ACCEPTED. In vitro interactions of micafungin with amphotericin B against. clinical isolates of Candida spp.

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1 AAC Accepts, published online ahead of print on January 00 Antimicrob. Agents Chemother. doi:./aac.0-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 0 In vitro interactions of micafungin with amphotericin B against clinical isolates of Candida spp. Carolina Serena, Marçal Mariné, Guillermo Quindós, Alfonso J. Carrillo, J. F. Cano, F. Javier Pastor and Josep Guarro * Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain. Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, Bilbao, España. Departamento de Microbiología, Asesoría Científica y de Investigación Aplicada, Barcelona, España. * Corresponding author. Mailing address: Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili. Carrer Sant Llorenç,.0 Reus, Spain. Phone -. Fax: -. josep.guarro@urv.cat Words Abstract= Words text= Downloaded from on October, 0 by guest

2 0 0 ABSTRACT The in vitro activity of amphotericin B in combination with micafungin was evaluated against isolates representing species of Candida. Overall, the percentages of synergistic interactions were 0% and 0% when the MIC- and MIC-0 endpoint criteria, respectively, were used. Antagonism was not observed. Some of the interactions were confirmed by time-kill assays. Downloaded from on October, 0 by guest

3 0 INTRODUCTION Candida spp. are an important cause of nosocomial infections with high morbidity and mortality (). The spectrum of invasive candidiasis is changing, and the incidence of infections due to non-c. albicans species is rising (). Amphotericin B () is one of the antifungal agents most commonly used to treat most invasive mycoses but its toxicity limits its use. Fluconazole (FLC) is commonly used for the treatment of candidiasis, but its activity is variable against non-c. albicans species, especially C. glabrata, and nil against C. krusei (, ). Echinocandins are a new and unique class, in that they inhibit synthesis of a distinct major cell wall component, β- (,)-D-glucan, resulting in morphological changes to the cell wall. Micafungin () is a promising echinocandin that was recently approved by the FDA and has demonstrated activity against Candida species, although it has shown high MICs (> µg/ml) against isolates of some species such as C. parapsilosis (,). A combination of with, two drugs with different targets, could be of interest for improving clinical results, shortening the duration of treatment and reducing toxic drug doses, which is especially important for. This combination has been synergistic in vitro against Cryptococcus spp., Rhodotorula glutinis, Trichosporon asahii and Scedosporium spp. (,, ) and showed efficacy for the treatment of murine disseminated infection by Trichosporon asahii, C. glabrata and Aspergillus spp. (,,, ). Moreover, the combination of other echinocandins, e.g. caspofungin, with has shown positive in vitro and in vivo interactions against C. parapsilosis and C. glabrata (, ). We considered it interesting to evaluate if this combination could also be beneficial for the treatment of infections by Candida spp., testing its activity against isolates representing seven of the commonest species. Downloaded from on October, 0 by guest

4 0 We tested a total of clinical isolates [C. albicans (n=), C. dubliniensis (n=0), C. glabrata (n=), C. krusei (n=), C. lusitaniae (n=), C. parapsilosis (n=) and C. tropicalis (n=)]. In order to find out which species all the strains received as C. parapsilosis complex belonged to, the internal transcribed spacer (ITS) adjacent to the.s r RNA gene was amplified and sequenced using primers ITS and ITS (0). All the sequences obtained showed a 0% similarity with the sequence of the type strain of C. parapsilosis (CBS 0, GenBank accession number AJ). Drug interactions were assessed by a checkerboard microdilution method after h of incubation at ºC (, ). The drugs were obtained as pure powders. (USP, Rockville, MD) was diluted in dimethyl sulfoxide (DMSO) and (Astellas Pharma Inc., Tokyo, Japan), in sterile distilled water. The final DMSO concentration was %. Antifungal agents were placed in rows or in columns in the trays, with concentrations ranging from to 0.0 µg/ml for and from to 0.0 µg/ml for. One of the limitations of the checkerboard method for some antifungal combinations is deciding which end point has to be used to evaluate interactions between the drugs (). The recommended endpoints for and are different, being the lowest drug concentration to show 0% growth inhibition (MIC-0) for the former () and a prominent reduction in growth (MIC-) for the latter (), however, the endpoints used in the checkerboard procedure should be the same for the two drugs tested. Since it is difficult to decide which of the two mentioned endpoints should be used, we have used both for the two drugs when tested alone and in combination. We used the fractional inhibitory concentration (FIC) index to quantify and classify drug interaction (). Interaction was considered synergistic if the FIC index was 0., indifferent if it was > 0. and, and antagonistic if it was >. The procedure, conservation of the strains and quality controls have all been detailed previously (). Approximately 0% Downloaded from on October, 0 by guest

5 0 of the tests were repeated, and the results showed the same tendencies (data not shown). However, when the results did not coincide, the test was repeated and the mode of the three MIC values was considered. The MIC values were compared using the Mann-Whitney U test. Calculations were made using Graph Pad.0 and SPSS version.0 for Windows. In order to confirm the results obtained with the checkerboard method time-kill studies were performed using two strains (one that showed indifference, and one that showed synergism by the previous methodology at MIC- endpoint) belonging to the three most common species (C. albicans FMR 00 and FMR ; C. glabrata FMR and FMR ; and C. parapsilosis FMR 0 and FMR, respectively). Both and were used at X MIC and X MIC. The numbers of CFU were determined at 0,,,, and h. The limit of detection was 0 CFU/ml. Synergism and antagonism were defined, respectively, as an increase or decrease of log CFU/ml in antifungal activity compared with the most active single agent, while a change of < log CFU/ml was considered indifferent (). Table shows the in vitro results using MIC-0 or MIC- as endpoint for both drugs, respectively. MIC-0 and MIC- of were significantly different for all the species (P<0.0) with the exception of C. albicans (P=0.0), C. glabrata (P=0.) and C. lusitaniae (P=0.). The geometric mean MIC (MIC-) of was lower than µg/ml against all species tested with the exception of C. parapsilosis, which agrees with other authors who also obtained high MICs for this species (, ). Differences between MIC-0 and MIC- were lower for, being significant only for C. parapsilosis, C. tropicalis and C. dubliniensis (P<0.000, P= 0.00, and P=0.00, respectively). For every species there is more synergism when MIC- values rather than MIC-0 values are used as endpoint. Using MIC-, synergistic interactions were Downloaded from on October, 0 by guest

6 0 obtained for % (/) of the isolates, while using MIC-0 synergistic interactions were obtained for only % (/) of them. This combination could potentially be more useful in the case of those species with strains resistant to both antifungals such as C. parapsilosis. Although this species mainly showed very high MICs, these were reduced significantly when the two drugs were combined (P<0.000) showing 0-0% of synergistic interactions. The efficacy of the combination of with one echinocandin, caspofungin, has already been proved in a murine model of disseminated infection by C. parapsilosis (). A 0% of agreement was obtained between checkerboard and time-kill (at both concentrations) procedures for the six strains tested. Figure shows the time-kill curves of the three strains that showed synergism or indifference at X MIC. In summary, we have demonstrated that the combination plus has some synergistic effect against Candida species although with important differences between the two reading criteria used. Further studies with animal models are warranted to determine the efficacy of this combination and to determine which reading criteria is more predictive for clinical outcome. Acknowledgements This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 000). Downloaded from on October, 0 by guest

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10 Table. In vitro activities of micafungin and amphotericin B, alone and in combination, against clinical isolates of Candida spp. using MIC-0 or MIC- endpoint. Candida species (number of isolates) MIC MIC-0 endpoint (µg/ml) % isolates showing the following interactions: MIC- endpoint (µg/ml) % isolates showing the following interactions: / Synergism Indifference / Synergism Indifference C. krusei () GM / /0. Range / /0.0- C. albicans () GM / /0.0 Range / / C. parapsilosis () GM. 0./ / Range -> 0.-/ /0.0- C. tropicalis () GM.. 0./ /0.0 Range / / C. dubliniensis (0) GM / / Range > /0.0- < > / C. glabrata () GM / /0.0 Range / / C. lusitaniae () GM / / Range / /0.0- /, MICs of antifungal agents in combination; GM, geometric mean;, amphotericin B;, micafungin. Downloaded from on October, 0 by guest

11 (A) (B) Log CFU/ml Log CFU/ml Log CFU/ml (C) (E) C. albicans FMR Time (h) C. parapsilosis FMR Time (h) C. glabrata FMR Time (h) Figure. Time-kill studies conducted at X MIC for C. albicans, C. parapsilosis and C. glabrata. Synergistic interactions were observed for strains FMR, FMR and FMR (A), (C) and (E) and indifferent interactions were observed for strains (D) (F) FMR 00, FMR 0 and FMR (B), (D) and (F). Log CFU/ml Log CFU/ml Log CFU/ml C. parapsilosis FMR Hora C. albicans FMR Time (h) C. glabrata FMR 0 0 Time (h) Downloaded from on October, 0 by guest