Invasive aspergillosis (IA) has emerged as a major cause of morbidity. Aspergillus terreus

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1 1594 Aspergillus terreus An Emerging Amphotericin B Resistant Opportunistic Mold in Patients with Hematologic Malignancies Ray Y. Hachem, M.D. 1 Dimitrios P. Kontoyiannis, M.D., Sc.D. 1 Maha R. Boktour, M.D. 1 Claude Afif, M.D. 1 Catherine Cooksley, Ph.D. 2 Gerald P. Bodey, M.D. 1 Ioannis Chatzinikolaou, M.D. 1 Cheryl Perego, M.P.H., B.S. 1 Hagop M. Kantarjian, M.D. 3 Issam I. Raad, M.D. 1 1 Department of Infectious Diseases, Infection Control, and Employee Health, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. 2 Department of Biostatistics and Applied Mathematics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. 3 Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. Presented in part as an abstract at the 40th Annual Meeting of the Infectious Diseases Society of America, Chicago, IL, October 24 27, The authors thank Schering-Plough for providing the posaconazole used in the current study and Dr. Michael Rinaldi s group (Department of Veterans Affairs Mycology Reference Laboratory, Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX) for performing the susceptibility testing described herein. Claude Afif s current address: Department of Infectious Diseases and Infection Control, University of Balamand, Beirut, Lebanon. Address for reprints: Ray Y. Hachem, M.D., Department of Infectious Diseases, Infection Control, and Employee Health (Unit 402), The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; Fax: (713) ; rhachem@mdanderson.org Received January 16, 2004; revision received May 28, 2004; accepted June 30, BACKGROUND. Invasive aspergillosis (IA) has emerged as a common cause of morbidity and mortality among immunocompromised patients. At The University of Texas M. D. Anderson Cancer Center (Houston, TX), Aspergillus terreus is second to A. fumigatus as the most common cause of IA. In the current study, the authors compared the risk factors and outcomes associated with IA caused by A. terreus and IA caused by A. fumigatus. METHODS. The authors retrospectively reviewed the medical records of 300 patients who received care at our institution between 1995 and 2001 and who had cultures that were positive for Aspergillus infection, including 90 patients whose cultures were positive for A. fumigatus and 70 patients whose cultures were positive for A. terreus. RESULTS. Thirty-two patients with IA caused by A. terreus and 33 patients with IA caused by A. fumigatus were evaluated. The two groups were comparable in terms of age, gender, and underlying disease. Leukemia was the most common underlying malignancy (84%). More than 40% of patients in each group had undergone bone marrow transplantation. There was a trend toward a higher frequency of neutropenia among patients with IA caused by A. terreus (P 0.12). IA caused by A. terreus was considered to be nosocomial in origin significantly more frequently compared with IA caused by A. fumigatus (P 0.03). In vitro, A. terreus was found to be more resistant to amphotericin B (minimal inhibitory concentration [MIC 90 ], 4.0 g/ml) than to antifungal therapy (MIC 90, 1.0 Hg/mL) in the isolates that were tested ( 50% of all isolates). The overall rate of response to antifungal therapy was 39% for patients with A. fumigatus infection, compared with 28% for patients with A. terreus infection (P 0.43). CONCLUSIONS. Despite the decreased in vitro susceptibility of A. terreus (relative to A. fumigatus) to amphotericin B, the two groups within the current patient population had comparably poor responses to amphotericin B preparation and somewhat improved responses to posaconazole. Cancer 2004;101: American Cancer Society. KEYWORDS: Aspergillus terreus, Aspergillus fumigatus, patients, cancer, neutropenia, lipid preparation. Invasive aspergillosis (IA) has emerged as a major cause of morbidity and mortality among immunocompromised patients. 1 5 Aspergillus species are recognized causes of opportunistic pneumonia and sinusitis in patients with malignant disease and are associated with mortality rates as high as 85%. 2,6 A. fumigatus and A. flavus account for the majority of documented cases of IA. However, at some institutions, including ours (The University of Texas M. D. Anderson Cancer Center [MDACC], Houston, TX), A American Cancer Society DOI /cncr Published online 23 August 2004 in Wiley InterScience (

2 Aspergillus and Hematologic Malignancies/Hachem et al TABLE 1 Characteristics of Patients with Aspergillus fumigatus and Aspergillus terreus Infections Characteristic Aspergillus fumigatus a Aspergillus terreus b P value c Male gender 19 (58) 16 (50) 0.53 Mean age in yrs SD (range) (24 69) (24 67) 0.24 d Malignancy 0.37 Leukemia 22 (67) 27 (84) Lymphoma 9 (27) 4 (13) Solid tumor 2 (6) 1 (3) Received BMT 15 (45) 13 (41) 0.68 Developed GVHD 13 (87) 12 (92) Neutropenia (ANC 500 per mm 3 ) 18 (55) 24 (75) 0.12 ANC per mm 3 14 (42) 14 (44) ANC 100 per mm 3 4 (12) 10 (31) 0.06 Steroid use (prednisone equivalent in mg) mg 14 (42) 13 (41) 600 mg 15 (45) 15 (47) None 4 (12) 4 (12.5) 0.48 Type of infection Definite 12 (36) 9 (28) Probable 21 (64) 23 (72) 0.03 Nosocomial 6 (18) 14 (44) Community-acquired 27 (82) 18 (56) SD: standard deviation; BMT: bone marrow transplant; GVHD: graft-versus-host disease; ANC: absolute neutrophil count. a n 33. b n 32. c Chi-square or Fisher exact test. d Two-sample t test. terreus infection is a frequent cause of IA among patients with hematologic malignancies. 7 9 The reason for the relative frequency with which IA caused by A. terreus infection is observed at our center is not clear. Many factors, such as unique environmental exposure, specific host-related characteristics, and the extensive use of systemic antifungal agents as prophylactic and empiric therapy, could account for this finding In vitro, clinical isolates of A. terreus are typically resistant to antifungal agents, and especially to amphotericin B (AMB). 14,15 Due to a lack of effective antifungal therapy, one would expect a poorer outcome in patients with IA caused by A. terreus infection who received AMB than in those with infections caused by other Aspergillus species. Because A. terreus infection is a common cause of IA at our institution, we conducted the current study to compare the outcomes and risk factors associated with IA caused by A. terreus and IA caused by A. fumigatus. We excluded patients who did not have proven or probable IA (n 43), patients who had more than 1 Aspergillus species found in their culture specimens (n 17), and patients who had received 7 days of systemic antifungal therapy (n 35). We collected information on demographic characteristics, underlying malignancy, bone marrow transplantation (BMT), GVHD, severity of neutropenia, steroid use, and type of infection (Table 1). MATERIALS AND METHODS We reviewed the medical records of 300 patients who received care at our institution between 1995 and 2001 and who had Aspergillus species isolated from culture specimens. Patients were identified via microbiology, histopathology, and infection control databanks. We used the definition of definite or probable IA that was set forth by the Mycoses Study Group of the National Institute of Allergy and Infectious Diseases and the European Organization for Research and Treatment of Cancer. 16 Definitions Definite aspergillosis Infection was considered to be definite if tissue histopathology revealed the presence of septate, acute branching hyphae and a culture obtained from the same tissue specimen using an invasive procedure, such as transbronchial biopsy or percutaneous needle

3 1596 CANCER October 1, 2004 / Volume 101 / Number 7 aspiration, was found to be positive for Aspergillus infection. Probable aspergillosis IA was considered to be probable in patients with cancer and recipient of hematopoietic stem cell transplants in the context of neutropenia lasting for 10 days, or graft-versus-host disease (GVHD), or cytotoxic therapy for malignant or immunologic disease, or corticosteroid treatment (daily prednisone equivalent 10 mg) lasting for 3 weeks. In addition, patients with probable aspergillosis had one sputum culture specimen or one bronchoalveolar lavage (BAL) washing or brushing specimen from which an Aspergillus species had been isolated, as well as new infiltrates on chest computed tomography findings (e.g., halo signs, air crescent signs, or cavity within area of consolidation) that were consistent with the presence of Aspergillus infection. Neutropenia Neutropenia was defined by the detection of 500 neutrophils per mm 3 at the onset of infection. Response (complete or partial) Response was defined as the resolution or significant clinical improvement of all attributable signs and symptoms of IA along with an improvement of 50% in any radiologic abnormalities present at enrollment and the discontinuation of further systemic antifungal therapy. The end of the study was defined as the point at which antifungal therapy was discontinued due to success, progression of the infection, or death. Treatment failure (stabilization or deterioration) Treatment failure was considered to have occurred when the attributable symptoms and signs of IA deteriorated or did not improve or when radiographically or bronchoscopically detected abnormalities persisted and necessitated alternative antifungal therapy or resulted in death. Nosocomial IA IA was considered to be nosocomial in patients whose first signs and symptoms occurred 14 days after hospital admission. Microbiology Isolates were obtained from a variety of sources, including sputum, BAL, and tissue biopsy specimens. The isolation and identification of Aspergillus species were achieved using standard microbiologic procedures. 17 Aspergillus isolates collected from patients who met the eligibility criteria for the current study were sent to Dr. Michael G. Rinaldi (Department of Veterans Affairs Mycology Reference Laboratory, Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX) for susceptibility testing. Minimal inhibitory concentrations (for 50% inhibition [MIC 50 ] and 90% inhibition [MIC 90 ]) for AMB, itraconazole, and posaconazole were recorded. MICs were determined using the broth microdilution method according to the National Committee for Clinical Laboratory Standards M38-A method. 18 Statistical Analysis All comparisons were two sided. For comparisons involving gender, BMT, and neutropenia, Pearson and chi-square tests were used. For comparisons involving malignancy type and steroid use, the Fisher exact test was used when appropriate. RESULTS A total of 300 patients with IA were identified at our hospital. A. fumigatus, the most common source of infection, was isolated from 90 patients (30%). In terms of frequency of detection, A. fumigatus was followed by A. terreus, which was found in 70 patients (23%), A. flavus, which was found in 65 patients (22%), and A. niger, which was found in 30 patients (10%); other Aspergillus species were detected in 45 patients (15%). We identified 32 patients with IA caused by A. terreus and 33 patients with IA caused by A. fumigatus. Both groups were comparable in terms of age, gender, and underlying disease (Table 1). Leukemia, the most common underlying disease, was observed in 84% of all patients with IA caused by A. terreus and in 67% of all patients with IA caused by A. fumigatus. Only three patients had solid tumors. More than 40% of patients in each groups had undergone BMT within the preceding 24 months. There was a trend toward a higher incidence of neutropenia at the onset of infection in the A. terreus group (P 0.12), and especially in the subset of patients in that group who had absolute neutrophil counts (ANCs) 100 neutrophils per mm 3 (P 0.06; trend approaches significance). However, the duration of neutropenia was similar in both groups (A. terreus: median, 28 days [range, days]; A. fumigatus: median, 25 days [range, 5 76 days]; P 0.3). Adrenal corticosteroid use was similar in both groups. IA caused by A. terreus was nosocomial in origin more frequently than was IA caused by A. fumigatus (P 0.03). Overall rates of response to therapy were 39% (n

4 Aspergillus and Hematologic Malignancies/Hachem et al TABLE 2 Response to Antifungal Therapy in Patients with IA due to Aspergillus fumigatus or Aspergillus terreus Aspergillus fumigatus (n 33) Aspergillus terreus (n 32) Treatment Mean daily dose No. of patients No. of patients P value b ABLC 5 mg/kg 12 3 (25) 16 3 (19) 0.99 LAMB 5 mg/kg 11 4 (36) 11 2 (18) 0.64 ABCD 5 mg/kg 2 0 (0) 2 0 (0) Amphotericin B 1 mg/kg 3 1 (33) 2 0 (0) Itraconazole 400 mg 5 1 (20) 1 0 (0) Posaconazole 800 mg 8 a 4 (50) 9 a 4 (44) 0.99 IA: invasive aspergillosis; ABLC: amphotericin B lipid complex; LAMB: liposomal amphotericin B; ABCD: amphotericin B colloidal dispersion. a Patients experienced failure after receiving a lipid formulation of amphotericin B and were subsequently treated with posaconazole. b Two-sample t test. TABLE 3 Response to Therapy According to Neutropenia Status in Patients with IA Caused by Aspergillus fumigatus or Aspergillus terreus Aspergillus fumigatus (n 33) Aspergillus terreus (n 32) No. of patients No. of patients P value a All treated patients (39) 32 9 (28) 0.43 No neutropenia a 15 8 (53) 8 4 (50) 0.99 Persistent neutropenia 10 1 (10) 15 1 (6) 0.99 Neutropenia resolved 8 4 (50) 9 4 (44) 0.99 IA: invasive aspergillosis. a Fisher exact test. 13) among patients with IA caused by A. fumigatus and 28% (n 9) among patients with IA caused by A. terreus. Twenty-eight patients with IA caused by A. fumigatus and 31 patients with IA caused by A. terreus received an AMB preparation as primary therapy; the subsequent response rates were 29% and 16%, respectively. The study cohort was not large enough to allow the identification of significant differences in efficacy among the various AMB preparations. Only one of six patients treated with itraconazole experienced a response to treatment. Seventeen patients were enrolled in a limited-access protocol, sponsored by Schering-Plough (Kenilworth, NJ) and approved by the MDACC Institutional Review Board, that involved salvage therapy with posaconazole. Eight patients with IA caused by A. fumigatus infection and 9 patients with IA caused by A. terreus infection received salvage therapy with posaconazole for a median of 14 days (range, 7 30 days) after failing to experience a response to primary treatment with an AMB preparation. It is noteworthy that 4 of the 8 (50%) patients with IA caused by A. fumigatus infection and 4 of the 9 (44%) patients with IA caused by A. terreus infection had responses to posaconazole therapy (Table 2). Recovery from neutropenia had a significant effect on outcome for patients with IA, regardless of the infecting Aspergillus species or the type of antifungal therapy used (Table 3). Only 2 (8%) of 25 patients with persistent neutropenia had responses to treatment. However, 8 patients (47%) who recovered from neutropenia during treatment achieved a response. A similar proportion (52%) of the 23 patients without neutropenia survived. The difference in response rates between those who had persistent neutropenia (8%) and those who recovered from or never had neutropenia (50%) was statistically significant (P 0.004). Data on the antifungal activity of AMB, itraconazole, and posaconazole against 30 isolates of IA are shown in Table 4. Posaconazole was highly active in vitro against both A. fumigatus and A. terreus, with MIC 90 values of 0.12 g/ml and 0.06 g/ml, respectively, whereas AMB was active against A. fumigatus (MIC 90, 1 g/ml) and inactive against A. terreus (MIC 90,4 g/ml).

5 1598 CANCER October 1, 2004 / Volume 101 / Number 7 TABLE 4 In Vitro Susceptibility Data on 26 Clinical Isolates of Aspergillus fumigatus or Aspergillus terreus MIC ( g/ml) Range 50% 90% Aspergillus fumigatus a (16) Amphotericin B Itraconazole Posaconazole Aspergillus terreus a (14) Amphotericin B Itraconazole Posaconazole MIC: minimal inhibitory concentration. a Number of isolates is shown in parentheses. DISCUSSION The most common factors predisposing patients to IA were prolonged neutropenia and the receipt of highdose steroid treatment. Not surprisingly, in the current study, most patients who had IA caused by A. fumigatus (94%) or A. terreus (97%) had hematologic malignancies. In addition, the majority of patients who underwent BMT had GVHD during their infectious episodes with A. terreus (92%) or A. fumigatus (87%). There was no significant difference in underlying disease type between the two patient groups, although leukemia was somewhat more common among patients infected with A. terreus (Table 1). The association of A. terreus infection with leukemia has been described by others. 8,19 21 Neutropenia is known to be a major risk factor for the development of IA. 6,22,23 IA caused by A. terreus appears to be associated with neutropenia more frequently than does IA caused by A. fumigatus. Seventyfive percent of patients had neutropenia at the onset of A. terreus infection, compared with 55% of patients at the onset of A. fumigatus infection; however, this trend was not statistically significant (P 0.12), possibly due to the limited number of patients investigated. Nonetheless, more patients in the A. terreus group had ANCs 100 neutrophils per mm 3 (P 0.06). It is well known that Aspergillus infection is rare in patients with solid tumors. In the current study, the three patients with solid tumors who developed IA had been receiving high-dose adrenal corticosteroid therapy, which undoubtedly played a role in their infections. Schaffner 24 stressed that steroids impair the anticonidial activity of macrophages, rendering them unable to kill or inhibit conidium germination. IA is most often caused by A. fumigatus or A. flavus. Recovery from neutropenia in patients with leukemia and reduction of the intensity of immunosuppressive therapy in BMT recipients are key factors influencing the response of IA to antifungal therapy. 6,22,23 In the current study, we found that patients with IA and persistent neutropenia had the poorest rate of response to therapy (8% [2 of 25], compared with 47% for patients who had recovered from neutropenia). The reason for the high frequency of A. terreus infection at our institution is unclear, although a greater proportion of these infections were nosocomial, as has been reported by Iwen et al. 7 Efforts to identify a source within the hospital have been unsuccessful, although epidemiologic and geographic factors may have played a role. Baddley et al., 25 whose study was conducted in a similar geographic location to ours (University of Alabama Birmingham, Birmingham, AL), reported a significant increase in A. terreus infection relative to infections involving other Aspergillus species. In addition, Lass-Florl et al. 11 reported an association between A. terreus infection in patients with hematologic malignancies and the presence of potted plants in the hospital environment. Poor outcomes following polyene therapy for patients with IA caused by A. terreus have been reported previously. 6,8,20 Using a murine model of disseminated aspergillosis, Dannaoui et al. 26 demonstrated that the A. terreus strain was highly resistant to AMB and that mortality rates and survival durations were identical for AMB-treated mice and untreated mice. Furthermore, in a rabbit model of pulmonary aspergillosis, Walsh et al. 27 recently demonstrated that A. terreus was resistant to AMB in vitro and in vivo, whereas antifungal triazoles, such as itraconazole and posaconazole, were active in vitro and in vivo against this species. In a clinical study conducted by Lass-Florl et al., 28 the susceptibility of Aspergillus species to AMB in vitro was found to be correlated with clinical treatment outcome. The findings of the current study may suggest similar trends. Only 5 of 31 patients (16%) who had A. terreus infections experienced responses to polyene therapy, compared with 8 of 28 patients (29%) who had A. fumigatus infections. Although not statistically significant, this finding may indicate that A. fumigatus is more sensitive to polyene therapy, with this increased responsiveness possibly being related to the species heightened susceptibility to AMB. This pattern of susceptibility agreed with in vitro data obtained in the current study (Table 4) as well as with in vitro susceptibility results reported by other centers. 14,15 The development of resistance

6 Aspergillus and Hematologic Malignancies/Hachem et al to polyene therapy by several pathogenic fungi, including A. terreus, has become increasingly apparent, especially in immunocompromised patients. There is growing awareness of drug-resistant fungal species, including A. terreus, 29,30 and novel antifungal agents and treatment strategies are required to combat the emergence of such species. The newly developed triazoles voriconazole and posaconazole have exhibited notable therapeutic efficacy against clinical isolates of A. terreus in a number of immunocompromised animal models. 27,31 In the current study, the results of salvage treatment with posaconazole were encouraging, with this novel azole compound showing equal efficacy against IA caused by A. fumigatus and IA caused by A. terreus. The increasing incidence of AMB-resistant A. terreus infection is troubling. Further studies may affirm posaconzole s status as an alternative treatment option for patients with IA caused by A. terreus. Despite the decreased susceptibility of A. terreus (relative to A. fumigatus) to AMB, both groups in the current, highly immunocompromised patient population had comparably poor outcomes following antifungal therapy. For patients with neutropenia, recovery from this hematologic toxicity is a critical factor influencing response to antifungal therapy. REFERENCES 1. Denning DW. Invasive aspergillosis in immunocompromised patients. Curr Opin Infect Dis. 1994;7: Kontoyiannis DP, Bodey GP. Invasive aspergillosis in 2002: an update. Eur J Clin Microbiol Infect Dis. 2002;21: Denning DW. Invasive apergillosis. Clin Infect Dis. 1998;26: Andriole VT. Infections with Aspergillus species. Clin Infect Dis. 1993;17 Suppl 2:S481 S Wald A, Leisenring W, van Burik JA, Bowden RA. 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