Scedosporium Prolificans
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caxcan Repositorio Institucional de la Universidad Autónoma de Zacatecas Mycopathologia DOI 10.1007/s11046-017-0137-5 Comparative Pathogenicity of Lomentospora prolificans (Scedosporium prolificans) Isolates from Mexican Patients Mariana Elizondo-Zertuche . Alexandra M. Montoya . Efre´n Robledo-Leal . Idalia Garza-Veloz . Ana L. Sa´nchez-Nu´n˜ez . Raquel Ballesteros-Elizondo . Gloria M. Gonza´lez Received: 6 December 2016 / Accepted: 20 April 2017 Ó Springer Science+Business Media Dordrecht 2017 Abstract We identified 11 Lomentospora prolificans used as markers of pathogenicity. All of the strains isolates recovered from Mexican patients using phe- were resistant to every antifungal tested with MIC’s for notypic and molecular characteristics. The identifica- AmB (8–[8 lg/ml), VRC (16–[16 lg/ml), PSC (16– tion of isolates was assessed by internal transcribed [16 lg/ml), FLC (64–[64 lg/ml) and echinocandins spacer (ITS rDNA) sequencing. In vitro susceptibility with MICs C8 lg/ml. One hundred, ninety and sixty to amphotericin B, fluconazole, voriconazole, percent of the infected mice with the strains 07-2239, posaconazole, caspofungin, anidulafungin and mica- 11-2242 and 04-2673 died during the study, respec- fungin was determined according to Clinical and tively. Regarding tissue burden, the highest fungal load Laboratory Standards Institute (CLSI) procedures. of the infected mice was detected in brain followed by Three isolates (07-2239, 11-2242 and 04-2673) were spleen and kidney, regardless of the strain. used to induce systemic infection in immunocompe- tent ICR mice. Survival and tissue burden studies were Keywords Pathogenicity Á In vitro susceptibility Á Survival study Á Tissue burden Á Lomentospora M. Elizondo-Zertuche Á A. M. Montoya Á prolificans A. L. Sa´nchez-Nu´n˜ez Á G. M. Gonza´lez (&) Departamento de Microbiologı´a, Facultad de Medicina, Universidad Auto´noma de Nuevo Leo´n, Madero y Dr. Eduardo A. Pequen˜o s/n, Colonia Mitras Centro, 64460 Monterrey, Mexico Introduction e-mail: [email protected] Lomentospora prolificans (formerly Scedosporium E. Robledo-Leal prolificans)[1] is an opportunistic filamentous mold; Departamento de Microbiologı´a e Inmunologı´a, Facultad de Ciencias Biolo´gicas, Universidad Auto´noma de Nuevo it has a worldwide distribution and is frequently present Leo´n, San Nicola´s de los Garza, Mexico in soil, sewage and polluted waters. It is responsible for serious infections due to its high virulence, propensity I. Garza-Veloz for invasion, dissemination and multidrug resistance to Laboratorio de Medicina Molecular, Unidad Acade´mica de Medicina Humana y Ciencias de la Salud, Universidad antifungals [2, 3]. Infections caused by this organism Auto´noma de Zacatecas, Zacatecas, Mexico can be localized, extended to the surrounding tissues or disseminated to distant organs depending of the infec- R. Ballesteros-Elizondo tion route and immunological status. When L. prolif- Departamento de Histologı´a, Facultad de Medicina, Universidad Auto´noma de Nuevo Leo´n, Monterrey, icans infects immunocompetent individuals, usually Mexico having a prior trauma in the form of puncture wounds, 123 Mycopathologia skin ulcers or surgery, it produces localized infections assimilation was determined for sucrose, L-arabinose, that involve skin, bone and joints [4, 5]. In immuno- lactose, D-ribose and maltose according to a method compromised patients with cystic fibrosis, where the previously described [15]. Hydrolysis of urea was most important risk factor for the acquisition of this determined using Christensen’s urea agar which was pathogen is inhalation and especially in those suffering incubated at 30 °C for 4 days, cycloheximide toler- hematologic malignancies or organ-transplant recipi- ance was determined by the isolates’ development on ents receiving immunosuppressive treatment, lesions Mycosel agar incubated at 30 °C for 10 days, and the spread and they are usually fatal in less than a month. gelatinase activity was assessed in gelatin agar slants Johnson et al. reported in 2014 that Scedosporiosis after 20 days at 30 °C[16]. Aspartyl proteinase (Scedosporium apiospermum/Lomentospora prolifi- activity was evaluated using bovine serum albumin cans) followed lung transplantation in 55% of the cases medium [17]. Phospholipase activity was carried out as well as other organ transplants (multivisceral, 18%; on egg yolk medium [18]. Hemolytic activity was heart, liver and small intestine in 45%). Scedosporiosis assayed using Sabouraud blood medium [19]. In these was preceded by colonization in 36% of the reports. three last tests, plates were incubated at 30 °C for Diseases included pneumonia (64%), mediastinitis 7 days and activity was expressed in terms of presence (18%) and fungemia/disseminated infections (18%) or absence. Thermotolerance was studied by incubat- [5, 6]. It is well documented that an altered immune ing the isolates on PDA for two weeks at 30, 37 and status of the host seems to be the main cause of invasion 40 °C[16]. Candida albicans ATCC 90028 was used by this mold, which mimics most of the pathologic as the quality control for all enzymatic activity aspects of other opportunistic fungi such as Fusarium determinations. spp., [7] Acremonium spp., [8]orAspergillus fumigatus In vitro susceptibility testing was performed by [9]. In these cases, the infection is probably acquired by the broth microdilution method for filamentous fungi inhalation of conidia and the fungus is able to develop in according to the CLSI document M38-A2 [20]. The practically any body organ. On the other hand, L. assayed antifungal agents were: amphotericin B prolificans shows universal in vitro and in vivo resis- (AmB) (Bristol-Myers Squibb, Princeton, NJ), flu- tance to available antifungal drugs [5, 10–14] and conazole (FLC) (Pfizer, Inc., New York, NY), recovery from neutropenia has been considered a posaconazole (PSC) (Merck, Rahway, NJ) and mandatory prerequisite for resolving the infection voriconazole (VRC) (Pfizer, Inc., New York, NY), irrespective of the antifungal treatment used [10]. which were obtained as pure reagent-grade powders; In this study, we identified 11 clinical isolates of L. caspofungin (CSF), anidulafungin (ANF) and mica- prolificans by morphological and molecular criteria. fungin (MCF) were purchased as Cancidas (Merck Furthermore, we evaluated the pathogenicity of three Sharp & Dohme), Eraxis (Pfizer, Inc.) and Myca- isolates of L. prolificans in an immunocompetent mine (Astellas Pharma, Inc.), respectively. The final murine model of disseminated infection. concentrations of the drugs ranged from 0.125 to 64 lg/ml for FLC, from 0.03 to 16 lg/ml for VRC, PSC and AmB and from 0.015 to 8 lg/ml for Materials and Methods echinocandins. The microplates were incubated at 35 °C for 72 h, and the MICs for all antifungals Strains Identification were read visually. Amphotericin B, voriconazole and posaconazole end points were determined as the A total of 11 isolates collected over a period of first clear well showing no growth, whereas flucona- 10 years (2003–2013) of L. prolificans were recovered zole was read at C50% inhibition compared to the from a broad spectrum of clinical presentations and control well. For echinocandins, MECs were deter- identified based on their macroscopic and microscopic mined microscopically as the lowest concentration morphologies and ITS rDNA sequencing. Regarding of drug promoting the growth of small, round, the phenotypic approach, clinical isolates were grown compact hyphae relative to the appearance of the at 30 °C on potato dextrose agar (PDA) for 2 weeks. filamentous forms seen in the control wells. Assays For microscopic observation, slide cultures were made were done in duplicate using Candida parapsilosis and incubated at 30 °C for 7 days. Carbohydrates ATCC 22019 and Paecilomyces variotii MYA 3630 123 Mycopathologia as quality control organisms. For in vitro suscepti- bility testing, we determined the geometric mean and range of MICs and MECs. For ITS rDNA sequencing, the DNA of the isolates was extracted using the method described by Gonza´lez Outcome after diagnosis et al. [21]. Ribosomal DNA ITS domains were acute myelogenous amplified using the forward primer ITS-5 (50- AML GGAAGTAAAAGTCGTAACAAGG-30) and reverse primer ITS-4 (50-TCCTCCGCTTATTGATATGC-30) [22]. Amplifications were performed in a final volume treatment of 30 ll containing 19 GoTaq Colorless Master Mix (Promega, Madison, WI), 200 nM of each primer and 20 ng of DNA. The thermocycling conditions were: 94 °C for 4 min, followed by 35 cycles of 94 °C for bronchoalveolar lavage, 30 s, 53 °C for 30 s and 72 °C for 30 s, with final extension at 72 °C for 3 min. The final products were BAL electrophoresed in 1.5% agarose gels and stained with SYBRÒ Green I Gel Nucleic Acid Gel Stain (Invit- rogenTM, Eugene, OR). HyperLadder I (Bioline USA Inc.) was used as a molecular weight marker for size determinations. The pattern of amplified bands was photographed and analyzed with the UVP Bioimaging System, EpiChemi III Darkroom. PCR products were L. prolificans purified using the commercial Wizard SV Gel and bone marrow transplantation, PCR Clean-Up system (Promega, Madison, WI). Sequencing was performed utilizing the BigDye BMT Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster city, CA) following the manufac- turer’s directions. Reactions were run and analyzed in Gender Underlying disease Site of isolates Antifungal an Avant 3100