An Azole-Resistant Candida parapsilosis Outbreak: Clonal Persistence in the Intensive Care Unit of a Brazilian Teaching Hospital

The incidence of candidemia by the Candida parapsilosis complex has increased considerably in recent decades, frequently related to use of indwelling intravascular catheters. The ability of this pathogen to colonize healthcare workers (HCW)' hands, and to form biofilm on medical devices has been associated with the occurrence of nosocomial outbreaks and high mortality rates. Fluconazole has been the leading antifungal drug for the treatment of invasive candidiasis in developing countries. However, azole-resistant C. parapsilosis isolates are emerging worldwide, including in Brazil. Few studies have correlated outbreak infections due to C. parapsilosis with virulence factors, such as biofilm production. We thus conducted a microbiological investigation of C. parapsilosis complex isolates from a Brazilian teaching hospital. Additionally, we identified a previously unrecognized outbreak caused by a persistent azole-resistant C. parapsilosis (sensu stricto) clone in the intensive care unit (ICU), correlating it with the main clinical data from the patients with invasive candidiasis. The molecular identification of the isolates was carried out by PCR-RFLP assay; antifungal susceptibility and biofilm formation were also evaluated. The genotyping of all C. parapsilosis (sensu stricto) was performed by microsatellite analysis and the presence of ERG11 mutations was assessed in the azole non-susceptible isolates. Fourteen C. parapsilosis (sensu stricto) isolates were recovered from patients with invasive candidiasis, eight being fluconazole and voriconazole-resistant, and two intermediate only to fluconazole (FLC). All non-susceptible isolates showed a similar pattern of biofilm formation with low biomass and metabolic activity. The A395T mutation in ERG11 was detected exclusively among the azole-resistant isolates. According to the microsatellite analysis, all azole non-susceptible isolates from the adult ICU were clustered together indicating the occurrence of an outbreak. Regarding clinical data, all patients infected by the clonal non-susceptible isolates and none of the patients infected by the susceptible isolates had been previously exposed to corticosteroids (p = 0.001), while the remaining characteristics showed no statistical significance. The current study revealed the persistence of an azole non-susceptible C. parapsilosis clone with low capacity to form biofilm over two years in the adult ICU. These results reinforce the need of epidemiological surveillance and monitoring antifungal susceptibility of C. parapsilosis isolates in hospital wards

Análise das espécies do complexo candida haemulonii, cryptococcus spp e paracoccidiodes sp por esctrometria de massas maldi-tof

AIMS: 1.) Describe the identification performance of the platforms and assess to the ribosomal protein expression for the species of the complex C. haemulonii. 2.) Analyze the interference of polysaccharide capsule for the identification of Cryptococcus species. 3.) Analyze the expression of ribosomal proteins between the species of Paracoccidíoides. Methods: For the complex of C. haemuloníi 29 clinical samples were analyzed, they were subjected to sequencing of the ITS region and extraction of ribosomal proteins with formic acid and acetonitrile for analysis by MALDITOF. The sequence data were compared with the NCBI BLAST platform site. Data extraction were compared with Biotyper database, and analyzed by FlexAnalysis and ClinProTools. The analysis ofthe interference ofthe polysaccharide capsule was done with eight genotypes of Cryptococcus, cultivated in different media (inductor capsule growth medium Sabouraud agar and reducing medium capsule) and analyzed the thickness of each genotype capsule in each type of culture medium and correlated with the identification of such genotypes by MALDI-TOF. The differentiation of the species of Paracoccidioides analyzes were performed by MALDI-TOF after extraction of ribosomal proteins through changes in the extraction protoco!. Results: Ali 29 samples of the complex C. haemulonii were correctly identified by sequencing and were not , correctly identified by MALDI-TOF. The species C. haemulonii and C. haemulonii var vulnera are very similar and few representative variations in their mass spectra. The thickness of the polysaccharide capsule of Cryptococcus genotypes is relevant to the quality of the spectra obtained by MALDI-TOF and the correct identification of the species. Paracoccidíoides species were correctly identified MALDI-TOF and differences were identified from the mass spectra between species. Conclusions: The sequence is the only way to correct identification of the species of the complex C. haemulonii. The distinctions between the species C. haemulonií and C. haemulonii var vulnera were scarce, but present. The reduction of Cryptococcus capsule was necessary for the correct identification of the Cryptococcus species. There were significant differences between mass spectra among Paracoccidioides species and the correct identification by MALDI-TOF was obtained.Objetivos: 1.) Descrever a performance de plataformas de identificação e avaliar a expressão proteica ribossomal para as espécies do complexo C. haemulonii. 2.) Analisar a interferência da cápsula de polissacarídeo para a correta identificação das espécies de Cryptococcus. 3) Analisar a expressão de proteínas ribossomais entre as espécies de Paracoccidioides. Métodos: Para o complexo de C. haemulonii foram estudadas 29 amostras clínicas, que foram submetidas ao sequenciamento da região ITS e extração das proteínas ribossomais com ácido fórmico e acetonitrila para análise por MALDI-TOF. Os dados do sequenciamento foram comparados com a plataforma BLAST do site NCBI. Os dados da extração foram comparados com o banco de dados Biotyper, e foram analisados pelo FlexAnalysis e ClinProTools. Para a análise da interferência da cápsula de polissacarídeos, os oito genótipos de Cryptococcus foram cultivados em meios de cultura diferentes (indutor do crescimento da cápsula, meio Sabouraud ágar e meio redutor de cápsula) e foi analisado a espessura da cápsula de cada ge~ótipo em cada tipo de meio de cultura e correlacionada com a identificação desses genótipos através de MALDI-TOF. Para a diferenciação das espécies de Paracoccidioides foram feitas análises por MALDI-TOF após extração das proteínas ribossomais através de modificações no protocolo de extração. Resultados: Todas as 29 amostras do complexo C. haemulonii foram identificadas corretamente pelo sequenciamento e não foram corretamente identificadas por MALDI-TOF. As espécies C. haemulonii e C. haemulonii var vulnera são muito semelhantes e com poucas variações representativas em seus espectros de massas. A espessura da cápsula de polissacarídeos dos genótipos de Cryptococcus é relevante na qualidade dos espectros obtidos por MALDI-TOF e na correta identificação das espécies. As espécies de Paracoccidioides foram corretamente identificadas MALDI-TOF e foram identificadas diferenças nos espectros de massas entre as espécies. Conclusões: O sequenciamento é a única forma de correta identificação entre as espécies do complexo C. haemulonii. As distinções entre as espécies C. haemulonii e C. haemulonii var vulnera são pouco relevantes, mas presentes. A redução da cápsula de Cryptococcus é necessária para a correta identificação das espécies de Crypiococcus. Há distinção entre os espectros deDados abertos - Sucupira - Teses e dissertações (2013 a 2016