/cm2.Conclusions: These results indicate that the reproducible focal damage of platelet mtDNA by RF/UVA may have a considerably greater effect upon platelet in vivo circulatory and hemostatic functions than does the introduction of random mtDNA cross-linking produced by AMT/UVA treatment, possibly because of the discrete reproducible defect caused by the RF/UVA on the platelet mtDNA. These data nonetheless support the hypothesis that platelet mtDNA damage produced by pathogen inactivation methodology is associated with and may affect subsequent in vivo platelet function following platelet transfusion to a recipient.

Viability and function of 8‐day‐stored apheresis platelets

Methods of bacterial detection and pathogen inactivation of platelets (PLTs) may allow extended storage of PLTs as long as PLT quality is maintained. Twenty normal volunteers had their PLTs collected with an apheresis machine (Haemonetics Corp.). A variety of in vitro PLT function and metabolic assays were performed both on Day 0 and after 8 days of storage. On Day 8, a small blood sample was drawn from each donor to obtain fresh PLTs. The fresh and stored autologous PLTs were labeled with either (51)Cr or (111)In, and the radiolabeled PLTs were transfused. Posttransfusion serial blood samples were drawn to determine the relative posttransfusion recoveries and survivals of the fresh versus the stored PLTs. Although the in vitro assays showed some differences between the two trial sites, the results were generally within the ranges expected for fresh and stored PLTs. Overall, PLT recoveries averaged 66 +/- 16 percent versus 53 +/- 20 percent and survivals averaged 8.5 +/- 1.6 days versus 5.6 +/- 1.6 days, respectively, for fresh compared to 8-day-stored PLTs. There were no significant differences in the in vivo PLT data between the trial sites or based on the radiolabel used for the measurements. After 8 days of storage, the in vivo posttransfusion recovery and survival of autologous Haemonetics apheresis PLTs meet the proposed standards for poststorage PLT quality.

Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis

Ultraviolet-C (UVC) irradiation is a pathogen inactivation method used for disinfection of pharmaceutical products derived from human blood. Previous studies have shown that UVC can potentially damage proteins through photolysis or can generate reactive species resulting in protein thiol oxidation. In this study, two fluorescence-based quantitative proteomic approaches were used to assess the effects of a novel UVC-disinfection strategy on human plasma fractions. We show minimal changes in protein content, but gross alterations in protein thiol reactivity, indicative of oxidative damage. We identify a number of the damaged proteins by mass spectrometry, including serum amyloid P component, and further demonstrate UVC-induced photolysis of its disulphide bond.

Consideration in Hemophilia Therapy Selection

The risk of pathogen transmission via clotting factor therapies has been reduced over the last two decades through the development of effective and progressively more sensitive pathogen screening and inactivation methods and the introduction of recombinant clotting factors for hemophilia, beginning with recombinant factor VIII (FVIII) in 1992. However, new understanding about the potential for transmission of an emerging infectious agent through blood and blood products has renewed concerns about vulnerabilities that remain in plasma-derived and some recombinant clotting therapies that still use plasma components during some stages of the manufacturing process. In the 1980s, patients with hemophilia became "canaries in the coal mine" for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in the blood supply. Moving forward, healthcare providers must continue to take a proactive role in educating themselves about new information regarding emerging pathogens and develop approaches to discussing this risk with their patients as part of their therapy selection process.

Effective use of optimized, high-dose (50 kGy) gamma irradiation for pathogen inactivation of human bone allografts

The safety of tissue allografts has come under increased scrutiny due to recent reports of allograft-associated bacterial and viral infections in tissue recipients. We report that 50kGy of gamma irradiation, nearly three times the dose currently used, is an effective pathogen inactivation method when used under optimized conditions that minimize damage to the tissue. Cancellous bone dowels treated with a radioprotectant solution and 50kGy of optimized irradiation had an ultimate compressive strength and modulus of elasticity equal to conventionally irradiated (18kGy) and non-irradiated control bone grafts. We subjected bone dowels treated with this pathogen inactivation method to an in vitro cytotoxicity test using three different mammalian cell lines and concluded that the treated grafts were not cytotoxic. The log reduction of nine pathogens spiked into radioprotectant-treated bone irradiated to 50kGy was also tested. We achieved 4.9logs of inactivation of a model virus for HIV and hepatitis C and 5logs inactivation of a model virus for human parvovirus B-19. Complete inactivation (6.0–9.2logs) of seven clinically relevant microorganisms was demonstrated. The results show that a combination of radioprotectants and optimized, high-dose gamma irradiation is a viable method for producing safer cancellous bone grafts that have the mechanical strength of existing grafts.

Improving the bacteriological safety of platelet transfusions

Despite the increased application of aseptic techniques for blood collection and the preparation of platelet concentrates, morbidity and mortality arising from the transfusion of bacterially contaminated allogeneic platelet products persist. This problem exists because stored platelet concentrates represent a nearly ideal growth medium for bacteria and because they are stored at temperatures (22° ± 2°C) that facilitate bacterial growth. The presence of bacteria in blood components including platelets has been a problem for many decades and currently is the most common microbiological cause of transfusion-associated morbidity and mortality. A variety of strategies have been devised and/or proposed in an attempt to try to reduce the risk of transfusion-associated sepsis. These include pretransfusion bacterial detection, efforts to reduce the likelihood of bacterial contamination, the optimization of blood product processing and storage, reducing recipient exposure, and the introduction of pathogen inactivation methodology. With regard to doing bacterial detection, a number of automated detection systems have become available to test for contaminated platelet components, but their utility to some extent is restricted by the time they take to indicate the presence of bacteria and/or their lack of sensitivity to detect initially low bacterial loads. A variety of other approaches has been shown to reduce the risk of bacterial contamination and include filtration to remove leukocytes and bacteria, diversion of the initial aliquot of blood during donation, and improved donor skin disinfection. Platelet pathogen inactivation methods under investigation include the addition of L-carnitine, γ-irradiation, riboflavin plus UVA irradiation, and amotosalen HCl plus UVA irradiation. The latter process is licensed for clinical use with platelets in some countries in Europe. All of these approaches, either collectively or individually, hold considerable promise that the prevalence of adverse events associated with bacteria in platelet products will decline significantly in the very foreseeable future.

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

Inactivation of viruses, bacteria, protozoa, and leukocytes in platelet concentrates: Current research perspectives

LTHOUGH increased safety of blood components has been achieved through continued improvements in donor testing, concern remains about the safety of blood products. A recent government report estimated that 2.7 of every 1,000 patients who receive a transfusion of 5 units of blood components are at risk of acquiring an infectious disease. 1 Currently, prevention of infectious disease transmission associated with the transfusion of cellular components rests largely on pretransfusion donor evaluation and laboratory testing. Despite improvements in testing, the aggregate risk of acquiring a viral infectious disease from a donor passing all current serological testing remains 1 per 34,000 donbr exposures. 2 Transfusion of cellular components has been implicated in transmission of viral, bacterial, and protozoan diseases. 3 Although it is commonly recognized that hepatitis B virus (HBV), hepatitis (2 virus (HCV), cytomegalovirus (CMV), and the retroviruses, such as human immunodeficiency virus (HIV) and the human lymphotrophic viruses (HTLV), can be transmitted through cellular components, other pathogens are emerging as potentially significant transfusion-associated infectious agents. For example, transmission of protozoan infections attributable to trypanosomes 4-6 and babesia have been reported. 7,8 In addition to viral and protozoal infectious agents, bacterial contamination o f platelet concentrates continues to be reported 9,1~ and may be underreported. 11 More importantly, new infectious agents, for example, hepatitis G virus, may enter the donor population long before tests adequate to maintain consistent safety of the blood supply can be implemented. 12 Even when identified early, the clinical significance of new pathogens may not be fully recognized until large epidemiological studies are completed. 13 Over the past 10 years, investigation of methods for inactivation of infectious pathogens in cellular

Disinfection of effluent from fish slaughteries

The purpose of this study was to evaluate methods for inactivation of fish pathogens in outlet water from fish slaughteries. The effect of three disinfectants, namely sodiumhypochlorite, heat and photozone, were tested on two fish pathogens, Vibrio anguillarum and Yersinia ruckeri. Outlet water was incubated with the test bacteria for 24 h prior to use, and contained in the area of 10 6–10 9 CFU (Colony Forming Units) per ml. The following doses gave 100% inactivation of the test bacteria: 30 min exposure to 250 ppm sodiumhypochlorite, 60°C for 2 min, 72°C for 15 s and a photozone generated redox potential of approximately 550 mV for 30 min. The following doses resulted in 100% inactivation of all bacteria in the outlet water: 30 min exposure to 350 ppm sodiumhypochlorite, 72°C for 15 s and a photozone generated redox potential of approximately 550 mV for 30 min.

Seroprevalence of cytomegalovirus antibodies among blood donors in Aminu Kano Teaching Hospital, Kano, Nigeria

Introduction: Transfusion transmissible infections (TTIs) may potentially lead to serious harm to recipients of blood transfusion. Cytomegalovirus (CMV) can be transmitted through blood transfusion, and its impact is the most severe among immunocompromised recipients. Its transmission can be reduced through screening of blood donors, blood and blood products for CMV antibodies, leuco-depletion or pathogen inactivation. Objective: This was a cross-sectional study conducted among 250 blood donors between October and December 2012 in Aminu Kano Teaching Hospital, Kano, Nigeria. To determine the prevalence of human immunodeficiency virus (HIV) I and II antibodies, HBsAg, hepatitis C virus (HCV) antibodies, syphilis and anti-CMV IgM and IgG antibodies. In addition, the type of donation and donation patterns were also assessed. Materials and Methods: Five milliliters were collected from the participants. One milliliter was dispensed into ethylenediaminetetraacetic acid (EDTA) container and mixed gently, the remaining 4 ml were dispensed into a plain bottle and was centrifuged; serum obtained was divided into two one was stored at − 20°C which was later used to test for the anti-CMV IgG/IgM antibodies, whereas the other portion was used for routine screening of TTIs. The EDTA sample was used to assess haemoglobin concentration. An interviewer administered simplified questionnaire was used to obtain information on demographic and other variables related to donation habits. Association between the socio-demographic characteristics of the blood donors and the anti-CMV antibodies was analysed. Similarly, association between the anti-CMV antibodies and the other routinely screened TTIs was assessed. Results were tested for evidence of statistical association. Results: Majority of the blood donors were males 242 (96.8%). One hundred and seven (42.6%) of the blood donors were within the age group of 25–34 years. Only 10.8% of the donors were voluntary donors whereas the remaining were family replacement donors. Prevalence rates of 100% and 4.4% were found for anti-CMV IgG and anti-CMV IgM antibodies, respectively. HBsAg, HIV I/II, anti-HCV antibodies and syphilis were prevalent in 6.8%, 1.2%, 0.8% and 0% of the donors, respectively. Conclusion: Seroprevalence of anti-CMV IgG and anti-CMV IgM antibodies among blood donors in this study was found to be 100% and 4.4%, respectively. Therefore, routine predonation screening for anti-CMV antibodies may not be cost effective. Leucocyte depletion and pathogen inactivation methods are better options. However, these can better be achieved when centralized system of blood transfusion is adopted. Therefore, there is a need to enhance the functional capacity of our national blood transfusion service.