Efficacy of amphotericin B encapsulated in liposomes (AmBisome) in ...

Journal of Antimicrobial Chemotherapy (1991) 28, Suppl. B, 73-82

Efficacy of amphotericin B encapsulated in liposomes (AmBisome) in the treatment of invasive fungal infections in immunocompromised patients O. Ringdtn', F. Marnier*, J. ToDemar", P. Rkcf, S. Tura', E. Kuse', M A. VIvianF, N. C. GoruV, J. Hasterskv*, P. Fenaux\ H. G. Prentice' and G. Ksionskr* 'Departments of Clinical Immunology and Transplantation Surgery, Huddinge Hospital, Sweden;b European Organization for Research and Treatment of Cancer (EORTC), Brussels, Belgium; 'Istituto di Ematologia, Bologna, Italy; 'Medizinische Hochschule Hannover, Hannover, Germany; 'Istituto Igiene, Milano, Italy;JServices des Maladies au Sang, Universiti Pierre et Marie Curie, Paris; 'Institut Jules Bordet, Brussels, Belgium; kService des Maladies du Sang, Lille, France; 'Department of Haematology, Royal Free Hospital, London, UK, and kVestar Inc., San Dimas, CA USA One hundred and twenty-six patients were treated for 137 episodes of fungal infection with liposomal amphotericin B (AmBisome) at 43 investigational centres. Among the patients were 72 with malignancies, 17 organ transplant recipients, 20 patients with immunological disorders and 17 others. AmBisome treatment was instituted after toxicity from previous amphotericin B treatment in 49 cases, nephrctoxicity or renal insufficiency in 40 and failure of previous antifungal treatment in 41. One hundred and eight episodes were clinically evaluable; among these 52 were caused by Candida spp. and 34 by Aspergillus spp. Ninety-nine patients were treated for at least eight days with a maximum dose of 0-7-5 mg/kg/day. Among 64 cases with proven invasive fungal infection 58% were cured. Fungi were eradicated in 35 of 54 (65%) mycologicaUy evaluable cases. The cumulative dose was 3-2 ±3-2 (mean ± S.D.) in casu where fungi were eradicated in comparison with 3-3 ± 2-3 g in cases where fungi persisted. The eradication rate was 83% for Candida spp. compared with 41% for Aspergillus spp. (P < 0-01). Among 24 cases with presumptive invasive fungal infections 14 (58%) were cured. Candida spp. were eradicated in seven of ten of these cases. Among 11 cases with superficial fungal infections eight were cured and three improved. Candida spp. were eradicated in four offivepatients. It is concluded that AmBisome is an effective antifungal agent in a majority of patients with invasive or superficial fungal infections. Introduction Opportunistic fungal infections, mainly caused by Candida and Aspergillus spp. may be life-threatening in severely immunocompromised patients (Myerowitz, Pazin & Allen, 1977; Schroter et al., 1977; Solomkin & Simmons, 1980; Meunier-Carpentier, Kiehn & Armstrong, 1981; Burchard et al., 1983; Clift, 1984; Hawkins & Armstrong, 1984; Colonna et al., 1988; Tollemar et al., 1989b). Patients at high risk for disseminated fungal infections include those with haematological malignancies treated with intensive 'Address for correspondence: Dr Oik Ringden, Department of Clinical Immunology, Huddinge Hospital, S-141 86 Huddinge, Sweden. 0305-7453/91/28B073+10 $02.00/0

73 © 1991 The British Society for Antimicrobial Chemotherapy

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chemotherapy, organ and bone marrow transplant recipients receiving immunosuppressive therapy, aged patients, patients with diabetes mellitus, those with malnutrition, and patients with indwelling catheters who have undergone multiple surgical procedures. The high mortality of deep disseminated fungal infections is due to frequent late diagnosis, a previous lack of effective non-toxic therapy and uncontrolled underlying disease. For a long time amphotericin B has been the drug of choice for these infections because of its potential fungicidal effect at therapeutic levels (Medoff & Kobayashi, 1980). However, the therapeutic use of amphotericin B has been limited by its acute toxicity, including headache, chills, fever, nausea, vomiting, diarrhoea, anorexia, malaise and muscle and joint pain. Other side effects include phlebitis at the infusion site, hypokalaemia, anaemia, bronchospasm and arrhythmias. A major side effect is nephrotoxicity (Utz et al., 1964; Maddux & Barriere, 1980). In transplant recipients treated with the immunosuppresive drug cyclosporin, therapy with amphotericin B is difficult because of a synergistic nephrotoxicity (Shulman et al., 1981). Recently amphotericin B has been encapsulated in liposomes; this reduces toxicity and allows the use of larger therapeutic doses (New, Chance & Heath, 1981; Taylor et al., 1982). Liposomal amphotericin B was shown to be effective in the treatment of experimental fungal infections (Graybill et al., 1982; Lopez-Berestein et al., 1983) and in clinical trials in cancer patients (Lopez-Berestein et al., 1985, 1987, 1989; Sculier et al., 1988). However, none of the previous preparations of liposomal amphotericin B could be stored for any appreciable amount of time; they were prepared at the investigational centres shortly before therapy and are not commercially available. AmBisome is a liposomal amphotericin B preparation, consisting of small, unilamellar vesicles, which has a desirable degree of stability and can be stored in lyophilized form. Here we report the clinical efficacy of AmBisome in the treatment of invasive fungal infections in a compassionate phase EI-III multicentre study. Materials and methods In total, 126 patients were enrolled in the study. Four patients were treated twice, one patient was enrolled four times and four patients had two simultaneous fungal infections, so that a total of 137 episodes of infection by an individual fungus were included. There were 79 males and 47 females. The median age was 35 years (range 4-87). Among the patients were 72 with malignancies (55 leukaemia, 9 lymphoma, 2 ovarian cancer, 4 multiple myeloma, 1 teratoma, 1 myelodysplastic syndrome). Seventeen patients had received the following organ transplants: liver (11), pancreas and kidney (2), kidney (2), pancreas (1), and heart (1). Among 20 patients with immunological disorders were 12 with AIDS, three with aplastic anaemia and one each with myeloid metaplasia, lupus erythematosus, sarcoidosis, chronic granulomatosis and metachromatic leukodystrophy. Among the patients were 25 recipients of allogeneic or autologous bone marrow transplants. Among 17 with other disorders were five with diabetes mellitus, two who had undergone heart valve replacement surgery, three without specific disorder and one each with diverticulosis, acute pancreatitis, pancreatic cysts, neutropenia, Fanconi anaemia, surgical wound dehiscence, and renal insufficiency. Patients were treated at 43 investigational sites in Austria, Belgium, France, West Germany, Italy, the Netherlands, Portugal, Sweden, the United Kingdom, Canada and the United States.

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Reasons for enrolment and termination In 49 cases AmBisome treatment was instituted because of nephrotoxicity of previouslyadministered conventional amphotericin B. In seven cases, nephrotoxicity was due to other causes, and 33 further cases had renal insufficiency. In 41 cases AmBisome therapy was given because previous antifungal treatment had failed. In three cases AmBisome was instituted for other reasons. The most common reasons for discontinuation of AmBisome were completion of the course of treatment (57 cases) and patient death (41 cases). (Six additional patients died within four weeks after discontinuation of AmBisome.) Other causes were withdrawal by investigator (8), other intercurrent illness (6), patient request (5), persistence of infection (3) and other (11). In two cases AmBisome was withdrawn because of adverse events. Administration and dosage Each AmBisome vial contained a sterile pyrogen-free lyophilized powder providing SO mg of amphotericin B encapsulated in liposomes. These small unilamellar vesicles consisted of hydrogenated soy phosphatidylcholine, cholesterol and distearoylphosphatidylglycerol. AmBisome was reconstituted in cold (2°-8°Q sterile water; 12 mL refrigerated sterile water for injection was added to each vial to form a suspension containing 4-0 g/L amphotericin B. This suspension was shaken for 1-2 min, incubated in a water bath at 65° C for 10 min and allowed to cool to room temperature. The suspension was then diluted with 5% dextrose to a final concentration of 0-5 g/L of amphotericin B as AmBisome. This was infused intravenously over a 30-60 min period. Dosing was begun at 0-5 or 1-0 mg/kg, and the dose was then increased in a step-wise fashion. The maximum daily dose ranged from 0-5 to 5 mg/kg (median 2-2 mg/kg). The total number of days during which AmBisome was given ranged from 1 to 97 (median 18) with a median cumulative dose of lS48mg (range 50-16,775) or 27 mg/kg (range 0-74-215-06). Clinical efficacy evaluation

Clinical efficacy was evaluated in 99 cases treated for at least eight days with AmBisome. The cases were divided into three groups, 64 with proven invasive infections, 24 with presumed invasive infections and 11 with superficial fungal infections. The identified pathogens are given in Table I. Among the 64 cases with proven invasive infections the fungi were identified by culture only (31), microscopy only (7), culture and microscopy (13), culture and serology (10), and microscopy and serology (3). Among the cases with presumptive infections diagnosis was identified by culture only (8), serology only (4), culture and microscopy (2), culture and serology (2), and presumptive (8). Among the 11 patients with superficial infections fungi were identified by culture only (5), microscopy only (1), culture and microscopy (2), culture and serology (2) and presumptive (1). The sites of infection in cases with proven invasive aspergillosis were pulmonary (25), pulmonary aspergilloma (1), hepatic (1), endocardial (1), and air sinus (1). Among the cases with invasive candidosis the sites of infection were blood (13), hepatic (4), abdominal (4), pulmonary (1), mediastinal (1), air sinus and eye (1) and nasopharynx and face (1). There were seven cases of cryptoccocosis, with positive CSF and one case with a positive blood culture. One patient had pulmonary mucormycosis and one had

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Table I. Aetiology of fungal infections in various patient groups Mycosis Proven Presumed invasive invasive Superficial Aspergillus spp. Candida spp. Cryptococcus neoformaru Coccidioides immitis Mucor spp. Saccharomyces cerevisiae Trichosporon capitation Fusarhm port Madwella grisea Not identified

29 25 7 1 1

Total

64

3 17

0 6

1 1 4 24

1 2 1 11

pulmonary coccidioidomycosis. There was also one patient with Trichosporon capitation septicaemia. Among the patients with presumptive fungal infections there were four with suspected pulmonary aspergillosis and 15 with suspected pulmonary candidosis. These patients lacked a proven diagnosis of fungus from a deep site. Two cases had a non-specified pulmonary fungal infection. In addition there was one case with a diagnosis of suspected disseminated candidosis made by positive serology, but no proven microscopy or culture of Candida spp. There was one patient with presumptive abdominal candidosis and one with a suspected fungal brain infection. Among the 11 cases with non-invasive infections were six with Candida spp. of which three were oropharyngeal, two gastro-intestinal and one oesophageal. In addition there were four with mycetoma and one with a Saccharomyces cerevisiae pulmonary infection. The duration and dosage of AmBisome treatment in the three patient groups are given in Table II. Cure was denned as disappearance of all symptoms, such as clearance of infiltrates on chest-films, disappearance of fever, healing of infected wounds, etc. Improvement was judged by the treating physician as clearance of some symptoms, but not complete cure. Mycological efficacy could be evaluated in cases where fungus was identified Table IL Duration and dose of AmBisome in various groups of patients with fungal infections, treated for at least 8 days Mycosis proven invasive presumed invasive Number of cases Duration (days) Dosage (mg/kg/day) Cumulative dose (g)

64 29±18 (8-97) 2-4 ±0-8 (0-9-4-0) 3-5 ± 3 1 (0-6-16-8)

24 23 ±14 (8-65) 2-4±ll (0-7-5^)) 2-8 ±2-2 (CM-9-1)

superficial 11 22±17 (8-65) 21 ±0-9 (1-0-4-0) 1-8 ±1-9 (02-7-2)

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before therapy with AmBisome and where samples to identify fungi were taken during and/or after therapy. Such cases were evaluated as eradication or persistence of fungus. Results Efficacy evaluation in cases with proven invasive infections Of the 64 cases with proven invasive infections, 37 (58%) were cured, 12 (19%) improved and 15 (23%) failed to respond (Table III). The cure rate in patients with Candida infections was 19/25 (76%) compared with 9/28 (32%) in aspergillus infections (P < 0.01, Fisher's exact test). All mycoses other than those caused by Candida or Aspergillus spp. were clinically cured or improved. Fifty-four cases with proven invasive infection were mycologically evaluable. Fungi were eradicated in 35 (65%) of these. In cases with aspergillus infection 9/22 (41%) were eradicated in comparison with 19/23 (83%) of proven invasive Candida infections (P < 0-01, Fisher's exact test). The eradication rate in fungal infections other than candidosis or aspergillosis was 7/9 (78%). Among the nine evaluable patients were six patients with AIDS and cryptococcal meningitis. Cryptococcus neoformans was eradicated in five patients who were cured. One patient improved clinically but cryptococci persisted in the CSF despite 42 days of treatment with a cumulative dose of 5-6 g AmBisome. Proven invasive fungal infections that were mycologically eradicated (n = 35) were treated for 28 ± 20 (mean ± S.D.) days, with a cumulative dose of 3-2 ± 3-2 g in comparison with 25 ± 14 days and a cumulative dose of 3-3 ±2-2 g in cases where fungi persisted (« - 19). Fungj were eradicated in 14/22 patients with leukaemia, 5/8 with immunodeficiencies, 9/10 with organ transplants, 1/3 with malignancies other than leukaemia and 6/11 others. In patients with leucocyte counts below 0-5 x lO'/L the eradication rate was 7/12 (58%) in comparison with 28/40 (70%) in patients with leucocyte counts above 0-5 x lO'/L (ns). Table HL Clinical efficacy of AmBisome Cured

Improved

Failed

Proven invasive aspergillosis candidosis other total

9 19 9 37

(32%) (76%) (82%) (58%)

8 2 2 12 (19%)

11 4 0 15 (23%)

Presumed invasive aspergillosis candidosis other total

2 10 (59%) 2 14 (58%)

2 3 1 6 (25%)

0 4 0 4 (17%)

0 3 3

0 0 0

Superficial candidosis other total

6 2 8 (73%)

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Responses in cases with presumptive fungal infections Among 24 cases with presumptive invasive fungi, 14 were cured (58%), six improved and four failed following treatment with AmBisome (Table HI). Among 17 cases with presumptive candidosis ten were judged to be cured, three improved and four failed. Among cases with presumptive aspcrgillosis two were cured, two improved and none failed. In other fungal infections two were cured, one improved and none failed. Candida was eradicated in seven of ten cases who were mycologically evaluable. Clinical and mycological efficacy in non-invasive fungal infections Overall eight cases were cured and three improved and there was no failure in the 11 cases with superficial infections (Table III). All six Candida infections were judged to be cured. Among fungal infections caused by other species two were cured and three improved. Six of the cases were mycologically evaluable. Candida was eradicated in four and persisted in one. One patient had a mycetoma due to Fusarium pori. The lesion improved, but cultures remained positive in spite of 65 days of treatment with 7-2 g AmBisome. Causes of death and autopsy findings Forty-seven patients died during or within four weeks of the AmBisome therapy. The major causes of death were persistent fungal infection and additional other complications (17), progression of the underlying disorder (10), persistent fungal infection alone Table IV. Ambisomc tissue concentrations from three autopsy cases Patient

no. 1

2

3

Total dose (mg) 3428

900

820

Tissue

Concentration (mg/kg tissue)

Total drug per organ (mg)

Percent of total dose

brain CSF* heart kidney liver lung spleen thyroid

215

3-07

009

0-56 7-83 50-00 291-30 45-39 163-30 19-84

2-52 14-50 474-82 43-26 2645

007 042

bone marrow heart kidney, right kidney, left Hver lung spleen

1-63 1-40 1059 10-75 143-06 4-54 150-20

bone marrow heart

26-80 3-61 7-87 92-80 0-55 4-04 29110

kidney tivcr lung muscle spleen

036 __ 035 2-63 2-67 202-43 3-60 23-28

116

13-85 1-26

077 001 _ 004 029 O30 22-49

O40 2-59

014 028

2-28 151-26

18-45

052 —

006 —

4716

5-75

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(8), bacterial septicaemia (3), multiple organ failure (2), heart failure (2), cytomegalovirus (CMV) infection (1), Pneumocystis carinii pneumonia (1), cerebral haemorrhage (1), rupture of a cerebral aneurysm (1), and septicaemia of unknown origin (1). Among the patients who died 23/47 were examined for fungal pathogens in samples taken at autopsy. Fungal pathogens were undetectable in 6/23 evaluable cases (26%). The mean length of treatment in cases where fungi were eradicated was 31 ± 37 days (mean ± s.D.) in comparison with 15 ± 8 days in cases where fungi persisted (ns). The mean cumulative dose of AmBisome was 5-3 ± 6-4 g and l-8±l-2g for the two groups, respectively (ns). In three autopsy cases, the concentrations of amphotericin B were determined in various tissues. The highest concentrations were detected in liver and spleen (92-291 mg/kg tissue). Lower and variable concentrations were found in kidneys, heart, bone marrow, lung and brain (Table TV). Discussion

EflBcacy evaluation of AmBisome is complicated by difficulties in the diagnosis of fungal infections, the patients' poor condition and frequent concomitant infections. However, in this heterogeneous population there were 64 cases of confirmed invasive fungal infections treated for longer than eight days, and 58% of these cases were clinically cured. Improvement of some, but not all, symptoms was seen in 19% of the cases. However, this response is not well defined and no real conclusion can be based on these cases. Of 54 clinically evaluable cases where mycological efficacy could be determined, pathogens were eradicated in 65%. The low mycological response rate in patients with aspergjllosis (41%) in comparison with candidosis (83%) (P < 0-01) may have occurred because the majority of aspergillus infections affected the lung, and lung tissue contained a low concentration of amphotericin B in two of three patients (Table TV). The patient with a high concentration in the lung had received almost four times the dose given to the other two patients. This may indicate that pulmonary infections require higher dosage of AmBisome. When all evaluable cases with invasive fungi were included, the cure rate was 32% for aspergillosis and 76% for candidosis (P < 0-01). The cumulative dose of AmBisome and the length of treatment had no influence on the eradication rate of fungi. The reason for this may be that patients with poorer prognoses and those with a slower response rate were treated with higher doses and for a longer period of time. In comparison with the previously observed response rate in similar patients treated with conventional amphotericin B, there is a clear improvement with AmBisome, which is in agreement with previous studies using other formulations of Uposomal amphotericin B (Lopez-Berestein etal., 1983, 1985, 1987). Diagnosis in these 64 cases can be considered solid since the presence of fungi in deep sites was indicated by one or two diagnostic criteria. Fungal infections may be overdiagnosed when only serologjcal criteria are used (Tollemar et al., 1989a). Therefore cases with positive serology as the only diagnostic criterion were included in the group with presumptive fungal infections. The largest group of presumptive cases comprised 15 patients with suspected Candida pneumonia. These were patients with infiltrates, typical of fungal infection, on chest radiographs. In addition Candida was isolated from sputum or bronchial lavage in many of these patients. It is possible that Candida spp. from the oropharynx contaminated these cultures. Therefore, these cases were considered presumptive, even if Candida was obtained from bronchial lavage. The cure rate in patients with presumptive fungal infections was the same as in patients with proven invasive infections, 58%. This suggests that most of these patients suffered from fungal

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infections and benefited from AmBisome treatment AmBisome was also eflFective in the treatment of 11 superficial fungal infections and Candida was eradicated in four of five such infections. This is in contrast to a report on eight transplanted patients with invasive fungi where superficial colonization by fungi was unaffected by treatment with AmBisome (Tollemar, Ringden & Tyden, 1990). Forty-seven of the 126 patients (37%) died within four weeks of AmBisome therapy, emphasising the poor prognosis in these conditions. Fungal infection was the cause of death, or contributed to death, in 25 patients. These patients might possibly have responded if antifungal therapy had been instituted earlier. Because of the difficulties in diagnosing deep disseminated fungal infections, AmBisome may be suitable for prophylactic use. Alternatively, AmBisome may be instituted for treatment of fever of unknown origin non-responsive to antibiotics in patients at high risk of invasive fungal infections. High-risk patients, such as bone marrow transplant recipients during the leucopenic phase, are often beyond therapy by the time the diagnosis of fungaemia is made (Clift, 1984; Tollemar et al, 1989A). Liposomes carrying amphotericin B may be entrapped in the organs of the reticuloendothelial system such as liver, spleen and lung. The high concentrations of amphotericin B in liver and spleen of the three autopsy cases treated with AmBisome, suggests this possibility (Table TV). Lung tissue showed marked variation in drug concentrations, with low concentrations in two cases and an intermediate concentration in one. This could be due to loss of the drug from autopsy material or entrapment of liposomes in liver and spleen rather than in the reticuloendothelial system of the lung. MycologicaL but not clinical, response rates were worse in pulmonary aspergillosis: therefore, concentrations of amphotericin B obtained following AmBisome treatment may have been inadequate in some cases. However, despite high tissue concentrations of amphotericin B (200mg/L) clear failures and fungal growth was reported (Christiansen etal., 1985; Colette et al., 1989). Therefore, the sensitivity to amphotericin B of various fungal strains must also be taken into consideration. The few side-effects encountered with AmBisome (Meunier, Prentice & Ringddn, 1991, this supplement) provide a contrast to the situation with conventional amphotericin B where acute toxicity and side-effects occur in a majority of patients even with doses as low as 0-3 mg/kg/day. With AmBisome treatment, doses up to 3-5 mg/kg/day were tolerated. Comparative studies of AmBisome would be desirable in immunocompromised patients who do not have renal impairment and who are not treated with nephrotoxic drugs. In this setting, AmBisome may be compared with conventional amphotericin B or other antifungal agents such as fluconazole or ketoconazole (Jones et al., 1984; Shepp et al., 1985; Hansen et al., 1987). Because of the difficulty of diagnosing deep fungal infections, prophylactic trials with AmBisome are warranted in patients with a high incidence of life-threatening fungal infections such as bone marrow and liver transplant recipients (Tollemar et al., 1989A; Tollemar & Ericzon, 1991). Prophylaxis or early therapy on suspicion of fungaemia is justified by the good patient tolerance of AmBisome. Acknowledgements We wish to thank Deborah A. Hein, and Dwain K. Allen for computer analysis of the data in this study. We also thank the following for contributing patients to this study: R. Hay, D. Huang, A. Goldstone, P. Jumaa, A. Bryceson, E. Leonard, London; R.

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Zittoun, B. DuPont, S. Blanche, Paris; N. Clumeck, Brussels; C. Stoutenbeeek, S. Rodenhuis, Amsterdam; S. de Marie, Rotterdam; A. Gabbas, Nuoro; F. Fraschini, Milan; P. Martino, Rome; M. Abecassis, A. Gouveia, J. Atouguia, Lisbon; W. Swinger, R. Zirkulnig, M. Kronawetter, Graz; R. Waldner, N. Vetter, Vienna; A. Lindemann, Mainz; R. Joeres, Wurzburg; H. link, Hannover; H. Pohle, Berlin; P. Meusers, Essen; W. Schroyens, Giessen; P. Pierce, Washington; R. Jenkins, Hollywood; R. Alemeleh, Brooklyn; P. Phillips, Vancouver, A. Rachlis, Toronto. Dr O. Ringden was supported by grants from the Children's Cancer Foundation (8403), The Swedish Medical Research Council (16X-05971) and the Swedish Cancer Society (77O-B9O-O3XQ. References Burchard, K. W., Minor, L. B., Slotman, G. J. & Gann, D. S. (1983). Fungal sepsis in surgical patients. Archives of Surgery 118, 217-21. Christiansen, K. J., Bernard, E. M., Gold, J. W. M. & Armstrong, D. (1985). Distribution and activity of amphotericin B in humans. Journal of Infectious Diseases 152, 1037-43. Qift, R. A. (1984). Candidiasis in the transplant patient. American Journal of Medicine 77, Suppl.

4D.34S. Colette, N., van der Auwera, P., Lopez, A. P., Heymans, C. & Meunier, F. (1989). Tissue concentrations and bioactivity of amphotericin B in cancer patients treated with amphotericin B-deoxycholate. Antimicrobial Agents and Chemotherapy 33, 362-8. Colonna, J. O., Winston, D. J., Brill, J. E., Goldstein, L I., Hoff, M. P., Hiatt, J. R. et al. (1988). Infectious complications in liver transplantation. Archives of Surgery 123, 360-4. Graybill, J. R., Craven, P. C , Taylor, R. L., Williams, D. M. & Magee, W. E. (1982). Treatment of murine cryptococcosis with h'posome-associated amphotericin B. Journal of Infectious Diseases 145, 748-52. Hansen, R. M., Reinerio, N., Sohnle, P. G., Abrams, R. A., Ritch, P. S., Libnoch, J. A. et al. (1987). Ketoconazole in the prevention of candidiasis in patients with cancer. A prospective, randomized, controlled, double-blind study. Archives of Internal Medicine 147, 710-2. Hawkins, C. & Armstrong, D. (1984). Fungal infections in the immunocompromised host. Clinics in Haematology 13, 599-630. Jones, P. G., Kauflman, C. A., McAuliffe, L. S., Liepman, M. K. & Bergman, A. G. (1984). Efficacy of ketoconazole versus nystatin in prevention of fungal infections in neutropenic patients. Archives of Internal Medicine 144, 549-51. Lopez-Berestein, G., Bodey, G. P., Fainstein, V., Keating, M., Frankel, L. S., Zeluff, B. et al. (1989). Treatment of systemic fungal infections with liposomal amphotericin B. Archives of Internal Medicine 149, 2533-6. Lopez-Berestein, G., Bodey, G. P., Frankel, L. S. & Mehta, K. (1987). Treatment of hepatosplenic candidiasis with liposomal amphotericin B. Journal of Clinical Oncology 5, 310-7. Lopez-Berestein, G., Fainstein, V., Hopfer, R., Mehta, K., Sullivan, M. P., Keating M. et al. (1985). Liposomal amphotericin B for the treatment of systemic fungal infections in patients with cancer a preliminary study. Journal of Infectious Diseases 151, 704-10. Lopez-Berestein, G., Mehta, R., Hopfer, R., Mehta, K., Hersh, E. M. & Juliano, R. (1983). Effects of sterols on the therapeutic efficacy of liposomal amphotericin B in murine candidiasis. Cancer Drug Delivery 1, 37-42. Maddux, M. S. & Barriere, S. L. (1980). A review of complications of amphotericin-B therapy: recommendations for prevention and management Drug Intelligence and Clinical Pharmacy 14, 177-81. Medoff, G. & Kobayashi, G. S. (1980). Strategies in the treatment of systemic fungal infections. New England Journal of Medicine 302, 145-55. Meunier, F., Prentice, H. G. & Ringden, O. (1991). Liposomal amphotericin B (AmBisome): safety data from a phase II/III clinical trial. Journal of Antimicrobial Chemotherapy 28, Suppl. 5,83-91.

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Mcunicr-Carpcntier, F., Kiehn, T. E. & Armstrong, D. (1981). Fungcmia in the immunocompromised host Changing patterns, antigcnemia, high mortality. American Journal of Medicine 71, 363-70. Myerowitz, R. L., Pazin, G. J. & Allen, C. M. (1977). Disseminated candidiasis. Changes in incidence, underlying diseases, and pathology. American Journal of Clinical Pathology 68, 29-38. New, R. R. C , Chance, M. L. & Heath, S. (1981). Antileishmanial activity of amphotericin and other antifungal agents entrapped in liposomes. Journal of Antimicrobial Chemotherapy 8, 371-81. SchrSter, G. P. J., Hoelscher, M., Putnam, C. W., Porter, K. A. & Starzl, T. E. (1977). Fungus infections after Hver transplantation. Annals of Surgery 186, 115-22. Sculier, J. P., Coune, A., Meunier, F., Brassinne, C , Laduron, C, Hollaert, C. et al., (1988). Pilot study of amphotericin B entrapped in sonicated liposomes in cancer patients with fungal infections. European Journal of Cancer and Clinical Oncology 1A, 527-38. Shepp, D. H., Klosterman, A., Siegel, M. S. & Meyers, J. D. (1985). Comparative trial of ketoconazole and nystatin for prevention of fungal infection in neutropenic patients treated in a protective environment. Journal of Infectious Diseases 152, 1257—63. Shulman, H., Striker, G., Deeg, H. J., Kennedy, M., Storb, R. & Thomas, E. D. (1981) Nephrotoxicity of cydosporin A after allogeneic marrow transplantation: glomerular thromboses and tubular injury. New England Journal of Medicine 305, 1392-5. Solomkin, J. S. & Simmons, R. L. (1980). Candida infection in surgical patients. World Journal of Surgery 4, 381-94. Taylor, R. L., Williams, D. M., Craven, P. C , Graybill, J. R., Drutz, D. J. & Magee, W. E. (1982). Amphotericin B in liposomes: a novel therapy for histoplasmosis. American Review of Respiratory Disease 125, 610-1. Tollemar, J. & Ericzon, B.-G. (1991). Invasive Candida albicans infections in orthotopic liver graft recipients. Incidence and risk-factors. Clinical Transplantation, in press. Tollemar, J., Holmberg, K., Ringden, O. & Lonnqvist, B. (1989a). Surveillance tests for the diagnosis of invasive fungal infections in bone marrow transplant recipients. Scandinavian Journal of Infectious Diseases 21, 205-12. Tollemar, J., Ringden, O., Bostrom, L., Nilsson, B. & Sundberg, B. (1989*). Variables predicting deep fungal infections in bone marrow transplant recipients. Bone Marrow Transplantation 4,635-41. Tollemar, J., Ringden, O. & Tyden G. (1990). Liposomal amphotericin-B (AmBisome) treatment in solid organ and bone-marrow transplant recipients. Efficacy and safety evaluation. Clinical Transplantation 4, 167-75. Utz, J. P., Bennett, J. E., Brandriss, N. W., Butler, W. T. & Hill, G. J. (1964). Amphotericin B toxidty. Combined clinical staff conference at the National Institutes of Health. Annals of Internal Medicine 61, 334-54.