JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1992, p. 3290-3293

Vol. 30, No. 12

0095-1137/92/123290-04$02.00/0 Copyright © 1992, American Society for Microbiology

Aspergillus quadrilineatus, a New Causative Agent of Fungal Sinusitis I. POLACHECK,' A. NAGLER,2 E. OKON,3 P. DRAKOS,2 J. PLASKOWITZ,4 AND K. J. KWON-CHUNGS* Departments of Clinical Microbiology,1 Bone Marrow Transplantation,2 and Pathology,3 Hadassah Medical Center, Jerusalem, Israel; Systematic Botany, Mycology and Nematology Laboratory, U.S. Department ofAgriculture, Beltsville, Maryland 207054; and Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 208925 Received 24 June 1992/Accepted 14 September 1992

AspergiUus qudrineatus was found to be the etiologic agent of pansinusitis in a patient suffering from acute nonlymphoblastic leukemia and who had undergone allogeneic bone marrow transplantation. A. quadrilineatus was cultured from biopsy specimens of the maxillary sinus, and tissue sections with fungal stains showed a necrotic area containing dichotomously branching septate hyphae, which is morphologically consistent with Aspergilus species. The patient was successfully treated with a combination of surgical debridement, granulocyte transfusions, and intravenous administration of amphotericin B-cholesterol sulfate colloidal dispersion. This is the first report of an infection caused by A. quadrilineatus.

Aspergilli are among the most ubiquitous fungi in the world. Since the genusAspergillus was established by Micheli in 1729, numerous species have been added to it. Raper and Fennell's monograph of the genus Aspergillus, published in 1965, recognized 132 species and 18 varieties (11). More than 35 species have been described since 1965, and, at present, the total number of species in the genus is close to 200 (7). Aspergillus fumigatus is the most common cause of both invasive and noninvasive aspergillosis worldwide. Aspergillus flavus is the second most common species isolated from immunosuppressed patients with invasive aspergillosis as well as from lesions originating in the nasal sinuses (7). Other, less common, pathogenic Aspeigillus species include A. niger, A. nidulans, and A. terreus. Invasive aspergillosis has become the second most prevalent mycosis in immunocompromised hosts, particularly in patients with leukemia or lymphoma, usually after a prolonged granulocytopenia, and especially in those having received corticosteroid or cytotoxic chemotherapy (1, 2, 6). A. quadrilineatus is reported here for the first time as an etiologic agent of aspergillosis in a human. The patient, who had undergone bone marrow transplantation in Israel, developed pansinusitis caused by this fungus and was successfully treated with a combination of surgical debridement, granulocyte transfusions, and systemic treatment with amphotericin B-cholesterol sulfate colloidal dispersion (ABCD). Report of a case. A 28-year-old female with acute nonlymphoblastic leukemia underwent allogeneic bone marrow transplantation. Transplant conditioning included fractionated total body irradiation followed by cyclophosphamide, melphalan, and etoposide administration and total lymphoid irradiation. The posttransplantation period of neutropenia was remarkable only for fever, which was treated successfully with broad-spectrum antibiotics and empiric amphotericin B (Fungizone) for 10 days (1 mg/kg of body weight every other day, with a total dose of 0.25 g). The engraftment was normal, with a peripheral blood granulocyte count of 0.5 x *

Corresponding author. 3290

109/liter on day 17, a leukocyte (WBC) count of >1.0 x 109/liter on day 19, and a platelet count of 50 x 109/liter on day 22 after bone marrow transplantation. The patient was discharged on day 26 in good general condition. Two months later, she was readmitted because of fever (37.5°C) and mild erythema, with pain and tenderness under the right orbit. Her WBC count was 1.4 x 109/liter, with 95% lymphocytes. Computerized tomographic (CT)-Scan of the orbits and sinuses disclosed frontal, maxillary, and ethmoid sinusitis, mainly on the right side, with orbital involvement. The patient underwent diagnostic maxillary sinus drainage, from which cultures were sterile. Because of fever and absolute neutropenia, the patient was started on mezlocillin, gentamicin, and amphotericin B (1 mg/kg daily). Because of the low total WBC count and the possibility of slow rejection, the patient was also started on intravenous cyclosporine and granulocyte macrophage-colony stimulating factor. The symptoms worsened, and a new CT-Scan showed progression of the pansinusitis and of the right periorbital swelling. Extensive surgical debridement (right Caldwell-Luc procedure and right external ethmoidectomy) was performed. The sinus mucosa had thickened, and the sinus was filled with mucopurulent exudate. The lacrimal bone (posterior aspect of ethmoid sinus) was destroyed, and infection was observed to have penetrated into the orbit. A connection between the posterior ethmoid sinus and the right maxillary sinus was also observed. A frozen section of maxillary sinus mucosa revealed invasion by septate hyphae ranging from 3 to 5 ,um in diameter. Cultures of biopsy specimens taken from the maxillary sinus were positive forAspergllus organisms after 1 week of incubation at 30°C. Since the patient developed toxic side effects to amphotericin B which included fever, flushing, rash, and chills, ABCD (Liposome Technology, Inc., Menlo Park, Calif.) was administered at an initial dose of 0.5 mg/kg. Daily granulocyte transfusions were concomitantly given for 10 days. Amphotericin B was also administered locally daily via the draining catheter into the ethmoid and maxillary sinuses. The ABCD doses were increased gradually by 0.5 mg/kg every 3 days until a maximal dose of 2.5 mg/kg was reached. The concomitant administration of ABCD and granulocyte

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FIG. 1. Section of a biopsy specimen from the maxillary sinus showing fungal growth in the necrotic center (hematoxylin and eosin; magnification, x200) and dichotomously branching septate hyphae (inset) (periodic acid-Schiff stain; magnification, x740).

transfusion proceeded uneventfully. There were no toxic effects noted as a result of ABCD administration, and no premedication was needed. Empiric broad-spectrum antibiotic therapy was discontinued. Clinically, the patient improved gradually but steadily, with resolution of the fever and suborbital erythema, swelling, and tenderness. The results of ophthalmologic examination including fundoscopy 1 month later were normal. CT-Scan of the sinuses and orbit performed 3 months after initiation of ABCD therapy showed marked improvement of the pansinusitis. The patient has remained afebrile since then, with no recurrence of the pansinusitis. The WBC count rose to 2.4 x 109/liter, and the platelet count rose to 120 x 109/liter. The patient received a total of 6.5 g of ABCD over 3-1/2 months, with no side effects and no signs of organ toxicity. Laboratory studies. Biopsy specimens taken from the maxillary sinus were cultured on chocolate agar, modified Sabouraud dextrose agar, and brain heart infusion agar containing chloramphenicol (50 ,ug/ml) and gentamicin (5 p,g/ml). The cultures were incubated at 30 and 37°C. Cultures were positive within a week in all three media, yielding many colonies of a fungus which had morphological characteristics of the A. nidulans group. In addition, portions of these biopsy specimens were processed for histological study and stained with hematoxylin and eosin, periodic acid-Schiff stain, and Gomori's methenamine silver stain. The hematoxylin-and-eosin-stained sections showed dense growth of unstained hyphae in a necrotic center (Fig. 1). The hyphae stained with periodic acid-Schiff and Gomori's methenamine silver stains were 3 to 5 p.m in diameter, with septation and dichotomous branching (Fig. 1, inset), which is consistent with the morphology of Aspergillus hyphae in tissue. Mycology. The fungus belonging to the A. nidulans group recovered from the biopsy specimens was subcultured on Czapek's solution agar and 2% malt extract agar and incu-

bated at 25, 30, and 37°C for 10 days. The growth was faster on malt extract agar than on Czapek's agar at all three temperatures. The colony grew faster at 30 and 37°C than at 25°C (Fig. 2) on both media. The growth rate at 37°C was similar to that at 30°C. On Czapek's solution agar, colonies were plain, with fimbriated margins when grown at 25°C (2.5 cm in diameter) (Fig. 2A) and 30°C (7.5 cm in diameter) but with floccose centers at 37°C (8 cm in diameter) (Fig. 2C). The color of colonies grown at 25°C was olive green with white margins (Fig. 2A), while the color of colonies was grayish purple with olive green margins when colonies were grown at 30 or 37°C (Fig. 2C). The colony reverse was purple at all three temperatures. The colonies grown on malt extract agar at 25°C were thin and plain (5 cm in diameter), olive green, and with fimbriated margins (Fig. 2B). The colonies grown at 30 and 37°C filled the plates within 1 week. The morphology of conidial structures, including the markings of the conidial surfaces, (Fig. 3) was not significantly different from that of A. nidulans (11). Cleistothecia enveloped by hulle cells were light brown and were produced on both media, most abundantly on malt extract agar at 37°C (Fig. 2D). Ascospores were purple-red, lenticular, and smooth walled and measured 4.0 to 4.5 ,um by 3.5 to 4.0 ,um, with four narrow equatorial crests. Scanning electron microscopy showed two pleated crests (0.5 ,um) and a secondary pair which were reduced in width (Fig. 3). This characterization of ascospores allowed us to identify the fungus as A. quadrilineatus. A. quadrilineatus is a member of the A. nidulans group (11). Other members of the group which have been isolated from cases of human infection are A. nidulans (3, 5, 9, 10, 12), A. nidulans var. echinulatus (15), and A. unguis (13). These fungi have rarely been reported to be involved in cases of invasive systemic disease. The four

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well-documented previous systemic cases due to the species A. nidulans occurred in patients with childhood chronic granulomatous disease (3, 15) or in children whose case histories suggest strongly that they may have had childhood chronic granulomatous disease (5, 12). The infection in these cases was confined to the lung (3, 5, 12) or the lung and thoracic vertebral bodies (15) and responded well to antifungal therapy (3, 5, 15). A. nidulans has been reported to be involved in cases of mycetoma in patients from Sudan (9, 10). A. unguis was associated with infection in toenails (13). A. quadrilineatus (teleomorph, Emericella quadrilineata) is a soil fungus that seemingly occurs most commonly in dry, warm soils such as those collected from the southwestern United States (11) and Saudi Arabia (4). It is not surprising to find a case in Israel because of the similar soil and climate. The conidial structures of A. quadrilineatus are not significantly different from those of A. nidulans, although the length of conidiophores may tend to be slightly shorter in the former species. This characteristic, however, is unreliable for the differentiation of the two species, since the ranges of the conidiophore lengths for the species overlap. Furthermore, we have observed that the range of conidiophore length became longer in A. quadrilineatus after the species was repeatedly subcultured on malt extract agar. The only reliable characteristic for the separation of the two species is the number of equatorial crests on the ascospores (11). The ascospores ofA. nidulans have two pleated equatorial crests ranging from 0.5 to 1.3 pLm in width (11), while those of A.

quadrilineatus have four crests of 0.5 ,um or less in width. According to the monograph by Raper and Fennell, in certain isolates of A. quadnlineatus some ascospores have four very much reduced crests while others have no trace of such ornamentation (11). Our isolate was also shown to produce some ascospores with very much reduced crests, so that the crests-especially those of the second pair-were not clearly discemable under a compound microscope. Scanning electron microscopy was helpful in revealing all four crests. Scanning electron microscopy of the ascospores produced by other species of the A. nidulans group showed no trace of the second pair of crests (14). Invasive aspergillosis of paranasal sinuses may be acute or chronic (7). Acute infection occurs in severely immunosuppressed patients, and the infection extends rapidly to contiguous structures (1, 2, 7, 8), as was the case in our patient. Surgical debridement is an important adjunct for the treatment of aspergillosis of the paranasal sinus (7). This is the first report of successful treatment of invasive aspergillosis of the paranasal sinuses by the combination of amphotericin B, ABCD, surgery, and granulocyte transfusions. The success of the treatment may have also been associated with the etiologic agent. Infection due to the members of A. nidulans group may be more successfully treated than those cases due to A. fumigatus or A. flavus, the two most common agents of invasive aspergillosis. Species identification, therefore, may be important to assess the outcome of the treatment.

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FIG. 3. Scanning electron microscopy of ascospores showing four equatorial crests (two major and two minor). The arrow indicates the rugous conidia mixed in with the ascospores (+). Bar = 1 ,um. REFERENCES 1. Axeisson, H., B. Carloo, J. Weibnng, and B. Winblad. 1978. Aspergillosis of the maxillary sinus. Acta Oto-Laringol. 86:303308. 2. Bodey, G. P., and S. Vartivarian. 1989. Aspergillosis. Eur. J. Clin. Microbiol. 8:413-437. 3. Bujak, J. S., K. J. Kwon-Chung, and M. J. Chusid. 1974. Osteomyelitis and pneumonia in a boy with chronic granulomatous disease of childhood caused by a mutant strain of Aspergillus nidulans. Am. J. Clin. Pathol. 61:361-367. 4. Centraalbureau voor Schimmelcultures. 1987. List of cultures, 31st ed. Baarn-Delft, The Netherlands. 5. Goldschmied-Reouven, A., B. Weisselberg, and E. Herczeg. 1987. Invasive pulmonary aspergillosis in an apparently nonimmunocompromised child. Topics in Mycology, 2nd Symposium onAspergillus and Aspergillosis, p. 131. Janssen Research Foundation, Antwerp, Belgium. 6. Klimowski, L. L., C. Rostein, and K. M. Cummings. 1989. Incidence of nosocomial aspergillosis in patients with leukemia over a twenty-year period. Infect. Control Hosp. Epidemiol. 10:299-305.

7. Kwon-Chung, K. J., and J. E. Bennett. 1992. Medical mycology, p. 207-247. Lea & Febiger, Philadelphia. 8. Lowe, J., and J. Bradley. 1986. Cerebral and orbital Aspergillus infection due to invasive aspergillosis of ethmoid sinus. J. Clin. Pathol. 39:774-778. 9. Mahgoub, E. S. 1971. Maduromycosis caused by Aspergillus nidulans. J. Trop. Med. Hyg. 74:60-61. 10. Mahgoub, E. S. 1973. Mycetomas caused by Culvularia lunata, Madurella grisea, Aspergillus nidulans, and Nocardia brasiliensis in Sudan. Sabouraudia 11:179-182. 11. Raper, K. B., and D. I. Fennell. 1965. The genus Aspergillus, p. 686. The Williams & Wilkins Co., Baltimore. 12. Redmond, A., J. J. Carre, J. D. Biggart, and D. W. R. Mackenzie. 1965. Aspergillosis (Aspergillus nidulans) involving bone. J. Pathol. Bacteriol. 29:391-395. 13. Schonborn, C., and J. Jahn. 1970. Untersuchungen uber Schim-

melpilzinfektionen der Zehennagel. Mykosen 13:253-272. 14. Udagawa, S. E., and Y. Hone. 1976. A new species of Emericella. Mycotaxon 4:535-539. 15. White, C. J., K. J. Kwon-Chung, and J. I. Gallin. 1988. Chronic granulomatous disease of childhood. Am. J. Clin. Pathol. 90: 312-316.

Aspergillus quadrilineatus, a new causative agent of fungal sinusitis.

Aspergillus quadrilineatus was found to be the etiologic agent of pansinusitis in a patient suffering from acute nonlymphoblastic leukemia and who had...
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