In vitro activities of eight antifungal drugs against 70 clinical and environmental isolates of Alternaria species

Sir, Melanized fungi of the order Pleosporales are emerging as causing opportunistic, cutaneous infections in immunocompromised patients mostly resulting from traumatic inoculation of contaminated plant debris. Among the most common aetiological agents of skin disease in the latter disorder are species of the genus Alternaria.1 Disseminated infection is uncommon but increasing, particularly among severely immunocompromised individuals.2 Recommendations for therapy are based on case reports and small in vitro studies. New antifungal agents with a better activity may help to improve the management of these infections. We aimed at evaluating the in vitro susceptibility of a collection of Alternaria spp. to amphotericin B, five triazoles and two echinocandins. A total of 70 strains of Alternaria (CBS Fungal Biodiversity Centre, The Netherlands) consisted of the following isolates: Alternaria infectoria (n = 50), Alternaria alternata (n = 7) and Alternaria malorum (n = 13). Correct identification of isolates was done by sequence analysis of ribosomal DNA. Except for enhancement of sporulation (1 week of incubation at 23°C under an alternating light/dark cycle consisting of 8 h of cool-white fluorescent daylight and 16 h of darkness), antifungal susceptibility testing was performed according to CLSI M38-A2.3 Amphotericin B (Bristol–Myers Squibb, Woerden, The Netherlands), fluconazole, voriconazole, anidulafungin (Pfizer Central Research, Sandwich, UK), itraconazole (Janssen Research Foundation, Beerse, Belgium), posaconazole (Schering–Plough, Kenilworth, NJ, USA), isavuconazole (Basilea Pharmaceutica Ltd, Basel, Switzerland) and caspofungin (Merck Sharp & Dohme BV, Haarlem, The Netherlands) were obtained as reagent-grade powders.3 The antifungal agents were dispensed into microdilution trays at a final concentration of 0.016–16 mg/L for amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin, 0.063–64 mg/L for fluconazole and 0.008–8 mg/L for isavuconazole and anidulafungin. Inoculum suspensions were prepared from 7 to 14 day cultures on potato carrot agar, by slightly scraping the surface of mature colonies with a sterile cotton swab wetted with sterile saline including Tween 40 (0.05%). The supernatants were adjusted spectrophotometrically at a wavelength of 530 nm to an optical density (OD) that ranged from 0.25 to 0.3 (2 × 104–7 × 104 cfu/mL).3 Microdilution plates were incubated at 30°C for 48 h (plates with insufficient growth were incubated for 72 h) and read visually after agitation. Table 1 summarizes the results of all in vitro activities of the eight antifungal drugs. Both environmental and clinical Alternaria isolates showed an MIC range from 0.125 to 1 mg/L for amphotericin B. We found the widest range and the highest MICs for fluconazole (range 4–32 mg/L). Surprisingly, most isolates of A. infectoria and A. alternata had high MICs of voriconazole, although the environmental isolates of A. malorum (a non-human pathogenic species) had lower MICs. Isavuconazole had high MICs similar to voriconazole with complete inhibition endpoints exhibiting MIC50 and MIC90 values of 4 mg/L against all Alternaria isolates. Itraconazole demonstrated better in vitro activity than voriconazole and isavuconazole with an MIC90 of 1 mg/L. Posaconazole had the lowest MIC90 (0.25 mg/L) of all the azoles. The echinocandin drugs caspofungin and anidulafungin both demonstrated in vitro activity against A. infectoria and A. alternata, with anidulafungin exhibiting the lowest MEC90 (0.008 mg/L) compared with caspofungin (MEC90 1 mg/L); MEC stands for minimal effective concentration. Both echinocandins had no activity against A. malorum (MEC90 > 8–16 mg/L). More than 150 cases of infection have been described in the world literature, with the skin being the most involved organ.1 Severe deep infections are rare complications and optimal treatment is not known.2 Various antifungal drugs have been used in the treatment of Alternaria infection, such as amphotericin B, flucytosine, fluconazole, miconazole and nystatin.1 There are few published data available concerning the in vitro antifungal susceptibility with microdilution techniques of Alternaria species;4,5 the largest collection included just 13 strains.4 Reported MIC50 and MIC90 values of posaconazole, itraconazole and amphotericin B are comparable to our results. Among the azoles, fluconazole showed uniform low activities against Alternaria species in this and previous studies.4,5 Except one report involving 11 isolates,5 itraconazole, voriconazole and posaconazole had good in vitro activity. Recently, the new azole isavuconazole was tested and generated MIC50 and MIC90 values of 1 mg/L.6 We describe lower in vitro activities (MIC90 4 mg/L) of voriconazole and isavuconazole. There was no isolate with an MIC of isavuconazole >4 mg/L, suggesting a potential role in therapy and prophylaxis if adequate drug levels are achieved at the site of infection. In contrast, posaconazole demonstrated potent activity (MIC90 0.25 mg/L) against all clinical and environmental isolates of Alternaria, but it remains to be confirmed that high enough drug levels can be reached at a remote site of infection. Isavuconazole has not been used clinically for Alternaria infections, but posaconazole was the only clinically active antifungal drug in the treatment of a severe invasive Alternaria infection in an immunocompetent patient.1 In the present study, the antifungal activities of posaconazole and anidulafungin were superior against clinical isolates of A. infectoria. Furthermore, we demonstrated better activity of anidulafungin compared with that of caspofungin, but it is not known whether these in vitro differences are clinically significant. In conclusion, posaconazole and anidulafungin demonstrated the highest in vitro antifungal activity against Alternaria species, including those clinical and environmental isolates that had higher MICs of voriconazole and isavuconazole; their clinical effectiveness in the treatment of Alternaria infection remains to be determined. In vitro susceptibility of Alternaria species to eight antifungal drugs E, environmental; C, clinical. For caspofungin and anidulafungin, MICs are read as MECs. In vitro susceptibility of Alternaria species to eight antifungal drugs E, environmental; C, clinical. For caspofungin and anidulafungin, MICs are read as MECs. This study was partially supported by an unrestricted grant from Basilea Pharmaceutica Ltd, Basel, Switzerland. H. B. was funded by the Ministry of Health and Medical Education of the Islamic Republic of Iran (no. 13081). J. F. M. has undertaken consultancy and/or received travel grants from Merck and Co. and its subsidiary Merck, Sharpe & Dohme, Basilea Pharmaceutica Ltd, Zeneus Pharmaceutica, Gilead, Pfizer and Schering–Plough. Other authors: none to declare.