Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil.

OBJECTIVES: The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS: The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS: Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS: Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

Comparison of commercial methods and the CLSI broth microdilution to determine the antifungal susceptibility of Candida parapsilosis complex bloodstream isolates from three health institutions in Rio de Janeiro, Brazil.

Two commercial methods, the Etest and Vitek 2, were compared with the Clinical and Laboratory Standards Institute broth microdilution method to determine the susceptibility of Candida parapsilosis complex to amphotericin B, caspofungin, fluconazole, voriconazole, and itraconazole. One-hundred bloodstream isolates of C. parapsilosis complex from three hospitals in Rio de Janeiro city, Brazil, between 1998 and 2006 were analyzed. C. parapsilosis sensu stricto (61 %) was the predominant species, followed by C. orthopsilosis (37 %) and C. metapsilosis (2 %). Most isolates were susceptible to the tested drugs. However, one C. parapsilosis sensu stricto isolate was considered resistant for amphotericin B. The essential agreement was 100 % between the methods, except for itraconazole (96.3 %). The categorical agreement varied for fluconazole and itraconazole by Etest and for amphotericin B and fluconazole by Vitek 2. This study reinforces the suitability of the commercial methods in routine clinical microbiology laboratories for antifungal susceptibility testing.

Phenotypical properties associated with virulence from clinical isolates belonging to the Candida parapsilosis complex.

The production of virulence attributes in three reference strains and 11 clinical isolates primarily identified as Candida parapsilosis was evaluated. Morphological and phenotypical tests were not able to discriminate among the three species of the C. parapsilosis complex; consequently, molecular methods were applied to solve this task. After employing polymerase chain reaction-based methods, nine clinical strains were identified as C. parapsilosis sensu stricto and two as C. orthopsilosis. Protease, catalase, and hemolysin were produced by all 14 strains, while 92.9% and 78.6% of strains secreted, respectively, esterase and phytase. No phospholipase producers were detected. Mannose/glucose, N-acetylglucosamine, and sialic acid residues were detected at the surface of all strains, respectively, in high, medium, and low levels. All strains presented elevated surface hydrophobicity and similar ability to form biofilm. However, the adhesion to inert substrates and mammalian cells was extremely diverse, showing typical intrastrain variations. Overall, the strains showed (1) predilection to adhere to plastic over glass and the number of pseudohyphae was more prominent than yeasts and (2) the interaction process was slightly enhanced in macrophages than fibroblasts, with the majority of fungal cells detected inside them. Positive/negative correlations were demonstrated among the production of these virulence traits in C. parapsilosis complex.