Experimental Murine Scedosporiosis: Histopathology and Azole Treatment

Abstract

The histopathology of clinical isolates of Scedosporium apiospermum, Scedosporium boydii, and Scedosporium aurantiacum in immunosuppressed mice was evaluated. The organs most affected were the brain, kidneys, and spleen. S. aurantiacum produced more tissue damage than the other two species. Amphotericin B (AMB) was ineffective in the treatment of murine infections caused by such isolates, and posaconazole and voriconazole showed efficacy that correlated with the in vitro susceptibility data.

Pseudallescheria boydii sensu lato is a complex of species, some of which are involved in severe infections (6, 14). The most clinically relevant species of the complex are Scedosporium boydii (anamorph of Pseudallescheria boydii), Scedosporium apiospermum (anamorph of Pseudallescheria apiosperma), and Scedosporium aurantiacum (1, 10, 11, 15). Despite aggressive therapy, invasive scedosporiosis causes a high mortality rate (6, 14). Few data exist on the antifungal response of the current species of the complex (12). Amphotericin B (AMB) has generally demonstrated poor in vitro and in vivo activity (4, 5, 7, 8, 14, 17). Voriconazole (VRC) and posaconazole (PSC) are active in vitro (7, 8, 14) and have shown efficacy in animal models (3, 5, 13) and in clinical trials and case reports (2, 16, 18, 20, 22). We have compared the virulence of S. apiospermum, S. boydii, and S. aurantiacum, and their responses to PSC and VRC, in disseminated murine infection.

We tested six strains, two representing each of the species mentioned above (Table 1). Their in vitro susceptibilities to the antifungals AMB, VRC, and PSC were determined by a reference method (19). AMB was not active against any of the isolates tested. VRC and PSC were active in vitro against most of the strains tested, except for one strain of S. aurantiacum against which these drugs were inactive (Table 2). Male OF1 mice were used. All animal care procedures were supervised and approved by the Universitat Rovira i Virgili Animal Welfare Committee. Mice were immunosuppressed by a single intraperitoneal (i.p.) injection of 200 mg of cyclophosphamide/kg of body weight plus intravenous administration of 5-fluorouracil at 150 mg/kg of body weight 1 day prior to the infection. The isolates were subcultured on potato dextrose agar (PDA) prior to testing. The inoculum size was determined from hemacytometer counts and conidial viability. Animals were infected with 1 × 10⁴ CFU/mouse in 0.2 ml of sterile normal saline injected via a lateral tail vein. For all the strains tested, this inoculum was able to produce an acute infection, with 100% of the animals dying within 6 to 10 days. For histopathological studies, groups of 10 animals were randomly established for each strain and were sacrificed on day 4 postinfection. The lungs, brain, spleen, liver, and kidneys were aseptically removed. Cultures of the homogenates of half of each organ were performed on PDA incubated at 30°C. The numbers of CFU per gram of tissue were calculated. For the histological study, half of each organ was fixed with 10% buffered formalin.

TABLE 1.

Strains of the P. boydii complex included in this study

Strain	Species	Origin
FMR 6694	S. boydii	Cerebral abscess, Barcelona, Spain
FMR 8627	S. boydii	Clinical, Madrid, Spain
CBS 117407	S. apiospermum	Keratitis, São Paulo, Brazil
IHEM 14464	S. apiospermum	Cystic fibrosis, Tours, France
CBS 116910	S. aurantiacum	Ankle ulcer, Santiago de Compostela, Spain
IHEM 15458	S. aurantiacum	Cystic fibrosis, Giens, France

TABLE 2.

In vitro antifungal activities of AMB, VRC, and PSC against two strains each of S. boydii, S. apiospermum, and S. aurantiacum

Species and strain	MIC (μg/ml)
	AMB	VRC	PSC
S. boydii
FMR 6694	32	1	1
FMR 8627	8	0.5	1
S. apiospermum
CBS 117407	8	0.5	1
IHEM 14464	32	0.25	0.5
S. aurantiacum
CBS 116910	32	32	32
IHEM 15458	32	1	2

In the treatment studies, the drugs tested were AMB, given at 1.5 mg/kg/day i.p.; PSC, at 10 or 20 mg/kg twice a day orally (p.o.); and VRC, at 20 or 40 mg/kg/day orally. From 3 days prior to infection, the mice that received VRC and the control group were given grapefruit juice instead of water in order to block VRC metabolism (9, 21). Groups of 10 mice were randomly established for each strain and each treatment. Treatments began 24 h after challenge and lasted for 10 days in survival studies; animals were checked daily for 20 days after challenge. For tissue burden studies, groups of 10 mice were also established, and the animals were sacrificed on day 4 after infection. The kidneys and brain were removed aseptically and were gently homogenized in 2 ml sterile saline. Dilutions of the homogenates were plated on PDA.

Mean survival time was estimated by the Kaplan-Meier method and was compared among groups by using the log rank test. Colony counts in tissue burden studies were analyzed by Kruskal-Wallis and Mann-Whitney U tests, and the Bonferroni correction was used when required.

Fungi were present in all organs tested. The three species tested showed marked brain tropism. The brain was most affected, followed by the kidney, spleen, liver, and lung (Fig. 1). Histological studies revealed focal infiltration by hyphae in blood vessels and in parenchyma, though only in the brains and kidneys of mice infected with S. boydii or, especially, with S. aurantiacum, which in addition produced edema and tubular necrosis in kidneys. In mice infected with S. apiospermum, abscesses were observed in the brain parenchyma and in the kidney, with abundant inflammatory cells but without the presence of hyphae (Fig. 2).

FIG. 1.

Quantitative fungal recovery from different organs of immunosuppressed mice infected with S. boydii FMR 6694 (A) or FMR 8627 (B), S. apiospermum IHEM 14464 (C) or CBS 117407 (D), or S. aurantiacum CBS 116910 (E) or IHEM 15458 (F). Superscript letters indicate significant differences from values for the lung (P < 0.005) (^a), liver (P < 0.005) (^b), kidney (P < 0.05) (^c), or spleen (P < 0.05) (^d). Horizontal lines indicate means.

FIG. 2.

Histological findings for S. boydii (A and B), S. apiospermum (C and D), and S. aurantiacum (E and F) in mice. (A) Fungal infiltration with abundant presence of hyphae in the kidney. (B) Brain section with invasion by hyphae but with no necrosis or inflammatory response. (C) Kidney showing an abscess with abundant inflammatory cells. (D) Microabscess in the brain parenchyma. (E) Kidney section showing hyphae invading the parenchyma, edema, and tubular necrosis. (F) Brain section showing hyphae invading a blood vessel lumen. The stains used are PAS (A, B, and E), hematoxylin and eosin (C and D), and Grocott strain (F).

AMB was not able to improve the survival of mice. All the doses of PSC and VRC tested prolonged survival in all cases (Fig. 3). The highest doses of the two azoles (40 mg/kg) showed similarly high efficacies. VRC showed a dose-response efficacy. S. aurantiacum strain CBS 116910 responded least to the highest doses of VRC and PSC. For this strain, PSC and VRC showed the highest MICs (32 μg/ml).

FIG. 3.

Cumulative mortality of mice infected with S. boydii FMR 6694 or FMR 8627, S. apiospermum IHEM 14464 or CBS 117407, or S. aurantiacum CBS 116910 or IHEM 15458. Mice were treated with AMB, PSC, or VRC. AMB 1.5, amphotericin B administered at 1.5 mg/kg/day i.p.; PSC 20, posaconazole administered at 10 mg/kg p.o. twice a day; PSC 40, posaconazole administered at 20 mg/kg p.o. twice a day; VRC 20, voriconazole administered at 20 mg/kg/day p.o.; VRC 40, voriconazole administered at 40 mg/kg/day p.o. Superscript letters indicate significant differences (P < 0.05) from values for the control (^a), AMB 1.5 (^b), PSC 20 (^c), or VRC 20 (^d).

In all cases, AMB was also ineffective at reducing the fungal load (Fig. 4). The highest doses (40 mg/kg) of VRC and PSC reduced tissue burdens in all cases except for S. aurantiacum strain CBS 116910. Lower doses of both drugs were less effective at reducing the fungal load.

FIG. 4.

Effects of the antifungal treatment on colony counts of S. boydii FMR 6694 and FMR 8627, S. apiospermum IHEM 14464 and CBS 117407, and S. aurantiacum CBS 116910 and IHEM 15458 in the brains and kidneys of mice. AMB 1.5, amphotericin B administered at 1.5 mg/kg/day i.p.; PSC 20, posaconazole administered at 10 mg/kg p.o. twice a day; PSC 40, posaconazole administered at 20 mg/kg p.o. twice a day; VRC 20, voriconazole administered at 20 mg/kg/day p.o.; VRC 40, voriconazole administered at 40 mg/kg/day p.o. Horizontal lines indicate means. Superscript letters indicate significant differences (P < 0.05) from values for the control (^a), AMB (^b), PSC 20 (^c), and VRC 20 (^d).

This study confirms the higher virulence of S. aurantiacum, which was already observed in a previous study, although that study tested only the survival rate of mice (10). In general, in vivo results correlated with in vitro data, since VRC and PSC displayed good in vitro activity, showed efficacy in prolonging survival, and reduced S. apiospermum and S. boydii burdens in tissues. In the case of S. aurantiacum, VRC and PSC at high doses were more effective at prolonging survival and reducing fungal loads in the brains and kidneys of mice challenged with the strain that showed lower MICs than in those infected with the strain that had higher MICs.

The different pathological behaviors of the species of the complex, together with their different responses to antifungals, seem to suggest that infections caused by S. aurantiacum could be more severe and more difficult to treat than those caused by other species. VRC and PSC may have a clinical role in the treatment of disseminated scedosporiosis caused by S. boydii, by S. apiospermum, and by strains of S. aurantiacum with low VRC and PSC MICs.