Cytomegalovirus Infection in a Volunteer Blood Donor Population

Transcription

1 INFECTION AND IMMUNrrY, Apr. 1975, p Copyright American Society for Microbiology Vol. 11, No. 4 Printed in U.S.A. Cytomegalovirus Infection in a Volunteer Blood Donor Population ROBERT C. KANE,' WYATT E. ROUSSEAU,2 GARY R. NOBLE,3 GARY E. TEGTMEIER, HERTA WULFF,3 H. BROOKS HERNDON, TOM D. Y. CHIN,4 AND WILLIAM L. BAYER* Community Blood Center of Greater Kansas City, Kansas City, Missouri 64111,* and the Center for Disease Control, Kansas City, Kansas Received for publication 18 November 1974 Among 223 volunteer blood donors who were studied for evidence of cytomegalovirus (CMV) infection, 58% had complement-fixing antibody and 59% had indirect hemagglutinating antibody to CMV. No virus was isolated from any donor's washed leukocytes or leukocyte-rich plasma in fibroblast monolayer culture. In seven asymptomatic donors (3%), CMV was recovered from urine cultures obtained at the time of blood donation. However, at the time of reexamination, viruria was no longer present and serum antibody titers had not changed. In the three patients studied who received blood from three of the cytomegaloviruric donors, serological evidence of CMV infection developed (fourfold or greater indirect hemagglutinating antibody rise), and one recipient also developed cytomegaloviruria; no illnesses were associated with these infections. Further study is needed to establish that the detection of viruria in donors may identify potentially infective blood. Among blood transfusion recipients, a spectrum of responses to cytomegalovirus (CMV) infection has been observed. These include a serological response in the absence of symptoms, the post-transfusion mononucleosis syndrome (11), polyneuritis (4), hepatitis (19), and pericarditis (12). Among organ transplant recipients and immunosuppressed patients, fatal CMV infections occur (5). The pathogenesis and the epidemiology of these infections have not yet been defined, but blood has been strongly implicated as a vehicle of CMV transmission. However, CMV infection in the transfused patient might also result from reactivation of latent endogenous infection or from exogenous nosocomial sources unrelated to blood products. Henle et al. (9) estimated that 5 to 12% of blood donors were carriers of the virus based on serological responses in recipients, and they also found, as confirmed by Prince et al. (17), a positive correlation between the volume of blood transfused and the risk of CMV seroconversion. Direct evidence for blood as the source IPresent address: National Cancer Institute, Bethesda, Md Present address: University of Texas Health Science Center at Dallas, Dallas, Tex Present address: Center for Disease Control, Atlanta, Ga Present address: Department of Human Ecology, University of Kansas Medical Center, Kansas City, Kan of CMV was reported by Diosi et al. (6), who isolated CMV from peripheral leukocyte cultures of two of 35 asymptomatic nonviruric donors in Rumania. However, Mirkovic et al. (15) were not able to isolate CMV from leukocytes of any of 290 random healthy blood donors in Texas, and Wentworth and Alexander (22) reported no virus isolations from blood specimens of 300 donors in Seattle. While methodological differences may account for some of the disparity, the conclusion (21) that CMV is present in the blood of apparently healthy adults lacks confirmation by virus isolation techniques. As part of a larger study to define further the epidemiology of CMV infections among blood transfusion recipients, we have examined the prevalence of CMV antibodies, viremia, and viruria among apparently healthy volunteer blood donors and have observed the recipients of blood from infected donors. MATERIALS AND METHODS Study population. Two hundred and twenty-three volunteer blood donors at the Community Blood Center of Greater Kansas City were chosen for study on the basis of their willingness to provide a sample of blood obtained during their donation, a urine specimen, and their assent to a possible need for a follow-up sampling. Informed consent was obtained from approximately eight donors per week, from June 1972 to February When a donor was identified as -19

2 720 KANE ET AL. INFECT. IMMUN. infected based on recovery of CMV from a cultured specimen, the donor and the recipient of the unit of blood were studied 6 to 16 weeks later. Collection and inoculation of specimens. (i) Cell culture system. Human fetal tonsil fibroblasts (23) were grown in Eagle minimal essential medium with 10% fetal calf serum, 200 U of penicillin per ml, and 100 ug of streptomycin per ml. Specimens were inoculated into roller tube cultures of fetal tonsil fibroblast monolayers maintained on the same medium with 2% fetal calf serum. Uninoculated control tissue culture tubes were followed in parallel with inoculated tubes. All inoculated tubes were incubated overnight on a stationary rack, after which the maintenance medium was changed, and the tubes were placed on roller drums and incubated at 37 C. Maintenance medium was changed weekly, and tubes were examined for cytopathology twice a week for 6 weeks. Blind passage of negative specimens was not performed. Isolates were identified as CMV by their characteristic cytopathic effect in serial passage in human fibroblast cell strains and by demonstration of nuclear inclusions in cells stained by the May Griinwald-Giemsa technique. (ii) Blood culture. Twenty milliliters of anticoagulated blood (acid/citrate/dextrose solution A) was collected and allowed to settle for 2 h at a 450 slant. The leukocyte-rich plasma layer was withdrawn, and 0.1 ml of leukocyte-rich plasma was inoculated into each of three roller tubes. The remaining leukocyte-rich plasma was centrifuged, and the supernatant plasma was stored at -20 C for antibody determinations. The pellet of white cells was suspended and washed twice in minimal essential medium, then resuspended in 1 ml of minimal essential medium; 0.3 ml of the washed leukocyte suspension was inoculated into each of three tubes of preformed fetal tonsil fibroblast monolayers. Approximately 106 white blood cells were inoculated into each tube. Greater numbers of leukocytes were toxic to the fibroblasts. Urine culture. Two thousand units of penicillin and 1,000 Ag of streptomycin were added to 10 ml of freshly voided urine, and 0.2 ml of the urine was inoculated into each of three tubes of fetal tonsil fibroblasts. The remainder of the specimen was stored at 4 C. Serological procedures. All plasma specimens were stored at -20 C and later tested simultaneously in duplicate under code with the same CMV antigen preparation. The method of Bernstein and Stewart (1) with minor modifications (personal communication, John A. Stewart) was used for the preparation of antigen (AD 169 strain) and for the measurement of indirect hemagglutination (IHA) antibody. Complement-fixing (CF) antibody was measured by the Laboratory Branch microtiter CF technique (2). CMV-specific immunoglobulin M (IgM) was measured by John A. Stewart in serum fractions collected after centrifugation of selected sera through sucrose gradients (3). Blood morphology. Peripheral blood smears were made whenever blood was obtained and differential leukocyte counts were read under code. RESULTS Blood donors. The 223 donors studied were representative of the Blood Center's donor population, which is entirely volunteer. Their ages ranged from 19 to 62 years with a mean of 37 years; 77% were males and 98% were Caucasian. During the 10-month period, there were no isolations of CMV from cultures of leukocyterich plasma or washed leukocytes from any donor. CMV was isolated from the urine specimens of seven donors (3%). In each case the CMV isolation was confirmed by reisolation from the original urine specimen. Six of these seven viruric donors (Table 1) were reexamined 6 to 14 weeks later, but CMV was not recovered from these subsequent urine or blood cultures. In addition, although all seven had detectable initial antibody titers by both CF and IHA, their titers did not distinguish them from the remainder of the donors, no changes in antibody titer occurred, and none of them gave any history of illness in the month preceding the blood donation or during the follow-up period. Six of the seven viruric donors were men. CMV CF plasma antibody was present at titers of 1:8 or greater in 58% of the donors TABLE 1. Laboratory data on seven blood donors with cytomegaloviruria Reciprocal anti- Donor Age Sex Time urine body (years) Se ie culture titer CF IHA M Donation +a (23)b weeks later Oc M Donation + (22) weeks later M Donation + (13) weeks later M Donation + (27) weeks later M Donation + (21) F Donation + (21) weeks later M Donation + (31) weeks later a +, CMV isolated. 'Number of days in culture when viral cytopathogenicity was first observed. " 0, CMV not isolated.

3 VOL. 11, 1975 CMV IN VOLUNTEER BLOOD DONORS 721 (Table 2). IHA antibody titers of 1:10 or greater were present in 59% (Table 3). With each method, the prevalence of antibody increased with age and the average net rate of acquisition of antibody by this donor group was 2% per year of age, up to age 50. In Table 4, the titers obtained by the two methods of antibody determination are compared for each donor, and a direct correlation is obtained. Only six sera had antibody detected by one technique but not by the other. All the donors had normal differential leukocyte counts. Viruric and nonviruric donors could not be distinguished by their peripheral blood morphology, and in no case did the number of atypical lymphocytes exceed 4% of the total leukocyte count. Transfusion recipients. Six of the seven units of blood collected from the seven CMV donors were transfused. Three recipients died before follow-up could be arranged, and three have been studied (Table 5). Two of the recipients had coronary artery bypass surgery, and the third had a gun shot wound repair. Each of the three studied recipients demonstrated serologic evidence of CMV infection by fourfold or greater IHA titer rises 13 to 16 weeks after transfusion. In addition, CMV-specific IgM antibodies were present in the post-transfusion sera of two patients; no post-transfusion serum was available for testing in the third patient (Table 5). No CF antibody titer rises occurred in the two patients with pre- and TABLE 2. CF antibody titers of 223 blood donors No. of donors with Age No. of % Posi- titer of: (years) donors tivea _ < aantibody titer. 1:8. TABLE 3. IHA titers of 223 blood donors Age No. % Posi- No. of donors with titer of: (years) donors tive < > aantibody titer > 1:10. Oi L TABLE 4. Distribution of 223 donors by IHA and CF antibody Reciprocal IHA Reciprocal CF titer titer < < > post-transfusion sera available for testing. CMV was isolated from the urine of one of these three recipients 14 weeks after transfusion. No illness related to CMV infection was noted. While we did not examine recipients of blood from the nonviruric donor group, during the same period 35 heart surgery patients were observed for 7 to 21 weeks (mean 13 weeks) after transfusion of blood from 154 volunteer donors from the same Community Blood Center. Three of these 35 patients (9%) developed serological evidence of CMV infection in contrast to the occurrence of CMV infection in all three recipients of blood from the three viruric donors. DISCUSSION The present study was undertaken to define further the epidemiology of CMV infection among a volunteer blood donor population, since volunteer donors may be expected to provide the major source of most blood transfusion requirements. In our sample, CMV antibody was present in 58% of donors by CF and in 59% by IHA determination. Discordance between the two tests (Table 4) was small. This close agreement provides additional validity for the IHA technique (1) in the assessment of CMV infection. The prevalence of antibody in the donors compared closely with another volunteer donor population in Seattle (22), in which seropositivity by CF titer was 52%. In a donor population in Bristol, England, CMV CF antibody was present in 55% (16). In addition, the higher prevalence of antibody with increasing age, as noted by others (22), suggests ongoing antigenic experience with CMV in adults. Since the majority of volunteer blood donors have serological evidence of prior exposure to CMV, donor serology is not a sensitive predictor of post-transfusion CMV infection (12). Blood transfusion has been established as a vehicle of transmission of certain bacterial,

4 722 KANE ET AL. INFECT. IMMUN. TABLE 5. Laboratory data on three recipients of Wlood from cytomegaloviruric donors Recipient (years) Age Sex Time Virus culture Reciprocal CMV antibody titer Blood Urine CF IHA IHMc 025R 61 F Pre-Txa _b < 8 9 weeks later Od weeks later , R 48 M Pre-Tx < 8 13 weeks later QNS' 198R 38 M Pre-Tx - - QNS 40 QNS 14 weeks later 0 +e a Tx, Transfusion. -, Not performed. c IHA antibody titer of serum fraction containing IgM. d 0, CMV not isolated. e +, CMV isolated. ' QNS, Quantity not sufficient. parasitic, and viral infections. In addition to transfusion, important risk factors of infection in recipients may also include severe underlying diseases, nosocomial exposure, concurrent use of drugs which may depress host defenses, and the possible effects of multiple transfusions on host immunity. The risk of CMV infection has previously been correlated with the number of units of blood transfused, but the identification of donors whose blood may transmit CMV has not previously been accomplished. We were unable to isolate CMV from freshly obtained specimens of leukocytes or leukocyte-rich plasma from 223 asymptomatic volunteer blood donors, even though 59% had serological evidence of prior exposure to CMV by both CF and IHA. While CMV has been recovered from blood cultures of patients with illnesses such as transfusion-associated CMV mononucleosis (7), congenital cytomegalic inclusion disease (14), and underlying diseases such as leukemia (8), only Diosi (6) has reported the isolation of CMV from blood of healthy individuals. In the description of his method, approximately 1.5 x 108 leukocytes from each donor were inoculated onto each human embryonic fibroblast culture; we inoculated 3 x 108 leukocytes from each donor onto fetal tonsil fibroblast cultures. Mirkovic et al. (15) failed to isolate CMV from donors' blood, although they incubated their cultures with phytohemagglutinin, which may have affected the growth and recovery of CMV in culture. In Wentworth and Alexander's study (22), techniques similar to ours were used, and no CMV was recovered from blood. Success with our culture methods was demonstrated during the study period by the isolation of CMV from leukocyte cultures of two patients with spontaneous CMV mononucleosis (10). If CMV is present in the blood of healthy donors, our inability to isolate the virus suggests either that the virus is present in very low titer, below the sensitivity of the culture system used, or that the virus is present in a latent or an incomplete form in blood, requiring for its expression a suitable recipient host. The latter possibility is consistent with the data of Prince et al. (17), in which immunosuppressed recipients had more than twice the incidence of post-transfusion seroconversion (52%) than non-immunosuppressed recipients (21%). Although CMV was not recovered from any of the donor's blood samples, CMV was isolated from the urine of seven (3%) of the donors at the time of blood donation; none of these donors had evidence of recent illness and repeat urine cultures were negative in each case. To our knowledge the prevalence of cytomegaloviruria in healthy adults has not previously been established. While Stern (18) found no viruria among 402 adults over 15 years of age, a majority of the samples were obtained from hospitalized persons with acute illnesses and thus do not strictly pertain to a blood donor population. The blood donor study by Perham et al. (16) did not include urine culture at the time of donation. Each of the three recipients of blood from three of the viruric donors developed serologic evidence of asymptomatic CMV infection, in the presence of preexisting antibody, and cytomegaloviruria occurred in one recipient. The lack of CF antibody titer rise in two of the recipients is unexplained, but it may reflect differences in antibody specificity, increased sensitivity of the IHA test, and/or antigenic

5 VOL. 1 l, 1975 CMV IN VOLUNTEER BLOOD DONORS 723 heterogeneity among the infecting virus strains (20, 23). Detection of CMV-specific IgM in the post-transfusion sera of two patients also indicates that reinfection may stimulate IgM production. In contrast with the finding of CMV infection in these three recipients of blood from viruric donors, CMV infection occurred in only three of 35 multitransfused patients studied during the same period. The evidence suggests that CMV was transmitted in blood from the cytomegaloviruric donors, although further study is needed to establish that the detection of viruria may be of value in identifying potentially infective blood donors. ACKNOWLEDGMENT This work was conducted under Public Health Service contract RFP-NHLI-72-3 from the National Institutes of Health. LITERATURE CITED 1. Bernstein, M. T., and J. A. Stewart Indirect hemagglutination test for detection of antibodies to cytomegalovirus. Appl. Microbiol. 21: Casey, H. L U. S. Public Health Service Public Health Monograph 74, p. 31. U. S. Department of Health, Education and Welfare, Washington, D. C. 3. Center for Disease Control Serodiagnosis of: toxoplasmosis, rubella, cytomegalic inclusion disease, herpes simplex, p Immunology Series No. 5, Procedural Guide. Center for Disease Control, Atlanta, Ga. 4. Constantino, T., and A. Weintraub The Guillain- Barre syndrome as a complication of the postperfusion syndrome. Am. Heart J. 84: Craighead, J. E Immunologic response to cytomegalovirus infection in renal allograft recipients. Am. J. Epidemiol. 90: Diosi, P., E. Moldovan, and N. Tomescu Latent cytomegalovirus infection in blood donors. Br. Med. J. 4: Foster, K. M., and I. Jack Isolation of cytomegalovirus from the blood leucocytes of a patient with post-transfusion mononucleosis. Australas. Ann. Med. 17: Harnden, D. G., T. R. Elsdale, D. E. Young, and A. Ross The isolation of cytomegalovirus from peripheral blood. Blood 30: Henle, W., G. Henle, M. Scriba, C. R. Joyner, F. S. Harrison, Jr., R. von Essen, J. Paloheimo, and E. Klemola Antibody responses to the Epstein-Barr virus and cytomegaloviruses after open-heart and other surgery. N. Engl. J. Med. 282: Jordan, M. C., W. E. Rousseau, J. A. Stewart, G. R. Noble, and T. D. Y. Chin Spontaneous cytomegalovirus mononucleosis: clinical and laboratory observations in nine cases. Ann. Intern. Med. 79: Kaariainen, L., E. Klemola, and J. Paloheimo Rise of cytomegalovirus antibodies in an infectiousmononucleosis-like syndrome after transfusion. Br. Med. J. 1: Klemola, E., L. Kiaariainen, R. von Essen, K. Haltia, A. Koivuniemi, and C. von Bonsdorff Further studies on cytomegalovirus mononucleosis in p.eviously healthy individuals. Acta Med. Scand. 182: Klemola, E., R. von Essen, J. Paloheimo, and U. Furuhjelm Cytomegalovirus antibodies in donors of fresh blood to patients submitted to open-heart surgery. Scand. J. Infect. Dis. 1: Lang, D. J., and B. J. Noren Cytomegaloviremia following congenital infection. J. Pediat. 73: Mirkovic, R., J. Werch, M. A. South, and M. Benyesh- Melnick Incidence of cytomegaloviremia in blood-bank donors and in infants with congenital cytomegalic inclusion disease. Infect. Immun. 3: : Perham, T. G. M., E. 0. Caul, P. J. Conway, and M. G. Mott Cytomegalovirus infection in blood donors-a prospective study. Br. J. Haematol. 20: Prince, A. M., W. Szmuness, S. J. Millian, and D. S. David A serologic study of cytomegalovirus infections associated with blood transfusions. N. Engl. J. Med. 284: Stem, H Isolation of cytomegalovirus and clinical manifestations of infection at different ages. Br. Med. J. 1: Toghill, P. J., M. E. Bailey, R. Williams, R. Zeegan, and R. Bown Cytomegalovirus hepatitis in the adult. Lancet 1: Waner, J. L., T. H. Weller, and S. V. Kevy Patterns of cytomegaloviral complement-fixing antibody activity: a longitudinal study of blood donors. J. Infect. Dis. 127: Weller, T. H The cytomegaloviruses: ubiquitous agents with protean clinical manifestations. N. Engl. J. Med. 285: , Wentworth, B. B., and E. R. Alexander Seroepidemiology of infections due to members of the herpesvirus group. Am. J. Epidemiol. 94: Wentworth, B. B., and L. French Plaque assay of cytomegalovirus strains of human origin. Proc. Soc. Exp. Biol. Med. 135: