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Proceedings of the 4th National Symposium on Biosafety
Defining the Risks and the Risk Reduction Strategies
Marian Michaels, MD, MPH
Children’s Hospital of Pittsburgh
Division of Pediatric Infectious Diseases
3705 Fifth Avenue
Pittsburgh, PA 15213-2583
412-692-7232Recently, xenotransplantation has re-emerged as an experimental option for organ, bone marrow and tissue transplantation. While the idea of putting animal organs and tissues into humans might seem rather bizarre when one first thinks about it, and even thinks about it a second time, the concept of having more than one species inhabit the same body is hardly new. It has been conceived of for generations as illustrated by the appearance of the Sphinx from ancient Egypt.
Allotransplantation has become well accepted as an effective treatment for a number of end-stage organ disorders. Improvements in surgical techniques and the development of more select immunosuppressive regiments has led to an increased number of potential beneficiaries of these procedures. However, the number of donors has not increased. In 1993 there were approximately 33,000 people on the waiting list for organs in the United States, but only 7,500 who had donated organs. These numbers and problems have led people who are in the field of transplantation to consider other possibilities such as animal sources for organs. In addition, there has been a recognition that certain animals appear to be resistant to some infections that afflict humans. For example, baboons appear to be resistant to hepatitis B virus infection. This led to baboon liver xenotransplants in two patients with end-stage liver disease from hepatitis B virus.
A more recent and widely publicized case was based on similar logic in the decision to perform a baboon bone marrow xenotransplantation. This experiment was based on the findings that baboon hematopoietic cells appear resistant to HIV-type I infections. Accordingly, a xenotransplant was performed in an attempt to reconstitute the immune system of person with advanced HIV infection.
Much of the xenotransplant research that has occurred to date has been related to the technical problems of putting an animal organ into a human body and the immunologic barriers which must be broached prevent rejection. Less information is known about the potential for infections to be transmitted from the animal to humans through these transplants. This audience is well aware of the potential for zoonotic infections; humans can be infected with microbial agents after direct or indirect contact with animal secretions. Dr. Richard Hueneke defined zoonoses yesterday during his talk: zoonoses are infections which are transmitted from animals to humans under natural" conditions, with an emphasis on the word natural. With the advent of xenotransplant experiments and manipulation of ?natural contacts' we felt that this term was no longer adequate and coined the term xenozoonoses to describe the new potential for the transmission of animal infectious agents that accompany the animal organ or tissues into the new human host.
Dr. Jonathan Allan will address infections specific to baboons and their associated problems, Dr. Jay Fishman will concentrate on problems and infections from swine, and Dr. Chapman will deal with the public health issues of xenotransplantation. During this presentation I will describe how and why some microorganisms might be xenozoonotic and consider some practical issues regarding needed areas of research to understand xenotransplant infections. I will also cover what is known and more importantly what is not known as well as some of the pitfalls in trying to plan to minimize the risk of xenozoonoses.
Because the true infectious risk from xenotransplantation is unknown at this time, it is necessary to start with a model where information is known – such as infections which occur after human to human allotransplantation. The major risk factor for these infections is the use of immunosuppression. While transplant researchers are actively pursuing methods which will enable transplantation to be performed without the need for immunosuppression it has not as yet been reliably achieved. Currently, immunosuppression is required and undoubtedly will continue to be required with xenotransplantation in the near future. To understand potential transplant infections consideration must also be given to the source of infectious agents. For example, endogenous flora can be a source of infection; people with cystic fibrosis undergoing lung transplantation remain colonized with bacteria associated with cystic fibrosis. These pseudomonas species can become a problem after allotransplantation leading to recurrent pneumonia or tracheitis. The environment is another source of infection for patients who undergo transplantation; examples include aspergillus or legionella exposure from the hospital environment. Finally, the donor organ can itself be a source of new infection in the recipient and will be the focus of xenotransplant infections.
The first two sources, namely the host and the environmental pathogens will be present regardless of whether a person undergoes an allo or xenotransplant procedure. Review of the early studies of xenotransplantation when Drs. Keith Reemstma and Thomas Starzl reported their series of six chimpanzee and baboon kidney xenotransplants respectively, reveal that five of the six deaths were associated with infection. In these patients the bacterial infections were bacterial and as such, were most likely due to surgical complications and the need for immunosuppression rather than being donor transmitted. Similarly, a patient who underwent a baboon liver xenotransplant in 1992 in Pittsburgh died from disseminated aspergillus. Again this occurred because of the need for immunosuppression and was an infection derived from the environment as opposed to a xenozoonoses. The second patient who underwent a baboon liver xenotransplant in Pittsburgh died with bacterial peritonitis after multiple surgical complications. For both of these procedures the infectious complications were not derived from the animal organ.
The source of microorganisms which we need to concentrate on are really those that are donor-associated infections. These agents will be related to the particular type of donor that is used, be it a baboon, a swine, or another species and how the animal was raised. Using the allotransplant model it is recognized that most donor transmitted microorganisms have a latent or intracellular phase. Because they are often asymptomatic or cause only minor symptoms in the donor they were not considered to be pathogens until they entered the new immunosuppressed recipient environment. Herpesviruses, in particular CMV and EBV, retroviruses, and intracellular parasites are exaples of types of infectious agents which can be problematic. All of these microorganisms are recognized as causing donor transmitted infections after human–> human transplantation.
Similar types of microorganisms might be able to gain access to the human host through xenotransplantation, thus it is helpful to review possible mechanisms which might be involved. First, microorganisms might be pathogenic for both the donor and the host species. Second, a microbe in the animal donor might be similar enough to an analogous human microbe that it is recognizable by human cell receptors to which it has access after transplantation. A non-pathogenic organism might become problematic in a patient who is immunosuppressed. The possibility of recombination of an animal virus with a human virus is of great concern. Finally, there is a chance that an animal virus might not be able to infect human cells and therefore be less of a risk after xenotransplantation. However, it is possible that it could still reactivate in the animal organ and lead to graft failure.
An example of an organism which can be pathogenic for both humans and animal donors is Toxoplasma gondii. The definitive host for this ubiquitous protozoa is the cat. Animals and humans become infected from ingestion of sporulated oocysts. Animals acquire the infection grazing in contaminated soil. Human ingestion can occur during cleaning of the kitty litter box, gardening without gloves, or eating unwashed fruits or vegetables that have fallen on the ground. Humans can also develop toxoplasmosis after consuming inadequately cooked meat containing latent T. gondii cyst. While anyone can become infected with this parasite it does not usually cause serious disease except in specific situations. It has long been recognized that infection in a woman during pregnancy can lead to the organism crossing the placenta and cause substantial disease in the developing fetus. We also recognize that reactivation of latent toxoplasma cysts can lead to severe disease in HIV infected people.
Early in my experience working with heart transplant recipients at Children's Hospital of Pittsburgh I learned that primary toxoplasma infection, presumably from the donor, could cause profound disease in previously naive recipients. One young teen, two months after transplant developed an illness characterized by fever, malaise and myocardial dysfunction. Toxoplasma gondii was found on his cardiac biopsy. Another youngster several months after heart transplant presented with blindness and left hemiparesis. Ophthalmologic examination revealed diffuse severe retinitis. A CT scan of her head, shown on this slide, revealed large brain lesions which eventually proved fatal. This child was also found to have primary toxoplasmosis with concurrent EBV infection. These cases illustrate several critical points. First, it should not be surprising that heart transplant recipients are at the highest risk of all organ transplant recipients because T. gondii has a natural propensity to become latent in muscle, including heart muscle. Accordingly, if a donor is seropositive for this agent, latent cysts can be present and carried along with the new organ. This can occur regardless of whether the donor is a human, a swine or a baboon. Finally, these cases teach us that protocols for caring for patients must have flexibility and be modified as new information is learned. Prior to these two cases of toxoplasmosis, donors at our institution were not screened. Now however, with understanding the disease transmission we do screen all cardiac donors and institute prophylaxis to prevent disease if the donor is found to be seropositive. Undoubtedly, this experience will not be unique to allotransplantation. As new infectious agents are recognized with xenotransplantation, researchers will have to be able to incorporate them into protocols for study, prevention and treatment.
Herpesviruses represent a class of viruses that are well recognized as causing disease after transplantation. Additionally, some herpesviruses illustrate problems which can occur when virus crosses species lines. This is probably best recognized with the alpha-herpesvirus of macaques, Herpes B. This virus is relatively benign in its normal host, but when inadvertently inoculated into a human, by a scratch or a bite from an actively shedding animal, it is anything but benign. The virus leads to a devastating acute ascending myelitis which almost always is fatal if prompt treatment is not initiated. Pigs likewise have an a-herpesviruses, pseudorabies. This virus similarly causes severe, fatal disease if injected into another species. The disease, "mad itch", acquired its name because of the accompanying intense pruritus. Nonfatal transmission of pseudorabies to humans has been anecdotally reported in the literature. Other alpha-herpesviruses such as H. saimiri of the squirrel monkey can induce malignant lymphomas in other primate species. These examples highlight the finding that benign 'species specific' viruses can be oncogenic or fatal when introduced across the species line into a new host. Alpha-herpesviruses are usually latent within neuronal tissue and as such are unlikely to be transmitted through transplantation of cells or organs which do not harbor the virus. On the other hand a ß-herpesvirus, cytomegalovirus, is recognized as one of the most important and common donor-associated infections after allotransplantation. Also the g-herpesvirus, Epstein-Barr virus, is recognized as being donor transmitted. This has been shown by epidemiologic evidence, and even more definitively with genetic DNA finger printing. EBV can cause serious disease. Post transplant lymphoproliferative disorders are often reversible with reduction of immune-suppression plus/minus the addition of other therapies such as antiviral agents, immunoglobulin or interferon. However, at other times, the disease will disseminate despite therapeutic maneuvers and lead to fatal disease. These classes of ß-herpesviruses, and g-herpesviruses, can be latent within passenger cells and/or organs that are being transplanted. Analogous viruses in the animal donors being considered for xenotransplant do exist. So it will be important to study their potential to cross species lines.
The Retroviridae family of viruses are a final major group to consider with xenotransplantation. Too often in the history of transfusion medicine and transplantation we have unwittingly transmitted infections such as HIV and the hepatitides. The majority of times this occurred because we did not have the ability to detect and thereby screen prospective donors. But even with routine screening in place, transmission of HIV can occur. R.J. Simonds and colleagues at the Center for Disease Control and Prevention, reported in the New England Journal in 1992, a case where HIV transmission occurred from a single donor to all four recipients of his organs and three of the four recipients of his bone grafts. This occurred despite screening having been performed. Most likely this donor had recent acquisition of HIV infection and therefore had not yet developed antibodies against the virus. This highlights a limitation found with all screening protocols. Xenotransplant actually offers the value of permitting repeated screening over time which might have prevented the use of an infected donor as in the above situation.
Not many years ago we were uncertain as to whether retro-viruses of primates could be transmitted to human beings. Clearly the answer is yes. Anonymous seroprevalence surveys of primate animal care workers found 3 of 472 workers to be seropositive for simian immunodeficiency virus (SIV). Two workers, who might have also been included in the above study, were known and studied in more detail. One had a non-persistent infection while the other has continued to have persistent infection. So retroviruses can be transmitted, at least across primates species. We know a fair amount about retroviruses in human and non-human primates but do not have as much data about exogenous retroviruses of other potential donor animals such as the pig. Endogenous retroviruses, as the name implies, have been incorporated into the genetic make up of the host animal. Without genetic engineering we cannot screen out this type of virus from a donor population. The risk of disease from these viruses after xenotransplantation is completely unknown. We know that both baboons and swine harbor endogenous retroviruses.
So what needs to be done? Well one way to attempt to prevent xenozoonoses is to have as clean an animal as possible, such as with raising animals in germ free environments. Most germ free environments have been developed for research on small laboratory animals such as mice and rats. Pigs have also been raised germ free, but only until a few months of age. Pigs are not unreasonable candidates. They have a large litter. They have a well-defined, short gestational period and the newborns can self feed at a relatively early age. The problems start when the animal is about 3 or 4 months of age. The amount of waste build up is logistically difficult to handle and likewise the animals do not thrive well. While germ free environments are burdensome for raising swine they are almost impossible to consider for primates. Primates have a long gestation period. They usually have one offspring per pregnancy and are completely dependent during infancy. A specific pathogen free (SPF) environment is more logical to consider but still logistically difficult. The animals would still have to be isolated at birth, screened, hand raised and it would take years to get a breeding colony. It is important to realize that neither germ free nor specific pathogen environments will prevent vertical transmission of infections in pigs or baboons nor the presence of endogenous retroviruses. Also critical to consider when thinking about SPF environments is what the term truly stands for; namely freedom from the pathogens which someone has decided upon ahead of time.
In order to develop either germ free or specific pathogen free conditions we need to start with donor screening. The ideal screening should consider organisms that are known to be zoonotic for humans especially under immunosuppression. It should take into account microorganisms not considered classically zoonotic but which might be present within organs or tissues and thus xenozoonotic and also consider microorganisms that might have a high possibility for recombination. The ideal screening should be extremely sensitive, cheap and easily reproducible. Unfortunately, I don't think the ideal screening exists. When considering individual infectious agents as potential xenozoonoses one can consider whether the microorganism is likely to be found in a particular donor species or unlikely but of extreme importance if it is found. For example, it is unlikely to find SIV in a baboon population however, it would be critical to look for this in the donor screening because if it is present it could be transmitted to humans with dire consequences. It is also worthwhile to track microorganisms of low or uncertain risks to humans as a marker of transmission across species lines such as looking for baboon or swine endogenous retroviruses.
Researchers from the University of Oklahoma performed screening on ten piglets that were raised in a SPF environment at 3 different time points. They looked for evidence of bacteria and parasites, and performed viral serology. After evaluation the researchers concluded that the animals were adequately clean to be used for xenotransplantation. However, of interest, two animals initially had positive serologic tests against HIV. The reason for this false positive result is unclear but deserves further investigation.
In anticipation of baboon xenotransplantation we performed serologic screening on 31 adult male baboons from Southwest Foundation for Biomedical Research. The animals were raised in the United States in an outdoor six acre corral. To increase sensitivity we sent paired specimens to two different primate laboratories which used different testing methods for the same viruses. When available the laboratories used serologic testing directed specifically against the primate virus, but when not available they used serology aimed at the analogous human virus. Review of results for herpesviruses was of interest. Tere was reasonable concordance in serologic tests directed against CMV and SA-8, the baboon -herpesvirus. However there was striking difference in the two laboratories' results for Epstein-Barr virus, Both laboratories relied on cross reactivity with commercial kits directed against human Epstein-Barr virus. The first laboratory used an ELISA directed against EBV nuclear antigen (EBNA), which turns out to be divergent across species. The second laboratory used a test directed against human EBV viral capsid antigen which is a more robustly conserved area of the virus. Hence the second laboratory had a higher likelihood of finding a positive animal. This occurrence highlights the need to develop tests that are directed against the specific donor organism rather than relying on cross reactivity with human viruses and shows some of the limitations which exist in screening protocols.
Tests which cross react with animal and human viruses will also present a limitation in being able to determine whether animal viruses have crossed into the new human host after xenotransplantation. Tests directed against baboon cytomegalovirus illustrate this problem. Because current primate CMV tests cross react with human CMV it was important to develop methods to distinguish these related organisms. Both human and baboon CMV cause similar cytopathic effects on human fibroblasts at the same rate and thus cannot be differentiated by cell culture. In order to distinguish the viruses from each other I developed PCR primers which would specifically amplify baboon CMV. More molecular studies are required to distinguish other related animal viruses from human viruses.
The human candidate needs to have routine screening performed as well. Similar to allotransplantation, screening should include retroviruses, hepatitides, and herpesviruses. Additionally, screening for primate or swine microorganisms should be included as a baseline to identify any cross reactivity present. Archiving serum, plasma and peripheral blood lymphocytes should be mandatory to use as baseline negative controls for surveillance studies following xenotransplantation.
Recipient follow-up must be carefully designed and strictly followed. There should be serial serologic screening and surveillance viral cultures at predetermined time points. If fevers develop more intense screening should be performed along with studies dictated by the clinical scenario. A gastric biopsy does not have to be performed if the person has an upper respiratory infection but studies obtained should be broad. They should include looking for viruses, fungi and bacteria including mycobacteria. Serology should be performed, but with the recognition of its limitations in an immunosuppressed patient who might not be capable of developing a detectable antibody response to a new antigen. Accordingly, molecular diagnostics should be used for primate or swine microorganisms whenever possible. For example in the patient who underwent a baboon bone marrow xenotransplantation for advanced HIV infection, quantitative HIV studies were included in the post-transplant screening protocols. Additionally, collaborators at the CDC developed molecular techniques to look for evidence of endogenous retroviruses, as well as RT activity which might signify other retrovirus activity.
The major risks of any experimental procedure are to the individual recipient. For this reason institutional review boards have developed over time to help ensure that the prospective candidate understand the risk-benefit ratio. Informed consent procedures exist so that patients can be told about the potential benefits of xenotransplantation but also apprised of its highly experimental nature and the risks involved. There are risks of the immunosuppressive drugs, risks of surgery, and finally risks of infection. Informed consent allows the individual to make the decisionto accept these risks or not. With xenotransplantation we're also being confronted with the potential risk of transmission of a novel infection to others, particularly hospital staff and close contacts. It is unlikely that microorganisms transmitted via xenotransplantation would be aerosolized. Rather infections that are donor-derived will more likely be in blood or secretions. Therefore, universal precautions must be strictly enforced within the hospital setting and consideration given for other types of isolation requirements in individual cases. Contact monitoring will be important but brings up an unchartered area regarding obtaining informed consent from contacts of the patients. Dr. Chapman will go into these issues in more detail.
Xenotransplantation is a new and rapidly expanding field which may benefit patients suffering from end-stage organ disorders and patients with other illnesses such as parkinson's disease, diabetes and perhaps even HIV. Infectious risks will undoubtedly be real but their precise nature is at this point in time still unknown. We must learn from past experiences with allotransplantation and attempt to determine which organisms might be able to cross species lines. It is critical to develop logical, feasible approaches for screening donors and perform surveillance on recipients as well as on contacts if needed. Finally new more sensitive and specific diagnostic tests must be developed to keep pace with this emerging field and will require the collaboration of transplant researchers, veterinarians, infectious disease specialists, virologists and public health officials.
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Last Modified: 1/2/97
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