Morphological and ultrastructural changes in fungal agents after LASER application.

BACKGROUND: Onychomycosis is a fungal nail infection of difficult treatment due to the fungal survival capacity and reduced number of effective therapies. The present study aimed to isolate fungal agents that cause onychomycosis in immunocompetent patients and evaluate how LASER treatments affect the growth and ultrastructure of isolates. METHODS: In total, 21 patients with positive direct microscopic examination (DME) for onychomycosis had nail samples collected for cultivation and phenotypic identification of microorganisms. From these patients, 12 underwent LASER treatment, divided in Group 1 (n = 5) treated with Nd: YAG 1,064 nm, and Group 2 (n = 7) treated with Nd: YAG 1,064 nm + Er: YAG 2,940 nm + topical isoconazole. Transmission Electron Microscopy (TEM) was performed to evaluate ultrastructural changes after treatment. RESULTS: DME, cultivation, and phenotypic identification showed that the most identified fungus was Trichophyton rubrum spp. After LASER therapy, sample cultivation showed alterations in the fungal morphology with reduction of hyphae, conidia, and reproductive structures. Alterations in fungal cell wall structure, cytoplasm density, and organelles were observed by TEM. CONCLUSION: LASER irradiation causes changes in the fungal cells, especially in the number of hyphae and the presence of conidia. In addition, it affects fungal growth and reproduction capacity, which interferes with their infection ability and virulence.

Terpinen-4-ol inhibits the growth of Sporothrix schenckii complex and exhibits synergism with antifungal agents.

Aim: This study investigated the effect of terpinen-4-ol against Sporothrix schenckii complex and its interactions with antifungals. Materials & methods: The antifungal activity of terpinen-4-ol was evaluated by broth microdilution. The potential effect on cellular ergosterol concentration was evaluated by spectrophotometry. The antibiofilm activity was evaluated by violet crystal staining and XTT reduction assay. The potential pharmacological interactions with antifungals were evaluated by the checkerboard assay. Results: terpinen-4-ol (T-OH) showed minimal inhibitory concentrations ranging from 4 to 32 mg/l decreasing cellular ergosterol content and presented a SMIC ranging from 64 to 1024 mg/l for Sporothrix spp. The combinations of T-OH with itraconazole or terbinafine were synergistic. Conclusion: T-OH has antifungal activity against Sporothrix spp. and acts synergistically with standard antifungals.

Tacrolimus Increases the Effectiveness of Itraconazole and Fluconazole against Sporothrix spp.

Calcineurin inhibitors - such as the clinically used drug tacrolimus - are active against important fungal pathogens, particularly when combined with azoles. However, tacrolimus has not been tested against sporotrichosis, an endemic subcutaneous mycosis with worldwide distribution. Here, we evaluated the activity of tacrolimus and cyclosporine A in vitro - as monotherapy and in combination with itraconazole or fluconazole - against yeasts of Sporothrix brasiliensis and S. schenckii, the main sporotrichosis agents in Brazil. We also analyzed the effect of tacrolimus treatment on intracellular neutral lipid levels, which typically increase after azole treatment. Tacrolimus inhibited the growth of yeasts from S. brasiliensis and S. schenckii reference isolates, with minimum inhibitory concentration (MIC) values (required for ≥50% growth inhibition) of 1 and 2 mg/L, respectively. Importantly, the combination of tacrolimus and azoles exhibited high synergy toward reference Sporothrix isolates. Tacrolimus combined with itraconazole significantly increased neutral lipid accumulation in S. brasiliensis, but not in S. schenckii. Clinical isolates of S. brasiliensis and S. schenckii were more sensitive to tacrolimus as monotherapy than feline-borne isolates, however, synergy between tacrolimus and azoles was only observed for feline-borne isolates. Cyclosporine A was effective against S. brasiliensis and S. schenckii as monotherapy (MIC = 1 mg/L), but exhibited no synergy with itraconazole and fluconazole. We conclude that tacrolimus has promising antifungal activity against sporotrichosis agents, and also increases the activity of the current anti-sporotrichosis therapy (itraconazole and fluconazole) in combination assays against S. brasiliensis feline-borne isolates.

Paracoccidioidomycosis infection in domestic and wild mammals by Paracoccidioides lutzii.

Paracoccidioidomycosis (PCM) is a systemic mycosis that occurs in several Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus Paracoccidioides spp. Serological studies to detect animal infection represent an excellent strategy for data on the agent's ecology. Although the state of Rio Grande do Sul (RS) is an endemic area for PCM in humans, there is scarce information available on the ecology of the agent in the region. This study aimed to investigate the infection by Paracoccidioides lutzii in animals living in RS, Brazil. A total of 85 wild mammals, 200 horses and 196 domestic dogs, previously tested for infection by P. brasiliensis, were included in this study. Serum samples from the animals were tested by ELISA to detect anti- P. lutzii antibodies. From the 481 animals tested, 105 (21.8%) were seropositive for IgG anti-P. lutzii. Of these, 54 were also positive for P. brasiliensis. A total of 11 horses (10.5%), 30 dogs (28.8%) and 10 wild mammals (9.5%) were positive only for P. lutzii (n=51). The detection of anti-P. lutzii antibodies in animals of RS suggests that the fungus can be found in southern Brazil, despite being described mainly in the midwest and southeast of the country.

Δ(24)-Sterol Methyltransferase Plays an Important Role in the Growth and Development of Sporothrix schenckii and Sporothrix brasiliensis.

Inhibition of Δ(24)-sterol methyltransferase (24-SMT) in Sporothrix schenckii sensu stricto and Sporothrix brasiliensis was investigated in vitro. The effects on fungal growth and sterol composition of the 24-SMT inhibitor 22-hydrazone-imidazolin-2-yl-chol-5-ene-3ß-ol (H3) were compared to those of itraconazole. MIC and MFC analysis showed that H3 was more effective than itraconazole against both species in both their filamentous and yeast forms. H3 showed fungistatic activity in a time-kill assay, with inhibitory activity stronger than that of itraconazole. GC analysis of cell sterol composition showed that sterols present in control cells (ergosterol and precursors) were completely replaced by 14α-methylated sterols after H3 exposure. Itraconazole only partially inhibited ergosterol synthesis but completely arrested synthesis of other sterols found in control cells, promoting accumulation of nine 14α-methyl sterols. Based on these results, we propose a schematic model of sterol biosynthesis pathways in S. schenckii and S. brasiliensis. Effects on cell morphology due to 24-SMT inhibition by H3 as analyzed by SEM and TEM included irregular cell shape, reduced cytoplasmic electron-density, and reduced thickness of the microfibrillar cell wall layer. Moreover, 24-SMT inhibition by H3 promoted mitochondrial disturbance, as demonstrated by alterations in MitoTracker(®) Red CMXRos fluorescence intensity evaluated by flow cytometry. When used in conjunction with itraconazole, H3 enhanced the effectiveness of itraconazole against all tested strains, reducing at least half (or more) the MIC values of itraconazole. In addition, cytotoxicity assays revealed that H3 was more selective toward these fungi than was itraconazole. Thus, 24-SMT inhibition by H3 was an effective antifungal strategy against S. schenckii and S. brasiliensis. Inhibition of the methylation reaction catalyzed by 24-SMT has a strong antiproliferative effect via disruption of ergosterol homeostasis, suggesting that this enzyme is a promising target for novel antifungal therapies against sporotrichosis, either as sole treatments or in combination with itraconazole.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.

Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification.

Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and guiding antifungal therapy. In areas of limited resources where sporotrichosis is endemic, high-throughput detection methods that are specific and sensitive are preferred over phenotypic methods that usually result in misidentification of closely related Sporothrix species. We sought to establish rolling circle amplification (RCA) as a low-cost screening tool for species-specific identification of human-pathogenic Sporothrix. We developed six species-specific padlock probes targeting polymorphisms in the gene encoding calmodulin. BLAST-searches revealed candidate probes that were conserved intraspecifically; no significant homology with sequences from humans, mice, plants or microorganisms outside members of Sporothrix were found. The accuracy of our RCA-based assay was demonstrated through the specificity of probe-template binding to 25 S. brasiliensis, 58 S. schenckii, 5 S. globosa, 1 S. luriei, 4 S. mexicana, and 3 S. pallida samples. No cross reactivity between closely related species was evident in vitro, and padlock probes yielded 100% specificity and sensitivity down to 3 × 10(6) copies of the target sequence. RCA-based speciation matched identifications via phylogenetic analysis of the gene encoding calmodulin and the rDNA operon (kappa 1.0; 95% confidence interval 1.0-1.0), supporting its use as a reliable alternative to DNA sequencing. This method is a powerful tool for rapid identification and specific detection of medically relevant Sporothrix, and due to its robustness has potential for ecological studies.

Human sporotrichosis beyond the epidemic front reveals classical transmission types in Espírito Santo, Brazil.

Sporotrichosis has emerged as the main subcutaneous mycosis of humans and animals around the world. With particular differences in frequency, the major species includes Sporothrix brasiliensis, S. schenckii, S. globosa and S. luriei. In Brazil, the main aspect of this epidemic is based on the zoonotic transmission through the scratches and bites of diseased cats contaminated with S. brasiliensis. Areas free of feline sporotrichosis are poorly characterised in Brazil. We investigated by molecular tools the epidemiology of human sporotrichosis in the Espírito Santo (ES) state, an area adjacent to Rio de Janeiro where is the epicentre of the long-lasting outbreak of cat-transmitted sporotrichosis. The human cases in the ES state reveal the prevalence of classical transmission types where subjects are mainly infected by accidental traumatic inoculation during manipulation of contaminated plant material. In agreement with an environmental source, Sporothrix schenckii was the major aetiological agent in the classical transmission. Unlike Rio de Janeiro, this study shows that cat-transmitted epidemic in Espírito Santo is still scanty, although the geographic proximity and similar climatic features. Sporothrix brasiliensis was the agent in the feline-transmitted cases. Sporothrix globosa was isolated from a patient with fixed cutaneous lesions that did not report any contact with diseased animals. In conclusion, beyond the borders of Rio de Janeiro epidemic, agents of sporotrichosis in Espírito Santo show a scattered occurrence with high species diversity.

Low-level laser therapy to the mouse femur enhances the fungicidal response of neutrophils against Paracoccidioides brasiliensis.

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

Chromosomal polymorphism in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (ß-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil.

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceará, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceará, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1b and 1c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1-5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceará have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglobulin G2 antibodies directed against carbohydrate epitopes.

Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM). The ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results. The aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos). We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting. Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P. brasiliensis antigen used in the ID test. IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgG1, and high levels of IgG2 compared with IDpos sera. The antibodies present in IDneg sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity. These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.

Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis.

Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus.

Molecular identification of Paracoccidioides brasiliensis by 5' nuclease assay.

A PCR assay based on the 5' nuclease assay using a fluorescent probe derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis. The assay could detect at least 10 copies of this DNA sequence, providing efficient accuracy to be useful for diagnosis of paracoccidioidomycosis.