Abstract
The fungus Saprochaete capitata causes opportunistic human infections, mainly in immunocompromised patients with hematological malignancies. The best therapy for this severe infection is still unknown. We evaluated the in vitro killing activity and the in vivo efficacy of posaconazole at 5, 10, or 20 mg/kg twice a day (BID) in a murine neutropenic model of systemic infection with S. capitata by testing a set of six clinical isolates. Posaconazole showed fungistatic activity against all of the isolates tested. The different doses of the drug, especially the highest one, showed good efficacy, measured by prolonged survival, reduction of (1-3)-β-d-glucan levels in serum, tissue burden reduction, and histopathology.
INTRODUCTION
Saprochaete capitata, formerly known as Trichosporom capitatum, Geotrichum capitatum, and Blastoschizomyces capitatus, is an uncommon clinical fungus belonging to the phylum Basidiomycota, but it is able to cause fatal fungemia in immunocompromised patients, especially in those with hematological malignancies (1–6). The therapeutic options against these infections are limited, because S. capitata is considered intrinsically resistant to the echinocandins (7–10). Currently, there are no recommendations for the management of infections caused by S. capitata, although amphotericin B is the drug most commonly used in the clinical setting, followed by itraconazole and voriconazole (9, 11–14). The use of these compounds is supported by the in vitro antifungal susceptibility of S. capitata to such drugs. However, despite treatment, the mortality rate still remains high, at around 60% (5, 15–19), making it necessary to explore new therapeutic approaches. In previous studies conducted on mice, high doses of fluconazole demonstrated higher efficacy than amphotericin B, flucytosine, and voriconazole (20). Posaconazole has not been evaluated against this fungal species before but has shown efficacy in experimental infections against a wide range of opportunistic fungi, such as Aspergillus spp., Curvularia spp., Rhizopus oryzae (21, 22, 23), and others, including Trichosporon asahii, which is taxonomically related to S. capitata (24). In the present study, we evaluated the in vitro killing activity of posaconazole against this fungus as well as its in vivo efficacy in a neutropenic murine model of systemic infection by S. capitata.
MATERIALS AND METHODS
Strains and inocula.
Six clinical strains of S. capitata (IHEM 5665, IHEM 5666, IHEM 5091, IHEM 6803, IHEM 16105, and IHEM 16109) were included in this study. The inocula were prepared from potato dextrose agar (PDA) cultures by flooding the plates with 3 ml of sterile saline solution and scraping the surface of the colonies with a loop in order to obtain a conidial suspension. To remove hyphal fragments and clumps of agar, the resulting suspension was filtered twice through sterile gauze and then adjusted by hemocytometer counts to the desired concentrations. Viability of inocula was determined by placing 10-fold dilutions of the conidial suspension on PDA plates.
In vitro studies.
Pure posaconazole powder provided by Schering-Plough (Kenilworth, NJ) was used in the in vitro study following the reference microdilution method according to CLSI document M27-A3 (25). Time-kill curves were determined as previously described (26). In brief, 2-fold serial dilutions, ranging from 64 to 0.06 μg/ml of posaconazole, were assayed. At predetermined time points (0, 4, 8, 24, and 48 h), aliquots of 100 μl were removed, serially diluted in sterile water, placed onto PDA plates, and incubated at 35°C for 24 to 48 h in order to determine the CFU/ml. This procedure allowed a limit of detection of 33 CFU/ml. All assays were carried out in duplicate, and the geometric mean and standard deviation were calculated. A CFU count reduction of ≥99.9% or of 3 log10 compared to the starting inoculum was considered indicative of fungicidal activity, while a CFU count reduction of <99. 9% was considered fungistatic (27).
In vivo studies.
Four-week-old OF-1 male mice (Charles River, Criffa Sa, Barcelona, Spain) weighing 28 to 30 g were used. All animals included in the study were immunosuppressed by intraperitoneal administration of a single dose of 200 mg/kg of cyclophosphamide (Genoxal; Laboratorios Funk Sa, Barcelona, Spain) 2 days prior to the infection and then every 5 days until the end of the experiment (28). In order to prevent bacterial infections, all animals received 5 mg/kg/day of ceftazidime subcutaneously. Mice were inoculated intravenously (i.v.) with 2 × 106 CFU/animal of each fungal strain in 0.2 ml of sterile saline solution into the lateral tail vein. This inoculum has previously proven appropriate for producing an acute infection (20). Animals were housed under standard conditions, and care procedures were supervised and approved by the Universitat Rovira i Virgili animal welfare committee. The efficacy of posaconazole was evaluated by prolongation of survival, (1-3)-β-d-glucan levels in serum, reduction of tissue burden, and histopathologic features.
Groups of 13 mice were randomly established: 5 for survival and 8 for tissue burden and determination of (1-3)-β-d-glucan levels in serum samples.
In a preliminary study, animals were challenged with the S. capitata strains IHEM 5666 and IHEM 16105, and the efficacy of posaconazole was assayed at increasing doses of 5, 10, and 20 mg/kg twice a day (BID) orally by gavage for 6 days starting 24 h after infection to determine the most effective dose. The doses were selected from time-kill results and previous drug pharmacodynamics studies (29, 30). Since posaconazole at 10 mg/kg already showed good efficacy, this dose was chosen to be tested against the four remaining strains in a second study.
Determination of glucan and drug levels and fungal load.
Control and treated mice from the tissue burden study group were anesthetized by inhalation of sevoflurane (Sevorane; Abbott, Madrid, Spain) on day 7 postinfection; 12 h after the last dose was administered, 1 ml of blood from each mouse was extracted by cardiac puncture. Animals were then euthanized by cervical dislocation. Serum samples were obtained by centrifugation of the blood at 3,500 rpm and were stored at −20°C until use. Serum levels of (1-3)-β-d-glucan were determined using the Fungitell kit (Associates of Cape Cod, East Falmouth, MA, USA) following the manufacturer's instructions, and serum levels of drug were determined by bioassay, as previously described (31). The liver, spleen, lungs, kidneys, and brain of animals were aseptically removed, and approximately one-half of each organ was weighed and mechanically homogenized in 1 ml of sterile saline solution. Homogenates were serially diluted (1:10), placed onto PDA plates, and incubated for 48 h at 35°C for fungal load calculation (CFU/g of tissue).
Histopathology.
The other half of each organ was fixed with 10% buffered formaldehyde. Samples were embedded in paraffin, stained with hematoxylin-eosin, periodic acid-Schiff (PAS), or Grocott methenamine silver (GMS), and examined in a blinded fashion by light microscopy.
Statistical analysis.
The mean survival times were estimated by the Kaplan-Meier method and compared among groups using the log-rank test. Results from the tissue burden studies were analyzed using the Mann-Whitney U test, and the Kolmogorov-Smirnov test was carried out to determine the normal distribution of (1-3)-β-d-glucan levels in serum with GraphPad Prism 6.0 for Microsoft Windows (GraphPad Software, San Diego, CA, USA). A P value of ≤0.05 was considered statistically significant.
RESULTS
Posaconazole showed fungistatic activity against the six strains of S. capitata tested, with a reduction in the viability of ≤0.14 log10 CFU/ml. Figure 1 illustrates the time-killing kinetic assay against strain IHEM 16105 as representative of all of the strains assayed. Additionally, the MIC value was 0.25 μg/ml against all strains.
FIG 1.
Time-killing kinetic assay of posaconazole against S. capitata strain IHEM 16105.
The dose escalation study showed the efficacy of posaconazole 10 and 20 mg/kg against the two strains tested in this first study in comparison to the control group (P ≤ 0.016), while posaconazole 5 mg/kg BID showed efficacy against only one of the two strains (IHEM 16105) (P = 0.029) (Fig. 2). The dose of 10 mg/kg BID was chosen for the second study for treating infections by the six strains, and this dose significantly prolonged the survival with respect to the control group (P ≤ 0.048) (Table 1).
FIG 2.
Survival of neutropenic mice infected intravenously with 2 × 106 CFU of S. capitata IHEM 5666 and IHEM 16105. Animals were treated for 6 days with posaconazole (PSC) at 5 mg/kg BID (PSC 5), 10 mg/kg BID (PSC 10), or 20 mg/kg BID (PSC 20). a, P value of ≤0.02 versus control; b, P value of 0.008 versus PSC 5.
TABLE 1.
Survival of mice infected with 6 different S. capitata strains and treated with PSC 10a
| Strain | MST (days) (95% CI)b |
P | |
|---|---|---|---|
| Control | PSC 10 | ||
| IHEM 5665 | 9.2 (6.9–11.42) | 17.8 (8.8–26.73) | 0.0039 |
| IHEM 5666 | 7.2 (5.58–8.81) | 14.20( 9.6–18.70) | 0.0025 |
| IHEM 5091 | 7.6 (5.71–9.48) | 17 (7.8–26.17) | 0.0039 |
| IHEM 6803 | 9.4 (7.7–11.07) | 15.40 (10.70–20.10) | 0.0048 |
| IHEM 16105 | 8.8 (7.18–10.42) | 11.8 (8.46–15.13) | 0.0027 |
| IHEM 16109 | 8.6 (5.88–11.32) | 13.4 (8.70–18.10) | 0.0480 |
PSC 10, posaconazole 10 mg/kg BID.
MST, mean survival time; CI, confidence interval.
The six strains tested caused a high fungal load in all organs, and the kidneys and brain generally were the most affected (mean log10 CFU/g tissue, ≥7.41 and ≥7.31, respectively). For the strains IHEM 5666 and IHEM 16105, any dose of posaconazole significantly reduced the fungal load in comparison to the control in all organs studied (P ≤ 0.0079) (Fig. 3), and posaconazole 10 mg/kg BID did so against the rest of the strains (P ≥ 0.0079) (Table 2). The reduction of CFU/g in animals receiving posaconazole 10 mg/kg BID, with respect to the control animals, ranged from 1.83 to 3.42 log10; the highest reduction observed was in the liver (mean ± standard deviation [SD] log10 reduction, 2.9 ± 0.41), and the lowest was in the brain (mean log10 reduction, 2.13 ± 0.21). Twelve hours after the end of the treatment with posaconazole at 5, 10, and 20 mg/kg, mean (± SD) serum levels of drug were 5.76 ± 0.5, 6.48 ± 0.75, and 7.46 ± 0.70 μg/ml, respectively; all levels were above the MIC values. At day 7 postinfection, the (1-3)-β-d-glucan levels in the serum of the controls ranged from 360 to 503 pg/ml. Posaconazole at 5, 10, or 20 mg/kg BID was able to reduce the (1-3)-β-d-glucan concentrations in serum in comparison with those of the untreated group, although reduction was not below the cutoff for positivity in human infections, which is 80 pg/ml (32) (Fig. 4).
FIG 3.
Effects of antifungal treatment on colony counts of neutropenic mice infected with 2 × 106 CFU of S. capitata in liver, lung, kidney, brain, and spleen and treated for 6 days with posaconazole (PSC) at 5 mg/kg BID (PSC 5), 10 mg/kg BID (PSC 10), or 20 mg/kg BID (PSC 20). a, P value of ≤0.007 versus control; b, P value of ≤0.007 versus PSC 5; c, P value of ≤0.014 versus PSC 10.
TABLE 2.
Effects of antifungal treatment on colony counts in the liver, lung, kidney, brain, and spleen of neutropenic mice infected with 2 × 106 CFU of S. capitata
| Strain | Treatmentb | Log10 CFU/g of tissue (95% CI)a |
||||
|---|---|---|---|---|---|---|
| Liver | Lung | Kidney | Brain | Spleen | ||
| IHEM 5665 | Control | 6.31 (5.88–6.92) | 6.76 (6.11–7.11) | 7.88 (7.75–8.10) | 8.01 (7.69–8.45) | 6.17 (5.80–6.45) |
| PSC 10 | 2.89 (2.48- 3.27)c | 3.69 (2.90–4.50)c | 5.52 (5.25–5.90)c | 6.00 (5.65–6.68)c | 3.69 (3.30–4.17)d | |
| IHEM 5666 | Control | 6.24 (5.78–6.82) | 6.35 (5.95–6.94) | 8.00 (7.44–8.93) | 7.51 (6.98–8.01) | 6.23 (5.45–6.86) |
| PSC 10 | 3.85 (3.27–4.57)d | 4.04 (3.49–4.70)c | 5.75 (5.00–6.35)c | 5.48 (4.45–6.62)c | 3.84 (3.1–4.75)c | |
| IHEM 5091 | Control | 6.11 (5.25–7.28) | 6.12 (5.58–6.62) | 7.68 (7.34–8.00) | 7.31 (6.62–7.81) | 6.04 (5.60–6.40) |
| PSC 10 | 2.89 (2.20–3.42)c | 2.97 (2.41–3.30)c | 5.07 (4.00–5.88)c | 5.20 (4.10–5.92)c | 3.61 (3.00–4.15)c | |
| IHEM 6803 | Control | 6.35 (5.80–6.73) | 6.68 (6.10–7.10) | 8.07 (7.78–8.50) | 7.54 (7.20–7.82) | 6.47 (5.57–7.74) |
| PSC 10 | 3.31 (3.10–3.61)c | 3.43 (2.80–4.25)c | 5.35 (4.9–5.96)c | 5.55 (4.90–5.96)c | 3.23 (2.90–3.39)c | |
| IHEM 16105 | Control | 6.71 (5.97–7.67) | 6.68 (6.19–7.26) | 7.78 (7.53–8.17) | 7.64 (6.99–8.17) | 6.85 (6.64–7.18) |
| PSC 10 | 4.24 (4.05–4.39)c | 4.85 (4.42–5.41)c | 5.37 (4.71–5.80)c | 5.08 (4.30–5.64)c | 4.57 (4.29–5.10)c | |
| IHEM 16109 | Control | 6.09 (5.88–6.26) | 6.29 (5.12–6.99) | 7.41 (6.98–7.86) | 7.51 (6.70–8.01) | 6.98 (6.3–6.46) |
| PSC 10 | 3.21 (2.30–4.07)c | 3.53 (2.80–4.37)c | 5.29 (4.62–5.75)c | 5.41 (5.02–5.83)c | 3.65 (3.10–4.32)c | |
CI, confidence interval.
PSC 10, posaconazole at 10 mg/kg BID for 6 days.
P value of 0.0002 compared to control group.
P value of 0.007 compared to control group.
FIG 4.
(1-3)-β-d-Glucan levels in the serum of mice infected with S. capitata strains IHEM 5666 and IHEM 16105 (group control, PSC 5, 10, or 20 mg/kg BID) (A) or strains IHEM 5665, IHEM 5091, IHEM 6803, and IHEM 16109 (group control, PSC 10 mg/kg BID) (B). Horizontal line indicates the cutoff positive (80 pg/ml). a, P value of ≤0.028 versus control; b, P value of ≤0.028 versus PSC 5; c, P value of ≤0.028 versus PSC 10.
The histopathologic studies also confirmed that kidney and brain were the most affected organs in untreated animals. Presence of necrotic and hemorrhagic foci with no inflammatory response and abundant fungal structures located in the parenchyma and associated with angioinvasion were observed in all of the studied organs (Fig. 5). In mice receiving the different doses of posaconazole, the presence of fungal cells was reduced in a dose-dependent manner; the presence was observed focally in the parenchyma, with no sign of necrosis or angioinvasion.
FIG 5.
Histological findings in kidneys of immunosuppressed mice infected with S. capitata (strain IHEM 16105) 7 days postinfection. (A, B) Control mice showing massive invasion of renal parenchyma by hyphae without inflammatory response or necrosis. (C, D) Mice treated with PSC 10 showing decrease of hyphae in renal parenchyma level. (E, F) Mice treated with PSC 20 showing less presence of hyphae within renal tubules. Stain for A, C, and E was PAS. Stain for B, D, and F was GMS. Magnification, ×400.
DISCUSSION
S. capitata causes serious opportunistic infections in patients with hematological malignancies, especially in those with acute leukemia, and is associated with a poor outcome (2, 4, 5). Although an improvement in neutropenia in patients with systemic infections by S. capitata leads to a better prognosis, it is not enough to cure the infection (2, 4–6).
In this study, we evaluated the efficacy of posaconazole against an unusual number of clinical strains for this type of study, trying to evaluate the possible intraspecies variability in antifungal response, as there is in many species. Therefore, we selected six clinical strains of S capitata with identical MICs and similar time-kill kinetics of posaconazole. Other authors have reported that posaconazole is active in vitro against S. capitata, with MICs ranging from 0.016 to 1 μg/ml (3, 5, 9) although MICs of ≥4 μg/ml have occasionally been reported (18).
In this study, posaconazole at any dose prolonged the survival of the animals compared to the control group, and the best results were obtained with posaconazole 10 and 20 mg/kg BID. In addition, posaconazole at any dose significantly reduced the fungal burden in all the studied organs as well as reduced the (1-3)-β-d-glucan levels in serum. This reduction was dose dependent and correlated with the levels of drug detected in serum after the end of the treatment. The (1-3)-β-d-glucan marker is a cell wall component that is common in the fungal kingdom, easily detectable, and quantifiable in serum and body fluids, and it is used as a marker of disseminated fungal infections, including those caused by S. capitata (9, 33). We found a correlation between the decrease in the marker, the fungal load, and the dose administered. Until now, the detectable levels of (1-3)-β-d-glucan have been used for diagnosis, although the relationship between such antigen levels and the fungal load found in the present study and in previous studies on animal models (9, 33) seems to indicate that (1-3)-β-d-glucan levels might be useful for evaluating prognosis in infections with S. capitata. Further studies are needed to confirm this finding.
As indicated above, the efficacy of other drugs, such as amphotericin B, flucytosine, voriconazole, and fluconazole, was previously evaluated in systemic infection by S. capitata using a murine model which showed that fluconazole at a high dose (80 mg/kg) was the most effective in prolonging the survival of mice and reducing the fungal burden in the liver, spleen, and kidney (20). This was similar to posaconazole activity in the present study. Despite good results obtained with fluconazole in the treatment of the experimental infection by S. capitata, an important limitation to its use is the reported resistance to that drug in vitro (1). Other reports indicate the lack of susceptibility of S. capitata to fluconazole and/or echinocandins, given that these antifungal compounds are administered empirically to prevent or treat infections by other fungi in patients with hematological malignancies (9). Considering the little experience in the management of systemic infections with S. capitata and the risk of acquired resistance to fluconazole and echinocandins, our results are of special interest. Posaconazole, which is a well-tolerated drug treatment, could be another useful tool in our fight against this difficult-to-treat infection, especially when other therapeutic options fail.
ACKNOWLEDGMENTS
We thank the Institute of Hygiene and Epidemiology (IHE) located in Brussels for the strains used in this study.
This project received funding from the European Union's Seventh Framework Programme for research, technological development, and demonstration under grant agreement HEALTH-2013-INNOVATION-2-601963.
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