No abstract

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...

The sensitivity of the double agar gel immunodiffusion test is about 90% in patients with untreated paracoccidioidomycosis (PCM), but it is much lower in cases of relapse. In addition, serum from patients with PCM caused by Paracoccidioides lutzii, frequent in the Midwest region of Brazil, do not react with the classical antigen obtained from Pb B-339. These findings showed the need for alternative diagnostic methods, such as biological markers through proteomics. The aim of this study was to identify biomarkers for the safe identification of PCM relapse and specific proteins that could distinguish infections caused by Paracoccidioides brasiliensis from those produced by Paracoccidioides lutzii. Proteomic analysis was performed in serum from 9 patients with PCM caused by P. brasiliensis, with and without relapse, from 4 patients with PCM produced by P. lutzii, and from 3 healthy controls. The comparative evaluation of the 29 identified plasma proteins suggested that the presence of the immunoglobulin (Ig) alpha-2 chain C region and the absence of Ig heavy chain V-III TIL indicate infection by P. lutzii. In addition, the absence of complement factor B protein might be a predictor of relapse. The evaluation of these proteins in a higher number of patients should be carried out in order to validate these findings.