BackgroundParacoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques.Methods/Principal FindingsAerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts.Conclusions/SignificanceData may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts.

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far.

Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001). Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.

Inteins are invasive intervening sequences that perform an autocatalytic splicing from their host proteins. Among eukaryotes, these elements are present in many fungal species, including those considered opportunistic or primary pathogens, such as Candida spp. Here we reviewed and updated the list of Candida species containing inteins in the genes VMA, THRRS and GLT1 and pointed out the importance of these elements as molecular markers for molecular epidemiological researches and species-specific diagnosis, since the presence, as well as the size of these inteins, is polymorphic among the different species. Although absent in Candida albicans, these elements are present in different sizes, in some environmental Candida spp. and also in most of the non-albicans Candida spp. considered emergent opportunistic pathogens. Besides, the possible role of these inteins in yeast physiology was also discussed in the light of the recent findings on the importance of these elements as post-translational modulators of gene expression, reinforcing their relevance as alternative therapeutic targets for the treatment of non-albicans Candida infections, because, once the splicing of an intein is inhibited, its host protein, which is usually a housekeeping protein, becomes non-functional.

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Corn cob is an agricultural byproduct that produces an estimated waste burden in the thousands of tons annually, but it is also a good source of xylan, an important bioactive polysaccharide. Silver nanoparticles containing xylan (nanoxylan) were produced using an environmentally friendly synthesis method. To do this, we extracted xylan from corn cobs using an ultrasound technique, which was confirmed by both chemical and NMR analyses. This xylan contained xylose, glucose, arabinose, galactose, mannose, and glucuronic acid in a molar ratio of 50:21:14:9:2.5:2.5, respectively. Nanoxylan synthesis was analyzed using UV–vis spectroscopy at kmax = 469 nm and Fourier transform infrared spectroscopy (FT-IR), which confirmed the presence of both silver and xylan in the nanoxylan product. Dynamic light scattering (DLS) and atomic force microscopy (AFM) revealed that the nanoxylan particles were ~102.0 nm in size and spherical in shape, respectively. DLS also demonstrated that nanoxylan was stable for 12 months and coupled plasma optical emission spectrometry (ICP-OES) showed that the nanoxylan particles were 19% silver. Nanoxylan reduced Leishmania amazonensis promastigote viability with a half maximal inhibitory concentration (IC50) value of 25 μg/mL, while xylan alone showed no effective. Additionally, nanoxylan exhibited antifungal activity against Candida albicans (MIC = 7.5 μg/mL), C. parapsilosis (MIC = 7.5 μg/mL), and Cryptococcus neoformans (MIC = 7.5 μg/mL). Taken together, these data suggest that it is possible to synthesize silver nanoparticles using xylan and that these nanoxylan exert improved antileishmanial and antifungal activities when compared to the untreated polysaccharide or silver nitrate used for their synthesis. Thus, nanoxylan may represent a promising new class of antiparasitic agents for use in the treatment of these microorganisms.

Background Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis. Methodology Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum . The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H . capsulatum cells and directly assayed with no previous DNA extraction. Results Both assays were able to detect down to 1 fg/μL of H . capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL. Conclusion ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.

Cryptococcus neoformans and Cryptococcus gattii are the etiologic agents of cryptococcosis, whose suitable treatment depends on rapid and correct detection and differentiation of the Cryptococcus species.