Juliana Siqueira Argenta

In Vitro Activities of Voriconazole, Itraconazole, and Terbinafine Alone or in Combination against Pythium insidiosum Isolates from Brazil

ABSTRACT We evaluated the in vitro activities of voriconazole, itraconazole, and terbinafine against 30 clinical isolates of Pythium insidiosum using a checkerboard macrodilution method. The combined activity of terbinafine plus itraconazole or plus voriconazole was synergic against 17% of the strains. Antagonism was not observed.

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.

In Vitro Activity of Terbinafine Combined with Caspofungin and Azoles against Pythium insidiosum

ABSTRACT In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin, miconazole, ketoconazole, and fluconazole against 17 Pythium insidiosum strains by using the microdilution checkerboard method. Synergistic interactions were observed with terbinafine combined with caspofungin (41.2% of the strains), fluconazole (41.2%), ketoconazole (29.4%), and miconazole (11.8%). No antagonistic effects were observed. The combination of terbinafine plus caspofungin or terbinafine plus fluconazole may have significant therapeutic potential for treatment of pythiosis.

Diversity of yeasts from bovine mastitis in Southern Brazil.

Mastitis is one of the most serious problems in the dairy cattle farms. The great majority of the cases are caused by bacteria, but lately there have been an increasing number of reports about cases of mycotic etiology. The objective of this work was to characterize the yeasts and yeast-like fungi associated with milk of cows with mastitis. Milk samples (n = 248) from a dairy belt situated around the region of Passo Fundo, hinterland of the state of Rio Grande do Sul, Southern Brazil, were analyzed. Aliquots of 0.1 ml of milk were inoculated on yeast extract-malta agar with chloramphenicol. After a period of incubation of 3-5 days at 22-25 degrees C, the counting of the morphologically distinct colonies was performed, as well as the isolation and identification through phenotypical and physiological criteria. It was possible to isolate 68 yeast species from 43 (17.3%) of the samples. The most frequent genera were Candida (37.9%), Pichia (19.1%), Cryptococcus (10.3%) and Rhodotorula (10.3%).

In vitro and in vivo susceptibility of two-drug and three-drug combinations of terbinafine, itraconazole, caspofungin, ibuprofen and fluvastatin against Pythium insidiosum.

The present study investigated the in vitro inhibitory activity of terbinafine, itraconazole, caspofungin, fluvastatin and ibuprofen against 15 isolates of Pythium insidiosum in double and triple combinations and determined in vivo correlations using rabbits with experimental pythiosis. The minimal inhibitory concentration (MIC) was determined in accordance with the Clinical and Laboratory Standards Institute M 38-A2 protocol (2008), and the in vitro interactions were evaluated using a checkerboard microdilution method. For the in vivo study, 20 rabbits inoculated with P. insidiosum zoospores were divided into four groups: group 1 was treated with terbinafine and itraconazole; group 2 was treated with terbinafine, itraconazole and fluvastatin; group 3 was treated with terbinafine and caspofungin; and group 4 was the control group. Combinations of terbinafine with caspofungin or ibuprofen were synergistic for 47% of the isolates, and antagonism was not observed in any of the double combinations. The triple combinations were mostly indifferent, but synergism and antagonism were also observed. In the in vivo study, the histological aspect of the lesions was similar among the groups, but group 2 showed the lowest amount of hyphae and differed significantly from the other groups.

Zoosporogênese in vitro entre isolados do oomiceto Pythium insidiosum

Pythium insidiosum is an aquatic oomycete and the etiology of a chronic disease called pythiosis, commonly found in the skin of horses. The production of mobile zoospores by this microorganism is the determinant factor of this disease. This study evaluated the zoosporogenesis and quantification of zoospores in 32 samples of Pythium insidiosum isolated from horses with pythiosis. The assay used culture of the Pythium insidiosum in Corn Meal Agar plus grass blades for 5 days at 37°C. The grass blades were incubated in Induction Medium at 37°C for 24 hours. The findings showed 16 samples (50%) yielded 20,000 zoospores mL-1, 12 (37.5%) samples yielded over 20,000 zoospores mL-1 and 4 samples (12.5%) yielded less than 20,000 zoospores mL-1. The zoospores production was higher between 6 and 8 hours of incubation. The protocol used in the zoosporogenesis induction was efficient and represents an important tool for Pythium insidiosum identification and the attainment of zoospores in adequate amounts for inoculation in experimental animals and application in the development of susceptibility tests.

In vitro paradoxical growth of Pythium insidiosum in the presence of caspofungin

Pythium insidiosum is a zoosporic organism which causes pythiosis in humans and animals. This study aimed to report the paradoxical growth of Brazilian P. insidiosum strains when submitted to in vitro susceptibility tests with caspofungin. The growth at concentrations above the minimal inhibitory concentration (MIC) ranged from 16 to 128 μg/ml and it was observed in 50% of the isolates tested. This paradoxical growth in the presence of caspofungin has been observed with Candida and Aspergillus strains, however, the phenomenon involving oomycetes was described here for the first time.