Microbiologic profile of intra-abdominal infections at Belo Horizonte, Brazil.

Intra-abdominal infections (IAIs) represent one of the most common clinical problems in hospital practice, especially in surgical areas and centers of intensive care. The treatment of IAIs generally involves the draining of abscesses and empirical antimicrobial therapy. In this study, among 150 patients suffering from IAI, 106 (70.7%) yielded samples that presented microbial growth. Polyinfection was detected in 51.9% of the cases and varied from 2 to 9 distinct microbes per specimen. The overall mean number of micro-organisms isolated per patient was 2.17. Aerobic bacteria (as strict aerobes and facultative anaerobes), strict anaerobic bacteria, and fungi of the genus Candida represented 93.4%, 30.2%, and 13.2% of the cases positive for micro-organisms, respectively. The most common aerobic bacteria were those of the genera Staphylococcus, Escherichia, Proteus, and Streptococcus. Despite the frequent prior use (52%) with antimicrobials of recognized action against strict anaerobes, these micro-organisms constituted 30.9% of the total isolates, and the most frequently found were of the Bacteroides fragilis group and Prevotella species. The high prevalence of anaerobes in the specimens obtained from IAI demonstrates the need to give greater importance to these micro-organisms by making available material and human resources to carry out culture of the anaerobes as part of routine hospital procedures.

Epidemiological aspects of bovine parasitic otitis caused by Rhabditis spp. and/or Raillietia spp. in the state of Minas Gerais, Brazil.

The occurrence of bovine external otitis in tropical regions is predominantly assigned to parasitic infections by rhabditiform nematodes and infestations by mites of the genus Raillietia. This research had the purpose of investigating the prevalence of parasitic otitis in the dry and rainy seasons of Minas Gerais and correlating it with variables age, breed and the presence of horns. The secretion or cerumen of 981 cattle was collected with sterile swabs, placed in test tubes and exposed to sunlight for the identification of clinical infections caused by rhabditiform nematodes. The ear canals of 109 animals were flushed to study the occurrence of mites of the genus Raillietia. Clinical otitis caused by rhabditiform nematodes was observed only in animals of the Gyr breed, affecting 169 (60.1%) of the total (278) of these cattle. The prevalence of otitis in this breed was significantly higher for mature adult cows (P<0.001) and in cows with horns (P<0.001). In a total of nine animals suspected of raillietiosis, the examinations revealed the presence of Raillietia spp. in two Dutch cows. These results demonstrate that treatment must be focused on adult Gyr cattle with horns. This and future studies with an emphasis on epidemiological aspects may contribute to development of alternative approaches to reduce disease.

Biochemical analysis of the methylic antigen of Paracoccidioides brasiliensis.

Yeast forms of five strains of Paracoccidioides brasiliensis (SN, 2, 18, 192 and JT-1) were cultured in a synthetic medium for obtaining methylic antigens. These antigens were lyophilized and studied for each strain, to determine their partial biochemical composition, through measurements of total lipid, protein and carbohydrate contents. Lipids of methylic antigens were purified and analysed for sterols, phospholipids, glycolipids, lipoproteins, and partial characterization of sterols. Significant differences were found among antigenic preparations derived from distinct P. brasiliensis strains, in relation to the quantitative determinations. On the other hand, sterol analysis revealed the presence of ergosterol, lanosterol and squalene in all samples. The diversity verified in the biochemical characteristics of antigens derived from different P. brasiliensis strains, confirm the need of using a pool of fungal samples in order to produce antigen preparations for serological procedures without hampering their sensitivity.

A case of mycotic keratitis caused by Fusarium solani.

A 36-year-old black man, without history of systemic disease or ocular trauma developed a corneal infection in his left eye. He was treated with antibacterial antibiotic and corticosteroids for one month prior to diagnosis. Fungal hyphae and chlamydospores were found in a KOH preparation of the corneal scrapings, and positive cultures for Fusarium solani were obtained in Sabouraud dextrose agar. It is emphasized the cautious use of antibiotics and steroids in corneal diseases, and the need of considering the involvement of opportunistic fungi in the etiology of these infections.

[The microhemagglutination reaction for the routine serologic diagnosis of syphilis]. / A reação de microhemaglutinação para sífilis no diagnóstico sorológico de rotina.

The authors present the results of VDRL reactions (qualitative and quantitative) and microhaemagglutination assays (MIPHA) on 305 serum samples. The samples selected among the sera asked for syphilis serology at a clinical laboratory, were those which the results on VDRL reactions were observed to be reactive at low titles, not reactive or inconclusive. From these 305 samples, 87 (28.5 por 100) were also processed by the FTA-Abs test. The reactivity concordance between the tests was compared and the efficiency of the MIPHA as confirmatory diagnostic test was discussed. The conclusions prompt to the usefulness of the MIPHA on syphilis serodiagnosis routine as a function of its sensitivity, specificity, low costs and technical simplicity.

In vitro activities of four antifungal drugs against Trichophyton rubrum isolates exhibiting resistance to fluconazole.

Thirty-two clinical isolates of Trichophyton rubrum exhibiting resistance to fluconazole [minimum inhibitory concentrations (MICs) > or = 64 microg ml(-1)] were selected to test the antifungal activity of ketoconazole, itraconazole, griseofulvin and terbinafine. We followed the guidelines of the National Committee for Clinical Laboratory Standards for testing filamentous fungi. The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051) and Trichophyton mentagrophytes (ATCC 40004) were included for quality control. The microdilution plates were incubated at 28 degrees C and were read visually after 7 days of incubation and endpoint determination readings were performed visually. The MIC ranges for the four antifungals were: 0.0625-2 microg ml(-1) for ketoconazole, 0.25-2.0 microg ml(-1) for griseofulvin, < or =0.031-1.0 microg ml(-1) for itraconazole and < or =0.031 microg ml(-1) for terbinafine (for all tested isolates). Terbinafine was the most potent drug against T. rubrum, in vitro, followed by itraconazole, ketoconazole and griseofulvin. Much work is still needed to correlate the MICs of these drugs with clinical outcomes to develop interpretative breakpoints for T. rubrum and other dermatophytes.

In vitro antifungal oral drug and drug-combination activity against onychomycosis causative dermatophytes.

We present the results of studies of the in vitro susceptibility of 52 isolates of Trichophyton rubrum and 40 of Trichophyton mentagrophytes to griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole and cyclopiroxolamine. All test strains were recovered from patients with toe nail onychomycosis and the minimum inhibitory concentration (MIC) of each antifungal against both species was individually assessed. In addition, we investigated the MIC of the combination of cyclopiroxolamine and itraconazole and cyclopiroxolamine and ketoconazole. The NCCLS approved procedure M38-A as modified by Santos and Hamdan was employed. The studies of the two drug combinations were conducted with a checkerboard design. Analysis of the data revealed that terbinafine was the most effective in vitro against all isolates, followed in order by itraconazole, cyclopiroxolamine, ketoconazole and fluconazole. We observed no significant difference in the in vitro susceptibility profiles between either species to any of the antifungals (P<0.05). Our in vitro results confirm that terbinafine is the most effective of the antifungals included in this study. Furthermore, synergistic interactions were found in the two drug combinations with all of the dermatophyte test isolates. The latter results are in agreement with clinical data that show synergism between oral and topical antifungals in the treatment of onychomycosis.

Susceptibility of yeast isolates from cattle with otitis to aqueous solution of povidone iodine and to alcohol-ether solution.

Lipid-dependent Malassezia species, Candida spp. and Rhodotorula mucilaginosa have been associated with bovine parasitic otitis. This paper evaluated the susceptibility of 63 yeast isolates from cattle with otitis to a povidone iodine aqueous solution (1% and 0.5% v/v) and to an alcohol-ether solution (1:1 v/v). The effectiveness of these antiseptics was assessed using the European suspension test. Products achieving equal to or greater than 5-log reduction in numbers of the challenge organism after 5 min contact are considered to have as acceptable microbicidal effect (ME). The two antiseptic solutions achieved ME greater than 5, when tested at 1 and 5 min contact time, against the majority of yeast strains. The exceptions were alcohol and ether solution against two Candida tropicalis strains. Urea broth macrodilution method was used to determine the minimum inhibitory concentration (MIC), defined as the lowest concentration that resulted in a visually negative urease test or, in the case of Candida spp., turbidity inhibition when compared with that produced by the growth control. Analysis of the results for all 63 isolates showed Malassezia sympodialis and Rhodotorula mucilaginosa to be more susceptible to povidone iodine and Malassezia furfur strains to be less susceptible. Malassezia sympodialis was significantly more susceptible to alcohol-ether solution than other species. This study showed the in vitro efficacy of alcohol-ether solution and povidone iodine and proposes the need for clinical evaluation of the topical treatment and control of bovine otitis with these antiseptics and their effects on the ear microbiota and the ear canal.

Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes.

A total of 92 clinical isolates of dermatophytes (52 of Trichophyton rubrum and 40 of Trichophyton mentagrophytes) were selected for testing with six antifungal drugs (terbinafine, griseofulvin, clotrimazole, miconazole, isoconazole, and fluconazole) and two pairs of drug combinations (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine). Two methods of inoculum preparation for susceptibility testing were evaluated that used (i) inocula consisting only of microconidia of dermatophytes filtered in Whatman filter model 40 and (ii) unfiltered inocula consisting of hyphae and microconidia. We followed the recommendations of approved document M38-A of CLSI (formerly NCCLS) with some adaptations, including an incubation period of 7 days and an incubation temperature of 28 degrees C. Reference strains of Candida parapsilosis, Candida krusei, Trichophyton rubrum, and Trichophyton mentagrophytes were included as quality-control strains. MICs were consistently higher (usually 1 to 2 dilutions for drugs tested individually) when nonfiltered inocula were tested (P < 0.01) except for terbinafine. Larger MICs were seen when testing drugs with nonfiltered inocula. The curves of drug interaction were used to analyze the reproducibility of the test, and it was shown that high levels of reproducibility were achieved using the methodology that included the filtration step. The standardization of methodologies is the first step to yield reliability of susceptibility testing and to proceed with clinical laboratory studies to correlate MICs with clinical outcomes.

Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum.

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35 degrees C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35 degrees C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.

Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis.

Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC.

An atypical isolate of Paracoccidioides brasiliensis.

An atypical strain of Paracoccidioides brasiliensis was obtained from a patient with disseminated paracoccidioidomycosis. This isolate, which has been maintained in the laboratory for 4 years by successive subcultures (30-40 days) in Fava Netto medium, produces cerebriform colonies characteristic of the yeast phase of the fungus when left at room temperature. Microscopy shows a predominance of yeasts with multiple buds, side by side with some branching filamentous structures presenting intercalary chlamydospores. The strain does not develop well at 37 degrees C, suggesting heat sensitivity. Storage at 4 degrees C does not change its macro- or microscopic characteristics. These findings support the hypothesis raised by others that the dimorphism of P. brasiliensis may not be exclusively heat dependent. Studies are currently being developed in our laboratory to better characterize this isolate in terms of biochemical peculiarities, degree of virulence and susceptibility/resistance to antifungal drugs.

In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs.

A total of 53 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates, were tested for their susceptibilities to amphotericin B, 5-flucytosine, ketoconazole, fluconazole, and itraconazole. The tests were performed according to the National Committee of Clinical Laboratory Standards recommendations (document M27-P). In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. This paper represents the first contribution in which susceptibility data for Brazilian C. neoformans isolates are provided.

Effects of three azole derivatives on the lipids of different strains of Cryptococcus neoformans.

The comparative effects of ketoconazole, itraconazole and fluconazole on the lipids of four Cryptococcus neoformans strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectrum. Growth of the cryptococcal isolates in the presence of the azoles derivatives concentrations below the minimum inhibitory concentration resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act through the inhibition of ergosterol biosynthesis, with resultant accumulation of lanosterol. Ketoconazole was found to be the least effective drug, as determined from a comparison of the effect of the three azoles on the sterol content of the four strains. Itraconazole showed to be the most effective drug, probably because of its high lipophilicity, which allows the drug to penetrate into fungi cells more efficiently.

In vitro susceptibilities of Paracoccidioides brasiliensis yeast form to antifungal drugs.

A total of 12 Paracoccidioides brasiliensis yeast form strains, including clinical and environmental isolates, were tested for their susceptibilities to amphotericin B, ketoconazole, fluconazole, and itraconazole. The tests were determined using a broth macrodilution procedure. In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates.

Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii.

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7-57.6%) and sterols (48.5-96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.

Otitis in cattle, an aetiological review.

Otitis externa in cattle has a significant impact in tropical and subtropical regions, and the aetiological agents are predominantly rhabditiform nematodes and mites of the genus Raillietia. Its prevalence is higher in mature and Zebu cattle. In advanced clinical cases there can be irreversible and fatal neural lesions. Ear infection in calves has been associated with concurrent respiratory diseases and mixed infection. The principal reported agents of otitis in calves are bacteria such as Actinomyces spp., Corynebacterium pseudotuberculosis, Escherichia coli, Haemophilus somnus, Pasteurella multocida, Mannheimia haemolytica, Pseudomonas spp., Streptococcus spp. and Mycoplasma bovis. The control and treatment of bovine otitis is not standardized and there is little evidence-based support for the diverse treatments available in the literature.

Effect of amphotericin B on the lipids of five different strains of Cryptococcus neoformans.

Cells of five strains of Cryptococcus neoformans were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectra. Such determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimal inhibitory concentration) levels. Marked alterations of the lipid and sterol contents were observed in the amphotericin B-treated cells. Moreover, ergosterol disappeared in these antibiotic-exposed cells. It is concluded that amphotericin B altered the lipid profiles, especially sterols of C. neoformans.

Effects of three azole derivatives on the lipids of different strains of Sporothrix schenckii.

The comparative effects of ketoconazole, itraconazole, and fluconazole on the lipids of five Sporothrix schenckii strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols, by thin-layer chromatography and ultraviolet spectrophotometry. Growth of the S. schenckii isolates in the presence of azole derivative concentrations below the minimum inhibitory concentration (MIC) resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol. Concerning the MIC values, fluconazole was found to be the least effective drug. On the other hand, as determined from a comparison of the effects of the three azoles on the sterol content of the strains studied, no significant differences in efficacy were found among the tested drugs.

Lipid composition and effect of amphotericin B on yeast cells of Paracoccidioides brasiliensis.

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 microgram/mL.