SummaryVesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans, raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a transcell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans, Candida parapsilosis, Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein-coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein-coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.

Endemic mycoses can be challenging to diagnose and accurate interpretation of laboratory data is important to ensure the most appropriate treatment for the patients. Although the definitive diagnosis of histoplasmosis (HP), one of the most frequent endemic mycoses in the world, is achieved by direct diagnosis performed by micro and/or macroscopic observation of Histoplasma capsulatum (H. capsulatum), serologic evidence of this fungal infection is important since the isolation of the etiologic agents is time-consuming and insensitive. A variety of immunoassays have been used to detect specific antibodies to H. capsulatum. The most applied technique for antibody detection is immunodiffusion with sensitivity between 70 to 100 % and specificity of 100%, depending on the clinical form. The complement fixation (CF) test, a methodology extensively used on the past, is less specific (60 to 90%). Detecting fungal antigens by immunoassays is valuable in immunocompromised individuals where such assays achieve positive predictive values of 96-98%. Most current tests in diagnostic laboratories still utilize unpurified antigenic complexes from either whole fungal cells or their culture filtrates. Emphasis has shifted, however, to clinical immunoassays using highly purified and well-characterized antigens including recombinant antigens. In this paper, we review the current conventional diagnostic tools, such as complement fixation and immunodiffusion, outline the development of novel diagnostic reagents and methods, and discuss their relative merits and disadvantages to the immunodiagnostic of this mycosis.

BackgroundThere have been several recent changes in the taxonomy of Sporothrix schenckii as well as new observations regarding the clinical aspects of sporotrichosis. In this study, we determined the identification of the Sporothrix species associated with both classic and unusual clinical aspects of sporotrichosis observed in the endemic area of sporotrichosis in Rio de Janeiro, Brazil.Methodology/Principal FindingsTo verify whether S. brasiliensis is associated with clinical manifestations of sporotrichosis, a cross-sectional study was performed in which Sporothrix isolates from 50 patients with different clinical manifestations were analyzed and their isolates were studied by phenotypic and genotypic methods. Data from these patients revealed a distinct clinical picture and therapeutic response in infections caused by Sporothrix brasiliensis (n = 45) compared to patients with S. schenckii sensu stricto (n = 5). S. brasiliensis was associated with disseminated cutaneous infection without underlying disease, hypersensitivity reactions, and mucosal infection, whereas patients with S. schenckii presented with less severe and more often localized disease, similar to the majority of previously described sporotrichosis cases. Interestingly, S. brasiliensis-infected patients overall required shorter durations of itraconazole (median 16 weeks) compared to the individuals with S. schenckii (median 24 weeks).Conclusions/SignificanceThese findings suggest that Sporothrix species are linked to different clinical manifestations of sporotrichosis and that S. brasiliensis is effectively treated with oral itraconazole.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

BackgroundHistoplasma capsulatum comprises a worldwide complex of saprobiotic fungi mainly found in nitrogen/phosphate (often bird guano) enriched soils. The microconidia of Histoplasma species may be inhaled by mammalian hosts, and is followed by a rapid conversion to yeast that can persist in host tissues causing histoplasmosis, a deep pulmonary/systemic mycosis. Histoplasma capsulatum sensu lato is a complex of at least eight clades geographically distributed as follows: Australia, Netherlands, Eurasia, North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B) and Africa. With the exception of the Eurasian cluster, those clades are considered phylogenetic species.Methodology/Principal FindingsIncreased Histoplasma sampling (n = 234) resulted in the revision of the phylogenetic distribution and population structure using 1,563 aligned nucleotides from four protein-coding regions. The LAm B clade appears to be divided into at least two highly supported clades, which are geographically restricted to either Colombia/Argentina or Brazil respectively. Moreover, a complex population genetic structure was identified within LAm A clade supporting multiple monophylogenetic species, which could be driven by rapid host or environmental adaptation (~0.5 MYA). We found two divergent clades, which include Latin American isolates (newly named as LAm A1 and LAm A2), harboring a cryptic cluster in association with bats.Conclusions/SignificanceAt least six new phylogenetic species are proposed in the Histoplasma species complex supported by different phylogenetic and population genetics methods, comprising LAm A1, LAm A2, LAm B1, LAm B2, RJ and BAC-1 phylogenetic species. The genetic isolation of Histoplasma could be a result of differential dispersion potential of naturally infected bats and other mammals. In addition, the present study guides isolate selection for future population genomics and genome wide association studies in this important pathogen complex.

Heat shock proteins with molecular masses of ∼60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungus Histoplasma capsulatum displays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) to H. capsulatum Hsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected with H. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modify H. capsulatum pathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Sporotrichosis has significantly increased in Brazil in the last decade, particularly in the state of Rio de Janeiro, with the occurrence of an epidemic related to zoonotic transmission from cats to humans. Recently, four new phylogenetic species were incorporated into the Sporothrix species complex based on the phenotypic and molecular characteristics, and a new species name (Sporothrix brasiliensis) was proposed for some of the Sporothrix isolates from this epidemic. This study describes the characterization of 246 isolates obtained from patients attending the Laboratory of Infectious Dermatology, IPEC-FIOCRUZ, between 1998 and 2008, together with one environmental sample. Two hundred and six of the isolates (83.4%) were characterized as S. brasiliensis, 15 (6.0%) as S. schenckii, and one (0.5%) as S. mexicana. Twenty-five isolates (10.1%) could not be identified according to their phenotype and were classified as Sporothrix spp. The calmodulin gene was sequenced to confirm the identity of these isolates. The molecular analysis demonstrated that 24 of the isolates were S. brasiliensis, with the remainder being a S. globosa isolate. The isolate characterized phenotypically as S. mexicana was clustered on the S. schenckii clade. The correlation between molecular data and phenotypic characteristics described in this study is fundamental to the identification of the Sporothrix complex.