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Coelomycetous fungi, those that produce their conidia (mitospores) within asexual fruiting structures known as conidiomata, are ubiquitous in nature, inhabiting the twigs, branches, and leaves of various host plants. Despite their prevalence, only a limited number of genera have been documented as agents of human disease, including Colletotrichum species, Coniothyrium fuckelii, Lasiodiplodia theobromae, Nattrassia mangiferae, Phoma species, Phomopsis species, Pleurophoma pleurospora, Pleurophomopsis lignicola, Pseudochaetosphaeronema larense, Pyrenochaeta species, and Sphaeropsis species, to name a few (1-4, 7, 10, 13, 15, 18, 22, 23; this study). Coelomycetes previously referred to as the Melanconiales or Sphaeropsidales produce conidiomata known either as acervuli (open and cup-shaped) or pycnidia (closed with ostioles), respectively, or structures sharing characteristics between the two. We report a pycnidial fungus, a Phomopsis species, that was isolated from the finger of a diabetic female undergoing chronic corticosteroid therapy for rheumatoid arthritis. The fungus was most likely introduced through frequent contact with plants and the soil. Salient features of this fungus will be reviewed and compared to those of the similar, more common coelomycetous taxon, Phoma.

Case report. At the time of clinical presentation, the patient was a 61-year-old African-American female with a history of diabetes and rheumatoid arthritis. She had been treated with 5 to 30 mg of prednisone per day and with weekly doses of methotrexate. She was also an urban gardener. In July or August 1996, she noted several small, painless, red lumps on the palmar aspect of the third and fourth fingers of her right hand. Most of these lumps resolved spontaneously after several weeks, but a lump on the distal palmar surface of the fourth finger of her right hand gradually became larger. She came to medical attention in December 1996 after this nodule had enlarged and had become quite erythematous and extremely painful. She was seen by her primary care physician, who detected a dime-sized pustular lesion. This abscess was incised and drained, and a large amount of purulent material was produced. Exploration of the wound revealed extension of the abscess down to the bone. Gram stain of this material revealed abundant polymorphonuclear neutrophils, moderate fungal hyphae, and no bacteria. Subsequent cultures grew abundant colonies of a mould that was initially identified as a Chrysosporium species. The patient was started empirically on 400 mg of fluconazole (FLU) daily. In addition to the clinical findings, osteomyelitis was confirmed radiographically by the presence of lytic bone changes on plain X-rays and abnormally increased radiotracer uptake in the distal phalanx on bone scans (Fig. 1). Despite 4 weeks of therapy, she had persistent erythema and a tender pea-sized nodule over the old surgical site. Treatment with FLU was discontinued, and the patient was begun on 200 mg of itraconazole (ITRA) per os twice a day. The isolate from the site was sent to the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio, under UTHSC accession no. 97-62, for identification and antifungal susceptibility testing. After 3 weeks of ITRA therapy, the patient experienced significant clinical improvement, with resolution of the painful nodule over the distal phalanx. She completed a 6-month course of ITRA without incident while continuing daily prednisone and weekly methotrexate therapy. There was no evidence of recurrence at 16 months of follow-up.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   X-ray revealing osteopenia of the right fourth distal phalanx (arrow).

Mycological studies. The culture, which was submitted to the Fungus Testing Laboratory on Sabouraud dextrose agar (Becton Dickinson Microbiology Systems, Cockeysville, Md.), was initially white and woolly; however, after being maintained at room temperature (25°C) for 4 weeks, slightly greenish areas developed. Subcultures onto potato flakes agar (PFA) (17), prepared in-house, were buff and sterile with a few black sclerotia after 4 weeks at 25°C. The prominent microscopic features of the culture on PFA were the chains of chlamydoconidia that formed throughout the culture. Temperature studies on PFA revealed good growth at 25 and 35°C but no growth at 42°C after 10 days of incubation. Subcultures onto Czapek Dox agar (Remel, Lenexa, Kans.) also remained sterile after 4 weeks. Subcultures onto carnation leaf agar (12) used for Fusarium identification produced dark brown to black pycnidia after 2 weeks of incubation at 25°C and are shown in Fig. 2 at 4 weeks. Mature pycnidia were teased from the carnation leaves, placed in 10% formalin, sectioned, and stained with a fungal periodic acid-Schiff (PAS) stain. Stained sections revealed conidiomata that were ostiolate (had openings at the top), thick walled (Fig. 3a), immersed (Fig. 3b), multilocular (had more than one cavity) (Fig. 3a and c), and measured 101 to 106 µm long by 196 to 274 µm wide. Crushed conidiomata revealed short, ellipsoidal alpha conidia that were 2.0 to 2.5 µm long by 5.8 to 6.0 µm wide (Fig. 4) and long, filamentous beta conidia that were 0.4 to 0.5 µm long by 18 to 22 µm wide (Fig. 5). For comparison, a thin-walled pycnidium from a Phoma species is shown in Fig. 6. It is important to also note in Fig. 6 the alternarioid, intercalary chlamydoconidium, which is frequently associated with Phoma species.

View larger version (115K): [in this window] [in a new window]   FIG. 2.   Pycnidial formation on carnation leaf agar, 25°C, 4 weeks.

View larger version (84K): [in this window] [in a new window]   FIG. 3.   (a) Thick-walled, multilocular pycnidium. PAS stain. Magnification, ×230. (b) Thick-walled, immersed pycnidium with conidiogenous cells lining the cavity. PAS stain. Magnification, ×430. (c) Thick-walled, multilocular pycnidium. PAS stain. Magnification, ×360.

View larger version (154K): [in this window] [in a new window]   FIG. 4.   Alpha conidia and phialides of Phomopsis. Magnification, ×920.

View larger version (173K): [in this window] [in a new window]   FIG. 5.   Ellipsoidal alpha and hamate beta conidia of Phomopsis. Magnification, ×920.

View larger version (120K): [in this window] [in a new window]   FIG. 6.   Phoma species showing thin-walled pycnidia, as well as an alternarioid, intercalary chlamydoconidium (arrow). Magnification, ×370.

In vitro antifungal susceptibility testing. The case isolate was tested to determine its susceptibilities to antifungal agents. Tests were performed by previously described macrodilution methods (11). Briefly, the case isolate and the Paecilomyces control strain (UTHSC 90-459) were grown on PFA, which was prepared in-house, for 14 days at 25°C. Because no conidia were produced on PFA after 2 weeks of incubation, the inoculum was standardized spectrophotometrically. The PFA slant was flooded with sterile distilled H₂O, adjusted to 95% transmission at 530 nm, and then diluted 1:10 in medium to provide a final inoculum concentration of 1.0 × 10⁴. Final drug concentration ranges were as follows: for amphotericin B (AMB; E. R. Squibb & Sons, Princeton, N.J.), 0.03 to 16 µg/ml; for FLU (Pfizer, Inc., New York, N.Y.), 0.125 to 64 µg/ml; and for ITRA (Janssen Pharmaceutica, Titusville, N.J.), 0.015 to 8 µg/ml. AMB was tested in antibiotic medium 3 (Difco, Detroit, Mich.); other antifungal agents were tested in RPMI 1640 with L-glutamine and morpholinepropanesulfonic acid (MOPS) buffer at a concentration of 165 mM and without sodium bicarbonate (American Biorganics, Inc., Niagara Falls, N.Y.). Previously prepared, frozen drug tubes containing 0.1 ml of drug were allowed to thaw and were inoculated with 0.9 ml of the hyphal medium suspension. A drug-free growth control tube was included with the tubes containing the case isolate and control organism. The tubes were incubated at 35°C, and MICs were determined at the first 24-h interval when growth was observed in the drug-free growth control tube (24 and 48 h). MICs were defined in terms of the first tube that gave a score of 0 (optically clear) for AMB and a score of 2 (reduction in turbidity of 80% in contrast to that of the drug-free control tube) for FLU and ITRA. The results of in vitro susceptibility tests, based on drug concentrations normally achievable in patients receiving recommended dosages, indicated that the isolate appeared susceptible to AMB, FLU, and ITRA. These data are shown in Table 1.

Discussion. Coelomycetous fungi have been traditionally referred to as the Melanconiales (producing acervuli or open cup-shaped conidiomata) and the Sphaeropsidales (producing pycnidia or closed conidiomata with ostioles), although these distinctions are losing favor (20). These fungi are being identified with increasing frequency in the Fungus Testing Laboratory, particularly in immunosuppressed hosts. The most common presentation has been skin and soft tissue infection (1, 18, 23), although one report suggests their role in systemic disease (10). These fungi are presumably introduced through some type of traumatic implantation, and this appears to be the method of acquisition of the Phomopsis species in the present case. The patient was an urban gardener with frequent contact with plants and soil. Her history of chronic immunosuppression due to diabetes and treatment of rheumatoid arthritis with prednisone and methotrexate was a likely risk factor for opportunistic phaeohyphomycotic osteomyelitis.