Daniel Assis Santos

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.

Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections

Skin mycoses are caused mainly by dermatophytes, which are fungal species that primarily infect areas rich in keratin such as hair, nails, and skin. Significantly, there are increasing rates of antimicrobial resistance among dermatophytes, especially for Trichophyton rubrum, the most frequent etiologic agent worldwide. Hence, investigators have been developing new therapeutic approaches, including photodynamic treatment. Photodynamic therapy (PDT) utilizes a photosensitive substance activated by a light source of a specific wavelength. The photoactivation induces cascades of photochemicals and photobiological events that cause irreversible changes in the exposed cells. Although photodynamic approaches are well established experimentally for the treatment of certain cutaneous infections, there is limited information about its mechanism of action for specific pathogens as well as the risks to healthy tissues. In this work, we have conducted a comprehensive review of the current knowledge of PDT as it specifically applies to fungal diseases. The data to date suggests that photodynamic treatment approaches hold great promise for combating certain fungal pathogens, particularly 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.

Pyridine-derived thiosemicarbazones and their tin(IV) complexes with antifungal activity against Candida spp.

[(n-Bu)Sn(2Ac4oClPh)Cl2] (1), [(n-Bu)Sn(2Ac4oFPh)Cl2] (2), [(n-Bu)Sn(2Ac4oNO2Ph)Cl2] (3), [(n-Bu)Sn(2Bz4oClPh)Cl2] (4), [(n-Bu)Sn(2Bz4oFPh)Cl2] (5) and [(n-Bu)Sn(2Bz4oNO2Ph)Cl2] (6) were obtained by reacting [(n-Bu)SnCl3] with 2-acetylpyridine-N4-orthochlorophenyl thiosemicarbazone (H2Ac4oClPh), 2-acetylpyridine-N4-orthofluorphenyl thiosemicarbazone (H2Ac4oFPh), 2-acetylpyridine-N4-orthonitrophenyl thiosemicarbazone (H2Ac4oNO2Ph), and with the corresponding 2-benzoylpyridine-derived thiosemicarbazones (H2Bz4oClPh, H2ABz4oFPh and H2Bz4oNO2Ph). The antifungal activity of the studied compounds was evaluated against several Candida species. Upon coordination of H2Bz4oNO2Ph to tin in complex (6) the antifungal activity increased three times against Candida albicans and Candida krusei and six times against Candida glabrata and Candida parapsilosis. The minimum inhibitory concentration (MIC) values of H2Ac4oNO2Ph and its complex (3) against C. albicans, C. parapsilosis and C. glabrata are similar to that of fluconazole. All studied compounds were more active than fluconazole against C. krusei.

The role of oxidative and nitrosative bursts caused by azoles and amphotericin B against the fungal pathogen Cryptococcus gattii

OBJECTIVES Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined. METHODS We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger. RESULTS The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger. CONCLUSIONS Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death

OBJECTIVES Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. METHODS We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. RESULTS The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. CONCLUSIONS Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.

Dynamic interaction between fluconazole and amphotericin B against Cryptococcus gattii

ABSTRACT Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii. Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii.

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.

Fluconazole Alters the Polysaccharide Capsule of Cryptococcus gattii and Leads to Distinct Behaviors in Murine Cryptococcosis

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Influence of Different Media, Incubation Times, and Temperatures for Determining the MICs of Seven Antifungal Agents against Paracoccidioides brasiliensis by Microdilution

ABSTRACT MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28°C] and 37°C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37°C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25°C (mycelial form) than at 37°C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides.