Effects of the essential oil of Thymus vulgaris L. Against Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast found in the environment, responsible for causing of meningoencephalitis in patients with a compromised immune system. In Brazil, cryptococcosis is the second cause of death among systemic mycoses. The limited efficacy of the available antifungal drugs used in its treatment has encouraged the search for therapeutic alternatives, such as medicinal plants. Thymus vulgaris, popularly known as thyme, is an aromatic plant whose essential oil (EO) possesses antifungal properties. The aim of this study was to assess the action of T. vulgaris EO on C. neoformans clinical isolates. This oil was analyzed by gas chromatography/mass spectrometry (GC/MS), which showed that its main components were thymol, ρ-cymene and linalool. Microdilution broth tests showed that this EO was effective against fungal isolates, with minimum inhibitory concentrations (MICs) ranging from 32 to 128 µg/mL. In vitro interaction tests between this oil and fluconazole (FCZ) showed no potentiation of the antifungal action of this drug. Its effect on mitochondrial metabolism of fungal cells was also evaluated and results demonstrated alterations on the mitochondrial enzyme activity of fungal cells only at concentrations >1,024 µg/mL. The results of the action of this EO on human erythrocytes indicated that it has low cytotoxic activity at MIC values. This investigation describes the antifungal action of T. vulgaris, showing its potential in the development of alternatives in the treatment of C. neoformans

Clinical and microbiological features of cryptococcal meningitis.

INTRODUCTION: In this study, the clinical features, underlying diseases and clinical outcomes of patients with cryptococcosis were investigated. In addition, a molecular analysis of the Cryptococcus neoformans species complex isolated from these patients was performed. METHODS: A prospective study of 62 cases of patients with cryptococcal infection was conducted at the Hospital de Doenças Tropicais de Goiás Dr. Anuar Auad from 2009-2010. Cryptococcal meningitis cases were diagnosed by direct examination and cerebrospinal fluid (CSF) sample culture. The profiling of these patients was assessed. The CSF samples were submitted to India ink preparation and cultured on Sabouraud dextrose agar, and C. neoformans was identified by the production of urease, a positive phenoloxidase test and assimilation of carbohydrates. C. neoformans and C. gattii isolates were distinguished by growth on L-canavanine-glycine-bromothymol blue medium, and molecular analysis was conducted via PCR fingerprinting reactions using M13 and (GACA)4 primers. RESULTS: From the 62 patients with cryptococcosis, 71 isolates of CSF were obtained; 67 (94.4%) isolates were identified as C. neoformans var. grubii/VNI, and 4 (5.6%) were identified as C. gattii/VGII. Of these patients, 53 had an HIV diagnosis. The incidence of cryptococcosis was higher among patients 20-40 years of age, with 74.2% of the cases reported in males. Cryptococcus-related mortality was noted in 48.4% of the patients, and the symptoms were altered sensorium, headache, fever and stiff neck. CONCLUSIONS: The high morbidity and mortality observed among patients with cryptococcosis demonstrate the importance of obtaining information regarding the epidemiological profile and clinical course of the disease in the State of Goiás, Brazil.

Effects of Pimenta pseudocaryophyllus (Gomes) L. R. Landrum, on melanized and non-melanized Cryptococcus neoformans.

In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5 times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8 times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p < 0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.

Molecular analysis and dimorphism of azole-susceptible and resistant Candida albicans isolates.

INTRODUCTION: Candida albicans is responsible for superficial or systemic infections known as candidiasis, which may be found in infected tissue as unicellular budding yeasts, hyphae, or pseudohyphae. In this study, the effects of both fluconazole and itraconazole antifungal agents on the hyphal formation and genotypic characterization of C. albicans isolates classified as either susceptible or resistant were investigated. METHODS: The hyphal production of five C. albicans isolates under the action of antifungal agents was investigated by culturing yeast on growth medium and on hyphal induction medium. The genotypic characterization was carried out for 13 isolates of C. albicans using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. RESULTS: The dimorphism analysis showed that the hyphal formation was higher in resistant than in the susceptible isolates to both azoles. The RAPD-PCR method identified the formation of two different groups. In group A, four resistant and two susceptible isolates were clustered, and in group B, one resistant and six susceptible isolates were clustered. CONCLUSIONS: Considering that hyphal formation was higher in resistant isolates in the presence of azole drugs, we confirmed that the hyphal production is closely related to susceptibility to azoles. These drugs may affect the morphogenesis of C. albicans depending on their susceptibility to these drugs. In relation to RAPD-PCR, most resistant isolates classified in group A and susceptible isolates in group B demonstrated that this method presented a similar standard between the two groups, suggesting that by this technique, a strong correlation between genotypes and fluconazole-resistant samples may be found.

Effects of antifungal agents in sap activity of Candida albicans isolates.

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base-bovine serum albumin (YCB-BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.

Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods.

The purpose of this study was to compare the agar dilution and broth microdilution methods for determining the minimum inhibitory concentration (MIC) of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine for 60 dermatophyte samples belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The percentage agreement between the two methods, for all the isolates with < 2 dilutions that were tested was 91.6% for ketoconazole and griseofulvin, 88.3% for itraconazole, 81.6% for terbinafine and 73.3% for fluconazole. One hundred percent agreement was obtained for Trichophyton mentagrophytes isolates evaluated with ketoconazole and griseofulvin. Thus, until a reference method for testing the in vitro susceptibility of dermatophytes is standardized, the similarity of the results between the two methods means that the agar dilution method may be useful for susceptibility testing on these filamentous fungi.

[Tinea capitis in children from Goiânia, Brazil]. / Tinha do couro cabeludo em crianças de Goiânia, Brasil.

During the period January 1999 to July 2002 a total de 164 cases of Tinea capitis were diagnosed by mycological examination in Instituto de Patologia Tropical e Saúde Pública of the Universidade Federal de Goiás. Of the 164 patients 94 (57.3%) were males, with an age of 3 months to 13 years. Laboratory studies were performed by direct examination with 20% KOH and cultivated on Mycobiotic agar medium and Sabouraud dextrose agar with chloramphenicol. The following species were identified: Microsporum canis (71.3%), Trichophyton tonsurans (11%), Trichophyton mentagrophytes (7.9%), Trichophyton rubrum (6.7%), and Microsporum gypseum (3%). Our study showed that the most frequent rise of scalp infection was a zoophylic fungi, called Microsporum canis.

[Epidemiology and etiology of dermatophytosis in Goiânia, GO, Brazil]. / Epidemiologia e etiologia das dermatofitoses em Goiânia, GO, Brasil.

Dermatophytes are a group of closely related fungi that have the capacity to invade keratinized tissue (skin, hair, and nails) of humans and animals to produce infections called dermatophytosis. In order to review the etiology and epidemiology of dermatophytosis in Goiânia, GO, 1955 specimens with diagnostic suspicion of dermatophytic lesions, were collected from January to December, 1999, from the Mycology Laboratory in the Institute of Tropical Pathology and Public Health, Federal University of Goiás. A total of 445 (22.8%) samples were positive for dermatophytes and Trichophyton rubrum was the most frequently isolated species (49.4%) followed by Trichophyton mentagrophytes (30.8%) and Microsporum canis (12.6%). Concerning the location of the lesions, the inferior limbs, feet and nails together with tinea capitis were the most frequently found clinical pattern in the majority of patients. Correlation between sex, age, location of the lesions and etiologic agents is considered in the study.