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Elastase production in clinical isolates of Aspergillus
DIAGNMICROBIOLINFECTDIS 1988;10:165-170
165
MYCOLOGY
Elastase Production in Clinical Isolates of A s p e r g i l l u s I Judith C. Rhodes, Robert B. B o d e , 2 and Constance M. McCuan-Kirsch
Clinical isolates of Aspergillus species were tested, both retrospectively and prospectively, for elastase activity using rose bengal-elastin agar plates. Patient records were then reviewed to determine the clinical diagnosis including the presence or absence of aspergillosis. All isolates that caused invasive aspergillosis produced elastase, but not all isolates producing elastase were associated with invasive disease.
INTRODUCTION Aspergillosis, an infection caused by species of the ubiquitous fungus Aspergil/us, is an increasingly prevalent infectious complication of hospitalized patients. The incidence of aspergillosis has almost doubled between 1976 and 1980-1982, and the number of patients suffering from the underlying conditions that predispose towards infection, e.g., hematologic malignancies and other immunosuppressive disorders, has continued to increase (Fraser et al., 1979; Reingold et al., 1986). The increased incidence, coupled with a mortality rate approaching 90% in some series (Fisher et al., 1981), points to the urgent need to improve the speed and accuracy with which we can make the diagnosis of aspergillosis. The ubiquity of the aspergilli can make interpretation of positive cultures from surveillance sites difficult (Yu et al., 1986). The report by Kothary et al. (1984) correlating production of an enzyme with elastase activity by environmental isolates of A. fumigatus with virulence, i.e., production of a rapidly fatal pneumonia in mice, suggests one factor that may be important in the pathogenesis of aspergillosis. Therefore, we examined, both retrospectively and prospectively, elastase activity in Aspergillus species isolated from patients at University Hospital. We
1Presented, in part, at the 85th Annual Meeting of the American Society for Microbiology, Las Vegas, NV, March 3-7, 1985. 2Present address: 1913 Chester Blvd., Richmond, IN 47324 From the Department of Pathology and Laboratory Medicine (J.C.R., C.M.McC.-K.) and Department of Medicine (R.B.B.), University of Cincinnati Medical Center, Cincinnati, Ohio. Address reprint request s to: Dr. Judith C. Rhodes, Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0529. Received July 5, 1988; accepted August 29, 1988. © 1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/88/$3.50
166
J.C. Rhodes et al.
hoped not only to determine the distribution of elastase activity in clinical isolates of Aspergillus, but also to see if an organism's elastase phenotype might provide additional information to help in predicting the clinical significance of the isolate. MATERIALS AND METHODS Isolates
Primary isolates of Aspergillus were subcultured on Czapek solution agar for 2-7 days at 30°C prior to identification. Isolates were identified by their macroscopic appearance and microscopic morphology according to standard methods (Raper and Fennell, 1965). Prospectively, all Aspergillus species isolated between April and November 1984 were examined. In addition, six isolates from documented cases of aspergillosis in the University Hospital Mycology Laboratory stock culture collection were included. Elastase Production
All organisms were screened for elastase production on solid medium, the rose bengal-elastin agar plates described by Kothary et al. (1984). Organisms were classified as elastase positive if dissolution of elastin particles was seen within 7 days of incubation at 37°C. Efficacy of each new batch of medium was tested with appropriate positive and negative controls. All isolates listed in Table 2 and isolates 2, 4, 5, 9, and 11 in Table 1 were tested within 1 wk of isolation. The remaining isolates were retrieved from storage in distilled water prior to testing Patient Data
Patient charts and autopsy records, when available, were reviewed by one of us (R.B.B.) for the following: 1) immunocompromise, e.g. chemotherapy, steroids, malignancy, et al.; 2) neutropenia, less than 1,000 neutrophils per ram3; 3) fever; 4) antibiotic usage; 5) Aspergillus as a pathogen, i.e., organ system involved; and 6) therapy with amphotericin B. For this study, confirmed cases of aspergillosis were defined as patients with a tissue diagnosis or a positive culture or KOH of an abscess or other normally sterile body site. Those patients in which the primary contribution to their diagnosis could not be attributed with certainty to Aspergillus, and those in whom the isolation of Aspergillus appeared to represent contamination of specimen or colonization were combined in the category of colonization and contamination. RESULTS Of 38 isolates of Aspergillus examined, 25 produced elastase activity when they were tested on rose bengal-elastin agar plates. All 11 patients with confirmed cases of aspergillosis (Table 1) were infected with isolates of Aspergillus (three fumigatus, seven flavus, and one terreus) that produced elastase. Eight of the 11 patients had either leukemia or a solid tumor as their underlying illness. Based on our definition of confirmed cases, 27 patients were excluded because the isolation of Aspergillus could not be proven to represent anything more than colonization of the patient or contamination of the specimen. Thirteen of the isolates from those patients were negative for elastase production and 14 were positive (Table 2). Among the patients with documented infections, greater than 50% (6/11) and
Aspergillus Elastase in Clinical Isolates
167
TABLE 1. Elastase Activity of Isolates from Patients w i t h Confirmed Aspergillosis Patient
Organism
1 2
A. fumigatus A. flavus
3
Source
Elastase°
Diagnosis (confirmation)
Sputum
+
Sputum
+
A. flavus
Sputum
+
4
A. flavus
Sinus
+
5
A. flavus
Sinus
+
6
A. flavus
Lung
+
7
A. flavus
Lung
+
8
A. fumigatus
Lung
+
9
A. flavus
Abdominal abscess
+
10
A. fumigatus
Empyema
+
11
A. terreus
Peritoneal fluid
+
Lung cancer, empyema (+ biopsy) AML,b pulmonary infiltrates (+ KOH for septate hyphae) ALL,c pulmonary infiltrate (autopsy) Aspergillus invasive sinusitis ( + biopsy) AMLb with Aspergillus invasive sinusitis ( + biopsy) AML,b bone marrow transplant with disseminated aspergillosis (autopsy) AML,b pulmonary infiltrate (autopsy) Esophageal cancer, pulmonary infiltrate (autospy) AMLb with abdominal abscess (+ KOH for septate hyphae) Empyema with bronchopulmonary fistula (+ KOH for septate hyphae, + serology) Continuous ambulatory peritoneal dialysis for chronic renal failure.
°Elastase activity was tested on rose bengal-elastin agar. bAML,acute myelogenous leukemia. CALL,acute lymphocytic leukemia. profound neutropenia, w h i c h p l a y e d a p r o m i n e n t role in their invasive aspergillosis. Seven of the patients h a d unequivocal invasive disease, confirmed by b i o p s y or autopsy. In this group, there were four cases of invasive p u l m o n a r y aspergillosis (patients 1, 3, 7, and 8), two cases of invasive Aspergillus sinusitis (patients 4 and 5), and one case of d i s s e m i n a t e d aspergillosis (patient 6). Two patients had Aspergillus species as the sole isolates from closed b o d y spaces, e m p y e m a and aspirate from a subcutaneous abscess. One patient with acute myelogenous l e u k e m i a and p u l m o n a r y infiltrates, w h i c h i m p r o v e d on a m p h o t e r i c i n B, h a d septate h y p h a e in sputum. The final patient, who was undergoing chronic a m b u l a t o r y peritoneal dialysis, had A. terreus as the sole isolate from two cultures of peritoneal fluid. The patient received broad spectrum antibiotics for 6 days w i t h o u t improvement; antibiotics were discontinued, and the patient i m p r o v e d after institution of a m p h o t e r i c i n B.
None of the patients in Table 2 had significant n e u t r o p e n i a as a part of their clinical illnesses. Six of the patients had solid tumors (three p r i m a r y lung cancers, one renal cell carcinoma, and two metastatic a d e n o c a r c i n o m a s of u n k n o w n primary). Thirteen of the other patients had chronic p u l m o n a r y disease or asthma.
168
J.C. Rhodes et al.
TABLE 2. Elastase Activity of Aspergillus Isolates Representing Patient C o n t a m i n a t i o n or Colonization Organism
Elastasea
No.
A. fumigatus
+
6
A. fumigatus A. flavus
+
1 7
A. flavus A. versicolor
+ -
1 6 2 1 1 1 1
A. niger
A. candidus A. oryzae A. nidulans A.
Sources Wound, sputum, bronchial washings Bronchial washings Wound, sputum, bronchial washings, burn Sputum Sputum, bronchial washings Wound, sputum Sputum Sputum .Sputum Sputum
ochraceous °Elastase activity tested on rose bengal-elastin agar.
DISCUSSION Aspergillosis is an increasingly c o m m o n infection in i m m u n o c o m p r o m i s e d patients, a n d the case:fatality ratio is high due to difficulty in t i m e l y diagnosis and profound n e u t r o p e n i a (Fraser et al., 1979; Reingold et al., 1986; Rinaldi, 1983). Surveillance cultures have been a d v o c a t e d as a means to p r o v i d e early evidence of infection (Aisner et al., 1979). Unfortunately, the ubiquity of Aspergillus species can make positive cultures difficult to interpret; positive cultures for Aspergillus from nonsterile sites (sputum, nares, skin, etc.) m a y represent invasive disease, colonization, or contamination. In their review of respiratory isolates of Aspergillus, Yu et al. (1986) f o u n d that n e u t r o p e n i a was a significant predictor of invasive disease and a d v o c a t e d e m p i r i c a m p h o t e r i c i n B in n e u t r o p e n i c patients from w h o m A. fumigatus or A. flavus were isolated. N e u t r o p e n i a was certainly an i m p o r t a n t u n d e r l y i n g factor in the cases of aspergillosis in our patients, In a d d i t i o n to the described host factors that a p p e a r to be predictive of invasive disease, we w i s h e d to e x a m i n e w h e t h e r factors inherent in the organisms themselves might predict clinical significance. In previous studies, elastase activity has been detected in a n u m b e r of both pathogenic and n o n p a t h o g e n i c fungi, excluding Aspergillus, but an association w i t h virulence, although suggested, has not been s h o w n (Hopsu-Havu et al., 1979; Nziramasanga a n d Lupan, 1985; R i p p o n and Varadi, 1968). However, proteinase activity in Candida albicans and elastase activity in Pseudom o n a s aeruginosa have been d e m o n s t r a t e d to p l a y roles in the virulence of those organisms (Kwon-Chung et al., 1985; Wretlind and Pavlovskis, 1983). Elastase activity in e n v i r o n m e n t a l isolates of A. fumigotus is p o s i t i v e l y correlated w i t h mouse virulence, and the histopathologic picture of invasive aspergillosis, i.e., fungal penetration of b l o o d vessels and bronchi, is compatible w i t h the elaboration of an elastinolytic e n z y m e (Kothary et al., 1984; Rinaldi, 1983). In this study, a collection of clinical isolates of Aspergillus was tested for elastase activity, and patients' records were r e v i e w e d to determine the significance of the isolates. We reasoned that if elastase activity were an i m p o r t a n t virulence m e c h a n i s m in man, the elastase p h e n o t y p e of the organism m i g h t be helpful in p r e d i c t i n g its significance. Eleven of 38 isolates e x a m i n e d were involved in cases of aspergillosis; all 11 isolates p r o d u c e d elastase. However, of the 27 isolates not i n v o l v e d with clinical aspergillosis, 14 were positive for elastase activity in vitro. Therefore, all
Aspergillus Elastase in Clinical Isolates
169
isolates that caused aspergillosis p r o d u c e d elastase, but not all isolates p r o d u c i n g elastase were associated with invasive disease. Elastase p r o d u c t i o n was also e x a m i n e d on a species basis. Over 90% of the isolates of A. fumigatus and A. flavus, the most c o m m o n pathogens in our series and in others (Rinaldo, 1983; Yu et al., 1986), p r o d u c e d elastase activity. That the two elastase negative isolates of A. fumigatus and A. flavus were not associated w i t h invasive disease suggests that lack of elastase activity in these two species m a y be predictive. Isolates of A. versicolor, w h i c h is rarely i m p l i c a t e d in invasive aspergillosis and does not grow well at 37°C, were uniformly negative. The percentage of clinical isolates of A. fumigatus producing elastase was similar to the percentage obtained from environmental isolates, a situation analagous to that seen with elastase p r o d u c t i o n in P. aeruginosa (Nicas and Iglewski, 1986). Therefore, selection for elastase p r o d u c t i o n in the clinical setting does not a p p e a r to occur, but rather the clinical isolates represent the distribution of p h e n o t y p e in the environment. In conclusion, our results on elastase activity in clinical isolates of Aspergillus are compatible w i t h the concept that the enzyme may p l a y a role in the pathogenesis of aspergillosis. For that reason, we are continuing to screen clinical isolates of c o m m o n l y pathogenic species to increase our data base and to study the e n z y m e to d e t e r m i n e its p h y s i o c h e m i c a l properties. At this time, however, the utility of detecting elastase activity in isolates in an effort to predict their clinical significance m a y be limited to instances in w h i c h the c o m m o n l y pathogenic species do not d i s p l a y elastase activity. This research was supported in part by Public Health Service Grant AI20992 from the National Institute of Allergy and Infectious Diseases. We would like to thank M. D. Bruno and the staff of the University Hospital Mycology Laboratory for technical assistance, and Naomi Hayes for manuscript preparation.
REFERENCES Aisner J~ Murillo J, Schimpff SC, Steere AC (1979) Invasive aspergillosis in acute leukemia: correlation with nose cultures and antibiotic use. Ann Intern Med 90:4. Fisher BD, Armstrong D, Yu B, Gold JWM (1981) Invasive aspergillosis. Progress in early diagnosis and treatment. Am J Med 71:571. Fraser DW, Ward JI, Ajello L, Plikaytis BD (1979) Aspergillosis and other systemic mycoses. The growing problem. JAMA 242:1631. Hopsu-Havu VK, Sonck CE, Tunnela E (1979) Production of elastase by pathogenic and nonpathogenic fungi. Mykosen 15:105. Kothary MH, Chase T Jr, Macmillan JD (1984) Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect Immun 43:320. Kwon-Chung KJ, Lehman D, Good C, Magee PT (1985) Genetic evidence for role of extracellular proteinase in virulence of Candida albicans. Infect Immun 49:571. Nicas TI, Iglewski B (1986) Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeruginosa. J Clin Microbiol 23:967. Nziramasanga P, Lupan DM (1985) Elastase activity of Coccidioides immitis. J Med Microbiol 19:109. Raper KB, Fennell DI (1965) The Genus Aspergillus. Baltimore: The Williams and Wilkins Company. Reingold AL, Lu XD, Plikaytis BD, Ajello L (1986) Systemic mycoses in the United States, 1980-1982. J Med Vet Mycol 24:433. Rinaldi MG (1983) Invasive aspergillosis. Rev Infect Dis 5:1061. Rippon JW, Varadi DP (1968) The elastases of pathogenic fungi and actinomycetes. J Invest Dermatol 50:54.
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J . C . R h o d e s et al.
Wretlind B, Pavlovskis OR (1983) Pseudomonas aeruginosa elastase and its role in pseudomonas infection. Rev Infect Dis 5(Suppl. 5):998. Yu VL, Muder RR, Poorsattar TA (1986) Significance of isolation of Aspergillus from the respiratory tract in the diagnosis of invasive pulmonary aspergillosis. Results from a threeyear prospective study. Am J Med 81:249.
165
MYCOLOGY
Elastase Production in Clinical Isolates of A s p e r g i l l u s I Judith C. Rhodes, Robert B. B o d e , 2 and Constance M. McCuan-Kirsch
Clinical isolates of Aspergillus species were tested, both retrospectively and prospectively, for elastase activity using rose bengal-elastin agar plates. Patient records were then reviewed to determine the clinical diagnosis including the presence or absence of aspergillosis. All isolates that caused invasive aspergillosis produced elastase, but not all isolates producing elastase were associated with invasive disease.
INTRODUCTION Aspergillosis, an infection caused by species of the ubiquitous fungus Aspergil/us, is an increasingly prevalent infectious complication of hospitalized patients. The incidence of aspergillosis has almost doubled between 1976 and 1980-1982, and the number of patients suffering from the underlying conditions that predispose towards infection, e.g., hematologic malignancies and other immunosuppressive disorders, has continued to increase (Fraser et al., 1979; Reingold et al., 1986). The increased incidence, coupled with a mortality rate approaching 90% in some series (Fisher et al., 1981), points to the urgent need to improve the speed and accuracy with which we can make the diagnosis of aspergillosis. The ubiquity of the aspergilli can make interpretation of positive cultures from surveillance sites difficult (Yu et al., 1986). The report by Kothary et al. (1984) correlating production of an enzyme with elastase activity by environmental isolates of A. fumigatus with virulence, i.e., production of a rapidly fatal pneumonia in mice, suggests one factor that may be important in the pathogenesis of aspergillosis. Therefore, we examined, both retrospectively and prospectively, elastase activity in Aspergillus species isolated from patients at University Hospital. We
1Presented, in part, at the 85th Annual Meeting of the American Society for Microbiology, Las Vegas, NV, March 3-7, 1985. 2Present address: 1913 Chester Blvd., Richmond, IN 47324 From the Department of Pathology and Laboratory Medicine (J.C.R., C.M.McC.-K.) and Department of Medicine (R.B.B.), University of Cincinnati Medical Center, Cincinnati, Ohio. Address reprint request s to: Dr. Judith C. Rhodes, Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0529. Received July 5, 1988; accepted August 29, 1988. © 1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
0732-8893/88/$3.50
166
J.C. Rhodes et al.
hoped not only to determine the distribution of elastase activity in clinical isolates of Aspergillus, but also to see if an organism's elastase phenotype might provide additional information to help in predicting the clinical significance of the isolate. MATERIALS AND METHODS Isolates
Primary isolates of Aspergillus were subcultured on Czapek solution agar for 2-7 days at 30°C prior to identification. Isolates were identified by their macroscopic appearance and microscopic morphology according to standard methods (Raper and Fennell, 1965). Prospectively, all Aspergillus species isolated between April and November 1984 were examined. In addition, six isolates from documented cases of aspergillosis in the University Hospital Mycology Laboratory stock culture collection were included. Elastase Production
All organisms were screened for elastase production on solid medium, the rose bengal-elastin agar plates described by Kothary et al. (1984). Organisms were classified as elastase positive if dissolution of elastin particles was seen within 7 days of incubation at 37°C. Efficacy of each new batch of medium was tested with appropriate positive and negative controls. All isolates listed in Table 2 and isolates 2, 4, 5, 9, and 11 in Table 1 were tested within 1 wk of isolation. The remaining isolates were retrieved from storage in distilled water prior to testing Patient Data
Patient charts and autopsy records, when available, were reviewed by one of us (R.B.B.) for the following: 1) immunocompromise, e.g. chemotherapy, steroids, malignancy, et al.; 2) neutropenia, less than 1,000 neutrophils per ram3; 3) fever; 4) antibiotic usage; 5) Aspergillus as a pathogen, i.e., organ system involved; and 6) therapy with amphotericin B. For this study, confirmed cases of aspergillosis were defined as patients with a tissue diagnosis or a positive culture or KOH of an abscess or other normally sterile body site. Those patients in which the primary contribution to their diagnosis could not be attributed with certainty to Aspergillus, and those in whom the isolation of Aspergillus appeared to represent contamination of specimen or colonization were combined in the category of colonization and contamination. RESULTS Of 38 isolates of Aspergillus examined, 25 produced elastase activity when they were tested on rose bengal-elastin agar plates. All 11 patients with confirmed cases of aspergillosis (Table 1) were infected with isolates of Aspergillus (three fumigatus, seven flavus, and one terreus) that produced elastase. Eight of the 11 patients had either leukemia or a solid tumor as their underlying illness. Based on our definition of confirmed cases, 27 patients were excluded because the isolation of Aspergillus could not be proven to represent anything more than colonization of the patient or contamination of the specimen. Thirteen of the isolates from those patients were negative for elastase production and 14 were positive (Table 2). Among the patients with documented infections, greater than 50% (6/11) and
Aspergillus Elastase in Clinical Isolates
167
TABLE 1. Elastase Activity of Isolates from Patients w i t h Confirmed Aspergillosis Patient
Organism
1 2
A. fumigatus A. flavus
3
Source
Elastase°
Diagnosis (confirmation)
Sputum
+
Sputum
+
A. flavus
Sputum
+
4
A. flavus
Sinus
+
5
A. flavus
Sinus
+
6
A. flavus
Lung
+
7
A. flavus
Lung
+
8
A. fumigatus
Lung
+
9
A. flavus
Abdominal abscess
+
10
A. fumigatus
Empyema
+
11
A. terreus
Peritoneal fluid
+
Lung cancer, empyema (+ biopsy) AML,b pulmonary infiltrates (+ KOH for septate hyphae) ALL,c pulmonary infiltrate (autopsy) Aspergillus invasive sinusitis ( + biopsy) AMLb with Aspergillus invasive sinusitis ( + biopsy) AML,b bone marrow transplant with disseminated aspergillosis (autopsy) AML,b pulmonary infiltrate (autopsy) Esophageal cancer, pulmonary infiltrate (autospy) AMLb with abdominal abscess (+ KOH for septate hyphae) Empyema with bronchopulmonary fistula (+ KOH for septate hyphae, + serology) Continuous ambulatory peritoneal dialysis for chronic renal failure.
°Elastase activity was tested on rose bengal-elastin agar. bAML,acute myelogenous leukemia. CALL,acute lymphocytic leukemia. profound neutropenia, w h i c h p l a y e d a p r o m i n e n t role in their invasive aspergillosis. Seven of the patients h a d unequivocal invasive disease, confirmed by b i o p s y or autopsy. In this group, there were four cases of invasive p u l m o n a r y aspergillosis (patients 1, 3, 7, and 8), two cases of invasive Aspergillus sinusitis (patients 4 and 5), and one case of d i s s e m i n a t e d aspergillosis (patient 6). Two patients had Aspergillus species as the sole isolates from closed b o d y spaces, e m p y e m a and aspirate from a subcutaneous abscess. One patient with acute myelogenous l e u k e m i a and p u l m o n a r y infiltrates, w h i c h i m p r o v e d on a m p h o t e r i c i n B, h a d septate h y p h a e in sputum. The final patient, who was undergoing chronic a m b u l a t o r y peritoneal dialysis, had A. terreus as the sole isolate from two cultures of peritoneal fluid. The patient received broad spectrum antibiotics for 6 days w i t h o u t improvement; antibiotics were discontinued, and the patient i m p r o v e d after institution of a m p h o t e r i c i n B.
None of the patients in Table 2 had significant n e u t r o p e n i a as a part of their clinical illnesses. Six of the patients had solid tumors (three p r i m a r y lung cancers, one renal cell carcinoma, and two metastatic a d e n o c a r c i n o m a s of u n k n o w n primary). Thirteen of the other patients had chronic p u l m o n a r y disease or asthma.
168
J.C. Rhodes et al.
TABLE 2. Elastase Activity of Aspergillus Isolates Representing Patient C o n t a m i n a t i o n or Colonization Organism
Elastasea
No.
A. fumigatus
+
6
A. fumigatus A. flavus
+
1 7
A. flavus A. versicolor
+ -
1 6 2 1 1 1 1
A. niger
A. candidus A. oryzae A. nidulans A.
Sources Wound, sputum, bronchial washings Bronchial washings Wound, sputum, bronchial washings, burn Sputum Sputum, bronchial washings Wound, sputum Sputum Sputum .Sputum Sputum
ochraceous °Elastase activity tested on rose bengal-elastin agar.
DISCUSSION Aspergillosis is an increasingly c o m m o n infection in i m m u n o c o m p r o m i s e d patients, a n d the case:fatality ratio is high due to difficulty in t i m e l y diagnosis and profound n e u t r o p e n i a (Fraser et al., 1979; Reingold et al., 1986; Rinaldi, 1983). Surveillance cultures have been a d v o c a t e d as a means to p r o v i d e early evidence of infection (Aisner et al., 1979). Unfortunately, the ubiquity of Aspergillus species can make positive cultures difficult to interpret; positive cultures for Aspergillus from nonsterile sites (sputum, nares, skin, etc.) m a y represent invasive disease, colonization, or contamination. In their review of respiratory isolates of Aspergillus, Yu et al. (1986) f o u n d that n e u t r o p e n i a was a significant predictor of invasive disease and a d v o c a t e d e m p i r i c a m p h o t e r i c i n B in n e u t r o p e n i c patients from w h o m A. fumigatus or A. flavus were isolated. N e u t r o p e n i a was certainly an i m p o r t a n t u n d e r l y i n g factor in the cases of aspergillosis in our patients, In a d d i t i o n to the described host factors that a p p e a r to be predictive of invasive disease, we w i s h e d to e x a m i n e w h e t h e r factors inherent in the organisms themselves might predict clinical significance. In previous studies, elastase activity has been detected in a n u m b e r of both pathogenic and n o n p a t h o g e n i c fungi, excluding Aspergillus, but an association w i t h virulence, although suggested, has not been s h o w n (Hopsu-Havu et al., 1979; Nziramasanga a n d Lupan, 1985; R i p p o n and Varadi, 1968). However, proteinase activity in Candida albicans and elastase activity in Pseudom o n a s aeruginosa have been d e m o n s t r a t e d to p l a y roles in the virulence of those organisms (Kwon-Chung et al., 1985; Wretlind and Pavlovskis, 1983). Elastase activity in e n v i r o n m e n t a l isolates of A. fumigotus is p o s i t i v e l y correlated w i t h mouse virulence, and the histopathologic picture of invasive aspergillosis, i.e., fungal penetration of b l o o d vessels and bronchi, is compatible w i t h the elaboration of an elastinolytic e n z y m e (Kothary et al., 1984; Rinaldi, 1983). In this study, a collection of clinical isolates of Aspergillus was tested for elastase activity, and patients' records were r e v i e w e d to determine the significance of the isolates. We reasoned that if elastase activity were an i m p o r t a n t virulence m e c h a n i s m in man, the elastase p h e n o t y p e of the organism m i g h t be helpful in p r e d i c t i n g its significance. Eleven of 38 isolates e x a m i n e d were involved in cases of aspergillosis; all 11 isolates p r o d u c e d elastase. However, of the 27 isolates not i n v o l v e d with clinical aspergillosis, 14 were positive for elastase activity in vitro. Therefore, all
Aspergillus Elastase in Clinical Isolates
169
isolates that caused aspergillosis p r o d u c e d elastase, but not all isolates p r o d u c i n g elastase were associated with invasive disease. Elastase p r o d u c t i o n was also e x a m i n e d on a species basis. Over 90% of the isolates of A. fumigatus and A. flavus, the most c o m m o n pathogens in our series and in others (Rinaldo, 1983; Yu et al., 1986), p r o d u c e d elastase activity. That the two elastase negative isolates of A. fumigatus and A. flavus were not associated w i t h invasive disease suggests that lack of elastase activity in these two species m a y be predictive. Isolates of A. versicolor, w h i c h is rarely i m p l i c a t e d in invasive aspergillosis and does not grow well at 37°C, were uniformly negative. The percentage of clinical isolates of A. fumigatus producing elastase was similar to the percentage obtained from environmental isolates, a situation analagous to that seen with elastase p r o d u c t i o n in P. aeruginosa (Nicas and Iglewski, 1986). Therefore, selection for elastase p r o d u c t i o n in the clinical setting does not a p p e a r to occur, but rather the clinical isolates represent the distribution of p h e n o t y p e in the environment. In conclusion, our results on elastase activity in clinical isolates of Aspergillus are compatible w i t h the concept that the enzyme may p l a y a role in the pathogenesis of aspergillosis. For that reason, we are continuing to screen clinical isolates of c o m m o n l y pathogenic species to increase our data base and to study the e n z y m e to d e t e r m i n e its p h y s i o c h e m i c a l properties. At this time, however, the utility of detecting elastase activity in isolates in an effort to predict their clinical significance m a y be limited to instances in w h i c h the c o m m o n l y pathogenic species do not d i s p l a y elastase activity. This research was supported in part by Public Health Service Grant AI20992 from the National Institute of Allergy and Infectious Diseases. We would like to thank M. D. Bruno and the staff of the University Hospital Mycology Laboratory for technical assistance, and Naomi Hayes for manuscript preparation.
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