Cytomegalovirus infection and disease after solid-organ transplantation: Epidemiology, prevention, and therapy

Cytomegalovirus Infection and Disease After Solid-Organ Transplantation: Epidemiology, Prevention, and Therapy Marc E. Uknis and David L. Dunn Disease caused by cytomegalovirus (CMV) infection remains one of the largest health concerns in clinical transplantation. CMV (transmitted to recipients before transplantation from exogenous sources, including blood transfusions, or during transplantation through the transmission of dormant virus within the transplanted organ) may replicate subsequent to either primary infection or reactivation. During and subsequent to host immunosuppression. CMV disease often manifests as fever and leukopenia; viral invasion of tissue, with involvement of more than I organ, is not uncommon. Formerly, CMV disease was associated with serious morbidity and a substantial decrease in allograft and patient survival rates. Fortunately, thanks to the advent of effective anti viral agent prophylaxis and therapy, CMV disease is lethal rarely today. However, even though the epidemiologic characteristics of disease caused by this viral pathogen have changed over the past 10 to 15 years, it remains a signifiEant cause of morbidity, as shown by rehospitalizations and use of anti viral agent prophylaxis and therapy, which result into increased health care costs. We review current epidemiology, prophylaxis, and therapy of CMV disease in solid-organ transplant recipients.

Copyright © 2000 by W.B. Saunders Company

or more than 3 decades, disease caused by cytomegalovirus (CMV) infection has been one of the largest health concerns in clinical transplantation. A ubiquitous opportunistic pathogen that affects all organ transplant recipients, CMV is a member of the Betaherpesvirinae subfamily of Herpesviridae DNA viruses and can remain latent after primary host infectionJ CMV is transmitted to transplant recipients before transplantation from exogenous sources, including blood transfusions, or during transplantation through the transmission of dormant virus within the transplanted organ. CMV may replicate subsequent to primary infection or reactivation, especially during and subsequent to host immunosuppression. However, only a portion of infected patients manifest systemic illness, or CMV disease, which results in a self-limiting flu-like syndrome in immunocompetent hosts. Unfortunately, complications related to CMV disease can be serious for immunocompromised patients. In this patient

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From theDepartmentofSurgery, UniversityofMinnesota,Minneapolis, AIN. Address reprint requeststo David L. Dunn, MD, PhD,Jay PhiUips Professorand Chairman of Surgery, Departmentof Surgery, Universityof Minnesota, Box 195 Mayo Building, 420 Delaware St SE, Minneapolis, MN 55455. Copyright© 2000~, W.B. SaundersCompany 0955-470X/00[1404-0004510.00/0 doi:l O.1053/trre.2000.16516

population, CMV disease often manifests as fever and leukopenia, and viral tissue invasion with involvement of more than 1 organ is common. 2-4In addition, increasing evidence points to an association of CMV infection and chronic allograft rejection among solidorgan transplant recipients, with the virus showing tropism for many different types of allografts.5,6 CMV disease formerly was associated with serious morbidity and a substantial decrease in allograft and patient survival rates. 7,aBefore the advent of effective antiviral agents, it was common for patients to die of CMV-related disease. 9 Fortunately, today CMV disease is only rarely fatal, l° However, although the epidemiologic characteristics of disease caused by tiffs viral pathogen have changed during the past 10 to 15 years, it remains a significant cause of morbidity, denoted by rehospitalization and the use of antiviral agent prophylaxis and therapy, all of which translate into increased health care costs. This indicates the need for better methods of early diagnosis, prevention, and treatment of CMV infection and disease.

Molecular Virologic and Immunologic Characteristics of CMV Similar to all members of the Herpesviridae family, CMV has a capsid, tegument, and envelope. The genome is composed of 230-kilobase pairs encoding

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approximately 200 genes. The genome is divided into a long (L) and short (S) segment flanked by terminal repetitive sequences. II Internal repeat (IR) sequences also are associated with the L and S segments; these sequences are designated IRL and IRs. Within the IR sequences are unique (U) sequences associated with both the L (Ut.) and S (Us) segments. ~2 Reflective of temporal gene expression, immediate-early, early, and late genes are expressed as a result of viral replication. )3 Capsid proteins are processed within the nucleus of infected cells and eventually become filled with viral DNA. Not surprisingly, antibodies to any of the 3 major capsid proteins (pUL86, pUL46, and pUL80a) do not show virusneutralizing capacity, likely the result of a protective effect of the CMV glycoprotein envelope. Thus, the major capsid proteins currently have little significance for diagnosing or treating CMV infection.14 The CMV tegument is formed by at least 7 proteins, the significance of which has yet to be fully elucidated. Similar to capsid proteins, there is no significant humoral host response to the tegument proteins. However, the pp65 (65 kd) protein, the product of a late gene, is expressed on the cell surface of infected cells both before and during active viral replication. This protein has been identified as a significant antigenic target for cytotoxic T lymphocytes (CTLs), which may be responsible for keeping viral load in check through the destruction of infected cells expressing the pp65 antigen. 15Therefore, a significant functional or numeric reduction in the host T-lymphocyte population, such as during immunosuppression with calcineurin inhibitors (eg, cyclosporine or tacrolimus) or antilymphoc3¢e antibody preparations, effectively diminishes surveillance and control of CMV-infected host cells, facilitating CM-V disease in the infected host. Although immunity to CMV appears to be primarily cell mediated, a humoral response directed mainly against the envelope glycoproteins occurs. Two of the major envelope glycoproteins (gps) are gpUI_55 (gB complex) and gpUL75 (gH), which are detectable on the surface of infected host cells. 16 Both gB and gH are recognized by neutralizing antibodies from the serum of CMV-seropositive individuals. This neutralizing capability may be responsible for the efficacy of pooled hyperimmune globulin preparations in the prevention of CMV disease, as noted by some investigators.17 The presence of neutralizing antibodies also may explain why seropositive individuals receiving transplants develop CMV disease less frequently than seronegative individuals.-9

Clearly, anti-CMV host responses involve both humoral and cell-mediated immunity'. However, the cellular limb of the immune system is the most important: CD8 + CTLs destroy infected host cells, and CD4 + T helper subtype 2 cells recognize CMV antigens in a major histocompatibility complexrestricted fashion, enhancing both the CTL response and humoral immunity through cytokine release. The calcineurin inhibitors selectively prevent the proliferation ofT cells by inhibiting the production of interleukin-2 receptors and the mitogenic cytokine, interleukin-2, preventing allograft rcjection. Unfortunately, this T-cell portion of host immunity also is responsible for maintaining the latency of CMV. Additionally, antilymphocyte preparations decrease T-cell counts and induce the release of such inflammatory cytokines as tumor necrosis factor-eq which has the capacity to induce viral replication in infected host cells. Thus, immunosuppression that includes calcineurin inhibitors, antilymphocyte preparations, or both types of agents not only decreases the effective numbers of antiviral T lymphocytes, but also functionally impairs them and induces viral replication. Therefore, until induction ofallograft tolerance without such agents becomes possible, it is highly likely that CMV x4ill remain the most prevalent opportunistic infection in solid-organ transplant recipients.

Epidemiologic Characteristics, Risk Factors, and CMV Disease Manifestations CMV is a ubiquitous pathogen in humans; historically, the prevalence ofserologic evidence of infection in adults has ranged from 40% to 50% to nearly 100%, depending on geographic area and socioeconomic status (eg, those in developing countries have a greater incidence).ts.19Horizontal transmission commonly occurs by person-to-person contact, usually through secretions, or through blood transfusions or organ transplantation. The most likely reservoir for the virus is in peripheral-blood lymphocytes and monocytes; however, the CMV genome has also been identified in polymorphonuclear leukocytes and endothelial cells of infected patients. ~° Recent studies indicate 50% to 75% of solid-organ transplant recipients develop CMV infection, and 15% to 25% (approximately one third of the infected patients) develop CMV disease. 2,)° In addition, transplant recipients previously treated for CMV disease have a

CMV and Transplantation

25% to 30% recurrence rate after completing an initial course ofantiviral agent therapy.2~ CMV infection and disease occur in close temporal association with maximal host immunosuppression and thus are frequent during the first months after transplantation, with a peak incidence between 2 and 4 months, although disease can occur years after transplantation. Risk factors for CMV disease include: (1) receiving an organ from a CMVseropositive donor (D+; CMV serologic status is denoted in relation to D and recipient [R]; D+/Rpatients are at substantially greater risk than D+/R + patients); this factor alone increases the risk for developing CMV disease by approximately 5 to 7 times; (2) receiving an organ from a cadaver; (3) undergoing treatment for acute rejection, particularly if an antilymphocyte antibody agent is used or several courses of antirejection therapy occur in close temporal association; (4) being older than 55 years; and (5) receiving a pancreas allograft (alone or in addition to a kidney). It is highly likely that factors 2 through 5 are related to the impact of immunosuppression on the host rather than other disparate causes. Thus, CMV disease is approximately 2 times more frequent among renal transplant recipients who receive an organ from a cadaver compared with a close relative and 2 to 3 times more common after antirejection therapy. Of note, it is also associated with a greater incidence of concurrent infection caused by other viruses and fungi. 22 Three types of CMV infection can occur: primary, secondary (or reactivation), and superinfection, any of which can provoke CMV disease. 23 Primary infection invariably occurs after blood transfusion or organ transmission of CMV from a seropositive donor (D +) to a seronegative recipient (R-). This type of infection causes CMV disease with greater frequency than reactivation infection, such that approximately 50% to 75% of the patients develop symptomatic disease. This type of CMV infection is the most difficult to prevent, is more frequently associated with severe clinical disease manifestations, recurs more commonly even after initial successful therapy, and still causes death on occasion, compared with secondary infection and disease. 24 Secondary infection is caused by reactivation of the virus strain that caused the initial infection in the recipient and entered into an episomic latent state within the immunosuppressed host, becoming active subsequent to the suppression of host defenses. Superinfection also can occur after a patient has experienced CMV infection caused by 1 strain of

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virus and is inoculated with a second strain. If this second strain is sufficiently different from the first and host defenses are severely diminished, CMV disease may occur because of reactivation, superinfection, or both. 25 In composite, secondary infection and superinfection can lead to clinical manifestations of CMV disease in approximately 10% to 20% of the transplant recipients, who show generally milder symptoms than those caused by primary infection. Intriguingly, nearly twice as many D+/R + patients develop CMV disease compared with D / R + patients, perhaps indicating that concurrent reactivation infection and superinfection are not uncommon events after solid-organ transplantation. Most patients who develop symptomatic CMV infection show mild symptoms of fever, myalgias, malaise, lethargy, and mild dyspnea, often accompanied by leukopenia. Although increasingly uncommon, more serious manifestations include pneumonitis or pneumonia, gastrointestinal bleeding, nephritis, hepatitis, pancreatitis, and severe leukopenia, which can occur alone or in combination. Progression to fatal CMV syndrome, consisting of severe hypoxia and respiratory failure caused by progressive pneumonitis, as well as hypotension, disseminated intravascular coagulation, massive gastrointestinal hemorrhage, multiple system organ failure, and death, was common but occurs rarely today. Not surprisingly, before the advent of suitable antiviral agents for prophylaxis, CMV disease was associated with a significant decrease in allograft survival in renal transplant recipients. During the past several decades, the impact of CMV infection and disease on allograft and patient survival has diminished. For example, studies conducted during the 1970s showed that CMV nearly halved allograft survival rates in renal transplant recipients. 7,8 More recently, Schnitzler et al26 provided evidence that patients receiving CMV-seronegative kidney allografts had significantly greater 3-year allograft and patient survival rates than recipients of CMVseropositive allografts, regardless of pretransplantation recipient serologic test results, although the impact was markedly less than noted in the earlier studies. However, Dunn et all0 examined the impact of CMV in a prospective, randomized trial involving 266 patients, all administered some type of antiviral agent prophylaxis. They found no significant detrimental effect on allograft or patient survival, even among patients in whom prophylaxis failed who developed CMV disease and underwent antiviral therapy. Dunn et all0 noted primarily an economic

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impact related to rehospitalization and the need for ganciclovir therapy. Other investigators have confirmed these findings.27 The clinical manifestations of CMV can be grouped into 3 categories. First, mild disease manifests as fever with leukopenia and generalized malaise, probably as a result of viremia without tissue invasion at a specific site or sites. Second, it is apparent that CMV has a tropism toward the allograft itself, such that nephritis occurs in some infected kidney transplant recipients, hepatitis in liver transplant recipients, coronary artery disease in heart transplant recipients, pneumonitis in lung transplant recipients, pancreatitis in pancreas transplant recipients, and enteritis in bowel transplant recipients. Last, tissue-invasion at 1 or many sites is the likely result of untreated viremia that progressed to more severe disease. Distant tissue-invasive disease most commonly manifests as diffuse pneumonitis or pneumonia but may involve any part of the gastrointestinal tract, often mimicking erosive gastritis or peptic ulcer disease, and as noted, hepatitis and pancreatitis can occur in all types of solid-organ transplant recipients. Also, a rare but ominous site of distant disease in the transplant recipient is the retina, in which bilateral involvement is the rule. Unfortunately, for some patients, permanent blindness results. Tissueinvasive disease may imitate allograft rejection and be indistinguishable from other forms ofinfectious or ulcerogenic entities in other organ systems. The safest approach when such a process is identified is to obtain tissue for diagnosis. In addition, we routinely seek a thorough ophthalmologic examination at the time of CMV disease diagnosis, regardless of the site, to prevent retinal complications. A strong index of suspicion fueled by knowledge of risk factors is necessary to prevent serious complications of CMV disease, as discussed next.

Diagnosis of CMV Suspicion of CMV disease in transplant recipients generally is based on the presence of fever with constitutional symptoms (fatigue, malaise, and anorexia) and nonspecific laboratory findings (eg, leukopenia). Body fluid or tissue should be sent to the laboratory to confirm or rule out active CMV disease. Advances in immunologic and molecular virologic testing have led to more sensitive and specific methods for diagnosing CMV infection, such that a number of laboratory methods serve to diagnose active CMV infection. However, their sensitivity, specificity,

and Predictive values vary tremendously, as does the information each test provides. The diagnostic methods described next were predated by the biopsy examination, which is still used to identify tissue-invasive disease. Patients with esophagitis, gastroenteritis, hepatitis, nephritis, or pneumonitis should have tissue sampled, if possible without compromising their overall condition. Described by most surgical pathologists as oak-eye or owl-eye cells, CMV-infected tissue shows a cytopathologic state known as cytomegalic inclusion disease. The biopsy has low sensitivity, primarily because of sampling error. However, a positive biopsy result has a very high predictive value (nearly 100%), especially when correlated with signs and symptoms related to the potential site of disease (eg, infiltrates on chest radiograph, melena, elevated fiver function test results, and elevated creatinine level). Quantitation of anti-CMV immunoglobulin G (IgG) and IgM is appropriate in nonimmunocompromised hosts, but as a diagnostic test, it is not sufficiently sensitive or specific to provide diagnostic benefit among immunocompromised patients. Depression of humoral immunity in immunosuppressed patients results in higbly unpredictable serologic antibody measurements. Because serologic tests yield retrospective information, we recommend donor and recipient CMV serologic status assessment only before transplantation. Donor and recipient serologic tests allow the clinician to estimate a relative risk for developing CMV disease. As mentioned, the D+/Rcombination places the recipient at a relative risk nearly 5 to 7 times that of all other transplant recipients with other equivalent risk factors; only rarely do D-/R- patients develop CMV disease. Thus, serologic data can help guide decisions about CMV prophylaxis and risk. However, if CMV disease is suspected, more direct determination of active infection should be undertaken. Conventional culture methods have been used to confirm clinical suspicions and the negative predictive value of other assays. Tissue or body fluid is inoculated onto fibroblast monolayers in the presence ofappropriate medium, and the cells are periodically checked for cytopathic effect. Unfortunately, such a long process is of little value to acutely ill patients. Currently, the clinical use of such cultures is quite limited unlike bacterial cultures (often confirmatorywithin 2 to 3 days), CMV cultures require 2 to 6 weeks of incubation. The reported sensitivity of conventional cultures for diagnosing CMV disease is less than 50%.28 However, this assay remains useful

CMV and Transplantation

on occasion for the isolation of viable virus for later assessment, eg, for patients who do not respond to initial therapy or who develop recurrent disease caused by drug-resistant strains of CMV. Shell vial culture is more commonly used to identify the presence of active CMV infection. Specimens are inoculated into shell vials that include a coverslip ofa fibroblast monolayer. Approximately 18 to 24 hours after incubation, the monolayers are exposed to murine monoclonal antibody directed against CMV immediate-early antigen, and binding is observed through immunofluorescent microscopy. The presence of immediate-early antigen on the cell surface of fibroblasts confirms active CMV replication and implies the presence of CMV virions in the specimen. The drawback to this method is a sensitivity for diagnosing CMV disease of less than 65%.29'30 Another assay now commercially available is the CMV rapid antigen assay, which can only be pet'formed on specimens of a cellular nature (eg, blood cells). Peripheral-blood leukocytes (PBLs) are separated from whole blood and exposed to anti-pp65 murine monoclonal antibody. Antibody binding is determined using immunofluorescent microscopy. The pp65 protein, a product of the UL83 gene, is a tegument protein. Expression of pp65 on the surface of PBLs indicates active in situ viral replication. If greater than 10 of 50,000 PBLs stain positive for pp65, the positive predictive value for the presence of CMV disease approaches 100%.31 This assay is more sensitive than the shell vial culture, although the latter may be performed on acellular fluid samples.3'z33 Unfortunately, both the shell vial culture and rapid antigen assay require immediate processing of the specimen, and a delay of even 24 hours after obtaining the specimen can dramatically decrease sensitivity and specificity.34 This is a drawback for patients treated at institutions that send specimens to diagnostic centers. Polyrnerase chain, reaction (PCR)-based assays hold great promise for improving the sensitivity and specificity of resuhs when diagnosing CMV disease. 28 Using this type of test, viral DNA can be detected in tissue, PBLs, or body fluid (eg, urine or bronchoalveolar lavage). Interestingly, specimens need not be processed immediately and often can be frozen or fixed before PCR assessment. Briefly, a segment of the major immediate-early and/or late gene is amplified using available primers. The presence of the amplified sequence is confirmed on agarose gel electrophoresis with appropriate controls. This method, known as qualitative PCR, is very sensitive

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for detecting the presence of viral DNA; however, such sensitivity is not always advantageous because asymptomatic viral shedding or latent virus may be detected in otherwise heahhy patients, so results must be correlated with the patient's clinical course. According to 1 study, surveillance qualitative PCR had a positive predictive value for CMV disease of only 55%; conversely, there was a 100% negative predictive value. However, the positive predictive value increased to 76% among D+/R- patients. 35Such information might prove valuable as an indication to initiate treatment before the onset of symptomatic disease (preemptive therapy). Although some investigators have advocated this approach, no large-scale, randomized trial has assessed its efficac3, and costeffectiveness compared with prophylractic antiviral therapy (vide infra). Quantitative PCR provides the advantage of measuring viral copies by amplifying a specific sequence and determining the exact number of DNA copies per milliliter in a dilution-type assay. Qualitative PCR may only determine the presence (or absence) of CMV virus, but quantitative PCR can show the number of CMV DNA copies (viral load), which correlates with viral replication and predates the onset of CMV disease. 3638However, precise standardization of this test and correlation with objective clinical parameters of disease remain to be established. For example, greater than 100,000 viral copies/ 106 leukocytes has been considered indicative of impending or present CMV disease, whereas other investigators have reported results as number of copies per milliliter of solution.39 Clearly, methods and reporting must be standardized before quantitative PCR can be widely applied clinically. As molecular techniques become less costly and more commonly available, qualitative PCR may become the standard method for establishing the diagnosis of CMV disease. Until that time, the diagnostic method ofchoice among patients who develop typical signs and symptoms of CMV disease remains either the shell vial culture or rapid antigen test. For now, PCR assays are reserved for assessing the presence of antiviral agent resistance gene sequences or confirming a negative shell vial cuhure or rapid antigen test result among patients in whom a high index of suspicion for the presence of CMV disease exists.

Prevention of CMV Disease In 1996, a conference on the management of CMV disease was held; approximately 20 transplant pro-

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grams were represented, a° Participants discussed their institution's method of preventing CMV disease in organ transplant recipients; interestingly, each method was unique, although most of the specialists agreed that antiviral drug prophylaxis should be administered. However, there was no consensus about duration, type of agent, or the question of whether only a subset of transplant recipients should be administered prophylaxis. The investigators concluded that the optimal anti-CMV regimen had yet to be defined, and 4 years later, this conclusion is still true. Nonetheless, the basic principles of an optimal prophylactic regimen remain the same: (1) efficacy, not only against CMV, but also against other Herpesviridae pathogens; (2) ease of administration, preferably an oral preparation; (3) minimal adverse drug interactions; (4) minimal patient toxicity; and (5) cost-effectiveness. 41 Intuitively, immunization appears to be an optimal way to prevent CMV disease. However, both active and passive immunization protocols have met with limited success. In the early 1980s, the University of Minnesota studied the effects of vaccination using a live attenuated form of CMV, the Towne strain. Among the cohort of patients administered the vaccine before kidney transplantation, the incidence of CMV disease after transplantation was no different compared with the cohort administered placebo. However, the severity of CMV disease significantly differed between the 2 cohorts, being ameliorated among the D+/R- patients.4~ Other investigators duplicated these initial results with the Towne vaccine, leading to attempts to develop more potent vaccines. Currently, the effects of subunit vaccines derived from the envelope glycoproteins, gB and gH, are being studied. The results of a single phase I trial indicate that a recombinant gB analogue is safe and stimulates antibody production in healthy volunteers. 43 However, given the 10% to 20% incidence of CMV disease among CMV-seropositive transplant recipients (ie, endogenous anti-gB or gH IgG), it is difficult to imagine that such subunit vaccines will be more efficacious than the Towne vaccine. Passive immunization has been studied extensively, using either nonspecific immunoglobufin preparations or hyperimmune or pooled immunoglobulin preparations from CMV-positive donors (CMVIg). CMVIg contains 5 to 8 times the concentration of anti-CMV immunoglobulins of nonspecific immunoglobulin preparations and effectivelyprevents serious CMV disease.44 CMVIg carries an indication from the US Food and Drug Administration (FDA) for

preventing CMV disease in seronegative recipients of a kidney from a seropositive donor (D+/R-). FDA approval was based on a placebo-controlled study by the Boston CMVIG Study Group of 59 D+/R- kidney transplant recipients, the authors noted a significant reduction in CMV disease rates (CMVIg, 21% v placebo, 60%; P < .01), but not in seroconversion (71% v 77%; P = not significant).45A similar study of liver transplant recipients showed no benefit in relation to CMV disease prevention for D+/R- patients; however, the CMVIg-treated cohort had an increased l-year survival rate compared with patients administered placebo (86% v 72%; P = .029).46 Several studies provided evidence that the use of acyclovir and CMVIg or nonspecific immunoglobulin preparations reduced the incidence of CMV in moderate-risk patients. 47,a0 However, Rostaing et a149 showed that prophylactic use of this agent in addition to acyclovir did not provide additional benefit in preventing CMV compared with acyclovir alone in D+/R- renal transplant recipients. Unfortunately, CMVIg is expensive, requires intravenous administration, and appears to provide merely an incremental benefit in cost-effectiveness analyses.5° Clearly, additional studies are.needed to establish whether this agent is a suitable choice for prophylaxis and for which groups of patients. During the past 2 decades, the capacity of antiviral agent prophylaxis to prevent the development of CMV disease after solid-organ and bone-marrow transplantation has been intensively investigated. Before the introduction of ganciclovir, acyclovir was the sole available antiviral agent that had a degree of activity against CMV. In 1989, a landmark study indicated that acyclovir prophylaxis (800 to 3,200 mg/d for 12 weeks) led to a significant reduction in the incidence of CMV disease among renal transplant recipients compared with placebo (acyclovir, 7.5% v placebo, 29%; P = .002).5' Unfortunately, not all subsequent studies were able to duplicate these findings; thus, the efficacy of this drug was questioned, particularly in light of its relatively low degree of in vitro CMV activity. In the late 1980s, ganciclovir, 50 times more active against CMV isolates in vitro than acyclovir, became available.5~ New trials focused on the use of prophylactic intravenous ganciclovir because an oral formulation was not available. Because CMV disease remains a substantial cause of morbidity and mortality among bone-marrow transplant recipients, initial trials using ganciclovir focused on this patient population. For example, Winston et als3 showed a significant incidence of CMV

CMV and Transplantation

infection among patients administered 120 days of ganciclovir after transplantation versus placebo (20% v 56%, respectively; P < .001); the incidence of CMV disease also was reduced, although not to a significant degree (10% v 24%; P = .09).53 Similarly, a randomized comparison of intravenous ganciclovir (6 mg/kg/d for 100 days) versus oral acyclovir (3,200 mg/d for 100 days) showed that ganciclovir reduced the incidence of CMV infection dramatically during the first 3 months posttransplantation (5% v 40%; P < .0001). 54-55 Likewise, other investigators showed the efficacy ofganciclovir in reducing the incidence of CMV disease in heart transplant recipients, particularly D+/R- individuals.56These results were encouraging; however, issues of cost, the need for established intravenous access, and the difficulty of intravenous administration made ganciclovir the prophylactic drug of choice only in the immediate perioperative period. At many transplant centers, acyclovir remained the drug used for CMV prophylaxis after hospital discharge. Given the proven efficacy of both antiviral drugs, most institutions, including our own, adopted protocols of sequential prophylaxis based on intravenous ganciclovir for 1 to 2 weeks postoperatively, followed by 10 to 12 weeks of oral acyclovir, because a limited course of intravenous ganciclovir alone was not effective.I°,57 Unfortunately, the 1980s saw the emergence of acquired immunodeficiency syndrome (AIDS) and introduced another population of patients at risk for serious opportunistic infections. It was in patients with AIDS that the first trials of the oral formulation of ganciclovir showed promising results, and the use of oral ganciclovir was approved by the FDA as maintenance therapy for patients with AIDS with CMV retinitis. A subsequent muhicenter trial of liver transplant recipients showed that oral ganciclovir was efficacious in preventing CMV compared with placebo (4.8% v 18.9%;P < .05).58 The most obvious shortcoming of this, trial was its failure to compare oral ganciclovir with the current standard of care, oral acyclovir. Oral ganciclovir has poor bioavailability (-8% to 9%) and is considerably more expensive than acyclovir. The results of a direct comparison of oral acyclovir with oral ganciclovir after preemptive ganciclovir therapy for CMV disease among 41 kidney and fiver transplant recipients administered antilymphocyte antibodies indicated a similar incidence of CMV disease (5%) in both groups. 59 Another concurrent study provided similar results, although D+/R- patients also were administered CMVIg during this trial.6° We are in the process of

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completing a 350-patient, randomized, prospective, controlled trial comparing the efficacy of initial intravenous ganciclovir followed by either oral ganciclovir or oral acyclovir for CMV prophylaxis in kidney, kidney-pancreas, and pancreas transplant recipients. The primary dilemma with the use of both acyclovir and ganciclovir has been their limited bioavailability compared with the respective intravenous formulations. However, acyclovir and ganciclovir have been reformulated to include the L-valylester and are now also produced as oral valacyclovir and valganciclovir. The addition of the valyl ester increases their bioavailability 3 to 5 times and establishes serum levels of either drug equivalent to those after intravenous administration. Recently, the results ef a trial examining the efficacy ofvalacyclovir for CMV prophylaxis in kidney transplant recipients were published. Valacyclovir prophylaxis reduced the incidence of CMV disease significantly among D+/R- patients compared with placebo (16% v 45%; P < .001).61 However, because patients in the control group were administered placebo rather than acyclovir and patients were administered an extremely high dose of valacyclovir (8 g/d for 90 days), the superiority of this agent to oral acyclovir or ganciclovir remains to be established. Clinical trials examining the impact of valganciclovir in reducing the incidence and severity of CMV disease have been implemented. Finally, several recent reports indicate some benefit (primarily cost-effectiveness) may be achieved by CMV surveillance using PCR-based tests and the implementation of intravenous ganciclovir therapy.28,59,62"66Aside from cost issues, a major impetus for the use of this modality has been concern over the development ofstrains of CMV resistant to drugs used for prophylaxis, which has yet to be substantiated. Ganciclovir-resistant CMV usually has a mutation within the UL97 codon, which encodes for a phosphotransferase necessary for activating ganciclovir, rendering it easily identifiable using available PCR techniques. We have been unable to identify the emergence of resistance during antiviral agent prophylaxis in large numbers of patients, 67 although we continue to monitor for such an event. On this basis, patients at our institution not entered into clinical trials are administered oral acyclovir prophylaxis for 12 weeks if the calculated risk for CMV disease does not exceed 2 times; patients with a risk analysis greater than 2 times are administered oral ganciclovir. Thus, all D+/R- patients, those administered antilymphocyte antibody induction or antirejection

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therapy, pancreas and bowel transplant recipients, and those with several compounding risk factors are administered oral ganciclovir. We do not currently use surveillance techniques or preemptive therapy. The following issues remain unresolved in relation to CMV disease in solid-organ transplant patients: (1) whether a form of prophylaxis should be used because some investigators generally observe mild and readily treatable CMV disease manifestations; (2) whether to use rapid antigen or PCR-based testing surveillance as an indicator to implement preemptive therapy; and (3) if prophylaxis is used, whether all patients or only selected subgroups of high-risk patients should be administered prophylaxis, which agent is optimal based on both efficacy in reducing CMV disease and cost, and what the optimal duration of prophylaxis is.

Treatment o[ CNiV Disease Ganciclovir and foscarnet are the drugs of choice for treating CMV disease, but foscarnet is rarely administered to solid-organ transplant patients because of such serious side effects as nephrotoxicity. As noted, ganciclovir, a nucleoside analogue, shows superior in vitro activity compared with acyclovir against CMV. The UL97 open-reading frame of CMV encodes a kinase that phosphorylates it within infected host cells to produce ganciclovir-triphosphate. The triphosphate product inhibits CMV replication by competitively inhibiting the incorporation of deoxygnanidine triphosphate into CMV DNA. This results in premature termination of viral DNA synthesis, inhibiting viral replication. Initial reports indicated considerable benefit derived from the use of this agent to treat CMV disease.2 Ganciclovir is entirely eliminated by the kidneys; the typical treatment dose of 5 mg/kg twice daily requires reduction for patients with diminished creatinine clearance. The parenteral formulation achieves serum concentrations 10-fold greater than the oral drug. For this reason, it is recommended that the intravenous formulation be used for treating CMV disease. We treat CMV disease using 2 weeks of intravenous ganciclovir followed by 10 weeks of oral therapy. In selected instances, mild CMV disease and mild rejection can be successfully treated concurrently.~ We believe that evidence of significant tissue-invasive disease (eg, gastrointestinal ulceration and bleeding, hepatitis, pancreatitis, severe pneumonitis) warrants 3 to 4 weeks of intravenous ganeiclovir followed by oral drug for 8 to 10 weeks.

Severe retinitis also may be treated with intravenous and intravitreal ganciclovir injection. It was recently suggested that qualitative PCR testing may serve to determine the adequacy of therapy.69 Life-threatening CMV disease should be treated with a combination of intravenous ganciclovir, CMVIg, and a concomitant reduction or cessation of immunosuppression. The use of CMVIg should be limited to transplant recipients with severe disease because its therapeutic benefit has not yet been evaluated in recent, large-scale, randomized, controlled clinical trials. However, uncontrolled trial data suggest that it may be useful in treating severe CMV disease in liver transplant recipients. 7° Adverse effects caused by ganciclovir occur among 5% to 15% of the patients and consist of bone marrow suppression (eg, neutropenia and/or thrombocytopenia, generally mild), nausea, diarrhea, fever, rash, elevated liver function test results, nephrotoxicity, and mental status changes. Neutropenia and thrombocytopenia probably are not solely related to the marrow-suppressive properties of ganciclovir, but also to additive similar effects caused by mycophenolate mofetil, azathioprine, or trimethoprim, and usually are reversible with drug cessation. Ganciclovir therapy is appropriate for CMV disease associated with mild to moderate leukopenia, but care must be taken to establish the diagnosis to ensure that another cause of marrow suppression does not exist. Thus, if neutropenia exists as a result of CMV disease, we consider 1 of 2 approaches: (1) the use of CMVIg alone ifthe diagnosis is in question, or (2) the concomitant use of recombinant granulocyte colonystimulating factor with ganciclovir, the latter only if the diagnosis of CM'V disease has been established unequivocally by standard tests. Concurrently, we cease the administration ofother potentially marrowsuppressive agents. If the former approach is used, ganciclovir therapy is implemented after the absolute neutrophil count increases to greater than 500 neutrophils//zL. Foscarnet (trisodium phosphonoformate hexahydrate), an inhibitor of viral DNA polymerase, has excellent in vitro activity against CMV. Its toxicity profile, although similar to ganciclovir, may be more severe and thus limits its clinical use. Nonetheless, it should be considered for transplant recipients with CMV disease unresponsive to ganciclovir. Additionally, 1 study indicates a synergism with ganciclovir in vitro.71 Finally, it bears note that a number of novel agents are being developed for use in the treatment of CMV disease. Currently under clinical investiga-

CMV and Transplantation

tion, the valyl ester o f ganciclovir (valganciclovir) possesses increased bioavailability; thus, as an oral formulation, it m a y be as efficacious as intravenous ganciclovir. O t h e r agents include such nucleoside analogues as cidofovir, b e n z i m i d a z o l e riboside compounds that potently inhibit C M V replication, C M V protease inhibitors, and antisense C M V D N A agents. H o w e v e r , large-scale, r a n d o m i z e d , prospective trials have not provided evidence o f suitable efficacy of these agents in prophylactic or t h e r a p e u t i c regimens. U n t i l such time, intravenous ganciclovir r e m a i n s the most suitable d r u g for the t r e a t m e n t of C M V disease a m o n g solid-organ transplant recipients.

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CMV and Transplantation

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