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CYTOMEGALOVIRUS INFECTION FOLLOWING EXTRACORPOREAL CIRCULATION IN CHILDREN
Saturday CYTOMEGALOVIRUS INFECTION FOLLOWING EXTRACORPOREAL CIRCULATION IN CHILDREN A
Prospective Study
J. A. EMBIL M.D., B.Sc. Havana
D. F. FOLKINS M.D., B.Sc. Dalhousie, F.R.C.P.C.
ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY
PÆDIATRICS AND MEDICINE
E. V HALDANE M.B., B.Sc. Edin.
C E. VAN ROOYEN M.D.,D.Sc.,F.R.C.P.,F.R.C.P.C.
RESEARCH FELLOW, DEPARTMENT OF MICROBIOLOGY
PROFESSOR AND HEAD OF DEPARTMENT OF MICROBIOLOGY
LECTURER IN
From the
Faculty of Medicine, Dalhousie University, and the Children’s Hospital, Halifax, Nova Scotia, Canada
Cytomegalovirus infection was sought in 16 patients before and after extracorporeal circulation during heart surgery. The cytomegalovirus complement-fixing-antibody titre rose postoperatively in 7, and in 3 of them cytomegalovirus was isolated in urine and/or throat swabs. 5 showed features of the postperfusion syndrome; the other 2 were symptom-free. The findings suggest that the infection was acquired during or soon after operation. The source of infection is not but evidence the known, presented suggests that cytoin transfused blood. be transmitted megalovirus may Summary
30 November
1968
transfusion of fresh blood during surgery. All had an infectious-mononucleosis-type syndrome, and in 1 of 2 who had undergone open-heart surgery a cytopathic agent with the characteristics of a cytomegalovirus was isolated from the urine. Toghill et al. (1967) described an adult patient in whom jaundice and splenomegaly developed 13 days after open-heart surgery, with subsequent rise in titre of C.M.V.C.F.A. and isolation of the virus in urine culture. In 4 cases of post-perfusion syndrome, Lang et al. (1968) found high titres of the antibody and isolated the virus from throat swabs, urine, and blood. We isolated cytomegalovirus in the urine of 4 children who had undergone open heart surgery during 1965 and early 1966 and who were referred to us by The Children’s Hospital cardiac follow-up clinic; 3 of them had unexplained hepatosplenomegaly. As a result of these observations we designed this prospective study to search for virological and serological evidence of cytomegalovirus infection both before and after operation. Patients and Methods
The 17 children who took part in this investigation were selected from 39 who underwent open-heart surgery with extracorporeal circulation between July, 1966, and January, 1968. Apart from the nature of the cardiac defect, all clinical details were withheld from the virus laboratory until after completion of its studies.
reported are: hepatomegaly, malaise, an erythematous papular rash, and, less consistently, leucocytosis, eosinophilia, lymphadenopathy, anaemia, jaundice, and abnormal liver function. The Paul-Bunnell heterophil-antibody test is usually negative, but otherwise the syndrome strikingly resembles the typhoidal " variety of infectious
The criteria for acceptance in the study were as follows: (1) fresh blood had been transfused during cardiac surgery; (2) a sample of urine and/or throat swab and a specimen of blood, for virological and serological studies, were obtained shortly before or immediately after operation; (3) at least two specimens of urine and/or throat swabs and blood were obtained later, one of them at least 8 weeks after the patient’s discharge from hospital. (The frequency and timing of the later specimens varied, depending upon the appointments arranged for the follow-up clinic and the ability to obtain specimens. Cases were unselected, but inevitably the children who recovered more slowly in the immediate postoperative period, whose convalescence was complicated, or who required further operation stayed in hospital longer or came to follow-up clinic more frequently, and therefore provided more specimens.) 17 patients fulfilled these criteria, but 1 of the 17 (case 4) could not be classified and was deleted from the final analysis. This case is described separately. The other 22 were excluded through death in the immediate postoperative period (8) or through failure to satisfy the criteria (14). 2 children required further operations: 1 died immediately after the third operation (case5) and 1 immediately after the second (case 13).
mononucleosis. Seaman and Starr (1962) thought that the transfusion of fresh blood might be the cause of the syndrome, there being no constant factor in blood additives, pumppriming substances, or the type of pump or oxygenator. In 1964 Engle et al. reported finding " a salivary gland virus in 1 of their patients with this syndrome. In 1966 Kaariainen and his colleagues (Kaariainen et al. 1966 a, b) described 4 patients with high titres of complement-fixing antibody to cytomegalovirus (C.M.V.C.F.A.) following the
Blood had been donated not more than 24 hours before transfusion and was collected by Canadian Red Cross teams in acid-citrate-dextrose solution. Approximately 2000-4000 ml. of blood was infused via the pump reservoir, and more fresh blood was later transfused directly, during the operation or shortly afterwards. Thus, each patient received blood from 5-10 donors. The blood in the pump was diluted to 500 with lactated Ringer’s solution and or 5°n dextrose in water, sometimes with fresh-frozen plasma. Heparin, calcium bicarbonate, and sodium bicarbonate were added as required, and the heparin was neutralised with protamine at the end of the
Introduction IN 1960 Kreel et al. reported a
syndrome of fever, and atypical lymphocytes, splenomegaly in patients to circulation during open-heart subjected extracorporeal surgery 3 to 6 weeks before. The clinical features of this post-perfusion syndrome were further delineated by Perillie and Glenn (1962), Seaman and Starr (1962), Wheeler et al. (1962), Anderson and Larsson (1963), Holswade et al. (1963), Smith (1964), Bastin et al. (1965), Horton (1966), Reyman (1966), and Riemenschneider and Moss (1966). The reported incidence in children and adults ranges from 3 to 11 % of patients perfused during surgery. In addition to the three main features, others
"
"
7579
1152
procedure. All patients were given ampicillin prophylactically for 8 days from 1 day before operation. An American Optical DeBakey-type roller pump with Grosstype reservoir and filters, Kay-Cross disc oxygenator, and Harrison Brown heat exchanger, was used. Cytomegalovirus Isolation and Identification Freshly voided urine was collected in a sterile container, and throat swabs were immersed in tissue-culture-maintenance medium (Eagle’s MEM Diploid medium). Cell cultures.-Human-embryo fibroblasts from skin, muscle, and lung, and human neonatal foreskin fibroblasts were used. Monolayers of these cells were prepared in our laboratory according to the technique of Embil and Faulkner (1964); 3 embryo-cell lines and 12 foreskin-cell lines were used during the study. Urine.-In the early part of the study, specimens were prepared by the technique of Embil et al. (1965). Later, the supernatant fluid was filtered through a ’ Millipore’ membrane, fitted to a Swinney syringe filter-holder, before inoculation. Throat swabs.- The maintenance medium the fibroblast monolayer.
was
inoculated
directly
on to
Specimens were examined twice a week for cytopathic effect for up to 60 days. One or two blind passages of scraped cells and tissue-culture fluid were made before any specimen was judged to be negative and was discarded. The isolated virus was identified as cytomegalovirus if it produced the morphology typical of cytopathic effect in human fibroblasts-i.e., focal necrosis, inclusions, and cytomegaly, as described by Rowe et al. (1958) and Weller and Rowe (1964). Serology samples were screened for complement-fixing cytomegalovirus (C.M.V.C.F.A.) by micro-complement fixation tests, as described by Hanshaw (1966). The antigen was prepared in our laboratories from the AD 169 strain of cytomegalovirus. The sera from each patient were tested simultaneously. The Paul-Bunnell heterophil-antibody test The
serum
antibody
to
(P.B.H.A.) and Davidsohn’s differential guinea-pig-kidney absorption test (G.P.K.A.) were performed to search for evidence of infectious mononucleosis.
Case-reports Full details of clinical and laboratory data marised in table I.
are sum-
Case 1 A 6-year-old boy with tetralogy of Fallot underwent total correction on July 12, 1966. X-ray films 3-5 days postoperatively showed infiltration of the lower lobe of the right lung. Fever of 38°C (100-4°F), without obvious cause, persisted from the 7th to the 23rd day. He was discharged on Aug. 2. Examination on Nov. 8, 1966, April 27 and Oct. 26, 1967, and Feb. 22, 1968, revealed no hepatomegaly, spleno-
megaly, rash,
or
lymphadenopathy.
Laboratory studies.-Cytomegalovirus was isolated from urine specimens at 3, 64, and 80 weeks. The c.M.v.c.F.A. titre rose from < 1/4 at 3 weeks to 1/32 at 16 weeks, and was 1/16 at 40 and 80 weeks. Serum was tested for evidence of infectious mononucleosis at 3, 16, 40, and 80 weeks; P.B.H.A. and G.P.K.A. tests were consistently negative. Case 2 A
4-year-old boy had resection of infundibular and valvular pulmonary stenosis on Sept. 8, 1966. Hepatomegaly was noted on Sept. 14, and he was mildly febrile without obvious cause from Sept. 20 to 30. He was discharged on Oct. 7. When he was seen in clinic on Feb. 23, 1967, the liver was palpable, although cardiac failure was absent. (Enlargement of the liver and spleen had been noted on April 12, 1966, before operation.) Laboratory studies.-Cytomegalovirus
was not
isolated from urine
throat swabs. The C.M.V.C.F.A. titre rose from < 1/4 before and 3 weeks after operation to 1/64 at 23 weeks. P.B.H.A. was negative before operation but present in a titre of 1/80 at 3 weeks, falling to 1/40 at 23 weeks. G.P.K.A. was negative throughout.
or
TABLE I--CASES WITH EVIDENCE OF CYTOMEGALOVIRUS INFECTION
1153 Case 3 A 4-year-old boy underwent open-heart surgery on Sept. 13, 1966, when a sinus-venosus type of atrial septal defect and anomalous pulmonary venous drainage from the upper and middle lobes of the right lung were corrected with a pericardial patch. No complications developed during the immediate postoperative period, but at 10 days the rectal temperature was 37-9°C (100-2°F). He was discharged on Sept. 30. When seen in clinic on March 28, 1967, he had had a " chest cold and mumps ". The liver was firm and enlarged 1 fingerbreadth, and the spleen was enlarged 2 fingerbreadths. He was seen again Sept. 26, 1967, and underwent surgical repair of coarctation of the aorta on Jan. 10, 1968. In Sept. 1967, and again when he was seen in clinic on March 21, 1968, there was neither splenomegaly nor hepatomegaly. was not isolated from urine throat swabs. The C.M.V.C.F.A. titre rose from <1/4 before and 2 weeks after operation to 1/128 at 28 weeks, and had fallen to 1/32 at 79 weeks. P.B.H.A. and G.P.K.A. were consistently negative.
Laboratory studies.-Cytomegalovirus
or
Case 6 A 3-year-old boy underwent total correction of tetralogy of Fallot on Nov. 15, 1966. Transient pulmonary infiltration and atelectasis occurred within the lst week but cleared quickly. The rectal temperature rose suddenly to 39-6°C (103-3°F) on the 24th day and persisted at 38°C (100’4OF) until he was discharged on Dec. 21, 1966. Coarse rhonchi which persisted during this time were found to be due to subglottic stenosis, for which tracheostomy was performed on Jan. 28, 1967. Splenomegaly was not noted. Hepatomegaly was noted on only one occasion, in association with acute respiratory insufficiency, due to retained secretions, and ventilation difficulty: this was immediately before the subglottic stenosis was discovered and was probably due to acute right-sided heart-failure.
Laboratory studies.-Cytomegalovirus was isolated from urine and throat swabs obtained at 10 weeks and from urine at 20 weeks. The c.M.v.c.F.A. titre rose from < 1/4 before and at 4 weeks to 1/128 at 10 weeks, then fell 1/64 at 13 weeks and to 1/16 at 120 weeks. P.B.H.A. titre before operation and at 4, 10, and 13 weeks was 1/160, 1/160, 1/80, and 1/160 respectively. G.P.K.A. titres were negative throughout. Case 8 A 9-year-old girl underwent closure of a secundum-type atrial septal defect on Dec. 13, 1966. The incision was reopened on Dec. 14 to remove blood-clot from the right pleural space. Recovery was otherwise straightforward, except for an unproductive cough which persisted until she was discharged on Dec. 23; this was attributed to irritation by the orotracheal airway during surgery. She was seen in clinic on Jan. 12 and July 11, 1967; fever, rash, hepatomegaly, and splenomegaly were absent. was not isolated from urine throat swabs. The C.M.V.C.F.A. titre rose from < 1/4 before and 11/2 and 4 weeks after operation to 1/32 at 30 weeks. P.B.H.A. and
Laboratory studies.-Cytomegalovirus
or
G.P.K.A. were
consistently negative.
Case 12 A 15-year-old girl underwent closure of an atrial septal defect of secundum type on April 18, 1967. There were no complications except for mild right-sided basal pneumonitis a week after operation. She was discharged on May 7. When she was seen in clinic on Sept. 2, 1967, no history of any interim illness was obtained, and the liver and spleen were not enlarged.
Laboratory studies.-Cytomegalovirus or
throat swabs. The
was not
isolated from urine
C.M.V.C.F.A. titre was 1/8 before operation and to 1/256 at 21 weeks. The P.B.H.A. titre was
1/4 at 3 weeks, rising 1/160 before operation, and 1/320 and 1/160 at 2 and 21 weeks respectively. The G.P.K.A. titre was negative before operation; because of insufficient
serum
the test could not be
repeated
later.
Case 17 A 6-year-old boy had closure of a secundum-type atrial septal defect on Jan. 25, 1968. The left atrium was accidentally torn during surgery, leading to considerable blood-loss and necessitating the transfusion of 6500 ml. of fresh blood. The patient had a low-grade fever 6 to 13 days after operation, and a morbilliform rash was noted 6 to 8 days postoperatively.
Hepatomegaly was noted at 15 days, and this persisted, increasing to 4 cm. below the costal margin. There was no splenomegaly, but atypical lymphocytes were noted in blood smears taken at 31 and 46 days. During this period, X-ray films showed of the cardiac silhouette; this was attributed to with effusion due to the post-pericardotomy syndrome. He was discharged on March 10, 1968, and was seen in clinic on April 30, when the liver was still palpable, 3 cm. below the right costal margin, but without causing symptoms.
enlargement
pericarditis
was isolated from urine 10 weeks. The C.M.V.C.F.A. titre rose from < 1/4 at 1 week at 10 weeks. P.B.H.A. and G.P.K.A. titres were negative
Laboratory studies.-Cytomegalovirus voided
at
1/256 throughout.
to
Results Patients with Evidence of Cytomegalovirus Infection In the 7 cases reported in detail the development of
cytomegalovirus infection postoperatively was demonstrated by increasing titres of cytomegalovirus complement-fixing antibody in the blood (see figure). In 3
Complement-fixing antibodies in 7 cases with evidence megalovirus infection (semilogarithmic graph).
of cyto-
cytomegalovirus was isolated from urine and throat swabs also. 5 of the patients had symptoms and 2 were symptom-free. cases,
clinical finding postin all of the 5 patients who operatively, being present had symptoms; it occurred in 4 cases during the 2nd and 3rd weeks and in 1 case during the 4th week, and lasted an average of 8 days (range 1-16 days). This compares with the findings in other series of the post-perfusion syndrome, in which fever is described in almost every case, most commonly starting at 2-4 weeks and lasting on average 7-28 days (Perillie and Glenn 1962, Seaman and Starr 1962, Holswade et al. 1963, Smith 1964, Bastin et al. 1965, Reyman 1966, Riemenschneider and Moss Fever
was
the most
common
1966).
Hepatomegaly occurred in 4 of the 5 cases; in 1 (case 2) it was of doubtful significance, as the liver was palpable before operation. In case 17 hepatomegaly was found at 2 weeks. In cases 3 and 6 it developed later and was absent at time of discharge from hospital at 3 and 5 weeks respectively, but was present on return to clinic, in case 3 at 28 weeks and in case 6 at 10 weeks. Hepatomegaly was not a prominent feature in other series of the syndrome and, when present, was usually mild. In only two other series was it noted in a significant proportion of patients: Holswade et al. (1963) found it in 5 of 6 cases, and Riemenschneider and Moss (1966) in 3 of 4. Splenomegaly developed in only 1 patient (case 3). As with his hepatomegaly, it was absent when he was discharged from hospital at 3 weeks and was first noted during his follow-up at 28 weeks; this development
1154 a rise in C.M.V.C.F.A. titre from < 1/4 to throat swabs, and the titre of C.M.V.C.F.A. did not increase 1/128. In other series, splenomegaly usually occurred (table 11). Splenomegaly, atypical lymphocytes, and rash in a larger proportion of cases (Perillie and Glenn 1962, were absent in all 5 patients (cases 9, 10, 11, 15, and 16) Seaman and Starr 1962, Smith 1964, Reyman 1966, were symptom-free and their postoperative course was Riemenschneider and Moss 1966). clinically normal. Fever developed in 2 cases (7 and 13). In case 7 it Atypical lymphocytes were detected in only 1 patient occurred on the 3rd and 4th days only, and coincided (case 17), and this was also the only patient in whom a with an erythematous rash of 3 days’ duration and mild rash developed. Whereas the rash and fever occurred early (6th day), the atypical cells were not seen until the hepatomegaly which disappeared between the 5th and 31st day; they were still present in a blood-smear taken 6th days postoperatively; 3 weeks later the titre of on the 46th day. Hepatomegaly also developed, being heterophil antibody had increased, but thereafter the first noted on the 15th day. 10 weeks after operation he postoperative course was uncomplicated. (This patient had cytomegalovirus in the urine, and the C.M.V.C.F.A. did not completely fulfil the criteria of the study and was titre had increased from < 1/4 preoperatively to 1/256. included because of the clinical findings in the early 1 patient (case 13) had mild Like splenomegaly, atypical lymphocytes were not a postoperative period.) fever until intermittent the 21st day; heart-failure in our whereas were noted series, they prominent finding 3 weeks after and during the next developed operation, very frequently in others (Perillie and Glenn 1962, to treatment. 9 months his condition responded poorly Holswade et al. 1963, Smith 1964, Bastin et al. 1965, Numerous of and blood throat swabs, urine, specimens Reyman 1966, Riemenschneider and Moss 1966). Rash no of showed evidence infection with cytomegalovirus. was reported only by Smith (1964). He died 2 days after a further cardiac operation. 8 2 and the In the 12) symptom-free patients (cases Hepatomegaly occurred in 3 cases (5, 7, and 14). In C.M.V.C.F.A. titre rose without clinical symptoms or signs, cases 5 and 14 it was associated with persistent rightwhich seems to confirm that subclinical cytomegalovirus sided heart-failure and pleural effusion. In case 5, three infection may occur in such patients (Kaariainen cardiac operations were performed within 15 months and et al. 1966b). the patient died immediately after the last. In case 14 Heterophil antibodies were present in 3 cases. The the heart-failure, pleural effusion and hepatomegaly guinea-pig-absorption test was consistently negative in all. persisted for 4 months and then cleared. Patients without Evidence of Cytomegalovirus Infection Heterophil antibodies were detected in the serum in 3 9 of the 16 fully documented cases showed no evidence cases (7, 14, and 15). In case 15 the rising P.B.H.A. titre of infection with cytomegalovirus—i.e., the virus could occurred simultaneously with increase in G.P.K.A. titre, not be isolated from repeated culture of urine and/or but the patient had no symptoms.
coincided with
TABLE II—CASES WITHOUT EVIDENCE OF CYTOMEGALOVIRUS
*All laboratory studies for cytomegalovirus
were
negative.
INFECTION*
1155
Unclassified Case 4 could not be classified. This 6-year-old North American Indian girl underwent repair of an atrial septal defect, with cardiopulmonary bypass, on Oct. 12, 1966. No specimens were obtained before operation. Cultures of urine obtained at 2 and 4 weeks grew cytomegalovirus, but that voided at 67 weeks did not. The titre of C.M.V.C.F.A. was 1/8 at 2 weeks and 67 weeks. Jaundice was said to have developed 2 weeks after her discharge from hospital on the 10th day; a brother and a sister had had jaundice just before her admission. Hepatomegaly was present when she was seen 67 weeks after operation. Discussion
This prospective study presents two important points for comparison in the 16 cases fully documented. First, a benign illness developed 2-4 weeks after operation in 5 of 7 patients who had had no evidence of cytomegalovirus infection before or immediately after cardiac surgery. Fever occurred in 5 and hepatomegaly in 3; and in 1 a rash, atypical lymphocytes, and splenomegaly developed simultaneously with evidence of recently acquired infection with cytomegalovirus. 2 patients with evidence of infection remained free of symptoms. Secondly, of 9 patients who showed no evidence of cytomegalovirus infection either before or after operation, 5 were completely symptom-free. Hepatomegaly, attributed to right-sided heart-failure, developed in 2; and in 1 in whom fever, rash, and hepatomegaly developed, the illness occurred early (3rd day) and lasted only 2-3
days.
duction of antibodies rather than passive transmission from donated blood. Since none of the patients without evidence of cytomegalovirus infection had had antibodies initially, there was no evidence that a positive antibody titre to cytomegalovirus preoperatively protected against infection. In only 1 of the 16 patients were complement-fixing antibodies detected before operation (case 12: titre 1/8), and in this patient the possibility of reactivation of virus or reinfection at this time could not be excluded. Although considerable evidence has been adduced for a temporal relationship between cytomegalovirus infection and the post-perfusion syndrome, there is no absolute proof that the two are causally related. Since the syndrome has been shown to develop after the transfusion of fresh blood, isolation of the virus from donated blood before its administration, with subsequent demonstration of cytomegalovirus infection in recipients in whom the
post-perfusion syndrome develops, evidence of causal relationship. This investigation was supported research grant no. 602-7-97.
would
provide major
by Canadian Public Health
The authors gratefully acknowledge the interest and encouragement of Dr. R. L. Ozere, Dr. D. L. Roy, Dr. D. A. Gillis, Dr. F. G. Dolan, Dr. C. E. Kinley, Dr. K. Majid-Uddin, and Miss F. Cook.
Requests for reprints should be addressed to J. A. E. at the Departof Microbiology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, N.S., Canada. ment
REFERENCES
Anderson, R., Larsson, O. (1963) Lancet, ii, 947. Bastin, R., Lapresle, C., Dufrène, F. (1965) Presse méd. 73, 63. Bergström, I., Dahlström, G. (1957) Acta tuberc. scand. 34, 132. Embil, J. A., Jr., Faulkner, R. S. (1964) Can. J. publ. Hlth, 55, 111. Ozere, R. L., van Rooyen, C. E. (1965) Can. med. Ass. J. 93, 1268. Engle, M. A., Ehlers, K. H., Ito, T. (1964) Adv. Pediat. 13, 65. Hanshaw, J. B. (1966) New Engl. J. Med. 275, 476. Harnden, D. G., Elsdale, T. R., Young, D. E., Ross, A. (1967) Blood, 30, 120. Holswade, G. R., Engle, M. A., Redo, S. F., Goldsmith, E. I., Barondess, J. A. (1963) Circulation, 27, 812. Horton, E. H. (1966) Br. med. J. i, 1419. Jack, I., Todd, H., Turner, E. J. (1968) Med. J. Aust. i, 210. Kääriäinen, L., Klemola, E., Paloheimo, J. (1966a) Br. med. J. i, 1270. Paloheimo, J., Klemola, E., Mäkelä, T., Koivuniemi, A. (1966b) Annls Med. exp. Biol. Fenn. 44, 297. Kreel, I., Zaroff, L. I., Canter, J. W., Krasna, I., Baronofsky, I. D. (1960) Surgery Gynec. Obstet. 111, 317. Lang, D. J., Scolnick, E. M., Willerson, J. T. (1968) New Engl. J. Med. 278, 1147. Perillie, P. E., Glenn, W. W. L. (1962) Yale J. Biol. Med. 34, 625. Reyman, T. A. (1966) Am. Heart J. 72, 116. Riemenschneider, T. A., Moss, A. J. (1966) J. Pediat. 69, 546. Rowe, W. P., Hartley, J. W., Cramblett, H. G., Mastrota, F. M. (1958) Am. J. Hyg. 67, 57. Seaman, A. J., Starr, A. (1962) Ann. Surg. 156, 956. Smith, D. R. (1964) Br. med. J. i, 945. Stulberg, C. S., Zuelzer, W. W., Page, R. H., Taylor, R. E., Brough, J. A. (1966) Proc. Soc. exp. Biol. Med. 123, 976. Toghill, P. J., Bailey, M. E., Williams, R., Zeegen, R., Brown, R. (1967) Lancet, i, 1351. Weller, T. H., Rowe, W. P. (1964) in Diagnostic Procedures for Viral and Rickettsial Diseases (edited by E. H. Lennette and N. J. Schmidt); p. 707. New York. Wheeler, E. O., Turner, J. D., Scannell, J. G. (1962) New Engl. J. Med. 266, 454. —
Fresh-blood transfusion appears to be the only common factor in all the series describing a comparable syndrome, both -after cardiac surgery with extracorporeal circulation and other operations with transfusion (Bergstrom and Dahlstrom 1957, Kaariainen et al. 1966 a, b, Reyman 1966, Lang et al. 1968). Kaariainen et al. (1966b) and Lang et al. (1968) also isolated cytomegalovirus and found a high titre of complement-fixing antibody to cytomegalovirus after open-heart surgery. The occurrence of cytomegaloviraemia is well documented. Cytomegalovirus was cultured from the buffy coat of the blood of a 4-month-old infant with hepatitis (Stulberg et al. 1966), from the washed leucocyte fraction of peripheral blood of an 8-year-old boy with acute lymphatic leukaemia (Harnden et al. 1967) and a 12-yearold boy with acute myeloid leukaemia (Jack et al. 1968), and from the washed leucocytes in 3 cases of post-perfusion syndrome (Lang et al. 1968). Also, we have cultured cytomegalovirus from blood obtained by direct heart puncture 6 hours post mortem from a 5-week-old infant who died from congenital cytomegalovirus infection. Large inclusion-bearing cells typical of cytomegalovirus were seen in the endothelium and free in the lumen of the pulmonary blood-vessels (unpublished observation
1968). In the present series antibody titres started to rise only several weeks after operation (see figure). In the 3 patients from whom virus was recovered (cases 1, 6, and 17) this presumably indicates active production of antibodies by the patient’s own immune mechanism. A similar pattern of dynamics in the antibody levels of the 4 patients from whom virus was not isolated (cases 2, 3, 8, and 12) argues in favour of a similar active pro-
—
Addendum
A further case has been studied during the preparation of this report: the titre of complement-fixing antibody to cytomegalovirus rose from < 1/4 a week before operation to 1/32 8 weeks postoperatively. This 2-year-old girl underwent closure of a ventricular septal defect, after which she had persistent fever up to 39 8°C (103-6°F) for 10 days which was attributed to an Escherichia coli urinarytract infection. There was progressive hepatomegaly, which reached 4 cm. below the right costal margin by the 16th day and was still present at 10 weeks.
Prospective Study
J. A. EMBIL M.D., B.Sc. Havana
D. F. FOLKINS M.D., B.Sc. Dalhousie, F.R.C.P.C.
ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY
PÆDIATRICS AND MEDICINE
E. V HALDANE M.B., B.Sc. Edin.
C E. VAN ROOYEN M.D.,D.Sc.,F.R.C.P.,F.R.C.P.C.
RESEARCH FELLOW, DEPARTMENT OF MICROBIOLOGY
PROFESSOR AND HEAD OF DEPARTMENT OF MICROBIOLOGY
LECTURER IN
From the
Faculty of Medicine, Dalhousie University, and the Children’s Hospital, Halifax, Nova Scotia, Canada
Cytomegalovirus infection was sought in 16 patients before and after extracorporeal circulation during heart surgery. The cytomegalovirus complement-fixing-antibody titre rose postoperatively in 7, and in 3 of them cytomegalovirus was isolated in urine and/or throat swabs. 5 showed features of the postperfusion syndrome; the other 2 were symptom-free. The findings suggest that the infection was acquired during or soon after operation. The source of infection is not but evidence the known, presented suggests that cytoin transfused blood. be transmitted megalovirus may Summary
30 November
1968
transfusion of fresh blood during surgery. All had an infectious-mononucleosis-type syndrome, and in 1 of 2 who had undergone open-heart surgery a cytopathic agent with the characteristics of a cytomegalovirus was isolated from the urine. Toghill et al. (1967) described an adult patient in whom jaundice and splenomegaly developed 13 days after open-heart surgery, with subsequent rise in titre of C.M.V.C.F.A. and isolation of the virus in urine culture. In 4 cases of post-perfusion syndrome, Lang et al. (1968) found high titres of the antibody and isolated the virus from throat swabs, urine, and blood. We isolated cytomegalovirus in the urine of 4 children who had undergone open heart surgery during 1965 and early 1966 and who were referred to us by The Children’s Hospital cardiac follow-up clinic; 3 of them had unexplained hepatosplenomegaly. As a result of these observations we designed this prospective study to search for virological and serological evidence of cytomegalovirus infection both before and after operation. Patients and Methods
The 17 children who took part in this investigation were selected from 39 who underwent open-heart surgery with extracorporeal circulation between July, 1966, and January, 1968. Apart from the nature of the cardiac defect, all clinical details were withheld from the virus laboratory until after completion of its studies.
reported are: hepatomegaly, malaise, an erythematous papular rash, and, less consistently, leucocytosis, eosinophilia, lymphadenopathy, anaemia, jaundice, and abnormal liver function. The Paul-Bunnell heterophil-antibody test is usually negative, but otherwise the syndrome strikingly resembles the typhoidal " variety of infectious
The criteria for acceptance in the study were as follows: (1) fresh blood had been transfused during cardiac surgery; (2) a sample of urine and/or throat swab and a specimen of blood, for virological and serological studies, were obtained shortly before or immediately after operation; (3) at least two specimens of urine and/or throat swabs and blood were obtained later, one of them at least 8 weeks after the patient’s discharge from hospital. (The frequency and timing of the later specimens varied, depending upon the appointments arranged for the follow-up clinic and the ability to obtain specimens. Cases were unselected, but inevitably the children who recovered more slowly in the immediate postoperative period, whose convalescence was complicated, or who required further operation stayed in hospital longer or came to follow-up clinic more frequently, and therefore provided more specimens.) 17 patients fulfilled these criteria, but 1 of the 17 (case 4) could not be classified and was deleted from the final analysis. This case is described separately. The other 22 were excluded through death in the immediate postoperative period (8) or through failure to satisfy the criteria (14). 2 children required further operations: 1 died immediately after the third operation (case5) and 1 immediately after the second (case 13).
mononucleosis. Seaman and Starr (1962) thought that the transfusion of fresh blood might be the cause of the syndrome, there being no constant factor in blood additives, pumppriming substances, or the type of pump or oxygenator. In 1964 Engle et al. reported finding " a salivary gland virus in 1 of their patients with this syndrome. In 1966 Kaariainen and his colleagues (Kaariainen et al. 1966 a, b) described 4 patients with high titres of complement-fixing antibody to cytomegalovirus (C.M.V.C.F.A.) following the
Blood had been donated not more than 24 hours before transfusion and was collected by Canadian Red Cross teams in acid-citrate-dextrose solution. Approximately 2000-4000 ml. of blood was infused via the pump reservoir, and more fresh blood was later transfused directly, during the operation or shortly afterwards. Thus, each patient received blood from 5-10 donors. The blood in the pump was diluted to 500 with lactated Ringer’s solution and or 5°n dextrose in water, sometimes with fresh-frozen plasma. Heparin, calcium bicarbonate, and sodium bicarbonate were added as required, and the heparin was neutralised with protamine at the end of the
Introduction IN 1960 Kreel et al. reported a
syndrome of fever, and atypical lymphocytes, splenomegaly in patients to circulation during open-heart subjected extracorporeal surgery 3 to 6 weeks before. The clinical features of this post-perfusion syndrome were further delineated by Perillie and Glenn (1962), Seaman and Starr (1962), Wheeler et al. (1962), Anderson and Larsson (1963), Holswade et al. (1963), Smith (1964), Bastin et al. (1965), Horton (1966), Reyman (1966), and Riemenschneider and Moss (1966). The reported incidence in children and adults ranges from 3 to 11 % of patients perfused during surgery. In addition to the three main features, others
"
"
7579
1152
procedure. All patients were given ampicillin prophylactically for 8 days from 1 day before operation. An American Optical DeBakey-type roller pump with Grosstype reservoir and filters, Kay-Cross disc oxygenator, and Harrison Brown heat exchanger, was used. Cytomegalovirus Isolation and Identification Freshly voided urine was collected in a sterile container, and throat swabs were immersed in tissue-culture-maintenance medium (Eagle’s MEM Diploid medium). Cell cultures.-Human-embryo fibroblasts from skin, muscle, and lung, and human neonatal foreskin fibroblasts were used. Monolayers of these cells were prepared in our laboratory according to the technique of Embil and Faulkner (1964); 3 embryo-cell lines and 12 foreskin-cell lines were used during the study. Urine.-In the early part of the study, specimens were prepared by the technique of Embil et al. (1965). Later, the supernatant fluid was filtered through a ’ Millipore’ membrane, fitted to a Swinney syringe filter-holder, before inoculation. Throat swabs.- The maintenance medium the fibroblast monolayer.
was
inoculated
directly
on to
Specimens were examined twice a week for cytopathic effect for up to 60 days. One or two blind passages of scraped cells and tissue-culture fluid were made before any specimen was judged to be negative and was discarded. The isolated virus was identified as cytomegalovirus if it produced the morphology typical of cytopathic effect in human fibroblasts-i.e., focal necrosis, inclusions, and cytomegaly, as described by Rowe et al. (1958) and Weller and Rowe (1964). Serology samples were screened for complement-fixing cytomegalovirus (C.M.V.C.F.A.) by micro-complement fixation tests, as described by Hanshaw (1966). The antigen was prepared in our laboratories from the AD 169 strain of cytomegalovirus. The sera from each patient were tested simultaneously. The Paul-Bunnell heterophil-antibody test The
serum
antibody
to
(P.B.H.A.) and Davidsohn’s differential guinea-pig-kidney absorption test (G.P.K.A.) were performed to search for evidence of infectious mononucleosis.
Case-reports Full details of clinical and laboratory data marised in table I.
are sum-
Case 1 A 6-year-old boy with tetralogy of Fallot underwent total correction on July 12, 1966. X-ray films 3-5 days postoperatively showed infiltration of the lower lobe of the right lung. Fever of 38°C (100-4°F), without obvious cause, persisted from the 7th to the 23rd day. He was discharged on Aug. 2. Examination on Nov. 8, 1966, April 27 and Oct. 26, 1967, and Feb. 22, 1968, revealed no hepatomegaly, spleno-
megaly, rash,
or
lymphadenopathy.
Laboratory studies.-Cytomegalovirus was isolated from urine specimens at 3, 64, and 80 weeks. The c.M.v.c.F.A. titre rose from < 1/4 at 3 weeks to 1/32 at 16 weeks, and was 1/16 at 40 and 80 weeks. Serum was tested for evidence of infectious mononucleosis at 3, 16, 40, and 80 weeks; P.B.H.A. and G.P.K.A. tests were consistently negative. Case 2 A
4-year-old boy had resection of infundibular and valvular pulmonary stenosis on Sept. 8, 1966. Hepatomegaly was noted on Sept. 14, and he was mildly febrile without obvious cause from Sept. 20 to 30. He was discharged on Oct. 7. When he was seen in clinic on Feb. 23, 1967, the liver was palpable, although cardiac failure was absent. (Enlargement of the liver and spleen had been noted on April 12, 1966, before operation.) Laboratory studies.-Cytomegalovirus
was not
isolated from urine
throat swabs. The C.M.V.C.F.A. titre rose from < 1/4 before and 3 weeks after operation to 1/64 at 23 weeks. P.B.H.A. was negative before operation but present in a titre of 1/80 at 3 weeks, falling to 1/40 at 23 weeks. G.P.K.A. was negative throughout.
or
TABLE I--CASES WITH EVIDENCE OF CYTOMEGALOVIRUS INFECTION
1153 Case 3 A 4-year-old boy underwent open-heart surgery on Sept. 13, 1966, when a sinus-venosus type of atrial septal defect and anomalous pulmonary venous drainage from the upper and middle lobes of the right lung were corrected with a pericardial patch. No complications developed during the immediate postoperative period, but at 10 days the rectal temperature was 37-9°C (100-2°F). He was discharged on Sept. 30. When seen in clinic on March 28, 1967, he had had a " chest cold and mumps ". The liver was firm and enlarged 1 fingerbreadth, and the spleen was enlarged 2 fingerbreadths. He was seen again Sept. 26, 1967, and underwent surgical repair of coarctation of the aorta on Jan. 10, 1968. In Sept. 1967, and again when he was seen in clinic on March 21, 1968, there was neither splenomegaly nor hepatomegaly. was not isolated from urine throat swabs. The C.M.V.C.F.A. titre rose from <1/4 before and 2 weeks after operation to 1/128 at 28 weeks, and had fallen to 1/32 at 79 weeks. P.B.H.A. and G.P.K.A. were consistently negative.
Laboratory studies.-Cytomegalovirus
or
Case 6 A 3-year-old boy underwent total correction of tetralogy of Fallot on Nov. 15, 1966. Transient pulmonary infiltration and atelectasis occurred within the lst week but cleared quickly. The rectal temperature rose suddenly to 39-6°C (103-3°F) on the 24th day and persisted at 38°C (100’4OF) until he was discharged on Dec. 21, 1966. Coarse rhonchi which persisted during this time were found to be due to subglottic stenosis, for which tracheostomy was performed on Jan. 28, 1967. Splenomegaly was not noted. Hepatomegaly was noted on only one occasion, in association with acute respiratory insufficiency, due to retained secretions, and ventilation difficulty: this was immediately before the subglottic stenosis was discovered and was probably due to acute right-sided heart-failure.
Laboratory studies.-Cytomegalovirus was isolated from urine and throat swabs obtained at 10 weeks and from urine at 20 weeks. The c.M.v.c.F.A. titre rose from < 1/4 before and at 4 weeks to 1/128 at 10 weeks, then fell 1/64 at 13 weeks and to 1/16 at 120 weeks. P.B.H.A. titre before operation and at 4, 10, and 13 weeks was 1/160, 1/160, 1/80, and 1/160 respectively. G.P.K.A. titres were negative throughout. Case 8 A 9-year-old girl underwent closure of a secundum-type atrial septal defect on Dec. 13, 1966. The incision was reopened on Dec. 14 to remove blood-clot from the right pleural space. Recovery was otherwise straightforward, except for an unproductive cough which persisted until she was discharged on Dec. 23; this was attributed to irritation by the orotracheal airway during surgery. She was seen in clinic on Jan. 12 and July 11, 1967; fever, rash, hepatomegaly, and splenomegaly were absent. was not isolated from urine throat swabs. The C.M.V.C.F.A. titre rose from < 1/4 before and 11/2 and 4 weeks after operation to 1/32 at 30 weeks. P.B.H.A. and
Laboratory studies.-Cytomegalovirus
or
G.P.K.A. were
consistently negative.
Case 12 A 15-year-old girl underwent closure of an atrial septal defect of secundum type on April 18, 1967. There were no complications except for mild right-sided basal pneumonitis a week after operation. She was discharged on May 7. When she was seen in clinic on Sept. 2, 1967, no history of any interim illness was obtained, and the liver and spleen were not enlarged.
Laboratory studies.-Cytomegalovirus or
throat swabs. The
was not
isolated from urine
C.M.V.C.F.A. titre was 1/8 before operation and to 1/256 at 21 weeks. The P.B.H.A. titre was
1/4 at 3 weeks, rising 1/160 before operation, and 1/320 and 1/160 at 2 and 21 weeks respectively. The G.P.K.A. titre was negative before operation; because of insufficient
serum
the test could not be
repeated
later.
Case 17 A 6-year-old boy had closure of a secundum-type atrial septal defect on Jan. 25, 1968. The left atrium was accidentally torn during surgery, leading to considerable blood-loss and necessitating the transfusion of 6500 ml. of fresh blood. The patient had a low-grade fever 6 to 13 days after operation, and a morbilliform rash was noted 6 to 8 days postoperatively.
Hepatomegaly was noted at 15 days, and this persisted, increasing to 4 cm. below the costal margin. There was no splenomegaly, but atypical lymphocytes were noted in blood smears taken at 31 and 46 days. During this period, X-ray films showed of the cardiac silhouette; this was attributed to with effusion due to the post-pericardotomy syndrome. He was discharged on March 10, 1968, and was seen in clinic on April 30, when the liver was still palpable, 3 cm. below the right costal margin, but without causing symptoms.
enlargement
pericarditis
was isolated from urine 10 weeks. The C.M.V.C.F.A. titre rose from < 1/4 at 1 week at 10 weeks. P.B.H.A. and G.P.K.A. titres were negative
Laboratory studies.-Cytomegalovirus voided
at
1/256 throughout.
to
Results Patients with Evidence of Cytomegalovirus Infection In the 7 cases reported in detail the development of
cytomegalovirus infection postoperatively was demonstrated by increasing titres of cytomegalovirus complement-fixing antibody in the blood (see figure). In 3
Complement-fixing antibodies in 7 cases with evidence megalovirus infection (semilogarithmic graph).
of cyto-
cytomegalovirus was isolated from urine and throat swabs also. 5 of the patients had symptoms and 2 were symptom-free. cases,
clinical finding postin all of the 5 patients who operatively, being present had symptoms; it occurred in 4 cases during the 2nd and 3rd weeks and in 1 case during the 4th week, and lasted an average of 8 days (range 1-16 days). This compares with the findings in other series of the post-perfusion syndrome, in which fever is described in almost every case, most commonly starting at 2-4 weeks and lasting on average 7-28 days (Perillie and Glenn 1962, Seaman and Starr 1962, Holswade et al. 1963, Smith 1964, Bastin et al. 1965, Reyman 1966, Riemenschneider and Moss Fever
was
the most
common
1966).
Hepatomegaly occurred in 4 of the 5 cases; in 1 (case 2) it was of doubtful significance, as the liver was palpable before operation. In case 17 hepatomegaly was found at 2 weeks. In cases 3 and 6 it developed later and was absent at time of discharge from hospital at 3 and 5 weeks respectively, but was present on return to clinic, in case 3 at 28 weeks and in case 6 at 10 weeks. Hepatomegaly was not a prominent feature in other series of the syndrome and, when present, was usually mild. In only two other series was it noted in a significant proportion of patients: Holswade et al. (1963) found it in 5 of 6 cases, and Riemenschneider and Moss (1966) in 3 of 4. Splenomegaly developed in only 1 patient (case 3). As with his hepatomegaly, it was absent when he was discharged from hospital at 3 weeks and was first noted during his follow-up at 28 weeks; this development
1154 a rise in C.M.V.C.F.A. titre from < 1/4 to throat swabs, and the titre of C.M.V.C.F.A. did not increase 1/128. In other series, splenomegaly usually occurred (table 11). Splenomegaly, atypical lymphocytes, and rash in a larger proportion of cases (Perillie and Glenn 1962, were absent in all 5 patients (cases 9, 10, 11, 15, and 16) Seaman and Starr 1962, Smith 1964, Reyman 1966, were symptom-free and their postoperative course was Riemenschneider and Moss 1966). clinically normal. Fever developed in 2 cases (7 and 13). In case 7 it Atypical lymphocytes were detected in only 1 patient occurred on the 3rd and 4th days only, and coincided (case 17), and this was also the only patient in whom a with an erythematous rash of 3 days’ duration and mild rash developed. Whereas the rash and fever occurred early (6th day), the atypical cells were not seen until the hepatomegaly which disappeared between the 5th and 31st day; they were still present in a blood-smear taken 6th days postoperatively; 3 weeks later the titre of on the 46th day. Hepatomegaly also developed, being heterophil antibody had increased, but thereafter the first noted on the 15th day. 10 weeks after operation he postoperative course was uncomplicated. (This patient had cytomegalovirus in the urine, and the C.M.V.C.F.A. did not completely fulfil the criteria of the study and was titre had increased from < 1/4 preoperatively to 1/256. included because of the clinical findings in the early 1 patient (case 13) had mild Like splenomegaly, atypical lymphocytes were not a postoperative period.) fever until intermittent the 21st day; heart-failure in our whereas were noted series, they prominent finding 3 weeks after and during the next developed operation, very frequently in others (Perillie and Glenn 1962, to treatment. 9 months his condition responded poorly Holswade et al. 1963, Smith 1964, Bastin et al. 1965, Numerous of and blood throat swabs, urine, specimens Reyman 1966, Riemenschneider and Moss 1966). Rash no of showed evidence infection with cytomegalovirus. was reported only by Smith (1964). He died 2 days after a further cardiac operation. 8 2 and the In the 12) symptom-free patients (cases Hepatomegaly occurred in 3 cases (5, 7, and 14). In C.M.V.C.F.A. titre rose without clinical symptoms or signs, cases 5 and 14 it was associated with persistent rightwhich seems to confirm that subclinical cytomegalovirus sided heart-failure and pleural effusion. In case 5, three infection may occur in such patients (Kaariainen cardiac operations were performed within 15 months and et al. 1966b). the patient died immediately after the last. In case 14 Heterophil antibodies were present in 3 cases. The the heart-failure, pleural effusion and hepatomegaly guinea-pig-absorption test was consistently negative in all. persisted for 4 months and then cleared. Patients without Evidence of Cytomegalovirus Infection Heterophil antibodies were detected in the serum in 3 9 of the 16 fully documented cases showed no evidence cases (7, 14, and 15). In case 15 the rising P.B.H.A. titre of infection with cytomegalovirus—i.e., the virus could occurred simultaneously with increase in G.P.K.A. titre, not be isolated from repeated culture of urine and/or but the patient had no symptoms.
coincided with
TABLE II—CASES WITHOUT EVIDENCE OF CYTOMEGALOVIRUS
*All laboratory studies for cytomegalovirus
were
negative.
INFECTION*
1155
Unclassified Case 4 could not be classified. This 6-year-old North American Indian girl underwent repair of an atrial septal defect, with cardiopulmonary bypass, on Oct. 12, 1966. No specimens were obtained before operation. Cultures of urine obtained at 2 and 4 weeks grew cytomegalovirus, but that voided at 67 weeks did not. The titre of C.M.V.C.F.A. was 1/8 at 2 weeks and 67 weeks. Jaundice was said to have developed 2 weeks after her discharge from hospital on the 10th day; a brother and a sister had had jaundice just before her admission. Hepatomegaly was present when she was seen 67 weeks after operation. Discussion
This prospective study presents two important points for comparison in the 16 cases fully documented. First, a benign illness developed 2-4 weeks after operation in 5 of 7 patients who had had no evidence of cytomegalovirus infection before or immediately after cardiac surgery. Fever occurred in 5 and hepatomegaly in 3; and in 1 a rash, atypical lymphocytes, and splenomegaly developed simultaneously with evidence of recently acquired infection with cytomegalovirus. 2 patients with evidence of infection remained free of symptoms. Secondly, of 9 patients who showed no evidence of cytomegalovirus infection either before or after operation, 5 were completely symptom-free. Hepatomegaly, attributed to right-sided heart-failure, developed in 2; and in 1 in whom fever, rash, and hepatomegaly developed, the illness occurred early (3rd day) and lasted only 2-3
days.
duction of antibodies rather than passive transmission from donated blood. Since none of the patients without evidence of cytomegalovirus infection had had antibodies initially, there was no evidence that a positive antibody titre to cytomegalovirus preoperatively protected against infection. In only 1 of the 16 patients were complement-fixing antibodies detected before operation (case 12: titre 1/8), and in this patient the possibility of reactivation of virus or reinfection at this time could not be excluded. Although considerable evidence has been adduced for a temporal relationship between cytomegalovirus infection and the post-perfusion syndrome, there is no absolute proof that the two are causally related. Since the syndrome has been shown to develop after the transfusion of fresh blood, isolation of the virus from donated blood before its administration, with subsequent demonstration of cytomegalovirus infection in recipients in whom the
post-perfusion syndrome develops, evidence of causal relationship. This investigation was supported research grant no. 602-7-97.
would
provide major
by Canadian Public Health
The authors gratefully acknowledge the interest and encouragement of Dr. R. L. Ozere, Dr. D. L. Roy, Dr. D. A. Gillis, Dr. F. G. Dolan, Dr. C. E. Kinley, Dr. K. Majid-Uddin, and Miss F. Cook.
Requests for reprints should be addressed to J. A. E. at the Departof Microbiology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, N.S., Canada. ment
REFERENCES
Anderson, R., Larsson, O. (1963) Lancet, ii, 947. Bastin, R., Lapresle, C., Dufrène, F. (1965) Presse méd. 73, 63. Bergström, I., Dahlström, G. (1957) Acta tuberc. scand. 34, 132. Embil, J. A., Jr., Faulkner, R. S. (1964) Can. J. publ. Hlth, 55, 111. Ozere, R. L., van Rooyen, C. E. (1965) Can. med. Ass. J. 93, 1268. Engle, M. A., Ehlers, K. H., Ito, T. (1964) Adv. Pediat. 13, 65. Hanshaw, J. B. (1966) New Engl. J. Med. 275, 476. Harnden, D. G., Elsdale, T. R., Young, D. E., Ross, A. (1967) Blood, 30, 120. Holswade, G. R., Engle, M. A., Redo, S. F., Goldsmith, E. I., Barondess, J. A. (1963) Circulation, 27, 812. Horton, E. H. (1966) Br. med. J. i, 1419. Jack, I., Todd, H., Turner, E. J. (1968) Med. J. Aust. i, 210. Kääriäinen, L., Klemola, E., Paloheimo, J. (1966a) Br. med. J. i, 1270. Paloheimo, J., Klemola, E., Mäkelä, T., Koivuniemi, A. (1966b) Annls Med. exp. Biol. Fenn. 44, 297. Kreel, I., Zaroff, L. I., Canter, J. W., Krasna, I., Baronofsky, I. D. (1960) Surgery Gynec. Obstet. 111, 317. Lang, D. J., Scolnick, E. M., Willerson, J. T. (1968) New Engl. J. Med. 278, 1147. Perillie, P. E., Glenn, W. W. L. (1962) Yale J. Biol. Med. 34, 625. Reyman, T. A. (1966) Am. Heart J. 72, 116. Riemenschneider, T. A., Moss, A. J. (1966) J. Pediat. 69, 546. Rowe, W. P., Hartley, J. W., Cramblett, H. G., Mastrota, F. M. (1958) Am. J. Hyg. 67, 57. Seaman, A. J., Starr, A. (1962) Ann. Surg. 156, 956. Smith, D. R. (1964) Br. med. J. i, 945. Stulberg, C. S., Zuelzer, W. W., Page, R. H., Taylor, R. E., Brough, J. A. (1966) Proc. Soc. exp. Biol. Med. 123, 976. Toghill, P. J., Bailey, M. E., Williams, R., Zeegen, R., Brown, R. (1967) Lancet, i, 1351. Weller, T. H., Rowe, W. P. (1964) in Diagnostic Procedures for Viral and Rickettsial Diseases (edited by E. H. Lennette and N. J. Schmidt); p. 707. New York. Wheeler, E. O., Turner, J. D., Scannell, J. G. (1962) New Engl. J. Med. 266, 454. —
Fresh-blood transfusion appears to be the only common factor in all the series describing a comparable syndrome, both -after cardiac surgery with extracorporeal circulation and other operations with transfusion (Bergstrom and Dahlstrom 1957, Kaariainen et al. 1966 a, b, Reyman 1966, Lang et al. 1968). Kaariainen et al. (1966b) and Lang et al. (1968) also isolated cytomegalovirus and found a high titre of complement-fixing antibody to cytomegalovirus after open-heart surgery. The occurrence of cytomegaloviraemia is well documented. Cytomegalovirus was cultured from the buffy coat of the blood of a 4-month-old infant with hepatitis (Stulberg et al. 1966), from the washed leucocyte fraction of peripheral blood of an 8-year-old boy with acute lymphatic leukaemia (Harnden et al. 1967) and a 12-yearold boy with acute myeloid leukaemia (Jack et al. 1968), and from the washed leucocytes in 3 cases of post-perfusion syndrome (Lang et al. 1968). Also, we have cultured cytomegalovirus from blood obtained by direct heart puncture 6 hours post mortem from a 5-week-old infant who died from congenital cytomegalovirus infection. Large inclusion-bearing cells typical of cytomegalovirus were seen in the endothelium and free in the lumen of the pulmonary blood-vessels (unpublished observation
1968). In the present series antibody titres started to rise only several weeks after operation (see figure). In the 3 patients from whom virus was recovered (cases 1, 6, and 17) this presumably indicates active production of antibodies by the patient’s own immune mechanism. A similar pattern of dynamics in the antibody levels of the 4 patients from whom virus was not isolated (cases 2, 3, 8, and 12) argues in favour of a similar active pro-
—
Addendum
A further case has been studied during the preparation of this report: the titre of complement-fixing antibody to cytomegalovirus rose from < 1/4 a week before operation to 1/32 8 weeks postoperatively. This 2-year-old girl underwent closure of a ventricular septal defect, after which she had persistent fever up to 39 8°C (103-6°F) for 10 days which was attributed to an Escherichia coli urinarytract infection. There was progressive hepatomegaly, which reached 4 cm. below the right costal margin by the 16th day and was still present at 10 weeks.