Evaluation of Candida peritonitis with underlying peritoneal fibrosis and efficacy of micafungin in murine models of intra-abdominal candidiasis

Abstract

Candida peritonitis is a crucial disease, however the optimal antifungal therapy regimen has not been clearly defined. Peritoneal fibrosis (PF) can be caused by abdominal surgery, intra-abdominal infection, and malignant diseases, and is also widely recognized as a crucial complication of long-term peritoneal dialysis. However, the influence of PF on Candida peritonitis prognosis remains unknown. Here, we evaluated the severity of Candida peritonitis within the context of PF and the efficacy of micafungin using mice. A PF mouse model was generated by intraperitoneally administering chlorhexidine gluconate. Candida peritonitis, induced by intraperitoneal inoculation of Candida albicans, was treated with a 7-day consecutive subcutaneous administration of micafungin. Candida infection caused a higher mortality rate in the PF mice compared with the control mice on day 7. Proliferative Candida invasion into the peritoneum and intra-abdominal organs was confirmed pathologically only in the PF mice. However, all mice in both groups treated with micafungin survived until day 20. Micafungin treatment tends to suppress inflammatory cytokines in the plasma 12 h after infection in both groups. Our results suggest that PF enhances early mortality in Candida peritonitis. Prompt initiation and sufficient doses of micafungin had good efficacy for Candida peritonitis, irrespective of the underlying PF.

Figure 1

Schematic study design of the present study and histopathological evaluation of PF. (A) Mice received 9 intraperitoneal injections of 0.1% CG or a vehicle (15% ethanol) at a dose of 0.2 mL/mouse in 3 weeks, followed by one injection with 1 mL of the C. albicans cell suspension (5.0 × 10⁷ CFU/mouse). Micafungin dissolved in sterile saline was subcutaneously administered at a dose of 5 mg/kg daily for 7 days, beginning 2 h after the infection. (B) Masson’s trichrome staining of peritoneal tissues at 100-fold magnification. Bars indicate the thickness of the submesothelial compact zone. In the control group, the monolayer of mesothelial cells covered the entire surface of the peritoneum. In the PF group, the submesothelial compact zone was markedly thickened. Bar graph shows the thickness of the submesothelial compact zones in the control and PF groups. The PF group showed significant thickening compared to the control group. An asterisk indicates p < 0.0001 (unpaired t test). Both control and PF groups in this assay were not inoculated with Candida. These results were confirmed on two separate occasions and representative data are shown. PF, peritoneal fibrosis; CG, chlorhexidine gluconate; SS, sterile saline; i.p., intraperitoneal injection; and s.c., subcutaneous injection.

Figure 2

Evaluation of survival assay and the fungal burden in the liver, spleen, and kidneys. (A) Survival curves were plotted for the following four groups (n = 8 for each group); control without micafungin treatment (open circles); PF without micafungin treatment (open triangles); control with micafungin treatment (filled circles); and PF with micafungin treatment (filled triangles). Without micafungin treatment, the PF mice had significantly higher mortality than the control mice on day 7 (p = 0.013, Kaplan-Meier log-rank test), although both groups showed no significant difference on day 20 (p = 0.29). All the PF and control mice treated with micafungin survived until the end of the experiment (day 20) (vs. untreated group, p < 0.01 each). Similar results were obtained in three different experiments and representative data are shown. (B) Fungal burden in the liver, spleen, bilateral kidneys, and blood was evaluated 6 h, 12 h, and 7 days after the infection (n = 10 for each group). In the control mice, fungal burden in the three target organs at 6 and 12 h, and that in spleen and kidneys on day 7 was significantly reduced by micafungin treatment. In the PF mice, there was significant difference in the fungal burden between micafungin-treated and -untreated groups at 12 h, but not at 6 h. Fungal burdens of all the three organs in the control mice and two organs (liver and kidneys) in the PF mice were significantly reduced by micafungin treatment on day 7 compared with the results at 12 h. Asterisks indicate p < 0.0083 (Mann-Whitney U test adjusted with Bonferroni correction). PF, peritoneal fibrosis.

Figure 3

Histopathological examination of the peritoneum, liver and spleen with H & E and Grocott staining. Histopathological examination was performed 12 h after the infection. (A,B) Abscess formation with Candida cells in the thickened submesothelial zone of peritoneum was observed with Grocott staining only in the PF mice without micafungin treatment. (C) Numerous inflammatory cells were also observed with H & E staining in the same sections. (D) Candida proliferation was not detectable in the PF mice with micafungin treatment with Grocott staining. Proliferative Candida infiltration into the subcapsular structure inside the liver (E,F) and spleen (H,I) were observed with Grocott staining only in the PF mice without micafungin treatment. (G,J) Numerous inflammatory cells were also observed with H & E staining in the same sections. The photographs are representative of two independent examinations. Magnification, A × 40, B × 400, C × 400, D × 400, E × 100, F × 400, G × 400, H × 100, I × 400, and J × 400.

Figure 4

Evaluation of the cytokines in the plasma. (A) The plasma cytokine concentrations of IFN-γ, IL-10, IL-17A, IL-1β, IL-6, and TNF-α were quantified by enzyme-linked immunosorbent assay in the following groups: control group (control) and PF group (PF) just before infection (0 h); and infected with or without micafungin administration, in both PF and control groups at 12 h after the infection (four mice each). All the six cytokines at 12 h after the intraperitoneal inoculation of C. albicans tended to be suppressed with micafungin treatment in both PF and control groups. (B) The plasma TNF-α concentrations of LPS-exposed mice instead of C. albicans inoculation were quantified by enzyme-linked immunosorbent assay, and no significant difference was observed between micafungin-treated and -untreated mice in both control and PF groups. Similar results were obtained in two different experiments and representative data are shown. Asterisks indicate p < 0.05 (Mann-Whitney U test). IFN-γ, interferon-gamma; IL-10, interleukin-10; IL-17A, interleukin-17A; IL-1β, interleukin-1 beta; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; PF, peritoneal fibrosis; and LPS, lipopolysaccharide.