Microbiological safety of xenotransplantation compared with allotransplantation

Transmission of viral, bacterial, parasitic, and fungal infections via organ allografts is uncommon but may be associated with life‐threatening disease. Internationally, programs for screening of human organ donors for infectious risk are non‐uniform and vary with national standards and the availability of screening assays. Further, the failure to recognize and/or to report transmission events limits the utility of available data regarding the incidence of allograft‐associated disease transmission. Advances in xenotransplantation biology have allowed some limited clinical trials with the prospect for increased opportunities for clinical xenotransplantation. As with human allotransplantation, the examination of infectious risk has been a central theme in these studies. Significant advances have been made in the breeding and screening of swine for preclinical studies including the identification of novel, potential human pathogens derived from source animals. Thus far, “expected” xenograft‐derived pathogens such as porcine cytomegalovirus (PCMV) have become activated in immunosuppressed primates but have not resulted in systemic infection outside the xenograft. PCMV has been bred out of swine herds by early weaning strategies. Conversely, host pathogens such as primate‐derived cytomegalovirus (CMV) have become activated and have produced serious infectious complications. These infections are preventable using antiviral prophylaxis. Xenogeneic tissues appear to be relatively resistant to infection by common human pathogens such as HIV, HTLV and the hepatitis viruses. Concerns regarding the potential activation of latent porcine retroviruses from xenograft tissues have resulted in the development of novel assays for xenotropic porcine endogenous retrovirus (PERV). PERV transmission to primate xenograft recipients or to human cells in in vivo models has not been detected. Multiple intrinsic cellular mechanisms appear to be active in the prevention of infection of human cells by PERV. Further, PERV appears to be susceptible to available antiretroviral agents. Thus, while the absolute risk for such infections remains unknown in the absence of human studies with prolonged graft survival in immunosuppressed xenograft recipients, the risk of transmission to human recipients appears limited. Some general principles have been developed to guide clinical trials: Outcomes of xenotransplantation trials, including any infectious disease transmissions, should be reported in the scientific literature and to appropriate public health authorities. Surveillance programs should be developed to detect known infectious agents as well as previously unknown or unexpected pathogens in the absence of recognizable clinical syndromes. Standardization of procedures and validation by expert and/or reference laboratories are needed for microbiological assays. Such validation may require international collaboration. Repositories of samples from source animals and from recipients prior to, and following xenograft transplantation are essential to the investigation of possible infectious disease events. Infection is common in allograft recipients. Thus, in advance of clinical trials, policies and procedures should be developed to guide the evaluation of any infectious syndromes that may develop. (e.g. fever of unknown origin [FUO], leukocytosis, leukopenia, graft dysfunction, pneumonia, hepatitis, abscess formation) in xenograft recipients. Based on preclinical experience these procedures will include: (i) Exclusion of infectious syndromes commonly associated with allotransplantation (e.g. CMV, bacterial pneumonia); (ii) Evaluation of PERV infection by serologic and NAT testing; (iii) Assessment of other recipients of xenografts derived from the same herd or source of swine; and (iv) Evaluation of social contacts of the recipient. Consideration of investigation of xenograft recipients for unknown pathogens may require application of advanced research technologies, possibly including use of broad‐range molecular probes, microarrays or high throughput pyrosequencing. References: 1. Meije Y, TÖnjes RR, Fishman JA. Retroviral restriction factors and infectious risk in xenotransplantation. Amer J Transplant 2010; 10: 1511–1516. 2. Fishman JA, Scobie L, Takeuchi Y. Xenotransplantation‐associated infectious risk: A WHO consultation. Xenotransplantation 2012; 19: 72–81.     

Infectious disease risks in xenotransplantation

Hurdles exist to clinical xenotransplantation including potential infectious transmission from nonhuman species to xenograft recipients. In anticipation of clinical trials of xenotransplantation, the associated infectious risks have been investigated. Swine and immunocompromised humans share some potential pathogens. Swine herpesviruses including porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesvirus (PLHV) are largely species‐specific and do not, generally, infect human cells. Human cellular receptors exist for porcine endogenous retrovirus (PERV), which infects certain human‐derived cell lines in vitro. PERV‐inactivated pigs have been produced recently. Human infection due to PERV has not been described. A screening paradigm can be applied to exclude potential human pathogens from “designated pathogen free” breeding colonies. Various microbiological assays have been developed for screening and diagnosis including antibody‐based tests and qualitative and quantitative molecular assays for viruses. Additional assays may be required to diagnose pig‐specific organisms in human xenograft recipients. Significant progress has been made in the evaluation of the potential infectious risks of clinical xenotransplantation. Infectious risk would be amplified by intensive immunosuppression. The available data suggest that risks of xenotransplant‐associated recipient infection are manageable and that clinical trials can be performed safely. Possible infectious risks of xenotransplantation to the community at large are undefined but merit consideration.     

Xenotransplantation-associated infectious risk: a WHO consultation

Xenotransplantation carries the potential risk of the transmission of infection with the cells or tissues of the graft. The degree of risk is unknown in the absence of clinical trials. The clinical application of xenotransplantation has important implications for infectious disease surveillance, both at the national and international levels. Preclinical data indicate that infectious disease events associated with clinical xenotransplantation from swine, should they occur, will be rare; data in human trials are limited but have demonstrated no transmission of porcine microorganisms including porcine endogenous retrovirus. Xenotransplantation will necessitate the development of surveillance programs to detect known infectious agents and, potentially, previously unknown or unexpected pathogens. The development of surveillance and safety programs for clinical trials in xenotransplantation is guided by a "Precautionary Principle," with the deployment of appropriate screening procedures and assays for source animals and xenograft recipients even in the absence of data suggesting infectious risk. All assays require training, standardization and validation, and sharing of laboratory methods and expertise to optimize the quality of the surveillance and diagnostic testing. Investigation of suspected xenogeneic infection events (xenosis, xenozoonosis) should be performed in collaboration with an expert data safety review panel and the appropriate public health and competent authorities. It should be considered an obligation of performance of xenotransplantation trials to report outcomes, including any infectious disease transmissions, in the scientific literature. Repositories of samples from source animals and from recipients prior to, and following xenograft transplantation are essential to the investigation of possible infectious disease events. Concerns over any potential hazards associated with xenotransplantation may overshadow potential benefits. Careful microbiological screening of source animals used as xenotransplant donors may enhance the safety of transplantation beyond that of allotransplant procedures. Xenogeneic tissues may be relatively resistant to infection by some human pathogens. Moreover, xenotransplantation may be made available at the time when patients require organ replacement on a clinical basis. Insights gained in studies of the microbiology and immunology of xenotransplantation will benefit transplant recipients in the future. This document summarizes approaches to disease surveillance in individual recipients of nonhuman tissues.     

Miniature swine as organ donors for man: Strategies for prevention of xenotransplant-associated infections

Abstract: Xenotransplantation could potentially overcome limitations in organ transplantation resulting from a shortage of donor organs. Transplantation from miniature swine raises concerns over the potential introduction of new infectious agents into humans. These "direct zoonotic infections" or "xenoses" derived from swine fall into four categories: (1) microbial agents known to cause infection in humans (traditional zoonoses);(2) "species specific" organisms restricted to infection of the donor xenograft tissues by the absence of receptors or other factors needed for growth in human cells;(3) organisms of broad "host range" capable of infection of tissues outside the xenograft in the immunocompromised transplant recipient;and (4) organisms, such as retroviruses, of unknown quantity and with unmeasured capacity for causing disease in humans. The behavior of donor-derived xenoses in immunocompromised xenotransplant recipients cannot be predicted. The ability of human-derived microbial agents to infect porcine xenografts, and the capacity of the human immune system to inhibit such infections, also merit investigation. Experimental assessment of the infectious disease risks associated with xenotransplantation is important and practical.     

Infectious risk and retroviral restriction factors in xenotransplantation

The clinical application of xenotransplantation evokes immunological and microbiological as well as virological challenges. Porcine pathogens that do not show any symptoms in their natural host could exhibit a risk of fatal infections to humans. The presence of pig infectious agents including zoonotic and dissimilar agents should be reduced by specific pathogen free (spf) breeding of donor animals. However, the genetic information of porcine endogenous retroviruses (PERV) is integrated in the pig genome and can not be eradicated by spf breeding. The concerns about PERV for human xenograft recipients are based on data of in vitro replication of PERV in some human cell lines. So far, viral replication of PERV has been difficult to demonstrate in non‐human primate cell lines and in preclinical studies of non‐human primates receiving porcine xenografts, respectively. In this regard, natural and effective mechanisms of human and porcine cells counteracting productive infections caused by PERV are important to investigate. Intracellular proteins and components of the innate immune system including endogenous “antiretroviral restriction factors” act at various steps in retroviral replication. The cellular front is composed by several constitutively expressed genes which prevent or suppress retroviral infections. Some of these factors such as members of the tripartite motif (TRIM) and the apolipoprotein B mRNA‐editing polypeptide (APOBEC) families as well as tetherin and zinc‐finger antiviral protein (ZAP) could be useful in the management of PERV in xenotransplantation. The risks of infection and the potential role of antiretroviral restriction factors in xenotransplantation are presented in detail.     

Retroviral Restriction Factors and Infectious Risk in Xenotransplantation

The clinical application of xenotransplantation poses immunologic, ethical, and microbiologic challenges. Significant progress has been made in the investigation of each of these areas. Among concerns regarding infectious risks for human xenograft recipients is the identification in swine of infectious agents including porcine endogenous retroviruses (PERV) that are capable of replication in some human cell lines. PERV replication has, however, been difficult to demonstrate in primate‐derived cell lines and in preclinical studies of non‐human primates receiving porcine xenografts. Endogenous ‘retroviral restriction factors’ are intracellular proteins and components of the innate immune system that act at various steps in retroviral replication. Recent studies suggest that some of these factors may have applications in the management of endogenous retroviruses in xenotransplantation. The risks of PERV infection and the potential role of retroviral restriction factors in xenotransplantation are reviewed in detail.     

Donor‐derived infections and infectious risk in xenotransplantation and allotransplantation

Post‐transplantation infections are common in allograft recipients and should be expected in all immunocompromised hosts. Based on the need for immunosuppression in xenotransplantation, procedures developed to enhance safety in allotransplantation can be applied in future xenotransplantation clinical trials. Standardized approaches can be developed to guide the evaluation of common infectious syndromes in xenograft recipients. The opportunity created by screening of swine intended as xenograft donors has equal applicability to allotransplantation—notably broader screening strategies for allograft donors such as use of advanced sequencing modalities including broad‐range molecular probes, microarrays, and high‐throughput pyrosequencing. Considerations in management of allotransplant‐ and xenotransplant‐associated infections are largely the same. Experience in xenotransplantation will continue to inform thinking regarding donor‐derived infections in allotransplantation. We expect that experience in managing complex allotransplant recipients will similarly inform clinical trials in xenotransplantation.     

Infection in xenotransplantation

Advances in transplantation immunology have enhanced the possibility of xenotransplantation as a therapeutic option for end-stage organ failure. The potential spread of animal-derived pathogens to the recipient and to the general population, termed "xenosis," is a potential complication of interspecies transplantation. Recognition of such novel infections may be complicated by infections due to altered microbiologic behavior and clinical symptomatology of these organisms, particularly in the immunocompromised xenograft recipient. Particular concern exists over the activation of latent viruses, including retroviruses, from xenograft tissues. Based on experience with human allogeneic transplantation, those pathogens considered most likely to cause human disease can be excluded prospectively from herds of animals developed for organ donation. Research is needed into the activation and behavior of retroviruses and other potential pathogens in xenotransplantation. Xenotransplantation may also provide unique opportunities not only for the care of patients with organ failure, but in the therapy of individuals with chronic infections to which the xenograft may be resistant. Clinical protocols must be developed so as to enhance the safety of the recipient and of the community-at-large.     

Can human viruses infect porcine xenografts?

Millard AL, Mueller NJ. Can human viruses infect porcine xenografts? Xenotransplantation 2010; 17: 6–10. © 2010 John Wiley & Sons A/S. Abstract: Xenotransplantation exposes the recipient to known and unknown pathogens of the donor pig (donor‐derived xenosis). A major effort has been undertaken to minimize the risk of transmission from the donor using specialized breeding techniques. With the exception of endogenous retroviruses and porcine lymphotropic herpesvirus, exclusion of known pathogens was successful and has eliminated a majority of donor pathogens. In the recipient, enhanced replication of many pathogens will be stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Infection of the graft may occur with unpredictable consequences due to the cross‐species situation. Infectivity may be decreased as entry or replication is altered by missing receptors or inability to use the cellular machinery. Replication of organisms in the xenograft and the inability of the human host to respond to human pathogens in the context of a xenograft infection due to immune suppression, or the presentation of such pathogens in the context of pig instead of human major histocompatibility complex (MHC) could impair control of such infections. Recent data suggest that some human herpesviruses infections, such as human cytomegalovirus, may infect porcine tissue and are associated with a pro‐inflammatory phenotype. This review focuses on human or recipient‐derived pathogens and their potential harmful role in xenograft infection.     

Infection in renal transplant recipients

Renal transplant recipients are susceptible to infection by a wide array of pathogens. Impaired inflammatory responses due to immunosuppressive therapies suppress clinical and radiologic findings engendered by microbial invasion. As a result, patients are often minimally symptomatic and evaluation and diagnosis are delayed. Specific microbiologic diagnosis is essential both for the optimization of antimicrobial therapy and to avoid unnecessary drug toxicities. Differential diagnosis is guided by knowledge of organisms commonly involved in infection in immunocompromised hosts and understanding of the limitations of prophylactic strategies. The risk of infection in the organ transplant recipient is determined by the interaction between the individual's epidemiologic exposures and net state of immunosuppression. Epidemiology includes environmental exposures in the community and hospital, organisms derived from donor tissues and latent infections activated in the host during immune suppression. The net state of immune suppression is determined by the interaction of all factors contributing to infectious risk. Routine antimicrobial prophylaxis is aimed at common infections and unique risk factors in individual patient groups. This includes trimethoprim-sulfamethoxazole (for Pneumocystis, Toxoplasma, most Nocardia and Listeria, common urinary pathogens), perioperative (eg, anti-fungal prophylaxis for pancreas transplants), or antiviral (for herpesviruses in high risk recipients).     

Xenotransplantation: Infectious Risk Revisited

Xenotransplantation is a possible solution for the shortage of tissues for human transplantation. Multiple hurdles exist to clinical xenotransplantation, including immunologic barriers, metabolic differences between pigs--the source species most commonly considered--and humans, and ethical concerns. Since clinical trials were first proposed almost 10 years ago, the degree of risk for infection transmitted from the xenograft donor to the recipient has been extensively investigated. A number of potential viral pathogens have been identified including porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV). Sensitive diagnostic assays have been developed for each virus. Human-tropic PERV are exogenous recombinants between PERV-A and PERV-C sequences and are present in only a subset of swine. Porcine cytomegalovirus can be excluded from herds of source animals by early weaning of piglets. In contrast, the risks associated with PLHV remain undefined. Microbiologic studies and assays for potential xenogeneic pathogens have furthered understanding of risks associated with xenotransplantation. Thus far, clinical xenotransplantation of pig tissues has not resulted in transmission of viral infection to humans; significant risks for disease transmission from swine to humans have not been confirmed. If immunologic hurdles can be overcome, it is reasonable to initiate carefully monitored clinical trials.     

Control of microbial contamination during surgical harvest of pig renal xenografts

Abstract:  The pig has been identified as the most likely source of xenograft material for clinical use and studies are ongoing to overcome the immunological hurdles of pig-to-human transplantation. Attention is now being focussed on identifying and reducing the potential microbiological hazards associated with this technique. Studies have primarily addressed issues surrounding the production and health monitoring of xenograft source pigs and none have so far specifically evaluated the possible risks of microbial contamination during xenograft harvest. In this report, we evaluate the possible routes for contamination of a pig kidney xenograft during organ harvest and describe approaches to the control of these hazards, including the novel use of a custom designed airtight surgical canopy. A standard procedure for microbiological monitoring during xenograft harvest was devised and evaluated. This allowed the rapid identification and anti-microbial sensitivity testing of any isolated organisms. This would enable an early and appropriate pre-emptive treatment of infection because of transmission of pig micro-organisms.     

Infectious Diseases of the Lower Gastrointestinal Tract

Gastrointestinal (GI) infections are a major cause of morbidity and mortality worldwide. Although infectious organisms are often recovered by microbiological methods, surgical pathologists play an invaluable role in diagnosis. The lower GI tract, including the appendix, large bowel, and anus, harbors a wide variety of pathogens. Some infections are part of disseminated disease, whereas others produce clinicopathologic scenarios that are specific to the lower GI tract. This review focuses on selected infectious disorders of the lower GI tract that may be encountered by the general surgical pathologist, including viral, bacterial, fungal, and parasitic organisms, and including infections caused by food- and water-borne pathogens. Diagnostic gross and histologic features are discussed, as well as useful clinical features and ancillary diagnostic techniques. Pertinent differential diagnoses are also emphasized, including other inflammatory conditions of the gut (such as ischemia or idiopathic inflammatory bowel disease) that can be mimicked by lower GI infections.     

Diagnosis and Treatment of Culture-Negative Periprosthetic Joint Infection

Identification of the causative organism(s) in periprosthetic joint infection (PJI) is a challenging task. The shortcomings of traditional cultures have been emphasized in recent literature, culminating in a clinical entity known as “culture-negative PJI.” Amidst the growing burden of biofilm infections that are inherently difficult to culture, the field of clinical microbiology has seen a paradigm shift from culture-based to molecular-based methods. These novel techniques hold much promise in the demystification of culture-negative PJI and revolutionization of the microbiology laboratory. This article outlines the clinical implications of culture-negative PJI, common causes of this diagnostic conundrum, established strategies to improve culture yield, and newer molecular techniques to detect infectious organisms.     

Infectious complications in renal transplant recipients

Renal transplantation has become a well-established therapeutic option for end-stage renal disease, but infectious diseases remain a significant cause of morbidity and mortality. Although a wide variety of pathogens may cause infection, viral ones must be regarded as the single most important class of infections. Progress has been made both in the prevention and the early recognition treatment of infections that are closely linked to rejection. Immunosuppressive therapy is central to the pathogenesis of both. Because of the particular characteristics of transplant recipients, it is desirable to establish a close collaboration between nephrologists, surgeons, and infectious disease specialists for the management of these patients. In this article, we describe the different kinds of infectious disease that may affect patients with kidney transplant and the fundamental principles of clinical management, particularly our experience in Polyoma virus (BK) infection.     

Herpesvirus infections in xenotransplantation: pathogenesis and approaches

Infectious risk remains an important consideration in the clinical application of xenotransplantation. Vascularized xenografts create unique immunological niches in which bidirectional transmission of pathogens between donor and recipient may occur. Enhanced replication of many pathogens is stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Herpesviruses are the prototype viruses that are activated during immunosuppression. Quantitative diagnostic molecular assays have been developed for the known herpesviruses causing infection in pigs. Recent data suggest that some herpesviral infections, such as porcine cytomegalovirus, may be excluded from swine used as source animals by careful breeding, while others will require novel strategies for control. This review focuses on porcine and baboon herpesviruses in pig-to-non-human primate solid organ xenotransplantation including direct effects (tissue damage), indirect effects (coagulopathy, rejection), and possible approaches to these infections.     

The production of transgenic pigs for potential use in clinical xenotransplantation: microbiological evaluation

Debate over the infection hazards of pig-to-human xenotransplantation has focused mainly on the porcine endogenous retroviruses (PERV). However, hazards of exogenous infectious agents possibly associated with the xenograft have also been evaluated (Xenotransplantation 2000; 7: 143). We report the results of a health monitoring program demonstrating the exclusion of more than 80 potential pathogens from nine cohorts of pigs reared in a high welfare bioexclusion facility as potential xenograft source animals. A dynamic bacterial flora of pigs reared under barrier conditions was characterized, emphasizing the significance of monitoring for multiresistant antimicrobial sensitivity patterns. Evidence was found for exclusion of two commonly residual exogenous viruses, porcine cytomegalovirus and porcine lymphotropic herpesviruses, among a proportion of the cohorts tested. Finally, there was histopathological evidence for low grade pneumonitis among sentinel pigs, likely to have been associated with the use of quaternary ammonium disinfectants during the production process, indicating a need for review of toxicology data for disinfectant agents used in such bioexclusion systems. Intensive health monitoring programs, based upon regularly updated recommendations from the microbiological research community, will enable significant reductions in the potential hazards associated with pig-to-human xenotransplantation.     

Screening of deceased organ donors: no easy answers

Transmission of infection to recipients of solid organs is uncommon but well documented. Improved technologies for the diagnosis of infectious diseases suggest possible changes to paradigms used in the screening of organ donors to prevent disease transmission with transplantation. Available microbiologic assays, including molecular tests, are generally designed for use as diagnostic tools in individuals believed to have a specific infection based on clinical or epidemiological criteria. By contrast, these assays often lack the performance characteristics required for screening of deceased organ donors. This challenge is apparent with the analysis of assays for human T-cell lymphotropic virus-I and -II in low-risk populations. Changing epidemiologic patterns associated with the spread of novel pathogens or altered patterns of immigration will necessitate flexibility in the "list" of potential pathogens. Individual benefits from transplantation generally outweigh the risk of transmission of infection. However, this favorable experience will not obviate the need to continuously improve screening practices.     

Intra‐abdominal infections in solid organ transplant recipients: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice

This new guideline from the AST IDCOP reviews intra‐abdominal infections (IAIs), which cause substantial morbidity and mortality among abdominal SOT recipients. Each transplant type carries unique risks for IAI, though peritonitis occurs in all abdominal transplant recipients. Biliary infections, bilomas, and intra‐abdominal and intrahepatic abscesses are common after liver transplantation and are associated with the type of biliary anastomosis, the presence of vascular thrombosis or ischemia, and biliary leaks or strictures. IAIs after kidney transplantation include renal and perinephric abscesses and graft‐site candidiasis, which is uncommon but may require allograft nephrectomy. Among pancreas transplant recipients, duodenal anastomotic leaks can have catastrophic consequences, and polymicrobial abscesses can lead to graft loss and death. Intestinal transplant recipients are at the highest risk for sepsis, infection due to multidrug‐resistant organisms, and death from IAI, as the transplanted intestine is a contaminated, highly immunological, pathogen‐rich organ. Source control and antibiotics are the cornerstone of the management of IAIs. Empiric antimicrobial regimens should be tailored to local susceptibility patterns and pathogens with which the patient is known to be colonized, with subsequent optimization once the results of cultures are reported.     

Opportunistic infections in renal allograft recipients

Two major factors for successful organ transplantation are better control of rejection and better prevention and treatment of infections. In renal allograft recipients, immunosuppressive drug therapy is the major cause of immunocompromised status and occurrence of infections, which arise most commonly as a result of invasion by endogenous opportunists. It may also follow colonization by exogenous environmental organisms and via transfer of cytomegalovirus along with the transplanted kidney. The overall incidence of opportunistic infections varies from center to center; up to 15% of renal transplant recipients die of these infections. Clinical signs and symptoms of infection in immunocompromised patients may be concealed or imitated by the underlying disease, and a high index of clinical suspicion is vital. The unusual pathogens encountered in these patients demand thorough investigation. A total of 84 opportunistic infections encountered in renal allograft recipients during histopathologic and cytopathological evaluation of various specimens during the last 15 years is presented in this report. Invasive fungal infections were the most common pathogens, amounting to 55% of all infections. The dramatic increase in the diversity and number of opportunistic infections detected in these patients is not only due to an increasing population of susceptible individuals but also due to an improved recognition by advanced laboratory diagnostic techniques. The success of management of opportunistic infections depends on strong clinical suspicion, early diagnosis, and prompt treatment. The challenges of early diagnosis of opportunistic infections and prompt treatment are great; the rewards are even greater.