Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and the etiologic agents of paracoccidioidomycosis, a disease with multiple clinical presentations, prolonged evolution, and high rates of mortality and morbidity (1-4). Until now, a lack of routine tests has made it difficult to determine whether patients who did not respond to treatment were infected with resistant strains (5-8). Amphotericin B (AMB) is still the first-choice drug despite its high toxicity, and itraconazole (ITZ) is effective for the mild and moderate forms of the disease (9, 10). A long course of therapy and sometimes even lifelong secondary prophylaxis are required, especially in AIDS patients, who have a high incidence of relapse (11). A more effective drug with a better safety profile than the currently available drugs would greatly improve the treatment of dimorphic fungal infections (10,12).CLSI (Clinical and Laboratory Standards Institute) document M27-A3 has proposed a method for determining the in vitro susceptibility of yeasts to different drugs. However, no standardized method is available for dimorphic fungi because of the difficulty in culturing them. For these reasons, more reliable assays are needed for use with the genus Paracoccidioides. The ability to determine the MICs of different compounds would inform clinicians in the choice of an antifungal agent and the management of therapy, as well as facilitating screens of new molecules for their antifungal activity.However, the microdilution method used today presents difficulties in visually reading the MIC because there is a lack of significant fungal growth. Nevertheless, this test has been used in studies in the literature (12-15). In contrast, the microplate alamarBlue assay (MABA) has been evaluated by several authors in Aspergillus fumigatus (16) The aim of our study was to compare the reference broth macrodilution microplate assay (MMA) and microdilution microplate assay (MMI) methods with the MABA to measure the presence of fungi of the Paracoccidioides genus and the activity of two drugs, AMB and ITZ.The MMI and MMA were performed according to docu- .0015 mg/liter, respectively, after the addition of the inoculums. The antifungal activity was analyzed against the P. brasiliensis phylogenetic species S1 isolates 18 (São Paulo), D03, and 339 (São Paulo), S2 isolate 02 (Venezuela), and PS3 isolate Epm83 (Colombia) and P. lutzii strain 01 (ATCC MYA-826/ Goiânia) and isolates EE (Mato Grosso) and 8334MMT (Goiânia). All the strains were isolated from human patien...

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“…Additionally, these values are similar to those described in the literature (12)(13)(14)(15)(21)(22)(23)(24)(25)(26).…”

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confidence: 89%

Microplate alamarBlue Assay for Paracoccidioides Susceptibility Testing

et al. 2013

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“…Determination of the MFC. The minimum fungicidal concentration was determined by following Takahagi-Nakaira et al (53). The Paracoccidioides species cells were incubated with argentilactone, which was serially diluted while maintaining the same concentrations and culture conditions used in the MIC test.…”

Section: Methodsmentioning

confidence: 99%

Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics

Silva

Tauhata

Baeza

et al. 2018

Antimicrob Agents Chemother

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Paracoccidioidomycosis (PCM) is the cause of many deaths from systemic mycoses. The etiological agents of PCM belong to the genus, which is restricted to Latin America. The infection is acquired through the inhalation of conidia that primarily lodge in the lungs and may disseminate to other organs and tissues. The treatment for PCM is commonly performed via the administration of antifungals such as amphotericin B, co-trimoxazole, and itraconazole. The antifungal toxicity and side effects, in addition to their long treatment times, have stimulated research for new bioactive compounds. Argentilactone is a compound that was isolated from the Brazilian savanna plant, and it has been suggested to be a potent antifungal, inhibiting the dimorphism of and the enzymatic activity of isocitrate lyase, a key enzyme of the glyoxylate cycle. This work was developed due to the importance of elucidating the putative mode of action of argentilactone. The chemoproteomics approach via affinity chromatography was the methodology used to explore the interactions between proteins and argentilactone. A total of 109 proteins were identified and classified functionally. The most representative functional categories were related to amino acid metabolism, energy, and detoxification. Argentilactone inhibited the enzymatic activity of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase. Furthermore, argentilactone induces the production of reactive oxygen species and inhibits the biosynthesis of cell wall polymers.

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“…Immune system alterations have been described in patients with the severe forms of PCM, including hypergammaglobulinemia, a high level of specific antibodies, eosinophilia, and depressed general and antigen-specific cellular immunity (10).…”

mentioning

confidence: 99%

Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections

Bagatin

Pimentel

Biavatti

et al. 2017

Antimicrob Agents Chemother

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This work evaluated new potential inhibitors of the enzyme homoserine dehydrogenase (HSD) of Paracoccidioides brasiliensis, one of the etiological agents of paracoccidioidomycosis. The tertiary structure of the protein bonded to the analogue NAD, and L-homoserine was modeled by homology. The model with the best output was subjected to gradient minimization, redocking, and molecular dynamics simulation. Virtual screening simulations with 187,841 molecules purchasable from the Zinc database were performed. After the screenings, 14 molecules were selected and analyzed by the use of absorption, distribution, metabolism, excretion, and toxicity criteria, resulting in four compounds for in vitro assays. The molecules HS1 and HS2 were promising, exhibiting MICs of 64 and 32 g · ml Ϫ1 , respectively, for the Pb18 isolate of P. brasilensis, 64 g · ml Ϫ1 for two isolates of P. lutzii, and also synergy with itraconazole. The application of these molecules to human-pathogenic fungi confirmed that the HSD enzyme may be used as a target for the development of drugs with specific action against paracoccidioidomycosis; moreover, these compounds may serve as leads in the design of new antifungals.

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Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

Cited by 6 publications

References 27 publications

Microplate alamarBlue Assay for Paracoccidioides Susceptibility Testing

Microplate alamarBlue Assay for Paracoccidioides Susceptibility Testing

Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics

Targeting the Homoserine Dehydrogenase of Paracoccidioides Species for Treatment of Systemic Fungal Infections

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