In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii.

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.

Paracoccidioidomycosis and AIDS: report of the first two Colombian cases.

The records of the first two Colombian patients with AIDS and paracoccidioidomycosis are presented. Both patients were males and had no known risk factors for HIV although in the past they had worked in the field where they could have been infected with the fungus. They exhibited the juvenile type of disease with multiple organ system involvement and symptoms of short duration. They were deeply immunodepressed as indicated by less than 100 CD4 T lymphocytes per mL; however, serologic tests revealed circulating anti-Paracoccidioides brasiliensis antibodies and in one patient the first diagnostic clue came from such tests. In one case, the mycosis preceded the AIDS diagnosis while in the other, both pathologies were discovered simultaneously. Antimycotic therapy with itraconazole was administered for over 10 months, with an initial dose of 200 mg/day followed by 100 mg/day; marked improvement of the mycotic signs and symptoms was soon noticed an there have been no signs of relapse. The patients improvement was also due to the combined retroviral treatment that was instituted. In spite of the rarity of the AIDS-paracoccidioidomycosis association, physicians practicing in endemic areas should consider the presence of the mycosis in immunosuppressed patients, since a prompt diagnosis and institution of combined antimycotic-anti-retroviral treatments would result in patient improvement and survival. It appears possible that the longer survival time of today's AIDS patients would give the quiescent fungus the opportunity to revive, multiply and cause overt disease.

Maxillary sinusitis caused by Schizophyllum commune and experience with treatment.

A case of sinusitis caused by the basidiomycete Schizophyllum commune is reported in a 36-year-old female with a history of allergic rhinitis and dermatitis. The patient presented with sudden nasal obstruction, purulent nasal discharge, headache and general discomfort. Computer tomography revealed extensive opacity of the left maxillary sinus as well as erosion of the nasal wall and maxillary bone. Mycological examinations of nasal discharges and material aspirated during anthrostomy showed hyaline, septate hyphae with rare spicules. Primary isolation yielded a white, woolly mould which demonstrated clamp connections and basidiocarp primordia but these characteristics were lost in subculture. Identification was confirmed by vegetative compatibility studies. The patient was treated with itraconazole to avoid possible postsurgical dissemination. Three months after cessation of therapy, no recurrence of infection had occurred.

In vitro susceptibility testing of Fonsecaea pedrosoi to antifungals.

Based on the difficulties experienced in the treatment of chromoblastomycosis, 12 primary human isolates of F. pedrosoi, were tested for their in vitro susceptibility to various antimycotics. We adapted the recommendations of the NCCLS for yeasts and followed the indications for mold testing from other authors in order to determine their MIC's and the MLC's. It was found that a significant proportion of the isolates were resistant to 3 of the 4 antimycotics tested, as revealed by high MIC values, as follows: 33% were resistant to amphotericin B (AMB), 58.3% to 5 fluocytosine (5 FC) and 66.7% to fluconazole (FLU). Contrarywise, none of the isolates proved resistant to itraconazole (ITZ). Determination of the MLC's revealed that a larger proportion of the isolates were not killed by AMB, 5 FC (91.7%), FLU (100%) or even, ITZ (41.7%). These data indicate that it would be desirable to determine the susceptibility of F. pedrosoi before initiating therapy, in order to choose the more effective antifungal and avoid clinical failure.

Treatment of chromoblastomycosis with itraconazole.

The results of long-term itraconazole therapy in 10 patients with active chromoblastomycosis due to F. pedrosoi were reported. Therapy consisted of 100 or 200 mg/day of itraconazole, the length of therapy depending on the patient's response (12 to 24 months). This new triazole proved effective in reducing the number, size, and severity of the lesions in nine of the patients. Those patients with minor involvement profited more from therapy and were cured; patients with moderate involvement achieved either minor or major improvement. In most cases, signs and symptoms began to improve after 6 months of therapy. Mycological tests (in which tissue samples were treated with potassium hydroxide and cultured) became negative in six patients, but the fungus was eradicated in only three patients. Itraconazole produced no side effects. In spite of the need for long-term therapy, this new azole derivative effectively controls the disease.

Water as a substrate for the development of Paracoccidiodes brasiliensis mycelial form.

Two isolates of P. brasiliensis in the mycelial form were studied for their capacity to survive and grow in sterile distilled water (SDW). Inoculum for the experiments consisted of a spectrophotometrically-standardized suspension of washed and homogenized mycelial fragments; these had been obtained from 2-week old cultures grown in a synthetic medium (SM). Series of tubes with SDW and SM were incubated with the above suspension and kept stationary for 6 months at either 4 degrees C or room temperature (RT). Growth was measured by dry weight (DW) and turbidity (OD) determinations; additionally, CFU and ultrastructural appearance by transmission electron microscope (TEM) were assessed for one of the isolates. In general, cultures in SM at RT, grew exponentially after 2 weeks, becoming stationary for 7 weeks and then, declining abruptly. In SDW, fungal development was slow for 5 months when an increase in mass was recorded. When incubated at 4 degrees C, both SDW and SM cultures required longer time to develop but mass also increased. Morphologically, mycelial elements in SDW at RT exhibited increased lipid vacuoles and glycogen deposits but were otherwise normal up to 6 weeks when they presented the inter-hyphae-hyphae phenomenum. In SDW P. brasiliensis appears to utilize debris from its degenerated fungal partners to continue growing.

Murine model of paracoccidioidomycosis. Production of fatal acute pulmonary or chronic pulmonary and disseminated disease: immunological and pathological observations.

A model of fatal acute pulmonary or chronic pulmonary and disseminated paracoccidioidomycosis was developed by intranasal challenge of young (3-4-week-old) mice with high doses (2.5-10 X 10(7) units) or low doses (0.1-1 X 10(7)) of yeast-phase Paracoccidioides brasiliensis. Fatal acute paracoccidioidomycosis was dose dependent; 10, 5, and 2.5 X 10(7) viable units of P. brasiliensis produced 100%, 66%, and 17% mortality, respectively, within 11 days. The pathologic picture was that of consolidation with a neutrophil infiltrate. Infection of adult (7-8-week-old) mice with even 10 X 10(7) units did not produce acute fatal paracoccidioidomycosis. The model of fatal acute paracoccidioidomycosis lends itself well to studies of therapeutic intervention. Increasing degrees of chronic pulmonary-disseminated paracoccidioidomycosis were produced by pulmonary infection of young mice with 0.1-1 X 10(7) viable units of P. brasiliensis. Although mice in all groups appeared healthy, pathology in lungs, liver, and spleen was evident at 7 and 10 weeks postinfection. Histopathologic observation revealed acinonodular granulomatous inflammation. At 12 weeks postinfection, there was evidence of less pathology, and of clearing of abscesses. In contrast to the pathology produced by 0.1 X 10(7) P. brasiliensis in young mice, no pathology evident on gross examination was produced by a similar dose in adult mice. Immunological evaluations of mice with chronic pulmonary-disseminated disease showed that spleen cells, but not lymph node cells, had significantly depressed blastogenic responses to concanavalin A (ConA) early after infection. However, at 12 weeks postinfection, when pathological examination indicated beginning resolution of infection, spleen cell responses to ConA were normal. The model of chronic disease is of interest for further immunological studies as well.

Comparison of various techniques for determining viability of Paracoccidioides brasiliensis yeast-form cells.

The viability of Paracoccidioides brasiliensis yeast-form cells was determined by colony-forming units, direct fluorescent staining, and production of germ tubes in slide culture. The first procedure was unreliable and time consuming; the latter two showed better correlation with hemacytometer total cell counts and required significantly less time.

Recovery of Paracoccidioides brasiliensis from a partially calcified lymph node lesion by microaerophilic incubation of liquid media.

Surgical specimens were obtained from a patient who had had life-threatening paracoccidioidomycosis 5 years previously. Residual lesions were found in the mesenteric lymph nodes; there were areas of caseous necrosis separated from the surrounding tissue by a fibrous capsule made of connective tissue. Abundant P. brasiliensis cells, many of which appeared degenerated, were observed in the necrotic material. Primary isolation was possible only under the microaerophilic conditions offered by fluid media kept stationary at 36 degrees C. Cultures in solid media were obtained after serial passages and gradual accommodation of the strain to aerobic incubation. It is apparent that P. brasiliensis yeast cells can become accustomed to reduced oxygen tension in the tissues. In this way, they are able to withstand the passing of time without completely losing their viability.

Survival of Paracoccidioides brasiliensis yeast cells under microaerophilic conditions.

The ability of P. brasiliensis yeast cells to withstand microaerophilic conditions was investigated in a liquid medium distributed in tall columns in screw-capped tubes. Young cells of three isolates were inoculated on top of the medium, and the tubes were incubated aerobically and anaerobically at 36 degrees C for 28 days. The viability of cells that had sedimented to the bottoms of the tubes was studied by fluorescent microscopy and by their capacity to resume growth when transferred to fresh medium under continuous agitation. The proportion of viable cells in the sediments diminished with time of incubation. However, after 28 days, 27% of the cells were still viable and fully capable of active growth when placed under adequate aeration. On the other hand, drastic reduction of oxygen access elicited an accelerated death rate, with no survival after 7 days of incubation.