THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.6. DECEMBER 1977 © 1977 by the University of Chicago. All rights reserved.

A Prospective Analysis of Interstitial Pneumonia and Opportunistic Viral Infection among Recipients of Allogeneic Bone Marrow Grafts From the Departments of Medicine, Laboratory Medicine, and Epidemiology, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, Washington

Paul E. Neiman, William Reeves, George Ray, Nancy Flournoy, Kenneth G. Lerner, George E. Sale, and E. Donnall Thomas

Allogeneic bone marrow transplantation is a useful new modality for the treatment of lethal marrow diseases such as acute leukemia and aplastic anemia. Increasing numbers of marrow graft recipients with durable remissions of these diseases have been described [1-4]. Several problems and complications attendant to marrow transplantation limit the fraction of recipients who derive long-term benefits from this procedure. Some of these, such as opportunistic bacterial and fungal infections in the period preceding adequate marrow graft function and graft-vs.-host (GVH) disease following successful engraftment, were anticipated and have been described [5-7]. Acute

respiratory failure from severe interstitial pneumonia was not anticipated as a major obstacle. However, as described in a retrospective analysis of our first 50 marrow transplant recipients [8], this syndrome was the largest single determinant of mortality in the first few months following successful engraftment. Because the problem was unexpected, there were many deficiencies in the available retrospective data. This initial analysis did, however, suggest that disseminated infection with cytomegalovirus (CMV) and perhaps the immunologic response to that infection might play an important role in a large fraction of the cases. Other opportunistic pathogens such as Pneumocystis carinii were observed in a smaller number of cases [9], and many cases could not be associated with any known pathogen. The role of other potential contributing factors, such as the type of disease for which the marrow transplantation was carried out, the method of marrow graft preparation, and GVH disease and its treatment, remained to be defined precisely. In an attempt to describe the nature of this problem more completely and to uncover its principal determinants, we prospectively studied a group of 80 patients with either aplastic anemia or hematologic malignancy who received allogeneic marrow grafts at our marrow transplant

Received for publication March 24, 1977, and in revised form July 22, 1977. This investigation was supported by grants no. CA 15704 and CA 18029 from the National Cancer Institute and by grants no. AM 01000 and RR 05714 from the National Institutes of Health. Dr. Neiman is a scholar of the Leukemia Society of America. Drs. Lerner and Sale are supported in part by junior faculty fellowships from the American Cancer Society, and Dr. Thomas is the recipient of research career development award no. AI 02425 from the National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. Paul E. Neiman, Division of Oncology, Fred Hutchinson Cancer Research Center, 1I24 Columbia Street, Seattle, Washington 98104.

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A prospective study of 80 bone marrow transplant recipients with acute leukemia and aplastic anemia employed serial viral cultures, determination of complement-fixing antibody to cytomegalovirus (CMV), and study of material obtained from open lung biopsy and autopsy. There were 43 episodes of interstitial pneumonia, 28 of which were fatal. About 40% of the cases were idiopathic. CMV was the most common candidate pathogen, present in 47% of affected lungs. By a median of 53 days following transplantation, 46% of the recipients were shedding CMV from some site. This event was three times more frequent among recipients who had positive titers of antibody to CMV before transplantation than among seronegative recipients. Failure to respond serologically to CMV infection markedly increased the hazard of dying of interstitial pneumonia. Graft-vs.-host disease significantly increased the incidence and lethality of interstitial pneumonia. The presence of leukemia (rather than aplastic anemia) and/or certain factors in the technique of preparation for engraftment may have been significant.

Interstitial Pneumonia after Transplant

center over an I8-month period ending in January 1976.

755

Table 1. Methods of preparation of patients for bone marrow engraftment. Group, treatment

Materials and Methods

54

4

16 3 22 5 2

2 26

19 7

NOTE. BCND = 1,3-bis(2-chloroethyl)-1-nitrosourea; and ATG = antiserum to human thymocyte globulin.

disease or, in the case of ATG and procarbazine, marrow graft rejection. Allogeneic marrow for engraftment was in every case obtained by established techniques [10] from sibling donors genotypically matched with the recipient at the serologically detected and lymphocyte-detected major histocompatibility loci. Management after grafting included immunosuppression with methotrexate for 100 days to prevent and/or modify GVH disease [II] and additional courses of ATG for GVH disease when it occurred [12, 13J. All patients received red blood cells and platelets until marrow graft function was adequate and intensive treatment with broad-spectrum antibiotics for presumed and documented bacterial infection, a virtually uniform occurrence in the first month after grafting. In addition, 36 patients received multiple therapeutic and/or prophylactic infusions of granulocytes (buffy coat cells), and 14 were housed in laminar air flow ultraisolation facilities supplemented by nonabsorbable antibiotic regimens for gut sterilization and sterile food diets during the first 50 days following engraftment. Serial studies were carried out until either death or discharge from the care of the transplantation center, generally about three months following marrow infusion. Follow-up of clinical status including episodes of interstitial pneumonia has been continuous to the present. Serologic studies. Samples of serum were ob-

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Patient population. During the period of this study, 82 patients new to the Acute Leukemia Center of the University of Washington and the Fred Hutchinson Cancer Research Center were entered into the allogeneic marrow transplantation program. With the exception of two terminally ill patients who died within 48 hr of transplantation, the group was entered consecutively into the study. As has been described in detail [1, 3], the 54 patients with hematologic malignancy (20 with acute lymphoblastic leukemia, 24 with acute myeloblastic leukemia, four with acute undifferentiated leukemia, two with blastic crisis of chronic myelogenous leukemia, three with malignant lymphoma, and one with myeloma) were end-stage patients unresponsive to the extensive chemotherapy they had all received. The remaining 26 patients with aplastic anemia had severe bone marrow failure. Virtually all of the patients had received multiple transfusions of fresh and stored blood products. They ranged in age from five to 55 years (median, 24.4 years). All but five patients survived the first month following marrow transplantation. Method of transplantation. The techniques of marrow transplantation and management after transplantation for these groups of recipients have been described [6]. Briefly, the patients with hematologic malignancy were prepared for engraftment with 1,000 rads of total body 60CO irradiation preceded by 120 mg of cyclophosphamide/kg (60 mg/kg on each of two consecutive days). In all but four instances, large doses of one of a number of other cytotoxic drugs were administered just preceding cyclophosphamide. Recipients with aplastic anemia were prepared with 200 mg of cyclophosphamide/kg (50 uxg] kg on each of four consecutive days), and in some instances with additional ATG (globulin fraction of antiserum to human thymocytes) and procarbazine. The distribution of preparative regimens is given in table 1. The variation of preparative regimens reflected specific protocols in force during the period of the study which attempted to reduce the hazard of either relapse of malignant

Hematologic malignancy Cyclophosphamide plus total body irradiation Alone Plus daunomycin Plus adriamycin Plus BCND Plus cytosine arabinoside Plus nitrogen mustard Plus ATG and procarbazine Aplastic anemia Cyclophosphamide Alone Plus ATG and procarbazine

No. of patients

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I

Table 2. Incidence of interstitial pneumonia among allogeneic bone marrow graft recipients.

Group Hematologic malignancy Marrow aplasia Total

No. of transient cases

No. of lethal cases (%)

Total no. of cases (%)

12* 3 15

22 (65) 6 (67) 28 (65)

34 (62) 9 (35) 43 (52)

*One patient had two distinct episodes.

invasive technique because of our unsatisfactory experience with the diagnostic adequacy of various closed biopsy procedures. Our judgment in this matter corresponds closely with that described recently by Michaelis et al. [15]. An adequate sample of tissue from open biopsy and/or autopsy was obtained in all but five of the 43 cases of pneumonia. Routine studies of tissue samples included bacterial, fungal, and viral cultures, extensive examination of sections for viral inclusions, and methenamine silver stains for P. carinii. Autopsy materials from patients dying from other causes were examined in the same fashion. Results

Interstitial pneumonia. Table 2 summarizes the incidence of interstitial pneumonia. About half of the marrow graft recipients developed interstitial pneumonia, but the incidence was clearly higher among recipients with hematologic malignancy than among patients with marrow aplasia. In both groups the pneumonia was fatal in about two-thirds of the cases. In eight cases there were other major contributing causes of death (bacterial and fungal sepsis, renal failure, and gastrointestinal hemorrhage). The remaining 20 fatalities were attributable solely to progressive pneumonia. Interstitial pneumonia occurred principally during the first three months following transplantation and only rarely thereafter (figure I). The median day of onset was about day 50 following transplantation. Time of onset did not differ between lethal and transient cases of pneumonia or between recipients with different diagnoses. Figure 2 describes the rate of occurrence of interstitial pneumonia calculated by an actuarial method. This rate reached a maximum of 1.65% per day between days 60 and 70. Episodes lasted for two to 45 days (median, 11 days). The duration was slightly but signifi-

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tained from donors and recipients prior to transplantation and then weekly from transplant recipients. Titers of antibody to CMV were determined by a microtiter CF test as previously described [14]. Positive sera were reactive at a dilution of ~1:8, and a minimum of a fourfold rise in titer above base-line values was required to identify seroconversion. Virologic studies. Standard diagnostic viral isolation techniques in routine use for detecting cytopathic viral pathogens were employed. CMV and other herpesviruses were isolated on human embryonic tonsil (HET) fibroblasts. In addition, cultures of primary monkey kidney cells, Vero cells, and certain heteroploid cell lines (Hep-2 and HL) were used to isolate other viral agents. Urine specimens and throat swabs from donors and rectal, throat, and urine specimens from recipients were cultured before and every two weeks following transplantation. These cultures were supplemented by clinically indicated cultures of suspicious lesions, biopsy material, and tissues obtained at autopsy (lung, liver, spleen, kidney, and gut). In addition, buffy coat cultures for CMV were carried out on a subset (30 recipients with hematologic malignancy and seven with marrow aplasia) of the patients. The buffy coat layer from 2 ml of whole blood was suspended in 0.2 ml of Hanks' medium, inoculated on the HET cultures, and left in contact for 4 hr. The medium was then changed, and the cultures were observed, as were all of the diagnostic cultures, for a period of three weeks. Buffy coat cultures were repeated every two weeks following transplantation. Finally, a sample from the donor marrow aspirate was cultured for CMV in 28 instances. Interstitial pneumonia and diagnostic studies. The radiologic and clinical features of interstitial pneumonia and histologic observations from lung tissues obtained at autopsy from fatal cases have been detailed [8, 9]. Briefly, cases were recognized by a constellation of findings consisting of tachypnea, fever, hypoxia, and interstitial (with or without patchy alveolar) pulmonary infiltrates on chest X ray. Affected patients produced scanty nonpurulent sputum which was negative in culture for bacterial and fungal pathogens. Wherever possible, definitive diagnoses were established by open lung biopsy through a small anterior thoracotomy incision. We used this

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Interstitial Pneumonia after Transplant

8 Figure 1. Occurrence of interstitial pneumonia following marrow transplantation. Each square is a single case. The heavy line encloses the fatal cases. The apparent etiology of each case is indicated by C (cytomegalovirus), P (Pneumocystis carinii) , H (herpes simplex virus), and I (idiopathic).

.---I

.----

r--

~ P

f--

I I

r--

I

C

f---

I

P

c;H I

C

C+P C+P C+P C+P

H

C

H

I

C

C

C

P

C

roc

C

C

C

I

I

I

c c+pl I 11

I

I

C

20

40

60

80

C

l

100

rn120

rIlJpl 190

Days after transplantation

Figure 2. Incidence (percentage) per day ("hazard") of interstitial pneumonia. The data in figure 1 were reanalyzed with respect to the number of living patients at risk for 10-day periods following transplantation to give an estimate of the daily incidence of this complication. The shaded plot shows the rate of pneumonia in which cytomegalovirus was isolated from the lungs.

S

"0

1.8

~

1.6

Q..

nificant, and the precision of this comparison is further compromised by the five transient pneumonias for which biopsies were not performed. The absence of positive cultures and seroconversion, which were present in the biopsy-proven, transient, CMV-associated pneumonias (see sections on virologic findings and on serologic findings), suggests, but does not prove, the absence of CMV in these four apparently idiopathic transient pneumonias. In the ultimately fatal cases of interstitial pneumonia in which premortem open lung biopsies revealed typical ClVIV inclusion bodies, CMV was also cultured from samples of lung tissue. Examination of the lungs at autopsy yielded the same findings. Furthermore, inclusion bodies were invariably present in all of the pneumonias in which CMV was the only pathogen cultured. Inclusions were not observed in two individuals who died with lungs heavily packed with P. carinii. CMV was cultured from the lungs of both of these patients although viral inclusions could not be identified. These individuals were grouped with three others with typical viral inclusions as having mixed CMV and pneumocystis pneumonia. To determine whether CMV was present in

_ 1.4 c ~

1.2

~

1.0 Q.. 0.8 Q)" -0 0 .6 .... 0.4

~ 0.2~1 _..m!~~ .E ~

0

20

40

60

80

100

Days after transplantation

120

140

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=

candy shorter (P 0.006, Student's t-test) for transient cases of pneumonia (median, seven days; range, two to 21 days) than for lethal episodes (median, 13 days; range, two to 45 days). Whereas clinical symptoms and measurements of blood gases improved rapidly in the nonfatal episodes, X-ray abnormalities often slowly resolved over a period of months. Pathogens associated with interstitial pneumonia. Candidate pathogens associated with involved lungs are listed in table 3. In five instances patients with rapidly resolving pneumonia were not biopsied. Noninvasive cultural and serologic findings were negative in four instances and positive for CMV infection in the fifth case. Four cases are listed in the table as idiopathic and the fifth as related to CMV. The rest of the idiopathic cases were negative in all diagnostic studies. On this basis 60% of the pneumonias were associated with a short list of opportunistic pathogens. Most prevalent was CMV which was isolated from 47% of the involved lungs. In 3170 of the pneumonias, CMV was the only pathogen isolated. Eighty percent of the CMV-associated pneumonias were lethal as opposed to 52% of those without CMV present. The number of cases is, however, too small for this difference to be sig-

Neiman et al.

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Table 3. Pathogens observed in lungs of patients with transient or fatal interstitial pneumonia. No. of cases with Hematologic malignancy Organism *

Fatal

2t 2 0 0 0

9 2 4 1 0

8:1:

6

Aplasia Transient Fatal 2 0 0 0 0 1§

1

0 1 1 1 2

Total

14 4 5 2 1 17

*CMV = cytomegalovirus; and HSV = herpes simplex virus. tNo biopsy was performed in one of these cases. :j:No biopsy was performed in three of these cases. § No biopsy was performed.

the lungs of marrow graft recipients without pneumonia, we studied 22 patients who died of causes other than pneumonia during the first three months after transplantation. Five of these individuals had positive pulmonary cultures for CMV. One had a focal pneumonia with typical inclusion bodies, which was not manifested clinically. The other four lacked histologic evidence of either active pneumonia or inclusion bodies. Two of these individuals had recovered from a recent episode of biopsy-proven interstitial pneumonia which was associated with CMV inclusions and positive cultures. The pneumonitis and viral

HERPES SIMPLEX VIRUS

Figure 3. Time of first isolation of DNA viruses. Each square is a single patient. Herpes simplex virus was invariably first isolated from the mouth. Site of first isolation of cytomegalovirus is given as U (urine), T (throat swab), and Be (buffy coat).

CYTOMEGALOVIRUS B.C. BOU

BC T

-10

o

BC

40

U

T

U

T

U

U

U

60

80

Days after transplantation

100

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CMV Pneumocystis carinii CMV + P. carinii . HSV HSV+ CMV Idiopathic

Transient

cytopathic change of these patients was resolved despite persistence of virus. Thus two of 19 recipients who never showed evidence of pneumonia had CMV in their lungs. P. carinii was a less common pathogen than CMV and was present in 21% of the pneumonias and the only recognized agent in 9%. Since P. carin ii would have been present in the five unbiopsied patients with transient pneumonia, this estimate might be low. In two instances P. carinii were observed in early open lung biopsies (one mixed with CMV). These patients developed progressive pneumonia despite treatment with pentamidine isethionate and a combination of trimethoprim and sulfamethoxazole. No P. carinii were seen in the advanced pneumonia present at postmortem examination. Herpes simplex virus (HSV) was cultured from three patients with lethal pneumonia. The virus was also recovered from more than one other deep organ in each case including brain, liver, bone marrow, lymph nodes, and kidney. Virologic findings. The only two viruses isolated with any frequency were CMV and HSV. Figure 3 shows the time of first isolation and the site of isolation for CMV. Twenty-three of the patients (29%) had positive cultures for HSV primarily associated with oral and facial lesions appearing within the first two weeks following transplantation. Two patients also had HSV-positive rectal lesions. Although these lesions caused

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Interstitial Pneumonia after Transplant

Table 4. Sites of isolation of cytomegalovirus from allogeneic bone marrow graft recipients. Group, diagnosis (no. of patients)

Urine culture

Throatj Buffy sputum coat

Any site

No pneumonia Hematologic malignancy (20) Aplastic anemia (17) Total (37)

3 4 7

0 3 3

0 0 0

5 6 11 (30)

Transient pneumonia Hematologic malignancy (12) Aplastic anemia (3) Total (15)

2 2 4

3 1 4

0 0 0

4 2 6 (43)

Lethal pneumonia Hematologic malignancy (22) Aplastic anemia (6) Total (28) All groups Hematologic malignancy (54) Aplastic anemia (26) Total (80)

6 1 7

11 7 18

1 2 3

4 6 10

5 0 5

5 0 5

15 5 20 (71)

25 12 37 (46)

NOTE. Data represent numbers of individuals with positive cultures (percentage). Some individuals were culturepositive from several sites, and others yielded virus only from specimens obtained at autopsy and biopsy.

positivity rate, about half the incidence of positive urine cultures. Serologic observations. Serologic responses to CMV were evaluated with regard to levels of antibody before transplantation, seroconversion following transplantation, and antibody titers of donors. Table 5 shows the relationship of titers in recipients before transplantation to subsequent events. The patients are divided into three groups: 44 with negative titers, 27 with low to moderate levels (1:8-1:32), and nine with high titers (1:64->1:512). The frequencies of both CMV infection and CMV -associated pneumonia were strikingly increased in the group with low to moderate initial titers. CMV was also isolated from more than half of those with high titers, but CMV-associated pneumonia occurred in only one of these individuals. This patient had a heavy pneumocystis pulmonary infection, and lacked viral inclusions in the lung despite positive cultures for CMV from both autopsy and biopsy specimens. This apparent correlation between initial antibody to CMV and viral shedding following transplantation has also been observed among reci pients of renal transplants [16]. We also assessed the effect of immunosuppressive therapy used to prepare patients for transplantation on persistence of initial titers of CF antibody (data not shown). Those patients with initial titers of ~1:64 sometimes showed a rapid twofold drop in titer in the first two weeks following transplantation but more often did not. In either case the titers usually remained stable over the subsequent three months (unless seroconversion occurred). Two of the five patients with initial titers of > I :64 actually had further rises in titer after an initial twofold drop in the Table 5. Effect of titer of CF antibody to cytomegalovirus (CMV) before transplantation in recipients of allogeneic bone marrow grafts on subsequent events.

CF antibody titer (no. of patients) Negative (44) 1:8-1 :32 (27) >1:64 (9)

No. with interstitial pneumonia Total

24 16 2

Lethal

13 14 1

No. with pneumonia with CMV isolated from lungs Total

7 13 1

No. with

CMV

isolated from Lethal any site

4 12 1

10 20 7

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annoying symptoms and interfered with nutrition, they did not lead to disseminated infection. Only one of the three patients with HSV pneumonia and other deep visceral involvement had early oral activation. Initial isolations of CMV from screening cultures of throat, urine, and buffy coat occurred from the period of marrow graft preparation to 130 days following engraftment (median, day 53). These cultures were positive in 27 individuals (34%), and CMV was isolated from biopsy and/or autopsy specimens of the lung and other organs in another 10 patients with negative screening cultures. Table 4 lists the number of individuals who had positive cultures from various sites. Isolation of CMV from any site appeared to be associated with an increased incidence of pneumonia. The group of culturepositive patients with lethal pneumonia includes all of the recipients with positive buffy coat cultures. These were five patients with leukemia who died with lethal CMV-associated pneumonia following transplantation. Since buffy coat cultures were carried out systematically in only 37 of these recipients, this figure represents a 13.5'10

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Neiman et at.

Table 6. Effect of seroconversion of titer of CF antibody to cytomegalovirus (CMV) on prognosis of pneum onia.

Seraconversion (no. of patients) Yes (65) No (15)

No. with interstitial pneumonia Total

Lethal

34 9

23 5

No. with pneumonia with CMV isolated from lungs Total 13 7

No. with pneumonia with CMV alone isolated from lungs

Lethal Total Lethal 13 3

9 5

9 1

NOTE. Seraconversion was identified as a fourfold or greater rise in titer after transplantation.

59 recipients without a serologic response to CMV, although this population includes many individuals not challenged by active CMV infection. 1£ one subtracts from this serologically unresponsive group the 42 patients from whom CMV. was not isolated, the figures for the incidence of lethal CMV-associated pneumonia jump to 77% and 53% for total and "pure" CMV-associated pneumonia, respectively. Although the latter calculations might exclude some patients with active CMV infection missed by the serial screening cultures and thus be overestimated values, the data suggest that serologic unresponsiveness to CMV infection increased the hazard of dying of interstitial pneumonia associated with CMV infection of the lung. The effect of the donor's titer of CF antibody to CMV for all but four of the marrow graft donors was also assessed. Although there was a numerical increase in the incidence of CMV infection in recipients of marrow from seropositive donors as compared with recipients of marrow from seronegative donors (35% vs. 52%), the difference was not statistically significant. Furthermore, there was no correlation with interstitial pneumonia per se (data not shown). The effect of supportive measures. An important unanswered question concerns the source of CMV infection in marrow graft reci pients. All cultures of urine and marrow from the donors were negative. All marrow graft recipients received numerous transfusions with fresh blood products before and after marrow engraftment. We have previously reported that there was no significant difference in the numbers of such exposures for recipients with or without CMV infection and interstitial pneumonia [8]. Granulocyte transfusion represents a particularly massive exposure to a potential exogenous source of CMV. However, the incidence of CMV infection among the 36 recipients of granulocyte transfusions was only slightly and not significantly higher (44%) than that among the 44 patients who did not receive such transfusions. (34%). U nfortunately, the titers of CF antibody to CMV in granulocyte donors were not determined. Confinement to rooms with "sterile" laminar air flow environments did not confer protection against interstitial pneumonia and its associated pathogens. Among the 14 patients who received transplants in the sterile environment and were kept

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first week after immunosuppression. Thus, despite the massive immunosuppression, it appeared that CF antibody to CMV continued to be produced, although the source (donor or host) could not be determined directly. Fifteen marrow graft recipients (eight with aplastic anemia) seroconverted a median of 55 (13-120) days following transplantation. All rises in titer were to levels of ~1:64 (~1:512 in seven instances). In 14 of these patients, CMV was isolated from one or more sites. The correlation between serologic response to CMV and CMV-associated pneumonia is shown in table 6. The incidence of pneumonia, particularly that associated with CMV, was increased in the group with serologic responses to CMV. The most striking finding, however, was that the prognosis for CMV-associated pneumonia was worse in patients who failed to seroconvert. All 13 such patients with CMV-associated pneumonia whose titer of CF antibody to CMV did not rise died of this complication. The mortality rate was considerably lower (20% for pneumonias associated with CMV alone and 42% for all pneumonias associated with CMV pulmonary infection) among those who did respond serologically to their pulmonary infection than those who did not. Furthermore, of the 21 patients who began the transplantation procedure with a high CF titer of antibody to CMV (~1 :64) and/or seroconverted to a similar titer, only three (14

A prospective analysis interstitial pneumonia and opportunistic viral infection among recipients of allogeneic bone marrow grafts.

THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.6. DECEMBER 1977 © 1977 by the University of Chicago. All rights reserved. A Prospective Analysis of...
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