In vitro antifungal susceptibility of Scopulariopsis brevicaulis isolates

Medical Mycology, 2014, 52, 723 727 doi: 10.1093/mmy/myu039 Advance Access Publication Date: 21 July 2014 Original Article Original Article In vitro antifungal susceptibility of Scopulariopsis brevicaulis isolates Magdalena Skóra 1,, Anna B. Macura 1 and Małgorzata Bulanda 2 1 Department of Mycology and 2 Department of Infection Epidemiology, Chair of Microbiology, Jagiellonian University Medical College, Kraków, Poland *To whom correspondence should be addressed. Magdalena Skóra Tel: + 48126332567;Fax:+ 48124233924. E-mail: magdalena.skora@uj.edu.pl Received 17 January 2014; Revised 25 April 2014; Accepted 7 May 2014 Abstract In humans, Scopulariopsis is mainly associated with onychomycoses, rarely with cutaneous infections or with invasive mycoses. However, during the last two decades, deep infections caused by members of this genus have been increasing. Scopulariopsis brevicaulis is the most common species described as an etiologic agent of human disease. Previous antifungal susceptibility studies indicate that this species is resistant in vitro to the broad-spectrum antifungal agents that are available today. Here, we describe the antifungal activity of amphotericin B, terbinafine, ciclopirox, itraconazole, ketoconazole, and voriconazole against 40 S. brevicaulis isolates. Antifungal susceptibility tests were performed using a modified Clinical and Laboratory Standards Institute M38-A2 procedure. The results showed that itraconazole had the highest minimal inhibitory concentration (MIC) of >16 mg/l; amphotericin B, voriconazole, and ketoconazole MICs were ranging from 4 to >16 mg/l, 8 to >16 mg/l, and 8 to >16 mg/l, respectively; and the best activity was found with terbinafine and ciclopirox with MICs ranging from 0.5 to 16 mg/l and 1 to 8 mg/l, respectively. Key words: Scopulariopsis brevicaulis, antifungals, antifungal susceptibility testing. Introduction Scopulariopsis can cause superficial, subcutaneous, and invasive infections in humans. In immunocompetent patients, it has mainly been associated with superficial mycoses, predominantly onychomycoses, but rarely with cutaneous infections. Reports of invasive infections are sparse. However, during the past two decades, the number of descriptions of invasive infections has been increasing, particularly in immunocompromised patients. Scopulariopsis was found to be an etiological agent of infection of the following: eye (keratitis, endophthalmitis, and retinal and choroidal inflammation), ear, sinuses, oral maxilla, nasal septum, brain, meninges, lungs, bronchi, endocardium, peritoneum, liver, muscles, and tendons, as well as disseminated mycoses [1 3]. There have been reports of infections caused by the anamorphic form of these fungi (Scopulariopsis) and also by the teleomorphic form (Microascus), but the asexual form is most often identified as the cause of disease [1]. The species identified in human infections include S. acremonium, S. americana, S. brevicaulis, S. brumptii, S. candida, S. fusca, S. hominis, and M. cinereus, M. cirrosus, C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 723

724 Medical Mycology, 2014, Vol. 52, No. 7 M. manginii, and M. trigonosporus [4,5]. The species most frequently isolated from clinical specimens is S. brevicaulis. S. brevicaulis infections are difficult to treat, and invasive mycoses are usually fatal. No treatment regimens have been developed for these infections, and there is little information on the in vitro susceptibility of S. brevicaulis to antifungal drugs. The studies conducted to date indicate that this species is resistant to commonly used antimycotics, with high minimal inhibitory concentrations (MICs) for amphotericin B, flucytosine, and azole compounds [6 8]. Therefore, it is necessary to identify drugs that are active against S. brevicaulis and that enable the effective treatment of infections caused by this species. Our aim in this study was to evaluate the in vitro antifungal activity of six antifungals against S. brevicaulis isolates with the determination of MICs. Materials and methods Strains A total of 40 S. brevicaulis strains (36 clinical strains originated from Poland and 4 from the CBS (Centraalbureau voor Schimmelcultures) collection, CBS 112377, CBS 119549, CBS 147.41, CBS 398.54) were evaluated. All clinical isolates were obtained from human nails or skin collected at the Department of Mycology, Chair of Microbiology, Jagiellonian University Medical College, Kraków, Poland. Identification was based on the macroscopic and microscopic characteristics of the isolates in culture [9]. Preparation of inocula Inocula were prepared from 14-day-old cultures on Czapek- Dox agar (BIOCORP Polska Sp. z o.o., Poland) by covering slants with 2 ml of sterile saline. The inoculum size was adjusted to 0.5 McFarland scale and later diluted 1:50 in Roswell Park Memorial Institute (RPMI)-1640 medium with L-glutamine, without sodium bicarbonate (Sigma-Aldrich Sp. z o.o., Poland), buffered to ph 7 with morpholinepropanesulphonic acid (MOPS; 0.165 mol/l; Sigma-Aldrich). The final inoculum densities were 0.3 0.4 10 4 cfu/ml. Antifungal agents Antifungal agents included amphotericin B, terbinafine, ciclopirox, itraconazole, ketoconazole, and voriconazole. They were obtained as standard powders from Sigma- Aldrich. The stock solutions were prepared in 100% dimethyl sulfoxide (POCH S.A., Poland) and later diluted 1:50 in RPMI-1640 medium with L-glutamine, without sodium bicarbonate (Sigma-Aldrich) and then buffered to ph 7 with MOPS (0.165 mol/l; Sigma-Aldrich). Antifungal susceptibility testing Antifungal susceptibility tests were performed using a modified broth microdilution Clinical and Laboratory Standards Institute M38-A2 method [10]. The lower incubation temperature was used 27 Cinsteadof35 C, which provided better growth of fungi. After 48 h and 72 h incubation, the MICs were read with the help of a mirror. Final MICs were defined as the lowest concentrations of the antifungal agents that completely inhibited fungal growth after 72 h incubation. Results and discussion Here, we present the antifungal susceptibility test results for 40 S. brevicaulis isolates (36 clinical strains originated from Poland and 4 strains from the CBS collection). Table 1 lists the individual susceptibility profiles of each strain tested. Table 2 contains MIC ranges, MIC modes, MIC 50,and MIC 90 (concentration of antifungal agent able to inhibit 50% and 90% of strains tested) for the six antifungal drugs tested against clinical S. brevicaulis isolates. Our study showed low activity of itraconazole, ketoconazole, and voriconazole against S. brevicaulis. Itraconazole MICs exceeded tested concentrations (>16 mg/l), while the MICs for ketoconazole and voriconazole ranged from 8 mg/l to >16 mg/l, with most strains (91.7%) having MICs >16 mg/l. Our results are consistent with data from the literature. Aguilar et al. [11], studying the sensitivity of five S. brevicaulis strains, showed that the MIC for itraconazole was >16 mg/l and the MIC for ketoconazole ranged from 1 to >16 mg/l. Cuenca-Estrella et al. [8] examined itraconazole and voriconazole MICs and found them to be >8 mg/l and 16 64 mg/l, respectively. They confirmed high voriconazole and itraconazole MICs against S. brevicaulis in later studies (voriconazole MIC 50 and MIC 90 >8 mg/l and itraconazole MIC 50 and MIC 90 >8 mg/l) [7,12,13]. Carrillo-Muñoz et al. [14] studied the antifungal activity of voriconazole against 11 S. brevicaulis strains and found high MICs for this antifungal; the MICs ranged from 1 to 16 mg/l and both the MIC 50 and MIC 90 were 16 mg/l. In this study, terbinafine and ciclopirox exhibited the best antifungal activity against S. brevicaulis, with MICs ranging from 0.5 to 16 mg/l for terbinafine and from 1 to 8 mg/l for ciclopirox. Mode, MIC 50, and MIC 90 were identical for both drugs and amounted to 4 mg/l. Other researchers have obtained comparable MICs for terbinafine. For example, Carrillo-Muñoz et al. [15] evaluated terbinafine activity against five S. brevicaulis strains

Skóra et al. 725 Table 1. Amphotericin B, terbinafine, and ciclopirox minimal inhibitory concentrations against 36 clinical Scopulariopsis brevicaulis isolates and 4 S. brevicaulis strains from the CBS collection. Strain number Origin Year of isolation Minimal inhibitory concentration (mg/l) Amphotericin B Terbinafine Ciclopirox 06/1174 Toenails 2006 8 1 4 06/1193 Toenails 2006 8 2 4 06/610 Toenails 2006 >16 4 4 06/777 Fingernails 2006 >16 8 2 07/1014 Toenails 2007 8 0.5 4 07/1187 Toenails 2007 16 2 8 07/392 Fingernails 2007 8 4 4 07/507 Toenails 2007 4 4 2 07/521 Toenails 2007 8 4 4 07/701 Toenails 2007 8 4 4 08/108 Skin 2008 16 2 4 08/1323 Toenails 2008 8 4 2 08/1356 Toenails 2008 16 1 4 08/479 Toenails 2008 8 4 4 08/585 Toenails 2008 16 2 8 08/699 Toenails 2008 8 4 2 08/958 Toenails 2008 16 4 2 08/D1 Toenails 2008 >16 4 4 08/D2 Toenails 2008 >16 4 4 09/1194 Fingernails 2009 16 1 2 09/1316 Toenails 2009 16 4 4 09/205 Toenails 2009 8 2 4 09/351 Fingernails 2009 8 4 2 09/456 Toenails 2009 16 2 2 09/600 Toenails 2009 16 8 4 10/1161 Foot interdigital spaces 2010 8 2 4 10/1229 Toenails 2010 8 2 2 10/1313 Toenails 2010 >16 2 1 10/820 Fingernails 2010 8 4 4 10/874 Toenails 2010 16 4 2 10/976 Toenails 2010 16 16 4 11/555 Toenails 2011 >16 4 2 11/649 Toenails 2011 8 1 2 HIV 115/10 Foot interdigital spaces 2010 >16 4 4 HIV 12/09 Foot interdigital spaces 2009 >16 1 4 HIV 147/11 Foot interdigital spaces 2011 8 4 2 CBS 112377 Indoor environment >16 4 2 CBS 119549 Skin biopsy 16 2 4 CBS 147.41 Nail 4 4 2 CBS 398.54 Toenail 8 1 4 Table 2. Ranges, modes, MIC 50,andMIC 90 for antifungal agents tested against 36 clinical isolates of Scopulariopsis brevicaulis. Minimal inhibitory concentration (mg/l) Antifungal agent Amphotericin B Terbinafine Ciclopirox Itraconazole Ketoconazole Voriconazole Range 4 to >16 0.5 to 16 1 to 8 >16 8 to >16 8 to >16 Mode 8 4 4 >16 >16 >16 MIC 50 16 4 4 >16 >16 >16 MIC 90 >16 4 4 >16 >16 >16 MIC 50 /MIC 90, concentration of antifungal agent able to inhibit 50% and 90% of strains tested.

726 Medical Mycology, 2014, Vol. 52, No. 7 and found MICs ranging from 0.01 to 16 mg/l, with a geometric mean of 1.38 mg/l. Garcia-Effron et al. [16] obtained similar results when they tested 21 S. brevicaulis strains; terbinafine MICs ranged from 1 to 16 mg/l and the MIC 90 was 16 mg/l. Cuenca-Estrella et al. [7,8] also studied this drug activity and found terbinafine MICs ranging from 4to>16 mg/l. We believe the present study is the first to test a large number of S. brevicaulis strains against ciclopirox. The drug appears to be active against S. brevicaulis and exhibits comparable activity against terbinafine. This study confirms our previous research in which in vitro antifungal susceptibility testing of S. brevicaulis against ciclopirox by an agar diffusion method (Neo-Sensitabs, Rosco, Denmark) was evaluated [17]; all S. brevicaulis strains tested had large inhibition zones for both terbinafine and ciclopirox. Hryncewicz-Gwóźdź et al.[18] performed a similar study and also noted that terbinafine and ciclopirox were highly effective.pawlik and Macura [19] evaluated the susceptibility of 11 S. brevicaulis strains against ciclopirox and found MICsthatrangedfrom1to10mg/l,MIC 50 = 5 mg/l, and MIC 90 = 10 mg/l. Gupta and Kohli [20] evaluated the in vitro susceptibility of ciclopirox against one S. brevicaulis strain using the M27-A procedure; the ciclopirox MIC was 0.125 mg/l. Strassmann [21] also studied the antifungal effect of ciclopirox by testing 25 S. brevicaulis isolates; ciclopirox MICs ranged from 2 to 8 mg/l and MIC 50 and MIC 90 were 4 mg/l and 8 mg/l, respectively. Jo Siu et al. [22], in a study comparing in vitro antifungal activities of efinaconazole and other antifungal agents, used four S. brevicaulis isolates and reported ciclopirox MICs ranging from 0.5 to 1 mg/l. Amphotericin B MICs found in this study were lower than those found for itraconazole, ketoconazole, and voriconazole but higher than those for ciclopirox and terbinafine. The MICs for amphotericin B ranged from 4 mg/l to >16 mg/l. Amphotericin B MICs determined by Aguilar et al. [11] and by Szekely et al. [23] against S. brevicaulis strains were within the range of 1 to >16 mg/l and 2 to 16 mg/l, respectively. Cuenca-Estrella et al. obtained comparable MICs in several studies as follows: 8 to >16 mg/l, MIC 50 = 16 mg/l, MIC 90 >16 mg/l [7]; 4 to 16 mg/l [8], MIC 50 = 4 mg/l, and MIC 90 = 16 mg/l [12]; and MIC 50 = 8 mg/l, MIC 90 >8mg/l[13]. Interpretative breakpoints for susceptibility testing of S. brevicaulis are not available to date, and clinical studies have also not been reported. However, high MICs for many antifungal drugs obtained in most studies, including the present study, allow us to suggest that this species is multiresistant. Here, we report some antifungal activity of terbinafine and ciclopirox against S. brevicaulis. The other antifungal agent with potent activity against this species is amorolfine. Clayton [24] and Strassmann [21] studied the antifungal effect of amorolfine against S. brevicaulis. Amorolfine MICs ranged from 0.03 to 0.12 mg/l and 0.01 to 0.5 mg/l, respectively. Jo Siu et al. [22] obtained similar results; amorolfine MICs against four S. brevicaulis isolates ranged from 0.063 to 0.13 mg/l. There is also information about the effective treatment of onychomycosis caused by S. brevicaulis with oral terbinafine and topical 5% amorolfine nail lacquer [25]. Because S. brevicaulis is multidrug resistant, the infections caused by this species, especially invasive mycoses, might not respond to standard antifungal treatment. 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