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Purpose: The purpose of this study was to assess the influence of cleaning pits and fissures with an aluminum oxide air abrasion system an the detection of occlusal caries in primary teeth using laser fluorescence (LF) and visual examination. Methods: The sample comprised 65 pit and fissure sites on extracted primary teeth suspected to be carious. The sites were submitted to 2 visual examinations (examiner JAR) and 2 LF readings (examiner TMV). Next, the occlusal surfaces were air-abraded and re-examined thereafter using both methods. The teeth were sectioned, and the histological analysis of the sites with a stereoscopic magnifying lens at X32 magnification was used as the gold standard. Results: Cohen's kappa statistic for LF and visual examination were, respectively, 0.282/0.884 before and 0.896/0.905 after air abrasion. LF showed a sensitivity of 0.28 increasing to 0.49 and a specificity of 0.50 increasing to 0.92. Visual examination showed sensitivity of 0.78 and specificity of 0.73. Bath increased after air abrasion. Conclusion: The findings suggest that cleaning pits and fissures with aluminum oxide air abrasion increased the accuracy of LF and visual examination for detection of occlusal caries in primary teeth. [ABSTRACT FROM AUTHOR]

Rabies is an endemic disease in Brazil, where it is considered a serious public health problem. Although the number of human and dog-transmitted cases has declined in recent decades, rabies in wildlife has emerged considerably. Among the sylvatic animals, wild canids have been considered important hosts of the rabies virus. We performed a retrospective study of reported cases of rabies in wild canids and human victims in Ceará state (Northeast Brazil) during 2003 to 2013. Information was provided by governmental laboratories involved in rabies detection and by the Ministry of Health. From January 2003 to December 2013, a total of 11 931 animal samples were examined for rabies. Positivity were detected in 438 samples (3.67%), of which 229 (52.28%) were domestic animals, 105 (23.97%) wild canids and 104 (23.74%) other wild animals (bats, marmosets and raccoons). Approximately 33% of wild canids surveyed (n = 317) were positive for rabies. During the studied period, a total of 1923 attacks on humans by wild canids were registered. Males (n = 1405) were more affected than females (n = 520; 72.98% versus 27.01%), and the median age of all cases was 36.5 years. Injuries to individuals up to 19 years old corresponded to approximately 30% (n = 565) of all cases. Most of the victims lived in rural areas (72.46%; n = 1395), and the majority showed bites (81.13%; n = 1677) or scratches (12.23%; n = 253). Injuries were considered profound (52.1%; n = 1003), superficial (40.91; n = 788) or multiple with severe laceration (6.98%; n = 134). Only 1300 (67.53%) victims were enrolled for the complete rabies post-exposure prophylaxis scheme. Data from the present study confirm that wild canids are important hosts of rabies virus in northeastern Brazil and jeopardize rabies control in this area. Local authorities should focus their efforts in education of health professionals. In addition, strategies should be formulated to preserve wildlife., (© 2016 Blackwell Verlag GmbH.)

Coccidioidomycosis is a potentially severe infection caused by dimorphic fungi Coccidioides immitis and Coccidioides posadasii. Although guidelines are well established, refractory disease is a matter of concern in the clinical management of coccidioidomycosis. In the present study three isoniazid-derived hydrazones N'-[(E)-1-(4-methoxyphenyl)ethylidene]pyridine-4-carbohydrazide, N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide, and N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide were synthesized and evaluated for antifungal activity against C. posadasii. Susceptibility assays were performed by macrodilution testing. Interactions between the hydrazones and amphotericin B or itraconazole were evaluated by the checkerboard method. We also investigated the impairment of such compounds on cell ergosterol and membrane integrity. The synthesized molecules were able to inhibit C. posadasii in vitro with MIC values that ranged from 25 to 400 μg/mL. Drug interactions between synthesized molecules and amphotericin B proved synergistic for the majority of tested isolates; regarding itraconazole, synergism was observed only when strains were tested against N'-[(E)-1-(phenyl)ethylidene]pyridine-4-carbohydrazide. Reduction of cellular ergosterol was observed when strains were challenged with the hydrazones alone or combined with antifungals. Only N'-[(E)-1-(4-methylphenyl)ethylidene]pyridine-4-carbohydrazide altered membrane permeability of C. posadasii cells. Isoniazid-derived hydrazones were able to inhibit C. posadasii cells causing reduction of ergosterol content and alterations in the permeability of cell membrane. This study confirms the antifungal potential of hydrazones against pathogenic fungi., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1μgmL(-1) for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4μgmL(-1)and from 0.0625 to 1μgmL(-1) for filamentous and yeast phase, respectively. Concerning the antifungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125μgmL(-1) and from 0.0039 to 0.0312μgmL(-1) for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p<0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum., (Copyright © 2016. Published by Elsevier Editora Ltda.)

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To investigate the isolation of enterobacteria associated with Macrobrachium amazonicum (M. amazonicum) farming and evaluate the in vitro antimicrobial susceptibility of Vibrio strains., Methods: Strains were isolated from female M. amazonicum prawns and environmental and hatchery water. Biochemical assays were used to identify bacterial genera and those belonging to the genus Vibrio were submitted to further analyses for species identification, through Vitek 2 automated system and serotyping. Susceptibility test was performed according to Clinical Laboratory Standards Institute., Results: The following genera of enterobacteria were recovered: Enterobacter (n = 11), Citrobacter (n = 10), Proteus (n = 2), Serratia (n = 2), Kluyvera (n = 2), Providencia (n = 2), Cedecea (n = 1), Escherichia (n = 1), Edwardsiella (n = 1) and Buttiauxella (n = 1). As for Vibrio, three species were identified: Vibrio cholerae non-O1/non-O139 (n = 4), Vibrio vulnificus (V. vulnificus) (n = 1) and Vibrio mimicus (n = 1). Vibrio spp. showed minimum inhibitory concentrations values within the susceptibility range established by Clinical Laboratory Standards Institute for almost all antibiotics, except for V. vulnificus, which presented intermediate profile to ampicillin., Conclusions: Enterobacteria do not seem to be the most important pathogens associated with M. amazonicum farming, whereas the recovery of Vibrio spp. from larviculture, with emphasis on Vibrio cholerae and V. vulnificus, deserves special attention due to their role as potentially zoonotic aquaculture-associated pathogens. Furthermore, the intermediate susceptibility of V. vulnificus to ampicillin reflects the importance of monitoring drug use in prawn farming., (Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the antifungal susceptibility of Candida spp. recovered from tortoises (Chelonoidis spp.) and sea turtles (Chelonia mydas, Caretta caretta, Lepidochelys olivacea, Eretmochelys imbricata). For this purpose, material from the oral cavity and cloaca of 77 animals (60 tortoises and 17 sea turtles) was collected. The collected specimens were seeded on 2% Sabouraud dextrose agar with chloramphenicol, and the identification was carried out by morphological and biochemical methods. Sixty-six isolates were recovered from tortoises, out of which 27 were C. tropicalis, 27 C. famata, 7 C. albicans, 4 C. guilliermondii and 1 C. intermedia, whereas 12 strains were obtained from sea turtles, which were identified as Candida parapsilosis (n = 4), Candida guilliermondii (n = 4), Candida tropicalis (n = 2), Candida albicans (n = 1) and Candida intermedia (n = 1). The minimum inhibitory concentrations for amphotericin B, itraconazole and fluconazole ranged from 0.03125 to 0.5, 0.03125 to >16 and 0.125 to >64, respectively. Overall, 19 azole-resistant strains (14 C. tropicalis and 5 C. albicans) were found. Thus, this study shows that Testudines carry azole-resistant Candida spp.

In recent years, the search for drugs to treat systemic and opportunistic mycoses has attracted great interest from the scientific community. This study evaluated the in vitro inhibitory effect of the antituberculosis drugs isoniazid and ethionamide alone and combined with itraconazole and fluconazole against biofilms of Cryptococcus neoformans and Cryptococcus gattii. Antimicrobials were tested at defined concentrations after susceptibility assays with Cryptococcus planktonic cells. In addition, we investigated the synergistic interaction of antituberculosis drugs and azole derivatives against Cryptococcus planktonic cells, as well as the influence of isoniazid and ethionamide on ergosterol content and cell membrane permeability. Isoniazid and ethionamide inhibited both biofilm formation and viability of mature biofilms. Combinations formed by antituberculosis drugs and azoles proved synergic against both planktonic and sessile cells, showing an ability to reduce Cryptococcus biofilms by approximately 50%. Furthermore, isoniazid and ethionamide reduced the content of ergosterol in Cryptococcus spp. planktonic cells and destabilized or permeabilized the fungal cell membrane, leading to leakage of macromolecules. Owing to the paucity of drugs able to inhibit Cryptococcus biofilms, we believe that the results presented here might be of interest in the designing of new antifungal compounds.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

The antifungal activity of some statins against different fungal species has been reported. Thus, at the first moment, the in vitro antifungal activity of simvastatin, atorvastatin and pravastatin was tested against Candida spp. and Cryptococcus spp. Then, in a second approach, considering that the best results were obtained for simvastatin, this drug was evaluated in combination with antifungal drugs against planktonic growth and tested against biofilms of Candida spp. and Cryptococcus spp. Drug susceptibility testing was performed using the microdilution broth method, as described by the Clinical and Laboratory Standards Institute. The interaction between simvastatin and antifungals against planktonic cells was analyzed by calculating the fractional inhibitory concentration index. Regarding biofilm susceptibility, simvastatin was tested against growing biofilm and mature biofilm of one strain of each tested yeast species. Simvastatin showed inhibitory effect against Candida spp. and Cryptococcus spp. with minimum inhibitory concentration values ranging from 15.6 to 1000 mg L(-1) and from 62.5 to 1000 mg L(-1), respectively. The combination of simvastatin with itraconazole and fluconazole showed synergism against Candida spp. and Cryptococcus spp., while the combination of simvastatin with amphotericin B was synergistic only against Cryptococcus spp. Concerning the biofilm assays, simvastatin was able to inhibit both growing biofilm and mature biofilm of Candida spp. and Cryptococcus spp. The present study showed that simvastatin inhibits planktonic cells and biofilms of Candida and Cryptococcus species., (Copyright © 2015 Elsevier Editora Ltda. All rights reserved.)

The aims of the present study were to isolate and identify clinical and environmental strains of Aeromonas spp. by means of biochemical tests and the automated method VITEK 2 and to investigate the presence of the virulence genes cytotoxic enterotoxin (act), hemolysin (asa-1), and type III secretion system (ascV), and also the in vitro antimicrobial susceptibility of the strains. From the clinical isolates, 19 Aeromonas hydrophila, 3 Aeromonas veronii bv. sobria, and 1 Aeromonas caviae were identified, while from the environmental strains, 11 A. hydrophila, 22 A. veronii bv. sobria, 1 A. veronii bv. veronii, and 1 A. caviae were recovered. The gene act was detected in 69.5% of clinical isolates, asa-1 in 8.6%, and ascV in 34.7%. In the environmental strains, the detection rates were 51.4%, 45.7%, and 54.2% for the genes act, asa-1, and ascV, respectively. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was observed in 15 and 3 clinical strains, respectively, and resistance to ceftazidime, meropenem, imipenem, ciprofloxacin, and trimethoprim-sulfamethoxazole was observed in 1 strain for each drug. Resistance to amoxicillin-clavulanate and piperacillin-tazobactam was detected in 17 and 1 environmental strain, respectively. Higher resistance percentages were observed in clinical strains, but environmental strains also showed this phenomenon and presented a higher detection rate of virulence genes. Thus, it is important to monitor the antimicrobial susceptibility and pathogenic potential of the environmental isolates.

Tyrosol is a quorum-sensing molecule of Candida albicans able to induce hyphal development in the early and intermediate stages of biofilm growth. In the present study, we evaluated the effect of high concentrations of exogenous tyrosol on planktonic cells and biofilms of C. albicans (n = 10) and C. tropicalis (n = 10), and investigated whether tyrosol could be synergic to antifungals that target cellular ergosterol. Antifungal susceptibility and drug interaction against planktonic cells were investigated by the broth microdilution method. Tyrosol was able to inhibit planktonic cells, with MIC values ranging from 2.5 to 5.0 mM for both species. Synergism was observed between tyrosol/amphotericin B (11/20 strains), tyrosol/itraconazole (18/20 strains) and tyrosol/fluconazole (18/20 strains). Exogenous tyrosol alone or combined with antifungals at both 10 × MIC and 50 × MIC were able to reduce biofilm of both Candida species. Mature biofilms were susceptible to tyrosol alone at 50 × MIC or combined with amphotericin at both 10 × MIC and 50 × MIC. On the other hand, tyrosol plus azoles at both 10 × MIC and 50 × MIC enhanced biofilm growth., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study evaluated the maintenance of Sporothrix spp. (6 Sporothrix brasiliensis; 6 S. schenckii; 5 S. mexicana, and 3 S. globosa) in saline at 4°C, and in 10% glycerol plus either 10% lactose or 10% sucrose, at -20°C and -80°C. Viability was assessed after 3, 6, and 9 months of storage, through the recovery of strains on potato dextrose agar and analysis of macro- and micromorphological features. Conidium quantification was performed before and after storage, at 3, 6 and 9 months. 100% viability was observed, regardless of storage conditions or time period. Storage at 4°C and at -20°C did not alter the number of conidia, but lower conidium counts were observed at -80°C. This study shows that the combination of glycerol with lactose or sucrose is effective to maintain Sporothrix spp. at freezing temperatures.

Sporotrichosis is a subacute or chronic subcutaneous infection, caused by the fungus Sporothrix schenkii complex, occurring in human and animal tissues. Potassium iodide and itraconazole have been used as effective therapy for first-choice treatment, while amphotericin B may be indicated for disseminated infection. However, the adverse effects of potassium iodide and amphotericin B or the long duration of therapy with itraconazole often weigh against their use, leading to the search for alternatives for the treatment of severe infections. Terpinen-4-ol and farnesol are components of essential oils present in many plant species and have been described to have antifungal activity against microorganisms. In this study, 40 strains of Sporothrix spp. were tested for the susceptibility to terpinen-4-ol and farnesol. Changes in cytoplasmic membrane permeability were also investigated. Terpenes inhibited all Sporothrix strains with MIC values ranging from 87.9 to 1,429.8 μg/ml for terpinen-4-ol and from 0.003 to 0.222 μg/ml for farnesol. The MFC values ranged from 177.8 to 5,722.6 μg/ml and from 0.027 to 0.88 μg/ml, respectively, for terpinen-4-ol and farnesol. Farnesol was the most active compound for the Sporothrix strains. Significant loss of 260 and 280 nm-absorbing material did not occur after treatment with concentrations equivalent to the MIC and sub-MIC of the tested terpenes, when compared to corresponding untreated samples. The failure of terpenes to lyse Sporothrix cells suggests that their primary mechanism of action is not by causing irreversible cell membrane damage. Thus, new studies are needed to better understand the mechanisms involved in the antifungal activity., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 μg/ml to 2 μg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 μg/ml to 1 μg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25-2 μg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 μg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo.

This study aimed at investigating the in vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against Burkholderia pseudomallei in planktonic and biofilm forms, through broth microdilution and resazurin-based viability staining, respectively. In planktonic growth, the strains were susceptible to the drugs, while in biofilm growth, significantly higher antimicrobial concentrations were required, especially for ceftazidime and imipenem, surpassing the resistance breakpoints. These results highlight the importance of the routine evaluation of biofilm antimicrobial susceptibility.

The objective of this study was to establish the phenotypical and molecular patterns of clinical isolates of Trichophyton tonsurans circulating in the state of Ceará, northeastern Brazil. For this purpose, 25 T. tonsurans strains isolated from independent cases of tinea capitis in children were phenotypically evaluated regarding their macro- and micro-morphological characteristics, vitamin requirements, urease production, and antifungal susceptibility. The molecular characterization was carried out with random amplified polymorphic DNA molecular markers and M13 fingerprinting. The presence of the genes CarbM14, Sub2, CER, URE, ASP, PBL, and LAC, which encode enzymes related to fungal virulence, was also evaluated. Finally, melanin production was assessed through specific staining. The data obtained demonstrated that these T. tonsurans strains have considerable phenotypical variation, although they showed a low degree of genetic polymorphism according to the markers used. The genes CarbM14, Sub2, CER, and URE were detected in all the analyzed strains. The gene LAC was also identified in all the strains, and melanin synthesis was phenotypically confirmed. The strains were susceptible to antifungals, especially itraconazole (GM = 0.06 μg/mL) and ketoconazole (GM = 0.24 μg/mL). Therefore, T. tonsurans strains can present great phenotypical heterogeneity, even in genetically similar isolates. Moreover, the presence of the LAC gene indicates the possible participation of melanin in the pathogenesis of these dermatophytes.

Respiratory infections are a common problem among equines and occur with variable rates of morbidity and mortality. Although some fungal species are considered primary agents of respiratory tract infections in several mammals, their relevance in respiratory diseases of equines is frequently neglected. In the present study, we performed an active search for Candida spp. in the nasal cavity of horses. The presence of Candida spp. was investigated through the use of nasal swabs that were streaked on culture media. These yeasts were identified through physiological testing and their in vitro antifungal susceptibility were also characterized. The analysis of the material from the nasal cavity of 97 randomly chosen horses resulted in the isolation of Candida spp. from 35 animals (36.08%), out of which 18 (32.14%) were C. famata, 14 (25%) C. parapsilosis, 12 (21.42%) Meyerozyma guilliermondii (C. guilliermondii), 11 (19.64%) C. tropicalis and 1 (1.78%) Wickerhamomyces anomalus (C. pelliculosa). The minimum inhibitory concentration (MIC) values ranged from 0.03125-1 μg/ml for amphotericin B; and from 0.03125-> 16 μg/ml and 0.125 to > 64 μg/ml for itraconazole and fluconazole, respectively. Resistance to fluconazole and itraconazole was observed among C. tropicalis (n = 3) and C. guilliermondii (n = 1). The data show a predominance of non-C. albicans Candida species in the nasal microbiota of healthy equines, including antifungal resistant isolates, reiterating the importance of monitoring fungal pathogens in these animals.

Studies of the genetic variation within populations of Coccidioides posadasii are scarce, especially for those recovered from South America. Understanding the distribution of genotypes among populations is important for epidemiological surveillance. This study evaluated the genetic diversity of 18 Brazilian strains of C. posadasii through the sequencing of the 18-28S region of nuclear rDNA, as well as through RAPD and M13-PCR fingerprinting techniques. The sequences obtained were compared to Coccidioides spp. previously deposited in GenBank. The MEGA5 program was used to perform phylogenetic analyses. Within the C. posadasii clade, a single cluster was observed, containing seven isolates from Ceará, which presented a single nucleotide polymorphism. These isolates were from the same geographical area. The strains of C. posadasii showed a lower rate of genetic diversity in the ITS1 and ITS2 regions. The results of M13 and RAPD-PCR fingerprinting indicated a similar electrophoretic profile. No differences between clinical and environmental isolates were detected. This was the first study assessing the genetic variability of a larger number of C. posadasii isolates from Brazil.

Coccidioidomycosis is a systemic mycosis caused by the dimorphic fungi Coccidioides spp. The treatment for chronic and/or disseminated coccidioidomycosis can be prolonged and complicated. Therefore, the search for new drugs is necessary. Farnesol is a precursor in the sterol biosynthesis pathway that has been shown to present antifungal activity. Thus, the objective of this study was to evaluate the in vitro antifungal activity of farnesol alone and in combination with antifungal agents against clinical and environmental strains of Coccidioides posadasii as well as to determine their effect on the synthesis of ergosterol and on cell permeability. This study employed the broth macrodilution method to determine the MIC of farnesol against 18 strains of C. posadasii. Quantification of ergosterol was performed with 10 strains of C. posadasii after exposure to subinhibitory concentrations of farnesol. Finally, the activity of farnesol was evaluated in the presence of osmotic stress, induced by the addition of NaCl to the culture medium, during the susceptibility tests. The results showed that farnesol exhibited low MICs (ranging from 0.00171 to 0.01369 mg/liter) against all tested strains. The combination of farnesol with the antifungals showed synergistic effects (fractional inhibitory concentration index [FICI] ≤ 0.5). As for the ergosterol quantification, it was observed that exposure to subinhibitory concentrations of farnesol decreased the amount of ergosterol extracted from the fungal cells. Furthermore, farnesol also showed lower MIC values when the strains were subjected to osmotic stress, indicating the action of this compound on the fungal membrane. Thus, due to the high in vitro antifungal activity, this work brings perspectives for the performance of in vivo studies to further elucidate the effects of farnesol on the host cells.

Trying to surpass host defenses, fungal infections alter the immune response. Components from nonpathogenic fungi present therapeutic anti-inflammatory and immunomodulating activities. This study reveals that proteins present in a Coccidioides posadasii extract provide anti-inflammatory benefit in experimental arthritis. Zymosan was given intra-articularly to rats and mice, and groups were pretreated with C. posadasii extract either per os or intraperitoneally. Controls received the vehicle. Acute hypernociception was evaluated using articular incapacitation and von Frey methods. Cell influx and cytokine levels were assessed in joint exudates. Joint damage was evaluated by histopathology and determination of glycosaminoglycan content of the cartilage. Synovia was evaluated for cell death and inducible nitric oxide synthase (iNOS) expression using TUNEL and immunohistochemistry, respectively. Pretreatment with C. posadasii extract significantly inhibited acute and chronic cell influx, hypernociception, and provoked reduction of glycosaminoglycan loss while reducing chronic synovitis, cell death, and iNOS expression. Reduction/alkylation of C. posadasii extract abrogated these effects. C. posadasii administration did not alter TNF-α, IL-1β, IL-17, and γ-interferon levels, whereas IL-10 levels were significantly reduced. Data reveal that a C. posadasii extract reduces iNOS expression that is associated with inhibition of synovial apoptosis and decrease in IL-10 levels released into zymosan-inflamed joints. Characterization of active components excluded charged carbohydrates while pointing to a protein as responsible for these effects. In summary, systemic administration of components from a pathogenic fungus provides anti-inflammatory effects, being species-independent and orally active. Besides adding to understand host response against fungi, the results may lead to therapeutic implications.

Farnesol is a sesquiterpene alcohol that modulates cell-to-cell communication in Candida albicans. In recent years, several studies have shown that this molecule presents inhibitory effects against non-albicans Candida species, Paracoccidioides brasiliensis and bacteria. The present study aimed at determining the effect of farnesol on the growth of strains of the Cryptococcus neoformans species complex, through microdilution assays. In addition, the effect of farnesol on the synthesis of phospholipase and protease - important virulence-associated enzymes - by C. neoformans and Cryptococcus gattii was also investigated. A total of 36 strains were studied, out of which 20 were from veterinary sources, 8 were from human cases and 8 were from a reference collection. The minimum inhibitory concentrations (MICs) were determined in accordance with the M27-A3 protocol as described by the CLSI and farnesol was tested at a concentration range of 0.29-150 μM. Phospholipase and protease activities were evaluated through growth on egg yolk agar and spectrophotometry, respectively, after pre-incubating the strains at different farnesol concentrations (MIC/4, MIC/2 and MIC). It was observed that farnesol presents an inhibitory activity against C. neoformans and C. gattii (MIC range: 0.29-75.0 μM). Although farnesol did not significantly alter phospholipase activity, a tendency to decrease this activity was observed. Concerning protease, no statistically significant differences were observed when comparing the production before and after pre-incubation at different farnesol concentrations. Based on these findings, it can be concluded that farnesol has in vitro inhibitory activity against C. neoformans and C. gattii, but has little impact on the production of the analyzed virulence factors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Coccidioidomycosis is a systemic mycosis with a variable clinical presentation. Misdiagnosis of coccidioidomycosis as bacterial pneumopathy leads to inappropriate prescription of antibiotics and delayed diagnosis. This report describes an outbreak among armadillo hunters in northeastern Brazil in which an initial diagnosis of bacterial pneumonia was later confirmed as coccidioidomycosis caused by Coccidioides posadasii. Thus, this mycosis should be considered as an alternative diagnosis in patients reporting symptoms of pneumonia, even if these symptoms are only presented for a short period, who are from areas considered endemic for this disease.

The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. The protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a β-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.

To analyze the eco-epidemiologic aspects of Histoplasma capsulatum in Brazil, we tested 83 bats for this fungus. Although H. capsulatum was not isolated, Coccidioides posadasii was recovered from Carollia perspicillata bat lungs. Immunologic studies detected coccidioidal antibodies and antigens in Glossophaga soricina and Desmodus rotundus bats.

The aim of the present study was to evaluate the effect of cotrimoxazole on the in vitro susceptibility of Coccidioides posadasii strains to antifungals. A total of 18 strains of C. posadasii isolated in Brazil were evaluated in this study. The assays were performed in accordance with the Clinical and Laboratory Standards Institute guidelines and the combinations were tested using the checkerboard method. The minimum inhibitory concentrations were reduced by 11, 2.4, 4.3 and 3.5 times for amphotericin B, itraconazole, fluconazole and voriconazole, respectively. Moreover, it was seen that cotrimoxazole itself inhibited C. posadasii strains in vitro. The impairment of folic acid synthesis may be a potential antifungal target for C. posadasii.

This study evaluated in vitro interactions of antituberculosis drugs and triazoles against Histoplasma capsulatum. Nine drug combinations, each including an antituberculosis drug (isoniazid, pyrazinamide, or ethambutol) plus a triazole (itraconazole, fluconazole, or voriconazole), were tested against both growth forms of H. capsulatum. Stronger synergistic interactions were seen in isoniazid or pyrazinamide plus triazoles for the mold form and ethambutol plus voriconazole for the yeast-like form. Further studies should evaluate these combinations in vivo.

This study evaluated the in vitro effect of sulfamethoxazole-trimethoprim against Histoplasma capsulatum var. capsulatum isolated from HIV-positive patients. The drugs were tested by microdilution testing in accordance with the CLSI guidelines. All of the strains were inhibited by sulfamethoxazole-trimethoprim, with MIC ranges of 0.039 (sulfamethoxazole)/0.0078 (trimethoprim) mg/ml to 0.625/0.125 mg/ml for mycelial forms and 0.0025/0.0005 to 0.02/0.004 mg/ml for yeast-like forms. However, in vivo studies are necessary to evaluate the significance of these results.

Despite the substantial literature on mycology, there are still limited reports of the interaction between fungi and human hosts in the postmortem period. Thus, the main goal of this study was to investigate the in vitro perforation test using Trichophyton mentagrophytes on hair from adult corpses in the postmortem period (gaseous period). The protocol was carried out with positive (prepubescent children's hair) and negative controls (healthy adult hair) as well. One strain of Trichophyton rubrum was also used as a negative perforation control. Perforations were found in all the hair samples from corpses and prepubescent children after 12–14 days exposure to T. mentagrophytes and were absent in the hair samples of healthy adults. Furthermore, hair perforation was not observed with T. rubrum. Our preliminary findings suggest the use of T. mentagrophytes as a potential marker of the death interval in forensic science.

Coccidioidomycosis is an endemic infection in the Americas caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii. Although the disease occurs in Brazil in sporadic form, little information about these cases is available. In this study, we summarize the most important clinical, epidemiologic, and diagnostic features of coccidioidomycosis in Ceará State (Northeast Brazil) during the past 12 years. In this period, 19 cases of coccidioidomycosis were diagnosed. All the patients were young males and came from semiarid areas of the state. The majority of cases were associated to armadillo hunting, and pulmonary disease was the most common clinical presentation. In our laboratory, coccidioidomycosis was confirmed by culture, serology, and polymerase chain reaction tests, which together were very suitable for the diagnosis of this disease. Based on our local experience, we believe many cases of this disease are misdiagnosed or not diagnosed in our region. Therefore, some strategies for improvement of diagnosis should be encouraged by local authorities.

Melioidosis is an infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Interest in the molecular identification of B. pseudomallei has increased after its classification as a category B agent by the US Centers for Disease Control and Prevention. The present article reports a diagnosis of B. pseudomallei directly in a bronchoalveolar lavage by polymerase chain reaction amplification. The results obtained show that direct detection of the 16-23s spacer sequence in bronchoalveolar lavage is a quick and specific test to diagnose melioidosis.

The aim of the present study was to evaluate the in vitro interactions of antituberculous drugs (ATDs) with antifungals against Coccidioides posadasii. Eighteen drug combinations, formed by an ATD (isoniazid, pyrazinamide or ethambutol) plus an antifungal (amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole or caspofungin), were tested using the checkerboard method. All the antimicrobial combinations inhibited C. posadasii strains and synergistic interactions were observed for 10 combinations. Antagonism between the tested drugs was not observed. Stronger synergistic interactions were seen in the combinations formed by triazoles plus ethambutol as well as itraconazole plus pyrazinamide. Further studies in animal models are needed to confirm the usefulness of these combinations.

Coccidioidomycosis is a systemic infection caused by Coccidioides spp. The disease is endemic in Brazil but its incidence is underreported as it is not a notifiable disease. This article presents the results of a serologic survey carried out with 229 volunteers in northeast Brazil by the immunodiffusion (ID) test with commercial Coccidioides spp. antigens. The commercial ID test detected 15 individuals without clinical diagnosis of the disease and two individuals in treatment for coccidioidomycosis. Regarding the epidemiological data, most of the positive individuals were male, between 18 and 65 years of age and were engaged in armadillo hunting. Three women who had never participated in armadillo hunts also displayed positive results for coccidioidal antibodies. Besides armadillo hunts, exposure to environmental dust in endemic areas may account for the serologic response detected in the study. The data from this study suggest the importance of performing epidemiological surveys for coccidioidomycosis in order to understand the prevalence of this disease in Brazil.

Onychomycosis is a fungal infection of nails caused by dermatophytes, yeasts and moulds, accounting for about 50% of onychopathies. A high frequency of onychomycosis caused by Candida species has been reported during the last few years in northeast Brazil, as well as in other regions of the world. A clinical diagnosis of onychomycosis needs to be confirmed through laboratory exams. We evaluated the importance of serial repetition of direct microscopic exams and fungal culture for the diagnosis of onychomycosis in the city of Fortaleza, Ceará, in northeast Brazil. We first made a retrospective study of 127 patients with onychomycosis, identifying the fungi that had been isolated from fingernails and toenails. We then made a prospective study of 120 patients, who were submitted to three successive mycological examinations. Ungual residues were scraped off and directly examined with a microscope and fungal cultures were made. In the retrospective study, in which only one sample was analyzed, the incidence of onychomycosis was 25.0%. In our prospective study, in which we had data from successive mycological examinations, 37.8% had onychomycosis. The most commonly isolated fungi in both studies were yeasts from the genera Candida, especially C albicans, C. parapsilosis and C. tropicalis. We found a high proportion of onychomycosis caused by Candida species. We also concluded that serial repetition of direct microscopic examination and fungal culture, with intervals of 2-5 days improved the diagnosis of onychomycosis. We suggest that this laboratorial strategy is necessary for accurate diagnosis of this type of mycosis, especially when the standard procedures fail to diagnose fungal infection, despite strong clinical suspicion.

The aim of this study was to perform a retrospective analysis of cases of candidemia in a Brazilian hospital in the city of Fortaleza, Ceará. A total of 50 blood cultures were analyzed from 40 candidemic patients. The mycological diagnosis was based on the phenotypical analysis and the patients' data were recorded in appropriate files. The most frequent species were Candida parapsilosis (n = 18), followed by C. albicans (n = 14), C. tropicalis (n = 8), C. guillermondii (n = 6), C. glabrata (n = 2), and Candida spp. (n = 2). A detailed descriptive study was undertaken with 21 patients whose medical records were complete. The candidemia episodes occurred in eight male patients and 13 female patients. The most representative risk factors implicated in candidemia were prior antibiotic therapy, central venous catheters, parenteral nutrition, gastric probes and mechanical ventilation. Death occurred in 13 of the 21-candidemic patients. This study demonstrated the emergence of candidemia caused by C. parapsilosis in a Brazilian hospital in the city of Fortaleza, Ceará.