Ana Carolina Remondi Souza

Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus.

The significant increase in the use of antifungal agents, both for the treatment of candidiasis and invasive aspergillosis and as azole fungicides in agricultural crop protection has resulted in the emergence of resistant clinical isolates, particularly to triazoles and echinocandins. Notably, among isolates that were primarily sensitive to fluconazole such as Candida parapsilosis and Candida tropicalis have witnessed an emerging resistance development. Also for echinocandins, the occurrence of Candida isolates with lower susceptibility to these drugs has been reported, which is possibly due to its broad clinical use. Triazole resistance among Aspergillus fumigatus and other Aspergillus species is commonly found in European and Asian countries. Specific mutations are associated with azole resistance in A. fumigatus and these mutations are now reported globally from six continents. Therefore, we highlight the need to conduct antifungal resistance surveillance studies using clinical isolates of Candida and Aspergillus in different geographical regions and monitoring of the infection rates in distinct population groups for early detection of resistance to these drugs and implementation of efficient policies for infection control and treatment.

Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

ABSTRACT We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-d-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Candida parapsilosis Resistance to Fluconazole: Molecular Mechanisms and In Vivo Impact in Infected Galleria mellonella Larvae

ABSTRACT Candida parapsilosis is the main non-albicans Candida species isolated from patients in Latin America. Mutations in the ERG11 gene and overexpression of membrane transporter proteins have been linked to fluconazole resistance. The aim of this study was to evaluate the molecular mechanisms in fluconazole-resistant strains of C. parapsilosis isolated from critically ill patients. The identities of the nine collected C. parapsilosis isolates at the species level were confirmed through molecular identification with a TaqMan qPCR assay. The clonal origin of the strains was checked by microsatellite typing. The Galleria mellonella infection model was used to confirm in vitro resistance. We assessed the presence of ERG11 mutations, as well as the expression of ERG11 and two additional genes that contribute to antifungal resistance (CDR1 and MDR1), by using real-time quantitative PCR. All of the C. parapsilosis (sensu stricto) isolates tested exhibited fluconazole MICs between 8 and 16 μg/ml. The in vitro data were confirmed by the failure of fluconazole in the treatment of G. mellonella infected with fluconazole-resistant strains of C. parapsilosis. Sequencing of the ERG11 gene revealed a common mutation leading to a Y132F amino acid substitution in all of the isolates, a finding consistent with their clonal origin. After fluconazole exposure, overexpression was noted for ERG11, CDR1, and MDR1 in 9/9, 9/9, and 2/9 strains, respectively. We demonstrated that a combination of molecular mechanisms, including the presence of point mutations in the ERG11 gene, overexpression of ERG11, and genes encoding efflux pumps, are involved in fluconazole resistance in C. parapsilosis.

Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

ABSTRACT Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.

Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms.

The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients. It has also been shown that different fungal strains produce gp43 with at least four isoform profiles. In this study, different isoform profiles from gp43, with pIs ranging from 5.8 to 8.5, were affinity purified from various P. brasiliensis (B-339, S.S., 1925 and I9) exoantigens. Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients. By using a specific and sensitive method for detection of human IgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody. Our results showed that recognition of pI8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339. On the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patients sera reacted similarly. The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P. brasiliensis strains.

Inhibition of bacterial and fungal pathogens by the orphaned drug auranofin

BACKGROUND We identified auranofin as an antimicrobial compound utilizing a high-throughput screen using a Caenorhabditis elegans-Staphylococcus aureus infection model. Results/methodology: Treatment of infected nematodes with auranofin resulted in a prolonged survival rate of 95%, reached with 0.78 μg/ml. Further investigation of the antimicrobial activity of auranofin found inhibition against S. aureus, Enterococcus faecium and Enterococcus faecalis. Importantly, the fungal pathogens Cryptococcus neoformans was also effectively inhibited with an MIC at 0.5 μg/ml. Auranofin appears to target the thioredoxin system. CONCLUSION This work provides extensive additional data on the antibacterial effects of auranofin that includes both reference and clinical isolates and reports a novel inhibition of fungal pathogens by this compound.

Revisiting Species Distribution and Antifungal Susceptibility of Candida Bloodstream Isolates from Latin American Medical Centers

The epidemiology of candidemia varies geographically, and there is still scarce data on the epidemiology of candidemia in Latin America (LA). After extensive revision of medical literature, we found reliable and robust information on the microbiological aspects of candidemia in patients from 11 out of 21 medical centers from LA countries and 1 out of 20 from Caribbean countries/territories. Based on 40 papers attending our search strategy, we noted that C. albicans remains the most common species causing candidemia in our region, followed by C. parapsilosis and C. tropicalis. In Argentina, Brazil, and Colombia, a trend towards an increase in frequency of C. glabrata candidemia was observed. Although resistance rates to fluconazole is under 3%, there was a slight increase in the resistance rates to C. albicans, C. parapsilosis and C. tropicalis isolates. Echinocandin resistance has been reported in a few surveys, but no single study confirmed the resistant phenotype reported by using molecular methods. We highlight the importance of conducting continuous surveillance studies to identify new trends in terms of species distribution of Candida and antifungal resistance related to episodes of candidemia in LA. This information is critical for helping clinicians to prevent and control Candida bloodstream infections in their medical centers.

Clinical impact of Candida spp. biofilm production in a cohort of patients with candidemia

Persistent candidemia refers to the continued isolation of the same Candida species in the blood of a candidemic patient despite appropriate therapy. Despite the clinical importance of persistent candidemia, studies have superficially addressed the biological conditions behind this phenomenon. The aim of this study was to evaluate the correlation between the biofilm-forming ability by Candida bloodstream isolates and the persistence of infection. A total of 55 isolates of Candida were tested and characterized in two groups: (i) group I, which included seven patients with persistent candidemia, and (ii) group II, which included 18 patients with nonpersistent candidemia. Microorganisms were identified at the species level by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Biofilm quantification was evaluated by the crystal violet staining method and confocal scanning laser microscopy (CSLM). Molecular tests confirmed the identification of Candida albicans (92% group I and 94% group II) and Candida dubliniensis isolates (8% group I and 6% group II). All 55 isolates were able to form biofilms, but a higher biofilm mass was produced by C. albicans/C. dubliniensis strains cultured from the persistent group (P < .05). Our data suggest that Candida sp. biofilm production should be considered a relevant biologic variable in explaining patients who fail to clear a bloodstream infection despite adequate antifungal treatment with triazoles.

Pathogenesis of the Candida parapsilosis Complex in the Model Host Caenorhabditis elegans

Caenorhabditiselegans is a valuable tool as an infection model toward the study of Candida species. In this work, we endeavored to develop a C. elegans-Candida parapsilosis infection model by using the fungi as a food source. Three species of the C. parapsilosis complex (C. parapsilosis (sensu stricto), Candida orthopsilosis and Candida metapsilosis) caused infection resulting in C. elegans killing. All three strains that comprised the complex significantly diminished the nematode lifespan, indicating the virulence of the pathogens against the host. The infection process included invasion of the intestine and vulva which resulted in organ protrusion and hyphae formation. Importantly, hyphae formation at the vulva opening was not previously reported in C. elegans-Candida infections. Fungal infected worms in the liquid assay were susceptible to fluconazole and caspofungin and could be found to mount an immune response mediated through increased expression of cnc-4, cnc-7, and fipr-22/23. Overall, the C. elegans-C. parapsilosis infection model can be used to model C. parapsilosis host-pathogen interactions.

High rate of Candida deep-seated infection in patients under chronic hemodialysis with extended central venous catheter use.

BACKGROUND Hemodialysis has been described as an important risk factor for the development of candidemia in patients suffering from chronic renal failure. AIMS The aim of this study was to evaluate the epidemiology of candidemia in outpatients with renal replacement therapy (RRT) by hemodialysis where the fungemia clearly represents a healthcare-associated infection. METHODS We retrospectively collected clinical and laboratory data from patients undergoing at least 3 months of RRT by hemodialysis who developed candidemia within 48h of hospital admission. RESULTS We identified 14 patients with candidemia with central venous catheters (CVC) in place for 11-277 days before developing fungemia. Deep-seated infection was documented in 6 out of 14 candidiasis cases (43%), including 5 cases of endocarditis (36%). CONCLUSIONS CVC in patients under RRT should be promptly replaced by fistulas and grafts to avoid bloodstream infections. Facing a case of candidemia, adequate source control and prompt initiation of antifungal therapy are mandatory to avoid morbidity and mortality.