Aspectos microbiológicos e moleculares de isolados clínicos pertencentes ao complexo Candida haemulonii
Data
2023-07-25
Autores
Lima, Soraia Lopes 
Orientadores
Colombo, Arnaldo Lopes
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O complexo de espécies Candida haemulonii compreende leveduras patogênicas oportunistas emergentes, representadas por C. haemulonii sensu stricto, C. haemulonii var. vulnera, C. duobushaemulonii, C. vulturna e C. khanbhai. Este complexo de espécies é capaz de causar infecções superficiais e profundas, incluindo candidemia, especialmente em neonatos, pacientes com câncer e/ou diabetes mellitus, bem comopacientes críticos expostos a procedimentos médicos invasivos e antibióticos, e são intimamente relacionadas as espécies C. pseudohaemulonii e C. auris. Isolados clínicos desse táxon têm sido descritos como patógenos multidroga-resistentes (MDR), exibindo elevados valores de concentração inibitória mínima (CIM) aos azólicos e anfotericina B. Atualmente, os métodos fenotípicos comerciais amplamente utilizados na rotina clínicolaboratorial, não permitem a identificação acurada destes agentes, dificultando sua caracterização epidemiológica e vigilância contínua em quadros de IFIs. Além disso, dada a atual instabilidade taxonômica em que as espécies do compl. C. haemulonii são encontradas, é relevante realizar investigações moleculares e biológicas para a classificação deste táxon. Artigo 1: Considerando a falta de dados clínicos e epidemiológicos em relação às infecções causadas pelas espécies do compl. C. haemulonii, o objetivo deste estudo foi investigar tendências históricas na prevalência de leveduras multirresistentes do complexo armazenadas em uma coleção leveduras obtidas durante 11 anos, incluindo amostras coletadas de pacientes em 12 centros médicos e seu perfil de susceptibilidade in vitro. Os isolados foram identificados a partir do sequenciamento da região ITS do DNA ribossomal. Os testes de susceptibilidade aos antifúngicos para anfotericina B, voriconazol, fluconazol, anidulafungina e 5- fluorocitosina foram realizados de acordo com o documento CLSI M27A3. Das 3799 leveduras avaliadas, 49 isolados (1,3%) foram identificados como C. haemulonii ss (n= 21; 43%), seguido por C. haemulonii var. vulnera (n= 15; 30,5%), e C. duobushaemulonii
(n= 13; 26,5%). A maior parte destes isolados foram recuperados de infecções invasivas (n= 38; 77,5%). A prevalência de espécies do complexo C. haemulonii aumentou de 0,9% no primeiro período para 1,7% no segundo período de análise [P1 (2008-2013): n=18 de 1931 isolados vs. P2 (2014-2019): n= 31 de 1868 isolados; p= 0,047]. Todos os isolados apresentaram altos valores de CIM para anfotericina B e fluconazol, permanecendo susceptíveis a 5-fluorocitosina e anidulafungina. Artigo 2: O objetivo deste estudo foi avaliar a heterogeneidade molecular e biológica do complexo C. haemulonii e seu potencial impacto na taxonomia de suas espécies. Foram selecionados 75 isolados clínicos e cepas referência. A caraterização molecular foi realizada por sequenciamento das regiões ITS e D1/D2 do DNA ribossomal e AFLP-fingerprinting. Já a caracterização biológica contou com a avaliação da capacidade de formação de biofilme e testes de susceptibilidade in vitro para 4 classes de antifúngicos. Na análise da região D1/D2 do DNAr, os isolados de C. haemulonii ss (n=34) e C. haemulonii var. vulnera (n=17) apresentaram 100% de similaridade sendo agrupados no mesmo cluster. Por outro lado, agrupamentos da região ITS do DNAr e do AFLP mostraram elevada heterogeneidade entre os isolados de C. haemulonii ss e C. haemulonii var. vulnera, sugerindo a presença de outras variedades associadas à espécie SS. Não foi caracterizada diversidade intraespecífica entre os 18 isolados de C. duobushaemulonii testados, que apresentaram maior similaridade genética com C. pseudohaemulonii (taxas de similaridade: ITS= 93%;
D1/D2=97%; ALPF= 95,3% ) do que com C. haemulonii ss (taxas de similaridade:ITS=86%; D1/D2= 90%; AFLP 62.1%). Quanto aos aspectos biológicos avaliados, isolados de C. duobushaemulonii apresentaram menor capacidade de formação de biofilme e maiores valores de MIC para AMB (média geométrica= 3,76 μg/ml; MIC90 16 μg/ml; p= <0,05) em comparação com C. haemulonii ss e C. haemulonii var. vulnera
(GM= 1.67 e 1.32 μg/ml; CIM90= 2 e 2 μg/ml, respectivamente). Não houve diferenças estatísticas no perfil de susceptibilidade in vitro entre C. haemulonii ss e C. haemulonii var. vulnera (p= >0,05).
The Candida haemulonii species complex comprises emerging opportunistic yeast pathogens represented by C. haemulonii sensu stricto, C. haemulonii var. vulnera, C. duobushaemulonii, C. khanbhai and C. vulturna that are closely related to C. pseudohaemulonii and C. auris. Clinical isolates of this taxon have been described as multidrug-resistant (MDR) pathogens, exhibiting high values of minimum inhibitory concentration (MIC) to azoles and amphotericin B. Currently, commercial phenotypic methods used in clinical and laboratory routine do not allow the accurate identification of these agents, making their epidemiological characterization and surveillance difficult in cases of IFIs. Futhemore, it is relevant to carry out molecular and biological investigations for the classification of this taxon duo to the current taxonomic instability in which the species of compl. C. haemulonii are found. Article 1: We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto (n = 21), followed by C. haemulonii var. vulnera (n=15) and C. duobushaemulonii (n=13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis (p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections. Article 2: The aim of this study was to evaluate the molecular and biological heterogeneity of C. haemulonii complex and the potential impact on their taxonomy. Seventy-five isolates comprising clinical and reference strains were selected. Molecular characterization was performed by sequencing ITS and D1/D2 regions of ribosomal DNA and AFLP-fingerprinting. Biological characterization included the assessment of biofilm formation and in vitro susceptibility tests for 4 antifungal classes. In the analysis of D1/D2 region of rDNA, the isolates of C. haemulonii ss (n=34) and C. haemulonii var. vulnera (n=17) showed 100% similarity being grouped in the same cluster. On the other hand, clusters of ITS region of rDNA and AFLP showed high heterogeneity between the isolates of C. haemulonii ss and C. haemulonii var. vulnera, suggesting the presence of higher intraspecific diversity within SS species. Intraspecific diversity was not characterized among the 18 isolates of C. duobushaemulonii tested, which showed greater genetic similarity with C. pseudohaemulonii clade (similarity rates: ITS= 93%; D1/D2=97%; ALPF= 95.3%) than C. haemulonii ss clade (similarity rates: ITS=86%; D1/D2=90%; AFLP 62.1%). Regarding biological aspects, isolates of C. duobushaemulonii showed lower capacity for biofilm formation and higher MIC values for AMB (geometric mean = 3.76 μg/ml; MIC90 16 μg/ml; p= <0.05) when compared to C. haemulonii ss/C. haemulonii var. vulnera clade (GM= 1.67 and 1.32 μg/ml; MIC90= 2 and 2 μg/ml, respectively). No statistically significant difference of in vitro susceptibility profile between C. haemulonii ss and C. haemulonii var. vulnera (p=>0.05) were found.
The Candida haemulonii species complex comprises emerging opportunistic yeast pathogens represented by C. haemulonii sensu stricto, C. haemulonii var. vulnera, C. duobushaemulonii, C. khanbhai and C. vulturna that are closely related to C. pseudohaemulonii and C. auris. Clinical isolates of this taxon have been described as multidrug-resistant (MDR) pathogens, exhibiting high values of minimum inhibitory concentration (MIC) to azoles and amphotericin B. Currently, commercial phenotypic methods used in clinical and laboratory routine do not allow the accurate identification of these agents, making their epidemiological characterization and surveillance difficult in cases of IFIs. Futhemore, it is relevant to carry out molecular and biological investigations for the classification of this taxon duo to the current taxonomic instability in which the species of compl. C. haemulonii are found. Article 1: We checked for historical trends in terms of prevalence rates and antifungal susceptibility of the Candida haemulonii species complex in our yeast stock culture collected during the last 11 years. The isolates were identified by sequencing the rDNA internal transcribed spacer (ITS) region, and antifungal susceptibility tests for amphotericin B, voriconazole, fluconazole, anidulafungin, and 5-fluorocytosine were performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. A total of 49 isolates were identified as Candida haemulonii sensu stricto (n = 21), followed by C. haemulonii var. vulnera (n=15) and C. duobushaemulonii (n=13), including 38 isolates cultured from patients with deep-seated Candida infections. The prevalence of the C. haemulonii species complex increased from 0.9% (18 isolates among 1931) in the first period (December 2008 to June 2013) to 1.7% (31 isolates among 1868) in the second period (July 2014 to December 2019) of analysis (p = 0.047). All isolates tested exhibited high minimum inhibition concentrations for amphotericin B and fluconazole, but they remained susceptible to 5-fluorocytosine and anidulafungin. We were able to demonstrate the increased isolation of the multiresistant Candida haemulonii species complex in our culture collection, where most isolates were cultured from patients with deep-seated infections. Article 2: The aim of this study was to evaluate the molecular and biological heterogeneity of C. haemulonii complex and the potential impact on their taxonomy. Seventy-five isolates comprising clinical and reference strains were selected. Molecular characterization was performed by sequencing ITS and D1/D2 regions of ribosomal DNA and AFLP-fingerprinting. Biological characterization included the assessment of biofilm formation and in vitro susceptibility tests for 4 antifungal classes. In the analysis of D1/D2 region of rDNA, the isolates of C. haemulonii ss (n=34) and C. haemulonii var. vulnera (n=17) showed 100% similarity being grouped in the same cluster. On the other hand, clusters of ITS region of rDNA and AFLP showed high heterogeneity between the isolates of C. haemulonii ss and C. haemulonii var. vulnera, suggesting the presence of higher intraspecific diversity within SS species. Intraspecific diversity was not characterized among the 18 isolates of C. duobushaemulonii tested, which showed greater genetic similarity with C. pseudohaemulonii clade (similarity rates: ITS= 93%; D1/D2=97%; ALPF= 95.3%) than C. haemulonii ss clade (similarity rates: ITS=86%; D1/D2=90%; AFLP 62.1%). Regarding biological aspects, isolates of C. duobushaemulonii showed lower capacity for biofilm formation and higher MIC values for AMB (geometric mean = 3.76 μg/ml; MIC90 16 μg/ml; p= <0.05) when compared to C. haemulonii ss/C. haemulonii var. vulnera clade (GM= 1.67 and 1.32 μg/ml; MIC90= 2 and 2 μg/ml, respectively). No statistically significant difference of in vitro susceptibility profile between C. haemulonii ss and C. haemulonii var. vulnera (p=>0.05) were found.