Morphological and ultrastructural changes in fungal agents after LASER application.

BACKGROUND: Onychomycosis is a fungal nail infection of difficult treatment due to the fungal survival capacity and reduced number of effective therapies. The present study aimed to isolate fungal agents that cause onychomycosis in immunocompetent patients and evaluate how LASER treatments affect the growth and ultrastructure of isolates. METHODS: In total, 21 patients with positive direct microscopic examination (DME) for onychomycosis had nail samples collected for cultivation and phenotypic identification of microorganisms. From these patients, 12 underwent LASER treatment, divided in Group 1 (n = 5) treated with Nd: YAG 1,064 nm, and Group 2 (n = 7) treated with Nd: YAG 1,064 nm + Er: YAG 2,940 nm + topical isoconazole. Transmission Electron Microscopy (TEM) was performed to evaluate ultrastructural changes after treatment. RESULTS: DME, cultivation, and phenotypic identification showed that the most identified fungus was Trichophyton rubrum spp. After LASER therapy, sample cultivation showed alterations in the fungal morphology with reduction of hyphae, conidia, and reproductive structures. Alterations in fungal cell wall structure, cytoplasm density, and organelles were observed by TEM. CONCLUSION: LASER irradiation causes changes in the fungal cells, especially in the number of hyphae and the presence of conidia. In addition, it affects fungal growth and reproduction capacity, which interferes with their infection ability and virulence.

Candida species causing fungal keratitis: molecular identification, antifungal susceptibility, biofilm formation, and clinical aspects.

The study aimed to evaluate the clinical aspects, molecular identification, biofilm formation, and antifungal susceptibility profile of Candida species isolated from fungal keratitis. Thirteen Candida isolates from 13 patients diagnosed with Candida keratitis were retrieved and grown in pure culture. Species identification was performed by micromorphology analysis and ITS-rDNA sequencing. The broth microdilution method tested the minimum inhibitory concentration (MIC) of four antifungal drugs (fluconazole, amphotericin B, voriconazole, and anidulafungin). The biofilms were cultured and incubated with antifungal drugs for 24 h. The XTT reduction assay measured the biofilm activity. Biofilm MICs were calculated based on a 50% reduction in metabolic activity compared with the activity of the drug-free control. Among isolates, two were C. albicans, 10 were C. parapsilosis (sensu stricto), and one was C. orthopsilosis. All isolates were classified as susceptible or intermediate to all four antifungal drugs. Four isolates were very low biofilm producers (30%). Nine isolates were biofilm producers, and all biofilm samples were unsusceptible to all drugs tested. Previous ocular surgery was the most common underlying condition for fungal keratitis (84.6%), and C. parapsilosis was the most frequent Candida species (76.9%). Four patients (30.7%) needed keratoplasty, whereas two (15.3%) required evisceration. The biofilm formation ability of Candida isolates decreased antifungal susceptibility compared with planktonic cells. Despite in vitro antifungal susceptibility, almost half of the patients were unresponsive to clinical treatment and needed surgery.

Trends towards lower azole susceptibility among 200 Candida tropicalis bloodstream isolates from Brazilian medical centres.

OBJECTIVES: Candida tropicalis is one of the three most frequent species causing candidaemia in Latin America. Despite the high prevalence of C. tropicalis in candidaemia cases in Brazil, little is known about the trends in fluconazole susceptibility over time. The objective of this study was to evaluate temporal trends in azole resistance rates among C. tropicalis bloodstream isolates from patients treated in six Brazilian medical centres over a 12-year period. METHODS: We selected 200 C. tropicalis bloodstream isolates from six medical centres in Brazil collected between 2007 and 2018. Species identification was confirmed by MALDI-TOF/MS. Antifungal susceptibility testing for four antifungal agents was performed by the Clinical and Laboratory Standards Institute (CLSI) microbroth method. RESULTS: Overall, rates of non-susceptibility were 4% and 3.5% to fluconazole and voriconazole, respectively. All isolates were susceptible to amphotericin B and only one isolate was resistant to echinocandins. CONCLUSION: Although we failed to demonstrate statistical differences in the rates of azole resistance documented during the period of analysis, trends towards lower susceptibility to fluconazole and voriconazole were shown.

Candidemia surveillance in Brazil: evidence for a geographical boundary defining an area exhibiting an abatement of infections by Candida albicans group 2 strains.

Prospective population surveillance has been conducted for candidemia in Brazil (A. L. Colombo, M. Nucci, B. J. Park, et al., J. Clin. Microbiol. 44:2816-2823, 2006). In the present study, a total of 63 isolates from 61 patients, representing 11 medical centers from nine geographic regions, were characterized by multilocus sequence typing (MLST). A total of 48 unique profiles or diploid sequence types (DSTs) were observed, with nine new sequence types (STs) and 32 new DSTs. There were no apparent correlations between center/region and DST patterns. Subtypes were compared to those in a known characterized reference set, including a large database of strains obtained worldwide. Significantly, only one C. albicans group 2 isolate was found in our collection, although isolates from this particular group are commonly found worldwide. These data, combined with information from other previously reported studies, establish a statistically significant diminishment of group 2 strains in Central and South America, including Mexico and portions of the Southwestern United States.

Multilocus sequence typing of sequential Candida albicans isolates from patients with persistent or recurrent fungemia.

Multilocus sequence typing (MLST) is a useful tool to explore the phylogenetics and epidemiology of Candida albicans isolates recovered from cases of invasive candidiasis. The goal of this study was to determine whether the same or different strains were responsible for persistent or recurrent fungemia through the use of MLST and ABC typing on sequential C. albicans isolates from the same patient. We applied both typing methods to 21 C. albicans strains recovered from 8 patients with persistent or recurrent candidemia. The isolates were collected during a multicenter surveillance study in four public tertiary care hospitals in Brazil. Persistent candidemia was defined as two or more blood cultures positive for C. albicans on 2 or more separate days. Recurrent candidemia was defined as an episode of candidemia occurring at least 1 month after the apparent complete resolution of an infectious episode caused by Candida species. We observed that, except for one patient, all strains from the first and second samples of the same patient showed the same MLST diploid sequence type (DST), ABC type and susceptibility profile to antifungals. Three distinct strains, well discriminated by MLST, were found in the seven samples collected sequentially over 10 days from one patient. The strains from the first four samples were indistinguishable, the fifth and sixth were also indistinguishable but different from the first four and seventh samples. Significantly, the seventh strain was the only C. albicans clade 2 isolate found in our total collection involving 61 patients, although clade 2 is commonly found worldwide. To the best of our knowledge, this is the first study describing the recovery of three distinct C. albicans strains in the same patient with a persistent blood stream infection within a short period of time.

Clinical and microbiological aspects of candidemia due to Candida parapsilosis in Brazilian tertiary care hospitals.

In order to characterize the epidemiology, microbiology and outcome of candidemia due to Candida parapsilosis, we examined a database of 282 episodes of candidemia prospectively collected from four tertiary care hospitals in São Paulo, Brazil between March 2002 and February 2003, and compared the characteristics of patients with candidemia due to C. parapsilosis (n=64) with those caused by Candida albicans (n=107). C. parapsilosis candidemia was associated with neutropenia (p=0.005), tunneled central venous catheter (p=0.005) and cancer chemotherapy (p=0.03). By multivariate analysis, candidemia due to C. parapsilosis was associated with the presence of a tunneled central venous catheter (relative risk 3.71, 95% confidence interval 1.28-10.70). Except for a single isolate of C. parapsilosis that exhibited MIC >1 microg/ml to amphotericin B, no resistance was observed in 166 isolates tested against fluconazole, itraconazole, 5-flucytosine and amphotericin B. The caspofungin MIC values of C. parapsilosis isolates were significantly higher than those exhibited by C. albicans isolates (p<0.001). The overall mortality of patients with candidemia due to C. parapsilosis was significantly lower (45% vs. 62%, p=0.03). The association between C. parapsilosis candidemia and a tunneled central venous catheter supports the idea that the main mode of acquisition of C. parapsilosis is from an external source.

Epidemiology of candidemia in Brazil: a nationwide sentinel surveillance of candidemia in eleven medical centers.

Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Brazil to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for fluconazole-resistant Candida species. Prospective laboratory-based surveillance was conducted from March 2003 to December 2004 in 11 medical centers located in 9 major Brazilian cities. A case of candidemia was defined as the isolation of Candida spp. from a blood culture. Incidence rates were calculated per 1,000 admissions and 1,000 patient-days. Antifungal susceptibility tests were performed by using the broth microdilution assay, according to the Clinical and Laboratory Standards Institute guidelines. We detected 712 cases, for an overall incidence of 2.49 cases per 1,000 admissions and 0.37 cases per 1,000 patient-days. The 30-day crude mortality was 54%. C. albicans was the most common species (40.9%), followed by C. tropicalis (20.9%) and C. parapsilosis (20.5%). Overall, decreased susceptibility to fluconazole occurred in 33 (5%) of incident isolates, 6 (1%) of which were resistant. There was a linear correlation between fluconazole and voriconazole MICs (r = 0.54 and P < 0.001 [Spearman's rho]). This is the largest multicenter candidemia study conducted in Latin America and shows the substantial morbidity and mortality of candidemia in Brazil. Antifungal resistance was rare, but correlation between fluconazole and voriconazole MICs suggests cross-resistance may occur.