Júnia Soares Hamdan

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

ABSTRACT 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°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.

Disseminated paracoccidioidomycosis: correlation between clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole

The present study relates to a case of subacute multifocal paracoccidioidomycosis where the upper intestinal tract is involved. The involvement of the upper digestive tract is uncommon. The recommended therapeutic treatment plans and the difficulty in the treatment of paracoccidioidomycosis are discussed in association with susceptibility tests to antifungal drugs in vitro. This is the first report available in the literature showing, in parallel, clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole, studied during the course of the disease.

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.

Randomly amplified polymorphic DNA as a valuable tool for epidemiological studies of Paracoccidioides brasiliensis.

ABSTRACT Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.

In Vitro Comparison of Activities of Terbinafine and Itraconazole against Paracoccidioides brasiliensis

ABSTRACT In vitro, terbinafine is highly active against a broad spectrum of pathogenic fungi. We evaluated the activities of terbinafine and itraconazole against 31 isolates of Paracoccidioides brasiliensis. The tests were conducted by using a broth macrodilution procedure. MICs, in micrograms per milliliter, were as follows: terbinafine, 0.015 to 1.0 (geometric mean, 0.1188); itraconazole, 0.007 to 0.5 (geometric mean, 0.03165). The usual therapy for paracoccidioidomycosis is sulfonamides, amphotericin B, and azole derivatives (ketoconazole, itraconazole, and fluconazole). In comparison to amphotericin B, azole derivatives allow shorter treatment courses, can be administered orally, and are equally effective. Itraconazole has as high efficacy as ketoconazole, but with superior tolerance. It is the current drug of choice for treatment of paracoccidioidomycosis. The data obtained in this study indicate that terbinafine is active against P. brasiliensis in vitro and suggest that this allylamine can be considered a new option as drug therapy for paracoccidioidomycosis.

In Vitro Susceptibilities of Isolates of Sporothrix schenckii to Itraconazole and Terbinafine

ABSTRACT Thirty isolates of the yeast form of Sporothrix schenckii were evaluated for in vitro susceptibility to itraconazole and terbinafine by the recommended NCCLS modified technique (M27-A2). The MICs of itraconazole obtained oscillated between 0.062 and 4.0 μg/ml, and those of terbinafine oscillated between 0.007 and 0.50 μg/ml; therefore, terbinafine showed greater in vitro activity.

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) ≥ 64 μg 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 °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 μg ml−1 for ketoconazole, 0.25–2.0 μg ml−1 for griseofulvin, ≤0.031–1.0 μg ml−1 for itraconazole and ≤0.031 μg 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.

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.

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

ABSTRACT 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.

Antifungal susceptibility of clinical and environmental isolates of Cryptococcus neoformans to four antifungal drugs determined by two techniques.

A total of 64 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates var. neoformans and var. gattii, were tested for susceptibility to amphotericin B, 5‐flucytosine, fluconazole and itraconazole. The tests were performed according to the recommendations of National Committee of Clinical Laboratory Standards and the method of macrodilution in liquid medium of Shadomy et al. [Manual de Microbiologia Clínica, 4th edn. Buenos Aires: Editorial Medica Panamericana, 1987: 1229–38]. For most drugs there was a significant difference between the readings taken at 24 and 48 h with both methods. When the minimum inhibitory concentrations obtained by the two techniques were compared, significant differences were observed for amphotericin B and fluconazole. Overall, differences in drug susceptibility with respect to the origin of the isolates or the variety of the fungus were not observed. As an exception, the gattii variety exhibited a high resistance rate to amphotericin B when the technique of Shadomy et al. was applied, a fact possibly related to the greater difficulty for treatment of the disease caused by this fungal variety.