Different scenarios for Candida parapsilosis fungaemia reveal high numbers of mixed C-parapsilosis and Candida orthopsilosis infections

Nosocomial fungal bloodstream infections (BSI) are increasing significantly in hospitalized patients and Candida parapsilosis has emerged as an important pathogen responsible for numerous outbreaks. The objective of this study was to evaluate C. parapsilosis sensu lato infection scenarios, regarding species distribution and strain relatedness. One hundred isolates of C. parapsilosis sensu lato derived from blood cultures and catheter tips were analysed by multiplex microsatellite typing and by sequencing D1/D2 regions of the ribosomal DNA. Our results indicate that 9.5 % of patients presented infections due to C. parapsilosis and Candida orthopsilosis, 57.1 % due to C. parapsilosis, 28.3 % due to C. orthopsilosis and 4.8% due to Candida metapsilosis. Eighty per cent of the C. parapsilosis BSIs were due to a single strain that was also identified in the catheter, but in 10% of the cases C. parasilosis was identified in the catheter but the BSI was due to C. orthopsilosis. There is a significant probability that C. parapsilosis isolates collected from the same patient at more than 3 months interval are of different strains (P=0.0179). Moreover, several isolates were identified persistently in the same hospital, infecting six different patients. The incidence of polyfungal BSI infections with C. parapsilosis and C. orthopsilosis is reported herein for the first time, emphasizing the fact that the species identified in the catheter is not always responsible for the BSI, thus impacting the treatment strategy. The observation that strains can remain in the hospital environment for years highlights the possible existence of reservoirs and reinforces the need for accurate genotyping tools, such as the markers used for elucidating epidemiological associations and detecting outbreaks.Financial support was provided by CAPES Foundation (BEX 19194/12-9), Ministry of Education of Brazil, Brasilia (DF 70.040-020), by FEDER through POFC-COMPETE and by Portuguese funds from FCT (PEst-OE/BIA/UI4050/2014). R.M.Z.-O. is supported in part by CNPq (350338/2000-0) and FAPERJ E (26/103.157/2011). We are grateful to Ronaldo Rozembaun from HSE and SAM and Andrea Pussenti Derossi from HUPE for providing the Candida isolates and technical assistance in sampling. Automated sequencing was done using the genomic platform/DNA sequencing platform at Fundacao Oswaldo Cruz, PDTIS/FIOCRUZ (RPT01A), Brazil. The authors declare that they have no conflict of interest.info:eu-repo/semantics/publishedVersio

Características fenotípicas associadas à virulência e ao perfil de suscetibilidade aos antifúngicos em isolados clínicos do complexo Candida glabrata

Made available in DSpace on 2018-06-25T11:52:28Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) maria_carvalho_ini_dout_2017.pdf: 12451752 bytes, checksum: 013fad1a745fd54310b64c8b7a729e47 (MD5)Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil.Candida glabrata é um patógeno emergente nos hospitais públicos e privados brasileiros. Baseado em análises moleculares, Candida nivariensis e Candida bracarensis foram descritas como duas novas espécies filogeneticamente relacionadas à C. glabrata, formando o complexo C. glabrata. O objetivo deste trabalho foi estudar as características fenotípicas associadas à virulência e ao perfil de suscetibilidade aos antifúngicos em isolados clínicos previamente identificados como C. glabrata oriundos de pacientes com quadro de candidíase entre 1998 e 2015 em dois hospitais públicos no município do Rio de Janeiro. Um total de 92 isolados clínicos foi submetido à análise molecular com base na amplificação e sequenciamento da região ITS1-5.8S-ITS2 do DNA ribossomal. A produção de enzimas hidrolíticas foi avaliada em placas ou tubos contendo meios ou reagentes específicos e a formação de biofilme foi determinada pelo método do cristal violeta e pelo ensaio de redução do XTT. O perfil de suscetibilidade aos antifúngicos in vitro foi determinado pelo método da microdiluição em caldo (CLSI, M27-A3). Candida glabrata stricto sensu foi a espécie predominante (n=91), seguida por C. nivariensis (n=1) a qual foi pela primeira vez descrita no Brasil. C. bracarensis não foi encontrada neste estudo. Em geral, os isolados de C. glabrata stricto sensu foram bons produtores de catalase, aspártico protease, esterase, fitase e hemolisina. Entretanto, não foram detectadas atividades in vitro de caseinase e fosfolipase. Além disso, esses isolados foram capazes de formar biolfime. Todos os isolados de C. glabrata stricto sensu foram suscetíveis à 5-fluorocitosina Entretanto, esses isolados apresentaram resistência à anfotericina B (9,9%), fluconazol (15,4%), itraconazol (5,5%), caspofungina (8,8%) ou micafungina (15,4%). Alguns isolados foram classificados como tipo não-selvagem para o voriconazol (33,0%) e para o posaconazol (4,4%). Anfotericina B e micafungina foram mais eficazes do que o itraconazol e a fluorocitosina frente aos isolados clínicos de C. glabrata stricto sensu na presença do biofilme. Relações estatisticamente significativas foram encontradas (i) entre o perfil de suscetibilidade da micafungina e a produção de esterase, bem como entre o perfil de suscetibilidade do fluconazol, itraconazol, micafungina e a atividade hemolítica; e (ii) entre a produção de diferentes enzimas hidrolíticas, esterase e hemolisina, e a formação de biofilme (p<0,05). O isolado de C. nivariensis foi excelente produtor de aspártico protease e catalase, e um bom produtor de fitase, porém, nenhuma atividade in vitro foi detectada para as demais enzimas testadas. Este isolado também foi capaz de formar biofilme. O isolado de C. nivariensis foi suscetível à anfotericina B, caspofungina e 5-fluorocitosina, porém suscetível dose-dependente ao itraconazol e resistente ao fluconazol e à micafungina. Para voriconazol e posaconazol, este isolado foi classificado como tipo selvagem e tipo não-selvagem, respectivamente. Este estudo reforça o potencial de virulência dessas espécies e destaca o perfil de resistência de alguns isolados aos principais fármacos geralmente usados no tratamento da candidíase.Candida glabrata is an emerging pathogen in public and private Brazilian hospitals. Based on molecular analysis, two new species phenotypically closely related to resemble C. glabrata have been described: Candida nivariensis and Candida bracarensis. This study evaluated the phenotypic characteristics associated with virulence and the antifungal susceptibility profile in clinical isolates previously identified as C. glabrata from patients with candidiasis between 1998 and 2015 in two public hospitals in the city of Rio de Janeiro. A total of 92 clinical isolates were submitted to molecular analysis based on the amplification and sequencing of the ribosomal DNA ITS1-5.8S-ITS2 region. The production of hydrolytic enzymes was evaluated in plates or tubes containing specific media or reagents and biofilm formation was evaluated by the crystal violet method and the XTT reduction assay. The in vitro antifungal susceptibility profile was determined by the broth microdilution method (CLSI, M2-A3). Candida glabrata stricto sensu was the predominant species (n = 91), followed by C. nivariensis (n = 1), which was for the first time described in Brazil. Candida bracarensis was not found in this study. Overall, C. glabrata stricto sensu isolates were good producers of catalase, aspartic protease, esterase, phytase, and hemolysin. No in vitro activities were detected for caseinase and phospholipase Moreover, these isolates were able to produce biolfim. All isolates of C. glabrata stricto sensu were susceptible to flucytosine. However, these isolates showed resistance to amphotericin B (9.9%), fluconazole (15.4%), itraconazole (5.5%), caspofungin (8.8%), or micafungin (15.4%). Some isolates were classified as non-wild-type for voriconazole (33.0%) and posaconazole (4.4%). Amphotericin B and micafungin were more effective than itraconazole and fluorocytosine against to clinical isolates of C. glabrata stricto sensu forming biofilms. Statistically significant correlations were identified between (i) micafungin minimum inhibitory concentration (MIC) and esterase production, as well as between fluconazole and micafungin MIC and hemolytic activity, and between amphotericin B MIC and phytase production, and between (ii) the production of different hydrolytic enzymes, esterase and hemolysin, and biofilm formation (p<0.05). The C. nivariensis isolate was an excellent producer of aspartic protease and catalase, and a good phytase producer. No in vitro activity was detected for the other enzymes tested. This isolate was also able to form biofilm, susceptible to amphotericin B, caspofungin and flucytosine, but susceptible dose-dependent to itraconazole, and resistant to fluconazole and micafungin. For voriconazole and posaconazole, this isolate was classified as wild type and non-wild type, respectively. This study reinforces the virulence potential of these species and highlights the resistance profile of some isolates to the main drugs commonly used for candidiasis management

Determinação da susceptibilidade aos fármacos antifúngicos de amostras do complexo de espécies de Candida parapsilosis isoladas de pacientes com fungemia

Made available in DSpace on 2014-12-22T16:37:26Z (GMT). No. of bitstreams: 2 maria_carvalho_ipec_mest_2012.pdf: 3003635 bytes, checksum: 510838807146764c9ee0f462f0cf0ea7 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014-10-07Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas, Rio de Janeiro, RJ, Brasil.Dentre as espécies de Candida não-albicans, Candida parapsilosis vem emergindo como importante patógeno de infecções fúngicas invasivas com disseminação hematogênica nas últimas décadas em diferentes continentes, principalmente, na Europa e na América Latina. C. parapsilosis foi considerada por muito tempo um complexo de três grupos distintos nomeados I, II e III. Tavanti et al (2005), baseado na tipagem da sequência multilocus (MLST), propôs o reconhecimento dos grupos II e III, como duas novas espécies: C. orthopsilosis e C. metapsilosis, respectivamente, mantendo o grupo I como C. parapsilosis stricto sensu. Até agora só tem sido possível distinguir essas três espécies por análise molecular. Métodos comerciais para testes de susceptibilidade aos antifúngicos vêm sendo utilizados para avaliar o comportamento de Candida spp. frente às drogas de uso clínico, incluindo o Etest® e o sistema Vitek® 2. Neste trabalho, estes dois métodos foram comparados ao método de referência de microdiluição em caldo pelo CLSI (M27-A3) para determinar a susceptibilidade in vitro de isolados clínicos do complexo psilosis aos fármacos antifúngicos anfotericina B, fluconazol, voriconazol e itraconazol. Um total de 53 isolados do complexo psilosis oriundos de hemoculturas obtidas de pacientes hospitalizados no município do Rio de Janeiro, entre 1998 e 2006, associados a episódios de fungemia, foram analisados Cinquenta e um isolados foram discriminados pela PCR, utilizando primers espécie-específicos, e dois isolados, pelo sequenciamento, sendo caracterizados como C. parapsilosis stricto sensu (75,4%), C. orthopsilosis (20,8%) e C. metapsilosis (3,8%). Os testes de susceptibilidade aos antifúngicos indicaram que a maioria dos isolados de C. parapsilosis stricto sensu foi sensível a todos os fármacos testados. Entretanto, um único isolado de C. parapsilosis stricto sensu apresentou uma CIM = 2 [g/mL para a anfotericina B. Três isolados de C. orthopsilosis apresentaram CIM entre 2 e 8 [g/mL para o fluconazol pelo Etest® e Vitek® 2 e CIM entre 0,19 e 0,25 [g/mL para o itraconazol pelo Etest®. Os isolados de C. metapsilosis foram sensíveis a todos os fármacos testados. A concordância essencial entre Etest® ou Vitek® 2 com CLSI foi excelente (100%), exceto para o itraconazol (90,9%). Por outro lado, a concordância categórica foi 72,7% para o itraconazol pelo Etest® e 100% para os outros fármacos por ambos os métodos. Para anfotericina B, a concordância categórica foi de 100% para o Etest® e de 97,5% pelo Vitek® 2 em relação ao CLSI. Este estudo reforça a importância dos métodos Etest® e Vitek® 2 que podem ser empregados nos laboratórios rotineiros de microbiologia clínica para monitorar e detectar diferenças no perfil de susceptibilidade aos antifúngicos dos isolados de C. parapsilosis stricto sensu, C. orthopsilosis e C. metapsilosisAmong non-albicans Candida species, Candida parapsilosis has emerged as an important agent of invasive fungal infections, and several cases associated with fungemia have been reported worldwide in last decade, mostly in Europe and Latin America. For many years, C. parapsilosis has been characterized as a complex composed of three genetically distinct groups (groups I, II, and III). Tavanti et al. (2005) based on multilocus sequence typing (MLST) technique, proposed the recognition of groups II and III as two different species: C. orthopsilosis and C. metapsilosis, respectively, maintaining the group I as C. parapsilosis sensu stricto. Up to now only has been possible to distinguish these three species just by molecular analysis. Commercial antifungal susceptibility methods including Etest® and Vitek® 2 system have been used to test the antifungal susceptibility of Candida spp. In this study, these methods were compared to the CLSI broth microdilution (BMD) reference method to determine in vitro susceptibility of clinical C. parapsilosis complex isolates to amphotericin B, fluconazole, voriconazole, and itraconazole. A total of 53 C. parapsilosis complex isolates from blood cultures obtained of patients who were hospitalized in the city of Rio de Janeiro between 1998 and 2006 associated with episodes of fungemia were analysed. Fifty-one isolates were discriminated by PCR using species-specific primers and two isolates by sequencing, being characterized as C. parapsilosis sensu stricto (75.4%), C. orthopsilosis (20.8%), and C. metapsilosis (3.8%). Antifungal susceptibility tests indicated that most of C. parapsilosis sensu stricto isolates were susceptible to all tested drugs. However, a single C. parapsilosis sensu stricto isolate presented MIC = 2 [g/ml for amphotericin B. Three C. orthopsilosis isolates showed MIC between 2-8 [g/ml for fluconazole by Vitek® 2 and MIC between 0.19-0.25 [g/ml for itraconazole by Etest®. C. metapsilosis isolates were susceptible to all tested drugs. The essential agreement between the Etest® or Vitek® 2 with the CLSI BMD for all drugs was excellent (100%), except for itraconazole (90.9%). The categorical agreement was 72.7% for itraconazole by Etest®, 97.5% for amphotericin B by Vitek® 2, and 100% for the other drugs by both methods compared with CLSI BMD. This study reinforces the importance of Etest® and Vitek® 2 methods in routine clinical microbiologycal laboratories to survey and detect differences in the profile of the antifungal susceptibility of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis isolates

Comparison of four molecular approaches to identify Candida parapsilosis complex species

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories

Evaluation of melanin production by Sporothrix luriei

<div><p>There is a paucity of studies on the cell biology of Sporothrix luriei, the less common of the pathogenic Sporothrix species worldwide. The production of DHN-melanin, eumelanin, and pyomelanin were evaluated on the mycelial and yeast forms of the S. luriei ATCC 18616 strain. The mycelial form of this species produced only pyomelanin, which protected the fungus against environmental stressors such as ultraviolet light, heat, and cold. The yeast form was unable to produce any of the tested melanin types. The lack of melanin in the parasitic form of S. luriei may be an explanation for its low frequency in human infections.</p></div