Promising application of the SsCBF ELISA test to monitor the therapeutic response of feline sporotrichosis caused by Sporothrix brasiliensis from Brazilian epidemics.

Sporotrichosis zoonotic transmission by cats has obtained hyperendemic magnitude in Rio de Janeiro, Brazil. Atypical cases, relapses, and reinfections as well as reduced diagnostic sensitivity of conventional methods have been reported. Previously, the anti-SsCBF enzyme-linked immunosorbent assay (ELISA) test was shown to be useful as a diagnostic tool for human sporotrichosis. Effective diagnosis and treatment are critical to interrupt the chain of transmission of this major pathogen in Brazilian Public Health. To evaluate its applicability for feline sporotrichosis diagnosis and/or therapeutic follow-up, 15 domestic cats from Rio de Janeiro were clinically and laboratory monitored by cytopathology, culture, Sporothrix genotyping, and anti-SsCBF IgG levels. Subsequently, animals were divided into satisfactory and non-satisfactory therapeutic responders. Averages of antibody serum levels obtained for diagnosis (first consultation) compared with the levels found after follow-up (last consultation) were significantly different in both groups (p = 0.0002 and p = 0.038, respectively). We conclude that the SsCBF ELISA test can predict feline sporotrichosis therapeutic responses even for animals with distinct clinical evolutions.

Coinfection of domestic felines by distinct Sporothrix brasiliensis in the Brazilian sporotrichosis hyperendemic area.

Microbial interactions may impact patient's diagnosis, prognosis and treatment. Sporotrichosis is a hyperendemic neglected zoonosis in Brazil, caused by Sporothrix brasiliensis. Four pairs of clinical isolates of Sporothrix were recovered from four diseased cats (CIM01-CIM04, two isolates per animal) raising the possibility of coinfection in a sporotrichosis hyperendemic area, Brazil. Each isolate of the pair had distinct pigmentation in mycological culture, and was designated as "Light" or "Dark", for low and high pigmentation, respectively. Dark isolates reacted strongly with monoclonal antibodies to melanin (p ≤ 0.05) by both ELISA and FACS quantitation, and displayed a ring pattern with some regions exhibiting higher punctuated labeling at cell wall by immunofluorescence. In turn, Light isolates reacted less intensely, with few and discrete punctuated labeling at the cell wall. PCR identified all isolates as S. brasiliensis, MAT1-2 idiomorph. Sequencing of ß-tubulin and calmodulin genes followed by phylogenetic analysis placed all eight isolates within the same cluster as others from the Brazilian hyperendemic area. The ability of these strains to stimulate cytokine production by human PBMCs (Peripheral blood mononuclear cells) was also analyzed. CIM01 and CIM03 Light and Dark isolates showed similar cytokine profiles to the control strain, while CIM02 and CIM04 behaved differently (p < 0.001), suggesting that differences in the surface of the isolates can influence host-fungus interaction. MICs for amphotericin B, terbinafine, caspofungin, micafungin, itraconazole, fluconazole, and voriconazole were obtained (CLSI M38-A2/M27-A3). Pairwise comparisons showed distinct MICs between Sporothrix Light and Dark isolates, higher than at least two-fold dilutions, to at least one of the antifungals tested. Isolates from the same pair displayed discrepancies in relation to fungistatic or fungicidal drug activity, notably after itraconazole exposure. Since S. brasiliensis Light and Dark isolates show disparate phenotypic parameters it is quite possible that coinfection represents a common occurrence in the hyperendemic area, with potential clinical implications on feline sporotrichosis dynamics. Alternatively, future studies will address if this specie may have, as reported for other fungi, broad phenotypic plasticity.

Correction to: Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

In the article mentioned above an author's name was misspelled. The correct author name reads as follows: Leila Maria Lopes-Bezerra. We apologize for the inconvenience.

Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

Sporotrichosis is an infection of the skin caused by traumatic inoculation of the fungus Sporothrix schenckii. Definitive diagnosis relies on direct visualization of the fungus or its isolation on culture medium, although both have low sensitivity. Alternatively, the detection of the antibody response offers a more rapid alternative for diagnosis. Although the available immunoassays possess good sensitivity and specificity, cross-reactivity is still a problem. This study aimed to evaluate the effect of sodium metaperiodate and 6 M urea solutions on the serological diagnosis of sporotrichosis using an enzyme-linked immunosorbent assay (ELISA) test. Ninety-six-well plates were sensitized with exoantigens from the yeast phase of S. schenckii. Sera of patients with confirmed sporotrichosis, sera of patients with paracoccidioidomycosis, and sera of individuals with a sporotrichin-negative skin test were tested. Two strategies were used; the first consisted of treating the antigen with sodium metaperiodate solution for different incubation times, and the second consisted of treating the serum with 6 M urea solution for different incubation times. ROC curve analysis revealed that the best discrimination parameters were obtained using 6 M urea solution incubated for 5 min and serum dilution at 1/600. The use of 6 M urea solution improves the performance of the ELISA test in the diagnosis of sporotrichosis.

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

BACKGROUND: Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans. METHODOLOGY: We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20). PRINCIPAL FINDINGS: Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis. CONCLUSIONS: A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

Peroxide sensing and signaling in the Sporothrix schenckii complex: an in silico analysis to uncover putative mechanisms regulating the Hog1 and AP-1 like signaling pathways.

In order to understand how fungal pathogens can survive inside the host, we must analyze how they evade the fungicidal mechanisms mounted by the host's immune system, such as generation of toxic reactive oxygen species. Studies have shown that infections caused by Sporothrix brasiliensis can be more aggressive than those due to Sporothrix schenckii. Therefore, we propose to analyze and compare the ability of these two pathogenic species to counteract oxidative stress, which, as noted, can be relevant in the host response to infection. We have shown that S. brasiliensis is more resistant to different oxidants, such as H2O2 and menadione, when compared with S. schenckii. Furthermore, our results suggest that the molecular mechanisms by which Sporothrix spp. AP-1 like transcription factors are regulated probably differs from the one seen in other fungal pathogens. Interestingly, comparison between sequences of SbHog1 and SsHog1 stress activated protein kinases suggest that S. brasiliensis Hog1 display mutations that could account for the differences seen in stress sensitivities of these two species. In summary, this is the first study to our knowledge to investigate oxidative stress responses of Sporothrix spp. and provided a model that can be employed in vivo to address how these fungal pathogens can surmount the oxidative stress generated by the host.

Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine.

The in vitro activity of the antifungal agents amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), and terbinafine (TRB) against 32 Brazilian isolates of Sporothrix brasiliensis, including 16 isolates from a recent (2011-2012) epidemic in Rio de Janeiro state, was examined. We describe and genotype new isolates and clustered them with 16 older (from 2004 or earlier) S. brasiliensis isolates by phylogenetic analysis. We tested both the yeast and the mycelium form of all isolates using broth microdilution methods based on the reference protocols M38-A2 and M27-A3 (recommended by the Clinical and Laboratory Standards Institute). Considering minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), TRB was found to be the most active drug in vitro for both fungal forms, followed by PSC. Several isolates showed high MICs for AMB and/or ITC, which are currently used as first-line therapy for sporotrichosis. VRC displayed very low activity against S. brasiliensis isolates. The primary morphological modification observed on treated yeasts by transmission electron microscopy analysis was changes in cell wall. Our results indicate that TRB is the antifungal with the best in vitro activity against S. brasiliensis and support the use of TRB as a promising option for the treatment of cutaneous and/or lymphocutaneous sporotrichosis.

Asexual propagation of a virulent clone complex in a human and feline outbreak of sporotrichosis.

Sporotrichosis is one of the most frequent subcutaneous fungal infections in humans and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrix species found in Brazil have been considered asexual as no sexual stage has ever been reported in Sporothrix schenckii, Sporothrix brasiliensis, or Sporothrix globosa. We have identified the mating type (MAT) loci in the S. schenckii (strain 1099-18/ATCC MYA-4821) and S. brasiliensis (strain 5110/ATCC MYA-4823) genomes by using comparative genomic approaches to determine the mating type ratio in these pathogen populations. Our analysis revealed the presence of a MAT1-1 locus in S. schenckii while a MAT1-2 locus was found in S. brasiliensis representing genomic synteny to other Sordariomycetes. Furthermore, the components of the mitogen-activated protein kinase (MAPK)-pheromone pathway, pheromone processing enzymes, and meiotic regulators have also been identified in the two pathogens, suggesting the potential for sexual reproduction. The ratio of MAT1-1 to MAT1-2 was not significantly different from 1:1 for all three Sporothrix species, but the population of S. brasiliensis in the outbreaks originated from a single mating type. We also explored the population genetic structure of these pathogens using sequence data of two loci to improve our knowledge of the pattern of geographic distribution, genetic variation, and virulence phenotypes. Population genetics data showed significant population differentiation and clonality with a low level of haplotype diversity in S. brasiliensis isolates from different regions of sporotrichosis outbreaks in Brazil. In contrast, S. schenckii isolates demonstrated a high degree of genetic variability without significant geographic differentiation, indicating the presence of recombination. This study demonstrated that two species causing the same disease have contrasting reproductive strategies and genetic variability patterns.

PCR as a screening test for invasive aspergillosis in haematological patients: a pilot study.

Invasive aspergillosis is a leading cause of morbidity and mortality in immunocompromised patients, particularly in individuals with haematological malignancy and in haematopoietic stem cell transplant recipients. Nowadays, the galactomannan (GM) assay has been widely used as an indication of invasive aspergillosis, even though the test is known to generate false-positive results. The aim of this study was to compare the performance of GM and real-time PCR (qPCR) to detected Aspergillus in blood samples obtained from high-risk haematological patients. Haematological patients were screened twice weekly with GM testing, which was performed by the Platelia ELISA kit. An additional sample of whole blood (4 ml) was obtained for the purpose of qPCR testing. Sixty-four samples from 12 patients with haematopoietic stem cell transplant or haematological malignancy were studied. The overall accordance between GM and qPCR tests was 96.9 % (62 samples). Only two samples showed contradictory results, with positive GM test and negative real-time PCR results. Based on the high concordance between GM and qPCR in terms of negative results, the main utility of qPCR could be in the confirmation of positive results seen with GM testing.

Pulmonary cavitation and skin lesions mimicking tuberculosis in a HIV negative patient caused by Sporothrix brasiliensis.

A 32-year-old HIV negative male presented with multiple pulmonary cavitation and skin abscesses up to 15 cm in diameter mimicking tuberculosis. Sporothrix brasiliensis was isolated and patient responded well to amphotericin B followed by itraconazole, except the skin lesions that had to be surgical drained to obtain cure.

Sporotrichosis in pregnancy: case reports of 5 patients in a zoonotic epidemic in Rio de Janeiro, Brazil.

Five cases of sporotrichosis occurring in pregnant women in a zoonotic epidemic in Rio de Janeiro, Brazil, are described. The main clinical features, as well as the challenging therapeutic choices for this specific group of patients, are discussed.

Esporotricose na gestação: relato de cinco casos numa epidemia zoonótica no Rio de Janeiro, Brasil / Sporotrichosis in pregnancy: case reports of 5 patients in a zoonotic epidemic in Rio de Janeiro, Brazil

Os autores apresentam cinco casos de esporotricose em gestantes numa epidemia zoonótica no Rio de Janeiro. São discutidos principalmente os aspectos clínicos e as dificuldades na escolha terapêutica desse grupo específico de pacientes.

Five cases of sporotrichosis occurring in pregnant women in a zoonotic epidemic in Rio de Janeiro, Brazil, are described. The main clinical features, as well as the challenging therapeutic choices for this specific group of patients, are discussed.

Serodiagnosis of sporotrichosis infection in cats by enzyme-linked immunosorbent assay using a specific antigen, SsCBF, and crude exoantigens.

The main objective of this study is to standardize an ELISA for the diagnosis of feline sporotrichosis. Sporothrix schenckii is the etiological agent of human and animal sporotrichosis. Cats may act as reservoirs for S. schenckii and can transmit the infection to humans by a bite or scratch. There are few methods for the serological diagnosis of fungal diseases in animals. In this paper, an ELISA test for the diagnosis of cat sporotrichosis is proposed, which detects S. schenckii-specific antibodies in feline sera. Two different kinds of antigens were used: "SsCBF", a specific molecule from S. schenckii that consists of a Con A-binding fraction derived from a peptido-rhamnomannan component of the cell wall, and a S. schenckii crude exoantigen preparation. The ELISA was developed, optimized, and evaluated using sera from 30 cats with proven sporotrichosis (by culture isolation); 22 sera from healthy feral cats from a zoonosis center were used as negative controls. SsCBF showed 90% sensitivity and 96% specificity in ELISA; while crude exoantigens demonstrated 96% sensitivity and 98% specificity. The ELISA assay described here would be a valuable screening tool for the detection of specific S. schenckii antibodies in cats with sporotrichosis. The assay is inexpensive, quick to perform, easy to interpret, and permits the diagnosis of feline sporotrichosis.

The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules.

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients. Interactions with endothelium are thought to be essential for systemic infections. In the present work, we studied the interaction between S. schenckii and human umbilical vein endothelial cells (HUVECs). S. schenckii interacts with HUVECs in a time-dependent manner. Morphological analysis showed that yeasts locate to interendothelial junctions. Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection. The viability of infected HUVECs was confirmed by the MTT assay. When HUVECs were treated with different concentrations of Interleukin-1beta or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed. The preliminary analysis of the endothelial surface ligands for S. schenckii cells revealed two major molecules, with Mr of approximately 90 and 135 kDa. The interaction of endothelial cell surface molecules with S. schenckii yeast cells was modulated by divalent cations. This is the first demonstration that S. schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity. Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process.

Humoral immune response in aspergillosis: an immunodominant glycoprotein of 35 kDa from Aspergillus flavus.

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity.