Lucas Xavier Bonfietti

Prevalence, distribution and antifungal susceptibility profiles of Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis bloodstream isolates

The Candida parapsilosis group encompasses three species: C. parapsilosis, Candida orthopsilosis and Candida metapsilosis. These species are phenotypically indistinguishable, and molecular methods are needed for their detection. We analysed 152 unique blood culture isolates of the C. parapsilosis group obtained during 1997-2011. The isolates were screened by PCR amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Isolates with RFLP patterns distinct from those of the C. parapsilosis group were characterized as C. parapsilosis sensu stricto (90.8 %), C. orthopsilosis (8.6 %) and C. metapsilosis (0.6 %). Antifungal susceptibility tests indicated that all isolates were susceptible to itraconazole, amphotericin B and caspofungin. Although C. orthopsilosis and C. metapsilosis isolates were susceptible to fluconazole, higher MICs (≥2 mg l(-1)) were observed for C. orthopsilosis. Three isolates (2.0 %) of C. parapsilosis sensu stricto were resistant to voriconazole. Five C. parapsilosis isolates (3.3 %) were intermediate, and a single isolate (0.7 %) was resistant (MIC 16 mg l(-1)) to fluconazole. These data were confirmed using reference strains. It was observed that C. parapsilosis isolates were less susceptible to all triazoles, and this finding deserves further attention to assess the appearance of cross-resistance phenomena. In conclusion, C. metapsilosis and C. orthopsilosis are involved in a small but significant number of invasive infections in Brazil.

SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY

SUMMARY During recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

Susceptibility to antifungal agents and genotypes of Brazilian clinical and environmental Cryptococcus gattii strains.

There are few reports concerning the in vitro antifungal susceptibility of clinical and environmental Cryptococcus gattii isolates. In this study, we performed polymerase chain reaction-restriction fragment length polymorphism to investigate the molecular subtypes of 50 clinical and 4 environmental Brazilian isolates of C. gattii and assessed their antifungal susceptibility for fluconazole (FLU) and amphotericin B (Amb) according to recent recommendations proposed for antifungal susceptibility testing of nonfermentative yeasts. Time-kill curve studies were performed using RPMI 1640 medium to analyze the fungicidal effect of AmB. We found 47 VGII (94%) molecular types and 3 VGI (6%) types among the clinical isolates. The environmental isolates were VGII (75%) subtype and VGI (25%) subtype. The FLU-MIC ranged from 1 to 64 mg L(-1), and MIC(50)/MIC(90) values were, respectively, 8/16 mg L(-1). For AmB, the MICs were low and homogeneous, ranging from 0.12 to 0.5 mg L(-1), for VGI or VGII. The time required to reach the fungicidal end point (99.9% killing) was 6 h for the majority of strains (64%), but viable cells of VGII were still present after 48 h of exposition. We pointed out the occurrence of high FLU-MICs for C. gattii isolates with highest values for VGII. Our data also suggest that the rate of killing of C. gattii by AmB is strain dependent, and viable cells of VGII genotype strains were still observed after an extended incubation time, addressing future studies to determine whether the in vitro fungicidal activity could be clinically relevant.

Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time–kill methodology

The in vitro activities of amphotericin B (AmB) were evaluated against 40 isolates of Cryptococcus neoformans using time-kill curves. The isolates were obtained from 20 AIDS patients with cryptococcal meningitis submitted to AmB therapy. Isolates were exposed in vitro to 1 microg/mL of AmB that represents a serum concentration of AmB, and the viable colony counts were determined over time. AmB exhibited fungicidal activity at 6 and 12 h for 70.6% of isolates, at 24 h for 7.3%, and at 48 h for 22% of isolates, respectively. This effect was not maximized when the test drug concentration was up to 4 times the AmB MIC for the isolates. Regrowth was observed in 17.5% of the isolates after fungicidal endpoint. With standard in vitro susceptibility testing, this tolerance phenomenon could not be assessed, and thus, these tests may underestimate the resistance of C. neoformans to AmB in vivo. AmB is the first-choice drug for the treatment of cryptococcosis in Brazil, and future studies using time-kill methodology are needed to estimate the predictive value of this test in the clinical failure.

Population Genetic Analysis Reveals a High Genetic Diversity in the Brazilian Cryptococcus gattii VGII Population and Shifts the Global Origin from the Amazon Rainforest to the Semi-arid Desert in the Northeast of Brazil

Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence.

Species distribution and antifungal susceptibility profile of Candida isolates from bloodstream infections in Lima, Peru.

Yeast identification and in vitro susceptibility testing provide helpful information for appropriate administration of antifungal treatments; however, few reports from the Latin American region have been published. The aim of this study was to identify the species present in isolates from bloodstream infections diagnosed in nine hospitals in Lima, Peru and to determine their in vitro susceptibility to four antifungal drugs. We tested and identified 153 isolates collected between October 2009 and August 2011 using standard methods. PCR and PCR-RFLP assays were performed to distinguish Candida albicans from Candida dubliniensis and to identify species of the Candida parapsilosis and Candida glabrata complexes. Antifungal susceptibility testing for fluconazole, anidulafungin and voriconazole was performed using the CSLI M27-A3 method, and amphotericin B susceptibility was determined using the Etest method. The most frequently isolated species were: C. albicans (61; 39.9 %), C. parapsilosis (43; 28.1 %), C. tropicalis (36; 23.5%) and C. glabrata (8; 5.2 %). The overall susceptibility rates were 98.0 %, 98.7 %, 98.0 % and 97.4 % for amphotericin B, fluconazole, voriconazole and anidulafungin, respectively. No isolate was resistant to more than one drug. These results showed that the rate of resistance to four antifungal drugs was low among Candida bloodstream isolates in Lima, Peru.

Comparison of the broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing and the Clinical Laboratory Standards Institute BMD method for non-Candida albicans and non-C. tropicalis bloodstream isolates from eleven tertiary hospitals in São Paulo state, Brazil

We aim in this study to provide levels of susceptibility of 162 bloodstream isolates of non-Candida albicans and non-C. tropicalis species from a sentinel program conducted in 11 hospitals in Brazil. Additionally, we compared the broth microdilution (BMD) method of the European Committee of Susceptibility Testing (EUCAST) with Clinical Laboratory Standards Institute (CLSI) BMD method for fluconazole, itraconazole, voriconazole, and amphotericin B. The study included 103 C. parapsilosis, 38 C. glabrata, 8 C. orthopsilosis, and 7 C. krusei isolates, and single isolates of Pichia anomala, C. famata, C. lusitaniae, C. kefyr, C. guilliermondii, and C. metapsilosis. Of note, we observed cross-resistance between fluconazole and voriconazole for two isolates being one C. parapsilosis and one C. glabrata. Good essential agreement (EA) was observed between the EUCAST and the CLSI results for C. parapsilosis and for fluconazole, itraconazole, voriconazole, and amphotericin B, respectively: 98%, 99%, 98%, and 97%. Otherwise, for C. glabrata, the EA for fluconazole was 84.2% and for voriconazole 89.4%. Because data from Brazil are scarce, our results contribute to the consolidation of the database of candidemia agents and monitoring of trends in the profile of drug resistance.

Onychomycoses in a Military Population in Brazil

Purpose of ReviewThis study aimed to isolate and characterize filamentous fungi onychomycosis agents in a military population assisted at a hospital outpatient clinic.Recent FindingsIn onychomycosis, the fungi colonize the subungual region causing thickening, discoloration, or cracking of the nail bed. Samples were collected from patients with clinical sights of onychomycosis.SummaryAmong 80 samples collected, 50 (62.5%) had positive culture. Isolated dermatophytes (86%) were Trichophyton rubrum (21; 42%), T. mentagrophytes var. interdigitale (19; 38%), and Microsporum gypseum (3; 6%) and non-dermatophyte molds were Fusarium spp. (1; 2%), Scytalidium spp. (1; 2%), and Chaetomium globosum (5; 10%). Minimal inhibitory concentrations (mg/L) of terbinafine, itraconazole, and fluconazole necessary to inhibit 50/90% of the isolates were respectively 0.015/0.06, 0.06/0.12, and 32/32. Etiological agents of onychomycosis in a military hospital are similar as reported in studies for the general population. High prevalence of non-dermatophytic agents was observed, especially for Chaetomium globosum.