Cytomegalovirus Infection in Dialysis Patients and Personnel NINA E. TOLKOFF-RUBIN, M.D.; ROBERT H. RUBIN, M.D., F.A.C.P.; EVELYN E. KELLER, M.A.; GEORGE P. BAKER, M.D., F.A.C.P.; JOHN A. STEWART, M.D.; and MARTIN S. HIRSCH, M.D.; Boston, Massachusetts
In a 12-month prospective study of cytomegalovirus infection on an acute hemodialysis unit, 10 of 8 0 patients ( 1 3 % ) and none of 26 staff developed active cytomegalovirus infection. Seven infections were coincidental with renal allograft rejection; three occurred 3 to 6 weeks after the transfusion of multiple units of conventional blood into seronegative patients. No person-to-person transmission was documented. In contrast to the effects of transfusing conventional blood, all 2 1 patients who entered dialysis without detectable cytomegalovirus antibody and received 2 to 10 U of frozen deglycerolyzed erythrocytes (total of 157 U) remained seronegative. Transmission of cytomegalovirus infection with transfusion with conventional blood is probably secondary to passage of leukocyte-borne virus that is lost during the freezing and deglycerolization procedure. Frozen erythrocytes prepared by cytoagglomeration procedures appear to be free of viable leukocytes and appear to carry a minimal risk of transmitting cytomegalovirus infection.
for children with cytomegalovirus infection than among offspring of nurses not similarly exposed. Consistent with this is the report by Yeager (21) that noted a 4.1% to 7.7% per year rate of cytomegalovirus seroconversion among previously seronegative pediatric nurses in contrast to no documented seroconversion among 27 seronegative nurses without exposure to patients excreting this agent, who were followed for an average of 29 months. The Massachusetts General Hospital (MGH) Hemodialysis Unit afforded an excellent opportunity for study of the infection, as its close affiliation with an active renal transplant service assured the constant introduction of patients excreting cytomegalovirus, and the acute nature of the unit provided a constant influx of new patients with renal failure of diverse origins. In addition, the availability of a variety of blood products for transfusion, including frozen deglycerolized erythrocytes prepared by the Huggins technique of cytoagglomeration (22), provided the opportunity to assess the role of different forms of blood transfusion in the transmission of this agent. Materials and Methods
T H E FREQUENCY, importance, and mode of spread of cytomegalovirus infection among hemodialysis patients and personnel are incompletely understood (1-9). Some workers (9, 10) have suggested that the infection has an epidemiologic pattern similar to that well documented for hepatitis-B infection. On the other hand, person-toperson contact on a dialysis unit has been suggested in at least one epidemic of cytomegalovirus infection among transplant patients (11). The possibility of dialysis-acquired cytomegalovirus infection is of considerable concern for both patients and staff. The serious consequences of the infection among renal transplant patients have been increasingly recognized (3, 12-19). The potential risk of congenital abnormalities in the offspring of dialysis nurses and technicians with overt or covert cytomegalovirus infection is also appreciated. In an analagous situation, Haldane and associates (20) have reported a significantly higher incidence of congenital defects among the offspring of nurses caring • From the Department of Medicine, Harvard Medical School; The Infectious Disease and Hemodialysis Units of the Medical Service of the Massachusetts General Hospital; Boston, Massachusetts; and the Laboratory Branch of the Center for Disease Control; Atlanta, Georgia.
Annals of Internal Medicine 89 (Part l):625-628, 1978
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All hemodialysis patients and staff of the MGH Hemodialysis Unit during the period 1 March 1975 to 28 February 1976 were enrolled in the study. Serum specimens were drawn from all patients and personnel at monthly intervals throughout their tenure on the unit. Samples were stored at — 20 °C and later tested for cytomegalovirus antibodies by complement fixation and indirect hemagglutination with antigens derived from the AD-169 strain of the virus (23). Titers equal to or greater than 1:8 were considered positive. All hemodialysis patients were checked weekly for serum glutamic oxalacetic transaminase (SGOT) abnormalities, and monthly for the presence of hepatitis B surface antigen (HBsAg). Specimens were obtained from dialysis personnel at monthly intervals for SGOT and HBsAg measurements. All patients and personnel who had abnormal liver function test results or an unexplained febrile illness of greater than 2 days' duration, and those entering onto dialysis because of renal allograft rejection, underwent the following: [1] A complete blood count and differential, erythrocyte sedimentation rate, SGOT, alkaline phosphatase, bilirubin and lactic dehydrogenase tests were done on at least two occasions 7 to 10 days apart; [2] blood samples for cytomegalovirus antibody levels were drawn 2 weeks apart, in addition to the usual monthly surveillance specimens; [3] cultures for cytomegalovirus done on buccal swabs and clean-voided urine specimens when available, on two separate occasions, 3 weeks apart. For virologic studies, each of three tubes of human embryon© 1 9 7 8 American College of Physicians
625
Table 1. Incidence of P personnel develop!ed active cytomeigalovirus infection during the period of study, t Stable titers of >1:8 by complement fix*ition and indirec t hemagglutination assays.
ic lung fibroblasts was inoculated with 0.3 ml of undiluted urine, which had been treated with antibiotics, and the pH adjusted to neutrality. Buccal swabs were placed in Eagle's media containing serum and antibiotics. Specimens were clarified by filtration or centrifugation, and 0.3 to 1.0 ml was inoculated into three tubes each of human embryonic lung, primary human embryonic kidney, and primary rhesus monkey kidney cells. Human embryonic lung cell cultures were maintained for 42 days; human embryonic kidney and rhesus monkey kidney cultures were maintained for 21 days. Virus isolates were identified by their characteristic cytopathic effects and host cell range. Hemodialysis was done using coil dialyzers via arteriovenous shunts or fistulae. Dialysis tanks were routinely cleansed with Chlorox . All patients with active cytomegalovirus infection or liver function test abnormalities were isolated during dialysis, and their dialysis tanks were soaked in formaldehyde for at least 2 h. All dialysis nurses were required to wear operating room scrub suits. Sterile gloves were worn whenever blood access sites were handled. Although the patients (particularly surgical patients with acute renal failure) had often received a wide variety of blood products before their entrance onto dialysis, once dialysis was instituted only frozen erythrocytes were used for transfusion. This blood had been stored at — 65 °C for variable periods of time, and then thawed and deglycerolized by the Huggins technique of cytoagglomeration before transfusion (22). Cytomegalovirus infection was diagnosed on the basis of either isolation of the virus or a greater than fourfold increase in the titer of antibody to the virus by both complement fixation and indirect hemagglutination techniques.
after massive blood transfusion. None of these three postsurgical patients had any documented contact with the seven post-transplant patients, as they were dialyzed by different nurses in different rooms with different machines at different times. In no instance could transmission of the virus to other dialysis patients or to dialysis unit personnel be documented. All 11 surgical patients plus five medical patients who had received 3 U or more of conventional blood before the initiation of dialysis (for a total of 168 U) had positive serologic tests for cytomegalovirus. Thirteen of these patients were antibody-positive when first tested approximately 1 month after conventional blood transfusion, and their titer of antibody did not change on serial testing. Three patients, all of whom were excreting the virus, seroconverted. In contrast, only 42 of 64 patients (66%) who received no blood or only frozen erythrocytes before dialysis had positive serologies (JP < 0.01). The antibody titers of the seropositive patients remained stable during the course of the study. Fifty of these 64 patients were older than 50 years of age, with the remaining 14 being older than age 20. There was no significant difference in ages between the medical and surgical patients. Twenty-one medical patients entered dialysis with negative cytomegalovirus serologies, and subsequently received 2 to 10 U of frozen erythrocytes during the course of the study (total of 157 U). None of these 21 patients receiving frozen blood developed positive cytomegalovirus serologies, abnormal liver function tests, or unexplained febrile illnesses. Twenty-six staff members were studied for evidence of cytomegalovirus infection: 18 permanent staff at monthly intervals for a year and eight fellows who rotated onto the unit for periods of 2 months each (Table 1). There was no evidence of active cytomegalovirus infection among any of these persons, all of whom had close, direct, personal contact with the patients. Particularly noteworthy are the negative serologies on all six dialysis nurses, all of whom had been working on the unit for a period of longer than 2 years, and four of them for longer than 5 years.
Results
Eighty hemodialysis patients were followed for a mean period of 3.4 months (range, 2.5 to 11.0) during the course of the study. Ten of these 80 patients (13%) had evidence of active cytomegalovirus infection. All 10 excreted the virus in their oral secretions or their urine, or both, and showed a greater than fourfold rise in their cytomegalovirus antibody titers. Any patient with a positive serology by the complement fixation test was invariably positive by the indirect hemagglutination technique. Seven of the 10 patients with active infection were renal allograft recipients who required dialysis because of acute graft rejection. All seven were shown to be excreting virus in specimens obtained on the first day of dialysis. The remaining three patients with active infection required dialysis because of acute renal failure that occurred in the setting of major surgical procedures requiring more than 10 U of conventional blood transfusion (total of 42 U). Active infection in these patients developed 3 to 6 weeks 6 2 6
November 1978
Discussion
Our study attempts to define the mode of spread of cytomegalovirus infection among hemodialysis patients and staff. Although 13% of patients undergoing treatment on the unit during this 12-month period were shown to have active cytomegalovirus infection, no person-to-person transmission could be found. These findings are further confirmed by the persistently negative cytomegalovirus status of the six dialysis nurses, four of whom had been working on the unit for longer than 5 years. Yeager (21) showed an approximately 5% per year rate of cytomegalovirus seroconversion among pediatric nurses working with virus-excreting infants. The difference in the rate of cytomegalovirus acquisition by personnel in dialysis and neonatal units may be related to isolation techniques or to a higher communicability by infected infants. Moreover, handling of excretions is under-
• Annals of Internal Medicine • Volume 89 • Number 5 (Part 1)
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standably less satisfactory with neonates. Whether such freedom from person-to-person spread of cytomegalovirus infection occurs on other dialysis units with less rigid techniques for isolation and handling of blood access sites remains to be seen. Of particular interest to this study is the differential role of a variety of blood products in transmitting this virus. Sixteen patients (all 11 surgical patients plus five medical patients) who had received more than 3 U of conventional blood before dialysis (total of 168 U) had positive serologic tests for cytomegalovirus; in contrast, only 42 of 64 patients (66%) who had received no blood or only frozen erythrocytes had positive cytomegalovirus serologies (P < 0.01). This figure of 66% positivity corresponds to the occurrence of cytomegalovirus antibodies among comparably aged normal populations in the U.S. (24-26). Most striking is the failure of 157 U of frozen deglycerolized blood prepared by the Huggins technique of cytoagglomeration to produce a single seroconversion among 21 seronegative patients. In contrast, the three surgical patients who were originally seronegative all became seropositive after transfusion of more than 10 U of conventional blood each (total of 42 U) and developed clinical evidence of active cytomegalovirus infection. Units of blood from an estimated 2.7% to 12% of all blood donors are thought to be capable of transmitting cytomegalovirus on transfusion (27-29). If these figures are accurate, then our experience with the frozen blood is even more dramatic, and a comparison of the characteristics of this blood product with others should provide important information on the pathogenesis of post-transfusion cytomegalovirus infection. The major difference between frozen erythrocytes thawed and deglycerolized by the Huggins technique of cytoagglomeration from other preparations appears to lie in the absence of viable leukocytes in the frozen blood. Leukocytes retrieved from this product have been found to be nonviable when assessed by vital staining and blastogenic culture assays, using phytohemagglutinin, concanavalin-A, pokeweed mitogen, and allogeneic lymphocytes as stimulants (Fuller TC: personal communication). In contrast, conventional blood (whether whole blood or even washed, packed erythrocytes) contains relatively large numbers of viable leukocytes. Since Kaariainen, Klemola, and Paloheimo (30) first suggested that cytomegalovirus was the major cause of the post-transfusion heterophile-negative mononucleosis syndrome, a number of attempts have been made to culture the virus from specific blood fractions. With few exceptions (31-33) this has not been possible, (24-26, 3335), and it would appear that in the great majority of instances cytomegalovirus is not transmitted in an infectious form. Recent studies of patients with active cytomegalovirus mononucleosis have shown that in such patients virus may be recovered from either or both the mononuclear and polymorphonuclear leukocyte cell populations (3, 36). Lang and others (37-40) have suggested that latent virus may be present in leukocytes of persons with previous cytomegalovirus infection and, after transfusion, may be activated to produce active infection. Re-
cently, the human transfusion experience has been duplicated in a murine model of cytomegalovirus (41). Transfusion of blood from latently infected mice into allogeneic hosts resulted in activation of the virus; similarly, transfusion from uninfected donors into latently infected recipients resulted in viral activation. Thus blastogenic transformation that follows the transfusion of viable leukocytes to an immunocompetent host may induce cytomegalovirus from a latent state within otherwise normal leukocytes (42). Clinical experience is consistent with these experimental findings. It appears that conventional blood containing large numbers of viable leukocytes carries a relatively high risk of initiating cytomegalovirus infection (27, 28). A recent report by Lang and colleagues (37) suggests that the transfusion of leukocyte-poor blood results in a lower rate of cytomegalovirus conversion. Our data suggest that frozen erythrocytes free of viable leukocytes carry a minimal risk of transmitting cytomegalovirus infection. If these data can be confirmed, the routine use of leukocytefree blood in situations where post-transfusion cytomegalovirus infection is an important cause of morbidity would be a significant advance. ACKNOWLEDGMENTS: Grant support: in part by Research Grant CA 12464 and Contract N I H 43222 from the U.S. Public Health Service. Presented in part at the Sixteenth Interscience Conference on Antimicrobial Agents and Chemotherapy; 27 October 1976; Chicago, Illinois. • Requests for reprints should be addressed to Robert H. Rubin, M.D., F.A.C.P.; Infectious Disease Unit, Massachusetts General Hospital; Fruit Street; Boston, MA 02114. Received 12 June 1978; revision accepted 15 August
References 1. C U T F O R T H R, M I T C H E L L RM, M U N D Y G R : Cytomegalovirus mono-
nucleosis following renal haemodialysis. Med J Aust 2:1103-1104, 1968 2. N A K A O T, I D E D A S, SHIONO H, SUZUKI M: Cytomegalovirus infection
in patients receiving long-term hemodialysis. JAMA
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3. F I A L A M, P A Y N E JE, B A R N E TV, M O O R E TC, H E N L E W, M O N T G O M ERIE JZ, C H A T T E R J E E SN, G U Z E LB: Epidemiology of cytomegalovirus
infection after transplantation and immunosuppression. / Infect Dis 132:421-433, 1975 4. PABICO RC, H A N S H A W JB, Y A K U B YN, T A L L E Y TE, F R E E M A N RB:
Cytomegalovirus infections in chronic hemodialysis patients. I. Initial observations. Proc Clin Dial Transplant Forum 1:117-118, 1971 5. B E T T S R F , D O U G L A S RG, PABICO RC, T A L L E Y TE, Y A K U B YN,
FREEMAN RB: Shedding patterns of Herpes simplex virus and cytomegalovirus among chronic dialysis patients. Trans Am Soc Artif Intern Organs 18:262-265, 1972 6. K A N T O R GL, JOHNSON BL JR: Incidence of cytomegalovirus infection in uremic patients. Antimicrob Agents Chemother 8:347-349, 1969 7. SPENCER ES: Cytomegalovirus antibody in uremic patients prior to renal transplantation. Scand J Infect Dis 6:1-4, 1974 8. B E T T S R F , F R E E M A N RB, D O U G L A S R G J R , T A L L E Y TE, R U N D E L L B:
Transmission of cytomegalovirus infection with renal allograft. Kidney Int 8:385-392, 1975 9. R Y T E L MW, BALAY J: Cytomegalovirus infection and immunity in renal allograft recipients: assessment of the competence of humoral immunity. Infect Immun 13:1633-1637, 1976 10. CRAIGHEAD JE: Immunologic response to cytomegalovirus infection in renal allograft recipients. Am J Epidemiol 90:506-513, 1969 11. COULSON AS, LUCAS ZJ, C O N D Y M, C O H N R: Forty-day fever. An
epidemic of cytomegalovirus disease in a renal transplant population. West J Med 120:1-7, 1974 12. SIMMONS RL, L O P E Z C, BALFOUR H J R , K A L I S J, R A T T A Z Z I LC, N A -
JARIAN JS: Clinical virological correlation in renal transplant recipients. Ann Surg 180:623-634, 1974 13. F I N E RN, G R U S H K I N CM, M A L E K Z A D E H M, W R I G H T H T J R : Cyto-
megalovirus syndrome following renal transplantation. Arch 105:564-570, 1972
Surg
14. B E T T S R F , F R E E M A N RB, D O U G L A S R G J R , T A L L E Y TE: Clinical Tolkoff-Rubin
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manifestations of renal allograft derived primary cytomegalovirus infection. AmJDis Child 131:759-763, 1977 15. SUWANSIRIKUL S, R A O N, D O W L I N G JN, H O M: Primary and secondary cytomegalovirus infection: Clinical manifestations after renal transplantation. Arch Intern Med 137:1026-1029, 1977
28. P R I N C E AM, SZMUNESS W, M I L L I A N SJ, D A V I D DS: A serologic study
of cytomegalovirus infections associated with blood transfusions. N Engl / M e d 284:1125-1131, 1971 29. A R M S T R O N G JA, TARR GC, Y O U N G B L O O D LA, D O W L I N G JN, S A S -
LOW AR, LUCAS JP, H O M: Cytomegalovirus infection in children undergoing open-heart surgery. Yale J Biol Med 49:83-91, 1976
16. R U B I N RH, COSIMI AB, T O L K O F F - R U B I N NE, R U S S E L L PS, H I R S C H
MS: Infectious disease syndromes due to cytomegalovirus and their significance among renal transplant recipients. Transplantation 24:458-464, 1977
30. K A A R I A I N I N L, K L E M O L A E, P A L O H E I M O J: Rise of cytomegalovirus
17. BALFOUR H H J R , S L A D E MS, K A L I S JM, H O W A R D RJ, SIMMONS RL,
31. A R M S T R O N G D, BALAKRISHNAN SL, STEGER L, Y U B, S T E N Z E L K H :
NAJARIAN JS: Viral infections in renal transplant donors and their recipients: a prospective study. Surgery 81:487-492, 1977 18. BETTS RF, H A N S H A W JB: Cytomegalovirus in the compromised host(s). Ann Rev Med 28:103-110, 1977 ciation of renal allograft rejections with virus infections. Am J Med 56:280-289, 1974
Cytomegalovirus infections with viremia following renal transplantation. Arch Intern Med 127:111-115, 1971 32. ARMSTRONG D, E L Y M, STEGER L: Post-transfusion cytomegaloviremia and persistence of cytomegalovirus in blood. Infect Immun 3:159163, 1971 33. DIOSI P, MOLDOVAN E, TOMESCU N: Latent cytomegalovirus infection in blood donors. Br Med J 4:660-662, 1969
20. H A L D A N E EV, V A N R O O Y E N CE, E M B I L JA, T U P P E R WC, G O R D O N
34. M I R K O V I C R, W E R C H J, S O U T H MA, B E N Y E S H - M E L N I C K M: Inci-
PC, WANKLIN JM: A search for transmissible birth defects of virologic origin in members of the nursing profession. Am J Obstet Gynecol 105:1032-1040, 1969 21. YEAGER AS: Longitudinal serological study of cytomegalovirus infections in nurses and in persons without patient contact. / Clin Microbiol 2:448-452, 1975 22. HUGGINS CE: Practical preservation of blood by freezing, in Red Cell Freezing: A Technical Workshop Presented by Committee on Workshops of the American Association of Blood Banks. Washington, D.C., American Association of Blood Banks 1973, pp. 31-54
dence of cytomegaloviremia in blood-bank donors and in infants with congenital cytomegalic inclusion disease. Infect Immun 3:45-50, 1971
19. L O P E Z C, SIMMONS RL, M A U E R SM, N A J A R I A N JS, G O O D RA: Asso-
antibodies in an infectious-mononucleosis-like syndrome after transfusion. Br Med J 1:1270-1272, 1966
35. P E R H A M TG, C A U L EO, C O N W A Y PJ, M O T T M G : Cytomegalovirus
infection in blood donors—a prospective study. Br J Haematol 20:307320, 1971 36. R I N A L D O CR J R , BLACK PH, H I R S C H MS: Virus-leukocyte interactions
in cytomegalovirus mononucleosis. J Infect Dis 136:667-678, 1977 37. L A N G DJ, E B E R T PA, R O D G E R S BM, BOGGESS HP, R I X S E RS: Reduc-
Manual
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of Clinical Immunology, edited by ROSE NR, FRIEDMAN H. American Society of Microbiology, Washington, D.C., 1976, pp. 423-427 24. W E N T W O R T H BB, ALEXANDER ER: Seroepidemiology of infections due to members of the herpesvirus group. Am J Epidemiol 94:496-507, 1971
39.
23. W A N E R JL, W E L L E R TH, S T E W A R T JA: Cytomegalovirus, in
25. K A N E RC, ROUSSEAU WE, N O B L E GR, T E G T M E I E R GE, W U L F F H, H E R N D O N HB, C H I N TDY, BAYER WL: Cytomegalovirus infection in a
volunteer blood donor population, infect Immun 11:719-723, 1975 26. BAYER WL, TEGTMEIER GE: The blood donor: detection and magnitude of cytomegalovirus carrier states and the prevalence of cytomegalovirus antibody. Yale J Biol Med 49:5-12, 1976 27. H E N L E W, H E N L E G, SCRIBA M, JOYNER CR, H A R R I S O N FS J R , V O N ESSEN R, P A L O H E I M O J, K L E M O L A E: Antibody responses to the Ep-
stein-Barr virus and cytomegaloviruses after open-heart and other surgery. TV Engl J Med 282:1068-1074, 1970
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tion of post-perfusion cytomegalovirus infections following the use of leukocyte depleted blood. Transfusion 17:391-395, 1977. LANG DJ: Cytomegalovirus infections in organ transplantation and post-transfusion: an hypothesis. Arch Gesamte Virusforsch 37:365-377, 1972 LANG DJ: Transfusion and perfusion-associated cytomegalovirus and Epstein-Barr virus infection: current understanding and investigation, in Transmissible Disease and Blood Transfusion, edited by G R E E E N W A L D TS, JAMIESON GA. New York, Grune and Stratton, 1975, pp. 153-169 RINALDO CR JR, HIRSCH MS, BLACK PH: Activation of latent viruses following bone marrow transplantation. Transplant Proc 8:669-762, 1976 C H E U N G KS, LANG DJ: Transmission and activation of cytomegalovirus with blood transfusion: a mouse model. J Infect Dis 135:841-845, 1977.
42. SCHECHTER GP, S O E H N L E N F, M C F A R L A N D W: Lymphocyte response
to blood transfusion in man. N Engl J Med 287:1169-1173, 1972.
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In a 12-month prospective study of cytomegalovirus infection on an acute hemodialysis unit, 10 of 8 0 patients ( 1 3 % ) and none of 26 staff developed active cytomegalovirus infection. Seven infections were coincidental with renal allograft rejection; three occurred 3 to 6 weeks after the transfusion of multiple units of conventional blood into seronegative patients. No person-to-person transmission was documented. In contrast to the effects of transfusing conventional blood, all 2 1 patients who entered dialysis without detectable cytomegalovirus antibody and received 2 to 10 U of frozen deglycerolyzed erythrocytes (total of 157 U) remained seronegative. Transmission of cytomegalovirus infection with transfusion with conventional blood is probably secondary to passage of leukocyte-borne virus that is lost during the freezing and deglycerolization procedure. Frozen erythrocytes prepared by cytoagglomeration procedures appear to be free of viable leukocytes and appear to carry a minimal risk of transmitting cytomegalovirus infection.
for children with cytomegalovirus infection than among offspring of nurses not similarly exposed. Consistent with this is the report by Yeager (21) that noted a 4.1% to 7.7% per year rate of cytomegalovirus seroconversion among previously seronegative pediatric nurses in contrast to no documented seroconversion among 27 seronegative nurses without exposure to patients excreting this agent, who were followed for an average of 29 months. The Massachusetts General Hospital (MGH) Hemodialysis Unit afforded an excellent opportunity for study of the infection, as its close affiliation with an active renal transplant service assured the constant introduction of patients excreting cytomegalovirus, and the acute nature of the unit provided a constant influx of new patients with renal failure of diverse origins. In addition, the availability of a variety of blood products for transfusion, including frozen deglycerolized erythrocytes prepared by the Huggins technique of cytoagglomeration (22), provided the opportunity to assess the role of different forms of blood transfusion in the transmission of this agent. Materials and Methods
T H E FREQUENCY, importance, and mode of spread of cytomegalovirus infection among hemodialysis patients and personnel are incompletely understood (1-9). Some workers (9, 10) have suggested that the infection has an epidemiologic pattern similar to that well documented for hepatitis-B infection. On the other hand, person-toperson contact on a dialysis unit has been suggested in at least one epidemic of cytomegalovirus infection among transplant patients (11). The possibility of dialysis-acquired cytomegalovirus infection is of considerable concern for both patients and staff. The serious consequences of the infection among renal transplant patients have been increasingly recognized (3, 12-19). The potential risk of congenital abnormalities in the offspring of dialysis nurses and technicians with overt or covert cytomegalovirus infection is also appreciated. In an analagous situation, Haldane and associates (20) have reported a significantly higher incidence of congenital defects among the offspring of nurses caring • From the Department of Medicine, Harvard Medical School; The Infectious Disease and Hemodialysis Units of the Medical Service of the Massachusetts General Hospital; Boston, Massachusetts; and the Laboratory Branch of the Center for Disease Control; Atlanta, Georgia.
Annals of Internal Medicine 89 (Part l):625-628, 1978
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All hemodialysis patients and staff of the MGH Hemodialysis Unit during the period 1 March 1975 to 28 February 1976 were enrolled in the study. Serum specimens were drawn from all patients and personnel at monthly intervals throughout their tenure on the unit. Samples were stored at — 20 °C and later tested for cytomegalovirus antibodies by complement fixation and indirect hemagglutination with antigens derived from the AD-169 strain of the virus (23). Titers equal to or greater than 1:8 were considered positive. All hemodialysis patients were checked weekly for serum glutamic oxalacetic transaminase (SGOT) abnormalities, and monthly for the presence of hepatitis B surface antigen (HBsAg). Specimens were obtained from dialysis personnel at monthly intervals for SGOT and HBsAg measurements. All patients and personnel who had abnormal liver function test results or an unexplained febrile illness of greater than 2 days' duration, and those entering onto dialysis because of renal allograft rejection, underwent the following: [1] A complete blood count and differential, erythrocyte sedimentation rate, SGOT, alkaline phosphatase, bilirubin and lactic dehydrogenase tests were done on at least two occasions 7 to 10 days apart; [2] blood samples for cytomegalovirus antibody levels were drawn 2 weeks apart, in addition to the usual monthly surveillance specimens; [3] cultures for cytomegalovirus done on buccal swabs and clean-voided urine specimens when available, on two separate occasions, 3 weeks apart. For virologic studies, each of three tubes of human embryon© 1 9 7 8 American College of Physicians
625
Table 1. Incidence of P personnel develop!ed active cytomeigalovirus infection during the period of study, t Stable titers of >1:8 by complement fix*ition and indirec t hemagglutination assays.
ic lung fibroblasts was inoculated with 0.3 ml of undiluted urine, which had been treated with antibiotics, and the pH adjusted to neutrality. Buccal swabs were placed in Eagle's media containing serum and antibiotics. Specimens were clarified by filtration or centrifugation, and 0.3 to 1.0 ml was inoculated into three tubes each of human embryonic lung, primary human embryonic kidney, and primary rhesus monkey kidney cells. Human embryonic lung cell cultures were maintained for 42 days; human embryonic kidney and rhesus monkey kidney cultures were maintained for 21 days. Virus isolates were identified by their characteristic cytopathic effects and host cell range. Hemodialysis was done using coil dialyzers via arteriovenous shunts or fistulae. Dialysis tanks were routinely cleansed with Chlorox . All patients with active cytomegalovirus infection or liver function test abnormalities were isolated during dialysis, and their dialysis tanks were soaked in formaldehyde for at least 2 h. All dialysis nurses were required to wear operating room scrub suits. Sterile gloves were worn whenever blood access sites were handled. Although the patients (particularly surgical patients with acute renal failure) had often received a wide variety of blood products before their entrance onto dialysis, once dialysis was instituted only frozen erythrocytes were used for transfusion. This blood had been stored at — 65 °C for variable periods of time, and then thawed and deglycerolized by the Huggins technique of cytoagglomeration before transfusion (22). Cytomegalovirus infection was diagnosed on the basis of either isolation of the virus or a greater than fourfold increase in the titer of antibody to the virus by both complement fixation and indirect hemagglutination techniques.
after massive blood transfusion. None of these three postsurgical patients had any documented contact with the seven post-transplant patients, as they were dialyzed by different nurses in different rooms with different machines at different times. In no instance could transmission of the virus to other dialysis patients or to dialysis unit personnel be documented. All 11 surgical patients plus five medical patients who had received 3 U or more of conventional blood before the initiation of dialysis (for a total of 168 U) had positive serologic tests for cytomegalovirus. Thirteen of these patients were antibody-positive when first tested approximately 1 month after conventional blood transfusion, and their titer of antibody did not change on serial testing. Three patients, all of whom were excreting the virus, seroconverted. In contrast, only 42 of 64 patients (66%) who received no blood or only frozen erythrocytes before dialysis had positive serologies (JP < 0.01). The antibody titers of the seropositive patients remained stable during the course of the study. Fifty of these 64 patients were older than 50 years of age, with the remaining 14 being older than age 20. There was no significant difference in ages between the medical and surgical patients. Twenty-one medical patients entered dialysis with negative cytomegalovirus serologies, and subsequently received 2 to 10 U of frozen erythrocytes during the course of the study (total of 157 U). None of these 21 patients receiving frozen blood developed positive cytomegalovirus serologies, abnormal liver function tests, or unexplained febrile illnesses. Twenty-six staff members were studied for evidence of cytomegalovirus infection: 18 permanent staff at monthly intervals for a year and eight fellows who rotated onto the unit for periods of 2 months each (Table 1). There was no evidence of active cytomegalovirus infection among any of these persons, all of whom had close, direct, personal contact with the patients. Particularly noteworthy are the negative serologies on all six dialysis nurses, all of whom had been working on the unit for a period of longer than 2 years, and four of them for longer than 5 years.
Results
Eighty hemodialysis patients were followed for a mean period of 3.4 months (range, 2.5 to 11.0) during the course of the study. Ten of these 80 patients (13%) had evidence of active cytomegalovirus infection. All 10 excreted the virus in their oral secretions or their urine, or both, and showed a greater than fourfold rise in their cytomegalovirus antibody titers. Any patient with a positive serology by the complement fixation test was invariably positive by the indirect hemagglutination technique. Seven of the 10 patients with active infection were renal allograft recipients who required dialysis because of acute graft rejection. All seven were shown to be excreting virus in specimens obtained on the first day of dialysis. The remaining three patients with active infection required dialysis because of acute renal failure that occurred in the setting of major surgical procedures requiring more than 10 U of conventional blood transfusion (total of 42 U). Active infection in these patients developed 3 to 6 weeks 6 2 6
November 1978
Discussion
Our study attempts to define the mode of spread of cytomegalovirus infection among hemodialysis patients and staff. Although 13% of patients undergoing treatment on the unit during this 12-month period were shown to have active cytomegalovirus infection, no person-to-person transmission could be found. These findings are further confirmed by the persistently negative cytomegalovirus status of the six dialysis nurses, four of whom had been working on the unit for longer than 5 years. Yeager (21) showed an approximately 5% per year rate of cytomegalovirus seroconversion among pediatric nurses working with virus-excreting infants. The difference in the rate of cytomegalovirus acquisition by personnel in dialysis and neonatal units may be related to isolation techniques or to a higher communicability by infected infants. Moreover, handling of excretions is under-
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standably less satisfactory with neonates. Whether such freedom from person-to-person spread of cytomegalovirus infection occurs on other dialysis units with less rigid techniques for isolation and handling of blood access sites remains to be seen. Of particular interest to this study is the differential role of a variety of blood products in transmitting this virus. Sixteen patients (all 11 surgical patients plus five medical patients) who had received more than 3 U of conventional blood before dialysis (total of 168 U) had positive serologic tests for cytomegalovirus; in contrast, only 42 of 64 patients (66%) who had received no blood or only frozen erythrocytes had positive cytomegalovirus serologies (P < 0.01). This figure of 66% positivity corresponds to the occurrence of cytomegalovirus antibodies among comparably aged normal populations in the U.S. (24-26). Most striking is the failure of 157 U of frozen deglycerolized blood prepared by the Huggins technique of cytoagglomeration to produce a single seroconversion among 21 seronegative patients. In contrast, the three surgical patients who were originally seronegative all became seropositive after transfusion of more than 10 U of conventional blood each (total of 42 U) and developed clinical evidence of active cytomegalovirus infection. Units of blood from an estimated 2.7% to 12% of all blood donors are thought to be capable of transmitting cytomegalovirus on transfusion (27-29). If these figures are accurate, then our experience with the frozen blood is even more dramatic, and a comparison of the characteristics of this blood product with others should provide important information on the pathogenesis of post-transfusion cytomegalovirus infection. The major difference between frozen erythrocytes thawed and deglycerolized by the Huggins technique of cytoagglomeration from other preparations appears to lie in the absence of viable leukocytes in the frozen blood. Leukocytes retrieved from this product have been found to be nonviable when assessed by vital staining and blastogenic culture assays, using phytohemagglutinin, concanavalin-A, pokeweed mitogen, and allogeneic lymphocytes as stimulants (Fuller TC: personal communication). In contrast, conventional blood (whether whole blood or even washed, packed erythrocytes) contains relatively large numbers of viable leukocytes. Since Kaariainen, Klemola, and Paloheimo (30) first suggested that cytomegalovirus was the major cause of the post-transfusion heterophile-negative mononucleosis syndrome, a number of attempts have been made to culture the virus from specific blood fractions. With few exceptions (31-33) this has not been possible, (24-26, 3335), and it would appear that in the great majority of instances cytomegalovirus is not transmitted in an infectious form. Recent studies of patients with active cytomegalovirus mononucleosis have shown that in such patients virus may be recovered from either or both the mononuclear and polymorphonuclear leukocyte cell populations (3, 36). Lang and others (37-40) have suggested that latent virus may be present in leukocytes of persons with previous cytomegalovirus infection and, after transfusion, may be activated to produce active infection. Re-
cently, the human transfusion experience has been duplicated in a murine model of cytomegalovirus (41). Transfusion of blood from latently infected mice into allogeneic hosts resulted in activation of the virus; similarly, transfusion from uninfected donors into latently infected recipients resulted in viral activation. Thus blastogenic transformation that follows the transfusion of viable leukocytes to an immunocompetent host may induce cytomegalovirus from a latent state within otherwise normal leukocytes (42). Clinical experience is consistent with these experimental findings. It appears that conventional blood containing large numbers of viable leukocytes carries a relatively high risk of initiating cytomegalovirus infection (27, 28). A recent report by Lang and colleagues (37) suggests that the transfusion of leukocyte-poor blood results in a lower rate of cytomegalovirus conversion. Our data suggest that frozen erythrocytes free of viable leukocytes carry a minimal risk of transmitting cytomegalovirus infection. If these data can be confirmed, the routine use of leukocytefree blood in situations where post-transfusion cytomegalovirus infection is an important cause of morbidity would be a significant advance. ACKNOWLEDGMENTS: Grant support: in part by Research Grant CA 12464 and Contract N I H 43222 from the U.S. Public Health Service. Presented in part at the Sixteenth Interscience Conference on Antimicrobial Agents and Chemotherapy; 27 October 1976; Chicago, Illinois. • Requests for reprints should be addressed to Robert H. Rubin, M.D., F.A.C.P.; Infectious Disease Unit, Massachusetts General Hospital; Fruit Street; Boston, MA 02114. Received 12 June 1978; revision accepted 15 August
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