Cytomegalovirus Disease Occurring Before Engraftment in Marrow Transplant Recipients

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1 830 Cytomegalovirus Disease Occurring Before Engraftment in Marrow Transplant Recipients Ajit P. Limaye, Raleigh A. Bowden, David Myerson, and Michael Boeckh From the Department ofmedicine, Division ofinfectious Diseases, University of Washington School ofmedicine; and the Fred Hutchinson Cancer Research Center, Seattle, Washington Little information is available regarding the incidence, clinical course, and response to treatment of cytomegalovirus (CMV) disease that occurs before engraftment in marrow transplant recipients. We identified 25 patients over a 12.5 year period who developed CMV disease before achieving engraftment. Twelve cases were diagnosed during life, and 13 cases were diagnosed at autopsy. The lung was the site most commonly involved (92% of patients), and most of the patients (92%) were CMV seropositive. Significant copathogens were identified in 45% of the patients. All nine patients with CMV pneumonia died within 6 weeks after the diagnosis was made, and one of two patients with gastrointestinal disease also died 6 weeks after the diagnosis was made despite the administration of antiviral therapy. Surveillance cultures were not helpful in identifying patients at risk for disease. Histopathological examination of the lungs of patients with early CMV pneumonia only infrequently showed typical CMV lesions. In conclusion, CMV disease occurring before engraftment in CMVseropositive recipients was uncommon, was frequently associated with the presence of other opportunistic pathogens, and was associated with a high fatality rate. Better diagnostic methods to identify patients at risk are required in the preengraftment period. Cytomegalovirus (CMV) disease is an important infectious cause of morbidity and mortality following marrow transplantation. Before the advent of preventive strategies, CMV disease typically occurred between 30 and 100 days (median interval, 55 days) after marrow transplantation [1]. Various preventive strategies such as the use of CMV-negative or filtered blood products for seronegative patients [2], administration of prophylactic ganciclovirto all CMV-seropositive patients at engraftment [3, 4], or early administration of ganciclovir at the first sign of CMV infection [5, 6] have led to a significant reduction in the incidence of CMV disease and CMV-related mortality [6-8]. See the editorial response by Fischer and Masur on pages Since the most widely used preventive strategies involve administration of an agent with significant marrow toxicity (i.e., ganciclovir), they are usually implemented only after marrow engraftment has occurred. Thus, these strategies have no impact on CMV disease that develops before engraftment. Received 15 May 1996; revised 25 November The guidelines of the institutional review board of the Fred Hutchinson Cancer Research Center were followed in the conduct of this study. This work was supported in part by the National Institutes of Health (CA 18029, CA 15704, and HL 36444) Correspondence: Dr. Ajit P. Limaye,Universityof Washington MedicalCenter, Box , 1959 Pacific Avenue N.E., Seattle, Washington Reprints: Dr. Michael Boeckh, Fred -Hutchinson Cancer Research Center, Program in Infectious Diseases, 1124 Columbia Street, Seattle, Washington Clinical Infectious Diseases 1997;24: by The University of Chicago. All rights reserved /97/ $02.00 Cases ofcmv disease that occurred before engraftment have been reported [9], but to our knowledge no detailed review of the frequency of this complication or of the response to treatment is available. We reviewed all cases of CMV disease that occurred before marrow engraftment over a 12.5-year period at Fred Hutchinson Cancer Research Center (Seattle), determined the incidence ofthe disease, identified associated factors, reviewed the histopathological findings, and described the response to treatment. Methods Patientselection. We included patients who had undergone marrow transplantation over a 12.5-year period (1 January June 1995) at Fred Hutchinson Cancer Research Center and who had CMV disease (as determined by biopsy or bronchoalveolar lavage [BAL]) before engraftment (i.e., an absolute neutrophil count of >500/mm 3 for 2 days). Evaluations for CMV disease before transplantation included routine chest radiography and appropriate diagnostic studies (endoscopy with biopsy, BAL, or open lung biopsy) when a patient had gastrointestinal or pulmonary complaints. We excluded patients who had CMV disease before marrow transplantation. Definitions. CMV disease was defined as demonstration of CMV in visceral biopsy specimens (i.e., from lung, gastrointestinal, or kidney tissue) by culture, histology, or by culture or immunofluorescence assay of BAL fluid with use of monoclonal antibodies [3]. CMV pneumonia was diagnosed at autopsy if CMV was isolated from lung tissue and concurrent pneumonitis was detected on histopathological examination. CMV-related mortality was defined as death that occurred within 6 weeks after the diagnosis of CMV disease was made

2 cm 1997;24 (May) CMV Disease Before Engraftment 831 [7, 10]. A copathogen was defined as any viral, fungal, bacterial, or parasitic pathogen that was detected by histopathology, culture, or immunofluorescence assay at the same site(s) where CMV was demonstrated. Virologic testing and antiviral prophylaxis and treatment. Serologies, tube cultures, fluorescent antibody assays, and shell vial cultures were performed as previously described [11, 12]. Between January 1986 and June 1992, all CMV-seropositive recipients ofallogeneic marrow received high-dose intravenous acyclovir (500 mg/m" every 8 hours) as previously described [13]. Between January 1987 and August 1993, CMV-seropositive autologous transplant recipients received high-dose intravenous acyclovir (500 mg/rrr' every 8 hours) from day 5 before transplantation until day 30 after transplantation [13, 14]. Patients who were seropositive for herpes simplex virus received low-dose intravenous acyclovir (250 mg/rrr' every 12 hours) from day 5 before transplantation until day 30 after transplantation. Neither foscamet nor ganciclovir was given prophylactically before engraftment. Patients with CMV disease were treated with either ganciclovir or foscamet, with or without intravenous immunoglobulin. Surveillance cultures. A surveillance culture was defined as a viral culture of the blood, urine, or throat that was obtained between 14 days before marrow infusion and 1 day before the onset of disease. Protocols for obtaining viral cultures varied during the study period. Before 1993, blood, urine, and throat cultures were done before transplantation and weekly thereafter. After 1993, routine surveillance cultures (of blood only) were done weekly starting on day 11 after transplantation. Statistical evaluation. We used the two-sided Fisher's exact test to compare the incidence of CMV disease between groups. Results Incidence ofcmvdisease before engraftment. From 1 January 1983 to 30 June 1995,3,554 allogeneic transplants (1,795 in CMV-seropositive recipients and 1,759 in CMV-seronegative recipients) and 770 autologous transplants (425 in CMVseropositive recipients and 345 in CMV-seronegative recipients) were performed. During this 12.5-year period, 27 patients had CMV isolated before engraftment from biopsy specimens or BAL fluid specimens or at autopsy. Two patients (both of whom had CMV disease diagnosed at autopsy) were excluded because neither concurrent histopathological evidence ofpneumonitis nor radiographic evidence ofpneumonia were present before death. The remaining 25 patients comprised the study group: 12 had CMV disease diagnosed during life, and 13 had CMV disease diagnosed at autopsy. Twenty cases of CMV disease occurred before engraftment in 1,795 CMV-seropositive patients who received allogeneic grafts, yielding an estimated incidence of 1.1%. The incidence of CMV disease was 1.7% (6 of 354) among recipients of HLA (human leukocyte antigen)-mismatched, related marrow; 1.6% (6 of 383) among recipients of matched unrelated marrow; and 0.8% (8 of 1,012) among recipients of HLA-matched, related marrow (P =.15 for recipients in the mismatched-related group vs. those in the matched-relatedgroup;p = 0.2 for recipientsin the matched unrelated group vs. those in the matched-related group). Two cases of disease occurred in seronegativerecipients who received marrow from a seronegativedonor (2 [0.1%] of 1,759recipients). The incidence of CMV disease before engraftment among patients who received autologous grafts was 0.7% (3 of 425) for seropositive recipients; no cases occurred in seronegative autograft recipients. The calculated incidence of preengraftment CMV disease did not change over the period of the study (incidences during three 4-yearperiods beginning in 1984 were 1.7%, 0.8%, and 1.3%; P = NS for all comparisons). Halfofthe total number ofcases of CMV disease that occurred before engraftment were diagnosed at autopsy. The overall autopsy rate at our center was 58%, and this rate did not change significantly throughout the study period. Also, the autopsy rate was similar for patients who died either before or after engraftment. Thus, the reported incidences may represent slight underestimates of the true incidence of CMV disease before engraftment. Characteristics of patients with CMV disease before engraftment. Table 1 shows the characteristics ofthe 25 patients who developed CMV disease before engraftment; CMV disease was diagnosed during life in 12 ofthese patients, while it was diagnosed at autopsy in 13. The lung was the site most commonly involved (92% ofpatients), followed by the esophagus (12%). Two patients with CMV disease diagnosed at autopsy had evidence ofdisseminated disease. Copathogens were identified in 45% ofpatients and included Aspergillus species (3 patients [12%]), Pneumocystis carinii (2 [8%]), bacteria (2 [8%]), Candida species (4 [16%]), or other viruses (2 [8%]). There were no statistically significant differences between patients with CMV disease diagnosed at autopsy and those with CMV disease diagnosed during life (P >.05 for all variables). Surveillance cultures. At least one surveillance culture of blood, urine, or a throat specimen was performed between 14 days before transplantation and the onset of disease for all 13 patients with CMV disease diagnosed at autopsy. Seven (54%) ofthese 13 patients had CMV isolated from urine and/or throat specimens before they underwent transplantation; three had positive surveillance cultures between transplantation and the onset ofdisease (two ofthese patients also had positive cultures before they underwent transplantation). Surveillance cultures were performed for nine of the 12 patients with CMV diagnosed during life. Of these, three had CMV isolated from throat specimens before transplantation. One additional patient had a positive throat culture between transplantation and the onset ofdisease (table 1). Histopathologicalfindings. Slides were available for review of the histopathological findings for 16 patients with CMV pneumonia (four of whom had the disease diagnosed

3 832 Limaye et a1. cm 1997;24 (May) Characteristics of 25 patients who developed cytomegalovirus disease before marrow en Table 1. graftment. No. (%) of patients with indicated characteristic CMV disease diagnosed CMV disease diagnosed Variable during life (n = 12) at autopsy (n = 13) Total Median age in y (range) 23 (5-51) 33 (11-51) 26 (5-51) Male gender 6 (50) 11 (85) 17 (72) Underlying disease Chronic myelogenous leukemia 4 (33) 3 (23) 7 (28) Acute myelogenous leukemia 2 (17) 2 (15) 4 (16) Acute lymphoblastic leukemia 4 (33) 5 (39) 9 (36) Aplastic anemia 1 (8.5) 1 (8) 2 (8) Lymphoma 1 (8.5) 2 (15) 3 (12) Disease status Remission 3 (25) 2 (15) 5 (20) Advanced or relapse 8 (67) 9 (70) 17 (68) Not applicable 1 (8) 2 (15) 3 (12) Type of graft Allogeneic, MR 3 (25) 6 (46) 9 (36) Allogeneic, MMR 3 (25) 4 (31) 7 (28) Allogeneic, MUR 4 (33) 2 (15) 6 (24) Autologous 2 (17) 1 (8) 3 (12) Conditioning regimen Cyclophosphamide 12 (100) 12 (92) 24 (96) Total body irradiation 10 (83) 12 (92) 22 (88) CMV serostatus Seropositive recipient 11 (92) 12 (92) 23 (92) Seronegative recipient 1(8) 1 (8) 2 (8) High-dose acyclovir therapy* 6 (50) 3 (23) 9 (36) GVHD, grade ~2t 7 (70) 3 (33) 10 (50) Median day of CMV disease diagnosis (range) 16 (5-28) 17(1-34) 17 (1-34) CMV disease site! Lung 10 (83) 13 (100) 23 (92) Esophagus 2 (17) 1 (8) 3 (12) Excretion of CMV Blood Urine 0 6 (43) 6 (27) Throat 4 (44) 7 (53) 11 (50) Any site 4 (44) 8 (62) 12 (54) NOTE. CMV = cytomegalovirus; GVHD = graft versus host disease; MMR = mismatched, related; MR = matched, related; MUR = matched, unrelated. * Intravenous acyclovir at a dosage of500 mg/nr' q8h beginning 5 days before marrow infusion. t GVHD diagnosed either before or within 3 days of the onset of CMV disease. t Some patients had CMV disease at more than one site. Only includes patients who had at least one set of surveillance cultures of blood, urine, or throat specimens performed before the onset of CMV disease. by biopsy during lifetime and 12 of whom had the disease diagnosed at autopsy) and for two patients with gastrointestinal disease. Three of the 16 patients with pneumonia had changes typical of CMV disease (i.e., diffuse foci [two patients] or necrotizing foci [onelwith inflammation of the intervening lung tissue). Slight changes suggestive of CMV disease were observed in four patients, while no specific changes were observed in seven (the pathology could not be evaluated in two patients because ofthe presence ofmassive fungal infection). Diffuse alveolar hemorrhage was seen in eight of 16 patients. Examination ofa sample from one oftwo patients with gastrointestinal disease showed typical esophageal lesions; a sample from the other patient was insufficient for review. CMV inclusions were seen in samples from three of 16 patients with pneumonia and samples from the one evaluable patient with gastrointestinal disease. Treatment andoutcome ofpreengraftment CMVdisease following marrow transplantation. Table 2 shows the treatment regimens used and the outcomes for the patients with CMV disease

4 cm 1997;24 (May) CMV Disease Before Engraftment 833 diagnosed during life. Ofthe 12 patients with CMV disease diagnosed during life, 10 had CMV pneumonia and two had esophageal disease. Only seven (60%) of 12 patients lived long enough after the diagnosis of CMV disease to achieve marrow engraftment (five of them were receiving ganciclovir), The other five patients died between 3 days and 22 days after the diagnosis of CMV disease and never achieved engraftment. All but two ofthe patients were treated; ofthose who received treatment, all received an antiviral agent (either ganciclovir or foscamet) with or without immunoglobulin. Both ofthe untreated patients died within 2 weeks after CMV disease was diagnosed. Of the 10 treated patients, nine died within 6 weeks after CMV disease was diagnosed, yielding a fatality rate of 90%. The only survivor was a patient who received ganciclovir for CMV esophageal disease. The time from diagnosis ofcmv disease until death ranged from 3 days to 37 days (median time, 17 days). Autopsy findings were available for nine of the 11 patients with CMV disease diagnosed during life. CMV was either the primary cause of death or a contributory cause of death (as stated in the autopsy report) for nine ofthe 11 patients. One patient did not undergo autopsy, and the other patient died of recurrent lymphoma 6 weeks after undergoing. marrow transplantation. Discussion Table 2. The results of our study indicate that CMV disease occurs before engraftment predominantly in seropositive marrow recipients. The incidence of CMV disease is 1.1% among allograft recipients and 0.7% among autograft recipients; this infection is often associated with other opportunistic pathogens and responds poorly to treatment. Neither baseline patient characteristics nor virological surveillance cultures appear to be helpful in identifying patients at risk for CMV disease before engraftment. Excretion ofcmv in urine and in the throat before the onset of CMV disease was detected in 54% of patients (table 1). However, since excretion of CMV in the urine or in the throat before engraftment is relatively common among seropositive allogeneic marrow transplant recipients (15%-20% of such recipients; [15]) the predictive value of this finding is low. Thus, it does not appear helpful for identifying patients at high risk for developing early CMV disease. None of the patients who excreted CMV before they developed CMV disease had the virus isolated from blood, probably because of the inefficiency of isolating CMV from blood with few circulating mononuclear cells. Perhaps a more sensitive technique, such as PCR assay of plasma, which could be performed even in the absence of peripheral WBCs, would have identified more patients with active CMV infection in the blood who were at high risk ofdeveloping early CMV disease [16-18]. An evaluation of this technique during the preengraftment period is warranted. One of the striking findings of our study was the fatality rate of nearly 100% despite treatment among patients who had preengraftment CMV disease diagnosed during life; this finding was probably related to the complete absence of host cellular Treatments and outcomes for patients with preengraftment cytomegalovirus disease diagnosed during life. Time Day of from High-dose Day of Site of engraftment diagnosis Patient Type of graft, acyclovir Grade of diagnosis after CMV Copathogens after until no. HLA match therapy GVHD transplantation disease isolated transplantation Treatment Outcome death (d) 1 Allogeneic, MUR Yes 3 13 Esophagus None 27 Fsc; Gcv + Ig Died 25 2 Autologous No 28 Lung None 37 Gcv + Ig Died 37 3 Autologous Yes 5 Lung None 31 Gcv; Fsc + Ig Died 35 4 Allogeneic, MMR No 2 12 Lung None Fsc + Ig Died 10 5 Allogeneic, MUR No 12 Lung Aspergillus Fsc + Ig Died 6 fumigatus 6 Allogeneic, MMR Yes 31 Lung Pneumocystis None Died 12 carin ii, RSV 7 Allogeneic, MR No 3 23 Lung None Gcv Died 22 8 Allogeneic, MR No 2 19 Lung None 22 Gcv + Ig Died 16 9 Allogeneic, MUR Yes 10 Lung Xanthomonas None Died 3 maltophilia 10 Allogeneic, MUR Yes 3 28 Lung None 30 Gcv + Ig Died Allogeneic, MR Yes 2 13 Lung Parainfluenza 20 Fsc; Gcv + Ig Died 17 I virus 12 Allogeneic, MMR No 2 23 Esophagus None 25 Gcv Survived NOTE. CMV = cytomegalovirus; Fsc = foscamet; Gcv = ganciclovir; GVHD = graft versus host disease; Ig = immunoglobulin; MMR = mismatched, related; MR = matched, related; MUR = matched, unrelated; RSV = respiratory syncytial virus.

5 834 Limaye et al. em 1997;24 (May) immunity. This response rate appears to be worse than that for patients who develop CMV disease after engraftmentresponse rates of 35%-70% have been reported for these patients [7, 10, 19-21]. In addition to the absence ofhost cellular immunity in the preengraftment period, the relatively high incidence of copathogens or the high proportion of patients with hyperacute graft versus host disease (tables 1 and 2) requiring treatment with high-dose steroids may also have contributed to the high fatality rate. The relatively high proportion of patients for whom diagnoses were made only at autopsy was an unexpected finding. One possible reason was that CMV infection was not considered in the differential diagnosis of pulmonary infiltrates occurring in the preengraftment period because the high-risk period for CMV disease is generally considered to be days after marrow transplantation. On the other hand, many of these patients were found during life to be infected with other pathogens, and these pathogens were believed to be the source of the pulmonary infiltrates and/or hypoxia. The finding that another pathogen was isolated (from the lung, blood, or another site) in 11 of the 13 patients with CMV disease diagnosed at autopsy supports the latter explanation. Whether CMV or the coisolate was pathogenic in these cases cannot be determined from our data. Unfortunately, there is currently no test that definitively establishes the causative.role of one particular pathogen in a setting of several potential pathogens. However, we used criteria for the definition ofcmv disease [3] that is in agreement with widely accepted criteria [22]. In addition, the fact that the patients infected with CMV alone had clinical pictures that were identical to those of patients infected with copathogens supports a pathogenic role for CMV in the pneumonitis in these patients. Furthermore, there was no difference in survival between those patients infected with CMV alone and those infected with CMV plus additional pathogens at the same site, both in our relatively small study and in an analysis ofsurvival in a recently completed randomized trial [23] (M. Boeckh, unpublished data). Thus, it is likely that CMV played a significant role, and more consideration should be given to the pathogenicity of CMV during the preengraftment period. The lung pathology observed in patients who had CMV pneumonia before engraftment was generally typical of CMV lung infections, but there were important differences. The typical CMV lesion is a diffuse interstitial thickening associated with moderate numbers of inflammatory cells, mostly lymphocytes. CMV-infected cells or cytomegalic cells may be present in a scattered distribution [24]. In some patients' lungs, CMVinfected cells cluster into foci in which an increased number of inflammatory cells are present focally ("diffuse focus"), whereas in other lungs the foci ate associated with significant tissue destruction ("necrotizing focus"), often in a miliary distribution [25]. We observed nothing more than focal or multifocal hemorrhage and little or no interstitial disturbance in many of the lungs from patients with CMV pneumonia before engraftment. Diffuse alveolar hemorrhage was sometimes seen, while typical CMV lesions were observed infrequently. The hemorrhage that was observed may also have been due to the presence of concurrent etiologic agents. Frequently, a diagnosis ofcmv infection could not be determined from the histology but was confirmed by the results of the viral culture. The decision to administer antiviral prophylaxis must be based on the relative incidence of CMV disease, the outcome once the diagnosis has been established, and the safety of the available antiviral agents. Prophylactic options for CMV disease that occurs before engraftment include high-dose acyclovir [8, 13, 26], foscamet [27, 28], or ganciclovir [4]. In the present study, the incidence of CMV disease before engraftment (data not shown) and the survival rate (table 2) appeared similar before and during the periods in which highdose acyclovir was routinely administered. Foscamet, which has infusion-related toxicity and renal toxicity but lacks marrow toxicity, was given throughout the preengraftment period in two studies [27, 28]. Ganciclovir has been given in a 7-day course before transplantation [4, 29], but it is usually not given during the interval between transplantation and engraftment because of its high potential for marrow toxicity [3, 4]. Whether either foscamet or a pretransplantation course of ganciclovir are effective in preventing disease cannot be determined from these studies because of the small sample sizes. Given the low incidence of CMV disease before engraftment, the toxicity associated with the currently available antivirals, and the lack of identifiable risk factors for CMV disease, a targeted approach based on a virological marker (i.e., a PCR assay of plasma) may be the most promising way to prevent CMV disease before engraftment, as has been reported for CMV disease following engraftment [5, 6]. In summary, we have described our experience with CMV disease before engraftment over a 12.5-year period at a single institution. We found that CMV disease occurred before engraftment mainly in CMV-seropositive marrow transplant recipients and was frequently associated with the presence of other pathogens. The fatality rate among these patients was nearly 100% despite the administration ofantiviral therapy and specific therapy for infections due to copathogens. As antiviral agents that are more effective and less toxic to the marrow become available, identification of patients who are at particularly high risk for developing CMV disease before engraftment by methods such as PCR assay ofplasma may help in decreasing the mortality associated with this infectious complication of marrow transplantation. Acknowledgment The authorsthank JenniferJames for assistance in the collection of data.

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