CASE REPORT

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A 26-year-old Syrian male who was known to have type I (insulin-dependent) diabetes mellitus was admitted to Mubarak Al-Kabeer Hospital in Kuwait in November 1996. He complained of general fatigue, tiredness, a productive cough, especially at night, and numbness in his fingertips and toes. He had lost 20 kg of weight during the previous year. The patient gave a history of pulmonary tuberculosis in his country of origin 4 years earlier, for which he received treatment for 6 months. On physical examination, the patient was conscious and fully alert. His temperature was 36.9°C, his pulse was 80 beats/min, and his blood pressure was 120/80 mmHg. He had no lymphadenopathy. There was clubbing of his fingers and toes. Examination of the chest revealed the presence of a pleural rub and anterior and posterior bilateral basal crepitations. Examination of the heart and abdomen revealed no abnormality. Central nervous system examination showed evidence of polyneuropathy of the gloves-and-stocking pattern. Nerve conduction studies of both the hands and feet showed the presence of moderately severe axonal peripheral neuropathy secondary to diabetes. Laboratory investigations showed markedly raised blood sugar levels (21 mmol/liter) with normal renal function. His white blood cell count was 10.8 × 10/liter with 61% neutrophils. Examination of the urine showed high levels of glucose (56 mmol/liter), but no protein or red blood cells were observed. A chest X ray and a computed tomography (CT) scan showed the presence of a "fungal ball" in the right and left apical regions (Fig. 1 and 2). It also showed in the upper zones the presence of fibronodular infiltrations, which were probably due to the old tubercular infection. Pulmonary function tests showed an obstructive pattern which improved with bronchodilators and a decreased total lung capacity which suggested a restrictive pattern. Microscopic examination of bronchoalveolar lavage specimens showed many pus cells, a number of fungal elements, gram-positive cocci, and gram-negative bacilli. A potassium hydroxide-calcofluor mount confirmed the presence of septate, dichotomously branched hyphae (Fig. 3). Culture yielded Haemophilus influenzae, beta-hemolytic streptococci, and Streptococcus pneumoniae and many colonies of a mold. Repeat culture of sputum specimens yielded the same fungus. Since the conidial heads were not observed on the initial examination of the fungal isolate, it was provisionally identified as a Scedosporium species due to the presence of thick-walled, single-celled conidia that arose directly from the hyphae or that developed at the tip of the conidiogenous cells and that were mistaken for annelloconidia. The isolate was later confirmed to be an atypical strain of Aspergillus terreus. The patient's serum was tested for precipitins against Aspergillus (poly) antigen (Meridian Diagnostics, Inc., Cincinnati, Ohio) and against culture filtrate antigens of Aspergillus fumigatus and Aspergillus flavus prepared in our laboratory (11), but it was found to be negative. However, when culture filtrate and mycelial antigens prepared from the patient's isolate were used, 3 and 1 precipitin lines, respectively, were obtained in an immunodiffusion test (Fig. 4). The culture filtrate antigen was prepared by growing the isolate in glucose asparagine broth as a stationary culture at 28°C for 3 weeks (11). The filtrate was dialyzed and concentrated 15 times by evaporation. The mycelial antigen was prepared by grinding 10 g of the separated mycelial mat in 20 ml of phosphate-buffered saline (0.05 M; pH 7.4) with glass beads in a pestle and mortar. The slurry so obtained was centrifuged at 15,000 × g for 15 min. The supernatant was filtered through a Nalgene filter unit (pore size, 0.45 µm), dialyzed, and concentrated about 10 times for use as an antigen. Since the patient had a chest infection, he was treated with ampicillin at 1 g every 6 h and cefotaxime at 1 g every 8 h. His diabetes was controlled with higher doses of insulin until a satisfactory level of blood sugar was achieved. He was discharged after 1 week of antibacterial therapy. No surgical intervention or specific antifungal therapy was planned since he had no hemoptysis or other aspergilloma-associated symptoms. The patient has since been lost for follow-up.

View larger version (126K): [in this window] [in a new window]   FIG. 1.   Posterioanterior chest radiograph showing cavitary lesions in both the lung fields with aspergilloma in the right upper zone (arrow).

View larger version (133K): [in this window] [in a new window]   FIG. 2.   Aspergilloma shown by CT scan in a cavity of the left lung (arrow).

View larger version (110K): [in this window] [in a new window]   FIG. 3.   Potassium hydroxide-calcofluor mount showing septate, dichotomously branched hyphae of A. terreus in bronchoalveolar lavage sediment. Magnification, ×400.

View larger version (118K): [in this window] [in a new window]   FIG. 4.   Ouchterlony's double diffusion test. The central well (G) contained the patient's serum, and peripheral wells contained culture filtrate (A) and mycelial antigens (B, C, and D) prepared from the A. terreus isolate. Peripheral wells contained culture filtrate antigens of A. fumigatus and A. flavus (E and F, respectively). Note the precipitin bands against A. terreus antigens.

Aspergillus species are widely distributed in the environment, and infection is acquired primarily through the respiratory tract (19). Although over 200 different species of Aspergillus are known, only a few are recognized human pathogens (3). A. fumigatus and A. flavus are the most frequent etiologic agents of pulmonary and systemic aspergillosis, whereas infection with A. terreus is rarely reported (3). So far, A. terreus has been incriminated as a causative agent in allergic bronchopulmonary aspergillosis (12, 13), invasive aspergillosis (4, 9, 10, 30), cardiac infections (6, 22, 23), osteomyelitis (25), and septic arthritis (27). Cases of pulmonary aspergilloma due to A. terreus have rarely been reported (12). Here, we describe a case of bilateral pulmonary aspergilloma in a 26-year-old diabetic patient caused by an atypical strain of A. terreus.

Description of isolate. The isolate was moderately fast growing on Sabouraud dextrose agar (Difco Laboratory, Detroit, Mich.), attaining a diameter of 60 mm in 10 days at 28°C with entire margins. The color was variable in shades of yellow but became salmon to cinnamon colored on aging and produced diffusible pigment. The isolate was able to grow on Mycobiotic agar (containing chloramphenicol and cycloheximide; Difco Laboratories) at 37°C reaching a diameter of 50 mm in 7 days (Fig. 5). Microscopically, hyphae were septate and hyaline, producing smooth, thick-walled, spherical to pear-shaped accessory conidia (formerly referred to as aleurioconidia), which mostly occurred singly and which measured 3 to 5 µm in diameter (Fig. 6). The isolate failed to produce fruiting structures on Sabouraud dextrose agar, Czapek agar, and malt yeast extract agar. The isolate was forwarded to the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center (UTHSC) at San Antonio, for further characterization and was accessioned into the stock collection as strain UTHSC 98-403. There the isolate was subcultured onto potato flake agar (PFA) prepared in-house and was incubated at 25°C (20). The isolate grew rapidly, producing deep orange colonies that had a lemon yellow diffusing pigment and that were similar to those produced on Sabouraud dextrose agar. A slide culture prepared on PFA produced the thick-walled globose cells but failed to produce diagnostic fruiting structures. After 10 weeks of incubation, stereoscopic examination of the colonies revealed small, strongly columnar fruiting structures consistent with an Aspergillus species. The microscopic morphology of these structures, when examined by tape mounts, revealed globose vesicles up to 15 µm in diameter, a biseriate arrangement with crowded metulae and phialides, and small, globose conidia (1.5 to 2.0 µm in diameter), all features characteristic for A. terreus (Fig. 7). This atypical strain of A. terreus has been deposited with the American Type Culture Collection under accession no. 201901. The isolate appeared to be resistant to amphotericin B, ketoconazole, and fluconazole and to be susceptible to itraconazole and flucytosine when tested on buffered RPMI 1640 medium with Etest strips (AB Biodisk, Solna, Sweden), on the basis of the National Committee for Clinical Laboratory Standards interpretive criteria for yeast fungi (17).

View larger version (116K): [in this window] [in a new window]   FIG. 5.   Colony morphology of A. terreus isolate on Mycobiotic agar after 7 days of incubation at 37°C.

View larger version (96K): [in this window] [in a new window]   FIG. 6.   Slide culture of A. terreus isolate on Sabouraud dextrose agar showing globose, thick-walled accessory conidia (aleurioconidia), Magnification, ×1,000.

View larger version (113K): [in this window] [in a new window]   FIG. 7.   Conidial head of the atypical isolate of A. terreus produced on PFA after 10 weeks of incubation at 25°C.

Discussion. A. terreus is widespread in the environment and particularly in warm arable soils. Although ubiquitous and sometimes disregarded as a mere colonizer, a review of recent English-language medical literature (Medline search) suggests that invasive infections due to A. terreus are being reported with increasing frequency (10; D. M. Flynn, B. G. Williams, S. V. Hetherington, B. F. Williams, M. A. Giannini, and T. A. Pearson, Letter, Infect. Control. Hosp. Epidemiol. 14:363-365, 1993). Furthermore, infection with this species may be associated with higher rates of morbidity and mortality compared to those associated with infection with other species of the genus, despite amphotericin B therapy (10, 14). Colonization of preexisting lung cavities with A. terreus has rarely been reported (12), and the case that we describe appears to be only the second one reported in the English-language medical literature. This case also underscores the variability that may be seen in isolates of A. terreus. The designations for several species have been reduced to synonyms under the species as the organisms seem to represent only cultural variants. The case isolate, on the basis of its colonial morphology, appears to be similar to A. terreus var. boedijni, described by Thom and Raper (29) (and since reduced), due to its bright orange-brown colonies. It also resembles two currently accepted varieties, A. terreus var. africanus and A. terreus var. aureus, in that it displays small, sclerotial-like masses, as in A. terreus var. africanus, and areas of golden yellow hyphae and a much greater abundance of accessory conidia both in the vegetative mycelium and in the aerial mycelium, as in A. terreus var. aureus (18). Sterile variants of Aspergillus have occasionally been isolated from patients with chronic cases of aspergillosis; these variants sometimes appear highly atypical and are difficult to identify (15; L. Sigler, M. A. Viviani, U. Margini, R. Epis, V. Fregoni, A. Grancini, and A. Pastorini, Abstr. 13th Int. Soc. Hum. Anim. Mycol., p. 396, 1997). A sterile white mold which is able to grow at 45°C, especially if it is isolated from a respiratory specimen, may be suspected to be A. fumigatus because of its thermotolerant nature. However, to our knowledge sterile variants of A. terreus have not been reported previously. Application of the exoantigen test may be helpful in the identification of such atypical isolates (24).