Jânio Morais Santúrio

In vitro susceptibility of fluconazole-susceptible and -resistant isolates of Malassezia pachydermatis against azoles

OBJECTIVES The first aim of this study was to evaluate the in vitro efficacies of fluconazole, ketoconazole, itraconazole and voriconazole on M. pachydermatis growth inhibition. This study also evaluated M. pachydermatis azole cross-resistance, comparing wild clinical isolates and the same isolates with in vitro-induced fluconazole resistance. METHODS Two techniques were used: (1) a broth microdilution method based on protocol M27-A3 from the Clinical and Laboratory Standards Institute to determine the minimum inhibitory concentration (MIC) and (2) the Fekete-Forgács method to induce fluconazole resistance in vitro. The isolates were divided into two groups: group 1 included fluconazole-susceptible clinical isolates (n=30) and group 2 contained the same isolates with in vitro-induced fluconazole resistance (n=30). RESULTS The two groups exhibited differences in susceptibility (p<0.001). Group 1 isolates were susceptible to azoles: ketoconazole (MIC 0.01-1.0 μg/mL), itraconazole (MIC 0.01-1.0 μg/mL), voriconazole (MIC 0.01-4.0 μg/mL), and fluconazole (MIC 0.01-4.0 μg/mL). Group 2 isolates demonstrated a wider range of MICs to azoles: ITZ (MIC 0.06-64.0 μg/mL), KTZ (MIC 0.25-32.0 μg/mL), VRZ (MIC 2.0-128.0 μg/mL), and FLZ (MIC 64.0-128.0 μg/mL). CONCLUSIONS It was shown that FLZ-resistant M. pachydermatis isolates exhibit cross-resistance to other azoles, reinforcing the importance of susceptibility tests as a guide for the therapeutic prescription of antifungals in medical and veterinary mycology.

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.

In vitro activity of carvacrol and thymol combined with antifungals or antibacterials against Pythium insidiosum.

We describe the in vitro activities of the combinations of carvacrol and thymol with antibiotics (azithromycin, clarithromycin, minocycline and tigecycline) and antifungal agents (amphotericin B, caspofungin, itraconazole and terbinafine) against 23 isolates of the oomycete Pythium insidiosum. The assays were based on the M38-A2 technique and checkerboard microdilution. Based on the mean FICI values, the main synergies observed were combinations of carvacrol+itraconazole and thymol+itraconazole (96%), thymol+clarithromycin (92%), carvacrol+clarithromycin (88%), thymol+minocycline (84%), carvacrol+minocycline (80%), carvacrol+azithromycin (76%), thymol+azithromycin (68%), carvacrol+tigecycline (64%) and thymol+tigecycline (60%). In conclusion, we found that combinations of carvacrol or thymol with these antimicrobial agents might provide effective alternative treatments for cutaneous pythiosis due to their synergistic interactions. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.

Insights into the pathophysiology of iron metabolism in Pythium insidiosum infections

Pythium insidiosum causes life-threatening disease in mammals. Animals with pythiosis usually develop anemia, and most human patients are reported to have thalassemia and the major consequence of thalassemia, iron overload. Therefore, this study evaluated the iron metabolism in rabbits experimentally infected with P. insidiosum. Ten infected rabbits were divided into two groups: one groups received a placebo, and the other was treated with immunotherapy. Five rabbits were used as negative controls. The hematological and biochemical parameters, including the iron profile, were evaluated. Microcytic hypochromic anemia was observed in the infected animals, and this condition was more accentuated in the untreated group. The serum iron level was decreased, whereas the transferrin level was increased, resulting in low saturation. The level of stainable iron in hepatocytes was markedly decreased in the untreated group. A high correlation was observed between the total iron binding capacity and the lesion size, and this correlation likely confirms the affinity of P. insidiosum for iron. The data from this study corroborate the previous implications of iron in the pathogenesis of pythiosis in humans and animals.

Carbohydrate assimilation profiles of Brazilian Candida dubliniensis isolates based on ID 32C system

The purpose of the present study was to evaluate the identification of 19 Brazilian C. dubliniensis based on the biochemical profile exhibited when tested by the commercial identification kit ID 32C (bioMerieux). Thirteen of the isolates were rigorously identified as C. dubliniensis and the remaining isolates (six) were considered as having a doubtful profile but the software also suggested that there was 83.6% of chances for them to be C. dubliniensis. As well as pointed by the literature the identification obtained by phenotypic tests should be considered presumptive for C. dubliniensis due to variability of this new species.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Efeitos in vitro de ocratoxina A, deoxinivalenol e zearalenona sobre a viabilidade celular e atividade de E-ADA em linfócitos de frangos de corte¹

Micotoxinas representam um vasto grupo de contaminantes quimicos naturais originados a partir do metabolismo secundario de fungos filamentosos patogenicos. Elas sao produzidas, principalmente, pelos generos Fusarium, Alternaria, Aspergillus e Penicillium, os quais podem contaminar graos e cereais, como trigo, milho e soja. Conforme sua natureza e niveis de concentracao, micotoxinas podem induzir efeitos toxicos em animais de producao e humanos. Um estudo in vitro foi realizado para avaliar a susceptibilidade das celulas linfocitarias de frangos de corte a diferentes concentracoes de ocratoxina A, deoxinivalenol e zearalenona. Cada micotoxina foi adicionada ao meio celular em diferentes concentracoes (0,001; 0,01; 0,1 e 1μg/mL). A viabilidade celular e atividade de ecto-adenosina desaminase foram analisadas em 24, 48 e 72 horas atraves de ensaios colorimetricos. Para isso, foram utilizados 0,7x105 linfocitos/mL em meio RPMI 1640, suplementado com 10% de soro fetal bovino e 2,5 UI de penicilina/estreptomicina por mL, incubados em atmosfera de 5% de CO2 a 37 °C. Todos os experimentos foram realizados em triplicata e os resultados foram expressos como media e erro padrao da media. Os resultados obtidos demonstraram que tanto ocratoxina A como deoxinivalenol induziram proliferacao linfocitaria e baixa atividade enzimatica in vitro (P 0,05). Foi possivel correlacionar os dados referentes a viabilidade celular e atividade de ecto-adenosina desaminase, sugerindo que, em concentracoes minimas, as micotoxinas testadas nao estimularam a atividade da enzima, que possui acao pro-inflamatoria e contribui para o processo de imunossupressao e, portanto, evitando um decrescimo na viabilidade celular. Este e o primeiro estudo feito com OCRA, DON e ZEA sobre linfocitos de frangos de corte em cultivos in vitro na avaliacao desses parâmetros.

Susceptibility to heat and antifungal agents of Cryptococcus neoformans var. neoformans (Serotype D) isolated from Eucalyptus spp in Rio Grande do Sul, Brazil

In this work we studied the susceptibility to heat and antifungal agents of the first strains of environmental Cryptococcus neoformans var neoformans (serotype D) isolated in the state of Rio Grande do Sul, Brazil. In order to achieve a rigorous analysis, we employed the methodology recommended by NCCLS, Yeast Nitrogen Base (YNB) proposed by Ghannoum et al (YNB-1), Antibiotic medium 3 (AM3) indicated by others, YNB adjusted to the NCCLS methodology (YNB-2) and Etest. Our results indicate that all strains were susceptible to amphotericin B (0.0625 – 0.5 μg/mL), fluconazole (0.125 – 8.0 μg/mL), itraconazole (0.031 – 0.25 μg/mL) and flucytosine (0.125 – 4.0 μg/mL). The C. neoformans serotype D strains were more susceptible to heat (47oC / 30 min) than C. neoformans serotype A.

Enzimas de função hepática na aflatoxicose aguda experimental em frangos de corte

The aim ofthis study is to evaluate ihe hepatic function of experimentally intoxicated broilers by aflatoxin with and without sodium bentonite. Forty Ross mole broilers, were used divided into 4 groups of 10 birds, and such groups have been submitted to the following treatments: T1- control (feed without aflatoxin or sodium bentonite), T2- feed containing 5ppm of aflatoxin, T3- feed containing 5ppm of aflatoxin and 0.5% of sodium bentonite and T4- feed containing 0.5% of sodium bentonite. Ali these treatments have been appiied from the lst to the 42nd day of lif e. On the days 21, 35 and 42, the serum leveis of the enzimes aspartate aminotransferase (AST), alanino aminotransferase (ALT), lactate dehydrogenase (LDH) and gamma glutamiltransferase (GGT). The analysis of variance showed an interaction between treatments and time of blood sampling for the following variables: AST, LDH and GGT. Tukey test was used to compare significant averages among treatments. It was observed that the enzimes AST, ALT and GGT have not shown significam diferences among treatments. However, in treatment 2, AST showed a linear increase (p < 0.01) along the experiment, more pronounced in treatment 2. The ALT had no variation in any treatment during the total period. The GGT enzyme in treatments 2 and 3 had a linear increase (p < 0.05) for ali periods. The LDH leveis, at the 21st day, was higher for treatments 2 and 3 compared to treatments l and 4. According to the results, it can be concluded that is possible to detect the hepatotoxicity of aflatoxin in through monitoring of enzimes AST and GGT leveis, in the serum of birds between 21 and 42 days of age, and by evaluation ofLDH at 21 days.

Phylogenetic analysis of Pneumocystis from pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil

The Pneumocystis genus is comprised of pathogens dwelling in the lungs of terrestrial, aerial, and aquatic mammals. Occasionally they induce severe pneumonitis, particularly in hosts with severe impairment of the immune system and progressively may fill pulmonary alveolar cavities causing respiratory failure. Molecular genetic studies revealed that Pneumocystis gene sequences present a marked divergence with the host species concerned. In the present study, the genetic diversity of Pneumocystis obtained from lungs of swines was examined by analyzing mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences. The samples were obtained from two slaughterhouses located in two Brazilian states. Phylogenetic analysis demonstrated that genetic groupings within Pneumocystis organisms were in accordance with those of the corresponding hosts and that two clusters were formed. In conclusion, these data show that there are genetically distinct porcine Pneumocystis genotypes with at least two separate clusters in Brazil.