Potential synergistic activity of antimycotic substances in combination with human platelets against Aspergillus fumigatus

Abstract

Sir, Invasive aspergillosis is increasingly recognized in immunocompromised hosts. Patients with prolonged and deep granulocytopenia following chemotherapy for haemato-oncological disorders or allogeneic bone marrow transplant recipients are particularly at risk. Platelets are second in abundance to red blood cells in the circulation, with 1.5–4.0×10 platelets/mL of blood in healthy individuals. They are essential components in haemostasis, but they also play an important role in antimicrobial host defence. Recently, we observed that platelets released serotonin after contact with aspergilli. Furthermore, platelets attenuated the virulence of Aspergillus spp. as the cell wall compound galactomannan was significantly decreased. Several in vitro studies have indicated that immune effector cells in combination with antifungals exert synergistic effects on aspergilli. However, no data are available on the effects of platelets and antimycotics on aspergilli. Two clinical Aspergillus fumigatus isolates and four antifungal agents were studied: amphotericin B (Sigma–Aldrich, Vienna, Austria); voriconazole (kindly provided by Pfizer, Vienna, Austria); posaconazole (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ, USA); and caspofungin (MSD, Vienna, Austria). The MICs of all drugs used in this study were 0.5 mg/L for both isolates. Based on the MICs, drug concentrations of 0.5 and 1 mg/L were used for the platelet/drug studies. Fresh platelets were provided from the local Department of Immunology and Blood Transfusion. Platelets were collected from healthy donors by thrombocytopheresis using an Amicus cell separator (Baxter, Vienna, Austria). One hundred microlitres each of platelets (1×10/mL) and conidia (1×10/mL) in an effector/target ratio of 100:1 and specific concentrations of antifungals (0.5 and 1 mg/L) were inoculated into microwell plates (Greiner, Vienna, Austria) and incubated at 378C. The morphology of the A. fumigatus strains treated either with the combination of platelets plus antifungals or with each substance or platelets alone was investigated by assessing the germination rate and hyphal elongation, as described previously. Hyphal damage was assessed by a colorimetric assay with the dye 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2-H-tetrazolium-5carboxynilide sodium salt (XTT; Sigma–Aldrich) plus 40 mg/mL coenzyme Q (Sigma–Aldrich) and antifungal activity was calculated as the percentage of hyphal damage. Each experiment was performed with platelets from one donor using duplicate or quadruplicate wells for each condition and strain, and repeated six times. Differences between mean values of both strains were statistically evaluated by repeated-measures analysis of variance followed by Dunnett’s correction for multiple comparisons. A twosided P value of ,0.05 indicated statistical significance. Human platelets plus amphotericin B achieved significantly greater inhibition of the germination rate (P,0.05) than did amphotericin B or platelets alone (Figure 1a). The effect was additive. These results were found with both tested amphotericin B concentrations, although 1 mg/L revealed a better inhibitory effect than the lower concentration (Figure 1a). With caspofungin, the germination rate was not significantly reduced (Figure 1b). When azoles were used in combination with platelets, the inhibitory effect was found to be additive in comparison with either azole or platelets alone (Figure 1c and d). As found for the germination rate, human platelets plus amphotericin B achieved significantly greater inhibition of hyphal elongation (P,0.05) than amphotericin B or platelets alone. Hyphal elongation was significantly reduced in all tested aspergilli, either under platelet or caspofungin treatment. When used in combination, the effect was found to be additive. The combination of platelets plus either azole at any concentration tested did not significantly enhance the reduction of hyphal elongation. Platelets decreased the ability of hyphae to reduce XTT; however, the combination of platelets plus antimycotics had no additive effect on hyphal damage (data not shown). Our findings indicate that platelets in combination with antimycotics exert additive effects in reducing the germination rate and hyphal elongation of A. fumigatus in vitro. Among the tested antimycotic substances, amphotericin B revealed the best results in combination with human platelets. However, platelets plus antimycotics were not additive for hyphal damage. In the immunocompromised patient, inhaled Aspergillus conidia germinate into hyphae, the growing and invading structures of filamentous fungi. Consequently, blocking fungal germination and delaying hyphal growth is crucial in preventing invasive disease. In our study, the combination of platelets plus amphotericin B synergistically enhanced the antifungal activity (P,0.05) in reducing germination rate and hyphal elongation. Amphotericin B is known to complex with sterols in the fungal cell membrane, leading to pore formation and