Browsing by Author "Sampaio, Paula"
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- Genotipagem de Candida parapsilosis com marcadores de DNA microssatélite: uma ferramenta para o estudo e controlo das infeções hospitalaresPublication . Sabino, Raquel; Sampaio, Paula; Veríssimo, Cristina; Pais, Célia
- Isolates from hospital environments are the most virulent of the Candida parapsilosis complexPublication . Sabino, Raquel; Sampaio, Paula; Carneiro, Catarina; Rosado, Laura; Pais, CéliaBackground: Candida parapsilosis is frequently isolated from hospital environments, like air and surfaces, and causes serious nosocomial infections. Molecular studies provided evidence of great genetic diversity within the C. parapsilosis species complex but, despite their growing importance as pathogens, little is known about their potential to cause disease, particularly their interactions with phagocytes. In this study, clinical and environmental C. parapsilosis isolates, and strains of the related species C. orthopsilosis and C. metapsilosis were assayed for their ability to induce macrophage cytotocixity and secretion of the pro-inflammatory cytokine TNF-a, to produce pseudo-hyphae and to secrete hydrolytic enzymes. Results: Environmental C. parapsilosis isolates caused a statistically significant (p = 0.0002) higher cell damage compared with the clinical strains, while C. orthopsilosis and C. metapsilosis were less cytotoxic. On the other hand, clinical isolates induced a higher TNF-a production compared with environmental strains (p < 0.0001). Whereas the amount of TNF-a produced in response to C. orthopsilosis strains was similar to the obtained with C. parapsilosis environmental isolates, it was lower for C. metapsilosis strains. No correlation between pseudo-hyphae formation or proteolytic enzymes secretion and macrophage death was detected (p > 0.05). However, a positive correlation between pseudo-hyphae formation and TNF-a secretion was observed (p = 0.0119). Conclusions: We show that environmental C. parapsilosis strains are more resistant to phagocytic host defences than bloodstream isolates, being potentially more deleterious in the course of infection than strains from a clinical source. Thus, active environmental surveillance and application of strict cleaning procedures should be implemented in order to prevent cross-infection and hospital outbreaks.
- New polymorphic microsatellite markers able to distinguish among Candida parapsilosis sensu stricto isolatesPublication . Sabino, Raquel; Sampaio, Paula; Rosado, Laura; Stevens, David A.; Clemons, Karl V.; Pais, CéliaAmong the Candida species causing bloodstream infections, Candida parapsilosis is one of the most frequently isolated. The objective of the present work was the identification of new microsatellite loci able to distinguish among C. parapsilosis isolates. DNA sequences with trinucleotide repeats were selected from the C. parapsilosis genome database. PCR primer sets flanking the microsatellite repeats were designed and tested with 20 independent isolates. On the basis of the amplification efficiency, specificity, and observed polymorphism, four of the sequences were selected for strain typing. Two hundred thirty-three independent C. parapsilosis sensu stricto isolates were genotyped by using these markers. The polymorphic loci exhibited from 20 to 42 alleles and 39 to 92 genotypes. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99. Reproducibility was demonstrated by submission of subcultures of 4 isolates each, in triplicate, interspersed with unique numbers among a group of 30 isolates for blind testing. Comparison of the genotypes obtained by microsatellite analysis and those obtained by randomly amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, and internal transcribed sequence grouping was performed and showed that the microsatellite method could distinguish individual isolates; none of the other methods could do that. Related species, C. orthopsilosis and C. metapsilosis, were not confused with C. parapsilosis sensu stricto. These new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis sensu stricto strains, vital in epidemiology to answer questions of strain relatedness and determine pathways of transmission.