RESUMEN
The 21st Congress of the European Society of Organ Transplantation (ESOT), held on September 17-20th, 2023, in Athens, Greece, was a pivotal event in transplantation, focusing on the theme "Disruptive Innovation, Trusted Care." The congress attracted a global audience of 2 826 participants from 82 countries, emphasizing its international significance. Machine perfusion, as a groundbreaking technology in organ transplantation, was one of the central focuses of the conference. This year's meeting had a remarkable increase in accepted abstracts on machine perfusion, evidencing its growing prominence in the field. The collective findings from these abstracts highlighted the efficacy of machine perfusion in improving organ viability and transplant outcomes. Studies demonstrated improvements in graft survival and reduction in complications, as well as novel uses and techniques. Furthermore, the integration of machine perfusion with regenerative medicine and its application across multiple organ types were significant discussion points. The congress also highlighted the challenges and solutions in implementing machine perfusion in clinical settings, emphasizing the importance of practical training and international collaboration for advancing this technology. ESOT 2023 served as a crucial platform for disseminating scientific advancements, fostering practical learning, and facilitating international collaborations in organ transplantation. The congress underscored the evolution and importance of machine perfusion technology, marking a significant step forward in enhancing patient outcomes in the field of organ transplantation.
Asunto(s)
Preservación de Órganos , Trasplante de Órganos , Perfusión , Humanos , Europa (Continente) , Supervivencia de Injerto , Preservación de Órganos/métodos , Trasplante de Órganos/métodos , Perfusión/métodos , Perfusión/instrumentación , Sociedades MédicasRESUMEN
The American Transplant Congress (ATC) 2023, held in San Diego, California, emerged as a pivotal platform showcasing the latest advancements in organ machine perfusion, a key area in solid organ and tissue transplantation. This year's congress, attended by over 4500 participants, including leading experts, emphasized innovations in machine perfusion technologies across various organ types, including liver, kidney, heart, and lung. A total of 85 abstracts on organ machine perfusion were identified. Noteworthy advancements included the use of normothermic machine perfusion in mitigating ex-situ reperfusion injury in liver transplantation, the potential of biomarkers in assessing organ quality, and the impact of machine perfusion on graft survival and ischemic cholangiopathy incidence. Kidney transplantation saw promising developments in novel preservation methods, such as subzero storage and pulsatile perfusion. Heart and lung sessions revealed significant progress in preservation techniques, including metabolic alterations to extend organ preservation time. The conference also highlighted the growing interest in machine perfusion applications in pediatric transplantation, multi-visceral organ recovery, Vascularized Composite Allotransplantation, and discussions on novel technologies for monitoring and optimizing perfusion protocols. Additionally, ATC 2023 included critical discussions on ethical concerns, legal implications, and the evolving definition of death in the era of machine preservation, illustrating the complex landscape of transplantation science. Overall, ATC 2023 showcased significant strides in machine perfusion and continued its tradition of fostering global knowledge exchange, further cementing machine perfusion's role as a transformative force in improving transplant outcomes and expanding the donor pool.
Asunto(s)
Preservación de Órganos , Trasplante de Órganos , Perfusión , Humanos , Preservación de Órganos/métodos , Preservación de Órganos/instrumentación , Trasplante de Órganos/métodos , Perfusión/métodos , Perfusión/instrumentaciónRESUMEN
Transplantation has generally been focused on end-stage organ disease in adults. Scientists from Japan are now focusing on in utero transplantation in fetuses with congenital anomalies.
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Trasplante de Tejido Fetal , Humanos , Femenino , Embarazo , Anomalías Congénitas/cirugía , Feto/cirugía , Enfermedades Fetales/cirugía , Trasplante de Órganos/métodosRESUMEN
OBJECTIVE: The mismatch between the demand for and supply of organs for transplantation is steadily growing. Various strategies have been incorporated to improve the availability of organs, including organ use from patients receiving extracorporeal membrane oxygenation (ECMO) at the time of death. However, there is no systematic evidence of the outcome of grafts from these donors. DESIGN: Systematic literature review (Scopus and PubMed, up to October 11, 2023). SETTING: All study designs. PARTICIPANTS: Organ recipients from patients on ECMO at the time of death. INTERVENTION: Outcome of organ donation from ECMO donors. MEASUREMENTS AND MAIN RESULTS: The search yielded 1,692 publications, with 20 studies ultimately included, comprising 147 donors and 360 organ donations. The most frequently donated organs were kidneys (68%, 244/360), followed by liver (24%, 85/360). In total, 98% (292/299) of recipients survived with a preserved graft function (92%, 319/347) until follow-up within a variable period of up to 3 years. CONCLUSION: Organ transplantation from donors supported with ECMO at the time of death shows high graft and recipient survival. ECMO could be a suitable approach for expanding the donor pool, helping to alleviate the worldwide organ shortage.
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Oxigenación por Membrana Extracorpórea , Donantes de Tejidos , Obtención de Tejidos y Órganos , Humanos , Oxigenación por Membrana Extracorpórea/métodos , Oxigenación por Membrana Extracorpórea/tendencias , Obtención de Tejidos y Órganos/métodos , Trasplante de Órganos/tendencias , Trasplante de Órganos/métodosRESUMEN
It is some 50 years since the first published reports appeared of ex vivo preservation of organs for transplantation. Over the intervening decades, organ preservation strategies have become one essential component of world-wide clinical transplant services. In the formative years, translational research in organ hypothermic preservation was grappling with the questions about whether static or dynamic storage was preferable, and the practical implications of those choices. Those studies were also informing the newly expanding clinical transplant services. During the middle years, both preservation modalities were practiced by individual group choices. By the 2000s, the shift in donor demographics demanded a re-evaluation of organ preservation strategies, and now a new era of research and development is promoting adoption of new technologies. In this review we outline many important academic studies which have contributed to this successful history, and give profile to the increasing innovative approaches which are being evaluated for the future. Doi.org/10.54680/fr24310110112.
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Criopreservación , Preservación de Órganos , Preservación de Órganos/métodos , Humanos , Criopreservación/métodos , Historia del Siglo XX , Trasplante de Órganos/métodos , Historia del Siglo XXIRESUMEN
PURPOSE OF REVIEW: This timely review delves into the evolution of multivisceral transplantation (MVT) over the past six decades underscoring how advancements in surgical techniques and immunosuppression have driven transformation, to provide insight into the historical development of MVT, shedding light on its journey from experimentation to a valuable clinical approach. RECENT FINDINGS: The review presents contemporary enhancements in surgical methods within the context of intestinal transplantation. The versatility of MVT is emphasized, accommodating diverse organ combinations and techniques. Both isolated intestinal transplantation (IIT) and MVT have seen expanded indications, driven by improved parenteral nutrition, transplantation outcomes, and surgical innovations. Surgical techniques are tailored based on graft type, with various approaches for isolated transplantation. Preservation strategies and ostomy techniques are also covered, along with graft assessment advancements involving donor-specific antibodies. SUMMARY: This review's findings underscore the remarkable evolution of MVT from experimental origins to a comprehensive clinical practice. The progress in surgical techniques and immunosuppression has broadened the spectrum of patients who can benefit from intestinal transplant, including both IIT and MVT. The expansion of indications offers hope to patients with complex gastrointestinal disorders. The detection of donor-specific antibodies in graft assessment advances diagnostic accuracy, ultimately improving patient outcomes.
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Trasplante de Hígado , Trasplante de Órganos , Trasplante de Páncreas , Humanos , Intestinos/trasplante , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Terapia de InmunosupresiónRESUMEN
PURPOSE OF REVIEW: Normothermic regional perfusion (NRP) is a novel procurement technique for donation after circulatory death (DCD) in the United States. It was pioneered by cardiothoracic surgery programs and is now being applied to abdominal-only organ donors by abdominal transplant programs. Multiple technical approaches can be used for abdominal-only NRP DCD donors and this review describes these techniques. RECENT FINDINGS: NRP has been associated with higher utilization of organs, particularly liver and heart grafts, from DCD donors and with better recipient outcomes. There are lower rates of delayed graft function in kidney transplant recipients and lower rates of ischemic cholangiopathy in liver transplant recipients. These benefits are driving increased interest from abdominal transplant programs in using NRP for DCD procurements. SUMMARY: This paper describes the technical aspects of NRP DCD that allow for maximization of its use based on different donor and policy characteristics.
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Trasplante de Hígado , Preservación de Órganos , Perfusión , Donantes de Tejidos , Humanos , Perfusión/métodos , Perfusión/efectos adversos , Perfusión/instrumentación , Donantes de Tejidos/provisión & distribución , Preservación de Órganos/métodos , Preservación de Órganos/efectos adversos , Trasplante de Hígado/métodos , Trasplante de Hígado/efectos adversos , Obtención de Tejidos y Órganos , Trasplante de Órganos/métodos , Selección de Donante , Trasplante de Riñón/métodos , Trasplante de Riñón/efectos adversosRESUMEN
In view of rapid advancements in the field of transplantology, emerging solutions in tissue procurement for transplantation became a crucial area of research. Tissue transplantation plays a notable role in improving the quality of life for patients afflicted with various ailments, and the increasing number of transplants necessitates the exploration of innovative procurement methods. This study examines a new direction in transplantology, placing focus on innovative approaches to tissue procurement and discussing the commonly used method of "ex mortuo," i.e., retrieving organs from deceased donors. Given the growing demand for organs, the paper discusses the innovative approach slowly emerging as 3D bioprinting. The paper discusses the key challenges associated with the use of this method in transplantology, including issues of biocompatibility, vascularization, and integration with the immune system. The paper also discusses the latest scientific achievements in the field, such as the first transplants of bioprinted organs, demonstrating the practical application of this technology in medicine. It is also the analysis of the ethical, social, and legal aspects related to these new solutions. The article provides a comprehensive overview of the latest trends in transplantology and presents a holistic view of the current state of knowledge and prospects for development in this pivotal area of medicine.
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Obtención de Tejidos y Órganos , Humanos , Obtención de Tejidos y Órganos/métodos , Trasplante de Órganos/métodos , Trasplante de Órganos/tendencias , Impresión Tridimensional , Bioimpresión , Donantes de TejidosRESUMEN
Implementation of the HIV Organ Policy Equity (HOPE) Act marks a new era in transplantation, allowing organ transplantation from HIV+ donors to HIV+ recipients (HIV D+/R+ transplantation). In this review, we discuss major milestones in HIV and transplantation which paved the way for this landmark policy change, including excellent outcomes in HIV D-/R+ recipient transplantation and success in the South African experience of HIV D+/R+ deceased donor kidney transplantation. Under the HOPE Act, from March 2016 to December 2018, there were 56 deceased donors, and 102 organs were transplanted (71 kidneys and 31 livers). In 2019, the first HIV D+/R+ living donor kidney transplants occurred. Reaching the full estimated potential of HIV+ donors will require overcoming challenges at the community, organ procurement organization, and transplant center levels. Multiple clinical trials are ongoing, which will provide clinical and scientific data to further extend the frontiers of knowledge in this field.
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Transmisión de Enfermedad Infecciosa/estadística & datos numéricos , Infecciones por VIH/epidemiología , VIH , Trasplante de Órganos/métodos , Donantes de Tejidos/provisión & distribución , Receptores de Trasplantes , Comorbilidad , Salud Global , Infecciones por VIH/transmisión , HumanosRESUMEN
Stimulator of interferon genes (STING) is an innate immune sensor of cytoplasmic dsDNA originating from microorganisms and host cells. STING plays an important role in the regulation of murine graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be similarly activated during other transplantation modalities. In this review, we discuss STING in allo-HSCT and its prospective involvement in autologous HSCT (auto-HSCT) and solid organ transplantation (SOT), highlighting its unique role in nonhematopoietic, hematopoietic, and malignant cell types.
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Trasplante de Células Madre Hematopoyéticas , Proteínas de la Membrana/inmunología , Trasplante de Órganos , Animales , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Proteínas de la Membrana/metabolismo , Trasplante de Órganos/métodos , Transducción de Señal , Trasplante Homólogo/métodosRESUMEN
The increased procurement of organs from donors with risk factors for blood-borne diseases and the expanding syphilis epidemic have resulted in a growing number of organs transplanted from donors with reactive syphilis serology in our center. Based on guidelines, recipients typically receive therapy shortly after the transplant, but data on outcomes are limited. The primary objective of this study was to determine syphilis seroconversion rates at three months post-transplant in recipients of solid organs procured from donors with reactive syphilis serology. Organ donors and recipients were tested for syphilis antibody; positive results were confirmed with Treponema pallidum Particle Agglutination (TPPA). Eleven donors with reactive syphilis antibody donated organs to 25 syphilis negative recipients. Three recipients seroconverted at post-transplant month 3. All of them had received therapy shortly after transplant. TPPA was negative in all 3. Despite post-transplant treatment, 3 of 25 (12%) syphilis negative recipients of organs from syphilis positive donors seroconverted at 3 months. All remained TPPA negative possibly reflecting passive antibody transfer or differing test sensitivity to low level treponemal antibodies. Further studies are needed to assess optimal syphilis transmission prevention strategies and follow up recipient testing in organ transplantation.
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Trasplante de Órganos , Sífilis , Humanos , Sífilis/diagnóstico , Sífilis/epidemiología , Estudios Retrospectivos , Estudios de Seguimiento , Treponema pallidum , Donantes de Tejidos , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Receptores de Trasplantes , AnticuerposRESUMEN
Solid organ transplant recipients (SOTRs) remain at high risk for infection throughout their post-transplant course. Dosing of immunosuppressive medications, strategies that prevent infection, and choice of empiric antimicrobial treatment could be optimized by a better understanding of an individual patient's risk for infectious complications. Diagnostic tests that qualitatively or quantitatively measure the function of the immune system and/or its response to infection may be useful for individualized management decisions. Numerous studies have identified an association between infectious outcomes after solid organ transplantation (SOT) and the results of a variety of non-pathogen-specific or "pathogen-agnostic" immune monitoring tests. These biomarkers include humoral immune markers, functional or quantitative assessments of cellular immunity, transcriptomic-based diagnostics, and replication of viruses within the human virome, which have been used to predict or diagnose a variety of different infectious diseases complicating SOT. In this narrative review, we discuss several host-derived immune biomarkers that show promise for either predicting or diagnosing infection among SOTRs. However, additional studies are needed to determine the optimal use of immune response testing. Whether immune biomarkers contribute added benefits to current standard clinical care has not yet been determined. Testing must be validated across a range of clinical scenarios, including surveillance to predict infection risk and diagnosis of active infection at various time points post transplant.
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Infecciones , Trasplante de Órganos , Humanos , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Infecciones/etiología , Receptores de Trasplantes , BiomarcadoresRESUMEN
Recent advances in antimicrobial resistance detection have spurred the development of multiple assays that can accurately detect the presence of bacterial resistance from positive blood cultures, resulting in faster institution of effective antimicrobial therapy. Despite these advances, there are limited data regarding the use of these assays in solid organ transplant (SOT) recipients and there is little guidance on how to select, implement, and interpret them in clinical practice. We describe a practical approach to the implementation and interpretation of these assays in SOT recipients using the best available data and expert opinion. These findings were part of a consensus conference sponsored by the American Society of Transplantation held on December 7, 2021 and represent the collaboration between experts in transplant infectious diseases, pharmacy, antimicrobial and diagnostic stewardship, and clinical microbiology. Areas of unmet need and recommendations for future investigation are also presented.
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Antiinfecciosos , Enfermedades Transmisibles , Trasplante de Órganos , Sepsis , Humanos , Antibacterianos/uso terapéutico , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Farmacorresistencia Bacteriana , Antiinfecciosos/uso terapéutico , Receptores de Trasplantes , Sepsis/tratamiento farmacológicoAsunto(s)
Xenoinjertos , Trasplante de Órganos , Porcinos , Obtención de Tejidos y Órganos , Trasplante Heterólogo , Animales , Humanos , Trasplante de Órganos/ética , Trasplante de Órganos/métodos , Trasplante de Órganos/tendencias , Obtención de Tejidos y Órganos/ética , Obtención de Tejidos y Órganos/métodos , Obtención de Tejidos y Órganos/tendencias , Xenoinjertos/trasplante , Trasplante Heterólogo/ética , Trasplante Heterólogo/métodos , Trasplante Heterólogo/tendenciasRESUMEN
Partial heart transplantation is a new type of transplant that delivers growing heart valve replacements for babies. Partial heart transplantation differs from orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. It also differs from homograft valve replacement because viability of the graft is preserved by tissue matching, minimizing donor ischemia times, and recipient immunosuppression. This preserves partial heart transplant viability and allows the grafts to fulfill biological functions such as growth and self-repair. These advantages over conventional heart valve prostheses are balanced by similar disadvantages as other organ transplants, most importantly limitations in donor graft availability. Prodigious progress in xenotransplantation promises to solve this problem by providing an unlimited source of donor grafts. In order to study partial heart xenotransplantation, a suitable large animal model is important. Here we describe our research protocol for partial heart xenotransplantation in nonhuman primates.
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Trasplante de Corazón , Trasplante de Órganos , Trasplantes , Animales , Trasplante Heterólogo/métodos , Primates , Trasplante de Órganos/métodos , Rechazo de InjertoRESUMEN
Organ transplant is one of the best options for many medical conditions, and in many cases, it may be the only treatment option. Recent evidence suggests, however, that the COVID-19 pandemic might have detrimentally affected the provision of this type of healthcare services. The main purpose of this article is to use Data Envelopment Analysis and the Malmquist Index to assess the impact that the pandemic caused by the novel coronavirus SARS-CoV-2 had on the provision of solid organ transplant services. To this purpose, we use three complementary models, each focusing on specific aspects of the organ donation and transplantation process, and data from Brazil, which has one of the most extensive public organ transplant programs in the world. Using data from 17 States plus the Federal District, the results of our analysis show a significant drop in the performance of the services in terms of the organ donation and transplantation process from 2018 to 2020, but the results also indicate that not all aspects of the process and States were equally affected. Furthermore, by using different models, this research also allows us to gain a more comprehensive and informative assessment of the performance of the States in delivering this type of service and identify opportunities for reciprocal learning, expanding our knowledge on this important issue and offering opportunities for further research.
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COVID-19 , Trasplante de Órganos , Obtención de Tejidos y Órganos , Humanos , SARS-CoV-2 , Pandemias , Trasplante de Órganos/métodosRESUMEN
UTx is performed to address absolute uterine infertility in the presence of uterine agenesis, a nonfunctional uterus, or after a prior hysterectomy. After the initial success of UTx resulting in a livebirth (2014) in Sweden, there are over 70 reported UTx surgeries resulting in more than 40 livebirths worldwide. Currently, UTx has been performed in over 10 countries. As UTx is transitioning from an "experimental procedure" to a clinical option, an increasing number of centers may contemplate a UTx program. This article discusses essential steps for establishment of a successful UTx program. These principles may be implemented in cis- and transgender UTx candidates.
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Infertilidad Femenina , Trasplante de Órganos , Anomalías Urogenitales , Femenino , Humanos , Histerectomía , Infertilidad Femenina/cirugía , Trasplante de Órganos/métodos , Planificación Estratégica , Útero/cirugíaRESUMEN
PURPOSE OF REVIEW: To review recent medical literature addressing antimicrobial stewardship in solid organ transplant (SOT) recipients. RECENT FINDINGS: Few studies with a strict focus on SOT patients have been published to date. Team-based antimicrobial stewardship interventions in SOT populations do have beneficial impacts on antimicrobial stewardship-relevant outcomes. Several studies have compared SOT-specific antibiograms to widely accepted hospital antibiograms; these studies all have found important differences in resistance rates among bacterial pathogens. Novel biomarkers like virome monitoring for assessing the net state of immunosuppression hold promise for individualized antimicrobial stewardship interventions. SUMMARY: SOT patients are an understudied population with respect to antimicrobial stewardship interventions. Current antimicrobial stewardship interventions for SOT patients are largely extrapolated from studies in general patient populations and may not accurately reflect SOT-specific infection risks or outcomes. Antimicrobial stewardship interventions do impact SOT-relevant care goals but require significantly more research to achieve the depth and reach of antimicrobial stewardship interventions developed for general populations.
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Programas de Optimización del Uso de los Antimicrobianos , Infecciones , Trasplante de Órganos , Programas de Optimización del Uso de los Antimicrobianos/métodos , Humanos , Pruebas de Sensibilidad Microbiana , Trasplante de Órganos/efectos adversos , Trasplante de Órganos/métodos , Receptores de TrasplantesRESUMEN
BACKGROUND AND AIMS: Kupffer cells (KCs) are the resident intravascular phagocyte population of the liver and critical to the capture and killing of bacteria. Calcineurin/nuclear factor of activated T cells (NFAT) inhibitors (CNIs) such as tacrolimus are used to prevent rejection in solid organ transplant recipients. Although their effect on lymphocytes has been studied extensively, there are limited experimental data about if and how CNIs shape innate immunity, and whether this contributes to the higher rates of infection observed in patients taking CNIs. APPROACH AND RESULTS: Here, we investigated the impact of tacrolimus treatment on innate immunity and, more specifically, on the capability of Kupffer cells (KCs) to fight infections. Retrospective analysis of data of >2,700 liver transplant recipients showed that taking calcineurin inhibitors such as tacrolimus significantly increased the likelihood of Staphylococcus aureus infection. Using a mouse model of acute methicillin-resistant S. aureus (MRSA) bacteremia, most bacteria were sequestered in the liver and we found that bacteria were more likely to disseminate and kill the host in tacrolimus-treated mice. Using imaging, we unveiled the mechanism underlying this observation: the reduced capability of KCs to capture, phagocytose, and destroy bacteria in tacrolimus-treated animals. Furthermore, in a gene expression analysis of infected KCs, the triggering receptor expressed on myeloid cells 1 (TREM1) pathway was the one with the most significant down-regulation after tacrolimus treatment. TREM1 inhibition likewise inhibited KC bacteria capture. TREM1 levels on neutrophils as well as the overall neutrophil response after infection were unaffected by tacrolimus treatment. CONCLUSIONS: Our results indicate that tacrolimus treatment has a significant impact directly on KCs and on TREM1, thereby compromising their capacity to fend off infections.
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Bacteriemia/etiología , Inmunosupresores/efectos adversos , Macrófagos del Hígado/efectos de los fármacos , Trasplante de Órganos/efectos adversos , Infecciones Estafilocócicas/etiología , Tacrolimus/efectos adversos , Animales , Femenino , Citometría de Flujo , Humanos , Inmunosupresores/uso terapéutico , Macrófagos del Hígado/fisiología , Masculino , Staphylococcus aureus Resistente a Meticilina , Ratones , Persona de Mediana Edad , Trasplante de Órganos/métodos , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Estudios Retrospectivos , Tacrolimus/uso terapéuticoRESUMEN
BACKGROUND: Several molecular platforms now exist for early detection of infectious pathogens. In this review, we present the currently available literature summarizing clinical outcomes using rapid diagnostic tests (RDTs) in the solid organ transplant (SOT) population. We also present potential benefits and drawbacks of these technologies for SOT patients. METHODS: We completed a PubMed search querying for 31 specific RDTs AND ("SOT"). We also queried PubMed for studies on RDT outcomes in the general population. References of the resulting relevant studies were reviewed and incorporated if the study population included at least one SOT patient. All culture specimen types were included. Only full-text peer-reviewed publications in English were examined. RESULTS: Our search yielded eleven studies. Across these studies, integrating RDTs with ASP intervention led to faster species identification and susceptibility results, faster time-to-optimal therapy, decreased hospital length-of-stay and costs, and decreased mortality. Potential drawbacks of RDTs in the SOT population included: overdiagnosis due to increased sensitivity (i.e., Clostridium difficile), decreased yield of tests for respiratory pathogens, and lack of identification of important pathogens in this population such as, Aspergillus species. CONCLUSIONS: Although there is a scarcity of studies involving SOT patients, current available data suggests that the use of RDTs helps improve patient outcomes and minimizes inappropriate antimicrobial use when coupled with proactive ASPs. Future studies should focus on clinical outcomes in SOT patients specifically, as well as how to optimize the use of RDTs in conjunction with traditional microbiology methods.