Rosane Christine Hahn

Is the geographical origin of a Paracoccidioides brasiliensis isolate important for antigen production for regional diagnosis of paracoccidioidomycosis

In this study, exoantigens produced from two Paracoccidioides brasiliensis strains isolated in two different geographical areas were compared in terms of sensitivity and specificity in relation to paracoccidioidomycosis (PCM) diagnosis. Exoantigens from P. brasiliensis 550B (Ag 550B) isolated in the central‐west region of Brazil (Mato Grosso State) and exoantigen produced from P. brasiliensis B‐339 (Ag B‐339) used in reference laboratories were compared by immunodiffusion (ID) tests. When Ag 550B was used in ID test against sera of patients from Mato Grosso and São Paulo, positivity was 92.3% and 41.3%, respectively. On the other hand, when Ag B‐339 was tested with the same sera, positivity was 26.2% and 100%, respectively. These results suggest that differences in the antigenic composition probably related to phylogenetic peculiarities in P. brasiliensis isolates from the central‐western region of Brazil should be considered in the diagnosis of PCM.

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.

Serology of paracoccidioidomycosis due to Paracoccidioides lutzii.

Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.

Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

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.

Case Report: Fatal Fungemia due to Paracoccidioides lutzii

We report the first case of fungemia caused by Paracoccidioides lutzii in a 51-year-old male farm worker from the central-west region of Brazil. The fungus was isolated from blood cultures and the species was confirmed by phylogenetic identification. Despite specific treatment and intensive care, the patient died 39 days after admission.

Candida isolated from vaginal mucosa of mothers and oral mucosa of neonates : Occurrence and biotypes concordance

Background: The common occurrence of Candida spp. on the vaginal mucosa of pregnant women suggests this as the source of neonatal candidiasis. Methods: This study investigated the occurrence of yeasts on the vaginal mucosa of 100 mothers at the time of birth, and on the oral mucosa of their respective neonates, all full-term, on the 1st, 3rd, and 9th days after birth by vaginal (72 cases) and cesarean (28 cases) routes. In each case where concordance at the level of species was found between the isolate from the mother and that from the neonate, tests were made to check for concordance between the genotypic and phenotypic profiles (susceptibility to killer toxins, serotyping, proteinase and phospholipase production, and susceptibility to antifungal agents). Results: For the vaginal-route group, yeasts were recovered from the vaginal mucosa of 47.2% of the mothers and from 25% of the neonates. For the cesarean-route group, these rates were 46.4% and 3.6%, respectively. Species found most frequently in the samples from the mothers and the neonates were, respectively C. albicans and C. guilliermondii. For the vaginal-route group, the rate of mother/neonate concordance at the level of species was 23.5% and no cases of concordance for the cesarean births. Of these cases with species concordance, there was concordance between the genotypic and phenotypic profiles in 6% (2 cases). Conclusion: The vaginal mucosa was not the main route of transmission of the Candida species to the neonate, because there was concordance between the genotypic and phenotypic profiles in only 6% (2 cases).

Serological and antigenic profiles of clinical isolates of Paracoccidioides spp. from Central Western Brazil

Clinical Paracoccidioides spp. isolates from patients with paracoccidioidomycosis (PCM) in Mato Grosso, Brazil exhibit different patterns of serologic reactivity. The results observed for reactions of radial immunodiffusion against the commonly used exoantigens containing a 43‐kDa glycoprotein (gp43) suggest that this fungus exhibits major antigenic variability by geographic region. There is a phylogenetic gap between Paracoccidioides spp. isolates among different regions of Latin America. In particular, those from the central region of Brazil (i.e. Mato Grosso state) exhibit a lower rate of genetic similarity. We aimed at investigating the phylogenetic classification of clinical isolates of Paracoccidioides spp. in Central Brazil and the different antigenic profiles that produce. Exoantigens were obtained from five clinical isolates: two P. brasiliensis (Pb166 and Pb2880) and three P. lutzii (PL2875, PL9840, and PL2912). The protein/glycoprotein profiles of P. lutzii exoantigens were different from each other. Isolate PL9840 exhibited the most distinct bands, and isolates PL2875 and PL2912 exhibited more diffuse bands and a very intense band between 50 and 60 kDa. P. brasiliensis isolates had similar protein profiles, exhibiting a low‐intensity band at 220 kDa and a diffuse band between 50 and 60 kDa. P. lutzii isolates exhibit high species‐specific antigen variability, which we have already been assessed in proteomic studies.

Aspectos micológicos e suscetibilidade in vitro de leveduras do gênero Candida em pacientes HIV-positivos provenientes do Estado de Mato Grosso

INTRODUCTION Candidiasis is one of the most common fungal infections among patients infected by human immunodeficiency virus. The present study aimed to characterize yeasts of the genus Candida from distinct clinical samples from HIV-positive patients and determine the in vitro susceptibility profile to five antifungal drugs. METHODS Characterization of Candida sp was achieved using the classic methodology: biochemical (zymogram and auxanogram) and micromorphology (germinative tube growth test and slide microculture) tests. Genotypic technique (PCR) and identification by the commercial method API 20C AUX (Biomeriéux) were also performed. To determine the in vitro susceptibility profile, five antifungal drugs were used (ketoconazole, fluconazole, itraconazole, voriconazole and amphotericin-B) following a commercially available method, the Etest. RESULTS The procedure isolated 105 yeasts of the genus Candida from 102 HIV-infected patients. Of these, 82 (78.1%) were characterized as Candida albicans, 8 (7.6%) as C. parapsilosi s, 8 (7.6%) C. tropicalis, 4 (3.8%) C. krusei, 2 (1.9%) C. glabrata, and 1 (1%) as C. guiilliermondii. CONCLUSIONS Considering the general profile of sensitivity, 60% of isolates were susceptible to all the antifungal drugs tested; however, the species C. tropicalis and C. krusei showed a tendency toward higher MICs to azoles than those obtained for C. albicans, suggesting resistance.